Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-21 to 2018-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2017-05-08
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25°C in a tightly closed original container, stored in a dry, cool and well venitlated place.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at first administration: males: 74 days; females: 75 days
- Weight at first administration: males: 407.0 g - 477.5 g; females: 242.5 g - 278.0 g
- Housing (with the exception of the mating period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg)
- Diet (ad libitum): certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Food quality: no contaminants above the limitations were noted.
Water quality: no contaminants above the limitations were noted.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 15 % (maximum range)
- Air changes: 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
According to international guidelines.
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to provide dose concentrations of 37.5, 125 or 375/250 mg/mL
The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.

- Administration volume: 2 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approx. 15 mL were taken at the following times and stored at ≤-20°C ± 10% until analysis:
1) At start of the treatment period (first dosing day):
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples / dose level group).
- homogeneity: immediately after preparation, during (middle) administration and before administration to the last animal/dose level group (3 samples / dose level group plus one sample/control).

2) At dose change (highest dose group, males)
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples of highest dose group).
- homogeneity: immediately after preparation, during (middle) administration and before administration to the last animal/dose level group (3 samples of highest dose group).

3) towards the end of the treatment period (when the majority of animals was dosed):
- analysis of concentration: during treatment always before administration to the last animal/dose level group (1 sample / group; low and intermediate dose groups; 2 samples from highest dose group: 1 sample from the male and 1 sample from the female group).
Duration of treatment / exposure:
- males: 35 treatment day (2 weeks prior to mating (from test day 15 until test day 29), during the mating period (14 days at maximum) and during the post-mating period until one day before sacrifice)
- females: 50 to 62 treatment days (2 weeks prior to mating, during the mating period (14 days at maximum) and during the lactation period until lactation day 13).
Frequency of treatment:
once daily
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
administered to both sexes, but due to the premature death of 2/10 males, the dose level was reduced from 750 to 500 mg/kg bw/day on test day 38 for the males.
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
starting on test day 38, this dose was administered to males instead of the 750 mg/kg bw/day dose due to premature deaths of the males in the high dose group.
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on available toxicological data and a 21-day dose range finding study (please refer to section 7.5.1 Repeated dose toxicity: oral: s_Hansen_2018_DRF). In the 21-day dose range finding study, cesium tetrafluoroaluminate was administered orally to male and female rats at dose levels of 2, 10, 50, 100, 200, 600 or 1000 mg/kg bw/day for 3 weeks from test days 1 to 21.
At 1000 mg/kg bw/day one of 5 male animals died prematurely (found dead on the day of necropsy on test day 22) after 21 days of dosing. A post-dosing salivation (salivation started immediately to 5 minutes after dosing and disappeared again between 20 and 60 minutes after dosing) was noted from a dose level of 50 mg/kg bw/day onwards. The observation of salivation was related to the dose level (1 of 5 affected male animal at a dose level of 50 mg/kg bw/day and 5 of 5 affected male and female animals from 200 mg/kg bw/day onwards).
A reduced body weight and body weight gain was noted for the male animals at 1000 mg/kg bw/day but not for the female animals.
After 21 days of dosing statistically significant changes were noted for one or both sexes for several haematological parameters at 100, 200, 600 and 1000 mg/kg bw/day. In detail, slightly to moderately decreased levels were noted for the haemoglobin concentration, the number of red blood cells, the haematocrit value and the mean corpuscular volume.
A moderate to marked increase was noted for the number of white blood cells at dose levels of 600 and 1000 mg/kg bw/day.
The macroscopic examination at necropsy after 21 days of dosing revealed test item-related changes at 1000 mg/kg bw/day for the male animals in the form of small testes (1 of 5 males) and stomach changes (thickened mucosa, ulcera; 3 of 5 males).

- Selection criteria for females to be included in the study: during the 14-day pre-exposure period (TD 1 to TD 14), the estrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal estrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study. Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to 5 day cycles during the 14-day pre-exposure period.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
clinical signs: before and after dosing at each time of dosing as well as regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approx. 3:30 p.m.
mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays, a similar procedure was followed with a final check at approx. 3.30 p.m.
- Cage side observations checked: behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity as well as mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (performed at least 2 hours after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations:
1) Males: weekly starting during the premating period and at sacrifice

2) Females:
premating period: weekly
gestation period: gestation days 0, 7, 14 and 20
lactation period: lactation days 1,4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg bw/day) was determined using the following
formula: Relative food consumption = (total food given (g) - total food left (g))/number of animal days* x body weight (kg)
*the term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

Food residue (or total food left) was weighed and recorded as followed:
- Pre-mating period
males and females: TD8 and TD15
- Mating period:
males and females: none
- Gestation period
males: not applicable
females: GD7, GD14 and GD21
- Lactation period
males: not applicable
females: LD1, LD8 and LD12

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: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual appraisal throughout the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the premating period on test day 29
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 animals/sex/group
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes as well as large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the premating period on test day 29
- Animals fasted: Yes, overnight
- How many animals: 5 animals/sex/group
- Parameters checked: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), blood urea, calcium, chloride, potassium, sodium, sodium/potassium ratio, blood urea/creatinine ratio, lactate dehydrogenase, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at least two hours after dosing and before any blood sampling for laboratory examinations:
males: test day 48
females: test day 64, 65, 66, 70 or 73
- Dose groups that were examined: all dose groupy (5 animals/sex/dose)
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed at the following times:
- males: on test day 50
- dams (surviving dams): on lactation day 14
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS.
The weight of the following organs of all adult male and female animals was determined before fixation (where applicable): adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testicles, thyroid, thymus, prostate, seminal vesicles with coagulating glands, uterus including cervix

HISTOPATHOLOGY: Yes
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin: epididymis, testicles, gross lesions observed, ovaries and oviducts, prostate, seminal vesicles with coagulating glands, thyroid (including parathyroids), uterus (including cervix) and vagina
Any other organs displaying macroscopic changes were also preserved.
5 male and female animals from each group were selected for histopathology examination and the following organs from the selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination: eye with optic nerve (2), epididymis (2), testicle (2), adrenal gland (2), bone, bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem (pons)), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), intestine, large (colon, rectum), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion, lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal), nerve (sciatic), oesophagus, ovary and oviduct, pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 sections), spleen, stomach, thyroid (including parathyroids), thymus, tissue masses or tumors including regional lymph nodes), tongue (including base), trachea (including larynx), urinary bladder, uterus (including cervix) and vagina.

Full histopathology was performed on the preserved organs of the selected parental animals of the control group and the highest dose group The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of the control group and the highest dose group following H-E and PAS staining.
Statistics:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant
effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi² test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs:
1) Males:
- 750/500 mg/kg bw/day: a haemorrhagic nose/snout for 3/8 surviving animals was observed on several test days (considered to be test item-related).

2) Females:
-750 mg/kg bw/day: piloerection was noted for all 4 surviving females during the lactation period on altogether 36 test days (considered to be test item-related).
- premature deaths (750 mg/kg bw/day): the observation of piloerection that was noted for 5 of the 6 prematurely deceased animals was also noted for all 4 surviving animals and therefore considered to be test item-related and not as a pre-mortal symptom
Mortality:
mortality observed, treatment-related
Description (incidence):
1) Males:
750/500 mg/kg bw/day: 2/10 male animals died prematurely, which was considered to be test item-related due to changes in the laboratory parameters, reductions in body weight and changes in organ weight.

2) Females:
750 mg/kg bw/day: 6/10 females died during the lactation period between test days 54 to 64 (corresponding to lactation days 1 to 9). Due to the high incidence of prematurely deceased high dosed females, these premature deaths were considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 750/500 mg/kg bw/day: a statistically significantly reduced body weight in comparison to the control group was noted from test day 29 until sacrifice on test day 50 (considered to be test item-related). In detail, on test day 29 the body weight of the males was 7.7 % below the value of the control group (p ≤ 0.05). Thereafter the difference in body weight slightly increased. On test day 49 (last day of dosing) the body weight of the animals was 9.5% below the value of the control group (p ≤ 0.01) and at sacrifice (test day 50) the body weight of the male animals was 11.0 % below the control value (p ≤ 0.01). The percentage of body weight gain was decreased in the high dose group in comparison to the control group and the 75 and the 250 mg/kg bw/day dose groups.
The body weight at autopsy for the 8 surviving male animals of the 750/500 mg test item/kg bw/day dose group was 10.7 % below the value of the control group (p ≤ 0.01). This was in the range of the difference that was noted for the live animals on the day of sacrifice (11.0 % below the control value) and considered to be test item-related.
- premature death (750/500 mg/kg bw/day): one male showed a marked reduction in body weight before death (minus 30.6 % between test day 15 and test day 29

2) Females:
- 750 mg/kg bw/day: a statistically significantly (p ≤ 0.05 or 0.01) reduced body weight was noted from gestation day 14 onwards until the end of the lactation period. This reduction in body weight in comparison to the control group was considered to be test item-related. In detail, the mean body weight of the females was 7.5 % and 13.9 % below the control value on gestation days 14 and 20, respectively (p ≤ 0.05 or p ≤ 0.01). At the beginning of the lactation period (lactation day 1) the mean body weight of the 9 living females (one female died during littering on lactation day 1) was 14.4 % below the control value (p ≤ 0.01). During the further course of the lactation period 5 further females of the treatment group died and the mean body weight of the 4 surviving animals on lactation day 13 was 21.9 % below the value of the control group (p ≤ 0.01). In accordance with the reduced body weights that were noted during the gestation and the lactation period, body weight gain for these periods was reduced at the high dose level in comparison to the control group.
A slight and statistically not significant reduction in body weight at autopsy (9.6% below the value of the control group) was noted for the 4 surviving female animals on lactation day 14. This was in agreement with the test item-related reduction in live body weight that was noted on lactation day 13 (21.9 % below the control value).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1) Females:
- 750 mg/kg bw/day: during the second week of the lactation period a reduction in food consumption (54.0% below the value of the control group, p ≤ 0.01) was noted for the 4 surviving female animals of the treatment group. This may be caused by the damaged teeth that were noted for the 4 surviving females at necropsy and/or the poor health condition of these 4 animals in general, as demonstrated by the reduced body weight or the poor results of the neurological screening. Due to these reasons, the reduced food consumption of the 4 surviving animals during the second week of the lactation period is considered to be test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 250 mg/kg bw/day: a slight and statistically not significant increase was noted for the number of white blood cells (47.5 % above the value of the control group). Regarding the subunits of the white blood cells, statistically significant increases were noted for the numbers of neutrophilic and eosinophilic granulocytes (p ≤ 0.01 and p ≤ 0.05, respectively). The increases in the number of neutrophilic and eosinophilic granulocytes were considered to be test item-related, as more pronounced increases were noted at the750/500 mg/kg bw/day dose level (dose-response relationship).
- 750/500 mg/kg bw/day: the increase in the number of white blood cells was more pronounced as at the 250 mg/kg bw/day dose level and statistically significant (143.3 % above the value of the control group; p ≤ 0.05). The cell numbers of nearly all subunits of the white blood cells (cell numbers of lymphocytes, monocytes, neutrophilic, eosinophilic and basophilic granulocytes, with the exception of the small population of large unstained cells) were markedly increased (statistically significant or not; p ≤ 0.01 or 0.05)). All changes that were noted at the 750/500 mg/kg bw/day dose level for the above mentioned parameters were considered to be test item-related. However, it has to be mentioned that a high variability was noted for the above mentioned parameters at the 750/500 mg test item/kg bw/day) dose level. Markedly increased numbers of white blood cells, lymphocytes, monocytes, neutropholic, eosinophilic and basophilic granulocytes were especially noted for the 2 prematurely deceased animals (one animal died 1 day after blood withdrawal on test day 30 and one animal died 7 days after blood withdrawal on test day 36) and one surviving animal. In contrast, the values of the above mentioned parameters for other two surviving animals were in the range of the control group (only for the number of neutrophilic granulocytes an increased value was noted for one animal).

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 250 and 750 mg/kg bw/day: increased numbers of neutrophilic granulocytes, eosinophilic granulocytes and monocytes were noted at the 250 and the 750 mg/kg bw/day dose levels (considered to be test item-related). The increases in the above mentioned cell numbers were statistically significant at p ≤ 0.05 / 0.01 or not statistically significant in comparison to the control group and slightly more pronounced at the 750 mg/kg bw/day dose level as at the 250 mg/kg bw/day dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 250 mg/kg bw/day: a slight but statistically significantly decreased potassium concentration was noted (p ≤ 0.01). Though all individual values were clearly within the laboratory background data, they were all slightly below the individual values of the control group. This small reduction was considered to be test item-related due to the test item-related reductions that were noted for the potassium concentrations at the 750/500 mg/kg bw/day dose levels of the male and more pronounced of the female animals.
- 750/500 mg/kg bw/day: test item-related changes were noted for the potassium concentration, the aspartate aminotransferase activity, the albumin and globulin concentration and the urea in blood concentration. The findings are described in detail below.
A statistically significantly decrease in potassium concentration was noted in males (p ≤ 0.01). A marked decrease in the potassium concentration was noted for one surviving male animal and one prematurely deceased animal (values below or at the lower range of laboratory background data), whereas the potassium concentrations of the remaining 3 animals remained at the control level. Though only 2 of 5 animals showed a decreased potassium concentration at the high dose group, this was considered to be test item-related, as a decreased potassium level was also noted for the female animals of the high dose group.
Furthermore, a statistically significantly increase in aspartate aminotransferase activity was observed at the high dose group (p ≤ 0.01). An increased aspartate aminotransferase activity that was above the upper range of the background data was noted for 4 of the 5 examined male animals. of the high dose group.
A slight but statistically significant decrease was noted for the protein (total) concentration and thus for the albumin and globulin concentrations (p ≤ 0.05 or p ≤ 0.01). All individual values for protein (total) and globulin were within the laboratory background data, whereas two individual values for albumin were slightly below the background data.
A moderately but statistically not significant increase was noted for the urea in blood concentration. Though this increase was mainly due to a markedly increased level of one prematurely deceased animal, it was considered to be test item-related as the urea in blood concentration of the high dosed females was also increased.

2) Females:
- 750 mg/kg bw/day: test item-related changes were noted for the electrolyte levels (potassium, chloride and calcium concentration), the urea in blood concentration, the cholesterol concentration and the aspartate aminotransferase activity. In detail, a statistically significantly decrease was noted for the potassium and chloride concentration and a statistically significantly increase for the calcium concentration (p ≤ 0.05 or p ≤ 0.01). Especially the effect on the potassium concentration was highly significant. All individual values were markedly below the lowest value of the other test groups (control, 75 and 250 mg/kg bw/day group) and at the lower range of thelaboratory background data.
A clear effect was also noted for the chloride concentration. Though the difference was rather small (3.3 % below the control value; p ≤ 0.01), 4 of 5 individual values of the high dose group were below the lower value of the control group (only the value of one 750 mg/kg bw/day dose group female was at the same level as the lowest value of the control group). Furthermore, all values were below or at the lower range of background data.
A statistically significantly increase was noted for aspartate aminotransferase activity (p ≤ 0.01). However, a markedly increased aspartate aminotransferase activity was only noted for one female, whereas the aspartate aminotransferase activities of the other high dosed females were only slightly increased, but within the laboratory background data. However, due to the more pronounced increased aspartate aminotransferase activity that was noted for the male animals, this was considered to be test item-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
1) Females:
- 750 mg/kg bw/day: an adverse effect was noted on some of the examined motoric skills (limb rotation, wire maneuver, positional passivity, positive geotropism, righting reflex) for the 4 surviving female animals that were used for the observational screening tests (considered to be test item-related). In addition, a statistically significantly reduced fore- and hindlimb grip strength was noted for the 4 surviving females of the 750 mg test item/kg bw/day dose group (p ≤ 0.01 and p ≤ 0.05), respectively). The grip strength values of the individual animals were below or near the lower range of laboratory background data. The decreased grip strength was considered to be test item-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 250 and 750/500 mg/kg bw/day: statistically significantly decreased relative and absolute thymus weights (p ≤ 0.01) were noted at the 250 mg/kg bw/day dose level and at the 750/500 mg/kg bw/day dose level. The reduced thymus weight that was noted at the 750/500 mg/kg bw/day dose level is in consistence with the observation of thymus atrophy that was noted during the histopathological examination. The decrease of thymus weight was test item- and dose-related.
A highly significant effect was noted at the 750/500 mg/kg bw/day dose level in the form of increased organ weights of the testes (absolute and relative), which was correlated with the macroscopic finding of enlarged testes at necropsy. In detail, 7/8 surviving male animals of the 750/500 mg/kg bw/day dose group revealed a markedly increased relative and absolute organ weight of the left testis in comparison to the control group. This was correlated with the macroscopic finding of an enlarged left testis in 4 high dosed animals at necropsy. Increased relative and absolute organ weights were also noted for the right testes (statistically not significant), which was correlated with the macroscopic finding of an enlarged right testis in 2 high dosed males. However, 2 animals were noted with macroscopically small right testis. The relative and the absolute organ weights of the right testes of these 2 animals were below the lowest value of the other test groups (control, 75 and 250 mg/kg bw/day dose group). Both, the increased and the decreased testes weights were considered to be test item-related.
- premature death (750/500 mg/kg bw/day); one male): increased absolute organ weights of the adrenal glands (confirmed by the finding of macroscopically enlarged adrenal gland at necorpsy).

2) Females:
- 250 and 750 mg/kg bw/day: reduced relative and absolute thymus weights were noted at the 250 mg/kg bw/day and the 750 mg/kg bw/day dose level. Though they failed statistical significance, an atrophy of the thymus was noted for all 4 surviving females of the 750 mg/kg bw/day dose group during the histopathological examination. Hence, as the finding of a reduced thymus weight was in consistency with histopathological findings at the 750 mg/kg bw/day dose level, the reduced thymus weight that was noted at the 750 mg/kg bw/day dose level, though statistically not significant, was considered to be test item-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 750/500 mg/kg bw/day: different kind of testes changes (enlarged, small, marmorated and/or malpositioned) were noted for 4/8 surviving male animals. One of these 4 animals (enlarged and malpositioned left and right testis) was examined microscopically and revealed degenerated/atrophied and dilated tubuli in both testes.
These testes changes were considered to be test item-related.

2) Females:
- 750 mg/kg bw/day: the teeth of the lower row of all 4 surviving females were pale and broken off (considered to be test item-related as all surviving females were affected). Furthermore, the thickened stomach mucosa that was noted for one female was considered to be spontaneous, as only one animal was affected.
- premature deaths (750 mg/kg bw/day): stomach observations at necropsy in the form of a reddened fundus and a reddened or detached mucosa were correlated with microscopic findings in the form of inflammation and ulceration and considered to be test item-related. The other necropsy observations were considered to be unspecific or considered to be related to the mode of death (e.g dark-red discoloured lungs that were correlated with the finding of lung congestion (agonal) at microscopy.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 750/500 mg/kg bw/day:
Adrenals:
- a slight to marked hypertrophy of the cortex was noted for 3 of 5 male animals (one suriving male and the two prematurely deceased animals; considdered to be test item-related).

Heart:
- a slight to moderate necrosis/inflammation was noted for 3 of 5 male animals (one surviving animal and the two prematurely deceased animals; considered to be test item-related).

Kidneys:
- several changes were noted for all examined animals (e.g. basophilic tubules in the cortex, degeneration of the proximal and distal tubules, glomerulonephritis, oil red staining; considered to be test item-related).

Lymph node (mesenteric):
- a sinus histocytosis was noted for 2 of 5 animals (one surviving animal and one prematurely deceased animal; considered to be test item-related).

Skeletal muscle:
- a muscle degeneration together with a granulomatous inflammation was noted for 2 of 5 male animals (one surviving male and one prematurely deceased animal; considered to be test item-related).

Stomach:
- several findings were noted for altogether 4 of 5 dosed animals (e.g. acute inflammation of the mucosa/submucosa, ulcertion of the mucosa; considered to be test item-related).

Thymus:
- a marked to severe atrophy was noted for 4 of 5 dosed males (three surviving animals and one prematurely deceased animal; considered to be test item-related).

Tests:
- two males of the 750/500 mg/kg bw/day dose group revealed degenerated/atrophied and dilated left and right testes during the microscopic examination. In case of one male the microscopic testes findings were correlated with macroscopic findings in the form of an enlarged and malpositioned left and right testis.

2) Females:
Note: all 10 females of the 750 mg/kg bw/day dose group were examined histopathologically: 6 prematurely deceased females and the 4 surviving females. The 4 surviving females were included in the 5 selected 750 mg/kg bw/day dose females that were selected for the histopathological examination at the begin of the study.

- 750 mg/kg bw/day:
Adrenal gland:
- a slight hypertrophy of the cortex was noted for 2/10 female animals (two prematurely deceased animals; considered to be test item-related).

Heart:
- a minimal or moderate necrosis/inflammation was noted for 4 of 10 female animals (prematurely deceased females; considered to be test item-related).

Kidneys:
- several changes were noted for 8/10 examined females (e.g. basophilic tubules in the cortex, degeneration of the proximal and distal tubules, oil red staining). With the exception of oil red staining (7 of 10), all other kidney observations were only noted for 1 or 2 different females of the high dose group. Findings were considered test item-related.

Lungs:
- Granulomas were noted for 2 of 10 animals (considered to be test item-related)

Lymph nodes:
- a sinus histocytosis was noted in the mesenteric lymph node from 7/10 females. Also 7/10 females revealed a plasmacytosis in the cervical lymph node. Findings were considered test item-related.

Skeletal muscle:
- a muscle degeneration together with a granulomatous inflammation was noted for 5 of 10 female animals (considered to be test item-related)

Spleen:
- an atrophy of the white pulp was noted for 5 of 10 females (considered to be test item-related).

Stomach:
- several findings were noted for altogether 9 of 10 females (e.g. erosions of the glandular mucosa, acute inflammation of the mucosa/submucosa, ulcertion of the mucosa; considered to be test item-related).

Thymus:
- a marked to severe atrophy was noted for all (10 of 10) females (considered to be test item-related).

Urinary bladder:
- a hyperplasia of the urothelial cells was noted for 3 of 10 females (considered to be test item-related).

Uterus:
- changes were noted in the uterus from 3 of 10 females in the form of an inflammation and / or an haemorrhagic endometrium. A slight to moderate mucification of the vaginal mucosa was noted for 4 of 10 females. These findings are considered to be test item-related.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS/DETAILED CLINICAL SIGNS:
Clinical signs:
1) Males:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 mg/kg bw/day: no changes in behaviour, the external appearance or the faeces were noted that were of toxicological relevance. However, a slight salivation was noted for 2/9 surviving animals on one day each. Such a post-dosing salivation that started briefly after dosing and lasted for a short time is often observed in gavage studies and maybe a reaction to the taste or irritation of the test article, rather than an indication of toxicity
- 250 mg/kg bw/day: salivation was observed in 6/10 animals (not toxicologically relevant). Furthermore, haemorrhagic urine was noted for one animal on 4 consecutive test days. As only 1 animal was affected this observation was considered to be spontaneous.
- 750/500 mg/kg bw/day: salivation was noted for all 8 surviving animals (not toxicologically relevant).
- premature death (75 mg/kg bw/day; one male): the observations that were noted a few days before death during the daily cage side observations (e.g. prone position) were considered to be caused by the misgavage and the following poor health state of the animal.

2) Females:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 and 250 mg/kg bw/day: no observations were noted that were considered to be of toxicological relevance. In the low dose group, slight to moderate salivation was observed in 2/9 animals during the gestation period. In the 250 mg/kg bw/day dose group, slight to pronounced salivation was observed in 1/10, 7/9 and 4/9 animals during the premating/mating, gestation and lactation period, respectively.
- 750 mg/kg bw/day: salivation was noted in 5/10, 9/10 and 2/4 animals during the premating/mating, gestation and lactation period, respectively (not toxicologically relevant).
- premature deaths (750 mg/kg bw/day): pre-mortal symptoms were noted during the daily cage side observations for all prematurely deceased animals in the form of convulsions, tremors, lateral position, reduced motility, breathing sounds and or a haemorrhagic nose/snout.

Start and duration of observations:
- salivation: with only a few exceptions, salivation started immediately to 5 min after administration and disappeared again between 20 and 60 min after administration.
- reduced motility: a reduced motility was only recorded for 2 animals on their day of death (premature death (one male and one female; high dose group).
For the male animal a reduced motility was soon noted in the morning at work begin on test day 30 and lasted until its premature death on test day 30. Due to the poor health condition of the male no administration of test item was carried out anymore on this day. Hence, no correlation of the symptoms to the time point of administration was possible on this day. In case of the female animal a reduced motility was noted immediately to 5 min after administration in the morning of lactation day 9 and persisted until its premature death during the further course of lactation day 9.
- prone position: prone position was noted for the prematurely deceased male of the 75 mg/kg bw/day dose group on test day 47 and on test day 48. It started between 20 and 60 min after administration on test day 47 and persisted until the premature death on the following day (test day 48).
- convulsions: convulsions were noted for 4 of 6 prematurely deceased high dosed females one day before and/or on their day of death. It started immediately to 2 hours after administration and disappeared between 5 min and 2 hours after administration.
- tremors: tremors were noted for prematurely deceased female of the 750 mg/kg bw/day dose group, on lactation day 1, one day before the animal was found dead on lactation day 2. Tremors were noted immediately to 5 min after administration and disappeared between 20 and 60 min after administration.

Detailed clinical signs:
1) Males:
- 0, 75, 250 and 750/500 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the male animals of the treatment groups and the control group.

2) Females:
- 0, 75, 250 and 750 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the female animals of the treatment groups and the control group.

MORTALITY:
1) Males:
- 0, 75 and 250 mg/kg bw/day: no test item-related death was noted. One male of the 75 mg/kg bw/day dose group died during test day 48 (two days before
scheduled removal on test day 50). The premature death of the animal was considered to be caused by a misgavage a few days before death and not test item-related.

2) Females:
- 0, 75 and 250 mg/kg bw/day: no test item-related premature death was noted.

BODY WEIGHT AND WEIGHT CHANGES:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the dose groups. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treamtent groups.

FOOD CONSUMPTION:
1) Males (Pre-mating period):
- 75, 250 and 750/500 mg/kg bw/day: during the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the rats of the control group and the rats of the treatment groups.

2) Females (Pre-mating, gestation and lactation period):
- 75 and 250 mg/kg bw/day: no test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period.
- 750 mg/kg bw/day: no test item-related differences in food consumption were noted during the pre-mating period, the gestation period and the first week of the lactation period (lactation days 1 to 8).

DRINKING WATER CONSUMPTION:
Males and females:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related changes in the consumption of drinking water was noted for the male and female rats.

HAEMATOLOGICAL FINDINGS:
1) Males.
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 and 750/500 mg/kg bw/day: slightly decreased haemoglobin contents were noted (statistically significant at the 250 mg/kg bw/day dose level, p ≤ 0.05). However, as the changes were only slight and showed no dose-response relationship, they were considered to be spontaneous.
- 75 and 750/500 mg/kg bw/day: slightly decreased MCH and MCV values were noted (statistically significant for the MCH value at the 75 mg/kg bw/day dose level (p ≤ 0.05) and for the MCH value and the MCV value at the 750/500 mg/kg bw/day dose level (p ≤ 0.01)). However, no decreased MCH and MCV values were noted at the 250 mg/kg bw/day dose level. As the MCH and MCV values at the 250 mg/kg bw/day dose level were at the level of the control group, a dose-response relationship from the 75 mg/kg bw/day to the 750/500 mg/kg bw/day dose level was missing. Hence, the changes that were noted for the MCH and MCV values were considered to be spontaneous.

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 mg/kg bw/day: a slight but statistically significantly decrease of activated partial thromboplastin time (p ≤ 0.01) was noted, whereas the activated partial thromboplastin time at the 750 mg/kg bw/day dose level was again in the range of the control group. As no dose-response relationship was noted, this observation was considered to be spontaneous.

CLINICAL BIOCHEMISTRY FINDINGS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.
- 250 and 750 mg/kg bw/day: the slightly statistically significantly decreased protein (total) and globulin concentrations that were noted at the 250 and the 750 mg/kg bw/day dose group were considered to be spontaneous. All values were in the range of the mean value of the laboratory background data. The significant differences between the control group and the 250 and the 750 mg/kg bw/day dose group may be due to an increased protein (total) concentration at the control group. This assessment is in contrast to those that was made for the male animals for these parameters. However, whereas all protein (total) values from the 750 mg/kg bw/day dosed females were near the mean value of the laboratory background data, a higher variability was noted for the male animals and 2 of 5 values from the 750/500 mg/kg bw/day dosed males were below the lower range of the background data.
- 75, 250 and 750 mg/kg bw/day: the statistically significantly decreased glucose concentrations that were noted were due to an increased glucose concentration at the control group and considered to be spontaneous. All mean values of the treatment groups were at the same level and in the range of the mean value of the laboratory background data.
- 250 and 750 mg/kg bw/day: slightly decreased alanine aminotransferase activities were noted (statistically significant or not). However, increased activities are an indicator for organ damage (alanine aminotransferase release from destroyed cells), whereas decreased activities were not of toxicological relevance and could be considered as spontaneous.

NEUROBEHAVIOURAL EXAMINATION:
1) Males:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted for the male animals of all treatment groups. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the treated male animals. Lastly, no test item-related differences in spontaneous motility were noted between the control group and the treatment groups. However, statistically significantly reduced numbers of movements were noted at the low dose level (57.1 % below the control value; p ≤ 0.05) and the high dose level (70.2 % below the control value; p ≤ 0.01). These observations were considered to be spontaneous and not test item-related due to the following reasons: a high standard deviation was noted for all groups (including the control group) and nearly all individual values were in the range of laboratory background data (only one animal of the high dose group with only 26 movements in 12 minutes was slightly below the lower laboratory background range).

2) Females:
Note: at the 750 mg/kg bw/day dose level only 4 surviving female animals were still available at the time point of the neurological screening.
- 75 and 250 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the female animals of the low and the intermediate dose group.
- 75 and 750 mg/kg bw/day: the slight but statistically significantly increased hind leg splay that was noted for the animals of the 75 and the 750 mg/kg bw/day dose level (13.4 % and 17.4 % above the control group; p ≤ 0.05) was not considered to be test item related. No dose response relationship was noted and all individual values were in the range of laboratory background data or only slightly above (one animal of the low dose group with a hindleg splay of 10.5 cm).
- 75, 250 and 750 mg/kg bw/day: no test item-related differences in spontaneous motility were noted between the control group and the treatment groups.

ORGAN WEIGHTS INCLUDING ORGAN7BODY WEIGHT RATIOS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes were noted. The statistically significantly increased relative organ weight of the left kidney (p ≤ 0.05) that was noted revealed no dose-response relationship and was considered to be spontaneous.
- 250 mg/kg bw/day: slight, but statistically significantly decreased organ weights were noted for the absolute organ weights of the left and right epididimydes (p ≤ 0.01 and p ≤ 0.05, respectively). As the changes were only small and no statistically significant changes were noted for the relative organ weights of the left and right epididymis, these changes were considered to be spontaneous.
- 750/500 mg/kg bw/day: more pronounced changes as at the 250 mg/kg bw/day dose level (statistically significant decreased; at p ≤ 0.01) were noted for the absolute organ weights of the left and right epididymis, the combined prostate and seminal vesicle and the liver. However, these changes were considered to be due to the reduced body weight of the 750/500 mg/kg bw/day dosed animals, as no statistically significant changes were noted for the relative organ weights of these organs .
- 750/500 mg/kg bw/day: the slight but statistically significantly increased relative organ weight of the brain (p ≤ 0.01) that was noted was also due to the reduced body weight at the dose level, as the absolute organ weight of the brain was in the range of the control group.

2) Females:
- 75 mg/kg bw/day: no test item-related influence on the relative and the absolute organ weights was noted.
- 750 mg/kg bw/day: the statistically significantly decreased absolute organ weights of the heart and the liver (p ≤ 0.01) were considered to be due to the reduced body weight of the 4 surviving female animals, as no statistically significant decrease was noted for the relative organ weights.

GROSS PATHOLOGICAL FINDINGS:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the surviving male animals. However, different observations were noted at the two dose level. These observations were considered to be spontaneous, as they were only noted for 1 (e.g. soft and enlarged left kidney) or 2 animals (observed for 2 animals: an urinary bladder which was filled with content, noted in animals of the low dose group). However, the urinary bladder and the kidneys were considered as target organs during the histopathological examination of the animals of 750/500 dose group.
- premature death (75 mg/kg bw/day; one male): the macroscopic findings at necropsy were considered to be due to the misgavage (abdomen filled with clear liquid or considered to be spontaneous (prostate findings). The observation of soft kidneys was correlated with microscopic kidney findings (e.g. basophilic tubules in the cortex) which were noted for the animal.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the female animals. The stomach changes in the form of haemorrhagic foci that were noted for 1 female of the 75 mg/kg bw/day dose group and 1 female animal of the 250 mg/kg bw/day dose group were considered to be spontaneous, as only 1 animal per group was affected. However, due to microscopic and related macroscopic findings at the 750 mg/kg bw/day dose level, the stomach was considered as a target organ.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
Critical effects observed:
not specified
Conclusions:
In this combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, the repeated dose oral administration of cesium fluoroaluminate to male and female CD rats caused adverse toxcicological effects in the haematological parameters and clinical biochemistry at the 250 mg/kg bw/day dose level. Based on these findings the NOAEL is determined to be 75 mg/kg bw /day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

Cesium fluoroaluminate was tested in a repeated dose toxicity study with reproductive toxicity screen (OECD 422, GLP) in rats via oral administration at dose levels of 75, 250 or 750 mg test item/kg bw/day. Due to the premature death of 2 male animals the dose level of the high dosed male animals was reduced to 500 mg Cesium fluoroaluminate/kg bw/day on test day 38. The dose level for the female animals of the high dose group remained at 750 mg Cesium fluoroaluminate/kg bw/day. Mortality in female animals was seen during the lactation period (between lactation days 1 and 9).

A NOAEL of 75 mg/kg bw/day was identified based on statistical significant changes in haematological and clinical biochemistry at the 250 mg/kg bw/day.

 

Justification for classification or non-classification