Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
Principles of method if other than guideline:
Mutant cells deficient in Hprt enzyme activity in the HPRT test are resistant to the cytostatic effects of the purine analogue 6-thioguanine (TG). The Hprt proficient cells are sensitive to TG, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain the Hprt enzyme, are not.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: HPRT test

Test material

Test material form:


Target gene:
Hypoxanthine-guanine phosphoribosyl transferase enzyme locus.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
Lot. No.: 12G006
Supplier: ECACC (European Collection of Cell Cultures)

Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of phenobarbital and β-naphthoflavone induced rat liver.
Test concentrations with justification for top dose:
5-hour treatment period without S9-mix: 125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix: 62.5, 125, 250, 500 and 1000 μg/mL
The concentrations were chosen based on the cytotoxicity and the maximum recommended concentration.
Vehicle / solvent:
The test item was suspended in DMSO.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
A pre-test on toxicity was performed to select treatment concentrations for the mutation assay.

A 5-hour treatment in the presence and absence of S9-mix was performed. For the 5-hour treatment, 5 x10E6 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survived, cell number was adjusted to 10E5 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

The pH and osmolality of the negative (solvent) control and test item solutions were determined in Experiment 1 and Experiment 2.
Plating for survival: Following adjustment of the cultures to 10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL.
A total of 5 mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies. Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.
Expression of the mutant phenotype: During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10E6 cells were taken on days 1, 3, 6, and evaluated on day 8.
Selection of the mutant phenotype: At the end of the expression period, cultures from each dose level were adjusted to 2 x 10E5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).
Plating for viability: At the end of the expression period cell number in the samples were adjusted to 2 × 10E5 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.
Fixation and staining of colonies: After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.
Rationale for test conditions:
See above.
Evaluation criteria:
Calculation of mutation frequency: The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10E6 cells: 5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10E6 clonable cells.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Clear treatment solutions were obtained and no precipitation in the medium was noted at the test item concentrations used. No relevant changes in pH or osmolality were found after treatment with the test item.

On Day 1, there was a clear evidence of toxicity with the test item in the presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
In the absence of the metabolic activation, no cytotoxicity was observed after test item treatment up to the highest applied concentration, confirming the response seen in the dose selection cytotoxicity assays.
There were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation.
There were no significant differences between treatment and control groups and no dose-response relationships were noted.
All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by a significant increase in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.
The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.

Applicant's summary and conclusion

The test item was not mutagenic in this in vitro mammalian cell gene mutation test.
Executive summary:

The test item, applied up to the maximum recommended concentration (2000 μg/mL) concentration without metabolic activation system and up to the cytotoxic concentration with metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.