Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 February 2015 to 09 April 2015
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 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)
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals / tissue source

Species:
chicken
Strain:
other: Ross 308
Details on test animals or tissues and environmental conditions:
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.

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, 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.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. 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.

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 isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. 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, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 µL/eye
Duration of treatment / exposure:
10 seconds and then rinsed with physiological saline
Duration of post- treatment incubation (in vitro):
30, 75, 120, 180 and 240 minutes after post-treatment rinse.
Number of animals or in vitro replicates:
3 eyes for test material and positive control
1 eye for negative control
Details on study design:
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within approximately 45 minutes before the start of application. Slight changes in thickness (-1.6% to 0.0%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test item was applied onto the centre of the cornea. The test item was applied in a volume of 30 μL onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 μL benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 μL of physiological saline (Salsol solution 0.9 %). One eye was treated with physiological saline, three eyes with the test item and another three with benzalkonium chloride solution 5 % (w/v).

The time of application was observed, then after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum.

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. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 minutes
Value:
0.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class I
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 minutes
Value:
0.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class I
Irritation parameter:
cornea opacity score
Value:
0.67
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class II
Irritation parameter:
fluorescein retention score
Value:
2.17
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class III

Any other information on results incl. tables

Treatment

Replicate

Corneal thickness (instrumental units)/Corneal swelling (%)

Corneal opacity score

Fluorescein retention

Observation time (min)

Observation time (min)

Maximum change in opacity

Observation time (min)

Change in fluorescein retention

0

30

75

120

180

240

0

30

75

120

180

240

0

30

Physiological saline

A

60

60/0.0

60/0.0

60/0.0

60/0.0

60/0.0

0

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.0

DMP tech

A

64

64/0.0

64/0.0

64/0.0

64/0.0

63/‑1.6

0

0.5

0.5

1.0

1.0

1.0

1.0

0

2

2.0

B

61

62/1.6

62/1.6

62/1.6

62/1.6

62/1.6

0

0.5

0.5

0.5

0.5

0.5

0.5

0

2

2.0

C

61

61/0.0

61/0.0

60/‑1.6

60/1.6

60/‑1.6

0

0.5

0

0.5

0.5

0.5

0.5

0.5

3

2.5

Mean

 

-/0.5

-/0.5

-/0.0

-/0.0

-/0.5

 

0.67

 

2.17

Benzalkonium chloride

A

64

67/4.7

69/7.8

72/12.5

76/18.8

78/21.9

0

4

4

4

4

4

4.0

0

3

3.0

B

64

67/4.7

70/9.4

72/12.5

77/20.3

79/23.4

0

4

4

4

4

4

4.0

0.5

3

2.5

C

62

64/3.2

66/6.5

68/9.7

72/16.1

76/22.6

0

3

4

4

4

4

4.0

0

3

3.0

Mean

 

-/4.2

-7.9

-/11.6

-/18.4

-/22.6

 

4.0

 

2.83

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The test material treated eyes showed no corneal swelling, a mean maximum change in opactiy score of 0.67 and a mean change in fluorescein of 2.17. The test item could not be classified as either a severe irritant or as a non-irritant on the basis of these data.
Executive summary:

An ex vivo eye irritation study has been conducted in isolated chicken's eyes with the submission substance in accordance with OCED guideline 438. Zero reference values were taken for corneal thickness, corneal opacity and fluorescein retention for each eye before 10 µL of test material, physiological saline (negative control) or benzalkonium chloride (positive control) was added. The test material and positive control were tested in triplicate. The negative control was tested in a single eye. After a 10 second exposure, the eyes were rinsed with saline. Corneal swelling, corneal opacity change and fluorescein retention change was noted for all eyes. The test material treated eyes showed no corneal swelling, a mean maximum change in opactiy score of 0.67 and a mean change in fluorescein of 2.17. The test item could not be classified as either a severe irritant or as a non-irritant.