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Diss Factsheets

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
Study period:
2010-03-17 to 2010-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
other: OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method (BCOP)), adopted 2009-09-07
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium cyanamide
EC Number:
205-861-8
EC Name:
Calcium cyanamide
Cas Number:
156-62-7
Molecular formula:
CN2.Ca
IUPAC Name:
calcium cyanoazanediide
impurity 1
Chemical structure
Reference substance name:
Calcium oxide
EC Number:
215-138-9
EC Name:
Calcium oxide
Cas Number:
1305-78-8
Molecular formula:
CaO
IUPAC Name:
oxocalcium
impurity 2
Chemical structure
Reference substance name:
Carbon
EC Number:
231-153-3
EC Name:
Carbon
Cas Number:
7440-44-0
Molecular formula:
C
IUPAC Name:
carbon
impurity 3
Reference substance name:
Hematite (Fe2O3)
EC Number:
215-275-4
EC Name:
Hematite (Fe2O3)
Cas Number:
1317-60-8
Molecular formula:
Fe2O3
IUPAC Name:
diiron oxide
impurity 4
Chemical structure
Reference substance name:
Urea
EC Number:
200-315-5
EC Name:
Urea
Cas Number:
57-13-6
Molecular formula:
CH4N2O
IUPAC Name:
urea
impurity 5
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
dioxosilane
impurity 6
Chemical structure
Reference substance name:
Trisilicon tetranitride
EC Number:
234-796-8
EC Name:
Trisilicon tetranitride
Cas Number:
12033-89-5
Molecular formula:
N4Si3
IUPAC Name:
trisilicon tetranitride
impurity 7
Chemical structure
Reference substance name:
Calcium dihydroxide
EC Number:
215-137-3
EC Name:
Calcium dihydroxide
Cas Number:
1305-62-0
Molecular formula:
CaH2O2
IUPAC Name:
calcium dihydroxide
impurity 8
Chemical structure
Reference substance name:
Aluminium oxide
EC Number:
215-691-6
EC Name:
Aluminium oxide
Cas Number:
1344-28-1
Molecular formula:
Al2O3
IUPAC Name:
aluminium oxide
impurity 9
Chemical structure
Reference substance name:
Cyanoguanidine
EC Number:
207-312-8
EC Name:
Cyanoguanidine
Cas Number:
461-58-5
Molecular formula:
C2H4N4
IUPAC Name:
2-cyanoguanidine
impurity 10
Chemical structure
Reference substance name:
Calcium acetylide
EC Number:
200-848-3
EC Name:
Calcium acetylide
Cas Number:
75-20-7
Molecular formula:
C2Ca
IUPAC Name:
calcium ethynediide
impurity 11
Reference substance name:
unknown
IUPAC Name:
unknown
Test material form:
solid: particulate/powder
Details on test material:
Kalkstickstoff (calcium cyanamide, technical grade)

Test animals / tissue source

Species:
other: Bovine corneas
Strain:
not specified
Details on test animals or tissues and environmental conditions:
isolated corneas from the eyes of cows and bulls aged between 18-20 month and free of macroscopic visible defects.
Supplier: Slaughterhouse Klaus Grandits, Ungerbachstr. 10, A-2860 Kirchschlag, Austria.

Test system

Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
750 µL test substance were introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed during exposure (close-chamber method).
Duration of treatment / exposure:
The corneas were exposed in a horizontal position for 4 hours while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min/max thermometer, was 30.9°C - 33.5°C.
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control
Details on study design:
Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour to achieve normal metabolic activity. After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to the test substance, the negative control and the positive control for 4 hours. Then the substances were removed and the corneas were accurately washed and the opacity and permeability of each cornea were recorded. Opacity was measured quantitatively with the aid of an opacimeter. Permeability was determined by the amount of sodium fluorescein dye that penetrated all cornea layers. For this purpose fluoresceine solution was filled into the anterior chambers of the cornea holders followed by an incubation period of 90 minutes. The amount of sodium fluoresceine that crossed into the posterior chambers was quantitatively measured with a spectrophotometer at OD490. Using opacity and permeability data an IVIS was calculated. The positive and negative control groups were simultaneously used for the concurrently performed studies.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
BCOP
Value:
443.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The negative and positive control substance values are valid.
The opacity value of "Calcium cyanamide" was 356.6, 15 x Permeability value was 86.835 which resulted in an IVIS of 443.4.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The opacity value of calcium cyanamide was 356.6, 15 x Permeability value was 86.835 which resulted in an IVIS of 443.4. Thus calcium cyanamide is regarded to be an ocular corrosive or severe irritant according to the OECD Guideline 437 for the testing of chemical "Bovine Corneal Opacity and Permeability test Method for identifying Ocular Corrosives and Severe Irritants".
Executive summary:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of calcium cyanamide.

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µL of the test substance preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 h and the final opacity was measured. Then 1 mL of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances (negative control: sterile aqua dest., positive control: 20% imidazole). Finally the IVIS (in Vitro Irritancy Score) was calculated as follows: IVIS = mean opacity value + (15 x permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS >= 55.1 is defined as ocular corrosive or severe irritant.

The IVIS for calcium cyanamide was 443.4. IVIS of the negative control was 4.9 and for the positive control 123.7, thus demonstration validity of the experiment. According to the results of this study and the OECD Guideline 437, the test substance calcium cyanamide is considered to be an ocular corrosive or severe irritant.