Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 437 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(2010)
Deviations:
no
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material in report: IFF 09-0035
- Physical state: Clear liquid
- Stability at higher temperatures: Yes, maximum temperature: 40°C, maximum duration: 60 minutes
- Storage condition of test material: At room temperature protected from light

Test animals / tissue source

Species:
other: Bovine eyes from young cattle
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands). Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: positive control: 10% (w/v) Benzalkonium Chloride
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
120 minutes
Number of animals or in vitro replicates:
Three corneas / substance or control.
Details on study design:
Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Selection of corneas and opacity readings
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.Three corneas were selected at random for each treatment group.

Treatment of corneas
The medium from the anterior compartment was removed and 750 μL of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

Histopathology
Not applicable.

Evaluation of results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numericalopacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substancetreated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Permeability measurement
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

In vitro irritancy score
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

Visual observation
After the 10 minutes exposure, possible pH effects of the test substance on the corneas were recorded. After the 120 minutes incubation period, Each cornea was inspected visually for dissimilar opacity patterns.

Results and discussion

Results of in vivo study
Irritation parameter:
overall irritation score
Remarks:
(In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 10 minutes
Score:
1.1
Reversibility:
other: Not applicable
Irritant / corrosive response data:
Corneal epithelium condition
The corneas were clear after the 10 minutes of treatment with IFF 09-0035 and the negative control. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment.
In vitro irritancy scores:
The in vitro irritancy score for the test item was 1.1
The in vitro irritancy score for the negative control was 0.0
The in vitro irritancy score for the positive control was 178.3
Other effects:
No other effects were observed.

Any other information on results incl. tables

Criterion for an acceptable test

The positive control in vitro irritancy score was within the range of 174.9 to 180.5. The positive control acceptance criterion was therefore satisfied.

The corneas treated with IFF 09-0035 showed opacity values ranging from -0.3 to 3.7 and permeability values ranging from -0.009 to 0.018. The corneas were clear after the 10 minutes of treatment with IFF 09-0035. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.5 to 3.9 after 10 minutes of treatment with IFF 09-0035.

Opacity score

Eye

Opacity

before treatment

Opacity

after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Opacity

Negative control

1

2

5

3

0.7

0.0

2

0

3

3

0.7

3

3

4

1

-1.3

 

Mean final opacity: 2.3

 

Positive control

4

2

79

77

74.7

85.3

5

2

108

106

103.7

6

2

82

80

77.7

IFF 09-0035

7

0

6

6

3.7

1.0

8

0

2

2

-0.3

9

3

5

2

-0.3

1 Final Opacity = Opacity after treatment – Opacity before treatment.

2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

Permeability score individual values (uncorrected)

Eye

Dilution factor

OD490

1

OD490

2

OD490

3

Average OD

Final OD

Mean final negative control

Negative control

1

1

0.015

0.015

0.012

0.014

0.014

0.007

2

1

0.004

0.001

0.001

0.002

0.002

3

1

0.006

0.006

0.004

0.005

0.005

Positive control(Benzalkonium Chloride)

 

4

6

1.126

1.116

1.119

1.120

6.722

 

5

6

0.854

0.854

0.845

0.851

5.106

 

6

6

1.143

1.133

1.173

1.150

6.898

 

IFF 09-0035

 

7

1

0.022

0.032

0.020

0.025

0.025

 

8

1

0.001

-0.003

-0.003

-0.002

-0.002

 

9

1

0.011

0.011

0.024

0.015

0.015

 

Permeability score individual values (corrected)

Eye

Dilution factor

Negative control corrected OD49011

Negative control corrected OD49021

Negative control corrected OD49031

Negative control corrected OD490

Average

Negative control corrected final

OD490

Average OD

Negative control

1

1

0.008

0.008

0.005

0.007

0.007

0.000

2

1

-0.003

-0.006

-0.006

-0.005

-0.005

3

1

-0.001

-0.001

-0.003

-0.002

-0.002

Positive control(Benzalkonium Chloride)

4

6

1.119

1.109

1.112

1.113

6.679

6.199

5

6

0.847

0.847

0.838

0.844

5.063

6

6

1.136

1.126

1.166

1.143

6.855

IFF 09-0035

7

1

0.015

0.025

0.013

0.018

0.018

0.006

8

1

-0.006

-0.010

-0.010

-0.009

-0.009

9

1

0.004

0.004

0.017

0.008

0.008

1 OD490values corrected for the mean final negative control permeability (0.007).

In Vitro irritancy score

Eye

Negative control correctedFinal Opacity

Negative control correctedFinal OD490

In vitroIrritancy Score1

Negative control

1

0.7

0.007

0.8

2

0.7

-0.005

0.6

3

-1.3

-0.002

-1.4

Positive control(Benzalkonium Chloride)

4

74.7

6.679

174.9

5

103.7

5.063

179.6

6

77.7

6.855

180.5

IFF 09-0035

7

3.7

0.018

3.9

8

-0.3

-0.009

-0.5

9

-0.3

0.008

-0.2

1  In vitroirritancy score (IVIS) = opacity value + (15 x OD490value).

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant, as IFF 09-0035 induced an IVIS ≤ 3.
Executive summary:

The eye irritancy potential of the test substance was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and GLP principles.

IFF 09 -0035 did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment.

As IFF 09 -0035 induced an IVIS ≤ 3, the substance is considered not to be an ocular corrosive or severe irritant, according to EU CLP criteria.