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Toxicological information

Genetic toxicity: in vitro

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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

Referenceopen allclose all

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 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 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 Type:
secondary source
Title:
Annual Plan Fiscal Year 1986
Author:
NTP
Year:
1986
Bibliographic source:
US NTP, p.74
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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