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EC number: 207-312-8 | CAS number: 461-58-5
Endpoint summary
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
- 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
- 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
- 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
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- 5th bacteria strain is TA1538 (according to old guidelines)
- Qualifier:
- according to guideline
- 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
- 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
- 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
- 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
- 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".
- 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
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)
- Qualifier:
- equivalent or similar to guideline
- 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.
Referenceopen allclose all
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
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
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.
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
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
