Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 15 July 2013 (study plan approval by the study director) to the 21 March 2014 (date of the test report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
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 : Lithium cryolite
- Molecular formula : Li3AlF6
- Molecular weight : 162
- Physical state: White powder
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: rats were obtained from a colony maintained under SPF-conditions at Harlan Laboratories, the Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: The body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights just before exposure on day 0 were 280 and 187 g for males and females of the range finding study, and 250 and 160 g for males and females of the main study, respectively.
- Fasting period before study: none
- Housing: The rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the studies. The animals were housed in macrolon cages with a bedding of wood shavings, and a wooden block and strips of paper as environmental enrichment. After allocation, the animals were housed three (range finding study) or five (main study) animals to a cage. During exposure, the rats were housed individually in the exposure unit and had no access to feed or water. Immediately after each exposure, the animals were returned to their home cages.
- Diet (e.g. ad libitum): Feed was provided ad libitum from the arrival of the animals until the end of the study, except during exposure and the fasting period prior to scheduled sacrifice. The animals received a cereal-based rodent diet from a commercial supplier. Each batch of diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled up as needed.
- Water (e.g. ad libitum): Drinking water was provided ad libitum from the arrival of the animals until the end of the study, except during exposure. Each cage was supplied with domestic mains tap-water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.
- Acclimation period: 8 days for the range-finding study and 7 days for the main study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45 - 65 % (except during short periods during which the relative humidity exceeded the upper limit, reaching maxima up to 73%. In addition, the relative humidity was above 65% in the range finding study on 8 August 2013 for about 9 hours (maximum 81%) and below 45% in the main study on 15 October 2013 for about 2.5 hours (minimum 42%)).
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark

IN-LIFE DATES:
- Range finding study :
Arrival of the animals : 17 July 2013
First exposure of the animals : 31 July 2013
Sacrifice of the animals : 7 August 2013
- Main study:
Arrival of the animals : 2 October 2013
First exposure of the animals : 11 October 2013
Sacrifice of the animals : 8 November 2013

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The average (+/- standard deviation) mass median aerodynamic diameter (MMAD) of the low, mid and high concentration test atmospheres were 1.06 (+/- 0.08), 1.39 (+/- 0.13) and 1.47 (+/- 0.05) µm, with corresponding average geometric standard deviations (+/- standard deviation) of 1.93 (+/- 0.10), 1.97 (+/- 0.19) and 1.91 (+/- 0.20), respectively .
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
The animals were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical polypropylene column (group 1) or a aluminium column (groups 2, 3 and 4) surrounded by a transparent cylinder. The columns had a volume of 39 (group 1) or 37 litres (groups 2-4) 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. The rodent tube sections had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling, and measurement of temperature and relative humidity. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.

- Method of holding animals in test chamber:
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. Animals were rotated weekly with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012).
By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided.

- Source and rate of air / Method of conditioning air / System of generating particulates/aerosols:
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmospheres for groups 3 and 4 were generated using a turntable dust feeder and an eductor. Test material was aerosolized in the eductor which was supplied with humidified compressed air. The resulting aerosol was led through a glass cyclone, which was used to remove the largest particles from the aerosol, and was subsequently introduced at the top inlet of the mid or high concentration exposure units. From there, the aerosol was directed downward and led to the noses of the animals. At the bottom of the units, the test atmosphere was exhausted.
The test atmosphere for group 2 was generated by extracting and diluting part of the test atmosphere of group 3 using an eductor. From the eductor, the test atmosphere was transported towards the top inlet of the exposure chamber of group 2, where it was mixed with a stream of humidified compressed air. The flow of this dilution air was controlled using a reducing valve and monitored using a mass view meter. Though the mass view meter was mounted during both the range finding and the main study, the dilution air was not needed during the main study (dilution using the eductor was enough to reduce the concentration to the target concentration of group 2).
The eductors were calibrated by measuring the total air flow at a range of driving air pressures encompassing the driving pressures used during the study. To calculate the dilution factor, the eductor used for group 2, was calibrated with and without the aspiration air flow.
The exposure chamber for the control animals (group 1) was supplied with a stream of humidified air only, which was controlled using a reducing valve and monitored using a mass view meter. At the settings of the eductors and mass view meters – recorded hourly during exposure – the total airflow through the exposure units was at least 13, 35, 98 and 84 L/min for the control, low, mid and high concentration exposure units during the range finding study, and at least 18, 21, 97 and 91 L/min for the control, low, mid and high concentration exposure units during the main study, respectively.

- Temperature, humidity, pressure in air chamber:
The temperature and relative humidity were recorded hourly during exposure. Temperature and relative humidity were measured using an RH/T device.
The mean temperature (+/- standard deviation) during exposure was 22.0 (+/- 0.3) for the control, low and mid concentration, and 22.1 (+/- 0.3) for the high concentration group.
The mean relative humidity (+/- standard deviation) during exposure was 38.3 (+/- 1.2), 42.6 (+/- 1.1), 43.5 (+/- 1.2), and 43.2 (+/- 1.1) % for the control, low, mid and high concentration groups, respectively.

- Air flow rate:
The total airflow was recorded hourly during exposure. Airflow was recorded by recording the readings of the mass view meters and the driving air pressure of the eductors as a measure for the flow. The mean chamber airflows (+/- standard deviation) were 22.5 (+/- 0.8), 97.8 (+/- 0.9) and 91.8 (+/- 0.0) L/min for the low, mid and high concentration groups, respectively. The mean chamber airflow for the control group was 20.5 (+/- 1.1) L/min.

- Air change rate: no data

- Method of particle size determination:
Particle size distribution measurements were carried out using a 10-stage cascade impactor once weekly and at least once during preliminary generation of the test atmosphere for each exposure concentration. Because of the relatively low target concentration of group 2, particle size measurement of this group was spread over 2 exposure days (a minimum amount of test atmosphere should be sampled to allow for an accurate measurement). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated (Lee, 1972).

- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used:
The actual concentration of Lithium Cryolite in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled amounts of 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. Samples were taken at least three times per day for each exposure condition, except for group 2. Since a minimum amount of test material should be present on each filter to allow accurate weighing, the low target concentration for this group meant that only one sample – encompassing almost the complete exposure period – could be taken during each exposure.

- Samples taken from breathing zone: yes

VEHICLE : not applicable
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
range finding study : 7-day period
28-day main study : 28-day period
Frequency of treatment:
range finding study : exposure for 6 hours per day, 5 days per week (i.e. 5 exposure days in total).
28-day main study : exposure for 6 hours per day, 5 days per week (i.e. 20 exposure days in total).
Doses / concentrationsopen allclose all
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
range-finding study.
mean actual concentration = 2.05 (+/-0.11) mg/m3
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
range-finding study.
mean actual concentration = 9.98 (+/-0.97) mg/m3
Dose / conc.:
50 mg/m³ air (nominal)
Remarks:
range-finding study.
mean actual concentration = 49.55 (+/-1.02) mg/m3
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
Main study, low dose.
mean actual concentration = 1.01 (+/-0.07) mg/m3
Dose / conc.:
3 mg/m³ air (nominal)
Remarks:
Main study, mid dose.
mean actual concentration = 3.34 (+/-0.24) mg/m3
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
Main study, high dose.
mean actual concentration = 10.22 (+/-0.38) mg/m3
No. of animals per sex per dose:
range finding study : 3 male and 3 female rats each
main study : 5 male and 5 female rats each
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The target concentrations were selected on the basis of a 7-day range finding study in which animals were exposed to target concentrations of 0, 2, 10 or 50 mg/m3 Lithium Cryolite. Exposure at 50 mg/m3 induced epithelial degeneration in the nose, increased weight of the lungs, alveolitis, accumulation of alveolar macrophages and enlarged tracheobronchial lymph nodes. At 10 mg/m3, increased lung weights and epithelial degeneration in the nose were still observed, while no clear exposure-related effects were observed at 2 mg/m3.

- Rationale for animal assignment: random
one day before start of exposure, the animals were allocated to the various exposure groups by computer randomisation proportionally to body weight (males and females separately)

- Rationale for selecting satellite groups: except blank control, none
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On exposure days, each animal was observed daily in the morning, prior to exposure, and checked again after exposure. During exposure, a group-wise observation was made about halfway through the 6-hour exposure period. In weekends and on public holidays only one check per day was carried out. During exposure, when observation was limited due to the animals’ stay in restraining tubes, attention was directed towards any breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.
- Cage side observations checked in table were included: a table is available in the test report on all the parameters checked, but no clinical abnormalities were observed.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals of the range finding study were weighed prior to exposure on the first day (day 0), on day 3 and on day 7 (day of scheduled sacrifice).
The animals of the main study were weighed prior to exposure on the first day (day 0) and then twice weekly (Mondays and Fridays) for the first two weeks (on days 3, 7, 10 and 14). Thereafter, the frequency was reduced to once weekly (Fridays only; days 21 and 27), because there were no statistically significant effects on body weight in the first two weeks. All animals of the main study were also weighed on their scheduled sacrifice date (day 28) in order to calculate the correct organ to body weight ratios.

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken at necropsy from the abdominal aorta of whilst under . EDTA was used as anticoagulant.
- Anaesthetic used for blood collection: Yes, pentobarbital anaesthesia
- Animals fasted: Yes, overnight fasted rats (water was freely available)
- How many animals: all animals of the main study
- Parameters checked in table were examined: a table is available in the test report on all the parameters checked, they cover at least the recommendation of the OECD TG 412.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical chemistry was conducted at the end of the treatment period in all animals of the main study at the same time blood samples for haematology were collected. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation.
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table were examined: a table is available in the test report on all the parameters checked, they cover at least the recommendation of the OECD TG 412.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals in the range-finding study and in the main study 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 (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.

Organ weights:
The following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights were calculated from the absolute organ weight and the terminal body weight.
- adrenals
- brain (only in the main study)
- heart
- kidneys
- liver
- lungs with trachea and larynx
- spleen
- testes
- thymus (ony in the main study)

HISTOPATHOLOGY: Yes

Tissue preservation:
For histopathological examination, the tissues and organs mentioned above (and the nose for the range-finding study) of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation.

In the main study, the following additionnal organs and tissues were analysed and the carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded:
- bone marrow
- esophagus
- ovaries
- seminal vesicles
- spinal cord (cervical, mid-thoracic, and lumbar)
- thyroid
- tracheobronchial lymph nodes
- uterus
- complete respiratory tract including nasopharyngeal tissues
- all gross lesions

The tissues to be subjected to histopathological examination were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. The nose was decalcified before embedding in paraffin.

Histopathological examination in the range-finding study:
The nose, larynx, trachea and lungs of all animals of the control group and the high concentration group, and the nose (levels 4 through 6) and lungs of the
intermediate concentration groups were processed for histopathological examination. These tissues were embedded in paraffin wax, sectioned at 5 μm and
stained with haematoxylin and eosin and subsequently examined histopathologically (by light microscopy).
The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994) in the control and high concentration groups, with one level to include the
nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT). In the intermediate concentration groups, the nasopharyngeal tissues were examined at three levels (levels 4 through 6). The larynx was examined at three levels (one level to include the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and each lung lobe at one level.

Histopathological examination in the main study:
All preserved tissues of all animals of the control and high concentration group were examined histopathologically (by light microscopy). Unless required for histopathological examination, the tissues of the animals of the low and mid concentration groups (groups 2 and 3) were not processed, except for the nose which was decalcified and embedded in paraffin concurrently with the nose of the animals of groups 1 (control) and 4 (high concentration). The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994; see Annex 10) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis), the trachea at three levels (including the bifurcation, and one longitudinal section through the carina), and each lung lobe at one level. Since treatment-related changes were observed in animals of the high concentration group, histopathological examination of the nasal tissues (levels 5 and 6), lungs and tracheobronchial lymph nodes was extended to animals of low and mid concentration groups.
Statistics:
The statistical procedures for analysis of data are described below.

- Post-treatment body weight data: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ as data transformation method (abbreviation AUTO).
Day 0 body weight data were used as covariate in the analysis of the corresponding posttreatment data unless removed during data preprocessing.

- Pretreatment body weight, organ weight, haematology and clinical chemistry data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method (abbreviation AUTO).

- Food consumption: no statistics were applied on food intake (only one cage/sex).

- Incidences of histopathological changes: Fisher’s exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical abnormalities were observed in response to exposure to Lithium Cryolite. One male of the low concentration group and one female of the high concentration group had a missing tip of the tail. These findings were not related to the exposure.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences in body weights between the test groups and the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable among the groups throughout the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant changes in red blood cell and clotting variables. There were no statistically significant differences in total white blood cell counts. The percentage of neutrophils was statistically significantly increased in males of the high concentration group. This finding was, however, not reflected in significant changes in absolute neutrophil count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The plasma cholesterol concentration was statistically significantly decreased in females of the high concentration group. There were no other statistically significant differences in clinical chemistry variables, apart from an increased plasma concentration of in inorganic phosphate in males of the mid concentration group which, in the absence of a dose-response relationship, was considered a chance finding.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A concentration-dependent increase in absolute and relative weight of the lungs was observed upon exposure to the test material, which reached the level of statistical significance in males and females of the mid and high concentration groups, and - for relative lung weight - also in males of the low concentration group.
The relative weight of the spleen was statistically significantly lower in females of the mid and high concentration group when compared to controls.
There were no significant weight differences in other organs, apart from an incidental increase in absolute brain weight in males of the mid concentration group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination at the end of the exposure period revealed no treatment related gross lesions. The macroscopic findings were considered unremarkable and part of background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Lungs
Microscopic examination of the lungs revealed a concentration-dependent increase in the number of enlarged/activated macrophages. In the low concentration group, a minimal increase was noted in all males and three females. In the mid concentration group, a minimal increase was noted in 2 males and 4 females while in the remaining 3 males and 1 female the degree was mild. In the high concentration group, a mild increase in the number of enlarged/activated macrophages was noted in all animals.
Minimal to mild perivascular mononuclear inflammation was observed in one female animal of the low concentration group, in 3/5 males and 3/5 females of the mid concentration group, and in 4/5 males and 1/5 females of the high concentration group.
- Nose
Microscopic examination of the nasal tissues revealed metaplasia of the olfactory epithelium into respiratory epithelium (at level 4, 5 and 6) in the mid concentration and high concentration groups. In addition, ulceration was found in the nose of four females of the mid concentration, and in two males and three females of the high concentration group. One female of the high concentration group also showed synechia.
- Tracheobronchial lymph nodes
Multifocal accumulations of macrophages were found in the tracheobronchial lymph nodes of all animals – except one female – of the high concentration group.
The remaining histopathological findings were considered unremarkable and part of background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1.01 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse changes in the nose, lungs and tracheobronchial lymph nodes.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
3.34 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
lungs
nasal cavity
trachea
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Discussion and conclusion:

In the 28-day main study, no exposure-related clinical signs or changes in growth, food consumption or haematology were observed. Clinical chemistry showed a decreased plasma cholesterol concentration in females of the high concentration group. Because this finding occurred in females only, and other blood lipids were not significantly affected, it was considered of doubtful, if any, toxicological concern.

The relevance of the decrease in the relative weight of the spleen in females of the mid and high concentration group is unclear. This finding was not associated with histopathological changes.

The exposure to Lithium Cryolite resulted in treatment-related changes in the nose, lungs, and tracheobronchial lymph nodes. The nasal changes consisted of ulceration and metaplasia of the olfactory epithelium in the posterior part of the nose, and were observed in animals of the mid and high concentration groups. Changes in tracheobronchial lymph nodes – accumulation of macrophage aggregates – were limited to animals of the high concentration group. Changes in the lungs consisted of increased numbers of enlarged/activated macrophages and perivascular inflammation, accompanied by a concentration-related increase in weight of the lungs. Most of these findings were confined to the mid and/or the high concentration groups. At the low concentration, treatment-related changes were limited to a minimal increase in the number of alveolar macrophages and a slightly elevated lung weight, which reached the level of statistical significance only for relative lung weight in males. The slight increase in lung weight may well be associated with, and secondary to, the accumulation of alveolar macrophages. It cannot be completely excluded that the presence of increased numbers of alveolar macrophages in animals exposed to the low concentration would eventually result in more chronic pathology. However, in the absence of any sign of an inflammatory reaction or any other indication for adverse changes in the respiratory tract, the increased number of alveolar macrophages at the low concentration is considered to represent a physiological adaptation to exposure to the particulate test material indicating enhanced alveolar clearance activity (ECETOC, 2002; Burger et al., 1989).

Applicant's summary and conclusion

Conclusions:
Exposure to Lithium Cryolite at concentrations of 3.34 mg/m3 or higher resulted in adverse changes in the nose, lungs and tracheobronchial lymph nodes. Changes observed upon exposure to the low concentration of 1.01 mg/m3 Lithium Cryolite – i.e. accumulation of alveolar macrosphages and an associated increased relative lung weight in males – were considered to represent a non-adverse adaptive response to the exposure. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) of Lithium Cryolite in rats exposed for 6 hours/day, 5 days/week for a period of 28 days was considered to be 1.01 mg/m3.
Executive summary:

The inhalation toxicity of Lithium Cryolite was studied in a sub-acute (28-day) inhalation toxicity study in Wistar rats performed in accordance with the OECD Testing Guideline No 412 and the GLP.

Four groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (control), 1, 3 or 10 mg/m3 Lithium Cryolite for 6 hours/day, 5 days/week over a 28-day period, with a total of 20 exposure days. The animals were sacrificed on the day after the last exposure.

To investigate the toxicity, data on clinical observations, body weight, food consumption, haematology and clinical chemistry were collected. At necropsy, the animals were examined for gross macroscopic abnormalities, organs were weighed and a selection of organs and tissues (including the complete respiratory tract with nasal passages) was examined microscopically.

The concentration levels were selected on the basis of a 7-day range finding study (5 exposure days in total) in which groups of 3 male and 3 female rats were exposed to target concentrations of 0, 2, 10 or 50 mg/m3. In this range finding study, exposure at 50 mg/m3 induced epithelial degeneration in the nose, increased weight of the lungs, alveolitis, accumulation of alveolar macrophages and enlarged tracheobronchial lymph nodes. At 10 mg/m3, increased lung weights and epithelial degeneration in the nose were still observed, while no clear exposure-related effects were observed at 2 mg/m3.

In the main study, the mean actual concentrations (± standard deviation) of Lithium Cryolite in the various test atmospheres – based on gravimetric analysis – were 1.01 (+/- 0.07), 3.34 (+/- 0.24) or 10.22 (+/- 0.38) mg/m3 for the low, mid and high concentration levels, respectively. The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 1.06 µm (with a geometric standard deviation of 1.93), 1.39 µm (gsd of 1.97) and 1.47 µm (gsd of 1.91) for the low, mid and high concentration test atmospheres, respectively.

There were no treatment-related clinical abnormalities and no changes in growth or food consumption in the main study. Haematology and clinical chemistry, conducted in all rats at necropsy, did not reveal any treatment-related changes. A decrease in plasma cholesterol concentration in females of the high concentration group was considered of doubtful, if any toxicological significance.

The weight of the lungs showed a concentration-dependent increase, which reached the level of statistical significance at the mid and high concentration, and for relative lung weight in male animals also at the low concentration. The relative weight of the spleen was decreased in females of the mid and high concentration group. Other significant changes in organ weights were not found.

Macroscopic examination at the end of the exposure period revealed no treatment-related gross lesions.

Microscopic examination of the lungs revealed a concentration-dependent increase in the number of enlarged/activated alveolar macrophages in all exposed groups. In addition, perivascular mononuclear inflammation was observed in the lungs of one female of the low concentration, in three males and three females of the mid concentration, and in four males and one female of the high concentration group.

In the treacheobronchial lymph nodes, accumulation of macrophage aggregates was noted at the high concentration level.

Microscopic examination of the nose revealed ulceration and metaplasia of the olfactory epithelium in posterior part of the nasal cavity of animals of the mid and high concentration group.

Exposure to Lithium Cryolite at concentrations of 3.34 mg/m3 or higher resulted in adverse changes in the nose, lungs and tracheobronchial lymph nodes. Changes observed upon exposure to the low concentration of 1.01 mg/m3 Lithium Cryolite –i.e. accumulation of alveolar macrophages and an associated increased relative lung weight in males – were considered to represent a non-adverse adaptive response to the exposure. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) of Lithium Cryolite in rats exposed for 6 hours/day, 5 days/week for a period of 28 days was considered to be 1.01 mg/m3.