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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:
28 Jan - 03 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline Study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
Due to small amount of the test item no retention sample is stored at the test facility.
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- name of test material (as cited in study report): Reaction mass of tetra (potassium and sodium) (2R,3R)-2-hydroxy-3-(phosphonatooxy)succinate and tetra (potassium and sodium) (2S,3S)-2-hydroxy-3-(phosphonatooxy)succinate
- physical state: White to slightly yellowish powder of crystalline appearance

Test animals / tissue source

Species:
other: cattle
Strain:
other: bovine eyes
Details on test animals or tissues and environmental conditions:
Identification of the source of the eyes, storage and transport conditions:
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, Lindern, Germany
- Donor animals: age: 6 to 12 months
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) containing penicillin (100 IU/mL) and streptomycin (100 μg/mL)
Preparation of the eyes (before exposure)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders with anterior (epithelium) and posterior (endothelium) chambers.
- Test medium and temperature conditions used in the cornea holder:
Eagle´s Minimum Essential Medium (EMEM) with and without phenol red; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacity of 3 (Note: Corneas with an initial opacity above 7 in the opacitometer or with any macroscopic defects were discarded.)
Determination of the initial opacity:
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany
- 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution)
- microplate reader: Tecan Sunrise Magellan Version 6.4, Tecan Deutschland GmbH, 74564 Crailsheim, Germany

Test system

Vehicle:
physiological saline
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
20% solution in 0.9% sodium chloride solution (w/v)
Duration of treatment / exposure:
4 h at 32 ± 1 °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: closed-chamber method
The controls or test substance were added to completely cover the corneas's epithelium in the anterior chamber. Corneas were exposed for 4 h with the test substance or the controls.
Post-exposure treatment:
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed at least three times.
- Medium for washing the corneas: EMEM containing phenol red
- Medium for final rinsing: EMEM without phenol red
Determination of the final opacity:
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling fresh EMEM without phenol red in the anterior chamber the final opacity was measured.
- Specification of the device: BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany
Determination of the corneal permeability:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. 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; dissolved in 0.9% sodium chloride solution)
- Incubation time: 90 ± 5 min at 32 ± 1 °C
- Treatment for measuring: OD490 in a microplate.
- Specification of the spectrophotometer: Tecan Sunrise, Tecan Deutschland GmbH, 74564 Crailsheim, 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:
< 0.01
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:
< 0.01
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:
< 0.01
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:
ca. 73.413
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:
ca. 0.808
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:
ca. 85.528
Reversibility:
other: not applicable
Remarks on result:
other: positive control
Irritant / corrosive response data:
The acceptance criteria for the solvent control are met.

The calculated IVIS value for the positive control was within two standard deviations of the current historical mean and well above the cut-off value of 55. Thus, the acceptance criteria for the test were fulfilled.

The calculated IVIS value for the test substance is below the cut-off value of 3 (UN GHS no category). Thus, the test substance is not classified for eye irritation or serious eye damage using the BCOP test method.
Other effects:
not applicable

Any other information on results incl. tables

Table 1: Opacity Values

 

Cornea No.

Opacity [Opacity Units]

Corrected Opacity

 

Mean of group

Standard deviation

0.9% NaCl

1

1.594

1.408

1.408

0.227

2

1.155

3

1.474

20% Imidazol

4

70.877

69.469

73.413

6.455

5

71.315

69.907

6

82.270

80.862

PWS

(20% w/v)

7

0.677

-0.731

-1.634

1.330

 

 

Table 2: In vitro irritancy score (IVIS)

 

Cornea No.

Opacity

Permeability

IVIS

Per Cornea

Per Group

Mean

SD

0.9% NaCl

1

1.594

0.020

1.894

1.733

0.333

2

1.155

0.013

1.350

3

1.474

0.032

1.954

20% Imidazol

4

69.469

0.814

81.679

85.528

6.265

5

69.907

0.816

82.147

6

80.862

0.793

92.757

PWS (20% w/v)

7

-0.731

-0.005

-0.806

-1.664

1.351

8

-1.010

0.003

-0.965

9

-3.161

-0.004

-3.221

 

 

Applicant's summary and conclusion

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