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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb to May 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Test material form:
Specific details on test material used for the study:
Stability in the solvent was analytically proved.


Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: PAA Ready Mix (PAA, Paching, Austria), consists of MEM, Earle, with supplements.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
To keep the number of spontaneous 6-TG resistant mutants at a low level, cell cultures were subcloned at least every two weeks.
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of male Aroclor 1254 induced Sprague-Dawley rats.
Test concentrations with justification for top dose:
Both for 1st experiment and independent repeat: 30, 80, 120, 240, 480, 960 µg/mL with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In this solvent the test substance was soluble up to 200 mg/mI.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: ethyl methanesulphonate, dimethylbenzanthracene
ethyl methanesulfonate used without S9-mix (900 µg/mL); dimethylbenzanthracene used with S9-mix (20 µg/mL).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; In all parts of this study incubation was performed at 37 °C in a humidified atmosphere with about 5 % CO2.
The method is based on the publication of Myhr and DiPaolo (Cancer Res. 38, 2539-2543, 1978).
Treatment Protocol without MetabolicActivation: Exponentially growing V79 cells were plated in culture medium at a final volume of 20 ml in two 75 cm² flasks per concentration (4x10exp6 per flask) including all control groups. After attachment (16-24 hours later), the cells were exposed for 5 hours in 20 ml culture medium with reduced serum content (2 %). The corresponding controls were incubated under the same conditions. Thereafter, cell monolayers were washed with PBS, trypsinized and replated in 20 ml culture medium using 1.5x10exp6 cells per 75 cm² flask and in 5 ml culture medium using 200 cells per Petri dish (diameter 60 mm). Per culture one flask and 3 Petri dishes ware used. The Petri dishes were incubated (normally 6 days) to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment ("Survival to Treatment").
Cells in 75 cm² flasks were incubated to permit growth and expression of induced mutations. Cells were subcultured (= count 1, normally after 3 days) by reseeding 1.5x10exp6 cells into 20 ml medium in 75 cm² flasks. At the end of the expression period (= count 2, normally a total of 6 days), cultures were reseeded in Petri dishes (diameter 100 mm) at 3x10exp5 cells per dish (8 dishes per culture) in 20 ml culture medium without
hypoxanthine but containing 10 µg/ml 6-TG for selection of mutants. In addition, 200 cells per dish (diameter 60 mm, 3 dishes per culture) were seeded in 5 ml culture medium to determine the absolute cloning efficiency for each concentration. After incubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG resistant colonies in the 'mutation assay dishes and the number of colonies in the cloning efficiency dishes.
At least two trials will be performed.
Treatment Protocol with Metabolic Activation: The activation assay was performed independently. The procedure was identical to the nonactivation assay except for the addition of 59 mix. In these experiments 19 instead of 20 ml culture medium and additionally 1 ml of 59 mix were added to the
flasks for the treatment period, resulting in a concentration of 5 % S9-mix in the cultures. The number of 6-TG resistant mutants and viability were determined as described above.

- Exposure duration: 5 hours.
- Expression time (cells in growth medium): normally 6 days
- Selection time (if incubation with a selection agent): 6 to 8 days, then colonies were fixed.

DETERMINATION OF CYTOTOXICITY: A preliminary cytotoxicity test was conducted without and with metabolic activation using concentrations ranging from 7.8 µg/mL to 2000 µg/ml. Result: No cytotoxic effects above 80 % were induced by the used concentrations in the pretest. However, precipitation of the test item in the culture medium started at 500 µg/ml without S9 mix and at 1000 µg/ml with S9 mix. Due to these findings, the test concentrations for the main testing ranged from 30 µg/ml to 960 µg/ml both without and with metabolic activation
- Method: Cytotoxicity was expressed by comparison of colonies in treated cultures versus negative control cultures (relative cloning efficiency).
Evaluation criteria:
Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10 % or greater.
A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result.
A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.
However, these criteria may be overruled by good scientific judgement.
The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. Mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10 % are not included in the statistical analysis.
The two mutant frequency values obtained per group are, although somewhat related, considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses will be run for each trial in order to examine the consistency of the results.
All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal significance level of a = 0.05 using the Dunnett test. The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, untreated and negative controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Mutation Assay without Metabolic Activation: Relevant test substance induced increases in mutant frequencies could not be found. In addition, the overall statistical analysis reveals no statistically significant increases.
Mutation Assay with Metabolic Activation: The test substance induced no relevant increases in mutant frequencies. In addition, the overall statistical analysis reveals no statistically significant increase.

For the test substance treated cultures no cytotoxic effects of 80 % to 90 % were induced. However, the substance was tested up to its limits of solubility in the solvent.

- Effects of pH: Concentrations of up to 2000 µg/ml test substance did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2000 µg/ml.
- Precipitation: Without S9 mix substance precipitation occurred in the medium at the concentration 480 µg/ml and above. With 59 mix precipitation was noted at the concentration 960 µg/ml.

Applicant's summary and conclusion

Executive summary:

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476 in concentrations up to and including 960 µg/ml, without and with a metabolising system (S9 mix). The substance induced no decreases in survival to treatment or in relative population growth, without and with S9 mix. However, precipitation of the substance in the culture medium was observed without S9 mix at 480 µg/ml and above and with S9 mix at 960 µg/ml. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls. The positive controls ethyl methanesulfonate and dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix. Based on these results, the substance is considered to be non-mutagenic in the V79/HPRT.