Caesium nitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro and in vivo tests with cesium nitrate and structural analogue substances showed that cesium nitrate is neither considered mutagenic nor clastogenic: Ames test The test item 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 tests The cesium hydroxide monohydrate and cesium iodide were used as read-across substances to determine the genotoxic potential of cesium nitrate. Both read-across substances were not mutagenic in an in vitro mammalian cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells. Based on these results cesium nitrate is considered to be not mutagenic. Chromosome aberration test (in vivo) The test item cesium hydroxide monohydrate was used as read-across substance to determine the genotoxic potential of cesium nitrate. 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 nitrate is not considered to be clastogenic also. An in vivo MN Test on cesium chloride supports the consideration. Chromosome aberration test (in vitro) Cesium hydroxide induced chromosomal aberration and numerical aberrations in CHL cells after 48 hour treatment without metabolic activation under the test conditions. Therefore an in vivo chromosome aberration test which did not confirm the clastogenic potential was conducted. REC assay (disregarded study) Cesium nitrate showed a positive result in the Bacillus subtilis REC assay and is thus rated mutagenic in this system, whereas Cs(NO3)3 was negative. Although this literature result with a non standard test is classified as acceptable, negative standard assays (see key studies) showed the absence of mutagenic potential for cesium nitrate.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-10 until 2011-05-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and Guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

, adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

, adopted 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and ß-naphthoflavone (BNF) induced rat liver S 9-mix

Test concentrations with justification for top dose:

5000; 1581; 500; 158; 50 and 15.8 μg/plate.

Vehicle / solvent:

- Vehicle/solvent used: distilled water
- Justification for choice of solvent/vehicle: This vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: 4-nitro-1,2-phenylene-diamine (TA98); Sodium azide (TA100); 9-aminoacridine (TA1535); Methyl-methanesulfonate (WP2). With metabolic activation: 2-aminoanthracene (all strains); Benzo(a)pyrene (all strains).

Details on test system and experimental conditions:

Standard plate incorporation procedure was performed, as an initial mutation test. Bacteria (cultured in Nutrient broth No.2) were exposed to the test item, both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per controls or concentration levels). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared fresh and added to the overlay (45 °C).
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions (without S9 Mix and with addition of S9 Mix) and each of them with the addition of negative and positive controls.
For the pre-incubation method, before overlaying the test item, the bacterial culture and the S9 Mix or phosphate buffer was added into the appropriate tubes, providing direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 ºC using a shaker. After the incubation the content of the tubes was added to the molten top agar prior to pouring onto the surface of minimal agar plates.
After solidification the plates were inverted and incubated at 37 °C for at least 48 hours in the dark.
The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Statistics:

not applicable

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
no signs of probable inhibitory, toxic effect of the test item were observed in the pre-experiment.
Based on the results of preliminary tests the highest dose was 5000 μg test item/plate in the final treatment mixture under the actual conditions of the test at the start of the experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item 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.

Executive summary:

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 nitrate. 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 nitrate 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 nitrate is considered non-mutagenic in this bacterial reverse mutation assay.

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 nitrate and structural analogue substances showed that cesium nitrate is neither considered mutagenic nor clastogenic:

Ames test

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 nitrate. 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 nitrate 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 nitrate is considered non-mutagenic in this bacterial reverse mutation assay.

HPRT tests

Read-across to cesium hydroxide monohydrate

The test item cesium hydroxide monohydrate was used as read-across substance to determine the genotoxic potential of cesium nitrate 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 nitrate is not considered to be mutagenic also.

Read-across to cesium iodide

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

The read-across substance 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 nitrate is not considered to be mutagenic also.

Chromosome aberration test (in vivo)

Read-across with cesium hydroxide

The test item cesium hydroxide monohydrate was used as read-across substance to determine the clastogenic potential of Cesium nitrate 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 test 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 potential observed in the in vitro chromosome aberration test with cesium hydroxide monohydrate (see below).

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

In vivo data:Micronucleus test:

Read-across with cesium chloride

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

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 cesium chloride. 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 mg/kg 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 levels 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. Based on these results cesium nitrate is not considered to be genotoxic also.

Chromosome aberration test (in vitro)

Read-across with cesium hydroxide

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

REC assay (disregarded study)

In a non standard Bacillus subtilis recombination assay (REC assay) in bacteria, strains H17 (Rec+, arg- try-) and M45 (Rec-, arg- try-) of B. subtilis were exposed to cesium nitrate and Cs(NO3)3 in water at concentrations ranging from 0.005 to 5 M in the absence of mammalian metabolic activation.

Cesium nitrate showed a positive result in the Bacillus subtilis REC assay and is thus rated mutagenic in this system, whereas Cs(NO3)3 was negative. No real positive controls were used, but many other positive chemicals were tested in parallel and induced the appropriate responses in the corresponding strains.

Although this literature result with a non standard test is classified as acceptable, negative standard assays (see key studies) showed the absence of mutagenic potential for cesium nitrate.

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo studies cesium nitrate 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 and in vivo micronucleus test.