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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In vitro and in vivo tests with cesium iodide and structural analogue substances showed that cesium iodide is neither considered mutagenic nor clastogenic: Ames test Cesium iodide was investigated for its mutagenic potential in vitro via an Ames assay with and without metabolic activation. Based on the results, the test item is considered to be non-mutagenic for bacteria. HPRT test Cesium iodide was not mutagenic in an in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells. Chromosome aberration test (in vivo) The test item cesium hydroxide monohydrate was used as read-across substance to determine the genotoxic potential of cesium iodide. According to the results achieved in this in vivo mammalian bone marrow chromosome aberration test cesium hydroxide monohydrate is not considered to be clastogenic. Thus, the in vitro results (see below) were not confirmed. Based on these results cesium iodide is not considered to be clastogenic also. Chromosome aberration test (in vitro) Cesium hydroxide and cesium chloride induced chromosomal aberration and numerical aberrations in CHL cells after 48 hour treatment without metabolic acitivation under the test conditions. Therefore an in vivo chromosome aberration test which didn't confirm the clastogenic potential of cesium hydroxide was conducted (see above). Micronucleus test (in vivo) The test item cesium chloride was used as read-across substance to determine the genotoxic potential of cesium iodide. According to the results achieved in this in vivo bone marrow micronucleus test cesium chloride is not considered to be genotoxic. Based on these results cesium iodide is not considered to be clastogenic also.
Link to relevant study records
Reference
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
Reference:
Composition 0
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 information:
Composition 1
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells
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
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro and in vivo tests with cesium iodide and structural analogue substances showed that cesium iodide is neither considered mutagenic nor clastogenic:

Ames test

In compliance with the OECD Guideline No. 417 and EU Method B.13/14, five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of cesium iodide. Following concentrations were tested in two independent experiments composed of an initial mutation test (plate incorporation test) and a conformation mutation test (pre-incubation test): 5000, 1581, 500, 158, 50 and 15.8 μg test item/plate. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the respective positive and negative controls, were tested in triplicate.

No substantial increases or decreases were observed in revertant colony numbers of any of the five test strains following treatment with cesium iodide at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values and/or revertant colony numbers above the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

Thus, it can be concluded that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the cesium iodide is considered non-mutagenic in this bacterial reverse mutation assay.

HPRT test

The test item, cesium iodide was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. 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.

Chromosome aberration test (in vivo)

The test item cesium hydroxide monohydrate was used as read-across substance to determine the clastogenic potential of Cesium iodide in an in vivo Mammalian Bone Marrow Chromosome Aberration Test with in the Rat (Hsd.Brl.Han: Wistar rats) according to OECD guideline No. 475 and EU method B.11. The doses of the test item for the study was determined according to a preliminary oral toxicity study, the doses selected were 200, 400 and 800 mg /kg body weight. The measured concentrations of dosing solutions ranged from 95 % to 96 % of nominal concentrations at the analytical control. The measured concentrations of blood serum ranged from 25.2 to 112 mg Cs/L at the analytical control. Vehicle control and a positive control group were included. The test item and the positive control item Cyclophosphamide were dissolved in Aqua ad injectabilia. Treatment was carried out with the test item and the vehicle, once by the oral route with a constant treatment volume (10 mL/kg bw). Cyclophosphamide was administered once, intraperitoneally with a treatment volume of 1 mL/kg bw. In the vehicle, low, mid and high dose groups the sampling from bone marrow was performed twice, about at 18 and 42 hours after treatment. In animals treated with Cyclophosphamide (25 mg/kg bw), the sampling was performed only at 18 hours. Five animals per dose group were used on each occasion. 100 well spread metaphase cells were analysed for structural aberrations from each animal. 1000 nucleated cells were examined for measure of cytotoxicity from each animal. The frequencies of bone marrow cells showing structural chromosome aberrations for the vehicle, and positive control rats were within acceptable ranges and compatible with the historical control data for this laboratory. The positive control values showed a large, statistically significant increase over the negative control values, demonstrating the sensitivity of the test. The single oral administration of 200 mg/kg bw, 400 mg/kg bw and 800 mg/kg bw of the test item did not induce statistically or biologically significant increases in the number of bone marrow cells showing structural chromosome aberrations compared to concurrent controls. The frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, with harvest at 18 or 42 hours following treatment start. Also, the single oral administration of 200 mg/kg bw, 400 mg/kg bw and 800 mg/kg bw of the test item not induce statistically significant decrease in the number of mitotic cells compared to concurrent controls. Under the described test conditions the tets item did not induce chromosome aberrations in the bone marrow of Hsd.Brl.Han: Wistar rats. Therefore, according to the results achieved in this in vivo mammalian bone marrow chromosome aberration test the test item cesium hydroxide monohydrate is not considered to be clastogenic and does therefore not confirm the clastogenic ptential observed in the in vitro chromosome aberration test with cesium hydroxide monohydrate (see below).

Based on these results cesium iodide is not considered to be clastogenic also.

Chromosome aberration test (in vitro)

The test item cesium hydroxide was used as read-across substance to determine the genotoxic potential of cesium iodide. The effect of cesium hydroxide on the chromosomal aberration was investigated using Chinese hamster lung fibroblasts (CHL cells), employing Mitomycin C (MMC) and Cyclophosphamide (CPA) as the positive controls for the tests without and with metabolic activation.

The inhibition test on cell growth and cell division was carried out to determine the dose levels of the test substance. From the result of this test, chromosomal aberration test was carried out using 375, 750 and 1500 µg/mL of the test substance for 24 hours treatment and 48 hours treatment by the direct method (without metabolic activation) and the metabolic activation method. MMC was employed at 0. 05 µg/mL for 24 hours treatment and 48 hours treatment by the direct method, and CPA at 10 µg/mL by the metabolic activation method.

The test substance induced no chromosomal aberration for 24 hours treatment by the direct method and by the metabolic activation method, however it showed structural chromosomal aberrations and numerical aberrations suspect positive effect at maximum dose of 1500 µg/mL in 48 hours treatment by the direct method.

Therefore, a re-examination test of chromosome aberration was carried out to confirm the structural chromosomal aberration and the numerical aberration suspect positive effect at the dose level of 750, 1125 and 1500 µg/mL for 48 hours treatment by the direct method. On the other hand, MMC and CPA induced evident chromosomal aberrations.

It is concluded that cesium hydroxide induced chromosomal aberration and numerical aberration by the direct method under the conditions tested. Therefore an in vivo chromosome aberration test was conducted in order to evaluate the clastogenic potential of cesium hydroxide.

Micronucleus test (in vivo)

Potential mutagenic activity of the structural analogous substance cesium chloride was examined in bone marrow of male and female NMRI BR mice. The frequencies of MPCEs for the untreated control mice were within an acceptable range and compatible with the historical control data for this labaratory. Cyclophosphamide treated mice (60 mg/kg bw) showed a large, statistically significant increase in the MPCEs number compared to the vehicle control, demonstrating an acceptable sensitivity of the test.

Slight, statistically significant (p<0.05) differences were observed in the number of MPCE in the male mice at 24 hours after the last treatment with 500 and 1000 mg/kg bw/day of cesiumchloride. However, the values observed in these groups were well below the mean historical control value of this laboratory, consequently they were considered to be of no biologically significance.

The intraperitoneal administration on two occasions, 24 hrs apart of 250 mg/kg bw/day, 500 mglkg bw/day and 1000 mg/kg body weight/day of cesium chloride did not induce any biologically relevant increase in the frequency of MPCEs in male or female mice at 24 after the last treatment compared to the vehicle control.

The percentage of PCE among total (polychromatic and normachromatic) erythrocytes in 1000 mg/kg bw/day dose groups of male was slightly lower than concurrent negative control value, but there was no clear evidence for test item induced hone marrow toxicity.

Under the conditions of this assay the test item cesium chloride did not induce any biologically relevant increase in the number of micronucleated polychromatic erythrocytes at dose Ievels of 250, 500 and 1000 mg/kg body weight after intraperitoneal administration on two occasions, 24 hrs apart in NMRI BR mice. Based on these results cesium iodide is also considered to be non-genotoxic.


Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study with test item

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo studies cesium iodide is not considered to be genotoxic/mutagenic or clastogenic and thus has not to be classified according to Regulation (EC) No 1272/2008. With respect to the read-across substance, the positive findings in the in vitro chromosome aberration test were not regarded to be biologically relevant considering the negative result of the in vivo chromosome aberration assay.