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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium [[N,N'-ethylenediylbis[N-(carboxylatomethyl)glycinato]](4-)-N,N',O,O',ON,ON']zincate(2-)
EC Number:
EC Name:
Disodium [[N,N'-ethylenediylbis[N-(carboxylatomethyl)glycinato]](4-)-N,N',O,O',ON,ON']zincate(2-)
Cas Number:
Molecular formula:
Zinc(2+) ion disodium 2-({2-bis(carboxylatomethyl)amino]ethyl} (carboxylatomethyl) amino)acetate
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Description: off-white powder (determined at NOTOX)
Batch: CFC-8826 (205S7001)
Purity/Composition: ~89%
Test substance storage: at room temperature in the dark
Stability under storage conditions: stable
Expiry date: 1 July 2014
pH 6-7 (1% solution)

No correction was made for the purity/composition of the test substance.

Test animals / tissue source

other: in vitro

Test system

unchanged (no vehicle)
Amount / concentration applied:
Since no workable suspension of EDTA-ZnNa2 in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas.

Duration of treatment / exposure:
See below
Observation period (in vivo):
See below
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Negative control: a negative control, physiological saline (Merck, Darmstadt, Germany) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Test System: bovine eyes were used as soon as possible after slaughter on the same day.
Rationale: in the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: eyes were collected and transported in physiological saline in a suitable container.

All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32 +/- 1 degrees C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 degrees C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 degrees C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

The medium from the anterior compartment was removed and 750 uL of the negative control, 20% (w/v) Imidazole solution (positive control) or 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1 degrees C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1 degrees C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 uL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
240 min
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
EDTA-ZnNa2 was tested tested as it is. Table 1 summarizes the opacity, permeability and in vitro irritancy scores of EDTA-ZnNa2 and the controls. The individual in vitro irritancy scores for the negative controls were -1.2. The individual positive control in vitro irritancy scores ranged from 105 to 118. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with EDTA-ZnNa2 showed opacity values ranging from 4 to 7 and permeability values ranging from 1.375 to 3.264. The corneas were hazy after the 240 minutes of treatment with EDTA-ZnNa2. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 27 to 53 after 240 minutes of treatment with EDTA-ZnNa2.

Any other information on results incl. tables

Table1            Summary of opacity, permeability andin vitroscores






Mean In vitro Irritation Score1, 2

Negative control




Positive control









1     Calculated using the negative control mean opacity and mean permeability values.

2     In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

Table 2            Historical control data for the BCOP studies



Negative control

Positive control




In vitro Irritancy Score

In vitro Irritancy Score


-5 – 1

-0.03 – 0.059

-4.7 – 1.1

70 – 180
















SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2009 to January 2012.



Applicant's summary and conclusion

Interpretation of results:
other: slightly irritating
The test substances was slightly irritating in OECD test 437.
Executive summary:

This report describes the ocular irritation properties of EDTA-ZnNa2 on an isolated bovine cornea. The possible ocular irritancy of EDTA-ZnNa2 was tested through topical application for approximately 240 minutes.

The study procedures described in this report were based on the most recent OECD and EC guideline.

Batch CFC-8826 (205S7001) of EDTA-ZnNa2 was an off-white powderwith a purity of ~89%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (305 to 319 mg).

The mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas, except the opacity of one of the corneas. However, since the opacity values of the other two corneas were within the historical control data range, the validity of the test was considered not affected.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 111 and within the historical positive controldata range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EDTA-ZnNa2induced ocular irritation through the permeability endpoint, resulting in a mean in vitro irritancy score of 45 after 240 minutes of treatment. Since the mean in vitro irritancy score for EDTA-ZnNa2 was below 55.1 after 240 minutes treatment EDTA-ZnNa2 is considered to be not severe irritant or corrosive.

Finally, it is concluded that this test is valid and that EDTA-ZnNa2 is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.