Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Remarks:
.
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No deviations from the study plan were observed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 2017: “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
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Conformity All procedures according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: not applicable
- Activation: not required
water as a additive
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Species Bos primigenius Taurus (fresh bovine corneas)
Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old:
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 within 1 hour 15 minutes.
The following Guideline was applied: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “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
Additional information was taken from:
• “Bovine Corneal Opacity and Permeability (BCOP) Assay”, SOP of Microbiological Associates Ltd., UK, Invittox (UK) protocol no. 124, Procedure Details, April 1997, last update Aug. 1999; based on Gautheron et al. (1992), refined by Vanparys et al. (1994)
• “The Bovine Corneal Opacity and Permeability Assay – Method of Gautheron”; Invit-tox (UK) protocol no. 98, April 1996
• Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.47 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, adopted 14. Feb. 2017


Test system

Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a liquid substance. It was tested directly, without dilution or preparation of a solution. The test item was shaken before use.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Chemicals
• Minimum Essential Medium (MEM) without phenol red
• Minimum Essential Medium (MEM) with phenol red
• L-glutamine
• Sodium bicarbonate NaHCO3
• 1% fetal calf serum
• cMEM without phenol red:
495 mL MEM without phenol red are supplemented with 5 mL FCS, 146 mg L-Glutamine and 0.55 g NaHCO3
248 mL MEM without phenol red are supplemented with 2.5 mL FCS, 73 mg L-Glutamine and 0.28 g NaHCO3
(as soon as the MEM without phenol red is supplemented with FCS and L-Glutamine and NaHCO3, it is designated in the following as cMEM (= complete MEM) without phenol red)
• cMEM with phenol red:
495 mL MEM with phenol red are supplemented with 5 mL FCS and 146 mg L-Glutamine (as soon as the MEM with phenol red is supplemented with FCS and L-Glutamine, it is designated in the following as cMEM (= complete MEM) with phenol red)
• Sodium fluorescein (C20H11O5Na)
• Hank’s Balanced Salt Solution (HBSS) with Penicillin-Streptomycin solution (transport medium)
• Hank’s Balanced Salt Solution (HBSS) without Penicillin-Streptomycin solution (negative control)
• Penicillin-Streptomycin solution
Duration of treatment / exposure:
1 h + 10 min
Duration of post- treatment incubation (in vitro):
90 min
Number of animals or in vitro replicates:
3
Details on study design:
Purpose and Principle of the Study
Thisin vitro study was performed to assess the corneal damage potential of test item by quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was performed for regulatory purposes.
The 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, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an “In Vitro Irritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).
The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is 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. Test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
Mean Opacity Difference corrected
Run / experiment:
Opacity Values Test Item and Positive Control: Mean Opacity Difference corrected
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Hank’s Balanced Salt Solution (HBSS) 0.96 Mean Opacity Difference
Positive controls validity:
valid
Remarks:
Dimethylformamide (DMF) 71.35 Mean Opacity Difference corrected
Remarks on result:
other: Detailed results not available at the moment
Irritation parameter:
other: Optical density
Run / experiment:
Optical density at 492 nm
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0052
Positive controls validity:
valid
Remarks:
1.3221
Remarks on result:
other: Detailed results not available at the moment
Remarks:
Detailed results not available at the moment
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean IVIS +/- Relative Standard Deviation IVIS
Value:
ca. 0
Negative controls validity:
valid
Remarks:
Mean IVIS 1.03 +/- 8.73 %
Positive controls validity:
valid
Remarks:
Mean IVIS 91.18 +/- 15%
Remarks on result:
other: Detailed results not available at the moment
Other effects / acceptance of results:
For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables

Any other information on results incl. tables

Findings and Results

Opacity and Permeability Values

Theilluminance(unit: LUX) values which were measured before and after exposure are given in the following table:

Table9.1‑a      Illuminance Values

Parameter

Negative Control

Test Item

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

938

952

994

958

978

1010

993

989

957

(I) Measured values after exposure

917

934

972

292

282

342

377

342

386

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation:

Opacity Values Negative Control

Parameter

Negative Control

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

5.89

5.22

3.34

Opacity after exposure

6.93

6.08

4.31

Opacity Difference

1.04

0.86

0.97

Mean Opacity Difference

0.96

Rep. = Replicate

Opacity Values Test Item and Positive Control

Parameter

Test Item

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep.

2. Rep.

3. Rep.

Opacity before exposure

4.94

4.04

2.66

3.38

3.55

4.99

Opacity
after exposure

106.13

111.29

84.85

73.31

84.85

70.68

Opacity
Difference

101.18

107.25

82.19

69.93

81.29

65.69

Opacity
Difference

corrected

100.23

106.30

81.23

68.98

80.34

64.74

Mean Opacity
Difference

corrected

95.92

71.35

Rep. = Replicate

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter

cMEM without phenol red

1. Measurement

0.035

2. Measurement

0.033

3. Measurement

0.032

Mean

0.033

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

Parameter

Negative Control

Test Item

Positive Control

1. Rep.

2. Rep.

3. Rep.

1. Rep. *

2. Rep. *

3. Rep. *

1. Rep.

2. Rep.

3. Rep.

1. Measurement

0.040

0.044

0.032

0.711

0.684

0.670

1.789

1.484

0.777

2. Measurement

0.039

0.039

0.034

0.701

0.680

0.671

1.837

1.475

0.780

3. Measurement

0.041

0.042

0.036

0.705

0.679

0.670

1.842

1.489

0.773

 

1. Measurement – blank

0.0067

0.0107

-0.0013

0.6777

0.6507

0.6367

1.7557

1.4507

0.7437

2. Measurement – blank

0.0057

0.0057

0.0007

0.6677

0.6467

0.6377

1.8037

1.4417

0.7467

3. Measurement – blank

0.0077

0.0087

0.0027

0.6717

0.6457

0.6367

1.8087

1.4557

0.7397

Mean of each replicate

0.0067

0.0083

0.0007

0.6723

0.6477

0.6370

1.7893

1.4493

0.7433

Mean of the

3 replicates

0.0052

--

--

Corrected

--

--

--

3.3564

3.2331

3.1798

1.7841

1.4441

0.7381

Corrected mean of the

3 replicates

--

3.2564

1.3221

Rep. = Replicate

* Note: All values of the three replicates of the test item were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

 IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table9.2‑a      IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

1.14

1.03

8.73%

0.99

0.98

Test Item

150.57

144.77

9.58%

154.79

128.93

Positive Control
DMF undiluted

95.74

91.18

15.00%

102.00

75.81

Assessment

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

Table9.4‑a      Classification Scheme

IVIS

UN GHS Category Eye Damage

≤ 3

No category

> 3 and≤ 55

No prediction can be made

> 55

Eye damage Category I

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 test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 144.77.

 The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the conditions of this test, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 144.77.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
Executive summary:

In a Study following OECD Guideline 437, cornea of bovine eyes were incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and exposed to the test item for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In VitroIrritancy Score) is 1.03.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 91.18.

 

Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 144.77.

 

According to OECD Guideline 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I