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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
Study period:
2010-06-22 - 2010-09-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline no. 437 and under GLP conditions.
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD Guideline 437: "Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants", 2009
Principles of method if other than guideline:
The BCOP (Bovine Corneal Opacity and Permeability) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. The BCOP test uses isolated corneas from freshly slaughtered cattle. Damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an in vitro irritancy score (IVIS), which is used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of a test substance.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals / tissue source

other: not applicable (this is an in vitro test)
other: not applicable (this is an in vitro test)
Details on test animals or tissues and environmental conditions:
not applicable (this is an in vitro test)

Test system

unchanged (no vehicle)
not required
Amount / concentration applied:
mean: 289 mg of crinipan were applied
Duration of treatment / exposure:
4 h at 32 °C
Observation period (in vivo):
not applicable
Opacity of the corneas is measured at 570 nm, using a spectral photometer.
Permeability is determined by the amount of sodium fluorescein dye that penetrates all corneal cell layers. The fluorescein permeability values are measured as optical density at 490 nm using a spectral photometer.
Number of animals or in vitro replicates:
not applicable
Details on study design:
For the BCOP test with crinipan bovine corneas from freshly slaughtered cattle aged 12 to 60 months were used. The eyes of the cattle were transported from the slaughterhouse within 4 h to the test facility in Hank's balanced salt solution supplemented with 0.01% streptomycin and 0.01% penicillin. The corneas were examined and only corneas which were free from defects were used. The dissected corneas were transferred to a cornea holder and incubated with complete minimum essential medium (cMEM) at 32 ± 1°C for 1 h. After the initial incubation, the medium was changed and the baseline opacity was determined.
The test item crinipan was not soluble in any appropriate solvent for the BCOP test and therefore was used neat. For each treatment group (negative control, positive control, test item) three replicates were used. The test item crinipan was applied directly using a weight board. According to the guideline, the "open chamber method" for non-surface-active solids was performed. Averagely, 0.2893 g of the test item was applied on the epithelium in a way that as much as possible of the cornea was covered with test item. The exposition time on the corneas was 10 min at 32 °C. After thorough rinsing with cMEM a post-incubation time of 2 h at 32 °C followed. Then the opacity value of each cornea was determined and the permeability with sodium fluorescein was measured.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
BCOP max. score: 80
Vehicle controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Calculated IVIS:

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group



Relative Standard Deviation IVIS

Negative Control 0,9% NaCl






Crinipan AD






Positive Control 20% Imidazole






Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
It can be stated that in this study and under the experimental conditions reported, climbazole did not exhibit any ocular irritating potential in vitro.
Executive summary:

The BCOP test was performed to assess the corneal irritation and damage potential of Crinipan AD by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item Crinipan AD was brought onto the cornea of a bovine eye which previously had been incubated with cMEM ( = complete Minimum essential medium) without Phenol red at 32±1°C for one hour and whose opacity had been determined. The test item was incubated on the cornea for four hours at 32±1°C. After removal of the test item, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control, imidazole (20% solution in 0,9% sodium chloride solution) was used as positive control. The positive control induced severe irritation on the cornea, mean IVIS was 74.42. The negative control showed no irritation, mean IVIS was 1.119. The test item was tested neat. A mean IVIS of -0.561 = 0.00 was calculated, corresponding to a classification as not eye irritant.