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:
06 November 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:
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)
Version / remarks:
Adopted 26 July 2013
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state/Appearance: Colourless to pale yellow liquid, slightly viscous.
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable under normal temperatures and pressure

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 were supplied by XXX.
- Characteristics of donor animals (e.g. age, sex, weight): Spring chickens (Gallus Gallus e.g. Ross 308 Broiler) male or female, weighed approximately 3kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue: 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. All eyes fall within the acceptance criteria identified in the test guideline. The temperature of the chambers was at 32 ±1.5 °C.
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.

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): 30 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of 0.9% (w/v) sodium chloride solution (pre-warmed to approximately 32 °C).
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
- 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 ±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.
- 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: 2, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 5% Benzalkonium chloride

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 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. 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.

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.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1.
- 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.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. 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.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- 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
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
- Pitting, loosening (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.

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
Run / experiment:
1
Value:
>= 0.5 - <= 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
1
Value:
>= 3.74 - <= 13.08
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 2.0 (180-240 min.), corresponding to the ICE class III;
- mean score of fluorescein retention: 0.7 corresponding to the ICE class II;
- maximal mean corneal swelling: 13.08% (75 min.) corresponding to the ICE class III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

HISTORICAL CONTROL DATA:
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 7.3.2/5: 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

3A

0.5

0.5

1

2

2

2

6A

0.5

0.5

2

2

2

2

8A

0.5

0.5

0.5

1

2

2

Mean

0.5

0.5

1.2

1.7

2.0

2.0

ICE Class

III

Fluorescein Retention

3A

 

1

 

 

 

 

6A

 

0.5

 

 

 

 

8A

 

0.5

 

 

 

 

Mean

 

0.7

 

 

 

 

ICE Class

II

Corneal Thickness

3A

0.72

0.70

0.84

0.77

0.78

0.76

6A

0.72

0.78

0.80

0.78

0.76

0.76

8A

0.70

0.76

0.78

0.84

0.74

0.70

Mean

0.71

0.75

0.81

0.80

0.76

0.74

Mean Corneal Swelling (%)

 

4.67

13.08

11.68

6.54

3.74

ICE Class

III

Corneal Epithelium Condition

3A

N

N

N

N

N

N

6A

N

N

N

N

N

N

8A

N

N

N

N

N

N

ICE Classes Combined:

1 x II, 2 x III

Classification:

No prediction can be made

Table 7.3.2/6: Individual scores and Mean scores for Corneal Effects - positive control

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

2A

0.5

3

3

4

4

4

5A

0.5

3

4

4

4

4

7A

0.5

4

4

4

4

4

Mean

0.5

3.3

3.7

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

2A

 

3

 

 

 

 

5A

 

3

 

 

 

 

7A

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

2A

0.68

0.80

0.88

0.84

0.80

0.82

5A

0.72

0.83

0.88

0.92

0.94

0.94

7A

0.72

0.90

0.90

0.97

0.90

0.92

Mean

0.71

0.84

0.89

0.91

0.88

0.89

Mean Corneal Swelling (%)

 

19.34

25.47

28.77

24.53

26.42

ICE Class

III

Corneal Epithelium Condition

2A

N

N

N

N

N

N

5A

N

S

S

S

S

S

7A

N

N

N

N

N

N

ICE Classes Combined:

2 x IV, 1 x III (sloughing in 1 eye)

Classification:

Category 1

Table 7.3.2/7: Individual scores and Mean score for Corneal effects - Negative controlthe test item

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

1A

0.5

0.5

0.5

0.5

0.5

0.5

4A

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.5

0.5

0.5

0.5

0.5

0.5

ICE Class

I

Fluorescein Retention

1A

 

0.5

 

 

 

 

4A

 

0.5

 

 

 

 

Mean

 

0.5

 

 

 

 

ICE Class

I

Corneal Thickness

1A

0.68

0.70

0.70

0.68

0.70

0.70

4A

0.72

0.71

0.74

0.72

0.70

0.72

Mean

0.70

0.71

0.72

0.70

0.70

0.71

Mean Corneal Swelling (%)

 

0.71

2.86

0.00

0.00

1.43

ICE Class

I

Corneal Epithelium Condition

1A

N

N

N

N

N

N

4A

N

N

N

N

N

N

ICE Classes Combined:

3 x I

Classification:

No Category

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions, no prediction can be made for the test substance according to the Annex VI of 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 ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item was applied, as supplied, at the dose of 30 µL, to 3 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: 2.0 (180-240 min.), corresponding to the ICE class III;

- mean score of fluorescein retention: 0.7 corresponding to the ICE class II;

- maximal mean corneal swelling: 13.08% (75 min.) corresponding to the ICE class III.

 

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, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions, no prediction can be made for the test substance according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for in vitro eye irritation endpoint.