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Toxicological information

Repeated dose toxicity: inhalation

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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:
supporting study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given

Data source

Reference
Reference Type:
publication
Title:
Experimental Study on the Fibrogenic Properties of Different Types of Alumina
Author:
Ess et al.
Year:
1993
Bibliographic source:
Am Ind Hyg Assoc J 1993; 54(7): 360-370.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Although the non-physiologic modes of administration used in this study limit its utility for the derivation of a dose-descriptor, the results clearly show the importance of the physical characteristics of the alumina dust for the biological response. The intratracheal instillation doses overloaded clearance mechanisms in all cases. Only one dose was used. Two species were investigated, animal weights were monitored, cytological and biochemical analyses of BALF were undertaken in addition to histopathological examinations of lung tissue.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test materials were five fine alumina dusts obtained from sieving raw alumina samples, a chemical grade alumina (Dust (6)) and an alumina produced in the laboratory (Dust (7)). Dust(1) Source: Rotary kiln (Sandy Gove, Australia)α content: 8% Specific surface BET (m²/g): 43X-ray diffraction (XRD): α, γ, traces of β-Na.Al7O11Size < 11μm: 77%Dust(2)Source: Fluid bed (Lurgi) (Sandy Interalumina, Venezuelaα content: 54%Specific surface BET (m²/g): 15XRD: α, δ, gibbsiteSize < 11μm: 89%Dust(3)Source: Fluid bed (Alcoa) (Sandy Kwinana, Australia)α content: 18%Specific surface BET (m²/g): 39XRD: α, γ, hydrargilliteSize < 11μm: 69%Dust(4)Source: Fluid flash (Sandy Distomon, Greece)α content: 79%Specific surface BET (m²/g): 7.4XRD: α, (traces of δ and γ)Size < 11μm: 45%Dust(5)Source: Rotary kiln (Floury Gardanne, France)α content: 71%Specific surface BET (m²/g): 0.5XRD: α, β-Na.Al7O11, SiO2 (quartz and cristobalite)Size < 11μm: 67%Dust(6) Source:Chemical grade Aluminium oxide C (Degussa, Germany) formed by flame hydrolysis of anhydrous AlCl3 “Aerosil”, α content: 0%Specific surface BET (m²/g): 100, highXRD: almost amorphous, probably δ and χ.Size < 11μm: 100%Median particle size: 0.008 µm (0.005-0.02 µm)Dust(7) Source: Laboratory produced alumina, Produced in the Vereinigte Aluminum-Werke laboratoty (Germany) from aluminium hydroxide by calcinations at 750 °C for 5 hours.α content: 0% Specific surface BET (m²/g): 110, highXRD: almost amorphous, probably δ and χ.Size < 11μm: 100%, Median particle size: 1.3 µm, all diameters < 5 µm.The articles reported chemical analyses of the dust samples. Levels of SiO2 were less than 600 ppm, Na2O levels were 1000-6000 ppm except for sample 6 (< 10 ppm) and levels of oxides of V, Ti, Zn, Ni, Mg, Mn and Cr were all less than 100 ppm.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Ivanovas, Kisslegg, Gremany- Age at study initiation: 2 month- Weight at study initiation: 200 - 250 g- Diet: Standard diet ad libitum- Water: ad libitum

Administration / exposure

Route of administration:
other: Intratracheal Instillation
Type of inhalation exposure:
not specified
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: No information was provided on the MMAD and GSD. The % of particles with sizes less than 11 μm was reported. Further detail on particle characteristics:Additionally, alumina dusts (1) to (5) had particle size distributions similar to those in air of aluminium smelters with prebake pots (smelter-grade aluminas). Alumina dust (6) contained particle sizes unlikely to occur in potroom air. Alumina dust (7) had a particle size distribution typical of the inhalable fraction of potroom dust.
Details on inhalation exposure:
The dusts were administered without prior sterilization in order to minimize any modifications to their physical characteristics that might influence the biological effects. Sterile isotonic saline was injected into a vial containing a pre-weighed sample of dust. The suspension was shaken vigorously by hand and then using a Vortex shaker until the animal was ready for instillation. Rats were lightly anaesthetized with methoxyflurane and instilled intratracheally with 0.1 mL of the fresh suspension or, for the negative controls, sterile saline. The instillation was guided using a laryngoscope.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Two weeks
Frequency of treatment:
5 injections.
Doses / concentrations
Remarks:
Doses / Concentrations:Basis:other: A total dose of 50 mg of the alumina were administered to the rats by 5 injections over a 2 week period. The total dose for quartz was 25 mg.
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
No information was provided on the method used to allocate the animals to groups.
Positive control:
Positive control: Quartz (25 mg) Size: median particle size 4.76 µm Specific surface BET (m²/g ): 3.1 m²/g

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed:Animal weights were recorded as an index of general health.Bronchoalveolar lavage:- BAL fluid was obtained by flushing the lungs of the sacrificed animals with 8 to 10 mL of physiological saline. The saline was introduced over the period of a minute, allowed to remain for a minute and then withdrawn. Flushing was carried out twice. The authors reported that 80 to 85% of the lavage fluid was recovered. The following parameters were determined in the BALF:- lactate dehydrogenase activity (LDH) in the cell-free supernatant (Bergmeyer and Bernt, pp. 574-579 in Methods of Enzymatic Analysis, Vol. 2, ed. By H.U. Bergmeyer. New York, Academic Press, 1974);- total protein (method of Bradford, Analyt Biochem 1976; 72: 248-254);- Cytology: cell counts (using a Neubauer’s hemocytometer chamber) and types (in a “cytocentrifuged stained preparation”).- Lung tissue pathology (methods reported below).Frequency of the observations and examinations:Weights were recorded each week.Observations were made at 4 timepoints; 60, 90, 180 and 361 days after exposure.
Sacrifice and pathology:
Animals were anaesthetized with sodium-pentobarbital and sacrificed by exsanguination. Blood was aspirated from the peritoneal cavity with a Pasteur pipette. Histopathological examinations of lung tissue were conducted using sections embedded in paraffin. Five sections were taken; at least one from each lobe that were stained with hematoxylin-eosin and van Gieson (to show connective tissue and collagen fibres).
Other examinations:
A separate experiment was conducted in male, one month old, NMRI mice. The mice were injected intra-peritoneally with a 0.5 mL volume of 1% suspension of dust in sterile isotonic saline. Five mice were sacrificed at 30, 90 and 180 days and 10 mice were examined at 360 days. Nodules formed by the dust on the anterior wall of the abdomen and over the peritoneal viscera were examined macroscopically and histopathology using formalin-fixed, paraffin sections stained with hematoxylin, eosin and also van Gieson stain to identify connective tissue and collagen fibres.
Statistics:
Non-parametric tests were used to compare LDH activity levels and protein content in BALF between the treated groups and the controls.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
for mortality data, no data for clinical signs
Mortality:
mortality observed, treatment-related
Description (incidence):
for mortality data, no data for clinical signs
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Rats (Intratracheal Instillation)Weight: The best weight gains were observed for control animals and animals dosed with alumina Dust(2). A relative reduction in weight gain (15%) was observed in animals dosed with quartz or alumina Dust(1).Cytotoxicity:The level of LDH activity in the saline control was 38 ± 28 mU/mL. At 60 days and 90 days post-instillation, LDH activity was significantly higher than control (p < 0.05) in all dusts; the highest value was observed in the quartz positive control. At 180 days, the results for Dust (6) were not significantly different than in the controls. For, all other dusts, the LDH activity at days 180 and 360 was significantly higher than control values (p < 0.05). In summary, persistent increases throughout the duration of the experiment were observed for Dust (1) and Dust (3) (ca. 7-8x higher). Increases were also observed for the other dusts, with the exception of Dust (6), but the magnitude was smaller. Dust (6) showed the lowest values (44 – 101) and quartz (positive control) the highest values (20-30x higher than the control). The results suggest the persistence of the inflammatory response in animals dosed with alumina.Total soluble protein levels in the saline control were 0.19 ± 0.08 mg/mL. Statistically significant increases were observed for all dusts. Dusts (5), (6) and (7) were the lowest (2-4 times higher than the control, Dust(1) was 6x higher at 180 days, Dust (2) & Dust (4) 3-4 times higher and Dust (3) 7 times higher at 360 days. The positive control, quartz, had levels 10-20 times higherCytology:Alveolar macrophages (AM). In the saline control, 98% of all nucleated cells were AM. For Dusts (1) to (7) the AM decreased in percentage, increased in size and many were vacuolated.Polymorphonuclears were increased in all alumina-treated animals; 10-15% were multinuclear (except Dust (5) which had 3-5% multinuclear). The increase persisted.Histopathology1 year post ITI:The histological findings for Dusts (1) to (5) were similar with a mild to severe alveolar reaction, moderate levels of aggregates of dust-laden macrophages but with no collagen. Regional lymph nodes contained dust-laden phagocytes but also no collagen.In animals instilled with Dust (6), the chemical grade “Aerosil”, an alveolar reaction was almost absent, dust-laden interstitial macrophages were very abundant and collagen was present in considerable amounts. Regional lymph nodes contained dust-laden phagocytes and some collagen fibres.Animals dosed with Dust (7) showed a mild alveolar reaction, abundant dust-laden interstitial macrophages and some collagen. Some collagen fibres were present in regional lymph nodes in addition to dust-laden phagocytes. The response was intermediate between the response to the smelter-grade alumina and Dust (6), the chemical grade product. Animals dosed with quartz had a very severe alveolar reaction, with very abundant aggregates of dust-laden interstitial macrophages and abundant collagen. Whorls of collagen were observed in regional lymph nodes.The saline controls exhibited normal lung pathology.Mice (intraperitoneal injection)The macroscopic examination of organs in the animals dosed with aluminas (1) to (5) typically found nodules on the liver, spleen and omentum. The nodules were less numerous in smelter-grade alumina (Dust (1) to (5))-treated animals compared with quartz-treated animals. Histopathology examinations of nodules showed a progression of responses between quartz (most severe), the Chemical (6) and Lab (7) grade alumina and smelter-grade alumina (Dusts (1) to (5) – least severe). In the animals dosed with Dusts (1) to (5), there were some phagocytes and fibroblasts and cell-free dust agglomerates at 30 days. At 360 days post-instillation, some collagen fibres were found encapsulating the dust but the inflammatory reaction was absent. In the animals dosed with Dusts (6) & (7), at 30 days the response consisted of mostly dust-laden macrophages, some free dust particles; a response consistent with a mild inflammatory reaction. At 90 days there were more fibroblasts and some collagen fibres. At 180 and 360 days, many aggregations showed an acellular central part made of free dust and dense collagen fibres. In the positive control animals dosed with quartz, at 30 days the cells were mostly macrophages with some fibroblasts and PMN. By 90 days, there were some collagen fibres at the core of aggregations, but most nodules were still highly acellular. At 180 and 360 days, collagen fibres were abundant. In summary, exposure to quartz, Dust (6) or Dust (7) resulted in nodules with dense collagen cores. This did not occur on exposure to the smelter-grade aluminas.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The results from this study provide clear evidence for an extensive and inflammatory reaction in the positive control (quartz-treated) animals. In contrast, dusts (1) to (5), the smelter-grade aluminas, showed no evidence of a progressive fibrotic effect. Dusts (6) and (7), the chemical and laboratory grade aluminas, respectively, showed evidence of a fibrotic effect. The response for dust (7) was intermediate between the smelter-grade aluminas and the chemical-grade dust (6).
Executive summary:

Ess et al. (1993) investigated the fibrogenic potential of alumina samples from different sources and with different physical properties. The test materials were five fine alumina dusts obtained from sieving raw alumina samples (Dusts (1) to (5)), a chemical grade alumina (Dust (6)) and an alumina produced in the laboratory (Dust (7)). The alumina dusts (1) to (5) had particle size distributions similar to those in air of aluminium smelters with prebake pots. Alumina Dust (6) contained particle sizes unlikely to occur in potroom air and the laboratory grade dust (Dust (7)) had a particle size distribution typical of the inhalable fraction of potroom dust. Two experiments were conducted; one in female Sprague-Dawley rats with administration by intratracheal instillation and the other in male NMRI mice with administration of the dusts by intraperitoneal injection. In the rat study, a total dose of 50mg was administered to the lightly anaesthetized animals using 5 injections of well-shaken suspensions (0.1 mL in sterile isotonic saline) of non-sterilized dust over a two week period. Animal weights were recorded weekly and five animals were sacrificed 60, 90, 180 and 361 days after exposure. Quartz was included as a positive control (total dose: 25 mg) and sterile isotonic saline as a negative control. However, it is not clear whether negative controls were included for every observation time point. Bronchoalveolar lavage fluid (BALF) was collected using two flushes and subjected to cytological and biochemical (lactate dehydrogenase (LDH), total protein) analyses. Histopathological examinations of lung tissue were undertaken staining to show general histology (hematoxylin-eosin) and also connective tissue and collage (van Gieson).

 

Weight gains were highest in control animals and animals dosed with alumina Dust (2). A reduction in weight gain (15%) was observed in animals dosed with quartz or alumina Dust (1). Biochemical analyses of the BALF showed significantly higher LDH activity in treated versus control animals at 60 and 90 days post-instillation for all the dusts with the highest value observed in the quartz positive control. At 180 days, the results for Dust (6) were not significantly different from the controls. For, all other dusts, the LDH activity at days 180 and 360 was significantly higher than control values (p<0.05). In summary, persistent increases throughout the duration of the experiment were observed, most notably for Dust (1)and Dust (3)(ca. 7-8x higher). Statistically significant increases in total protein were observed for all dusts. Dusts (5), (6) and (7) were the lowest (2-4 times higher than the control, Dust (1) was 6x higher at 180 days, Dust (2) & Dust (4) 3-4 times higher and Dust (3) 7 times higher at 360 days. The positive control, quartz, had levels 10-20 times higher. Cytological analyses showed that 98% of all nucleated cells were AM in the saline control. For Dusts (1) to (7) the AM decreased in percentage, increased in size and many were vacuolated. Polymorphonuclears were increased in all alumina-treated animals; 10-15% were multinuclear (except Dust (5)which had 3-5% multinuclear). The increase persisted. The histological findings for Dusts (1) to (5) one year post-intratracheal instillation were similar with a mild to severe alveolar reaction, moderate levels of aggregates of dust-laden macrophages but with no collagen. Regional lymph nodes contained dust-laden phagocytes but also no collagen. In animals instilled with Dust (6),the chemical grade “Aerosil”, an alveolar reaction was almost absent, dust-laden interstitial macrophages were very abundant and collagen was present in considerable amounts. Regional lymph nodes contained dust-laden phagocytes and some collagen fibres. Animals dosed with Dust (7) showed a mild alveolar reaction, abundant dust-laden interstitial macrophages and some collagen. Some collagen fibres were present in regional lymph nodes in addition to dust-laden phagocytes. The response for Dust (7) was intermediate between the response to the smelter-grade alumina and dust (6), the chemical grade product. Animals dosed with quartz had a very severe alveolar reaction, with very abundant aggregates of dust-laden interstitial macrophages, interstitial nodules and abundant collagen. Whorls of collagen were observed in regional lymph nodes. The saline controls exhibited normal lung pathology.

  

The mice were injected intra-peritoneally with a 0.5 mL volume of 1% suspension of dust in sterile isotonic saline. Five mice were sacrificed at 30, 90 and 180 days and 10 mice at 360 days. Nodules formed by the dust on the anterior wall of the abdomen and over the peritoneal viscera were examined macroscopically and histopathology using formalin-fixed, paraffin sections stained with hematoxylin, eosin and also van Gieson stain to identify connective tissue and collagen fibres. Nodules were observed typically on the liver, spleen, and omentum of the treated mice. The nodules were less numerous in smelter-grade alumina (Dust (1) to (5))-treated animals compared with quartz-treated animals. Histopathology examinations of nodules showed a progression of responses between quartz (most severe; progressive fibrotic), the chemical-grade Dust (6) and laboratory grade Dust (7) and then the smelter-grade alumina (Dusts (1) to (5) – least severe).

 

Overall, all dust samples produced an inflammatory alveolar reaction on intratracheal instillation at these doses. The smelter-grade dusts, dusts (1) to (5) did not show evidence for a fibrotic effect in the rats’ lungs during the period of a year following intratracheal instillation. In contrast, Dust (6), the chemical grade, ultrafine non-alpha alumina and Dust (7), the laboratory grade alumina, showed evidence of definite fibrotic changes. The doses that were instilled into the rats overloaded clearance mechanisms – yet there was a difference in intensity of response between the dusts.

Although the modes of administration used in this study limit its utility for the derivation of a dose-descriptor, the results clearly show the importance of the physical characteristics of the alumina dust on the biological response. The intratracheal instillation doses overloaded clearance mechanisms in all cases. Only one dose was used. Two species were investigated, animal weights were monitored, cytological and biochemical analyses of BALF were undertaken in addition to histopathological examinations of lung tissue. The study was well-reported, described the test-items and methods adequately providing reliable but non-guideline information useful for the risk assessment.