Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
15 October to 17 November 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The test substance was aluminium trifluoride. The test material was supplied by the sponsor in a plastic bottle, containing 1.5 kg of the substance. The substance was described as a white powder, with a purity of 90%, the batch number was July 2004 (date of production), the melting point was 1278°C, and the vapour pressure was 1.3 hPa at 1238°C. The material was stored at ambient temperature in a safety cupboard, an expiry date of 31/12/2008 was provided.
A read across is proposed from aluminium trifluoride to calcium fluoride.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The animals were young SPF male and female Wistar derived rats (Crl:(WI)WU BR), obtained from Charles River Deutschland. They were 6 weeks old on arrival. The rats were checked for overt signs of ill health on arrival, and then quarantined. The microbiological status of the rats was checked by conducting serological controls in a few randomly chosen animals. Following negative results in the microbiological tests, quarantine was raised and the rats were acclimatised to the laboratory conditions for 7 days.
Rats were housed in same sex groups of 5 in macrolon cages with wood shavings as bedding. The animal room was maintained at a temperature of 22±3°C, relative humidity, 30-70%, artificial ighting was provided on a 12 hour light/dark cycle and there were approximately 10 air changes per hour.
Food (Rat & Mouse No. 3 Breeding Diet, SDS, UK) and tap water were provided ad libitum, except during exposure. Certificates of analysis were retained with the study records.
Rats were allocated to treatment groups by weight using a computer randomisation program. Weight variation was < 20% of the mean weight for each sex. Individuals were identified by ear tattoo.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Particle size distribution measurements were carried out during the range finding study and the 28 day study, using a 10-stage Andersen casacade impactor. The MMAD was calculated as the 50th percentile of the distribution and the mean GSD was calculated as the square root of the ratio of the 84th percentile to the 16th percentile of the distribution.
Details on inhalation exposure:
Animals were exposed in nose-only inhalation units. They were secured in plastic animal holders (Battelle), positioned radially around the central column. A positive pressure was maintained in the central column to prevent leakage of test atmosphere, ensuring a constant concentration in the breathing zone of the rats. The placing of animals within the chambers was rotated on a weekly basis.
The test material was too coarse aerosolised in the required 1-3 µm range (MMAD), therefore the material was ground before use with a ball mill. The ground test material was transported using miniature screw conveyors to eductors in which the test material was aerosolised. Temperature, relative humidity, input pressures of the eductor and rotameter readings (flow) were recorded 3 times per exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the test atmosphere was determined by gravimetric analysis. The test atmosphere was sampled in the breathing zone of the animals.
Duration of treatment / exposure:
6 hours/day, 5 days/week for 28 days (resulting in a total number of 20 exposure days)
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 7 and 50 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes
Details on study design:
Controls were similarly exposed to humidified air only.
The concentrations used in the 28 day study were based on a 7 day range finding study conducted with exposure concentrations of 0, 4, 20 and 100 mg/m³. Increased lung weights were seen in rats exposed to 100 mg/m³, therefore 50 mg/m³ was chosen as the high dose for the 28 day study.
Positive control:
A positive control was not included.

Examinations

Observations and examinations performed and frequency:
Each animal was observed daily in the morning by cage-side observation, and if necessary handled to detect signs of toxicity. A group-wise observation was made once, approximately halfway during each days exposure. On working days, all cages were checked again in the afternoon, particularly for dead or moribund animals. At weekends (non-exposure days) only 1 check was carried out.
Body weights were recorded once during acclimatisation, then on day -1, day 0 (initiation of treatment), and once weekly thereafter. The animals were also weighed on the day before overnight fasting and on their scheduled sacrifice date.
Food consumption was measured weekly by cage, and efficiency of food utilisation was calculated and expressed in g weight gain per g food consumed.
Sacrifice and pathology:
Scheduled necropsy was conducted on all surviving animals at the end of the exposure period. Rats were fasted overnight prior to necropsy, and blood samples were taken from the abdominal aorta whilst under anaesthesia. Haematology parameters determined were: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume, mea corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Clinical chemistry parameters determined were: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate.
At necropsy, each rat was killed by exsanguination from the abdominal aorta under general anaesthesia, and the examined grossly for pathological changes. The following organs were weighed: adrenals, heart, kidneys, liver, lungs with trachea and larynx, spleen and thymus.
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The tissues marked with * were preserved but not processed for histopathological examination, because histopathology was not considered necessary on the basis of gross observations: adrenals, bone marrow, brain. caecum*, colon*, epididymides, eyes*, heart, kidneys, liver, lungs including trachea and larynx, lymph nodes, mammary glands (females)*, nasal passages, ovaries, sciatic nerve, prostate, seminal vesicles* and coagulating gland*, small intestines*, spinal cord, spleen, stomach*, testes, thymus, thryoids, parathyroids, urinary bladder, uterus including the cervix, vagina*, al gross lesions.
The tissues retained for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Histopathological examination was performed on all animals of the control and high dose groups. Since treatment related histopathological changes were observed in the lungs and tracheobronchial lymph nodes of animals of the high-concentration group, histpathology of the lungs and lymph nodes was extended to include the low and mid-concentration groups.
Other examinations:
No other examinations reported.
Statistics:
ANOVA (and ANCOVA) followed by Dunnett's multiple comparison test, or Kruskal-Wallis followed by Mann-Whitney U-tests as appropriate, and Fischer's exact probability test.

Results and discussion

Results of examinations

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:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
No signs of toxicity were observed. One female in the mid concentration group had a swollen leg during the first 6 exposure days, but this was not considered to be treatment related. There were no statistically significant differences in body weight gain between the groups. Food consumption and food conversion efficiency were not statistically different between groups. The concentration of red blood cells was significantly increased in mid-dose females, but there were no dose-relationship therefore this finding was not considered related to treatment. The concentration of neutrophils and the contribution in terms of percentage of neutrophils to the number of white blood cells was significantly increased in the high-dose males.
The albumin/globulin ratio in plasma was significantly decreased in high-dose males, indicating an increase in the globulin concentration as calculated from the difference of the total protein and albumin concentrations. An increase in bilirubin concentration was seen in the low-dose females, but there was no dose-relationship therefore this finding was not considered to be treatment related. There was an increase in absolute and relative lung weight in high-dose males and females. Absolute and relative weight of the liver in high-dose males was increased, and absolute and relative weight of the heart was increased in mid-dose males. The latter finding was not considered to be treatment related in the absence of a dose-relationship.
Gross pathology did not reveal any treatment related findings. Histopathological changes were seen in the lungs of all exposed rats and in the tracheobronchial lymph nodes of the high-dose group. In the lungs of the high-concentration group, multifocal alveolar 'pigment' macrophages with particulate material in their cytoplasm were accompanied by inflammatory changes. The inflammatory changes consisted of multifocal very slight polymorphonuclear cell infiltration in the alveoli and slight to moderate multifocal perivascular mononuclear cell infiltrates. In two males of this group, hyperplasia of BALT tissue was seen. In the tracheobronchial lymph nodes of all rats of this group, the pigment was seen as deposits of (phagocytised) particulate material in the cortex and medulla. Some of the macrophages with phagocytised material exhibited aggregation. Pigment deposits and macropages aggregates were not seen in the tracheobronchial lymph nodes of animals of the mid and low-dose groups. In the lungs of these rats only focal (low-dose group) or multifocal (mid-dose group) macrophages containing a single or a few pigments were seen. Since phacocytosis of particulate material was seen in all exposed rats, the statistically significant decreased incidence of focal accumulation of alveolar macrophages in the exposed males, compared to controls, is of not toxicological relevance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
7 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased liver weights and associated clinical chemistry. Increased lung weights and histopathology consistent with particle overload at 50 mg/m3.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The averages of the daily mean concentrations and their standard deviations were 1.01±024 mg/m³, 7.0±0.6 mg/m³ and 50.0±1.8 mg/m³. The averages of the daily nominal concentrations were 3.2, 16.9 and 170.6 mg/m³, hence average generation efficiencies of 32, 41 and 29% were obtained for the low, mid and high concentration test atmospheres, respectively. These efficiences are within the range expected for generation of a dust.

The average MMAD's (± standard deviation) were 1.2 (±0.2) µm, 1.6 (±0.1) µm and 1.7 (±0.1) µm for the low, mid and high concentration test atmospheres, respectively. The corresponding average gsd's (± standard deviation) were 3.7 (±0.1), 3.1 (±0.0) and 3.5 (±0.2). In the exposure chambers, the temperature was maintained between 20.8 -23.2°C, and relative humidity was maintained between 30-52%.

Applicant's summary and conclusion

Conclusions:
The 28 day inhalation NOAEL for aluminium trifluoride was 7 mg/m³.
Executive summary:

The inhalation toxicity of an aerosol of aluminium trifluoride was investigated in a sub-acute 28 day study in Wistar rats. Groups of 5 rats/sex were exposed to target concentrations of 0 (control), 1, 7 or 50 mg/m³, for 6 hours/day, 5 days/week for 28 days (a total of 20 exposures). No treatment-related abnormalities were noted during exposure. There were no treatment-related effects on food consumption, food conversion efficiency and body weight gain. In the high-dose males there was a relative and absolute increase in the concentration of neutrophils, an increase of the globulin concentration, and an increase in both absolute and relative liver and lung weights. The increase in absolute and relative lung weights was also seen in high-dose females. There were treatment-related histopathological changes in the lungs of all exposed animals and in the tracheobronchial lymph nodes of high-dose males and females. In the lungs of animals from the high-dose group, multifocal alveolar 'pigment' macrophages with particulate material in their cytoplasm were accompanied by inflammatory changes. In the tracheobronchial lymph nodes of all rats in the high-dose group, the pigment was seen as deposits of (phagocytised) particulate material in the cortex and medulla. In the lungs of the low and mid-dose group, only very slight focal (low-dose) or very slight multifocal (mid-dose) macrophages containing a single or a few pigments were seen. Since phagocytosis and processing of foreign material is a normal function of alveolar macrophages, the presence of pigment macrophages in the lungs of the animals of the low and mid-dose groups was not considered to be an adverse toxicological effect. The 28 day inhalation NOAEL for aluminium trifluoride was 7 mg/m³. The effects of the substance on the liver are not consistent with fluoride toxicity, but are consistent with the effects of aluminum. It can therefore be concluded that there is no evidence of systemic fluoride toxicity in this study.