Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Cesium hydroxide was judged to have no reverse mutagenic potenial in an Ames test with and without metabolic activation. The test item cesium hydroxide monohydrate was not mutagenic in a in vitro mammalian cell gene mutation test (HPRT) performed with CHO-K1 (Chinese hamster ovary) cells. Cesium hydroxide induced chromosomal aberration and numerical aberrations in CHL cells after 48 hour treatment without metabolic acitivation. Therefore an in vivo chromosome aberration test was conducted in order to evaluate the clastogenic potential of cesium hydroxide. According to the results achieved in an in vivo mammalian bone marrow chromosome aberration test cesium hydroxide monohydrate is not considered to be clastogenic. Based on these results cesium hydroxide anhydrous is not considered to be clastogenic also.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-20 until 2012-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
, adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Version / remarks:
, adopted 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest Hungary
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 284 - 309 g
- Housing: Type III polypropylene/polycarbonate
- Diet: 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: 22 +/- 3°C
- Humidity: 30-70 %
- Air changes: 8-12 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours dark per day
Route of administration:
oral: gavage
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 two male and female rats were treated on one occasion by oral at dose levels of 200, 400, 800 and 1000 mg/kg bw. The treatment volume was 10 mL/kg bw. Animals were examined regularly for toxic signs and mortalities. No bone marrow smears were prepared from these rats. On the basis of results of preliminary toxicity test, doses for the Mammalian Chromosomal Aberration Test were: 200, 400 and 800 mg/kg bw.
The main test was performed using male rat because the toxic effect of the test item was similar in both sexes in the preliminary acute oral toxicity test.
The test/vehicle items were administered orally by gavage once. The treatment volume was 10 mL/kg bw. Animals were examined regularly for toxic signs and mortalities.
Duration of treatment / exposure:
The sampling was made twice about at 18 and 42 hours after treatment. Five male animals per dose group were used for sampling of chromosomal aberration on each occasion. Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume: 1 mL/kg bw. Sampling was performed about at 18 hours after the treatment and five male animals were used for sampling. Four hours before the sampling the animals were treated with Colchicine (2mg/kg bw). Colchicine was administered intraperitoneally with a treatment volume: 1 mL/kg bw/day.
Frequency of treatment:
Single applicaction
Post exposure period:
None
Remarks:
Doses / Concentrations:
200, 400, 800 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Pre-test: 2 male and 2 female animals/group, 4 dose groups.
Main-test: 5 male animals/group/time point, 3 dose groups and positive and vehicle control.
Two additional male animals / dose group and 2 additional male animals / vehicle group for serum analysis.
Two additional male rats were dosed in highest dose group in order to potentially replace any animals which die before the scheduled sacrifice time.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: intraperitoneally
- Doses / concentrations: 1 mL/kg bw
Tissues and cell types examined:
Bone marrow, origin: femur
Details of tissue and slide preparation:
Bone marrow was obtained from two exposed femurs of the animals from every time point immediately after sacrificing. The bone marrow was flushed with Hank’s solution (5 mL) prewarmed 37 °C. After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded.
Smears of the cell pellet were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (3:1) mixture of methanol:acetic-acid until the preparation becomes serum free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
Prior to microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code labels covered the original animal numbers to ensure that the slides were scored without bias.
– the mitotic index was determined as a measure of cytotoxicity in at least 1000 cells per animal for all treated animals (including positive control) and untreated negative control animals.
– 100 metaphase cells containing 2 n ± 2 centromeres were evaluated for structural aberrations from each animal. Chromatid-type and chromosome-type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally the number of polyploid end reduplicated cells was scored.
Evaluation criteria:
The Mammalian Bone Marrow Chromosome Aberration Test is considered acceptable if it meets the following criteria:
– the spontaneous frequency of cells with chromosome aberration(s) are 2 % or less.
– frequency of cells with chromosome aberration(s) found in the (negative) vehicle controls falls within the range of historical laboratory control data.
– the positive control item should produce biologically relevant increases in the number of cells with chromosome aberration(s).
– each sampling time point of test item treated and control group should include at least 5 analysable animals.

- The test item is classified as positive if the frequency of aberrant metaphase cells is significantly increased in at least one dose group at a single sampling time and if the increase is dose-dependent. If only one of these two conditions is met, the experimental results are classified as equivocal.
– Biological relevance of the results should be considered first.
– Statistical significance should not be the only determining factor for a positive response.
– Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
– The test item is considered as negative in this assay if none of the above criteria met.
– Positive results from the in vivo chromosome aberration test indicate that test item induces chromosome aberrations in the bone marrow of the species tested.
– Negative results indicate that, under the test conditions, the test item does not induce chromosome aberrations in the bone marrow of the species tested.

Statistics:
The statistical evaluation of appropriate data was performed with the Chi-square test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Dose dependent toxicity was observed from 400 to 1000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Clinical Signs and Mortality
No animal died during the main study.
No adverse reactions to treatment were observed in the rats of the vehicle and positive control groups. The rats dosed at the 200 mg/kg bw dose level were symptom-free during the study.
The rats dosed at 400 mg/kg body weight showed slight decrease in activity (6/10) between 3 and 5 hours after the treatment.
The animals dosed at 800 mg/kg bw showed a slight decrease in activity, piloerection (8/10) and humped position (7/10) between 1-2 and 5 hours as well as at 18 hours after the treatment. In this dose group moderate straub tail (6/10) and dyspnoea (2/10) were observed between 3 and 5 hours after the treatment.

Frequency of Bone Marrow Chromosome Aberrations
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 (see Appendix 3). 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 cesium hydroxide monohydrate 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.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different doses of cesium hydroxide monohydrate.

Mitotic Index
1000 nucleated cells were analysed for the percentage of mitotic cells as a measure of cytotoxicity. The single oral administration of 200 mg/kg bw, 400 mg/kg bw and 800 mg/kg bw of cesium hydroxide monohydrate did not induce statistically significant decrease in the number of mitotic cells compared to concurrent controls.

Formulation analysis
The measured concentrations of dosing solutions ranged from 95 % to 96 % of nominal concentrations at the analytical control.

Blood analysis
The measured concentrations of blood serum ranged from 25.2 to 112 cesium mg/L at the analytical control.





Conclusions:
Interpretation of results (migrated information): negative
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 hydroxide anhydrous is not considered to be clastogenic also.
Executive summary:

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 hydroxide anhydrous is not considered to be clastogenic also.

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

Additional information

Additional information from genetic toxicity in vivo:

Ames test:

The reverse mutation test with cesium hydroxide was performed on Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, and Escherichia coli strain WP2 uvrA using the pre-incubation method with and without metabolic activation up to a limit concentration of 5000 µg/plate.

The results showed that the number of revertant colonies was less than twice of the negative control for all strains at any dose level with or without metabolic activation. The growth inhibition was observed in all strains at 5000 µg/plate. The positive controls showed a distinct increase of revertant colonies, and the positive controls and the negative controls were within a range of the background data of the laboratory. There were no fluctuations which might have affected the test results because the sterility test confirmed the absence of micro organisms.

In conclusion, cesium hydroxide was judged to have no reverse mutagenic potential under the conditions tested.

HPRT test:

The test item, cesium hydroxide monohydrate 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, 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.

Cesium hydroxide monohydrate 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 hydroxide anhydrous is not considered to be mutagenic.

Chromosome Aberrations test (in vitro):

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.

Chromosome Aberrations test (in vivo):

Potential mutagenic activity of test item was tested in an in vivo Mammalian Bone Marrow Chromosome Aberration Test with cesium hydroxide monohydrate 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/kg bw cesium hydroxide monohydrate. 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 cesium hydroxide monohydrate 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, rhe single oral administration of 200 mg/kg bw, 400 mg/kg bw and 800 mg/kg bw of cesium hydroxide monohydrate 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 hyroxide monohydrate is not considered to be clastogenic.

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


Justification for selection of genetic toxicity endpoint
Four key studies all considered according to current guidelines and GLP are available.

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo studies neither cesium hydroxide monohydrate nor the anhydrous form is considered 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 the in vivo chromosome aberration assay.