Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

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 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Vanadium, oxalate complexes
- Trivial name: vanadyl oxalate
- Physical state: blue crystals, odourless
- Storage condition of test material: at room temperature in tightly closed container (further detailed: Harlan CCR SOP SUBST.doc)

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
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
A 20% (w/v) suspension of the test item in the vehicle was prepared using ultrasonic technique.
Duration of treatment / exposure:
240 ± 15 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s Ballanced Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 (© Biochrom) supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder as described in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
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). Each cornea with a value of the basal opacity > 7 was discarded and not used in the test.

OUTLINE OF STUDY
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl solution in deionised water (saline, produced in-house, lot no. 180113)) or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 180113)).
The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae. Since the test item could not be suspended homogeneously, each 0.75 mL of the so prepared stock was distributed to each cornea. Thereby it was taken care of that the corneae were evenly covered with the test item.
The test item, positive control and negative control were tested in triplicate.
The anterior compartment was then plugged again. The corneae were turned into a horizontal position and slightly rotated to ensure uniform covering of the corneae with the test or control items and were incubated in a water-bath in horizontal position at 32 ± 1 °C for 240 minutes.
After the incubation, the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water at least three times or until no visual evidence of the test substance was observed. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment, replacing the cMEM. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

CRITERIA FOR DETERMINATION OF A VALID TEST
According to the OECD 437 protocol, the test its considered acceptable if the positive control gives an In Vitro Irritation Score (IVIS) that falls within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Accordingly and based on historical control data, the test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

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 (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was 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 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 in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Results and discussion

In vivo

Results
Irritation parameter:
other: In vitro irritation score
Basis:
mean
Time point:
other: 240 minutes
Score:
69.31
Irritant / corrosive response data:
Relative to the negative control, exposure of the test item vanadium, oxalate complexes to the corneae did not induce an increase of the corneal permeability, but a clear increase of the opacity values occurred. The calculated mean in vitro irritation score was 69.31 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD guideline 437, the test item is classified as corrosive / severe irritant to the eye.

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 IrritationScore 

Mean in vitro irritation score± Standard Deviation

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative Control

5

2.00

0.052

0.055

5.78

2.83 ± 2.60

Non corrosive / non severe irritant

1

0.055

1.83

0

0.059

0.89

Positive Control

146.00*

0.001*

146.01*

213.03 ± 90.54

Corrosive / severe irritant

316.00*

0.002*

316.03*

177.00*

0.003*

177.04*

Vanadium, oxalate complexes

85.00*

0.044*

85.66*

69.31 ± 14.32

Corrosive / severe irritant

63.00*

0.018*

63.27*

59.00*

- 0.010*/** 

59.00*

 * Corrected values (subtraction of mean opacity/permeability of negative control from measured opacity/permeability of each replicate, respectively)

** Value was set to “0” for the calculation of the irritation score

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.83). The score is well within the concurrent negative control range, i.e. within two standard deviations of the current historical mean.

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacity of the corneae (mean in vitro irritation score 213.03) corresponding to a classification as corrosive/severe irritant to the eye (CLP/EPA/GHS (Cat 1)). The score is well within the concurrent positive control range, i.e. within two standard deviations of the current historical mean.

- Thus, based on historical positive and negative control data, this test meets the acceptability criteria in accordance with OECD 437.

Table 2: Historical data

 

Positive Control

Negative Control

Mean in vitro Irritation Score

176.71

1.78

Standard Deviation

42.65

0.75

Range of in vitro Irritation Scores

99.4 - 292.3

0.41 - 2.99

Values of 138 studies with solid test items performed between February 2007 and November 2012

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Vanadium, oxalate complexes is corrosive / severely irritating to the eye and should be classified and labelled as severe eye irritant according to Regulation (EC) No. 1272/2008 (Category 1).