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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:
18 March 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. Where a negative result is obtained, an in vivo test is subsequently required.

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)
Principles of method if other than guideline:
The study procedures described in the report are also based on the following documents:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 124. Bovine Opacity and Permeability Assay - SOP of Microbiological Associates Ltd., 1999.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-methoxy-1-oxohexadecane-2-sulphonate
EC Number:
EC Name:
Sodium 1-methoxy-1-oxohexadecane-2-sulphonate
Cas Number:
Molecular formula:
There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C17H34O5S.Na C17H33NaO5S C14H29CH(CO2CH3)SO3Na CH3(CH2)13CH(SO3Na)COOCH3
sodium 1-methoxy-1-oxohexadecane-2-sulfonate
Constituent 2
Chemical structure
Reference substance name:
Sodium methyl 2-sulphooctadecanoate
EC Number:
EC Name:
Sodium methyl 2-sulphooctadecanoate
Cas Number:
Molecular formula:
There are different notations of the molecular formula, dependent on whether it is depicted as protonated or not: C19H38O5S.Na or C19H37O5S.Na CH3(CH2)15CH(SO3Na)COOCH3
sodium methyl 2-sulphooctadecanoate
Test material form:
solid: flakes
Details on test material:
- Physical state: pale yellow flakes
- Storage condition of test material: In refrigerator (2-8 ºC) in the dark

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were used as soon as possible after slaughter on the same day.

Source: obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands) as soon as possible after slaughter, and transported in physiological saline

The isolated corneas were stored at 32 +/- 1ºC in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum).

Test system

physiological saline
yes, concurrent vehicle
Amount / concentration applied:
750 µl of a 10% (w/v) suspension
Duration of treatment / exposure:
10 ± 1 min
Observation period (in vivo):
120 ± 10 min
Number of animals or in vitro replicates:
1 cornea per treatment group
Details on study design:
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1ºC. After the incubation the control or test substance was removed and the epithelium was washed at least three times with cMEM. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1ºC. After the completion of the incubation period opacity determination was performed (opacitometer)

Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. For thta, the medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution and were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1ºC.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at
490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
mean of replicates
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
The pH of the 10% (w/v) solution of MIZULAN FL-80 was 5.5.
The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.2. The individual positive control in vitro irritancy scores ranged from 156 to 189 for Benzalkonium Chloride. The corneas treated with the positive control substances were turbid after the 10 minutes of treatment.

The corneas treated with MIZULAN FL-80 showed opacity values ranging from 0 to 2 and permeability values ranging from 1.122 to 2.304. The corneas were clear after the 10 minutes of treatment with MIZULAN FL-80. Hence, the in vitro irritancy scores ranged from 17 to 37 after 10 minutes of treatment with MIZULAN FL-80.

Any other information on results incl. tables







Mean In vitro Irritation Score1, 2

Negative control




Positive control

(Benzalkonium Chloride)









1          Calculated using the negative control mean opacity and mean permeability values.

2           In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).


Applicant's summary and conclusion

Interpretation of results:
other: not irritating
It is concluded that this test is valid and that MIZULAN FL-80 is a non irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.