Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-amino-4-(ethylamino)-9,10-dihydro-9,10-dioxoanthracene-2-carbonitrile
EC Number:
263-606-6
EC Name:
1-amino-4-(ethylamino)-9,10-dihydro-9,10-dioxoanthracene-2-carbonitrile
Cas Number:
62570-50-7
Molecular formula:
C16H14N2O2
IUPAC Name:
1-amino-4-(ethylamino)-9,10-dioxo-9,10-dihydroanthracene-2-carbonitrile
Test material form:
solid: particulate/powder
Details on test material:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99 %
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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 a physiological saline in a suitable container under cooled conditions.The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from physiological saline and holding them in the light. Those exhibiting defects were discarded.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
318-391 mg of Disperse Blue 359
Duration of treatment / exposure:
Corneas were first incubated in cMEM for a minimum of 1 hour at 32 ± 1 °C, the corneas were then incubated with the tests material in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.
Number of animals or in vitro replicates:
9 samples in total, 3 replicates each for Disperse Blue 359, the positive control and the negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. -0.7
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
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.
Positive controls validity:
valid
Remarks:
The mean in vitro irritancy score of the positive control was 159 and within two standard deviations of the current historical positive control mean. It was therefore concluded that test conditions were adequate and that test system functioned properly.
Remarks on result:
no indication of irritation

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.8 to -0.9. The individual positive control in vitro irritancy scores ranged from 140-187. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with Disperse Blue 359 showed opacity values ranging from -1.2 to -0.4 and permeability values ranging from 0 to 0.0037. The corneas were clear were clear after the 240 minutes of treatment with Disperse Blue 359. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.2 to 0.2 after 240 minutes of treatment with Disperse Blue 359.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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 159 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. Disperse Blue 359 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment. Since Disperse Blue 359 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Study describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Disperse Blue 359 was tested through the topical application for approximately 240 minutes. Procedures were based on the most recent OECD guideline. Since no workable suspension of test material in physiological saline solution could be obtained, the test item was used as delivered and added pure on top of the corneas.

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 159 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. Disperse Blue 359 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment. Since Disperse Blue 359 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.