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Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Exposure to multi-walled carbon nanotubes results in aggravation of airway inflammation and remodeling and in increased production of epithelium-derived innate cytokines in a mouse model of asthma
Author:
Ronzani C, Casset A and Pons F
Year:
2013
Bibliographic source:
Arch. Toxicol., DOl 10.1007/s00204-013-1116-3. Published online: 15 August 2013.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The most frequent respiratory allergen so far associated with asthma in humans, house dust mite (HDM) was used to trigger allergen-specific systemic immune response and airway inflammation and remodeling in the mouse. The effects of MWCNT were investigated on these responses and examined the production of the epithelium-derived innate cytokines TSLP, IL-25, lL-33, and GM-CSF.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Tangled Multi-Walled Carbon Nanotubes
EC Number:
701-160-0
Cas Number:
7782-42-5
Molecular formula:
Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms
IUPAC Name:
Tangled Multi-Walled Carbon Nanotubes
Constituent 2
Reference substance name:
GRAPHISTRENGTH C100
IUPAC Name:
GRAPHISTRENGTH C100
Test material form:
solid: nanoform

Test animals

Species:
mouse
Strain:
other: BALB/cByJ
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Saint-Germain-sur-l'Arbresle, France)
- Age at study initiation: no data
- Weight at study initiation: 10 weeks
- Housing: 4 mice per polycarbonate exhaust ventilated cage
- Diet: standard diet 4RF21, Mucedola, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50± 10
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Route of induction exposure:
other: intranasal (25 µL)
Vehicle:
other: synthetic lung surfactant
Concentration:
A Dermatophagoides pteronyssinus (HDM) extract was purchased from GREER® Laboratories Inc., as a lyophilized preparation (Lenoir, NC). This extract was dissolved in saline, and its content in the major allergen Der p 1 (210 µg/mL) and in endotoxin (12.5 ng/mL, i.e., 59 pg endotoxin per µg Der p 1) was determined using a Der p 1 ELISA (Indoor Biotechnologies, Charlottesville, VA) and the Chromo LAL assay (Associates of Cape Cod Inc., East
Falmouth, CA), respectively.
No. of animals per dose:
6
Details on study design:
6 groups of 6 animals:
- vehicle alone (Control group),
- HDM extract alone 2 µg Der p 1/administration (HDM group)
- MWCNT (total dose 75 µg)
- MWCNT (total dose 225 µg)
- HDM (2 µg Der p 1/administration) and MWCNT (75 µg)
- HDM (2 µg Der p 1/administration) and MWCNT (225 µg)
HDM extract or vehicle was administered on days 0, 7, 14, and 21 of the protocol.
MWCNT (25 µg/administration) +/- HDM were administered on days 0, 7, and 14 MWCNT (75 µg) or every other day from day 0 to day 18 MWCNT (225 µg).
All animals were used on day 23.
Positive control substance(s):
none
Negative control substance(s):
none

Results and discussion

Results:
MWCNT Graphistrength C100 increase in dose-dependent manner systemic immune response and airway inflammation, mucus production, and fibrotic response induced by HDM in the mouse. These effects are associated with an increased production of the epithelium-derived innate cytokines TSLP, IL-33, and IL-25.

Any other information on results incl. tables

Effect of MWCNT on serum levels of total and allergen-specific lgG1:

Mice exposed to HDM alone exhibited significant levels of specific lgG1 in serum when compared to control animals (p< 0.01), demonstrating sensitization to mite allergens (Fig. l b). Specific IgG1 levels were significantly increased in HDM + 75 µg MWCNT (p < 0.01) and HDM + 225 µg MWCNT (p < 0.001) mice when compared to control mice, and in HDM + 225 µg MWCNT (p < 0.05) mice when compared to HDM mice (Fig. 1b). Furthermore, total lgG1 levels were significantly increased in the HDM + 225 µg

MWCNT group when compared to control (p < 0.001) or HDM (p < 0.01) group (Fig. l a).

Effect of MWCNT on allergen-induced airway inflammation:

Although not significant, an inflammatory cell infiltrate was observed in BALF of mice that received 75 or 225 µg MWCNT when

compared to controls (Fig. 2a). Likewise, a nonsignificant cell infiltrate was observed in mice exposed to HDM alone (Fig. 2a). By contrast, exposure to HDM + MWCNT caused a massive influx of inflammatory cells into mouse airways. Indeed, the total number of cells was significantly greater in BALF from HDM + 75 µg MWCNT (p < 0.01) and HDM + 225 µg MWCNT (p < 0.001) mice compared to control mice, and in BALF from HDM + 225 µg MWCNT animals (p < 0.001) compared to HDM animals.

As well, the numbers of eosinophils, neutrophils, and lymphocytes were significantly greater in HDM + 75 µg MWCNT (p < 0.01, p < 0.001, and p < 0.01, respectively) and HDM + 225 µg MWCNT (p < 0.001, p < 0.001, and p < 0.01, respectively) groups when compared to the control group, and in HDM + 225 µg MWCNT mice (p < 0.001, p < 0.001, and p < 0.05, respectively) when compared to HDM mice. Histological analysis showed peribronchial and perivascular inflammatory infiltrates in the lung of mice

exposed to 225 µg MWCNT (Fig. 2c), HDM (Fig. 2d), and HDM + 225 µg MWCNT (Fig. 2e) when compared to control animals (Fig. 2b). These infiltrates were the most important in animals exposed to HDM + MWCNT.

Effect of MWCNT on allergen-induced airway remodeling:

Total soluble collagen and TGF-b1 levels were significantly increased in mice exposed to HDM alone (p < 0.01 and p < 0.001, respectively) or HDM + 75 µg MWCNT (p < 0.001) when compared to control mice, and even more in mice exposed to HDM + 225 µg MWCNT compared to control (p < 0.001) or HDM (p < 0.05) group (Fig. 3a, b). An increased collagen deposition was seen around bronchioles and vessels of mice exposed to 225 µg MWCNT (Fig. 3d), HDM (Fig. 3e), and HDM + 225 µg MWCNT (Fig. 3f) when compared to control animals (Fig. 3c ).

Mice exposed to 225 µg MWCNT (p < 0.05) or to HDM (p < 0.001) alone exhibited mucus hyperproduction when compared to control mice, as evidenced by increased mucin levels in lung homogenates (Fig. 4a). Mucin levels were further increased in lung homogenates of mice exposed to HDM + 75 µg MWCNT or to HDM + 225 µg MWCNT (p < 0.05 and p < 0.001, respectively) when compared to HDM animals (Fig. 4a). Mucus hyperplasia was observed on lung sections from mice that received 225 µg MWCNT (Fig. 4c), HDM (Fig. 4d), or HDM + 225 µg MWCNT (Fig. 4e) when compared to control animals (Fig. 4b ).

Effect of MWCNT on the production of allergic and epithelium-derived innate cytokines:

In mice exposed to MWCNT alone, levels of allergic cytokines were not different from control mice, but levels of TSLP and IL-25 were significantly increased (p < 0.01 and p < 0.001, respectively) in the case of animals exposed to 225 µg MWCNT. Eotaxin,

TARC, IL-25, and IL-33 were significantly increased in mice exposed to HDM (p < 0.001, p < 0.01, p < 0.05, and p < 0.001, respectively) compared to control mice, whereas TSLP and GM-CSF were unchanged. But all allergic and epithelium-derived cytokines were significantly increased in the lung of mice exposed to HDM + 225 µg MWCNT when compared to control (p < 0.001 for all cytokines) or to HDM animals (p < 0.05 for TARC; p < 0.01 for IL-13 and GM-CSF; p < 0.001 for eotaxin, TSLP, IL-25, and IL-33).

Applicant's summary and conclusion

Interpretation of results:
other: Graphistrength C 100 aggravates allergen-induced systemic immune response, as well as airway inflammation and remodeling in a dose-dependent manner
Conclusions:
Graphistrength C100 increase in dose-dependent manner systemic immune response and airway inflammation, mucus production, and fibrotic response induced by HDM in the mouse. These effects are associated with an increased production of the epithelium-derived innate cytokines TSLP, IL-33, and IL-25.
Executive summary:

The effect of MWCNT (Graphistrength C 100) on systemic immune response and airway inflammation and remodeling induced by the most frequent allergen so far associated with asthma, house dust mite (HDM) were investigated in the mouse. The production of the innate cytokines thymic stromal lymphopoietin (TSLP), IL-25, IL-33, and GM-CSF was evaluated. Mice exposed to HDM exhibited specific IgG1 in serum and inflammatory cell infiltration, and increased Th2 cytokine production, mucus hyperproduction, and collagen deposition in the airways when compared to naive animals. Levels of total IgG 1 and HDM-specific IgG I, influx of macrophages, eosinophils and neutrophils, production of collagen, total tumor growth factor TGF-b1, and mucus, as well as levels of IL-13, eotaxin, and TARC, were dose-dependently increased in mice exposed to HDM and MWCNT compared to HDM alone. These effects were associated with an increased production of TSLP, IL-25, IL-33, and GM-CSF in the airways. MWCNT increase in a dose-dependent manner systemic immune response, as well as airway allergic inflammation and remodeling induced by HDM in the mouse. The data suggest also a role for airway epithelium and innate cytokines in these effects.