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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 December 2018 - 19 December 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

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)
Version / remarks:
Adopted October 09, 2017
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
liquid: viscous
Specific details on test material used for the study:
Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light
Test Facility test item number: 209996/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
Test System:

Bovine eyes were used as soon as possible after slaughter.


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.


Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

physiological saline
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 uL of undiluted substance, positive control (Ethanol) and negative control (physiological saline).
Duration of treatment / exposure:
10 +/- 1 minutes.
Duration of post- treatment incubation (in vitro):
210 minutes.
Number of animals or in vitro replicates:
Details on study design:
Experimental design:

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 °C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading was determined and recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements:

The medium from the anterior compartment was removed and 750 uL of either the negative control, positive control (Ethanol) or substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the substance over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1 °C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the substance on the corneas were recorded. 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 +/- 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

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 (TECAN Infinite® M200 Pro Plate 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 substance was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. Altough one of the negative control treated corneas was translucent, the mean results are within the acceptability range therefore this has no impact on the study result. The mean in vitro irritancy score of the positive control (Ethanol) was 47 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

Any other information on results incl. tables

Summary of Opacity, Permeability and In Vitro Scores.






Mean In vitro Irritation Score1, 2

Negative control




Positive control









1    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The results of an in vitro eye corrosion/irritation study employing isolated bovine cornea exposed to undiluted substance for 10-minutes indicate that it is a non-irritant to the eye.
Executive summary:

In a reliable in vitro eye irritation study, conducted 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’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined.


The substance was applied undiluted at 750 µL onto corneas (n=3) for a period of 10 minutes, followed by rinsing of the substance and further incubation for 2 hours. The opacity and permeability of the corneas were determined after 90 minutes incubation with fluorescein.

The substance did not induce ocular irritation according to opacity and permeability, resulting in a mean in vitro irritancy score (IVIS) of -0.7. In conclusion, since the substance induced an IVIS ≤ 3, the substance is considered to be a non- irritant to the eye and not expected to cause serious eye damage.