5-Amino-N,N'-Bis(2,3-Dihydroxypropyl)-2,4,6-Triiodoisophtalamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. –12.12. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

474-080-4

EC Name:

-

Cas Number:

76801-93-9

Molecular formula:

C14H18I3N3O6

IUPAC Name:

5-amino-N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-1,3-dicarboxamide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00104016
- Expiration date of the lot/batch: 12/2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: Stable

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

- Time interval prior to initiating testing:
The time interval between collection of the eyes and use of corneas in the BCOP was minimized, so the collected eyes were processed on the same day.
All eyes used in the assay were from the same group of eyes collected on a specific day.

- indication of any existing defects or lesions in ocular tissue samples:
Only corneas from eyes free of defects including scratched, and neovascularisation were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

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): 750 μl of suspension (2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution).

Duration of treatment / exposure:

4 hrs

Duration of post- treatment incubation (in vitro):

1.5 hr

Number of animals or in vitro replicates:

Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 1, 4, 6)
Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14)
Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9)

Details on study design:

SELECTION OF CORNEAS
Only corneas from eyes free of defects including scratched, and neovascularisation were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

PREPARATION OF CORNEAS
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist
in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
Each test group (test item, concurrent negative and positive controls) consisted of the three corneas.

NUMBER OF REPLICATES
Exposed group (test substance) - 3 corneas (No. 1, 4, 6)
Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14)
Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9)

NEGATIVE CONTROL USED
0.9% NaCl
POSITIVE CONTROL USED
20%w/v Imidazole in 0,9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 µL of application form (2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution), 4 hrs

TREATMENT METHOD: closed-chamber method

REMOVAL OF TEST SUBSTANCE
After the exposure period, the negative and positive control substances and the test item were removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

POST-EXPOSURE INCUBATION: application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C)

METHODS FOR MEASURED ENDPOINTS:
OPACITY: the diminution of lights passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. Corneal opacity (as value of illuminance) was measured quantitatively with the aid of an opacitometer (Opacitometer, Kit OP3,0 – Duratec, Analysertechnik – Germany). Resulting values of illuminance was converted to opacity values (OP-kit).
PERMEABILITY: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows: IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
≥ 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.23

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study met all the acceptance criteria for positive and negative controls. The In Vitro Irritancy Score (IVIS) for ATIBA-A was 0.23.
This value of IVIS is ≤ 3 therefore the classification of test item effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Executive summary:

The test item, ATIBA-A, was tested for the potential to cause ocular corrosivity or severe irritancy, as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to OECD Test Guideline No. 437 (2017) and Method B.47 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, Commission Regulation (EU) 2017/735, Adopted 14th February 2017.

The testing was performed on three groups of corneas: test item treatment group (20% in NaCl), positive control group (20% w/v imidazole in 0.9% NaCl solution) and negative control group (0.9% NaCl solution). Three corneas per group were used.

The closed-chamber method was used, because the test item solution was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The study met all the acceptance criteria for positive and negative controls.

The In Vitro Irritancy Score (IVIS) for ATIBA-A was 0.23.

This value of IVIS is ≤ 3 therefore the classification of test item effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.