Cesium potassium fluoroaluminate

Endpoint summary

Description of key information

Oral exposure to cesium potassium fluoroaluminate (OECD 422, GLP) resulted in treatment-related changes in the stomach at all dose levels (local NOAEL <10 mg/kg bw) and in decreased plasma levels of total protein, albumin and urea and an increase in total cholesterol in the high-dose group (systemic NOAEL 30 mg/kg bw).

Based on read-across from multiconstituent aluminium potassium fluoride, the  most critical effects of cesium potassium fluoroaluminate after repeated inhalation exposure are expected to be effects on the lungs. Based on the available study, the overall NOAEC for local effects is 1.21 mg/m3. The overall systemic NOAEC is >3.08 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 Jan 2016 to 09 Sept 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

03 November 2015

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

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

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

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

Observations and examinations performed and frequency:

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

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

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

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

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

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

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

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

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

Sacrifice and pathology:

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

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

Other examinations:

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

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

Local

Effect level:

< 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

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

Executive summary:

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

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

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

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

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

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 Oct 2003 to 23 Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

19 August 2004

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: males: 208 g; females: 157 g
- Housing: five rats/cage, in macrolon cages with bedding based on corn cobs or wood shavings.
- Diet: ad libitum, RM3 breeding diet (Special Diets Services)
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (+/-3)
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: low exposure: 2.2 µm / 2.1
mid exposure: 2.6 µm / 2.1
high exposure: 2.6 µm / 2.0

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only inhalation unit
- Method of holding animals in test chamber: restraining tube
- Source and rate of air: compressed air
- Method of conditioning air: humidification
- System of generating particulates/aerosols: A test atmosphere was generated in a base inhalation unit by passing test material to a Vac-eductor - which aerosolizes the test material - using a dry material feeder. From the base inhalation unit, parts of the test atmosphere were extracted using eductors to dilute and transport the test atmospheres to the various exposure units.
- Temperature, humidity, pressure in air chamber: Mean temperature: 21.2, 22.4, 22.1, 23.6 °C; mean relative humidity: 46.4, 48.0, 45.6, 34.8%; for the control, low-, mid-, and high-concentration exposure, respectively. Pressure: generally +10 Pa
- Air flow rate: 59.7- 72.1 l/min
- Method of particle size determination: a 10-stage cascade impactor

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Once daily in low- and mid-concentration groups, two times a day in the high-concentration group.
- Representative test atmosphere samples were obtained from the animals' breathing zone by passing test atmospheres through fibre glass filters. The filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again after sampling. The concentration of test substance was calculated by dividing the amount of test material present on each filter by the volume of the test atmosphere taken.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week, 6 hours/day

Remarks:

Doses / Concentrations:
0.3, 1, 3 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0.32 (0.04), 1.21(0.18), 3.08 (0.26) mg/m3 (mean (SD))
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: based on 2 previously performed 28-day toxicity studies
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 2 recovery groups (control and high dose) were kept for post-exposure observation for 60 days
- Section schedule rationale (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes. Twice/day on workdays, once/day during the weekend

DETAILED CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatization, one day before the first treatment, at initiation of treatment, once per week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes

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: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of treatment (all animals) and during the last week of exposure in the control and high dose group

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necopsy
- Anaesthetic used for blood collection: Yes, Nembutal
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study groups
- Haematology parameters: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count. Calculated: mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration. In addition: total white blood cell count, differential white blood cell count, red blood cell count, haemoglobin, packed cell volume and thrombocyte count were determined in (overnight fasted) females at the end of the recovery period.
- Clinical chemistry parameters: ALP activity, ASAT activity, ALAT activity, GGT activity, bilirubin total, cholesterol, triglycerides, total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, phospholipids, Ca, Na, K, Cl, inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: day before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: Main study (all groups except recovery): volume, creatinine, fluoride content in control and high-concentration groups, and in females of the low- and mid-concentration groups. Recovery groups: urinary volume in males and females; creatinine, fluoride content in females

Sacrifice and pathology:

- GROSS PATHOLOGY: Yes, complete gross necropsy. Organs weighed: adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, thyroid with parathyroids, lungs with trachea and larynx, ovaries, uterus, epididymides

- HISTOPATHOLOGY: Yes
Tissues examined: Main study: respiratory tract , teeth in control and high exposure groups. Lungs and larynx in low and mid exposure groups. Recovery groups: lungs and larynx
The histopathological examination was extended to include all organs and tissues of the animals of the control and high concentration groups.

Statistics:

Bodyweight: one-way analysis of covariance followed by Dunnett's multiple comparison tests and by Student's t-tests in case of recovery groups
Food consumption/efficiency: one-way ANOVA followed by the Least Significant Difference test
Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, urinalysis, organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests
Reticulocytes and relative differential white blood cell counts: Kruskal-Walllis non-parametric ANOVA followed by Mann-Whitney U-tests
Histopathology: Fisher's exact probability test
All tests were two-sided. probability values of p<0.05 were considered significant.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No treatment-related clinical signs or mortality were detected. Two males and two females of the control group died, most probably due to hypothermia.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related changes were observed.

FOOD INTAKE / FOOD CONVERSION: No treatment-related changes were observed.
HAEMATOLOGY: Increases in absolute and relative numbers of neutrophils in females of the high exposure group, only absolute number was statistically significant. At the end of the recovery, absolute and relative numbers were still higher. The increase in percentage of neutrophils was accompanied by a decrease in percentage of lymphocytes.

CLINICAL CHEMISTRY: No treatment-related changes were observed.
URINALYSIS: Urinary volume, and total (16-hour) creatinine and fluoride excretion were higher in females of the high exposure group. At the end of the recovery period, no changes were observed.

ORGAN WEIGHTS: Absolute and relative lung weights were higher in females of the high exposure group, only absolute weight was statistically significant. No changes were observed at the end of the recovery period.

GROSS NECROPSY: No treatment-related changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: Concentration-related changes in the lungs: typical alveolar macrophage accumulations in animals of the mid and high concentration group. A tissue reaction was absent. The macrophage accumulations persisted after the recovery period. The macrophages were somewhat smaller in size when compared to those in animals of the high concentration group at the end of the exposure period, but more conspicuous because their cytoplasm was darkly stained. Despite the persistent presence of the macrophages, a tissue reaction was still absent.
The presence of these macrophages are considered a physiological response to the exposure and therefore not considered adverse as such.

Microscopic examination of the non-respiratory tract organs and tissues did not reveal treatment-related changes in the animals of the high exposure group at the end of the treatment period. The histopathological changes observed are common findings in rats of this strain and age. Furthermore, they were about equally distributed amongst the different treatment groups or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Key result

Dose descriptor:

NOAEC

Remarks:

(local effects)

Effect level:

1.21 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEC

Remarks:

(systemic effects)

Effect level:

> 3.08 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects occurred

Critical effects observed:

not specified

Conclusions:

From the results of the present study in rats, it was concluded that the most critical effects of the substance after repeated inhalation exposure were effects on the lungs. The overall NOAEC for local effects is 1.21 mg/m3. The overall systemic NOAEC is >3.08 mg/m3

Executive summary:

The inhalation toxicity of multiconstituent aluminium potassium fluoride was studied in a sub-chronic (90-day) study in Wistar rats. Groups of 10 male and 10 female rats were exposed to target concentrations of 0 (control), 0.3, 1, or 3 mg/m3 for six hours a day, 5 days a week during a period of 90 days, with a total of 62 - 63 exposure days. The rats were necropsied the day after the last exposure. Satellite groups of 10 male and 10 female animais each, similarly exposed to target concentrations of 0 or 3 mg/m3 of the test substance were kept for an additional recovery period of 60 days. To examine the toxicity of the test material clinical signs, ophthalmology, body weights, food consumption, food conversion efficiency, haematology, clinical chemistry, and urinalysis were used. In addition, a full necropsy was performed and the respiratory tract was examined microscopically.

The concentration levels used during the 90-day study were based on a previously performed 28-day study with a total of 20 exposure days. The level of 1 mg/m3 was considered to be a Minimal-Observed-Adverse-Effect Level (MOAEL). This was based on the presence of typical alveolar macrophage accumulations with cellular debris/material lying freely in the alveolar lumen, suggesting impairment or insufficient clearance capacity of the alveolar macrophages.

In the present study, the mean actual concentrations of multiconsituent aluminium potassium fluoride in the test atmospheres, based on gravimetrical analysis were 0.32, 1.21, and 3.08 mg/m3 , for the low, mid, and high concentration, respectively. Mean Mass Median Aerodynamic Diameters (MMAD) were 2.2, 2.6 and 2.6 μm with corresponding geometric standard deviations of 2.1, 2.1 and 2.0, respectively.

No treatment-related abnormalities were observed. Two male and two female animals of the control group were found dead during exposure on day 58 of the study most probably due to hypothermia. Another similarly affected control male recovered within 3 days. No treatment-related changes in body weight gain were observed. No treatment-related changes in food intake and food conversion efficiency were observed. No treatment-related changes in red blood cell variables were observed. Increases in both absolute and relative numbers of neutrophils were observed in females of the high concentration group at the end of the exposure period, but a statistical significant degree was reached in absolute number only. At the end of the recovery period, absolute and relative neutrophil counts were still higher in females. The increase in the percentage of neutrophils in these females was accompanied by a decrease in the percentage of lymphocytes at that time. No treatment-related changes in clinical chemistry were observed.

Absolute and relative lung weights were higher in females of the high concentration at the end of the exposure period; a statistical significant degree was observed in absolute weight only. In males no such changes were seen. At the end of the recovery period, no changes in lung weights were observed.  Macroscopic examination at necropsy did not reveal treatment-related changes. Microscopic examination of the respiratory tract at the end of the 90-day exposure period revealed a concentration-related change in the lungs consisting of typical alveolar macrophage accumulations in animals of the mid and high concentration group. A tissue reaction appeared absent. The macrophage accumulations persisted after a recovery period of 60 days. The macrophages were somewhat smaller in size when compared to those in animals of the high concentration group at the end of the exposure period, but more conspicuous because their cytoplasm was darkly stained. Despite the persistent presence of the macrophages, a tissue reaction was still absent. The presence of the macrophages are considered a physiological response to the exposure and therefore not considered adverse as such. Therefore, 1.21 mg/m3 is considered a NOAEC for local effects. The overall systemic NOAEC is >3.08 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

3.08 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

System:

respiratory system: lower respiratory tract

Organ:

lungs

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 Oct 2003 to 23 Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

19 August 2004

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: males: 208 g; females: 157 g
- Housing: five rats/cage, in macrolon cages with bedding based on corn cobs or wood shavings.
- Diet: ad libitum, RM3 breeding diet (Special Diets Services)
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (+/-3)
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: low exposure: 2.2 µm / 2.1
mid exposure: 2.6 µm / 2.1
high exposure: 2.6 µm / 2.0

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only inhalation unit
- Method of holding animals in test chamber: restraining tube
- Source and rate of air: compressed air
- Method of conditioning air: humidification
- System of generating particulates/aerosols: A test atmosphere was generated in a base inhalation unit by passing test material to a Vac-eductor - which aerosolizes the test material - using a dry material feeder. From the base inhalation unit, parts of the test atmosphere were extracted using eductors to dilute and transport the test atmospheres to the various exposure units.
- Temperature, humidity, pressure in air chamber: Mean temperature: 21.2, 22.4, 22.1, 23.6 °C; mean relative humidity: 46.4, 48.0, 45.6, 34.8%; for the control, low-, mid-, and high-concentration exposure, respectively. Pressure: generally +10 Pa
- Air flow rate: 59.7- 72.1 l/min
- Method of particle size determination: a 10-stage cascade impactor

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Once daily in low- and mid-concentration groups, two times a day in the high-concentration group.
- Representative test atmosphere samples were obtained from the animals' breathing zone by passing test atmospheres through fibre glass filters. The filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again after sampling. The concentration of test substance was calculated by dividing the amount of test material present on each filter by the volume of the test atmosphere taken.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week, 6 hours/day

Remarks:

Doses / Concentrations:
0.3, 1, 3 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0.32 (0.04), 1.21(0.18), 3.08 (0.26) mg/m3 (mean (SD))
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: based on 2 previously performed 28-day toxicity studies
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 2 recovery groups (control and high dose) were kept for post-exposure observation for 60 days
- Section schedule rationale (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes. Twice/day on workdays, once/day during the weekend

DETAILED CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatization, one day before the first treatment, at initiation of treatment, once per week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes

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: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of treatment (all animals) and during the last week of exposure in the control and high dose group

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necopsy
- Anaesthetic used for blood collection: Yes, Nembutal
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study groups
- Haematology parameters: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count. Calculated: mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration. In addition: total white blood cell count, differential white blood cell count, red blood cell count, haemoglobin, packed cell volume and thrombocyte count were determined in (overnight fasted) females at the end of the recovery period.
- Clinical chemistry parameters: ALP activity, ASAT activity, ALAT activity, GGT activity, bilirubin total, cholesterol, triglycerides, total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, phospholipids, Ca, Na, K, Cl, inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: day before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: Main study (all groups except recovery): volume, creatinine, fluoride content in control and high-concentration groups, and in females of the low- and mid-concentration groups. Recovery groups: urinary volume in males and females; creatinine, fluoride content in females

Sacrifice and pathology:

- GROSS PATHOLOGY: Yes, complete gross necropsy. Organs weighed: adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, thyroid with parathyroids, lungs with trachea and larynx, ovaries, uterus, epididymides

- HISTOPATHOLOGY: Yes
Tissues examined: Main study: respiratory tract , teeth in control and high exposure groups. Lungs and larynx in low and mid exposure groups. Recovery groups: lungs and larynx
The histopathological examination was extended to include all organs and tissues of the animals of the control and high concentration groups.

Statistics:

Bodyweight: one-way analysis of covariance followed by Dunnett's multiple comparison tests and by Student's t-tests in case of recovery groups
Food consumption/efficiency: one-way ANOVA followed by the Least Significant Difference test
Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, urinalysis, organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests
Reticulocytes and relative differential white blood cell counts: Kruskal-Walllis non-parametric ANOVA followed by Mann-Whitney U-tests
Histopathology: Fisher's exact probability test
All tests were two-sided. probability values of p<0.05 were considered significant.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No treatment-related clinical signs or mortality were detected. Two males and two females of the control group died, most probably due to hypothermia.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related changes were observed.

FOOD INTAKE / FOOD CONVERSION: No treatment-related changes were observed.
HAEMATOLOGY: Increases in absolute and relative numbers of neutrophils in females of the high exposure group, only absolute number was statistically significant. At the end of the recovery, absolute and relative numbers were still higher. The increase in percentage of neutrophils was accompanied by a decrease in percentage of lymphocytes.

CLINICAL CHEMISTRY: No treatment-related changes were observed.
URINALYSIS: Urinary volume, and total (16-hour) creatinine and fluoride excretion were higher in females of the high exposure group. At the end of the recovery period, no changes were observed.

ORGAN WEIGHTS: Absolute and relative lung weights were higher in females of the high exposure group, only absolute weight was statistically significant. No changes were observed at the end of the recovery period.

GROSS NECROPSY: No treatment-related changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: Concentration-related changes in the lungs: typical alveolar macrophage accumulations in animals of the mid and high concentration group. A tissue reaction was absent. The macrophage accumulations persisted after the recovery period. The macrophages were somewhat smaller in size when compared to those in animals of the high concentration group at the end of the exposure period, but more conspicuous because their cytoplasm was darkly stained. Despite the persistent presence of the macrophages, a tissue reaction was still absent.
The presence of these macrophages are considered a physiological response to the exposure and therefore not considered adverse as such.

Microscopic examination of the non-respiratory tract organs and tissues did not reveal treatment-related changes in the animals of the high exposure group at the end of the treatment period. The histopathological changes observed are common findings in rats of this strain and age. Furthermore, they were about equally distributed amongst the different treatment groups or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Key result

Dose descriptor:

NOAEC

Remarks:

(local effects)

Effect level:

1.21 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEC

Remarks:

(systemic effects)

Effect level:

> 3.08 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects occurred

Critical effects observed:

not specified

Conclusions:

From the results of the present study in rats, it was concluded that the most critical effects of the substance after repeated inhalation exposure were effects on the lungs. The overall NOAEC for local effects is 1.21 mg/m3. The overall systemic NOAEC is >3.08 mg/m3

Executive summary:

The inhalation toxicity of multiconstituent aluminium potassium fluoride was studied in a sub-chronic (90-day) study in Wistar rats. Groups of 10 male and 10 female rats were exposed to target concentrations of 0 (control), 0.3, 1, or 3 mg/m3 for six hours a day, 5 days a week during a period of 90 days, with a total of 62 - 63 exposure days. The rats were necropsied the day after the last exposure. Satellite groups of 10 male and 10 female animais each, similarly exposed to target concentrations of 0 or 3 mg/m3 of the test substance were kept for an additional recovery period of 60 days. To examine the toxicity of the test material clinical signs, ophthalmology, body weights, food consumption, food conversion efficiency, haematology, clinical chemistry, and urinalysis were used. In addition, a full necropsy was performed and the respiratory tract was examined microscopically.

The concentration levels used during the 90-day study were based on a previously performed 28-day study with a total of 20 exposure days. The level of 1 mg/m3 was considered to be a Minimal-Observed-Adverse-Effect Level (MOAEL). This was based on the presence of typical alveolar macrophage accumulations with cellular debris/material lying freely in the alveolar lumen, suggesting impairment or insufficient clearance capacity of the alveolar macrophages.

In the present study, the mean actual concentrations of multiconsituent aluminium potassium fluoride in the test atmospheres, based on gravimetrical analysis were 0.32, 1.21, and 3.08 mg/m3 , for the low, mid, and high concentration, respectively. Mean Mass Median Aerodynamic Diameters (MMAD) were 2.2, 2.6 and 2.6 μm with corresponding geometric standard deviations of 2.1, 2.1 and 2.0, respectively.

No treatment-related abnormalities were observed. Two male and two female animals of the control group were found dead during exposure on day 58 of the study most probably due to hypothermia. Another similarly affected control male recovered within 3 days. No treatment-related changes in body weight gain were observed. No treatment-related changes in food intake and food conversion efficiency were observed. No treatment-related changes in red blood cell variables were observed. Increases in both absolute and relative numbers of neutrophils were observed in females of the high concentration group at the end of the exposure period, but a statistical significant degree was reached in absolute number only. At the end of the recovery period, absolute and relative neutrophil counts were still higher in females. The increase in the percentage of neutrophils in these females was accompanied by a decrease in the percentage of lymphocytes at that time. No treatment-related changes in clinical chemistry were observed.

Absolute and relative lung weights were higher in females of the high concentration at the end of the exposure period; a statistical significant degree was observed in absolute weight only. In males no such changes were seen. At the end of the recovery period, no changes in lung weights were observed.  Macroscopic examination at necropsy did not reveal treatment-related changes. Microscopic examination of the respiratory tract at the end of the 90-day exposure period revealed a concentration-related change in the lungs consisting of typical alveolar macrophage accumulations in animals of the mid and high concentration group. A tissue reaction appeared absent. The macrophage accumulations persisted after a recovery period of 60 days. The macrophages were somewhat smaller in size when compared to those in animals of the high concentration group at the end of the exposure period, but more conspicuous because their cytoplasm was darkly stained. Despite the persistent presence of the macrophages, a tissue reaction was still absent. The presence of the macrophages are considered a physiological response to the exposure and therefore not considered adverse as such. Therefore, 1.21 mg/m3 is considered a NOAEC for local effects. The overall systemic NOAEC is >3.08 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.21 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

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

A sub-acute toxicity study by the oral route is available to assess the toxicity of cesium potassium fluoroaluminate after repeated exposure. Since no sub-chronic toxicity study is available for cesium potassium fluoroaluminate, the results from the structural analogue multiconstituent aluminium potassium fluoride are used instead (for details see Read-across justification as attached in section 13). The studies are described below.

Oral

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

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

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

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

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

Inhalation

The inhalation toxicity of aerosols of multiconstituent aluminium potassium fluoride was studied in a subchronic (90-day) study in rats (performed according to OECD guideline 413 and under GLP). Groups of 10 male and 10 female rats were exposed to test substance concentrations of 0 (control), 0.32, 1.21, or 3.08 mg/m3.

Increases in both absolute and relative numbers of neutrophils were observed in females of the high concentration group at the end of the exposure period, but a statistical significant degree was reached in absolute number only. At the end of the recovery period, absolute and relative neutrophil counts were still higher in females. The increase in the percentage of neutrophils in these females was accompanied by a decrease in the percentage of lymphocytes at that time. Absolute and relative lung weights were higher in females of the high concentration at the end of the exposure period; a statistical significant degree was observed in absolute weight only. In males no such changes were seen. At the end of the recovery period, no changes in lung weights were observed.  Macroscopic examination at necropsy did not reveal treatment-related changes. Microscopic examination of the respiratory tract at the end of the 90-day exposure period revealed a concentration-related change in the lungs consisting of typical alveolar macrophage accumulations in animals of the mid and high concentration group. A tissue reaction appeared absent. The macrophage accumulations persisted after a recovery period of 60 days. The macrophages were somewhat smaller in size when compared to those in animals of the high concentration group at the end of the exposure period, but more conspicuous because their cytoplasm was darkly stained. Despite the persistent presence of the macrophages, a tissue reaction was still absent. The presence of the macrophages are considered a physiological response to the exposure and therefore not considered adverse as such. Therefore, 1.21 mg/m3 is considered a NOAEC for local effects.The overall systemic NOAEC is 3.08 mg/m3. These values will be taken forward to risk assessment.

Justification for classification or non-classification

In a sub-chronic inhalation study with the read-across candidate multiconsituent aluminium potassium fluoride a NOAEC of 1.21 mg/m3 was established based on local effects in the lungs. Therefore the substance cesium potassium fluoroaluminate needs to be classified as STOT Rep. Exp. Cat. 1; H372 for the inhalation route according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.