Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of Dicyandiamide was evaluated in an in vitro gene mutation study in bacteria (Ames test) according to OECD 471, both in the presence and the absence of metabolic activation, and in a second Ames test conducted by El-Tarras et al. (1989) using metabolic activation by the S-14 plant fraction.
Clastogenicity of DCD in CHO cells was assessed in an in vitro Chromosome aberration test equivalent to OECD 473 with and without metabolic activation. A forward mutation (HGPRT) assay in mammalian CHO cells (OECD 476) was conducted in the presence or absence of metabolic activation. In addition a rat hepatocyte unscheduled DNA synthesis assay equivalent to OECD 482 was conducted.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 1984 - May 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no specification of test compound
GLP compliance:
yes
Remarks:
No GLP compliance statement, and no information on a GLP certificate are available. But according to laboratory procedures study is GLP.
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable;
cell line used: ATCC (American Type Culture Collection) CCL 61 (ovary, Chinese hamster, CHO K1)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 medium with 10 % fetal calf serum (FCS) and frozen stocks are held in a liquid nitrogen storage tank
- Properly maintained: yes
- Cells: K-1, Number CCL61, ontained from American Type Culture Collection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
20, 67, 200, 670, and 2000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
control for experiment without metabolic activation
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
control for experiment with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (Ham's F-12 medium)


DESIGN AND DOSING
- 5 concentrations of Dicyandiamid were prepared to contain the test dose in either a 0.025 or 0.05 ml volume
- highets dose tested both with and without metabolic activation was approx. 2,000 µg/ml with subsequent concentrations of 670, 200, 67, and 20 µg/ml

- Initial assay: twenty-eight 75 cm² tissue culture flasks (three hour sampling)and sixty 25 cm² tissue culture flasks ( 8 + 12 hour sampling) were seeded with 7.5 x 10^5 and 5.0 x 10^5 cells, respectively
- The non-activation part of the assay was repeated and all cell cultures were seeded with 7.5 x 10^5 CHO cells in 75 cm² flasks
- In all cases, 25 cm² flasks contained 5 ml Ham's F-12 medium and 75 cm² flasks contained 10 ml Ham's F-12

- 24 hours after culture initiation, the medium, S-9 mix, and test solution were dispensed into the appropriate flasks in duplicate
- Each flask was mixed, tightly capped, and incubated at 37 +/- 0.5 °C
- Following 2 hour exposure, flasks labeled 16 and 23-44 containing mitomycin C (16), and S-9 mix (23-44) were rinsed with a balanced salt solution, 5 or 10 ml Ham's F-12 medium added to each flask, and the flasks incubated again at 37 +/- 0.5 °C

- Approx. 1, 6, or 10 hours after addition of the test metarial, colcemid (0.2 µg/ml) was added to the appropriate flasks to arrest cells in metaphase; without metabolic activation: 3, 8, or 12 hours


PREPARATION OF CHO CHROMOSOMES
- Metaphases were collected by miotic shake-off two hours after the addition colcemid
- Each flask was firmly tapped and the media was poured into appropriately labeled 15 ml centrifuge tube
- The cells were washed once in 0.075 M KCl, and washed twice in an acetic alcohol fixative (3:1, methanol : acetic acid)


PREPARATION OF SLIDES
- Four slides were prepared for each treatment group, two from each duplicate flask
- Slides were prepared by dropping cells onto clean wet slides and air drying


SLIDE STAINING
- Slides were stained for 10 minutes in 2 % Giemsa in phosphate buffer (pH 6.8), rinsed twice in glass distilled H2O, and air dried.
- The slides were mounted with glass coverslips using Cover Bond


SLIDE RANDOMIZATION
- Flask numbers on each slide were covered with masking tape and each was assigned a temporary slide number
- The coding was performed by another individual not involved in evaluation of slides
- The code was not broken until all slides had been analyzed


CYTOGENTIC ANALYSIS
- 100 cells in metaphase were examined for each group, 50 from each duplicate flask, whenever possible
- The slides were scanned with a low power objective (10 or 25X) to find cells in metaphase stage and the chromosomes were analyzed with high power oil immersion lens (100x)
- Only those cells in the metaphase stage of mitosis were analyzed for the presence of cytogentic abnormalities
- Numbers and types of chromosomal aberrations, modal number and the vernier location of each metaphase containing damage were recorded
Evaluation criteria:
- statistically significant increase in the frequency of chromosomal aberrations compared to control values for the levels tested at the sampling times
- aneuploid or polyploid cells?
- statistically significant increase in aberration frequency for positive control substances
Statistics:
Upon completion of the scoring, the slides were decoded and the data were entered into the appropriate group for statistical analyses.
The percent aberrant cells for each treatment group were analyzed using a CHI-Square test for multiple proportions. The mean number of aberrations per cell for each group was analyzed by Analysis of Variance (ANOVA) with individual group comparison performed by Dunnett's t-test. All data were analyzed at the 95 % confidence interval (p < 0.05) by a one-tailed test.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No statistically significant increase in the frequency of chromosomal aberrations compared to control values for any of the levels tested at any of the sampling times.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 2: Summary of Aberration Data – in vitro Chromosomal Aberration (CHO) Assay

 

Dicyandiamide – Without Activation – 3 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

1

Media Control

100

4

4.0

-

8

0.08

-

2

Solvent Control – (DMSO)

100

6

6.0

-

7

0.07

-

3

Dicyandiamide (2000 µg/ml)

100

4

4.0

NS

6

0.06

NS

4

Dicyandiamide (670 µg/ml)

100

3

3.0

NS

6

0.06

NS

5

Dicyandiamide (200 µg/ml)

100

6

6.0

NS

9

0.09

NS

6

Dicyandiamide (67 µg/ml)

100

5

5.0

NS

6

0.06

NS

7

Dicyandiamide (20 µg/ml)

100

5

5.0

NS

8

0.08

NS

 

Dicyandiamide – With Activation – 3 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

23

Media Control

100

1

1.0

-

2

0.02

-

24

Solvent Control – (DMSO)

64

1

1.6

-

1

0.02

-

25

Dicyandiamide (2000 µg/ml)

100

3

3.0

NS

4

0.04

NS

26

Dicyandiamide (670 µg/ml)

100

2

2.0

NS

2

0.02

NS

27

Dicyandiamide (200 µg/ml)

100

1

1.0

NS

1

0.01

NS

28

Dicyandiamide (67 µg/ml)

100

0

0

NS

0

0

NS

29

Dicyandiamide (20 µg/ml)

100

1

1.0

NS

1

0.01

NS

 

Dicyandiamide – Without Activation – 8 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

8

Media Control

100

3

3.0

-

6

0.06

-

9

Solvent Control – (DMSO)

100

2

2.0

-

3

0.03

-

10

Dicyandiamide (2000 µg/ml)

100

2

2.0

NS

2

0.02

NS

11

Dicyandiamide (670 µg/ml)

100

6

6.0

NS

7

0.07

NS

12

Dicyandiamide (200 µg/ml)

100

2

2.0

NS

3

0.03

NS

13

Dicyandiamide (67 µg/ml)

100

3

3.0

NS

8

0.08

NS

14

Dicyandiamide (20 µg/ml)

100

3

3.0

NS

5

0.05

NS

 

Dicyandiamide – With Activation – 8 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

30

Media Control

100

0

0

-

0

0

-

31

Solvent Control – (DMSO)

100

2

2.0

-

12

0.12

-

32

Dicyandiamide (2000 µg/ml)

100

3

3.0

NS

6

0.06

NS

33

Dicyandiamide (670 µg/ml)

100

3

3.0

NS

6

0.06

NS

34

Dicyandiamide (200 µg/ml)

100

2

2.0

NS

4

0.04

NS

35

Dicyandiamide (67 µg/ml)

100

2

2.0

NS

4

0.04

NS

36

Dicyandiamide (20 µg/ml)

100

3

3.0

NS

5

0.05

NS

 

Dicyandiamide – Without Activation – 12 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

15

Media Control

100

2

2.0

-

3

0.03

-

16

Positive Control – (80 µg/ml)

36

17

47.2

S

86

2.39

S

17

Solvent Control – (DMSO)

100

3

3.0

-

4

0.04

-

18

Dicyandiamide (2000 µg/ml)

100

2

2.0

NS

3

0.03

NS

19

Dicyandiamide (670 µg/ml)

100

4

4.0

NS

5

0.05

NS

20

Dicyandiamide (200 µg/ml)

100

2

2.0

NS

2

0.02

NS

21

Dicyandiamide (67 µg/ml)

100

1

1.0

NS

2

0.02

NS

22

Dicyandiamide (20 µg/ml)

100

2

2.0

NS

3

0.03

NS

 

Dicyandiamide – With Activation – 12 Hour Sampling

Group Number

Treatment

Number of Metaphases Analyzed per Group

Number of Aberrant Cells per Group

Percent Aberrant Cells per Group

Statistical Significance

(p < 0.05)

Total Number of Aberrations per Group

Average Number of Aberrations per Cell

Statistical Significance

(p < 0.05)

37

Media Control

100

2

2.0

-

4

0.04

-

38

Positive Control – (CP 140 µg/ml)

100

43

43.0

S

163

1.63

S

39

Solvent Control – (DMSO)

100

0

0

-

0

0

-

40

Dicyandiamide (2000 µg/ml)

100

3

3.0

NS

5

0.05

NS

41

Dicyandiamide (670 µg/ml)

100

0

0

NS

0

0

NS

42

Dicyandiamide (200 µg/ml)

100

3

3.0

NS

5

0.05

NS

43

Dicyandiamide (67 µg/ml)

100

1

1.0

NS

2

0.02

NS

44

Dicyandiamide (20 µg/ml)

100

0

0

NS

0

0

NS

NS     =        Not Significant

S        =        Significant

CP      =        Cyclophosphamide

Mito    =        Mitomycin C

Conclusions:
The exposure of CHO cells to 2000, 670, 200, 67, and 20 µg/ml of Dicyandiamide produced no significant increases in the frequency of chromosomal aberrations, with or without metabolic activation.
Therefore, under the conditions of this study, Dicyandiamide is considered not to be clastogenic.
Executive summary:

Dicyandiamide, identified as a white solid, was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary (CHO) cells in vitro with and without extrinsic metabolic activation (S9). The compound was dissolved in DMSO and tested at five concentrations: 2000, 670, 200, 67, and 20 µg/ml for two hours in the presence of metabolic activation followed by a 1, 6, or 10 hour expression period, and for 3, 8, or 12 hours in the absence of metabolic activation.

Results show that no statistically significant increase in the frequency of chromosomal aberrations was seen for any of the levels tested at any of the sampling times, either with or without metabolic activation.

Therefore, under the conditions of this study, Dicyandiamide is considered not to be clastogenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
5th bacteria strain is TA1538 (according to old guidelines)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
5th bacteria strain is TA1538 (according to old guidelines)
GLP compliance:
yes
Remarks:
The study was carried out under conditions of good laboratory practice. Within reason there have been no circumstances that might have affected the quality and integrity of the study.
Type of assay:
bacterial reverse mutation assay
Target gene:
- genes involved in histidine synthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requiring
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine requiring
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0 - 3.1 - 9.3 - 27.8 - 83.3 and 250.0 mg/ml (highest soluble amount)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test solutions have been diluted in water immediately before use to obtain test concentrations (see above)
Untreated negative controls:
yes
Remarks:
solvent/vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0 mg/ml
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg per 0.1 ml water per plate; for strains TA 1535 and TA 100 without S-9 mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg per 0.1 ml DMSO per plate; for strain TA 1537 without S-9 mix
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1.0 µg per 0.1 ml DMSO per plate for strain TA 1538 and 2.0 µg per 0.1 ml DMSO per plate for strain TA 98 without S-9 mix
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.0 µg per 0.1 ml DMSO per plate for all strains in the presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: three

Evaluation criteria:
In case of questionable or positive results the plate incorporation assay is partly repeated and/or a number of revertant colonies is checked for histidine requirements and for other strain characteristics if appropriate.
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
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No additional information

Table 2: Results of the Salmonella / Microsome Mutagenicity Test with Dicyanodiamide (DCD)

 

 

 

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Sample

Dose

mg/pl

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

 

 

 

 

DCD

 

 

 

 

0.00

 

 

 

 

mean

sd

51

65

64

 

56

7

15

18

21

 

18

3

9

12

15

 

12

3

14

16

26

 

19

6

15

24

30

 

23

8

45

45

51

 

47

3

21

24

29

 

25

4

51

53

55

 

53

2

151

152

169

 

157

10

158

170

173

 

167

8

 

 

 

 

DCD

 

 

 

 

0.31

 

 

 

 

mean

sd

37

52

53

 

47

9

17

21

24

 

21

4

14

15

15

 

15

1

13

13

20

 

15

4

12

19

25

 

19

7

39

44

50

 

44

6

25

28

37

 

30

6

27

36

41

 

35

7

156

162

171

 

163

8

158

162

170

 

163

6

 

 

 

 

DCD

 

 

 

 

0.93

 

 

 

 

mean

sd

39

39

40

 

39

1

19

21

31

 

24

6

16

18

20

 

18

2

13

14

27

 

18

8

16

21

33

 

20

4

42

42

50

 

45

5

27

29

33

 

30

3

44

62

64

 

57

11

125

174

181

 

160

31

146

148

158

 

151

6

 

 

 

 

DCD

 

 

 

 

2.78

 

 

 

 

mean

sd

43

48

51

 

47

4

17

18

19

 

18

1

7

9

12

 

9

3

8

13

27

 

16

10

14

21

25

 

20

6

40

48

55

 

48

8

21

24

38

 

28

9

43

52

53

 

49

6

129

151

156

 

145

14

148

157

163

 

156

8

 

 

 

 

DCD

 

 

 

 

8.33

 

 

 

 

mean

sd

40

41

43

 

41

2

14

20

25

 

20

6

7

12

14

 

11

4

15

15

25

 

18

6

18

19

25

 

21

4

43

44

60

 

49

10

27

29

32

 

29

3

40

64

65

 

56

14

173

182

183

 

179

6

150

153

172

 

158

12

 

 

 

 

DCD

 

 

 

 

25.0

 

 

 

 

mean

sd

49

50

52

 

50

2

16

17

22

 

18

3

7

13

13

 

11

3

9

17

18

 

15

5

9

24

25

 

19

9

38

43

56

 

46

9

24

30

33

 

29

5

39

41

48

 

43

5

153

157

161

 

157

4

153

168

168

 

163

9

 

 

 

 

pos. Controls

 

 

 

 

*****

 

 

 

 

mean

sd

257

265

290

 

271

17

464

491

502

 

486

20

1520

1576

1802

 

1633

149

148

153

163

 

155

8

665

779

850

 

765

93

1246

1524

1517

 

1429

1259

932

1057

1145

 

1045

107

985

1001

1134

 

1040

82

307

337

435

 

360

67

1455

1490

1580

 

1508

64

 

 

 

 

VC

(10E7/ml)

 

 

 

 

mean

sd

68

76

 

72

6

37

44

 

41

5

40

41

 

41

1

49

52

 

51

2

37

45

 

41

6

 

MEAN: average number of revertants per plate

SD:     standard deviation

VC:     visible counts

S9:      liver homogenate from rats treated with Aroclor

*****:    for actual concentrations see page 5 of original report or section “Controls” in this dossier

Conclusions:
From the findings of this study it is concluded that Dicyanodiamide did not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 of S. typhimurium were exposed to Dicyanodiamide. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at six concentrations in the range of 0.00 to 25.0 mg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of metabolic activation system with a suitable positive control.

The experiments with and without metabolic activation were performed as standard plate incorporation assay.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of Dicyanodiamide led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Based on the results of these experiments, it is concluded that Dicyanodiamide and its metabolites did not induce gene mutations in the strains of S. typhimurium used.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No info on GLP; no specification of test compound; 5th bacteria strain is TA1538 (according to old guidelines); only TA98 & TA100 were tested with S9 mix. S9 mix from mice, not rats, no indication if induced. This study is listed in OECD SIDS of DCD (3)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 were tested with and without metabolic activation. Concentrations without metabolic activation: 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate;wWith metabolic activation (mouse S-9 mix): 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate, (maize fraction S-14): 0.1, 1.0, 5.0, 10.0 mg/plate
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
other: histidine auxotrophy
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix, S-14 mix
Test concentrations with justification for top dose:
Without metabolic activation: 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate,
With metabolic activation (mouse S-9 mix): 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate,
(maize fraction S-14): 0.1, 1.0, 5.0, 10.0 mg/plate
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity

This substance reduced the numbers of bacterial colonies in plates with 10, 5 or 2.5 mg/plate, respectively. It did not exert mutagenic effects on tester strains incubated was observed in strain TA1535 when tested with 0.1, 0.5 and 1.0 mg per plate.

Conclusions:
negative without metabolic activation
negative with metabolic activation S-9 mix
ambiguous with metabolic activation S-14 fraction, only TA98
Cyanoguanidine became not mutagenic in all of the tested strains of Salmonella typhimurium without metabolic activation and with metabolic activation by the S-9 mix.
Cyanoguanidine became mutagenic for Salmonella strain TA98 after metabolic activation by the S-14 plant fraction.
Comment: the mutagenic effect for Salmonella strain TA98 after metabolic activation by the S-14 plant fraction is not relevant in regards with toxic/mutagenic effects in humans because of different enzymes (plant-human).
Executive summary:

The mutagenic activity of Cyanoguanidine was tested in reversion mutagnicity assays with a set of histidine auxotrophic strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538). A possible metabolic activation of the test substance by cell free fractions from maize-seedlings (S-14-fraction) and for comparison from mouse liver (s-9 mix) was examined. Following concentrations were used: Without metabolic activation: 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate, With metabolic activation (mouse S-9 mix): 0.1, 0.5, 1.0, 2.5, 5.0 mg/plate, (maize fraction S-14): 0.1, 1.0, 5.0, 10.0 mg/plate.

Cyanoguanidine did not exert mutagenic activity in the bacterial strains without metabolic activation. Cyanoguanidine became mutagenic for Salmonella strain TA98 after metabolic activation by the S-14 plant fraction. Cyanoguanidine was not mutagenic in the presence of S-9 mix made from mouse liver.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 1984 - February 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
No GLP compliance statement, and no information on a GLP certificate are available. But according to laboratory procedures study is GLP.
Type of assay:
mammalian cell gene mutation assay
Target gene:
X-linked HGPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line: subclone of D1 of Kao and Puck's Chinese hamster ovary cells, clone K1 (CHO-K1-BH4)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
5000, 4000, 3000, 2000, and 1000 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Medium control: Ham's F-12 medium with and without metabolic activation
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (Ham's F-12)

TEST MATERIAL
Solubility:
- In a solubility test, Dicyandiamide was found to be insoluble at 50 mg/ml in culture medium. In DMSO, however, Dicyandiamide was slightly insoluble at 500 mg/ml. Therefore, 500 mg/ml in DMSO was prepared to serve as the 100 x stock concentration yielding 5000 µg/ml high dose level in the Toxicity Evaluation

Toxicity Evaluation:
- dose levels for the toxicity test ranged from 5000 to 50 µg/ml both in the presence and the absence of S-9 metabolic activation

Compound preparation for the Forward Mutation Assay:
- Dicyandiamide did not exhibit > 90 % toxicity at the limit of solubility (5000 µg/ml] in the Toxicity test => thid dose served as high dose level for the forward mutation assay


FORWARD MUTATION ASSAY
- on the day prior to treatment, log phase cells were trypsinized and 5 x 10^5 CHO cells were inoculated into each 25 cm² flask for each test dose
- the cultures were incubated overnight at 37 +/- 1 °C

- on the next day the culture medium was replaced with either 4 ml of serum free medium in cultures scheduled for s9 metabolic activation or in 5 ml in cultures not scheduled for metabolic activation
- cultures were returned to the incubator for an acclimation period of at least 30 minutes
- after acclimation, 1 ml of the S9 mixture was aliquoted to each flask in the activation group
- 50 µl of the appropriate medium control, solvent control, positive control, or test material were added to each flask
- the cultures were returned to the incubator fo a 5 hour exposure
- the treatment was terminated by aspirating off the test solution, rinsing cultures two times and finally adding 5 ml of growth medium
- the cultures were incubated overnight for recovery from treatment

- on day 1 (day after treatment) two dilutions of each treated culture were prepared
- first dilution: for determination of the initial toxicity by plating a specific number of cells on triplicate plates for each duplicate dose
- after incubation the resulting colonies were stained and counted
- the toxicity of each dose was calculated by comparing the mean number of colonies per 200 cells in the treated series to the mean number of colonies per 200cells in the solvent control and expressing the result in percent

- the second dilution was prepared to initiate the 8-day growth period to express any genetic damage occuring at the HGPRT locus resulting from treatment with the test material
- to maintain an actively growing culture during this period, confluence is avoided be periodic dilutions and dividing cells
- in this assay, cells from each culture were transferred to a 100 mm diameter plate on day 1 with subsequent culture dilutions on day 3 and day 6

- on day 8 the cultures were prepared for 6-thioguanine selection of mutant colonies
-each culture was trypsinized and diluted to 12 ml containing 10^5 cells per ml
2 ml of this culture were selected, a parallel set of three dishes were each seeded with 200 cells for each dose
- the assay was placed in a humidified, 5 % CO2 in air, 37 +/- 1°C tissue culture incubator to allow growth of mutant cells into macroscopic colonies

- the selection of viability cultures were incubated until the colonies reached macroscopic size after which they were rinsed with saline, fixed with methyl alcohol and stained with crystal violet
- mutant and viable colonies were counted, data recorded and the mutation frequency calculated
Evaluation criteria:
- no significant increase in the mutation frequency in either the presence or absence of metabolic activation at any dose level of Dicyandiamide selected compared to the DMSO solvent control
- both activated (MCA) and nonactivated (EMS) positive controls induced a significant (p < 0.01) increase in the mean mutation frequnecy compared to the solvent control
Statistics:
Statistical significance was calculated on the mean of duplicate doses using mutation frequencies that were transformed with the power transfomation Y = (mutation frequency + 1)^0.15 to ensure that statistical assumptions of homogenous variance and normal distribution of experimental errors were satisfied. Analysis of variance of the test compound doses were used to test for the presence of a dose-response relationship. The average response at each test dose level was compared to that in the solvent control using Student's t-test at a 99 % confidence level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with and without metabolic activation
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
For detailed results please refer to box "Any other information on results incl. tables".

CHO/HGPRT Forward Mutation Assay – Dicyandiamide –Without Metabolic Áctivation

Cytotoxicity

Mutagenicity

Test 

Material

Dose 

[µg/ml]

Cells

Plated/

Dish

Mean Colonies/ Dish

RelativePE

%

Mean

Viable

Count

Mean

Mutant Count

MF

Mean MF

Statistics**

DMSO

NA

NA

NA

200

200

200

149

158    158*

166

 

100

111

120

114

0.0

1.4

0.0

0.0

11.7

0.0

 

3.9

NA

DCD

5000

5000

200

200

185

172

117

109

142

144

1.4

1.2

9.9

8.3

9.1

NS

DCD

4000

4000

200

200

172

181

109

115

107

143

0.0

2.4

0.0

16.8

8.4

NS

DCD

3000

3000

200

200

174

166

110

105

144

167

2.4

1.2

16.7

7.2

12.0

NS

DCD

2000

2000

200

200

146

142

92

90

196

138

0.0

3.8

0.0

27.5

13.8

NS

DCD

1000

1000

200

200

145

145

92

92

188

141

2.4

1.6

12.8

11.3

12.1

NS

EMS

300

300

300

200

200

200

43

42

36

27

27

23

157

92

106

18.2

27.4

28.4

115.9

297.8

267.9

227.2

S

p = 0.0041

F12

NA

NA

NA

200

200

200

129

157

143

NA

NA

NA

175

179

108

0.2

0.0

1.6

1.1

0.0

14.8

5.3

NA

 

CHO/HGPRT Forward Mutation Assay – Dicyandiamide –With Metabolic Áctivation

Cytotoxicity

Mutagenicity

Test 

Material

Dose 

[µg/ml]

Cells

Plated/

Dish

Mean Colonies/ Dish

RelativePE

%

Mean

Viable

Count

Mean

Mutant Count

MF

Mean MF

Statistics**

DMSO

NA

NA

NA

200

200

200

151

143    139*

123

100

219

181

222

0.0

2.0

1.0

0.0

11.0

4.4

5.1

NA

DCD

5000

5000

200

200

92

85

66

61

224

160

0.4

0.2

1.8

1.3

1.6

NS

DCD

4000

4000

200

200

110

85

79

61

234

206

3.8

1.8

16.2

8.7

12.5

NS

DCD

3000

3000

200

200

98

100

71

72

171

201

1.6

1.6

9.4

8.0

8.7

NS

DCD

2000

2000

200

200

118

113

85

81

170

156

0.0

0.6

0.0

3.8

1.9

NS

DCD

1000

1000

200

200

102

112

73

81

199

161

1.6

1.2

8.0

7.5

7.8

NS

MCA

4.0

4.0

4.0

200

200

200

100

112

95

72

81

68

168

235

275

22.4

23.2

24.8

133.3

98.7

90.2

107.4

S

p = 0.0049

F12

NA

NA

NA

200

200

200

130

146

148

NA

NA

NA

232

173

213

0.6

1.6

0.4

2.6

9.2

1.9

4.6

NA

 

* Mean number used to calculate relative plating efficiency

** Calculation are performed with transformed data

 

DCD = Dicyandiamide

DMSO = Dimethylsulfoxide

EMS= Ethyl methanesulfonate

MCA = 3-Methylcholanthrene

NS = Not significant

S = Significant

MF = Mutation frequency per 106viable cells

NA = Not applicable

Conclusions:
No significant (p < 0.01) increase in the mutation frequency was observed at any dose level tested either in the presence or absence of metabolic activation. Therefore, under the test conditions, Dicyandiamide is considered negative in the CHO/HGPRT Forward Mutation Assay.
Executive summary:

In a mammalian cell gene mutation assay Chinese hamster ovary (CHO) cells cultured in vitro were exposed to Dicyandiamide dissolved in DMSO at concentrations of 5000, 4000, 3000, 2000, and 1000 µg/ml in the presence and absence of mammalian metabolic activation (S-9 mix) for the induction of 6-thioguanine-resistant mutations.

The positive controls did induce the appropriate response. No significant (p< 0.01) increase in the mutation frequency was observed at any dose level tested either in the presence or absence of metabolic activation. Therefore, under the test conditions, Dicyandiamide is considered negative in the CHO/HGPRT Forward Mutation Assay.

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

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 798.5550 (DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Species / strain / cell type:
hepatocytes: from male Fischer 344 rats
Test concentrations with justification for top dose:
5000, 2500, 1000, 500, 250, 100, 50, 10, 5 and 1 µg/ml
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
Remarks:
solvent: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Species / strain:
hepatocytes: male Fischer 344 rats
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Dicyandiamide did not induce a significant positive response at any dose level tested. Therefore, under the conditions of this assay, Dicyandiamide is negative in the Rat Hepatocyte Unscheduled DNA Synthesis Assay.
Executive summary:

In an unscheduled DNA synthesis assay, primary rat hepatocyte cultures were overnight exposed to Dicyandiamide in DMSO at concentrations of 0, 5000, 2500, 1000, 500, 250, 100, 50, 10, 5, 1 and 0 (control) µg/ml /ml.  

Dicyandiamidewas tested up to limit concentration, 5000 µg/ml. The positive controls induced the appropriate response. 

There was no evidence (or a dose related positive response) that unscheduled DNA synthesis, as determined by grain counts was induced.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5550; OECD 482 for other genotoxic mutagenicity data. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Dicyandiamide was tested in an in vitro gene mutation assays in bacteria (Ames test), an in vitro chromosome aberration test in CHO cells (cytogenicity), and an in vitro gene mutation study in mammalian cells (CHO/HPRT). This set of studies covers the standard information requirements of REACH. In addition a Rat Hepatocyte Unscheduled DNA Synthesis Assay was conducted. All studies but one Ames test gave negative results. The single positive results in Salmonella strain TA98 after metabolic activation by the S-9 plant fraction is not relevant with regards to mutagenic effects in humans because of the different enzyme present in plants. The overall conclusion is that Dicyandiamide is non-mutagenic.

Justification for classification or non-classification

No evidence of mutagenic potential in an appropriate test battery.