N,N-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September- 26 September

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

The following deviation from the study plan occured:

The positive control group had an overall IVIS of 133.2 which was slightly higher than the defined criteria range for the test acceptability. Due to the amount that it was exceeded by being so small ,the result was accepted on the basis that the positive control group still fulfilled its purpose of showing sensitivity of the test system to a known occular irritant.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3)

EC Number:

302-766-4

EC Name:

N,N-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3)

Cas Number:

94134-01-7

Molecular formula:

C6H9NO6.3C2H7NO

IUPAC Name:

2-[bis(carboxymethyl)amino]acetic acid; tris(2-aminoethan-1-ol)

Test material form:

solid: bulk

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Cattle (12 to 60 months old)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes excised from freshly slaughtered cattle were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100IU/ml and streptomycin at 100uh/ml) and transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were refrigerated on arrival and used within 24 hours of receipt.
- indication of any existing defects or lesions in ocular tissue samples: None reported
- Indication of any antibiotics used: penicillin at 100IU/ml and streptomycin at 100µg/ml

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Concentration: The test item was prepared as a 20% w/v solution in Sodium chloride 0.9% w/v

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Lot/batch no. (if required): 3012487
- Purity: 0.9%

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

none

Number of animals or in vitro replicates:

Three

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : Eyes from adult cattle typically 12 to 60-month-old were obtained from a local abattoir as a by-product from freshly slaughtered animals.The eyes were excised by an abattoir employee post slaughter, placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/ml and stretomycin at 100ug/ml). they were transported on the same day of slaughter to the test facility on ice packs.The corneas were refrigerated on arrival and used within 24 hours of receipt. All eyes were visibly inspected before and after dissection. Only corneas free from damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS : The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders The front and back chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES : 3 for Test item, 3 for postive control, 3 for negative control.

NEGATIVE CONTROL USED : Sodium Chloride 0.9% w/v.

POSITIVE CONTROL USED : Imidazole 20% w/v.

APPLICATION DOSE AND EXPOSURE TIME : 0.75ml for 4 hours

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Post exposure, the test and control items were removed from the front chamber and the cornea was rinsed 3 times with fresh EMEM containing phenol red prior to a final rinse with complete EMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Change in opacity for each cornea (including the negative control) calculated via substraction of the initial opacity reading from the final opacity reading. Subsequently these values were corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492) using the Labtech LT-4500 microplate reader.
- Others histopathology: Corneas were retained after testing for possible conduct of histopathology.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) : The formula used to determine the In Vitro Irritancy Score was as follows: In vitro Irritancy score= mean opacity value + (15 X mean permeability OD492 value).

DECISION CRITERIA:The decision criteria indicated in OECD TG437 was used.
IVIS= mean opacity value + (15 X mean permeability OD492 value).
Opacity and permeability values were evaluated individually to determine if the test item produced a response through only one of the two endpoints. Cornea condition post treatment was assessed macroscopically. Classification of the test item was made in accordance with the following prediction model:

IVIS Classification:
≤3: No Category. Not requiring classification to UN GHS or EU CLP;
≥3: ≤ 55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

14.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were opaque post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was marginally outside the range of 71.2 to 132.9. The positive control acceptance criterion was therefore not strictly satisfied and is reported as a deviation. However, therefore the difference of a 0.3 IVIS value is considered negligible.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The eye damage potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was evaluated. Based on the results, no conclusion can be made regarding the capacity of the substance to induce eye damage or eye irritation.

Executive summary:

An in vitro GLP compliant test was performed in accordance with OECD TG 437 to assess the eye damage potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3). Quantitative measurements of changes in corneal opacity and permeability are used to assess damage by the test item. N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for four hours. Both positive (Imidazole 20% w/v.) and negative (Sodium Chloride 0.9% w/v.) controls were tested simultaneously. Two endpoints namely opacity and permeability were used to generate an In Vitro Irritancy Score (IVIS). The IVIS for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was found to be 14.6 and for the positive and negative controls the scores were 133.2 and 1.0 respectively. In accordance with the decision criteria guidance table provided in OECD TG 437 as the IVIS score obtained for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was ≥3 and ≤ 55 no prediction could be made on the eye irritation potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3).