Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 to 19 January 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 guideline
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

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Batch number: E010016621
Purity: Not indicated
Specific details on test material used for the study:
Batch number: E010016621
Purity: Not indicated

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 311.9 to 329.7 mg
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
Three corneas each group
Details on study design:
Treatment of corneas and opacity measurements
The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (311.9 to 329.7 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Permeability determinations
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 has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.9. The individual positive control in vitro irritancy scores ranged from 101 to 154. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test substance showed opacity values ranging from -0.8 to 0.0 and permeability values ranging from 0.000 to 0.013. The corneas were clear after the 240 minutes of treatment with the test substance. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.8 to 0.2 after 240 minutes of treatment with the test substance.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Since the test substance induced an IVIS≤3, no classification is required for eye irritation or serious eye damage.