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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Data source

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:
Limit test:

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): cryolite
- Supplier: Bayer AG, Leverkusen, Germany
- Analytical purity: 98.9%
- Lot/batch No.: Partie no. 2

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles river Limited (Manston Road Margate, UK)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 142-146 g
- Housing: 5 or 6 of the same sex in a cage
- Diet: ad libitum, standard quality-controlled laboratory rat food
- Water: ad libitum, tap water

- Temperature (°C): 21±3
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12 hours

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The particulate aerosols employed in the study contained 86 to 89% respirable particles with diameters < 7 µm. The MMAD ranged from 1.9-2.8 for the different exposure concentrations.
Details on inhalation exposure:
- Exposure apparatus: dust generator (to produce an aerosol from the powder supplied)
- Exposure chamber: ADG snout-inhalation chamber
- Method of holding animals in test chamber: rat restraining tubes
- System of generating particulates/aerosols: Wright Dust Feed (WDF) mechanism
- Method of particle size determination: Marple Model 296 Personal Cascade impactor sampler
- Air supply: each exposure system was operated with an air extract attached to the base of the inhalation chamber. A supply of clean, dried compressed air was used to operate the WDF. A supplementary air supply was used to balance the chamber air flows. Air supplies were provided by a compressor, the air was filtered to remove any residual particulate and was dried, air extract was provided by vacuum pumps.

- Brief description of analytical method used: gravimetric determination
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Gravimetric determination. Sampling (2-3 times per exposure day): collection using Whatman GF/A glass fibre filters (air was drawn through each filter in its holder using a vacuum pump). Volumes of each sample removed were measured by an in-line Wet-type gas meter.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week, 6 hours/day
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.2, 1.0 and 5.0 mg/m3
other: target concentration
Doses / Concentrations:
0.21, 1.04, 4.6 mg/m3
analytical conc.
Doses / Concentrations:
0.85, 4.4, 23.3 mg/m3
nominal conc.
No. of animals per sex per dose:
Control animals:
Details on study design:
Rats in the air control group, sodium fluoride dose group, and high dose cryolite group were maintained in their holding cages for a 13 week period following the last exposure.

An additional group of rats was exposed to a (study mean analysed) concentration of 5.7 mg/m3 sodium fluoride, as a comparative control.


Observations and examinations performed and frequency:
- Time schedule: twice a day

- Time schedule: at least once each week

- Time schedule for examinations: once a week

- Food consumption determination: weekly

- Time schedule for examinations: daily

- Time schedule for examinations: once

- Time schedule for collection of blood: once in week 13
- Anaesthetic used for blood collection: Yes --> ether
- Animals fasted: No
- How many animals: all

- Time schedule for collection of blood: once in week 13
- Animals fasted: No
- How many animals: all

- Time schedule for collection of urine: once in week 13 and once in week 26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Sacrifice and pathology:
Macroscopic examination and organ weights: Yes
Microscopic pathology: Yes
Other examinations:
Urinary inorganic fluoride and aluminium analysis: following urinanalysis during week 13, the residual individual samples were pooled for 5 rats of the same sex in each group. During week 26 (week 13 of withdrawal) urine samples were collected from all withdrawal rats and pooled for 5 rats of the same sex in each group.
All statistical analyses were carried out separately for males and females.Food and water consumption was analysed using cage mean values.
For all other parameters the analyses were carried out using individual animal as the experimental unit. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data.
If the data consist predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
Bartlett's test was applied to test for heterogeneity of variance between treatments; where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
Except for pre-exposure data, analyses of variance were followed by a Student’s t test and Williams' test for a dose-related response, although only Williams' test was reported. The Kruskal-Wallis analyses were followed by Shirley's test, the non-parametric equivalent of the t test and Williams' tests.
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight dat4 the final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might influence the organ weights.
For microscopic findings Fisher’s exact test was employed to detect treatment-related differences.

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:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
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:
At termination, increased inorganic fluoride concentrations in urine, bones, and teeth were evident for rats in the high dose cryolite (4.6 mg/m3) and the sodium fluoride dose group. Aluminium concentrations in the urine were increased in both sexes in the high and mid dose cryolite group, and in females in the low dose cryolite group. However, a dose relationship was not evident for this effect. Fluoride concentrations in bones and in tooth samples were increased in rats of both sexes of high dose cryolite group. Analysed values for aluminium in bones and teeth were below the limit of detection for the method of analysis used. After 13 weeks of recovery the fluoride levels and the aluminium concentrations in the urine and the fluoride concentration in the teeth of all groups returned to the control range, whereas the fluoride concentration in bones remained unchanged compared to terminal concentrations (no evidence for recovery was seen). Since the increased aluminium and inorganic fluoride excretion via urine could not be correlated to toxic effects, these findings were not considered to be toxicologically adverse.

At termination, increased lung weights were present in rats of both sexes of the high dose cryolite group. A similar but less obvious effect was present following 13 weeks of recovery.

The necropsy protocol was in line with recommendations in OECD TG 413. However, bones and teeth, considered likely to be target organs, were excluded from light microscopic examination. Pulmonary inflammatory lesions were observed in a majority of animals receiving cryolite at the high dose, and to a lesser degree, in some animals from the mid dose group. In the majority of animals from the high dose cryolite group, treatment-related findings in the lungs have comprised varying degrees of macrophage aggregation which contained brown pigmented material around alveolar ducts and alveolitis with thickening of alveolar duct walls. In addition, perivascular inflammatory infiltration with increased collagen in the alveolar duct walls and extension of bronchiolar epithelium into alveolar ducts were observed. Macrophages containing brown pigmented material were also present in the tracheobronchial and mediastinal lymph nodes of the high dose cryolite rats. The observed lung changes in cryolite exposed rats were typical of a non-specific reaction over time to a particulate with irritant properties, and attempts at clearance of deposited material via the lung macrophage/lymph node routs. No treatment-related laryngeal changes were seen in cryolite exposed animals. The treatment-related changes had, with the exception of the increased lung weights and the presence of small foci of brown pigmented alveolar macrophages, resolved after the recovery period.

Following exposure to sodium fluoride at 5.7 mg/m3, 6 out of 19 animals exhibited aggregations of alveolar macrophages in the lung parenchyma and around the alveolar ducts, 16 out of 19 animals had laryngeal epithelial hyperplasia and 9 out of 19 animals had subepithelial inflammation of the larynx. No treatment-related changes were seen in the lymph nodes of sodium fluoride exposed animals.

Overall, the response of respiratory tract inhalation exposure to sodium fluoride differed from the response to exposure to cryolite at a similar concentration and particle size. In rats exposed to sodium fluoride, lesions were noted in the larynx, whereas in rats exposed to cryolite, lesions were in the lungs. The reasons for the differences in localisation of the respiratory tract lesions may be related to the relative solubility of cryolite and sodium fluoride. Sodium fluoride is more soluble than cryolite and may not remain in the lungs in particulate form for a period of time sufficient to cause the degree of response seen with cryolite.

No effects of treatment were evident in clinical signs, bodyweight gain, food or water consumption. Haematological, biochemical and urinalysis parameters did not indicate findings considered of toxicological significance.

For cryolite, the NOAEC for systemic effects in male and female rats was 4.6 mg/m3 and the NOAEC for local toxic effects on the respiratory tract in rats was 0.21 mg/m3.

Effect levels

Dose descriptor:
Effect level:
0.21 mg/m³ air
Basis for effect level:
other: pulmonary inflammatory lesions

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion