Registration Dossier

Toxicological information

Respiratory sensitisation

Currently viewing:

Administrative data

respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Well-documented publication on respiratory sensitisation

Data source

Reference Type:
Inhalation exposure to nanosized and fine TiO2 particles inhibits features of allergic asthema in a murine model.
Rossi, E. M. et al.
Bibliographic source:
Particle and Fibre Toxicology, 7: 35

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Non-allergic (healthy) and ovalbumin-sensitized (asthmatic) female mice were exposed via inhalation to two different sizes of titanium dioxide particles, nanosized and fine , for 2 hours a day, three days a week, for four weeks at a concentration of 10 mg/m^3. Different endpoints were analysed to evaluate the immunological status of the mice.
GLP compliance:
not specified

Test material

Test material form:
other: micro- and nanoparticles
Details on test material:
- Name of test material (as cited in study report): titanium dioxide (two different types of TiO2: nanosized and coarser fine-sized)
1) Fine rutile particle (fTiO2)
- Product number: 224227 (Sigma-Aldrich, Steinheim, Germany)
- Initial particle size: under 5 µm
- Specific surface area: 2 m^2/g
- Phase structure: rutile
- Composition: Ti . O.

2) Nanosized rutile (nTiO2)
- Product number: 637262 (Sigma-Aldrich)
- silica coated, needle-like and ca. 10 × 40 nm in size
- Specific surface area: 132 m^2/g (130 - 190 m^2/g)
- Phase structure: rutile
- Composition: Ti . O.; SiO2 coating (<5%)

Test animals

Details on test animals and environmental conditions:
- Source: Scanbur AB, Sollentuna, Sweden
- Age at study initiation: 7 week old
- Acclimation period: 1 week

Test system

Route of induction exposure:
other: intraperitonal
Route of challenge exposure:
other: phosphate-buffered saline (PBS)
10 ± 2 mg/m^3
No. of animals per dose:
8 female mice
Details on study design:
The animals were exposed using a solid particle dispenser (Rotating Brush Generator, RBG 1000, Palas, Karlsruhe, Germany) as described by Rossi et al.(2010)*.

The aerosol size distributions were measured from 10 nm to 1000 nm with a scanning mobility particle sizer (SMPS) consisting of a 63Ni bipolar aerosol neutralizer, a Vienna type differential mobility analyzer (DMA) with length of 28 cm and TSI model 3010 condensation particle counter (CPC) (Wang, 1990)*. Aerosol aerodynamic size distribution was measured from 15.9 nm to 10 μm with an electronic low pressure impactor (ELPI, Dekati) (Marjamäki, 2000). The aerosol mass concentration was determined gravimetrically after collection on nitrocellulose filters (Millipore). Particle concentration inside the particle dispenser chamber was also monitored using a personal DataRAM (online mass concentration meter, pDR1000AN, Waltham, MA, USA).
Aerosolized particles occured as agglomerates. Specific surface area of nTiO2 is 132 m^2/g and 2 m^2/g for fTiO2. The aerodynamic number size distributions indicate that nTiO2 dispersed in air using the solid particle dispenser occurred mostly as agglomerates of 100 nm at a number concentration of 69 000 cm^-3 whereas fTiO2 occurred as agglomerates of 1 μm at a number concentration of 8000 cm^-3. fTiO2 distribution consist mainly of agglomerates of 0.1 - 2 μm as measured using ELPI. It can be concluded that in particle number concentration most fTiO2 are below 1 μm in size but particle mass concentration relies on over 1 μm particles. Analysis of the hydroxyl radical formation capacity using the benzoic acid probe showed that neither of the used particles produced •OH-radicals in a systematic dose-response pattern (Rossi et al., 2010)*.

- Induction exposure
Mice sensitized intraperitonally with 20 μg of ovalbumin (OVA) in alum (Sigma-Aldrich, St Louis, MO) in 100 μl of phosphate-buffered saline (PBS) on days 1 and 14 of the experiment. Control group was given alum in 100 μl of PBS. Exposure groups were exposed three times a week for 2 hours for the duration of the four week experiment. The exposure concentration was 10 ± 2 mg/m^3 in all tests.

- Challenge exposure
On days 25-27 all mice were challenged with 1% ovalbumin solution via the airways for 20 minutes administered using the ultrasonic nebulizer (DeVilbiss, Glendale Heights, IL).

A. Measurement of airway responsiveness
Airway responsiveness was measured on day 28 using a single chamber, whole-body plethysmograph system (Buxco, Troy, NY) as described in Hamelmann et al. (1997)*. Mice were exposed to increasing concentrations (1, 3, 10, 30 and 100 mg/ml) of metacholine (Sigma Aldrich) in PBS delivered via an AeroSonic 5000 D ultrasonic nebulizer (DeVilbiss, ITW, Glendale Heights, IL). Before metacholine exposure the baseline is measured for three minutes. After baseline measurements the metacholine is nebulized for 1.5 minutes and airway reactivity is measured for 5 minutes per concentration. Lung reactivity parameters were expressed as Penh (enhanced pause) values. After measurement of lung responsiveness, the mice were sacrificed using an overdose of isoflurane and samples were collected for analysis.

B. Collection of biological samples and preparation
Sample collection and preparation were done as described by Rossi et al. (2010). The following samples were collected: blood serum for antibody
analysis, bronchoalveolar lavage (BAL) cells and supernatant for May Grünwald-Giemsa (MGG) staining and protein analysis and lung samples for RNA isolation and hematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS) staining. The spleens were also dissected from the mice onto 6-well plates with PBS for spleen cell stimulations.

C. Spleen cell stimulations
Spleens were mechanically broken down into paste and filtered to remove larger pieces. Cells were resuspended in RPMI media, counted under light microscopy and plated at a concentration of one million cells/mL. Cells to be stimulated were plated in RPMI containing 100 μg/ml ovalbumin. All cells were incubated for 48 hours after which the supernatant was collected and stored at -70°C prior to analysis.

D. mRNA and protein analyses
- RNA isolation from the lung tissues
The lung samples were homogenized in a FastPrep FP120 (BIO 101, Thermo Savant, Waltham, Mass. USA) -machine and RNA was extracted using the FastRNA Pro Green Kit (Qbiogene/MP Biomedicals, Illkirch, France) and its instructions.
- cDNA synthesis
cDNA was synthesized from 1 μg of total RNA in a 25 ul reaction using MultiScribe Reverse Transcriptase and random primers (The High-Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA) using the manufacturer’s protocol. The synthesis was performed in a 2720 Thermal Cycler (Applied Biosystems, Carlsbad, California, USA) starting with 25°C for 10 minutes and continuing with 37°C for 120 minutes.
- Polymerase chain reaction (PCR) amplification
PCR primers and probes were ordered as pre-developed assay reagents (18 S rRNA, TNFa, IL-1b, IL-4, IL-13, IL-10, foxp3, CCL3, CXCL5 and CXCL2) from Applied Biosystems (Carlsbad, California). The real-time quantitative PCR was performed as described by Rossi et al. (2010).
- Enzyme-linked immunosorbent assay (ELISA)
TNF-a and IL-13 protein levels from spleen cell supernatants were determined using ELISA kits (eBioscience, San Diego, CA, US) used according to manufacturer’s instructions. An ELISA plate microtiter reader (Multiskan MS, Labsystems, Helsinki, Finland) was used to read the results.
- Luminex
For analysis of TNF-a and IL-13 proteins in BAL fluid supernatants a Milliplex Mouse Cytokine/Chemokine Immunoassay (Millipore Corporation, Billerica, MA) was used according to the manufacturers’ protocol. 3% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO) in PBS was added at a concentration of 0.5% to samples, controls and standards to ensure sufficient protein amounts for the assay. Assay was performed using Luminex xMAP Technology (Bio-Plex 200 System, BioRad, Hercules, CA).

E. Measurement of serum antibodies
For the analysis of OVA-specific IgE, ELISA plates were coated with Purified Rat Anti-mouse IgE antibody (BD Biosciences, San Jose, CA) and incubated overnight. On the second day the plates were blocked with 3% BSA in PBS, serum samples (1:10 and 1:20 dilution) were added to the plate and incubated overnight. Biotinylated OVA was added and streptavidin horseradish peroxidase (Pharmingen 13047E, BD Biosciences) followed by ABTS solution (ABTS Microwell Peroxidase Substrate System, KPL, Gaithersburg, MD). Absorption was read at 405 nm with ELISA plate absorbance reader (Multiskan
MS, Labsystems, Helsinki, Finland).
For OVA-specific IgG2a analysis, ELISA plates were coated with ovalbumin (BD Biosciences) and incubated overnight. On the second day the plates were blocked with 3% BSA in PBS, serum samples (1:60, 1:180, 1:540 and 1:1620 dilution) were added to the plate and incubated overnight. Biotinylated anti-mouse IgG2a (Pharmingen 02012 D, BD Biosciences) was added and streptavidin horseradish peroxidase (BD Biosciences). ABTS solution and peroxidase were mixed 50:50 and added before reading absorption at 405 nm with ELISA plate absorbance reader.

F. Analysis of mucus producing cells
The lung tissue was analysed from formalin-fixed, paraffin-embedded sections. These sections were stained for mucus secreting goblet cells using Periodic acid-Schiff (PAS) -stain. The data was analyzed with Leica Image Manager IM50 version 4.0 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). PAS+ cells were counted as an average of PAS+ cells found in 100 μm of bronchus counted from three bronchioles of similar size per mouse (n = 8 mice per group).

G. Hydroxyl radical (•OH) formation capacity
The •OH formation capacity was determined using the benzoic acid probe as described by Rossi et al. (2010).

H. Statistical analysis
The toxicological data was analyzed with the Graph-PadPrism software (GraphPadPrism Software, Inc., San Diego, CA). For all statistical analysis an analysis of variance using one-way ANOVA (nonparametric Kruskal-Walles test) was performed first and when the ANOVA was positive post-testing was performed. The different mice groups were compared using nonparametric Mann-Whitney U - test. P-values less than 0.05 were considered statistically significant.

- Rossi EM, Pylkkanen L, Koivisto AJ, Vippola M, Jensen KA, Miettinen M, Sirola K, Nykasenoja H, Karisola P, Stjernvall T, Vanhala E, Kiilunen M, Pasanen P, Mäkinen M, Hämeri K, Joutsensaari J, Tuomi T, Jokiniemi J, Wolff H, Savolainen K, Matikainen S, Alenius H: Airway Exposure to Silica-Coated TiO2 Nanoparticles Induces Pulmonary Neutrophilia in Mice. Toxicol Sci 2010, 113:422-433.
- Wang SC, Flagan RC: Scanning Electrical Mobility Spectrometer. Aerosol Science and Technology 1990, 13:230.
- Marjamäki M, Keskinen J, Chen D-R, Pui DYH: Performance evaluation of the Electrical Low-Pressure Impactor (ELPI). Journal of Aerosol Science 2000, 31:249-261.
- Hamelmann E, Schwarze J, Tadeka K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW: Noninvasive Measurement of Airway Responsiveness in Allergic Mice Using Barometric Plethysmography. Am J Respir Crit Care Med 1997, 156:766-767.
Challenge controls:
Please refer to the field "Details on study design" above
Positive control substance(s):
not specified
Negative control substance(s):
not specified

Results and discussion

- asthmatic mice demonstrate increased numbers of eosinophils and lymphocytes in the airways compared to healthy controls.
- in healthy mice (PBS) there was a significant 4.6-fold increase in the influx of neutrophils following exposure to nTiO2 whereas the numbers of macrophages, eosinophils and lymphocytes remained unaffected.
- exposure to fTiO2 had no effects on any cells in the BAL of healthy mice.
- numbers of eosinophils and lymphocytes were dramatically reduced in asthmatic mice (OVA) after exposure to both nTiO2 and fTiO2. Macrophages were reduced in half following nTiO2 and increased 1.7-fold following fTiO2 exposure in asthmatic mice. Changes in numbers of pulmonary neutrophils remained non-significant.
- healthy mice showed clear lungs with no mucus secreting cells whereas PAS+ cells were abundant in the bronchial epithelium of allergic mice.
- both TiO2 particles caused a drastic reduction in the numbers of PAS+ cells in the allergic mice.
- both exposed groups showed a statistically significant decrease in goblet cell numbers in the epithelium after the particle exposure.
- exposure of OVA sensitized and challenged mice to nTiO2 reduced airway hyperreactivity (AHR) to the level of healthy mice.
- exposure to fTiO2 slightly increased the reactivity of the lungs, showing modest exacerbation of asthmatic symptoms.
- PBS groups were exposed to TiO2 particles but no difference on airway reactivity to inhaled methacholine was found between them and unexposed PBS groups.
- mRNA expression of proinflammatory cytokines, IL-1b and TNF-a, was downregulated after particle exposure to about half and third respectively in asthmatic mice when compared to the non-exposed control. IL-1b and TNF-a levels were also reduced in half in healthy (PBS) mice following exposure to fTiO2. Th2 type cytokines IL-4 and IL-13, which are present in asthmatic but not in healthy mice, were significantly diminished after exposure to both nTiO2 and fTiO2.
- fTiO2 exposure reduced IL-10 levels by 2.5-fold and Foxp3 levels by 4.5-fold.
- nTiO2 exposure reduced IL-10 levels by over 8-fold and Foxp3 levels by 2.3-fold.
- reduction in the protein levels of both TNF-a and IL-13 in the BAL from asthmatic mice exposed to TiO2 particles was observed with statistical significance (one-way ANOVA) in IL-13 levels.
- mRNA expression of proinflammatory CCL3 and neutrophil attracting CXCL5 and CXCL2 was decreased in asthmatic mice after exposure to both used particles.
- in healthy mice exposure to nTiO2 caused an almost 5-fold elevation of CXCL5 levels, corresponding to high levels of neutrophils seen in the BALF of the same mice.
- spleen cells from asthmatic nTiO2 exposed mice were either stimulated with OVA or left untreated. OVA stimulated cells express TNF-a and IL-13, but when the mice had been exposed to nTiO2 the protein levels were reduced amply.
- OVA sensitized mice show high levels of OVA specific IgE, but exposure to fTiO2 reduces the levels of OVA specific IgE significantly.
- no significant changes in the levels of OVA specific IgE could be seen after exposure to nTiO2 particles.
- levels of OVA specific IgG2a were low and unaffected by particle exposure.
Positive control results:
no data
Negative control results:
no data

Applicant's summary and conclusion

Interpretation of results:
not sensitising
According to the authors, healthy mice elicited pulmonary neutrophilia accompanied by increased chemokine CXCL5 expression when exposed to nTiO2. No such response was observed in animals exposed to pigment sized titanium dioxide. Allergic pulmonary inflammation was dramatically suppressed in asthmatic mice which were exposed to nTiO2 or fTiO2 particles - i.e. the levels of leucocytes, cytokines, chemokines and antibodies characteristic to allergic asthma were substantially decreased.
The authors mentioned that the results suggest that repeated airway exposure to TiO2 particles modulates the airway inflammation depending on the immunological status of the exposed mice.