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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to 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:
according to guideline
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
C21 H18 Cl F
Test material form:

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals:
- Characteristics of donor animals: age: 15 - 29 months, cornea diameter: 24 – 26 mm
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: Corneas were prepared immediately after delivery of the eyes to the laboratory, testing started on the day of collection
- Indication of any existing defects or lesions in ocular tissue samples: Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin were added in transport medium (5 mL/ 500 mL HBBS).
- Selection and preparation of corneas: The corneas were carefully removed from the eyes but a rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in incubation medium (EMEM, pre-warmed at 32 +/- 1 °C). Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O -ring) of the posterior part of the holder. The anterior part of the holder was positioned on top of the cornea. Both compartments of the holder were filled with incubation medium (EMEM). The posterior compartment was filled first to return the cornea to its natural convex form. For equilibration, the corneas in the holder were incubated (incubator: Grumbach BSS 160) in a vertical position at 32 ± 1 °C for about one hour. At the end of the incubation period, the incubation medium (EMEM) was removed from both compartments and replaced by fresh incubation medium (EMEM).
- Quality check of the isolated corneas: The baseline opacity was determined with an opacitometer (BASF-OP2.0). The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value is converted into an opacity value. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

Test system

physiological saline
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 750 µL (i.e. 150mg/750µL), The test item was prepared as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

NEGATIVE / VEHICLE CONTROL: 750 µL, 0.9 % sodium chloride solution

POSITIVE CONTROL: 750 µL, Imidazole was dissolved with 0.9 % sodium chloride solution to a concentration of 20 % (w/v).
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
Incubation with fluorescein solution: 90 min
Number of animals or in vitro replicates:
3 replicates in all groups
Details on study design:
TREATMENT METHOD: closed chamber / open chamber, not specified

- Number of washing steps after exposure period: 3

- Corneal opacity: The opacity was determined with an opacitometer (BASF-OP2.0). The change in opacity was calculated by subtracting the initial baseline opacity from the post treatment opacity. In addition, the opacity values of treatment and positive control groups were corrected for the man negative control opacity value.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (BioTek ELx800) at 490 nm (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)


Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

- Acceptance criteria met for negative control: After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 0.7 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20 % Imidazole) the calculated IVIS was 114.6 and, thus, within two standard deviations of the current historical mean of the positive control (IVIS: 81.5 – 132.9).

Any other information on results incl. tables

per corneaper group
(mean value)
Negative control0.9 % NaCl Solution1.5-0.0031.4550.70.7
Positive control20 % Imidazole solution81.52.182114.230114.63.7
Test itemTest item1.70.0021.7300.11.5


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item did not show an eye hazard potential.
Executive summary:

A study according to OECD TG 437 was conducted to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 0.7 (study acceptance criteria range: -1.5 – 3.2). Treatment with the positive control revealed an IVIS of 114.6 (study acceptance criteria range: 81.5 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 0.1 and, thus, lower than 3, i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category). Under the conditions of the present study, the test item did not show an eye hazard potential.