1,3,5-triazine-2,4,6-triamine phosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restricitions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Responses of the L5178Y tk+/tk- Mouse lymphoma cell forward mutation assay III. 72 Coded Chemicals.

Author:

McGregor DB, Brown A, Cattanach P, Edwards I, McBride D, Riach C, Caspary W

Year:

1988

Bibliographic source:

Environ. Mol. Mutagen., 12, 85-154

Reference 2

Reference Type:

secondary source

Title:

Predicition of chemical carcinogenicity in rodents from in vitro genotoxcity assays

Author:

Tennant R.W

Year:

1987

Bibliographic source:

Science, 236, 933-941

Reference 3

Reference Type:

secondary source

Title:

Induction of forward mutations at the thymidine kinase locus of mouse lymphoma cells: evidence for electrophilic and non-electrophilic mechanisms

Author:

Henry B. et al.

Year:

1998

Bibliographic source:

Mutation Research, 397, 313-335

Reference 4

Reference Type:

secondary source

Title:

Annual Plan Fiscal Year 1986

Author:

NTP

Year:

1986

Bibliographic source:

US NTP, p.74

Reference 5

Reference Type:

secondary source

Title:

Melamine

Author:

Trochimowicz H. J. et al.

Year:

2001

Bibliographic source:

Patty´s Toxicology, 5th Edition, Vol.4

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Method: other: Clive and Spector (1975); Clive et al. (1979) with modifications;
performed according to NTP-Standards (US NTP Study ID 091419)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

1,3,5-triazine-2,4,6-triamine

Details on test material:

- Analytical purity: 99%
The chemical was supplied from the US National Toxicology Program Chemical Repository , Radian Corporation, Austin, TX 78766 (USA)

Method

Target gene:

tk

Species / strain

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

Controls

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) .


The tk+/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen. Thawed samples were cultured and used for up to 3 months, then discarded. Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37°C on gyratory tables. Fischer's medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10P). 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 F1OP containing THMG (thymidine, 6 mg/mL hypoxanthine, 5 mg/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

Results and discussion

Test results

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

Any other information on results incl. tables

Results tables were not included in the publication. They can be viewed at

http://ntp-apps.niehs.nih.gov/ntp_tox/index.cfm?fuseaction=ntpsearch.searchresults&searchterm=108-78-1

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative