Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

EC Number:

268-018-3

EC Name:

Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Cas Number:

67989-88-2

Molecular formula:

C10H12CuN2O8.(NH4)2

IUPAC Name:

copper(2+) ion 2-({2-[(carboxylatomethyl)(carboxylmethyl)amino]ethyl}(carboxymethyl)amino)acetate diamine

Test material form:

other: solution

Details on test material:

Description Clear blue liquid
Batch CFC-10635 (555H1011)
Purity/Composition ~55%
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 01 September 2013

General information Avoid contact with aluminium, nickel, zinc, copper and copper alloys.
Store in PVC, PP, PE, Bituminized tanks
pH 6-8 (1% solution)





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

Test animals / tissue source

Species:

other: in vitro

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

The test substance was tested undiluted.

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: 10% (w/v) Benzalkonium Chloride (Sigma-Aldrich Chemie GmbH, Germany) [CAS Number 63449-41-2] 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: eEyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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 neovascularisation 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, 10% (w/v) Benzalkonium Chloride or test substance were introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1degrees C (protected from light). After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 degrees C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

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, Darmstadt, Germany) 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 4 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

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

10 min

Value:

-2.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

EDTA-Cu(NH4)2 was tested neat. Table 1 summarizes the opacity, permeability and in vitro irritancy scores of EDTA-Cu(NH4)2 and the controls. The individual in vitro irritancy scores for the negative controls ranged from -3.1 to 2.3. The individual positive control in vitro irritancy scores ranged from 108 to 122 for Benzalkonium Chloride (Table 2). The corneas treated with the positive control substance were turbid after the 10 minutes of treatment.

The corneas treated with EDTA-Cu(NH4)2 showed opacity values ranging from -3 to -1 and permeability values ranging from -0.013 to -0.003. The corneas were clear after the 10 minutes of treatment with EDTA-Cu(NH4)2. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -3.0 to -1.0 after 10 minutes of treatment with EDTA-Cu(NH4)2.

Any other information on results incl. tables

Table1            Summary of opacity, permeability andin vitroscores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control (Benzalkonium Chloride)	82	2.339	117
EDTA-Cu(NH4)2	-2	-0.006	-2.1

 

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
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-1 – 1	-0.020 – 0.014	-1.1 – 1.2	93 – 189
Mean	0	0.00	0.3	141
SD	0.45	0.00	0.48	21
n	80	85	85	81

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: not irritating

Conclusions:

The test substances was not irritating in OECD test 437.

Executive summary:

This report describes the ocular irritation properties of EDTA-Cu(NH4)2 on an isolated bovine cornea. The possible ocular irritancy of EDTA-Cu(NH4)2 was tested through topical application for 10 ± 1 minutes.

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

Batch CFC-10635 (555H1011) of EDTA-Cu(NH4)2 was a clear blueliquid with a purity of ~55%. The test substance was applied as it is (750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas,except the response of one cornea. However since the opacity and permeability value were just above the upper limit of the range and all other values were less than the upper limits of the laboratory historical range, this deviation in the opacity and the permeability score had no effect on the results of the study.

The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 117 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EDTA-Cu(NH4)2 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -2.1 after 10 minutes of treatment.

Finally, it is concluded that this test is valid and that EDTA-Cu(NH4)2 is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.