Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study (OECD 437/ EC 440/2008).

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

not specified

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: The Ocular Toxicity Working Group (OTWG) of the ICCVAM and the NICEATM, BRD: current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method March 2006.

Deviations:

not specified

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

not specified

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

EC Number:

205-381-9

EC Name:

Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

Cas Number:

139-89-9

Molecular formula:

C10H18N2O7.3Na

IUPAC Name:

trisodium [{2-[bis(carboxylatomethyl)amino]ethyl}(2-hydroxyethyl)amino]acetate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: HEDTA-Na3
- Physical state: white crystalline powder
- Analytical purity: 88.6% (as anhydrous form)
- Composition of test material, percentage of components: HEDTA-NA3 x 2.5H2O) = 100.2% (calculated)
- Purity test date: 17 October 2012
- Lot/batch No.: CFC 7826
- Expiration date of the lot/batch: 30 October 2015
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals / tissue source

Species:

other: cow

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelcos 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 under cooled conditions.
- Preparation of corneas: 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 degreeC 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 degree C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 degree C.

- All other template details: Not applicable

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 +/- 1 degree C
- All other template details: Not applicable

IN-LIFE DATES: Not applicable

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 ul
- Concentration (if solution): 40% w/w, and 100%

VEHICLE
- Source: Merck, Darmstadt, Germany
- All other template details: Not reported

Duration of treatment / exposure:

4 hours (240 minutes), +/- 10 minutes

Observation period (in vivo):

Immediately after exposure period

Number of animals or in vitro replicates:

3 eyes per treatment

Details on study design:

CORNEA SELECTION AND OPACITY READING
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.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 l of either the negative control, positive control (20% (w/v) Imidazole solution) or 40% (w/w) test substance was introduced onto the epithelium of the cornea. In addition HEDTA-Na3 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (302 to 313 mg).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C. After the incubation the solutions and the test compound 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.

OPACITY MEASUREMENT
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.

APPLICATION OF SODIUM FLOURESCEIN
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 degree C.

PERMEABILITY DETERMINATIONS
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 l 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.

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
- Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.
- The following in vitro classification system was used for the ocular irritation properties of the test substance as described reference 7.
- A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. The recommended decision criteria for using the BCOP to identify non-severe irritants are provided in Table 1.

Table 1 Overall BCOP classification criteria
In vitro score range Ìn vitro classification
0 - 3 Non irritant (1)
3.1 - 25 Mild irritant (2)
25.1 - 55 Moderate irritant (3)
≥ 55.1 Severe irritant (4)

(1) EPA Category IV; GHS Not classified; EU Not labelled
(2) EPA Category III; GHS Category 2B; EU Category R36
(3) EPA Category II; GHS Category 2A; EU Category R36
(4) EPA Category I; GHS Category 1; EU Category R41

TOOL USED TO ASSESS SCORE: opacitometer for opacity, fluorescein and microplate reader for permeability

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

58.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

other:

Remarks on result:

other: 100% HEDTA-Na3

Other effects / acceptance of results:

See Table 2 for Summary of opacity, permeability, and in vitro scores.
The corneas were slightly hazy after the 240 minute exposure to HEDTA-Na3 as a 100% solution.

Any other information on results incl. tables

The corneas treated with HEDTA-Na3 ‘as it is’ showed opacity values ranging from -1 to 7 and permeability values ranging from 2.214 to 4.998. The corneas were slightly hazy after the 240 minutes of treatment with HEDTA-Na3 ‘as it is’. Although on one cornea a part of the cornea was clear. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 37 to 82 after 240 minutes of treatment with HEDTA-Na3 ‘as it is’.

Table 2. Summary of opacity, permeability, and in vitro scores.

Treatment	Mean Opacity	Mean Permeability	MeanIn vitroIrritation Score^1, 2
Negative control	0	0.000	0.0
Positive control	81	2.592	120
HEDTA-Na3 ‘as it is’	3	3.708	58.6

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:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Undiluted HEDTA-Na3 (as is) induced severe ocular irritation mainly through the permeability endpoint, resulting in a mean in vitro irritancy score of 59 after 240 minutes of treatment. Since undiluted HEDTA-Na3 induced an IVIS ≥ 55.1, it is concluded that undiluted HEDTA-Na3 tested ‘as is’ is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test and should be labelled (GSH) as Category 1.

Executive summary:

Screening for the eye irritancy potential of HEDTA-Na3 has been undertaken using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the most recent OECD and EC test guidelines. The possible ocular irritancy of undiluted HEDTA-Na3 (as is) were tested through topical application for approximately 240 minutes.

 

Undiluted HEDTA-Na3 (as is) induced severe ocular irritation mainly through the permeability endpoint, resulting in a mean in vitro irritancy score of 59 after 240 minutes of treatment. Since undiluted HEDTA-Na3 induced an IVIS≥55.1, it is concluded that HEDTA-Na3 tested ‘as is’ is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test and should be labelled (GSH) as Category 1 according to the criteria of Regulation (EC) No. 1272/2008.