Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 2015; signature: November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light, container flushed with nitrogen. Use amber glassware or wrap container in
aluminum-foil
- Other: Colourless to pale yellow liquid

Test animals / tissue source

Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse.
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 ±1 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND 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. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Earle’s Minimum Essential Medium (cMEM) and the holders were incubated at 32 ± 1 ºC for a minimum of 1 hour. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Only corneas with opacity ≥ 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol ; > 99.9% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. Following exposure the holders were incubated, for 120 ± 10 minutes at 32 ± 1°C. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
- POST-EXPOSURE INCUBATION: Following the final opacity measurement the posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: 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). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were turbid post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:
1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean. ACTUAL: PC IVIS range of 34.7 to 78.2. Mean = 48.0.
2. Physiological saline solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤3.0 and for permeability ≤0.042. ACTUAL: PC IVIS = 1.1, opacity ≤ 1.8 and permeability ≤ 0.007.

Any other information on results incl. tables

Table 1 Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity #1

Mean Permeability #1

Mean In vitro Irritation Score #1 , 2

Negative Control

1.2

-0.001

1.1

Positive Control

(ethanol)

22

1.778

48.0

Test item

0.1

0.004

0.2

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2 Opacity score

Eye

Opacity before treatment

Opacity after treatment

Final opacity #1

Negative control corrected final opacity #2

Mean Opacity

Negative Control

1

2.9

4.7

1.8

-

1.2

2

4.0

4.4

0.4

-

3

3.1

4.4

1.3

-

Positive Control

4

3.9

30.9

27.0

26

22

5

3.6

23.7

20.1

19

6

1.8

22.8

21.0

20

Test item

7

3.8

5.2

1.4

0.3

0.1

8

2.6

3.2

0.6

0.5

9

3.0

4.8

1.8

0.7

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

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

 

Table 3 Permeability score individual values (uncorrected)

Eye

Dilution Factor

OD490 1

OD490 2

OD490 3

Average OD490

Final OD

Mean Final Negative Control

 

Negative Control

 

1

1

-0.011

-0.004

-0.011

-0.009

-0.009

0.000

2

1

0.002

0.007

0.013

0.007

0.007

3

1

-0.003

-0.004

-0.002

-0.003

-0.003

 

Positive Control

 

4

1

1.320

1.317

1.331

1.323

1.323

5

6

0.332

0.332

0.339

0.334

2.006

6

6

0.339

0.322

0.332

0.331

1.986

 

Test substance

7

1

0.0028

0.013

0.019

0.02

0.020

8

1

-0.007

-0.006

-0.008

-0.007

-0.007

9

1

-0.004

-0.004

-0.007

-0.005

-0.005

 

Table 4 In vitro irritancy score

Eye

Negative control corrected final opacity

Negative control corrected Final OD490

In vitro Irritancy Score #1

Negative Control

1

1.8

-0.009

-0.1

2

0.4

0.007

0.0

3

1.3

-0.003

-0.9

Positive Control

4

26

1.324

46

5

19

2.015

49

6

20

1.995

50

Test substance

7

0.3

0.021

0.6

8

-0.5

-0.006

-0.6

9

0.7

-0.004

0.6

#1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria not met
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be irritating to the eye.
Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the test item was placed the cornea. The negative control group received physiological saline and the positive control group received neat ethanol. For each group the corneas were incubated for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) 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. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 48.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from -0.5 to 0.7 and permeability values ranging from -0.006 to 0.021 and in vitro irritancy scores ranged from -0.6 to 0.6. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 10 minutes of treatment. Based on these results the test item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.