Cyanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyanamide was evaluated for its mutagenic and genotoxic potential in vitro and in vivo. Cyanamide showed no mutagenic activity in bacterial cells. In mammalian cells cyanamide showed a ambiguous (due to effects of osmolarity) weak positive result in the mouse lymphoma test at the thymidine kinase locus at the maximum concentrations and no mutagenic effects at the HPRT-locus in V79 at the maximum concentrations.
DNA damage and repair was investigated in vitro in CHO cells (Sister Chromatid Exchange) and in the UDS test in primary rat hepatocytes. In both experiments there was no indication of DNA damage and repair caused by cyanamide. Two chromosome aberrations tests in CHO cells and in human lymphocytes showed distinct clastogenic effects of cyanamide.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable (no target gene)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from Aroclor 1254 induced male Sprague-Dawley rat liver

Test concentrations with justification for top dose:

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.
+S9: 438- 1310 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

80 ng/mL for the non-activation set

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

17.5 and 50 µg/mL requiring activation

Details on test system and experimental conditions:

Cells were harvested at 10 (with S9 mix) and 20 hours (with and without S9 mix), respectively after treatment had commenced. For the final 2.5 hours prior to harvest, cultures were exposed to colcemide. These test conditions had been selected on the basis of a preliminary range-finding test.
Following harvest, cells were fixed on slides, stained and examined for chromosomal aberrations. 100 cells from each duplicate culture were analysed, except at 283 µg/mL without and 1.310 µg/mL with metabolic activation at the 20 hour preparation interval where only 50 cells were investigated.

Statistics:

Fishe´s Exact Test (p<0.01)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

4 concentrations were analysed beginning at 42.4 up to 283 µg/mL. A significant increase was obtained at 141 and 283 µg/mL and a dose-response was established.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A complete toxicity at 424 and 565 µg/mL and a reduction in observable mitotic cells was obtained at 283 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

At 20 h preparation interval (438, 875 and 1.310 µg/ml) a significant dose-dependent increase in aberrant cells was obtained beginning at the lowest concentration. In the 10 hour preparation interval a slight but not significant abberations increase.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

complete toxicity was obtained at 1.750 µg/mL and few mitotic cells were discernible at 1.310 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the preliminary concentration finding test, CHO cells were exposed to culture medium and to various concentrations of hydrogen cyanamide ranging from 17.7 µg/mL to 5.300 µg/mL in the absence and in the presence of S9 mix. Without metabolic activation a significant cell cycle delay was obtained beginning 53.0 and 177 µg/mL. Complete toxicity was obtained above concentrations of 530.0 µg/mL without S9 mix. Therefore, a 20 hour preparation interval was selected for testing a dose range of 28.2 µg/mL to 565 µg/mL without metabolic activation.
In the presence of metabolic activation complete toxicity was observed at 1750 µg/mL. A cell cycle delay was obtained at 530 µg/mL. Therefore, two harvest times were selected for the aberration assay with a dose range of 437 to 1750 µg/mL at the 20 hour preparation interval and a dose range of 107 µg/mL to 428 µg/mL at 10 hour preparation interval.

Table 1: Chromosome aberrations in Chinese hamster ovary (CHO) cells:

Test groups/Concentrations	Cells scored	% Cells with aberrations without gaps
Fixation interval 20 h after treatment without S9 mix
Negative and Solvent control	200	2.0
Mitomycin C 80 ng/ml	25	28.0*
42.4	200	1.0
56.5	200	1.5
141.0	200	33.5*
283.0	50**	96.0*
424	-	-
Fixation interval 20 h after treatment with S9 mix
Negative and Solvent control	200	1.0
Cyclophosphamide 17.5 µg/ml	25	36.0*
438.0	200	6.0*
875.0	200	42.0*
1310.0	50**	94.0*
1750.0	-	-
Fixation interval 10 h after treatment with S9 mix
Negative and Solvent control	200	1.5
Cyclophosphamide 17.5 µg/ml	25	16.0*
321.0	200	1.5
428.0	200	5.0

* statistically significant by Fisher's Exact Test (p<0.01)

** highly toxic effects

- no scorable metaphases

 

 

Conclusions:

Under the conditions of the assay described in this report, Hydrogen cyanamide induced an increase in structural chromosome aberrations in CHO cells and should be considered as clastogenic in this test system.
It is concluded, that cyanamide has a clastogenic potential in vitro. However, the clasogenic effects observed in vitro could not be detected in vivo. Therefore, cyanamide is considered to be not clastogenic in vivo.

Executive summary:

Hydrogen cyanamide was assessed for its potential to induce structural chromosome aberrations in Chinese hamster ovary (CHO) cells in vitro. Hydrogen cyanamide was tested in the presence and absence of metabolic activation (S9 mix prepared from Aroclor 1254 induced male Sprague-Dawley rat liver). The test article was dissolved in culture medium. 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. Cells were harvested at 10 (with S9 mix) and 20 hours (with and without S9 mix), respectively after treatment had commenced. For the final 2.5 hours prior to harvest, cultures were exposed to colcemide. These test conditions had been selected on the basis of a preliminary range-finding test which was also described in the original report. Following harvest, cells were fixed on slides, stained and examined for chromosomal aberrations. 100 cells from each duplicate culture were analysed, except at 283 µg/mL without and 1.310 µg/mL with metabolic activation at the 20 hour preparation interval where only 50 cells were investigated. Mitomycin C (80 ng/mL for the non-activation set) and cyclophosphamide (17.5 and 50 µg/ml, respectively requiring activation) served as positive control substances. An untreated negative control (culture medium) was also included in the testing.

In the main experiment without metabolic activation there was complete toxicity at 424 and 565 µg/mL and a reduction in observable mitotic cells was obtained at 283 µg/ml. 4 concentrations were analysed beginning at 42.4 up to 283 µg/ml. A significant increase was obtained at 141 and 283 µg/mL and a dose-response was established. With metabolic activation complete toxicity was obtained at 1.750 µg/mL and few mitotic cells were discernible at 1.310 µg/ml. Three concentrations (438, 875 and 1.310 µg/ml) were evaluated at the 20 hour preparation interval. In this experiment a significant dose-dependent increase in aberrant cells was obtained beginning at the lowest concentration. In the 10 hour preparation interval 2 concentrations 321 and 428 µg/mL were evaluated with no observable toxicity at the highest concentration. In this experiment a slight but not significant increase in cells with chromosomal aberrations was obtained at the highest investigated concentration.

Under the conditions of the assay described in this report, Hydrogen cyanamide induced an increase in structural chromosome aberrations in CHO cells and should be considered as clastogenic in this test system.

 

It is concluded, that cyanamide has a clastogenic potential in vitro. However, the clastogenic effects observed in vitro could not be detected in vivo. Therefore, cyanamide is considered to be not clastogenic in vivo.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC 84/449 (1984)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable (no specific target gene, the assay detects chromosome abberations)

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from Aroclor 1254 induced male Sprague-Dawley rat liver

Test concentrations with justification for top dose:

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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL for the non-activation assay

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

15 µg/mL requiring activation

Details on test system and experimental conditions:

- Since no preliminary range-finding test was performed a variety of concentrations in an appropriate range were chosen
- For both assays, cells were harvested at 24 hours after treatment had commenced. For the final 3 hours prior to harvest, cultures were exposed to colcemide.
- Following harvest, cells were fixed on slides, stained and examined for chromosomal aberrations. 100 cells from each duplicate culture were analysed.

Statistics:

Chi-square test criteria, p<0.05 or p<0.01, p<0.001.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

A statistically significant increase in numerical chromosomal aberrations (exclusive gaps) was observed in the absence and in the presence of S9 mix at the highest concentration (33.3 µg/mL) without S9 mix and at 33.3 and 333.3 µg/mL with S9 mix.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Since no preliminary range-finding test was performed a variety of concentrations in an appropriate range were chosen. In the absence of S9 mix the mitotic index was reduced by 46 % at 33.3 µg/mL and in the presence of S9 mix at 333.3 µg/mL by 61 %. Based on these observations the following doses were selected for the scoring of chromosome aberrations: 3.3, 10.0 and 33.3 µg/mL without S9 mix and 10.0, 33.3, 100.0 and 333.0 µg/mL in the presence of S9 mix.

Conclusions:

Under the conditions of the assay described in this report, Hydrogen cyanamide induced a significant increase in structural chromosome aberrations with nonactivation and metabolic activation in human lymphocytes in vitro and should be considered as clastogenic in this test system.

Executive summary:

Hydrogen cyanamide was assessed for its potential to induce structural chromosome aberrations in cultured human lymphocytes in vitro. Hydrogen cyanamide was tested in the presence and absence of metabolic activation (S9 mix prepared from Aroclor 1254 induced male Sprague-Dawley rat liver). The test article was diluted in culture medium. 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. For both assays, cells were harvested at 24 hours after treatment had commenced. For the final 3 hours prior to harvest, cultures were exposed to colcemide. An appropriate range and number of dose levels was chosen, instead of a preliminary range-finding test. Following harvest, cells were fixed on slides, stained and examined for chromosomal aberrations. 100 cells from each duplicate culture were analysed. Mitomycin C (0.1 µg/mL for the non-activation assay) and cyclophosphamide (15 µg/mL requiring activation) served as positive control substances. An untreated solvent control (culture medium) was also included in the testing. Since no preliminary range-finding test was performed a variety of concentrations in an appropriate range were chosen. In the absence of S9 mix the mitotic index was reduced by 46 % at 33.3 µg/mL and in the presence of S9 mix at 333.3 µg/mL by 61 %. Based on these observations the following doses were selected for the scoring of chromosome aberrations: 3.3, 10.0 and 33.3 µg/mL without S9 mix and 10.0, 33.3, 100.0 and 333.0 µg/mL in the presence of S9 mix. A statistically significant increase in numerical chromosomal aberrations (exclusive gaps) was observed in the absence and in the presence of S9 mix at the highest concentration (33.3 µg/ml) without S9 mix and at 33.3 and 333.3 µg/mL with metabolic activation. The positive control substances revealed statistically significant increases in chromosome aberration rate and demonstrated the sensitivity of the assay. Under the conditions of the assay described in this report, Hydrogen cyanamide induced a significant increase in structural chromosome aberrations with nonactivation and metabolic activation in human lymphocytes in vitro and should be considered as clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

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:

, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC 87/301 (1987)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA FIFRA 40 CFR

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT-locus in V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix the concentration range was 31.3 to 1000.0 µg/mL in experiment 1 and 15.6 to 500.0 µg/mL in experiment 2. Concentrations from 1.25 to 30.0 µg/mL (experiment 1) and from 2.0 to 250.0 µg/mL (experiment 2) were investigated with metabolic activation.
Experiment 1:
+S9: 1.25-30.0 µg/mL
-S9: 31.3-1000 µg/mL
Experiment 2:
+S9: 2.0-250 µg/mL
-S9: 15.6-500 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Not requiring activation

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Acting only after metabolic activation

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

after 4 h treatment: moderate toxic effects at 1000 µg/mL observed., after 24 h treatment strong toxic effects at 250.0 and 500.0 µg/mL. These effects were more distinct at the highest concentration where the cultures could not be continued.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong toxic effects occurred at 62.5 µg/mL and above, with more distinct effects at 125 and 250 µg/mL (maximum concentration) where the cultures could not be continued.

Additional information on results:

- In a preliminary cytotoxicity assay (by means of a growth inhibition test), the test substance was applied at a maximum concentration of 1000.0 µg/mL (10 mM) and 8 lower concentrations. According to the results of the pre-experiment the top concentration in experiment I (4 h treatment interval) were selected to be 1000.0 µg/mL (without S9 mix) and 30 µg/mL (with S9 mix), respectively.

According to the results of the pre-experiment the top concentration in experiment I (4 h treatment interval) were selected to be 1000.0 µg/mL (without S9 mix) and 30 µg/mL (with S9 mix), respectively. Without metabolic activation moderate toxic effects, evidenced by a reduction of the cell density at the first subcultivation were observed at the maximum concentration. However, in the experiment with metabolic activation, in contrast to the pre-experiment no relevant toxic effects occurred up to the highest concentration. Therefore, this experiment has to be repeated.

The second experiment (24 h treatment interval) without S9 mix was carried out with 500 µg/mL as a top concentration. Strong toxic effects were obtained at 250.0 and 500.0 µg/ml. These effects were more distinct at the highest concentration where the cultures could not be continued.

The repeat experiment with metabolic activation was performed with 250 µg/mL as the maximum concentration. In this experiment strong toxic effects occurred at 62.5 µg/mL and above, with more distinct effects at 125 and 250 µg/mL where the cultures could not be continued.

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.

Conclusions:

In conclusion, it can be stated 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.

Executive summary:

The potential mutagenic effect of Cyanamid L500 in mammalian cells was examined by assaying the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells. In a preliminary cytotoxicity assay (by means of a growth inhibition test), the test substance was applied at a maximum concentration of 1000.0 µg/mL( 10 mM) and 8 lower concentrations. Thereafter, four independent main assays were conducted. The treatment period was 4 h in the first experiment with and without S9 mix and 24 h in the second experiment without S9 mix. A third experiment using 4 h treatment in the presence of S9 mix was required since the toxic concentration range was not reached. This experiment has to be repeated due to technical reasons (experiment four). Without S9 mix the concentration range was 31.3 to 1000.0 µg/mL in experiment 1 and 15.6 to 500.0 µg/mL in experiment 2. Concentrations from 1.25 to 30.0 µg/mL (experiment 1) and from 2.0 to 250.0 µg/mL (experiment 2) were investigated with metabolic activation. S9 mix was obtained from the liver of rats induced with phenobarbital and b -naphthoflavone. The test substance was dissolved in deionised water which was also used as solvent control, whereas the positive control substances ethylmethane sulfonate (EMS, not requiring activation) and 7,12-dimethylbenz(a)anthracene (DMBA, acting only after metabolic activation) were diluted in water and DMSO respectively. The effects of treatment were examined at expression time of 7 days.

According to the results of the pre-experiment the top concentration in experiment I (4 h treatment interval) were selected to be 1000.0 µg/mL (without S9 mix) and 30 µg/mL (with S9 mix), respectively. Without metabolic activation moderate toxic effects, evidenced by a reduction of the cell density at the first subcultivation were observed at the maximum concentration. However, in the experiment with metabolic activation, in contrast to the pre-experiment no relevant toxic effects occurred up to the highest concentration. Therefore, this experiment has to be repeated.

The second experiment (24 h treatment interval) without S9 mix was carried out with 500 µg/mL as a top concentration. Strong toxic effects were obtained at 250.0 and 500.0 µg/ml. These effects were more distinct at the highest concentration where the cultures could not be continued.

The repeat experiment with metabolic activation was performed with 250 µg/mL as the maximum concentration. In this experiment strong toxic effects occurred at 62.5 µg/mL and above, with more distinct effects at 125 and 250 µg/mL where the cultures could not be continued.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The clastogenic effects observed in vitro could not be detected in vivo (in three micronucleous assays). Therefore, cyanamide is considered to be not clastogenic in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

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

Duration of treatment / exposure:

Exposure for 24, 48 and 72 hours at all dose levels

Frequency of treatment:

Daily

Post exposure period:

No post exposure period

Dose / conc.:

35 mg/kg bw/day (nominal)

Remarks:

the dosis analytical found was 31.44 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

the dosis analytical found was 157.4 mg/ kg bw/day

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

the dosis analytical found was 330.5 mg/kg bw/day

No. of animals per sex per dose:

Five mice/sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

A positive control triethylenemelamine

Tissues and cell types examined:

Polychromatic erythrocytes and normochromatic erythrocytes from the bone marrow

Details of tissue and slide preparation:

Bone marrow smear slides were prepared and stained. Approximately 1000 polychromatic erythrocytes (PCE’s) were examined for the presence of micronuclei. The ratio of poly- to normochromatic erythrocytes was determined to assess inhibition of erythropoesis.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

At the high dose group 3 male animals were found dead after 5 and 20 hours, respectively. All males at this dose level had ruffled coats throughout the duration of the study. All other animals were apparently healthy until the appropriate sacrifice time.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A dose rangefinding study was carried out to assess toxicity with dose levels of 75, 150, 300, 450 and 600 mg/kg bw. Based upon the results of this trial the dose levels selected for the micronucleus assay were 35, 175 and 350 mg/kg bw.

The stability of the test substance throughout the study period was shown by reanalysis. The homogeneity was guaranteed by mixing before preparations of the test solutions. The stability of the test substance in water was verified analytically and the following concentrations were determined: 31.44, 157.4 and 330.5 mg/kg bw.

Conclusions:

Hydrogen cyanamide did not induce micronuclei in polychromatic erythrocytes of mice when orally (by gavage) treated up to 350 mg/kg body weight.

Executive summary:

The ability of hydrogen cyanamide to cause chromosomal damage in vivo was investigated in the mouse micronucleus assay. Male and female ICR mice were dosed orally by gavage with a aqueous solution of Hydrogen cyanamide. Based upon the results of the range finding trial the dose levels selected for the micronucleus assay were 35, 175 and 350 mg/kg bw. The vehicle control as well as the positive control triethylenemelamine were also tested. The stability of the test substance throughout the study period was shown by reanalysis. The homogeneity was guaranteed by mixing before preparations of the test solutions. The stability of the test substance in water was verified analytically and the following concentrations were determined: 31.44, 157.4 and 330.5 mg/kg.

Five mice/sex were exposed for 24, 48 and 72 hours at all dose levels. Only one sampling time, 24 hours after treatment, was performed with the negative and the positive control. A second group of animals was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group.

Bone marrow smear slides were prepared and stained. Approximately 1000 polychromatic erythrocytes (PCE’s) were examined for the presence of micronuclei. The ratio of poly- to normochromatic erythrocytes was determined to assess inhibition of erythropoesis and all animals were examined after dosing and periodically throughout the duration of the study for toxic effects and/or mortalities.

At the high dose group 3 male animals were found dead after 5 and 20 hours, respectively. All males at this dose level had ruffled coats throughout the duration of the study. All other animals were apparently healthy until the appropriate sacrifice time. No significant changes in the ration of NCE´s to PCE´s were observed. The test substance, hydrogen cyanamide, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the negative controls in either sex or at any sampling interval. The positive control, triethylenmelamine, induced significant increases in micronucleated PCEs in both sexes.

It can be concluded that hydrogen cyanamide did not induce micronuclei in polychromatic erythrocytes of mice when orally (by gavage) treated up to 350 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Cyanamide was evaluated for its mutagenic and genotoxic potential in vitro and in vivo. Both Ames-Tests were negative indicating no mutagenic activity in bacterial cells. Two gene mutation assays using mammalian cells were carried out one using mouse lymphoma cells and the other V79-cells. A weak positive result was obtained in the mouse lymphoma test at the thymidine kinase locus at the maximum concentrations (1600 and 1000 µg/mL, respectively), in experiment 1 and 2 without metabolic activation. However, the biological relevance of the observed slight increases is ambiguous as they were obtained at concentration above and at the level of 10 mM, the limit for mutagenicity tests carried out in cell cultures in order to avoid false positive results due to effects of osmolarity. Therefore the weak mutagenicity in this assay is ambiguous. Furthermore, no mutagenic effects were obtained at the HPRT-locus in V79 with the maximum concentrations of 1000 and 500 µg/mL without S9 mix and 30 and 250 µg/mL with S9 mix.

The chromosome aberrations test in CHO cells and in human lymphocytes showed distinct clastogenic effects of cyanamide. In the assay with CHO cells a significant dose-dependent increase in cells with aberrations was obtained with and without metabolic activation at the 20 hour preparation interval. A statistically significant increase in numerical chromosomal aberrations (exclusive gaps) was obtained at the maximum concentrations (33.3 µg/ml) without S9 mix and at 33.3 and 333.3 µg/mL in the presence of S9 mix in human lymphocytes. Both assays indicate a clastogenic response of cyanamide in vitro.

Two micronucleus assays were carried out in order to investigate the potential of cyanamide to cause chromosomal damage in vivo. The studies where performed with male and female ICR mice and Swiss mice, respectively. In the two assays carried out in mice the test substance (hydrogen cyanamide and cyanamide Colme, respectively) was administered via gavage and no induction of micronuclei was obtained up to the highest investigated dose (350 and 247 mg/kg bw, respectively). DNA damage and repair was investigated in vitro in CHO cells (Sister Chromatid Exchange) and in the UDS test in primary rat hepatocytes. In both experiments there was no indication of DNA damage and repair caused by cyanamide. An in vivo UDS test was not considered necessary to perform as the weak positive and questionable response in the mouse lymphoma gene mutation test was attributed to clastogenic effects and not to point mutations. This assumption is supported by the negative result of the HPRT-Test in V79 cells.

It is concluded, that Cyanamide has a clastogenic potential in vitro. However, the clastogenic effects observed in vitro could not be detected in vivo. Therefore, cyanamide is considered to be not clastogenic in vivo. Results of the mutagenicity studies are summarised in the following table:

 

Study type	Cells / species	Test conditions	Results	Reference
Ames test	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538	+ S9:20 – 2540 µg/plate - S9: 20 – 2540 µg/plate   experiment 1 and 2	Negative	Jagannath, Myhr, B.C., 1987 557-001
Ames test	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538   E.coli strains GY 4015 and GY 5027	+ S9:10 – 5000 µg/plate - S9: 20 – 5000 µg/plate	Negative	Cadena et al., 1984 592-007
HPRT test	Chinese hamster V79 cells	+ S9:1.25 – 30.0 µg/mL - S9:31.3 – 1000 µg/mL Experiment 1 + S9: 2.0 – 250 µg/mL -       S9: 15.6 – 500 µg/mL Experiment 2	Negative	Wollny, Arenz, 2000 550-001
Point mutation test at the Thymidin kinase locus	Mouse lymphoma cells L5178Y	+ S9:     5 – 35 µg/mL - S9:50 – 1600 µg/mL Experiment 1 + S9: 5.0 – 22.5 µg/mL - S9: 50 – 1000 µg/mL Experiment 2	Weakly positive at concentrations at and above 10 mM	Enninga, 1988 557-008
in vitro cytogenetic cells	Chinese hamster ovary cells (CHO)	+ S9: 438 – 1310 µg/mL 20 h preparation interval + S9: 321 and 428 µg/mL 10 h preparation interval - S9:   42.4 – 283 µg/mL 20 h preparation interval	Positive	Ivett, 1987 557-010
in vitro cytogenetic cells	Human lymphocytes	+ S9: 10 – 333 µg/mL - S9: 3.3 - 33.3 µg/mL	Positive	Enninga, van de Waart, 1988 557-007
in vitro UDS test	primary rat hepatocytes	5.95 – 190 µg/mL	Negative	Cifone, 1987 557-002
in vivo micronucle-us test	ICR mice	35 - 350 mg/kg bw treatment intervals 24, 48, and 72 hours	Negative	Ivett, 1987 557-003
in vivo micronucle-us test	Swiss mice	10 – 247 mg/kg bw treatment 48 hours	Negative	Menargues et al., 1984 592-008

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on Cyanamide showed clastogenic potential in vitro. However, the clastogenic effects observed in vitro could not be detected in vivo. Therefore, cyanamide is considered to be not clastogenic in vivo. Other studies indicated that cyanamide induces no mutagenic activity and no DNA damage and repair therefore cyanamide does not require classification as mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.