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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-03-20 until 2012-04-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant. An experimental study was performed with a structural analogous read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
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

Test material

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

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Dose dependent toxicity was observed from 400 to 1000 mg/kg bw
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
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.





Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
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. Based on these results cesium nitrate is not considered to be clastogenic also.
Executive summary:

The test item cesium hydroxide monohydrate was used as read across substance to determine the clastogenic potential of cesium iodide in a 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. Based on these results cesium iodide is not considered to be clastogenic also.