Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-20 to 2012-01-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals / tissue source

Species:
other: not applicable
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
not applicable

Test system

Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
750 microL of the undiluted test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 10 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.
Details on study design:
Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals
(from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse 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 (MC2, Clermont, France)
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 in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed
into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1
was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the
measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer
was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated.
The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity
measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity
readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of
each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed.
The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL 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 opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with
fresh complete RPMI. 1 mL of a 4 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 control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard
deviations of the current historical mean.

Evaluation of Results:
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 mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500),
were taken into account by multiplying the OD490 value of the dilution by the dilution factor. 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 score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
140.44
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The in vitro 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.

Any other information on results incl. tables

Opazität
Cornea-No. Test Item Opacity Opacity Change of Corrected
    blank value post dose opacity values opacity values
1 Negative 3 4 1  
2 Control 3 2 -1  
3   3 4 1  
MV   3.00 3.33 0.33  
4 Positive 3 80 77 76.67
5 Control 3 67 64 63.67
6   3 65 62 61.67
MV   3.00 70.67 67.67 67.33
7 Test item 2 110 108 107.67
8 Genamin 2 111 109 108.67
9 CH 020 2 113 111 110.67
MV   2.00 111.33 109.33 109.00

Permaebilität
Cornea-No. Test Item OD490 Corrected
      OD490 values
1 Negative 0.008  
2 Control 0.005  
3   0.005  
MV   0.006  
4 Positive 1.016 1.010
5 Control 0.967 0.961
6   1.432 1.426
MV   1.138 1.132
7 Test item 2.111 2.105
8 FHP-OHS 2.088 2.082
9   2.107 2.101
MV   2.102 2.096

in vitroScore
Cornea-No. Test Item Corrected Corrected  in vitro
    opacity value OD490 value Score
1 Negative 1 0.008  
2 Control -1 0.005  
3   1 0.005  
MV   0.33 0.006 0.42
4 Positive 76.67 1.010  
5 Control 63.67 0.961  
6   61.67 1.426  
MV   67.33 1.132 84.32
7 Test item 107.67 2.105  
8 FHP-OHS 108.67 2.082  
9   110.67 2.101  
MV   109.00 2.096 140.44

Applicant's summary and conclusion

Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the evaluation criteria the test item is classified as very severe eye irritant.
Executive summary:

The eye irritancy potential of the undiluted test substance was investigated in the bovine corneal opacity and permeability assay and the mean in vitro score was calculated to be 140.44. Based on the results of this study a mean in vitro score of 140.44 was obtained. The test substance therefore is classified as severe eye irritant. The in vitro score obtained with the positive control was within the current historical mean and therefore this assay is considered to be valid.