Aluminate(1-), tetrafluoro-, cesium (1:1), (T-4)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 July 1998 (Start of in-life phase) to 18 February 1999 (GLP compliance statement)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

: No satellite groups were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: No satellite groups were used.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

EC Number:

604-086-6

Cas Number:

138577-01-2

Molecular formula:

Al.Cs.F

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Cesium Fluoro Aluminate Complex
- Purity: 99.5%
- Batch: 604/013
- Physical state: White amorphous powder
- Storage condition of test material: At room temperature in the dark
- Expiration date of the lot/batch: 22 May 2001

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks old
- Weight at study initiation: 227 - 268 g (males) and 150-180 g (females). Body weight variation did not exceed +/- 20% of the sex mean.
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust (B.M.I. Helmond, The Netherlands) provided as bedding.
- Diet: Free access to pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Water: Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled at 21°C
- Humidity (%): controlled at 50 %
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Fluctuations from the optimal conditions were noted, but were considered not to have affected study integrity.

IN-LIFE DATES: From 27 July - 24 August 1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations, as a solution in 1% aqueous carboxymethyl cellulose. Formulations (w/w) were prepared daily within 4 hours prior to dosing. Samples of 5 ml were taken from representative dose formulations, prepared after termination of the study, as soon as possible after preparation, to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Testing for stability was considered not applicable since the composition of the test substance is complex of elements and considered not to be degradable. The analysis of formulations show that the values were within the range of 96 to 109% which was considered acceptable.

VEHICLE
- Concentration in vehicle: The concentration of the test substance in vehicle was varied to allow constant dosage volume in terms of mL/kg bw.
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at NOTOX.
- Lot/batch no. (if required): No data

MAXIMUM DOSE VOLUME APPLIED:
5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Detection method: Inductively coupled plasma atomic emission spectrometry (ICP-AES)
- Detection limits (LOD, LOQ): The limit of detection and determination were determined by analysing a blank solution six times. The limit of detection was 0.03 mg/L, the limit of determination was 0.10 mg/L.
- Reproducibility in %: The repeatability was determined by analysing a sample six times. The relative standard deviation of the six measurements was 2.9%.
- Linearity range: 2.5 - 10 mg/L Aluminium.
- Internal or external calibration: The calibration of Aluminium was based on an external standard solution using five calibration solutions containing between 0 and 10 mg/L Aluminium. The correlation coefficient was 0.998.
- Extraction recovery (indicate if results are corrected or not for recoveries): The recovery was checked at two levels (2.5 and 7.5 mg/L. The results show recoveries between 98 and 102%. The results obtained with the test samples have not been corrected for the recovery.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30, 150, 845 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30, 150, 750 mg/kg/day
Basis:
other: Nominal dose levels

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 5-day dose range-finding study.
No satellite groups were used.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Mortality/viability was checked twice daily. Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on days 8, 15 and 22 this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded on days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION:
- Food consumption was recorded weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: Subjective appraisal was maintained during the study but no quantative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Animals fasted: yes, overnight
- How many animals: all animals
- Parameters examined: Erythrocyte count, haematocrit, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, platelet count, red cell distribution, total leucocyte count, differential leucocyte count, prothrombin time, partial thomboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 immediately prior to scheduled post mortem examination.
- Animals fasted: yes, overnight
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), bilirubin, cholesterol, creatinine, glucose, urea, protein (total and albumin), alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, phosphorus.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: A battery of functional observational tests was performed during week 4 of treatment. The following tests were performed on all animals: hearing ability, pupillary reflex, static righting reflex, grip strength (score 0=normal/present, score 1=abnormal/absent), acitivity test (based on hourly data per animal).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving animals were deeply anaesthetised using ether vapour and subsequently exsanguinated. The animals were necropsied and descriptions of all macroscopic abnormalities recorded.

Organ weights:
The following organs weights were recoreded from the surviving animals on the scheduled day of necropsy:
- adrenal glands
- brain
- epididymides
- heart
- kidneys
- liver
- spleen
- testes
- thymus

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals and fixed in a 4% formaldehyde solution: adrenal glands, aorta, brain, caecum, (cervix), (clitoral gland), colon, duodenum, epididymides, (eyes with optic nerve and Harderian gland), (female mammary gland area), (femur including joint), heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung, lymph nodes, (nasopharynx), oesophagus, ovaries, pancreas, Peyer's patches (jejenum, ileum) if detectable, pituitary gland, (preputial gland), prostate gland, rectum, (salivary glands), sciatic nerve, (seminal vesicles), (skeletal muscle), (skin), spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid, (tongue), trachea, urinary bladder, uterus, (vagina)

For histopathological examination, all organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the mid and high dose groups as well as from all ainmals of all dose groups which died spontaneously or were sacrificed, and all gross lesions of all animals were examined by a pathologist. Based on treatment related morphological changes; stomach, spleen, kidneys and urinary bladder were also examined from all rats of the low dose group. Tissues mentioned between brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

None

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hunched posture, piloerection, staining of the fur, pale appearance and hypersensitivity to touch were observed in high dose group animals. Emaciation, lethargy, tremors and diarrhea were observed in single animals shortly before death. In the mid dose group temporary staining of the fur and a single case of piloerection and swelling of the throat during week 2 and/or 3 of treatment was observed. In the low dose group temporary staining of the fur and a single case of piloerection was observed during week 2 of treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality occurred in the low and mid dose and control animals during the study period. Treatment related mortality was seen among animals receiving 750 mg/kg/day during the study. The incidence of mortality for males was 4/5 and for females 3/5. The animals were found dead between days 15 and 29 (day of scheduled necropsy). A fourth animal died during blood sampling on day 29.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight or body weight loss, and consequent changes in body weight gain, were observed in animals receiving 750 mg/kg/day. Body weights and body weight gain of treated animals receiving 30 and 150 mg/kg/day remained in the same range as controls over the 4-week study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Excessive food spillage by the males and females receiving 150 or 750 mg/kg/day was observed on day 9 until day 15 of the study. The food consumption of the animals receiving 750 mg/kg/day was markedly reduced over weeks 1, 2 for males and over week 2 for females. There were no differences in food consumption between animals receiving 30 and 150 mg/kg/day and control animals.

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

Description (incidence and severity):

The mean corpuscular volume (MCV) was decreased and the red cell distribution width (RDW) was increased in the two surviving animals receiving 750 mg/kg/day. A marked increase in differential count for the neutrophils (SEG) was also observed in the high dose animals

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes were found in the values for aspartate aminotransferase, creatinine, urea and inorganic phosphate (increased values) and in the values for albumin and potassium (decreased values) in the two surviving females receiving 750 mg/kg/day. Similar changes were seen in the surviving male receiving 750 mg/kg/day. In the females receiving 150 mg/kg/day, the mean values for total protein and albumin were decreased in comparison to the controls. There were no differences noted between control and treated males receiving 150 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

A dose dependent decrease in (motor) activity recorded by the uppper sensors was observed in both males and females. In males, a tendency of a dose-dependent increase in activity recorded by the lower sensors was seen, resulting in a similar overall activity for each dose group, when compared to the controls. The overall motor activity of the females showed a dose dependent decrease. No effects were observed in the other functional observation tests.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No data for organ weights are available for 4/5 males and 4/5 males of the high dose group due to death prior to necropsy. Since only single organ weight values were available for this high dose group, no evaluation of these values was performed.
Statistically significant increases in weight of the kidneys and spleen were found in females of the mid-dose group. However, the organ:body weight ratios were within the range of those of controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The main macroscopic observations associated with treatment were in the stomach and spleen in the animals receiving 750 mg/kg/day and in the stomach only in the animals receiving 30 or 150 mg/kg/day. The findings comprised of haemorrhages in or red discolouration of the glandular mucosa, irregular surface of the forestomach and irregular surface, thickening or discolouration of the limiting ridge in the stomach and reduced size of the spleen. The incidence of the findings in the stomach showed a dose dependent relationship. Additionally, the thymus was reduced in size or haemorrhagic in 7/10 high dose animals, reduced size of the prostate, testes and seminal vesicles was found among the males and haemorrhagic contents of the duodenum and jejunum, black discolouration of the mesenteric lymph nodes and reduced size of caecum was found among females of the high dose group.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Stomach:
- A dose related granulocytic inflammation of the glandular mucosa, in association with foveolar apoptosis, was present in the low, mid and high dose groups.
- Mucosal erosion (males) and increased severity of forestomach inflammation (females) in the high dose group.
Heart:
- An increase in the incidence and severity of cardiac myofiber degeneration/necrosis in the high dose group.
Kidneys:
- Tubular regeneration in most males and single females of the mid and high dose groups.
Urinary bladder:
- Urothelial hyperplasia in some rats of both sexes of the mid dose group.
Testes:
- Tubular atrophy in two high dose males.
Spleen:
- Reduced splenic haemopoiesis and lymphoid atrophy occurred in the high dose group.
Lymph nodes:
- Sinusoidal macrophages in mesenteric and mandibular lymph nodes in the high dose group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

From the results of this study a NOAEL for cesium fluoro aluminate complex of 30 mg/kg/day was concluded.

Executive summary:

In a GLP compliant 28 day sub-acute study performed according to OECD 407, cesium fluoro aluminate complex was administered daily by oral gavage to Wistar rats. The animals (5 per sex per dose) were exposed to 0 (control), 30, 150 and 750 mg/kg/day.

Clinical signs, functional observations, body weight and food consumption were monitored during the study. Haematology and blood chemistry was evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination and a histopathological evaluation of tissues was performed.

No mortality occurred in the low and mid dose and control animals. Treatment related mortality was seen among animals receiving 750 mg/kg/day during the study. The incidence of mortality was 4/5 for males and 3/5 for females. Animals were found dead between days 15 and 29 (day of scheduled necropsy). A fourth female died during blood sampling on day 29. The lesions in the stomach of these animals were indicative of irritating properties of the test substance to the stomach and were considered to have contributed to the cause of death. Moreover, the animals showed uraemia, correlating the microscopic lesions in the kidneys and alterations in several blood parameters, and reduced haempoiesis in the spleen, correlating the reduced size of this organ seen at necropsy. The changes in clinical appearance, motor activity and body weights were secondary to the stomach, renal and/or splenic lesions and considered indicative of a poor condition of these animals. Macroscopic and/or microscopic changes were also found in the heart, testes and lymph nodes of the high dose animals. The fact that lesions were found in several organs may indicate that there is no specific target organ for or mechanism of toxicity of cesium fluoro aluminate complex. The observed effects in the rats treated at 150 mg/kg/day consisted of findings in the stomach, kidneys and spleen, which were similar to those found in the high dose animals, but at a lower incidence and/or severity. No correlating changes were found in the blood parameters, except for decreased serum protein concentrations in females.

The findings in the animals treated at 30 mg/kg/day comprised of a few clinical signs during week 2, which had disappeared after continuation of treatment, and effects in the stomach. The effects in the stomach were characteristic of an (adaptive) effect of the irritating properties of the test substance and are commonly seen after treatment by gavage. The symptoms were of minor severity and were not supported by organ dysfunction. No treatment related changes were found in body weights, food consumption, clinical laboratory investigations and organ weights.

From the results of this study a NOAEL for cesium fluoro aluminate complex of 30 mg/kg/day was concluded.