Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2016 - 30 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
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 July 26, 2013)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 03 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)
Specific details on test material used for the study:
No correction was made for the purity/composition of the test item. A solubility test in physiological saline was performed, but no workable suspension could be obtained.

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 341.4 to 386.9 mg per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
Appr. 240 minutes ± 10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing: yes (at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies)). The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM (MEM containing 1% L-glutamine and 1% FBS).
- Time after start of exposure: 240 minutes ± 10 minutes

SCORING SYSTEM:
- The opacity of a cornea was measured by the diminution of light passing through the cornea.
Following the final opacity measurement, the medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. Corneas were incubated with the sodium-fluorescein solution in a horizontal position for 90 ± 5 minutes at 32 ± 1°C. After the incubation period, the medium in the posterior compartment was removed and the optical density at 490 nm (OD490) was measured in triplicate.

- 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
Remarks:
(mean of 3)
Run / experiment:
Single run
Value:
2.5
Negative controls validity:
valid
Remarks:
-1
Positive controls validity:
valid
Remarks:
123.3
Other effects / acceptance of results:
The corneas treated with the test item showed opacity values ranging from 1.0 to 3.7 and permeability values ranging from -0.004 to 0.025. The corneas were slightly translucent with a small spot after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.9 to 3.7 after 240 minutes of treatment with disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate.
The individual in vitro irritancy scores for the negative controls ranged from -1.1 to -0.8. The individual positive control in vitro irritancy scores ranged from 93 to 154. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
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 123 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:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD gudieline and GLP principles, it is concluded that disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate is not irritant or corrosive for the eye.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate according to OECD guideline 437 and GLP principles. Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate was applied undiluted (341.4 to 386.9 mg per cornea, n=3). The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 123.3, and the mean in vitro irritancy score of the negative control (physiological saline) was -1.0. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate did not induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of 2.5 after 10 minutes of treatment. Since disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.