Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013-07-26
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Version / remarks:
April 1997
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): tin difluoride
- Physical state: solid, clear

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir (Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany)
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were transported to the laboratory in Hank’s Buffered Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. After removing the corneas from the eyes, they were also collected in Hank’s BSS supplemented with streptomycin / penicillin.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

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): 0.75 mL
- Concentration: 20% suspension (w/v) in vehicle
Duration of treatment / exposure:
240 ± 15 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The isolated corneas were also examined macroscopically for defects.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- each corneae with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative control or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 ± 15 minutes).
- after the incubation time, the test item or control items were rinsed off with saline
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240)
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with the spectrophotometer.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula was used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the individual IVIS values which was determined from each individual treatment and positive control cornea.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below)

DECISION CRITERIA:
The test was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Value:
440.06
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.24).
- the positive control (10% (w/v) Benzalkonium chloride in saline) showed distinct opacity of the corneae (mean IVIS = 117.97) corresponding to a classification as serious eye damaging (CLP (Cat 1)).

Please refer to the field "Any other information on results incl. tables" below.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Relative to the negative control, the test item caused a significant increase of the corneal opacity and permeability. In conclusion, according to the current study and under the experimental conditions reported, the substance is serious eye damaging (the calculated IVIS = 440.06). A substance with an in vitro irritation score ≥ 55 does require classification for eye irritation and serious eye damage according to OECD 437 (2013). According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1).