Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

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
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)
Version / remarks:
July 2013
according to
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder

Test animals / tissue source

other: isolated cornea from eyes of slaughtered cattle
not specified
Details on test animals or tissues and environmental conditions:
- Source: isolated cornea from eyes of slaughtered cattle from Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1 L containers with 500 mL HSS and 1 % penicillin I streptomycin solution, transport of the containers in coolers on ice
- Time interval prior to initiating testing: 1 day
- indication of any existing defects or lesions in ocular tissue samples: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.

Test system

physiological saline
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 750 µL of the test item preparation
- Concentration (if solution): The test item was suspended by rotation shortly prior to application in isotonic saline solution to achieve a concentration of 20% (w/v).
The preparation was visually described as suspension. The usage of isotonic saline solution for formulation of the test item is plausible based on the current knowledge of the sponsor. No objections were seen particularly in regard to stability.

- Concentration (if solution): 0.9% w/v sodium chloride solution
Duration of treatment / exposure:
4 h
Duration of post- treatment incubation (in vitro):
90 min fluorescein incubation
Number of animals or in vitro replicates:
each 3 corneas for test item, negative and positive control
Details on study design:
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32 °C(± 1 °C) for about 2 hours.
For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour in the incubator at 32 °C (± 1 °C).

After approximately 1 hour, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then.
The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± 1 standard deviation were selected for the actual test and assigned to the test groups.
The numbers of the selected corneas selected were documented in an appropriate protocol.
The holders were labelled with a new serial number for the further testing.

Sodium chloride 0.9% w/v


The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.
The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently.

POST-INCUBATION PERIOD: yes; 90 min fluorescein incubation

After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea.

- POST-EXPOSURE INCUBATION: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium in anterior chamber of each holder was replaced by 1mL of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes.

- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort V3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
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 GenS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD492 value) according to OECD guideline 437; additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints. Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
mean of three corneas
Vehicle controls validity:
Negative controls validity:
other: see vehicle control
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The negative control group had a mean opacity reading of -0.7 and mean permeability 0.007. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was 94.7. The positive control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
The study was conducted under GLP according to OECD guideline 437 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosion potential of the test item to the eye in vitro.
The mean IVIS of the test item is > 55 (231.6). Hence, the test item is classified as seriously damaging the eye (Eye Damage 1).
Executive summary:

This GLP study was performed to assess the corneal damage potential of the solid test item with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea. The study was conducted in accordance with international accepted Guidelines (OECD TG 437, EU Method B.47).

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.


20% (w/v) concentrations of the test item and the positive control imidazole in isotonic saline solution were tested on 3 bovine corneas each in comparison to the negative control, isotonic saline solution. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour treatment time.

Test items were applied to the epithelial surface of the cornea in a special corneal holder.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data:

IVIS (Test Item) = 231.6

IVIS (Negative Control) = -0.7

IVIS (Positive Control) = 94.7

In accordance with OECD TG 437 and the study results Hydroxytoluic acid was characterized as seriously damaging the eye (Eye Damage 1); in vitro irritancy score is >55 (231.6). Thus, no further test needs to be condcuted to assess the irritation potential of the substance.