Cyanamide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cyanamide

EC Number:

206-992-3

EC Name:

Cyanamide

Cas Number:

420-04-2

Molecular formula:

CH2N2

IUPAC Name:

cyanamide

Test material form:

other: aqueous solution

Details on test material:

- Test substance: Hydrogen cyanamide
- Purity: 50 % w/w (equivalent to 53 % w/v)
- Appearance: Clear, pale yellow liquid
- Batch: 02/10/86

Method

Target gene:

Not applicable (no target gene)

Species / strain

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

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

80 ng/mL for the non-activation set

Controls 3

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)

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.