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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Etablissement Brun, 33820 Etauliers, France
- Age/weight: That of spring chickens traditionally processed by a poultry alaughterhouse (i.e., approximately 7 weeks old, 1.5-2.5 kg).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads were collected on 26 April 2017 at 8:10 am and they were enucleated at 9:50 am.

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
- Concentration (if solution): Test item us such (sodium chlorite 9% water solution)
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyelids were carefully excised and further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut. The optic nerve was left attached. The nictitating membrance and the other connective tissue were finally cut away. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically and it was transferred to a chamber of the superfusion apparatus (32.0-32.4 ºC). The entire cornea was supplied with the physiological saline drip (0.1-0.15 mL/min). The eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was measured. Eyes with (i) a fluorescein retention scope of > 0.5, (ii) corneal opacy > 0.5, or (iii) any additional signs of damage were replaced. Moreover, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once the eyes were examined and approved, the eyes were incubated between 45 and 56 minutes to be equilibrated. Then, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e. time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Physiological saline (Dutscher Batch No. 3012316)

POSITIVE CONTROL USED: 5% Benzalkonium chloride (Sigma Batch No. BCBQ9761V)

APPLICATION DOSE AND EXPOSURE TIME : 30 µL and 10 seconds.

OBSERVATION PERIOD: 0, 30, 75, 120, 180 and 240 minutes after post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Eyes were rinsed with 20 mL of physiological saline at ambient temperature.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was not densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: Calculated for the 30 min observation time period only.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 9 1/2. equalling 0.095 mm.
- Macroscopic morphological damage to the surface: Including "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea.

SCORING SYSTEM:
- Mean corneal swelling (%) : [(Corneal thickness at time I - corneal thickness at time 0) / corneal thickness at time 0] x 100.
- Mean maximum opacity score: Based on the highest mean score at any time point, an overall category score was given:
0 - No opacity
0.5 - Very faint opacity
1 - Scattered or diffuse area; 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 visible
- 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 single cell staining
3 - Confluent large areas of the cornea retaining fluorescein.

DECISION CRITERIA: as indicated in the TG.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean 1-3
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean 1-3
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
percent corneal swelling
Run / experiment:
Maximal 1-3
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not stated.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (3 x I)
- Acceptance criteria met for positive control: Yes (3 x IV)

Any other information on results incl. tables

Endpoint

Eye no.

Time (min)

0

30

75

120

180

240

Corneal opacity

7

0

1

1

1

1

2

8

0

1

1

1

1

2

9

0

1

2

2

2

2

Mean

0.0

1.0

1.3

1.3

1.3

2.0

ICE class

III

Fluorescein retention

7

0.5

2

-

-

-

-

8

0.5

2

-

-

-

-

9

0.5

2

-

-

-

-

Mean

0.5

2.0

-

-

-

-

ICE class

III

Corneal thickness

7

0.59

0.64

0.64

0.65

0.65

0.66

8

0.60

0.60

0.60

0.60

0.60

0.62

9

0.64

0.64

0.64

0.64

0.64

0.64

Corneal swelling (%)

7

-

8

8

10

10

12

8

-

0

0

0

0

3

9

-

0

0

0

0

0

Mean

-

3

3

3

3

5

ICE class

I

Combination

2 x III, 1 x I

Classification

No prediction can be made

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
Conclusions:
Results obtained under the in-vitro Isolated Chicken eye test lead to the category "no prediction can be made" since the combination of the three endpoints for the test item was 2xIII, 1xI.
Executive summary:

The in-vitro Isolated Chicken Eye test was performed according to OECD Guideline 438 (GLP study). The test item was applied at the dose of 30 µL to 3 enucleated chicken eyes during 10 seconds and then, the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity and fluorescein retention at 30, 75, 120, 180 and 240 minutes after treatment. The ocular reactions observed in the eyes treated with the test item were: maximal mean score of corneal opacity of 2.0, corresponding to ICE class III; mean score of fluorescein retention of 2.0, corresponding to ICE class III and maximal mean corneal swelling of 5%, corresponding to ICE class I. The combination of the three endpoints was 2xIII, 1xI, which lead to the category "no prediction can be made" as defined by the OECD guideline. Bothe the positive and negative controls were acceptable.