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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997
according to
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
according to
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Specific details on test material used for the study:
- Physical state: liquid
- Analytical purity: 86.3%
- Purity test date: 2014-07-30
- Lot/batch No.: 140023P040
- Expiration date of the lot/batch: 2015-07-25
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stability was verified at room temperature in DMSO for at least 4h.


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 with or without (only during treatment with S9) FCS. All media were supported with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
First experiment:
Without S9: 0.23, 0.47, 0.94, 1.88, 3.75, 7.5, 15, 30µg/mL
With S9: 1.88, 3.75, 7.5, 15, 30, 60, 120µg/mL

2nd experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL
With S9: 2.19, 4.38, 8.75, 17.5, 35, 70, 140µg/mL

Third experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL

In all experiments, at least four concentrations were evaluated.
Vehicle / solvent:
Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:

- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 6-8 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine


- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 106 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Effects of pH: with S9 mix, the pH was lowered at concentrations at and above 3012.5µg/mL
- Precipitation: precipitation in the culture medium (but not in the vehicle) occured at concentrations of 1506.3µg/mL and above (with or without S9 mix)
These concentrations are well above those used in the study due to high cytotoxicity. No influence on the result is expected.

RANGE-FINDING/SCREENING STUDIES: The doses were selected to reduce cloning efficiency to about 20% or less.

The positive control (EMS) without S9 mix did not produce the expected increase in mutant colonies. That's why this experiment was repeated - designated experiment 3. All other positive and negative control values were within the historical range. Also, mutant frequencies of the treatment groups did not exceed the historical negative control data.

Any other information on results incl. tables

Experimenta result without S9
Experiment Test group Mutant frequency [per 106cells] Cytotoxicity CE1 [%] Cytotoxicity CE2 [%]
1 vehicle control 0.61 100.0 100.0
0.23 n.c. 97.7 n.c.
0.47 n.c. 102.4 n.c.
0.94 0.32 96.9 95.6
1.88 0.63 96.8 95.9
3.75 5.20 90.7 91.1
7.50 1.73 49.9 65.5
15.00 n.c. 0.1 n.c.
30.00 n.c. 0.0 n.c.
positive control 111.10 96.8 96.0
2 vehicle control 5.72 100.0 100.0
0.27 n.c. 95.3 n.c.
0.55 6.06 96.9 116.5
1.09 1.55 86.5 105.1
2.19 1.99 84.1 109.1
4.38 0.35 87.9 96.2
8.75 n.c. 1.1 n.c.
17.50 n.c. 0.3 n.c.
positive control 5.18 88.5 119.8
Positive control did not fulfill acceptance criteria
3 vehicle control 6.41 100.0 100.0
0.27 n.c. 86.5 n.c.
0.55 n.c. 97.2 n.c.
1.09 3.93 86.2 85.8
2.19 5.29 79.9 83.2
4.38 1.78 77.5 86.6
8.75 0.00 35.8 74.2
17.50 n.c. 0.0 n.c.
positive control 111.19 83.9 73.2

Experimenta result with S9
Experiment Test group Mutant frequency [per 106cells] Cytotoxicity CE1 [%] Cytotoxicity CE2 [%]
1 vehicle control 1.71 100.0 100.0
1.88 n.c. 102.5 n.c.
3.75 n.c. 99.6 n.c.
7.50 4.00 98.2 93.1
15.00 2.65 94.4 102.4
30.00 10.43 66.7 95.1
60.00 2.07 30.6 102.7
120.00 n.c. 0.0 n.c.
positive control 120.67 101.6 102.0
2 vehicle control 1.73 100.0 100.0
2.19 n.c. 94.0 n.c.
4.38 1.35 83.6 102.4
8.75 1.00 50.8 100.5
17.50 3.55 38.7 100.2
35.00 7.01 24.1 93.3
70.00 n.c. 0.2 n.c.
140.00 n.c. 0.2 n.c.
positive control 89.81 107.1 93.0

Vehicle controls (culture medium, DMSO, acetone, ethanol, tetrahydrofurane)

Without S9 With S9
Mutant frequency uncorrected corrected* uncorrected corrected*
Mean 2,51 3,09 2,29 2,84
Min 0,00 0,00 0,00 0,00
Max 13,33 16,43 14,44 16,12
SD 2,57 3,18 2,35 2,94
Number of experiments 131 239

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period

Applicant's summary and conclusion

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.