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Diss Factsheets

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
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
EC Number:
928-779-0
Molecular formula:
C18-H21-N-O4 x HBr
IUPAC Name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
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: 600CC
- Expiration date of the lot/batch: 28 February 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: Immediatley fter 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
Remarks:
0.9 % Sodium Chloride Solution (aq)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg of the test substance. 750 uL of the positive control.

VEHICLE
- Amount(s) applied (volume or weight with unit): Physiological saline 0.9% NaCl was applied as a moistner. Volumes not reported.
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. Eyes that were noted to have defects were discarded and not used on the study.

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 mg of the test item and 750 µl per control substance. Exposure 4 hours ± 5 minutes.

TREATMENT METHOD: Test substance (open-chamber method), control substance (closed-chamber method)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test item, 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:
12.64
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
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 resulted in opacity and permeability values which were less than the established upper limits for background bovine corneas treated with the respective negative control.
At the microscopic assessment, all of the 3 corneas treated with the test substance showed a slight opacity of the tissue and were reported to be distinctively different from controls. They were all consistently characterized by moderately increased cytoplasmic acidophilia (eosinophilia) of the most superficial layers of the corneal epithelium, sometimes submembranous, with occasional extension of this change in individual cells of the mid and basal layers. This main change was associated with slight to moderate desquamation and a brown cytoplasm involving the most superficial layers. Minimal to slight cytoplasmic vacuolation and cell swelling was also consistently noted. In the most affected cornea moderate desquamation was associated with slight epithelial erosion/ulcer, and slight necrosis of the corneal endothelium.

Any other information on results incl. tables

The mean in vitro irritation score was calculated to be 12.64. According to IVIS criteria, no predication could be made regarding the UN GHS classification of the test substance.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
No prediction can be made regarding the classification of the test substance according to the evaluation criteria. However, all 3 corneas treated with the substance showed a slight opacity of the tissue.
Based on a worst case decision, the substance will be classified for CLP Category 1 (H318).