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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: GLP guideline study. K2 score due to read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Thymidine kinase (TK) locus at chrmosome 11
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (With HEPES and L-Gln) from Bio Whitacker, Verviers, Belgium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.62 mmol/l, 1.25 mmol/l, 2.5 mmol/l, 4.9 mmol/l, 7 mmol/l, 10 mmol/l
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Culture medium without serum
- Justification for choice of solvent/vehicle: Identical with culture medium
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: MMS (methyl methanesulphonate). With metabolic activation: MCA (3-methylcholanthrene).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h
- Expression time (cells in growth medium): 2 d

NUMBER OF REPLICATIONS: 2 per dose

NUMBER OF CELLS EVALUATED: Without metabolic activation 3 million cells, with metabolic activation 5 million cells

DETERMINATION OF CYTOTOXICITY
- Method: Relative initial cell yield, relative suspension growth (RSG) and relative total growth (RTG).
Evaluation criteria:
Criteria used:
a) the average cloning efficiency of the negative controls should not be less than 60% or more than 140 %.
b) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT -resistant mutants per 1,000,000 clonable cells.
c) the mutant frequency of the positive controls should be higher than 400 TFTresistant mutants per 1,000,000 clonable cells, and should be at least twice that
of the corresponding negative control.
d) unless the material to be tested shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the
highest test substance concentration should result in a clear cytotoxic response. The RTG value of one ofthe data points should be between 10 and
20%, or one data point between 1 and 10% and another between 20 and 30%.

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.

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
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Not performed

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

L-valine is not mutagenic at the TK-locus of mouse Iymphoma L5178Y cells under the conditions used in this study. The maximum test concentration was 10 mmol/ l = 1171 mg/l.