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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
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

Test material

Constituent 1
Test material form:
solid: crystalline
Details on test material:
Identification: Nocolok Cs Flux (SM)
Chemical name (IUPAC), synonym or trade name: Cesium potassium fluoroaluminate
CAS Number: 566947-29-3
Molecular formula: K(x)Cs(y)AlF(z)
Appearance: White powder
Batch: MPA 22002a
Purity/Composition: >99%
Test item storage: At room temperature
Specific details on test material used for the study:
Stable under storage conditions until 31 July 2016 (expiry date)

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

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.