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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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:
2-N-(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 / strainopen allclose all
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 / 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 resultsopen allclose all
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
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.