Calcium cyanamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Since the charcoal did not dissolve in the vehicle used (DMSO) it was not possible to state that all of the calcium cyanamide had dissolved in the solution. Only 20 metaphases per slide were scored for sister chromatide exchange.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

The test substance did not dissolve completely; Only 20 metaphases per slide were scored

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

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

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

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

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

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

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

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

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

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

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)

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham´s F10 medium supplemented with 15% of newborn calf serum, 50 mg/L of streptomycin and 50 mg/L of sodium penicillin G
- After 30 - 40 passages cells were replaced by cells from a stock kept at -196°C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate

Test concentrations with justification for top dose:

10, 50, 250 and 500 mg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: and Trenimon (trifunctional alkylating agent, does not require metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Cells were plated in 75 cm2 flaks and to 250 mL of culture medium, 0.2 mL of test compound dissolved in DMSO, 0.5 mL of coenzyme solution in sodium phosphate buffer (pH 7.4) and 0.2 mL of S9- Aroclor 1254 were added; the latter two of which were added only in cases where the test compound was tested in the presence of metabolic activation.

DURATION
- Preincubation period: no preincubation; cells were allowed to attach themselves to the bottom of the culture flask for 4 hours
- Exposure duration: 1 hour
- Expression time (cells in growth medium): 24 hours
- Three hours before harvesting, colchicine was added to the cells at a concentration of 2 µg/10 mL. After harvesting, cells were treated for 7 minutes with a 0.075 M KCl solution, fixed with a 3:1 mixture of methanol and acetic acid and transferred to slides.

STAIN (for cytogenetic assays): Hoechst 33258 fluorochrome, Giemsa

NUMBER OF CELLS EVALUATED: One slide was prepared from the contents of each flask and the number of sister chromatid exchanges in 20 metaphases on each slide was scored.

Evaluation criteria:

No data

Statistics:

The coefficient of the calculated dose-response lines were determined.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at concentrations up to 100 mg/L

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Calcium cyanamide did not dissolve completely at 0.5 or 1% in DMSO, thus, the substance was tested at the maximum possible dose concerning solubility.

Remarks on result:

other: strain/cell type: as stated above

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Induction of sister chromatid exchange by Kalkstickstoff and the positive controls Trenimon and Cyclophosphamide

Kalkstickstoff [mg/L]	Mean number of sister chromatid exchange observed per 20 metaphases ± SD
	+ S9	- S9
0	12.05 ± 5.21	11.6 ± 3.61
10	12.85 ± 4.58	11.25 ± 4.55
50	13.95 ± 4.45	11.7 ± 4.94
250	13.85 ± 5.28	11.9 ± 5.02
500	11.5 ± 3.5	9.75 ± 3.54
Positive Controls*:
Trenimon (1.25 µg/L)	-	23.2 ± 6.92
Cyclophosphamide (5 mg/L)	50.6 ± 12.06	-

* for positive controls, only 10 metaphases were scored.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The results obtained indicate that calcium cyanamide technical grade (Kalkstickstoff) is negative in the sister chromatid exchange test in vitro.

Executive summary:

The study was conducted in order to evaluate the potential of calcium cyanamide technical grade (Kalkstickstoff) to induce sister chromatide exchanges in vitro.

Therefore, chinese hamster ovary cells were exposed up to the maximum possible dose of calcium cyanamide, concerning solubility, both in the absence and in the presence of metabolic activation. The highest dose tested (500 mg/L) represents 330 mg/L of calcium cyanamide and represents the maximal soluble concentration.

Results revealed that calcium cyanamide technical grade (Kalkstickstoff) did not induce an increase in the number of sister chromatide exchanges per metaphase at the dose levels tested: The coefficient of the calculated dose-response lines were negative and did not differ significantly from zero. The two positive control substances used both gave a clear effect.