Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD 471, GLP): non mutagenic

CA in vitro (OECD 473, GLP): non clastogenic

Genotoxicity in mammalian cells in vitro (similar to OECD 476, non GLP): non mutagenic (melamine and cyanurate tested separately)

Further in-vitro and in-vivo studies show absence of genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Adquately documented publication on cyanurate.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- name in publication: monosodium cyanurate
- supplier: Monsanto company
- composition: 77% cyanurate, 13% sodium and 10% water
- solubility in water: 0.7% w/v
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
liver S9 preparted from arochlor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
50 - 2000 μg/plate (toxicity test)
250, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/plate (mutagenicity test)
Vehicle / solvent:
cell culture medium (F10P) or S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: no data
- Exposure duration: 4h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): trifluorothymidine



NUMBER OF REPLICATIONS: triplicate


NUMBER OF CELLS EVALUATED: Clones growing from 200 cells/plate


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Statistic difference to vehicle control
Statistics:
not required
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Relative cloning efficiencies ranged from 86 - 111% and 88 - 106% for all incubations with and without metabolic activation, respectively.

Mutant frequency (10exp-6) Mutant frequency (10exp-6)
with S9 without S9
Solvent control 47 42
positive control 538 929
2000 48 58±9
1750 63±17 23±6
1500 39±18 46±9
1250 53±5 41
1000 52±5 40±2
750 48±11 culture lost
500 37±2 36±5
250 58±3 35±5
Conclusions:
Sodium cyanurate is not mutagenic in mammalian cells in vitro.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Adequately documented publication on cyanurate. Only 4 tester stains, little information on test item, vehicle not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only four tester strains used (outdated version of current guideline)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- name in publication: monosodium cyanurate
- supplier: Monsanto company
- composition: 77% cyanurate, 13% sodium and 10% water
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S9 preparted from arochlor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
10 - 10000 μg/plate
Vehicle / solvent:
not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene (TA 98, TA100 without S9), Sodium nitrite (TK 1535 without S9), 9-Aminoacridine (TA1537 without S9), Benzo[a]pyrene (TA98, TA100 with S9), 2-Aminoanthracene (TA 1535 and TA1537 with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate-incorporation

NUMBER OF REPLICATIONS: 3

A dose-range finding test was performed with TA 100 to determine the highest tolerable concentration.

Evaluation criteria:
Significant difference from control (p<0.01)
Statistics:
Bartlett's test for homogeneity. Comparison between control and treated groups were made with a one-sided t-test and, within levels, pooled variance.
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication on cyanurate with sufficient details on non-standard method.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
yes
Remarks:
limited reporting details
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- name in publication: monosodium cyanurate
- supplier: Monsanto company
- composition: 77% cyanurate, 13% sodium and 10% water
- solubility in water: 0.7% w/v
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 preparted from arochlor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
94, 187, 375, 750 and 1500 μg/plate
Vehicle / solvent:
culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethylnitrosamine
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP, QUA)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: US FDA Title 21 Code of Federal Regulations Part 58; US EPA (FIFRA), Title 40 Code of Federal Regulations Part 160; US EPA (TSCA), Title 40 Code of Federal Regulations Part 792
GLP compliance:
yes
Remarks:
OECD Principles of Good Laboratory Practice
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): FR-6120
- Physical state: white powder
- Analytical purity: ca. 99.5%
- Impurities (identity and concentrations): water (0.27%), Melamine (0.19%) and cyanuric acid (0.012%) (weight%)
- Purity test date: 04 May 2000
- Lot/batch No.: 37432-18-3
- Expiration date of the lot/batch: 22 May 2001
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature in the dark
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
from rat liver microsomal enzymes which were routinely prepared from adult male Wistar rats (obtained from Charles River, Sulzfeld, Germany).
Test concentrations with justification for top dose:
0, 3, 10, 33, 100, 333, 1000, 3330 or 5000 µg/plate (first experiment) or 0, 3, 10, 33, 100 or 333 µg/plate (second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
other: but the Ames test is regulary conducted in the testing facility (historical controls available)
Positive controls:
yes
Remarks:
with and without S9-mix (see below)
Positive control substance:
other: TA1535 (10 µg sodium azide [SA] in saline without S9-mix/2µg 2-aminoanthracene [2AA] in DMSO with S9-mix), TA1537 (60 µg 9-aminoacridine [9AC] in saline/ 1µg 2AA in DMSO), TA98 (4 µg daunomycine [DM] in saline/1µg 2AA in DMSO); continue below
Remarks:
positive control substance (continue): TA100 (650 µg methylmethanesulfonate [MMS] in DMSO/1µg 2AA in DMSO), and WP2uvrA (10 µg 4-nitroquinoline N-oxide [4-NQO] in DMSO/5µg 2AA in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine deprivation; after the incubation period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coil) were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: (a) the negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain; (b) the positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance; (c) the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
A test substance is considered negative (not mutagenic) in the test if: (a) the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation; (b) the negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if: (a) it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant; (b) the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
None necessary
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments (Table 1).
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test substance precipitated in the top agar at concentrations of 100 and 333 µg/plate. Precipitation of test substance on the plates was observed at the start of the incubation period at concentrations of 100 and 333 µg/plate. At the end of the incubation period, precipitation on the plates was observed at the concentration of 333 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- Precipitate: precipitation of test substance on the plates was observed at the start of the incubation period at concentrations of 100 µg/plate and upwards. At the end of the incubation period, precipitation on the plates was observed at concentrations of 333 µg/plate and upwards.
- Toxicity: to determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: the results were within the range of historical controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Mutagenic response of FR-6120 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - First experiment

 

Metabolic activation

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coil strain

TA1535

TA1537

TA98

TA100

WP2uvrA

 

 

 

 

Without S9-mix

Positive control

893 ± 40

253 ± 34

481 ± 57

621 ± 26

146 ± 29

Solvent control

17 ± 4

7 ± 1

21 ± 5

68 ± 7

10 ± 2

3

20 ± 2

6 ± 1

21; 21(1)

74 ± 9

13 ± 3

10

20 ± 1

5 ± 2

18; 23(1)

75 ± 11

7 ± 1

33

20 ± 1

5 ± 1

15 ± 3

65 ± 2

9 ± 3

100

19 ± 3

6 ± 2

19 ± 6

72 ± 9

12 ± 2

333sp

21 ± 2

6 ± 2

26 ± 3

78 ± 3

11 ± 5

1000sp

 

 

 

67 ± 7

9 ± 1

3330sp

 

 

 

78 ± 7

7 ± 2

5000sp

 

 

 

71 ± 7

10 ± 2

 

 

 

 

With S9-mix

Positive control

245; 284(1)

488 ± 134

847 ± 49

1112 ± 89

145 ± 1

Solvent control

13 ± 2

3 ± 1

24 ± 5

66 ± 5

8 ± 1

3

12 ± 1

4 ± 1

17 ± 4

74 ± 8

10 ± 1

10

15 ± 3

2 ± 1

23 ± 3

73 ± 4

7 ± 0

33

10 ± 4

2; 10(2)

25 ± 2

76 ± 11

11 ± 2

100

11 ± 3

2 ± 1

24 ± 8

86 ± 16

9 ± 3

333sp

12 ± 2

5 ± 2

29 ± 6

75 ± 13

7 ± 3

1000sp

 

 

 

79 ± 8

11 ± 1

3330sp

 

 

 

87 ± 6

12 ± 3

5000sp

 

 

 

74 ± 7

8 ± 2

(1): One plate infected with other bacteria; sp: slight precipitate

 

Table 1(continue): Mutagenic response of FR-6120 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Second experiment

 

Metabolic activation

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coil strain

TA1535

TA1537

TA98

TA100

WP2uvrA

 

 

 

 

Without S9-mix

Positive control

639 ± 156

270 ± 41

485 ± 51

564 ± 146

423 ± 119

Solvent control

11 ± 3

4 ± 1

14 ± 3

97 ± 3

8 ± 5

3

11 ± 3

3 ± 1

17 ± 2

93 ± 9

9 ± 1

10

15 ± 2

3 ± 1

18 ± 1

102 ± 6

9 ± 2

33

11 ± 1

3 ± 1

18 ± 4

79 ± 12

9 ± 2

100

13 ± 2

4 ± 1

21 ± 3

93 ± 7

11 ± 2

333sp

13 ± 2

4 ± 1

15 ± 3

86 ± 3

10 ± 2

 

 

 

 

With S9-mix

Positive control

263 ± 12

249 ± 28

472 ± 52

809 ± 136

340 ± 24

Solvent control

11 ± 1

6 ± 3

26 ± 1

88 ± 14

11 ± 4

3

14 ± 5

5 ± 3

18 ± 2

95 ± 10

12 ± 1

10

11 ± 3

3 ± 1

20 ± 2

84 ± 10

10 ± 3

33

12 ± 2

4 ± 1

19 ± 4

95 ± 20

11 ± 1

100

12 ± 4

4 ± 1

20 ± 2

100 ± 8

10 ± 2

333sp

14 ± 1

4 ± 2

19 ± 3

84 ± 18

9 ± 2

sp: slight precipitate

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study compliant to OECD guideline, performed under GLP and test design and reporting details adequate.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Name: Melamine-cyanurate
Batch No.: A91109
Aggregate State at RT: solid
Colour: white
Molecular Weight: 255.2
Purity: > 99.8%
Analysis No./Date: 2052920 March 03, 1990
Target gene:
None
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM + 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
- supplied by LMP, D-6100 Darmstadt, F.R.G.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Arcochlor-induced rat liver homogenate (S9-mix)
Test concentrations with justification for top dose:
with and without S9 mix;
7 h; 1.0; 2.0; 2.5; 3.0 mg/L
18 h: 0.1; 0.3; 1.0; 2.0; 2.5; 3.0 mg/L
28 h: 1.0; 2.0; 2.5; 3.0 mg/L

In the pre-experiment for toxicity the colony forming ability of the V79 cells was not reduced even after treatment with the highest concentration (3.0 mg/L) either in the absence or in the presence of S9 mix. However, higher concentration could not be dissolved in DMSO and precipitated in the culture medium.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (without S9) and cyclophosphamide (with S9)
Details on test system and experimental conditions:
Treatment interval was 4 hours. For each experimental point duplicate cultures were used. In both main experiments, in the presence and absence of S9 mix at each fixation interval samples after treatment with 3.0 mg/L as highest dose level were evaluated for cytogenetic damage. With this concentrations applied the mitotic index was not suppressed. At fixation interval 18 h, samples treated with 0.3 and 1.0 mg/L were chosen for the low and medium dose levels.

After 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 ml/L S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with phosphate-buffered saline.
5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 mg/ L culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. All two slides per group were prepared.
After fixation the cells were stained with giemsa .
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) was scored.

All incubations were done at 37° C in a humidified atmosphere with 4.5 % carbon dioxide.
Evaluation criteria:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 %.
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (P < 0.05) was evaluated by means of the chi square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

Melaminecyanurate is not clastogenic in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restricitions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Clive and Spector (1975); Clive et al. (1979) with modifications;
performed according to NTP-Standards
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Analytical purity: 99%
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mis from male rats injected with Aroclor 1254
Test concentrations with justification for top dose:
0; 10; 20; 40; 80; 100; 120; 140; 160 µg/ml;
Testing of melamine was limited to 160 µg/ml by its solubility in the exposure medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without MA: Ethylmethanesulfonate (EMS, 250 µg/ml); with MA: 3-methylcholanthren (3-MC, 2.5 µg/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension


DURATION
- Preincubation period: not specified
- Exposure duration: 4h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 days


SELECTION AGENT (mutation assays): trifluorothymidine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: not applicable; mutants producted from 10 exp 6 cells were counted.


DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency


OTHER: Cells were obtained from Dr . D. Clive, Burroughs Wellcome Co ., Research Triangle Park, NC 27709, and stored in liquid nitrogen. Absecne of mycoplasma contamination was confirmed. On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to Flop containing THMG (thymidine, 6 μg/ml hypoxanthine, 5 μg/ml, glycine, 7 .5 μg/ml and methotrexate, 0 .1 μg/ml), then for 3 days to Flop containing THG only, (i .e ., THMG without methotrexate) .
Evaluation criteria:
Positive reponse: Out of three trials, a positive trial was reproducible (The dose-related trend and the response at one of the three highest acceptable doses were statistically significant)
Negative response: Out of three trials, a positive response or a positive dose was not reproducible .
Statistics:
Chi-square test for consistency
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Melamine is not mutagnic in mammalian cells in vitro.
Endpoint:
genetic toxicity in vitro, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 97, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Melamine (MEL) and cyanuric acid (CYA) (1:1)
Bacterial toxicity: 1250 μg/plate (TA 98 and TA 100 with and without S9), > 625 μg/plate (TA102 with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard vehicle for studies of this type
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: DEXON, 2-aminoanthracene*
Species / strain:
other: TA 97, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Ames test (Liu et al., 2014)

 

Number of His* Revertant Colonies/plate (mean +/- S.D.)

Substance

Conc.

(µg/plate)

TA97

TA98

TA100

TA102

 

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

MC(1:1)

500

180.8±13.5

190.4±21.3

46.7±7.4

51.3±6.4

185.3±17.3

205.7±20.1

292.0±28.5

298.2±31.5

 

250

160.5±18.2

170.1±18.7

38.5±3.6

46.2±5.8

174.5±10.5

183.8±15.4

273.5±20.2

290.7±26.7

 

125

176.4+9.5

204.7±16.4

42.9±5.2

34.6±5.2

158.8+14.2

170.5+9.6

286.2+18.6

302.5±21.6

 

62.5

164.7+11.6

182.3±14.4

33.3±4.7

38.5±3.6

163.3±9.8

178.2±12.6

280.4+15.7

315.3±28.4

Revertants

0

156.7±15.4

161.5±13.6

30.8±6.3

28.2±7.2

152.5±6.3

170.6±14.5

258.6±24.0

265.2±32.5

DMSO

-

165.8±9.6

170.5±18.3

30.2±5.8

32.6±8.2

168.8±7.8

178.4±11.2

274.6±18.6

282.4±28.4

Dexon

50

2365.7±186.4

-

1028.5±56.4

-

-

-

752.8±40.2

-

Sodium azide

1.5

-

-

-

-

2695.2±95.4

-

-

-

2-Nitrofluorene

10

-

1325.6±38.6

-

5764.3±876.5

-

2784.5±157.3

-

-

Conclusions:
The 1:1 mixture of melamine and cyanuric acid is not mutagenic in bacteria.
Executive summary:

The combined gentoxicity of melamine (MEL) and cyanuric acid (CYA) (1:1) was investigated in a bacterial reverse mutation assay (Liu et al., 2014). According to the plate incorporation procedure described by OECD Test Guideline 471, the mutagenicity of MEL and CYA (1:1) was assessed at nontoxic concentrations ranging from 62.5 to 500 µg/plate in tester strains S. typhimurium TA 97, TA 98, TA 100, and TA 102 with and without metabolic activation. The bacteria grew normally in negative control. No genotoxicity was observed.

Positive control items induced the appropriate response over background.

The same results were observed for other mixtures of melamine and cyanuric acid (2:1, 1:2).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Reliable data is available regarding mutagenicity in bacteria: The study was performed following OECD testing guideline 471 and GLP. Melamine cyanurate was found to be non mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation.

Reliable data is also available regarding clastogenicity in mammalian cells in vitro. The study was performed following OECD testing guideline 473 and GLP: Melamine cyanurate was found to be non clastogenic in Chinese hamster lung fibroblasts (V79) with and without metabolic activation.

Reliable data regarding mutagenicity in mammalian cells in vitro has been published for the components melamine and cyanurate (McGregor 1988 and Hammond 1985). Both substances are of higher solubility than their 1:1 complex melamine cyanurate and both were found to be non mutagenic in the mouse lymphoma L5178Y cells both in the presence and absence of metabolic activation. Melamine cyanurate is therefore considered to be non mutagenic in mammalian cells in vitro.

Data on a micronucleus study in mice has been published for melamine and cyanurate (Shelby 1993 and Hammond 1985). Melamine was reported to be non genotoxic upon intraperitoneal application of up to 2000 mg/kg bw. Cyanurate was reported to be non genotoxic upon gavage application of up to 5000 mg/kg bw. Limited information is available concerning chromosome damage upon inhalation of melamine cyanurate (Babayan 1986). Chromosome damage was observed at lethal doses and was considered to be a secondary effect of the overall very poor condition. No genotoxicity was observed at lower doses.

In conclusion, melamine cyanurate is considered to be non genotoxic in vitro and in vivo.

According to supporting data (Liu et al., 2014), a 1:1 mixture of melamine and cyanurate was not genotoxic in an Ames test and in an in vivo mouse micronucleus test.


Short description of key information:
Melamine cyanurate was found to be non genotoxic in the Ames test (OECD 471) und in the chromosome aberration test in vitro (OECD 473). Both melamine and cyanurate are not genotoxic in mammalian cells in vitro and in the micronucleus test in vivo (McGregor 1988, Shelby 1993 and Hammond 1985). This data is acceptable to conclude on the absence of a genotoxicity hazard of melamine cyanurate.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.