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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-19 to 2016-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adpted July 26, 2013
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 09, 2010
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraethylammonium benzoate
EC Number:
240-957-3
EC Name:
Tetraethylammonium benzoate
Cas Number:
16909-22-1
Molecular formula:
C8H20N.C7H5O2
IUPAC Name:
tetraethylazanium benzoate
Test material form:
other: liquid (solution)
Details on test material:
tetraethylammonium benzoate manufactured by Evonik Degussa GmbH, batch BRA 1434, purity 94.4 w/w (excluding solvent)
Specific details on test material used for the study:
The test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% (w/v).

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial
contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL . Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
750 µl test item, 20% suspension in 0.9% sodium chloride solution (w/v)
750 µl solvent control item, 0.9% sodium chloride solution
750 µl positive control item, 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution
Duration of treatment / exposure:
Exposure period: 240 minutes
Duration of post- treatment incubation (in vitro):
After rinsing the corneas were incubated at 32°C ± 1°C for 90 ± 5 minutes. After this post incubation period, the corneas were examined.
Number of animals or in vitro replicates:
Three corneas were used for each treatment group (test item, solvent control and positive control).
Details on study design:
PREPARATION OF BOVINE EYES
- Corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium)
chambers
- The chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM)
- Corneal holder was equilibrated at 32 ± 1°C for at least one hour
- After equilibration period, fresh pre-warmed EMEM was added to both chambers
- Baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation,
neovascularisation) or an opacity >7 opacity units were discarded
- Mean opacity of all equilibrated corneas was calculated by use of an opacitometer
- A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas
- The remaining corneas were then distributed into treatment, solvent and positive control groups

ADMINISTRATION
- Three corneas were used for each treatment group
Negative control item: 0.9% sodium chloride solution
Positive control item: 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution
Test item: 20% suspension in 0.9% sodium chloride solution (w/v)
- Exposure period: 240 minutes
- After the exposure period of 240 minutes (the recommended exposure time for non-surfactant solids) the test item, the solvent control, the negative and positive controls, were removed from each chamber.
- Subsequently, the epithelium was washed with EMEM containing phenol red at least three times.
- Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.
- The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.
- The chamber was then filled with EMEM without phenol red.

EXAMINATION
- Corneal injury was assessed by evaluating the opacity and permeability of the cornea
- Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
resulting in opacity values measured on a continuous scale
- To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior
chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM
- The holder was incubated in a horizontal position at 32 ± 1°C for 90 ± 5 minutes
- Amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader
(Tecan Sunrise Magellan Version 6.412).
- Measurements at 490 nm were recorded as optical density (OD490).
- The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.






Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
BOVINE CORNEAL OPACITY AND PERMEABILITY TEST
Value:
0.302
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Any other information on results incl. tables

Opacity Values

 

Cornea No.

Opacity [Opacity Units]

Corrected Opacity

 

Mean of group

Standard deviation

0.9% NaCl

1

-0.877

-0.824

-0.824

0.092

2

-0.718

3

-0.876

20% Imidazol

 

4

88.566

89.390

78.354

20.510

5

53.865

54.689

6

90.160

90.984

20%

Test item

7

-0.518

0.306

0.452

0.476

8

0.160

0.984

9

-0.757

0.067

Permeability OD Values (490 nm)

 

Cornea no.

Permeability
[OD]

 

Mean of Tripli-

cates

Corrected Permeability [OD]

 

Per Cornea

Per Group

 

Mean

SD

Mean

SD

0.9% NaCl

1

0.030

0.030

-

 

 

0.025

0.010

0.029

-

 

 

0.030

-

0.030

0.001

2

0.013

0.013

-

0.013

0.001

0.013

-

0.012

-

3

0.030

0.032

-

0.032

0.002

0.033

-

0.033

-

20% Imidazol

 

4

1.582

1.601

1.557

1.576

0.017

1.736

0.139

1.604

1.579

1.616

1.591

5

1.840

1.835

1.815

1.810

0.014

1.820

1.795

1.846

1.821

6

1.832

1.847

1.807

1.822

0.022

1.838

1.813

1.872

1.847

20%

Test item

7

0.007

0.006

-0.018

-0.019

0.001

-0.010

0.017

0.006

-0.019

0.005

-0.020

8

0.004

0.004

-0.021

-0.021

0.001

0.005

-0.020

0.003

-0.022

9

0.036

0.035

0.011

0.010

0.002

0.036

0.011

0.032

0.007

SD : standard deviation

OD : optical density

In vitro irritancy score (IVIS)

 

Cornea No.

Opacity

Permeability

IVIS

Per Cornea

Per Group

Mean

SD

0.9% NaCl

1

-0.877

0.030

-0.427

-0.449

0.066

2

-0.718

0.013

-0.523

3

-0.876

0.032

-0.396

20% Imidazol

4

89.390

1.576

113.030

104.394

19.711

5

54.689

1.810

81.839

6

90.984

1.822

118.314

20% Test item

7

0.306

-0.019

0.021

0.302

0.332

8

0.984

-0.021

0.669

9

0.067

0.010

0.217

SD : standard deviation

Applicant's summary and conclusion

Conclusions:
Under the present test conditions test item, tested in the in vitro BCOP test method, had an IVIS value of 0.302, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.
Executive summary:

The purpose of this study was to determinea possible potency of test item of being'ocular corrosive and severe irritant'employing an in vitro system.

The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability inisolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was dissolved in a 0.9% sodium chloride solution with a final concentration of 20% test item as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with test item a mean opacity of 0.452 ± 0.476 and a mean permeability value of <0.01 compared to the negative control were determined. The calculated IVIS of 0.302 ± 0.332 is below the cut-off value of 3 (UN GHS no category). Hence,the test item did not show severely irritant or corrosive properties andconsequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

Conclusion

Under the present test conditions test item, tested in the in vitro BCOP test method, had an IVIS value of 0.302, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.