nanotechnology is one of the fastest emerging fields involving development and manipulation of materials 100  nm in size .
there are numerous potential perspectives for the applications arising from nanotechnology which include their use in a wide range of fields , for example , medicine , environment , occupational setting , chemistry , energy production , information and communication , heavy industry , and consumer goods .
human exposure to engineered nanomaterials ( enms ) can occur at different stages of their life cycle ( manufacture , use , and disposal ) and several concerns have been expressed about their potential to cause unanticipated adverse effects in humans .
the remarkable diversity of engineered nanomaterials ( enms ) , together with their unique properties and behaviour , complicates their risk assessment ; there are currently about 50,000 different types of carbon nanotubes obtained by different raw materials and production processes .
similar diversity applies to other types of enms , rendering ad hoc risk assessment of all of these materials an immense task [ 1 , 2 ] .
the reach regulation ( registration , evaluation , authorization , and restriction of chemicals ) is the current regulatory framework for chemical risk assessment and management in european union .
although reach should be applied to enms , the technical guidance documents   for preparing a risk assessment currently include very little reference to substances in particulate form , thus lacking in addressing specific characteristics of enms . in 2007 and 2009 , the european union ( eu ) scientific committee on emerging and newly identified health risks ( scenihr ) concluded the applicability of the current risk assessment approaches / methodologies to identify the enm - associated hazard , though pointing to main limitations , thus stressing the need for several adjustments [ 46 ] .
nevertheless , though particular enms properties and behaviour ( i.e. , translocation and capability to cross biological barriers ) have been described in several studies ( i.e. , mode of action , toxicity targets , dose - response relationships , and the potential to react with constituents of cells at the portal of entry and beyond ) , several aspects concerning np - associated risks are still unknown , and critical steps in the risk assessment of enms remain so far the same as those used for conventional chemicals .   furthermore , there are major challenges in assessing exposure ( both airborne / administered and internal dose , i.e. , particle deposition in lung ) .
enms are produced from many substances , in many forms and sizes , and with a variety of surface coatings .
the health risk assessment for such diverse materials requires validated analytical methods permitting their characterization in bulk samples and their detection and measurement in workplace air .
efforts are also needed for the identification of properties that trigger enms toxicity ( e.g. , contaminants , impurities , and defects ) .   today
, there are not enough studies to validate the potential hazards posed by these novel materials and hence the definitive conclusions and tools , technologies , systems , and methods to obviate the risks .
several tools for the assessment of risks are still in the conceptual stage , and further , there are considerable uncertainties on how to assess nanoparticles ( nps ) exposure ( i.e. , which metrics to adopt systematically ) and what methods to use to assess toxicity .
thus , being unable to perform a quantitative risk assessment for enms , due to the lack of sufficient data on exposure , biokinetics , and organ toxicity , it should be made mandatory to prevent exposure by appropriate precautionary measures and practicing best industrial hygiene to avoid future shock scenarios from environmental or occupational exposures . in the safety assessment area
, two basic questions still need to be addressed : ( i ) do nanomaterial properties necessitate a new toxicological science ? and ( ii ) what are the biological and biokinetic properties to be considered for toxicity testing ; in particular , can biology and certain mechanisms of effects of enms ( e.g. , proinflammatory action , oxidative stress , mitochondrial perturbation , generation of neoantigens and protein complexes in the body , enhanced protein degradation at the large surface area of nps complexes , etc . )
be used as a basis for studying nps toxicity and risk assessment ? addressing these questions is of crucial importance to define adequate strategies and establish whether enm - tailored testing methods should be added to conventional toxicity testing protocols to comply with regulatory demand and properly characterize the enms potential hazard . according to the major institutions [ 7 , 9 , 10 ] and international consensus meetings ,
the proposed multitiered testing protocols could be used to address toxicological research and health risk assessment for nps .
toxicity testing should firstly include a careful physicochemical characterization ( e.g. , particle size , particle size distribution , reactivity , surface area , particle mass , impurity , and aggregation tendency ) , assisted by using reference materials , as well as the use of acellular systems to explore the reactivity of the materials in acellular environments .
subsequent steps include the use of validated cellular ( in vitro ) models to support evidence - based testing process , followed by a limited series of in vivo studies guided by information generated from in vitro studies , especially the toxicological data relevant to ranking of enms , designing appropriate exposure concentrations and defining the critical health endpoints to be monitored .
biosafety should be evaluated by tests examining general toxicity , target organ toxicity , and biocompatibility in line with regulatory requirements , limiting the use of lab animals in toxicological research [ 12 , 13 ] , to identify molecular endpoints and multiple toxicity pathways .  
the present work represents an example of a tiered approach for toxicity testing , employing cnts as a practical model suitable to validate a two - level strategy combining in vitro and in vivo experiments .
such investigation is related to our recent studies on the pulmonary toxicity of a series of functionalized multiwalled carbon nanotubes ( mwcnts ) .
human a549 pneumocytes , selected as potential target cells during respiratory exposure associated with occupational setting and environment [ 1 , 7 , 1418 ] , have been used to assess in vitro the cytotoxicity of mwcnts , with different degrees of functionalization , using mtt assay and calcein / propidium iodide ( pi ) staining .  
administration of the previously reported type of cnts ( 1  mg / kg b.w . ) .
major endpoints tested included ( i ) histopathology of lung tissue ( haematoxylin / eosin staining ) , ( ii ) apoptotic / proliferating features examined by tunel and pcna immunostaining , and ( iii ) presence / distribution of ( 1 ) transforming growth factor - beta1 ( tgf1 ) , ( 2 ) interleukin-6 ( il-6 ) , and ( 3 ) collagen ( type i ) investigated by immunochemical methods , as markers of lung toxicity , inflammation , and fibrosis , respectively . among the several types of engineered nanoparticles
, cnts are emerging as one of the most promising and revolutionary nanomaterials , widely employed within commercial environmental and energetic sectors , due to their unusual one - dimensional hollow nanostructure and unique physicochemical properties [ 19 , 20 ] .
cnts have been also proposed in medicine as nanovectors for vaccine and drug delivery , nanodevices , or as substrates for tissue engineering [ 21 , 22 ] .
consequently , this expanding usage , paralleled by occupational exposure at all phases of the material life cycle , may lead to widespread human exposure via skin contact , inhalation , and via intravenous injection in medical applications .
moreover , in view of some similar aspects to fibers , such as structural characteristics , extreme aspect ratio , low specific density , and low solubility , cnts might exhibit toxicity similar to those observed with other fibrous particles such as asbestos .
further , their small size accompanied by high surface area defines the chemical reactivity of cnts inducing changes in permeability and conductivity of biological membranes ; in fact , a typical behaviour has been reported regarding translocation and distribution of certain enms in the body and their capability to cross internal barriers .
moreover , chemical functionalization not only extends the applications of cnts conferring them new functions that can not otherwise be acquired by pristine nanomaterials , but also impacts on biological response to cnts modifying their toxicological profile [ 22 , 23 ] .
thus , the growing use together with the suspense whether cnts have negative impact on human health and environment evokes concern by worldwide public and regulatory institutions .  
nowadays , a number of in vivo and in vitro studies have also been performed on cnt toxic effects , evaluating different mechanistic endpoints [ 14 , 2430 ] . however , the existing data are controversial and findings have been difficult to be interpreted in some cases .
toxicity and reactivity of cnts were shown to vary depending on characteristics of the specific type of material tested , such as fiber length , fiber diameter , surface area , tendency to agglomerate , dispersibility in media , and the method used for synthesis which can produce impurities and leave metal catalyst residues [ 3138 ] .
some scientists have suggested that metal traces associated with the commercial nanotubes are responsible for the cytotoxicity and that cnts show no signs of acute toxicity .
it has also to be taken into consideration that pristine cnts are insoluble in almost all solvents , and , effects of cnts can also be modified by chemical functionalization .
in certain studies , a high degree of functionalization was shown to mitigate toxic effects [ 39 , 40 ] , while , contrarily , in other studies , functionalization increased lung toxicity of cnts in mice exposed by inhalation .
further , dissimilar results from in vivo studies have been ascribed to differences in cnt size and surface area , rodent species used , route of exposure , and differences in observation period [ 4247 ] .
( brattleboro , usa ) and cb from aldrich - fluka ( milan , italy ) .
sio2 particles were kindly provided by degussa ag ( germany ) ( table 1 ) .
mwcnts with different functionalization were prepared as described by fagnoni et al . , 2009 .
the lab - made materials produced included carboxyl ( cooh ) functionalized mw - cooh and amine - containing nanotubes with low amino groups content ( mw - nh2 ) .   before the in vitro and in vivo exposure , exhaustive physicochemical characterization of differently functionalized cnts was performed by ir spectroscopy , thermogravimetric analysis ( tga ) , and by noncontact atomic force microscopy ( nc - afm ) .
it has been estimated that one functional chain is anchored each ~16 carbon atoms of the cnts surface for the mw - cooh and each ~100 carbon atoms of the cnts surface for the mw - nh2 .
all the three types of cnts had an outer diameter of 2030  nm and the wall thickness was 1 - 2  nm .
the particle length was 100300  nm for both functionalized cnts ( e.g. , mw - cooh and mw - nh2 ) and 5002000  nm for the pristine mwcnts .
the content of iron ( fe ) , cobalt ( co ) , and nickel ( ni ) in both pristine and functionalized mwcnts was determined by inductively coupled plasma optical emission spectrometry ( icp - oes ) analysis .
results indicated the presence of fe and ni impurities at concentrations of 0.25% and 1.5% , respectively , in pristine mwcnts .
previous to the experiments , a stock of 1  mg / ml and a suspension of 1  mg / kg b.w . of each test material ( i.e. , mwcnt , mw - cooh , mw - nh2 , sio2 , and cb )
were prepared in dmem plain or in nacl 0.9% for the in vitro expo pure and in vivo exposure , respectively . just before use
, these suspensions were sonicated for 15  min with an ultrasonic sonopuls ( bandelin electronics , berlin , germany ) in a short break every two minutes , also vortexing the suspension on ice to further force the cnt dispersion , avoiding the agglomeration and the formation of bundle - like structures .
a549 cells from a human caucasian lung adenocarcinoma with the alveolar type ii phenotype were obtained from ecacc ( sigma aldrich , milan , italy ) .
the cells were cultured in dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal calf serum ( fcs ) , 2  mm l - glutamine , 50  iu / ml penicillin , and 50  g / ml streptomycin , in a humidified atmosphere containing 5% co2 at 37c and grown to 80% confluence .
exposures to the different nanomaterials were done on subconfluent cells .   at the end of the incubation period ,
cell cultures were examined by phase contrast microscopy using a zeiss axiovert 25 light microscope .
cells were seeded in 96-well plates at a density of 1   10/cm in complete medium . after 24  h of cell attachment , plates were washed with 100  l / well of phosphate - buffered saline ( pbs ) .
cells were then exposed to suspensions ( 10  l ) of the test materials ( pristine mw - cnts , mw - cooh , mw - nh2 , cb , or sio2 ) at concentrations as 1 , 10 , and 100  g / ml , for 24 or 48  h. no fetal bovine serum was used in these preparations as it was proven to interact with nanotubes .
the doses tested ( 1100  g / ml ) and the time points of determinations ( 24 and 48  hr ) have been derived from previous studies showing different adverse effects ( e.g. , oxidative stress , inflammatory response , cell death , loss of cellular morphology , and gene expression levels changes ) caused by various carbon nanotubes .
six replicate wells were used on each 96-well plate for all the treatments , and each experiment was repeated independently three times .
significant effects ( p < 0.05 ) were determined by one - way analyses of variance ( anova ) ( software package spss inc .
data are presented as mean   sd over the mean experimental values of each of the three independent experiments .  
two dye - based methods , namely , the live ( calcein - green fluorescence)-dead ( propidium iodide - red fluorescence ) staining ( calcein / pi ) and the mtt assay were used to assess cytotoxicity .  
the live / dead viability test detects cell membrane integrity and measures the number of damaged cells .
fluorescence microscopy labels both live ( green colour ) and dead ( red colour ) cells simultaneously thereby permitting visualization and enumeration of all cells by a single counting procedure .
only active mitochondria contain dehydrogenases enzymes able to cleave the tetrazolium ring and reduce mtt to insoluble dark - blue formazan crystals and , therefore , the reaction only occurs in viable cells .  
absorbance , directly proportional to cell viability , was determined at 550  nm in a biorad microplate reader .
the absorbance values were normalized by the controls and expressed as percent viability . after the incubation period ,
all cultures were also examined and photographed in phase contrast using a zeiss axiovert 25 light microscope combined with a digital camera ( canon powershot g8 ) .
all experimental procedures involving animals were performed in compliance with the european council directive 86/609/eec on the care and use of laboratory animals .
adult sprague - dawley rats ( 25 males , 12 weeks old ) were purchased from charles river italia ( calco , italy ) at least 2 weeks before treatment and allowed to acclimatize for 3 weeks . throughout the experiment ,
animals were kept in an artificial 12  h light  :  12  h dark cycle with humidity at 50  10% .
animals were provided with rat chow ( 4rf21 diet ) and tap water ad libitum . for the treatment , groups of 6 rats
were anesthetized with pentobarbital sodium for veterinary use and were intratracheally instilled with mwcnts , pristine or functionalized , dispersed at a dose of 1  mg / kg b.w . in nacl 0.9%
sixteen days after the i.t . , treated and control rats were deeply anesthetized with an overdose i.p .
injection of 35% chloral hydrate ( 100  l/100  g b.w . ) ; lung preparation for microscopic evaluations was done by vascular perfusion of fixative .
the pulmonary artery was cannulated via the ventricle , and an outflow cannula was inserted into the left atrium . in quick succession , the tracheal cannula was connected to about 7  cm h2o pressure source to inflate the lungs with air , and clearing solution ( saline with 100  u / ml heparin , 350 mosm sucrose ) was perfused via the pulmonary artery .
after blood was cleared from the lungs , the perfusate was switched to fixative consisting of 4% paraformaldehyde in 0.1  m phosphate buffer ( ph 7.4 ) .
after fixation , the lungs were carefully removed , washed in nacl 0.9% , and postfixed by immersion for 7  h in 4% paraformaldehyde in 0.1  m phosphate buffer ( ph 7.4 ) ; then , the tissues were dehydrated through a graded series of ethanol and finally embedded in paraplast .
eight m thick sections of the samples were cut in the transversal plane and collected on silane - coated slides . to avoid possible staining differences due to small changes in the procedure ,
all the reactions were carried out simultaneously on slides of control and treated animals at all stages .  
immunohistochemistry was performed using commercial antibodies on rat lung specimens , to assess the presence and distribution of ( i ) transforming growth factor - beta1 ( tgf1 ) , ( ii ) interleukin-6 ( il-6 ) , ( iii ) collagen ( type i ) , and ( iv ) proliferating cell nuclear antigen ( pcna - pc10 ) , as typical markers of general lung toxicity , inflammation , fibrosis , and cell proliferation , respectively .
lung sections were incubated overnight at room temperature with ( i ) a primary rabbit polyclonal antibody against tgf1 ( santa cruz biotechnology , santa cruz , ca , usa ) diluted 1  :  100 , ( ii ) a primary rabbit polyclonal antibody against collagen ( type i ) ( chemicon , temecula , ca , usa ) diluted 1  :  400 , ( iii ) a primary goat polyclonal antibody against il-6 ( santa cruz biotechnology ) diluted 1  :  100 , ( iv ) a primary mouse monoclonal antibody against pcna ( american biotechnology , plantation , usa ) diluted 1  :  5 in pbs .
biotinylated anti - rabbit , anti - goat , and anti - mouse secondary antibodies and an avidin biotinylated horseradish peroxidase complex ( vector laboratories , burlingame , ca , usa ) were used to reveal the sites of antigen / antibody interaction .
louis , mo , usa ) was used as the chromogen , followed by a light counterstaining with haematoxylin .
then , the sections were dehydrated in ethanol , cleared in xylene , and finally mounted in eukitt ( kindler , freiburg , germany ) . in the case of negative controls , some sections were incubated with phosphate - buffered saline instead of the primary antibodies ; no immunoreactivity was observed in these conditions .
in addition to morphological criteria , the apoptotic cell death was assayed by in situ detection of dna fragmentation using the terminal deoxynucleotidyl - transferase ( tunel ) assay ( oncogene res .
the lung sections were incubated for 5  min with 20  g  ml proteinase - k solution at room temperature , followed by treatment with 3% h2o2 to quench endogenous peroxidase activity .
after incubation with the tunel solution ( 90  min with tdt / biotinylated dntp and 30  min with hrp - conjugate streptavidin ) in a humidified chamber at 37c , the reaction was developed using 0.05% 3-amino-9-ethylcarbazole ( aec ) in 0.1  m tris buffer ( ph 7.6 ) with 0.2% h2o2 ; in some specimens the reaction was developed using a 0.1% dab solution .  
as a negative control , the tdt incubation was omitted ; no staining was observed in these conditions .  
( a ) scoring different specimens , the immunostaining for il-6 , tgf1 , and collagen ( type i ) was evaluated in conventional brightfield microscopy by recording the localization and intensity of labelling according to a semiquantitative scale from absent / undetectable (  ) to maximal ( + + + + ) .
then , to assess the significance of the immunohistochemical results , a kruskal - wallis nonparametric analysis of the semiquantitative data was performed . a p value of < 0.05 was considered significant .
( b ) the evaluation of pcna- and tunel - cytochemically positive cells ( pcna l.i .
, tunel l.i . ) was calculated as the percentage ( labelling index ) of a total number ( about 500 ) of bronchiolar , alveolar , and macrophagic cells , for each animal and experimental condition , in different representative microscopic fields .
statistical analyses among the different biological situations were performed by the student 's t - test , and differences between medians were considered significant at * p < 0.05 .
the images were recorded with an olympus camedia c-2000 z digital camera and stored on a pc running olympus software .
laboratory - made functionalized multiwalled cnts ( mw - cooh and mw - nh2 ) were tested in comparison with pristine mwcnts in cultures of a549 pneumocytes .
carbon black ( cb ) and silica ( sio2 ) were also investigated as reference nanoparticles [ 50 , 51 ] .
purity and physicochemical properties of the tested nanomaterials are reported in table 1 . 
 in vitro cytotoxicity
was assayed in parallel by two classical dye - based cell viability assays , for example , mtt metabolism and live / dead - calcein / pi staining . in preparations treated with all nanotube types ( pristine and functionalized ) and cb the calcein / pi test indicated no loss of cell viability ( figure 2 ) , whereas mtt data of pristine mwcnts , mw - cooh , and mw - nh2 apparently showed cytotoxic response , occurring not dose - dependently at exceedingly low cnt concentrations ( 1  g / ml ) , with 50% viability loss at 10  g / ml and no further cell death increase at 100  g / ml ( figure 1 ) .
loss of cell viability was apparently more pronounced with cb compared to sio2 ( figure 1 ) .
notably , as shown in figure 2 , the data obtained by calcein / pi staining considerably differed from the mtt results in that ( i ) cell viability was unaffected after treatment with pristine mwcnts , functionalized mwcnts , or cb , even at the higher concentrations and for longer time of exposure ( i.e. 48  h ) ; ( ii ) loss of cell viability was noted in cells treated with sio2 with approximately 40% cell death occurring after 24  h exposure to 100  g / ml sio2 ( figure 2 ) .
pristine mwcnts , mw - cooh , and mw - nh2 were either insoluble or little soluble in polar solvents and difficult to evenly disperse in an aqueous matrix .
they were sonicated to force their dispersion in the medium before application to cell cultures . in cells incubated with these materials
cnts were shown to adhere to each other forming dense micron - sized assemblies completely covering the cell surface ( figure 3 ) .
agglomeration increased with increasing doses , at both incubation time points ( 24 and 48  h ) .
the dosimetry of these nanoparticles tested in vitro is a fundamental question , including not only amount and time but also primary particle characteristics : physical properties ( e.g. , size , shape , and agglomeration state ) , core particle , and surface chemistry .
the present data on ( i ) dose metric characteristics ( table 1 ) , ( ii ) agglomeration tendency ( figure 3 ) , and ( iii ) in vitro cytotoxicity suggest some considerations . specifically , overt in vitro toxic effects
were observed after exposure to sio2 ( viability results from both mtt and calcein / pi ) , while contrasting results were obtained from mtt and calcein / pi after pristine and functionalized mwcnts .
as far as physicochemical properties concern , although the functionalized nanomaterials possessed lower aggregate size and particle size ranges ( table 1 ) than those displayed by the pristine ones , these characteristics seemed not to markedly modify their cytotoxic potential when compared to that of pristine mwcnts .   further , according to the literature , reporting interactions between cnts and colorimetric dyes commonly used in classical dye - based cytotoxicity assays , such as mtt and neutral red assays [ 28 , 5154 ] , the present mtt data seemed more likely to reflect a false positive cytotoxicity signal possibly due to nonspecific cnt interaction with cell culture components or mtt formazan salt .  
subsequently , the second focused in vivo step addressed the pulmonary effects of the three different previously mentioned mwcnts ( pristine versus two diverse lab - made functionalized mwcnts , i.e. , mw - cooh and mw - nh2 ) on some cell kinetic ( i.e. , tunel and pcna ) and cytochemical parameters ( i.e. , il-6 , tgf1 , and collagen ) investigated in rats after 16 days from a single i.t . exposure ( 1  mg / kg b.w . ) .
exposure to all the three different cnts , lung morphology micrographs clearly showed a marked uptake of the cnts into the macrophages ( figure 4 ) .
noticeably , the widespread presence of dark , particulate - laden macrophages was evident as a consequence of the cnts engulfing at alveolar , stromal , and also bronchiolar levels in accordance with the poor solubility of the mwcnts ( pristine and lab - made mw - cooh and mw - nh2 ) already evidenced in the first in vitro step , as described previously and irrespectively of the nature of functionalization .  
histologically , alteration of lung architecture was also observed in several areas showing collapsed thick - walled alveoli and presence of microhaemorrhagic foci ( figure 4 ) , not accompanied by evident signs of fibrotic reaction .
occasionally , inflammatory shedding of leucocyte clusters characterized the parenchyma , even though the occurrence of true granulomas was never detected .
tunel and pcna staining , employed as typical markers of apoptosis and cell proliferation , respectively , showed a significant increase of reactivity in different cell populations ( i.e. , bronchiolar , alveolar cells and macrophages ) , as expression of ( i ) diverse sensitivity of different cell categories to the insult and ( ii ) an improved cellular turnover aimed at replacing damaged elements and finalizing a repair process ( figures 5 and 6 ) . in agreement with previous literature data reporting toxic pulmonary effects including inflammation ( characterized by an increase in alveolar cell number and in cytokines , e.g. , tnf - alpha and il-1 ) and oxidative stress [ 24 , 25 , 44 , 45 , 55 ]
, our experimental results showed an extensive spreading in the bronchiolar , alveolar , and stromal cells of pulmonary il-6 and tgf1 , evidencing the cellular inflammatory reaction to the cnts instillation ( table 2 and figure 7 ) , while , differently , evident changes for collagen were not detected .
the persistence of inflammation , still observable 16 days after instillation , could be explained as the consequence of failure to eliminate the inciting agent ( i.e. , different mwcnts ) , as demonstrated by the durability of fibrocarbonaceous nanomaterial internalized by macrophages .
differently , the lack of a marked collagen accumulation could be attributable to a physiological tissue repair in which the tissue remodelling occurred moderately .
these research studies on cnts clearly indicated that certain negative properties that are typical of classic cnts such as cytotoxicity , poor blood compatibility , inflammogenic effects , and target - organ toxicity are maintained to some extent in functionalized cnts , supporting the view that overcoming the safety problems remains a considerable challenge for these nanomaterials .
in summary , our first in vitro step investigation reported conflicting results on the cytotoxic effects of pristine and differently functionalized mwcnts ; specifically all the tested cnts exhibited mild to moderate cytotoxicity when tested using mtt assay , with changes occurring not dose - dependently already at very low cnt concentrations .
by contrast , the calcein / pi test showed that cell viability was unaffected by all tested cnts , even at the higher concentrations and for longer exposure time .
these diverging results highlighted some limitations intrinsical to classical in vitro cytotoxicity tests , applied to the study of the enms , due to the peculiar physicochemical characteristics of these new nanomaterials , fully supporting the notion [ 56 , 57 ] that a number of issues remain to be resolved before the exclusive use of in vitro studies can be accepted in the safety evaluation of nanomaterials , calling for research approaches complementary to the in vitro studies aimed at understanding effects at all physiological levels and predicting human health hazards .
strategies for the safety assessment of enms and agglomerates are under discussion and development in several national and international projects .
it has been suggested   to develop a tiered strategy of tests starting with an assessment of cell viability providing a first - stage attempt at screening particles , allowing their benchmarking , before choosing specific particles for further testing , perhaps in vivo .
since investigations in laboratory animals may give essential insight , the in vivo study , being the subsequent second essential stage , has provided fundamental information , contributing to clarifying the cnt toxicity mechanisms and to understanding the different cnt toxicity targets , at different pulmonary cytochemical levels , in order to improve the overall understanding of the possible adverse outcomes resulting from cnt exposure . in particular , our in vivo data demonstrated that intratracheal instilled mwcnts can induce lung toxicity associated with inflammation , even in absence of fibrosis , irrespective of nanotubes functionalization . this latter finding suggests caution before considering chemical functionalization as a broad way to improve biocompatibility and safety characteristics of cnts , being even more important for cnts that are proposed for diagnostic or therapeutic applications .
these overall experimental results further support the use of a multitiered strategy for the safety assessment of the human health impact of novel nanotechnology - based products .
 increasing application of engineered nanomaterials within occupational , environmental , and consumer settings has raised the levels of public concern regarding possible adverse effects on human health . 
 we applied a tiered testing strategy including ( i ) a first in vitro stage to investigate general toxicity endpoints , followed by ( ii ) a focused in vivo experiment . 
 cytotoxicity of laboratory - made functionalized multiwalled carbon nanotubes ( cnts ) ( i.e. , mw - cooh and mw - nh2 ) , compared to pristine mwcnts , carbon black , and silica , has been assessed in human a549 pneumocytes by mtt assay and calcein / propidium iodide ( pi ) staining . 
 purity and physicochemical properties of the test nanomaterials were also determined . 
 subsequently , pulmonary toxic effects were assessed in rats , 16 days after mwcnts i.t . 
 administration ( 1  mg / kg b.w . ) , investigating lung histopathology and monitoring several markers of lung toxicity , inflammation , and fibrosis . in vitro data : calcein / pi test indicated no cell viability loss after all cnts treatment ; mtt assay showed false positive cytotoxic response , occurring not dose dependently at exceedingly low cnt concentrations ( 1  g / ml ) . in vivo results demonstrated a general pulmonary toxicity coupled with inflammatory response , without overt signs of fibrosis and granuloma formation , irrespective of nanotube functionalization . 
 this multitiered approach contributed to clarifying the cnt toxicity mechanisms improving the overall understanding of the possible adverse outcomes resulting from cnt exposure . 
1. Introduction
2. Materials and Methods 
3. Results and Discussion
4. Conclusions 