Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-12-17 to 2018-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
EC Number:
609-276-2
Cas Number:
3663-80-7
Molecular formula:
C9H8O4
IUPAC Name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
Test material form:
solid
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Storage: The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum).
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 352.5 to 383.3 mg
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
three corneas for each treatment group.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
- Preparation of Corneas: The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) 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°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- Cornea Selection: 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. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS : The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES : Three corneas for each treatment group

NEGATIVE CONTROL USED : physiological saline

POSITIVE CONTROL USED : 20% (w/v) Imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
- Dose: 352.5 to 383.3 mg of the test item; 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control)
- Exposure time: 240 ± 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
251
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
271
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
297
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean opacity and mean permeability for the negative controls are 0.5 and 0.014, respectively, which are less than the upper limits of the laboratory historical range.
- Acceptance criteria met for positive control: The mean in vitro irritancy score for the positive controls is 138 that falls within two standard deviations of the current historical mean.

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.7. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 118 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 242 to 288 and permeability values ranging from 0.166 to 0.599. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 251 to 297 after 240 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, since the test substance induced an IVIS ≥55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test substance as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to 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).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.7. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 118 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 242 to 288 and permeability values ranging from 0.166 to 0.599. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 251 to 297 after 240 minutes of treatment with the test item. The test item induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 273 after 4 hours of treatment.

 

In conclusion, since the test substance induced an IVIS55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.