N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-04-04 to 2022-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

853-587-8

Cas Number:

199387-97-8

Molecular formula:

C17 H30 N2 O7

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Test material form: white to off-white crystalline powder
- Batch No.: LFND3B1003
- Storage conditions: 2 - 8 °C
- Expiry date: 01 August 2025

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: LFND3B1003
- Purity: 100% (HPLC)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was used as provided by the sponsor and moistened wit ha drop of physiological saline 0.9% NaCl.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir Vion Beef B.V., Buchloe, Germany

On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C to allow equilibration.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): enough test item to completely cover the cornea (due to the low density of the test material less than 750 mg sufficed for completely covering the cornea which is conformity to OECD guideline 437)
- Concentration (if solution): The test item was moistened with a drop of physiological saline

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 21412415

Duration of treatment / exposure:

4 hours ± 5 minutes

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

3 corneas each for the test item, solvent control and positive control

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline 0.9% NaCl

POSITIVE CONTROL USED: 20% imidazole

APPLICATION DOSE AND EXPOSURE TIME: enough test item to completely cover the cornea was applied; 4 hours ± 5 minutes incubation

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
After incubation the test substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium (without phenol red) and an illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (BASF-OP3.0, Duratec GmbH). Calibration was performed before the test and is documented in the raw data. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: Following treatment and washing steps, each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS) = mean opacity value + (15x mean permeability OD490 value)

DECISION CRITERIA: As indicated in the OECD TG 437:
- IVIS ≤ 3: No Category
- 3- >55: Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

0.34

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control (opacity 2.07; permeability 0.043).
- Acceptance criteria met for positive control: Due to the fact that the IVIS of the positive control did not fall within two standard deviations of the current historical mean in the first experiment, it was not considered to be valid. Therefore, the experiment was repeated. The results of both experiments are reported, noting that data from the first experiment is invalid and is not considered for experimental outcome in the present study. All data presented refers to the 2nd experiment. In the second experiment the in vitro irritation score obtained with the positive control fell within two standard deviations of the current historical mean and therefore this assay is considered to be valid (Range of historical mean IVIS Score of Positive Control: 83.91 - 155.02).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 0.34 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test item can be considered to be non-irritant and no classification for eye irritation/serious eye damage is warranted.

Executive summary:

The eye irritation potential of the test item was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (purity: 99%) was directly applied on the cornea and moistened with a drop of 0.9% NaCl. A mean in vitro irritation score of 0.34 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no classification for eye irritation/serious eye damage is warranted.