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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Principles of method if other than guideline:
The study was conducted in accordance with the Study Plan agreed upon. There were no major deviations from this Study Plan. However, the following minor deviations were noted:
-The following additional parameters were evaluated using the existing data following the recommendations of OECD guideline for testing of chemicals (No. 476, July 21, 1997 and the Mouse Lymphoma Workgroup, Aberdeen, 2003): suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG).
-In the presence of TFT the plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted and not 11 to 12 days as stated in the Study Plan caused by a typing error.
These minor deviations did not affect the validity of the scientific results obtained in this study.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas

Method

Target gene:
Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If the thymidine analogue 5-trifluoro-thymidine (TFT) is included in the growth medium, the analogue will be phosphorylated via the TK pathway and will cause cell death by inhibiting DNA synthesis. Cells which are heterozy¬gous at the TK locus (TK+/-) may undergo a single-step forward mutation to the TK-/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis may still proceed by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK-/- mutants is the lack of any ability to utilize toxic analogues of thymidine, which enable only the TK-/- mutants to grow in the presen¬ce of TFT. Cells which grow to form colonies in the presence of TFT are therefore assumed to have mutated, either spontaneously or induced by the test item, to the TK-/- genotype.
Two types of mutated cells can be distinguished, large, normal-growing colonies and small, slow-growing colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions or deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this system, gene mutations within the tk gene and chromosomal events involving the gene may be detected and distinguished.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
s9
Test concentrations with justification for top dose:
A preliminary cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiment. This study was performed without and with S9 metabolic activation.

Based on the results of the preliminary study five concentrations of 15.63, 31.3, 62.5, 125 and 250 µg POLYCAT 9 Catalyst/mL medium were employed in the mutagenicity tests.

The lowest separation factor of 2 as recommended by the guidelines was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.
Vehicle / solvent:
aqua ad iniectabilia (Injectable water)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
POLYCAT 9 Catalyst was completely dissolved in aqua ad iniectabilia. A correction factor of 1.02 was used as the purity of POLYCAT 9 Catalyst was 98.5% only. The vehicle aqua ad iniectabilia served as the negative control.

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 250 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mutation frequencies of the cultures treated with POLYCAT 9 Catalyst ranged from 59.06 to 106.72 per 106 clonable cells (3 hours exposure) and from 64.82 to 124.38 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 51.57 to 107.58 per 106 clonable cells (3 hours exposure, first assay) and from 50.87 to 107.94 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.
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

The mutation frequencies of the cultures treated with POLYCAT 9 Catalyst ranged from 59.06 to 106.72 per 106 clonable cells (3 hours exposure) and from 64.82 to 124.38 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 51.57 to 107.58 per 106 clonable cells (3 hours exposure, first assay) and from 50.87 to 107.94 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.