Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-02-01
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)
Version / remarks:
2013
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch Number: 159
- Expiration date of the lot/batch: 30 April 2017
- Purity test date:Not reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature
- Stability under test conditions:Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. The test substance was administered directly.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:The test substance was administered directly. Physiological saline (0.9% NaCl) was used as a moistner.
- Preliminary purification step (if any): Not reported.
- Final dilution of a dissolved solid, stock liquid or gel:Not reported.
- Final preparation of a solid:Not reported.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany (isolated corneas obtained as by-product from animals freshly slaughered)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS containing Pen/strep on ice to the laboratory
- Time interval prior to initiating testing: after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: eyes presenting defects were discarded
The corneas was excised leaving a 2 to 3 mm rim of sclera and stored in a petri dish containig HBSS and posterior mounted on corneal holders . The corneas (which are free of defects) were incubated for one hour at 32 °C +/-1.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- suspended with physiological saline 0.9% NaCl (B. Braun Melsungen lot no. 1406787) to gain a 20% concentration

Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 ±1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
Duration of post- treatment incubation (in vitro):
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C

QUALITY CHECK OF THE ISOLATED CORNEAS:
Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes (treated with physiological saline 0.9% NaCl)

POSITIVE CONTROL USED: yes (treated with imidazole 20% in physiological saline 0.9% NaCl )

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of the test item preparation (20% concentration) or the control substance. Exposure 4 hours ± 5 minutes

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times with MEM (either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red)

- POST-EXPOSURE INCUBATION: 90 minutes at 32 ± 1 °C


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry] (OD490)
- Others (e.g, pertinent visual observations, histopathology): pertinent visual observation and histopathology (the corneas of the test group and control groups were fixed in a 10% formalin-buffered solution for histopathological assessment; histological processing and evaluation of all preserved tissues to haematoxylin and eosin stained microscope slides (approximate thickness of 4 µm))


SCORING SYSTEM: In Vitro Irritancy Score (IVIS). This was detailed in the study report as follows:
= 3 = No UN GHS Category
> 3; = 55 = No UN GHS Prediction can be made
>55 = Category 1.
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.

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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
244.84
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
At the macroscopic assessment, all of the 3 corneas treated with the substance showed complete opacity of the tissue and were reported to be distinctively different from controls. They were all consistently characterized by diffuse moderate epithelial vacuolation, both intracellular
and extracellular located.
This vacuolation was consistently most pronounced in the mid-layers of the epithelium, associated with multifocal minimal to slight cleft/bulla (vesicle) formation. Vacuoles were either optically empty or filled with pale amphiphilic to blue grey amorphous to finely granular material

Any other information on results incl. tables

According to the evaluation criteria and the results of histopathology the test substance is classified into UN GHS Category 1 Eye Irritant.

 

Under the conditions of this study, the test substance (20%; in saline) caused moderate epithelial vacuolation in all corneas, moderately to markedly increased diffuse cytoplasmic acidophilia (eosinophilia) with marked brown cytoplasmic colour of the most superficial layers and in 1/3 corneas, slight cell swelling of the superficial corneal epithelial layers. The vacuolation was more pronounced in the mid-layers of the corneal epithelium, and was associated with multifocal minimal to slight bulla/cleft (vesicle) formation. Slightly increased cytoplasmic basophilia, along with minimal mitotic figures, were noted in the deep/basal layers of all corneal epithelium. Increased magnitude of multifocal SCN/apoptosis was also present in basal and mid-layers of the corneal epithelium in 2/3 corneas. Minimal and moderate endothelial necrosis and minimal to slight keratocyte SCN/apoptosis were also present in 2/3 corneas. In one cornea, there was also focal linear moderate ulcer that was considered pre-existing, but favoured higher degree of stromal and endothelial degenerative changes.

Major changes were noted in all layers of the positive control corneas, supporting the validity of the assay.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
According to the evaluation criteria and the results of histopathology, the test substance is classified as a Category 1 Eye Irritant accoring to CLP criteria.