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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March 2016
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
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)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Specific details on test material used for the study:
Batch /Lot /Notebook Ref: E00974-38D&F
Label Lot: E00350-288
Retest Date: 22 June 2018
Physical Description: Pale amber waxy solid
Purity: 100%
Storage Conditions: Ambient / dark

Test animals / tissue source

other: Bovine
Details on test animals or tissues and environmental conditions:
Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to the lab was on the same day as slaughter. Eyes were kept cool during transport. HBSS and transport materials were provided by the lab.

Corneas were prepared and used for testing on the day of collection. Upon receipt, eyes were rinsed with HBSS at ambient temperature and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of 2 to 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
75 mg per disc
Duration of treatment / exposure:
incubator set to maintain a temperature of 32C (actual incubator temperature 31.5+/-C) for 10 +/- 1 min
Duration of post- treatment incubation (in vitro):
2 h +/- 5 min in an incubator set to maintain a temperature of 32C (actual incubator temperature was 31.0C)
Number of animals or in vitro replicates:
3 replicate for negative and postive control
4 replicate for substance
Details on study design:
Bovine corneas were prepared and then mounted into corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed (ca 32ºC) Minimum Essential Medium (MEM) without phenol red. Holders were then allowed to equilibrate for =1 h in an incubator set to maintain a temperature of 32¿C. At the end of the equilibration period, baseline opacity readings were then taken using an Opacitometer. Damaged corneas (opacity >7 opacity units) were rejected from further use.

The negative control, physiological saline, was applied to the surface of three corneas, undiluted. Ethanol was used as a positive control and was applied to the surface of three corneas each, undiluted. The Substance was applied undiluted as a solid disc to 4 corneas. The dosed units were then returned to an incubator set to maintain a temperature at a temperature of 32¿C for 10 ¿ 1 min.

Following the exposure incubation, the chambers were opened and underwent a rinsing procedure. The corneas were then incubated for 2 h ¿ 5 min in an incubator maintained at a temperature of 32¿C and allowed to recover. Following the recovery period, the opacity of each cornea was measured. Gross damage or other changes were observed by visual assessment and recorded in the study files.

Following the measurement of opacity, a permeability test was conducted. The cornea were rotated to the horizontal position, and incubated for 90 ¿ 5 min in an incubator at a temperature of 32¿C

At the end of the permeability test, MEM from the posterior chambers was collected. These samples were stored in a refrigerator set to maintain a temperature of 4¿C overnight. Following overnight storage, triplicate aliquots from each sample were transferred into a 96 well plate, and analysed with a Multiskan Spectrum plate reader at 490 nm. Three blank wells containing MEM without phenol red were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein; 10 µg/mL).

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
ca. 6.07
Negative controls validity:
Positive controls validity:
Remarks on result:
not determinable

Any other information on results incl. tables

See attached background document

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
In conclusion, the BCOP assay was unable to categorise the Substance, giving a result of “No prediction can be made”.
Executive summary:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. The objective of this study was to evaluate the ocular irritation potential of substance using the Bovine Corneal Opacity and Permeability (BCOP) assay.


Potential for causing eye damage is an important safety evaluation for chemicals and products. Classification is based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), which differentiates severity of eye damage as follows: Serious Eye Damage is the production of tissue damage in the eye which is not fully reversible within 21 days of application, whereas Eye Irritation is the production of changes which are fully reversible within 21 days of application. Compounds causing irreversible eye damage are classified as GHS Category 1 and compounds causing reversible eye damage are classified as GHS Category 2. Compounds which cause no eye damage are Not Classified (NC).


Corneas were dissected from freshly obtained cow eyes, and mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated with the Substance (ca 75 mg), melted and weighed onto a filter paper, and tested by direct application to the cornea surface with saline (675 µL). The Substance was tested following the BCOP method for liquid and surfactant test items, with physiological saline and ethanol as negative and positive controls, respectively. The volume applied for the control treatments wasca 750µL.


Following exposure for 10 min ± 1 min, the test item filter paper discs were removed. The corneas were then rinsed. The corneas then underwent an additional recovery period of 2 h ± 5 min. The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The IVIS of the substance was determined to be 6.07, resulting in an outcome of “No prediction can be made”.


In conclusion, the BCOP assay was unable to categorise the substance, giving a result of “No prediction can be made”.