Caesium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

In vitro and in vivo tests with cesium chloride and structural analogue substances showed that cesium chloride is neither considered mutagenic nor clastogenic: Regarding the in vitro data two negative Ames tests and one positive chromosome aberration test with cesium chloride are available. No HPRT test on cesium chloride is available. Consequently, two negative tests with cesium iodide and cesium hydroxide monohydrate respectively were used. Two negative in vivo tests - a micronucleus test with cesium chloride and a chromosome aberration test with cesium hydroxide monohydrate - showed that the result from the in vitro chromosome aberration test with cesium chloride has no biological relevance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-12-04 to 2008-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EEC Directive 2000/32, L 136/50

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CRL:NMRI BR mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER (EUROPE) LABORATORIES INC. Toxi Coop Ltd. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult mice (8-9 weeks)
- Weight at study initiation: Preliminary test: Male: 27.5-30.8, Female: 23.2-25.7 g; main test (MNT): Male: 25.3-29.1 g; Female: 23.2-25.7 g
- Assigned to test groups randomly: yes: All animals were sorted according to body weight by computer and divided to weight ranges. There were equal number of animals from each weight group in each of the experimental groups assigned by randomisation.
- Housing: 5 animals/cage, expect of highest dose, where 7 animals/cage); cage type: II type polypropylene/polycarbonate
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 12 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 h light daily

IN-LIFE DATES: From: 04.12.2007 To: 03.01.2008

Route of administration:

intraperitoneal

Vehicle:

distilled water

Details on exposure:

A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary test also was used to determine whether there are large differences in toxicity between the sexes or not. Groups of three male and female mice were treated on two occasion, 24 hrs. apart by intraperitoneal injections at dose levels of 250, 500, 1000, 1250 and 1500 mg/kg bw. Animals were examined regularly for toxic signs and mortalities. The treatment volume was 10 mL/kg bw.

Duration of treatment / exposure:

The test/vehicle control items were administred by intraperitoneal injection on two occasions, 24 hrs. apart.

Frequency of treatment:

The test/vehicle control items were administred by intraperitoneal injection on two occasions, 24 hrs. apart.

Post exposure period:

Sampling was made once at 24 hours after final treatment.

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg bw /day on two occasions, 24 hrs apart, with a constant treatment volume of 10 mL/kg body weight
Basis:
other: intraperitoneal injection

No. of animals per sex per dose:

Five male and five female animals per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (positive control) was administered intraperitonealy on one occasion at a treatment volume of 10 mL/kg bw/day. Sampling was perfonned 24 hours after the treatment and five male and five female animals were used for sampling.

Tissues and cell types examined:

Bane marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to identify the appropriate maximum dose Ievel for the main test. Groups of three male and female mice
were treated on two occasions, 24 hrs apart with solutions of cesium chloride by intraperitoneal injection at dose Ievels of 250, 500, 1000, 1250 and 1500 mg/kg bw/day.

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet was made on standard microscope slides. Slides were then dried at room temperature. Subsequently the slides were stained as follow: 1. Fixed for a minimum of 5 miutes in methanol and allowed to air-dry. 2. Stained with Giemsa solution (99 mg/mL distilled water) for 25 minutes. 3. Rinsing in distilled water. 4. Drying at room temperature (at least 12 hours). 5. Coating with E-Z-Mount xylene (SHANDON).

METHOD OF ANALYSIS:
Prior to microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code Iabels were covered the
original animal numbers to ensure that the slides were scored without bias. Two thousand polychromatic erythrocytes (PCEs) was scored per animal to assess the micronucleated cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 200 immature erythrocytes.

Evaluation criteria:

The Micronucleus Test is considered acceptable if it meets the following criteria:
- The proportion of polychromatic erythrocytes among total erythrocytes in treated groups is not less than 20% ofthe control value.
- The frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls falls within the range of historical Iabaratory control data.
- The positive control item should produce biologically relevant increases in the number ofmicronucleated polychromatic erythrocytes.
- Each treated and control group should include at least 5 analysable animals per sex.

Statistics:

The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative control group. Statistically analysis was performed using Kruskall Wallis Non Parametrie ANOV A test. Data were analysed separately formale and female animals.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 1250 and 1500 mg/kg bw/day
- Clinical signs of toxicity in test animals: No adverse reactions to treatment were observed in the mice dosed 250 mg/kg body weight/day. The male and female animals dosed at 500 mg/kg body weight/day showed a slight decrease in activity, straub tail, salivation, diarrhoea, piloerection and dyspnoea. The symptoms were observed between 5 min. and 1 hour after the treatment. No symptoms were observed thereafter. In the 1000 mg/kg body weight dose group, moderate decreased activity, straub tail, salivation, diarrhoea, piloerection and dyspnoea were observed after the treatment. The symptoms were observed between 5 min. and 2 hours after the treatment. The mice dosed at the 1250 mg/kg body weight/day dose Ievel showed moderate decreased activity , salivation, diarrhoea, piloerection, straub tail and strong dyspnoea. The symptoms were observed between 5 min. and 2 hours after the treatment. In the group dosed at 1500 mg/kg body weight/day, moderate decreased activity, salivation, diarrhoea, piloerection, straub tail and strong dyspnoea. The death (one male and one female) was observed within halfhour after the first treatment. On the second day the death was observed (two males and two females) within a similar timeframe.
- Evidence of cytotoxicity in tissue analyzed: Necropsy in group 1500 mg/kg bw/day: In animals which died early ( 4 of 6), pale raised areas were found in the lungs.


RESULTS OF DEFINITIVE STUDY
-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.

Conclusions:

Interpretation of results (migrated information): negative
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.
Cesium chloride did not show any genotoxic activity in this Mouse Micronucleus Test.

Executive summary:

Potential mutagenic activity of 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 laboratory. 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.

Cesium chloride did not show any genotoxic activity in this Mouse Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro and in vivo tests with cesium chloride and structural analogue substances showed that cesium chloride is neither considered mutagenic nor clastogenic. Please refer to IUCLD section 13 for read-across justification.

In vitro data:

Ames:

Two negative bacterial reverse mutation tests are available for cesium chloride:

Key study: In compliance with the OECD Guideline No. 471 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 chloride. 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 chloride 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, cesium chloride is considered non-mutagenic in this bacterial reverse mutation assay.

Supporting study: The reverse mutation test of cesium chloride was performed in Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA using the pre-incubation method with and without metabolic activation system. The numbers of the revertant colonies for all strains in groups which were treated with the test substance were less than twice that of each negative control, with or without S9 Mix. The positive controls showed the distinct increase of revertant colonies, and the positive and the negative controls were within a range of the background data in our laboratories. There were no fluctuations which affected the test results because the sterility test confirmed the absence of micro-organisms. In conclusion, cesium chloride was judged to have no reverse mutagenic potential under the present test conditions.

Chromosomen Aberration:

The effect of cesium chloride on the chromosomal aberration was investigated using Chinese hamster lung fibroblasts (CHL cells), for the tests without and with metabolic activation, respectively.

Cell growth inhibition test and cell division inhibition test were carried out to determine the dose levels of the test substance From the results of these tests, chromosomal aberration tests were carried out using 425, 850 and 1700 µg/ml (10mM) of the test substance for the test without and with metabolic activation.

As a result, in 24 h treatment without metabolic activation, chromosomal aberration was not induced up to the highest concentration. On the other hand, in the 48 h treatment without metabolic activation, numerical aberration was induced only at the level of 1700 µg/ml. Therefore, re-examination in the 48 h treatment without metabolic activation was conducted by setting concentration at 850, 1275 and 1700 µg/ml and was carried out. Results showed dose dependent increases in the chromosomal aberration induction in the range of 1275 - 1700 µg/ml. In the cultures using positive controls, MMC and CPA processing markedly induced chromosomal aberrations. Cesium chloride was concluded to induce chromosomal aberration under the conditions of the present study, and D₂₀value of cesium chloride was 2000 µg/ml. Therefore in vivo data were taken into account to evaluate the clastogenic potential of cesium chloride.

HPRT:

No HPRT test on cesium chloride is available. Consequently, results of tests with cesium iodide and cesium hydroxide monohydrate were used in a weight of evidence approach.

The test item cesium iodide was used as read-across substance to determine the genotoxic potential of cesium chloride.

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.

Based on these results cesium chloride is not considered to be mutagenic also.

The test item cesium hydroxide monohydrate was used as read-across substance to determine the genotoxic potential of cesium chloride 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, 4200, 4600*, and 5000*µg/mL

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

625, 1250, 2500, 3125, 3750, 4000, 4200*, 4600*, and 5000*µg/mL

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

625, 1250, 2500, 3125, 3750, 4000, 4200, 4600*, and 5000*µg/mL

*These concentrations were very toxic and there was not enough cells start the phenotypic expression period after the treatment.  

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.

The read-across substance 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 hydroxide monohydrate was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Based on these results cesium chloride is not considered to be mutagenic also.

In vivo data: Micronucleus test:

Potential mutagenic activity of 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.

Cesium chloride did not show any genotoxic activity in this Mouse Micronucleus Test.

Chromosome aberration:

An in vivo micronucleus test but not an in vivo chromosame aberration test is available for cesium chloride. Consequently, results from cesium hydroxide monohydrate are used as supporting data.

Potential mutagenic activity of test item cesium hydroxide monohydrate was tested in an in vivo mammalian bone marrow chromosome aberration test. In the Rat in Hsd.Brl.Han: Wistar rats according to OECD guideline No. 475 and EU method B.11. The doses of the test item for the In Vivo Mammalian Bone Marrow Chromosome Aberration Test was determined according to a preliminary oral toxicity study, the doses selected were 200, 400 and 800 mg cesium hydroxide monohydrate 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 Iniectabilia. Treatment was carried out with test item cesium hydroxide monohydrate and the vehicle, once by the oral route with a constant treatment volume (10 mL/kg body weight). Cyclophosphamide was administered once, intraperitoneally with a treatment volume of 1 mL/kg body weight.

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 analyzed 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 did not induce statistically significant decrease in the number of mitotic cells compared to concurrent controls. Under the described test conditions cesium hydroxide monohydrate 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 cesium hydroxide monohydrate is not considered to be clastogenic.

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

Justification for selection of genetic toxicity endpoint
In vivo study according to GLP and OECD and EU guideline

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo with cesium chloride and structural analogue substance cesium chloride is considered not to be genotoxic/mutagenic or clastogenic and thus had not to be classified according to Regulation (EC) No 1272/2008. The positive findings in the in vitro chromosome aberration test were shown to be not biologically relevant based on the results of in vivo tests.