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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Reference substance name:
Aluminum potassium fluoride
EC Number:
EC Name:
Aluminum potassium fluoride
Cas Number:
Details on test material:
- Batch number: 5878
- Purity:
K: 29.1 %wt
Al: 16.9 %wt
F: 50.7 %wt
Ca: 0.003 %wt
Fe: 0.01 %wt

Test animals

Details on test animals or test system and environmental conditions:
- 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

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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: water
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:
- 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

- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
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
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.3, 1, 3 mg/m3
other: target concentrations
Doses / Concentrations:
0.32 (0.04), 1.21(0.18), 3.08 (0.26) mg/m3 (mean (SD))
analytical conc.
No. of animals per sex per dose:
Control animals:
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


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

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

- 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


- 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

- 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

- 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

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.
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
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.
Remark of the reviewer:
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.

Effect levels

open allclose all
Dose descriptor:
(local effects)
Effect level:
1.21 mg/m³ air
Basis for effect level:
other: see 'Remark'
Dose descriptor:
(systemic effects)
Effect level:
3.08 mg/m³ air
Basis for effect level:
other: no adverse systemic effects occurred

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion