Registration Dossier

Diss Factsheets

Toxicological information

Acute Toxicity: other routes

Currently viewing:

Administrative data

Endpoint:
acute toxicity: other routes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Coating carbon nanotubes with a polystyrenebased polymer protects against pulmonary toxicity
Author:
Tabet L, Bussy C, Setyan A, Simon-DeckersA, Rossi MJ, BoczkowskiJ, Lanone S
Year:
2011
Bibliographic source:
Particle and Fibre Toxicology 2011, 8:3 (http://www.particleandfibretoxicology.com/content/8/1/3)
Reference Type:
other: Thesis
Title:
Unnamed
Year:
2009
Report date:
2009
Reference Type:
publication
Title:
Coating With A Polystyren Polymer Protects Against Respiratory Toxicity Of Carbon Nanotubes In Vivo In Mice
Author:
Tabet L, Bussy C, Setyan A, Marcos E et al.
Year:
2010
Bibliographic source:
Am. J. Respir. Crit. Care Med.; 181: A3089.

Materials and methods

Principles of method if other than guideline:
Study of MWCNT-induced oxidative stress and inflammation in mice intratracheally instilled and followed up to 6 month postinstillation.
GLP compliance:
no
Limit test:
no

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:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier (Le Genest-St-Isle, France)
- Age at reception: 7-9 weeks
- Weight at reception: 22 ± 0.23 g,
- Fasting period before study:
- Housing: standard wiretopped cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

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

Administration / exposure

Route of administration:
other: intratracheal instillation
Vehicle:
other: Dulbecco’s modified Eagle’s medium (DMEM)
Details on exposure:
MWCNTs was suspended at 10 mg/ml in culture Dulbecco’s modified Eagle’s medium (DMEM), vortexed for 1 min, and then sonicated (Elma S30H, 50-60 Hz) for 30 min under cooling conditions, with 30-s interruption every 10 min for vortex at maximum speed.
The suspension was instilled in mice under anesthesia [1.6 mg ketamine (Merial, Lyon, France) plus 300 µg xylazine (Bayer, Puteaux, France)].
Doses:
10 or 100 µg/mouse
No. of animals per sex per dose:
6-8
Control animals:
yes
Details on study design:
- Positive controls:
Asbestos fibers crocidolite (80-nm diameter)
Nanosized carbon black (CB, FR103, 95-nm diameter; Degussa-Germany)

- Mice were sacrificed at 1, 7, 30, 90 or 180 days post-instillation.
Bronchoalveolar Lavage (BAL) and Lung were collected. The percentage of macrophages having internalized the different CNT was analyzed.
Quantification of the mRNA expression of different genes involved in oxidative stress (SOD-2 and HO-1) and inflammation (TNF-a and MIP-2) was performed by quantitative RT-PCR. In addition, expression of collagen-1 and -3 was analyzed, as markers of interstitial fibrosis.
Lung histological analysis was performed in a subset of animals different from that in which BAL analysis and lung gene expression were analyzed. The number and size of MWCNT agglomerates in the lungs of animals exposed for 24 hours, 1 month, or 6 months was measured in five representative animals per group. For each animal, five fields were randomly selected from top to bottom across the vertical diameter of the section.
Statistics:
Each value is the mean ± SEM of at least 4 different experiments (in vitro study) and 6-8 animals (in vivo study). The data were analyzed by one-way ANOVA or non-parametric tests as appropriate. For all tests, a p<0.05 was considered significant.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: MWCNT internalisation in macrophage
Effect level:
10 - 100 other: µg/mouse
Remarks on result:
other: between 1 day and 1 month after instillation
Sex:
male
Dose descriptor:
other: BAL fluid analysis
Effect level:
100 other: µg/mouse
Remarks on result:
other: increase in total cell count and influx of neutrophils and macrophages, only 24 h post-instillation.
Sex:
male
Dose descriptor:
other: Lung histology
Effect level:
10 - 100 other: µg/mouse
Remarks on result:
other: Clusters of cells surrounding visible MWCNTs agglomerates. No evidence of fibrosis
Sex:
male
Dose descriptor:
other: mRNA expression of genes implicated in oxidative stress, inflammation, and fibrosis (SOD-1 and -2, GPX-1, HO-1, TNF-a and -b, CXCL2, and collagen-1 and -3)
Effect level:
10 - 100 other: µg/mouse
Remarks on result:
other: No modifications in gene expression
Mortality:
None.
Other findings:
BAL analysis showed that exposure to NT1 induced a dose-dependent increase in total cell count (p<0.05 vs control) and a significant neutrophil influx (p<0.05 vs. control). These effects were observed only at 24 h postinstillation. NT1 was internalized in macrophages between 1 day and 1 month after instillation.
Histological analysis of the lungs at 1 day after instillation demonstrated the presence of micrometric agglomerates of NT1 at the level of the distal bronchi and the alveolar wall. The agglomerates were preferentially located in the distal bronchi. The late lung response resulted in
infiltration and encasement by macrophages to form a connective tissue rich granulomatous inflammation which is typical of the lungs response to an insoluble particle. These lesions were observed from 3 months after instillation and persisted at 6 months post-instillation. No fibrosis was observed.
mRNA expression of various genes implicated in oxidative stress, inflammation and fibrosis was quantified in lung homogenates. No modification in the expression of these genes was observed in animals exposed to NT1. No modification of GPX-1, SOD-1 or TGF-ß mRNA expression was observed.

Applicant's summary and conclusion

Executive summary:

The pulmonary response of mice was evaluated after exposure to multi-wall carbon nanotubes (MWCNTs, Graphistrength C100). The MWCNTs suspension in sterile Dulbecco’s modified Eagle’s medium (DMEM) was introduced into mice lungs by intratracheal administration. Male Balb/C mice were treated with either 10 or 100 µg of MWCNTs . Mice were sacrificed at 1, 7, 30, 90 or 180 days post-instillation. Bronchoalveolar Lavage (BAL) and lung were collected. The percentage of macrophages having internalized the different CNT was analyzed. Quantification of the mRNA expression of different genes involved in oxidative stress (SOD-2 and HO-1) and inflammation (TNF-a and MIP-2) was performed by quantitative RT-PCR. In addition, expression of collagen-1 and -3 was analyzed, as markers of interstitial fibrosis. Lung histological analysis was performed in a subset of animals different from that in which BAL analysis and lung gene expression were analyzed. The results showed significant cellular influx by a single exposure to MWCNTs. Yields of total cells and the number of neutrophyles in BAL cells were significantly elevated in MWCNT-treated mice post-treatment day 1. Histological analysis of the lungs at 1 day after instillation demonstrated the presence of micrometric agglomerates of MWCNTs at the level of the distal bronchi and the alveolar wall. The agglomerates were preferentially located in the distal bronchi. The late lung response resulted in infiltration and encasement by macrophages to form a connective tissue rich granulomatous inflammation which is typical of the lungs response to an insoluble particle. These lesions were observed from 3 months after instillation and persisted at 6 months post-instillation. No fibrosis was observed. mRNA expression of various genes implicated in oxidative stress, inflammation and fibrosis was quantified in lung homogenates. No modification in the expression of these genes was observed in animals exposed to MWCNTs. No modification of GPX-1, SOD-1 or TGF-ß mRNA expression was observed.