Calcium cyanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Calcium cyanamide technical grade (Kalkstickstoff) was evaluated for its mutagenic and genotoxic potential both in vitro and in vivo. The Ames tests performed in S. typhimurium strains for both Kalkstickstoff and calcium cyanamide were negative in the absence or presence of metabolic activation. A non-guideline bacterial mutation test with calcium cyanamide in Aspergillus nidulans was positive in the absence but negative in the presence of S9-mix (metabolic activation). Both mammalian chromosomal aberration test as well as the unscheduled DNA synthesis (UDS) test in a human cell line on Kalkstickstoff were shown to be negative. Also, in an in vitro mammalian celle gene mutation test (HPRT-locus) conducted with calcium cyanamide, purum, no mutagenic activity of calcium cyanamide was observed.

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

Not applicable

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

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.

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'.

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.

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.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Non-standard UDS test

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

cells were not blocked in S-phase but a pre-labelling was performed to distinguish cell cycle phases; 3H-TdR was added to the cells 1 hour after treatment with test substance; only 25 instead of 50 cells were counted per culture; no statistical analysis

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

Not applicable

Species / strain / cell type:

primary culture, other: human fibroblasts

Details on mammalian cell type (if applicable):

- Type and identity of media: HAM'S F10 medium without hypoxanthine and thymidine, supplemented with 15% fetal calf serum, L-glutamine (148 µg/mL), hypoxanthine (4 µg/mL), penicillin (200 U/mL) and streptomycine (100 µg/mL)
-Periodically checked for Mycoplasma contamination: yes, using the method of Peden (Peden,K. W. C., Experientia, Volume 31, p. 1111-1112, 1975)
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: HeLa, human cell line

Details on mammalian cell type (if applicable):

- Type and identity of media: HAM'S F10 medium without hypoxanthine and thymidine, supplemented with 10% fetal calf serum, L-glutamine (148 µg/mL), hypoxanthine (4 µg/mL), penicillin (200 U/mL) and streptomycine (100 µg/mL)
- Periodically checked for Mycoplasma contamination: yes, using the method of Peden (Peden,K. W. C., Experientia, Volume 31, p. 1111-1112, 1975)
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from male rat livers

Test concentrations with justification for top dose:

A dose range of 0 - 1000 µg/mL of calcium cyanamide (final concentration) was tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: for indirectly acting agents (+S9) and 4-nitro-quinoline-1-oxyde for directly acting agents (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 2x10E5 cells of both cell types were seeded in 6 cm petri-dishes and were allowed to attach by incubation for 48 hours at 37°C prior to treatment.
- Exposure duration: pre-labelling for 2 hours to distinguish S-phase cells from G1- and G2-phase cells; after washing, treatment with test substance for 1 hour at 37°C in medium without serum
- Expression time (cells in growth medium): labelling of the cells for three hours at 37°C with tritium-thymidine
- Fixation time (start of exposure up to fixation or harvest of cells): 20 minutes at room temperature with Buoin

STAIN: 0.02% Toluidine blue, 0.5 M citric acid buffer


NUMBER OF REPLICATIONS: two independent experiments performed


NUMBER OF CELLS EVALUATED: silver grains were scored over at least 25 nuclei of non-S-phase cells on each microscopic slide

Evaluation criteria:

Silver grains are scored over nuclei of non-S-phase cells. After counting, the data are processed by computer analysis and the frequency distribution of the number of grains found over the nuclei is plotted. The method is calibrated by performing simultaneously with the tests of the two reference compounds with known response in the UDS test (known genotoxic properties). The results obtained for the test substance, calcium cyanamide, was put in relation to the responses observed with the controls (positive control substances and vehicle control).

Statistics:

No data

Key result

Species / strain:

primary culture, other: human fibroblasts

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: cells showed poor morphology

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: HeLa, human cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: cells showed poor morphology

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Two independent experiments were performed. In one experiment, the cells were incubated with calcium cyanamide both in the presence and in the absence of metabolic activation (S9 mix). In the second experiment, a more extended range of concentrations was tested, but only in the absence of S9 mix.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
All cells treated at calcium cyanamide showed a poor morphology. If or how far the morphological changes might have interferred with the UDS cannot be decided from the results obtained in the present investigation. However, lethally treated cells have frequently been found to be very well able to perform UDS.

Remarks on result:

other: strain/cell type: as stated above

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results:
negative with and without metabolic activation

There was no induction of unscheduled DNA synthesis from exposure to calcium cyanamide in the primary human fibroblasts and in the human cell line, HeLa, both in the absence and in the presence of metabolic activation. All of the treated cells showed poor morphology, which might have influenced DNA repair. However, lethally treated cells have frequently been found to be very well able to perform UDS and, therefore, it is concluded that calcium cyanamide was negative in the UDS assay.

Executive summary:

The objective of the study was to test calcium cyanamide, technical grade (Kalkstickstoff) for possible mutagenic / carcinogenic activity by an indirect method which is based on the detection of repair of DNA damage that may be induced by the compound tested in cultured human cells.

DNA repair was studied by measuring the induction of unscheduled DNA synthesis (UDS) in normal human primary fibroblasts and in an established human cell line, HeLa.

Calcium cyanamide was tested at 0 - 1000 µg/mL with and without a rat liver homogenate (S9) for metabolic activation. As reference compounds with known positive reactions in UDS, 4 -nitro-quinoline-1-oxyde and benzo(a)pyrene were used for directly and indirectly acting agents, respectively.

Both the vehicle controls (assay without administration of test substance) and the positive control substances revealed the expected results.

Calcium cyanamide, technical grade (Kalkstickstoff) was negative in the UDS test at all applied doses in both cell types, when tested in the absence and in the presence of metabolic activation. The only effect noted at pronounced indication for cytotoxicity was poor morphology of the cells but which was considered not to have influenced the outcome of the study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The test substance was tested up to 1 mg/plate only.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The substances were not tested up to the maximum concentration of 5 mg/plate as recommended by the guideline.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth was inoculated with the frozen culture and grown up overnight at 37°C.
- The reversion properties were checked using the mutagens N-nitrosomorpholine and benzo(a)pyrene. In addition, the strains were checked for histidine requirement, sensitivity for crystal violet and deoxycholate as well as for resistance to ampicilline.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth was inoculated with the frozen culture and grown up overnight at 37°C.
- The reversion properties were checked using the mutagens N-nitrosomorpholine and benzo(a)pyrene. In addition, the strains were checked for histidine requirement, sensitivity for crystal violet and deoxycholate as well as for resistance to ampicilline.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate of Aroclor-induced rats

Test concentrations with justification for top dose:

4, 20, 100, 500 and 1000 µg/plate of calcium cyanamide and 0.004, 0.02, 0.1, 0.5 and 1 µL/plate of the 50% cyanamide solution.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: and N-nitrosomorpholine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
To 2.5 mL molten soft agar was added: 0.1 mL of a fully grown culture of one of the tester strains , 0.1 mL of the appropriate dilution of the test compound and the liver microsome system (S9) if required

DURATION
- Exposure duration: three days at 37°C.

SELECTION AGENT (mutation assays): Histidine requirement

NUMBER OF REPLICATIONS: All determinations were carried out in triplicate.

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

both for calcium cyanamide and the 50% cyanamide solution

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

not cytotoxic up to the maximum concentration tested (1000 µg calcium cyanamide)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Due to the variable results obtained with strain TA 1538, the test was repeated using 4 and 20 µg of calcium cyanamide

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: and in addition, strain TA 1538 was tested under the same conditions

Result of the mutagenic evaluation of calcium cyanamide in the Salmonella / microsome mutagenicity test

calcium cyanamide per plate[µg]**	S9-mix/plate [µL]	mean number of his+ revertants*
		TA 1535	TA 1537	TA 1538***	TA 98	TA 100
0	0	13	11.3	6; 15.3	14.7	34.7
4	0	12	8	6.7; 11.7	15.7	45.3
20	0	10.3	8	11.7; 5.7	15.7	33
100	0	10.3	6	8.7	13.3	40.7
500	0	12	8.7	6.3	12	45.3
1000	0	12.7	4.3	6.3	12.7	34
0	50	19.3	16.7	16.3; 11.7	28.3	44.3
4	50	11.7	9	3.0; 14.7	22.3	52
20	50	13	13	14.7	25.3	42
100	50	17	13.3	13.7	24.7	41
500	50	16.7	8.7	12	23	53.7
1000	50	18	7.3	10.3	17.3	40.3

* determinations were carried out in triplicate

** dissolved in 0.1 mL of DMSO

** in view of the variable results obtained with strain TA 1538, the test was repeated with 4 and 20 µg calcium cyanamide per plate.

Conclusions:

Interpretation of results:
negative without metabolic activation both for calcium cyanamide and the 50% cyanamide solution
negative with metabolic activation both for calcium cyanamide and the 50% cyanamide solution

From the present results it can be concluded that up to 1000 µg calcium cyanamide per plate or up to 1 µL cyanamide per plate did not reveal any mutagenic activity in the plate incorporation assay with S. typhimurium TA 1535, TA 1537, TA 1538, TA 100 or TA 98 both in the presence and in the absence of liver microsome activation system (S9 mix).

Executive summary:

A study was conducted to examine the mutagenic activity of calcium cyanamide technical grade and a 50% cyanamide solution in the Salmonella/microsome mutagenicity test.

A set of five histidine requiring mutants of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were treated at 4 - 1000 µg calcium cyanamide or 0.004 - 1 µL of cyanamide per plate both in the presence and in the absence of liver homogenate prepared from Aroclor-induced rats (S9 mix).

Incorporation of calcium cyanamide at levels up to 1000 µg/plate or 50% cyanamide at levels up to 1 µL/plate did not increase the number of his+ revertants in any of the five tester strains, either in the presence or in the absence of the liver microsome activation system. There were no signs that chemical toxicity interfered with the mutagenicity testing: background lawn of bacterial growth and number of revertants in control and test plates were comparable.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-19 to 2018-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Gü-AM 13/41
- Expiration date of the lot/batch: 05 February 2019



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature; protected from light
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior testing: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test DMSO was used as solvent. The solvent was compatible with the survival of the cells and the activity of the S9 mix. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 479 mOsm/kg.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (CCL-93)
- Suitability of cells: These cells are characterised by their high proliferation rate and their high cloning efficiency of untreated cells (usually more than 50%). These features of the cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: 12 - 14 h doubling time
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B; cultured at a humidified atmosphere of 5% CO2 and at 37 °C
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver

Test concentrations with justification for top dose:

Cytotoxicity of the test item was determined in a pre-experiment. In the pre-experiment it was observed that cytotoxic effects occurred with and without metabolic activation at concentrations of 4.5 and 10 mM. Based on these data the concentrations for the main experiment were selected.

The test item was tested at the following concentrations:
* without metabolic activation:
0.25, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 mM
* and with metabolic activation:
0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.25 and 1.5 mM

According to OECD test guideline 476, more than 4 concentrations may be particularly important for evaluating mutagenicity when using single cultures. Therefore, the following 5 concentrations were selected on the basis of cytotoxicity for evaluating mutagenicity.
* without metabolic activation: 0.50, 1.0, 1.5, 2.0, 2.5 mM
* with metabolic activation: 0.50, 0.75, 1.0, 1.25, 1.5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: treatment medium (MEM + 0 % FBS)
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test, the treatment medium was used as solvent. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 5E+06 cells per each concentration, negative and positive controls, were seeded in complete cell culture medium (MEM supplemented with 10 % FBS) in a 175 cm³ culture flask.

DURATION
- Preincubation period: 24 h after seeding
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7–9 days after treatment
- Selection time (if incubation with a selection agent): 9–11 days for mutant frequency and 6–8 days for cloning efficiency

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (TG)

NUMBER OF REPLICATIONS: 2 for cytotoxicity; 2–5 for mutagenicity

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 200 cells for cloning efficiency and 400000 cells for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The relative survival (indicator of cytotoxicity) was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined,
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test, and
- all results are inside the distribution of the historical negative control data.
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined,
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative control a concentration-related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the concentration groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest concentration for mutagenicity (2.5 mM)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For individual results see attachment "Illustration (picture/graph)".

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted at concentrations of 2.5 mM (with metabolic activation) and 1.5 mM (without metabolic activation).:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : See illustration (picture/graph)
Most of the mutant values are within the historical data base of the test facility. In the experiment with metabolic activation at two concentrations the mutant frequencies induced by the test item did show an increase beyond the historic control range of 9.6 - 44.0 mutants per 10^6 cells. At concentration 0.5 mM an additional statistically significant increase was observed. However, this effect was only observed at the lowest concentration evaluated and since no statistically significant concentration-response relationship was determined, these effects were considered as not biologically relevant.

Conclusions:

In an in vitro cell gene mutagenicity test, the test item calcium cyanamide (pure) is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Executive summary:

An in vitro mammalian cell gene mutation test (HPRT-locus) according to OECD guideline 476, V79 cells cultured in vitro were exposed to calcium cyanamide (pure) at concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5 mM in the absence and 0.5, 0.75, 1.0, 1.25 and 1.5 mM in the presence of metabolic activation.

Biologically relevant growth inhibition was observed in the experiment without metabolic activation. The relative survival was 33% for the highest concentration (2.5 mM) evaluated. The positive controls did not induce the appropriate response. In the experiment with metabolic activation, at a concentration 0.5 mM an additional statistically significant increase was observed. However, this effect was only observed at the lowest concentration evaluated and since no statistically significant concentration-response relationship was determined, these effects were considered as not biologically relevant.There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 or OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The results of an in vivo micronucleus test did not reveal any evidence for mutagenic activity of Kalkstickstoff in male or female rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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:

Animals were analysed for micronuclei six hours after the last treatment. Only one dosage was tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Animals were killed 6 hours after the last treatment; Only one dosage tested

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: 4-5 weeks
- Weight at study initiation: mean bodyweight for animals treated as Kalkstickstoff was 98 g for males and 82 g for females (animals of the water control: 99 g for males and 79 g for females)
- Assigned to test groups randomly: animals were assigned to groups according to bodyweight
- Fasting period before study: 14-15 hours
- Housing: screen-bottomed cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: yes; the duration of this period is not stated in the report

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24°C ± 1°C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

DIET PREPARATION
Aqueous solutions / suspensions of each of the test samples were administered twice with an interval of 24 hours by stomach tube at dose levels of 1/5 of the LD50 in 5 mL of water per kg body weight.

Duration of treatment / exposure:

Animals were exposed twice at 153 mg/kg bw of test substance with an interval of 24 hours. The dose level used was based on the LD50 for rats (765 mg/kg bw).

Frequency of treatment:

Twice; with an interval of 24 hours

Post exposure period:

Animals were killed by decapitation 6 hours after the last treatment.

Remarks:

Doses / Concentrations:
153 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Only one dosage applied (corresponding to 1/5 of the LD50 value); 5 animals per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Trenimon
- Justification for choice of positive control(s): Trenimon is a well-known mutagen in rats
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.0625 mg/kg bw in physiological saline; administered twice with an interval of 24 hours

Tissues and cell types examined:

A total of 400 erythrocytes per slide were examined for the incidence of micronucleated erythrocytes and the ratio of poly- and normochromatic erythrocytes.

Details of tissue and slide preparation:

The bone marrow of the femora was flushed into centrifuge tubes containing fetal calf serum and centrifuged. The cells were then resuspended by gentle mixing with a pasteur pipette. A drop of the solution was placed on a slide cleaned with methanol overnight and spread with a haemocytometer cover glass. Five slides were prepared for each animal. The smears were air-dried, fixed in methanol and stained according to May-Grünwald Giemsa.

Evaluation criteria:

No data

Statistics:

No data

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No mortality or abnormalities of condition or behaviour, attributable to treatment, were observed in any of the animals during the exposure period.
The incidence of micronucleated erythrocytes and the percentage of polychromatic erythrocytes in the controls (water) and the test group (calcium cyanamide) were comparable.

Mean numbers of micronucleated erythrocytes and percentage polychromatic erythrocytes in bone marrow of rats after treatment with "Kalkstickstoff"

Type of treatment	sex	Mean incidence of micronucleated cells per 2000 erythrocytes per rat (range)	Percentage of polychromatic erythrocytes ± SD
Vehicle control	male	4.4 (2–7)	74.2 ± 3.5
Kalkstickstoff		5.2 (4–7)	70.2 ± 7.5
Vehicle control	female	5.0 (2–8)	63.8 ± 5.1
Kalkstickstoff		4.8 (2–8)	61.9 ± 2.0
Positive control:
Solvent	male	3.8 (2–8)	69
Trenimon		22.6 (20–26)	42
Solvent	female	3.6 (3–5)	70
Trenimon		34.4 (27–38)	46

Conclusions:

Interpretation of results: negative
The results of the in vivo micronucleus test did not reveal any evidence for mutagenic activity of Kalkstickstoff in male or female rats.

Executive summary:

A study was conducted in order to evaluate the mutagenic potential of Calcium Cyanamide technical grade (Kalkstickstoff) in the in vivo micronucleus assay.

Therefore, rats were treated twice at 153 mg/kg bw with an interval of 24 hours and were examined for the incidence of micronucleated erythrocytes and the ratio of poly- and normochromatic erythrocytes six hours after the last treatment.

No mortality or abnormalities of condition or behaviour, attributable to treatment, were observed in any of the animals during the exposure period. The incidence of micronucleated erythrocytes and the percentage of polychromatic erythrocytes in the control and treated group were comparable. In contrast, the positive control Trenimon induced an increase in micronucleated erythrocytes and a decrease in the percentage of polychromatic erythrocytes. Thus, no evidence for mutagenic activity was observed for Kalkstickstoff and it was therefore concluded that Kalkstickstoff was negative in the in vivo micronucleus assay under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Calcium cyanamide technical grade (Kalkstickstoff) was evaluated for its mutagenic and genotoxic potential in vitro and in vivo.

 

Two Ames tests, performed in S. typhimurium strains according to OECD guideline 471, using Kalkstickstoff and calcium cyanamide, respectively, were negative both in the absence or presence of metabolic activation via rat liver homogenate. In another non-guideline bacterial mutation test, calcium cyanamide was tested for the induction of three different genetic events, mitotic gene conversion, mitotic crossing-over and mitotic non-disjunction, in Aspergillus nidulans. Results revealed genotoxic activity extending to different genetic events such as gene conversion and non-disjunction but did not induce crossing-over in the absence of S9 mix (metabolic activation). In the presence of metabolic activation, however, no increase in genetic activity was observed. As the results of the Ames tests did not show mutagenic activity and the non-guideline bacterial mutation test was as well negative in the presence of metabolic activation, which is more likely to represent the physiological (in vivo) conditions, the positive reaction in the absence of metabolic activation is considered to be not relevant for the assessment of the mutagenic potential of calcium cyanamide.

 

A chromosome aberration test in Chinese hamster ovary (CHO) cells was conducted to evaluate the potential of Kalkstickstoff to induce sister chromatid exchanges in vitro. Cells were exposed to the maximum possible Kalkstickstoff dosage (based on solubility), and results revealed that Kalkstickstoff did not induce an increase in the number of sister chromatid exchanges per metaphase in either the absence or presence of metabolic activation. The two positive control substances used both gave a positive effect. It was concluded that Kalkstickstoff was not mutagenic in the chromosome aberration test.

 

Kalkstickstoff was tested for possible mutagenic / carcinogenic activity by an indirect method based on the detection of repair of DNA damage in a cultured human cell line (HeLa) and human primary fibroblasts in a non-standard UDS (unscheduled DNA synthesis) assay. Kalkstickstoff was tested at 0 - 1000 µg/mL both in the absence and in the presence of S9 mix. Both the vehicle control and the positive control substances used revealed the expected results. Kalkstickstoff was negative at all applied dosages in both cell types in the absence or presence of metabolic activation. The only effect noted was poor morphology of the cells, which was considered not to have influenced the outcome of the study. Kalkstickstoff was considered negative for mutagenic activity in the UDS assay.

An in vitro mammalian cell gene mutation test (HPRT-locus) was conducted with calcium cyanamide, purum. In this study, V79 cells cultured in vitro were exposed at concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5 mM in the absence and 0.5, 0.75, 1.0, 1.25 and 1.5 mM in the presence of metabolic activation. Biologically relevant growth inhibition was observed in the experiment without metabolic activation. The relative survival was 33% for the highest concentration (2.5 mM) evaluated. The positive controls did not induce the appropriate response. There was no evidence of induced mutant colonies over background.

An in vivo micronucleus assay was conducted in male and female Wistar rats which were treated twice at 153 mg/kg bw of Kalkstickstoff with an interval of 24 hours and were examined for the incidence of micronucleated erythrocytes and the ratio of poly- and normochromatic erythrocytes six hours after the last treatment. No mortality or abnormalities in condition or behaviour were noted and the incidence of micronucleated erythrocytes and the percentage of polychromatic erythrocytes were comparable to the one in the control group. In contrast, the positive control substance induced an increase in micronucleated erythrocytes and a decrease in the percentage of polychromatic erythrocytes. Thus, no evidence for mutagenic activity was observed for Kalkstickstoff in this in vivo study.

 

It is concluded that calcium cyanamide (Kalkstickstoff) is not mutagenic in vitro or in vivo. The results of the mutagenicity studies are summarised in the following table:

Mutagenicity studies of Calcium cyanamide

Study Type	Cells/Species	Test conditions (test substance)	Result	Reference
Ames test	S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100	+ and – S9-mix: 4, 20, 100, 500 and 1000 µg/plate (Kalkstickstoff)	negative	Willems, M. I., 1978, report number 5707
Ames test	Aspergillus nidulans	+ and – S9-mix: 0.26, 0.44, 0.62 g (calcium cyanamide)	- S9: negative for crossing over; positive for gene conversion and non-disjunction + S9: negative	Vallini, G. et al, 1983, report number 1250/III
Ames test	S. typhimurium TA 1535, TA 1537, TA 98, TA 100	+ and – S9-mix: 10, 33, 100, 333, 1000, 3333 µg/plate (calcium cyanamide)	negative	Haworth, S. et al, 1983, report number 503-005
In vitro mammalian chromosome aberration	Chinese hamster ovary (CHO) cells	+ and – S9-mix: 10, 50, 250, 500 mg/L (Kalkstickstoff)	negative	De Raat, W. K., 1979, report number CL/78/120
UDS human cells	HeLa; Human primary fibroblasts	+ and – S9-mix: final concentration in the range of 0 – 1000 µg/mL (Kalkstickstoff)	negative	Lohman, P. H. M., 1981, report number SMC 1,2
In vitro mammalian cell gene mutation test	Chinese hamster V79 cells	+ and – S9-mix: final concentration in the range of 0.5 - 2.5 mM (without metabolic activation) 0.5 - 1.5 mM (with metabolic activation) (calcium cyanamide, purum)	negative	Voges, Y., 2018, report number 185088
In vivo micronucleus assay	Wistar rat	153 mg/kg bw applied twice with an interval of 24 hours (Kalkstickstoff)	negative	Willems, M. I., 1979, report number R 6012

Disregarded studies for cyanamide:

Cyanamide was also considered for its mutagenic and genotoxic potential in vitro (7 studies) and in vivo (2 studies). Even though calcium cyanamide can rapidly hydrolyse to cyanamide (e.g. in aqueous solutions), due to the differences in physicochemical properties (e.g. chemical composition) and dissolution kinetics (e.g. in the stomach under low pH conditions) as well as the dissimilar outcomes between cyanamide and calcium cyanamide observed in some of the toxicity studies (e.g. acute toxicity, developmental toxicity and carcinogenicity), the use of data from cyanamide as read-across or weight-of-evidence approach for evaluating the genotoxic potential of calcium cyanamide technical grade is not appropriate (for more information, see report "Scientific Rationale for not using Cyanamide as Read-Across Substance for Calcium Cyanamide on Toxicological Endpoints” in Section 13.2). Furthermore, there are existing genotoxicity studies of calcium cyanamide for its evaluation for this endpoint as well as a carcinogenicity study, which reported that calcium cyanamide technical grade was not carcinogenic for both rats and mice of either sex. Nevertheless, a summary of the genotoxicity studies on cyanamide is given in this section below to demonstrate the full consideration of the available toxicological data on cyanamide.

Two Ames tests performed in Salmonella typhimurium strains with or without metabolic activation (S9 mix from the liver of Aroclor 1254 induced male Sprague-Dawley rats). In the test with cyanamide, no bacteriotoxic effects were observed up to the highest investigated concentrations in all strains used with and without metabolic activation (TA98, TA100, TA1535, TA1537, TA1538). No relevant increase in revertant number was observed in any of the strains tested. Similar results were found for E. coli strains GY 4015 and GY 5027, and the test was confirmed with positive and negative controls. Hydrogen cyanamide was tested for mutagenicity in concentrations ranging from 0.1 to 15.0 µL/plate, corresponding to 20 to 2540 µg/plate with or without S9 mix in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538. Slightly reduced revertant rates were observed at the highest investigated concentration of 15 µL/plate, corresponding to 2540 µg/plate, without S9 mix. No relevant increase in the number of histidine (his+) revertants was observed in the bacterial strains TA98, TA100, TA 1535, TA1537 and TA1538 tested either with or without S9 mix. The sensitivity of the test system used was demonstrated by the induction of an increased number of revertants by the positive controls. It can be concluded that cyanamide did not exhibit mutagenic effects in bacterial reverse mutation assays with or without metabolic activation via S9 mix in all strains tested.

In a gene mutation test according to OECD guideline 476, Cyanamid L500's potential mutagenic effect was examined by assaying the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells. Assays were run for 4 and 24 hours, with or without metabolic activation by S9 mix, and Cyanamid L500 concentrations ranged from 1.20 to 250 ug/mL (with activation) and 15.6 to 1000 µg/mL (without activation). A comparison of the mutation rates found in the groups treated with Cyanamid L500 with the negative and solvent controls did not show any relevant increase of gene mutations. Cyanamid L500 did not induce a reproducible concentration-related increase in mutant colony numbers. EMS (0.3 mg/mL) and DMBA ( 2.5 µg/mL) were used as positive controls and showed distinct increases in induced mutant colony number. It can be concluded that in this mutagenicity assay and under the experimental conditions reported, Cyanamid L500 did not induce gene mutations at the HPRT-locus in V79 cells. By measuring unscheduled DNA synthesis (UDS) in rat primary hepatocytes in vitro, the potential of hydrogen cyanamide to cause DNA damage was assessed. Concentrations tested ranged from 5.95 to 190 µg/mL hydrogen cyanamide along with deionised water and 2-Acetyl aminfluorene (2-AAF) as the negative and positive controls, respectively. The highest concentration of hydrogen cyanamide was highly toxic and the remaining seven doses were slightly or not toxic (relative survival 53.3% to 100.8%). It did not significantly increase net nuclear grain counts at any of the treatment concentrations and no concentration response was observed. The positive control confirmed the sensitivity of the test system. Under the test conditions with rat primary hepatocytes, hydrogen cyanamide did not induce an UDS response at any dose level.

In one test, hydrogen cyanamide was assessed for its potential to induce structural chromosome aberrations in Chinese hamster ovary cells in vitro, in the presence or absence of metabolic activation (S9 mix from Aroclor 1254 -induced male Sprague-Dawley rat liver). Duplicate cultures of CHO cells were exposed to the test substance for 20 hours at concentrations of 42.4, 56.5, 141, 283 and 424 µg/mL in the non-activation assay and for 2 hours at 438, 875, 1310 and 1750 µg/mL (20 hours preparation interval) and 321 and 428 µg/mL (10 hours preparation interval) in the presence of metabolic activation and then exposed to colcemide 2.5 hours prior to harvest. With or without metabolic activation, in the 20-hour preparation experiment, complete toxicity was seen in the higher doses and a significant dose-dependent increase in aberrant cells was obtained. In the 10-hour preparation, no observable toxicity was seen at the highest concentration and a slight but insignificant increase in cells with aberrations was obtained at the highest evaluated concentration. Under the conditions of the assay described, hydrogen cyanamide induced an increase in structural chromosome aberrations in CHO cells. Another test evaluated the ability of hydrogen cyanamide to induce structural chromosome aberrations in human lymphocytes in vitro with or without metabolic activation (S9 mix).

Duplicate cultures of human lymphocytes cells were exposed to the test substance for 24 hours at concentrations of 1.0, 3.3, 10.0, 33.3, 100.0, 333.0, 1000.0, 3330 and 5000 µg/mL in the non-activation assay and for 2 hours at concentrations of 0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100.0, 333.0 1000.0, 3330.0 and 5000 µg/mL culture medium in the presence of metabolic activation. The cells were harvested after 24 hours with the application of colcemide 3 hours prior to harvest. A statistically significant increase in numerical chromosomal aberrations was observed in the absence and presence of S9 mix at the highest concentration of 33.3 ug/mL (without S9) and at 33.3 and 333.3 µg/mL (with S9). Hydrogen cyanamide can be considered clastogenic in this human lymphocyte assay. In a mouse lymphoma assay, the potential effect of hydrogen cyanamide was examined by assaying the induction of forward mutations at the thymidine kinase locus in L5178 cells. Without metabolic activation with S9 mix, strong toxic effects were obtained at the highest concentrations (1000 and 1600 µg/mL) and mutation factors slightly over 2 (2.1 and 2.4, respectively) were reported. There was a weak increase in the induction of gene mutations at this concentration. No increase in mutation frequency was obtained with metabolic activation. The maximum concentration of 1600 ug/mL in one experiment was slightly over the limit of 10 mM (~1000 µg/mL) for mutagenicity tests in cell cultures in order to avoid false positive results due to effects of osmolarity. Because the highest concentrations tested were very close to the osmolality limit, the reported weak mutagenic effect is ambiguous and cyanamide may or may not have a mutagenic potential in vitro according to this mouse lymphoma assay.

The ability of hydrogen cyanamide to cause chromosomal damage in vivo was investiagted in two mouse micronucleus assay studies. The dose levels tested were 35, 175, and 350 mg/kg bw and five mice per sex were exposed for 24, 48, and 72 hours via oral gavage. All animals appeared healthy until sacrifice time except for those in the highest dose level where mice had ruffled coats and 3 males were found dead within 24 hours. No significant changes in the NCE/PCE ratio was observed. No significant increases in micronucleated polychromatic erythrocytes over the levels observed in the negative controls were observed and the positive control triethylenmelamine induced significant increases in micronucleated polychromatic erythrocytes in both sexes. In a supporting mouse micronucleus assay study, the NCE/PCE ratio was significantly decreased in the animals treated via oral gavage with the highest tested dose of cyanamide (Colme; 247 mg/kg bw) but no dose tested induced a significant increase in micronucleated polychromatic and normochromatic erythrocytes over the levels observed in the negative controls. Based on these two studies, hydrogen cyanamide and cyanamide (Colme) do not induce micronuclei of mice up to 350 and 247 mg/kg bw, respectively.

Justification for classification or non-classification

Calcium cyanamide technical grade (Kalkstickstoff) did not show mutagenic activity in vitro or in vivo with the exception of one non-guideline bacterial mutation assay. This assay revealed a positive results only in the absence of metabolic activation, which is considered not to be of relevance as the presence of metabolic activation is more likely to represent the physiological (in vivo) situation in animals and humans. No indication for mutagenic effects were noted and all the other tests were negative, including an in vivo micronucleus test. Therefore, Kalkstickstoff is not classified according to Regulation (EC) No 1272/2008 (GHS/CLP).