Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February - 5 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance an OECD guideline by a GLP accredited laboratory.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD guideline 438 (7 September 2009)
Qualifier:
according to
Guideline:
other: EU Method B.48 (ICE test 8 December 2010)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tropicate
- Substance type: pure active substance
- Physical state: liquid

Test animals / tissue source

Species:
other: Spring chicken
Details on test animals or tissues and environmental conditions:
The eyes collected from chickens obtained from a slaughterhouse. Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with isotonic saline.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
Amount / concentration applied:
30 µl of pure test substance or control was put onto enucleated chicken eyes during 10s.
Duration of treatment / exposure:
The incubation period was 10±1 min at 32±1°C.
Observation period (in vivo):
Corneal opacity, swelling and morphological effects were evaluated at 30, 75, 120, 180 and 240 minutes post-dosing. Fluorescein retention was determined at pretreatment and 30 minutes post-dosing.
Number of animals or in vitro replicates:
3 eyes per dose group (except negative control: 1 eye).
Details on study design:
The eyelids were excised and dissected from the skull. The eyeball was pulled from the orbit and placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The cornea of the enucleated eye was positioned vertically by a steel clamp and placed in a superfusion apparatus in such a way that the entire cornea was supplied with the isotonic saline drip.
The eyes were then examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was measured at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. Individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
The approved eyes were incubated between 60 and 76 minutes prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline. The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
Following the zero reference measurements, the eye was removed from the superfusion apparatus, placed in a horizontal position. 30 μL of the test substance was applied to the cornea for 10 seconds, such that the entire surface of the cornea is evenly covered with the test item. The test substance was applied and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye was subsequently returned to the superfusion apparatus in the original upright position for further measurements.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
ICE class swelling
Basis:
mean
Score:
2
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
corneal opacity
Basis:
mean
Remarks:
maximum
Score:
0.5
Max. score:
4
Irritation parameter:
other: ICE class corneal opacity
Basis:
mean
Remarks:
maximum
Score:
1
Max. score:
4
Irritation parameter:
other: Fluorescein retention
Basis:
mean
Score:
1
Max. score:
3
Irritation parameter:
cornea opacity score
Remarks:
ICE class fluorescein retention
Basis:
mean
Score:
1
Max. score:
4
Irritant / corrosive response data:
The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as non corrosive/severe irritant.
The combination of the three endpoints for the positive control, 10% acetic acid solution, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant.
The combination of the three endpoints for the test substance was 1 x I and 2 x II.

Any other information on results incl. tables

Assessment eye corrosion/severe irritation of an eye exposed to physiological saline (negative control), 10% acetic acid (positive control) and the test substance.

    time / min
End-point 0 30 75 120 180 240
Negative
control
Corneal opacity 0 0 0 0 0 0
Fluorescein retention 0.5 0.5        
Corneal thickness 0.51 0.50 0.50 0.53 0.50 0.50
Positive
control
Corneal opacity 0 2.7 3.0 3.0 3.0 3.0
Fluorescein retention 0.5 3.0        
Corneal thickness - 56 45 43 47 40
Test
substance
Corneal opacity 0 0.5 0.5 0.5 0.5 0.5
Fluorescein retention 0 1        
Corneal thickness - 8 6 9 9 6

ICE classification of the negative control, positive control and the test substance.

  End-point ICE
Negative
control
Corneal opacity I (=1)
Fluorescein retention I (=1)
Corneal thickness I (=1)
Positive
control
Corneal opacity IV (=4)
Fluorescein retention IV (=4)
Corneal thickness IV (=4)
Test
substance
Corneal opacity I (=1)
Fluorescein retention II (=2)
Corneal thickness II (=2)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance must not be classified as causing “irreversible effects on the eye”.
Executive summary:

The eye irritancy of Tropicate was assessed according to the Isolated Chicken Eye Test as described in OECD method 438. 30ml test substance was applied to 3 enucleated chicken eyes during 10s. The eyes were subsequently rinsed with 10ml of physiological saline. Three other eyes were treated with a positive control and one eye with a negative control. The damage by the test substance was 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 the eyes treated with the test substance were slightly to moderate. Based on the results of the test, the test substance does not need to be classified as causing “irreversible effects on the eye”.