Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
The characterization of the test item was conducted in a GMP environment.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: 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. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
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

Vehicle:
unchanged (no vehicle)
Remarks:
Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 314.6 to 336.6 mg mg per cornea
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
None
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
TEST SITE
- Isolated bovine cornea
- Uniform distribution of the control or the test substance over the entire cornea was ensured by horizontal position of the corneas.
- Possible pH effects of the test substance on the corneas were recorded.

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by measurement of the opacity by the diminution of light passing through the cornea. Then, corneas were incubated in sodium-fluorescein solution for 90 ± 5 minutes at 32 ± 1°C after which optical density at 490 nm (OD490) of the medium in the posterior compartment was measured to determine permeability.

The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range ≤ 3 No Category
In vitro score range > 3; ≤ 55 No prediction can be made
In vitro score range >55 Category 1


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≥ 55, no prediction on irritant potency can be made.

The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from -1.1 to 0.6. The individual positive control in vitro irritancy scores ranged from 124 to 181. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Diona-R showed opacity values ranging from -1.4 to 1.6 and permeability values ranging from -0.004 to 0.017. The corneas were clear after the 240 minutes of treatment with Diona-R. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.5 to 1.7 after 240 minutes of treatment with Diona-R.

The 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. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 155 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Diona-R is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test .

Executive summary:

The eye hazard potential of Diona-R was assessed using the Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD/EC guidelines and GLP principles. The eye damage of Diona-R was tested through topical application for approximately 240 minutes. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 155 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Diona-R did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 240 minutes of treatment. Since Diona-R induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.