Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.07.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
26 July 2013
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: solid yellow powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Freshly isolated bovine cornea obtained as a by-product from animals freshly slaughtered
- Source: A. Moksel AG, Buchloe, Germany

Test system

Vehicle:
physiological saline
Remarks:
physiological saline 0.9% NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item, the negative or positive control
Duration of treatment / exposure:
4 hours ± 5 minutes at 32 ± 1 °C, washed at least three times with minimum essential medium MEM (containing phenol red)
Duration of post- treatment incubation (in vitro):
- After the illuminance measurement the corneas were incubated for 90 minutes with RPMI (Roswell Park Memorial Institute) medium and sodium fluorescein solution at 32 ± 1 °C for the optical density (OD) determination
Number of animals or in vitro replicates:
3 corneas/group
Details on study design:
Preparation of the Corneas:
On the test day, fresh eyes were collected from the slaughterhouse and were transported in Hanks' balanced salt solution (HBSS) containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws.
The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% fetal bovine serum FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.

750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and
the corneas were incubated for 90 minutes at 32 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

Validity of the assay:
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Evaluation of Results:
The following formula was used to calculate the opacity, whereas the values a and b are equipment specific variables empirically determined by the manufacturer:
Opacity= ( I0/I-b)/a; with a = 0.025 and b = 0.9894.
I = Illuminance

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneas / 4 h incubation time
Value:
95.93
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Any other information on results incl. tables

Evaluation of the BCOP Assay

IVIS (In Vitro Irritancy Score):

Cornea no. Test item Corrected opacity Corrected OD490 value IVIS
1 Negative control 0.62 0.005 1.08
2 1.16 0.019
3 0.88 0.014
MV 0.89 0.013
4 Positive control 110.84 1.757 137.91
5 108.08 1.837
6 96.22 2.977
MV 105.05 2.191
7 Test item 127.5 0.008 95.93
8 76.98 0.006
9 83.06 0.002
MV 95.85 0.006

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
A mean in vitro irritation score of 95.93 was calculated. According to the evaluation criteria the test item should be classified as having irreversible eye effects, category 1.
Executive summary:

The eye irritancy potential of test item Mordant Red 11 was investigated in the bovine corneal opacity and permeability assay according to OECD 437, in compliance to GLP.

All 3 corneas treated with the test item showed intense opacity and a rose discolouration of the tissue. The following mean in vitro irritation score was calculated: 95.93

It cannot be excluded that the high opacity score may be related to the rose discoloration of the tissue due to the intense red colour of the test item. Nevertheless due to the observed intense opacity of the treated corneas and the high rate of the score, an eye irritancy potential of the test item is assumed.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

According to the evaluation criteria the test item Mordant Red 11 should be considered as having irreversible eye effects, category 1.