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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:
2011 - 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - 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:
other: solid, melt
Details on test material:
Please refer to the section "confidential details on test material" below.

Method

Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I (S9 mix +/-): 39.4, 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (exposure period 4 h)
Experiment II (S9 mix -): 39.4, 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (exposure period 24 h)
Experiment II (S9 mix +): 39.4, 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (exposure period 4 h)
Vehicle / solvent:
- Vehicle/solvent used: deionised 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; 0.15 mg EMS/mL = 1.2 mM EMS
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation; 1.1 μg DMBA/mL = 4.3 μM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 1 day (following the expression time)
- Fixation time: 8 days

SELECTION AGENT: 6-thioguanine (6-TG)

STAIN: Methylene blue

NUMBER OF REPLICATIONS: 5 replicates (mutagenicity), 2 replicates (viability)

NUMBER OF CELLS EVALUATED: appr. 5 x 10E5 cells (mutagenicity), appr. 500 cells (viability)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

EXPERIMENTAL PERFORMANCE
Two to three days 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 PBS.
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA. Approximately 1.5 x 10E6 (single culture) and 5 x 10E2 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 50 μL/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" (pH 7.2).
In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1.5 x 10E6 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 – 5 x 10E5 cells each in medium containing 6-thioguanine. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective 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.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 1 x 10E6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

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 concen-trations 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.
Biological and statistical significance was 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:
cytotoxicity
Remarks:
in experiment II without metabolic activation at 1260 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and/or osmolality: No effects.
- Precipitation: No precipitation of the test item was observed up to the maximum concentration of all experiments.

RANGE-FINDING/SCREENING STUDIES
The range finding pre-experiment was performed using a concentration range of 9.8 to 1260 μg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.

COMPARISON WITH HISTORICAL CONTROL DATA
The mutant frequency remained well within the historical range of solvent controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test item did not induce point mutations at the HGPRT locus in V79 cells.

Applicant's summary and conclusion