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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:
2016-3-2 to 2016-7-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The Bovine Corneal Opacity and Permeability (BCOP) test method is an in vitro test method that can be used to classify substances as “ocular corrosives / severe irritants” and “non-irritants”. The BCOP is recommended for use as part of a tiered-testing strategy for regulatory classification and labelling within a specific applicability domain. Test substances can be classified as ocular corrosives / severe irritants or non-irritants without further testing in rabbits.

Data source

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

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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Cas Number:
1190427-50-9
Molecular formula:
C46H65Cl2N2PRuS
IUPAC Name:
[1,3-bis(2,4,6-trimethylphenyl)-4,5-dimethylimidazol-2-ylidene](2-thienylmethylidene)(tricyclohexylphosphine)ruthenium(II)dichloride
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
The assay used isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg

Duration of treatment / exposure:
4 hours ± 5 minutes
Number of animals or in vitro replicates:
3 corneas per test group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 > I(0)/1.1651 lux were used for the assay.

NUMBER OF REPLICATES:
3 corneas per test group

NEGATIVE CONTROL USED
750 μl physiological saline 0.9% NaCl

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
750 μl 20% imidazole in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 mg of the solid test substance was applied and moistened with physiological saline 0.9%. For controls, 750 μl of PBS or the control substanceswas introduced into the anterior chamber. Exposure time was 4 hours ± 5 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 4 at least


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lied in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
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. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in TG 437 were used (please see attached file for results and classification criteria).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
2.06
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Please see attached file for results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The following mean in vitro irritation score was calculated: 2.06
Therefore the test item was classified into UN GHS No Category.