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Toxicological information

Eye irritation

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Trialuminium bismuth hexaoxide
EC Number:
EC Name:
Trialuminium bismuth hexaoxide
Cas Number:
Molecular formula:
Test material form:

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
- Source: Obtained from abattoir local to the testing laboratory
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: Corneas prepared immediately on arrival from the abattoir
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

physiological saline
yes, concurrent vehicle
Amount / concentration applied:
Test substance prepared as a 20% w/v solution in sodium chloride (0.9% w/v).
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
Duration of post- treatment incubation (in vitro):
Number of animals or in vitro replicates:
Three corneas per negative and positive control groups and three allocated for the test substance.
Details on study design:
Preparation of corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of corneas and opacity reading:
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Treatment of corneas:
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of sodium fluorescein:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability determinations:
After incubation the medium in the posterior chamber of each holder was decanted and retained. Media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during a period for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.4 and for permeability ≤0.072.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 2.4 and permeability ≤ 0.072. The negative control acceptance criteria were therefore satisfied.

The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
The IVIS score for the test substance was calculated as 22.3. As this value is > 3 and