Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Dec. 07, 2017 to Dec. 07, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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)
Version / remarks:
Oct. 09, 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160
Version / remarks:
“Guidance Document On “The Bovine Corneal Opacity and Permeability (BCOP) and Isolated Chicken Eye (Ice) Test Methods: Collection of Tissues for Histological Evaluation and Collection of Data on Non-Severe Irritants”; 25. Oct. 2011
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:
Feb. 14, 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test Substance Name: Zinc acrylate
Batch no: SC1703131C
Test Facility Identification Number: 17102308G
Appearance: White powder
Composition: 99.5% Zinc acrylate
Purity: 99.5% Zinc acrylate
Homogeneity: Homogeneous
Expiry date: Sep. 09, 2018
Storage: Room emperature: (20 ± 5°C), keep away from humidity.

Test animals / tissue source

Species:
cattle
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Test System: Bos primigenius Taurus (fresh bovine corneas). Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the Day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1°C) without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1°C.

Test system

Vehicle:
Hank's balanced salt solution
Remarks:
Batch no.: 20171207, 10-fold concentrated, diluted in demineralised water (1:10)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test substance Preparation: The test substance was a solid non-surface-active substance. It was tested as a suspension with a concentration of 20% in HBSS. The suspension was prepared freshly on the Day of the assay.
Duration of treatment / exposure:
Incubation time: 4 h
Number of animals or in vitro replicates:
For each treatment group [negative control solution (HBSS), test substance and positive control (20% imidazole solution)], three replicates were used.
Details on study design:
OPACITY TEST
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, test substance suspension and positive control solution were applied to each replicate. The test or control preparations were given directly on the epithelium in such a manner that as much as possible of the cornea was covered with test or control substances. Exposure time on the corneas was 4 h at 32 ± 1°C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

PERMEABILITY TEST
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 min. at 32 ± 1°C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three replicates
Value:
ca. 121.95
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Remarks:
Relative Standard Deviation IVIS: 7.22%; The test substance induced serious eye damage on the cornea of the bovine eye.
Other effects / acceptance of results:
In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I.

Any other information on results incl. tables

Opacity and Permeability Values

 

The illuminance (unit: LUX) values measured before and after exposure

Parameter

Negative Control

Test substance

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

(I) Measured values before exposure

968

995

972

999

1022

975

994

989

979

(I) Measured values after exposure

969

938

926

250

243

267

358

390

352

Rep. = Replicate

 

Opacity Values Negative Control

Parameter

Negative Control

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

3.85

2.67

3.67

Opacity after exposure

3.80

5.23

5.81

Opacity Difference

-0.04

2.56

2.14

Mean Opacity Difference

1.55

 

Opacity Values Test substance and Positive Control

Parameter

Test substance

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

2.51

1.56

3.54

2.72

2.93

3.36

Opacity
after exposure

128.11

132.93

117.44

77.57

67.97

79.56

Opacity
Difference

125.60

131.37

113.90

74.85

65.04

76.20

Opacity Difference corrected

124.05

129.82

112.35

73.30

63.49

74.65

Mean Opacity Difference corrected

122.08

70.48

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value.

Optical density at 492 nm of Blank

Parameter

cMEM without phenol red

1. Measurement

0.041

2. Measurement

0.035

3. Measurement

0.042

Mean

0.039

 

Optical density at 492 nm of Negative Control, Test substance and Positive Control

Parameter

Negative Control

Test substance

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

1 Measurement

0.068

0.050

0.060

0.040

0.050

0.060

1.435

2.137

1.411

2. Measurement

0.064

0.047

0.054

0.036

0.048

0.057

1.463

2.140

1.413

3. Measurement

0.069

0.048

0.050

0.039

0.049

0.057

1.465

2.143

1.396

 

1. Measurement – blank

0.0287

0.0107

0.0207

0.0007

0.0107

0.0207

1.3957

2.0977

1.3717

2. Measurement – blank

0.0247

0.0077

0.0147

-0.0033

0.0087

0.0177

1.4237

2.1007

1.3737

3. Measurement – blank

0.0297

0.0087

0.0107

-0.0003

0.0097

0.0177

1.4257

2.1037

1.3567

Mean of each replicate

0.0277

0.0090

0.0153

-0.0010

0.0097

0.0187

1.4150

2.1007

1.3673

Mean of the 3 replicates

0.0173

--

--

Corrected

--

--

--

-0.0183

-0.0077

0.0013

1.3977

2.0833

1.3500

Corrected mean of the

3 replicates

--

-0.0082

1.6103


IVIS Value

The calculated IVIS for each replicate and the corresponding means

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

0.37

1.81

69.47%

2.69

2.37

Test substance
Zinc acrylate

123.78

121.95

7.22%

129.71

112.37

Positive Control
20% imidazole solution

94.27

94.64

0.35%

94.74

94.90

Validityof the Study: The validity criteria and findings

Parameter

Criterion

Found

Assessment

IVIS of negative control

HBSS

3

1.81

ok

IVIS of positive control
20% imidazole solution

70.92 – 157.64

94.64

ok

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under study conditions, the test substance was determined to induce serious eye damage on the cornea of the bovine eye.
Executive summary:

A study was conducted to determine the eye irritation potential of test substance using the in vitro Bovine Corneal Opacity and Permeability (BCOP) test method according to OECD Guideline 437, in compliance with GLP. The BCOP test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. Fresh bovine eyes were obtained from the slaughterhouse on the day of the test. The cattle were between 12 and 60 months old. The corneas were screened for test requirements, prepared and mounted in corneal holders. Prior to treatment, the test system was previously incubated with cMEM without phenol red at 32 ± 1°C for 1 h and Corneal opacity was determined. The test substance preparation [20% suspension in Hank’s Balanced Salt Solution (HBSS)] was incubated on the cornea for 4 h at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. For each treatment group [negative control solution (HBSS), test substance and positive control (20% imidazole solution)], three replicates were used. In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category I .The negative control and the positive control met the validity criteria. The calculated IVIS (In Vitro Irritancy Score) was 121.95. Under study conditions, the test substance was therefore determined to induce serious eye damage on the cornea of the bovine eye (Geitlinger, 2018).