Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
2013-07-26
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 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: white to yellow, powder
- Storage condition of test material: at room temperature

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:
physiological saline
Controls:
other: Control (saline) bovine corneae were used.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL of the prepared stock (test item as 20 % suspension (w/v) in vehicle)
(Note: the test item was crushed in mortar with a pistil to improve its consistency. The test item could not be suspended or solved homogeneously, therefore, the prepared stock was distributed to each cornea. Thereby it was taken care of that the corneae were evenly covered with the test item.)
Duration of treatment / exposure:
240 minutes
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 which 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 corneae.
- 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 suspension or negative control (saline) or positive control (10 % (w/v) benzalkonium chloride in saline) at a volume of 0.75 mL each on the surface of the corneae.
- the corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- the test item, positive control and negatice control were tested in triplicate.
- the test item or control items were rinsed off with saline.
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240)
- permeability of the corneae was determined.

PERMEABILITY MEASUREMENT
- after the final opacity measurement, the incubation 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 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, well mixed and the optical density at 490 nm was determined with a spectrophotometer.
- optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm.
- absorbance values were determined using the 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 (t240 – 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 category according to OECD 437 (please refer to table 1 in the field "Any other information on materials and methods incl. tables" below).

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vivo

Results
Irritation parameter:
other: in vitro irritancy score
Basis:
mean
Time point:
other: 240 minutes
Score:
2.62
Irritant / corrosive response data:
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 2.62 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is not categorized.

Any other information on results incl. tables

Table 1: Results after 240 minutes incubation time

Test group

Opacity value = Difference (t240 – t0) of opacity

Permeability at 490 nm (OD490)

In vitro irritancy score

Mean in vitro irritancy score

 

 

Mean

 

Mean

 

 

Negative control

1

 

0.33

0.087

 

0.087

2.31

 

1.64

0

0.087

1.31

0

0.088

1.32

Positive control

119.67*

0.033*

120.16

 

122.16

133.67*

0.036*

134.20

110.67*

0.097*

112.12

Test item

3.67*

0.034*

4.17

 

2.62

-0.33*

-0.006*

-0.43

2.67*

0.096*

4.10

* corrected values

- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.64).

- the positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 122.16) corresponding to a classification as serious eye damaging (CLP (Cat 1)).

Table 2: Historical data

 

Positive Control

Negative Control

Mean IVIS

116.58

1.38

Standard Deviation IVIS

9.22

0.35

Range of IVIS

98.49 – 128.43

0.88 – 2.04

Mean Opacity t240min

114.03

0.20

Standard Deviation

Opacity t240min

13.16

0.26

Range of Opacity t240min

91.67 – 159.00

0.00 – 0.67

Mean Permeability

0.196

0.130

Standard Deviation

Permeability

0.323

0.187

Range of Permeability

0.000 – 1.438

0.055 – 0.750

Values of 21 studies with solid test items performed from February 2015 (calendar week 7) until January 2016 (calendar week 3)

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 an increase of the corneal opacity or permeability. In conclusion, according to the current study and under the experimental conditions reported, the substance is not irritating to the eye (the calculated IVIS = 2.62). 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:

In thisin vitro study the substance was tested for its eye irritation potential according to the OECD guideline 437 (2013) using fresh bovine corneae. Three corneae were exposed to either the test item as 20 % suspension (w/v) (0.75 mL), the positive control ( 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline)); 0.75 mL), or the negative control (saline; 0.75 mL) for 240 minutes (incubation temperature: 32 ± 1 °C). After the incubation phase the substance, the positive control, and the negative control were each rinsed from the corneae. Next, the corneal opacity and permeability were measured and the In vitro Irritancy Score (IVIS) was calculated for the test item and the controls.

Relative to the negative control, the substance did not cause an increase of the corneal opacity or permeability.

The calculated mean IVIS was 2.62 (threshold for serious eye damage: IVIS≥ 55). According to OECD 437 (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 adaptations, the substance is not irritating to the eye.