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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)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency
Qualifier:
according to
Guideline:
other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare
Qualifier:
according to
Guideline:
other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry
GLP compliance:
yes (incl. certificate)
Remarks:
RCC Cytotest Cell Research GmbH (RCC-CCR), In den Leppsteinswiesen 19, D-64380 Rossdorf
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40'812/A
- Purity: Approx. 75%
- Lot/batch No.: WP 3/03
- Expiration date: 23 April 2010
- Stability in Solvent: 7 days in water, saline, polyethylene glycol, CMC, 1 day in vaseline, and FCA
- Storage conditions: At room temperature

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Before freezing, the levels of spontaneous mutants was depressed by treatment with HAT medium.
- Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I
without S9 mix: 37.5, 75, 150, 300, 450, 600 µg/mL
with S9 mix: 75, 150, 300, 600, 1200, 2400 µg/mL

Experiment II
without S9 mix: 25, 50, 100, 200, 400, 600 µg/mL
Vehicle / solvent:
water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

MAIN EXPERIMENT
Seeding: Three days old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen). Approximately 1.5E6 (single culture) and 5E2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent negative and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium in the absence of metabolic activation. The pH was adjusted to 7.2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment. Three days after treatment 1.5xE6 cells per experimental point were subcultivated in 175 cm2 flasks containing 30 mL medium. Following the expression time of 7 days, five 80 cm2 cell culture flasks were seeded with about 3 - 5E5 cells each in medium containing 6-TG. Two additional 25 cm2 flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures are incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt). The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Düsseldorf). Subculturing of a log-phase culture showed an initial spontaneous mutation rate at the beginning of the experiment of 8.0 mutant colonies (Experiment I) and 7.0 (Experiment II) mutant colonies per 1E6 cells.
Evaluation criteria:
- Acceptability of the assay: The gene mutation assay is considered acceptable if it meets the following criteria: the numbers of mutant colonies per 1E6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range; the positive control substances must produce a significant increase in mutant colony frequencies; the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.
- Evaluation of results: A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 31.8 mutants per 1E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiments up to the maximal concentration. All mutant frequencies remained well within the historical range of negative and solvent controls. At the concentration of 1200 µg/mL with metabolic activation a single increase of 37.0 mutant colonies/1E6 cells occured, exceeding the range of the historical data of the solvent control (0.7-24.9 mutant colonies/1E6). Since the induction factor of 3 times the corresponding solvent control was not reached and the effect was not observed in the parallel culture, this effect was judged to be biologically irrelevant.

TEST-SPECIFIC CONFOUNDING FACTORS:
- No precipitation of the test item was observed up to the maximal concentration in all experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In experiment I relevant toxic effects indicated by a strongly reduced relative clonong efficiency occured at 150 µg/mL and above in the absence and at 1200 µg/mL and above in the presence of metabolic activation (both cultures).
- In experiment II a strong toxic effect was observed at the highest concentration of 600 µg/mL at both cultures.
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 test substance is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 4 hours. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.