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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:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods, therefore the study is considered relevant, adequate and reliable for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Target gene:
hprt locus at the X-chromosome
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
*V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
* For subculturing, a trypsin (0.05%)-EDTA (ethylenediamine-tetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
* Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, by using the HOECHST stain 33258.
- The spontaneous mutation rate was continuously monitored.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary cytotoxicity experiment without and with metabolic activation: 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium
mutagenicity test without metabolic activation: 7.81,15.63, 31.3, 62.5 or 125 µg/mL medium
mutagenicity test with metabolic activation: 62.5, 125, 250, 500 or 1000 µg/mL medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate in DMSO
Remarks:
600 and 700 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl-1,2-benzanthracene in DMSO
Remarks:
20 and 30 µg/mL with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without S9-mix:4 hours (1st experiment) or 24 hours (2nd experiment); With S9-mix: 4 hours
- Expression time (cells in growth medium): until day 8 with one subcultivation on day 5
- Selection time (if incubation with a selection agent): about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL)

NUMBER OF REPLICATIONS:
cytotoxicity: triplicate
mutagenicity: for selection of mutants 5 replicate plates; for the estimation of plating efficiencies (PE) 3 replicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: other: relative plating efficiency was determined for each dose to obtain an accurate measure of the toxic effect of the chemical.
Evaluation criteria:
lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the compound is considered as negative in the test.
In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.
Equivocal results, if applicable are clarified by further testing, in agreement with Sponsor and Study Monitor.
Statistics:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion).

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 the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 125 or 1000 µg/mL in the absence and presence of metabolic activation, respectively.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Along with toxicity, changes in the pH of the test solutions was assessed. The pH was measured at the highest test item treatment level.
The pH of the vehicle control and the test item formulations in the medium were determined employing a digital pH meter type WTW pH 525 (series no. 51039051). No changes in the pH values in the medium were noted.
- Effects of osmolality:
Along with toxicity, the Osmolality of the test solutions was assessed. Osmolality determination were carried out in test solutions without target cells both in the presence and absence of metabolic activation. The Osmolality of the highest test item treatment condition, lowest precipitating test item level and the highest soluble test item level in test solution was measured.
- Precipitation:
Along with toxicity, the ability of the test item to cause precipitation in the test solution was assessed.


RANGE-FINDING/SCREENING STUDIES:
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 10 6 survivors in S9 activation solvent controls (BRADLEY, M. O., B. BHUYAN, M. C. FRANCIS, R. LANGENBACH, A. PETER¬SON and E. HUBERMANN. Mutagenesis by chemical agents in V79 Chinese hamster cells: a report and analysis of the literature. A report of the Gene-Tox Program. Mutation Research 87, 81 - 142 (1981)).
The background data obtained at LPT are given at the end of chapter 'Results and Discussion'. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. Aqua ad iniectabilia served as the vehicle control. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4150 µg/mL medium were employed. Pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 1000 µg in the experiments without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 125 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 1000 µg/mL in the presence of metabolic activation.

Main study 

Five concentrations 7.81,15.63, 31.3, 62.5 or 125 and 62.5, 125, 250, 500 or 1000 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.  

Cytotoxicity 

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 125 or 1000 µg/mL in the absence and presence of metabolic activation, respectively. 

 

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 15.47 and 17.27 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 7.81, 15.63, 31.3, 62.5 or 125µg test item/mL culture medium ranged from 3.20 to 12.62x 106clonable cells. These results are within the normal range of the vehicle controls.

 Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 14.29 and 14.32 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 or 1000 µg test item/mL culture medium ranged from 7.33 to 13.13 x 106clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 307.73 to 830.00 x 10-6clonable cells in the case of EMS and ranging from 319.22 to 909.47 x 10-6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPTranges from 1.30 to 38.36 x 10-6clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells for EMS and 130.0 to 2693.3 x 10-6clonable cells for DMBA.

Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.