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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 16, 2007 to April 08, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: Males: 163 – 207 g; Females: 125 – 167 g
- Fasting period before study: No
- Housing: 5 animals housed in a single solid floor polypropylene cage with softwood flakes
- Diet (e.g. ad libitum): Pelleted diet (Rodent 5LF2 (certified) Diet, BCM IPS Limited, London, UK), ad libitum
- Water (e.g. ad libitum): Drinking water in polycarbonate bottle attached to the cage, ad libitum
- Acclimation period: 9 d prior to study initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 ºC
- Humidity (%): 55±15 %
- Air changes (per hr): At least 15 air changes per h
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent light for continuous 12 h and 12 h darkness


Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Different concentrations of the test substance prepared by dissolving appropriate weighed quantities of the test substance in distilled water.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle:
Levels of the active ingredient in the vehicle are as follows:
15 mg/kg/day – 3 mg/mL
150 mg/kg/day – 30 mg/mL
500 mg/kg/day – 100 mg/mL

- Amount of vehicle (if gavage): Vehicle and test substance solutions gavaged at a rate of 5 mL/kg
- Lot/batch no. (if required): Not reported
- Purity: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance solutions in distilled water are determined by ion chromatography (IC) using an external standard technique.

Samples - Samples of the test substance and standard material prepared in RO water to a final concentration of 1 mg/ml.
IC system - Dionex ion chromatograph fitted with eluant de-gas module, conductivity detector, gradient pump and self regenerating suppression system
Column - IONPAC CS 16 (250 x 3 mm id) at 40°C
Mobile phase - 8 ml of (methane sulfonic acid 70 % wt solution in RO water) per l of RO water
Flow-rate – 0.7 ml/min
Detection system - Electronically suppressed conductivity detector
Retention time - ~ 5.4 mins
Homogeneity assessment done by visual inspection

For stability, samples analyzed immediately after sampling and again after 14 d of storage. Verification of dose/formulation concentrations done within three days of preparation.
The results indicate that the prepared formulations were within ±6 % of nominal values.
Duration of treatment / exposure:
Study animals treated with test substance for up to 28 consecutive days
Frequency of treatment:
Daily once
Remarks:
Doses / Concentrations:
15 mg/kg/day – 3 mg/mL 150 mg/kg/day – 30 mg/mL 500 mg/kg/day – 100 mg/mL
Basis:
nominal in water
No. of animals per sex per dose:
Five animals/sex/dose in both main and recovery groups
Control animals:
yes
Details on study design:
- Dose selection rationale:
Based on the results of a 14 d range-finding study at 500 and 1000 mg/kg/day. Mortality observed in high dose females, therefore dosing terminated on day 11 for this dose. Significant reduction in bodyweight observed in high dose males. Results suggested a 28-d repeated dose study with above-mentioned dose levels.
- Rationale for animal assignment (if not random): Animals randomly assigned to different groups such that the group mean body weights were similar.
- Rationale for selecting satellite groups: Satellite groups selected to observe any reversibility in the toxicity observed at high dose group
- Post-exposure recovery period in satellite groups: 14 d treatment-free recovery period
- Section schedule rationale (if not random): Not applicable

Positive control:
Not applicable

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily according to the following schedule:
-Pre-dose, immediately post-dose, one and five hour post-dosing on weekdays
-Pre-dose and one hour post-dosing on weekends
-Twice a day during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations:
Days 1 (prior to start of treatment), 8, 15, 22 and 28/29 (prior to terminal kill) for main groups, Days 36 and 43 for recovery groups

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time x 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Once weekly until week 3 then daily from week 3 to end of the recovery period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 for main groups, Day 42 for recovery groups
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All surviving animals from each group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 for main groups, Day 42 for recovery groups
- Animals fasted: No
- How many animals: All surviving animals from each group

URINALYSIS: No
- Time schedule for collection of urine: Not applicable
- Metabolism cages used for collection of urine: Not applicable
- Animals fasted: Not applicable

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
1. Behavioural assessment:

2. Functional performance tests:
-Prior to start of treatment and Week 4 for all groups
-Day 19 only for control and high dose groups during treatment period
-Final week of recovery period for the recovery groups

3. Sensory reactivity:
-Prior to start of treatment and Week 4 for all groups
-Day 19 only for control and high dose groups during treatment period
-Final week of recovery period for the recovery groups
-Additionally on Day 19 only for control and high dose groups during treatment period; Final week of recovery period

- Dose groups that were examined: All treatment groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Sensory activity, grip strength and motor activity

OTHER: None
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

-Surviving non-recovery animals at 500 mg/kg/day were sacrificed on Day 28
-All other non-recovery animals sacrificed at the end of the treatment period (Day 29)
-Recovery group animals sacrificed at the end of the 14-d recovery period.

Animals sacrificed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All major organs were observedfor gross lesions. Selected organs were weighed. A set of major organs were microscopically examined for pathological changes
Other examinations:
None
Statistics:
Non-recovery organ weight (absolute and relative to terminal bodyweight) and overall grip strength data assessed for dose-response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance.
Where variances were shown to be homogenous, pairwise comparisons conducted using Dunnett’s test. Where Levene’s test showed unequal variances, data analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
High dose animals showed tenseness, nervousness and hypersensitivity on days 15 to 17. No other treatment-related signs in the other dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality of two females on day 25 and two males on days 27 to 28 at 500 mg/kg/day. No mortality in any of the other treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in bodyweight gain in males during the treatment period and non-significant reduction in females during Weeks 3 and 4 at 500 mg/kg/day. Bodyweight loss for one male and two females during Week 4. Changes reversible during the recovery period. No treatment-related effects in the other dose groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight reduction in food consumption during treatment period at 500 mg/kg/day. Recovery in the treatment-free period. No treatment-related effects in the other dose groups.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Reduction in food efficiency at 500 mg/kg/day during the treatment period. Recovery in the treatment-free period. No treatment-related effects in the other dose groups.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water intake at 500 mg/kg/day from Week 3 until end of treatment and recovery period. No treatment-related effects in the other dose groups.
Ophthalmological findings:
not examined
Description (incidence and severity):
Not applicable.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced mean cell haemoglobin, mean cell volume, haemoglobin, erythrocyte and hematocrit counts at 500 mg/kg/day. Haemoglobin, erythrocyte and hematocrit counts still reduced in the recovery period. Increased reticulocyte counts at all dose levels, not reversed in the recovery period. Reduced platelet counts and clotting times in both sexes and reduced activated partial prothrombin time in males at 500 mg/kg/day. Increased leucocyte counts (especially neutrophil fraction) at 500 (both sexes) and 150 mg/kg/day (females). No recovery for high dose males during treatment-free period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased urea and glucose, decrease total protein and albumin at 500 mg/kg/day. Increased potassium and chloride ion levels at 150 (chloride: only females) and 500 mg/kg/day. Most effects still evident in treatment-free period. Increased AST and reduced AP, cholesterol and bilirubin at 500 mg/kg/day. Reduced ALAT at 500 and 150 (males) mg/kg/day. Most effects still evident in treatment-free period. No treatment-related effects at 15 mg/kg/day.
Urinalysis findings:
not examined
Description (incidence and severity):
Not applicable.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
-Behavioural assessments: higher incidences of urination and defecation at 500 mg/kg/day, still evident in males during recovery period. Additional clinical signs for males at this dose in Week 4, e.g. tail elevation, reduction in transfer arousal, respiratory pattern changes, pallor of extremities, hunched posture and high stepping gait. Tiptoe gait also evident in one female at this dose. Decreased respiration still seen in one male during the recovery period. No treatment-related effects in the other dose groups.
-Functional performance tests: increased overall activity at 500 mg/kg/day during Weeks 3 and 4 (statistically significant only in males). Decreased mean overall grip strength in males at this dose during Week 3 and in the final week of recovery period. No treatment-related effects in the other dose groups.
-Sensory reactivity assessments: higher incidences of vocalisation touch escape and tail pinch (males) at 500 mg/kg/day throughout the treatment period and following cessation of treatment. Higher startle reflex scores, grasp response and toe pinch at this dose during the treatment period. No treatment-related signs in the other dose groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
-Males: increases in relative adrenal and brain weight, increases in relative and absolute testis weight and reduction in absolute liver weights (absolute and relative) at 500 mg/kg/day. Changes in testis and liver weights not reversed in the recovery period, with males also displaying increased kidney weight. Reduced relative liver weight in males at 150 mg/kg/day. No treatment-related effects at 15 mg/kg/day
-Females: reduction in ovary weights during the treatment period at 500 mg/kg/day. No treatment-related effects in the other dose groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Probable signs of gastric irritation evident in the gross pathological observations at 500 mg/kg/day. Raised limiting ridge in glandular epithelium of stomach and sometimes reddened/thickened glandular gastric epithelium seen in animals found dead before study end and three males killed on Day 28. Other observation included dark or enlarged liver (males and females), small epidydime, prostate, seminal vesicle, changes in testis, dark pancreas and dark lungs (males). No treatment-related effects in remaining males at 500 mg/kg/day, females at this dose killed on Day 28 or animals of both sexes in the other dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
-Heart: increased incidence of myocarditis/myocardial fibrosis in males at 150 and 500 mg/kg/day, not reversed during recovery period. No effect in females or any animals at 15 mg/kg/day.
-Liver: vacuolation (males and females) and enlargement of hepatocytes (males) at 500 mg/kg/day. No regression of hepatocyte enlargement during the recovery period. No treatment-related effects at other dose levels.
-Spleen: lymphoid hyperplasia at 500 and 150 (only one animal per sex) mg/kg/day, reversed during treatment-free period. Marginally greater incidence of higher grade of haemosiderin accumulation for females at 500 and 150 mg/kg/day, not reversible at the highest dose. No treatment-related effects at 15 mg/kg/day.
-Cervical lymph node: slightly increased incidence of higher grades of severity of plasma cell density observed in males at 500 mg/kg/day with regression during the recovery period. No treatment-related effects in females or males at other dose levels.
-Kidney: hypertrophy of medullary tubule epithelium and basophilia/degeneration of cortical tubules at 500 mg/kg/day. Hypertrophy of medullary tubule epithelium still evident during recovery period in a few animals at a lower grade. No treatment-related effects at other dose levels.
-Adrenal gland: vacuolation of zona glomerulosa cells at 500 mg/kg/day in both sexes, with effect still evident during the recovery period. No treatment-related effects at other dose levels.
-Stomach: mucosal basophilia and acanthosis/hyperkeratosis of the limiting ridge at 500 mg/kg/day, along with focal congestion in superficial mucosa in three males and one female at 150 mg/kg/day. Effects regressed during the recovery period. No treatment-related effects at 15 mg/kg/day.
-Testes and epididymes: atrophy of the testes, dilatation of the seminiferous tubules, reduced spermatozoal content and cellular debris in males at 500 mg/kg/day. No regression of these effects during the recovery period. No treatment-related effects at other dose levels.
-Seminal vesicles: reduced secretory content in males at 500 mg/kg/day with regression during the recovery period. No treatment-related effects at other dose levels.
-Skeletal muscles: focal degeneration of the muscle fibers, frequently with associated mononuclear cell foci at 500 mg/kg/day, with partial regression during the recovery period. No treatment-related effects at other dose levels.

HISTORICAL CONTROL DATA:
Hematological, biochemical and organ weight data from the historical control animals used for comparison with the data obtained with the control animals of the study.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the study conditions the oral administration of the test substance by gavage to rats for a period of up to 28 d at 15, 150 and 500 mg/kg/day (incorporating a correction factor for a purity of 78.94 %) resulted in treatment-related effects at all doses. The results indicate that the prepared formulations were within ±6 % of nominal values. Effects detected at 15 mg/kg/day were not considered to be adverse. Therefore 15 mg/kg/day was selected as the NOAEL for this study.
Executive summary:

A study was conducted to determine the systemic toxicity of the test substance in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to 28 consecutive days, at dose levels of 0, 15, 150 and 500 mg/kg/day (incorporating a correction factor for 78.94 % purity). Stability and homogeneity of the test substance solutions in distilled water were determined by ion chromatography. Two recovery groups, each of five males and five females, were treated with the high dose (500 mg/kg/day) or the vehicle alone for up to 28 consecutive days and then maintained without treatment for a further fourteen days. Due to deaths encountered at the highest dose level, all females dosed with 500 mg/kg/day were treated for 25 days only. Non-recovery females and males were terminated on day 28. Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. The test substance treatment caused effects at all dose levels in the Sprague-Dawley rats. The target organs were considered predominantly to be heart, liver, spleen, central nervous system, skeletal muscles and reproductive organs changes in serum biochemistry were noted at high doses. Reticulocyte counts were increased at all doses. Under the study conditions the oral administration of the test substance by gavage to rats for a period of up to 28 d at 15, 150 and 500 mg/kg/day (incorporating a correction factor for a purity of 78.94 %) resulted in treatment-related effects at all doses. The results indicate that the prepared formulations were within ±6 % of nominal values. Effects detected at 15 mg/kg/day were not considered to be adverse. Therefore 15 mg/kg/day was selected as the NOAEL for this study (Dhinsa, 2008).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
21 September, 2015 to 15 December, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
Once taken up by the organism, cesium formate dissociates into the cesium cation (Cs) and the formate anion (HCO2 -). Toxicity is caused by the cesium cation, therefore conducting a study on cesium chloride as a surrogate for cesium formate is justified.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: RccHan™;WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited (formally Harlan (UK) Ltd)
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age of the main study and recovery animals at start of
treatment: 41 to 47 days.
- Weight range of the main study and recovery animals at the start of treatment: Males: 114 to 173 g, Females: 115 to 145 g
- Housing: Polycarbonate cages with a stainless steel mesh lid, changed at appropriate intervals. Five of the same sex per cage, unless reduced by mortality. Bedding: Wood based bedding which was changed at appropriate intervals each week
- Diet (e.g. ad libitum): Teklad 2014C Diet. Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 12 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: gavage
Details on route of administration:
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Volume dose: 10 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled
body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency: Once daily at approximately the same time each day.
Vehicle:
water
Details on oral exposure:
- Starting with the low concentration (1.3 mg/mL), the formulation was prepared by adding 90% of the final volume of vehicle to the test item. An initial mix, crushing any large particles using a hand spatula, was performed if required and the mixture was transferred to a larger container and mixed using a magnetic stirrer until dissolved. Once the material was fully dissolved the pH was measured. For all preparations, the pH was found to be between 5 and 7 and no adjustment was needed. The solution was transferred to a measuring cylinder and made up to volume before transfer to a suitable container and stirred using a magnetic stirrer. The procedure was repeated for the remaining groups in order of ascending concentration.
- Frequency of preparation: Weekly, and was prepared in advance of the first day of dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability of the test item in the liquid matrix.
- Assessment of homogeneity was not relevant as CsCl forms a solution in water.
- Achieved concentration: Samples of each formulation prepared for administration in Weeks 1, 4 and 13 of treatment were analyzed for achieved concentration of the test item.
- Analysis method: ICP-MS
Duration of treatment / exposure:
- Exposure: 90 days
- Recovery period: 16 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
13 mg/kg bw/day (actual dose received)
Remarks:
Equivalent to 15 mg cesium formate monohydrate/kg bw/day
Dose / conc.:
38 mg/kg bw/day (actual dose received)
Remarks:
Equivalent to 44 mg cesium formate monohydrate/kg bw/day
Dose / conc.:
127 mg/kg bw/day (actual dose received)
Remarks:
Equivalent to 147 mg cesium formate monohydrate/kg bw/day
Dose / conc.:
253 mg/kg bw/day (actual dose received)
Remarks:
Equivalent to 295 mg Cs/kg bw/day
No. of animals per sex per dose:
115 males and 54 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 13, 38, 127 and 253 mg CsCl/kg/day which correspond with 0, 10, 30 100 and 200 mg Cs/kg/day) were selected based on the findings of an oral gavage 90 day toxicity study of CsOH.H2O in the rat and an oral gavage reproduction/developmental screening study (56 days of treatment) of CsNO3 in the rat.
- Post-exposure recovery period in satellite groups: The recovery periods were initially intended to be 4 or 8 weeks duration. These were extended when the severity of effect on spermatogenesis and lack of recovery seen after 4 weeks recovery for animals at the high dose indicated a longer period of recovery would be needed.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or
marked.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

FOOD EFFICIENCY: No
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. Precise measurements were undertaken from Week 10 of treatment.
Water consumption recording: Weekly from Week 10 to the end of the treatment period. During Week 4, 8, 12 and 16 of recovery.
Recorded over a 3-day period on each occasion. In addition, water consumption was recorded for Group 5 males in Recovery Weeks 1, 2, 3 and 5. These measurements were not required by protocol.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatement: all animals; Week 12: all animals of Group 1 and 4
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
- Week 9, prior to premature termination: Group 5
- Week 13: All main study animals (Groups 1 to 4)
- Recovery Week 4: Group 5 Recovery Week 4 animals
- Recovery Week 8: Group 1 and 4 Recovery Week 8 animals
- Recovery Week 12: Group 5 Recovery Week 12 animals
- Recovery Week 12: Group 1 and 4 Recovery Week 12 animals (limited parameter list)
- Recovery Week 16: Group 1 and 4 Recovery Week 16 animals (five animals/group) (limited parameter list)
Blood sampling was performed on the morning after overnight collection of urine with the exception of Group 5 animals terminated early. Animals, were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined s using a Bayer Advia 120 analyzer.

CLINICAL CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
- Week 9, prior to premature termination: Group 5
- Week 13: All main study animals (Groups 1 to 4)
- Recovery Week 4: Group 5 Recovery Week 4 animals
- Recovery Week 8: Group 1 and 4 Recovery Week 8 animals
- Recovery Week 12: Group 5 Recovery Week 12 animals
- Recovery Week 12: Group 1 and 4 Recovery Week 12 animals (limited parameter list)
- Recovery Week 16: Group 1 and 4 Recovery Week 16 animals (five animals/group) (limited parameter list)
Blood sampling was performed on the morning after overnight collection of urine with the exception of Group 5 animals terminated early. Animals, were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.

URINALYSIS: Yes
Animals were placed in an individual metabolism cage, without water. Urine samples were collected overnight at the following occasions:
- Week 13: All main study animals (Groups 1 to 4)
- Recovery Week 4: Group 5 Recovery Week 4 animals
- Recovery Week 8: Group 1 and 4 Recovery Week 8 animals
- Recovery Week 12: Group 5 Recovery Week 12 animals (See Deviation from Protocol)
- Recovery Week 12: Group 1 and 4 Recovery Week 12 animals (limited parameter list)
- Recovery Week 16: Group 1 and 4 Recovery Week 16 animals (Urine sample frozen (nominally -20°C) pending any future requirement for analysis)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Battery of functions tested: sensory activity / grip strength / motor activity

Sensory reactivity and grip strength assessments were performed (before dosing) on all main study animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

During Week 12 of treatment (before dosing), the motor activity of each main study animal was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.

Schedule Main study animals of Groups 1 to 4 were killed following 13 weeks of treatment. Group 5 animals were killed prematurely in Week 9 for animal welfare reasons; females on
Day 59 and males on Day 63. Group 1 and 4 animals assigned to the recovery phase were
killed and after 8, 12 or 16 weeks of recovery, which followed 13 weeks of treatment. Group 5 animals were killed following 59 days of treatment and either 4 or 12 weeks of recovery. The necropsy and CASA investigations for Group 5 animals after 12 weeks of recovery were conducted on the same day as for Groups 1 and 4 after 8 weeks of recovery.

ORGAN WEIGHTS: Yes
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for main study and recovery animals killed at scheduled
intervals.

HISTOPATHOLOGY: Yes
Full List: All animals killed or dying prematurely. Main study animals of Group 5 killed after 8 weeks of treatment. Main study animals of Groups 1 and 4 killed after 13 weeks of treatment.
Male reproductive tissues (testes, seminal vesicles and epididymis), kidneys and macroscopic abnormalities only: All main study animals of Groups 2 and 3 killed after 13 weeks of treatment. All recovery phase animals, i.e. all Group 5 animals killed after 4 or 12 weeks of recovery and all Group 1 and 4 animals killed after 8, 12 or 16 weeks of recovery.
Tissues which were considered to exhibit a reaction to treatment in Group 4: adrenals, heart, salivary gland (submandibular and sublingual), stomach, spleen. Mammary tissue (males only): All main study animals of Groups 2 and 3 killed after 13 weeks of treatment. Recovery phase animals of Groups 1 and 4 killed after 8 weeks of recovery
Other examinations:
SPERM ANALYSIS

Immediately after scheduled sacrifice (main study and recovery) of each male, including Group 5 males at planned early termination, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
- Sperm motility - all groups. A sample of sperm was expressed from the vas deferens into prewarmed (approximately 37C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS Computer Assisted Sperm Analyser (CASA).
- Sperm morphology – all groups. A 200 µL aliquot of the sperm/medium mixture was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.
- Sperm count - all groups. The left cauda epididymis of each male was weighed and then the tunica was removed. The portion obtained was weighed then homogenised for at least thirty seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
- Homogenisation-resistant spermatid count – all groups. After removal of the tunica, the left testis of each male was homogenised for at least thirty seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.
Statistics:
The study report contains serial observations pertaining to all weeks of study completed, together with signs data collected during the necropsy period. In the case of clinical signs, body weight and food consumption data, only information from the final week of the acclimatization period are presented.
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data (except differential white blood cells and cited parameters in urinalysis). Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It was, therefore, not always possible to derive exact group values from the data presented in the appendices.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment was generally well tolerated up to Week 9, though animals from all treated groups, but particularly those receiving 127 or 253 mg/kg/day, became irritable and vocal from Week 7 when handled for dosing.

In Week 9 of treatment, animals receiving 253 mg/kg/day showed deterioration in condition. Convulsions were observed for two females and one male resulting in these animals dying or being killed for welfare reasons. Other animals receiving this dose level showed signs included hunched posture, partially closed eyelids, piloerection, tremors, dull eyes, rapid or irregular breathing, red staining on the nose and under- or over-activity. As this dose level was not tolerated, treatment was stopped in Week 9 (Day 58 for females and Day 59 for males).

From approximately Week 7, a higher incidence dorsal hair loss and of encrustations or skin abrasions, particularly on the lower jaw, head and paws were observed for animals receiving 127 or 253 mg/kg/day. Red or brown staining around the nose (chromodacryorrhea) was also observed for a few animals receiving 127 or 253 mg/kg/day from Week 7 reflecting the poorer health status of these animals.

A slightly high incidence of chin rubbing was observed immediately after dosing in Weeks 7 and 8, predominantly for animals receiving 253 mg/kg/day, but also for occasional animals of other treated groups. This sign is not uncommon in studies where the test material is administered by oral gavage and, as such, is considered of no toxicological importance. During the recovery period, the condition of the animals generally improved, though the deterioration of two males that previously received 253 mg/kg/day, three days following cessation of treatment (No. 108) and during Week 7 of recovery (No. 105) was attributed to previous treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
One male and three females receiving 253 mg/kg/day died or were killed during the treatment period and a further two males that received this dose level died or were killed during the
recovery period. These deaths were attributed to treatment or previous treatment with CsCl.

Female, No. 147 (253 mg/kg/day) was found dead at the 1-2 hour post-dose observation on Day 49, Week 7 of treatment. There was no signs seen ante-mortem. Macropathology findings at necropsy included blood in the thoracic cavity and pale frothy fluid in the trachea. At microscopic examination, treatment-related findings were seen in the adrenals, salivary glands, skin, skeletal muscle around the bone of the sternum, ovaries and uterus. Atrophy/mucification was also seen in the vagina.

Female No. 145 (253 mg/kg/day) had a convulsion prior to dosing on Day 58, Week 9 of treatment and died immediately after. The animal showed bodyweight loss during the week prior to death and was recorded as having thin build. Necropsy examination revealed dark areas on the stomach, the caecum was devoid of contents, the thymus was small and there were scabs on the forelimbs. Treatment-related microscopic findings were seen in the adrenals, salivary glands, skeletal muscle around the bone of the sternum, ovaries and uterus, skin and extremities. Atrophy/mucification was also seen in the vagina.

Female No. 141 (253 mg/kg/day) had a convulsion lasting approximately 2 minutes during the afternoon of Day 58 (Week 9). Following the convulsion the animal showed tremors, welfare reasons. The animal showed bodyweight loss during Weeks 7 and 8. Necropsy examination revealed abnormally coloured (bright red) blood, pale areas on the heart and kidneys, all lobes of the lungs were red, the spleen was an abnormal colour (brown), small thymus and scabs on the fore and hind-limbs. Treatment-related microscopic findings were seen in the adrenals, heart, salivary glands, stomach, ovaries, uterus and extremities.

Male No. 27 (253 mg/kg/day) had a convulsion lasting approximately 40 seconds prior to dosing on Day 60. Following the convulsion the animal showed tremors, rapid breathing, piloerection and salivation and was killed for welfare reasons. Necropsy examination revealed pale areas on the adrenals and kidneys, swollen epididymides and the stomach showed darkened areas and thickened glandular mucosa. Treatment-related microscopic findings were seen in the adrenals, heart, kidneys, salivary glands, skeletal muscle around the bone of the femur, mammary glands, spleen, stomach, epididymides and testes.

Male No. 108 (253 mg/kg/day) was found dead three days following cessation of treatment (Day 63). There were no signs observed ante-mortem. Necropsy examination revealed pale areas on the heart and kidneys, dark areas on the pituitary and stomach, swollen epididymides, distended urinary bladder and scabs on the forelimbs, jaw and tail. Treatment-related microscopic findings were seen in the adrenals, heart, salivary glands, epididymides, testes, skin and extremities.

Male No. 105 (253 mg/kg/day) was killed for welfare reasons during Week 7 of the recovery phase. Clinical signs included a hunched posture, irregular breathing, pallor and piloerection. Necropsy examination revealed abnormal (dark) contents in the cecum and jejunum, small epididymides and small and soft testes. At microscopic examination, treatment-related findings were seen in the salivary glands, adrenals, heart, kidneys, epididymides, testes, skin and extremities.

In addition, one male (No. 62) receiving the intermediate dose of 127 mg/kg/day was killed due to poor condition in Week 13 of treatment. The factor contributory to the death of this animal at microscopic examination was considered to be gastrointestinal tract lesions. Clinical signs comprised decreased activity, irregular breathing, thin build, hair loss, yellow and loose feces, partially closed eyelids with dull and dark eyes, abnormal (swaying) gait and hunched posture. Necropsy examination revealed a perforated and thickened jejunum with multiple firm dark masses, multiple firm pale masses and pale areas on the liver, pale areas on the kidneys, dark mesentery, peritoneum and mesenteric lymph nodes, pale payer’s patches, dark areas, thickened non-glandular mucosa and a depression on the glandular mucosa of the stomach, small and dark thymus, scabs on the lower jaw and hair loss. The animal had lost 37g in bodyweight in the week prior to dispatch. Treatment-related microscopic findings were seen in the adrenals, kidneys, heart, salivary glands, epididymides, testes and skin. Microscopic changes were also seen in the gastrointestinal tract, liver and some other tissues. The factor contributory to death was considered to be the gastrointestinal tract lesions. The degenerative and inflammatory responses seen were considered unrelated to treatment as they were only seen in this one animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain (Week 0-8) for animals receiving 253 mg/kg/day was statistically significantly lower than that of the controls (0.75X and 0.67X control for males and females, respectively). From Week 7, the effect on body weight became more pronounced, with females showing body weight losses and body weight gain of males falling to approximately half that of the controls.

Overall body weight gain (Weeks 0-13) for males receiving 127 mg/kg/day was also statistically significantly lower than that of the controls (0.89X control). Body weight gain for animals receiving 13 and 38 mg/kg/day and females receiving 127 mg/kg/day were unaffected by treatment.

Weight gain during the recovery period was markedly higher than controls for males which previously received 127 or 253 mg/kg/day, with the greatest gains occurring during the first 8 weeks following cessation of treatment (1.4X and 1.8X control for Weeks R0-R8 at 127 and 253 mg/kg/day, respectively). Absolute body weights for males receiving 127 mg/kg/day were also similar to those of the controls by Week 8 of recovery, indicating full recovery from the previous effect of treatment. Absolute body weight at the end of the 12-week recovery period for males previously receiving 253 mg/kg/day was still lower than controls (0.85X control value for Week R12) indicating partial recovery.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption (Weeks 1-8) for animals receiving 253 mg/kg/day was low when compared with that of the controls (0.89X and 0.90X control for males and females,
respectively).

Food consumption for males receiving 127 mg/kg/day was slightly low from Week 6 when compared to the controls resulting in a slightly low overall value, (0.95X control).

Food consumption for animals receiving 13 or 38 mg/kg/day and females receiving 127 mg/kg/day was not clearly affected by treatment.

During the recovery phase, food consumption for males previously receiving 127 or 253 mg/kg/day was similar to or slightly higher than that of the controls (1.1X and 1.4X controls for Weeks R1-R12, respectively), indicating recovery from the previous effect of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Visual observation of water consumption indicated a potential effect of treatment. Quantitative measurements were, therefore, undertaken from week 10 of treatment. Water consumption for males and females receiving 38 or 127 mg/kg/day and females receiving 13 mg/kg/day was high during Weeks 10 to 13, when compared with that of the controls. There was no clear dose-relationship, particularly in females where the highest consumption was observed in the low dose group. Males receiving 13 mg/kg/day were unaffected by treatment.

During the recovery period (up to Week 16 of recovery), water consumption for males previously treated with 127 mg/kg/day remained slightly higher than that of the Controls. After Week 4 of recovery, however, the amount of water consumed was lower than that measured during the treatment period, indicating at least partial recovery. Water consumption during the recovery phase for males which previously received 253 mg/kg/day was high at the start of the recovery period, reduced as the recovery period progressed, indicating recovery from the previous effects of treatment.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological investigation during Week 9 of treatment for males and females receiving 253 mg/kg/day, prior to early termination, revealed, when compared with the control values for samples obtained in Week 13, low haematocrit, hemoglobin concentration and erythrocyte count, associated with increased reticulocyte counts, in both sexes. There was also an increase of the red cell distribution width and a reduction in mean cell haemoglobin concentration in both sexes and high mean cell volume in males.

Total leucocyte counts were also high in Week 9 for males and females receiving 253 mg/kg/day. These were predominantly a consequence of increased neutrophil counts which were high in both sexes, though lymphocyte, eosinophil and monocyte counts were also high for females receiving this dose level.

The mean activated partial thromboplastin times were reduced, when compared with the controls, for males and females receiving 253 mg/kg/day. The hematological investigation during Week 13 of treatment for males and females receiving 127 mg/kg/day showed similar effects to those seen in the higher dose group after 9 weeks of treatment. Haematocrit, hemoglobin concentration and erythrocyte counts were low in both sexes, associated with increased reticulocyte counts. There was also an increase in red cell distribution width in both sexes and low mean cell hemoglobin and mean cell hemoglobin concentration in females. In addition, hemoglobin concentration for females receiving 38 mg/kg/day was slightly low when compared with the controls, attaining statistical significance.

Total leucocyte counts were increased in Week 13 for animals receiving 127 mg/kg/day, associated with increased neutrophil, lymphocyte, eosinophil and monocyte counts in both sexes. Prothrombin times were considered to be slightly increased in Week 13 of treatment, when compared with the controls, for animals receiving 127 mg/kg/day. Although the group mean prothrombin time for males receiving this dose was similar to that of the controls, the group mean control value was higher than expected due to a very high value for one animal (1M 43, 52.6 sec). When this anomalous value was excluded, the group mean was 22.8 sec for the controls compared with a group mean value of 26.0 sec for males receiving 253 mg/kg/day.

The mean activated partial thromboplastin times were reduced, when compared with the controls, for males receiving 38 or 127 mg/kg/day.

All other differences from controls observed during the treatment period were minor, lacked dose relationship or were confined to one sex and were therefore attributed to normal biological variation.

During Week 4 of recovery for males which previously received 253 mg/kg/day, effects on red blood cell parameters comprising low haematocrit, hemoglobin concentration and erythrocyte count, associated with increased reticulocyte counts, and increased red cell distribution width and mean cell volume in males were still apparent. White blood cell counts were also slightly high compared with the controls. The magnitude of effect for all parameters was less than that at Week 9 of treatment, indicating partial recovery from previous treatment.

During Week 8 of recovery for males which previously received 127 mg/kg/day, haematocrit and red blood cell counts were still slightly low and mean cell hemoglobin, mean cell hemoglobin concentration and mean cell volume slightly high, when compared with controls. White blood cell counts (including neutrophil, lymphocyte, eosinophil, basophil and monocyte counts) were still statistically significantly higher than controls. Prothrombin time was still increased compared to controls. The magnitude of effect was reduced compared with Week 13 of treatment, indicating partial recovery from previous treatment.

During Week 12 of recovery, there was almost complete recovery for males which previously received 127 mg/kg/day. There was still a statistically significant reduction in red blood cell count, and prothrombin time was still slightly increased for previously treated males compared to controls. All other parameters had recovered.

There were no changes in Week 12 of recovery for males which previously received 253 mg/kg/day that were attributed to previous treatment. During Week 16 of recovery, prothrombin time for males which previously received 127 mg/kg/day was still marginally higher than that of the controls, attaining statistical significance. This minor difference was considered not of toxicological importance. Red blood cell count for the previously treated males was similar to controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination during Week 9 of treatment for males and females receiving 253 mg/kg/day, prior to early termination, revealed, when compared with the control values for samples obtained in Week 13, an increase in aspartate amino-transferase and creatinine kinase activities for both sexes. Plasma urea was markedly increased in both sexes; glucose concentration was low in males only. Cholesterol concentration was reduced in both sexes and triglyceride concentration was increased for females only. Potassium was markedly reduced in both sexes. Phospholipids were reduced and chloride slightly reduced in both sexes. Magnesium was reduced in males only and bicarbonate in females only.

Total protein concentrations were markedly reduced in both sexes at 253 mg/kg/day; attributed to reductions of both the albumin and globulin fractions. There were also reductions in albumin-globulin ratio that was due to a proportionately greater reduction of albumin levels.

The biochemical examination of the blood plasma in Week 13 revealed, when compared with controls, an increase in aspartate amino-transferase and creatinine kinase activities for both sexes receiving 127 mg/kg/day.

There was a decrease of alanine amino-transferase activity for males receiving 127 mg/kg/day, though this is not considered of toxicological significance.

Plasma urea concentration was markedly higher than controls in Week 13 for animals receiving 127 mg/kg/day (1.64X and 1.29X control for males and females, respectively) and slightly high for males receiving 38 mg/kg/day (1.15X control). Creatinine concentrations were statistically significantly higher than those of the controls in Week 13 for males and females receiving 127 mg/kg/day (1.36X and 1.28X control, respectively).

Lactate was statistically significantly increased in males receiving 127 mg/kg/day; females were not clearly affected. Bicarbonate was slightly decreased in animals receiving 127 mg/kg/day, but only attaining statistical significance in the females.

Plasma cholesterol and phospholipid concentrations were low for males and females receiving 127 mg/kg/day and males receiving 38 mg/kg/day. There effect on cholesterol did not show a dose-relationship in males.

Potassium concentrations were low to very low, with a clear dose-relationship, for both sexes receiving 38 or 127 mg/kg/day. Magnesium concentrations were low for both sexes receiving 127 mg/kg/day and males receiving 38 mg/kg/day with a clear dose-effect relationship. Chloride concentration was marginally low for both sexes receiving 127 mg/kg/day and males receiving 38 mg/kg/day without clear dose-relationship.

Total protein concentrations were statistically significantly lower than controls in Week 13 at all doses in males. This was attributed to small reductions of both the albumin and globulin fractions. Conversely, in females receiving 127 mg/kg/day there was a slight increase in globulin levels resulting in a decrease in albumin-globulin ratio.

All other differences from controls observed during the treatment period were minor, lacked dose relationship or were confined to one sex and were therefore attributed to normal biological variation.
The biochemical examination during Week 4 of recovery for males which previously received 253 mg/kg/day indicated partial recovery. Many of the changes seen in Week 9 of treatment were still apparent, but to a lesser degree. These comprised increased aspartate amino-transferase activity and urea concentration and decreased cholesterol, phospholipid, potassium, chloride, magnesium and total protein concentrations, associated with reduced albumin and globulin fractions.

At the biochemical examination during Week 8 of recovery for males which previously received 127 mg/kg/day, many of the findings present at the end of treatment had recovered. Changes still evident comprised reduced potassium, and slightly reduced chloride and magnesium concentrations and whilst total protein for previously treated males was similar to controls, albumin concentration was still slightly reduced and globulin fractions were slightly high resulting in a statistically significant decrease in albumin-globulin ratio. In addition, glucose concentration was slightly but statistically significantly lower than controls, though this was not apparent at the end of the treatment period for animals of this dose group (but apparent at 253 mg/kg/day in Week 9).

For males which previously received 253 mg/kg/day, the biochemical investigations in Week 12 of recovery revealed, when compared to control animals sampled in Recovery Week 8, low triglyceride, phospholipid and potassium concentrations. Marginally low albumin and marginally high globulin fractions resulted in a low albumin-globulin ration.

Aspartate amino-transferase activity was still marginally higher than that of the controls, and lactate concentration was also high, though this had been similar to controls at the Recovery Week 4 investigations.

All blood chemistry parameters for males which previously received 127 mg/kg/day were considered to have recovered by Week 12 of recovery. A slightly low albumin value attained statistical significance, but this slight difference from controls was considered not to be of biological significance. Re-evaluation of this parameter in Week 16 of recovery confirmed that this change had completely resolved.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis investigations were not performed prior to early termination in Week 9 of animals receiving 253 mg/kg/day for welfare reasons.

The urinalysis investigation in Week 13 of treatment revealed, when compared with the controls, high urinary volume by males and females receiving 127 mg/kg/day and this associated with low specific gravity. Urinary sodium, potassium and chloride outputs were high for both sexes at 127 mg/kg/day. Magnesium, calcium and creatinine levels were also high in males, though the difference magnesium and creatinine did not attain statistical significance. Glucose output was low in both sexes at 127 mg/kg/day and total protein was low in males at 127 mg/kg/day. The pH of the urine was low for females receiving 127 mg/kg/day. In addition, blood in the urine (small to large amount) was recorded for 1/10 males and 2/10 females receiving 127 mg/kg/day, compared with none in the controls.

There were no other changes in the appearance and/or composition of the urine that were attributable to treatment. Low calcium output for females which received 127 or 253 mg/kg/day showed no dose-relationship and was in the opposite direction to the effect seen in males and, therefore was attributed to normal biological variation.

During Week 4 of recovery urinalysis investigations for males which previously received 253 mg/kg/day revealed (when compared with controls sampled in Week 13 of treatment which were performed on the same day) high urinary volume with associated low specific gravity, high urinary creatinine, magnesium, sodium, potassium and chloride outputs and low total protein output. The pH of the urine was also low. In addition, blood in the urine (moderate to large amount) was recorded for 2/10 males.

During Week 8 of recovery for males which previously received 127 mg/kg/day, urine volume was still higher than that of the controls and associated with low specific gravity. Blood in the urine was apparent (large amount) for 1/9 males and creatinine, magnesium and potassium outputs were also still slightly high compared with controls, though partial recovery was evident.
In Week 12 of recovery, blood was present in the urine (large amount) for one animal (No. 106) that previously received 253 mg/kg/day. There were no other changes in the urine that were attributed to previous treatment, demonstrating full recovery had occurred for the remainder of the animals.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
SENSORY REACTIONS

Sensory reactivity responses and grip strength were considered to be unaffected by treatment. Excessive vocalisation during the testing procedures was recorded for a small number of
females of all treated groups assessed (13, 38 or 127 mg/kg/day), reflecting the clinical observations during the study.

Group mean forelimb and hindlimb grip strength values for all treated male groups were high compared with Controls, with statistical significances attained in all treated groups for forelimb grip strength and at 127 mg/kg/day for hindlimb grip strength. All forelimb grip strength values, including those of the controls, were however, below the historical control data range (0.89 to 1.07 kg), except for males at 127 mg/kg/day, which was within the historical control range. Mean forelimb grip strength for females receiving 127 mg/kg/day was also slightly high compared with Controls and also above the historical control data range (0.73 to 0.94 kg for forelimbs and 0.33 to 0.46 kg for hindlimbs), but without statistical significance. One male and two females in the control group were reluctant to grip the forelimb bar and values for these animals were lower. The differences were, therefore, attributed to atypically low scores for the controls.

Mean hindlimb grip strength values for males receiving 38 or 127 mg/kg/day were slightly above the historical control data range (0.41 to 0.49 kg), attaining statistical significance at 127 mg/kg/day. Mean hindlimb grip strength values for treated females were similar to those of the controls, with mean values for both control and females receiving 127 mg/kg/day being marginally above the historical control data range (0.33 to 0.46 kg). These minor differences were considered not of toxicological significance.

MOTOR ACTIVITY

Motor activity in Week 12 was considered to be unaffected by treatment.

Group mean motor activity scores were similar to Controls at all doses in males. The majority of the low beam 6-minute interval scores and the total low beam scores (a measure of cage floor activity) for all treated females were slightly high compared with Controls, but attained statistical significance on just two occasions (12-minute interval for females receiving 38 or 127 mg/kg/day and at the 24-minute interval for low beams for females receiving 127 mg/kg/day). The high beam 6-minute interval and total high beam scores (a measure of rearing activity) were similar to those of the controls, the only statistical difference (36-minute interval for females receiving 127 mg/kg/day) being in the opposite direction to the low beam score. Considering that the differences from controls seen for the low beam scores were minimal, with no similar effect in high beam scores which are usually more sensitive to effects, and in one sex only, the differences were attributed to natural variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Analysis of organ weights for animals receiving 253 mg/kg/day was made difficult because of the bodyweight effect and lack of concurrent control animals. When compared with background data and control animals killed four weeks later, there were indications of low liver and thymus weights in both sexes and low epididymides and testes weights in males.

Analysis of organ weights for animals killed after 13 weeks of treatment revealed, when compared with the controls, low absolute and bodyweight-adjusted epididymides weights and low absolute testes weights for males at 127 mg/kg/day, increased bodyweight-adjusted kidney weights in both sexes at 127 mg/kg/day (attaining statistical significance in females only) and increased absolute kidney weights for females at 127 mg/kg/day, decreased absolute and bodyweight-adjusted liver weights for males at 13, 38 or 127 mg/kg/day; increased absolute and bodyweight-adjusted spleen weights for females at 127 mg/kg/day; decreased absolute and bodyweight-adjusted thymus weights in both sexes at 127 mg/kg/day.

In addition, the absolute and bodyweight-adjusted weights uterus and cervix weights were decreased in females at 127 mg/kg/day; though the differences did not attain statistical significance. Bodyweight-adjusted adrenal weights for males at 127 mg/kg/day were slightly high compared to controls, but did not attain statistical significance.

Analysis of organ weights following a 4-week recovery period for males given 253 mg/kg/day revealed (when compared with controls killed after 13 weeks of treatment which were performed on the same day) reduced absolute epididymides, liver, testes and thymus weights and increased kidney weights.

Following 8 weeks of recovery, absolute epididymides weights were still statistically significantly lower than those of the controls and absolute and bodyweight-adjusted kidney weights were still statistically significantly higher than those of the controls for males previously treated at 127 mg/kg/day. Absolute and bodyweight-adjusted liver and thymus weights and absolute testes weights were slightly lower than those of the controls, but the differences were minimal and did not attain statistical significance indicating partial recovery.

There were no statistically significant differences in organ weights between controls and males previously treated at 127 mg/kg/day following 12 weeks of recovery. For animals that previously received 253 mg/kg/day, absolute epididymides, liver, testes and thymus weights remained low following 12 weeks of recovery, at least in part due to the low bodyweights still evident in these animals.

There were no differences after 16 weeks of recovery that were attributed to previous treatment. A slight difference in kidney weights attained statistical significance after 16 weeks of recovery, but the difference was minimal and considering the lack of any difference after 12 weeks of recovery, this was considered due to normal biological variation.

A difference in brain weight for previously attained statistical significance but since no similar change was observed on completion of treatment or on any of the previous recovery occasions, this was considered a consequence of normal variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed after 9 or 13 weeks of treatment revealed: pale adrenals at 127 or 253 mg/kg/day; pale areas in the kidney at 253 mg/kg/day and males at 127 mg/kg/day; scabs and depressions on the skin and subcutis at 127 or 253 mg/kg/day; scabs on the forepaws at 253 mg/kg/day; darkening of the salivary glands in both sexes at 127 mg/kg/day and at 38 and 13 mg/kg/day in males; soft testes at 127 mg/kg/day and small testes and epididymides at 253 mg/kg/day; dark areas; depressions and thickening in the stomach at 253 mg/kg/day. Following 12 weeks of recovery, the changes in the mandibular salivary gland, epididymides and testes were still evident in a small number of animals that previously received 253 mg/kg/day, whilst all changes at 127 mg/kg/day had recovered. Following 16 weeks of recovery, treatment-related changes in the salivary gland and testes were evident for 1/5 males previously receiving 127 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes related to treatment were seen in the mandibular and sublingual salivary glands (degranulation and atrophy), heart (myocardial degeneration/inflammation), stomach (foveolar/mucosal hyperplasia), spleen (males only - increased extramedullary haemopoiesis), adrenals (diffuse hypertrophy of the zonal glomerulosa), mammary glands (males only – atrophy), skin and subcutis (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), epididymides (reduced sperm content and cell debris at 127 and 253 mg/kg/day and ductal atrophy and sperm granuloma at 127 mg/kg/day) and testes (tubular degeneration/atrophy) of animals treated at 253 mg/kg/day for 59 days or 127 mg/kg/day for 13 weeks. Additional changes were seen in the kidneys (males only, tubular basophilia), skeletal muscles (myofibre degeneration), extremities (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), ovaries (decreased corpora lutea) and uterus (atrophy) of animals treated at 253 mg/kg/day. At 38 mg/kg/day, treatment-related changes were seen in the adrenals (diffuse hypertrophy of the zonal glomerulosa) and testes (minimal tubular degeneration/atrophy). No treatment-related change was seen at 13 mg/kg/day. Full recovery was demonstrated in the kidneys, stomach, spleen, epididymides and mammary glands. Partial recovery was demonstrated in the salivary glands, heart, adrenals after 8 weeks of recovery and in the testes after 16 weeks of recovery.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
SPERM ANALYSIS

Treatment with cesium chloride at 13 mg/kg/day for 90 days produced no adverse effects on sperm motility, concentration or morphology.

Following 13 weeks of treatment with cesium chloride at 38 mg/kg/day, slight but statistically significant changes in sperm morphology, motility (increase of beat cross frequency only), and a reduction in sperm number in the cauda epididymis were seen.

Treatment at 127 mg/kg/day resulted in most males showing no motile or normal sperm and significant reductions in cauda epididymal sperm numbers compared to Control animals. Additionally, testicular sperm numbers were lower than Controls; this change was not statistically significant. Similar results were seen for sperm motility, morphology and cauda epididymal sperm following only 59 days treatment at 253 mg/kg/day, however effects on testicular sperm numbers were not apparent. Abnormalities seen at the two highest dose levels were mostly decapitate sperm with irregular and/or frayed midpiece and/or tails. A high incidence of flattened head was also apparent. Results suggest an effect on maturation of the sperm with most sperm breaking down within the epididymis.

Following a 4 week recovery period for animals which received 253 mg/kg/day, a progression of effects was apparent; all sperm were immotile and abnormal. Sperm numbers in both the cauda epididymis and testis were markedly lower than seen at the end of treatment with cesium chloride at 253 mg/kg/day.

Following an 8 week recovery period those males previously treated at 127 mg/kg/day showed some evidence of recovery, however values for motility, counts and morphology were still significantly lower than those of the Control males.

Following a 12 week recovery period males previously treated with cesium chloride at 127 mg/kg/day showed values for motility and morphology approaching Control levels. Epididymal sperm numbers were lower than Controls but this difference was not statistically significant. Additionally in males previously treated at 253 mg/kg/day, sperm motility and normal morphology values were low; this was reflected in the motion parameters, however some evidence of recovery was apparent.

Group mean results suggest that recovery was incomplete for males previously treated at 127 mg/kg/day following a 16 week off dose period, though all sperm parameters when compared with controls, with the exception of total sperm in the cauda epididymis, did not attain statistical significance. Examination of individual data, however, showed 4/5 males to exhibit values similar to Controls. Only one male in this group showed low sperm numbers, all of which were immotile and abnormal.
Key result
Dose descriptor:
NOAEL
Effect level:
13 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No change on sperm numbers, morphology or motility or any pathological changes. No indications of an effect on the kidney.
Remarks on result:
other: equivalent to 15 mg cesium formate monohydrate/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
38 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
other: sperm cell number and morphology
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the study conditions, the NOAEL for cesium chloride in rats was considered to be 13 mg/kg bw/day (equivalent to 15 mg cesium formate monohydrate/kg bw/day).
Executive summary:

A study was conducted to assess the systemic toxicity of cesium (Cs) in a 13 week oral gavage study in Han Wistar rats followed by a recovery period of up to 16 weeks, according to OECD 408, in compliance with GLP. This study was designed to investigate the relationship between the toxicity produced by Cs (e.g. uremia, hypokalemia, alkalosis) and male reproductive effects (e.g. decrease in sperm motility, decrease in spermatogenesis).

Four groups received the vehicle or cesium chloride (CsCl) at doses of 13, 38 and 127 mg/kg bw/day for 13 weeks, followed by an 8, 12 or 16 week recovery period. A fifth treated group received cesium chloride (CsCl) at 253 mg/kg bw/day for a reduced treatment period of 59 days because of excessive toxicity, followed by an approximate 4 or 12 week recovery period. During the study, clinical condition, body weight, food consumption, water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, sperm analysis, organ weight, macropathology and histopathology investigations were undertaken.

Three males and three females which received 253 mg/kg bw/day died or were killed prematurely due to poor condition during treatment or the subsequent recovery period. The histopathological examination did not identify any factors contributory to death, but the clinical deterioration of the animals was attributed to treatment with CsCl. In addition, one male receiving the intermediate dose of 127 mg/kg bw/day was killed due to poor condition in Week 13 of treatment. The factor contributory to the death of this animal at microscopic examination was considered to be gastrointestinal tract lesions and was not attributed to treatment due to the isolated incidence.

Treatment was generally well tolerated up to Week 9, though treated animals, particularly at 127 or 253 mg/kg bw/day were generally more irritable and vocal from Week 7. In Week 9 of treatment, animals receiving 253 mg/kg bw/day showed deterioration in condition. Convulsions were observed for two females and one male resulting in these animals dying or being killed for welfare reasons. Other animals receiving this dose level showed signs included hunched posture, partially closed eyelids, piloerection, tremors, dull eyes, rapid or irregular breathing, red staining on the nose and under- or over-activity. The dose of 253 mg/kg bw/day clearly exceeded the maximum tolerated dose and treatment at this dose level was stopped in Week 9.

Dorsal hair loss, red or brown staining around the nose (chromodacryorrhea) and encrustations or skin abrasions, particularly on the lower jaw, head and paws were observed for animals receiving 127 or 253 mg/kg bw/day. During the recovery period, the condition of the animals generally improved, though the deterioration of two males, necessitating premature sacrifice during the recovery period were attributed to previous treatment.

Sensory reactivity responses, grip strength and motor activity were considered to be unaffected by treatment.

Body weight gain and food consumption were low for males and females receiving 253 mg/kg bw/day. Bodyweight gain was also low for males receiving 127 mg/kg bw/day, whilst food consumption was only slightly lower than that of the controls. Recovery was demonstrated after cessation of treatment.

Water consumption was increased for males receiving 38 mg/kg bw/day or above and at all doses in females, without dose-relationship. Recovery was demonstrated after cessation of treatment, but intake remained slightly high in the off-dose period for males which had previously received 253 mg/kg bw/day.

There were no treatment-related ophthalmic findings.

Disturbances in red blood cell indices for animals receiving 127 or 253 mg/kg bw/day (Week 13 and 9 of treatment, respectively) included: low haematocrit, hemoglobin concentration and erythrocyte counts, associated with increased reticulocyte counts; increased red cell distribution width; low mean cell hemoglobin and mean cell hemoglobin concentration. A high mean cell volume was also apparent in males at 253 mg/kg bw/day. The only change at 38 mg/kg bw/day was slightly low hemoglobin concentration in females only. Recovery was demonstrated for all parameters by Week 16 of recovery.

Treatment-related findings in white blood cell parameters for animals receiving 127 or 253 mg/kg bw/day (Week 13 and 9 of treatment, respectively) included: increased total leucocyte counts, associated with increased neutrophil, lymphocyte, eosinophil and monocyte counts. All of these findings showed complete recovery by Week 12 of recovery.

Activated partial thromboplastin times were reduced for both sexes receiving 253 mg/kg bw/day and for males receiving 38 or 127 mg/kg bw/day. Recovery was demonstrated. Prothrombin times were slightly increased in Week 13 of treatment for animals receiving 127 mg/kg bw/day. Prothrombin time was still slightly lower than that of the controls at the end of the recovery period, but the increase was very mild and not of toxicological significance.

Treatment-related biochemical changes in blood plasma included: increased aspartate amino-transferase and creatinine kinase activities for both sexes at 127 or 253 mg/kg bw/day; decreased alanine amino-transferase activity for males receiving 127 mg/kg bw/day; increased urea in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day; increased creatinine concentrations in both sexes at 127 mg/kg bw/day; low glucose concentration in males at 253 mg/kg bw/day; reduced cholesterol concentration in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day, without dose-relationship; increased triglyceride concentration for females at 253 mg/kg bw/day; low phospholipid concentrations for animals of both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day; potassium concentrations were low to very low for both sexes receiving 38, 127 or 253 mg/kg bw/day with a clear dose-relationship; marginally reduced chloride in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day without clear dose-relationship; low magnesium for both sexes at 127 mg/kg bw/day and males receiving 253 or 38 mg/kg bw/day; increased lactate in males at 127 mg/kg bw/day; slightly low bicarbonate in females at 253 mg/kg bw/day and both sexes at 127 mg/kg bw/day. Total protein concentrations were markedly reduced in both sexes at 253 mg/kg bw/day and slightly reduced in males at all other doses; attributed to reductions of both the albumin and globulin fractions. A reduction in albumin-globulin ratio indicated that there was a proportionately greater reduction of albumin levels. In females at 127 mg/kg bw/day there was a slight increase in globulin levels resulting in a decrease in albumin-globulin ratio.

All of these findings showed complete recovery by Week 12 of recovery. Treatment-related changes in urinary parameters in Week 13 of treatment for animals receiving 127 mg/kg bw/day included: high urinary volume, associated with low specific gravity; high urinary sodium, potassium and chloride outputs in both sexes and high magnesium, calcium and creatinine levels in males (the difference in magnesium and creatinine did not attain statistical significance); low glucose output in both sexes and low total protein in males; low urine pH for female; blood in the urine was recorded for 2/10 males and 2/10 females at 127 mg/kg bw/day. Recovery was demonstrated.

Sperm analysis indicated a dose-dependent detrimental effect, predominantly on the epididymis with marked effects observed at the 127 and 253 mg/kg bw/day doses. Treatment for 13 weeks at 127 mg/kg bw/day resulted in most males showing no motile or normal sperm and significant reductions in cauda epididymal sperm numbers compared to Control animals. Additionally, testicular sperm numbers were lower than Controls; this change was not statistically significant. Similar results were seen for sperm motility, morphology and cauda epididymal sperm following 59 days treatment at 253 mg/kg bw/day, however effects on testicular sperm numbers were not apparent. Abnormalities seen at 127 and 253 mg/kg bw/day were mostly decapitate sperm with irregular and/or frayed midpiece and/or tails. A high incidence of flattened head was also apparent. Results suggest an effect on maturation of the sperm with most sperm breaking down within the epididymis. At 38 mg/kg bw/day, slight but statistically significant changes in sperm morphology, motility (increase of beat cross frequency only), and a reduction in sperm number in the cauda epididymis were seen. There were no adverse effects on sperm motility, concentration or morphology effects at 13 mg/kg bw/day. Partial recovery was evident following 12-weeks off dose for animals previously treated at 253 mg/kg bw/day. Following a 16 week off dose period for animals which received 127 mg/kg bw/day, 4/5 males showed complete recovery, whilst one male in this group still showed low sperm numbers, all of which were immotile and abnormal.

Analysis of organ weights for animals killed after 9 or 13 weeks of treatment revealed low epididymides and absolute testes weights at 127 or 253 mg/kg bw/day; increased kidney weights at 127 mg/kg bw/day; decreased liver weights for males at 13, 38 or 127 mg/kg bw/day and both sexes at 253 mg/kg bw/day; increased spleen weights for females at 127 mg/kg bw/day; decreased thymus weights at 127 or 253 mg/kg bw/day; slightly low uterus and cervix weights in females at 127 mg/kg bw/day (without statistical significance) and slightly high adrenal weights for males at 127 mg/kg bw/day (without statistical significance). Recovery was demonstrated.

The macroscopic examination performed after 9 or 13 weeks of treatment revealed: pale adrenals at 127 or 253 mg/kg bw/day; pale areas in the kidney at 253 mg/kg bw/day and males at 127 mg/kg bw/day; scabs and depressions on the skin and subcutis at 127 or 253 mg/kg bw/day; scabs on the forepaws at 253 mg/kg bw/day; darkening of the salivary glands in both sexes at 127 mg/kg bw/day and at 38 and 13 mg/kg bw/day in males; soft testes at 127 mg/kg bw/day and small testes and epididymides at 253 mg/kg bw/day; dark areas; depressions and thickening in the stomach at 253 mg/kg bw/day. Following 12 weeks of recovery, the changes in the mandibular salivary gland, epididymides and testes were still evident in a small number of animals that previously received 253 mg/kg bw/day, whilst all changes at 127 mg/kg bw/day had recovered.

Following 16 weeks of recovery, treatment-related changes in the salivary gland and testes were evident for 1/5 males previously receiving 127 mg/kg bw/day. Histopathological changes related to treatment were seen in the mandibular and sublingual salivary glands (degranulation and atrophy), heart (myocardial degeneration/inflammation), stomach (foveolar/mucosal hyperplasia), spleen (males only - increased extramedullary haemopoiesis), adrenals (diffuse hypertrophy of the zonal glomerulosa), mammary glands (males onlyatrophy), skin and subcutis (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), epididymides (reduced sperm content and cell debris at 127 and 253 mg/kg bw/day and ductal atrophy and sperm granuloma at 127 mg/kg bw/day) and testes (tubular degeneration/atrophy) of animals treated at 253 mg/kg bw/day for 59 days or 127 mg/kg bw/day for 13 weeks. Additional changes were seen in the kidneys (males only, tubular basophilia), skeletal muscles (myofibre degeneration), extremities (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), ovaries (decreased corpora lutea) and uterus (atrophy) of animals treated at 253 mg/kg bw/day.

At 38 mg/kg bw/day, treatment-related changes were seen in the adrenals (diffuse hypertrophy of the zonal glomerulosa) and testes (minimal tubular degeneration/atrophy). No treatment-related change was seen at 13 mg/kg bw/day. Full recovery was demonstrated in the kidneys, stomach, spleen, epididymides and mammary glands. Partial recovery was demonstrated in the salivary glands, heart, adrenals after 8 weeks of recovery and in the testes after 16 weeks of recovery.

In conclusion,oral administration of cesium chloride (CsCl) to Han Wistar rats at doses of 13, 38, 127 or 253 mg/kg bw/day resulted in pathological changes in the mandibular and sublingual salivary glands, heart, stomach, spleen, adrenals, mammary glands, skin and subcutis, epididymides and testes of animals treated at 253 mg/kg bw/day for 59 days or 127 mg/kg bw/day for 13 weeks. Additional changes were seen in the kidneys, skeletal muscles, extremities, ovaries and uterus of animals treated at 253 mg/kg bw/day. At 38 mg/kg bw/day, treatment-related changes were seen in the adrenals and testes. In addition, changes in the blood and urine indicative of an effect on kidney function were apparent at doses of 38 mg/kg bw/day and above; in particular, there was a clear increase in plasma urea and decrease in plasma potassium which was dose-related and marked at the 127 and 253 mg/kg bw/day dose levels. There was a dose-dependent detrimental effect on maturation of the sperm with most sperm breaking down within the epididymis. Treatment at 127 or 253 mg/kg bw/day resulted in most males showing no motile or normal sperm and significant reductions in sperm numbers. There was also a significant reduction in total sperm number in the cauda epididymis at 38 mg/kg bw/day and a slight effect on sperm morphology.

The dose of 253 mg/kg bw/day clearly exceeded the maximum tolerated dose and treatment at this dose level was stopped in Week 9. Recovery from all treatment-related changes was demonstrated, though one male receiving 127 mg/kg bw/day, still showed changes in the testes with associated low sperm numbers, all of which were immotile and abnormal after 16 weeks of recovery. No change on sperm numbers, morphology or motility or any pathological changes were seen at 13 mg/kg /day, nor were there any indications of an effect on the kidney. Consequently, based on these findings, the NOAEL was considered to be 13 mg/kg bw/day (equivalent to 15 mg cesium formate monohydrate/kg bw/day) (Webley, 2016).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
Once taken up by the organism, cesium formate dissociates into the cesium cation (Cs) and the formate anion (HCO2 -). Toxicity is caused by the cesium cation, therefore conducting a study on cesium chloride as a surrogate for cesium formate is justified.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crj:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
The barrier-system animal rooms were maintained at a stable temperature (21 -25°C) and relative
humidity (45-65%) with 10-15 air changes per hour and artificial light-dark cycle of 12-12 hours
(light on: 7:00 and light off: 19:00). The animals were housed in hanging stainless steel cages
with wire-mesh floor (165 Wx300 Dx150 H mm, TOKIWA KAGAKU K.IKAI) individually.
Undertrays were changed twice a week, cages were once a week and racks were every two
weeks. Racks and cages were identified by cards. The animals had free access to an MF
pelleted diet (Oriental Yeast Co., Ltd.) and water (chlorinated) from the Hita City supply via
sipper tubes from automatic waterer. The diet and housing materials were autoclaved at 121°C
for 30 minutes prior to use. Analysis of contaminants in both the diet and drinking water were
confirmed that they would not affect the test results.
No environmental factors which might affect the test results were noted during the quarantine,
acclirnation or study period.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d, with a 14 d recovery period at the end
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 per sex per group
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Before dosing: day -2 (at grouping)
During the dosing period: days 1 (at the start of dosing), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24,
26 and 28
During the recovery period: recovery days 1 (at the start of the recovery period), 3, 5, 8, 10,
12 and 14
In addition, immediately before necropsy, body weights were measured for calculation of
relative organ weights.

FOOD CONSUMPTION:
Before dosing then twice a week.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Except for dead or euthanized animals, all animals were fasted overnight (16 - 20 hours) at the end of the dosing and recovery periods, and blood samples were taken via abdominal aorta under ether anesthesia. Sodium citrate was used as an anticoagulant for prothrombin time and activated partial thromboplastin time, and EDTA-2K was used for other parameters.

CLINICAL CHEMISTRY: Yes
Sera separated from the blood samples, which were used in hematological examinations, were examined. Creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were examined with plama.

URINALYSIS: Yes
Sixteen-hour urine samples were collected from all animals (except for dead or euthanized
animals) by individual metabolic cages at day 28 and recovery day 14, and were examined for volume, calor and additional items such as pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen using a test paper (N-Multistix®, Bayer Yakuhin). Na, K and Cl were measured by PV A -alii.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
The following organs were weighed wet in all animals:
Brain, liver, spleen, kidneys, adrenals, testes (or ovaries)

HISTOPATHOLOGY: Yes
The following organs and tissues were collected from all animals and were preserved in 10 % formalin:
Brain (cerebrum, cerebellum), pituitary gland, eyeballs, thyroid (with parathyroid), heart,
lungs, liver, kidneys, spleen, adrenals, stomach, intestine (duodenum to rectum), testes (or
ovaries), urinary bladder, bone marrow (femur) and gross legions

Following organs and tissues were embedded in paraffm, sectioned, stained with hematoxylin and eosin and histopathologically examined by a light microscope. The liver of the male animal (no. 30) was stained with Oil red 0 and examined.

Statistics:
Data regarding body weights, food consumption, hematological examination, blood chemical examination, urine volume and organ weights were analyzed using Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. When there was a significant difference in this analysis, the differences between the vehicle control group and each treatment group were analyzed by Dunnett's test (equal number of data) or Scheffe's test (unequal number of data). If the variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test was used. When there was a significant difference in this test, the differences between the vehicle control group and each treatment group were analyzed by nonparametric Dunnett's test (equal number of data) or nonparametric Scheffe's test (unequal number of data).
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
Six males and three females in the 1000 mglkg group had died by day 8. Therefore, the other animals (6 males and 9 females) in the same dose group were enthanized on day 8. The body weights and food consumption were calculated statistically in the dead animals and euthanized animals but other parameters were not.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Conclusions:
Based on these results, the NOEL of cesium chloride under the conditions tested was considered to be 40 mg/kg/day.
Executive summary:

A study was conducted to determine the systemic toxicity of cesium chloride in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of six male and five female Cij :CD(SD) strain rats, for up to 28 consecutive days, at dose levels of 0, 8, 40, 200 and 1000 mg/kg bw/day. A 14 d recovery period for the dose groups of 0, 200 and 1000 mg/kg bw/day was also set. Clinical signs, bodyweight development and food consumption were monitored during the study. Haematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.In this study, many animals in the 1000 mg/kg bw/day group died in the dosing period and the other animals seemed not to survive for 28 days. Therefore, all remaining animals in the 1000 mg/kg bw/day group were euthanized. In the 1000 mg/kg bw/day group, salivation, decreased spontaneous locomotion, ptosis, decreased respiratory rate, gasping, hyper sensitivity, tremor, clonic convulsion, tonic convulsion, paleness and lateral position were observed in both sexes, and subnormal temperature was noted in the males. Six males and three females died by day 8 with suppressed body weight gains. In the pathological examinations, dehydration, congestion of the liver, necrosis of the tubules of the kidneys, congestion and edema, hemorrhage and congestion of the lungs were observed in both sexes of the dead animals. Congestion of the lungs, hyperplasia of the mucosal epithelium of the forestomach, congestion of mucosa of the glandular stomach and hyperplasia of the mucosal epithelium of the urine bladder were noted in males. Diffuse lipid droplet of the liver was observed in females. In the euthanized animals, fibrosis of the kidneys was observed in males, and calcium deposition of the kidneys, hemorrhage, hemorrhage and edema of the lungs were in females. Changes which were noted other than the high dose groups are following: Salivation, ptosis, reddish urine, reddish tear, reddish tear mark, staining around nose and mouth, staining lower abdomen and staining around anus were observed in males in the 200 mg/kg bw/day group during the dosing period. There were no abnormalities in body weight or food consumption during the dosing period. Hematological examinations showed no abnormalities at the end of dosing period. In the blood chemical examinations, GOT and blood urea nitrogen were increased and K was decreased in both sexes in the 200 mg/kg group and creatinine was increased in males and chloride was decreased in females in the same group. In the urinalyses, positive for occult blood was increased in males in the 200 mg/kg group at the end of dosing period. In the organ weights, kidney weights were increased in females in the 200 mg/kg bw/day group at the end of the dosing period. In the gross necropsy, enlargement of the kidneys, thickening of the wall of the stomach and calculis and thickening of the wall of the urinary bladder were observed in the males in the 200 mg/kg bw/day group at the end of the dosing period as a change which had a dose-dependency. In the histopathological examinations, following findings were observed in the 200 mg/kg bw/day group had a dose-dependency: male; basophilic change of the tubules, cyst formation, dilatation of the tubules and pelvic dilatation of the kidneys and hyperplasia of the mucosal epithelium of the urinary bladder, female; calcium deposition of the kidneys. In the recovery study, the body weights and food consumption were decreased significantly at the early stage of the recovery period in male in the 200 mg/kg bw/day group, however, these changes tended to recover by the end of the recovery period. The changes of the kidneys and urinary bladder remained at the end of the recovery period, and they became more severe. In males, an increase of white blood cell counts, anemia, and decreases of glucose, total protein, albumin, etc. were noted newly. Based on these results, the NOEL of cesium chloride under the conditions tested was considered to be 40 mg/kg/day (Yamane, 1995).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
Once taken up by the organism, cesium formate dissociates into the cesium cation (Cs) and the formate anion (HCO2 -). Toxicity is caused by the cesium cation, therefore conducting a study on cesium hydroxide as a surrogate for cesium formate is justified.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Hsd.Brl.Han: of Wistar origin
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

Number of animals:
- 20 males, 20 females,
- 5 animals/sex in the control and dose groups

Sex:
Males and nulliparous, non-pregnant females

Age of animals at starting:
- Male animals: 45 – 47 days old
- Female animals: 55 – 60 days old

Body weight range at starting:
- Male animals: 169 – 183 g
- Female animals: 138 – 162 g

Acclimatization time:
5 days

HUSBANDRY
Animal health:
Only healthy animals were used for the test.

Housing:
2 or 3 animals of the sex/ cage

Cage type:
Type II polypropylene/polycarbonate

Bedding:
Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany).

Light:
12 hours daily, from 6.00 a.m. to 6.00 p.m.

Temperature:
22 ± 3 °C

Relative humidity:
30 - 70 %

Ventilation:
8 – 12 air exchanges / hour by central air-condition system.
The temperature and relative humidity was checked and recorded once daily during the study.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 d
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first exposure and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 7, 14, 21 and 27.

FOOD CONSUMPTION :
- Time schedule for examinations: on Days 7, 14, 21 and 27 by reweighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes
Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment.
Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 ml 9NC Microtube, 0.106 mol/L, Sarstedt,), one for hematology (MiniCollect® EDTA tubes, spray-dried, 0.5 ml, Greiner Bio-One International AG), and the third one (VACUETTE® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
treatment. Animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® (details are presented in "Details of Other Materials").
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. The following organs/tissues were preserved in 4 % buffered formaldehyde solution except testes and epididymides, which were fixed in modified Davidson solution.
adrenals
aorta
bone marrow (femur)
brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
eyes (lachrymal gland with Harderian glands)
female mammary gland
gonads (testes with epididymides, ovaries, uterus with vagina)
gross lesions
heart
kidneys
large intestines (cecum, colon, rectum, including Peyer’s patches),
liver
immersion;)
lymph nodes (submandibular and mesenteric)
muscle (quadriceps)
esophagus
pancreas
pituitary
prostate
salivary glands (submandibular)
sciatic nerve
seminal vesicle with coagulating gland
skin
small intestines (representative regions: duodenum, ileum, jejunum)
spinal cord (at three levels: cervical, mid-thoracic and lumbar)
spleen
sternum
stomach
thymus
thyroid + parathyroid
trachea
urinary bladder

ORGAN WEIGHT
The following organ weights were determined and recorded:
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands;
With precision of 0.001g: adrenals, ovaries
Paired organs were measured together.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (group 1) and high dose (group 4) groups.
In groups 2 (25 mg/kg bw/day), and 3 (125 mg/kg bw/day), kidneys were also processed histologically in accordance with macroscopic observations for animal no. 424, (male) and animal no. 422 (male), respectively.
Statistics:
following data:
- body weight
- food consumption
- hematology
- clinical chemistry
- organ weight data
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in either group in the course of the entire observation period. The general physical condition and behavior of animals were considered to be normal throughout the entire observation period.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in the control, 25, 125 and 250 mg/kg bw/day groups during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day
In the male animals, the body weight gain was less than in the control group during the entire observation period with statistical significances on weeks 1, 2 and 4 (p<0.01, p<0.05), thus the total body weight gain remained below control value (p<0.01), too. The reduced body weight gain resulted in a lower body weight values on days 14, 21 and 27 with respect to controls (p<0.01, p<0.05).
In the female animals, the mean body weight gain was significantly less than in the control group between days 14 and 21 (p<0.01), however the body weight values and total body weight gain was comparable to the appropriate control value.
125 mg/kg bw/day
In the male animals, statistical significances were noted for a lower body weight gain with respect to the control on weeks 2 and 4 (p<0.05, p<0.01) and for the total body weight gain (p<0.05). The body weight was below the control value with statistical significance on day 27 (p<0.05).
In the female animals, there were no significant differences in mean body weight and body weight gain compared to the control value.
25 mg/kg bw/day
The mean body weight and body weight gain were similar to those of the control group in male animals throughout the treatment period.
In the female animals, the body weight gain remained below the control value (p< 0.05) on week 3.
In summary, Cesiumhydroxidmonohydrat 99.95 caused a reduction in body weight gain in male animals at 250 mg/kg bw/day and at 125 mg/kg bw/day doses resulting in a lower body weight at 250 mg/kg bw/day from day 14 up to the end of the treatment period and at 125 mg/kg bw/day on day 27.
The transiently lower body weight gain of female animals dosed with 25 and 250 mg/kg bw/day with respect to control was not considered toxicologically relevant as this did not induce changes in body weight and in total body weight gain and values were comparable with historical control data.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistical significances were noted for less mean daily food consumption at 250 mg/kg bw/day in male animals on week 1 and in female animals on week 3.
At 25 mg/kg bw/day, the mean daily food intake of female animals was less than in the control during weeks 1, 2 and 3.
In summary, the statistical significances indicated only slight differences between the control and test item treated groups and were not considered to be biologically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal test item related changes in the examined parameters.
Statistically significant differences were only noted for female animals with respect to controls in percent of eosinophil cells (EOS) and mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 250 mg/kg bw/day, in prothrombin time (PT) and activated partial thromboplastin time (APTT) at 125 mg/kg bw/day and in percent of eosinophil cells at 25 mg/kg bw/day.
All these values were with low degree and were within the historical control ranges (except MCHC) and in the lack of a dose-response relationship and any pathological changes these were not considered to be toxicologically significant. Mean values of MCHC were higher than the upper limit of historical control value in all groups including control. The slight difference in MCHC (2 %) between the control and 250 mg/kg bw/day had no biological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day
In the male animals, the activity of aspartate aminotransferase (AST) and albumin-globulin ratio (A/G) slightly exceeded the control value, while activity of alanine aminotransferase (ALT) and concentration of potassium (K+), chloride (Cl-), albumin (ALB) and total protein (TPROT) were less than in the control group.
In the female animals, an elevated level of creatinine (CREA) was observed. The mean activity of alanine aminotransferase and concentration of potassium, chloride as well as A/G ratio were less than in the control group.
125 mg/kg bw/day
In the male animals, statistical significances were noted for the lower activity of alanine aminotransferase and higher albumin-globulin ratio with respect to the controls.
In the female animals, the potassium and chloride concentrations and A/G ratio were below the control value.
25 mg/kg bw/day
There were no statistically significant differences in the examined clinical chemistry parameters between the control and test item treated groups (male and female animals).
In summary, the slightly elevated creatinine concentration in female animals treated with 250 mg/kg bw/day together with the slight changes in kidney weights might be indicative of a test item influence on the renal function. However the values were marginal to the historic control range and there were no related histopathological changes to substantiate their relevance. The lower potassium levels at 250 and 125 mg/kg bw/day might be due to the chemically similar structure of cesium and potassium. Potassium wasting without damage in renal tissue might refer to an altered renal function. Changes in creatinine and potassium levels in serum were considered to be signs of adaptation of the organ to the altered demands. Statistically significantly higher AST activity in male animals treated with 250 mg/kg bw/day remained well within the range of historical control and without supporting histopathological changes was judged to be toxicologically not significant.
Sporadic statistical differences (ALT, Cl-, ALB, TPROT and A/G) were considered to be of little or no biological significance. All values were within the historical control ranges except ALT however a decrease in enzyme activity of ALT has no biological significance in the lack of any related histological changes. A/G values changed in an opposite direction in male and female animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Positional struggle in a marked degree was found in the control (1/5, male), in 125 mg/kg bw/day (1/5, male) and in 250 mg/kg bw/day (1/5, male) group. The equilibrium test was negative in one female control (1/5) and in one (1/5) 250 mg/kg bw/day treated animal.
Functional battery observation determinations demonstrated no treatment-related differences with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period. Incidences and extent of reactions observed in groups of treated animals were comparable to those observed in control animals, and are, thus, without any toxicological significance. Variations in equilibrium test were within the normal biological variation, with respect to behavior, reactions to different type of stimuli or manipulations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
250 mg/kg bw/day
The absolute organ weights and organ weights relative to the body weight were not relevant for evaluation because of the lower mean body weight of male animals. Body weight relative to brain weight and organ weights relative to brain weight were less than in the control group in case of liver, heart and thymus.
In the female animals, kidney weights relative to body and brain weights were slightly higher than in the control group. (p<0.01 and p<0.05, respectively).
125 mg/kg bw/day
In the male animals, the body weight, weight of heart (absolute) and thymus (absolute and relative to the body and brain weights) and body weight relative to brain weight were slightly below the control value.
In the female animals, weights of all examined organs were similar to the control value.
25 mg/kg bw/day
No differences in the examined organ weights were noted with respect to controls in male and female animals.
In summary, evaluation of absolute and relative organ weights did not reveal clear evidence of test item related alterations. Slight but statistically significant changes in the kidney weights of female animals might be related to a test item influence on renal function taking into account the changes in creatinine and potassium levels. However the degree of all of these changes was small and there were no supporting histopathological findings therefore it was considered to be indication of the adaptation process and the toxicological significance is equivocal.
Changes in thymus weights relative to brain weight were not dose dependent in male animals, but a significant reduction (-32 % and -28 %, respectively) may be indicative of a test item influence at 250 and 125 mg/kg bw/day. There were no related changes in other organs and tissues of immune system and histopathological examination revealed a normal involution process of thymus therefore changes in thymus weights were not considered to be toxicologically significant.
Statistical significances noted for some other organs (liver and heart) were with small degree and were not dose dependent and were not associated with any clinical pathological or histopathological findings therefore were not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
250 mg/kg bw/day
Minimal alveolar emphysema (2/5 male, 1/5 female), minimal acute hemorrhage (1/5 male) and mild hyperplasia of bronchus associated lymphoid tissue (BALT; 1/5 male, 1/5 female) were observed in the lungs.
125mg/kg bw/day
No microscopic alterations were found in the kidneys of single male animals processed histologically.
25 mg/kg bw/day
In the kidney of male animal processed histologically, unilateral pyelectasia was noted.
Control group
Minimal or mild alveolar emphysema (2/5 male, 2/5 female), minimal acute hemorrhage (1/5 female) and mild hyperplasia of bronchus associated lymphoid tissue (1/5 male, 2/5 female) were observed in the lungs.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system was observed.
The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.
In summary, toxic or other test item related lesions detectable by histological examination of investigated organs were not found in the experimental animals.
The alveolar emphysema (minimal or mild degree) and minimal acute hemorrhage in the lungs occurred with similar incidence in the control and dose group and were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The sporadic hyperplasia of bronchus associated lymphoid tissue (BALT) without inflammatory lesions was considered as physiologic phenomenon.
The slight pyelectasia (one side) without degeneration, inflammation or fibrosis is a common finding in experimental rats, as individual disorder.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
Conclusions:
Under the study conditions, the test substance caused a slight depression in the body weight development at 250 and 125 mg/kg bw/day and in serum potassium levels at 250 mg/kg bw/day in male Hsd.Brl.Han: Wistar rats during the 28 d oral (by gavage) administration. In female animals, changes in serum potassium levels at 250 and 125 mg/kg bw/day, serum creatinine concentration and in kidney weights relative to body and brain weights at 250 mg/kg bw/day were indicative of a possible test substance influence however without any structural (histopathologic) alterations. Based on these observations, the 28 d NOAEL was determined to be 125 mg/kg bw/day for male and female animals (Szakonyiné, 2011).
Executive summary:

A study was conducted to determine the systemic toxicity of cesium hydroxide monohydrate 99.95 in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of five male and five female Hsd.Brl.Han strain rats, for up to 28 consecutive days, at dose levels of 0, 25, 125 and 250 mg/kg bw/day. Stability and concentration of test substance in distilled water were confirmed analytically beforehand. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of the study. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including hematology, and clinical chemistry were conducted at the termination of the treatment. Gross pathology was performed on all animals on the day following the last treatment. The absolute and relative organ weights of selected organs were determined. A full histopathology examination was performed on the preserved organs and tissues of the animals of control and high dose groups and on the kidneys with macroscopic findings of single male animals in the low and middle dose group.No mortality occurred during the observation period. Toxic signs related to the test substance were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery. The body weight gain was statistically significantly reduced at 250 mg/kg bw/day and at 125 mg/kg bw/day doses in male animals only resulting in a slightly lower body weight compared to control group at 250 mg/kg bw/day from Day 14 up to the end of the treatment period and at 125 mg/kg bw/day on Day 27. No significant differences were observed in the mean daily food consumption between the control and test substance treated groups (male and female) during the entire observation period. There were no test substance related alterations in the examined hematology and blood coagulation parameters. Clinical chemistry evaluation revealed test substance related changes in serum potassium levels at 250 mg/kg bw/day in male and female animals and at 125 mg/kg bw/day in females and in creatinine concentration at 250 mg/kg bw/day in female animals. Specific alterations related to treatment were not found during the terminal necropsy and at the histopathological investigations at 250 mg/kg bw/day dose. Slight, but statistically significant differences with respect to controls in kidney weights relative to body and brain weight in female animals dosed with 250 mg/kg bw/day might be indicative of a test substance influence on the renal function however values remained within the historical control ranges and were without any histopathological changes. Under the study conditions, the test substancecaused a slight depression in the body weight development at 250 and 125 mg/kg bw/day and in serum potassium levels at 250 mg/kg bw/day in male Hsd.Brl.Han: Wistar rats during the 28 d oral (by gavage) administration. In female animals, changes in serum potassium levels at 250 and 125 mg/kg bw/day, serum creatinine concentration and in kidney weights relative to body and brain weights at 250 mg/kg bw/day were indicative of a possible test substance influence however without any structural (histopathologic) alterations. Based on these observations, the 28 d NOAEL was determined to be 125 mg/kg bw/day for male and female animals (Szakonyiné, 2011).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
Justification for type of information: Once taken up by the organism, cesium formate dissociates into the cesium cation (Cs) and the formate anion (HCO2 -). Toxicity is caused by the cesium cation, therefore conducting a study on cesium hydroxide as a surrogate for cesium formate is justified.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Hsd.Brl.Han: Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sufficient stability of Cesiumhydroxidmonohydrat 99.95 in the chosen vehicle was analytically verified up front. Cesiumhydroxidmonohydrat 99.95 was stable for 7 days at room temperature and in a refrigerator. Concentrations of the test item in the dosing formulations varied from 95 % to 97 % of nominal concentrations at all analytical occasions, thereby confirming proper dosing.
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


FOOD CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No r:

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes

HISTOPATHOLOGY: Yes
Other examinations:
ESTROUS CYCLE: Yes

SPERM ANALYSIS: Yes
Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ESTROUS CYCLE: inconclusive

SPERM ANALYSIS: treatment-related effects
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
other: sperm mobility and motility
Conclusions:
Under the study conditions, the NOAEL for cesium hydroxide monohydrate was determined to be 25 mg/kg bw/day for male and female animals.
Executive summary:

A 90 d toxicity study was conducted to determine the toxicity of cesium hydroxide monohydrate in rats at three dose levels following 90 d oral administration. The study was performed according to OECD Guideline 408, EU Method B.26 and EPA Guideline OPPTS 870.3100, in compliance with GLP. The test substance was administered orally (by gavage) to Hsd. Brl. Han: Wistar rats (n=10 animals/sex/group) once a day at 0 (vehicle control), 250, 125 and 25 mg/kg bw/day. A sufficient stability of cesium hydroxide monohydrate in the chosen vehicle was analytically verified up front. Cesium hydroxide monohydrate was stable for 7 days at room temperature and in a refrigerator. Concentrations of the test substance in the dosing formulations varied from 95 to 97% of nominal concentrations at all analytical occasions, thereby confirming proper dosing. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including haematology and clinical chemistry were conducted one day after the last treatment. Gross pathology was performed on animals on the day following the last treatment. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups. Testes epididymides, prostate and seminal vesicles with coagulating gland were also evaluated histologically in all animals of the low and mid dose groups due to the necropsy and histopathological findings in these organs. The results of study were interpreted comparing test substance treated groups with respect to controls, which were administered concurrently with vehicle (distilled water) only.

There was no test substance related mortality. One male and one female animal from the high dose group (250 mg/kg bw/day) died in the course of the study. In the female animal, clinical signs (irritability, piloerection, decreased activity and dyspnea) and severe body weight loss were observed before death. Histological examination revealed serious involution of thymus and signs of suffocation indicative of an individual metabolic disorder. The male animal died due to over anesthesia at the blood sampling at the termination of the study. Toxic signs related to the test substance were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery. A test substance related depression of the body weight development was detected in male animals at 250 and at 125 mg/kg bw/day during the entire study. The less body weight gain resulted in a less body weight compared to control group at 250 mg/kg bw/day from Day 21 and at 125 mg/kg bw/day from Day 35 up to the end of the treatment period. The daily mean food consumption was reduced in male animals at 250 mg/kg bw/day during the entire observation period. There were no abnormalities in the eyes of animals in the control and high dose groups at termination of the treatment. A test substance influence on the estrous cycle cannot be excluded with the results of this study. None of high dose (250 mg/kg bw/day) treated animal had regular cycle however the number of control animals with regular cycle was also significantly less than in the historical control data. There was no significant difference in the number of animals with an irregular estrous cycle in the low dose group as compared to the control group. A slightly higher mean white blood cell count and higher mean percentage of reticulocytes were detected in male and female animals at 250 and 125 mg/kg bw/day with respect to control group. Blood coagulation. There were no test substance induced pathologic changes in the examined blood coagulation parameters. Clinical chemistry evaluation revealed an elevated activity of aspartate aminotransferase at 250 and 125 mg/kg bw/day (male and female), as well as higher concentrations of urea, creatinine and less mean glucose concentration at 250 mg/kg bw/day (male and female) and at 125 mg/kg bw/day (male). These changes were indicative of the altered metabolic processes as histopathological examinations did not point out a damage in the relevant organs or tissues. The testes and epididymides were judged to be smaller than normal in animals of 250 mg/kg bw/day group at the necropsy observations. Significant reduction of the mean testes weights at 250 mg/kg bw/day and in epididymides weights at 250 and 125 mg/kg bw/day reflected a test substance related influence. The significantly less liver weights in male animals administered with 250 or 125 mg/kg bw/day probably were in accordance with the body weight alteration. Slightly higher kidneys weights at 250 mg/kg bw/day (male and female), and at 125 mg/kg bw/day (female) with respect to controls were considered to be signs of adaptation of the organ to the altered demands. The mean thymus weight (absolute and relative to brain weight) in the male animals) and the absolute mean thymus weight in the female animals were less than the control in animals administered with 250 mg/kg bw/day. Sperm analysis revealed lack of the sperm cells at 250 mg/kg bw/day and damage of the motility and morphology of the sperm cells at 125 mg/kg bw/day dose. Histological examination detected decreased intensity of spermatogenesis, which was accompanied with lack of mature spermatozoa in the seminiferous tubuli in the testes and in the ductuli of epididymides, and decreased number of spermatids in a proportion of seminiferous tubuli in 250 mg/kg bw/day dose treated male animals.

Under the conditions of the present study, the 250 mg/kg bw/day dose of the test substance caused reduced body weight, body weight gain, and reduced food consumption (male), and changes in hematology parameters (white blood cell count and percentage of reticulocyte – male and female). Damage in spermatogenesis (smaller than normal testes, reduced weights of testes and epididymides, decreased intensity of spermatogenesis, accompanied with lack of mature spermatozoa in the seminiferous tubuli in the testes and in the ductuli of epididymides, and decreased number of spermatids in a proportion of seminiferous tubuli) after the 90 d oral (gavage) administration in Hsd. Brl. Han: Wistar rats was observed. These test substance-related changes were considered to be toxicologically relevant. A test substance influence on the estrous cycle could not be excluded as none of the female animals in the dose group of 250 mg/kg bw/day had a regular estrous cycle. At this dose level, slight changes in thymus weights were noted in male and female animals, which were associated with an accelerated involution process as compared with the controls. The toxicological significance of this finding was considered to be equivocal. At 125 mg/kg bw/day, depression of the body weight development (male), altered clinical pathology parameters (slightly higher mean white blood cell count and higher mean percentage of reticulocytes (male and female), reduced weight of epididymides and damage of sperm motility and morphology) were indicative of adverse test substance related effects. At 25 mg/kg bw/day, there were no test substance related toxic alterations. Based on these observations, the NOAEL for cesium hydroxide monohydrate was determined to be 25 mg/kg bw/day for male and female animals (TOXICOOP, 2012).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality dataset

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

28 day exposure

Cesium formate

A study was conducted to determine the systemic toxicity of the test substance in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to 28 consecutive days, at dose levels of 0, 15, 150 and 500 mg/kg/day (incorporating a correction factor for 78.94% purity). Stability and homogeneity of the test substance solutions in distilled water were determined by ion chromatography. Two recovery groups, each of five males and five females, were treated with the high dose (500 mg/kg/day) or the vehicle alone for up to 28 consecutive days and then maintained without treatment for a further fourteen days. Due to deaths encountered at the highest dose level, all females dosed with 500 mg/kg/day were treated for 25 days only. Non-recovery females and males were terminated on day 28. Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.The test substance treatment caused effects at all dose levels in the Sprague-Dawley rats. The target organs were considered predominantly to be heart, liver, spleen, central nervous system, skeletal muscles and reproductive organs changes in serum biochemistry were noted at high doses. Reticulocyte counts were increased at all doses.Under the study conditions the oral administration of the test substance by gavage to rats for a period of up to 28 d at 15, 150 and 500 mg/kg/day (incorporating a correction factor for a purity of 78.94%) resulted in treatment-related effects at all doses. The results indicate that the prepared formulations were within ±6% of nominal values. Effects detected at 15 mg/kg/day were not considered to be adverse. Therefore 15 mg/kg/day was selected as the NOAEL for this study (Dhinsa, 2008).

Cesium chloride

A study was conducted to determine the systemic toxicity of cesium chloride in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of six male and five female Cij :CD(SD) strain rats, for up to 28 consecutive days, at dose levels of 0, 8, 40, 200 and 1000 mg/kg bw/day. A 14 d recovery period for the dose groups of 0, 200 and 1000 mg/kg bw/day was also set. Clinical signs, bodyweight development and food consumption were monitored during the study. Haematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.In this study, many animals in the 1000 mg/kg bw/day group died in the dosing period and the other animals seemed not to survive for 28 days. Therefore, all remaining animals in the 1000 mg/kg bw/day group were euthanized. In the 1000 mg/kg bw/day group, salivation, decreased spontaneous locomotion, ptosis, decreased respiratory rate, gasping, hyper sensitivity, tremor, clonic convulsion, tonic convulsion, paleness and lateral position were observed in both sexes, and subnormal temperature was noted in the males. Six males and three females died by day 8 with suppressed body weight gains. In the pathological examinations, dehydration, congestion of the liver, necrosis of the tubules of the kidneys, congestion and edema, hemorrhage and congestion of the lungs were observed in both sexes of the dead animals. Congestion of the lungs, hyperplasia of the mucosal epithelium of the forestomach, congestion of mucosa of the glandular stomach and hyperplasia of the mucosal epithelium of the urine bladder were noted in males. Diffuse lipid droplet of the liver was observed in females. In the euthanized animals, fibrosis of the kidneys was observed in males, and calcium deposition of the kidneys, hemorrhage, hemorrhage and edema of the lungs were in females. Changes which were noted other than the high dose groups are following: Salivation, ptosis, reddish urine, reddish tear, reddish tear mark, staining around nose and mouth, staining lower abdomen and staining around anus were observed in males in the 200 mg/kg bw/day group during the dosing period. There were no abnormalities in body weight or food consumption during the dosing period. Hematological examinations showed no abnormalities at the end of dosing period. In the blood chemical examinations, GOT and blood urea nitrogen were increased and K was decreased in both sexes in the 200 mg/kg group and creatinine was increased in males and chloride was decreased in females in the same group. In the urinalyses, positive for occult blood was increased in males in the 200 mg/kg group at the end of dosing period. In the organ weights, kidney weights were increased in females in the 200 mg/kg bw/day group at the end of the dosing period. In the gross necropsy, enlargement of the kidneys, thickening of the wall of the stomach and calculis and thickening of the wall of the urinary bladder were observed in the males in the 200 mg/kg bw/day group at the end of the dosing period as a change which had a dose-dependency. In the histopathological examinations, following findings were observed in the 200 mg/kg bw/day group had a dose-dependency: male; basophilic change of the tubules, cyst formation, dilatation of the tubules and pelvic dilatation of the kidneys and hyperplasia of the mucosal epithelium of the urinary bladder, female; calcium deposition of the kidneys. In the recovery study, the body weights and food consumption were decreased significantly at the early stage of the recovery period in male in the 200 mg/kg bw/day group, however, these changes tended to recover by the end of the recovery period. The changes of the kidneys and urinary bladder remained at the end of the recovery period, and they became more severe. In males, an increase of white blood cell counts, anemia, and decreases of glucose, total protein, albumin, etc. were noted newly. Based on these results, the NOEL of cesium chloride under the conditions tested was considered to be 40 mg/kg/day (Yamane, 1995).

Cesium hydroxide

A study was conducted to determine the systemic toxicity of cesium hydroxide monohydrate 99.95 in rat according to OECD Guideline 407, in compliance with GLP. The test substance was administered by gavage to three groups, each of five male and five female Hsd.Brl.Han strain rats, for up to 28 consecutive days, at dose levels of 0, 25, 125 and 250 mg/kg bw/day. Stability and concentration of test substance in distilled water were confirmed analytically beforehand. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of the study. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including hematology, and clinical chemistry were conducted at the termination of the treatment. Gross pathology was performed on all animals on the day following the last treatment. The absolute and relative organ weights of selected organs were determined. A full histopathology examination was performed on the preserved organs and tissues of the animals of control and high dose groups and on the kidneys with macroscopic findings of single male animals in the low and middle dose group.

No mortality occurred during the observation period. Toxic signs related to the test substance were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery. The body weight gain was statistically significantly reduced at 250 mg/kg bw/day and at 125 mg/kg bw/day doses in male animals only resulting in a slightly lower body weight compared to control group at 250 mg/kg bw/day from Day 14 up to the end of the treatment period and at 125 mg/kg bw/day on Day 27. No significant differences were observed in the mean daily food consumption between the control and test substance treated groups (male and female) during the entire observation period. There were no test substance related alterations in the examined hematology and blood coagulation parameters. Clinical chemistry evaluation revealed test substance related changes in serum potassium levels at 250 mg/kg bw/day in male and female animals and at 125 mg/kg bw/day in females and in creatinine concentration at 250 mg/kg bw/day in female animals. Specific alterations related to treatment were not found during the terminal necropsy and at the histopathological investigations at 250 mg/kg bw/day dose. Slight, but statistically significant differences with respect to controls in kidney weights relative to body and brain weight in female animals dosed with 250 mg/kg bw/day might be indicative of a test substance influence on the renal function however values remained within the historical control ranges and were without any histopathological changes. Under the study conditions, the test substancecaused a slight depression in the body weight development at 250 and 125 mg/kg bw/day and in serum potassium levels at 250 mg/kg bw/day in male Hsd.Brl.Han: Wistar rats during the 28 d oral (by gavage) administration. In female animals, changes in serum potassium levels at 250 and 125 mg/kg bw/day, serum creatinine concentration and in kidney weights relative to body and brain weights at 250 mg/kg bw/day were indicative of a possible test substance influence however without any structural (histopathologic) alterations. Based on these observations, the 28 d NOAEL was determined to be 125 mg/kg bw/day for male and female animals (Szakonyiné, 2011).

 

90 day exposure

Cesium chloride

A study was conducted to assess the systemic toxicity of cesium (Cs) in a 13 week oral gavage study in Han Wistar rats followed by a recovery period of up to 16 weeks, according to OECD 408, in compliance with GLP. This study was designed to investigate the relationship between the toxicity produced by Cs (e.g. uremia, hypokalemia, alkalosis) and male reproductive effects (e.g. decrease in sperm motility, decrease in spermatogenesis).

Four groups received the vehicle or cesium chloride (CsCl) at doses of 13, 38 and 127 mg/kg bw/day for 13 weeks, followed by an 8, 12 or 16 week recovery period. A fifth treated group received cesium chloride (CsCl) at 253 mg/kg bw/day for a reduced treatment period of 59 days because of excessive toxicity, followed by an approximate 4 or 12 week recovery period. During the study, clinical condition, body weight, food consumption, water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, sperm analysis, organ weight, macropathology and histopathology investigations were undertaken.

Three males and three females which received 253 mg/kg bw/day died or were killed prematurely due to poor condition during treatment or the subsequent recovery period. The histopathological examination did not identify any factors contributory to death, but the clinical deterioration of the animals was attributed to treatment with CsCl. In addition, one male receiving the intermediate dose of 127 mg/kg bw/day was killed due to poor condition in Week 13 of treatment. The factor contributory to the death of this animal at microscopic examination was considered to be gastrointestinal tract lesions and was not attributed to treatment due to the isolated incidence.

Treatment was generally well tolerated up to Week 9, though treated animals, particularly at 127 or 253 mg/kg bw/day were generally more irritable and vocal from Week 7. In Week 9 of treatment, animals receiving 253 mg/kg bw/day showed deterioration in condition. Convulsions were observed for two females and one male resulting in these animals dying or being killed for welfare reasons. Other animals receiving this dose level showed signs included hunched posture, partially closed eyelids, piloerection, tremors, dull eyes, rapid or irregular breathing, red staining on the nose and under- or over-activity. The dose of 253 mg/kg bw/day clearly exceeded the maximum tolerated dose and treatment at this dose level was stopped in Week 9.

Dorsal hair loss, red or brown staining around the nose (chromodacryorrhea) and encrustations or skin abrasions, particularly on the lower jaw, head and paws were observed for animals receiving 127 or 253 mg/kg bw/day. During the recovery period, the condition of the animals generally improved, though the deterioration of two males, necessitating premature sacrifice during the recovery period were attributed to previous treatment.

Sensory reactivity responses, grip strength and motor activity were considered to be unaffected by treatment.

Body weight gain and food consumption were low for males and females receiving 253 mg/kg bw/day. Bodyweight gain was also low for males receiving 127 mg/kg bw/day, whilst food consumption was only slightly lower than that of the controls. Recovery was demonstrated after cessation of treatment.

Water consumption was increased for males receiving 38 mg/kg bw/day or above and at all doses in females, without dose-relationship. Recovery was demonstrated after cessation of treatment, but intake remained slightly high in the off-dose period for males which had previously received 253 mg/kg bw/day.

There were no treatment-related ophthalmic findings.

Disturbances in red blood cell indices for animals receiving 127 or 253 mg/kg bw/day (Week 13 and 9 of treatment, respectively) included: low haematocrit, hemoglobin concentration and erythrocyte counts, associated with increased reticulocyte counts; increased red cell distribution width; low mean cell hemoglobin and mean cell hemoglobin concentration. A high mean cell volume was also apparent in males at 253 mg/kg bw/day. The only change at 38 mg/kg bw/day was slightly low hemoglobin concentration in females only. Recovery was demonstrated for all parameters by Week 16 of recovery.

Treatment-related findings in white blood cell parameters for animals receiving 127 or 253 mg/kg bw/day (Week 13 and 9 of treatment, respectively) included: increased total leucocyte counts, associated with increased neutrophil, lymphocyte, eosinophil and monocyte counts. All of these findings showed complete recovery by Week 12 of recovery.

Activated partial thromboplastin times were reduced for both sexes receiving 253 mg/kg bw/day and for males receiving 38 or 127 mg/kg bw/day. Recovery was demonstrated. Prothrombin times were slightly increased in Week 13 of treatment for animals receiving 127 mg/kg bw/day. Prothrombin time was still slightly lower than that of the controls at the end of the recovery period, but the increase was very mild and not of toxicological significance.

Treatment-related biochemical changes in blood plasma included: increased aspartate amino-transferase and creatinine kinase activities for both sexes at 127 or 253 mg/kg bw/day; decreased alanine amino-transferase activity for males receiving 127 mg/kg bw/day; increased urea in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day; increased creatinine concentrations in both sexes at 127 mg/kg bw/day; low glucose concentration in males at 253 mg/kg bw/day; reduced cholesterol concentration in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day, without dose-relationship; increased triglyceride concentration for females at 253 mg/kg bw/day; low phospholipid concentrations for animals of both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day; potassium concentrations were low to very low for both sexes receiving 38, 127 or 253 mg/kg bw/day with a clear dose-relationship; marginally reduced chloride in both sexes at 127 or 253 mg/kg bw/day and males at 38 mg/kg bw/day without clear dose-relationship; low magnesium for both sexes at 127 mg/kg bw/day and males receiving 253 or 38 mg/kg bw/day; increased lactate in males at 127 mg/kg bw/day; slightly low bicarbonate in females at 253 mg/kg bw/day and both sexes at 127 mg/kg bw/day. Total protein concentrations were markedly reduced in both sexes at 253 mg/kg bw/day and slightly reduced in males at all other doses; attributed to reductions of both the albumin and globulin fractions. A reduction in albumin-globulin ratio indicated that there was a proportionately greater reduction of albumin levels. In females at 127 mg/kg bw/day there was a slight increase in globulin levels resulting in a decrease in albumin-globulin ratio.

All of these findings showed complete recovery by Week 12 of recovery. Treatment-related changes in urinary parameters in Week 13 of treatment for animals receiving 127 mg/kg bw/day included: high urinary volume, associated with low specific gravity; high urinary sodium, potassium and chloride outputs in both sexes and high magnesium, calcium and creatinine levels in males (the difference in magnesium and creatinine did not attain statistical significance); low glucose output in both sexes and low total protein in males; low urine pH for female; blood in the urine was recorded for 2/10 males and 2/10 females at 127 mg/kg bw/day. Recovery was demonstrated.

Sperm analysis indicated a dose-dependent detrimental effect, predominantly on the epididymis with marked effects observed at the 127 and 253 mg/kg bw/day doses. Treatment for 13 weeks at 127 mg/kg bw/day resulted in most males showing no motile or normal sperm and significant reductions in cauda epididymal sperm numbers compared to Control animals. Additionally, testicular sperm numbers were lower than Controls; this change was not statistically significant. Similar results were seen for sperm motility, morphology and cauda epididymal sperm following 59 days treatment at 253 mg/kg bw/day, however effects on testicular sperm numbers were not apparent. Abnormalities seen at 127 and 253 mg/kg bw/day were mostly decapitate sperm with irregular and/or frayed midpiece and/or tails. A high incidence of flattened head was also apparent. Results suggest an effect on maturation of the sperm with most sperm breaking down within the epididymis. At 38 mg/kg bw/day, slight but statistically significant changes in sperm morphology, motility (increase of beat cross frequency only), and a reduction in sperm number in the cauda epididymis were seen. There were no adverse effects on sperm motility, concentration or morphology effects at 13 mg/kg bw/day. Partial recovery was evident following 12-weeks off dose for animals previously treated at 253 mg/kg bw/day. Following a 16 week off dose period for animals which received 127 mg/kg bw/day, 4/5 males showed complete recovery, whilst one male in this group still showed low sperm numbers, all of which were immotile and abnormal.

Analysis of organ weights for animals killed after 9 or 13 weeks of treatment revealed low epididymides and absolute testes weights at 127 or 253 mg/kg bw/day; increased kidney weights at 127 mg/kg bw/day; decreased liver weights for males at 13, 38 or 127 mg/kg bw/day and both sexes at 253 mg/kg bw/day; increased spleen weights for females at 127 mg/kg bw/day; decreased thymus weights at 127 or 253 mg/kg bw/day; slightly low uterus and cervix weights in females at 127 mg/kg bw/day (without statistical significance) and slightly high adrenal weights for males at 127 mg/kg bw/day (without statistical significance). Recovery was demonstrated.

The macroscopic examination performed after 9 or 13 weeks of treatment revealed: pale adrenals at 127 or 253 mg/kg bw/day; pale areas in the kidney at 253 mg/kg bw/day and males at 127 mg/kg bw/day; scabs and depressions on the skin and subcutis at 127 or 253 mg/kg bw/day; scabs on the forepaws at 253 mg/kg bw/day; darkening of the salivary glands in both sexes at 127 mg/kg bw/day and at 38 and 13 mg/kg bw/day in males; soft testes at 127 mg/kg bw/day and small testes and epididymides at 253 mg/kg bw/day; dark areas; depressions and thickening in the stomach at 253 mg/kg bw/day. Following 12 weeks of recovery, the changes in the mandibular salivary gland, epididymides and testes were still evident in a small number of animals that previously received 253 mg/kg bw/day, whilst all changes at 127 mg/kg bw/day had recovered.

Following 16 weeks of recovery, treatment-related changes in the salivary gland and testes were evident for 1/5 males previously receiving 127 mg/kg bw/day. Histopathological changes related to treatment were seen in the mandibular and sublingual salivary glands (degranulation and atrophy), heart (myocardial degeneration/inflammation), stomach (foveolar/mucosal hyperplasia), spleen (males only - increased extramedullary haemopoiesis), adrenals (diffuse hypertrophy of the zonal glomerulosa), mammary glands (males only–atrophy), skin and subcutis (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), epididymides (reduced sperm content and cell debris at 127 and 253 mg/kg bw/day and ductal atrophy and sperm granuloma at 127 mg/kg bw/day) and testes (tubular degeneration/atrophy) of animals treated at 253 mg/kg bw/day for 59 days or 127 mg/kg bw/day for 13 weeks. Additional changes were seen in the kidneys (males only, tubular basophilia), skeletal muscles (myofibre degeneration), extremities (epidermal scabs/ulceration and hyperplasia, and dermal/ subcutaneous inflammation), ovaries (decreased corpora lutea) and uterus (atrophy) of animals treated at 253 mg/kg bw/day.

At 38 mg/kg bw/day, treatment-related changes were seen in the adrenals (diffuse hypertrophy of the zonal glomerulosa) and testes (minimal tubular degeneration/atrophy). No treatment-related change was seen at 13 mg/kg bw/day. Full recovery was demonstrated in the kidneys, stomach, spleen, epididymides and mammary glands. Partial recovery was demonstrated in the salivary glands, heart, adrenals after 8 weeks of recovery and in the testes after 16 weeks of recovery.

In conclusion, oral administration of cesium chloride (CsCl) to Han Wistar rats at doses of 13, 38, 127 or 253 mg/kg bw/day resulted in pathological changes in the mandibular and sublingual salivary glands, heart, stomach, spleen, adrenals, mammary glands, skin and subcutis, epididymides and testes of animals treated at 253 mg/kg bw/day for 59 days or 127 mg/kg bw/day for 13 weeks. Additional changes were seen in the kidneys, skeletal muscles, extremities, ovaries and uterus of animals treated at 253 mg/kg bw/day. At 38 mg/kg bw/day, treatment-related changes were seen in the adrenals and testes. In addition, changes in the blood and urine indicative of an effect on kidney function were apparent at doses of 38 mg/kg bw/day and above; in particular, there was a clear increase in plasma urea and decrease in plasma potassium which was dose-related and marked at the 127 and 253 mg/kg bw/day dose levels. There was a dose-dependent detrimental effect on maturation of the sperm with most sperm breaking down within the epididymis. Treatment at 127 or 253 mg/kg bw/day resulted in most males showing no motile or normal sperm and significant reductions in sperm numbers. There was also a significant reduction in total sperm number in the cauda epididymis at 38 mg/kg bw/day and a slight effect on sperm morphology.

The dose of 253 mg/kg bw/day clearly exceeded the maximum tolerated dose and treatment at this dose level was stopped in Week 9. Recovery from all treatment-related changes was demonstrated, though one male receiving 127 mg/kg bw/day, still showed changes in the testes with associated low sperm numbers, all of which were immotile and abnormal after 16 weeks of recovery. No change on sperm numbers, morphology or motility or any pathological changes were seen at 13 mg/kg /day, nor were there any indications of an effect on the kidney. Consequently, based on these findings, the NOAEL was considered to be 13 mg/kg bw/day (equivalent to 15 mg cesium formate monohydrate/kg bw/day) (Webley, 2016).

Cesium hydroxide

A 90 d toxicity study was conducted to determine the toxicity of cesium hydroxide monohydrate in rats at three dose levels following 90 d oral administration. The study was performed according to OECD Guideline 408, EU Method B.26 and EPA Guideline OPPTS 870.3100, in compliance with GLP. The test substance was administered orally (by gavage) to Hsd. Brl. Han: Wistar rats (n=10 animals/sex/group) once a day at 0 (vehicle control), 250, 125 and 25 mg/kg bw/day. A sufficient stability of cesium hydroxide monohydrate in the chosen vehicle was analytically verified up front. Cesium hydroxide monohydrate was stable for 7 days at room temperature and in a refrigerator. Concentrations of the test substance in the dosing formulations varied from 95 to 97% of nominal concentrations at all analytical occasions, thereby confirming proper dosing. Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including haematology and clinical chemistry were conducted one day after the last treatment. Gross pathology was performed on animals on the day following the last treatment. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups. Testes epididymides, prostate and seminal vesicles with coagulating gland were also evaluated histologically in all animals of the low and mid dose groups due to the necropsy and histopathological findings in these organs. The results of study were interpreted comparing test substance treated groups with respect to controls, which were administered concurrently with vehicle (distilled water) only.

There was no test substance related mortality. One male and one female animal from the high dose group (250 mg/kg bw/day) died in the course of the study. In the female animal, clinical signs (irritability, piloerection, decreased activity and dyspnea) and severe body weight loss were observed before death. Histological examination revealed serious involution of thymus and signs of suffocation indicative of an individual metabolic disorder. The male animal died due to over anesthesia at the blood sampling at the termination of the study. Toxic signs related to the test substance were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery. A test substance related depression of the body weight development was detected in male animals at 250 and at 125 mg/kg bw/day during the entire study. The less body weight gain resulted in a less body weight compared to control group at 250 mg/kg bw/day from Day 21 and at 125 mg/kg bw/day from Day 35 up to the end of the treatment period. The daily mean food consumption was reduced in male animals at 250 mg/kg bw/day during the entire observation period. There were no abnormalities in the eyes of animals in the control and high dose groups at termination of the treatment. A test substance influence on the estrous cycle cannot be excluded with the results of this study. None of high dose (250 mg/kg bw/day) treated animal had regular cycle however the number of control animals with regular cycle was also significantly less than in the historical control data. There was no significant difference in the number of animals with an irregular estrous cycle in the low dose group as compared to the control group. A slightly higher mean white blood cell count and higher mean percentage of reticulocytes were detected in male and female animals at 250 and 125 mg/kg bw/day with respect to control group. Blood coagulation. There were no test substance induced pathologic changes in the examined blood coagulation parameters. Clinical chemistry evaluation revealed an elevated activity of aspartate aminotransferase at 250 and 125 mg/kg bw/day (male and female), as well as higher concentrations of urea, creatinine and less mean glucose concentration at 250 mg/kg bw/day (male and female) and at 125 mg/kg bw/day (male). These changes were indicative of the altered metabolic processes as histopathological examinations did not point out a damage in the relevant organs or tissues. The testes and epididymides were judged to be smaller than normal in animals of 250 mg/kg bw/day group at the necropsy observations. Significant reduction of the mean testes weights at 250 mg/kg bw/day and in epididymides weights at 250 and 125 mg/kg bw/day reflected a test substance related influence. The significantly less liver weights in male animals administered with 250 or 125 mg/kg bw/day probably were in accordance with the body weight alteration. Slightly higher kidneys weights at 250 mg/kg bw/day (male and female), and at 125 mg/kg bw/day (female) with respect to controls were considered to be signs of adaptation of the organ to the altered demands. The mean thymus weight (absolute and relative to brain weight) in the male animals) and the absolute mean thymus weight in the female animals were less than the control in animals administered with 250 mg/kg bw/day. Sperm analysis revealed lack of the sperm cells at 250 mg/kg bw/day and damage of the motility and morphology of the sperm cells at 125 mg/kg bw/day dose. Histological examination detected decreased intensity of spermatogenesis, which was accompanied with lack of mature spermatozoa in the seminiferous tubuli in the testes and in the ductuli of epididymides, and decreased number of spermatids in a proportion of seminiferous tubuli in 250 mg/kg bw/day dose treated male animals.

Under the conditions of the present study, the 250 mg/kg bw/day dose of the test substance caused reduced body weight, body weight gain, and reduced food consumption (male), and changes in hematology parameters (white blood cell count and percentage of reticulocyte – male and female). Damage in spermatogenesis (smaller than normal testes, reduced weights of testes and epididymides, decreased intensity of spermatogenesis, accompanied with lack of mature spermatozoa in the seminiferous tubuli in the testes and in the ductuli of epididymides, and decreased number of spermatids in a proportion of seminiferous tubuli) after the 90 d oral (gavage) administration in Hsd. Brl. Han: Wistar rats was observed. These test substance-related changes were considered to be toxicologically relevant. A test substance influence on the estrous cycle could not be excluded as none of the female animals in the dose group of 250 mg/kg bw/day had a regular estrous cycle. At this dose level, slight changes in thymus weights were noted in male and female animals, which were associated with an accelerated involution process as compared with the controls. The toxicological significance of this finding was considered to be equivocal. At 125 mg/kg bw/day, depression of the body weight development (male), altered clinical pathology parameters (slightly higher mean white blood cell count and higher mean percentage of reticulocytes (male and female), reduced weight of epididymides and damage of sperm motility and morphology) were indicative of adverse test substance related effects. At 25 mg/kg bw/day, there were no test substance related toxic alterations. Based on these observations, the NOAEL for cesium hydroxide monohydrate was determined to be 25 mg/kg bw/day for male and female animals (TOXICOOP, 2012).

Justification for classification or non-classification

Based on the results of a 90 day repeated dose toxicity study in rat with cesium chloride, the substance is considered to warrant classification as STOT RE 2 - H373: may cause damage to organs (kidneys, adrenals, nervous system, blood) through prolonged or repeated exposure according to CLP (EC 1272/2008) criteria.