Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
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)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state / Appearance: White solid

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day (4th September 2017).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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. All eyes were placed in the superfusion chamber by 9.36 am (4th September 2017). 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.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g of test item (undiluted) was applied to the cornea
- For the purpose of this study the test item was used as supplied and was handled under safety lighting.
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
- Acceptable eyes 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 32.0 ± 1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp 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 minutes 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.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
0.03 mL of the negative control item, Sodium chloride 0.9% w/v, was applied to the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye.

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish.
- 0.03 g of test item was applied to the cornea and the test item remained in place for 10 seconds.

OBSERVATION PERIOD
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

METHODS FOR MEASURED ENDPOINTS:
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

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.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris 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 is 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 minute time interval only. These measurements are used for the overall classification for each test item.
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:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
10 seconds exposure
Value:
37.09
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class IV
Other effects / acceptance of results:
OCULAR REACTIONS
Corneal Opacity Scores: Complete corneal opacity; iris invisible was noted in all positive control treated eyes and all test item treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in all positive control treated eyes.
Fluorescein Retention Scores: Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes and all test item treated eyes. Very minor single staining was noted in both negative control treated eyes.

ACCEPTANCE OF RESULTS:
Acceptance criteria met for negative control: Yes; Maximal mean score for corneal opacity: 0.5; Mean score of Fluorescein Retention: 0.5; Maximal mean corneal swelling compared to time zero: 1.37%
Acceptance criteria met for positive control: Yes; Maximal mean score for corneal opacity: 4.0; Mean score of Fluorescein Retention: 3.0; Maximal mean corneal swelling compared to time zero: 40.47%
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Any other information on results incl. tables

Table 7.3.2/3: Individual Scores and Mean Scores for Corneal Effects – Test Item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3B

0.5

4

4

4

4

4

6B

0.5

4

4

4

4

4

8B

0

4

4

4

4

4

Mean

0.3

4.0

4.0

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

3B

 

3

 

 

 

 

6B

 

3

 

 

 

 

8B

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

3B

0.70

1.00

1.08

1.08

1.06

1.00

6B

0.70

0.78

0.86

0.84

0.86

0.90

8B

0.73

0.94

0.98

0.92

0.84

1.02

Mean

0.71

0.91

0.97

0.95

0.92

0.97

Mean Corneal Swelling (%)

 

27.70

37.09

33.33

29.58

37.09

ICE Class

IV

Corneal Epithelium Condition

3B

N

N

N

N

N

N

6B

N

N

N

N

N

N

8B

N

N

N

N

N

N

ICE Classes Combined:

3 x IV

Classification:

Category 1

N= Normal

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 

The test item was applied, as supplied, at 0.03 g onto the cornea of each of three enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 4.0, corresponding to the ICE class IV;

- mean score of fluorescein retention: 3.0, corresponding to the ICE class IV;

- maximal mean corneal swelling: 37.09% corresponding to the ICE class IV.

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, Imidazole, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye damage endpoint.