Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.05.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Specific details on test material used for the study:
purity: 100%

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:
0.9% NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 L
Duration of treatment / exposure:
- 4 hours ± 5 minutes at 32 ± 1 °C, washed at least three times with MEM (containing phenol red), finally rinsed with complete RPMI (without phenol red)
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
- After the illuminance measurement was performed the corneas were incubated for 90 minutes at 32 ± 1 °C
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 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% 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 L 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 theposterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneas / 4 h incubation time
Value:
357.21
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
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.

Deviations:
Deviations did not influence the quality or integrity of the present study

Any other information on results incl. tables

In Vitro Irritation Score

Cornea no. Test item Corrected opacity Corrected OD490 value IVIS
1 Negative control -0.04 0.006 0.67
2 0.15 0.015
3 1.35 0.015
MV 0.49 0.012
4 Positive control 76.72 0.951 97.7
5 82.82 0.932
6 88.91 1.094
MV 82.82 0.992
7 Test item 276.79 5.528 357.21
8 334.61 3.203
9 267.57 4.113
MV 292.99 4.281

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 357.21 was calculated.
According to the evaluation criteria the test item Ingrain Blue 2.2 is classified into UN GHS Category 1.
Executive summary:

The eye irritancy potential of Ingrain Blue 2.2 was investigated in the bovine corneal opacity and permeability assay.

The test was perforemd in accordance to the OECD TG 437 and in compliance to GLP.

All 3 corneas treated with Ingrain Blue 2.2 showed a very intense opacification and an orange discoloration of the tissue.

The following mean in vitro irritation score was calculated: 357.21

It cannot be excluded that the high opacity score may be related to the orange discolouration of the tissue but 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.

A mean in vitro irritation score of 357.21 was calculated.

According to the evaluation criteria the test item Ingrain Blue 2.2 is classified into UN GHS Category 1.