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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Potassium superoxide induction of rabbit corneal endothelial cell damage
Author:
Hull DS
Year:
1984
Bibliographic source:
Curr Eye Res. 3(11):1321-8

Materials and methods

Principles of method if other than guideline:
Perfusion of rabbit corneal endothelial cells with a Krebs Ringer bicarbonate solution to which the test substance had been added.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium superoxide
EC Number:
234-746-5
EC Name:
Potassium superoxide
Cas Number:
12030-88-5
Molecular formula:
KO2
IUPAC Name:
potassium superoxide
Test material form:
solid

Test animals / tissue source

Species:
rabbit
Strain:
other: albino
Details on test animals or tissues and environmental conditions:
Corneal endothelial cells mounted in a specular microscope

Test system

Vehicle:
other: Krebs Ringer bicarbonate solution containing 28 mM glucose
Remarks:
without adenosine and glutathione
Controls:
yes, concurrent vehicle
Amount / concentration applied:
Concentrations of 0.3, 0.5, 0.7 and 1.0 mM
Duration of treatment / exposure:
1 hour (at a perfusion rate of 1 mL/h) after a rapid perfusion with 10 mL of the solution
Duration of post- treatment incubation (in vitro):
one half hour
Number of animals or in vitro replicates:
5 corneas per experimental group
Details on study design:
Perfusion of rabbit corneal endothelial cells (at 37°C, 15 mm Hg,1 mL/h) with a Krebs Ringer bicarbonate solution (bubbled mixture of 97% oxygen - 3% CO2, mean osmolarity: 308mOsm, pH: 7.3) to which the test substance had been added (with in addition: SOD, catalase, ascorbic acid, D-mannitol, EDTA, DETAPAC, EDTA + FeCL2, or DMSO). Silicone oil (Dow Corning 360 Medical Fluid) was placed on the epithelial surface to prevent dehydration.

After an 1 hour stabilization period with a perfusion of KBR, the corneal thickness was recorded. KBR was then added to the pulverized potassium superoxide. Immediately after, corneas were perfused rapidly with 10 ml of the KO2 solution to completely replace the KBR into the microscope perfusing chamber. The perfusion with the KO2 solution continued for 1 hour at a rate of 1 ml/h and was followed by an one-half hour perfusion with KBR.

Tissues were prepared for either electron microscopy or determination of intracellular glutathione. The concentration of H2O2 was detrmined by spectrophotometric assay using dichlorophenol-indophenol. The reduction of nitroblue tetrazolium was used to establish the generation of oxygen free radicals by the KO2 solution.

The corneal swelling rate was determined by linear regression analysis. A comparison of experimental and control regression lines was made by analysis of covariance (Snedecor GW and Cochran WG 1967). Variance in the data was expressed as the mean +/- 95% confidence limits.

Results and discussion

In vitro

Results
Irritation parameter:
other: corneal swelling
Run / experiment:
corneal thickness measurement
Value:
0.5
Vehicle controls validity:
valid
Other effects / acceptance of results:
Perfusion with solutions containing different concentrations of the test substance led to O2- production and subsequent H2O2 generation through the dismutation reaction.
Concentrations of the test substance of 0.5 mM and higher resulted in severe anatomic and physiologic alteration (disrupted morphology) of endothelial cells that resulted in corneal swelling (dose- and time-dependent patterns). Perfusion with 0.3 mM test substance resulted in an initial corneal swelling the first one-half hour, but a recovery thereafter.
The addition of catalase to the perfusion medium containing KO2 prevented both anatomic alteration of endothelial cells and corneal swelling. The addition of SOD, ascorbic acid, D-mannitol, EDTA, DETAPAC, EDTA + FeCL2, or DMSO did not modify the corneal swelling rate induced by perfusion with the KO2 solution.
Endothelial intracellular glutathione levels and redox state were unaffected by perfusion with 0.3 mM of the test substance.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the study conditions, the test substance was found to be irritating to rabbit corneas.

Executive summary:

A study was conducted to evaluate the eye irritation potential of the test substance in rabbit corneas. The study was performed following the protocol of McCarey BE et al., 1973, i.e. ''Perfusion of ex vivo rabbit corneas''. Rabbit corneal endothelial cells were perfused with a Krebs Ringer bicarbonate solution to which the test substance (at 0.3 to 1.0 mM) had been added along with superoxide dismutase, ascorbic acid, DETAPAC, EDTA, EDTA-FeCl2, or DMSO. Concentrations of test substance of 0.5 mM and higher resulted in severe anatomic and physiologic alteration of endothelial cells that resulted in corneal swelling. Catalase offered protection whereas the toxic effect was unaltered by superoxide dismutase, ascorbic acid, DETAPAC, EDTA, EDTA-FeCl2, or DMSO. Under the study conditions, the test substance was found to be irritating to rabbit corneas (Hull, 1984).

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