Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01DEC2015 - 04DEC2015
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:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
(adopted July 26, 2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(09 December 2010)
Deviations:
no
Qualifier:
according to
Guideline:
other: as summarized under "Principles of method if other than guideline"
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: colourless liquid
- Storage conditions: at room temperature

Test animals / tissue source

Species:
cattle

Test system

Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 750 µL per cornea

NEGATIVE CONTROL
- Amount applied: 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
After exposure the cornea is thoroughly rinsed to remove the test substance followed by incubation for 120 ± 10 minutes in cMEM. Then, corneas were incubated in sodium-fluorescein solution for 90 ± 5 minutes (both incubations at 32 ± 1°C).
Number of animals or in vitro replicates:
3
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- 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).


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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-0.1
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks:
IVIS = 121
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on an BCOP assay conducted in accordance with OECD/EC guidance and GLP principles, DBI does not have irritant or corrosive effects on the eye.