Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-23 - 2017-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
negative control: 750 µL
positive control: 750 µL
test item: 150 mg
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse . To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.The quality of each cornea was also evaluated at later steps in the assay. 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.

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:
In a preliminary study, no homogeneous suspension of the test item could be achieved in a 0.9% sodium chloride solution due to the high viscosity of the test item. Hence, 150 mg Ukanol FR 70 (corresponding to 750 µL of the recommended concentration of 20% (w/v) by the test guideline 437 for non-surfactant solids) were applied to a plastic sheet prior to application to the cornea.The cornea was moistened with 600 µL 0.9% NaCl solution prior to application.

TREATMENT METHOD: [closed chamber / open chamber]:
The open-chamber method was used. The window-locking ring and glass window from the anterior chamber were removed prior to treatment. The control or test chemical was applied directly to the epithelial surface of the cornea using a micro-pipet or the plastic sheet with the test item-treated side, respectively. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE:
After the exposure period of 240 minutes (the recommended exposure time for non-surfactant solids) the test item, the solvent control, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was 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.
- Number of washing steps after exposure period:at least three times
- POST-EXPOSURE INCUBATION:
To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 6.4 ). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA:
A test item that induces an IVIS > 55 is defined as a corrosive or severe irritant.
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
Run / experiment:
test item
Value:
0.807
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
negative control
Value:
1.455
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
positive control
Value:
75.65
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions Ukanol FR 70 tested in the in vitro BCOP test method, had an IVIS value of 0.807, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.
Executive summary:

The purpose of this study was to determine a possible potency of Ukanol FR 70 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.In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability inisolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was 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.The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, solvent control and positive control). In a preliminary study, no homogeneous suspension of the test item could be achieved in a 0.9% sodium chloride solution due to the high viscosity of the test item. Hence, the test item was applied directly to the epithelial surface of the cornea. 150 mgUkanol FR 70 (corresponding to 750 µL of the recommended concentration of 20% (w/v) by thetest guideline 437 for non-surfactant solids) were applied to a plastic sheet prior to application to the cornea.The cornea was moistened with 600 µL0.9% NaCl solutionprior to application.0.9% NaCl solution was used as the solvent control and 20% Imidazole in0.9% NaCl solution as the positive control item.

750 µL of the control items as recommended as suitable test volume according to OECD TG 437 were added to completely cover the cornea’s epithelium. 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 open-chamber method was used.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with Ukanol FR 70 a mean opacity of 0.942±0.738 and a mean permeability value of <0.01compared to the negative control were determined. The calculated IVIS of 0.807±0.590 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 as a severe irritant and is not corrosive according to UN GHS classification.