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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting and completion date was 2010-01-29.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by Hessisches Minesterium für Umwelt, ländlichen Raum und Verbraucherschutz (2009-03-30)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bismuth(III)trinitrate, pentahydrate
- Molecular formula (if other than submission substance): Bi(NO3)3 * 5H2O
- Molecular weight (if other than submission substance): 485.07 g/mol
- Physical state: solid, colourless crystals
- Storage condition of test material: Keep container closed and store substance in a cool and dry place. The test item is hygroscopic.

Test animals / tissue source

Species:
other: in vitro testing with freshly isolated bovine cornea
Details on test animals or tissues and environmental conditions:
not applicable

Test system

Vehicle:
physiological saline
Controls:
yes
Amount / concentration applied:
Prior to the application the test item was suspended in saline (20% (w/v)).
Duration of treatment / exposure:
The incubation time lasted 240 minutes (± 5 minutes).
Observation period (in vivo):
direct after removal of test substance or positive control substance (t240)
Number of animals or in vitro replicates:
not applicable
Details on study design:
Total Number of Corneae: 9:
Number of Corneae per Group: 3
Number of Test Item Group: 1
Number of Negative Control Group: 1
Number of Positive Control Group: 1

OPACITY MEASUREMENT:
The opacitometer was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. Complete medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a water-bath at 32 ± 2 °C. Afterwards, the opacity was measured again.

PERMEABILITY DETERMINATION:
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 2 °C. Complete medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

SCORING SYSTEM:
0-3 = non eye irritant
3.1-25 = mild eye irritant
25.1-55 = moderate eye irritant
55.1-80 = severe eye irritant
>80 = very severe eye irritant

TOOL USED TO ASSESS SCORE:
- Opaccity: For measurement of the corneae opacity the OP_KiT opacimeter (Electro Design, 63 -Riom France) was used.
- Permeability of the cornea: measured spectrophotometrically at 490 nm

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: in vitro score
Basis:
mean
Time point:
other: after 240 minutes incubation
Score:
1.25
Max. score:
1.98
Reversibility:
not specified
Remarks on result:
other: in vitro score of the negative control
Irritation parameter:
other: in vitro score
Basis:
mean
Time point:
other: after 240 minutes incubation
Score:
181.26
Max. score:
187.78
Reversibility:
not specified
Remarks on result:
other: in vitro score of the positive control substance
Irritation parameter:
other: in vitro score
Basis:
mean
Time point:
other: after 240 minutes incubation
Score:
435.13
Max. score:
436.47
Reversibility:
not specified
Remarks on result:
other: in vitro score of bismuth(III)trinitrate, pentahydrate
Irritant / corrosive response data:
For information see table above.
Other effects:
With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 1.25.
The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The mean in vitro score was calculated as 181.26.
The test item Bismuth(III) trinitrate, pentahydrate caused opacity and permeability of the corneae compared with the results of the negative control. The calculated in vitro score was 435.13 and therefore, the test item was classified as very severe eye irritant.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Bismuth(III) trinitrate, pentahydrate caused opacity and permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 435.13 and therefore, the test item was classified as very severe eye irritant.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Bismuth(III) trinitrate, pentahydrate is considered to be a very severe eye irritant.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Bismuth(III) trinitrate, pentahydrate by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item Bismuth(III) trinitrate, pentahydrate, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 2 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °Cin a horizontal position. The liquid coming out was measured spectrophotometrically.

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant.

The test item Bismuth(III) trinitrate, pentahydrate caused opacity and permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 435.13 and therefore, the test item was classified as very severe eye irritant.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Bismuth(III) trinitrate, pentahydrate is considered to be a very severe eye irritant.