Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11

Test material

Reference
Name:
Unnamed
Type:
Constituent

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:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
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 BSS 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 incubation medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for about 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 undiluted test item or negative control (saline) 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 (incubation time: 10 minutes).
- the test item, positive control and negatice control were tested in triplicate.
- after the test item or control items were rinsed off with saline.
- 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) sodium 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 (software SoftMax Pro Enterprise, version 4.7.1).

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae is 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 is 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 is 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 IVIS values.
Depending on the score obtained, the test item was classified into the followingc ategory according to OECD 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

CRITERIA FOR DETERMINATION OF A VALID TEST
The test is acceptable if the IVIS of the positive control is ≥ 30 and the IVIS of the negative control is ≤ 3.

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
in vitro irritation score
Value:
0.08
Vehicle controls valid:
not examined
Negative controls valid:
no
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.08.

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 irritancy score

Mean in vitro irritancy score

 

 

Mean

 

Mean

 

 

Negative control

-1

 

-0.33

0.049

 

0.048

-0.27

 

0.38

0

0.048

0.72

0

0.046

0.69

Positive control

57.33

0.811

69.50

 

63.65

53.33

0.561

61.75

51.33

0.557

59.69

Test item

0.33

0.012

0.52

 

0.08

-0.67

0.007

-0.56

0.33

-0.003

0.29

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.38).

- The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS =63.65).

Table 2: Historical data

 

Positive Control

Negative Control

Mean IVIS

72.53

1.15

Standard Deviation

19.02

0.78

Range of IVIS

33.59 – 153.20

0.00 – 2.84

Values of 190 studies with solid test items performed from February 2007 until July 2013

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is not irritating to the eye (IVIS = 0.08).
According to OECD 437 (adopted July, 26th 2013) a chemical that induces an IVIS≤ 3 is considered as not requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item not causes serious eye damage.