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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:
2011-05-24 to 2011-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells
Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver)
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment period without and with S9 mix:
625, 1250, 2500, 3125, 3750, 4375, and 5000 µg/mL
Experiment 2, 20-hour treatment period without S9 mix:
625, 1250, 2500, 2750, 3000, 3250, 3500, 3750, and 4000 µg/mL
Experiment 2, 5-hour treatment period with S9 mix:
625, 1250, 2500, 3125, 3750, 4375, and 5000 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: Ham's F12 medium (cell growth medium)
- Justification for choice of solvent/vehicle: good solubility in aqueous solutions
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9 mix: 7,12-Dimethyl benzanthracene (DMBA); without S9 mix: Ethyl methanesulfonate (EMS)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 5 h (with and without S9 mix)
Experiment 2: 5 h (with S9 mix), 20 h (without S9 mix)
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT: 6-thioguanine (6 TG)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2
NUMBER OF EXPERIMENTS: 2

NUMBER OF CELLS EVALUATED: 1E6 cells: 5 plates at 2x1E5 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In pH no significant differences between treatment and control groups were observed.
- Effects of osmolality: In osmolality no significant differences between treatment and control groups were observed.
- Evaporation from medium: no evaporation expected
- Water solubility: A clear solution was obtained up to a concentration of 50 mg/mL.
- Precipitation: No precipitation in the medium was noted.

RANGE-FINDING/SCREENING STUDIES:
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays (Experiments 1 and 2), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The results obtained were used for dose selection of the test item in the Main Mutation Assays.

COMPARISON WITH HISTORICAL CONTROL DATA:
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 in the absence and in the presence and in Experiment 2 in the presence of metabolic activation the upper examined test item dose level selected was 5000 µg cesium iodide /mL. In Experiment 2 in the absence of metabolic activation the upper examined test item dose level was 4000 µg cesium iodide /mL.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item cesium iodide was not mutagenic in an in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.
Executive summary:

The test item, cesium iodide was tested in a HPRT Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476 and EU method B.17.

The test item was dissolved in Ham's F12 medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without and with S9 mix:

625, 1250, 2500, 3125, 3750, 4375, and 5000 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

625, 1250, 2500, 2750, 3000, 3250, 3500, 3750, 4000 µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

625, 1250, 2500, 3125, 3750, 4375, and 5000 µg/mL

In Experiment 1, 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 statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Cesium iodide tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Cesium iodide was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.