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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:
experimental study
Adequacy of study:
key study
Study period:
23 Apr - 14 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TK locus
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
(growth medium: medium supplemented with 0.05% Pluronic F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% by volume); treatment medium: growth medium without sodium pyruvate, gentamycin and fungizone; cleansing medium: growth medium supplemented with approx. 4.0E-05 M thymidine, 1.2E-04 M hypoxanthine, 3.3E-05 M glycine and 7.2E-07 M methotrexate; recovery medium: cleansing medium without methotrexate; selction medium: growth medium supplemented with 3 µg/mL TFT)
- 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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
15.63, 31.3, 62.5, 125, and 250 µg/mL with and without S9 mix
Vehicle / solvent:
- Vehicle used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Remarks:
-S9: methylmethansulfonate (MMS): 10 and 15 µL/mL; +S9: 3-methylcholanthrene (3-MC): 2.5 and 4.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 3 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-17 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and cloning efficiency, relative suspension growth and suspension growth

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.
Evaluation criteria:
- Minimum criteria for mutagenesis: mutant frequency is ≥ 2 times the concurrent background mutant frequency.
- The increase in mutant frequency should be concentration-related.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the top concentration of 250 µg/mL with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The concentrations employed in the mutagenicity test were based on the result of a cytotoxicity study: 25, 100, 250, 1000, 2500, and 5000 µg/mL test substance was tested with and without metabolic activation system. Pronounced to complete cytotoxicity (decreased survival) was noted starting at a concentration of 250 µg/mL. In addition, test item precipitation was noted starting at 1000 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean values of mutation frequencies of the negative controls in the experiments with and without metabolic activation were well within the historical data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plating efficiency of the negative control was >= 50%, the mean cloning efficiencies wthin the range of 65% to 120% two days after treatment, and the mean suspension growth within the range of 8 to 32 following 3 h treatments and between 32 and 180 following 24 h treatments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The mutation frequencies of the negative controls ranged from 70.23 to 103.77 per 1E06 clonable cells in the experiments without S9 mix and from 59.04 to 93.88 per 1E06 clonable cells in experiments with S9 mix. The mutation frequencies of the cultures treated with the test substance ranged from 60.48 to 91.55 per 1E06 clonable cells (3 h exposure) and from 65.72 to 95.59 per 1E06 clonable cells (24 h exposure) in the experiments without S9 mix. In the experiments with S9 mix, the mutation frequencies of the cultures treated with test substance ranged from 60.23 to 86.70 per 1E06 clonable cells (3 h exposure, first assay) and from 61.81 to 87.37 per 1E06 clonable cells (3 h exposure, second assay).

These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

No change was observed in the ratio of small to large mutant colonies ranging from 0.67 to 1.67 for treated cells and from 0.76 to 1.27 for the negative controls, whereas in the case of methylmethanesulfonate treated cells, the colony size ratio was moderately shifted towards an increase in small colonies. Thus, there is no indication for a clastogenic potential of the test substance.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the L5178Y TK +/- mammalian cell mutagenicity test, the test substance tested up to cytotoxic concentrations in the absence and presence of metabolic activation did neither induce mutations nor have any chromosomal aberration potential.