Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Apr 2018
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:
adopted: 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Expiry Date: 20 December 2020
Storage Conditions: at room temperature, protection from light
Description: Colourless to yellow liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Time interval prior to initiating testing: The corneas were incubated for one hour at 32 ± 1 °C
- Indication of any antibiotics used: Pen/Strep

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 μL of the test substance or the control substance was introduced into the anterior chamber.
Duration of treatment / exposure:
10 minutes incubation at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
- Illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.
Number of animals or in vitro replicates:
Triplicates
Details on study design:
- Preparation of the Corneas: 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.
- Calibration of the Opacitometer: The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux.
- 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 substance or the control substance was introduced into the anterior chamber. After 10 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 after 2 hours incubation at 32 ± 1°C. 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 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 (Jenway 6405 UV/VIS).
- 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. The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. 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 cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (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 irritation score (IVIS): IVIS = mean opacity value + (15 x mean permeability OD490 value). The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: ≤ 3: No Category; > 3 - ≤ 55: No prediction can be made; > 55: Category 1.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean based on #1, #2, #3
Value:
1.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Mean based on #1, #2, #3
Value:
1.81
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Mean based on #1, #2, #3
Value:
-0.014
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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 resulted 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

Table 1: Opacity

Cornea
No.

Test Item

Initial
Opacity

Final
Opacity

Change of Opacity Value

Corrected Opacity Value

1

 

1.11

1.61

0.50

 

2

Negative

1.11

1.87

0.75

 

3

Control

1.11

2.16

1.05

 

MV

 

1.11

1.88

0.77

 

4

 

3.19

34.99

31.80

31.03

5

Positive

2.16

29.80

27.64

26.87

6

Control

2.27

29.49

27.22

26.45

MV

 

2.54

31.43

28.89

28.12

7

 

0.29

2.46

2.17

1.41

8

Test Item

-0.38

1.83

2.21

1.44

9

 

-0.73

2.01

2.75

1.98

MV

 

-0.28

2.10

2.38

1.61

MV = mean value

Table 2: Permeability

Cornea
No.

Test Item

OD490

Corrected OD490 Value

1

 

0.022

 

2

Negative

0.045

 

3

Control

0.015

 

MV

 

0.027

 

4

 

0.985

0.958

5

Positive

1.166

1.139

6

Control

0.905

0.878

MV

 

1.019

0.991

7

 

0.014

-0.013

8

Test Item

0.017

-0.010

9

 

0.010

-0.017

MV

 

0.014

-0.014

 MV = mean value

Table 3: In Vitro Irritation Score

Cornea
No.

Test Item

Corrected Opacity

Corrected
OD490 Value

IVIS

1

 

0.50

0.022

 

2

Negative

0.75

0.045

 

3

Control

1.05

0.015

 

MV

 

0.77

0.027

1.18

4

 

31.03

0.958

 

5

Positive

26.87

1.139

 

6

Control

26.45

0.878

 

MV

 

28.12

0.991

42.99

7

 

1.41

-0.013

 

8

Test Item

1.44

-0.010

 

9

 

1.98

-0.017

 

MV

 

1.61

-0.014

1.41

MV = mean value

 

Table 4: Historical Mean In Vitro Irritation Score of the Positive Control

 

IVIS Positive Control - Ethanol 100 %

Mean Value (MV)

48.57

Standard Deviation (SD)

9.69

MV- 2xSD

29.20

MV+2xSD

67.94

Number of Replicates providing Historical Mean: 50

Positive controls are updated after every single experiment or at
least every 3 months
 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met