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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:
From January to February 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 29 July, 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material



Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. 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.
- Culturing: the CHO K1 cells for the 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 %).

- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
MAIN EXPERIMENT: 250, 500, 1000 and 2000 µg/ml, with and without metabolic activation.
PRELIMINARY EXPERIMENT for CYTOTOXICITY: 125, 250, 500, 1000, 1500 and 2000 µg/ml, with and without metabolic activation.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test. The suitability was confirmed with the available laboratory’s historical database.
Untreated negative controls:
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 x10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: 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.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

REPLICATES: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.

Following adjustment of the cultures to 2 x 10^5 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 4-6 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 MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 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 10^5 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 OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 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.

Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. During the Pre-test on Toxicity (cytotoxicity assay), proliferating cells were trypsinised and cell suspensions was prepared in Ham's F12-10 medium. Cells were seeded into petri dishes (tissue cultures quality: TC sterile) at 2.5x10^6 cells each and incubated with culture medium.
After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 ml/ml) and incubated at 37 °C for 5 hours.
After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration will be adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml will be plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then be fixed with methanol and will be stained with Giemsa and the colonies will be counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments.

A non GLP Preliminary Solubility Test was performed suspending the test item in DMSO. A homogeneous suspension was obtained up to a concentration of 100 mg/ml.

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The S9 Mix (with Rat Liver S9)
Before adding to the culture medium the S9 Mix will be kept in an ice bath.
S9 mix preparation (concentration in the mix): HEPES 0.2 ml/ml, KCl 0.1 ml/ml, MgCl2 0.1 ml/ml, NADP 0.1 ml/ml, D-Glucose-6-phosphate (Monosodium salt) 0.1 ml/ml, F12 medium 0.1 ml/ml and S9 0.3 ml/ml.

The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
all results are inside the distribution of the historical negative control data (based 95 % control limit).
Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks on result:
other: test still in progress

Any other information on results incl. tables

Preliminary experiment for cytotoxicity (5 hours treatment)

Test group S9-mix Treatment/time/ hour Number of colonies/200cells/dish Mean  Relativeasurvival (%)
Untreated Control  - 206 201 203 203.3 101
Solvent Control (DMSO) 5 202 200 204 202.0 100
Test item, 125µg/ml 5 203 199 203 201.7 100
Test item, 250µg/ml 5 205 202 203 203.3 101
Test item, 500µg/ml 5 199 203 204 202.0 100
Test item, 1000µg/ml 5 203 202 204 203.0 100
Test item, 1500µg/ml 5 194 200 197 197.0 98
Test item, 2000µg/ml 5 194 199 198 197.0 98
Untreated Control + - 201 201 203 201.7 103
Solvent Control (DMO) + 5 195 196 196 195.7 100
Test item, 125µg/ml + 5 191 190 195 192.0 98
Test item, 250µg/ml + 5 194 192 192 192.7 98
Test item, 500µg/ml + 5 152 158 157 155.7 80
Test item, 1000µg/ml + 5 135 139 140 138.0 71
Test item, 1500µg/ml + 5 128 132 127 129.0 66
Test item, 2000µg/ml + 5 124 126 122 124.0 63

* Relative to Solvent Control

Applicant's summary and conclusion

Substance not cytotoxic; main test still ongoing
Executive summary:

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure.