Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2017 to 2nd October 2017
1 (reliable without restriction)

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:

Test material

Test material form:
solid: particulate/powder

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
The test system (target tissue) was isolated bovine cornea obtained as a by-product from freshly slaughtered animals. They were obatined from the abattoir of J.W, Treuth & Sons Inc, Baltimore, MD. The eyes were excised by an abattoir employee (as soon after slaughter as possible) and held in HBSS on ice. Once the required number of eyes were obtained, they were transported to the lab. Immediately upon receipt of the eyes into the lab, preparation fo the corneas was initiated.

Test system

yes, concurrent positive control
Amount / concentration applied:
20% (w/v)
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Details on study design:
The solid test articles were tested as 20% (w/v) dilutions in sterile, deionized water. An aliquot of 750 µL of the test article, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. In the open chamber window technique, the glass window was removed from the anterior chamber immediately prior to treatment. Each cornea was completely covered with test article. After dosing, the glass window was replaced on the anterior chamber. The corneas were incubated in the presence of the test article, positive control or negative control at 32 ± 1ºC for approximately 4 hours. After the 4-hour exposure period, the control or test article treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). For the corneas treated using the open chamber window technique, the glass window was removed from the anterior chamber, and the test article was rinsed from the treated cornea and the anterior chamber with Complete MEM (with phenol red). Care was taken not to spray the corneas directly. The chamber windows were returned to the chambers when most or all of the test article had been removed. The rinsing process continued in the same manner as the positive and negative control corneas. The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 mL of a 5 mg/mL fluorescein solution. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered corresponding to chamber number. Aliquots of 360 µL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red). A 360 µL sample of each 1:5 dilution was transferred to its specified well on the 96-well plate. The plate was read again and the final reading was saved to a designated print file.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
ca. 100.4
Vehicle controls validity:
Negative controls validity:
not specified
Positive controls validity:
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Based on their in vitro scores and the following classification system, the test article is predicted to be severe irritant.
In vitro score:
Up to 4 = non-irritant
>4 -12 = slight irritant
>12.1 to 25 = mild irritant
25.1 to 55 = moderate irritant
55.1 and above = severe irritant