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Repeated dose toxicity: other routes

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short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Lung deposition and toxicological responses evoked by multi-walled carbon nanotubes dispersed in a synthetic long surfactant in the mouse
Ronzani C, Spiegelhalter C, Vonesch J-L, Lebeau L and Pons F
Bibliographic source:
Arch Toxicol, 86:137-149

Materials and methods

Principles of method if other than guideline:
The lung deposition and the inflammatory and remodeling responses evoked by surfactant-dispersed MWCNT were investigated in the mouse.
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Tangled Multi-Walled Carbon Nanotubes
EC Number:
Cas Number:
Molecular formula:
Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms
Tangled Multi-Walled Carbon Nanotubes
Constituent 2
Reference substance name:
Test material form:
solid: nanoform

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories (Saint-Germain-sur-l' Arbresle, France)
- Age at reception: nine-week-old
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: no data
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: intranasal instillation
other: see below
Details on exposure:
Preparation of the synthetic lung surfactant and other dispersion media:
The synthetic lung surfactant developed in the present study was prepared as follows. dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol (PG), cholesterol (Chol) and bovine serum albumin (BSA) (all reagents from Sigma-Aldrich, Saint-Quentin-Fallavier, France) were dissolved in chlorofonn-methanol (9:1) at a mass ratio of 70:10:10:10%. After evaporation of the solvent, the residue was suspended in 10 mM HEPES buffer containing 145 mM NaCl (pH 7.4) at a final DPPC concentration of 1 mg/mL. The resulting solution was sonicated in a bath (Bioblock Scientific, Strasbourg, France) at a power of 56 W and a frequency of 40 kHz for 10 s before use. Other dispersing media used for comparison included saline (0.9% NaCl) and saline containing 0.1% DPPC, 0.5% BSA, 1% Pluronic® F68 (BASF Corp, New Milford, CT) or 1% sodium dodecyl sulfate (SDS, Qbiogene, Strasbourg, France), as CNT dispersing media previously described in the literature.
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
on days 0, 7 and 14
Doses / concentrations
Doses / Concentrations:
1.5, 6.25 and 25 µg/mouse
No. of animals per sex per dose:
Control animals:
other: Control animals received instillations of the same volume of surfactant or saline
Details on study design:
Doses were administered under light anesthesia into the lung of mice by intranasal instillation of 25 µL of a 1 mg/4 mL dispersion (6.25 µg), a 4-time dilution of this dispersion (1.5 µg) or a 4 mg/4 mL preparation (25 µg). Importantly, at 4 mg MWCNT/4 mL surfactant, dispersion properties of surfactant were preserved. Control animals received instillations of the same volume of surfactant or saline.


Sacrifice and pathology:
Bronchoalveolar lavage fluid and tissue collection
The experiment was terminated on day 21 by i.p. injection of ketamine and xylazine. The trachea was cannulated to perform bronchoalveolar lavages. Lungs were lavaged by 6 instillations of 0.5 mL ice-cold saline supplemented with 2.6 mM EDTA (saline-EDTA). Bronchoalveolar lavage fluids (BALF) recovered from the two first instillations were centrifuged (200xg for 5 min at4 °C), and the resulting supernatant was stored at - 20°C until cytokine measurements. Cell pellets recovered from the 6 instillations were resuspended in saline-EDT A and used to determine total and differential cell numbers. After the collection of BALF, lungs were perfused through the pulmonary artery with ice-cold PBS, collected and either frozen in liquid nitrogen and stored at -80°C until cytokine and collagen assays, or fixed in 4% paraformaldehyde for histology.

Determination of total and differential cell counts in bronchoalveolar lavage fluids
BALF were centrifuged (200xg for 5 min at 4°C) to pellet cells, and erythrocytes were lysed by hypotonic shock. Cells were then resuspended in 500 µL ice-cold saline-EDTA, and total cell counts were determined using a Neubauer's chamber. Differential cell counts were assessed on cytologic preparations obtained by cytocentrifugation (Cytospin 4, Thermo Scientific, France) of 200 µL of diluted BALF (250,000 cells/mL in ice-cold saline-EDTA). Slides were stained with Microscopy Hemacolor® (Merck, Germany), and determinations were made by counting at least 400 cells for each preparation. Eosinophil, neutrophil, lymphocyte and macrophage numbers were expressed as absolute numbers from total cell counts. Moreover, the percentage of macrophages containing MWCNT was determined by optical microscopy.

Cytokine assay
Tumor necrosis factor (TNF)-a, keratinocyte-derived chemokine (KC), interleukin (lL)-17, eotaxin and total tumor growth factor (TGF)-ß1 were measured in bronchoalveolar lavage fluids by ELISA.

Collagen assay
Lung collagen content was assessed by quantifying total soluble collagen using the Sircol Collagen Assay kit (Biocolor, Carrickfergus, UK). Briefly, the frozen lungs were homogenized in 2 mL of PBS containing a protease inhibitor cocktail (Complete EDTA-free tablets, Roche, Germany) using an Ultra-Turrax homogenizer (T25, lka, Staufen, Germany). Homogenates were centrifuged (12,000xg, 15 min, 4°C) before collagen and protein assay, according to the manufacturer' s instructions and the Bradford method, respectively. Data were expressed as total soluble collagen per mg of lung protein.

Fixed lungs were rinsed in PBS, dehydrated and embedded in paraffin using standard procedures. Tissue sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E) for routine morphologic assessment, Masson's trichrome for the evaluation of collagen deposition and periodic acid-Schiff (PAS) for mucus visualization.

TEM on lung tissue
Freshly perfused lungs were cut into small pieces and fixed with 2.5% glutaraldehyde/4% formaldehyde in 0.1 M phosphate buffer. Tissues were then post-fixed with 1% osmium tetroxide, dehydrated and resin-embedded using an automated microwave embedding apparatus (AMW, Leica, Microsystems). Ultrathin tissue sections (60 nm) were collected on grids and observed by TEM using a CM12 Philips microscope operated at 80 kV. Image
acquisition was done with an Orins 1,000 CCD camera (GAT AN).
Data are presented as means ± SEM. Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's test, using the GraphPad Prism 4.0 software. Data were considered as significantly different when P < 0.05.

Results and discussion

Results of examinations

Clinical signs:
not examined
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Deposition of surfactant-dispersed MWCNT in the mouse airways:
Histology on lung tissue sections demonstrated a deposition of surfactant-dispersed MWCNT mainly in the alveoli in mice exposed to repeated administration of MWCNT. Importantly, no airway obstruction by MWCNT was noted in animals.
Microscopic examination of cells collected in BALF from mice exposed to repeated administrations of MWCNT showed macrophages loaded with MWCNT, with a percentage of MWCNT-containing macrophages of about 20%. TEM on lung sections showed MWCNT in alveolar macrophages, which was consistent with light microscopy observations of BALF cells. lt also revealed the presence of MWCNT entrapped in the mucus layer covering ciliated airway epithelial cells, as well as in neutrophils infiltrated in the airway lumen and in type 2 alveolar epithelial cells.

Lung inflammation in response to surfactant-dispersed MWCNT (Table 2):
Surfactant alone evoked no changes in BALF cellularity or cytokine content when compared to saline, suggesting surfactant biocompatibility.
In contrast, a neutrophil infiltrate (P < 0.01) was observed in BALF of mice that received MWCNT when compared to the surfactant group. As well, a marked increase in macrophages (P < 0.05) together with some neutrophil (P < 0.05) and eosinophil (P < 0.01) infiltrates was observed. Levels of KC were threefold higher (P < 0.001) in MWCNT-treated mice when compared to surfactant treated mice.

Histopathology of lung tissue from mice exposed to surfactant-dispersed MWCNT
Repeated administrations of synthetic surfactant alone evoked no histopathologic changes in the lung of mice. In contrast, histopathology of lung tissue from mice exposed to 6.25 µg of surfactant-dispersed MWCNT revealed the presence of peribronchial and perivascular cellular infiltrates, which confirmed the inflammatory response found in BALF. Moreover, repeated exposure to MWCNT resulted in the appearance, of some discrete granulomas. These granulomas were organized around MWCNT material and formed of macrophages and some infiammatory cells.

Airway remodeling in lung tissue from mice exposed to surfactant-dispersed MWCNT:
As shown in Table 2, the inflammatory response evoked by MWCNT was dose dependent. In these animals, fibrogenic response was first assessed quantitatively by measuring total soluble collagen in lung homogenates and total TGF-ß1 in BALF. Total soluble collagen levels were dose-dependently increased in mouse exposed to surfactant-dispersed MWCNT, with a significant elevation at the highest dose tested, namely 25 µg. Likewise, the highest dose of MWCNT induced a significant increase in total TGF-ß1 in BALF of mice. Collagen deposition induced by surfactant-dispersed MWCNT exposure was also assessed qualitatively by Masson's trichrome staining of lung tissue sections. A gentle increase in collagen deposition was seen around bronchioles and vessels of mice exposed to the highest dose ofMWCNT compared to control animals. These sites of deposition reminded the location of inflammatory cell infiltrates. Collagen deposition was not observed in alveolar tissue or within granulomas. In animals repeatedly exposed to increasing doses of surfactant-dispersed MWCNT, mucus production was also assessed by histology. In the line of the collagen findings, mucus hyperplasia was observed on lung sections from mice exposed to 25 µg of MWCNT

Effect levels

Dose descriptor:
Effect level:
6.25 other: µg/mouse
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dispersions of MWCNT in surfactant were analyzed by DLS. Particle size distribution indicated the presence of two populations of particles, with mean size of 150 and 500 nm, indicating polydispersity. TEM images of MWCNT dispersed in surfactant showed the presence of MWCNT bundles, as well as individual MWCNT, that could reflect the two particle size populations detected by

DLS. At 24 h, dispersions of MWCNT in surfactant remained stable, as observed visually. At last, to assess MWCNT integrity during dispersion in surfactant, size analysis of individual MWCNT was carried out on TEM images. This analysis gave an average

MWCNT outer mean diameter of 13 nm (data not shown) and a length distribution from 0.22 up to 5.30 µm with a median value of 0.95 µm

Applicant's summary and conclusion

Executive summary:

A synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), was elaborated as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (Graphistrength C100) in mice (1.5, 6.25 and 25 µg/mouse). Deposition of surfactant-dispersed Graphistrength C100 in the lung of BALB/c mice upon repeated administrations (on days 0, 7 and 14) was analyzed by histology and TEM on day 21. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-ß1 and collagen, and by histology. Surfactant dispersed Graphistrength C100 distributed all throughout the mouse airways and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. After repeated Graphistrength C100 administrations, increases in macrophage number, KC and TGF-ß1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed.