2,2'-iminobis[4,6-diamino-1,3,5-triazine]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May - 6 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-iminobis[4,6-diamino-1,3,5-triazine]

EC Number:

222-695-1

EC Name:

2,2'-iminobis[4,6-diamino-1,3,5-triazine]

Cas Number:

3576-88-3

Molecular formula:

C6H9N11

IUPAC Name:

N~2~-(4,6-diamino-1,3,5-triazin-2-yl)-1,3,5-triazine-2,4,6-triamine

Constituent 2

Reference substance name:

melam

IUPAC Name:

melam

Details on test material:

Name of test material (as cited in study report): Melam
Chemical name: 2,2'-iminobis[4,6-diamino-1,3,5-triazine]
Lot No.: ACES062008
CAS No.: 3576-88-3
Appearance: Crystalline powder
Molecular formula, Molecular weight: C6H9N11,235.21
Purity: 98.45%
pH: 9.6
Density: 1679 kg/m3 (at 20°C)
Melting point: > 300 °C
Particle size: 0.138 - 30.200 µm
Manufacturing date: Feb. 17, 2009
Expiry date: Feb. 16, 2010
Storage condition: Room temperature (21.7-24.9 °C)
Handling & notice: Routine hygienic procedures were used to ensure the health and safety of the personnel according to MSDS.
Receipt date: May 18,2009
Residue treatment: Disuse after completion of the study

Method

Target gene:

histidine- Salmonella typhimurium (TA98, TA100, TA1535, and TA1537)
tryptophan- Escherichia coli (WP2uvrA(pKM101)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth medium
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range finding test: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 µg/plate.
Main test: 313, 625, 1250, 2500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle used: water for injection
- Justification for choice of vehicle: From the information of solubility of the test substance provided by the Sponsor and the result of preliminary solubility test, the test substance was suspended in water for injection (50 mg/mL); therefore, water for injection was determined as the vehicle for this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle of test substance was used as the negative control.

True negative controls:

no

Positive controls:

yes

Remarks:

See: Any other information on materials and methods incl. tables

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: in a shaking water bath at 37°C for 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 8.5 hours


SELECTION AGENT (mutation assays):
Salmonella typhimurium: 0.5 mM L-Histidine/D-Biotin (Sigma-Aldrich, U.S.A.) in the ratio of 10 to 1
Escherichia coli: 0.5 mM L-Tryptophan (Sigma-Aldrich, U.S.A.) solution in the ratio of 10 to 1


NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the number of revertant colonies was automatically counted by the
colony counter (ProtoCOL, SINBIOSIS, UK). If automatic counting was considered not
to be accurate, the number of revertant colonies was counted macroscopically.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test substance was considered as positive if the following conditions were met:
- The test substance should be increased at least twice as compared with the negative control group at one or more doses in the number of relevant colonies per plate in at least one strain.
- The number of relevant colonies should be increased dose dependently.

Statistics:

Other than the calculations of individual plate count, the mean value and standard deviation of relevant colonies, statistical analysis was
not performed.

Results and discussion

Test results

Test results 1

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, but tested up to precipitating concentrations

Remarks:

The precipitation did not interfere with the colony counting.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

The precipitation did not interfere with the colony counting.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean number of revertant colonies for negative and positive controls was within the
range of the historical control data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance, Melam, did not show the mutagenic potential under the conditions of this study.

Executive summary:

This study was performed to determine the mutagenic potential for Melam using histidine-requiring Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and tryptophan-requiring Escherichia coli (WP2uvrA(pKM101)) strains.

In the dose range finding test, no growth inhibition was observed at any dose levels of all strains in the absence and presence of metabolic activation. The precipitation was observed more than 800 µg/plate in all strains in the absence of metabolic activation and more than 2000 µg/plate in all strains in the presence of metabolic activation, but it did not interfere with the colony counting. Therefore, 5000 µg/plate in all strains as the highest dose level of the main test was selected. And these were sequentially diluted by the geometric ratio of 2 to produce 4 additional lower dose levels (2500, 1250, 625, and 313 µg/plate). Also, the negative and positive control groups were set.

As a result of the main test, the mean number of revertant colonies was less than twice compared with the negative control values at all dose levels of the test substance in the absence and presence of metabolic activation without dose-related increase. The growth inhibition was not observed at any dose levels of all strains in the absence and presence of metabolic activation. The precipitation was observed more than 625 µg/plate in all strains

in the absence of metabolic activation and more than 1250 µg/plate in all strains in the presence of metabolic activation, but it did not interfere with the colony counting.

The mean number of colonies of positive control was distinctly increased as compared with negative control group.

In conclusion, the test substance Melam did not show a mutagenic potential under the conditions of this study.