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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Potassium tetrafluoroborate
- Physical state: White powder
- Analytical purity: 99.1%
- Storage condition of test material: At room temperature in the dark
- Lot/batch No.: BWF10626
- Expiration date of the lot/batch: 31 July 2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 weeks
- Weight at study initiation: 243 g (males); and 171 g (females)
- Fasting period before study: none
- Housing: macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and a wooden block and strips of paper (Enviro-dri) as environmental enrichment, 5 animals to a cage
- Diet: cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diet Services, Whitham, England), ad libitum
- Water: domestic mains tap-water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- 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
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 1.68 μm (with a geometric standard deviation (gsd) of 2.31), 2.57 μm (gsd of 2.57) and 2.52 μm (gsd of 2.54) for the low, mid and high concentration test atmospheres, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical polypropylene (group 1) or aluminium (groups 2-4) column, surrounded by a transparent cylinder. The column had a volume of approximately 50 litres and consisted of a top assembly with the entrance of the unit, a mixing chamber, a rodent tube section and at the bottom the base assembly with the exhaust port.
- Method of holding animals in test chamber: secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
- Source of air: fresh test atmosphere.
- System of generating particulates/aerosols: Since the aerodynamic particle size of the original test material (as delivered by the sponsor) was above the range of 1-3 μm recommended by OECD guideline 412, the test material was milled using a ball mill fitted with a agate beaker and 5 agate balls with a diameter of 20 mm. Among a few alternative scenarios, milling of batches of 5 gram of test material for a period of 30 minutes resulted in a powder that could be aerosolized with a MMAD (mass median aerodynamic particle size) below 3 μm. The test atmospheres for groups 3 and 4 were generated by aerosolization of the test material using a turntable dust feeder, an eductor and a jetmill. The eductor was supplied with humidified compressed air and operated at a pressure of 1.0 bar; the jetmill was supplied with dry compressed air and operated at 5.0 bar. The test atmosphere, exhausted from the jetmill at the top inlet of the exposure unit, was directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted. To generate the test atmosphere for group 2, part of the atmosphere of group 3 was extracted using an eductor. The eductor was mounted in the rodent tube section of the exposure chamber of group 3 and was supplied with humidified compressed air to dilute the test atmosphere. The resulting aerosol was directed towards the top inlet of the exposure chamber of group 2.
- Temperature and humidity in air chamber: controlled at a temperature of 22 ± 2°C and 30-70% relative humidity.
- Air flow rate: ≥1 litre/min for each rat.
- Method of particle size determination: carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

TEST ATMOSPHERE
- Brief description of analytical method used: by means of gravimetric analysis.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative test atmosphere samples were obtained from the animals’ breathing zone during the main study by passing 138, 46 and 18.4 Ln2 test atmosphere at 4.6 Ln/min through fiber glass filters. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.
Duration of treatment / exposure:
A total of 20 exposure days over a 28-day period.
Frequency of treatment:
6 hours/day, 5 days/week.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25, 75, 225 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
20.5 (± 1.8), 74.0 (± 5.1) and 224.2 (± 10.1) mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
38.4 (± 2.7), 107.3 (± 11.3) and 264.4 (± 12.9) mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on a 7-day range finding study in which no exposure-related changes were found up to a concentration of 104.4 mg/m3, the highest concentration tested.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway through each exposure day. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see cage side observations.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded two days before the start of exposure and prior to exposure on the first day (day 0). Subsequently, animals were weighed twice weekly for the first two weeks (on days 4, 7, 11 and 14). Thereafter, the frequency was reduced to once weekly (i.e. on days 21 and 27), because there were no statistically significant effects on body weight in the first two weeks. All animals were also weighed on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION: Yes
- Food consumption was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Consumption was measured over three 7-day periods, starting on the day of the first exposure, followed by a 6-day period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: 3 males and 5 females of each group (due to an error in handling of the blood samples, clotting occurred in 2 of 5 samples of the male animals of each group, preventing haematological analysis)
- Parameters examined: haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell count, prothrombin time, thrombocyte count. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate.

URINALYSIS: No (Samples of urine were stored for one year for possible future analysis)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
SACRIFICE: The animals were sacrificed on the day after the last exposure in such a sequence that the average time of killing was approximately the same for each group. Animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia.

GROSS PATHOLOGY: Yes.
HISTOPATHOLOGY: Yes
Other examinations:
None data.
Statistics:
- Body weight data: ‘Ancova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ as data transformation method (abbreviation AUTO).
- Haematology, clinical chemistry and organ weight data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method (abbreviation AUTO).
- Food consumption: no statistics were applied.
- Incidences of histopathological changes: Fisher’s exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The only clinical abnormalities observed during the study were sparsely haired areas and encrustations of the skin on the back of a few females in the last 1-2 weeks of the study. These findings, probably the results of some irritation due to the restraint in restraining tubes during exposure, were observed throughout the groups – including the control group – and were not related to exposure to the test material.

BODY WEIGHT AND WEIGHT GAIN
Male animals of the high concentration group showed a decreased body weight gain when compared to control animals, which reached statistical significance by day 21. On day 4 of the study, an increased body weight was observed in male animals of the low concentration group. Since this was an isolated finding, it was not considered to be treatment-related. No effects on growth were observed in female animals.

FOOD CONSUMPTION:
Slightly decreased consumption of food was observed in male animals of the high concentration group, which was most evident in the last week of the study. In female animals, food consumption was similar among the groups throughout the study period.

HAEMATOLOGY
Although in male animals only 3 out of 5 samples per group could be analyzed, it was clear that exposure-related changes in haematology parameters did not occur in male or in female animals.

CLINICAL CHEMISTRY
Clinical chemistry results indicated a dose-dependent increase in plasma activity of alkaline phosphatase (ALP) in male animals, which reached statistical significance at the mid and high concentration level. No such increase was observed in female animals. Decreased plasma concentrations of cholesterol and phospholipids were found in male animals of the mid concentration group. In the absence of a dose-response relationship and any corroborative changes in females, this was considered to be a change finding.

ORGAN WEIGHTS
Absolute weights of almost all organs were decreased in males of the high concentration group, when compared to unexposed controls. The decrease in brain and liver weight reached statistical significance. Although brain weight is usually relatively unaffected by body weight changes, it is highly likely that all absolute organ weight changes were a secondary effect associated with the growth retardation, since the changes occurred in almost all organs and relative organ weights were unaffected. No organ weight changes were observed in females.

GROSS PATHOLOGY
Macroscopic examination at the end of the exposure period revealed no treatment-related gross lesions. The abnormalities observed, mostly representing background pathology, were considered to be chance findings because they occurred only incidentally or at random incidences between the groups. As already mentioned, the sparsely haired areas and encrustions of the skin in a few females were probable the result of some irritation due to the restraint during exposure, and were not related to exposure to the test material.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histopathological changes in the respiratory tract were slight and focal, observed at similar incidences in exposed animals and controls, and were considered part of common background pathology. No visible test material residues were detected in the airways of animals exposed to the high concentration Potassium tetrafluoroborate. No microscopic abnormalities were observed in the thyroid, a possible target organ of the test material. Microscopic examination of the other organs and tissues did not reveal any treatment-related changes either; the histopathological changes observed occurred only incidentally and/or at similar incidences between the control and the high concentration group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
74 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Impaired growth and reduced food intake in male animals of the high concentration group.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A NOAEC of 74 mg/m3 was established in a 28-day inhalation study in rats.
Executive summary:

In a GLP compliant inhalation toxicity study performed according to OECD Guideline 412, potassium tetrafluoroborate was administered to Wistar rats. Four groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (control), 25, 75 or 225 mg/m3 potassium tetrafluoroborate for 6 hours/day, 5 days/week over a 28 -day period, with a total of 20 exposure days. The concentration levels were chosen based on the results of a 7 -day range finding study in which no exposure related changes were found up to a concentration of 104.4 mg/m3, the highest concentration tested. The mean actual concentrations (± standard deviation) of potassium tetrafluoroborate in the various test atmospheres – based on gravimetric analysis – were 20.5 (± 1.8), 74.0 (± 5.1) and 224.2 (± 10.1) mg/m3 for the low, mid and high concentration levels, respectively. The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 1.68μm (with a geometric standard derviation (gsd) of 2.31), 2.57μm (gsd of 2.57) and 2.52μm (gsd of 2.54) for the low, mid and high concentration test atmospheres, respectively. No treatment-related clinical abnormalities or mortality were observed in response to the exposure to potassium tetrafluoroborate. Decreased body weight gain was observed in males of the high concentration group. A concomitant reduction in food consumption was observed in males of this group. No other changes in body weight or food consumption were observed during the study. Hematology, conducted in all rats at necropsy, did not reveal any treatment-related abnormalities. Clinical chemistry, conducted in plasma obtained from all rats at necropsy, did not show any treatment-related changes. An elevated – although within the historical control range – alkaline phosphatase (ALP) activity in males of the mid and high concentration group was not corroborated by changes in other parameters and was therefore considered to be of no toxicological relevance. There were no treatment-related changes in absolute organ weights or in organ to body weight ratios. The slightly decreased absolute weights of almost all organs in males of the high concentration group – of which brain and liver weight reached statistical significance – were considered secondary to the growth impairment, as relative organ weight remained unaffected. Macroscopic examination at necropsy and histopathological examination of organs and tissues – including the complete respiratory tract – did not reveal any treatment-related changes. No visible residues of test material were detected in the airways during histopathological examination. No indications of impaired thyroid function – as judged by possible organ weight or microscopic changes – were observed. In conclusion, exposure to potassium tetrafluoroborate resulted in impaired growth and reduced food intake in male animals of the high concentration group. Therefore, the NOAEC in rats was placed at the mid concentration level of 74.0 mg/m3 potassium tetrafluoroborate.