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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 guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Purity: 100 % (according to data of the sponsor)
- Storage condition of test material: refrigerator

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarital/beta-naphthoflavone induced male rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I: 4.7, 9.4, 18.8, 37.5, 56.3, 75.0, 112.5, 150 µg/mL (- S9 mix) and 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 µL/mL (+ S9 mix)
Experiment II: 3.1, 6.3, 12.5, 25.0, 37.5, 50.0, 75.0 µg/mL (- S9 mix) and 23.4, 46.9, 93.8, 187.5, 375.0, 750.0, 1500.0 µg/mL (+ S9 mix)
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate (EMS; -S9 mix); 7, 12-dimethylbenzanthracene (DMBA; +S9 mix)
Details on test system and experimental conditions:
- Dose Selection:In the range finding pre-experiment test item concentrations between 0.04 µL/mL and 5.0 µL/mL were used to evaluate toxicity (4 hours treatment) in the presence and absence of metabolic activation. Relevant toxic effects were observed at 0.08 µL/mL and above in the absence of metabolic activation. In the presence of metabolic activation no relevant toxic effect occurred up to the maximum concentration. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 0.16 to 1.25 µL/mL in the absence of metabolic activation and at 0.08 to 1.25 µL/mL in the presence of metabolic activation . Phase separation occurred at 0.63 µL/mL and above in the absence and presence of metabolic activation. There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations generally spaced by a factor of 2 was used. Narrower spacing was used at high concentrations without metabolic activation to cover the
putative toxic range more closely.

- Culture Medium: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank's salts, neomycin (5 µg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

- Seeding: Two days (experiment I) or three days (experiment II) after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin). Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). Approximately 1.5x10exp6 (single culture) and 5x10exp2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.

- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". 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 1.5x10exp6 cells per experimental point were sub-cultivated 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 - 5x10exp5 cells each in medium containing 6-TG. Two additional 25 cm2 flasks were seeded with approx. 500 cells each in nonselective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
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 solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose-dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Precipitation occurred in the absence of metabolic activation at 37.5 µg/mL and above in experiment I and at 50.0 µg/mL and above in experiment II. In experiment I in the presence of metabolic activation phase separation was observed at 0.31 µg/mL. In experiment II phase separation occurred at 37.5 µg/mL and above in the absence of metabolic activation and at 93.8 µg/mL and above in the presence of metabolic activation.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation.

The dose range of both main experiments was limited by the solubility of the test item in aqueous medium. Precipitation or phase separation was noted in the first experiment at 0.31 µg/mL and above with and at 37.5 µg/mL and above without metabolic activation. In the second experiment precipitation or phase separation occurred at 93.8 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10exp6 cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 9.4, 37.5, and 56.3 µg/mL. However, the mutation frequency exceeded the historical range of solvent controls solely in the precipitation concentration range of 37.5 and 56.3 µg/mL . Since no comparable increase was observed in the parallel culture without metabolic activation or in the second experiment without metabolic activation, this isolated increase was judged as precipitation artefact.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency using SYSTAT® 11 statistics software. A single significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in the second culture of the first experiment without metabolic activation. However, the trend was judged as biologically irrelevant precipitation artefact as discussed above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.2 up to 25.1 mutants per 10exp6 cells; the range of the groups treated with the test item was from 7.6 up to 49.8 mutants per 10exp6 cells.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

The substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first and second experiment with and without metabolic activation. In both experiments no substantial and reproducible dose dependent increase of the mutation frequency was observed for the test item in the cultures with and without S9 mix when tested up to cytotoxic concentrations. Based on these results the test substance is considered to be non-mutagenic in the HPRT assay.