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

Endpoint summary

Description of key information

A NOAEL of 30 mg/kg bw/day was established in an oral 28-day repeated dose study in rats with the structural analogues cesium fluoro aluminate complex and cesium potassium fluoroaluminate.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: No satellite groups were used.

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

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

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

Remarks:

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

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

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.

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

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 Jan 2016 to 09 Sept 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

03 November 2015

Limit test:

no

Specific details on test material used for the study:

Stable under storage conditions until 31 July 2016 (expiry date)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Age at start pretest Females: approximately 10-12 weeks.
Age at start F0-treatment Males: approximately 10-12 weeks; Females: approximately 12-14 weeks.
- Housing:
- Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MI II type, height 18 cm).
- Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage.
Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: ad libitum
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: 25 Jan 2016 To: 26 April 2016

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.

VEHICLE: water
- Rationale: Based on trial formulations performed at facilities.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (04 March 2016), according to a validated method (Test Facility Study No.511280). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were exposed for 50-54 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were exposed for 42 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on results of a 28-day dose range finding study in which dose levels of 20, 100 and 500 mg/kg were tested and dose-limiting effects were noted at 500 mg/kg.
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Observations and examinations performed and frequency:

Mortality / Viability At least twice daily (early in the morning and close to the end of the working day)

CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations (clinical signs and arena) were conducted and functional observations were started at least 1 hour (± 30 min) after dosing.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4).
For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

FUNCTIONAL OBSERVATIONS: Yes
The following tests were performed on the selected 5 animals/sex/group:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

BLOOD: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

HAEMATOLOGY: The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
White blood cells (WBC), Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets

CHEMICAL BIOCHEMISTRY: Yes
Blood Sampling for Thyroid Hormone Analysis
F0-generation, males and females:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire).
Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution.
Identification marks: not processed (Nasopharynx), Adrenal glands (Esophagus), (Aorta), Ovaries, Brain -cerebellum, mid-brain, cortex (7-levels), (Pancreas), Caecum Peyer's patches [jejunum, ileum] if detectable Cervix Pituitary gland, Clitoral gland, Preputial gland, Colon, Prostate gland, Coagulation gland, Rectum, (Cowper’s gland), (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes (with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Mammary gland area (males and females), Spinal cord -cervical, midthoracic, lumbar Femur including joint Spleen, (Glans penis), Sternum with bone marrow, (Levator ani plus bulbocavernosus muscle, complex (LABC)), Stomach, Testes, Heart, Thymus, Ileum ,Thyroid including parathyroid if detectable, Jejunum, (Tongue), Kidneys, Trachea, (Lacrimal gland, exorbital), Urinary bladder, (Larynx), Uterus, Liver, Vagina, Lung, infused with formalin, All gross lesions, Lymph nodes - mandibular, mesenteric Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- Stomach and Adrenal glands of all selected 5 animals of Groups 2 and 3 (males and females), based on treatment-related changes in these organs in Group 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile (see table below) or which died before mating to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

Other examinations:

The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
For Selected 5 animals/sex/group:
Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix).
For all remaining animals:
Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid.
Absolute organ weights and organ to body weight ratios were reported.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled
variances. Individual values, means and standard deviations may have been rounded off before printing.
Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

The daily clinical observations showed no treatment-related findings.
The weekly observations outside the home cage in a standard arena did not show any additional clinical signs of behavioural changes in any of the animals of all dose groups.
Clinical findings noted incidentally occurred within the range of background findings to be expected for rat s of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be toxicologically relevant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters were not affected by treatment.
Statistically significant variations noted in a few haematology parameters at 100 mg/kg were unrelated to treatment due to the slight magnitude of the difference from controls (values in treated rats remained within normal limits).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Lower total protein and albumin in males at all dose levels. The differences from controls increased with dose.
- Lower urea in males at 100 mg/kg.
- Higher total cholesterol in both sexes at 100 mg/kg.
The other statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the difference from controls (values in treated rats remained within normal limits) and/or absence of a dose-related response.
Thyroid hormone analyses:
Serum levels of T4, measured in F0 males, were not affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between treated and control animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.
All organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related macroscopic findings in the stomach. Dark red/reddish foci in the glandular stomach were recorded at an increased incidence in males 100 mg/kg (microscopic correlate congestion/hemorrhage). This was recorded in 5/10 males at 100 mg/kg. In addition this was recorded in 1/10 males at 10 mg/kg, which is within background incidence.
The incidence and severity of the macroscopic findings recorded for the glandular stomach of females of all dose groups including controls (dark red/reddish foci and/or thickened and/or irregular surface) was above background severity, but as these findings didn’t show a dose relationship they were regarded unrelated to the treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted noted in the glandular stomach of both sexes and adrenal glands of females as described below.
Glandular stomach:
An increased incidence and severity of lymphogranulocytic inflammation was recorded in the glandular stomach of males and females starting at 10 mg/kg. This was accompanied by edema in some males at 100 mg/kg and females at 30 and 100 mg/kg.
Congestion/hemorrhage was recorded at an increased incidence and severity in males at 100 mg/kg.
The edema and congestion/haemorrhage of the glandular stomach recorded for females at 10 mg/kg, 30 mg/kg and for the control group were comparable in incidence and severity and therefore considered to be unrelated to the test item.
Adrenals:
In adrenal glands of females vacuolation of the zona glomerulosa, slightly above background, was recorded at 100 mg/kg.
The minimal vacuolation recorded for a single male at 10 mg/kg and a single female at 30 mg/kg was considered to be within background.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

Local

Effect level:

< 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

A NOAEL of 30 mg/kg bw for systemic effects and a NOAEL of 100 mg/kg bw for fertility and developmental toxicity was established in a combined oral repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422, GLP).

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of cesium potassium fluoroaluminate in rats by oral gavage was performed according to OECD 422 and under GLP conditions. Based on the results of a 28-day dose range finding study in which dose levels of 20, 100 and 500 mg/ kg were tested and dose-limiting effects were noted at 500 mg/kg, the dose levels for this combined 28- day oral gavage study with reproduction/developmental toxicity screening test were selected to be 10, 30 and 100 mg/kg.

The test item, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females with offspring were exposed for 50-54 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 42 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.

In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity and stability. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

There were no treatment-related changes in the in-life results, haematology parameters or organ weights. Clinical biochemistry parameters showed treatment-related decreases in total protein and albumin, dose-dependently, in males at 10 mg/kg and above, a decrease in urea in males at 100 mg/kg, and an increase in total cholesterol in both sexes at 100 mg/kg. The changes at 100 mg/kg were considered to be toxicologically relevant. Microscopic examination revealed local treatment-related changes in the glandular stomach suggestive of irritating properties of the test item. These changes consisted of an increased incidence and severity of lymphogranulocytic inflammation in both sexes starting at 10 mg/kg, accompanied by edema in some males at 100 mg/kg and some females at 30 and 100 mg/kg. Additionally, males at 100 mg/ kg showed congestion/haemorrhage which correlated with the macroscopically observed dark red/reddish foci in the glandular stomach of these males. No reproduction or developmental toxicity was observed up to the highest dose level tested (100 mg/kg).

Based on the study results, the NOAEL for systemic effects is 30 mg/kg based on changes in clinical biochemistry parameters at 100 mg/kg. The NOAEL for local effects is < 10 mg/kg based on histopathological changes in the glandular stomach at 10 mg/kg and above.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

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

No repeated dose toxicity studies are available on cesium tetrafluoroaluminate. However, Article 13 of the REACH legislation states that, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i.e., applying alternative methods such as in vitro tests, QSARs, grouping and read-across. Data on repeated dose toxicity are available on the structural analogues, cesium fluoro aluminate complex (CAS 138577-01-2) and cesium potassium fluoroaluminate (EC 944-087-1). The basic structure of the main constituent of the target substance – tetrafluoroaluminate anion – is identical to the main constituent of the source substances. In Aluminium cesium fluoride complex and Cesium potassium fluoroaluminate concentrations amount to 70 % and 88.5 %, respectively. Their other constituents are higher homological fluoroaluminates that dissociate to tetrafluoroaluminate. Thus, it is evident that the composition of the target and the source substances is very similar and therefore the resulting chemical reactivity and their (eco)toxicity can be expected to be very similar as well. Further information on the read-across jutification is included as attachment in Section 13.

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 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 systemic NOAEL for cesium fluoro aluminate complex of 30 mg/kg/day was concluded.

In a GLP compliant combined 28-day oral repeated dose toxicity study with reproduction/developmental toxicity screening test performed according to OECD 422, cesium potassium fluoroaluminate was administered daily by oral gavage to Wistar rats. The animals (10 per sex per dose) were exposed to 0 (control), 10, 30 and 100 mg/kg bw/day test substance dissolved in water.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females with offspring were exposed for 50-54 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 42 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.

In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity and stability. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

There were no treatment-related changes in the in-life results, haematology parameters or organ weights. Clinical biochemistry parameters showed treatment-related decreases in total protein and albumin, dose-dependently, in males at 10 mg/kg and above, a decrease in urea in males at 100 mg/kg, and an increase in total cholesterol in both sexes at 100 mg/kg. The changes at 100 mg/kg were considered to be toxicologically relevant. Microscopic examination revealed local treatment-related changes in the glandular stomach suggestive of irritating properties of the test item. These changes consisted of an increased incidence and severity of lymphogranulocytic inflammation in both sexes starting at 10 mg/kg, accompanied by edema in some males at 100 mg/kg and some females at 30 and 100 mg/kg. Additionally, males at 100 mg/ kg showed congestion/haemorrhage which correlated with the macroscopically observed dark red/reddish foci in the glandular stomach of these males. No reproduction or developmental toxicity was observed up to the highest dose level tested (100 mg/kg). Based on the study results, the NOAEL for systemic effects is 30 mg/kg based on changes in clinical biochemistry parameters at 100 mg/kg. The NOAEL for local effects is < 10 mg/kg based on histopathological changes in the glandular stomach at 10 mg/kg and above.

Justification for classification or non-classification

Based on a NOAEL of 30 mg/kg bw/day that was established in an oral 28-day repeated dose study in rats with the read-across candidates cesium fluoro aluminate complex and cesium potassium fluoroaluminate, cesium tetrafluoroaluminate is classified "May cause damage to organs through prolonged or repeated exposure" for Specific Target Organ Toxicity - Repeated exposure (Category 2, H373, oral route) according to the EC legislations (CLP regulation 1272/2008).