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Diss Factsheets

Administrative data

respiratory sensitisation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given

Data source

Reference Type:
Effects of Asian Sand Dust, Arizona Sand Dust, Amorphous Silica and Aluminium Oxide on Allergic Inflammation in the Murine Lung.
Ichinose et al.
Bibliographic source:
Inhal Toxicol 2008; 20: 685-694.

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
Examination of the absolute and relative numbers of different cell types and specific biochemical parameters in fluid obtained from bronchoalveolar lavage of animal lungs (BALF) following either intratracheal or inhalation exposure to a substance can provide information on the nature of the reaction of the tissue, i.e the mechanism of action. This information is useful to support and interpret histopathological observations and/or external observations of respiratory symptoms or changes in lung function. In this study, histopathology and BALF analyses were done in the the presence and absence of ovalbumin, a known inducer of eosinophilic inflammation, in order to provide further information on the nature of the mechanism of action (allergenic versus non-specific irritative).
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium oxide
EC Number:
EC Name:
Aluminium oxide
Cas Number:
Molecular formula:
aluminium oxide
Details on test material:
Name: Aluminium oxideBatch number: not providedCAS number: not providedDescription of the test item: not providedManufacture date: not providedExpiry date: not providedOrigin: Asian Sand Dust (SD): collected from surface soils in the Shapotou Desert; sieved to a diameter less than 10 microns; SEM measurements (of 600 particles) showed a peak between 4 and 6 microns; heated to 360ºC for 30 minutes in an electric heater to inactivate microbial components.Arizona SD: obtained from Powder Technology Inc. (Woonsocket, RI); sieved to a diameter less than 10 microns; SEM measurements (of 600 particles) showed a peak between 6.6 and 8.6 microns; heated to 360ºC for 30 minutes in an electric heater to inactivate microbial components.SiO2: amorphous silica from Sigma-Aldrich (0.5 to 10 microns with 80% between 1 and 5 microns).Al2O3: aluminium oxide from Strem Chemicals (Newburyport, MA); particle sizes approximately 1 to 5 microns.Purity: The particles were analysed for contaminants. The purity of the amorphous silica and aluminium oxide particles were reported as 99%. Asian SD was 60% silica, 11% aluminium oxide, 4.1% iron oxide, 1.8% disodium oxide, 9.0% calcium oxide, 2.5% magnesium oxide, 0.7% titanium dioxide, 2.2% potassium oxide (loss on ignition 8.7%). Arizona SD was 68 to 76% silica, 10 to 15% aluminium oxide, 2 to 5% iron oxide, 2 to 4% sodium oxide, 2 to 5% calcium oxide, 1 to 2% magnesium oxide, 0.5 to 1% titanium oxide, 2 to 5% potassium oxide (with 2 to 5% loss on ignition). Levels of liposaccharide and β-glucan in the SD particulates were assessed by using an Endospec ES test MK and Fungitec GtestMK assays (both Seikagaku Corp., Tokyo), respectively. The detection limits for the two assays were <0.001 EU/mL and 2 pg/mL, respectively.Storage conditions: Not stated.Safety precautions: Not stated.

Test animals

Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Japan, Inc. (Kanagawa, Japan)- Age at study initiation: 5 weeks at arrival; 6 weeks at testing- Weight at study initiation: 307-315 g- Housing: Plastic cages placed in a “conventional” room. Bedding with soft wood chips not otherwise specified.- Diet: commercial diet CE-2 obtained from CLEA Japan Inc., Tokyo, Japan. - Water: ad libitum- Acclimation period: 1 weekENVIRONMENTAL CONDITIONS- Temperature (°C): 23 °C- Humidity (%): 55-70%- Air changes (per hr): not reported- Photoperiod (hrs dark / hrs light): Artificial light 12 hours daily from 6 a.m. to 6 p.m.Justification of species and strain: ICR mice were chosen based on the results of an earlier study (Ichinose T et al., 2003; Toxicol Appl Pharmacol 187: 29-37) that showed that this species is “moderately” responsive to airway inflammation on exposure to OVA by intratracheal instillation (IT).

Test system

Route of induction exposure:
other: Intratracheal instillation
Route of challenge exposure:
other: not applicable
other: Normal saline
Negative vehicle control (saline),OVA alone,Asian SD alone, Arizona SD alone, SiO2 alone, Al2O3 alone,Asian SD + OVA,Arizona SD + OVA, SiO2 + OVA, Al2O3 + OVA.Concentration: 0.1 mg per mouseDeposition efficiencies were calculated using a tidal volume of 0.15 mL/mouse and a breathing rate of 200 breaths per minute. 0.1 mg/mouse was reported to be 111 times the weekly amount of particulate matter that would be deposited in the alveoli assuming 3% deposition (based on the ICRP model at 5.5 micron particle size) at an exposure level of 0.1 mg/m3.
No. of animals per dose:
16 animals/group- 8 animals used for pathology and 8 animals for BALF analyses.
Details on study design:
Preparation of Test Solution:In the absence of OVA, 5 mg of particulate was suspended in 2.5 or 5 mL of vehicle and sonicated for 5 minutes while being cooled (temperature not specified)For the combined OVA and particulate exposure, the OVA (100µg) was dissolved in 10 mL or 5 mL of saline vehicle. 2.5 mL of the OVA solution was then mixed with 2.5 mL of the particle suspension.Administration of Test Solution:Volume: 0.1 mLMethod: The suspension was intratracheally administered under anaesthesia (4% halothane, Takeda Chemical, Osaka, Japan) using a polyethylene tube.The animals were dosed 4 times in total. The interval between doses was 2 weeks. Further Study details:The animals were killed by exsanguination one day after the last intratracheal instillation when anaesthetised using an i.p. injection of pentobarbital. At this time, the animals were approximately 12 weeks of age.Observations:Pathology of Lung Tissue:Pathologic examinations were done on the lung tissue from 8 out of 16 mice/group. The lungs were fixed with 10% neutral phosphate-buffered formalin, stained with haematoxylin and eosin (H&E) to assess infiltration of eosinophils and lymphocytes and also, to assess proliferation of goblet cells, periodic acid-shiff (PAS). The evaluation was done by two pathologists independently.Bronchoalveolar fluid (BALF) analyses:Cell counts:The BALF of the remaining 8 mice was evaluated for cell counts. Slides were prepared using a Cytopsin (Sakura Co., Ltd., Tokyo, Japan) and were stained with Diff-Quik. 300 cells were counted.Levels of cytokines: The cytokines interleukin-5 (IL-5), IL-12, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), macrophage inflammatory protein-1alpha (MIP-1α), IL-13, and eotaxin were measured using ELISA.Levels of lactate dehydrogenase (LDH): Measured using a lactate dehydrogenase C II-Test Wako from Wako Chemicals Ltd. (Osaka, Japan).Antigen (OVA)-specific and total IgE and IgG1 antibodies: The antibodies were measured using commercial ELISA kits.Statistical Analyses:Group differences were assessed using ANOVA and the Fisher’s projected least significant differences (P/SD) test.
Challenge controls:
Not applicable.
Positive control substance(s):
Negative control substance(s):
not specified

Results and discussion

Pathology of Lung Tissue:Al2O3:The animals treated with aluminium oxide did not show a significantly increased proliferation of goblet cells or infiltration of lymphocytes when compared with the saline control.OVA + Al2O3:Lymphocyte infiltration was higher in the OVA+Al2O3 group compared with the control (p<0.001), and also when compared with OVA alone (p<0.01) and with Al2O3 alone (p<0.001).Eosinophil infiltration was higher in the OVA+Al2O3 group compared with the control (p<0.01) and also OVA+Al2O3 compared with Al2O3 alone (p<0.01).Goblet cell proliferation was higher in the OVA+Al2O3 group compared with the control (p<0.001), OVA+Al2O3 compared with OVA alone (p<0.05) and also when compared with Al2O3 alone (p<0.001).Among the groups exposed to the particulates alone, the greatest changes were observed in the SiO2-treated group. Among the groups treated with OVA+particulates, the greatest changes were observed in the OVA+SiO2 group followed by the OVA+Arizona SD group, the OVA+Asian SD group and last the OVA+Al2O3 group.Bronchoalveolar Lavage Fluid (BALF):Cell countsTotal cells:Among the groups exposed to particulates alone, only the SiO2-treated group had levels significantly greater than the control.In the combined exposure groups, OVA+Asian SD, OVA+Arizona SD, and OVA+SiO2 had levels greater than the control (p<0.001). Total cells in the OVA + Al2O3 group were also significantly greater than the control but not to the same extent (p<0.05). No groups had cell counts greater than that observed in the group with OVA alone. The highest numbers were observed in the OVA+SiO2 group.Macrophages: Among the groups exposed to particulates alone, only Arizona SD and SiO2 caused significant increases in macrophages compared with the control (p<0.01 and p<0.001, respectively). Among the combined exposure groups, OVA + Asian SD, OVA + Arizona SD, and OVA + SiO2 showed levels greater than the control. Numbers of macrophages in the OVA + Al2O3 group were greater than the control but not to the same extent (p<0.01). Only OVA+Arizona SD and OVA+SiO2 had greater numbers of macrophages than the OVA alone group (p<0.05 and p<0.001, respectively).Eosinophils:Particulates alone:No effects observed.Combined exposures:OVA+Al2O3
Positive control results:
Not applicable.
Negative control results:
Not applicable.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
When co-administered with OVA, all the tested materials showed an increase in eosinophils on H&E stained slides (OVA+SiO2>OVA+Arizona SD>OVA+Asian SD>OVA+Al2O3). Considering the results for Al2O3, specifically, in BALF, Al2O3 alone did not lead to significant increases in total cells, macrophages, eosinophils, neutrophils or lymphocytes. When Al2O3 was administered with OVA, small but statistically significant increases in total cells (p<0.05), macrophages (p<0.01) and lymphocytes (p<0.05) were observed relative to the control but not relative to the OVA alone group. Significant (p<0.05) increases in IFN-γ and TNF-α relative to the saline control were observed in BALF of Al2O3 treated animals. IL-5, IL-6, IL-12 and IL-13 did not show significant increases in the Al2O3-treated animals. In exposures combined with OVA, the only significantly elevated cytokine was IFN-γ. Overall, the results from the study showed that allergic inflammatory effects of atmospheric dusts are likely due to SiO2. Al2O3 was the least inflammatory material tested and led to only weak effects on the mouse lung.
Executive summary:

Ichinose et al. (2008) studied allergic inflammation after intratracheal instillation of Asian sand dust,sand dust, amorphous silica and Al2O3in 6-week old male ICR mice. Four instillations were performed at 2-week intervals. There were ten groups of animals (n=16 in each). One of these groups received Al2O3(particle size 1~5 µm), a dose of 0.1 mg suspended in saline. The control group received saline only (0.1 mL). The animals were killed one day after the last instillation. Eight out of 16 animals in each group were used for pathologic examination. The lung samples were stained with haematoxylin and eosin to evaluate the degree of infiltration of eosinophils or lymphocytes in the airways, and with periodic acid-shiff to evaluate the degree of proliferation of goblet cells in the bronchial epithelium. The other 8 mice were used for examination of free cell counts (total and differential), determination of levels of lactate dehydrogenase (LDH), cytokines (Interleukins – IL-5, IL-6, IL-12, IL-13, interferon-IFN-gand tumor necrosis factor- TNF-a) and chemokines in bronchoalveolar lavage fluids (BALF), and also total IgE in serum using enzyme-linked immunosorbent assays (ELISA). In the group of mice exposed to Al2O3, the levels of eosinophil and lymphocyte infiltration in the submucosa and proliferation of goblet cells in the airways, the level of LDH, chemokines and interleukins, number of cells in BALF and the level of IgE in serum were not significantly different from those in the control mice. The results suggest that intratracheal administration of Al2O3does not produce allergic inflammatory effects in the lungs of mice.