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Eye irritation

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (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:
EC Number:
Cas Number:
Molecular formula:
Test material form:

Test animals / tissue source

other: Assessment of the corneal damage potential of the test item with the BCOP test (exposure 10 min./rt followed by 2 hours incubation at 32 °C, subsequently measuring of corneal opacity).
Details on test animals or tissues and environmental conditions:
Eyes of slaughtered cattle were isolated and transferred in containers with Hank’s balanced salt solution (HBSS) supplemented with penicillin/streptomycin. During transport the containers were ice-cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers were stored in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.

Test system

unchanged (no vehicle)
other: for negative control: water (750 µL)
Amount / concentration applied:
750 μL per cornea and chamber
Duration of treatment / exposure:
10 min./32 °C
Observation period (in vivo):
post-exposure incubation: 2 hours (32 ° C)
Details on study design:
Corneal opacity is measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea before and after treatment.
Tests were performed in triplicates. Intact corneas were isolated and fixed in a cornea holder of an opacitometer chamber, that is filled with Minimal Essential Medium (MEM). After aspiration of the medium from the anterior side, 750 µL of the undiluted test substance was applied directly onto the epithelial surface of the corneas. Then, the cornea was incubated for 10 min. at 32 °C (± 1 °C). After exposure, the test substance was aspirated and the cornea rinsed several times. Then, fresh MEM medium was filled into the chambers and a further incubation period for about 2 hours at 32 °C (± 1° C) was conducted.
The measurements of opacity were carried out using an Opacitometer (BASF OP3.0). For determination of permeability sodium flourescein solution was applied to the epithelial surface for about 90 min./32 °C. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

Positive control: 1 % NaOH in distillated water (w/v; 750 µL)

Results and discussion

Any other information on results incl. tables

According to OECD TG 437 a substance is identified as inducer of serious eye damage if the IVIS (in vitro irritation score) value exceeds 55.

In vitro irritancy score:

   Cornea No. Opacity change  Permeability per cornea IVIS per cornea  IVIS mean  SD
 neg. control (water)  1  0.1  0.007  0.2    
   2  5.7  0.006  5.8  3.6  3
   3  4.9  0.005  5.0    
 pos. control (1 % NaOH)  4  112  0.614 118    
   5  106  0.967  117  126 15.2
   6  136  0.747  144    
 test item  7  0.3  0.007  -3.2    
 8  1.0  0.002  -2.5  -2.6  0.5
   9  1.3  0.003  -2.2    

No potential for serious eye damage was concluded from the study, as the IVIS was below 55 for the test item.

Applicant's summary and conclusion

Interpretation of results:
other: no potential for eye damage
Executive summary:

The test substance was investigated according to OECD TG 437 in the Bovine Corneal Opacity and Permeability (BCOP) test. This in vitro test is used to identify chemicals with a potential for serious eye damage.

In this study 750 µL of the unchanged test substance was applied on the epithelial surface of an extracted bovine cornea. Measurement of corneal opacity and permeability after a 10-minute exposure time and an additional 2-hour incubation time revealed an IVIS well below the cut-off value of 55, that is indicating a classification for serious eye damage according to OECD TG 437. The positive (1 % sodium hydroxide solution) and negative (water) controls confirmed the validity of the test.

Thus, under the conditions of this test no potential for serious eye damage can be concluded.

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