Cesium potassium fluoroaluminate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

other: 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

Test material information

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 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 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%.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug.
This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

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.

Duration of test:

29 days for males and 42 to 54 days for females.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

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.
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 bodyweight.

Examinations

Maternal examinations:

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:

Fetal examinations:

All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence no milk. If possible, defects or cause of death were evaluated.

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).

Indices:

Post-implantation survival index (%)
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%)
Viability index (%)
Lactation index (%)
Group mean values were calculated from individual litter values.
Sex ratio (percentage males)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Description (incidence and severity):

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

Food efficiency:

not examined

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, non-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.

Maternal developmental toxicity

Dead fetuses:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- Fertility and Conception index: Fertility and conception index were not affected by treatment.
One control female (no. 44, mated with male no. 4) and one female at 100 mg/kg (no. 80, mated with male no. 40) were not pregnant (as confirmed by negative Salewski staining). No abnormalities were seen in the reproductive organs which could account for their nonpregnancy.
In the absence of a dose-related incidence, this non-pregnancy was considered not to be related to treatment.
- Precoital time: Precoital time was not affected by treatment.
- Number of implantation sites: Number of implantation sites was not affected by treatment.
For female nos. 59 (10 mg/kg), 62 (30 mg/kg) and 71 (100 mg/kg), the number of pups born was slightly higher than the number of implantations. This was considered to be due to normal resorption of these a reas as these enumerations were performed on Day 16 of lactation.
- Microscopic examination of reproductive organs and spermatogenic profiling: There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

2

Key result

Dose descriptor:

NOAEL

Remarks:

Local

Effect level:

< 10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

other: the whole parameters

Results (fetuses)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- Live birth index
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
The slightly lower mean number of living pups observed at first litter check at 100 mg/kg was caused by the total litter loss in a single female (no.76) within that group. An incidental total litter loss is a normal biological event and based on its single occurrence within this study considered on toxicological significance. Dead pups at first litter check were also observed in a few other females within this and other group(s).
These number of dead pups were considered to be within the normal background incidence.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.

Other effects:

no effects observed

Description (incidence and severity):

- Viability index
The number of live offspring on Day 4 before culling compared to the number of live offspring on Day 1 was not affected by treatment.
One pup at 10 mg/kg (litter no. 56) was killed in extremis on lactation Day 1 because this pups had a broken neck. Two pups of the control group (litters no. 45 and 47) and three pups at 10 mg/kg (litter no. 60) went missing on Days 2 or 3 of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

- Lactation index
No pups were found dead or went missing between lactation Days 5 and 13. For each group, the number of live offspring on Day 13 after littering was the same as the number of live offspring on Day 4 (after culling), resulting in a lactation index of 100% for all groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: developmental toxicity

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

There were no treatment-related changes in the in-life parameters investigated (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption) and in the haematology parameters and organ weights in this study up to 100 mg/kg.
No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg).
No developmental toxicity was observed up to the highest dose level tested (100 mg/kg).

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Nocolok Cs Flux (SM) in rats by oral gavage was conducted according to the OECD guidelines 422 and 407 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 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. Parental results: There were no treatment-related changes the in-life results, haematology parameters or organ weights. Clinical biochemistry parameters showed treatment-related decreases in total protein and albumin, dosedependently, 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. Treatment-related vacuolation of the zona glomerulosa in the adrenal gland, slightly above background, was noted at 100 mg/kg in females. In the absence of any degenerative changes, this finding was considered to be non-adverse. No reproduction or developmental toxicity was observed up to the highest dose level tested (100 mg/kg).

Based on the above results, the following No Observed Adverse Effect Levels (NOAELs) were derived.

- Developmental NOAEL: at least 100 mg/kg.

- Maternal toxicity NOAEL: at least 100 mg/kg.