Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted to recognised test guidelines

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: ICCVAM, Invittox and Draft OECD methods
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): THPI D4 - 1,2,3,6 tetrahydrophthalimide
- Physical state: Solid
- Analytical purity: 97.91%
- Lot/batch No.: STI4208324
- Expiration date of the lot/batch: 2010-11-19
- Storage condition of test material: Room temperature, protected from light

Test animals / tissue source

other: Bovine corneas
Details on test animals or tissues and environmental conditions:
- Source: Bovine eyes obtained from cattle less than 12 months of age from Societe Vitreenne d'Abattage under licenced control by Prefecture d'Ille-et-Vilaine,

IN-LIFE DATES: From: 2009-06-29 To: 2009-07-06

Test system

physiological saline
other: 3 corneas
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 750 microlitres
- Concentration (if solution): 20% (w/w)
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
90 minutes
Number of animals or in vitro replicates:
3 corneas
Details on study design:
Preparation of corneas
Excised eyes were conditioned in Hank’s Balanced Salt Solution (HBSS) during transport and were used within 5 hours after slaughter of the cattle. On receipt, excised eyes are macroscopically examined for opacity, scratches, pigmentation and neo- vascularisation. Damaged eyes were discarded.
The cornea was carefully dissected from the eye such that at least 5 mm of sclera remained around the limbic zone. Corneas were held in Petri dishes containing HBSS with 1% penicillin/Streptomycin until placed into incubation chambers.
Each cornea was rinsed with phosphate buffered saline (PBS) and placed with the corneal endothelium against the posterior chamber of the incubation holder. The anterior chamber was placed over the cornea and both compartments of the holder fixed. Both compartments were filled with Eagle’s Minimal Essential Medium (MEM) supplemented with serum (1%), L-glutamine (1%) and sodium bicarbonate (2.2 g/L). The chambers were sealed and the cornea holders placed in a waterbath and incubated for at least 60 minutes at +32°C± 1°C with the corneas in a vertical position.

Opacity measurements – pre-treatment
After the incubation period the medium was removed from both anterior and posterior compartments of each cornea holder and replaced with Eagle’s MEM.
An opacimeter was used to determine the light transmission through the center of each cornea in the incubation chambers. Any comeas with an opacity unit between (-5) and (+5) were selected for use while the others were discarded. The mean value of the basal opacity, for all selected corneas, was then determined and any corneas deviating from this by more than 2 units were discarded.

The medium of each anterior chamber of the corneas forming the test group was removed and substituted for 7501μL of the test substance. In parallel, corneas were identically treated with the negative control (Eagle’s MEM) and the reference (positive control) substance, imidazole at 20% concentration. Comeas were incubated at 32±1°C for 240 minutes with the anterior chamber upwards so that corneas were uniformly covered with the substance. Following incubation, the substance of each chamber was removed and each cornea rinsed at least 3 times with Eagles’s MEM with phenol red supplemented with serum (1%), and sodium bicarbonate (2.2 g/L) to eliminate all residues. A final rinsing was performed with Eagle’s MEM.

Opacity measurements – post-treatment
The medium of each anterior chamber was replaced with Eagle’s MEM and the opacity of each cornea measured.

Permeability measurements
After the post-treatment opacity measurement the medium was removed from both anterior and posterior chambers. The posterior chamber was filled with Eagle’s MEM while the anterior chamber was filled with lmL of fluorescein at 5mg/mL in PBS. The corneas are placed in a horizontal position, with the anterior chamber upwards, and incubated at 32°C ±1°C for 90 minutes. After incubation, for each chamber, the medium of the posterior compartment was removed and transferred to identified test tubes for determination of optical density (OD). An aliquot of each identified solution was transferred into a cuvette with a 1 cm path length which was placed into a spectrophotometer set at 490 nm. A sample of Eagle’s MEM was used as a blank. Any solutions giving an OD beyond the maximum absorbanee limit was diluted in Eagle’s MEM and re-examined. This value is multiplied by its dilution factor to obtain the OD value of the original sample.

Expression and interpretation of the results
The individual value of the opacity measured at the end of the treatment was corrected against the basal opacity (determined before the treatment). Afterwards, the individual values obtained for the corneas treated with the test substance and the reference (positive control) substances were corrected according to the average of individual values obtained for corneas treated with the negative control using the following formula:
Corrected opacity = Individual opacity - average of negative control opacity
The mean corrected opacity was then obtained by calculating the average of the corrected values for each treatment.
The OD obtained for the corneas treated with the test and reference substances were corrected according to the average of the OD obtained for the corneas treated with the negative control. The mean corrected OD value is then obtained by calculating the mean of the corrected ODs for each treatment condition.

Determination of the final score
The final score was defined according the formula below:
IVIS = Mean corrected opacity value + (15 x Mean corrected OD value)

The irritant potential of the test substance was given by the following scale (OECD Guideline):
In-vitro score Classification
0 to 3 non irritant
3.1 to 25 Slightly irritant
25.1 to 55 Moderately irritant
≥ 55.1 Severely irritant

Results and discussion

In vivo

Irritation parameter:
Time point:
other: not applicable
Max. score:
not specified
Remarks on result:
other: In-vitro BCOP assay

Any other information on results incl. tables

Cornea No.

Corrected opacity score

Corrected OD score















In-Vitro Irritation Score = 5.84

Applicant's summary and conclusion

Interpretation of results:
other: Slightly irritant
Criteria used for interpretation of results: expert judgment
The test substance is not expected to be severly irritanmt or corrosive to the eye
Executive summary:

The test substance is not expected to be severly irritanmt or corrosive to the eye