Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with minor deviations to the OECD 437 (adopted July, 26th 2013) without effects on results: - IVIS cut-off values used for identifying test chemical were different, but no influence to the classification of the test item. - information on corneal diameter and time interval prior to initiating testing with the eyes were missing

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:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2009-09-07
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
, 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: solid, whitish to white crystals
- Storage condition of test material: ambient temperature, dark, dry, in original container

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
other: 0.9% (w/v) NaCl in deionised water
Controls:
other: Control (saline) bovine corneae were used.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 10 % (w/v) suspension in vehicle

Duration of treatment / exposure:
10 minutes (± 30 seconds)
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
COLLECTION OF BOVINE EYES
- isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir and were transported in Hank's buffered salt solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature.

PREPARATION OF CORNEAE
- all eyes were carefully examined macroscopically for defects.
- the cornea was carefully removed from the eye.
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with complete medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1 °C in a water-bath.
- at the end of the incubation period, the basal opacity was determined (t0) of all cornea.
- each corneae with a value of the basal opacity > 7 was discarded.

EXPOSURE OF THE TEST GROUPS TO THE CORNEAE
- the anterior compartment received the test item or negative control or positive control (2-Ethoxyethanol; tested neat) at a volume of 0.75 mL on the surface of the corneae.
- the corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
- the test item, positive control and negative control were tested in triplicate.
- the test item or control items were rinsed off with cMEM (complete medium).
- the corneae were then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).
- permeability of the corneae was determined.

PERMEABILITY MEASUREMENT
- after the final opacity measurement, the complete medium was removed from the anterior compartment and replaced by 0.5 % (w/v) fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm was determined with a spectrophotometer.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score (IVIS) of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the individual IVIS values.


CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if:
- in vitro irritation score of the positive control was ≥ 30
- in vitro irritation score of the negative control was ≤ 3

Results and discussion

Results of in vivo study
Irritation parameter:
other: in vitro irritancy score
Basis:
mean
Time point:
other: 10 minutes
Score:
0.55
Irritant / corrosive response data:
Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability.

Any other information on results incl. tables

Table 1: Results after 10 minutes incubation time

Test group

Opacity value = Difference (t130 – t0) of opacity

Permeability at 490 nm (OD490)

In vitro score

Mean in vitro irritation score

 

 

Mean

 

Mean

 

 

Negative control

-1

 

-0.67

0.064

 

0.058

-0.04

 

0.21

-1

0.055

-0.18

0

0.056

0.84

Positive control

63.67*

0.319*

68.45

 

76.72

62.67*

1.071*

78.73

60.67*

1.489*

83.00

Test item

0.67*

-0.003*

0.62

 

0.55

-0.33*

-0.012*

-0.52

1.67*

-0.008*

1.54

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.21).

- The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS =76.72).

Table 2: Historical data

 

Positive control

Negative control

Mean in vitro irritation score

78.94

1.09

Standard deviation

22.76

0.97

Values of 104 studies with liquid test items performed from 2007 until 2011

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Relative to the negative control, the substance did not cause any increase of the corneal opacity or permeability. Thus, the substance is not irritating to the eye (IVIS = 0.55) under the experimental conditions. A substance with an in vitro irritation score ≤ 3 does not require classification for eye irritation and serious eye damage according to OECD 437 (2013). According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not irritating to the eye.
Executive summary:

The eye irritation potential was examined according to the OECD guideline 437 (2009) . Three fresh bovine corneae were exposed to the substance for 10 minutes, rinsed and incubated for another 120 minutes. Corneal opacity and permeability were measured and the irritancy score was calculated. Relative to the negative control, the substance did not cause any increase of the corneal opacity or permeability. The mean irritance score amounts to 0.55. According to OECD 437 (2013), a substance with an in vitro irritance score ≤ 3 does not require classification for eye irritation and serious eye damage. According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not irritating to the eye.