Ethylenediamine-N,N'-di(acetic acid)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediamine-N,N'-di(acetic acid)

EC Number:

227-105-6

EC Name:

Ethylenediamine-N,N'-di(acetic acid)

Cas Number:

5657-17-0

Molecular formula:

C6H12N2O4

IUPAC Name:

2-({2-[(carboxymethyl)amino]ethyl}amino)acetic acid

Test material form:

solid: particulate/powder

Details on test material:

Chemical name: Ethylenediaminediacetic acid (ED2A-H2)
Batch/lot No.: FC-C 12874
Appearance: Yellow powder (visual)
Assay: 99.2% (HPLC)
pH: 4.7 (saturated solution)
Solubility in water: soluble in water at 1.0 g/L

Specific details on test material used for the study:

purity: 99.2%

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

30 uL negative control
30 mg NaOH (positive control)
30 mg ED2A-H2

Duration of treatment / exposure:

10 sec, then rinsing with 20 mL saline

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

negative control: 1
positive control and ED2A-H2: 3

Details on study design:

Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5-2.5 kg, were used as eye donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature. Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system (attached; see below). Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Value:

1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Value:

0.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Value:

0.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ED2A-H2 caused corneal effects leading to a “Not Classified” classification, consisting of no or very slight corneal swelling (mean of 1%), very slight or slight opacity (mean score of 0.7) and very slight fluorescein retention (mean score of 0.5).
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination of the corneas treated with ED2A-H2 revealed very slight erosion of the epithelium in one out of three corneas.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Applying the classification criteria of the ICE, the following irritation classifications can be assigned: ED2A-H2: NC:“Not Classified” (UN-GHS and EU-CLP classifications)

Executive summary:

ED2A-H2 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg of the test substance for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

ED2A-H2 caused corneal effects leading to a “Not Classified” classification, consisting of no or very slight corneal swelling (mean of 1%), very slight or slight opacity (mean score of 0.7) and very slight fluorescein retention (mean score of 0.5). Microscopic examination of the corneas revealed very slight erosion of the epithelium in one cornea.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, disorder of stromal fibres (3/3 corneas) and endothelial necrosis (3/3 corneas).

Applying the classification criteria of the ICE, the following irritation classification can be assigned: NC:“Not Classified” (UN-GHS and EU-CLP classifications).