Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Aug 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2014

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:
2013
Deviations:
yes
Remarks:
a photometer instead of a opacitometer was used.
Qualifier:
according to guideline
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. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): trade name given
- Substance type: white, crystalline powder
- Analytical purity: 99.5%
- Solubility: water: < 0.1 g/L
- Lot/batch No.: 1592ZG-076
- Expiration date of the lot/batch: Aug 2015
- Storage condition of test material: at room temperature 20 ± 5 °C

Test animals / tissue source

Species:
other: cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in 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 IVIS, which is used to classify the test item in the UN GHS System.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Müller Fleisch GmbH, Birkenfeld, Germany
- Donor animals: The cattle were between 12 and 60 months old.
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) on ice with 0.01% penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: 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 without phenol red was filled.
- Test medium and temperature conditions used in the cornea holder: Minimum Essential Medium (MEM) with and without phenol red; cMEM without phenol red supplemented with fetal calf serum, L-Glutamine and NaHCO3; cMEM with phenol red supplemented with fetal calf serum and L-Glutamine; prior to use: pre-warmed to 32±1 °C
- Equilibration time: 1 h at 32±1 °C
- Quality check of the equilibrated corneas: Yes; baseline opacity for each cornea was recorded. None of the corneas showed tissue damage. Please refer to Table 1 and 2.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by placing the holder with the cornea in a spectral photometer and recording the absorption at 570 nm. Opacity is calculated from the measured opacity following the equation:
Opacity = 10^A
A: Absorption at 570 nm
- Specification of the device: Spectral photometer, Specord 205, Analytik Jena, Germany

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 506 mg (mean)

POSITIVE SUBSTANCE
- Concentration: 20% imidazole solution in physiol. saline
- Amount(s) applied in the test: 750 µL

NEGATIVE CONTROL
- Physiological sodium chloride solution
Duration of treatment / exposure:
4 h ± 5 min at 32 °C
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
number of eyes for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: Open-Chamber method: In order to apply the test item, the nut was unscrewed to remove the glass disc. The controls or test substance were applied directly to the epithelial surface of the cornea. Corneas were exposed for 4 h ± 5 min with the test substance or the controls.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed by thorough rinsing.
- Medium for washing the corneas: cMEM containing phenol red
- Medium for final rinsing: cEMEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by measurement of the absorption at 570 nm in a spectral photometer and subsequent calculation using the following equation:
Opacity = 10^A
- Time of determination: directly after refilling fresh cMEM without phenol red in both chambers
- Specification of the device: Spectral photometer, Specord 205, Analytik Jena, Germany

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the front chamber of the cornea holder. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: 90 ± 5 min at 32 ± 1 °C
- Treatment for measuring: OD490 of an aliquot was determined in a cuvette with a path length of 0.2 cm.
- Specification of the spectrophotometer: Spectral photometer, Specord 205, Analytik Jena, Germany

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: opacity
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
0.285
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: permeability
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
0.031
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: IVIS
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
-0.585
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: opacity
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
101.116
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: permeability
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
1.247
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: IVIS
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
118.485
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: opacity
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
0.388
Reversibility:
other: not applicable
Remarks on result:
other: Negative control
Irritation parameter:
other: permeability
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
0.063
Reversibility:
other: not applicable
Remarks on result:
other: Negative control
Irritation parameter:
other: IVIS
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
1.333
Reversibility:
other: not applicable
Remarks on result:
other: Negative control
Irritant / corrosive response data:
The negative control (physiological sodium chloride solution) showed no irritating effect on the cornea.
The positive control (20% imidazole solution) induced serious eye damage on the cornea.
Under the conditions of the test system, the test substance showed no effects on the cornea of the bovine eye. The calculated In Vitro Irritancy Score (IVIS) is -0.585.

Any other information on results incl. tables

Table 1. Absorption and opacity values negative control.

Parameter

Negative control

Absorption before exposition

0.1636

0.1674

0.1628

Absorption after exposition

0.2477

0.2092

0.3342

Opacity before exposition

1.4575

1.4703

1.4548

Opacity after exposition

1.7689

1.6188

2.1587

Opacity Difference

0.3114

0.1485

0.7039

Table 2. Absorption and opacity values test substance and positive control.

Parameter

Test substance

Positive control

Absorption before exposition

0.1402

0.2017

0.1976

0.1309

0.168

0.1629

Absorption after exposition

0.2621

0.2345

0.2696

2.1271

1.8589

2.0059

Opacity before exposition

1.3810

1.5911

1.5762

1.3518

1.4723

1.4551

Opacity after exposition

1.8285

1.7159

1.8604

133.9985

72.2603

101.3678

Opacity Difference

0.4475

0.1248

0.2842

132.6468

70.7880

99.9127

Table 3. Optical density at 490 nm.

Repl.

Negative control

Test substance

Positive control

Meas.

0.0083

0.0151

0.0144

0.0070

0.0052

0.0063

0.2220

0.2637

0.2624

Corr.

0.0415

0.0755

0.0720

0.0350

0.0260

0.0315

1.1100

1.3185

1.3120

Mean

0.0630

0.0308

1.2468

Table 4. IVIS

Test group

IVIS

Mean IVIS

Relative standard deviation IVIS

Negative control

0.9% NaCl

0.934

1.333

32.1%

1.281

1.784

Test substance

- 0.360

-0.585

39.1%

- 0.818

- 0.576

Positive control
20% imidazole solution

147.964

118.485

24.8%

89.233

118.260

Table 5. Validity

Parameter

Criterion

Found

Assessment

IVIS of negative control 0.9% NaCl

0 -3

1.333

ok

IVIS of positive control 20% imidazole

35.6 – 129.2

118.458

ok

Table 6. Historical data (all experiments up to 13 May 2014).

Parameter

IVIS negative control

IVIS positive control

Substance

0.9% sodium chloride solution

20% imidazole solution

Mean

1.064

82.4

Standard Deviation

0.488

23.4

Range (min – max)

0.138 - 2.281

32.6 - 132.4

Range (validity)

0 -3

35.6 – 129.2

Study 14071601G850

1.333

118.458

All three values for negative and two values for positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified