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Toxicological information

Eye irritation

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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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
other: white to off-white powder
Details on test material:
- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no.
0010416
- Expiration date of the lot/batch:
April 2019
- Purity test date:
5 october 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

Test animals / tissue source

Species:
cattle
Strain:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS)
- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL .

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution):20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution):0.9% sodium chloride solution
- Lot/batch no. (if required): Batch no. 173568002; B. Braun Melsungen AG, 34212 Melsungen, Germany
Duration of treatment / exposure:
240 min
Number of animals or in vitro replicates:
Three corneas were used for each treatment group (test item, negative control and positive control).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The 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.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED 0.9% sodium chloride solution

POSITIVE CONTROL USED 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME 750 µL / 240 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

IVIS UN GHS
≤ 3 No Category
> 3 and ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean group
Run / experiment:
240 min exposure time
Value:
0.285
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
test material
Irritation parameter:
in vitro irritation score
Remarks:
cornea 7
Run / experiment:
240 min exposure time
Value:
0.41
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
test material
Irritation parameter:
in vitro irritation score
Remarks:
cornea 8
Run / experiment:
240 min exposure time
Value:
0.569
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
test material
Irritation parameter:
in vitro irritation score
Remarks:
cornea 9
Run / experiment:
240 min exposure time
Value:
-0.123
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
test material
Irritation parameter:
in vitro irritation score
Remarks:
mean group
Run / experiment:
240 min exposure time
Value:
0.442
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
vehicle 0.9% NaCl
Irritation parameter:
in vitro irritation score
Remarks:
mean group
Run / experiment:
240 min exposure time
Value:
89.075
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Positive control 20% Imidazol

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions IsoQure UR 300 tested in the in vitro BCOP test method, had an IVIS value of 0.285, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.
Executive summary:

The purpose of this study was to determine a possible potency of IsoQure UR 300 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 cornea in vitro. Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% IsoQure UR 300as recommended in the test guideline 437 for non-surfactant solids.0.9% NaCl solution was used as the solvent control and 20% Imidazole in0.9% 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 was 240 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 IsoQure UR 300 a mean opacity of 0.305±0.354 and a mean permeability value of <0.001 compared to the negative control were determined. The calculated IVIS of 0.285±0.362 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.