Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Justification for type of information:
The study procedures were based on the following documents:
- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for
Testing of Chemicals; Guideline no. 437: "Bovine corneal opacity and permeability (BCOP) test
method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009).
- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the
Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and
permeability method for identifying ocular corrosives and severe irritants ". Official Journal of
the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the
Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document
(BRD): current status of in vitro test methods for identifying ocular corrosives and severe
irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and
Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An
in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Principles of method if other than guideline:
Rationale : in the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence, a validated and accepted in vitro test for eye irritation should be performed before in vivo test are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
GLP compliance:

Test material

Test material form:
solid: flakes
Details on test material:
Beige flakes, stroed at room temperature, in the dark, stable.
No workable suspension of 852 imide brute in physiological saline has been obtained : the test substance was thus delivered by the sponsor and added pure on the top of the corneas.

Test animals / tissue source

other: bovine eye
other: cattle from a slaughter house (Vitelco, Hertogenbosch, The Netherlands)
Details on test animals or tissues and environmental conditions:
Eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
They were collected and transported in physiological saline in a suitable container.

Test system

unchanged (no vehicle)
Duration of treatment / exposure:
852 Imide brute was weighted in a bottle and applied directly on the corneas, in such a way that the cornea was completeley covered. The holder was slighlty rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in an horizontal position for 240 +/- 10 minutes at 32°C +/-1°C.
After the incubation, the solution (negative and positive controls) and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle's Minimum Essential Medium). Possible pH effects of the test substance on the corneas were recorded. The anteor and posterior compartments were refilled with fresh cMEM and an opacity determination was performed without any further incubation.
After the completion of the incubation period, each cornea was inspected visually for dissimilar opacity patterns and the opacity determination was performed.
Number of animals or in vitro replicates:
3 corneas / each treatment group (negative, positive controls and test item)
Details on study design:
A negative control (physiological saline) was included to detect no specific change in the system and to provide a baseline for the assay endpoint.
A positive control was also prepared with 20% (w/w) Imidazole ( CAS 288-32-4) solution, prepared in physiological saline.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
240 min
ca. 158
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Irritation parameter:
cornea opacity score
Run / experiment:
240 min
ca. 73
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Irritation parameter:
other: Mean Permeability
Run / experiment:
240 min
ca. 5.652
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:

In vivo

Irritant / corrosive response data:
opacity and permeability were evaluated.
the mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score (see attached report)

Applicant's summary and conclusion

Interpretation of results:
highly irritating
Migrated information Criteria used for interpretation of results: EU
852 Imide brute ( tetrahydrocyclopenta[c]pyrrole-1,3(2H,3aH)-dione) induced severe ocular irritation tough both endpoints resulting in a mean in vitro irritancy score of 158 after 240 minutes of treatment.
Since 852 Imide Brute induced an IVIS >55.1, it is concluded that 852 Imide brute is corrosive or severe irritant in the Bovine Corneal opacity and Permeability test under the experimental conditions described in the report.