Trisodium [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]manganate(3-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Members of the aminocarboxylic acid (ethylenediamine-based) chelants chemical category possess similar molecular structures that contain common functional groups. All members have a molecular structure with an ethylenediamine or a diethylenetriamine backbone, which has 4 or 5 acetic acid groups attached to the nitrogens. The diethylenetriamine struc¬tures contain five acetic acid groups (DTPA); the ethylenediamine structure has four acetic acid groups (EDTA).
Therefore, all category members have identical functional groups (DTPA and EDTA) and have an ethylene-diamine-backbone. It is the presence of multiple carboxylic acid groups on the amine that provides chelants with their unique metal ion chelating or sequestering properties. This common property is the important feature to consider in assessing the aquatic and mammalian toxicity of chelants and in justifying their consideration as a category.
The substantial body of evidence that chelants are not directly toxic to aquatic and mammalian organisms but exert their influence by affecting mineral balance, together with the fact that the backbone structures of the chelants in the category have qualitative similar affinities for metals supports the inclusion of these chelants in a category. Subtle differences in toxicity due to the presence of calcium, magnesium, manganese, ferric (or ferrous) iron, copper or zinc can be explained by their affinity towards these metals and their ability to supply or deny metals to organisms.
As tested substance EDTA-MnNa2 and target substance DTPA-MnNa3 have the same metal cation and the chelating agents (EDTA and DTPA) are very similar structure, the results obtained for EDTA-MnNa2 are also valid for DTPA-MnNa3.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

EC Number:

239-407-5

EC Name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']manganate(2-)

Cas Number:

15375-84-5

Molecular formula:

C10H12N2O8MnNa2

IUPAC Name:

Disodium; 2-[2-(bis(carboxylatomethyl)amino)ethyl-(carboxylatomethyl)amino]acetate; manganese(+2) cation

Details on test material:

Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Test animals / tissue source

Species:

other: bovine eyes from a slaughterhouse

Details on test animals or tissues and environmental conditions:

No animals used

Test system

Vehicle:

physiological saline

Controls:

other: negative and positive controls were used

Amount / concentration applied:

A 20% (w/w) solution of EDTA-MnNa2 was prepared in physiological saline (Merck, Darmstadt, Germany).

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.

Duration of treatment / exposure:

240 min

Observation period (in vivo):

no observation period

Number of animals or in vitro replicates:

3 eyes each were used for the negative and positive control and for the test sample

Details on study design:

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 3 were not used. Three corneas were selected at random for each treatment group.

The medium from the anterior compartment was removed and 750 µl 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). 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 was 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 dgerees 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 µl 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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). 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:

3 eyes each were used for the negative and positive control and for the tet treatment, duration of treatment 240 min.

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

physiological saline

Positive controls validity:

valid

Remarks:

20% Imidazole

Other effects / acceptance of results:

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 119 and 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-MnNa2 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 240 minutes of treatment.

Any other information on results incl. tables

Table            Summary of opacity, permeability and in vitro scores

 

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control	71	3.186	119
EDTA-MnNa2	2	0.025	2.4

 

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

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other:

Conclusions:

Finally, it is concluded that this test is valid and that EDTA-MnNa2 is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

This report describes the ocular irritation properties of EDTA-MnNa2 on an isolated bovine cornea. The possible ocular irritancy of EDTA-MnNa2 was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD guideline.

Batch CFC 9380 of EDTA-MnNa2 was an off-white powder.The test substance was applied as a 20% (w/w) solution (750 µl) directly on top of the corneas. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% w/v Imidazole) was 119 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-MnNa2 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 240 minutes of treatment. It was concluded that this test is valid and that EDTA-MnNa2 is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.