Inhalation Exposure to Carbon Nanotubes (CNT) and Carbon ...

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=uteb20

Download by: [University of Rochester] Date: 14 September 2015, At: 07:54

Journal of Toxicology and Environmental Health, Part BCritical Reviews

ISSN: 1093-7404 (Print) 1521-6950 (Online) Journal homepage: http://www.tandfonline.com/loi/uteb20

Inhalation Exposure to Carbon Nanotubes (CNT)and Carbon Nanofibers (CNF): Methodology andDosimetry

Günter Oberdörster, Vincent Castranova, Bahman Asgharian & Phil Sayre

To cite this article: Günter Oberdörster, Vincent Castranova, Bahman Asgharian & PhilSayre (2015) Inhalation Exposure to Carbon Nanotubes (CNT) and Carbon Nanofibers (CNF):Methodology and Dosimetry, Journal of Toxicology and Environmental Health, Part B, 18:3-4,121-212, DOI: 10.1080/10937404.2015.1051611

To link to this article: http://dx.doi.org/10.1080/10937404.2015.1051611

Published with license by Taylor & Francis©Günter Oberdörster, Vincent Castranova,Bahman Asgharian, and Phil Sayre

Published online: 11 Sep 2015.

Submit your article to this journal

Article views: 10

View related articles

View Crossmark data

http://www.tandfonline.com/action/journalInformation?journalCode=uteb20

http://www.tandfonline.com/loi/uteb20

http://www.tandfonline.com/action/showCitFormats?doi=10.1080/10937404.2015.1051611

http://dx.doi.org/10.1080/10937404.2015.1051611

http://www.tandfonline.com/action/authorSubmission?journalCode=uteb20&page=instructions

http://www.tandfonline.com/action/authorSubmission?journalCode=uteb20&page=instructions

http://www.tandfonline.com/doi/mlt/10.1080/10937404.2015.1051611

http://www.tandfonline.com/doi/mlt/10.1080/10937404.2015.1051611

http://crossmark.crossref.org/dialog/?doi=10.1080/10937404.2015.1051611&domain=pdf&date_stamp=2015-09-11

http://crossmark.crossref.org/dialog/?doi=10.1080/10937404.2015.1051611&domain=pdf&date_stamp=2015-09-11

Journal of Toxicology and Environmental Health, Part B, 18:121–212, 2015Published with license by Taylor & FrancisISSN: 1093-7404 print / 1521-6950 onlineDOI: 10.1080/10937404.2015.1051611

INHALATION EXPOSURE TO CARBON NANOTUBES (CNT) AND CARBONNANOFIBERS (CNF): METHODOLOGY AND DOSIMETRY

Günter Oberdörster1, Vincent Castranova2, Bahman Asgharian3, Phil Sayre4

1Department of Environmental Medicine, University of Rochester, Rochester, New York, USA2Formerly with the National Institute for Occupational Safety and Health, West Virginia UniversitySchool of Pharmacy, Morgantown, West Virginia, USA3Applied Research Associates, Raleigh, North Carolina, USA4Formerly with the U.S. Environmental Protection Agency, Washington, DC, USA

Dedicated to Professor Paul Morrow, 1922–2013In respectful and fond memory of a devoted scientist, inspirational

colleague, caring mentor, selfless friend, and in gratitudefor his lasting groundbreaking contributions to inhalation toxicology

Carbon nanotubes (CNT) and nanofibers (CNF) are used increasingly in a broad array ofcommercial products. Given current understandings, the most significant life-cycle exposuresto CNT/CNF occur from inhalation when they become airborne at different stages of theirlife cycle, including workplace, use, and disposal. Increasing awareness of the importance ofphysicochemical properties as determinants of toxicity of CNT/CNF and existing difficultiesin interpreting results of mostly acute rodent inhalation studies to date necessitate a reex-amination of standardized inhalation testing guidelines. The current literature on pulmonaryexposure to CNT/CNF and associated effects is summarized; recommendations and conclu-sions are provided that address test guideline modifications for rodent inhalation studies thatwill improve dosimetric extrapolation modeling for hazard and risk characterization based onthe analysis of exposure-dose-response relationships. Several physicochemical parametersfor CNT/CNF, including shape, state of agglomeration/aggregation, surface properties, impu-rities, and density, influence toxicity. This requires an evaluation of the correlation betweenstructure and pulmonary responses. Inhalation, using whole-body exposures of rodents, isrecommended for acute to chronic pulmonary exposure studies. Dry powder generator meth-ods for producing CNT/CNF aerosols are preferred, and specific instrumentation to measuremass, particle size and number distribution, and morphology in the exposure chambers

© Günter Oberdörster, Vincent Castranova, Bahman Asgharian, and Phil SayreThis is an Open Access article. Non-commercial re-use, distribution, and reproduction in any medium, provided the original work is

properly attributed, cited, and is not altered, transformed, or built upon in any way, is permitted. The moral rights of the named authorshave been asserted.

Address correspondence to Günter Oberdörster, DVM, PhD, Department of Environmental Medicine, University of Rochester,601 Elmwood Avenue, Med. Ctr. Box 850, Rochester, NY 14642, USA. E-mail: [email protected]

121

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

mailto:[email protected]

122 G. OBERDÖRSTER ET AL.

are identified. Methods are discussed for establishing experimental exposure concentrationsthat correlate with realistic human exposures, such that unrealistically high experimental con-centrations need to be identified that induce effects under mechanisms that are not relevantfor workplace exposures. Recommendations for anchoring data to results seen for positive andnegative benchmark materials are included, as well as periods for postexposure observation.A minimum data set of specific bronchoalveolar lavage parameters is recommended. Retainedlung burden data need to be gathered such that exposure-dose-response correlations may beanalyzed and potency comparisons between materials and mammalian species are obtainedconsidering dose metric parameters for interpretation of results. Finally, a list of researchneeds is presented to fill data gaps for further improving design, analysis, and interpretationand extrapolation of results of rodent inhalation studies to refine meaningful risk assessmentsfor humans.

Carbon nanotubes (CNT) and carbonnanofibers (CNF) are commonly used incommerce, and applications are expectedto increase in the near future (Zhao andCastranova 2011; De Volder et al. 2013). Sinceapproximately 2004, the U.S. EnvironmentalProtection Agency (EPA) has reviewed over60 notices for commercialization of these mate-rials under Section 5 of the Toxic SubstancesControl Act. Releases during the manufactureof these fibrous carbon nanomaterials, and dur-ing the incorporation of CNT/CNF into finishedproducts, coupled with results from experimen-tal animal studies showing asbestos-like effects,raised considerable human health concerns(Nowack et al. 2013). These exposures arecommonly in the form of CNT/CNF-containingaerosols, resulting in a need to monitor expo-sure and assess inhalation effects upon workers(National Institute for Occupational Safetyand Health [NIOSH] 2013). Available dataindicate that releases through use and disposalof products containing CNT/CNF are far lower(Kingston et al. 2014). In order to assess theinhalation toxicity of these fibrous carbonnanomaterials, it is critical to consider whetherand how testing of these materials differs frommethods recommended in existing standardtest guidelines for assessing effects of aerosols ofsoluble chemicals and larger solid particulates.

This review summarizes the effects ofCNT/CNF reported after dosing of the respira-tory tract and examines respiratory tract testingconducted in rodents to date for these mate-rials. It specifically addresses the challenges

posed by inhalation testing with CNT/CNF,including methods of particle generation,options for animal exposure systems, con-sideration of critical physicochemical proper-ties for characterization of the materials, andimportance to characterize exposures, deter-mine doses and evaluate responses whenexamining exposure-dose-response relation-ships. Modifications to existing standard testguidelines were recommended to accommo-date these challenges.

DOSING METHODS FOR THERESPIRATORY TRACT

Human exposure to CNT and CNF mayoccur throughout their life cycle, from theirmanufacture at the workplace to their finaldisposal, depending on whether processesalong the life cycle yield airborne inhalableor respirable particulate elements of CNT andCNF. Although dermal exposure and ingestionvia contaminated food and water may alsooccur, the major exposure route is inhalationwith the respiratory tract as portal of entry,which is the focus of this review. When assess-ing potential effects of airborne CNT and CNFin animal studies, equivalent human exposureconditions ideally need to be mimicked byconsidering exposure methods and mode anddosimetric aspects. Although dosing of the res-piratory tract might also be achieved using bolustype delivery methods, such as intratrachealinstillation (IT) or oropharyngeal aspiration ofmaterial suspended in a physiological medium

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

CNT AND CNF INHALATION: METHODOLOGY AND DOSIMETRY 123

such as 0.9% saline, inhalation is considered thegold standard.

Table 1 highlights some differencesbetween the different methods of dosing therodent respiratory tract with nanomaterials.A main difference is the delivery of mate-rial either as a bolus exposure (instillation,aspiration) or by inhalation, with the formerrepresenting a nonphysiological mode ofdelivery of the liquid suspended materialwithin a fraction of a second (very high doserate), whereas the latter physiological inhala-tion deposits aerosolized materials over anextended period of time (days, weeks, ormonths, termed low dose rate). The highdose rate issue for instillation or aspirationmay be mitigated by administering bolus-typedeliveries at lower doses over the course ofweeks. However, multiple exposures requiremultiple anesthetizations, which subsequentlyincrease stress to animals and require carefulanimal monitoring.

A high dose rate and high doses may over-whelm normal defense mechanisms and thusresult in significant initial pulmonary inflam-mation, and may also affect disposition ofthe administered material to secondary organs.In addition, the necessary pretreatment ofnanomaterials with dispersants to prevent theiragglomeration in the vehicle medium used forinstillation/aspiration delivery alters the par-ticle surface, which is likely to impact theirbiokinetics and induction of effects. Inhalationof pristine unmodified nanomaterials avoidsthese sources of potential introduction of arti-facts from surface modifications. The impactof high doses also needs to be consideredwhen the amounts exceed by orders of mag-nitude dose levels that are expected to bedeposited in the respiratory tract of humansunder realistic inhalation exposure scenarios.The selection of high bolus doses is often jus-tified by arguments that the delivered doseis the same—per unit alveolar surface area—as is deposited per unit alveolar surface areain humans exposed to occupational exposurelevels over a 40-yr working life. This ignorescompletely the effect of dose rate, that is, deliv-ery of the same dose over a long period (days,

months, years) versus within a fraction of a sec-ond (Oberdörster 2012; Baisch et al. 2014).Responses induced by such high doses are likelydue to mechanisms, such as particle overloador effects of homeostasis, that are not opera-tive at relevant low doses (Slikker et al. 2004).For example, overload doses of poorly solu-ble particles (PSP) of low toxicity overwhelmthe alveolar macrophage clearance function,which was shown to induce lung tumors inrats (International Life Sciences Institute [ILSI]2000).

Another disadvantage of bolus type deliveryis the uneven distribution of the administeredmaterial throughout the respiratory tract rela-tive to inhalation. A more central depositionof the delivered dose and its uneven distribu-tion in the lower respiratory tract occur (Brainet al. 1976; Pritchard et al. 1985; Dorries andValberg 1992; Driscoll et al. 2000). On theother hand, the great advantage of bolus-typedelivery is the ease of delivering material to thelung, requiring no special equipment. Resultsalso allow for the ranking of pulmonary toxicityof materials, and may be useful for qualitativerisk assessment, although results are inadequatefor purposes of quantitative risk assessment.Bolus delivery does not simulate normal humaninhalation exposure conditions, and thereforean important component for risk assessment,that is, exposure to defined airborne con-centrations over time, is missing. In general,instillation or aspiration may be consideredas “proof of principle studies” or “hypothesis-forming studies” for identifying mechanismsand obtaining information on biodistribution,which needs to be verified subsequently byinhalation.

Other bolus type delivery methods that arenot listed in Table 1 include laryngeal aspira-tion and intratracheal microspray of liquid sus-pended nanomaterials (NM) and intratrachealinsufflation of NM powders (Morello et al.2009; Penn Century [www.penncentury.com]).The latter two involve delivery of a preset dosein a syringe-type dispenser either as liquid sprayor as dry powder, with the tip of the cannula-type sprayer inserted into the rodent tracheaapproximately 3–5 mm above the main-stem

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

TABL

E1.

Met

hods

ofD

osin

gRo

dent

Low

erRe

spira

tory

Trac

twith

Parti

cles

Met

hod

Ane

sthe

siaD

epos

.all

Reg.

Resp

.Tr.

Even

ness

ofD

epos

ition

Phys

iol.

Mod

eD

ose

Rate

Use

for

Toxi

city

Rank

ing

Use

for

Qua

nt.R

iskAs

sess

m.

NP

Pre-

treat

men

tto

avoi

dAg

glom

er

Repe

at/

Long

-ter

mEx

posu

res

Anim

alSt

ress

atda

ilyEx

posu

re

Spec

ial

Expe

rtise

/

Equi

pmen

tRe

quire

dC

osts

Bolu

sTy

peIn

trana

sali

nstil

latio

nA

yes

yes

good

topo

orno

high

yes

noye

slim

ited

high

nolo

w

Intra

trach

inst

illat

ions

Aye

sN

ogo

odto

poor

nohi

ghye

sno

yes

limite

dhi

ghno

low

Oro

phar

.asp

iratio

nB

yes

nogo

odto

poor

nohi

ghye

sno

yes

limite

dhi

ghno

low

Inha

latio

nC

who

lebo

dyno

yes

exce

ll.ye

slo

wye

sye

sno

Dye

slo

wye

shi

ghno

seon

lyno

yes

exce

ll.ye

slo

wye

sye

sno

Dye

sm

edye

shi

gh

A-

sync

hron

ize

with

insp

iratio

n;B

-ca

veat

:sig

nific

anti

nflam

mat

ion

inra

ts;

C-

requ

ires

larg

eram

ount

sof

mat

eria

l:D

-m

osta

eros

oliz

atio

nm

etho

dsre

sult

insig

nific

ant,

yetr

ealis

tic,

aggl

omer

atio

nof

NPs

.

124

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


carina, to be delivered in synchrony with theinspiration of the animal. Advantages and dis-advantages of these are essentially the sameas those for other bolus type delivery systems,except that insufflation does not require disper-sant pretreatment of the dry NM.

Delivery of airborne engineered nanomate-rials (ENM) to the lung by inhalation is achievedin different ways, with the most common beingwhole-body and nose-only exposures. The for-mer requires individual placement of rodentsin compartmentalized areas in order to avoidhuddling of the animals and permits somefree movement, whereas their movement isseverely restricted by placing them in narrowtubes during nose-only exposure with the nosesprotruding into the plenum with continuouslysupplied aerosol. This induces significant stressin the animals (Phalen 2009), as evidencedby a retarded body weight gain during longerterm nose-only exposures (Coggins et al. 1993,2011; Pauluhn and Mohr 1999; Rothenberget al. 2000). In contrast, contamination of thefur by aerosolized materials is restricted to thehead and perhaps shoulder area, whereas dur-ing whole-body exposure aerosol depositionoccurs on the whole-body surface area, result-ing in some additional gastrointestinal (GI) tractexposure due to preening (see more discussionunder “Types of Inhalation”).

In addition to these two most commoninhalation methods, intratracheal inhalation hasbeen used, which prevents any external expo-sure of fur and allows simulation of differentbreathing scenarios, including breath holding.This requires less material to be aerosolized, butrequires anesthesia throughout the exposure(Oberdörster, Cox, and Gelein 1997; Kreylinget al. 2009).

RESPONSES TO CNT/CNF EXPOSURES

Since the mid 1990s, material scien-tists have developed and perfected meth-ods to arrange carbon atoms in a crys-talline graphene lattice with a tubular mor-phology, thus creating CNT. CNT may be pro-duced as a single tubular structure to form a

single-walled carbon nanotube (SWCNT), as atube within a tube forming a double-walled car-bon nanotube (DWCNT), or as multiple tubeswithin a tube forming a multiwalled carbonnanotube (MWCNT). SWCNT have a diameterof 1–2 nm, while MWCNT are synthesized withdiameters ranging from 10 to 80 nm depend-ing upon the number of encapsulated tubesforming the CNT structure. CNT range in lengthfrom 0.5 to over 30 μm. Therefore, CNT exhibithigh aspect ratios and may be classified asman-made fibrous materials, according to theWorld Health Organization (WHO) respirablefiber definition, that is, a particle longer than5 μm, <3 μm in diameter, and with an aspectratio of >3:1 (WHO 1981).

CNT (1) are resistant to acid or heat treat-ment, (2) exhibit high tensile strength, (3) pos-sess unique electrical properties, and (4) maybe easily functionalized. Therefore, applicationsas structural materials, electronics, heating ele-ments, in production of conductive fabric, forbone grafting and dental implants, and in drugdelivery systems are being developed. Recentin vitro findings regarding catalytic degrada-tion of carboxylated, but not pristine, SWCNThave also been verified for in vivo conditions(Allen et al. 2008; 2009; Liu, Hurt, and Kane2010a; Kotchey et al. 2013) With the antici-pated increase in the synthesis and commer-cialization of CNT, human and environmentalexposure during the life cycle of CNT, that is,production, distribution, use, and disposal, isanticipated. Therefore, it is critical to deter-mine the bioactivity of CNT and characterizethe dose and time dependence of possibleadverse health effects of exposure to informrisk assessment and development of preventionstrategies.

A complication in evaluating the poten-tial adverse health effects resulting from pul-monary exposure to CNT is the wide rangeof physicochemical properties among differentCNT. For example, CNT might be synthesizedby three distinctively different processes, result-ing in differences in chirality and degrees ofcatalyst contaminants, and exposures may beto raw CNT or CNT purified to remove cat-alytic metals. CNT may be produced with

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


a wide variety of fiber widths and lengths,and airborne CNT exhibit a wide range ofstructural sizes and shapes from micrometeragglomerates to nanometer nanoropes. Lastly,CNT may be functionalized to alter theirphysicochemical properties, such as hydropho-bicity versus hydrophilicity. In addition, dop-ing of MWCNT with nitrogen was foundto reduce their toxicity significantly (Carrero-Sanchez et al. 2006; Elias et al. 2007).

At present, data are inadequate to deter-mine to what degree biological responses toa given CNT and CNF can be generalizedas one nanoparticle class. Indeed, reportedeffects following exposure of rodents to CNT bybolus-type delivery or inhalation revealed thatalthough responses are qualitatively similar, themagnitude and time course of responses mayvary significantly, suggesting that quantitativedifferences in bioactivity are likely (Shvedovaet al. 2005; 2008; Ryman-Rasmussen, 2009b).Three recent 13-wk inhalation studies in ratswith two different types of MWCNT and one13-wk rat inhalation study with CNF showedsimilar results, yet different LOAEL and/orNOAEL were derived (Ma-Hock et al. 2009a;Pauluhn 2010; DeLorme et al. 2012; Kasaiet al. 2014). The results also suggested thatMWCNT and CNF might be generically placedinto the same hazard category. However, addi-tional subchronic inhalation studies with differ-ent types of MWCNT and CNF are urgentlyneeded to substantiate this suggestion. Indeed,the International Agency for Research onCancer (IARC) (Grosse et al. 2014) recognizedthis uncertainty when classifying only Mitsui-7 MWCNT as possibly carcinogenic to humans(Group 2 B) and other MWCNT and SWCNT asnot classifiable with respect to carcinogenicity(Group 3).

Indeed, a most recent key study involving 2year whole body inhalation exposures of rats tothree concentrations of Mitsui-7 MWCNT con-firmed a significant dose-dependent inductionof neoplastic lesions in the lung (adenoma andcarcinoma) at the medium concentration (0.2mg/m3; 26%) and high concentration (2 mg/m3;32%) in male rats and at only the high concen-tration (2 mg/m3; 22%) in female rats. The low

concentration (0.02 mg/m3; 4%) was not dif-ferent from controls (4–6 %). (Fukushima, per-sonal communication, 2015). A paper with fulldetails of this milestone study is in preparation.The apparently greater sensitivity of males vsfemales is opposite to earlier findings of chronicinhalation studies with poorly soluble particlesof low cytotoxicity. (Lee et al, 1985) With thisconfirmation of the carcinogenicity of inhaledMitsui-7 MWCNT the IARC classification islikely to change to: Probably carcinogenic tohumans (Group 2 A).

PULMONARY RESPONSE TO SWCNT

Due to the low density of CNT, it isanticipated that respirable particles may begenerated during manufacturing, as a resultof transfer, weighing, mixing, and blendingof CNT. Therefore, inhalation is considereda primary route for human exposure. Thepulmonary effects attributed to exposure ofrodents to SWCNT were first reported in2004. Warheit et al. (2004) exposed rats byIT instillation to high doses of a raw formof SWCNT (0.25–1.25 mg/rat), with the CNTsample containing 30–40% amorphous car-bon, 5% nickel, and 5% cobalt. Althoughsuspended in phosphate-buffered saline (PBS)containing 1% Tween 80, the SWCNT werehighly agglomerated. Pulmonary exposure toSWCNT resulted in a rapid but transient inflam-matory and injury response, as evidencedby increased levels of bronchoalveolar lavagefluid (BALF) neutrophils, lactate dehydrogenase(LDH) activity, and protein. Granulomas, pre-dominantly in the terminal bronchioles, werereported 1 wk postexposure and persistedthrough 3 mo postexposure. A 15% rise inmortality rate within 1 d postexposure wasnoted and attributed to physical blockage ofconducting airways by large SWCNT agglom-erates. Lam et al. (2004) also reported rapidand persistent granulomas in mice after ITinstillation of mice to very high doses of SWCNT(0.1–0.5 mg of SWCNT/mouse). These inves-tigators compared raw (containing 25% metalcatalyst) to purified (approximately 2% iron [Fe])

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


SWCNT and found the granulomatous reactionwas not dependent on metal contaminationof SWCNT. The high mass doses induced amaximal response, which may have masked anadditional effect of the higher Fe content.

Mangum et al. (2006) exposed rats bypharyngeal aspiration to purified SWCNT(0.5 mg/rat; 2.6% Co and 1.7% Mo) suspendedin 1% Pluronic. Data showed no apparentinflammatory responses. However, cell prolifer-ation and platelet-derived growth factor (PDGF)protein levels were significantly increased 1 dpostexposure and significant interstitial fibro-sis was noted at 21 d postexposure. Shvedovaet al. (2005) exposed mice by pharyngeal aspi-ration to purified SWCNT (10–40 μg/mouse).The suspended SWCNT preparation, sonicatedbut with no dispersant, contained micrometer-sized agglomerates as well as some smallernanorope structures. A rapid and transientinflammation and pulmonary damage werenoted. In addition, granulomatous lesions andinterstitial fibrosis within 7 d postexposure,which lasted through the 59-d course ofthe study, were observed. Granulomas wereassociated with the deposition of agglomer-ates in the terminal bronchioles and proximalalveoli, while interstitial fibrosis was associatedwith deposition of more dispersed SWCNTstructures in the distal alveoli. This distinctionbetween deposition site (proximal vs. distalalveoli) and response (granulomas vs. intersti-tial fibrosis) was confirmed using gold-labeledSWCNT, which allowed nanoropes structuresto be visualized in lung tissue (Mercer et al.2008). As in the Lam et al. (2004) study,the transient inflammatory/injury response andpersistent granulomatous and fibrotic lesionswere not markedly affected by the presenceor absence of catalytic metals using raw versuspure SWCNT (Shvedova et al. 2005; 2008).

Mercer et al. (2008) compared the pul-monary response of mice after pharyngealaspiration of a poorly dispersed versus well-dispersed (acetone-dispersed, washed, and sus-pended in PBS with sonication) preparation ofSWCNT. It was found that transient inflam-mation and persistent interstitial fibrosis werefourfold greater on an equal delivered mass

burden basis for well-dispersed SWCNT com-pared to poorly dispersed SWCNT. Shvedovaet al. (2008) reported the pulmonary responseof mice to inhalation of SWCNT (5 mg/m3,5 h/d, 4 d). Qualitatively, short-term inhala-tion produced pulmonary responses similarto bolus exposure by pharyngeal aspiration,that is, transient inflammation and damagebut persistent granulomas and interstitial fibro-sis. Aerosolization of dry SWCNT resultedin smaller structures (count median diameter[CMD] approximately 220 nm) than suspensionof SWCNT for aspiration (a mixture of μm sizeagglomerates and smaller nanorope structures).Quantitatively, SWCNT were estimated to befourfold more potent on an equal mass burdenbasis (5–10 μg/lung) in producing inflammationand fibrosis after inhalation (deposited lung bur-den was estimated from aerosol concentration,minute ventilation, duration of exposure, andestimated deposition fraction) than was aspira-tion of less dispersed SWCNT (Shvedova et al.2008). Of interest, the degree of the response toaspiration of well-dispersed SWCNT was similarto that for short-term inhalation, using a similartype of SWCNT (Mercer et al. 2008; Shvedovaet al. 2008), suggesting that aspiration exposuremay be appropriate for testing well-dispersedCNT for purposes of hazard identification.

Murray et al. (2012) compared the potencyof SWCNT to crocidolite asbestos after aspi-ration in mice. At 1 and 7 d postexposure,SWCNT (40 μg/mouse) were significantly morepotent in inducing transforming growth factor(TGF)-beta, a fibrogenic factor, than asbestos(120 μg/mouse). In addition, SWCNT weresubstantially more potent in inducing alveolarinterstitial fibrosis as evidenced by lung colla-gen and alveolar wall connective tissue thick-ness than asbestos at 28 d postexposure.Shvedova et al. (2014) confirmed the greaterfibrotic potency of SWCNT compared toasbestos at 1 year after aspiration in mice.The high bolus doses expressed by mass needto be considered which in addition raises thequestion about comparing responses by dif-ferent metrics: Would surface characteristicssuch as area, charge, and chemistry be moreappropriate?

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Recently, Shvedova et al. (2013) presenteddata suggesting that SWCNT may act as a can-cer promoter. In this study, mice were exposedto SWCNT (80 μg/mouse) or phosphate-buffered saline vehicle by pharyngeal aspira-tion. Two days after aspiration, mice were inoc-ulated (iv) with Lewis lung carcinoma cells.In vehicle control mice, inoculation with cancercells resulted in lung tumors 21 d after iv injec-tion. However, lung tumor growth was signifi-cantly greater in mice preexposed to SWCNT asmeasured by the following: (1) 5-fold increasein lung weight, (2) 2.5-fold elevation in thenumber of visible tumors, and (3) 3-fold risein the area of metastatic nodules. Data indi-cated that this tumor promotion was associatedwith SWCNT-induced upregulation of granu-locyte myeloid-derived suppressor cells, whichwould depress antitumor immunity. This maybe viewed as an important hypothesis-formingstudy. However, the extremely high bolus doseand dose rate need to be compared to dose-response relationships following realistic inhala-tion exposures.

In summary, pulmonary exposure of mice toSWCNT induced granulomatous lesions asso-ciated with deposition of micrometer-sizedagglomerates as well as rapid and progres-sive interstitial fibrosis associated with migrationof smaller SWCNT structures into the alveolarsepta. Although most studies were conducted inmice, exposure of rats to SWCNT also inducedgranulomas and fibrosis. It should be notedthat most of these SWCNT studies involvedbolus exposure, resulting in high lung burdensat a very high dose rate. The single inhala-tion study with SWCNT involved a relativelyhigh aerosol concentration (5 mg/m3) and 5-d exposure duration (Shvedova et al. 2008).Therefore, dose rate was still relatively high.Thus, there is the need for 13-wk inhala-tion studies in rodents. Lung burdens fromreported bolus exposure studies may be usedas guidance for determination of aerosol expo-sure concentration (ideally resulting in low,medium, and high doses). Current knowl-edge gaps include (1) determination of workerexposure to SWCNT (aerosol concentrationand size distribution of airborne structures),

(2) characterization of the effect of catalyticmetals on pulmonary responses to SWCNT,(3) determination of whether inhaled SWCNTcan translocate to the pleura or other organsand produce intrapleural or systemic effects,(4) determination of whether inhaled SWCNTinduce lung cancer, (5) evaluation of thedegree to which SWCNT agglomerates are dis-persed into smaller structures upon contactwith pulmonary surfactant or cellular enzymes,(6) determination of the biopersistence ofSWCNT, and (7) elucidation of mechanismsinvolved in these adverse responses.

PULMONARY RESPONSE TO MWCNT

Muller et al. (2005) exposed rats by ITinstillation to MWCNT (0.5–5 mg/rat) sus-pended in 1% Tween 80. Dose-dependentinflammation, granulomas, and fibrosiswere noted, with the unground MWCNT(6 μm length) being more potent thanground MWCNT (0.7 μm length). At 60 dpostexposure, 81% of unground MWCNTwere retained in the lung, as determined by0.5% Co impurities, compared to 36% forshort MWCNT. Li et al. (2007a) compared thepulmonary response of mice exposed to puri-fied MWCNT by IT instillation (50 μg/mousebolus dose) versus inhalation (32.6 mg/m3,6 h/d, for 5–15 d [estimated deposited doseof 70–210 μg]). MWCNT aerosolized from drymaterial were less agglomerated than whensuspended in 1% Tween 80. Intratrachealinstillation produced inflammation and severedestruction of alveolar structures, while inhala-tion predominately resulted in moderatepathology consisting of alveolar wall thickeningand cell proliferation but general alveolarstructure was retained. This study demon-strated significant differences in the type anddegree of pulmonary responses to MWCNTin mice between bolus-type IT instillation andinhalation, with higher doses deposited inlung by inhalation resulting in only moderateeffects compared to severe lesions inducedby instillation of lower doses. Yu et al. (2013)compared the influence of IT instillation of 100

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


μg pristine MWCNT to acid-treated MWCNTfor 6 mo and noted that pristine MWCNTinduced pulmonary autophagy accumulationand more potent tumorigenic responses thanthe acid-treated MWCNT, suggesting theimportance of physicochemical characteris-tics in toxicity outcome. Similarly, Kim et al.(2010a) found that pristine MWCNT producedmore severe acute inflammatory cell recruit-ment in BALF and multifocal inflammatorygranulomas compared to acid-treated MWCNTin mice IT instilled with 100 μg for 1 wk up to4 mo. In another study, exposure of rats by ITinstillation of high doses of purified MWCNT(0.25–1.75 mg/rat) suspended in 1% Tween80 produced a rapid but transient inflammatoryresponse and persistent alveolar wall thickening(Liu et al. 2008).

Kobayashi et al. (2010) exposed rats by ITinstillation to a well-dispersed suspension ofMWCNT (10–250 μg/rat). Transient inflamma-tion and damage and a granulomatous responsewas observed. No fibrosis was found, but a col-lagen stain for histopathology was not used.In contrast, Porter et al. (2010) reported a rapidand persistent fibrotic response in mice afteraspiration of a well-dispersed suspension ofpurified MWCNT (10–80 μg/mouse dispersedin a diluted artificial lung lining fluid), usingSirius red staining for collagen. In addition, tran-sient inflammation and damage with persistentgranulomas at sites of agglomerate depositionwere noted. Mercer et al. (2011) conductedmorphometric analysis of the interstitial fibroticresponse from the Porter et al. (2010) study.Analysis indicates a time- and dose-dependentfibrotic response. At 80 μg of MWCNT/mouse,interstitial fibrosis at 28 d postexposure wassignificantly above control measured as thethickness of alveolar wall collagen. At 56 dpostexposure, interstitial fibrosis at 40 μg ofMWCNT/mouse was significantly greater thancontrol at 40 μg of MWCNT/mouse, with non-significant trends observed at lower doses.

Similarly, Aiso et al. (2010) reported tran-sient inflammation and damage, and persis-tent granulomas and alveolar wall fibrosis inrats after IT instillation of different MWCNT(40–160 μg/rat). In a recent report, mice

were exposed by inhalation (5 mg/m3, 5 h/d,12 d) to Mitsui-7 MWCNT (lung burden of28 μg/lung) and alveolar interstitial fibrosiswas monitored for 336 d postexposure (Merceret al. 2013a). The thickness of alveolar sep-tal collagen increased 14 d postexposure andcontinued to progress to 70% above control by336 d postexposure.

Han et al. (2010; 2015) exposed miceby aspiration to carboxylic or hydroxylfunctionalized MWCNT (20–40 μg/mouse).These functionalized MWCNT were welldispersed in PBS. Similar to other reports,transient inflammation (1 and 7 d post), ele-vated cytokine production (1 day post), andlung injury (1 day post) were found at 20 and40 μg/mouse. Surface functionalization ofMWCNT was reported to affect bioactivity,with carboxylated or acid-treated MWCNTbeing less inflammatory and fibrotic whileamine-functionalized MWCNT were morepotent than unmodified MWCNT (Kim et al.2010a; Yu et al. 2013; Bonner et al. 2013;Li et al. 2013; Sager et al. 2014). Reportedbiodegradation of carboxylated SWCNT andMWCNT may be attributed to peroxidases(Zhao, Allen, and Star 2011; Kotchey et al.2013).

Pauluhn (2010) exposed male and femalerats by nose-only inhalation (0.1–6 mg/m3,6h/d, 5 d/wk, 13 wk) to Baytube MWCNT.Aerosolized structures were large, compactagglomerates—defined as assemblages by theauthor—with a mass median aerodynamicdiameter (MMAD) of approximately 3 μm.Subchronic inhalation resulted in persistentinflammation, lung damage, granulomas,alveolar wall thickening, and increased intersti-tial collagen staining at exposures of 0.4 mg/m3

or higher. Notably, Pauluhn (2010) did notgenerate significant amounts of fibrous orloosely agglomerated structures with BaytubeMWCNT and described them as assemblages.The non-ibrous morphology of these assem-blages was used as explanation to characterizeresponses as being due to volumetric over-load of alveolar macrophages phagocytizingthese structures (Pauluhn 2010). The lowestexposure concentration of 0.1 mg/m3 was

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


determined to be the NOAEL. Of specialimportance, the study by Pauluhn (2010) is theonly one to determine in a sufficiently longpostexposure period the retention halftime(T1/2) of the retained MWCNT (shown later inTable 3). This demonstrated a prolongation ofT1/2 with high lung burdens, similar to otherpersistent particles ranging from more benign(TiO2; carbon black [CB; [Morrow 1988;Elder et al. 2005]) to highly reactive knownhuman carcinogens (asbestos; crystalline SiO2[Bolton et al. 1983; Hemenway et al. 1990]).Inclusion of a sufficiently long postexposureperiod needs to be a mandatory compo-nent of a well-designed repeated inhalationstudy to determine retention kinetics andregression/progression of effects.

Ma-Hock et al. (2009a) reported pul-monary responses of male and femalerats to nose-only inhalation of MWCNT(0.1–2.5 mg/m3, 6 h/d, 5 d/wk, 13 wk;resultant burden 47–1170 μg/rat [crudelyestimated, no clearance assumed]). Theaerosolized MWCNT were well dispersed(count median diameter approximately60 nm). Pulmonary responses includedincreased lung weight, neutrophilic inflam-mation, and granulomatous inflammation.No apparent fibrosis was reported. However,Ma-Hock et al. (2009a) did not use a specificcollagen stain for histopathologic analysis;consequently, fibrosis may have been under-scored. To address this, Treumann et al. (2013)reported results of additional histopathologicalanalyses describing a slight increase of collagenfibers in the medium- and high-dose animalswithin the microgranuloma, yet no collagenfibers in alveolar walls and pleura. One rat ofthe lowest exposure group (0.1 mg/m3) devel-oped minimal granuloma. Data also showedevidence of MWCNT degradation in alveolarmacrophages. Overall, data demonstrated thatthe hallmark of asbestos exposure, pleuralinflammation and/or fibrosis, was absent. Thelowest exposure concentration of 0.1 mg/m3

was the LOAEL in this study.A multidose 2-wk whole-body inhalation

study in rats (6 h/d, 5 d/wk) with Mitsui-7MWCNT determined a NOAEL of 0.2 mg/m3,

whereas significant pulmonary dose-dependentinflammatory effects were seen in groupsexposed to 1 and 5 mg/m3 group (Umedaet al. 2013). Inflammatory indicators weredecreased at 4 wk postexposure, but par-tially still elevated. MWCNT translocated to theperitracheal lymph nodes, which increased inthe postexposure observation period. Goblet-cell hyperplasia induced in the 1- and 5-mg/m3-exposed rats did largely regress dur-ing the postexposure period. The retainedlung burden (43 μg in 5 mg/m3 group) atthe end of the 2-wk inhalation exposure didnot change significantly in the subsequent 4-wk postexposure period, and evidence indi-cated lengthy in vivo biopersistence of theseMWCNT.

In a subsequent whole-body inhalationstudy, Kasai et al. (2015)1 exposed femaleand male F-344 rats to Mitsui-7 MWCNTat 3 concentrations ranging from 0.2 to5 mg/m3 (MMAD = 1.4–1.6 μm) for 13 wk(6 h/d, 5 d/wk) using whole-body inhala-tion chambers. Retained burden in the leftlung, determined by a hybrid marker tech-nology (Ohnishi et al. 2013), ranged between2.3 and 120 μg/rat. Dose-dependent pul-monary inflammation, granulomas, and inter-stitial fibrosis were observed. Kasai et al. (2015)also observed MWCNT in the mesothelial liningof rodent diaphragms in the highest exposuregroup (5 mg/m3). In addition, Kido et al. (2014)subsequently noted that subchronic inhala-tion exposure to MWCNT resulted in system-atic inflammation as indicated by increases inmRNA expression in splenic macrophages. Thelowest exposure concentration of 0.2 mg/m3

was the LOAEL in this study, indicating thata 2-wk inhalation study where a NOAEL of0.2 mg/m3 was found for the same MWCNT intheir previous investigation (Umeda et al. 2013)was insufficient to derive a longer term NOAEL.It is to be expected that the NOAEL from achronic 2-yr study might be even lower.

1Data were furnished by the Japan Bioassay ResearchCenter to Dr. G. Oberdörster. This study was contracted andsupported by the Ministry of Health, Labor, and Welfare of Japan.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Ryman-Rasmussen et al. (2009b) exposedmice to MWCNT by nose-only inhalation (30 or1 mg/m3 for 6 h; MMAD 0.18 and 0.16 μm).Lung burdens were not quantified but esti-mated based on assumed deposition efficien-cies. Two to 6 wk postexposure, MWCNT inalveolar macrophages were detected in thesubpleural tissue and mesothelial cells lin-ing the lung, as well as subpleural fibrosisin the high-dose group only. Porter et al.(2010) also found MWCNT in the subpleuraltissue and subpleural lymphatics, with somefibers penetrating into the intrapleural space1–2 mo after aspiration of a well-dispersedsuspension of MWCNT (20–80 μg/mouse) sus-pended in diluted artificial lung lining fluid.Mercer et al. (2010) conducted morphomet-ric analyses of the Porter et al. (2010) dataand noted significant migration of MWCNTinto the intrapleural space as early as 1 dpostexposure and at doses of 20 μg/lung. Datademonstrated (using morphometric measure-ments) that 12,000 MWCNT penetrated intothe intrapleural space at 56 d after aspira-tion of 80 μg of MWCNT in a mouse model,with such penetrations being dose and timedependent at 20 μg/mouse or higher. Lianget al. (2010) found that MWCNT translo-cate to the lung and induce fibrosis 28 dafter intraperitoneal (ip) injection of a largedose of 250 μg MWCNT/mouse or greater.These MWCNT had been functionalized withphosphorylcholine to make them hydrophilic.The Sakamoto et al. (2009) observations alsosupport translocation of intrascrotally adminis-tered MWCNT to the thorax, where diaphrag-matic granulomatous lesions were found.Recently, Mercer et al. (2013b) reportedtranslocation of inhaled MWCNT (lung bur-den 28 μg/mouse) from the lung to the chestwall and diaphragm and observed MWCNT inintrapleural lavage fluid. In addition, low butprogressive translocation of MWCNT was notedfrom the lung to the lymphatics, liver, kidneys,heart, and brain over a 336-d postexposureobservation period.

Pauluhn and Rosenbruch (2015) comparedpulmonary effects and retention kinetics ofMWCNT (Baytubes) aerosols generated either

as dry powder aerosols or well dispersed from aliquid suspension. Rats were nose-only exposedonce for 6 h to large agglomerated aggregateswith MMAD of 2.6 μm from powder genera-tion or to smaller well-dispersed structures fromliquid nebulization with MMAD of 0.79 μm, athigh concentrations of 25–30 mg/m3. Markeddifferences were seen in terms of threefoldhigher initial lung burdens (lung burdenswere quantified by NIOSH method 5040 andDoudrick et al. 2013) and about twofold fasterclearance of wet-dispersed MWCNT in the3-mo postexposure period. Macroscopically,lungs from wet-dispersed MWCNT displayeda gray discoloration, which was also seenin the enlarged lymph nodes, whereas dryMWCNT-exposed lungs appeared normal.Histopathological examination revealed agreater alveolar macrophage response andminimal septal thickening in lungs from wet-exposed rats only. Pauluhn and Rosenbruch(2015) emphasized the importance of usingrelevant exposure scenarios of pristine nano-materials rather than artificial conditions whentesting nanomaterials, to avoid flawed con-clusions based on high-dose acute studies.This investigation also highlights the necessityof determining lung burdens rather than onlyknowing exposure concentrations.

A recent investigation indicated thatMWCNT act as a strong promoter of lungtumors (Sargent et al. 2014). In this study,B6C3F1 mice were injected ip with either cornoil (vehicle) or 10 μg/g bw of methylcholan-threne (an initiator). One week later, micewere then exposed by whole-body inhalation(5 h/d, 15 d) to either filtered air or MWCNT(Mitsui-7, 5 mg/m3, resulting lung burdenof 31 μg/lung) generated by an acousticalgenerator as described by McKinney, Chen,and Frazer (2009). At 17 mo postexposure,mice were euthanized and lung tissue wasexamined by a board-certified veterinarypathologist for lung tumor formation. MWCNTdid not significantly increase the incidenceof tumor formation (percent of mice with atumor) in mice receiving corn oil. However,MWCNT produced a marked rise in tumorincidence in mice treated with the initiator

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


chemical (initiator alone having a 50% inci-dence, while the initiator plus MWCNT groupdisplayed a 90% incidence). In addition,MWCNT increased multiplicity (number oftumors/lung) compared to the inducer alonefrom 1.4 to 3.3 tumors/lung, with tumors beingsignificantly larger in the initiator plus MWCNTgroup compared to initiator alone. Pathologicalanalysis indicated that tumors were both bron-choalveolar adenomas and bronchoalveolaradenocarcinomas, with significantly moremalignant tumors in the initiator plus MWCNTmice. This study is the first to demonstrate lungcancer promotion after inhalation of MWCNT.Although the experimental design of this studywas weakened by the absence of negativeand/or positive particle control groups, a mostrecent chronic rat inhalation study (see above)confirmed the induction of both adenomasand carcinomas in the lung. It is of interest thatpharyngeal aspiration of 10 μg MWCNT inmice followed by 56 d postexposure revealedsignificantly enhanced expression of a gene setrelated to human lung cancer, suggesting thistechnique may be utilized for early detectionof lung cancer (Guo et al. 2012).

In general, available studies indicate thatpulmonary exposure of mice or rats toMWCNT induce granulomas and interstitialfibrosis. Evidence also indicates that MWCNTdeposited in the lung migrate to the pleuraand diaphragm. Results are qualitatively similarfor both bolus exposure studies and inhala-tion studies. Results also appear to be simi-lar after inhalation exposure of both rats andmice when the same MWCNT (Mitsui-7) andaerosol dispersion (MMAD of approximately1.5 um) were employed, as in the Merceret al. (2013a); 2013b) and Kasai et al. (2015)studies. Knowledge gaps include (1) determi-nation of typical worker exposure to MWCNT(aerosol concentration and size distribution ofaerosolized structures), (2) evaluation of theeffects and biopersistence of catalytic metals,fiber dimensions, and surface functionaliza-tion on the pulmonary toxicity of MWCNT,(3) determination of the degree to whichdeposited MWCNT agglomerates disperse intosmaller structures upon contact with pulmonary

surfactant of cellular enzymes, (4) verificationof whether MWCNT can induce or promotelung cancer and mesothelioma, and (5) eluci-dation of mechanisms involved with adversepulmonary responses.

PULMONARY RESPONSES TO CNF

Only three studies evaluating responses fol-lowing exposures to CNF have been reported,two mouse aspiration studies and a 13-wk ratinhalation study. The first mouse study (Murrayet al. 2012) administered 120 μg of CNF(98.6% elemental carbon; 1.4% iron; 80 to160 nm thick; 5–30 μm long) to mice byoro-pharyngeal aspiration, and effects wereevaluated at 1, 7, and 28 d postexposure.The second mouse study used the same CNFand dosing, but followed the induced fibro-sis for 1 yr postaspiration (Shvedova et al.2014). The rat inhalation study involved 13-wk(5 d/wk; 6 h/d) inhalation of both male andfemale rats to 3 concentrations (0.54, 2.5, or25 mg/m3) and filtered air controls with evalu-ation of effects at 1 d and 13 wk postexposure(DeLorme et al. 2012). The CNF weredifferent (99.5% elemental carbon, 0.003%iron; 40–350 nm thick; 1–14 μm long) fromthose of Murray et al. (2012) or the Shvedovaet al. (2014) studies. Pulmonary responses inthe high bolus-dosed mice included inflamma-tion (lung lavage increased polymorphonuclearleukocytes [PMN], protein, and LDH activityat d 1, which were still significantly elevatedat 28 d postexposure); established oxidativestress responses; and elevated biochemical (col-lagen) and histochemical (Sirius red) indicatorsof fibrosis for as long as 1 yr after aspiration(Murray et al. 2012; Shvedova et al. 2014).Effects after inhalation exposure of rats includeddose-dependent elevation in lung weight, stillincreased in the highest dose group at 13 wkpostexposure; persistent inflammation (BALF,significant rise in BAL PMN and LDH at high-est dose) at 1 d and 17 wk postexposure;interstitial thickening; and proliferation ofType II cells in the subpleural, parenchymal,and bronchiolar region in the high-exposuregroup, which was persistent for the subpleural

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


region at 13 wk postexposure. Indicatorsof cardiovascular effects (C-reactive protein[CRP]; coagulation parameters; histopathology)were not markedly different from controls.A NOAEL of 0.54 mg/m3 was determined,and a LOAEL of 2.5 mg/m3 based onminimal inflammation in terminal bronchi-oles and alveolar ducts (DeLorme et al.2012).

In summary, pharyngeal aspiration of CNFin mice (high bolus dose) resulted in intersti-tial fibrosis. Using bolus dosing, the fibroticpotency of CNF was substantially lower thanthat of SWCNT (Murray et al. 2012; Shvedovaet al. 2013). Inhalation exposure of rats to CNFover 13 wk resulted in a far lower degree offibrosis. DeLorme et al. (2012) indicated thata lower, more worker-relevant dose rate in thisinhalation study might explain this difference.However, lung burden was not determined inthe inhalation study. The reported MMAD of3 μm suggests a lower alveolar deposition com-pared to a more respirable rat aerosol of ≤2 μmMMAD, explaining the low alveolar interstitialfibrosis. Therefore, there remains a need for asubchronic inhalation study to a CNF aerosol, inwhich retained lung burden in the lower respi-ratory tract needs to be measured. Knowledgegaps given for SWCNT and MWCNT are alsoapplicable to CNF.

RESPONSES TO INTRAPERITONEALINJECTION OF MWCNT

Takagi et al. (2008) reported that injec-tion of MWCNT (3 mg, 109 fibers/mouse)into the abdomen of p53± mice resulted inmesothelioma similar to the asbestos positivecontrol. The findings in this study have beenquestioned due to the extremely high doseof MWCNT used (Ichihara et al. 2008). Theaforementioned caveats associated with non-physiological bolus-type dosing need to beconsidered. However, a follow-up study alsofound induction of abdominal mesothelioma(25% incidence) after ip injection of 3 μg (106

fibers/mouse) of MWCNT (Takagi et al. 2012).Peritoneal and pleural metastatic mesothelioma

were also reported after intrascrotal administra-tion of MWCNT (1 mg/kg body weight) intorats, with the extent of response exceedingthat of asbestos when compared on the basisof delivered mass as dose metric (Sakamotoet al. 2009). Nagai et al. (2011) administeredto rats very high doses (1 and 10 mg/rat)of different MWCNT (agglomerated, nonag-glomerated, tangled, thin [50 nm], and thick[150 nm], including Mitsui MWCNT), whichresulted in high mortality of the 10-mg-dosedrats and peritoneal mesothelioma at 1 yr in thesurviving rats for certain groups. The nonag-glomerated thinner MWCNT, including Mitsui,produced greatest peritoneal inflammation andcarcinogenicity, which correlated with theircrystallinity, sharpness, and rigidity; these prop-erties facilitated piercing of cell membranesas observed in additional in vitro studies.Thick MWCNT produced less inflammationand carcinogenicity, and tangled MWCNT,even when thin, did not induce mesothe-lioma. Similarly, Muller et al. (2009) noted nomesothelioma over a 2-yr period after abdom-inal injection of very short MWCNT (<1 μm;20 mg/rat). These negative findings wouldhave been predicted by results of Poland andcoworkers (2008), who found that abdominalinjection of 50 μg of long MWCNT/mouseresulted in granulomatous lesions on thediaphragm within 2 wk of exposure, whileshort MWCNT were ineffective, as were tan-gled MWCNT. Murphy et al. (2011) reportedthat intrapleural injection of long MWCNT(5 μg/mouse), but not short MWCNT, resultedin persistent (24 wk) inflammation and fibro-sis of the parietal pleural surface similar to thatseen with long amosite.

Results of a large 2-yr ip injection studyinvolving 500 male Wistar rats given alow and high dose of 4 different types ofMWCNT (50 rats per dose) showed that allMWCNT dosed rats developed mesothelioma(Rittinghausen et al. 2014). Administration ofhigh ip doses by fiber number was the same forall MWCNT at 1 × 109 and 5 × 109 fibers/rat,equivalent to mass bolus doses ranging from50 μg to 3 mg. When amosite asbestos was

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


used as a positive control given as one 10-fold lower ip dose of 0.1 × 109 fibers/rat,66% mesothelioma incidence resulted, whereasonly 1 rat in medium-injected controls devel-oped mesothelioma (2%). Data indicated thatMWCNT were capable of inducing mesothe-lioma and point to the high durability of allCNT. In addition, a higher toxic and carcino-genic potency of straight and more needle-likeshaped MWCNT was noted and a lower poten-tial for curled, bent, or tangled MWCNT, whichis similar to the findings of Nagai et al. (2011)reported earlier.

While direct intracavitary injection ofMWCNT of straight fiber structures resulted inthe reported length-dependent inflammatoryand fibrosis responses, the occurrence of air-borne MWCNT as complex tangles raises ques-tions with regard to their translocation to thepleura upon inhalation and subsequent pleuraleffects. Appropriate inhalation studies need tobe designed. Such an inhalation studies wereconducted recently by Mercer et al. (2013a;2013b; Kasai et al. 2015). Mercer et al. (2013b)reported that MWCNT individual fibers wereobserved by enhanced dark-field microscopyin pleural lavagate, chest wall, and diaphragm1 d after inhalation of MWCNT (5 mg/m3,5 h/d, 12 d; lung burden of 28 μg/mousedetermined by an absorbance assay). The num-ber of MWCNT in the diaphragm significantlyincreased from 1 d to 336 d postexposure.Kasai et al. (2015) also observed by electronmicroscopy translocation of MWCNT to thediaphragm after a 13-wk inhalation of Mitsui-7 MWCNT in rats. Fibers were also detected inthe pleural lavagate of rats after administrationof two types of MWCNT (Mitsui-7 and NikkisoMWCNT) by intratracheal spraying of largedoses (250 μg 5 times over 9 d for a total of1.25 mg/rat) (Xu et al. 2012). Such high-bolus-dose pulmonary exposure to MWCNT resultedin an increase of inflammatory cells in the pleu-ral cavity, inflammatory and fibrotic lesions ofthe visceral pleura, and mesothelial hyperplasiain the visceral pleura as determined 6 h afterthe last dosing. No lesions were observed inthe parietal pleura. Xu et al. (2014) recently

also exposed rats by intratracheal spraying tolong-thick (8 μm × 150 nm) and short-thin(3 μm x 15 nm) MWCNT. Exposure was to125 μg/rat, 13 times over 24 wk for a totalvery high dose of 1.625 mg/rat. The short-thin MWCNT sample consisted of agglomer-ated tangles with few free fibers. Long, butnot short, MWCNT translocated to the pleu-ral cavity and deposited in the visceral andparietal pleura. Long MWCNT induced fibro-sis in the parietal and visceral pleura, andparietal mesothelial proliferative lesions werefound after this 24-wk treatment, confirming—as previously suggested—the importance offiber length for translocation from depositsin the lung to and effects at remote targetsites.

In summary, intracavitary high bolus injec-tion of MWCNT in mice and rats inducesmesothelioma. MWCNT were found in thepleural lavage, chest wall, and diaphragm afterinhalation exposure of mice or rats to MWCNT.Successive high-dose nonphysiological bolusexposures (over 9 d–24 wk; lung burdens of1.25–1.63 mg/rat) resulted in pleural inflam-mation and mesothelial hyperplasia. However,induction of lung tumors or mesotheliomahas not yet been demonstrated after realisticinhalation exposure in a noncompromisedrodent model. Thus, although the presentlyavailable studies confirm a carcinogenic hazardof MWCNT, data cannot be used for quantita-tive risk characterization of repeated inhalationexposures. The outcome of an ongoing 2-yrrat inhalation study with Mitsui-7 MWCNT(S. Fukushima et al. personal communication,2014) may provide essential information onthe carcinogenic potential of inhaled MWCNT.Other knowledge gaps include (1) deter-mination of dose and time dependence ofpleural effects, (2) examination of the impactof fiber dimensions (length and width) andagglomeration/aggregation state on pleuraleffects, (3) analysis of the effects of functional-ization on MWCNT potency, and (4) measuringtranslocation rates to other potential targetsites including liver, spleen, CNS, and bonemarrow.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


SYSTEMIC CARDIOVASCULAR ANDNEUROLOGICAL RESPONSES TOPULMONARY EXPOSURE TO CNT

Li et al. (2007b) observed that multipleaspirations of SWCNT (20 μg/mouse, every2 wk, for 2 mo) in Apo E -/- mice produced anincreased number of aortic plaques. Inhalationof MWCNT (26 mg/m3 for 5 h; calculatedlung burden of 22 μg) resulted in a depressionof the responsiveness of coronary arteriolesto dilators 24 h postexposure (Stapletonet al. 2011). Further, pharyngeal aspirationof MWCNT (80 μg/mouse) produced aninduction of mRNA for certain inflammatorymediators and markers of blood–brain barrierdamage in the olfactory bulb, frontal cortex,midbrain and hippocampus 24 h postexposure(Sriram et al. 2009). However, Han et al.(2015) demonstrated that pharyngeal aspira-tion of MWCNT (40 μg) induced pulmonaryinflammatory responses 7 d postexposurewith no progression of atherosclerosis inapolipoprotein-E-deficient mice.

A recent MWCNT inhalation study inrats reported 24 h after a 5-h inhalationto 5 mg/m3 pulmonary inflammation andtranslocation to systemic organs (Stapletonet al. 2012). Data also showed an impairmentof endothelium-dependent dilation in cardiaccoronary arterioles, which was not resolvedyet by 7 d postexposure, revealing the poten-tial of inhaled MWCNT not only to inducepulmonary inflammatory effects but also toproduce serious extrapulmonary effects after ashort-term exposure to a high concentration of5 mg/m3, resulting in an estimated lung burdenof 13 μg/rat. In contrast, Warheit, Reed, andDeLorme (2013) reported no histopathologicalchanges in heart muscle or cardiovasculartissue and no marked changes in C-reactiveprotein or coagulation parameters betweenCNF-exposed and air-exposed control rats aftera 90-d inhalation of CNF (25 mg/m3). It ispossible that differences of physicochemicalproperties of these fibrous nanomaterials(hollow vs. solid fibers; retained doses, fiberdimension, surface area, or fiber count/mass)may explain these results. In addition, it isuncertain how long cardiovascular responses

persist or adapt after initial exposure (90 d afterinitial exposure [Warheit, Reed, and DeLorme2013] vs. 1–7 d postexposure [Stapleton et al.2012]). Further, identifying cardiovasculareffects may require additional specific methodsto determine with high sensitivity effects on thecardiovascular system.

Several possible mechanisms have beenpostulated to explain systemic responses topulmonary exposure to CNT.

Translocation of CNT to Systemic SitesTranslocation of ip MWCNT from the

abdominal cavity to the lung was reported byLiang et al. (2010). However, thus far there is noapparent evidence that systemic effects notedearlier in this report were associated with CNTin the affected tissue. Indeed, aspirated gold-labeled SWCNT were not found in any systemicorgan 2 wk postexposure using transmissionelectron microscopy (TEM) (Mercer et al. 2009).However, the study by Stapleton et al. (2012)found low levels of systemic translocation ofinhaled MWCNT into liver, kidneys, and heart.DeLorme et al. (2012) also observed rareevents of translocated CNF in brain, heart, liver,kidneys, spleen, intestinal tract and mediastinallymph nodes, but no histopathological changeswere seen. Mercer et al. (2013b) documentedtranslocation of MWCNT to the lymphatics,liver, kidneys, heart, and brain after inhalationof MWCNT (lung burden of 28 μg/mouse).However, this systemic translocation at 336 dpostexposure was less than 0.25% of the initiallung burden.

Systemic InflammationErdely et al. (2009) reported that aspira-

tion of SWCNT or MWCNT (40 μg/mouse)induced a significant increase in blood neu-trophils and mRNA expression and protein lev-els for certain inflammatory markers in blood at4 h postexposure, but not at later times. Suchpulmonary CNT exposure also significantly ele-vated gene expression for mediators, such asHif–3α and S100a in the heart and aorta at4 h postexposure. Evidence also indicates thatpulmonary exposure to particles alters systemic

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


microvascular function by inducing activatedPMN to adhere to microvessel walls and releasereactive species that scavenge NO producedby endothelial cells (Nurkiewicz et al. 2006;2009). This decreased responsiveness of sys-temic muscle and coronary arterioles to dilatorswas observed 24 h after inhalation of nano-TiO2at a lung burden of 10 μg (Nurkiewicz et al.2008; LeBlanc et al. 2009) and after inhalationof MWCNT (Stapleton et al. 2012).

Kido et al. (2014) interpreted their findingof increased mRNA expression of inflammatorycytokines in splenic macrophages after 3 mo ofinhalation exposure of rats to MWCNT as indi-cators of systemic inflammation. TranslocatedMWCNT were noted in spleen of exposedrats, and both splenic macrophages and T-lymphocytes displayed increased expressionof cytokines/chemokines including interleukin(IL)-2, suggesting a potential impact on antitu-mor activities and general immunosurveillance.

Neurogenic SignalsIntratracheal instillation of SWCNT (250 μg)

to rats was found to elevate baroreflex functionby twofold (Legramante et al. 2009; Coppetaet al. 2007). Although data specifically for CNTare not yet available, pulmonary exposure toinhaled ultrafine TiO2 was reported to stim-ulate sensory neurons in the lung (Kan et al.2012). Scuri et al. (2010) demonstrated that invivo inhalation of TiO2 nanoparticles producedupregulation of lung neutrophins in weanlingand newborn rats but not in adults. Furtherinhibition of sympathetic input to systemic arte-rioles reversed the decreased responsivenessof microvasculature to dilators after pulmonaryexposure to ultrafine TiO2 (Knuckles et al.2012).

In summary, although a few studiesreported cardiovascular responses after pul-monary exposure to CNT, the results are farfrom complete. Dose and time-course relation-ships for a variety of cardiovascular endpointsneed to be determined. Mechanisms by whichpulmonary particles induce cardiovascularresponses require elucidation. AlthoughMWCNT have been observed in cardiovascular

tissue after pulmonary exposure, it is unclearwhether this tissue burden is sufficient toexplain cardiovascular reactions observed.Knowledge gaps concerning relationshipsbetween CNT/CNF dimensions, surface areaand surface activity, and possible cardiovascularresponses need to be addressed. The findingof translocation of inhaled MWCNT to thespleen and activation of splenic macrophagesand lymphocytes requires further mechanisticstudies regarding systemic inflammatory andimmunomodulatory effects.

Relationship Between PhysicochemicalProperties and ToxicityPhysicochemical properties may affect the

potential toxicity of nanomaterials. There areseveral extensive lists of properties that couldapply to nanomaterials, such as the 18 proper-ties noted by the OECD (2010). These prop-erties were examined as part of the OECDnanomaterial testing program, which examineda set of standardized nanomaterials for rele-vant physicochemical, fate, toxicity, and eco-toxicity endpoints. Other more focused listsare intended to correlate health effects withphysicochemical characteristics (Bernstein et al.2005). There have been efforts to take suchfocused lists for health effects and narrowthem even further to apply to nanomaterials ingeneral (Nanomaterial Registry 2014; Warheit2008; Nel et al. 2009).The list of propertiesrelevant to all nanomaterials may be narrowedwhen considering health effects of a particulargrouping of nanomaterials. For CNT and CNF,a possible reduced list of traits is contained inTable 2.

TABLE 2. Physicochemical Characteristics which influenceToxicity of CNTs and CNFs

1. Method of Generation2. Shape (length, width, morphology)3. Agglomeration/aggregation4. Surface Properties (area, charge, defects, coating,

reactivity)5. Impurities6. Density

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Method of GenerationThe manufacturing technique used to pro-

duce CNT or CNF may affect all resultantphysicochemical properties, and hence needsto be considered in order to understand therelative potency of the nanoparticle. Carbonnanotube production is accomplished by eitherchemical, physical, or other processes (Rafiqueand Iqbal 2011). Chemical vapor deposition(CVD) is a method for large-scale production ofCNT, involving use of a hydrocarbon gas such asethylene, methane, or acetylene; a process gas,either ammonia, nitrogen, or hydrogen; and asubstrate such as silicon, glass, or alumina withan associated catalyst particle such as Fe, Co,or Ni. The nanotube diameter is dependent onthe catalyst particle size. While most CVD pro-cesses produce random groups of CNT, othervariants of the process might yield verticallyaligned CNT and/or DWCNT. A second chemi-cal process, the high-pressure carbon monoxidereaction method (HiPco), is also suitable forlarge-scale synthesis of SWCNT. In this method,the catalyst (Fe pentacarbonyl, or a mixture ofbenzene and ferrocene; Ni may also be used)is introduced in the gas phase, with CO used asthe hydrocarbon gas. A third chemical processto produce SWCNT has been developed thatuses cobalt and molybdenum catalysts, a silicasubstrate, and CO gases (CoMoCAT). SWCNTmay be either metallic or semiconducting.These traits in turn influence their commercialapplications. Physical processes for CNT pro-duction include arc discharge and laser abla-tion. These methods are mostly used for exper-imental purposes, and produce CNT in highlytangled forms, mixed with carbon contaminantsand catalysts and therefore likely require purifi-cation, unlike CVD, which does not requireextensive posttreatment. Finally, there are mis-cellaneous processes that are used even less.These include helium arc discharge, electrol-ysis, and flame synthesis methods. Efforts arecurrently under way to produce chirality-pureSWCNT in a lab setting. This is of interestsince the electronic and optical characteristicsof SWCNT are dependent on chirality (Liu, Ma,and Zachariah 2012).

Carbon nanofiber synthesis methods havebeen summarized by Kim et al. (2013). CNFdiffer from CNT in that they are generallylarger in diameter and noncontinuous. CNFare produced via catalytic CVD using nano-sized metal particles such as Fe, dispersed ona ceramic substrate (similar to production ofCNT), or by electrospinning of organic polymerssuch as polyacrylonitrile followed by thermaltreatment. CNF have special desirable thermal,electrical, and mechanical properties. A float-ing catalyst method (Singh et al. 2002) was alsoestablished involving metallocenes solutions ascatalyst and benzene as feedstock sprayed intoa furnace at high temperature to produce CNFin a continuous process at high yield. CNFproduced by CVD consist of regularly stacked,truncated conical or planar layers along the fil-ament length, and differ from CNT, which arecomprised of concentric graphene tubes thatcontain an entire hollow core, and are flexible(Murray et al. 2012). In addition, such CNF usu-ally behave as semiconductors, and their layeredges are chemically active. CNF producedby electrospinning and thermal treatment con-sist of randomly arranged flat or crumpledgraphene sheets, with the long axis of thesheets running parallel to the fiber axis (Harris2005). Electrospinning of two immiscible poly-mer solutions may produce porous nanosizedfibrous carbon materials, and similar to CNT,CNF may be treated to reduce residual catalystmetals.

Shape (Length and Width)The fiber-like shape of CNT and CNF has

some resemblance to asbestos fibers, raisingconcerns that the well-known fibrogenic,carcinogenic, and mesotheliogenic effectsattributed to asbestos inhalation may alsobe associated with inhalation of CNT/CNF.However, amphibole asbestos are thicker, andconsist of straight fibers, many of which exceed15–20 μm in length. Inhalation of these fibersresults in “frustrated” phagocytosis by alveolarmacrophages, with associated inflammatorycytokine release and cell death. Serpentineasbestos (chrysotile) consists of more curly

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


thinner fibrils, and hence may be a bettercorrelate to CNT tangles. Detailed morpho-logical examination using TEM or scanningelectron microscopy (SEM) analysis needs to becompleted prior to testing.

Ranges of length and width for CNThave been summarized in Donaldson et al.(2006). The lengths of CNT are typically severalmicrometers, although significantly shorter andlonger fibers have been made. SWCNT diame-ters vary between 0.7 and 3 nm, while MWCNTouter diameters generally are in the 10 to200 nm range. Although some are straight, mostSWCNT and MWCNT are bent or curled toform clumps, assemblages, or ropes that arelonger and wider than the individual nanotubesinglets. While CNF might be produced by syn-thesis techniques similar to those of CNT andhave a filamentous shape, these differ in severalways from CNT: CNF are composed of regu-larly stacked truncated conical or planar layersthat run along the filament length and providechemically active end planes in the inner andouter fiber surfaces; unlike CNT, CNF are notcomprised of concentric tubes and may or maynot contain a hollow core (Kim et al. 2013).CNF are flexible, and have diameters around100 nm and an aspect ratio above 100 (Kimet al. 2013).

The size of CNT/CNF aerosols is deter-mined by their agglomeration state, whichaffects their deposition in all regions of the res-piratory tract. Depending on the deposited andretained dose, CNT/CNF may induce adversepulmonary effects and translocate to otherorgans and tissues. Fibrous particles/fibers aredefined as elongated particles with a length-to-diameter ratio (i.e., aspect ratio) equal toor greater than 3 to 1 and longer than 5 μm(EPA 2001; WHO 1981). Respirable (EPA 2001)infers that the particle in question might pen-etrate to the alveolar region upon inhalation.There are considerable differences in fiber res-pirability between lab rodents and humansas discussed later (Inhalable, Thoracic, andRespirable CNT and CNF Fractions) and inAppendix A. Translocation from depositionsites of inhaled MWCNT from the lungs of

mice to pleural sites, to local lymph nodes,and to secondary organs is dependent onagglomeration state and size (Mercer et al.2013b). In this study, translocation rates andamount translocated to secondary organs werelow less than 0.1% over a year, whereas migra-tion to bronchial-associated lymphatics wasmuch higher over 7%. Most of the MWCNT insecondary organs were found in liver, but tinyamounts of short single CNT structures werealso detected in brain, heart, and kidneys, inaddition to pleural sites. Whether such lowtranslocation of MWCNT might directly pro-duce adverse effects in these distal organs hasyet to be determined. Direct ip injection of highdoses induced inflammation, granulomas, andmesothelioma (Takagi et al. 2008; Poland et al.2008; Rittinghausen et al. 2014).

Several studies using intracavitary bolusinjection of MWCNT in rodents demonstratedthe length dependency of persistent inflamma-tory responses, which were inversely relatedto clearance at the parietal pleura (Donaldsonet al. 2010; Murphy et al. 2011). These resultsconfirmed earlier pioneering work with ip injec-tion of long and short asbestos demonstratingthat the size of lymphatic stomata on theparietal pleural of about 8–10 μm allowsefficient clearance of short but not of long(>10–15 μm) fibers (Moalli et al. 1987).Donaldson et al. (2010) and Murphy et al.(2011) also showed that inflammatory responseto MWCNT agglomerates or tangles was lessand resolved quickly. Similarly, Nagai et al.(2011) and Rittinghausen et al. (2014) reporteda low mesotheliogenic response to ip injectedtangled MWCNT. It should also be noted thateven short 5-μm MWCNT may be proinflam-matory when given at high bolus doses, andsimilarly that lymphatic clearance pathways inthe parietal pleura might be blocked by highbolus doses of such short fibers. While therelevance of direct injection of a high bolusdose of CNT into pleural or peritoneal cavityas surrogate for real-world inhalation exposuresis questionable, this method may be consid-ered as a proof-of-principle, which needs to beconfirmed by realistic inhalation exposures.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Agglomeration/AggregationThe International Organization for

Standardization (ISO 2009) definedagglomeration as a collection of weaklybound (e.g., van der Waals forces) particleswhere the resulting surface area is similar tothe sum of the surface areas of the individualcomponents. Aggregation is defined as stronglybound or fused particles where the resultingexternal surface area may be significantlysmaller than the sum of the individual sur-face areas (CEN ISO/TS 27687 2008; Jiang,Oberdörster, and Biswas 2009). Agglomerationand/or aggregation of CNT and CNF may haveimportant implications for the biodistributionand toxicity of these materials, since they maybe strongly influenced by the physicochemicalproperties of the agglomerate masses, inaddition to the properties of the individualsinglets that comprise the agglomerates. Forexample, the 12-d inhalation study by Merceret al. (2013b) using highly agglomeratedMWCNT aerosols exhibiting a wide range ofagglomerated structures revealed a significantincrease of singlet MWCNT in lymph nodesand extrapulmonary secondary organs, whichindicated preferential translocation of MWCNTas singlets, due to either a slow process ofdeagglomeration of agglomerates deposited inthe lung, or—if the agglomerate structure iskept intact—a slow translocation of MWCNTthat were initially deposited as singlets. Theformer scenario is more likely, given the lownumber of individual singlet MWCNT thatwere present in the aerosol.

Deposition of agglomerated MWCNTaerosols in the alveolar region of the lungand subsequent phagocytosis by alveolarmacrophages may result in a high burdenwhen expressed as volume. This volumetricload—if exceeding a threshold defined as lungparticle overload (Morrow 1988)—results in animpairment of alveolar macrophage clearancefunction as suggested by Pauluhn (2010). If sig-nificant aggregation is present, deaggregationand release of singlet MWCNT in the lungis not likely. The presence of aggregation or

agglomeration may affect the surface area ofthe MWCNT bundle, a dose metric that wassuggested as more appropriate to be used whencomparing effects—including those associatedwith lung particle overload—of a wide rangeof different particle sizes from nano to micro(Oberdörster et al. 1994b; Tran et al. 2000a;Stoeger et al. 2006). If particle surface areais used as the dose metric, the slightly lowersurface area of aggregated versus agglomeratedstructures needs to be considered as definedearlier.

Surface PropertiesSurface properties of nanomaterials

are important determinants of biological/toxicological activity. For example, surfacecharge may affect cellular uptake, and becauseit is the surface of poorly (in vivo) solublematerials that interacts with the organism,surface area (cm2/g material) as dose met-ric has been widely applied in nanoparticletoxicology. Yet this metric is often misunder-stood by assuming that all nanomaterials fiton a common dose-response relationshipif specific surface of each material is usedas metric. Rather, the surface area conceptimplies that expressing an effect—observed invitro or in vivo—as a function of surface areaof poorly soluble materials provides a moremeaningful toxicological dose metric for hazardranking than using mass or number of thetested nanomaterial (Donaldson, Beswick, andGilmour 1996; Tran et al. 2000b; Oberdörster,Oberdörster, and Oberdörster 2005a). If theonly difference is the particle size, with allother properties of a nanomaterial being thesame, a common surface area dose-responserelationship can be established for this material.Of interest in this regard, the fibrotic potencyof SWCNT > MWCNT CNF correlates withtheir increasing specific surface areas (Murrayet al. 2012; Mercer et al. 2011). An interestingfinding supporting the surface area concept is astudy by Timbrell et al. (1988) demonstratingthat the lung fibrosis score of asbestos in miners

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


correlated best with the retained asbestos fibersin lung tissue.

Solubility (static in vitro systems) and dis-solution (dynamic in vivo system) are impor-tant determinants of toxicity of nanomate-rials in general (Utembe et al., 2015), butsolubility/dissolution are not critical for poorlysoluble CNT/CNF. The surface area dose met-ric concept for particles is different from solublechemicals, where the dissolved mass is theappropriate dose metric. An expansion of thesurface area concept is to express the maximaleffect induced by each of a collection of differ-ent nanomaterials as effect per unit of their sur-face area derived from the steepest section oftheir dose-response relationships. This way thedegree of the effect-inducing potential of eachnanomaterial can be compared and rankedagainst each other on the basis of surface reac-tivity. The surface area normalized maximumresponses (per cm2) of different nanomaterialsthen may reflect their different hazard potentialfor purposes of hazard ranking (Rushton et al.2010; Duffin et al. 2007).

The plausibility of the surface area conceptis supported by findings that surface prop-erties determine effects upon contact withcells, receptors, organelles. Thus, Sager et al.(2014) found that altering surface propertiesof MWCNT resulted in different effects andbioactivity profiles in a mouse model. Thesurface area dose metric concept is also likelyto apply to the fibrous structure of CNT andCNF; however, when present as straight singlestructures, length and diameter also may needto be considered as determinants of theirbiological/toxicological effects, based on thethree-dimensional (3D) paradigm that is wellknown from asbestos and glass fiber toxicology(Broaddus et al. 2011), but that is unlikely tobe applicable for highly tangled/agglomeratedCNT.

Ideally, the bioavailable or effective surfacearea should be the metric for defining surfacereactivity (response per unit surface area). Thegas (mostly nitrogen) adsorption method afterBrunauer, Emmett, and Teller (BET) (Brunauer,Emmett, and Teller 1938) is generally usedto determine specific surface area, which,

however, needs to be considered only as asurrogate for a biologically available surface,whose real definition is still elusive. Donaldsonet al. (2013) noted the central importance of abiologically effective dose (BED) in toxicology,which drives toxic responses and includes par-ticle surface area as one metric. Peigney et al.(2001) developed a mathematical model toestimate the surface area of bundles of paral-lel CNT by accounting for the void spaces andalso the number of layers in MWCNT. Evidenceindicated that the calculated values are in goodagreement with measured data by BET. Giventhat CNT agglomerates do not usually consistof straight, well-aligned parallel tubes, thesetheoretical calculations are—like BET—not pro-viding information on bioavailable surface area.

Surface modification, for example,carboxylation, might affect biopersistence.Carboxylation of SWCNT and MWCNT makesthem more vulnerable to oxidative destruc-tion by peroxidases, which reduces theirbiopersistence significantly and may point tonew possibilities to produce less reactive CNTthrough surface modifications (Zhao, Allen, andStar 2011; Kotchey et al. 2013). Carboxylationalso makes the CNT more hydrophilic and lessagglomerated. Carboxylation may decreasethe ability of these nanoparticles to enterepithelial cells and cross cell membranes, enterthe alveolar interstitium, and produce fibrosis(Wang et al. 2011; Sager et al. 2014). TheROS-inducing potential of CNT expressed perunit surface area may also be determined asa measure to categorize CNT and CNF basedon their surface reactivity. Cell-free assays andelectron spin resonance (ESR) spectroscopywere shown to be effective methods to assessthis property (Rushton et al. 2010; Tsuruokaet al. 2013). In contrast to raw CNT, CNTpurified to remove catalytic metals do notgenerate high levels of ROS in cell-free systems(Kagan et al. 2006).

Other surface properties and modification,like zeta potential, electrostatic charge, coat-ings, and defects, may also significantly influ-ence their biological/toxicological responses(Fenoglio et al. 2008; Shvedova et al.2005; Tabet et al. 2011; Li et al. 2013;

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Salvador-Morales et al. 2008). Indeed, Li et al.(2013) observed that MWCNT functionalizedwith polyetherimide, which resulted in a strongpositive surface charge, were more fibrogenicthan bare MWCNT after aspiration in a mousemodel. In addition, surface charge influencesthe agglomeraton state of CNT/CNF, whichimpacts their aerodynamic behavior whenaersolized.

ImpuritiesImpurities that might affect pulmonary tox-

icity of CNT and CNF may be present andfall into three classes (Donaldson et al. 2006):support material, organics, and metals. Supportmaterial includes aluminates, silicates, andmagnesium oxide. Residual organics includeamorphous particles, or microstructured par-ticles such as graphite sheets, which mightarrange into carbon nanofibers or spheres.Little toxicity information is available that cor-relates adverse pulmonary effects with impurityamounts of either support material or residualorganics. The most commonly used metals inCNT synthesis include Co, Fe, Ni, and Mo. CNTmay be processed to remove most of the metalcatalysts, such as those encapsulated in layers ofamorphous soot or graphite.

Transition metal complexes and free Feand Ni are known catalysts of biological freeradical reactions. Therefore, where metal con-tent was attributed to effects seen, correla-tions need to be considered in light of metalbioavailability. The effects of Fe and Ni catalystson the pulmonary toxicity responses of CNTin three studies were recently reviewed byNIOSH (2013). Generally, higher Fe contentsup to 18% were not associated with a higherlung response (Shvedova et al. 2008; Merceret al. 2008), suggesting low bioavailability.In contrast, bioavailability of a high Ni- con-tent affected the outcome granuloma formation(Lam et al. 2004). Bioavailability of metal impu-rities is a key determinant, as is potential con-tamination with endotoxin (lipopolysaccharide,LPS) which needs to be carefully tested (Eschet al. 2010).

DensityThe relative density or specific gravity of

nanomaterials is often regarded as being oflittle or no importance for interpretation ofbiological/toxicological effects. However, mate-rial density and bulk density need to be con-sidered because inhalation is the main route ofhuman exposure to CNT and CNF, and aerosoldensity is an important property to predict andunderstand their aerodynamic behavior anddeposition fractions throughout the respiratorytract (Park et al. 2004). Further, because (1) cel-lular uptake of deposited materials, in particularby macrophages, results in a volumetric load-ing that was suggested to impair cell function ifexceeding approximately 6% of the normal cellvolume (Morrow 1988), and (2) volume anddensity are highly correlated, data on materialdensity and bulk density need to be includedin a listing of physicochemical properties forCNT/CNF characterization.

Material density of nanomaterials is specif-ically important for aerosols consisting of sin-glet particles in the aerodynamic size range,that is, above approximately 0.3 μm. In con-trast, for singlet nanoparticles below 100 nmin the aerosol the main mechanism for theirdeposition in the respiratory tract is by diffu-sion, for which particle density does not playa role. Because airborne nanomaterials rarelyoccur as singlets, the aerodynamic behaviorof their larger agglomerate sizes is determinedby the effective density of the agglomerates.Difficulties in determining the effective aerosoldensity were described for regular particles(Park et al. 2004; Ku et al. 2007; Liu, Ma,and Zachariah 2012; Miller et al. 2013),but solutions have not yet been proposedfor bundles/agglomerates of CNT and CNFaerosols. To overcome this problem, gas pyc-nometry was used to determine bulk densityof bundled MWCNT (Pauluhn and Rosenbruch2015) using the method of ISO 15901–1 (ISO2005a). Wang et al. (2015) derived an expres-sion for the effective density based on fractal-like structure of CNT and fit the equation tomeasured mass and mobility diameter to obtain

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


values for effective density as a function ofmobility or outer diameter of CNT agglomer-ates. There is limited applicability of the find-ings to the respiratory tract because calculationswere based on CNT transport in an electric fieldalone. Extension of this model to other externalforces, such as inertia and gravity, is not justifiedand requires parallel studies. Bulk density mayalso be assessed by pouring the agglomeratedor aggregated CNT or CNF into a graduatedcylinder and measuring volume and weight(DIN EN 725–9 2006–05(E) 2006). Dependingon conditions of undisturbed pouring (poureddensity) versus tapping the cylinder (tappingdensity), different values may occur. However,it needs yet to be determined whether theresults approximate the effective aerosol den-sity when using the poured density, whereasthe tapping density may approximate the some-what higher density present after uptake intocells for deriving the volumetric burden inmacrophages. Thus, validated methods to mea-sure aerosol densities of CNT still await generalacceptance.

Additional Physicochemical PropertiesOther physicochemical parameters

of CNT/CNF may influence biological/toxicological responses, yet respective infor-mation and convincing data are not available.These include chirality of MWCNT, their tipconfiguration, crystallinity, and fiber stiffness,strength, and hardness. An important param-eter may also be the degree of oxidation,which is rarely reported. New findings mayreveal other relevant properties. For exam-ple, what is the biological significance of therecent discovery of high-speed pulse-like watermovement through CNT, as reported by Sisanand Lichter (2014), using numerical simulationsof single-file water flow?

SummaryIn general, pulmonary exposure of rats

or mice by IT instillation, pharyngeal aspira-tion, or inhalation of SWCNT or MWCNT andCNF results in transient inflammation and lung

damage. Granulomatous lesions and interstitialfibrosis, which are of rapid onset and persistentin nature, have also been a common occur-rence. Benchmark dose analysis (NIOSH 2013)noted no striking differences in responsive-ness to CNT between rat and mouse models.Indeed, inhalation exposure of mice or ratsto the same type of CNT (Mitsui-7 MWCNT)resulted in similar responses, that is, pulmonaryinflammation, granulomas, interstitial fibrosis,and translocation of MWCNT to the pleura(Mercer et al. 2013a; 2013b; Kasai et al. 2015).The degree of agglomeration does affect depo-sition site and response. Large agglomeratestend to deposit more centrally at the terminalbronchioles and proximal alveoli and induce agranulomatous response, while more dispersedstructures may deposit more peripherally in dis-tal alveoli and produce interstitial fibrosis. Thelung most likely might be exposed to moredispersed CNT and CNF structures by inhala-tion than by bolus-type exposure to a sus-pension of CNT/CNF. When this is the case,the response to short-term inhalation may dif-fer from that of the same lung burden givenas a bolus dose. Thus far, reported data sug-gest that quantitative differences in bioactivitybetween different types of CNT are likely. Forexample, on an equal mass basis, SWCNTappear to be more potent than MWCNT inproducing alveolar interstitial fibrosis (Merceret al. 2011). Limited data available for CNFsuggests that the fibrotic potency of CNF isless than that of CNT (Murray et al. 2012).Mercer et al. (2011) proposed that specific sur-face area, dimensions, or hydrophobicity cor-relates with the rate of migration of fibrousnanomaterials to the interstitium. However, fur-ther investigation is required to resolve thisissue.

The influence of manufacturing methodsand surface modifications on physical–chemicalproperties of CNT/CNF needs to be consid-ered when analyzing the correlation betweenthese properties and toxicity. Understandingthese correlations may help predict potentialtoxicities and also for identifying data gapsand research needs to be addressed in futurestudies.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


CONSIDERATIONS AND CONCEPTSFOR REALISTIC RODENT INHALATION

Ideally, human exposure to CNT and CNFneeds to be simulated when performing inhala-tion studies in rodents, not only to identify apotential hazard that is associated with suchexposure but also to generate data that can beused for risk assessment. Thus, serious effortsneed to be undertaken to obtain knowledgeabout exposure concentrations, airborne par-ticle size, and size distribution, particularly atthe sites of manufacture but also along the lifecycle of CNT and CNF (packaging, distribu-tion, incorporation into products, application,disposal), for potential exposure of workers andconsumers. Such data are important for gener-ation of an equivalent exposure atmosphere forrodents, taking into consideration differencesbetween humans and rodents with respectto inhalability and respirability (see Inhalable,Thoracic, and Respirable CNT Fractions sub-section). Generation of appropriately realis-tic exposure conditions is critical for pur-poses of dosimetric extrapolation and for riskassessment.

Available Exposure DataThe first available datum concerning air-

borne levels of CNT in a workplace wasreported by Maynard et al. (2004). This studyevaluated aerosolization of SWCNT in a smalllab setting that simulated a workplace environ-ment. Results indicated that SWCNT may beaerosolized with sufficient agitation. Estimatesof the airborne concentration of SWCNT frompersonal air samples suggested that work-place respirable dust concentrations were lowerthan 53 μg/m3. Evaluation of activities ina lab handling MWCNT reported significantaerosolization during weighing, transferring,and sonication (Johnson et al. 2010). Particlecounts over the size range of 10–1000 nmwere 1,576 particles/ml during weighing and2776 particles/ml during sonication, with CNTstructures generated during sonication beingless agglomerated than those produced dur-ing weighing. Han et al. (2008) measured totalairborne dust levels in a lab producing MWCNT

in Korea. Processes such as weighing, blend-ing and spraying produced airborne dust lev-els as high as 400 μg/m3. However, not allthese particles were MWCNT, and the MWCNTstructures generated varied from agglomeratesto more dispersed individual fibers. Admixtureof background aerosol also needs to be con-sidered. Lee et al. (2010) recently monitoredworkplace total dust levels at seven MWCNTfacilities using personal samplers, area sam-plers, and real-time monitoring by SMPS, dustmonitor and aethalometer. Particle generationwas significant during oven opening, catalystpreparation, gel spraying, and preparation andsonication of MWCNT. The highest total par-ticle concentration was 320 μg/m3 with amean 106 μg/m3, with number concentrationsfrom approximately 7000 to 75,000 per cubiccentimeter. Diameters depended on manufac-turing process, with mode diameters rangingfrom 20 to 30 nm for catalyst preparation andfrom 120 to 300 nm for ultrasonic disper-sion. It should be noted that only a fractionof these airborne particle were MWCNT, withmetal nanoparticles being a significant con-tributor to the total dust aerosolized in theseworkplaces.

It should be anticipated that within groupsof CNT, for example, MWCNT, displaying sig-nificant differences between different typesof MWCNT with regard to their physical(tangles, agglomerates) and chemical (contam-inants, surface chemistry) characteristics, atleast quantitative differences of responses arelikely. Examples of response differences areapparent from the 13-wk inhalation studies ofPauluhn (2010) and Kasai et al. (2015). Thelatter study, using a more dispersed MWCNTaerosol, reported CNT-specific toxicity. In con-trast, the first study, using approximately 3-μmagglomerates, attributed pulmonary responsesto a nonspecific overload mechanism. Surfacefunctionalization may also affect the biologicalpotency of MWCNT. Indeed, Carrero-Sánchezet al. (2006) showed that nitrogen-dopedMWCNT given by different routes of admin-istration to mice produced significantly lesstoxicity than pure MWCNT. In other studies,the biopersistence of carboxylated SWCNT

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


and MWCNT was significantly reduced bybiodegradation via peroxidases, as determinedin vitro and in vivo, which reduced inflamma-tory potential (Allen et al. 2008; Liu, Hurt, andKane 2010a; Kotchey et al. 2013).

Han et al. (2008) measured size distribu-tions of airborne MWCNT in a research facilityand found a majority of non-fibrous particlesand CNT agglomerates of different sizes withfew single fibers (Figure 1a). Other measure-ments in commercial and research facilitiesconfirmed that only a few single fibers werefound, with the majority of the airborne struc-tures being agglomerates/clusters of differentsizes (Tsai et al. 2009; Methner et al. 2010) asillustrated in Figure 1b.

Less information is available for CNF expo-sure measurements. Measurements by Birchet al. (2011), Evans et al. (2010), and Dahmet al. (2012; 2013) showed varying airborneconcentrations, particle shapes, and aerody-namic sizes, depending on the processing sitewithin a manufacturing facility. Similar to thefindings with CNT, bundled and entangledstructures were mostly found (Figure 2), as

well as nonfibrous structures. Maximum con-centrations due to uncontrolled transfer andbagging reach 1.1 mg/m3 (Evans et al. 2010);the dominant mode by particle number ofCNF was between 200 and 250 nm for bothaerodynamic and mobility equivalent diame-ters, indicating an effective density close tounity. Depending on the purification process,significant concentrations of polycyclic aro-matic hydrocarbons (PAH) and Fe-rich sootwere detected in the CNF aerosol, which needsto be considered as causative/contributory fac-tors for potential toxicity. There is an urgentneed to obtain more information on the char-acteristics of CNT and CNF in workplace air.The suggestion that delivery of CNT and CNFto the respiratory tract of experimental ani-mals, by inhalation or by instillation/aspiration,needs to be done with well-dispersed mate-rials is not supported by presently publishedoccupational exposure data, which uniformlydemonstrate highly agglomerated structures inthe airborne state (Methner et al. 2010; Tsaiet al. 2009; Han et al. 2008). It may be possiblethat one aerosol generation system is equally

FIGURE 1a. MWCNT aerosols at a research facility.

Aerosols collected at a research facility during manufacture of MWCNT revealing a multiplicity of structures. (Han et al. 2008).Shown are samples collected in the facility after opening the furnace, demonstrating many non-fibrous structures in addition to somesingle fibers but mostly agglomerates of different sizes. Initially high concentrations could be significantly reduced following installation ofproper enclosure and ventilation systems.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 1b. MWCNT aerosols collected at a research facility.

Aerosols collected at a research laboratory generating MWCNT by chemical vapor deposition (A: Tsai et al. 2009) and several com-mercial and research facilities manufacturing MWCNT (B, C, D: Methner et al. 2010). In the research facility, mostly agglomerates wereseen and only some single filaments with numerous attached clusters of nanoparticles were found that consisted of carbon and includedFe from the catalyst (A). The aerosol in a commercial facility was described as “various forms like agglomerates, clusters, bundles, or nestsrather than individual fibers or spherical particles” (B, C, D). These were collected under different working tasks (e.g., sawing, dumping).An example of the appearance of the few individual fibers and spherical particles is shown in (C). Total carbon concentrations measuredon filters according to NIOSH NMAM Method 5040 (Issue 2, 1998) were equivalent to 1.8 mg/m3. In general, samples from both facilitiesindicate that long and individual fibers were hardly seen, in contrast to clusters/tangles of different sizes. The MWCNT aerosol generatedexperimentally for use in animal inhalation studies seems to be much better dispersed.

well suited for appropriate (workplace repre-sentative) aerosolization of all different typesof CNT and CNF, but this still needs to beverified.

Workplace MonitoringISO/TR12885 (ISO 2008) lists and dis-

cusses several air monitoring instruments andtechniques for monitoring nano-aerosol expo-sure at the workplace by airborne mass, num-ber, and size distribution, as discussed later.However, whereas for spherical particles thescanning mobility particle sizer (SMPS) andDifferential Mobility Analysing System (DMAS)provide valuable information of their airbornesize distribution as mobility diameter of thecharged particles, measurements of CNT/CNFof complex structures with these devices arehard to interpret. However, the following meth-ods are well suited for measuring airborneCNT/CNF size distribution.

The micro-orifice uniform deposition impactor(MOUDI) and nano-MOUDI measureaerodynamic diameter, down to 10 nm(at low pressure stages). This measure forCNT/CNF may be used as input into thepredictive multiple path particle dosimetry(MPPD) model for rats and humans if datafor the density of the airborne structuresare available. The advantage of the nano-MOUDI is that airborne particle structuresof nanosizes are determined based on theiraerodynamic deposition behavior achievedwith a low-pressure system. Particle mor-phology may then be examined (electron)microscopically on the individual stages.Further development into an electrical lowpressure impactor (ELPI) was achieved byadding a corona charging unit before theinlet, so that aerodynamically separated par-ticles can be measured by multichannel elec-trometers in real time based on their charge(Keskinen, Pietarinen, and Lehtimäki 1992).

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 2. CNF aerosols collected on impactor stages during thermal treatment.

Transmission electron microscopy images of airborne CNF size selected by impactors located in a thermal treatment processing areafor CNF. Impactor Stage A: 2.5–10 μm; Impactor Stage B: 1.0–2.5 μm; Impactor Stage C: 0.5–1.0 μm; Impactor Stage D: 0.25–0.5.Lacey carbon-coated Ni and SiO-coated Ni grids were used in impactors. (From: Birch et al. 2011).

Filter samples for gravimetric measurement(μg/m3): Care needs to be taken to adjustfilter to same humidity before and aftersampling to get net weight.

TEM/SEM: With the use of electrostatic precip-itator and filter samples length and diameterdistribution, agglomeration state/tangles canbe determined.

While the instruments just described provideaccurate measurements for aerosol size distri-bution, caution needs to be observed whenmeasuring CNT and CNF. A condensationnuclei counter (CNC)/condensation particlecounter (CPC) may safely be used to countthe number of CNT and CNF in the airbecause there is no size or shape effectinvolved in the counting process. The differ-ential mobility analyzer (DMA) and SMPS sep-arate submicrometer-sized particles based ontheir mobility in an electric field. It is conceiv-able that CNT and CNF of different shapesmight attain the same mobility and be sepa-rated together thus obscuring size classification.The same limitation holds for the aerodynamic

particle sizer (APS), which is based on the timeof flight of particles. Aerodynamic drag andtorque of CNT depend on the shape and ori-entation of CNT and CNF in the air. Differentsize and shape of CNT and CNF may yield sim-ilar drag and torque forces under certain flowconditions and hence be grouped together bythe instrument. Separation of CNT and CNF ina MOUDI or nano-MOUDI impactor is basedon their inertial forces, which is a functionof their orientation in the air. Thus, a shapecorrection needs to be applied when usingthese instruments. The most reliable techniquefor size measurements of CNT and CNF isTEM/SEM, which provides both size and shape.However, electron microscope (EM) sizing istime-consuming, provides no real-time mea-surement, and the measured dimensions (i.e.,CMD and geometric standard deviation [GSD])need to be converted to equivalent diame-ters if CNT and CNF deposition is going tobe calculated. This will be a most difficult taskbecause the density of the aerosol particleshas to be known. Given the wide range ofstructures/shapes of aerosolized CNT (Figures 1

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


and 2), it becomes apparent that it is notthe density of carbon per se that is importantas much as the effective density, which maybe similar to the packing density. Knowledgeregarding the actual density of the CNT aerosolin the workplace air may also be needed forusing nano-MOUDI results (MMAD and GSD)as input values for estimating/predicting depo-sition in rodent and human lung with the MPPDmodel. While it is presently not possible to inte-grate a Nano-MOUDI into a personal sampler,number concentration (CNC/CPC) and massconcentration of airborne CNT might be builtinto such personal sampler. Asbach et al. (2012)and Meier, Clark, and Riediker (2013) testedsmall samplers that might quality for use aspersonal samplers.

Inhalable, Thoracic, and Respirable CNTand CNF FractionsWhen evaluating the potential toxic and/or

carcinogenic effect of CNT/CNF in inhalationstudies in lab animals or by in vitro testing viaaerosol exposure of primary cells or cell lines,ideally comparable or identical exposure tohuman scenarios are required to deliver similardoses to target sites or cells of the respiratorytract. In essence, an equivalent human expo-sure concentration (HEC) needs to be definedbased on metrics of exposure. Calculations ofa HEC account for interspecies differences ininhalability, respirability, and lung depositionof CNT/CNF. CNT/CNF inhalability, or theinhalable fraction, is defined as the fractionof airborne CNT/CNF that penetrates theextrathoracic airways on inhalation. Similarly,the thoracic fraction is defined as the fractionof airborne CNT/CNF that travels through thehead airways and reach the lung. Finally, therespirable fraction is the portion of airborneparticles that reaches the alveolar regionof the lung. The American Conference ofGovernmental Industrial Hygienists (ACGIH2010) established sampling criteria to definethese three fractions in terms of induction ofhazard in the three regions of the respiratorytract and respective defined airborne particlesizes.

Calculation of CNT/CNF inhalability isthe first step in ensuring delivering compat-ible doses among exposure settings. WhileCNT/CNF below 10 μm aerodynamic sizes arecompletely inhalable in humans, only a fractionis inhalable in small animals. In addition, lossesof CNT/CNF in the extrathoracic passages ofeach species are different, which combinedwith inhalability differences results in differentdelivered doses (thoracic and respirable frac-tions) of CNT/CNF to the lower respiratorytract. Therefore, CNT/CNF inhalable, thoracic,and respirable fractions need to be examinedand accounted for when extrapolating results ofanimal inhalation studies to humans based onthe delivered dose of CNT/CNF, or when usingknown human exposure conditions to designrealistic rodent inhalation investigations.

There is a wealth of published lab dataon penetration of particles into the lungs ofhumans and animals (Aitken et al. 1999;Asgharian, Kelly, and Tewksbury 2003; Berryand Froude 1989; Breysse and Swift 1990;Hinds, Kennedy, and Tatyan 1998; Hsu andSwift 1999; Kennedy and Hinds 2002). In addi-tion, there are recent developments of semiem-pirical, predictive models of particle inhalabilityin humans and lab animals (Ménache, Miller,and Raabe 1995; Brown 2005; Asgharian,Kelly, and Tewksbury 2003). However, thereis a major data gap on inhalability and res-pirability of nonspherical and irregularly shapedparticles such as CNT and CNF. Becauseof their asymmetric geometry, the orientationof CNT/CNF affects penetration and depo-sition during inhalation in the lungs. Hence,inhalability and respirability models for CNTand CNF need to account for differences in sizecharacteristics and transport properties as com-pared to spherical particles. A more detaileddiscussion of inhalable, thoracic, and respirablefractions is provided in Appendix A.

Interspecies ExtrapolationInhalation studies are conducted in lab

animals to observe various biological end-points. Interpretations of the findings includeextrapolation to human exposure scenarios

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


for purposes of human risk assessment.Conversely, if available, human exposure datacan be extrapolated to animal exposures forthe design of inhalation studies to examinebiological/toxicological responses. Thus, inter-species data extrapolation based on variousdose metrics of exposure is needed to eitherdetermine a no-observed-adverse-effect level(NOAEL) as input for risk assessment mod-els, or to select realistic exposure levels foranimal studies. While dosimetry of extrapo-lation has often been based on the inhaleddose (inhalable, thoracic, and respirable frac-tions), it needs to be based upon the depositeddose because the biological outcome is mostclosely associated with the deposited and notinhaled dose. This is particularly importantfor purposes of extrapolation when depositedand retained taking into account clearancedoses are to be compared between humansand rodents. Figure 3 illustrates a conceptualapproach of dosimetric interspecies extrapola-tion. More information on extrapolation mod-eling is provided in Appendix B.

Types of Respiratory-Tract ExposuresRats or mice might be exposed to

CNT/CNF by IT instillation, (Warheit et al.2004), pharyngeal aspiration, (Porter et al.2010), or inhalation (Shvedova et al. 2008)(Table 1). The major advantages of IT instillationor pharyngeal aspiration are that exposureis technically simple and inexpensive, requir-ing little specialized instrumentation, and thelung burden is estimated simply from mass ofCNT/CNF delivered to the lungs by assum-ing that between 80 and 90% deposits inthe alveolar region (Mercer et al. 2010a). Themajor disadvantages of IT instillation and pha-ryngeal aspiration are nonuniform distributionof particles in the lung and the fact thatlung burden is delivered as a bolus dose ina fraction of a second rather than graduallyover time as with inhalation, that is, the doserate is many orders of magnitude different.Both IT instillation and pharyngeal aspirationrequire that CNT/CNF be suspended in a bio-compatible fluid. Therefore, agglomeration is

FIGURE 3. Dosimetric Extrapolation of Inhaled Particles from Ratsto Humans.

Concept of dosimetric extrapolation of effects of inhaled mate-rials in experimental animals to humans. A Human EquivalentConcentration (HEC) is derived based on study in rats (scenario2 described in text) by using the MPPD model for rats to esti-mate the inhaled (delivered) and daily deposited dose in ratsresulting from the specific experimental exposure concentrations.Knowing the material rat-specific clearance rates or retention half-time (T1/2) in the lung, the retained dose that has accumulatedover the duration in the lung can be calculated, but may alsobe determined by analytical methods at the end of exposure.Using then human-specific clearance rates—if available—andbreathing scenarios as inputs to the MPPD model for humansthe HEC can be estimated for either the MMAD and GSD ofthe experimental aerosol, or for an aerosol size distribution thatis known to be present for the human exposure scenario. TheHEC is equivalent to the rat exposure in terms of resulting inthe same deposited dose—or retained dose—in both species.Through using the MPPD model human and rat inhalability andrespirability differences are taken into consideration. Depositedand retained dose can be expressed either per unit of epithelialsurface in the respiratory tract—tracheobronchial or alveolar—,or per unit mass of the lung. In addition, the material based dose-metric can be mass (as indicated in the scheme), or surface area ornumber of CNT/CNF, if respective information is available. [From:(Oberdörster 1989)]

an issue, with micrometer-size agglomeratesbeing common when CNT/CNF are suspendedin phosphate-buffered saline (PBS; pH 7.4).To improve dispersion of CNT/CNF, 5–10%serum, 1% Pluronic, or 1% Tween 80 has beenemployed. However, as shown by Dutta et al.(2007) and Haniu et al. (2011), the use of suchdispersants significantly affects cellular uptakeand masks potential effects of CNT. Porter et al.(2008) argued that CNT/CNF would interactwith alveolar lining fluid when deposited inthe respiratory region of the lung. Therefore,

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


they used a dispersion medium that contained0.6 mg/ml species-specific serum albumin and0.01 mg/ml disaturated phosphatidyl choline(DSPC) in PBS, that is, albumin and DSPC lev-els found in the initial aliquot of rodent BALF.This artificial diluted alveolar lining fluid wasfound to effectively disperse MWCNT, and datasuggested that it did not appear to mask par-ticle surface activity. However, Gasser et al.(2010) noted that precoating of MWCNT withlipids from lung surfactant altered the bind-ing of plasma proteins on their surface. Thus,the potential impact of using dispersants whenassessing nanomaterial toxicity has to be criti-cally considered, since it affects interaction withcells and tissues. As with inhalation exposure, itis critical to qualitatively evaluate the structuresize distribution by electron microscopy ofCNT/CNF in suspensions delivered to the lung.The following sections focus on inhalationexposures as the physiologically most appro-priate method for delivery of CNT/CNF to therespiratory tract.

Requirements for an Inhalation SystemA critical component of acquiring scien-

tific knowledge is the ability for findings froma given lab to be reproduced and extendedby other labs. Therefore, simplicity, ease, andconsistency of operation in maintaining a sta-ble airborne concentration of CNT/CNF is animportant goal in developing aerosol genera-tion systems for CNT/CNF. McKinney, Chen,and Frazer (2009) reported the use of an acous-tical generation system for the aerosolization ofMWCNT. Automated computer control allowedgeneration of aerosols of stable concentrationsand particle size distribution with little opera-tor manipulation required. In contrast, Baronet al. (2008) reported a generation systemfor SWCNT that was capable of generatinga stable output, using a knife mill generator,but required continuous adjustment by a well-trained technician. This difference in complex-ity of operation of generation systems reflectsthe fact that SWCNT agglomerate more avidlythan MWCNT. In general, the greater the diam-eter of the CNT/CNF, the straighter and lesscurled the fibers, and the easier they are to

disperse. Because this principle also applies toworkplace exposures, it is important to producerealistic CNT/CNF aerosols with appropriategeneration systems.

Inhalation exposures to CNT/CNF using adry powder aerosolization system is the pre-ferred method that best simulates human expo-sures under real-world conditions. Generatingan aerosol from a liquid suspension might beproblematic, as dispersants may have to beadded to prevent excessive clumping. Sageret al. (2007) compared the efficiency of dispers-ing nanoparticles (NP) in PBS with and with-out BALF or in Dipalmitoylphosphatidylcholine(DPPC) as the major constituent of the alveolarlining fluid, with and without albumin. Basedon their finding that DPPC + albumin in PBSwas effective, although still less than BALF inPBS, for use during in vivo and in vitro stud-ies, Gasser et al. (2012) confirmed that usinga commercially available lung surfactant tocoat MWCNT needs to be considered for invitro studies. Schleh et al. (2013) agreed onthe importance of simulating in vivo condi-tions by coating NP with pulmonary surfactantfor in vitro investigations. With regard to sur-factant precoating of NP to be delivered tothe respiratory tract in suspension, inhalation,or instillation, it has to be considered thatadsorption of proteins/lipids is likely to be dif-ferent from that on pristine particles (Duttaet al. 2007; Haniu et al. 2011; Gasser et al.2010). Thus, inhalation studies need to aimto mimic real-world conditions by generatingaerosols in their original state as would beexperienced by humans (Fubini, Ghiazza, andFenoglio 2010). As discussed earlier, since NPaerosols—including CNT/CNF—are present inthe workplace as agglomerates, there is no needto add dispersants when dosing the respiratorytract by inhalation.

As pointed out earlier, it is important to takeinto consideration inhalability and respirabilityof rodents versus humans when reproduc-ing real-world human exposure conditions forexperimental exposures of rodents. This maybe difficult in cases where long CNT and aero-dynamically larger agglomerates are presentin the workplace aerosol that are respirableby humans depositing in human pulmonary

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


region, but are less or not respirable by rodents.Enrichment of the aerosol for such longerCNT might be considered to achieve com-parable lung burdens in rodents. Conversely,depending on the CNT aerosol generation sys-tem, large rodent nonrespirable agglomeratesmay be present in the experimental aerosolbut are not found at the workplace. Thesemay be removed by appropriate preseparators(cyclone; impactor) to avoid exposure to highmass concentrations (large agglomerates repre-sent most of the aerosol mass) that result inunrealistic high nasal deposits and irrelevantexposure-dose-response data. Use of grind-ing and milling may introduce surface arti-facts and contaminants, which need to becontrolled carefully. A prerequisite for establish-ing exposure-dose-response relationships andfor dosimetric extrapolation modeling is theavailability of reproducible methods to accu-rately (1) characterize and monitor exposureatmosphere and (2) determine deposited andretained CNT/CNF burdens in the respiratorytract and in extrapulmonary tissues/organs fornecessary biokinetic information.

Analysis of Exposure-Dose-ResponseRelationshipsExposure-dose-response relationships

established in rodent inhalation studies as abasis for deriving an HEC may be expressedwith different dose metrics, either as exposure-response or as dose-response relationships.The latter is either based on deposited dose or,preferably, on the retained dose in longer terminhalation studies (Figure 3). Comparing theserelationships between several materials usingdifferent dose metrics may provide informationabout the relative hazard ranking of these mate-rials. Figure 4 compares exposure-response anddose-response data of subchronic rat inhala-tion studies with MWCNT (Pauluhn 2010;Ma-Hock et al. 2009a; Kasai et al. 2015),carbon black (CB) (Elder et al. 2005), andCNF (DeLorme et al. 2012). The subchronicCB study was added as a comparison to awell-studied carbon nanomaterial, which isgenerally viewed as a poorly soluble particle(PSP) of low cytotoxicity that has been used in

a number of rodent inhalation studies relatedto the lung particle overload phenomenon(Morrow 1988). In order to compare theresponses to these different materials, the sameeffect endpoints were selected. Increased lungweight was a common endpoint in all five stud-ies for which exposure-response relationshipscan be established.

Another sensitive quantitative endpoint,pulmonary inflammation as determined bythe increase of polymorphonuclear leukocytes(PMN), was also used to compare exposure-response relationships, although for only fourof the subchronic inhalation studies; this end-point was not included in the MWCNT studyby Ma-Hock et al. (2009a). Of greater rel-evance, however, is a comparison of dose-response relationships, given that depositedand retained doses differ significantly betweendifferent investigations, because they dependon the size distributions of the inhaled aerosol.Two of the subchronic rat inhalation studies,Ma-Hock et al. (2009a) and DeLorme et al.(2012), did not determine retained lung bur-den, so approximate lung burdens have to beestimated based on aerosol sizes as input to theMPPD deposition model (Figure 4b).

Figure 4a shows that exposure-responsecorrelations for the three MWCNT based onincrease in lung weight are steeper than thosefor CB and CNF. Selection of another sensi-tive endpoint of particle-induced pulmonaryinflammation, that is, increase of neutrophils inlung lavage after 3 mo of exposure, demon-strates the steepest dose-response for the twoMWCNT, less for CB, and least for CNF. Resultsof only two MWCNT studies might be includedin this plot because no lung lavage data areavailable from Ma-Hock et al. (2009a).

However, as stated earlier, due to differ-ences in deposition and retention in the lungfor different inhaled particulate compounds,the actual retained lung burden rather thaninhaled airborne concentration is the preferredmetric. Further, expressing the retained lungburden by different metrics of mass, surfacearea, or volume of the retained particles mayprovide some mechanistic insight. Figure 4bshows these relationships for the endpoint lungweight for all studies. As explained earlier,

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 4. Exposure-Response and Dose-Response.

Exposure-Response and Dose-Response relationships of five 3-month inhalation studies in rats with MWCNT, CNF and CB. The carbonblack (CB) study is added as a well studied low toxicity particle for comparison.

Percent increase in lung lavage neutrophils (PMN) and retained lung dose were not reported for all of the five subchronic rat inhalationstudies. Thus, to approximate retained lung burden of the MWCNT study by Ma-Hock et al. and for the CNF study by DeLorme et al.,deposited and retained doses were calculated with the MPPD model, Version 2.90.3, using MMAD and GSD data and the packingdensity of the materials provided by the authors and a normal rat-specific pulmonary retention halftime (T1/2) of 70 days as model inputs.This T1/2 is appropriate for the rats exposed to the lower concentrations; for the high concentrations and resulting high lung burdens,a prolongation of T1/2 has to be expected (for example, as determined by Pauluhn et al., 2010, Table 3). Therefore, the lung burdenscalculated for the high dose groups of these two studies are likely underestimated. The x-axes have been truncated to better separate theindividual MWCNT/CNF data, although as a consequence the high exposure/date points could not be displayed.A: Lung weight and lung lavage neutrophil exposure-responses. B: Lung weight dose-responses based on retained lung burden expressedas mass, surface area and volume of the retained material.

- MWCNT (Pauluhn, 2010); - MWCNT (Ma-Hock et al., 2009a); - CNF (DeLorme et al., 2012); - CB (Elder et al., 2005);- MWCNT (Kasai et al., 2015).

the retained lung burden data for Ma-Hocket al. (2009a) and DeLorme et al. (2012) wereestimated. As shown in Figure 4, the metricretained particle surface area revealed that thedose-response curves for the three MWCNTand the CNF nanomaterials are considerablysteeper compared to CB. In contrast, whenretained doses are expressed as retained massor retained volume (volume based on bulk, or

packing densities), CNF and Mitsui-7 MWCNTwere not markedly different from CB.

The example of these studies (Figure 4) indi-cates the importance of including measurementof lung burden as endpoint in inhalation stud-ies. While exposure-response relationships pro-vide valuable information about the responseslope, it is only possible through the knowledgeof the actual retained lung burden at different

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


times during and after exposure to correlatedirectly a real dose with a measured responseand to obtain information about retention andclearance kinetics. At a minimum, four ani-mals per time point need to be scheduled fordosimetric analysis.

Multidose subchronic or chronic inhala-tion studies are designed to identify a no-observed-adverse-effect level (NOAEL) or alowest-observed-adverse-effect level (LOAEL).Because of the costs associated with a 2-yr chronic rat inhalation study, subchronicmultidose 90-d studies are preferred (Bernsteinet al. 2005). Shortening the exposure durationto 4 wk or even 5 d (Pauluhn 2009; Ma-Hocket al. 2009b) still needs to be validated as tothe reliability of obtaining sufficient informationregarding chronic toxicity in the primary organof entry as well as secondary remote organs.Mechanisms both of toxicity and of adaptiveresponses may not have fully developed atshorter exposure durations, such that—untilfurther validation data become available—thepresent standard of a 90-d assay is thus pre-ferred. In any case, it is mandatory to adda sufficiently long postexposure observationperiod such that persistence/resolution as wellas retention/clearance kinetics in the primaryand in secondary organs can be determined(Pauluhn 2009). Ideally, the observation periodneeds to approximate at least the normalpulmonary retention halftime (T1/2) of low-toxicity poorly soluble particles (PSP), which inthe rat is about 60–80 d. This—in conjunc-tion with toxicity endpoints—enables one toidentify a potential hazard vis-à-vis other PSP.

Expressing retained lung burden ofCNT/CNF with different metrics of mass orsurface area or volume (Figure 4b) facilitatesa comparison to a PSP such as CB based onthe magnitude of an effect per unit of thedose (Rushton et al. 2010). Using the retainedparticle volume as metric for dose is based onMorrow’s hypothesis (1988) that the clearancefunction of alveolar macrophages becomessignificantly impaired when the volume ofphagocytized particles of a poorly solubleparticle of low cytotoxicity exceeds 6% of thealveolar macrophage volume. With respect

to MWCNT, Pauluhn (2009; 2010) suggestedthat the volume of the CNT/CNF tangles(assemblages) might be used to calculatethe volumetric loading of the macrophages.Thus, not the material density of carbonbut the much lower effective density of theassemblages needs to be considered. On theother hand, it was suggested that the particlesurface area is the appropriate metric becauseit correlates better than particle volume withan effect on macrophage clearance functionand with alveolar inflammatory response instudies involving nanomaterials (Oberdörster,Ferin, and Lehnert 1994a; 1994b; Tran et al.2000a). Applying the volumetric macrophageoverload hypothesis assumes that nanomate-rials in general act like PSP particles, whereasthe particle surface area concept is based onthe understanding that the reactivity (e.g.,inflammatory potential) per unit of the materialsurface area is material dependent and usedas an indicator for toxicity ranking (Rushtonet al. 2010). As demonstrated in Figure 4b,retained nanomaterial surface area in the five3-mo inhalation studies supports the surfacearea concept of particle dose metric.

Of interest in the context of applying dosemetrics to evaluate the toxicity of fiber-shapedmaterials is a study by Timbrell et al. (1988)with asbestos: The exposure metric to charac-terize asbestos exposure is generally expressedas number of WHO-fibers per cubic centime-ter, yet when Timbrell et al. (1988) correlatedthe severity of fibrosis in asbestos miners withthe amount of asbestos fibers retained per unitweight of lung tissue, it turned out that surfacearea, but not number or mass, was the bestdose metric (Figure 5). Surface area-associatedreactivity of nano-sized particles was suggestedas metric (Dick et al. 2003; Hsieh et al. 2012;Borm et al. 2006; Donaldson, Beswick, andGilmour 1996). It needs to be realized, though,that surface area—generally measured by gasadsorption (BET)—needs to be viewed as asurrogate for a biologically active or availablesurface that is yet to be defined. As an exam-ple, the reactive oxygen specias (ROS)-inducingcapacity per unit surface area of a particle maybe considered (Rushton et al. 2010).

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 5. MWCNT aerosol size distribution in rodent inhalation study.

A: Subchronic inhalation of different types of cigarettes and air-exposed controls in nose-only tubes.B: Chronic inhalation of different types of cigarette smoke and diesel engine exhaust and air-exposed controls in nose-only tubes.Note: Effect of restraint stress on bodyweight in sham (clean air) exposed controls, highlighted in red.

Safety of Lab PersonnelRecommendations for engineering controls

to protect lab staff working with nanomateri-als have been provided by NIOSH (2012). Thetypes of engineering controls recommended(local exhaust ventilation, chemical hoods, andglove boxes) depend on the state of thenanomaterial (free or bound in a matrix; dryor in a liquid) and the energetics of the labprocess or activity. Personal protective equip-ment (PPE), including gloves, respirators, andprotective clothing, is discussed in the NIOSH(2012) publication. It is also of interest to con-sider NIOSH (2013), which is more specific toCNT and CNF.

STRATEGY/CONCEPTS OF INHALATIONTESTING FOR CNT/CNF TOXICITY

ObjectiveThe goal is to assess pulmonary and

systemic (secondary target organs) responsesto inhaled CNT/CNF in acute and repeat(subchronic; chronic) rodent inhalation stud-ies mimicking relevant exposure conditionsof humans. The primary target is the res-piratory tract, while secondary targets areextrapulmonary organs that are known orsuspected to be affected by nanomaterialtranslocation from the primary portal-of-entry,including pleura (still part of pulmonary),

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


cardiovascular, liver, spleen bone marrow, kid-neys, central nervous system (CNS), fetus, other(reproductive organs).

In order to identify secondary target sites,biokinetic studies are essential, includingretention, clearance, or accumulation of CNTin primary or secondary organs. For this, anappropriately sensitive label is needed todetermine the disposition of inhaled materialthroughout the body, including eliminationpathways. Caution needs to be exercised,since any label may alter the physicochemicalproperties and likely affect biodistribution.In addition, it is important that any label be sta-ble and not dissociate/leach. For carbon-basedaerosols, turbidity measurement of retainedmaterial in organs/tissue was used and foundto yield reproducible results (Elder et al. 2005).However, sensitivity to low concentrations maybe problematic, and NIOSH Method 5040(NIOSH 2013) may be an appropriate alterna-tive. Mercer et al. (2008) attached 10-nm goldNP or fluorescent quantum dots to SWCNTto determine their biodistribution followingaspiration in mice. However, such labelingis not feasible for large amounts neededfor inhalation exposures. Another method isslow thermal degradation and measurementof CO2 (Hyung et al. 2007; Peterson andRichards 1999; NIOSH Method 5040 withmodifications given in CIB 65 [NIOSH 2013];Watson, Chow, and Chen 2005). A modifiedthermal degradation method with increasedsensitivity was presented specifically foranalyzing CNT/CNF in biological samples.The key is a thorough two-step digestionof tissue to completely eliminate low levelsof interfering tissue carbon, yet avoidingharsh treatment to keep CNT/CNF intact(Westerhoff, Doudrick, and Herckes 2012;Doudrick et al. 2013). A promising sensitivemethod is the use of a hybrid marker (Ohnishiet al. 2013). Suggestions to use specific metallicimpurities embedded in the CNT to quantifytheir retention may be inaccurate due topossible leaching of such metals from CNT invivo and interference from background levelsin tissue. Ideally, 14C-labeled CNT may beused, but pose additional problems related to

radio-safety issues, particularly when used asaerosol.

When simulating human exposure condi-tions for designing appropriate CNT/CNF expo-sures, available human exposure data needto be adapted with regard to differences inrespirability and inhalability between humansand rodents. Three concentrations, logarith-mically spaced, are recommended, includingexpected/predicted human exposure levels.It is essential for performing risk assessment thatat least one dose (maximum tolerated dose,MTD) gives a significant response. If humanexposure data are not available, a worst-casescenario may be assumed by using a rat (rodent)respirable aerosol at several mg/m3 as high-est concentration. A short-term (approximate2–4 wk) pilot study is recommended as a rangefinder in order to estimate a maximal func-tionally tolerated dose (MFTD) and NOAEL forthe longer-term study, and also to verify properoperation of the exposure system and obtainbiokinetic data for identification of secondaryorgans and retained doses in these organs.

Methodology: Inhalation of CNTAerosolization of CNT/CNF from a dry

powder is preferred, rather than from a liq-uid suspension, in order to simulate condi-tions of human exposure to pristine CNT/CNF.Aerosolizing hydrophobic CNT/CNF as anaqueous suspension requires the use of adispersant, which would alter the surfaceof CNT/CNF and thereby potentially affectresponses and biodistribution (Pauluhn andRosenbruch 2015). This is particularly impor-tant if the goal is to test a specific CNT/CNFunder the same conditions to which humansare exposed. A detailed physicochemical char-acterization of bulk and aerosolized material isdesirable. Concerning the bulk CNT/CNF testmaterial, Table 2 provides some guidance.

The CNT/CNF aerosol generated and deliv-ered to the inhalation system requires specialattention. A qualitative estimation of structureand size distribution may be determined byelectron microscopic evaluation of filter sam-ples taken from the inhalation chamber using

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


47-mm polycarbonate filters at a samplingrate of 1 L/min. The filters are then ana-lyzed by TEM or SEM to determine the rangeof CNT/CNF structures and agglomerationin the breathing zone of the exposed ani-mals. Sampling artifacts on the filter haveto be considered. CNT/CNF diameter andlength may be quantified manually under elec-tron microscopy, although it may be difficultdepending on the degree of entanglement andpresence of curved structures. Thus, it is cur-rently not feasible to develop a number limitfor CNT/CNF due to the absence of count-ing rules, similar to those for asbestos. SinceCNT/CNF aerosols are a mixture of agglomer-ated structures containing various numbers ofindividual CNT/CNF, the issue is which struc-tures are bioactive and need to be counted,and which are too agglomerated and are lessbioactive and need not be counted. Are sin-glets more bioactive than large agglomerate?Do small agglomerates dissociate in the lung tosingle tubes? Is there an agglomerate size limitthat is too large to dissociate? Until these ques-tions are answered, development of meaningfulcounting rules for CNT/CNF is not possible.

Particle size distribution may be quan-tified using a micro-orifice uniform depositimpactor (MOUDI) and a nano-MOUDI, thusobtaining a mass median aerodynamic diame-ter (MMAD) and geometric standard deviation(GSD). However, as described earlier, appro-priate correction factors need to be applied toaccount for the shape and orientation effectsof CNT/CNF in the airborne state. The parti-cle count mode diameter might be determinedby visual inspection of filters from the vari-ous stages of the MOUDI and nano-MOUDIunder a SEM, and if particles on all stagesare counted then the count median diameterderived. However, this is a rather tedious pro-cess. Particle bounce from one stage to the nextmight be minimized by greasing alternate stagesof the impactor. The airborne concentrationof CNT/CNF generated within the exposurechamber may be assessed by gravimetric analy-sis of filter samples.

Impaction diameter for CNT/CNF canbe established by correlating measured

dimensions of CNT/CNF from filter samplesto measurements at different stages of theimpactor. Similar measurements in a diffu-sion battery and an elutriator are conductedto obtain equivalent diameters for diffusionand sedimentation. Alternatively, performingCNT/CNF aerosol size distribution with a nano-MOUDI and counting individual structures oneach stage by TEM/SEM might be used to plota count distribution and determine from thisplot a count median aerodynamic diameter(CMAD) and GSD. Scanning mobility particlesizers (SMPS) or differential mobility analyzers(DMA) provide count median diameters (CMD)based on the electrical mobility properties ofthe electrically charged CNT/CNF. However,it is difficult to interpret the results, which arebased on the electrical mobility of equivalentlycharged spherical particles. For purposes ofusing airborne size distribution to predictdeposition in the rodent and human respiratorytract (see MPPD model) the MMAD and GSDobtained with the nano-MOUDI seem to bebest suited for inertial losses, which occur inthe respiratory tract.

ISO Standard 10808 (ISO 2010a)describes requirements and proceduresfor monitoring/characterizing aerosolizedNP during inhalation exposures. Number-based particle size distribution and massconcentration measurements are described.Four prerequisites for proper measurement ofairborne particle number based size distributionshould be fulfilled:

(1) The method used shall be able to monitor parti-cle size distribution in a continuous manner duringparticle exposures with time resolution appropri-ate to checking the stability of particle size dis-tribution and concentration. (2) The measurablerange of particle sizes and concentrations in theanimal’s breathing zone shall cover those of thenanoparticle aerosols exposed to the test systemduring the toxicity test. (3) Particle size and con-centration measurements in the animal’s breathingzone should be accurate for nanoparticle toxicitytesting, and can be validated by means such as cali-bration against appropriate reference standards (seeISO/IEC 17025: ISO 2005b). (4) The resolution ofparticle sizing shall be accurate and the range ofparticle sizes measured shall be sufficiently broad topermit conversion from number-based distribution

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


to surface area-based or volume-based distribution.(OECD 2012)

These requirements in ISO 10808 are fol-lowed by a note that DMAS is the only cur-rently available method that meets all the aboverequirements in the size range below 100 nm(see ISO 15900: ISO 2009). However, ISO10808 also cautions with an important com-ment that “Estimation of particle size fromDMAS measurements can result in significanterror for non-spherical particles. Application ofDMAS for non-spherical particles is not recom-mended.”

With regard to measurement of airbornemass concentration, ISO 10808 stipulates thatthe method selected shall be accurate andsensitive, and defined by the limit of quan-tification. Suggested methods include hte betaattenuation monitor (BAM), tapered oscillatingbalance (TEOM), piezoelectric microbalance,filter gravimetric, and other methods basedon chemical analysis of particles collected onfilter media that meet requirements accord-ing to ISO/TR 27628 (ISO 2007). A note isadded stating that “Mass concentration can bederived from number-based size distributionmeasurement data by making an assumptionregarding particle density, particularly for spher-ical particles which may match bulk materialdensity (Ku and LaMora, 2009). However, sig-nificant errors in calculated mass concentrationmay result if particle density is inaccurate orunknown. Derived mass concentration fromnumber-based size distribution data shouldbe accepted only when no other acceptedmethods meet the measuring requirements.”In addition, an important caveat also statesthat “Mass concentration estimation by DMAs(Differential Mobility Analyzer) based on par-ticle size can produce error for non-sphericalparticles.” In case of highly agglomerated struc-tures the effective density will be different frommaterial density, as mentioned earlier.

Thus, an appropriate approach for charac-terization of CNT/CNF aerosols is the use ofa nano-MOUDI for determining aerodynamicsize distribution and a filter-based method(gravimetric, chemical) for exposure massconcentration. This would allow derivation of a

mass-based exposure limit. Monitoring in realtime the consistency of the aerosol concentra-tion throughout the exposure is necessary andcan be accomplished by a condensation par-ticle counter (CPC), SMPS, or optical particlecounter (e.g., Data-RAM). Although measure-ments would be best done in the animals’breathing zone, the possibility of confoundersfrom animal dander and/or bedding-generateddust needs to be considered, so the point ofmeasurement may be selected at the entranceof the CNT/CNF aerosol into the inhalationchamber.

Types of InhalationInhalation is considered the gold standard

for dosing the respiratory tract. The majoradvantage of inhalation exposure is that itmimics human pulmonary exposure by dis-tributing the inhaled material throughout thewhole respiratory tract in a physiological wayover time. It also provides a more even dis-tribution of particles in the lung than bolusexposures to particles in suspension. Lastly,agglomeration is less of a problem with air-borne CNT/CNF than when CNT/CNF aredelivered as a suspension in biocompatiblefluid; therefore, CNT structures and size distri-bution need to more closely mimic that foundin workplace aerosols. Available measurementsfrom workplaces show large diversity rang-ing from a few single CNT/CNF structures tomostly agglomerates/tangles, depending on thematerial (Maynard et al. 2009; Han et al. 2008;Tsai et al. 2009; Birch et al. 2011). The majordisadvantages of inhalation exposure are that itrequires specialized and costly instrumentationand technical expertise that are not universallyavailable in research institutions. Inhalation alsorequires greater quantities of the test materialthan bolus exposure.

There are two types of inhalation exposuresystems, nose-only and whole-body. Clearly,both modes of inhalation are superior to bolus-type exposures, as discussed earlier (Table 1).OECD Guidelines seem to favor nose-onlyexposure, but leave the choice of the modeof exposure to the investigators, as stated in

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


OECD GD 39 (OECD 2009b): “While nose-only is the preferred mode of exposure, specialobjectives of the study may give preference tothe whole-body mode of exposure.” The guide-lines also state: “When an animal is confined toa restraining tube the observation of its behaviorand physical condition is somewhat restricted.”

Several studies have addressed pros andcons of either mode of exposure. Phalen (2009)summarized specific advantages for whole-body exposures, such as minimal restraint,controllable environment, and better suitabilityfor chronic studies, and for nose-only expo-sures, less unintentional exposures of extra-respiratory-tract organs (skin, eyes) and loweramounts of material to be aerosolized. In termsof disadvantages, Phalen (2009) pointed outthe unavoidable additional routes of expo-sures (skin, eyes, oral), the inefficient use ofmaterial and higher costs for whole-body expo-sures versus discomforts due to heat buildup,restraint stress, and the need for extra han-dling (more labor intensive) associated withnose-only exposures. However, Phalen (2009)also suggested that well-designed nose-only sys-tems permit low stress exposure. For example,acclimatization of rodents prior to nose-onlyexposure reduces stress; however, as deter-mined by Narciso et al. (2003), full adaptationto restraint requires 14 d of fixed-duration dailyrestraint. In contrast, the usual length of theperiod of adaptation to nose-only tube expo-sure is 5 d. Mauderly (1986) reported alteredrespiration and minute volume in rats restrainedin nose-only tubes without adaptation.

In contrast to significant stress induced bynose-only tube exposure, the nonstressed ani-mals exposed in compartmentalized whole-body chambers tend to sleep and may covertheir noses when curling up and thus reduceaerosol inhalation. This should result in lowerretained lung burdens. Yeh et al. (1990) com-pared deposited lung burdens in male andfemale rats (8 groups total at 5 rats/group)either nose-only or whole-body exposed to thesame TiO2 and brass aerosols for 4 h. Theretained lung burdens at 1 h postexposure wereconsistently higher in all 4 whole-body exposedgroups (5 rats each of males and females)compared to the 4 nose-only groups. Although

this was significantly different only for the brass-aerosol-exposed rats, results of this study do notsubstantiate concern that whole-body expo-sures reduce aerosol intake. The differences inlung burden in the Yeh et al. study (1990) maybe due to differences in the breathing patternsobserved between nose–only and whole-bodyexposed rats: Miller et al. (2014), after thor-ough review of the literature, reported a 38%higher breathing rate in rats restrained in noseonly tubes compared to whole-body breathing.This well-demonstrated difference in breathingfrequency – most likely due to the higher stresslevel – between whole-body and nose-onlyexposures and associated effects on minuteventilation and tidal volume will predictablyaffect deposition efficiency and deposition sitesof inhaled particles in the respiratory tract.

In the context of the efficiency of aerosolsdepositing in the respiratory tract, an interestingquestion to consider relates to the relevancy oftypically used daytime exposures in nocturnalanimals. Indeed, a comparison of lung burdensbetween daytime- and nighttime-exposed ratsfor 11.2 h (Hesseltine et al. 1985) revealedan approximately 30% higher retained lungdose in the nighttime-exposed rats, which wasattributed to increased respiration accompa-nying nocturnal activity. Such exposure wouldbe comparable to human daytime exposure.However, exposing rodents during nighttimeby either mode of inhalation creates additionalproblems in terms of limiting food intake(nose-only system) and aerosol contaminationof feed (whole body system), and it also addsmore work.

In a study evaluating 6-h daily nose-only and whole-body inhalation in pregnantmice to air or water aerosols from gesta-tion day 6 through 15, Tyl et al. (1994)found—compared to whole-body air exposureas control—in both nose-only groups increasedmaternal toxicity, reduced body weights, andreduced maternal gestational weight gain.In the air nose-only exposed groups 13.3% ofthe pregnant females died. No significant dif-ferences in fetal malformations were found,and evidence indicated that restraint did notmarkedly affect normal embryo/fetal morpho-logic development but produced indications

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


of maternal toxicity. Thomson et al. (2009)interpreted their own findings of enrichmentof immune response, apoptosis, and signalingterms in nose-only air-exposed mice as beingconsistent with effects due to restraint stress.

An undesirable effect due to restraint stressin rats during subchronic nose-only exposuresis apparent from published body growth curvesshown in studies by Coggins et al. (1993;2011) and Stinn et al. (2005). These investi-gations demonstrated convincingly a retarda-tion of body weight gain, even in air-exposedcontrols, as evidenced by a rise in bodyweight in the postexposure phase immedi-ately following the termination of subchronic orchronic tube exposures (Figure 6). Rothenberget al. (2000) found that even short dailynose-only tube exposures of rats to air for42 d resulted in a 24% lower body weight,although no adverse effects on testes or repro-ductive performance was found. Pauluhn andMohr (1999) confirmed this effect of nose-only exposure on body weight reduction ina 4-wk study in rats. Restraint stress-relatedeffects from nose-only exposure in mice werereported by Van Eijl et al. (2006) to includereduced body weight gain and increasedadrenal weights in a 3-mo exposure comparedto unrestrained littermates. A pronouncedhypothermia was prevented in this study byheating the metallic nosepiece to 38C, which,however, did not reduce stress experiencedby animals. Other studies also demonstratedthat restraint or immobilization leads to a sig-nificant stress response involving activation ofthe hypothalamic–pituitary–adrenal (HPA) andneuroendocrine axes (Fawcett et al. 1994;Pare and Glavin 1986; Udelsman et al. 1993;Sistonen et al. 1992).

Restraint stress is a main concern for repeatnose-only exposures of rats. Stress-reducingtube designs, which permit easy draining ofurine and feces and facilitate dissipation of heatby placing the rat’s tail outside the restrainttube, need to be mandatory (Bernstein andDrew 1980). The rat’s tail is a crucial site forheat exchange; it lacks fur and is well vas-cularized with numerous arterio-venous anas-tomoses facilitating an increase of blood flowduring heat stress to dissipate heat via its high

surface area to volume ratio (Gordon 1993).Surrounding room air might be sufficient toaid cooling of rats. Rothenberg et al. (2000)suggested room air temperature of 64–70F toreduce thermal stress during nose-only expo-sures.

In addition, a multitube arrangement forexposures of many animals in several layersneeds to be designed as a flow past nose-only system such that each port has its ownaerosol delivery line and separate exhaust forthe exhaled air (Baumgartner et al. 1980;Cannon, Blanton, and McDonald 1983). Thisavoids rebreathing the exhalation of otheranimals, which is also a concern for verti-cal flow whole-body chambers consisting ofseveral layers. As suggested by Moss, James,and Asgharian (2006) based on model calcu-lations, the airflow directed to the animal’snose through the delivery line of Cannon-typeflow past nose-only tubes should be 2.5- to4-fold higher than the minute ventilation ofthe animal to minimize rebreathing exhaledair. Pauluhn and Thiel (2007) confirmed thissuggestion by actually measuring that underthese conditions there was no increase of CO2at the inhalation port of a flow-past nose-only system, verifying that no rebreathing doesoccur. Given that the additional stress fromrestraint may be a significant modifying factor ofaerosol-induced effects in a nose-only exposurestudy, the choice of either mode of exposureneeds to be carefully weighed against concernsthat with whole-body inhalation, exposure offur/skin and subsequent ingestion of mate-rial through preening is greater. However, withrespect to oral uptake of CNT, Matsumoto et al.(2012) found that daily gavage of rats to sin-gle and MWCNT for 28 d did not producedeath or toxicological effects, even at high-est daily doses (12.5 mg/kg BW, SWCNT; and50 mg/kg BW, MWCNT). Thus, lower inges-tion of CNT from repeat whole-body exposuresis not a concern. In contrast to the manypublications describing significant stress fromnose-only exposures, OECD (2009b) appearsto minimize the significance of restraint tube-induced stress when depicting it as “nose-onlymode of exposure-specific mild immobilizationstress.”

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 6. MWCNT aerosols collected in rodent inhalation study.

Aerosolized MWCNT collected on filter (A, B) and carbon-coated 200 mesh N: grids (C, D). The MWCNT were aerosolized usingan acoustic aerosol generation system (McKinney, Chen, and Frazer 2009) for dry powder generation. Large variations of the airbornestructures were observed, ranging from a few single tubes to differently-sized structures of agglomerated CNT to larger tangles resulting in anairborne size distribution as shown in Figure 5. The aerodynamic, settling and diffusional behavior cannot be inferred from these pictures;the large tangle in section D may not be inhalable by rodents and is likely not respirable, but can be estimated from their aerodynamicsize distribution. Such experimentally generated MWCNT aerosol for rodent exposures needs to be compared to atmospheres found atworkplaces. (See Figures 1, 2).

Provided that enough material is availablefor repeat long-term exposures, the low-stresswhole-body system is preferable. However,specific circumstances, including low amountsof material to be aerosolized or better res-pirability of dispersed long and straight asbestosfibers lining up in the narrow flow past deliv-ery line (D. Bernstein personal communica-tion 2015), might justify the use of nose-onlysystems.

As an alternative to whole-body or nose-only exposure, an IT inhalation system for ratswas used to deliver hazardous or radioactiveaerosols that prevents any contamination ofthe skin/fur of the animals (Oberdörster, Cox,and Gelein 1997; Kreyling et al. 2009). Lessmaterial is also consumed with this method,and eight or more rats may be exposed simul-taneously for several hours, mimicking differ-ent breathing scenarios. However, animals areanesthetized and repeat exposures may only

be done in weekly intervals, rather than ona daily basis, to minimize stress. Less stressfulwhole-body exposures need to use compart-mentalized systems with individual units foreach animal. The aerosol flow needs to beevenly distributed to all units, and daily/weeklyrotation of the animals is required if locationsare downstream or upstream relative to eachother.

Aerosol GeneratorsDifferent types of generation systems for

CNT/CNF have been used, involving liquid sus-pension or dry powder generators. As statedearlier, dry powder systems are preferredbecause they simulate more closely actualhuman exposures (unless there are situations ofreal-world exposures to CNT/CNF contained inliquid aerosols). Important for delivery of thegenerated aerosols to the animal exposure units

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


is their neutralization via a radioactive source(Phalen 2009).

Generators for droplet (liquid) and dry dust(powder) aerosols were reported (Phalen 2009;Wong 2007). The former include diverse jetand ultrasonic nebulizers, for solutes but also forpoorly or insoluble particles suspended in wateror physiological fluids (saline, PBS); the latterinclude various designs ranging from fluidizedbed to scrape or brush-type and venturi-typefeeder systems as described by Wong (2007)and Phalen (2009). Specific for metallic NP gen-eration, high-voltage electric sparking betweentwo opposing metal electrodes in argon gas,induction of vapor and recondensation ormetal evaporation, and subsequent condensa-tion in a high temperature tube furnace hasbeen used (Elder et al. 2006; ISO/FDIS10801:ISO 2010b). The electric spark method wasalso applied to generate carbon NP for usein inhalation studies in humans and rodents(Frampton et al. 2006; Oberdörster et al. 2004).However, while all of the listed methods canbe used to aerosolize spherical NP—albeitwith some limitations—CNT/CNF aerosols arebest generated by dry powder methods thatare not destructive yet deliver well-dispersedCNT/CNF that closely reproduce known orexpected human exposures. Currently, thereare no generally accepted methods or guide-lines specific for inhalation of nano-sized par-ticles. In the past 5 yr, extensive efforts havebeen applied to develop systems to generate anaerosol of CNT/CNF for animal inhalation stud-ies. Such generation systems include knife mill,acoustical energy, rotating brush, fluidized bed,jet nebulizer, and electrospray aerosolizationsystems.

Dry Powder SystemsBaron et al. (2008) described a system

to generate SWCNT. This system fed acousti-cally fluidized SWCNT through a knife mill tobreak up agglomerates. The output of the knifemill was fed through a settling chamber whereSWCNT were electrically discharged. At thispoint, the cutoff aerodynamic diameter wasapproximately 14 μm. The aerosol was then

passed through a cyclone with a 50% cutoffat 4 μm and a second cyclone with a cut-off at 2 μm. The generator system produced aSWCNT aerosol of 5.52 ± 0.25 mg/m3 overan 8-h period for 4 d. The output efficiencyof the generator was 10–12%. Mass medianaerodynamic diameter was 4 μm with a countmode aerodynamic diameter of approximately240 nm (Shvedova et al. 2008). By adjustingthe feed rate, this generation system might pro-duce outputs as high as 25 mg/m3. Althoughthis system produced target concentrations of adispersed aerosol of SWCNT, it required con-stant monitoring and periodic adjustment bya well-trained technician to maintain a stableoutput and size distribution. Since this systememployed a knife mill, there is concern thatSWCNT may be damaged or shortened duringgeneration. Great caution needs to be exer-cised because micronization, depending on themethod, may significantly increase CNT reactiv-ity, as reported by Ma-Hock et al. (2009a) whenball-milling MWCNT.

McKinney, Chen, and Frazer (2009)described a system that produced an aerosolof MWCNT using an acoustical generation sys-tem. Bulk MWCNT (5 g) were placed in a largecylindrical acrylic chamber (18 inches high and14 inches diameter) enclosed at both endswith flexible latex diaphragms (0.02 inchesthick) held tightly in place with rubber O-rings. The cylinder was lined with conductivefoil tape, which was grounded to preventstatic charge buildup. A 15-inch loudspeakerwas placed below the bottom diaphragm ofthe cylinder. The speaker was driven by acomputer-generated analog signal (a frequencysine wave swept back and forth from 10 to18 Hz over a 20-s period) through an audioamplifier, thus spanning the resonant frequencyof the chamber plus diaphragms. The outputof the generator was controlled by varying theamplitude of the signal driving the speaker.MWCNT, forming a fluidized cloud in thechamber, were collected from the chamber bypassing clean air into a port at the bottom edgeof the cylinder and collecting the aerosolizedMWCNT from a port at the opposite top edgeof the cylinder at an airflow of ≤15 LPM

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


(L/min). This MWCNT aerosol then entered anexposure chamber where concentration andparticle size distribution were monitored. Massoutput was 10.01 ± 0.18 mg/m3. Mass medianaerodynamic diameter was 1.5 μm with ageometric standard deviation of 1.7. Countmode aerodynamic diameter was ∼400 nm.Output of the generator was tightly controlledby three different computerized feedbackloops to control aerosol concentration by alter-ing power of the amplifier, trans-diaphragmpressure by altering airflow into the boxenclosing the cylinder chamber, and exposurechamber pressure by altering airflow out of theexposure chamber. It was stable over manyhours of operation. Larger agglomerates can beremoved by a 2-μm cutoff cyclone (Figure 7).

An improved dispersion method foraerosolizing MWCNT was developed byTaquahashi et al. (2013) with the goal ofgenerating predominantly single dispersedfibers by effectively removing agglomerateswithout changing length and diameter. Twopreparatory steps were involved, starting withsuspending MWCNT in tert-butyl alcohol(TB) followed by filtration through a vibratingmetal sieve of 25 μm, then snap-freezing byliquid nitrogen and vacuum sublimination,which removed all TB. Small cartridges loadedwith the prepared MWCNT were air pressureinjected at 6-min intervals into the inhalationchamber, which was continuously suppliedwith an airflow of 15 L/min. The resultingsawtooth-like concentration was very stableover 2 h at average concentrations of 1.3 and2.8 mg/m3, depending on the cartridge loads.

Another MWCNT aerosol generation sys-tem was developed by Kasai et al. (2014) forwhole-body rat inhalation exposure. This sys-tem, termed a “cyclone sieve method,” involvesdelivery of MWCNT from a hopper-type dustfeeder into a vertical cylindrical container inwhich an upward spiraling airstream of fil-tered air keeps the MWCNT in suspensionthrough gravitational and centrifugal forces.At the upper end of the cylinder a vibratingsieve (53 μm) limits passage only to smalleraerosols to the animal exposure chamber,whereas larger agglomerates settle down intoa collection flask at the bottom. This system

generates a well-dispersed MWCNT aerosolwithout larger agglomerates, one that main-tained a stable concentration over a 6-h expo-sure period. Moreover, particle size distributionwas similar for airborne mass concentrationsranging from 0.2 to 5 mg/m3, with MMADbetween 1.04 and 1.33 μm and GSD between2.7 and 3.4. This system was used successfullyin the recently finished 90-d (Kasai et al. 2015)and an ongoing 2-yr rat inhalation studies withMWCNT (Fukushima personal communication2014).

Myojo et al. (2009) described a genera-tion system for MWCNT employing a rotatingbrush and fluidized bed (RBG—1000 gen-erator, PALAS GmbH, Karlsruhe, Germany).MWCNT were fed (2–20 mm/h) to a rotat-ing brush (1200 rpm) and suspended intoan airstream (13.3 L/min). The aerosolizedMWCNT were fed into a two-componentfluidized bed (bed: irregular stainless-steelbeads) to remove agglomerates and stabilizeconcentration. MWCNT aerosol concentrationincreased linearly with feed rate to the rotat-ing brush (feed rate of 2–20 mm/h, resultingin an output of 5000–20,000 particles/cm3).The generator produced a well-dispersedaerosol of MWCNT, as evidenced by elec-tron micrographs of filter samples. However,MMAD or CMD were not provided. At afeed rate of 2 mm/h, MWCNT aerosol con-centration was 4 mg/m3. Calculated concen-tration of MWCNT surface area ranged fromapproximately 50 to 400 μm2/cm3. Generatorefficiency was 11–13%.

Ma-Hock et al. (2009a; 2009b) used apiston-fed rotating stainless-steel brush togetherwith a cyclone separator to aerosolize MWCNT:A high output of 30 mg/m3 with MMAD0.5–1.3 μm, and GSD 3.1–5.4; CMD 580 nm(Optical Particle Counter) and 60 nm (SMPS).Larger agglomerates were present. A similarpiston-fed rotating stainless steel brush was suc-cessfully used in an earlier study for generatingglass-fiber aerosols (Bernstein et al. 1995).

Fujitani, Furuyama, and Hirano (2009) useda sieve shaker generator to disperse MWCNTmixed with 5-mm stainless-steel balls in analuminum cyclone separator, followed by asecond cyclone. The maximal concentration

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 7. MWCNT aerosols collected in rodent inhalation study.

Aerosolized MWCNT collected on filter (A, B) and carbon-coated 200 mesh N: grids (C, D). The MWCNT were aerosolized using anacoustic aerosol generation system (McKinney et al., 2009) for dry powder generation. Large variations of the airborne structures wereobserved, ranging from a few single tubes to differently-sized structures of agglomerated CNT to larger tangles resulting in an airbornesize distribution as shown in Figure 8. The aerodynamic, settling and diffusional behavior cannot be inferred from these pictures; thelarge tangle in section D may not be inhalable by rodents and is likely not respirable, but the aerodynamic behavior can be estimatedfrom their aerodynamic size distribution. Such experimentally generated MWCNT aerosol for rodent exposures needs to be compared toatmospheres found at workplaces. (See Figures 1, 2).

was 1.2 mg/m3, with a CMD of 300 nmand an aerodynamic diameter of 1–2 μm; theaerosol was well dispersed and also had largeragglomerates. Concerns are the potential ofcontamination from steel balls, aluminum, andshortening of CNT.

Mitchell et al. (2007) used a screw feedersystem to feed MWCNT into a jet mill, andplaced a cyclone separator before inhalationchamber entry. Varying states of agglomerationwere seen, more with higher concentration of5 mg/m3. The MMAD ranged from 0.7 to1.8 μm, and GSD from 2 to 2.5; CMDmeasured by DMA was 350–400 nm; noinformation was provided concerning exposurestability.

For inhalation of Baytubes, a Wrightdust feeder was used. MWCNT were com-pressed into a Wright dust feeder anda slow-moving blade abrasion generatedan aerosol that passed through a cyclonebefore dilution and entering the exposuresystem (Pauluhn 2010). Specifically, Baytubes

(tight agglomerates of MWCNT) were firstmicronized, then packed into a dust feeder.Resulting MMAD values as measured byimpactor were 2.74–3.42, and GSD 2–2.14 forconcentrations of 0.4–5.68 mg/m3 and agglom-erate count by SMPS from 320 to 1763/cm3.

The CNF aerosols in the subchronic inhala-tion study by DeLorme et al. (2012) weregenerated by feeding dry CNF material into aglass tube into which a high-pressure air streamwas directed that carried the resulting CNFaerosol into the exposure chamber. Differentconcentrations were achieved by varying thefeed rate into the airstream and/or varying theon/off period of the feeder. Resulting MMADranged from 1.9 to 3.3 μm at concentrationsfrom 0.5 to 25 mg/m3, indicating agglomeratedCNF structures.

Li et al. (2007a) described an MWCNTaerosol generation system consisting of adry powder generator with two sequentialdepositor tiers to retain larger agglomeratesbefore the aerosol entered the animal exposure

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


chamber. The resulting MWCNT aerosol was“almost respirable,” but the concentrationdecreased continually over a period of 90 minso that the exposures had to be repeated4 times within a 6-h exposure session.

Table 3 compares design, exposures, andresults of the two 90-day inhalation studiesusing different MWCNT and one CNF inhala-tion study. The similarity in dosimetry andresponses is apparent, although there are sig-nificant differences in some of the histologicalfindings. A NOAEL of 0.1 mg/m3 was found byPauluhn (2010), whereas this level was a LOAELin the Ma-Hock et al. (2009a) study. Of interestis that the increase in lung weight as indica-tor of overall respiratory toxicity seems to besimilar between the Pauluhn (2010) and Ma-Hock et al. (2009a) investigations when basedon retained lung burden (Table 3). Exposure-response and dose-response relationships areshown in Figure 4.

Liquid CNT Suspension SystemsFunctionalized CNT/CNF that are well

dispersed in aqueous or physiological salinecan be dispersed using jet nebulizers. Carefulcharacterization with regard to size and sizedistribution and agglomeration state of thegenerated aerosol with appropriate instru-mentation is mandatory, as it is for thedry powder generation systems. CNT/CNFthat are not well dispersed in deionizedwater or saline require use of dispersants forliquid-phase aerosol generation, which posesthe same problem/concern as for bolus-typedosing by instillation or aspiration in termsof altering surface properties, with potentialconsequences for disposition and effects ofdeposited CNT/CNF in the respiratory tract.In addition, it needs to be considered thatdissolution/leaching of contaminants (catalysts)may occur in the suspension, which may alsoaffect surface properties. In general, disper-sion dryers before entry into exposure chamberneed to be used with CNT/CNF aerosol gener-ated from liquid suspensions.

Oyabu et al. (2011) described the gener-ation of a well-dispersed aerosol of MWCNT

using a nebulization system developed forNP by Shimada et al. (2009). Briefly, anaqueous suspension of MWCNT dispersed in0.5 mg/ml Triton X-100 was atomized bya pressurized nebulizer equipped with anejector to break liquid threads into droplets(Nanomaster, JSR Corp., Tokyo, Japan). Thenebulizer received clean air (35 L/min) at aninlet pressure of 0.25 MPa and an ejector pres-sure of 0.20 MPa. Generated droplets weredried in a heater (50C). The dried MWCNTwere then passed into the exposure chamberthrough a deionizer. The exposure concentra-tion was 0.37 ± 0.18 mg/m3. Although nei-ther MMAD nor CMD values were reported,scanning electron micrographs of filter sam-ples from the exposure chamber indicate thatthis system produced a well-dispersed aerosolof MWCNT with geometric mean diameter of63 nm and a geometric mean length of 1.1 μm.Use of surfactant may have changed surfaceproperties.

Kim et al. (2010b) modified an electro-spray nebulizer to generate a well-dispersedaerosol of MWCNT. The CNT were fed intoan electrospray capillary nozzle as a suspensionin ethanol at a low feed rate. A high-voltagefield between the capillary and a groundedorifice plate produced highly charged parti-cles, with the agglomeration state being con-trolled by the feeding rate. Two 210Po radia-tion sources reduced the charges subsequently,and the associated ethanol vapor was reducedefficiently by an activated carbon dryer beforeMWCNT entered the animal exposure system.Airborne size distribution as determined bySMPS showed a double peak, 150 nm (singlets)and 400 nm (bundles), finely dispersed. Theconcentration, using one nozzle, was approx-imately 2 mg/m3. No dissolution/leaching ofmetal catalyst or ethanol contamination was tobe expected. Several nozzles are required forhigher concentrations.

Ryman-Rasmussen et al. (2009a) useda Retsch mixer mill to grind MWCNT.Subsequently, MWCNT were suspendedin nonionic surfactant (1% pluronic) insterile DPBS. Generation of aerosol fornose-only exposure of rats was achieved

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 3. 90-Day Rat Inhalation Studies with MWCNT and CNF, Exposure-Dose-Response Comparison

Ma-Hock et al. (2009a) Kasai et al. (2015)

MaterialMWCNT (NanacylNC7000)

Pauluhn (2010) MWCNT(Baytubes)

(Hodogaya ChemicalCo., LTD; MWNT-7)

DeLorme et al. (2012)CNF (VGCF-H)

CharacterizationLength/diameter, nm 100–10,000 /5–15 nm 200–1000/10 nm

(micronization)2000–5700/40–90nm 1000–14000/40–350

nmAgglomeration Yes Yes (coiled structures) Yes YesDensity, g/cm3 Packing: 0.043 Bulk: 0.11 – 0.33 Bulk; 0.007(production

plant)Bulk: 0.077

Impurities 9.6% Al; <0.2% Co –0.5% Co 0.2 – 0.4%(notspecified)

0.003% Fe

BET surf area, m2/g 250 – 300 255 24–28 m2/g 13.8AnimalsRat strain Wistar; males/females Wistar; males/females F344, males/females Sprague-Dawley;

males/femalesBody weight (age) B.W. not reported (8–9

weeks)–228 g (m); 180 g (f); (–10

weeks)B.W. not reported

(6 wks)B.W. not reported(>5

weeks)ExposureExposure mode Nose-only Nose-only Whole body Nose-onlyGenerator Brush feed generator +

cycloneWright-Dust Feeder Cyclon-sieve generator Accurate Bin Feeder

Duration 90 days: 6 hr/d; 5d/wk 90 days: 6 hr/d; 5d/wk 90 days: 6 hr/d; 5d/wk 90 days: 6 hr/d; 5d/wkConctr, mg/m3 0; 0.1; 0.5; 2.5 0, 0.1; 0.4; 1.5; 6 0; 0.2; 1; 5 0.54; 2.5; 25MMAD μm (GSD) 2.0(2.8); 1.5(2.1);

0.7(3.6) (Berner L.P.Cascade Impactor)

3.05(1.98); 2.74(2.11);3.42(2.14) (MarpleCascade Impactor)

1.5 (2.8); 1.5 (2.8); 1.5(2.6) (Micro orificeUniform Depos.Impact.)

1.9(3.1); 3.2(2.1); 3.3(2.0) (Sierracyclone/cycloneimpactor)

Size distrib.nm(SMPS) 58 ± 1.8 – 64 ± 1.7 75; 320; 908; 1763 Not done Not donePost-expos. recovery No 6 mos. (males only) No 3 mos. (males, females;

0.25 mg/m3)Dosimetry/BiokineticsLung burden (90 days,

males)47; 243; 1170 μg

(deposited)a–5, 25; 100; 420 μg

(retained)b (alsopost-exposure)

9.69;63.60;360.90 μg(retained)c

Not determined

ExtrapulmonaryTranslocation

Mediastinal lymphnodes(inside granulomatousnodules formingacrophages)

Hilar lymphnodes; otherorgans not reported

Mediastinallymphnodes; parietalpleura (diaphragm,capillaries of muscle),spleem

Tracheo-bronchiallymphnodes; rarely inbrain, heart, liver,kidneys, spleen,intestinal tract,mediastinallymphnodes (noadverse histopath.)

Retention halftime(T1/2, days)

Not done 151; 350; 318; 375b Not done Not done

ResponseBody wt. change No significant difference No significant difference No significant

difference15% lower in lowest

conc. group; no sign.difference in othergroups

Lung weight (90 days) + 1%; + 23%; + 81%(males)

+0; + 12; +27; +61%(males)

+5;+17;+30% (males) –2; + 8; +22; (males)

Lymphnode wt. Increased size (highconc)

Increased wt (1.5 and 6mg/m3)

Not reported not reported

BAL–PMN (90 days) Not reported –0.5; 3.8; 13; 19% 0.6;13.5;45.7% 1.4; 2.7; 11.0%BAL-PMN

post-exposure (3mos)[6 mos] highdose only

Not done (22%) [19%] Not done (13%) [not done]

Neutrophilic,histiocytic inflam.

Yes Yes Yes Yes

(Continued)

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 3. (Continued)

Ma-Hock et al. (2009a) Kasai et al. (2015)

MaterialMWCNT (NanacylNC7000)

Pauluhn (2010) MWCNT(Baytubes)

(Hodogaya ChemicalCo., LTD; MWNT-7)

DeLorme et al. (2012)CNF (VGCF-H)

Granulomat. inflam. All concentrations 6 mg/m3 All concentrations Not reportedInterstitial fibrosis Fibrotic changes At 1.5 and 6 mg/m3

groupsAt 1 and 5 mg/m3

groupsInterstitial thickening, no

fibrosisAlv. lipoproteinosis Yes, 0.5; 2.5 No No NoHypercellularity Not reported Yes (0.4 and higher) Goblet cells in nasal

cavity, all conc.Yes (2.5 and higher

conc.)Lymphnode histology Granul. inflam, all conctr. Increased cellularity, all

conctr. (signif. at 0.4and higher)

Not reported Not reported

Visceral pleura Not reported Thickening, collagen at1.5 and 6 mg/m3

Inflamm. Infiltration(5 mg/m3)

Not reported

Blood neutrophilia Yes (highest conc.) Not reported Yes, females only, allconc.

Not reported

Systemic Toxicity Not detected Not detected Yes (increased mRNA ofinflammatorycytokines in spleen,all conc.)

Not detected

EvaluationNOAEL No 100 μg/m3 No 540 μg/m3

LOAEL 100 μg/m3 400 μg/m3 200 μg/m3 2500 μg/m3

a estimated by authors, no clearance considered; b based on retained Co in lung; c extrapolated from amount determined in left lungand using a left to right lung weight ratio of 1:2.

with six jet Collison nebulizers, followedby removal of large droplets by a particletrap, and water removal by silica gel dryers.MMAD was 714 nm, GSD approximately 2,and CMD 160 nm. High concentrations of103 mg/m3 were measured, consisting ofapproximately 2 μm agglomerates and indi-vidual CNT. Concerns are the grinding processand use of dispersant.

Pauluhn and Rosenbruch (2015) also used aCollison-type nebulizer to aerosolize MWCNTin a 6 h nose-only exposure of rats to 30 mg/m3

with sodium carboxymethyl-O-cellulose as dis-persant to compare effects of wet dispersionexposure to those of dry powder genera-tion of the same MWCNT. The smaller wet-generated aerosol (MMAD 0.79 μm) inducedgreater effects than larger pristine dry pow-der aerosols (MMAD 2.6 μm). It is worthwhilenoting that caution against using unrealistichigh-dose acute exposure leading to an incom-plete picture of potential pulmonary toxicities isrequired.

A novel approach was suggested by Ahn,Kim, and Yu (2011): Uncoated MWCNT werecontinuously sonicated in 80C hot deionized

water, which lowered surface tension and facil-itated dispersion and then nebulized via aCollison-type nebulizer with subsequent dry-ing of aerosol in a diffusion dryer. This systemproduced well-dispersed MWCNT without theuse of dispersant. Stable and high concentra-tions of 50 mg/m3 were obtained. Aerosolsize distributions were not determined, andadditional information about its use in actualinhalation studies should be forthcoming.

As pointed out before, for any generationmethod it is important to characterize the gen-erated CNT/CNF aerosol in sufficient detail inorder to ensure adequate dosing of the testanimals, in particular regarding the respirabilityof the CNT/CNF aerosols. Limitations of mea-sured data have to be critically considered. Forexample, the use of DMA to determine equiv-alent mobility diameters of CNT/CNF (Ku et al.2007) is based on separation by a mechanismthat does not occur in the respiratory tract (i.e.,electrical mobility), and DMA measurementsmay be in error if the size of tangled CNT/CNFexceeds submicrometers. The mobility diam-eters of doubly and triply charged CNT/CNFare different from singly charged CNT/CNF, on

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


which the DMA separation is based. Key shouldbe to replicate as best as possible the CNT/CNFaerosol characteristics that humans are exposedto with the proviso that differences in humanversus rodent respirability/dosimetry have to beconsidered. CNT/CNF may be aerosolized ina variety of shapes, which may be groupedto define several CNT/CNF shape categories.A different characterization may be neededfor each shape. Obtaining different equivalentdiameters for each shape requires the use ofdifferent instruments and might prove to bea challenge, given the availability of resourcesand time.

Endpoints and General ApproachBased on numerous studies showing the

potential of nano-sized particles to translocatebeyond the portal of entry, thereby poten-tially producing systemic effects and effects insecondary organs, and given MWCNT-specificresults indicating translocation to and effectsat pleural tissues, there is a need to expandpresently required endpoints (examination oforgans, tissues and effects) relative to existingguidelines (EPA 1998; OECD 2009a) and guid-ance (OECD, 2009b) for subchronic/chronicinhalation studies. These include incorporationof additional effect endpoints for CNT/CNFinhalation studies, as well as data for lungdosimetry and biokinetics.

Biokinetics necessitates the development ofappropriate sensitive methods/labels for quan-tifying biodistribution as briefly discussed pre-viously. To measure lung burdens, sensitivemethods are necessary to reliably measure evenlow levels of retained CNT/CNF (Doudricket al. 2013; Ohnishi et al. 2013). Becauseof the concern that CNT/CNF may behavelike asbestos, there needs to be a specificemphasis on pleural–lymphatic translocationand clearance pathways involving lymphaticstomata of the parietal pleura (Goodglick andKane 1996; Donaldson et al. 2010). Thus, har-vesting thoracic bifurcational lymph nodes assentinels for clearance of lung-deposited CNT,and superior mediastinal lymph nodes anddiaphragm as sentinels for clearance from thepleural space (Parungo et al. 2005), needs to

be part of the study design. Indeed, Bernsteinet al. (2011) reported that just a 5-d inhala-tion exposure of rats to a high concentra-tion of amosite and chrysotile asbestos wassufficient to demonstrate significant inflamma-tory, granulomatous, and fibrotic responses inlung and pleura, including translocation tothe pleura for amosite, whereas this was notobserved with the low-biopersistent chrysotile.In contrast, while pleural translocation ofaspiration-delivered well-dispersed MWCNTwas found (Mercer et al. 2010), and singleMWCNT at pleural sites and diaphragm of ratsfollowing inhalation exposure were observed(Mercer et al. 2013a,b), translocation of inhaledcomplex agglomerates of MWCNT to the pleu-ral space has still to be demonstrated andremains a research gap to be filled. Anotherimportant tissue to include is the bone marrow,which was found as a site for significant uptakeof NP (Bazile et al. 1992; Ballou et al. 2004;Cagle et al. 1999; Gibaud et al. 1994; 1996;1998; Rinderknecht et al. 2007).

To determine retained lung burdens andCNT/CNF translocated to extrapulmonary sec-ondary organs and target sites, sensitive ana-lytical methods are necessary to reliably quan-tify even low tissue levels of elemental carbon(EC) of retained/translocated CNT/CNF. Earlymethods developed for measuring retainedlung burdens in rats of diesel particles following22–32 wk of inhalation to high concentrationsof diesel exhaust used KOH/ethanol digestionof dried lungs, followed by sonication of thewater-resuspended pellet, and optical densityreading (Rudd and Strom 1981). This methodwas sufficient to measure the expected highlung burdens of 2–10 mg. NIOSH (2003) pub-lished a standard method to determine carbonfrom diesel exhaust on filter samples using athermal optical method involving two stageswith stepped-up temperature, first in heliumand then in an He/O2 mixture. This allowedseparate measurement of organic carbon (OC)and EC. Diesel concentrations ranging between6 and 630 μg/m3 ambient air could be mea-sured, but the method was not applied totissue samples. Mercer et al. (2013a) used athree-step process to determine lung burdensof MWCNT after exposure of mice. Dried

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 8.

lungs were digested in KOH/methanol, thenin HNO3/methanol, followed by resuspensionin dH2O with NP-40. Optical density in aspectrophotometer was read after sonication.Lung burdens of 3.125 μg/ml (sample size100 μl) (Dale Porter personal communication,2015) MWCNT were detected. Doudrick et al.(2013) developed a programmed thermal anal-ysis (PTA) method to quantify CNT in lungtissue. After examining eight different tissuedigestion methods, they noted that a two-step extraction/digestion method was optimal,consisting of solvable (mixture of 2.5% NaOH,2.5–10% N,N-dimethyldodecylamine N-oxide,2.5–10% polyethylene glycol trimethylnonylester) and proteinase K. Two types of MWCNwere used, with weak thermal stability (highdefect density) and strong thermal stability (lowdefect density). Similar to the NIOSH Manualof Analytical Methods (NMAM) (2003) method,PTA involved two heating steps: the first in100% helium to remove any remaining OCfrom tissue, and the second in 90% He/10% O2to develop the CNT thermogram and quantifythe carbon content after conversion to methaneand detection by flame ionization detec-tion (FID). Overall, recovery of MWCNT inspiked lung samples was 98% for lung burdensestimated between 1 and 10 μg in rats.

Ohnishi et al. (2013) established a differentapproach using a novel method to measurewith high sensitivity retained lung burden ofMWCNT-7 (Mitsui) following inhalation andIT instillation exposure of rats. Their conceptinvolved adsorption of a PAH marker ontoMWCNT followed by HPLC detection afterdesorption of the marker (Figure 8). Preparatorysteps involved overnight digestion of rat lungs inC99-K200 (lab bleach), which was significantlyimproved by prior formalin fixation of thetissue; centrifugation at 10,500 × g, additionof Tween 80 mixture (9.6% phosphate bufferedsaline with 0.1% Tween 80), and centrifugationfollowed by brief concentrated sulfuric acidfor complete digestion of undissolved tissuecomponents; centrifugation and washing indH2O and addition of the hybrid markerbenzo[ghi] perylene (B[ghi]P) for adsorptionto MWCNT; centrifugation and extractionof marker to acetonitrile; and filtration andquantitative examination of eluate by high-performance liquid chromatography (HPLC).Recovery of spiked lung samples was 92.5%,and the limit of quantitation 0.2 μg in thelung. This is the most sensitive quantitativemethod reported so far and is an importantcontribution to nanotoxicology, providing thescientific community with a tool to establish

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


essential dose-response relationships ofcarbonaceous NM.

As stated earlier, it is desirable to con-sider realistic human exposure conditionswhen designing rodent inhalation studies withCNT/CNF. Relevant exposure levels and mea-suring respective lung burdens need to beincluded to obtain exposure-dose-responserelationships. The analysis of the results needsto take into account dose metrics and responsemetrics when performing dosimetric extrap-olation to humans for hazard identificationand risk assessment purposes. For example,deposited and retained CNT/CNF doses in thelung can be expressed per unit alveolar ortracheobronchial surface or per unit lung tis-sue volume or gram lung tissue to normalizedoses, which will allow extrapolation betweenhumans and rats or mice. The selection of thisdose metric is associated with the mode ofaction; it needs to be closely related to effectsat the target site within the respiratory tract (i.e.,epithelial vs. interstitial). Care needs to be takenin the selection of the region associated withthe toxicological response, as dose per epithe-lial surface area will highly depend on the totalretained amount and total surface area of theselected region.

In addition to dose metric, exposure met-rics to characterize inhalation of particles areimportant. Exposure concentrations expressedas mass per cubic meter or number per cubicmeter or also surface area per cubic meterwere all used. Although for spherical NP ina well-dispersed state (singlets) these relation-ships can readily be determined, this is diffi-cult for the less uniform and rather complexstructures and tangles of CNT/CNF, exceptfor the mass/surface area correlation. Thus,knowing this relation, expressing exposure asmicrograms per cubic meter and determiningdeposited and retained mass allows one toconvert a tissue mass dose to particle surfacearea dose. Additional model development isrequired to estimate tissue dose from retaineddose on the surface of the airways.

In general, the use of tissue dose dependson CNT/CNF clearance rate into the inter-stitial space, including dissolution rateof contaminants/impurities, and possible

interaction of CNT/CNF with interstitial cells.Data with poorly soluble particles indicate thata far greater portion of deposited particles inthe lung migrates to the interstitium in primatesthan in rat (Nikula et al. 1997; 2001), whichis supported by findings in coal mine workersand humans exposed to radioactive aerosols(Kuempel et al. 2001; Gregoratto, Bailey, andMarsh 2011). However, there is some evidencethat—likely due to the hydrophobic natureof CNT—interstitialization of SWCNT andMWCNT may be substantial in rodents (Merceret al. 2008; 2011). Further studies with CNTand CNF are required to describe biokineticsfully.

When expressing the deposited dose perunit surface area (cm2) of airway epithelia, theinflation state of the lung needs to be consid-ered: for example, functional residual capac-ity (FRC) versus total lung capacity (TLC). Forexample, values for the human alveolar surfacevary widely, from approximately 60 m2 (EPA2004) to 140 m2 (Gehr, Bachofen, and Weibel1978). This range of published values reflectsdifferent inflation states of the lung when pre-pared for morphometric analyses, from FRC toTLC. A critical analysis and comparison of lit-erature data by Miller et al. (2011) resultedin more definite and authoritative values fortracheobronchial and alveolar surface areas ofhuman and rat lungs at FRC (Table 4).

Normal breathing starts at FRC such that theepithelial surface area available for depositionof inhaled particles is FRC plus the open-ing of additional surface by the tidal volume.Considering the tidal cycling of inhalation andexhalation during breathing, the approximateaverage epithelial surface area for particledeposition is determined by FRC + ½TV, which

TABLE 4. Surface Areas of Respiratory Tract Regions at FRC

Rat Human

cm2 % of total cm2 % of total

Nasal 18.5 0.75 210 0.03Trach-bronch 24 1.00 4149 0.65Alveolar 2422 98.25 634620 99.32

Keyhani et al., 1997; Kimbell et al., 1997; Miller et al., 2011.(Keyhani, Scherer, and Mozell 1997; Kimbell et al. 1997; Miller

et al. 2011).

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


is close to the surface of FRC, but lowerthan at TLC. Thus, lung inflation state at FRCis the more appropriate for dosimetric con-siderations and comparisons; it ranges fromabout 35 to 45% of TLC for different species.Table 4 shows values for the upper respira-tory tract reported by Keyhani, Scherer, andMozell (1997) and Kimbell et al. (1997). It isimportant for dosimetric comparison of exper-imental results of different rat inhalation stud-ies and for purposes of dosimetric extrapola-tions to humans that researchers base assess-ments on the same well-justified and acceptedvalues. Table 4 shows that for both speciesthe alveolar region has essentially the same(98.3–99.3%) relative surface area, whereasthere is a large difference between species forthe less than 1% overall surface area of the nasalarea.

Diverse methods to evaluate the biodis-tribution of CNT following different routes ofadministration have been used. These includeTEM, radioactive and stable isotope labeling,Raman spectroscopy, near-infrared (NIR) spec-troscopy, x-ray and other fluorescence, andmetal impurity analysis. Liu et al. (2010b),after reviewing these methods and discussingtheir advantages and disadvantages, concludedthat analytical methods for quantitation needto be improved and that optimization ofbiodistribution methods is urgently needed.The in vivo stability of any label has to becarefully confirmed. Other methods includethermo-optical analysis (Hyung et al. 2007;Peterson and Richards 1999; NIOSH Method5040: NIOSH 2013; Watson, Chow, and Chen2005; Westerhoff, Doudrick, and Herckes2012; Doudrick et al. 2013) and turbidity mea-surement of retained carbonaceous materialafter tissue digestion (Rudd and Strom 1981).The most sensitive method appears to be theuse of a hybrid marker, described by Ohnishiet al. (2013).

A minimum number of pulmonaryendpoints—in addition to lung weight—measured at various time points postexposureneeds to include an evaluation of inflamma-tion, lung damage, granulomatous lesions, andinterstitial fibrosis. Inflammation and damage

may be evaluated by analysis of fluid obtainedby BALF. Lung lavage after euthanasia may beperformed in situ (lavaging via tracheal cannulawithout opening thorax), after opening thethorax in situ (lung expands more easily) orafter excising the lungs and trachea en blocand lavaging outside (on wetted gauze withgentle massaging of the lungs). Three to fiverepeated lavages using physiological salineor PBS are usually performed. The excisedlung lavage results in significantly greaternumbers of lavaged cells, probably due toretrieving more cells from the lung periphery.Lung cell damage may be determined bymeasuring LDH activity in the acellular fluidobtained from the first or combined first andsecond lavage aliquot. Alveolar air–bloodbarrier damage/integrity may be measured asthe protein or albumin level in this acellularBALF. Selected cytokine and chemokine (IL–1,tumor necrosis factor [TNF] α, macrophageinflammatory protein [MIP]-1, etc.) levels in thefirst acellular BALF might also be measured asmarkers of inflammation. In addition, TGF-βlevels in the first acellular BALF would be usefulas an indicator of fibrogenic stimulation. TotalBALF cells and cell differentials, in particularneutrophil counts, would be a sensitive indi-cator of pulmonary inflammation. Oxidantstress may be monitored by measuring totalantioxidant levels in lung tissue. These assaysare described in mouse and rat models afterexposure to CNT (Lam et al. 2004; Warheitet al. 2004; Shvedova et al. 2005; Porter et al.2010).

Histopathological analysis, using both lightand electron microscopy, also needs to beconducted. Analyses needs to include aqualitative evaluation of the deposition sitesof CNT/CNF agglomerates and more dis-persed structures, pathological evaluation ofgranulomatous lesions (onset, distribution, andpersistence), and a quantification of interstitialfibrosis (collagen) in Sirius red-stained lung tis-sue determining onset, distribution, and pro-gression. Examples of such histopathologicalquantification for CNT were described byMercer et al. (2008) for SWCNT and Merceret al. (2010; 2011) for MWCNT.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Cell proliferation in response to CNT/CNFexposure is a useful indicator of hyperplasia fora potential pre-neoplastic response and maybe quantified using the BrDU method (Warheitet al. 2004; DeLorme et al. 2012). In addi-tion, histopathological analysis for bronchioleand alveolar epithelial and mesothelial hyper-plasia and hypertrophy is of value (Porter et al.2010).

Acute pulmonary exposure to high dosesof CNT were found to produce adversecardiovascular effects, including (1) oxidantstress in aortic and cardiac tissue, (2) an increasein aortic plaque lesions in ApoE-/-mice (Liet al. 2007b), and (3) inhibition of the abilityof coronary arterioles of rats to respond nor-mally to dilators (Stapleton et al. 2011; 2012).Therefore, systemic response to pulmonaryCNT/CNF exposure may be monitored by his-tological evaluation of aortic plaque formationin ApoE-/- (atherosclerotic sensitive) mice andby monitoring the responsiveness of the rattail artery to vasomodulating factors (Krajnaket al. 2009). In addition, systemic inflammatoryresponse to pulmonary exposure to CNT/CNFmay be monitored by measuring the ability ofperipheral blood PMN leukocytes to producereactive oxygen species (ROS) in response toex vivo stimulation (Nurkiewicz et al. 2006) ordetermining blood mRNA expression or proteinfor selected inflammatory markers (Erdely et al.2009). Blood fibrinogen and measurement ofplatelet activation may be used as markersfor potential thrombogenic effects (Khandogaet al. 2004). While some of these measure-ments require specific methodological expertiseand cannot easily be incorporated into a 90-dinhalation study, other endpoints determiningacute-phase systemic responses from analysisof blood samples (e.g., fibrinogen; IL-6; CRP)may readily be incorporated into the designof acute, subchronic, and chronic studies. Thetiming of blood sample biomarker assays needsto be carefully considered, as some biomarkersmay be expressed transiently.

There may be value in conducting genomicor proteomic analyses on lung tissue samplesto obtain mechanistic information concerningpathways involved in pulmonary responses.

This depends on the study objective; it wouldbe useful though to save specific organs forlater analysis. For example, lungs, bronchus-associated lymphatic tissue, lymph nodes, andother identified target tissues/organs could berapidly removed and frozen at –80C.

EvaluationAlthough dosing of the respiratory tract with

CNT/CNF can easily be achieved by bolus-typedelivery of liquid suspension, or insufflationas powder, directly into the trachea, this can-not mimic physiological inhalation exposuresin terms of disposition within the respiratorytract and with respect to the normally lowinhaled deposition rate in the lung. Therefore,inhalation exposures of lab animals are mostappropriate for toxicity testing. Selecting amethod for the generation of CNT/CNFaerosols that closely resembles exposures thatworkers/consumers may experience requires acareful evaluation of aerosol generator tech-nology, CNT/CNF aerosol characterization, anddosimetric issues. As indicated, a key con-sideration of an experimental exposure-dose-response design is to include doses and distri-bution in the respiratory tract of experimentalanimals that are predicted to be present inexposed humans.

The detailed discussion of inhalability andrespirability in this review should provide guid-ance in this respect. This enables one not onlyto establish exposure-response relationships—which are often not easy to interpret—but todevelop more comprehensive exposure-dose-response data. A critical evaluation of exposure-dose-response information from rodent inhala-tion studies may serve as a basis for risk assess-ment by considering different endpoints forpulmonary and secondary organ effects as wellas systemic effects.

In addition to the analysis of exposure-doseresponses, information about biokinetics pro-vides essential information to the overall assess-ment of potential adverse effects of CNT/CNF.However, as previously discussed, sensitivemethods to quantify low levels of CNT/CNF intissue still need to be developed and refined.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 5. Acute to Chronic Inhalation Exposures of Rodents to CNT/CNF for Toxicity Testing. (rat as preferred species; 4–6 hrs/day,5 d/wk; whole-body)

Acute/Subacute (14–28 days) Subchronic (90 days) Chronic (2 years)

• to obtain hazard ID and ranking(pos/neg control?)

• may be preceded by i.t. instillation or1 day inhalation with range of doses toestimate inhaled concentration withMPPD model

• assure and test rat respirable aerosolwith range of concentr.

• if available use workplace or consumerexposure data to inform aerosolgeneration

• to determine concentration for 90-day;(range-finding)

• to collect biokinetic data for portal ofentry, and possibly identification ofsecondary target organs, incl. pleura,and fetus

• to provide guidance for dose levels formechanistic in vitro testing, incl.secondary organs

• post-exposure observation perioddesirable (∼2 months)

• to derive NOAEL• use minimum 3 conc, including known

or expected human exposure levels;both sexes optional

• if no effect at 50mg/m3 rodentrespirable aerosol, then no need to dochronic study∗

• to identify hazard: total resp.tract;pleura, cardiovascular, CNS, bonemarrow

• identify target organs• to select conc, for chronic study• detailed biokinetics: retention,

clearance, organ accumulation,• predicting long-term effects?• to perform risk assessment by

extrapolation to human via dosimetricextrapolation

• post-exposure observation period forlonger-term effects to assessprogression-regression (∼3 months)

• to determine long latency effects(cancer); life shortening; extrapulmonarytarget organs

• 3 conc; based on 90-day orrange-finding study results; includehuman exposure level; high dose: MTD;low dose: no significant effect

• to assess total respiratory tract, pleuraand systemic effects, nose to alveoli;cardiovascular, CNS, bone marrow,others (reproductive?)

• to determine detailed biokinetics:resp.tract retention, clearance, organaccumulation

• to perform extrapolation to human forrisk assessment

• post-exposure observation period up toa total study duration of 30 months (ifsurvival of ≥20%)

∗This suggestion is based on the fact that such high concentrations will never be reached in a repeat exposure scenario, and it would bea waste of animals (ethical) and resources ($), (Bernstein et al. 2005) to design a chronic workplace study. It should even be considered tomove this suggestion to the acute/subacute category and not follow up with a subchronic study if an appropriate postexposure (∼60 d)observation period is included. Even shorter-term (5 d) inhalation studies with CNT/CNF less than 10 mg/m3 or a single 6-hr. inhalationat 30 mg/m3 have induced significant effects (Ma-Hock et al. 2009; Ryman-Rasmussen et al. 2009a; b; Stapleton et al. 2012; Shvedovaet al. 2008). Thus, a cutoff at 50 mg/m3 in a subchronic study is very conservative.

In the absence of such data, visualization ofCNT/CNF in lung and secondary organs by lightand electron microscopy together with markersof exposure, such as pleural plaque formationor molecular biology-based effects, providesqualitative or descriptive proof for the existenceof biokinetic pathways.

DISCUSSION

Types and Purpose of Inhalation ToxicityTestingAssessing the potential of CNT/CNF expo-

sure to induce adverse responses in the respira-tory tract and in secondary organs requires thedesign and use of inhalation exposures. Whiledosing of the respiratory tract by noninhalationmethods may be useful for purposes of toxi-city ranking, inhalation avoids potential prob-lems associated with bolus-type delivery or withpretreatment of the pristine CNT/CNF. Thus,

while one goal of mimicking human exposureto CNT/CNF aerosols is achieved by admin-istering them to rodents by inhalation, theappropriate methodology with respect to char-acterizing airborne properties of CNT/CNF alsoneeds to be considered in order to reproducethe equivalent for rodent exposures.

The durations of experimental inhalationstudies may range from acute short one-time to28-d exposures to chronic repeat daily expo-sures for up to 2 yr. The purpose and somedesign and methodological details of these dif-ferent exposures are summarized in Table 5.These range from using results of short-termstudies to establish long-term study concentra-tions via data for accumulation, retention, andclearance kinetics; to identification of potentialshort- and long-term hazards in the respira-tory tract, extrapulmonary tissues, and organs;to determination of a NOAEL; to collectionof biokinetic data; and to risk assessment anddosimetric extrapolation.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


General Guidelines for InhalationToxicity TestingGuidelines have been issued by the U.S.

EPA and OECD describing general require-ments for testing chemicals by inhalation,including animal selection, concentration levelselection, repeat exposure durations, exposurechambers and exposure conditions, data evalu-ation, quality control, data reporting, and manyother details (EPA 2000; OECD 2009a; 2009b).Both guidelines focus on 90-d subchronicinhalation studies and both suggest nose-onlyexposures as the preferred method over whole-body, to minimize secondary oral exposure dueto preening of the fur deposits. However, thestress induced by long-term nose-only expo-sure in restraining tubes is significant andaffects the animals’ growth and physiology (dis-cussed earlier, Types of Inhalation). An ear-lier EPA guideline (EPA 2001) that appliesto chronic toxicity/carcinogenicity testing offibrous particles allowed for either nose-only orwhole-body exposure.

Although these guidelines do notspecifically address nanotechnology-relatedtesting requirements, they are useful in termsof providing general recommendations forperforming a subchronic 90-d inhalation study.For example, the EPA (2001) test guidelinesprovided valuable guidance that are applicablefor testing NP, including concentrations, selec-tions, lung burden analysis, and BALF analysis.As another example, the introduction to theOECD (2009a) guideline states:

Subchronic inhalation toxicity studies are primarilyused to derive regulatory concentrations for assess-ing worker risk in occupational settings. They arealso used to assess human residential, transporta-tion, and environmental risk. This guideline enablesthe characterization of adverse effects followingrepeated daily inhalation exposure to a test arti-cle for 90 days (approximately 10% of the lifespanof a rat). The data derived from subchronic inhala-tion toxicity studies can be used for quantitative riskassessments and for the selection of concentrationsfor chronic studies. This Test Guideline is not specif-ically intended for the testing of nanomaterials.

The OECD (2009a) Summary reads:

This revised Test Guideline 413 (TG 413) has beendesigned to fully characterize test article toxicityby the inhalation route for a subchronic duration

(90 days), and to provide robust data for quantita-tive inhalation risk assessments. Groups of 10 maleand 10 female rodents are exposed 6 hours perday during a 90 day (13 week) period to a) thetest article at three or more concentration lev-els, b) filtered air (negative control), and/or c)the vehicle (vehicle control). Animals are gener-ally exposed 5 days per week, but exposure for7 days per week is also allowed. Males and femalesare always tested, but they may be exposed atdifferent concentration levels if it is known thatone sex is more susceptible to a given test arti-cle. This guideline allows the study director theflexibility to include satellite (reversibility) groups,interim sacrifices, bronchoalveolar lavage (BALF),neurologic tests, and additional clinical pathologyand histopathological evaluations in order to bettercharacterize the toxicity of a test article.

Some specific requirements listed in OECD(2009a) include the target concentrationsselected need to identify target organ(s) anddemonstrate a clear concentration response:

• The high concentration level should result intoxic effects but not produce lingering signsor lethality that would prevent a meaningfulevaluation.

• The intermediate concentration level(s)should be spaced to produce a gradation oftoxic effects between that of the low andhigh concentration.

• The low concentration level should producelittle or no evidence of toxicity and

A satellite (reversibility) study may be used toobserve reversibility, persistence, or delayed occur-rence of toxicity for a post-treatment period ofan appropriate length, but no less than 14 days.Satellite (reversibility) groups consist of 10 malesand 10 females exposed contemporaneously withthe experimental animals in the main study. Satellite(reversibility) study groups should be exposed tothe test article at the highest concentration leveland there should be concurrent air and/or vehiclecontrols as needed.

Another guidance document relates toacute inhalation toxicity testing of chemicals ingeneral (OECD 2009b). General recommenda-tions are presented, addressing the principleof the test, monitoring of exposure conditionsincluding inhalation chamber design, and sta-tistical analysis of data. One crude endpoint forthis acute test is mortality (LC50), although non-lethal points are also considered. The preferred

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 6. Percent Deposition of Inhaled Particles of Unit Density (ρ = 1 g/cm3) in Extrathoracic, Tracheobronchial, and Alveolar Regionsof Rodents

Mouse Rat

MMAD (GSD) ET TB Alv Total Lung ET TB Alv Total Lung

4 μm (1) 51.75 1.34 0.79 2.13 70.3 0.14 0.13 0.274 μm (2) 42.94 1.71 1.56 3.27 61.4 0.69 1.28 1.974 μm (3) 35.86 1.70 1.83 3.53 49.79 1.14 2.50 3.643 μm (1) 55.34 2.93 2.26 5.19 75.92 0.55 0.75 1.33 μm (2) 45.74 1.83 1.81 3.64 59.45 1.15 2.38 3.533 μm (3) 37.28 1.60 1.90 3.50 43.58 1.17 3.02 4.192 μm (1) 53.26 3.49 3.37 6.86 70.45 2.06 3.80 5.862 μm (2) 46.25 2.54 2.55 5.09 50.11 1.24 3.10 4.342 μm (3) 39.16 1.85 2.23 4.08 39.14 0.99 3.23 4.221 μm (1) 39.16 2.77 3.43 6.20 21.87 2.00 5.68 7.681 μm (2) 38.41 3.02 3.78 6.80 32.98 1.89 5.53 7.421 μm (3) 36.35 3.48 4.37 7.85 38.14 1.62 5.38 7.00

MPPD Model 2.90.3+ Inhalability AdjustmentRat: Asymmetrical Multiple PathMouse: Balb-CDefault settings (nasal breathing; FRC; tidal volume; breathing frequency; no pause)

exposure mode again is nose-only, although forspecial objectives and longer duration studieswhole-body chamber exposures may be givenpreference. Recommendations for aerosol sizeare given as MMAD between 1 and 4 μm andGSD 1.5–3.

As discussed earlier (Types of Inhalation),nose-only exposures may not be advisablefor exposures to NP aerosols because ofthe associated significant stress resulting inretarded body weight gain and other stress-related effects. Further, CNT ingested dueto fur preening appear to be cleared fromthe gut quickly without significant toxicity(Matsumoto et al. 2012). CNT/CNF inhala-tion studies should not be conducted with anMMAD of 4 μm, since this is close to theupper limit of the thoracic fraction for rodents(Figure 2). Specific guidelines for nanomateri-als should consider workplace exposure con-ditions and include inhalability and respirabil-ity differences between humans and rodents.Some of these items are already addressedin the first nanomaterial-specific guidanceissued by OECD (OECD 2012). Thus, lowerMMAD and particularly CMD up to severalhundred nanometers—depending on actualworkplace data—need to be included in futurenano-specific guidelines, including aerosols ofCNT/CNF.

A specific recommendation for optimalaerosol sizes for rodents would be an MMADup to 2 μm with a geometric standard deviationup to 3. Table 6 lists deposition efficiencies forrats and mice for MMAD from 1 μm to 3 μm,and geometric standard deviations from 1 to 3.The low deposition efficiency of 3-μm particlesbecomes obvious, considering that seeminglysmall differences in deposition fractions result inlarge differences of retained lung burdens dur-ing a subchronic or chronic exposure; retainedlung burdens would be even less for 4-μmMMAD particles, which would not be ideal forexposing rodents. Instrumentation to determinemedian aerodynamic diameters (low pressureand regular impactors) and count median diam-eters (diffusion or electrical mobility particlesize spectrometers) of the aerosols is used. Ratsare the preferred species for inhalation expo-sures to poorly soluble particles because oftheir greater sensitivity to detect fibrotic andcarcinogenic responses relative to mice (ILSI2000).

Nanotechnology-Specific GuidelinesPresently no guidelines specific for

CNT/CNF inhalation testing have beenissued; however, several newer guidelinesthat focus on nanotechnology should briefly

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


be mentioned for interested readers becauseof their close connection to the topic of thisdocument.

ISO Standard 10808 (ISO 2010a) “specifiesrequirements for, and gives guidance on, thecharacterization of airborne nanoparticles ininhalation chambers for the purpose of inhala-tion toxicity studies in terms of particle mass,size distribution, number concentration andcomposition.” It includes definitions of physicalcharacteristics of nanoparticles and emphasizesthe importance of precise characterization ofthe test material for establishing a quantitativerelationship between the observed toxicologicaloutcome and the dose metrics used. The phys-ical and chemical properties include medianand mean size, size distributions, numberand mass concentrations, composition, surfacearea, electrical charge, surface characterization,hygroscopicity, and shape. Obviously, shapebecomes very important for CNT/CNF but isnot further discussed in the guidance docu-ment. Agglomeration and aggregation state alsoneed to be added, as should be purity andcontaminants.

ISO 10808 further describes measuring sys-tems for airborne nanosized particles, describesgeneral and specific monitoring methods, anddiscusses assessment of results. Measurementsof number-based size distributions by DMAis suggested as the only method to fulfill allof the requirements for continuous monitoringand accuracy of size and concentration in theanimals’ breathing zone with high resolutionthat permits conversion from number-baseddistribution to surface-area-based or volume-based distribution. However, an importantcaveat is highlighted in the document for non-spherical particles by cautioning that DMA-derived particle size measurement might resultin significant error and, therefore, that DMAapplication for nonspherical particles is not rec-ommended.

With regard to mass-concentration mea-surements, gravimetric measurements of mate-rial collected on membrane filters, beta atten-uation mass monitor (BAM), tapered elementoscillating microbalance (TEOM), and piezo-electric microbalance are listed. Estimation ofmass by DMA is also mentioned for NP when

particle density is known (Ku and De La Mora2009). However, it is cautioned again thatfor nonspherical particles DMA-based massestimates might produce errors, and signifi-cant errors in calculated mass concentrationmay result if particle density is inaccurate orunknown.

Maynard et al. (2006) and Ku et al. (2006)used tandem mobility–mass analysis consistingof a DMA and aerosol particle mass analyzer(APM) to characterize shape and structure ofSWCNT and of CNF. However, while certainmorphological features correlated with masscan be assessed, this method is not suitable forroutine analyses. In particular, the DMA–APMsystem is useful for well-defined fibers/spheres,but not for larger, irregular complex structuresand tangles.

Specific recommendations regarding char-acterization of experimental CNT/CNF aerosolsare not given in ISO 10808. Gravimetricsampling for mass concentration data is areliable, though discontinuous, method. Themeaning of DMA and SMPS for continuousreal-time measurement of the electrical mobil-ity diameter of CNT/CNF is not clarified, asthere is no agreement how to interpret the data(Ku et al. 2007). As alternative, a micro-orificeuniform deposit impactor (MOUDI) providesan MMAD and the GSD for size distributionsdown to 0.1 μm; if used as a low pressureimpactor, or nano-MOUDI, aerodynamic sizedistributions to approximately 10 nm can bedetermined, although in a discontinuous mode(see Figure 9). The different shapes, structures,and agglomerate sizes are collected based onthe aerodynamic properties of the CNT/CNFaerosols. These data are useful for dosimetricextrapolation modeling.

ISO/FDIS10801 (ISO 2010b) is specific forgeneration of silver NP through an evaporation(heating of solid silver)–condensation methodand reiterates general principles of NP prop-erties, their measurements, monitoring, andexposure system operations.

Two ISO technical reports (TR) relate tohealth and safety assessment at the workplacerelevant to nanotechnologies. ISO/TR 27628(ISO 2007)—workplace atmospheres/ultrafinenanoparticle and nanostructured aerosols/

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE 9. MWCNT aerosol size distribution in rodent inhalation study.

MWCNT aerosol size distribution as determined by a nano-MOUDI through weighing of deposits on the individual stages. Theaerosol was generated with an acoustic aerosol generation system (Fig. 6) and collected with a 13-stage nano-MOUDI (NanoMoudi-IITM

Model 125A, MSP Corp., Shoreview, MN) for 10 minutes at a flow rate of 10 l/min. Lower stages (10–13) were removed because depositscould not be reliably weighed; they contained less than 2% of the total mass. Larger structures with aerodynamic sizes >5 μm contributemuch of the total mass concentration of the aerosol, but not to the deposition in the lower respiratory tract of rodents. Thus, describinga CNT/CNF aerosol exposure only by its total airborne mass concentration can be highly misleading when used as comparison to ahuman exposure concentration. Concepts of dosimetric rodent to human extrapolation discussed in this document have to be taken intoconsideration.

Placing a cyclone separator in line before the aerosol enters the inhalation chamber may eliminate the non-inhalable tangles. On theother hand, if such larger structures are still inhalable by humans and if they are present at the workplace, it could be considered touse intratracheal inhalation (Oberdörster et al., 1997) in rats to circumvent the upper respiratory tract. However, intratracheal inhalationrequires anesthesia of rats and is not recommended for day-to- day repeat exposures.

inhalation exposure characterization andassessment—contains sections on source andformation of occupational nano-aerosols, andstrategies for their exposure characterizationand exposure assessment, including measure-ment of airborne size distribution by electricalmobility analysis (SMPS) and by aerodynamicinertial impaction. Problems associated withthe interpretation of SMPS data when sam-pling nanofibers and nanotubes are pointedout. These are due to the ill-defined electri-cal charging of such structures, as has beendescribed in the OECD documents summarizedearlier.

Supplementing Current GuidelinesSeveral issues of importance for risk

assessment purposes have not been addressedin currently existing testing guidelines. Somehave been alluded to previously, but are

included here again with some additionaldetails.

Particle Size Distributions for Worst-CaseInhalation Testing As pointed out earlier inTable 6, optimal particle sizes for inhalationexposure of rodents to dose the alveolar regionof the rodent lung should not exceed an MMADof 2 μm with a GSD up to 3. It would in factbe ideal if experimental exposure atmosphereswere correlated to the aerosols to whichhumans are exposed under real-world occupa-tional or environmental conditions. However,most often these aerosols are of larger, agglom-erated sizes with MMAD that are signifi-cantly larger, approaching the limits of rodentrespirability. Micronization of the agglomeratedstructures as conducted by Pauluhn (2010) forexposing rats to MWCNT may be one solutionto make the aerosol rodent respirable. As indi-cated by Pauluhn (2010), care has to be taken

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


that the method of micronization does notalter other physicochemical properties, whichin many cases may be difficult to achieve. Still,the objective of a general testing strategy shouldbe to expose the experimental animals to mate-rials that are similar to the pristine materials towhich humans are exposed.

To verify appropriate dosing, sensitive ana-lytical methods for quantifying retained dosesof CNT/CNF in rodent lungs need to be avail-able (Doudrick et al. 2013; Ohnishi et al. 2013)so that exposure-dose-response relationshipscan be established (Figure 4). A minimum ofthree concentrations (plus zero control) shouldbe planned, with the highest at the maximumaerosol concentration (MAC) to deliver themaximum tolerated dose (MTD), and the low-est concentration, if possible, being at theNOAEL (Vu et al. 1996; Morrow et al. 1996;Oberdörster 1997). The MAC should inducesignificant toxicity without substantially alteringthe normal life span of the animals, whereasthe lowest concentration should not inducetoxicity, and some signs of toxicity should beinduced at an appropriately spaced interme-diate concentration. Measuring retained lungburdens following inhalation exposure of anyduration should be mandatory, to be deter-mined in the postexposure recovery phase todefine retention and clearance kinetics.

With regard to defining an MFTD for PSPof low cytotoxicity, Muhle et al. (1990) fol-lowed suggestions by Morrow (1986) and pro-posed to use a maximum functionally toler-ated dose (MFTD) for these types of parti-cles. Muhle et al. (1990) defined it—based onMorrow (1986)—as “the maximum lung bur-den above which macrophage-mediated lungclearance is significantly impaired,” and sug-gested as a reasonable criterion a two- to four-fold increase in the alveolar retention half timeof the tested or suitable surrogate particulatematerial. However, one has to be careful inapplying this concept of MFTD indiscriminatelyto any particulate nanomaterial without confir-mation that the material to be tested indeed isa PSP material. For example, exposure to crys-talline silica and amphibole asbestos—knownhuman carcinogens—also induces significantretardation of alveolar retention halftimes for

particles, easily exceeding an assumed MFTDbased on volumetric dust overloading of thelung. Although the MFTD concept still is mean-ingful for such highly reactive persistent par-ticles, it may well be that other endpointsof toxicity are more sensitive, showing signif-icant adverse effects (toxicity) before alveolarmacrophage mediated clearance function isaffected. Thus, before uncritically focusingsolely on this concept of MFTD for nanomate-rials, other sensitive endpoints also need to beconsidered.

Benchmark and Reference Materials Theavailability of benchmark and reference mate-rials will facilitate the interpretation of results ofrodent inhalation studies. Reference materialsare defined as “sufficiently homogeneous andstable with respect to one or more specifiedproperties . . . [and] established to be fit forits intended use in a measurement process”(ISO 2006). Benchmark materials should bewell characterized physicochemically andtoxicologically for a specific response end-point and serve as a point of reference forcomparing dose-response relationships for usein risk assessment (Oberdörster et al. 2005b;Kuempel et al. 2012). Although, referenceand benchmark materials may serve differentpurposes, ideally, a toxicologically well char-acterized benchmark material should also bemetrologically characterized as reference orcertified reference material.

Examples of traceable certified referencematerials for use in metrology of nanomaterialsthat are available at the National Institute forStandards and Technology (NIST) include gold(10, 30, 60 nm), polystyrene (60, 100 nm),TiO2, and SWCNT (NIST 2012). No MWCNTreference is yet available. Officially sanc-tioned benchmark materials for purposesof toxicological comparative hazard andrisk assessment have not been established.However, Figure 4 shows an example ofcomparing responses observed in 3-mo ratinhalation studies with CNT and CNF, usingas benchmark the data of a previously per-formed 3-mo rat inhalation study with particlesfor a rather benign material (CB, negativebenchmark control). Ideally, a highly toxicmaterial (e.g., crystalline SiO2, asbestos) could

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


be selected as a positive benchmark. However,it was not possible to identify a subchronic ratinhalation study with appropriate endpoints(lung lavage data, lung weight, multidose).If possible, rats would be exposed to positiveand/or negative control at the same time;however, given the extraordinary high demandon labor and costs, simultaneous exposuresare impractical and not recommended, so thesearch for previous appropriately performedinhalation studies is suggested. Preferably,benchmark materials should be of similartype and inhalation exposures be of thesame duration as used for the material tobe tested, but this is impossible for MWCNTgiven the lack of other prior multidosesubchronic MWCNT or CNF inhalationstudies.

Postexposure Observation Adding apostexposure recovery or observation period isessential for any type of inhalation exposure,whether subacute, subchronic, or chronic. Thisnot only enables one to obtain information onclearance of the retained lung burden, but alsoprovides evidence of progression or regressionof effects observed at the end of exposure. Theduration of the postexposure recovery shouldbe at least 1–2 mo for shorter exposures, andapproximately 3 mo for subchronic exposures,and longer for chronic 2-yr exposures. Theclearance kinetics of different retained lungburdens provide valuable information as towhether the alveolar macrophage clearancefunction for particles has been affected dose-dependently. Thus, a postexposure period of60–70 d—which is the physiological reten-tion half time for PSP of low cytotoxicity inrats—needs to be considered as minimumduration for the shorter exposures, and longerrecovery times for subchronic and chronicstudies. Knowledge about progression offibrotic or other health effects or their poten-tial revision over this time period is helpfulfor planning subsequent studies and for riskassessment process. Pauluhn (2010) addeda 6-mo postexposure observation period tothe 3-mo MWCNT rat inhalation study todetermine T½ in the different exposure groups(Table 3).

Monitoring Exposure Atmospheres Useof static size-selective samplers using inertialcascade impactors is discussed in some detailin ISO/TR 27628 (ISO 2007). Potential prob-lems due to overloading (altering impactorcharacteristics) and bouncing (skewing parti-cle size distribution) are identified. Of impor-tance for nano-aerosols including CNT/CNF,the use of low pressure impactors (nano-MOUDI) is acknowledged, yet longer samplingtimes for the expected low mass in the 10-to 100-nm particle sizes are required withthe potential of overloading the upper stages(>0.5–1 μm). An example of a lab-generatedMWCNT aerosol for toxicity testing is shown inFigure 9. Size selective pre-separators may benecessary to avoid upper-stage overloading.

The monitoring and characterizationby available instruments discussed in ISOTR27628 for monitoring nano-aerosol expo-sure are summarized in a table in this documentaccording to mass, number and surface areaas endpoints. This table is updated in a sec-ond workplace related ISO documentation,ISO/TR12885 (ISO 2008), which is directedat the protection of workers. A major aspectin this ISO guidance document is sampling ofparticles at the workplace and their charac-terization. Four tasks are listed to accomplishthis: (1) assessment of personal exposure forcompliance with regulations, (2) determinationof personal exposure for linking with potentialadverse health effects in epidemiological stud-ies, (3) identification of major emission sourcesfor establishing targeted control plan, and(4) assessment of efficiency of control systemsdeployed.

An additional purpose for workplace sam-pling and characterization that is not listed inthe document, but indirectly serves to pro-tect workers’ health and to prevent adverseeffects, is providing information on the designof relevant animal inhalation studies to assesspotential acute and long-term toxicity and thecorrelation with exposure metrics and dosemetrics. In the already-mentioned table of ISOTR 12885, the metrics of mass, number, andsurface area are separately grouped as bothdirectly measured and calculated by specific

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 7. Instruments and techniques for characterizing nano-aerosol exposure (modified from ISO TR12885)

Metric Devices RemarksCNT Monitoring ∗(M)and Quantitation (Q)

Mass directly Membrane filtersampling

Requires size selective inlet cutoff device to collect onlystructures <100 nm. However, most aerosols generatedfrom nanomaterials contain larger agglomerates andaggregates so a size limitation may not be meaningful.Gravimetric and chemical analyses can be used. Majorproblem: separation of background.

Yes(M + Q)(average/integrationover longer time)

Size selectivestatic sampler

The only devices offering a cut point around 100 nm arecascade impactors (Berne-type low pressure impactors, orMicroorifice impactors). Allows gravimetric and chemicalanalysis of samples on stages below 100 nm

Yes (M + Q)(average/integrationover longer time)

TEOM∗ Sensitive real-time monitors such as the Tapered ElementOscillating Microbalance (TEOM) might be useable tomeasure nanoaerosol mass concentration on-line, with asuitable size selective inlet.

Yes (M + Q)

Mass bycalculation

ELPITM Real lime size selective (aerodynamic diameter) detection ofactive surface-area concentration giving aerosol sizedistribution. Mass concentration of aerosols can becalculated, only if particle charge and density are assumedor known. Sue selected samples might be further analyzedoff-line (as above).

Yes (M)

No(Q)DMAS Real-time size-selective (mobility diameter) detection of

number concentration, giving aerosol size distribution. Massconcentration of aerosols can be calculated, only if particleshape and density are known or assumed.

Yes (M)

No (Q)Number directly CPC CPCs provide real-time number concentration measurements

between their particle diameter detection limits. Without ananoparticle preseparator, they are not specific to thenanometre size range. P-Trak has diffusion screen to limittop size to 1 μm.

Yes (M)

No(Q)Number directly DMAS Real-time size-selective (mobility diameter) detection of

number concentration, giving number-based sizedistribution.

Yes (M)

No (Q)Electron

MicroscopyOffline analysis of electron microscope samples can provide

information on size-specific aerosol number concentrationYes (M + Q) and

morphologyMass by

calculationELPITM Real-time size-selective (aerodynamic diameter) detection of

active surface-area concentration, giving aerosol sizedistribution, Data might be interpreted in terms of numberconcentration. Size-selected samples might be furtheranalyzed off-line.

Yes (M)

No (Q)Diffusion Charger Real-time measurement of aerosol active surface area. Active

surface area does not scale directly with geometric surfacearea for particles larger than 100 nm. Note that not allcommercially available diffusion chargers have a responsethat scales with particle active surface area for particlessmaller than 100 nm. Diffusion chargers are only specific tonanoparticles if used with an appropriate inlet preseparator.

No (M+Q)

Surface-areadirectly

ELPITM Real-time size-selective (aerodynamic diameter) detection ofactive surface-area concentration. Active surface area doesnot scale directly with geometric surface area for particleslarger than 100 nm.

No (M + Q)

ElectronMicroscopy

Off-line analysis of electron microscope samples can provideinformation on particle surface area with respect to size.TEM analysis provides direct information on the projectedarea of collected particles, which might be related togeometric area for some particle shapes.

Yes (M + Q)

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


instruments. This table is reproduced herewith an addition for applicability to CNT/CNFaerosols (Table 7).

Chapter 5 of ISO TR 12885, entitled“Exposure assessment to nanomaterials,” is auseful contribution to our discussion of char-acterizing CNT/CNF aerosols. This chapter notonly provides valuable suggestions for expo-sure assessment at workplaces, which is nec-essary complementary information for per-forming risk assessment together with resultsof well-designed animal studies, but theaerosol measurement methods discussed in ISOTR12885 can also be applied to characteriz-ing exposures of animal inhalation studies withCNT/CNF. Table 7 expands the table of thatISO document and is expanded to includeadditional information on the applicability ofmeasurement methods to CNT/CNF aerosols.Future developments of an ideal universal real-time monitoring device that simultaneouslymeasures all three exposure metrics (mass,number, surface area) as well as aerodynamicand mobility behavior may be envisioned(Maynard and Aitken 2007), but its realizationis unlikely, at least within the next decade.

Evaluation of Carcinogenicity andToxicityThe International Agency for Research on

Cancer (IARC) convened a Monograph meet-ing in October 2014, to evaluate—amongother compounds—evidence for labeling CNTwith respect to carcinogenicity (Grosse et al.2014). Based on the data of mesotheliomainduction by MWCNT-7 in several studiesin mice and rats, the Working Group con-cluded that there is sufficient evidence ofcarcinogenicity for MWCNT in animals, lim-ited evidence for two other types of MWCNT,and inadequate evidence for SWCNT. Giventhat there was inadequate evidence in humansand sufficient evidence in two animal species,MWCNT-7 were classified as “possibly car-cinogenic to humans” (Group 2B), and otherMWCNT and SWCNT as “not classifiable asto their carcinogenicity to humans” (Group3). Thus, the IARC Working Group acknowl-edged that differences in biological activities

between distinct MWCNT need to be consid-ered for carcinogen classification, given alsothat mechanistic evidence for carcinogenicityis not strong for any specific CNT. Since theIARC (2014) carcinogenicity classification forMWCNT is presently based on bolus-type ipinjection studies in rodents, the outcome of a 2-yr multidose rat inhalation study with MWCNT-7 with respect to their carcinogenicity will becrucial for a final classification of these MWCNTas either a Group 1 or Group 2A or 2B car-cinogen. The 2-yr inhalation study has beencompleted, and results will be published in thenear future (Fukushima et al. personal commu-nication).

Results of three of the subchronic inhala-tion studies in rats with MWCNT and CNF(Table 3) have been used to derive occupationalexposure levels (OEL) for workplace exposurebased on different methods of extrapolationmodeling of a NOAEL or LOAEL of toxicityobserved in the rat studies. NIOSH (2013)derived a Recommended Exposure Level of2 μg/m3 and 1 μg/m3 for MWCNT and CNFusing a Benchmark Dose (BMD) approach;Aschberger et al. (2010) used the approachbased on guidance by Registration, Evaluation,Authorization and Restriction of Chemicals(REACH) in Europe to derive OEL of 2 μg/m3

and 1 μg/m3 for two different MWCNT. In con-trast to these extremely low values, Pauluhn(2010), using dosimetric extrapolation and thevolumetric overload concept, derived specif-ically for the MWCNT Baytubes an OEL of50 μg/m3. Results of a 2-yr rat inhalation studywith MWCNT are forthcoming (Fukushimaet al. personal communication) and will beessential for deriving more definitely an OEL.

RECOMMENDATION/CONCLUSION

General• Inhalation is the preferred method to obtain

data necessary for risk assessment of pul-monary exposure to CNT/CNF. Intratrachealinstillation or pharyngeal aspiration maybe useful exposure techniques for hazardcharacterization and for screening varioustypes of CNT/CNF for relative toxicity.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


However, effects due to bolus delivery (veryhigh dose rate) need to be considered and—ifdispersants are used to suspend CNT/CNF ina liquid—responses and biokinetics may bealtered.

• The rat is the preferred rodent species. Ratshave been found in studies with fine particlesto be more sensitive regarding fibrosis andlung tumor responses than mice. In addition,the availability of larger tissues is of advantage(e.g., lavage of only the right lung and savethe left lung for histology/histochemistry).Rodent respirability (Table 6) has to be con-sidered to optimize deposition in the lowerrespiratory tract (MMAD ≤ 2 μm). One mayincrease sensitivity to observe lung canceror mesothelioma with KO mouse models,which would provide information concerningmechanisms of action. In general, mesothe-lioma induction by inhaled fibrous particlesin rats is a rare event, and hamsters wouldbe the preferred species for this endpoint(McConnell et al. 1999).

• Whole-body exposure is less stressful andthus preferred over nose-only exposure, inparticular for 90-d and longer inhalationstudies.

• At present, 90-d inhalation studies are pre-ferred (Bernstein et al. 2005; see objectivesin Table 5), with CNT/CNF aerosol charac-terization according to OECD/EPA generalguidelines for performing a subchronic study.In general, OECD test guidelines, guidance,and good laboratory practices for subchronicinhalation studies are applicable; however,modification noted here should be con-sidered. Physicochemical properties of theas-produced and as-administered materialshould be characterized (Table 8).

• Use a minimum of three concentrationsthat include a NOAEL and an MTD.Determination of retained lung burden isessential in order to express results basedon dose-response data rather than only asexposure-response relationship. Risk assess-ment prefers at least one dose that gives apositive response (e.g., MTD). However, useof a dose for this purpose far exceeding theMTD may be of little practical value and leadsto erroneous conclusions.

• Inclusion of a postexposure observation/

recovery period is highly desirable. Thiswould be specifically important for the high-est concentration to determine regression,persistence, or progression of effects, andto determine retention/clearance kinetics inprimary and secondary organs.

• A 28-d inhalation exposure may provide suf-ficient information with added postexposureobservation/recovery time. Shorter repeatexposures (5–10 d) designed to achieve thesame retained lung burdens as in a 90-dstudy by using higher concentrations need toconsider the potential impact of significantdifferences in dose rate.

• Precede the 90-d inhalation study with anacute 1- to 10-d inhalation study with 3 ormore concentrations in order to identifyeffects; estimate exposure concentrations forsubchronic 90-d study; and verify aerosolcharacteristics and evenness of chamber dis-tribution and general tolerance of the ani-mals to exposure. (Inhalation study maybe preceded by dose-response study withintratracheal instillation (rats), oropharyngealaspiration (mice), to identify types of effects.)

Sample Preparation of CNT/CNF forInhalationUse pristine material as experienced by

human inhalation exposures; no treatment withdispersant or sonication to mimic human expo-sure, but consider rodent/human differencesin respirability. Aerosolization from liquid sus-pension needs to consider likely impact ofalteration of pristine CNT/CNF. Micronizationof non-rat-respirable larger agglomerates needsto assure that no alteration of CNT or CNFsurfaces is introduced.

Aerosol Generation• Generators: Dry powder generators are

highly preferred. Sufficient agitation isrequired to achieve CNT/CNF aerosolsthat would be representative of the sizedistribution of CNT/CNF structures foundat human exposure sites. Need to avoidchanging CNT/CNF morphology (cutting,

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


TABLE 8. Recommendation for exposure characterization of sampled and airborne CNT/CNF (modified from Oberdörster et al., 2005)

Human exposure Rodent Exposure

Characterization Type Aerosol Supplied material Aerosol Filter samples

Size distribution (primary particles) E D E DAgglomeration/aggregation state E D E D∗Density D E D NShape E E E ESurface area O E D D∗Concentration, mass and number E − E EComposition E E E ESurface chemistry O E D DSurface contamination D E D ESurface charge – suspension/solution O O D ESurface charge – powder D O D ECrystal structure D E D EParticle physicochemical structure D E D ESolubility N E N EPorosity N E N NMethod of production E E E EPreparation process E E E EHeterogeneity D E E EPrior storage of material E E E E

E: These characterizations arc considered to be essential and to be available for each characterization at least once.D: These characterizations are considered to provide valuable information, but are not recommended as essential due to constraints

associated with complexity, cost and method availability.O: These characterizations are considered to provide valuable but non-essential information.N: These characterizations are not considered to be essential for all studies.∗The CNT/CNF property is assumed to be the same as in the supplied material and therefore no need to repeat measurement. If the

CNT/CNF material has been altered from that supplied (e.g., micronization), then the new properties as used in the rodent assay haveto be determined.

shearing, breaking) and producing CNT/CNFcontamination (addition of metals) from agenerator device. If a liquid suspension isused, consider alteration of surface propertiesof the pristine material due to dispersants.

• It is key to mimic human exposure in termsof aerosol characteristics and concentrations.Therefore, it is essential to obtain availableinformation about such human exposure atworkplaces and to consumers. However, toallow for adequate penetration of CNTs andCNFs into the lower respiratory tract, thegenerated aerosol of singlets, agglomeratedstructures, or aggregated structures should beadministered to rodents with an MMAD ofless than or equal to 2 μm, and GSD notexceeding 3.

• If no human exposure data are available, gen-erate a rodent-respirable CNT/CNF aerosol(mix of individual structures and agglomer-ated versus well-dispersed forms). In order

to optimize the composition of such mix,knowledge about effects of preferentially sin-gle CNT/CNF structures and of preferentiallytangles of CNT/CNF would be helpful (seeResearch Gaps).

• Consider including the use of positive(asbestos) and negative (CB) benchmark con-trols where feasible. Otherwise, consideringthe high cost of nanomaterial inhalationstudies, data on new CNT/CNF shouldbe anchored to preexisting studies withappropriate benchmark materials. (SeeResearch Gaps for identifying referenceCNT/CNF.)

CNT AerosolCharacterization/MonitoringEvaluate size distribution, mass, number

concentration, agglomeration/aggregation state(see later description), density, and other

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


physicochemical characteristics, such as sur-face properties, impurities, length, diameter,dissolution, and leaching (in vivo and in vitro)related to biodurability/biopersistence. Table 8lists recommendations for characterization ofCNT/CNF aerosols occurring for human expo-sures (workplace, consumer) and experimentalanimal exposures. They are identified by theiressentiality for CNT/CNF based on the generalrecommendations on nanomaterial characteri-zation by participants of a 2005 ILSI Workshop(Oberdörster et al. 2005b).

Endpoints• As listed in the OECD and EPA 90-

d inhalation guidelines, one should monitorconventional standard endpoints listed there.Of particular importance are lung weightchanges as integrating indicator of adverseeffects.

In addition, the following should be consid-ered as

a. Mandatory:• Pulmonary endpoints should include lung

lavage (BALF) analysis for lung damage(cellular, biochemical, cytokines) relatedto inflammation and oxidative stress.

• Retained lung burden at end of exposureand in postexposure period to establishexposure-dose-response relationships andclearance kinetics as essential data for pur-poses of risk assessment.

• Histopathology (see existing guidelines)should include specific staining for fibro-sis and analysis of granulomatous lesions,cell proliferation and pleural response.

b. Optional, evaluation of:• Cardiovascular effects (heart rate, blood

pressure, microvascular effects, levels ofclotting factors in the blood).

• Systemic effects (acute-phase proteins toassess systemic inflammation, e.g., fibrino-gen, CRP, IL-6).

• CNS effects (markers of inflammation anddamage; behavioral tests).

• Bone-marrow effects and effects in othersensitive organs.

• Dosimetry/biokinetics: Evaluation ofdeposition, translocation, accumulation,elimination/clearance (e.g., lung; pleura; liver;spleen; heart; CNS; bone marrow) and fordetermining pulmonary retention T½ shouldbe included as an important goal in thestudy design. This requires sensitive analyticalmethods for detecting retained CNT/CNF inthe primary and secondary targets. Analysis ofCNT/CNF retention separately for BALF cells,supernatant, and lung tissue and lymph nodes(bifurcational vs. supramediastinal) is desirable.

InstrumentationThe usefulness and limitations of

instruments for measuring size, mass, andnumber concentration needs to be understood(Table 7):

• Mass, gravimetric (filter; mass only; averagingover time): nano-MOUDI (aerodynamic sizedistribution and mass, see Figure 9, averagingover time); TEOM (real-time monitoring massonly); APS (optical short time, size, num-ber, and mass, but only above approximately300 nm).

• Number: CPC (real-time, no size informa-tion; okay for monitoring stability); SMPS;DMAS (size distribution with unknown rele-vancy; caveats).

• Morphology, shape: TEM; SEM; scanningtransmission electron microscopy (STEM).

• Surface area: BET.

Data AnalysisCould include:

• Interpretation of aerosol characterizationdata.

• Evaluation of exposure-dose-responsedose response, including the NOAELand postexposure time course (retention;persistence/resolution of effects).

• Considering the need for a risk assess-ment if the NOAEL is at a high exposureconcentration of rodent-respirable aerosol(>50 mg/m3) and results demonstrate high

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


lung burden (>2 mg/g) in a 28-d or even90-d study (including 90-d recovery period).

• Dosimetric extrapolation to humans(dose/alveolar epithelial surface area)of deposited dose (only short-term; noretention data needed) and retained dose(preferable, but retention T½ required).

Research Gaps/NeedsA number of publications have pointed

out potential risks of nanomaterials as dis-cussed at the beginning of this document.After reports described mesothelioma-inducingasbestos-like effects of MWCNT in 2008/2009,concerns increased and CNT/CNF-specificreviews (Donaldson et al. 2006; 2010) anddocuments (NIOSH Intelligence Bulletin, 2013)emphasized the need for preventive mea-sures, avoiding exposure, and called for moreresearch to understand the risk of CNT/CNFexposure. In fact, the NIOSH IntelligenceBulletin (2013) derived a recommended expo-sure level for CNT and for CNF of 1 μg/m3.

The ultimate goal is to develop a sci-entifically well supported realistic risk assess-ment of CNT/CNF inhalation exposure. Are allCNT/CNF to be treated the same in terms oftheir potential or potency of causing fibrosis,cancer, and diverse systemic adverse effects?Most likely there may be differences in bioac-tivity between SWCNT and MWCNT and CNF,as well as differences within each group (raw vs.purified CNT/CNF, functionalization, length orwidth, agglomerated vs. dispersed).

There is an urgent need to address thisusing inhalation as the most prominent routeof exposure, and carrying out appropriatelydesigned and executed studies is key. AlthoughIT instillation or pharyngeal aspiration studiesmay be useful in ranking the relative bioactivityof various CNT/CNF, they cannot replaceinhalation studies. A future goal is to movefrom reliance on in vivo studies toward accep-tance of alternative testing strategies includ-ing predictive validated in vitro screeningtests. This should be a high priority, requiringhighly focused research efforts and sufficientfunding.

The following list of research needs wasgenerated to fill data gaps for improving thedesign, analyses, interpretation, and extrapo-lation of results of inhalation studies in orderto refine our ability to perform meaningful riskassessments in the absence of human epidemi-ological data. Addressing these research needswill lead to the development of new conceptsfor assessing the biokinetics and toxicity ofinhaled CNT/CNF and for the understandingof results from acute and chronic inhalationstudies:

• Determine accumulation and clearancekinetics: T½ and translocation to secondarytarget organs (pleura; CNS; bone marrow).

• Compare results of inhalation exposureof individual versus heavily agglomeratedCNT/CNF: Is response different?

• Define inhalability and respirability of highlyvariable CNT/CNF aerosol shapes and incor-porate into existing predictive depositionmodels.

• Determine the suitability of a 28-d inhalationstudy plus postexposure period to replace 90-d subchronic study.

• Perform chronic inhalation study (1 yr orlonger + long postexposure period) todetermine tumorigenic (rat, lung tumor)and mesotheliogenic (hamster mesothelioma)potential of MWCNT that tested positive in ipstudies.

• Measure equivalent diameters in variousinstruments that simulate CNT/CNF deposi-tion in the lung, such as impactor (impactiondiameter), diffusion battery (diffusion diam-eter), elutriator (sedimentation diameter).(These diameters are needed to assess theCNT/CNF dose to the lung.)

• Determine in vivo translocation to pleura ofinhaled CNT/CNF tangles versus inhaled sin-gle CNT/CNF, considering also rigidity andflexibility, as well as length and diameter.

• Develop and optimize sensitive detection/

quantification methods in tissue fordeposited/translocated CNT/CNF.

• Develop a validated method to determineeffective density of CNT/CNF aerosol.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


• Identify reference material for CNT/CNF(positive and negative controls).

• Compare effects of liquid aerosol (+ surfac-tant) versus dry aerosol at the same depositeddose.

• Evaluate inhalation exposure-dose-responsedata with dose as mass, number, and surfacearea: impact of dose metric?

• Compare/correlate acute and long-term effects and biokinetics of instilled(single/repeat dosing) versus inhaledCNT/CNF.

• Correlate in vivo with in vitro response todevelop and validate simple predictive high-throughput assays, as part of a tiered test-ing approach incorporating in vitro (cell-freeand cellular) and in vivo (acute, repeat)studies emphasizing exposure-dose-responseanalysis.

• Develop a universal monitoring instrumentfor combined measurement of aerosol expo-sure characteristics for mass, number, surfacearea, volume distribution, and morphology(Wang et al. 2011).

• Determine exposure data during CNT/CNFlife cycle.

• Determine in vivo (lung, secondary tissues)adsorption of proteins and change in asso-ciated particle surface properties and theimpact on kinetics and effects in vivo.

• Identify underlying mechanisms of toxicityand its dependence on dose.

• Investigate stability of agglomerates deposit-ing in the lung: Does lung surfactant and/ortissue motion disperse CNT/CNF? Dispersionis likely to facilitate interstitial uptake.

• Investigate species differences in biologicalsensitivity to CNT/CNF between rodents andhumans.

ACKNOWLEDGMENTS

Portions of this work were funded by theU.S. Environmental Protection Agency, Officeof Pollution Prevention and Toxics (OPPT)under EPA contract EP-W-09-27 (GO, BA), sup-ported in part by NIEHS Grant P30 ESO1247(GO), and the National Science FoundationEPSCoR Research Infrastructure Improvement

Cooperative Agreement #1003907 (VC). Theauthors wish to thank Mrs. Judy Havalack foroutstanding editorial assistance in the prepara-tion of this article.

DISCLAIMER

Views expressed in this document are thoseof the authors and do not necessarily reflect EPAor NIOSH policy.

REFERENCES

ACGIH. 2010. 2010 TLVs®

and BEIs®, Threshold

limit values for chemical substances and phys-ical agents & biological exposure indices.ACGIH Publications, Cincinnati, OH.

Ahn, K.-H., S.-M. Kim, and I. J. Yu. 2011.Multi-walled carbon nanotube (MWCNT)dispersion and aerosolization with hot wateratomization without addition of any surfac-tant. Safety and Health at Work 2:65–69.doi:10.5491/SHAW.2011.2.1.65.

Aiso, S., K. Yamazaki, Y. Umeda, M. Asakura,T. Kasai, M. Takaya, T. Toya, S. Koda, K.Nagano, H. Arito, and S. Fukushima. 2010.Pulmonary toxicity of intratracheally instilledmultiwall carbon nanotubes in male Fischer344 rats. Industrial Health 48:783–95.doi:10.2486/indhealth.MS1129.

Aitken, R. J., P. E. J. Baldwin, G. C. Beaumont,L. C. Kenny, and A. D. Maynard. 1999.Aerosol inhalability in low air movementenvironments. Journal of Aerosol Science30:613–26. doi:10.1016/S0021-8502(98)00762-9.

Allen, B. L., P. D. Kichambare, P. Gou, I. I.Vlasova, A. A. Kapralov, N. Konduru, V. E.Kagan, and A. Star. 2008. Biodegradation ofsingle-walled carbon nanotubes throughenzymatic catalysis. Nano Letters 8:3899–903. doi:10.1021/nl802315h.

Allen, B. L., G. P. Kotchey, Y. Chen, N. V. K.Yanamala, J. Klein-Seetharaman, V. E. Kagan,and A. Star. 2009. Mechanistic investigationsof horseradish peroxidase-catalyzed degra-dation of single-walled carbon nanotubes.Journal of the American Chemical Society131:17194–205. doi:10.1021/ja9083623.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.5491/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesSHAW.2011.2.1.65

http://dx.doi.org/10.2486/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesindhealth.MS1129

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesS0021-\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes8502\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes(98)00762-9

http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesnl802315h

http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesja9083623


Asbach, C., H. Kaminski, D. Von Barany, T.A. J. Kuhlbusch, C. Monz, N. Dziurowitz,J. Pelzer, K. Vossen, K. Berlin, S. Dietrich,U. Götz, H.-J. Kiesling, R. Schierl, and D.Dahmann. 2012. Comparability of portablenanoparticle exposure monitors. Annals ofOccupational Hygiene 56:606–21.

Aschberger, K., H. J. Johnston, V. Stone,R. J. Aitken, S. M. Hankin, S. A. K.Peters, C. L. Tran, and F. M. Christensen.2010. Review of carbon nanotubes toxicityand exposure—Appraisal of human healthrisk assessment based on open literature.Critical Reviews in Toxicology 40:759–90.doi:10.3109/10408444.2010.506638.

Asgharian, B., W. Hofmann, and R. Bergmann.2001. Particle deposition in a multiple-pathmodel of the human lung. Aerosol Scienceand Technology 34:332–39. doi:10.1080/02786820119122.

Asgharian, B., J. T. Kelly, and E. W. Tewksbury.2003. Respiratory deposition and inhalabilityof monodisperse aerosols in Long-Evansrats. Toxicological Sciences 71:104–11.doi:10.1093/toxsci/71.1.104.

Asgharian, B., and O.T. Price. 2006. AirflowDistribution in the Human Lung and itsInfluence on Particle Deposition, InhalationToxicology 18:795–801.

Asgharian, B., and O. T. Price 2008. A predic-tive model of inhaled nanofibers/nanotubesdeposition in the human lung. Final reportsubmitted to CDC/NIOSH.

Baisch, B. L., N. M. Corson, P. Wade-Mercer,R. Gelein, A. J. Kennell, G. Oberdörster, andA. Elder. 2014. Equivalent titanium diox-ide nanoparticle deposition by intratrachealinstillation and whole body inhalation: Theeffect of dose rate on acute respiratory tractinflammation. Particle Fibre Toxicology 11:5.

Ballou, B., B. C. Lagerholm, L. A. Ernst, M.P. Bruchez, and A. S. Waggoner. 2004.Non-invasive imaging of quantum dots inmice. Bioconjugate Chemistry 15:79–86.doi:10.1021/bc034153y.

Baron, P. A., G. J. Deye, B. T. Chen, D.Schwegler-Berry, A. A. Shvedova, andV. Castranova. 2008. Aerosolizationof single-walled carbon nanotubes

for an inhalation study. InhalationToxicology 20:751–60. doi:10.1080/08958370801975303.

Baumgartner, H., and C. R. E. Coggins. 1980.Description of a continuous smoking inhala-tion machine for exposing small animals totobacco smoke. Beiträge zur TabakforschungInternational 10:169–74.

Bazile, D., C. Ropert, P. Huve, T. Verrecchia,M. Mariard, A. Frydman, M. Veillard, andG. Spenlehauer. 1992. Body distributionof fully biodegradable [14C]-poly(lacticacid) nanoparticles coated with albuminafter parenteral administration to rats.Biomaterials 13:1093–102. doi:10.1016/0142-9612(92)90142-B.

Bernstein, D., V. Castranova, K. Donaldson, B.Fubini, J. Hadley, T. Hesterberg, A. Kane,D. Lai, E. E. McConnell, H. Muhle, G.Oberdorster, S. Olin, and D. B. Warheit.2005. Testing of fibrous particles: Short-termassays and strategies. ILSI report. InhalationToxicology 17:497–537. doi:10.1080/08958370591001121.

Bernstein, D. M., and R. Drew. 1980.Experimental approaches for exposureto sized glass fibers. Environmental HealthPerspectives 34:47–57. doi:10.1289/ehp.803447.

Bernstein, D. M., R. A. Rogers, R. Sepulveda,K. Donaldson, D. Schuler, S. Gaering,P. Kunzendorf, J. Chevalier, and S. E.Holm. 2011. Quantification of the patho-logical response and fate in the lungand pleura of chrysotile in combinationwith fine particles compared to amosite-asbestos following short-term inhalationexposure. Inhalation Toxicology 23:372–91.doi:10.3109/08958378.2011.575413.

Bernstein, D. M., P. Thevenaz, H. Fleissner,R. Anderson, T. W. Hesterberg and R.Mast. 1995. Evaluation of the oncogenicpotential of man-made vitreous fibres: theinhalation model. Ann Occup Hyg 39(5):661–672.

Berry, R. D., and F. Froude 1989. An investi-gation of wind conditions in the workplaceto assess their effect on the quantity ofdust inhaled. HSE Internal Report IR/L/DS/

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.3109/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes10408444.2010.506638

http://dx.doi.org/10.1080/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes02786820119122

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes71.1.104

http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesbc034153y


http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes0142-9612(92)90142-B


http://dx.doi.org/10.1289/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesehp.803447



89/3, Health and Safety Executive, London,UK.

Birch, M. E., B.-K. Ku, D. E. Evans, and T.A. Ruda-Eberenz. 2011. Exposure and emis-sions monitoring during carbon nanofiberproduction—Part I: Elemental carbon andiron-soot aerosols. Annals of OccupationalHygiene 55:1016–36. doi:10.1093/annhyg/mer073.

Bolton, R. E., J. H. Vincent, A. D. Jones, J.Addison, and S. T. Beckett. 1983. An over-load hypothesis for pulmonary clearance ofUICC amosite fibres inhaled by rats. BritishJournal of Industrial Medicine 40:264–72.

Bonner, J. C., R. M. Silva, A. J. Taylor,J. M. Brown, S. C. Hilderbrand, V.Castranova, D. W. Porter, A. Elder, G.Oberdörster, J. Harkema, L. A. Bramble,T. J. Kavanagh, D. Botta, A. Nel, and K. E.Pinkerton. 2013. interlaboratory evaluationof rodent pulmonary responses to engi-neered nanomaterials: The NIEHS NanoGO Consortium. Environmental HealthPerspectives 121:676–82. doi:10.1289/ehp.1205693.

Borm, P. J. A., F. Kelly, N. Kunzli, R.P. F. Schins, and K. Donaldson. 2006.Oxidant generation by particulate mat-ter: From biologically effective dose toa promising, novel metric. Occupationaland Environmental Medicine 64(2): 73–74.doi:10.1136/oem.2006.029090

Brain, J. D., D. E. Knudson, S. P. Sorokin, andM. A. Davis. 1976. Pulmonary distribu-tion of particles given by intratrachealinstillation or by aerosol inhalation.Environmental Research 11:13–33.doi:10.1016/0013-9351(76)90107-9.

Breysse, P. N., and D. L. Swift. 1990.Inhalability of large particles into the humannasal passage: In vivo studies in still air.Aerosol Science and Technology 13:459–64.doi:10.1080/02786829008959460.

Broaddus, V. C., J. I. Everitt, B. Black, and A.B. Kane. 2011. Non-neoplastic and neoplas-tic pleural endpoints following fiber expo-sure. Journal of Toxicology and EnvironmentalHealth, Part B 14:153–78. doi:10.1080/10937404.2011.556049.

Brown, J. S. 2005. Particle inhalability atlow wind speeds. Inhalation Toxicology17:831–37. doi:10.1080/08958370500241296.

Brunauer, S., P. H. Emmett, and E. Teller. 1938.Adsorption of gases in multimolecular layersJ. Amer. Chem. Soc., 60:309 ContributionFrom the Bureau of Chemsitry and Soils andGeorge Washington University.

Cagle, D. W., S. J. Kennel, S. Mirzadeh, J.M. Alford, and L. J. Wilson. 1999. In vivostudies of fullerene-based materials usingendohedral metallofullerene radiotracers.Proceedings of the National Academy ofSciences of the United States of America96:5182–87. doi:10.1073/pnas.96.9.5182.

Cannon, W. C., E. F. Blanton, and K. E.McDonald. 1983. The flow-past chamber:An improved nose-only exposure systemfor rodents. American Industrial HygieneAssociation Journal 44:923–28. doi:10.1080/15298668391405959.

Carrero-Sánchez, J. C., A. L. Elías, R. Mancilla,G. Arrellín, H. Terrones, J. P. Laclette,and M. Terrones. 2006. Biocompatibilityand toxicological studies of carbonnanotubes doped with nitrogen. NanoLetters6:1609–16. doi:10.1021/nl060548p.

CEN ISO/TS 27687. 2008. Nanotechnologies—Terminology and definitions fornano-objects—Nanoparticle, nanofiberand nanoplate. International Organizationfor Standardization, Geneva, Switzerland.

Coggins, C. R. E., P. H. Ayres, A. T. Mosberg,J. W. Sagart, and A. W. Hayes. 1993.Comparative inhalation study in rats usingcigarettes containing tobacco expandedwith chlorofluorocarbon-11 (CFC-11) orhydrochlorofluorocarbon-123 (HCFC-123). Inhalation Toxicology 5:97–115.doi:10.3109/08958379309034496.

Coggins, C. R. E., J. S. Edmiston, A. M.Jerome, T. B. Langston, E. J. Senaa, D.C. Smith, and M. J. Oldham. 2011. Acomprehensive evaluation of the toxicologyof cigarette ingredients: Essential oils andresins. Inhalation Toxicology 23(S1): 41–69.doi:10.3109/08958378.2010.543188

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesannhyg/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesmer073


http://dx.doi.org/10.1136/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesoem.2006.029090

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes0013-9351(76)90107-9



http://dx.doi.org/10.1080/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes08958370\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes500\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes241296

http://dx.doi.org/10.1073/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyespnas.96.9.\gdef yesno\penalty -\@M \gdef \ \penalty -\@M \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty -\@M \gdef \ \penalty -\@M \gdef nono\gdef yesyes5182


http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesnl060548p




Coppeta, L., J. Legramante, A. Galante, A.J. Bergamashi, E. Bergamashi, A. Margrini,and A. Pietroiusti. 2007. Interaction betweencarbon nanotubes and cardiovascular auto-nomic nervous system regulation: Proposalof an animal model and preliminary find-ings. Giornale Italiano di Medicina del LavoroErgonomics 5:465–67.

Dahm, M. M., D. E. Evans, M. K. Schubauer-Berigan, M. E. Birch, and J. A. Deddens.2013. Occupational exposure assessmentin carbon nanotube and nanofiber primaryand secondary manufacturers: Mobile direct-reading sampling. Annals of OccupationalHygiene 57:328–44. doi:10.1093/annhyg/mes079.

Dahm, M. M., D. E. Evans, M. K. Schubauer-Berigan, M. E. Birch, and J. E. Fernback.2012. Occupational exposure assessmentin carbon nanotube and nanofiber primaryand secondary manufacturers. Annals ofOccupational Hygiene 56:542–56.

De Volder, M. F. L., S. H. Tawfick, R.H. Baughman, and A. J. Hart. 2013.Carbon nanotubes: Present and future com-mercial applications. Science 339:535–39.doi:10.1126/science.1222453.

DeLorme, M. P., Y. Muro, T. Arai, D. A.Banas, S. R. Frame, K. L. Reed, and D.B. Warheit. 2012. Ninety-day inhalationtoxicity study with a vapor grown car-bon nanofiber in rats. Toxicological Sciences128:449–60. doi:10.1093/toxsci/kfs172.

Dick, C. A. J., D. M. Brown, K. Donaldson,and V. Stone. 2003. The role of freeradicals in the toxic and inflammatoryeffects of four different ultrafine parti-cle types. Inhalation Toxicology 15:39–52.doi:10.1080/08958370304454.

DIN EN 725–9 2006–05(E). 2006. DeutschesInstitut fur Normung E.V. Advancedtechnical ceramics—Method of test forceramic powders—Part 9: Determinationof untapped bulk density. DAkkS (DeutscheAkkreditierungsstelle). Accessed July 15,2015. http://www.beuth.de/en/standard/din-en-725-9/88827999.

Donaldson, K., R. Aitken, L. Tran, V. Stone, R.Duffin, G. Forrest, and A. Alexander. 2006.

Carbon nanotubes: A review of their prop-erties in relation to pulmonary toxicologyand workplace safety. Toxicological Sciences92:5–22. doi:10.1093/toxsci/kfj130.

Donaldson, K., P. H. Beswick, and P. S.Gilmour. 1996. Free radical activity associ-ated with the surface of particles: A unify-ing factor in determining biological activity?Toxicology Letters 88:293–98. doi:10.1016/0378-4274(96)03752-6.

Donaldson, K., F. A. Murphy, R. Duffin,and C. A. Poland. 2010. Asbestos, car-bon nanotubes and the pleural mesothe-lium: A review and the hypothesis regard-ing the role of long fibre retention in theparietal pleura, inflammation and mesothe-lioma. Particle and Fibre Toxicology 7:5.

Donaldson, K., A. Schinwald, F. Murphy,W.-S. Cho, R. Duffin, L. Tran, and C.Poland. 2013. The biologically effective dosein inhalation nanotoxicology. Accounts ofChemical Research 46:723–32. doi:10.1021/ar300092y.

Dorries, A. M., and P. A. Valberg. 1992.Heterogeneity of phagocytosis for inhaledversus instilled material. American Reviewof Respiratory Disease 146:831–37.doi:10.1164/ajrccm/146.4.831.

Doudrick, K., N. Corson, G. Oberdörster, A.C. Eder, P. Herckes, R. U. Halden, and P.Westerhoff. 2013. Extraction and quantifica-tion of carbon nanotubes in biological matri-ces with application to rat lung tissue. ACSNano 02 September 2013. http://pubs.acs.org 7:8849–56. doi:10.1021/nn403302s.

Driscoll, K., D. Costa, G. Hatch, R. Henderson,G. Oberdörster, H. Salem, and R.Schlesinger. 2000. Intratracheal instillationas an exposure technique for the evaluationof respiratory tract toxicity: Uses and lim-itations. Toxicological Sciences 55:24–35.doi:10.1093/toxsci/55.1.24.

Duffin, R., L. Tran, D. Brown, V. Stone, and K.Donaldson. 2007. Proinflammogenic effectsof low-toxicity and metal nanoparticlesin vivo and in vitro: Highlighting therole of particle surface area and surfacereactivity. Inhalation Toxicology 19:849–56.doi:10.1080/08958370701479323.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesannhyg/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesmes079

http://dx.doi.org/10.1126/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesscience.1222453

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyeskfs172


http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyeskfj130


http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesar300092y

http://dx.doi.org/10.1164/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesajrccm/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes146.4.831

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes55.1.24


http://www.beuth.de/en/standard/din-en-725-9/88827999

http://www.beuth.de/en/standard/din-en-725-9/88827999

http://pubs.acs.org

http://pubs.acs.org


Dutta, D., S. K. Sundaram, J. G. Teeguarden,B. J. Riley, L. S. Fifield, J. M. Jacobs, S. R.Addleman, G. A. Kaysen, B. M. Moudgil,and T. J. Weber. 2007. Adsorbed proteinsinfluence the biological activity and molec-ular targeting of nanomaterials. ToxicologicalSciences 100:303–15. doi:10.1093/toxsci/kfm217.

Elder, A., R. Gelein, J. N. Finkelstein,K. E. Driscoll, J. Harkema, and G.Oberdörster. 2005. Effects of subchron-ically inhaled carbon black in three species.I. Retention kinetics, lung inflammation,and histopathology. Toxicological Sciences88:614–29. doi:10.1093/toxsci/kfi327.

Elder, A., R. Gelein, V. Silva, T. Feikert, L.Opanashuk, J. Carter, R. Potter, A. Maynard,Y. Ito, J. Finkelstein, and G. Oberdörster.2006. Translocation of inhaled ultrafine man-ganese oxide particles to the central nervoussystem. Environmental Health Perspectives114:1172–78. doi:10.1289/ehp.9030.

Elías, A. L., J. C. Carrero-Sánchez, H. Terrones,M. Endo, J. P. Laclette, and M. Terrones.2007. Viability studies of pure carbon- andnitrogen-doped nanotubes with Entamoebahistolytica: From amoebicidal to bio-compatible structures. Small 3:1723–29.doi:10.1002/(ISSN)1613-6829.

EPA. 1998. Health effects test guidelines,OPPTS870.3465 90-day inhalation toxicity.EPA 712-C-98-204, August.

EPA. 2000. OPPTS 870. 3465, US EPA healtheffects test guidelines, 90-day inhalation tox-icity. EPA 712-C-98-204. Washington, DC:Environmental Protection Agency.

EPA. 2001. OPPTS 870. 8355,US EPA healtheffects test guidelines, combined chronictoxicity/carcinogenicity testing of repirablefibrous particles. EPA 712-C01-352, JulyWashington, DC: Environmental ProtectionAgency.

EPA. 2004. Air quality criteria forparticulate matter. EPA/600/P-99/002aF; EPA/600/P-99/002bF, October.Washington, DC: Environmental ProtectionAgency.

Erdely, A., T. Hulderman, R. Salmen, A. Liston,P. C. Zeidler-Erdely, D. Schwegler-Berry,

V. Castranova, S. Koyama, Y.-A. Kim, M.Endo, and P. P. Simeonova. 2009. Cross-talk between lung and systemic circulationduring carbon nanotube respiratory expo-sure. Potential Biomarkers Nano Letters 9:36–43.

Esch, R. K., L. Han, K. K. Foarde, andD. S. Ensor. 2010. Endotoxin contam-ination of engineered nanomaterials.Nanotoxicology 4:73–83. doi:10.3109/17435390903428851.

Evans, D. E., B. K. Ku, M. E. Birch, and K.H. Dunn. 2010. Aerosol monitoring duringcarbon nanofiber production: Mobile direct-reading sampling. Annals of OccupationalHygiene 54:514–31. doi:10.1093/annhyg/meq015.

Fawcett, T. W., S. L. Sylvester, K. D. Sarge,R. I. Morimoto, and N. J. Holbrook. 1994.Effects of neurohormonal stress and agingon the activation of mammalian heat shockfactor 1. Journal of Biological Chemistry269:32272–78.

Fenoglio, I., G. Greco, M. Tomatis, J. Muller,E. Raymundo-Piñero, F. Béguin, A. Fonseca,J. B. Nagy, D. Lison, and B. Fubini.2008. Structural defects play a majorrole in the acute lung toxicity of mul-tiwall carbon nanotubes: Physicochemicalaspects. Chemical Research in Toxicology21:1690–97. doi:10.1021/tx800100s.

Frampton, M. W., J. C. Stewart, G. Oberdörster,P. E. Morrow, D. Chalupa, A. P. Pietropaoli,L. M. Frasier, D. M. Speers, C. Cox, L.-S.Huang, and M. J. Utell. 2006. Inhalationof ultrafine particles alters blood leuko-cyte expression of adhesion molecules inhumans. Environmental Health Perspectives114:51–58. doi:10.1289/ehp.7962.

Fubini, B., M. Ghiazza, and I. Fenoglio.2010. Physico-chemical features of engi-neered nanoparticles relevant to their toxic-ity. Nanotoxicology 4:347–63. doi:10.3109/17435390.2010.509519.

Fujitani, Y., A. Furuyama, and S. Hirano.2009. Generation of airborne multi-walledcarbon nanotubes for inhalation studies.Aerosol Science and Technology 43:881–90.doi:10.1080/02786820903002423.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyeskfm217

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestoxsci/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyeskfi327


http://dx.doi.org/10.1002/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes(ISSN)1613-6829


http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesannhyg/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesmeq015

http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestx800100s





Gasser, M., B. Rothen-Rutishauser, H. F. Krug,P. Gehr, M. Nelle, B. Yan, and P. Wick.2010. The adsorption of biomolecules tomulti-walled carbon nanotubes is influencedby bothpulmonary surfactant lipids and sur-face chemistry. Journal of Nanobiotechnology8:31.

Gasser, M., P. Wick, M. J. D. Clift, F.Blank, L. Diener, Y. Yan, P. Gehr, H. F.Krug, and B. Rothen-Rutishauser. 2012.Pulmonary surfactant coating of multi-walledcarbon nanotubes (MWCNT) influencestheir oxidative andpro-inflammatory poten-tial in vitro. Particle and Fibre Toxicology9:17.

Gehr, P., M. Bachofen, and E. R. Weibel. 1978.The normal human lung: Ultrastructureand morphometric estimation of diffusioncapacity. Respiration Physiology 32:121–40.doi:10.1016/0034-5687(78)90104-4.

Gibaud, S., J. P. Andreux, C. Weingarten,M. Renard, and P. Couvreur. 1994.Increased bone marrow toxicity of dox-orubicin bound to nanoparticles. EuropeanJournal of Cancer 30:820–26. doi:10.1016/0959-8049(94)90299-2.

Gibaud, S., M. Demoy, J. P. Andreux, C.Weingarten, B. Gouritin, and P. Couvreur.1996. Cells involved in the capture ofnanoparticles in hematopoietic organs.Journal of Pharmaceutical Sciences85:944–50. doi:10.1021/js960032d.

Gibaud, S., C. Rousseau, C. Weingarten,R. Favier, L. Douay, J. P. Andreux, andP. Couvreur. 1998. Polyalkylcyanoacrylatenanoparticles as carriers for granulocyte-colony stimulating factor (G-CSF). Journal ofControlled Release 52:131–39. doi:10.1016/S0168-3659(97)00194-6.

Goodglick, A., and A. B. Kane 1996. The roleof fiber length in crocidolite abestos toxicityin vitro and in vivo. VIIth InternationalPneumoconioses Conference Pittsburgh,Pennsylvania.

Gordon, C. J. 1993. Temperature regula-tion in laboratory rodents. New York, NY:Cambridge University Press.

Gregoratto, D., M. R. Bailey, and J. W. Marsh.2011. Particle clearance in the alveolar-interstitial region of the human lungs: Modelvalidation. Radiation Protection Dosimetry144:353–56. doi:10.1093/rpd/ncq314.

Grosse, Y., K. Z. Guyton, B. Lauby-Secretan,F. El Ghissassi, V. Bouvard, L. Benbrahim-Tallaa, N. Guha, C. Scoccianti, H. Mattock,and K. Straif, on behalf of the IARCMonograph Working Group. 2014.Carcinogenicity of fluoro-edenite, sili-con carbide fibres and whiskers, andcarbon nanotubes. Lancet/Oncology 15:1427–28.

Guo, N. L., Y.-W. Wan, J. Denvir, D. W.Porter, M. Pacurari, M. G. Wolfarth, V.Castranova, and Y. Qian. 2012. Multiwalledcarbon nanotube-induced gene signatures inthe mouse lung: Potential predictive valuefor human lung cancer risk and progno-sis. Journal of Toxicology and EnvironmentalHealth, Part A 75:1129–53. doi:10.1080/15287394.2012.699852.

Han, J. H., E. J. Lee, J. H. Lee, K. P.So, Y. H. Lee, G. N. Bae, S. Lee, J.H. Ji, M. H. Cho, and I. J. Yu. 2008.Monitoring multiwalled carbon nanotubeexposure in carbon nanotube researchfacility. Inhalation Toxicology 20:741–49.doi:10.1080/08958370801942238.

Han, S. G., R. Andrews, and C. G. Gairola.2010. Acute pulmonary response of mice tomulti-wall carbon nanotubes. InhalationToxicology 22:340–47. doi:10.3109/08958370903359984.

Han, S. G., D. Howatt, A. Daugherty,and G. Gairola. 2015. Pulmonary andatherogenic effects of multi-walled carbonnanotubes (MWCNT) in apolipoprotein-E-deficient mice. Journal of Toxicology andEnvironmental Health, Part A 78:244–53.doi:10.1080/15287394.2014.958421.

Haniu, H., N. Saito, Y. Matsuda, Y.-A.Kim, K. C. Park, T. Tsukahara, Y. Usui,K. Aoki, M. Shimizu, N. Ogihara, K.Hara, S. Takanashi, M. Okamoto, N.Ishigaki, K. Nakamura, and H. Kato. 2011.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015



http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesjs960032d

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesS0168-3659(97)00194-6

http://dx.doi.org/10.1093/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesrpd/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesncq314






Effect of dispersants of multi-walled car-bon nanotubes on cellular uptake and bio-logical responses. International Journal ofNanomedicine 6:3295–307.

Harris, P. J. F.. 2005. New perspectiveson the structure of graphitic carbons.Critical Reviews in Solid State andMaterials Sciences 30:235–53. doi:10.1080/10408430500406265.

Hemenway, D. R., M. P. Absher, L. Trombley,and P. M. Vacek. 1990. Comparativeclearance of quartz and cristobalite fromthe lung. American Industrial HygieneAssociation Journal 51:363–69. doi:10.1080/15298669091369790.

Hesseltine, G. R., R. K. Wolff, R. L. Hanson,R. O. McClellan, and J. L. Mauderly.1985. Comparison of lung burdens ofinhaled particles of rats exposed duringthe day or night. Journal of Toxicologyand Environmental Health 16:323–29.doi:10.1080/15287398509530744.

Hinds, W. C., N. J. Kennedy, and K. Tatyan.1998. Inhalability of large particles formouth and nose breathing. Journal ofAerosol Science 29(suppl. 1): S277–S278.doi:10.1016/S0021-8502(98)00416-9

Hsieh, S.-F., D. Bello, D. F. Schmidt, A. K. Pal,and E. J. Rogers. 2012. Biological oxidativedamage by carbon nanotubes: Fingerprintor footprint? Nanotoxicology 6:61–76.doi:10.3109/17435390.2011.553689.

Hsu, D.-J., and D. L. Swift. 1999. The mea-surements of human inhalability of ultralargeaerosols in calm air using mannikinS.Journal of Aerosol Science 30:1331–43.doi:10.1016/S0021-8502(99)00022-1.

Hyung, H., J. D. Fortner, J. B. Hughes, andJ.-H. Kim. 2007. Natural organic matter sta-bilizes carbon nanotubes in the aqueousphase. Environment Science & Technology41:179–84. doi:10.1021/es061817g.

Ichihara, G., V. Castranova, A. Tanioka,and K. Miyazawa. 2008. Letter to edi-tor, Induction of mesothelioma in p53+/- mouse by intraperitoneal applica-tion of multi-walled carbon nanotube.Journal of Toxicological Sciences 33:381–82.doi:10.2131/jts.33.381.

International Life Sciences Institute. 2000.The relevance of the rat lung response toparticle overload for human risk assess-ment: A workshop consensus report.Inhalation Toxicology 12:1–17. doi:10.1080/08958370050029725.

International Organization for Standardization.2005a. ISO 15901-1. Pore size distribu-tion and porosity of solid materials bymercury porosimetry and gas adsorption.Part 1: Mercury porosimetry. AccessedJuly 15, 2015. htttp://www.iso.org/iso/home

International Organization for Standardization.2005b. ISO/IEC 17025. General require-ments for the competence of testing andcalibration laboratories.

International Organization for Standardization.2006. ISO guide 35: 2006: Referencematerials—General and statisticalprinciples for certification. Geneva,Switzerland: International Organizationfor Standardization.

International Organization for Standardization.2007. ISO/TR 27628. Workplace atmo-spheresUltrafine, nanoparticle andnano-structured aerosols—Inhalationexposure characterization and assessment.

International Organization for Standardization.2008. ISO/TR12885. Technical Report:Nanotechnologies—Health and safety prac-tices in occupational settings relevant tonanotechnologies.

International Organization for Standardization.2009. ISO 15900. Determination ofparticle size distribution—Differentialelectrical mobility analysis for aerosolparticles.

International Organization for Standardization.2010a. ISO Standard 10808. ISO/TC229—Nanotechnologies—Characterization ofnanoparticles in inhalation exposurechambers for inhalation toxicity testing.

International Organization forStandardization. 2010b. ISO/FDIS10801.Nanotechnologies—Generation of metalnanoparticles for inhalation toxicity test-ing using the evaporation/condensationmethod.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015







http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyeses061817g

http://dx.doi.org/10.2131/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesjts.33.381


htttp://www.iso.org/iso/home

htttp://www.iso.org/iso/home


Jarabek, A. M., B. Asgharian, and F. J. Miller.2005. Dosimetric adjustments for inter-species extrapolation of inhaled poorlysoluble particles (PSP). Inhalation Toxicology17:317–34. doi:10.1080/08958370590929394.

Jiang, J., G. Oberdörster, and P. Biswas. 2009.Characterization of size, surface charge,and agglomeration state of nanoparticledispersions for toxicological studies.Journal of Nanoparticle Research 11:77–89.doi:10.1007/s11051-008-9446-4.

Johnson, D. R., M. M. Methner, A. J.Kennedy, and J. A. Steevens. 2010. Potentialfor occupational exposure to engineeredcarbon-based nanomaterials in environmen-tal laboratory studies. Environmental HealthPerspectives 118:44–54.

Kagan, V. E., Y. Y. Tyurin, V. A. Tyurin,N. V. Konduru, A. I. Potapovich, A.Osipov, E. R. Kisin, D. Schwegler-Berry,R. Mercer, V. Castranova, and A. A.Shvedova. 2006. Direct and indirect effectsof single walled carbon nanotubes onRAW 264.7 macrophages: Role of iron.Toxicology Letters 165:88–100. doi:10.1016/j.toxlet.2006.02.001.

Kan, H., Z. X. Wu, S.-H. Young, T.-H. Chen,J. L. Cumpston, F. Chen, M. L. Kashon,and V. Castranova. 2012. Pulmonary expo-sure of rats to ultrafine titanium diox-ide enhances cardiac protein phospho-rylation and substance P synthesis innodose ganglia. Nanotoxicology 6:736–45.doi:10.3109/17435390.2011.611915.

Kasai, T., K. Gotoh, T. Nishizawa, T. Sasaki,T. Katagiri, Y. Umeda, T. Toya, and S.Fukushima. 2014. Development of a newmulti-walled carbon nanotube (MWCNT)aerosol generation and exposure systemand confirmation of suitability for conduct-ing a single-exposure inhalation study ofMWCNT in rats. Nanotoxicology 8:169–78.doi:10.3109/17435390.2013.766277.

Kasai, T., Y. Umeda, M. Ohnishi, H. Kondo,T. Takeuchi, S. Aiso, T. Nishizawa, M.Matsumoto, and S. Fukushima. 2015.Thirteen-week study of toxicity of fiber-like multi-walled carbon nanotubes with

whole-body inhalation exposure in rats.Nanotoxicology 9:413–22.

Kennedy, N. J., and W. C. Hinds. 2002.Inhalability of large solid particles. Journalof Aerosol Science 33:237–55. doi:10.1016/S0021-8502(01)00168-9.

Keskinen, J., K. Pietarinen, and M. Lehtimäki.1992. Electrical low pressure impactor.Journal of Aerosol Science 23:353–60.doi:10.1016/0021-8502(92)90004-F.

Keyhani, K., P. W. Scherer, and M. M. Mozell.1997. A numerical model of nasal odoranttransport for the analysis of human olfaction.Journal of Theoretical Biology 186:279–301.doi:10.1006/jtbi.1996.0347.

Khandoga, A., A. Stampfl, S. Takenaka,H. Schulz, R. Radykewicz, W. Kreyling,and F. Krombach. 2004. Ultrafine par-ticles exert prothrombotic but notinflammatory effects on the hepaticmicrocirculation in healthy mice in vivo.Circulation 109:1320–25. doi:10.1161/01.CIR.0000118524.62298.E8.

Kido, T., M. Tsunoda, T. Kasai, T. Sasaki, Y.Umeda, H. Senoh, H. Yanagisawa, M.Asakura, Y. Aizawa, and S. Fukushima.2014. The increases in relative mRNAexpressions of inflammatory cytokinesand chemokines in splenic macrophagesfrom rats exposed to multi-walled carbonnanotubes by whole-body inhalation for13 weeks. Inhalation Toxicology 26:750–58.doi:10.3109/08958378.2014.953275.

Kim, J.-E., H.-T. Lim, A. Minai-Tehrani, J.-T. Kwon, J.-Y. Shin, C.-G. Woo, M. Choi,J. Baek, D. H. Jeong, Y.-C. Ha, C.-H.Chae, K.-S. Song, K.-H. Ahn, J.-H. Lee,H.-J. Sung, I.-J. Yu, G. R. Beck, and M.-H. Cho. 2010a. Toxicity and clearance ofintratracheally administered multiwalled car-bon nanotubes from murine lung. Journal ofToxicology and Environmental Health, Part A73:1530–43. doi:10.1080/15287394.2010.511578.

Kim, S. C., D.-R. Chen, C. Qi, R. M. Gelein,J. N. Finkelstein, A. Elder, K. Bentley, G.Oberdörster, and D. H. Y. Pui. 2010b.A nanoparticle dispersion method forin vitro and in vivo nanotoxicity study.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


http://dx.doi.org/10.1007/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyess11051-008-9446-4

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesj.toxlet.2006.02.001




http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes0021-8502(92)90004-F

http://dx.doi.org/10.1006/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesjtbi.1996.0347

http://dx.doi.org/10.1161/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes01.CIR.0000118524.62298.E8


http://dx.doi.org/10.1080/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes15287394.2010.\gdef yesno\penalty -\@M \gdef \ \penalty -\@M \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty -\@M \gdef \ \penalty -\@M \gdef nono\gdef yesyes511578


Nanotoxicology 4:42–51. doi:10.3109/17435390903374019.

Kim, Y. A., T. Hayashi, M. Endo, and M.Dresselhaus. 2013. Carbon nanofibers. InSpringer handbook of nanomaterials, R. Vajtaied., 233–62. Berlin, Germany: Springer-Verlag.

Kimbell, J. S., M. N. Godo, E. A. Gross,D. R. Joyner, R. B. Richardson, and K.T. Morgan. 1997. Computer simulationof inspiratory airflow in all regions ofthe F344 rat nasal passages. Toxicologyand Applied Pharmacology 145:388–98.doi:10.1006/taap.1997.8206.

Kingston, C., R. Zepp, A. Andrady, D. Boverhof,R. Fehir, D. Hawkins, J. Roberts, P. Sayre,B. Shelton, Y. Sultan, V. Vejins, and W.Wohlleben. 2014. Release characteristics ofselected carbon nanotube polymer com-posites. Carbon 68:33–57. doi:10.1016/j.carbon.2013.11.042.

Knuckles, T. L., J. Yi, D. G. Frazer, H. D.Leonard, B. T. Chen, V. Castranova, andT. R. Nurkiewicz. 2012. Nanoparticleinhalation alters systemic arteriolarvasoreactivity through sympathetic andcyclooxygenase-mediated pathways.Nanotoxicology 6:724–35. doi:10.3109/17435390.2011.606926.

Kobayashi, N., M. Naya, M. Ema, S. Endoh,J. Maru, K. Mizuno, and J. Nakanishi.2010. Biological response and morpholog-ical assessment of individually dispersedmulti-wall carbon nanotubes in the lungafter intratracheal instillation in rats.Toxicology 276:143–53. doi:10.1016/j.tox.2010.07.021.

Koblinger, L. 1985. Analysis of human lungmorphometric data for stochastic aerosoldeposition calculations. Physics in Medicineand Biology 30:541–56. doi:10.1088/0031-9155/30/6/004.

Kotchey, G. P., Y. Zhao, V. E. Kagan, andA. Star. 2013. Peroxidase-mediatedbiodegradation of carbon nanotubesin vitro and in vivo. Advanced DrugDelivery Reviews 65:1921–32. doi:10.1016/j.addr.2013.07.007.

Krajnak, K., S. Waugh, C. Johnson, R. Miller,and M. Kiedrowski. 2009. Vibration disruptsvascular function in a model of metabolicsyndrome. Industrial Health 47:533–42.doi:10.2486/indhealth.47.533.

Kreyling, W. G., M. Semmler-Behnke, J. Seitz,W. Scymczak, A. Wenk, P. Mayer, S.Takenaka, and G. Oberdörster. 2009. Sizedependence of the translocation of inhalediridium and carbon nanoparticle aggre-gates from the lung of rats to the bloodand secondary target organs. InhalationToxicology 21 (S1):55–60. doi:10.1080/08958370902942517

Ku, B. K., and J. F. De La Mora. 2009. Relationbetween electrical mobility, mass, and sizefor nanodrops 1–6.5 nm in diameter in air.Aerosol Science and Technology 43:241–49.doi:10.1080/02786820802590510.

Ku, B. K., M. S. Emery, A. D. Maynard, M.R. Stolzenburg, and P. H. McMurry. 2006.In situ structure characterization of airbornecarbon nanofibres by a tandem mobility–mass analysis. Nanotechnology 17:3613–21.doi:10.1088/0957-4484/17/14/042.

Ku, B. K., A. D. Maynard, P. A. Baron,and G. J. Deye. 2007. Observation andmeasurement of anomalous responses ina differential mobility analyzer caused byultrafine fibrous carbon aerosols. Journalof Electrostatics 65:542–48. doi:10.1016/j.elstat.2006.10.012.

Kuempel, E. D., V. Castranova, C. L. Geraci,and P. A. Schulte. 2012. Developmentof risk-based nanomaterial groups foroccupational exposure control. Journal ofNanoparticle Research 14:15. doi:10.1007/s11051-012-1029-8.

Kuempel, E. D., E. J. O’Flaherty, L. T. Stayner,R. J. Smith, F. H. Y. Green, and V. Vallyathan.2001. A biomathematical model of particleclearance and retention in the lungs of coalminers. I. Model development. RegulatoryToxicology and Pharmacology 34:69–87.doi:10.1006/rtph.2001.1479.

Lam, G.-W., J. T. James, R. McCluskey, and R. L.Hunter. 2004. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


http://dx.doi.org/10.1006/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyestaap.1997.8206

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesj.carbon.2013.11.042


http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesj.tox.2010.07.021

http://dx.doi.org/10.1088/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes0031-9155/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes30/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes6/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes004

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesj.addr.2013.07.007

http://dx.doi.org/10.2486/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesindhealth.47.533



http://dx.doi.org/10.1088/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes0957-4484/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes17/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes14/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes042

http://dx.doi.org/10.1016/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesj.elstat.2006.10.012


http://dx.doi.org/10.1006/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesrtph.2001.1479


after intratracheal instillation. ToxicologicalSciences 77:125–34.

LeBlanc, A. J., J. L. Cumpston, B. T. Chen,D. Frazer, V. Castranova, and T. R.Nurkiewicz. 2009. Nanoparticle inhala-tion impairs endothelium-dependentvasodilation in subepicardial arterioles.Journal of Toxicology and EnvironmentalHealth, Part A 72:1576–84. doi:10.1080/15287390903232467.

Lee, K. P., H. J. Trochimowcz, and C. F.Reinhardt 1985. Pulmonary response of ratsexposed to titanium dioxide (TiO2) by inhala-tion for two years. Toxicology and AppliedPharmacology 79: 179–192.

Lee, J. H., S.-B. Lee, G. N. Bae, K. S. Jeon,J. U. Yoon, J. H. Ji, J. H. Sun, B. G. Lee, J.H. Lee, J. S. Yang, H. Y. Kim, C. S. Kang,and I. J. Yu. 2010. Exposure assessmentof carbon nanotube manufacturing work-places. Inhalation Toxicology 22:369–81.doi:10.3109/08958370903367359.

Legramante, J. M., F. Valentini, A. Magrini, G.Palleschi, S. Sacco, I. Iavicoli, M. Pallante,D. Moscone, A. Galante, E. Bergamaschi,A. Bergamaschi, and A. Pietroiusti. 2009.Cardiac autonomic regulation after lungexposure to carbon nanotubes. Human& Experimental Toxicology 28:369–75.doi:10.1177/0960327109105150.

Li, J.-G., W.-X. Li, J.-Y. Xu, X.-Q. Cai, R.-L. Liu,Y.-J. Li, Q.-F. Zhao, and Q.-N. Li. 2007a.Comparative study of pathological lesionsinduced by multiwalled carbon nanotubesin lungs of mice by intratracheal instillationand inhalation. Environmental Toxicology22:415–21. doi:10.1002/(ISSN)1522-7278.

Li, R., X. Wang, Z. Ji, B. Sun, H. Zhang,C. H. Chang, S. Lin, H. Meng, Y.-P. Liao,M. Wang, Z. Li, A. A. Hwang, T.-B. Song,R. Xu, Y. Yang, J. I. Zink, A. Nel, and T.Xia. 2013. Surface charge and cellular pro-cessing of covalently functionalized multiwallcarbon nanotubes determine pulmonary tox-icity. ACS Nano 7:2352–68. doi:10.1021/nn305567s.

Li, Z., T. Hulderman, R. Salmen, R. Chapman,S. S. Leonard, A. Shvedova, M. I. Luster,and P. P. Simeonova. 2007b. Cardiovascular

effects of pulmonary exposure to single-wallcarbon nanotubes. Environmental HealthPerspectives 115:377–88. doi:10.1289/ehp.9688.

Liang, G., L. Yin, J. Zhang, R. Liu, T.Zhang, B. Ye, and Y. Pu. 2010. Effects ofsubchronic exposure to multi-walled carbonnanotubes on mice. Journal of Toxicology andEnvironmental Health, Part A 73:463–70.doi:10.1080/15287390903523378.

Liu, A., K. Sun, J. Yang, and D. Zhao. 2008.Toxicological effects of multi-wall carbonnanotubes in rats. Journal of NanoparticleResearch 10:1303–07. doi:10.1007/s11051-008-9369-0.

Liu, J.-H., S.-T. Yang, H. Wang, and Y.Liu. 2010b. Advances in biodistributionstudy and tracing methodology of car-bon nanotubes. Journal of Nanoscience andNanotechnology 10:8469–81. doi:10.1166/jnn.2010.2689.

Liu, Q., X. Ma, and M. R. Zachariah. 2012.Combined on-line differential mobility andparticle mass analysis for determination ofsize resolved particle density and microstruc-ture evolution. Microporous and MesoporousMaterials 153:210–16.

Liu, X., R. H. Hurt, and A. B. Kane.2010a. Biodurability of single-walled carbonnanotubes depends on surface functionaliza-tion. Carbon 48:1961–69.

Ma-Hock, L., S. Burkhardt, V. Strauss, A. O.Gamer, K. Wiench, B. Van Ravenzwaay, andR. Landsiedel. 2009b. Development of ashort-term inhalation test in the rat usingnano-titanium dioxide as a model substance.Inhalation Toxicology 21:102–18.

Ma-Hock, L., S. Trenmann, V. Strauss, S. Brill, F.Luizi, M. Mertler, K. Wiench, A. O. Gamer,B. Van Ravenzwaay, and R. Landsiedel.2009a. Inhalation toxicity of multiwall car-bon nanotubes in rats exposed for 3 months.Toxicological Sciences 112:468–81.

Mangum, J. B., E. A. Turpin, A. Antao-Menezes,M. F. Cesta, E. Bermudez, and J. C. Bonner.2006. Single-walled carbon nanotubes(SWCNT)-induced interstitial fibrosis in thelungs of rats is associated with increasedlevels of PDGF mRNA and the formation

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015




http://dx.doi.org/10.1002/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes(ISSN)1522-7278

http://dx.doi.org/10.1021/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesnn305567s




http://dx.doi.org/10.1166/\gdef yesno\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyes\gdef \ \gdef \ \ \gdef nono\gdef yesyes\penalty \z@ \gdef \ \penalty \z@ \gdef nono\gdef yesyesjnn.2010.2689


of unique intercellular carbon struc-tures that bridge alveolar macrophagesin situ. Particle and Fibre Toxicology3:15. doi:10.1186/1743-8977-3-15.

Matsumoto, M., H. Serizawa, M. Sunaga,H. Kato, M. Takahashi, M. Hirata-Koizumi,A. Ono, E. Kamata, and A. Hirose.2012. No toxicological effects on acuteand repeated oral gavage doses of single-wall or multi-wall carbon nanotube inrats. Journal of Toxicological Sciences 37:463–74.

Mauderly, J. L. 1986. Respiration of F344 rats innose-only inhalation exposure tubes. Journalof Applied Toxicology 6:25–30.

Maynard, A. D., and R. J. Aitken. 2007.Assessing exposure to airborne nanomate-rials: Current abilities and future require-ments. Nanotoxicology 1:26–41.

Maynard, A. D., P. A. Baron, M. Foley, A. A.Shvedova, E. R. Kisin and V. Castranova.2004. Exposure to carbon nanotube mate-rial: Aerosol release during the handlingof unrefined single-walled carbon nan-otube material. J. Toxicology & EnvironmentalHealth, Part A 67:87–107.

Maynard, A. D., P. A. Baron, M. Foley, A. A.Shvedova, E. R. Kisin, and V. Castranova.2009. Exposure to carbon nanotube mate-rial during the handling of unrefined singlewalled carbon nanotube material. Journalof Toxicology and Environmental Health A67:87–107.

Maynard, A. D., B. K. Ku, M. Emery,M. R. Stolzenburg, and P. H. McMurry.2006. Measuring particle size-dependentphysiochemical structure in airborne sin-gle walled carbon nanotube agglomer-ates. Journal of Nanoparticle Research 9:865–92.

McConnell, E. E., C. Axten, T. W. Hesterberg,J. Chevalier, J. Miiller, J. Everitt, G.Oberdörster, G. R. Chase, P. Thevenaz,and P. Kotin. 1999. Studies on the inhalationtoxicology of two fiberglasses and amositeasbestos in the Syrian golden hamster. PartII. Results of chronic exposure. InhalationToxicology 11:785–835.

McKinney, W., B. Chen, and D. Frazer.2009. Computer controlled multi-walled car-bon nanotube inhalation exposure system.Inhalation Toxicology 21:1053–61.

Meier, R., K. Clark, and M. Riediker. 2013.Comparative testing of a miniature diffusionsize classifier to assess airborne ultrafine par-ticles under field conditions. Aerosol Scienceand Technology 47:22–28.

Ménache, M. G., F. J. Miller, and O. G.Raabe. 1995. Particle inhalability curves forhumans and small laboratory animals. Annalsof Occupational Hygiene 39:317–28.

Mercer, R. R., A. F. Hubbs, J. F. Scabilloni,L. Wang, L. Battelli, D. Schwegler-Berry,V. Castranova, and D. W. Porter. 2010a.Distribution and persistence of pleural pen-etrations by multi-walled carbon nanotubes.Particle and Fibre Toxicology 7:28.

Mercer, R. R., A. F. Hubbs, J. F. Scabilloni, L.Wang, L. A. Battelli, S. Friend, V. Castranova,and D. W. Porter. 2011. Pulmonary fibroticresponse to aspiration of multi-walled car-bon nanotubes. Particle and Fibre Toxicology8:21.

Mercer, R. R., J. Scabilloni, L. Wang, E.Kisin, A. R. Murray, D. Schwegler-Berry,A. A. Shvedova, and V. Castranova. 2008.Alteration of deposition pattern and pul-monary response as a result of improveddispersion of aspirated single walledcarbon nanotubes in a mouse model.American Journal of Physiology: LungCellular and Molecular Physiology 294:L87–L97.

Mercer, R. R., J. F. Scabilloni, A. F. Hubbs, L.A. Battelli, W. McKinney, S. Friend, M. G.Wolfarth, M. Andrew, V. Castranova, and D.W. Porter. 2013a. Distribution and fibroticresponse following inhalation exposure tomulti-walled carbon nanotubes. Particle andFibre Toxicology 10:33.

Mercer, R. R., J. F. Scabilloni, A. F. Hubbs,L. Wang, L. A. Battelli, W. McKinney,V. Castranova, and D. W. Porter. 2013b.Extrapulmonary transport of MWCNT fol-lowing inhalation exposure. Particle and FibreToxicology 10:38.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.1186/1743-8977-3-15


Mercer, R. R., J. F. Scabilloni, L. Wang, L.A. Battelli, and V. Castranova. 2009. Useof labeled single walled carbon nanotubesto study translocation from the lungs.Toxicologist 108:A2192.

Mercer, R. R., J. F. Scabilloni, L. Wang, L.A. Battelli, and V. Castranova. 2010. Useof labeled single walled carbon modulatesarteriolar sympathetic constriction: Role ofnitric oxide, prostanoids, and α-adrenergicreceptors. Toxicologist 114:A1728.

Methner, M., L. Hodson, A. Dames, andC. Geraci. 2010. Nanoparticle EmissionAssessment Technique (NEAT) for theidentification and measurement of poten-tial inhalation exposure to engineerednanomaterials—Part B: Results from 12 fieldstudies. Journal of Occupational andEnvironmental Hygiene 7:163–76.

Miller, F. J., B. Asgharian, J. D. Schroeter,O. Price, R. A. Corley, D. R. Einstein, R.E. Jacob, T. C. Cox, S. Kabilan, and T.Bentley. 2014. Respiratory tract lung geom-etry and dosimetry model for male Sprague-Dawley rats. Inhalation Toxicology 26:524–44.

Miller, F. J., S. W. Kaczmar, R. Danzeisen, andO. R. Moss. 2013. Estimating lung burdensbased on individual particle density esti-mated form scanning electron microscopyand cascade impactor samples. InhalationToxicology 25:813–27.

Miller, F. J., J. S. Kimbell, R. J. Preston,J. H. Overton, E. A. Gross, and R. B.Conolly. 2011. The fractions of respira-tory tract cells at risk in formaldehydecarcinogenesis. Inhalation Toxicology 23:689–706.

Mitchell, L. A., J. Gao, R. V. Wal, A. Gigliotti,S. W. Burchiel, and J. D. McDonald.2007. Pulmonary and systemic immuneresponse to inhaled multiwalled carbonnanotubes. Toxicological Sciences 100:203–14.

Moalli, P. A., J. L. MacDonald, L. A. Goodglick,and A. B. Kane. 1987. Acute injury andregeneration of the mesothelium in responseto asbestos fibers. American Journal ofPathology 128:426–45.

Morello, M., C. L. Krone, S. Dickerson, E.Howerth, W. A. Germishuizen, Y. L. Wong,D. Edwards, B. R. Bloom, and M. K.Hondalus. 2009. Dry-powder pulmonaryinsufflation in the mouse for applicationto vaccine or drug studies. Tuberculosis89:371–77.

Morrow, P. E. 1986. The setting of particulateexposure levels for chronic inhalation toxic-ity studies. Journal of the American College ofToxicology 5:533–44.

Morrow, P. E. 1988. Possible mechanismsto explain dust overloading of the lungs.Fundamental and Applied Toxicology10:369–84.

Morrow, P. E., J. K. Haseman, C. H. Hobbs, K.E. Driscoll, V. Vu, and G. Oberdörster. 1996.Workshop overview—The maximum toler-ated dose for inhalation bioassays: Toxicityvs. overload. Fundamental and AppliedToxicology 29:155–67.

Moss, O. R., R. A. James, and B. Asgharian.2006. Influence of exhaled air on inhal-tion exposure delivered through a directed-flow nose-only exposure system. InhalationToxicology 18:45–51.

Muhle, H., B. Bellmann, O. Creutzenberg, R.Fuhst, W. Koch, U. Mohr, S. Takenaka, P.Morrow, R. Kilpper, J. MacKenzie, and R.Mermelstein. 1990. Subchronic inhalationstudy of toner in rats. Inhalation Toxicology2:341–60.

Muller, J., F. Huaux, N. Moreau, P. Misson, J.-F.Heilier, M. Delos, M. Arras, A. Fonseca, J.B. Nagy, and D. Lison. 2005. Respiratorytoxicity of multi-wall carbon nanotubes.Toxicology and Applied Pharmacology207:221–31.

Muller, J., M. Delos, N. Panin, V. Rabolli,F. Huaux, and D. Lison. 2009. Absenceof carcinogenic response to multiwall car-bon nanotubes in a 2-year bioassay in theperitoneal cavity of the rat. ToxicologicalSciences 110: 442–47.

Murphy, F. A., C. A. Poland, R. Duffin, K. T. Al-Jamal, H. Ali-Boucetta, A. Nunes, F. Byrna,A. Prina-Mello, Y. Volkov, S. Li, S. J. Mathor,A. Bianco, M. Prato, A. MacNea, W. A.Wallace, K. Kosturelos, and K. Donaldson.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


2011. Length-dependent retension of car-bon nanotubes in the pleural space of miceinitiates sustained inflammation and progres-sive fibrosis on the parietal pleura. AmericanJournal of Pathology 178:2587–600.

Murray, A. R., E. R. Kisin, A. V. Tkach,N. Yanamala, R. Mercer, S. H. Young, B.Fadeel, V. E. Kagan, and A. A. Shvedova.2012. Factoring-in agglomeration of carbonnanotubes and nanofibers for better predic-tion of their toxicity versus asbestos. Particleand Fibre Toxicology 9:10.

Myojo, T., T. Oyabu, K. Nishi, C. Kadoya, I.Tanaka, M. Ono-Ogaswara, H. Sakae, and T.Shirai. 2009. Aerosol generation and mea-surement of multi-wall carbon nanotubes.Journal of Nanoparticle Research 11:91–99.

Nagai, H., Y. Okazaki, S. H. Chew, N. Misawa,Y. Yamashita, S. Akatsuka, T. Ishihara, K.Yamashita, Y. Yoshikawa, H. Yasui, L. Jiang,H. Ohara, T. Takahashi, G. Ichihara, K.Kostarelos, Y. Miyata, H. Shinohara, and S.Toyokuni. 2011. Diameter and rigidity ofmultiwalled carbon nanotubes are criticalfactors in mesothelial injury and carcinogen-esis. Proceedings of the National Academyof Sciences of the United States of America108:1330–38.

Nanomaterial Registry. 2014. Accessed August15, 2014. https://www.nanomaterialregistry.org/about/MinimalInformationStandards.asppx.

Narciso, S. P., E. Nadziejko, L. C. Chen,T. Gordon, and C. Nadziejko. 2003.Adaptation to stress induced by restrainingrats and mice in nose-only inhalation hold-ers. Inhalation Toxicology 15:1133–43.

National Institute for Occupational Safety andHealth. 2003. Elemental carbon (dieselparticulate)—Method 5040. NIOSH manualof analytical methods, 4th ed., 2003 issue 3.NIOSH.

National Institute for Occupational Safety andHealth. 2012. General safe practices forworking with engineered nanomaterials inresearch laboratories. Accessed August 15,2014. http://www.cdc.gov/niosh/doc/2012.147/.

National Institute for Occupational Safetyand Health. 2013. Current IntelligenceBulletin 65: Occupational exposure to car-bon nanotubes and nanofibers. NIOSH/CDC/Department of Health and Human Services.Atlanta: NIOSH/CDC.

National Institute of Standards and Technology.2012. Presentation, BERM-13 meeting,Vienna, Austria, June 2012: Developmentof nanoscale reference materials at NIST,presented by Stephen Wise (NIST).

Nel, A. E., L. Mädler, D. Velegol, T. Xuia, E.M. V. Hoek, P. Somasundaran, F. Klaessig,V. Castranova, and M. Thompson. 2009.Understanding biophysicochemical interac-tions at the nano-bio interface. NatureMaterials 8:543–57.

Nikula, K. J., K. J. Avila, W. C. Griffith,and J. L. Mauderly. 1997. Lung tissueresponses and sites of particle retentiondiffer between rats and cynomolgus mon-keys exposed chronically to diesel exhaustand coat dust. Fundamental and AppliedToxicology 37:37–53.

Nikula, K. J., V. Vallyathan, F. H. Green, andF. F. Hahn. 2001. Influence of exposureconcentration or dose on the distributionof particulate material in rat and humanlungs. Environmental Health Perspectives109:311–18.

Nowak, B., R. David, H. Fissan, H. Morris, J.Shatkin, M. Stintz, R. Zepp, and D. Brower.2013. Potential release scenarios for carbonnanotubes used in composites. EnvironmentInternational 59:1–11.

Nurkiewicz, T. R., D. W. Porter, M. Barger, L.Millecchia, K. M. Rao, P. J. Marvar, A. F.Hubbs, and V. Castranova. 2006. Systemicmicrovascular dysfunction and inflamma-tion after pulmonary particulate matterexposure. Environmental Health Perspectives114:412–19.

Nurkiewicz, T. R., D. W. Porter, A. F. Hubbs, J.L. Cumpston, B. T. Chen, D. G. Frazer, andV. Castranova. 2008. Nanoparticle inhala-tion augments particle-dependent systemicmicrovascular dysfunction. Particle and FibreToxicology 5:1.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

https://www.nanomaterialregistry.org/about/MinimalInformationStandards.asppx



http://www.cdc.gov/niosh/doc/2012.147/

http://www.cdc.gov/niosh/doc/2012.147/


Nurkiewicz, T. R., D. W. Porter, A. F. Hubbs,S. Stone, B. T. Chen, D. G. Frazer, M.A. Boegehold, and V. Castranova. 2009.Pulmonary nanoparticle exposure disruptssystemic microvascular nitric oxide signaling.Toxicological Sciences 110:191–203.

Oberdörster, G. 1989. Dosimetric principles forextrapolating results of rat inhalation stud-ies to humans, using nickel as an example.Health Physics 57 (suppl. 1): 213–20.

Oberdörster, G. 1997. Pulmonarycarcinogenicity of inhaled particles andthe maximum tolerated dose. EnvironmentalHealth Perspectives 195(suppl. 5): 1347–56.

Oberdörster, G. 2012. Correspondence:Nanotoxicology: In vitro–in vivo dosimetry.Environmental Health Perspectives 120:A13.

Oberdörster, G., C. Cox, and R. Gelein. 1997.Intratracheal instillation versus intratrachealinhalation of tracer particles for measuringlung clearance function. Experimental LungResearch 23:17–34.

Oberdörster, G., J. Ferin, and B. E. Lehnert.1994a. Correlation between particle size,in vivo particle persistence, and lunginjury. Environmental Health Perspectives102(suppl. 5): 173–79.

Oberdörster, G., J. Ferin, S. C. Soderholm,R. Gelein, C. Cox, R. Baggs, and P. E.Morrow. 1994b. Increased pulmonary tox-icity of inhaled ultrafine particles: Due tolung overload alone? Annals of OccupationalHygiene 38(suppl. 1): 295–302.

Oberdörster, G., A. Maynard, K. Donaldson,V. Castranova, J. Fitzpatrick, K. D. Ausman,J. Carter, B. Karn, W. Kreyling, D. Lai, S.Olin, N. Monteiro-Riviere, D. Warheit, andH. Yang, A.A.R.F.I.R. Foundation. 2005b.Principles for characterizing the poten-tial human health effects from exposureto nanomaterials: Elements of a screen-ing strategy. Particle and Fibre Toxicology2:8.

Oberdörster, G., E. Oberdörster, and J.Oberdörster. 2005a. Invited review:Nanotoxicology: An emerging disciplineevolving from studies of ultrafine parti-cles. Environmental Health Perspectives113:823–39.

Oberdörster, G., Z. Sharp, V. Atudorei, A.Elder, R. Gelein, W. Kreyling, and C. Cox.2004. Translocation of inhaled ultrafine par-ticles to the brain. Inhalation Toxicology 16:437–45.

Organization for Economic Cooperation andDevelopment. 2009a. OECD Guideline forthe testing of chemicals, section 4: Healtheffects. Test no. 413: Subchronic inhalationtoxicity: 90-Day study. Test Guideline 413(TG 413), adopted 7 September.

Organization for Economic Cooperation andDevelopment. 2009b. Organization forEconomic Cooperation and Development.2009. Environment, health and safetypublications, series on testing and assess-ment, no. 39: Guidance document on acuteinhalation toxicity testing. ENV/JM/MONO28. 21 July.

Organization for Economic Cooperation andDevelopment. 2010. OECD Environment,health and safety publications—Series onthe safety of manufactured nanomaterials,no. 14: Guidance manual for the testing ofmanufactured nanomaterials: OECD’s spon-sorship programme.

Organization for Economic Cooperation andDevelopment. 2012. OECD Environment,health and safety publications—Series onthe safety of manufactured nanomaterials,no. 36. Guidance on sample preparaiton anddosimetry for the safety testing of manufac-tured nanomaterials.

Ohnishi, M., H. Yajima, T. Kasai, Y. Umeda,M. Yamamoto, S. Yamamoto, H. Okuda,M. Suzuki, T. Nishizawa, and S. Fukushima.2013. Novel method using hybridmarkers: Development of an approachfor pulmonary measurement of multi-walled carbon nanotubes. Journal ofOccupational Medicine and Toxicology8:30.

Oyabu, T., T. Myojo, Y. Morimoto, A.Ogami, M. Hirohashi, M. Yamamoto,M. Todoroki, Y. Mizuguchi, M. Hashiba,B. W. Lee, M. Shimada, W. N. Wang,K. Uchida, S. Endoh, N. Kobayashi, K.Yamamoto, K. Fujita, K. Mizuno, M. Inada,T. Nakazato, J. Nakanishi, and I. Tanaka.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


2011. Biopersistence of inhaled MWCNT inrat lungs in a 4-week well-characterizedexposure. Inhalation Toxicology 23:784–91.

Pare, W. P., and G. B. Glavin. 1986. Restraintstress in biomedical research: A review.Neuroscience and Biobehavioral Reviews10:339–70.

Park, K., D. B. Kittelson, M. R. Zachariah,and P. H. McMurry. 2004. Measurementof inherent material denisty of nanoparit-cle agglomerates. Journal of NanoparticleResearch 6:267–72.

Parungo, C. P., Y. L. Colson, S. M. Kim, S. Kim, L.H. Cohn, M. G. Bawendi, and J. V. Frangioni.2005. Sentinel lymph node mapping of thepleural space. Chest 127:1799–804.

Pauluhn, J. 2009. Comparative pulmonaryresponse to inhaled nanostructures:Considerations on test design and end-points. Inhalation Toxicology 21(suppl 1):40–54.

Pauluhn, J. 2010. Subchronic 13-week inhala-tion exposure of rats to multiwalled car-bon nanotubes: Toxic effects are deter-mined by density of agglomerate structures,not fibrillar structure. Toxicological Sciences113:226–42.

Pauluhn, J., and U. Mohr. 1999. Repeated4-week inhalation exposure of rats: Effectof low-, intermediate, and high-humiditychamber atmospheres. Experimental andToxicologic Pathology 51:178–87.

Pauluhn, J., and M. Rosenbruch. 2015. Lungburdens and kinetics of multi-walled car-bon nanotubes (Baytubes) are highly depen-dent on the disaggregation of aerosolizedMWCNT. Nanotoxicology 9:242–52.

Pauluhn, J., and A. Thiel. 2007. A simpleapproach to validation of directed-flow nose-only inhalation chambers. Journal of AppliedToxicology 27:160–67.

Peigney, A., C. Laurent, E. Flahaut, R. R. Bacsa,and A. Rousset. 2001. Specific surface areaof carbon nanotubes and bundles of carbonnanotubes. Carbon 39:507–14.

Peterson, M. R., and M. H. Richards 1999.Thermal-optical-transmittance analysis fororganic, elemental, carbonate, total carbon,

and OCX2 in PM2.5 by the EPA/NIOSHMethod—#83. Accessed August 15, 2014.www.rti.org/pubs/OC-EC_Paper_83_3b.pdf.

Phalen, R. F. 2009. Inhalation studies, founda-tions and techniques, 2nd ed. New York, NY:Informa Healthcare.

Poland, C. A., R. Duffin, I. Kinloch, A. Maynard,W. A. Wallace, A. Seaton, V. Stone, S.Brown, W. MacNee, and K. Donaldson.2008. Carbon nanotubes introduced into theabdominal cavity of mice show asbestos-like pathogenicity in a pilot study. NatureNanotechnology 3:423–28.

Porter, D., K. Sriram, M. Wolfarth, A. Jefferson,D. Schwegler-Berry, M. Andrew, and V.Castranova. 2008. A biocompatible mediumfor nanoparticle dispersion. Nanotoxicology2:144–54.

Porter, D. W., A. Hubbs, R. Mercer, N. Wu,M. G. Wolfarth, K. Sriram, S. Leonard, L.Battelli, D. Schwegler-Berry, S. Friend, M.Andrew, B. T. Chen, S. Tsuruoka, M. Endo,and V. Castranova. 2010. Mouse pulmonarydose-and time course-response induced byexposure to multi-walled carbon nanotubes.Toxicology 269:136–47.

Pritchard, J. N., A. Holmes, J. C. Evans, N.Evans, R. J. Evans, and A. Morgan. 1985.The distribution of dust in the rat lung fol-lowing administration by inhalation and bysingle intratracheal instillation. EnvironmentalResearch 36:268–97.

Raabe, O. G., H.-C. Yeh, G. M. Schum,and R. F. Phalen 1976. Tracheobronchialgeometry: Human, dog, rat, hamster, LF-53.Albuquerque, NM: Lovelace Foundation.

Rafique, M. M. A., and J. Iqbal. 2011.Production of carbon nanotubes by differentroutes - A review. Journal of Encapsulationand Adsorption Sciences 1:29–34.

Rinderknecht, A., A. Elder, R. Prud’homme,M. Gindy, J. Harkema, and G. Oberdörster.2007. Surface functionalization affects therole of nanoparticle disposition. AmericanJournal of Respiratory and Critical CareMedicine 175:A246.

Rittinghausen, S., A. Hackbarth, O.Creutzenberg, H. Ernst, U. Heinrich, A.Leonhardt, and D. Schaudien. 2014.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

www.rti.org/pubs/OC-EC_Paper_83_3b.pdf


The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) afterintraperitoneal injection in rats. Particle andFibre Toxicology 11:59.

Rothenberg, S. J., R. M. Parker, R. G. York, G. E.Dearlove, and M. M. Martin. 2000. Lack ofeffects of nose-only inhalation exposure ontesticular toxicity in male rats. ToxicologicalSciences 53:127–34.

Rudd, C. J., and K. A. Strom. 1981. A spec-trophotometric method for the quantitationof diesel exhaust particles in guinea piglung. Journal of Applied Toxicology 1:83–87.

Rudolf, G., J. Gebhart, J. Heyder, C. F. Schiller,and W. Stahlhofen. 1986. An empirical for-mula describing aerosol deposition in manfor any particle size. Journal of AerosolScience 17:350–55.

Rudolf, G., R. Kobrich, and W. Stahlhofen.1990. Modelling and algebraic formula-tion of regional aerosol deposition inman. Journal Aerosol Sciences 21(suppl. 1):S403–S406.

Rushton, E. K., J. Jiang, S. S. Leonard, S. Eberly,V. Castranova, P. Biswas, A. Elder, X. Han,R. Gelein, J. Finkelstein, and G. Oberdörster.2010. Concept of assessing nanoparticlehazards considering nanoparticle dosemet-ric and chemical/biological response-metrics.Journal of Toxicology and EnvironmentalHealth, Part A 73:445–61.

Ryman-Rasmussen, J. P., M. F. Cesta, A. R.Brody, J. K. Shipley-Phillips, J. I. Everitt, E.W. Tewksbury, O. R. Moss, B. A. Wang,D. E. Dodd, M. E. Anderson, and J. C.Bonner. 2009b. Inhaled carbon nanotubesreach the subpleural tissue in mice. NatureNanotechnology 4:747–51.

Ryman-Rasmussen, J. P., E. W. Tewksbury, O.R. Moss, M. F. Cesta, B. A. Wong, andJ. C. Bonner. 2009a. Inhaled multiwalledcarbon nanotubes potentiate airway fibrosisin murine allergic asthma. American Journalof Respiratory Cell and Molecular Biology40:349–58.

Sager, T. M., D. W. Porter, V. A. Robinson,W. G. Lindsley, D. E. Schwegler-Berry, andV. Castranova. 2007. Improved method to

disperse nanoparticles for in vitro and invivo investigation of toxicity. Nanotoxicology1:118–29.

Sager, T. M., M. W. Wolfarth, M. Andrew,A. Hubbs, S. Friend, T. Chen, D. W.Porter, N. Wu, F. Yang, R. F. Hamilton,and A. Holian. 2014. Effect of multi-walledcarbon nanotube surface modification onbioactivity in the C57BL/6 mouse model.Nanotoxicology 8:317–27.

Sakamoto, Y., D. Nakae, N. Fukumori, K.Tayama, A. Maekawa, K. Imai, A. Hirose,T. Nishimura, N. Ohashi, and A. Ogata.2009. Induction of mesothelioma by a sin-gle intrascrotal administration of multi-wallcarbon nanotube in intact male Fischer344 rats. Journal of Toxicological Sciences 34:65–76.

Salvador-Morales, C., E. V. Basiuk, V. A.Basiuk, M. L. H. Green, and R. B. Sim.2008. Effects of covalent functionalizationon the biocompatibility characteristics ofmulti-walled carbon nanotubes. Journalof Nanoscience and Nanotechnology8:2347–56.

Sargent, L., D. W. Porter, L. M. Staska, A.F. Hubbs, D. T. Lowry, L. Battelli, K. J.Siegrist, M. L. Kashon, R. R. Mercer, A. K.Bauer, B. T. Chen, J. L. Salisbury, D. Frazer,W. McKinney, M. Andrew, S. Tsuruoka, M.Endo, K. Fluharty, V. Castranova, and S. H.Reynolds. 2014. Promotion of lung adeno-carcinoma following inhalation exposure tomulti-walled carbon nanotubes. Particle andFibre Toxicology 11:3.

Scherer, P. W., L. H. Shendalman, andN. M. Greene. 1972. Simultaneous diffu-sion and convection in single breath lungwashout. Bulletin of Mathematical Biophysics34:393–412.

Schleh, C., W. Kreyling and C. M. Lehr. 2013.Pulmonary sufactant is indispensable in orderto simulate the in vivo situation. Part FibreToxicol 10: 6.

Scuri, M., B. T. Chen, V. Castranova, J. S.Reynolds, V. J. Johnson, L. Samsell, C.Walton, and G. Piedimonte. 2010. Effects oftitanium dioxide nanoparticle exposure onneuroimune responses in rat airways. Journal

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


of Toxicology and Environmental Health, PartA 73:1353–69.

Shimada, M., W.-N. Wang, K. Okuyama,T. Myojo, T. Oyabu, Y. Morimoto, I.Tanaka, S. Endoh, K. Uchida, K. Ehara, H.Sakurai, K. Yamamoto, and J. Nakanishi.2009. Development and evaluation ofan aerosol generation and supplying sys-tem for inhalation experiment of manufac-tured nanoparticles. Environmental Science &Technology 43:5529–34.

Shvedova, A. A., E. Kisin, A. R. Murray, V. J.Johnson, O. Gorelik, S. Arepali, A. F. Hubbs,R. R. Mercer, P. Keohavong, N. Sussman,J. Jin, S. Stone, B. T. Chen, G. Deye, A.Maynard, V. Castranova, P. A. Baron, andV. E. Kagan. 2008. Inhalation, versus aspi-ration of single walled carbon nanotubes inC57BL/6 mice: Inflammation, fibrosis, oxida-tive stress and mutagenesis. American Journalof Physiology: Lung Cellular and MolecularPhysiology 295:L552–L565.

Shvedova, A. A., E. R. Kisin, R. Mercer, A.R. Murray, V. J. Johnson, A. I. Potapovich,Y. Y. Tyurina, O. Gorelik, S. Arepulli, D.Schwegler-Berry, A. F. Hubbs, J. Antonini, D.E. Evans, B.-K. Ku, D. Ramsey, A. Maynard,V. E. Kagan, V. Castranova, and P. Buron.2005. Unusual inflammatory and fibrogenicpulmonary responses to single walled car-bon nanotubes in mice. American Journalof Physiology: Lung Cellular and MolecularPhysiology 289:L698– L708.

Shvedova, A. A., A. Tkach, E. R. Kisin, A.R. Murray, N. Yanamala, A. Hubbs, P.Keohavong, L. P. Sycheva, V. E. Kagan, andV. Castranova. 2014. Long-term effects ofcarbon containing engineered nanomateri-als and asbestos in the lung: One year postexposure comparisons. American Journal ofPhysiology: Lung Cellular and MolecularPhysiology 306:L170–L182.

Shvedova, A. A., A. V. Tkach, E. R. Kisin, T.Khaliullin, S. Stanley, D. W. Gutkin, A. Star,Y. Chen, G. V. Shurin, V. E. Kagan, and M.R. Shurin. 2013. Carbon nanotubes enhancemetastatic growth of lung carcinoma viaup-regulation of myeloid-derived suppressorcells. Small 9:1691–95.

Singh, C., T. Quested, C. B. Boothroyd, P.Thomas, I. A. Kinloch, A. I. Abou-Kandil, andA. H. Windle. 2002. Synthesis and character-ization of carbon nanofibers produced by thefloating catalyst method. Journal of PhysicalChemistry. B 106:10915–22.

Sisan, T. B., and S. Lichter. 2014. Solitonstransport water through narrow car-bon nanotubes. Physical Review Letters112:044501–05.

Sistonen, L., K. D. Sarge, B. Phillips, K.Abravaya, and R. I. Morimoto. 1992.Activation of heat shock factor 2 duringhemin-induced differentiation of human ery-throleukemia cells. Molecular and CellularBiology 12:4104–11.18

Slikker, W., Jr., M. E. Andersen, M. S.Bogdanffy, J. S. Bus, S. D. Cohen, R. B.Conolly, D. M. David, N. G. Doerrer, D.C. Dorman, D. W. Gaylor, D. Hattis, J. M.Rogers, S. R. Woodrow, J. A. Swenberg, andK. Wallace. 2004. Dose-dependent transi-tions in mechanisms of toxicity: Case stud-ies. Toxicology and Applied Pharmacology201:203–25.

Sriram, K., D. W. Porter, A. M. Jefferson, G.X. Lin, M. G. Wolfarth, B. T. Chen, W.McKinney, D. G. Frazer, and V. Castranova.2009. Neuro inflammation and blood–brain barrier changes following exposureto engineered nanomaterials. Toxicologist108:A2197.

Stahlhofen, W., G. Rudolf, and A. C. James.1989. Intercomparison of experimentalregional aerosol deposition data. Journal ofAerosol Medicine 2:285–308.

Stapleton, P. A., V. Minarchick, A. Cumpston,W. McKinney, B. T. Chen, D. Frazer, V.Castranova, and T. R. Nurkiewicz. 2011.Time-course of improved coronary arteri-olar endothelium-dependent dilation aftermulti-walled carbon nanotube inhalation.Toxicologist 120:A194.

Stapleton, P. A., V. C. Minarchick, A. M.Cumpston, W. McKinney, B. T. Chen, T.M. Sager, D. G. Frazer, R. R. Mercer, J.Scabilloni, M. E. Andrew, V. Castranova,and T. R. Nurkiewicz. 2012. Impairment ofcoronary arteriolar endothelium-dependent

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


dilation after multi-walled carbon nan-otube inhalation: A time-course study.International Journal of Molecular Sciences13:13781–803.

Stinn, W., A. Teredesai, E. Anskeit, K.Rustemeier, G. Schepers, P. Schgnell,H.-J. Haussmann, R. A. Carchman, C. R. E.Coggins, and W. Reininghaus. 2005. Chronicnose-only inhalation study in rats, comparingroom-aged sidestream cigarette smoke anddiesel engine exhaust. Inhalation Toxicology17:549–76.

Stoeger, T., C. Reinhard, S. Takenaka, A.Schroeppel, E. Karg, B. Ritter, J. Heyder,and H. Schulz. 2006. Instillation of six dif-ferent ultrafine carbon particles indicates asurface area threshold dose for acute lunginflammation in mice. Environmental HealthPerspectives 114:328–33.

Tabet, L., C. Bussy, A. Setyan, A. Simon-Deckers, M. J. Rossi, J. Boczkowski, and S.Lanone. 2011. Coating carbon nanotubeswith a polystyrene-based polymer protectsagainst pulmonary toxicity. Particle and FibreToxicology 8:3.

Takagi, A., A. Hirose, M. Futakuchi, H. Tsuda,and J. Kanno. 2012. Dose-dependentmesothelioma induction by intraperitonealadministration of multi-wall carbonnanotubes in p53 heterozygous mice.Cancer Science 103:1440–44.

Takagi, A., A. Hirose, T. Nishimura, W.Fukumori, A. Ogata, and N. Ohashi.2008. Induction of mesothelioma in p53+/- mouse by intraperitoneal applica-tion of multi-walled carbon nanotube.Journal of Toxicological Sciences 33:105–16.

Taquahashi, Y., Y. Ogawa, A. Takagi, M. Tsuji,K. Morita, and J. Kanno. 2013. An improveddispersion method of multi-wall carbon nan-otube for inhalation toxicity studies of exper-imental animals. Journal of ToxicologicalSciences 38:619–28.

Thomson, E. M., A. Williams, C. L. Yauk, andR. Vincent. 2009. Impact of nose-only expo-sure system on pulmonary gene expression.Inhalation Toxicology 21(S1): 74–82.

Timbrell, V., T. Ashcroft, B. Goldstein, F.Heyworth, L. O. Meurman, R. E. G.Randall, J. A. Reynolds, K. B. Shilkin, andD. Whitaker. 1988. Relationships betweenretained amphibole fibres and fibrosis inhuman lung tissue specimens. In: InhaledParticles VI. Annals of Occupational Hygiene32(suppl. 1): 323–40.

Tran, C. L., D. Buchanan, R. T. Cullen, A.Searl, A. D. Jones, and K. Donaldson. 2000a.Inhalation of poorly soluble particles. II.Influence of particle surface area on inflam-mation and clearance. Inhalation Toxicology12:1113–26.

Tran, C. L., D. Buchanan, B. G. Miller, and A.D. Jones. 2000b. Mathematical modeling topredict the responses to poorly soluble par-ticles in rat lungs. Inhalation Toxicology 12(Suppl. 3):403–09.

Treumann, S., L. Ma-Hock, S. Gröters, R.Landsiedel, and B. Van Ravenzwaay. 2013.Additional histopathologic examination ofthe lungs from a 3-month inhalation toxic-ity study with multiwall carbon nanotubes inrats. Toxicological Sciences 134:103–10.

Tsai, S. J., M. Hofmann, M. Hallock, E.Ada, J. Kong, and M. Ellenbecker.2009. Characterization and evaluationof nanoparticle release during the synthesisof single-walled and multiwalled carbonnanotubes by chemical vapor deposi-tion. Environmental Science & Technology43:6017–23.

Tsuruoka, S., K. Takeuchi, K. Koyama, T.Noguchi, M. Endo, F. Tristan, M. Terrones, H.Matsumoto, N. Saito, Y. Usui, D. W. Porter,and V. Castranova. 2013. ROS evaluationfor a series of CNT and their derivativesusing an ESR method with DMPO. Journal ofPhysics: Conference Series 429:012029.IOPPublishing

Tyl, R. W., L. Ballantyne, C. Fisher, D. L. Fait, T.A. Savine, I. M. Pritts, and D. E. Dodd. 1994.Evaluation of expousre to water aerosol orair by nose-only or whole-body inhationprocedures for CD-1 mice in developmen-tal toxicity studies. Fundamental and AppliedToxicology 23:251–60.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


Udelsman, R., M. J. Blake, C. A. Stagg, D.-G. Li, D. J. Putney, and N. J. Holbrook.1993. Vascular heat shock protein expres-sion in response to stress. Journal of ClinicalInvestigation 91:465–73.

Umeda, Y., T. Kasai, M. Saito, H. Kondo,T. Toya, S. Aiso, H. Okuda, T. Nishizawa,and S. Fukushima. 2013. Two-week toxi-city of multi-walled carbon nanotubes bywhole-body inhalation exposure in rats.Journal of Toxicology and Pathology 26:131–40.

Utembe W., K. Potgieter, A. B. Stefaniak,J. M. Gulumian. 2015. Dissolution andbiodurability: Important parametersneeded for risk assessment of nanoma-terials. Particle and Fibre Toxicology 12:11.doi:10.1186/s12989-015-0088-2.

Van Eijl, S., R. Van Oorschot, B. Olivier, F.P. Nijkamp, and N. Bloksma. 2006. Stressand hypothermia in mice in a nose-onlycigarette smoke exposure system. InhalationToxicology 18:911–18.

Vu, V., C. Barrett, J. Roycroft, L. Schuman,D. Dankovic, P. Baron, T. Martonen,W. Pepelko, and D. Lai. 1996. Chronicinhalation toxicity and carcinogenicitytesting of respirable fibrous particles.Regulatory Toxicology and Pharmacology 24:202–12.

Wang, J., C. Asbach, H. Fissan, T. Hülser, T. A. J.Kuhlbusch, D. Thompson, and D. Y. H. Pui.2011. How can nanobiotechnology oversightadvance science and industry: Examplesfrom environmental, health, and safety stud-ies ofnanoparticles (nano-EHS). Journal ofNanoparticle Research 13:1373–87.

Wang J., Y.K Bahk, S-.C. Chen, D.Y.H. Pui.2015. Characteristics of airborne fractal-likeagglomerates of carbon nanotubes. Carbon93: 441–450.

Warheit, D. B. 2008. How meaningful arethe results of nanotoxicity studies in theabsence of adequate material characteriza-tion? Toxicological Sciences 101:183–85.

Warheit, D. B., B. R. Laurence, K. L. Reed,D. H. Rouch, G. A. M. Reynolds, and T.R. Webb. 2004. Comparative pulmonarytoxicity assessment of single-wall carbon

nanotubes in rats. Toxicological Sciences77:117–25.

Warheit, D. B., K. L. Reed, and M. P. DeLorme.2013. Subchronic inhalation of carbonnanofibers: No apparent cross-talk betweenlocal pulmonary and cardiovascular/systemicresponses. Carbon 62:165–76.

Watson, J. G., J. C. Chow, and L.-W. A. Chen.2005. Summary of organic and elementalcarbon/black carbon analysis methods andintercomparisons. Aerosol and Air QualityResearch 5:65–102.

Weibel, E. R. 1963. Morphometry of the humanlung. Berlin, Germany: Springer Verlag.

Westerhoff, P., K. Doudrick, and P. Herckes2012. Detection of carbon nanotubes inenvironmental and biological matrices usingprogrammed thermal analysis. AmericanChemical Society Annual Conference, ,Philadelphia, PA, August 19–23.

WHO. 1981. Methods of monitoring and eval-uating airborne man-made mineral fibres.Report on a WHO Consultation. EUROReports and Studies 48, Copenhagen,Denmark, 29 April–1 May 1980.

Wong, B. 2007. Inhalation exposure systems:Design, methods and operation. ToxicologicPathology 33:3–14.

Xu, J., D. B. Alexander, M. Futakuchi, T.Numano, K. Fukamachi, M. Suzui, T.Omori, J. Kanno, A. Hirose, and H. Tsuda.2014. Size- and shape-dependent pleu-ral translocation, deposition, fibrogenesis,and mesothelial proliferation by multiwalledcarbon nanotubes. Cancer Science 105:763–69.

Xu, J., M. Futakuchi, H. Shimizu, D. B.Alexander, K. Yanagihara, K. Fukamachi,M. Suzui, J. Kanno, A. Hirose, A. Ogata,Y. Sakamoto, D. Nakae, T. Omori, andH. Tsurda. 2012. Multi-walled carbonnanotubes translocate into the pleural cavityand induce visceral mesothelial proliferationin rats. Cancer Science 103:2045–50.

Yeh, H. C., and G. M. Schum. 1980.Models of human lung airways and theirapplication to inhaled particle deposi-tion. Bulletin of Mathematical Biology 42:461–80.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

http://dx.doi.org/10.1186\gdef yesno$/$\gdef \ignorespaces $/$\gdef nono\gdef yesyes\gdef --\gdef \ignorespaces --\gdef nono\gdef yesyes$/$\gdef \ignorespaces $/$\gdef nono\gdef yesyess12989-015-0088-2.


Yeh, H. C., M. B. Snipes, A. E. Eidson, andC. H. Hobbs. 1990. Comparative evaluationof nose-only versus whole-body inhalationexpsoures for rats - Aerosol characteristicsand lung deposition. Inhalation Toxicology2:205–21.

Yu, C. P.1978. Exact analysis of aerosol depo-sition during steady state breathing. PowderTechnology 21:55–62.

Yu, K.-N., J. E. Kim, H. W. Seo, C. Chae, and M.-H. Cho. 2013. Differential toxic responsesbetween pristine and functionalized mul-tiwall naniotubes involve induction ofautophagy accumualtion in murine lung.Journal of Toxicology and EnvironmentalHealth, Part A 76:1282–92.

Zhang, L., and C. P. Yu. 1993. Empirical equa-tions for nasal deposition of inhaled parti-cles in small laboratory animals and humans.Aerosol Science and Technology 19:51–56.

Zhao, J., and V. Castranova. 2011. Toxicologyof nanomaterials used in nanomedicine.Journal of Toxicology and EnvironmentalHealth B 14:593–632.

Zhao, Y., B. L. Allen, and A. Star. 2011.Enzymatic degradation of multiwalled car-bon nanotubes. Journal of Physical Chemistry.A 115:9536–44.

APPENDIX A: RESPIRATORY-TRACTFRACTIONS

InhalabilityTo develop a model for CNT/CNF

inhalability, one must first characterize air-borne CNT/CNF. Unlike particles, which canbe represented by their diameters, CNT/CNFare described by several characteristics dimen-sions called equivalent diameters. Equivalentdiameters are identified with respect to thephysical mechanism(s) controlling the trans-port of CNT/CNF in the respiratory tract.A reduction in inhalability will result wheninhaled CNT/CNF are prevented from enter-ing the extrathoracic passages due to theirinertias. Therefore, inhalability of CNT/CNFdepends on their equivalent diameter forimpaction (or impaction diameter). The

following model illustrations are with theassumption of CNT/CNF being present assingle, straight fibers. Conditions (shape factor)will be different for complex structures of tan-gles, agglomerates, and aggregates. Once theshape of CNT/CNF is characterized, impactiondiameter is obtained by equating the drag andinertial forces of CNT/CNF for an equivalentspherical particle. For example, by assumingthat CNT/CNF are shaped as cylinders and arerandomly oriented in the air, an expression forequivalent diameter for impaction (dei) may befound as (Asgharian and Price, 2006)

dei = df

√√√√ρβ ln(β + √

β2 − 1)

√β2 − 1

(1)

where df , β, and ρ are the CNT/CNF diame-ter, aspect ratio, and mass density, respectively.Predictive models for the inhalability of parti-cles may be extended to CNT/CNF by replac-ing particle diameter with equivalent impactiondiameter. The majority of existing predictivemodels of particle inhalability are related toparticle diameter (Ménache, Miller, and Raabe1995; Brown 2005). However, particle iner-tia depends both on its diameter and travelingvelocity. Asgharian, Kelly, and Tewksbury (2003)developed a semiempirical expression for ratinhalability based on measurements obtainedfrom a series of brief nose-only inhalation stud-ies in which rats were exposed to titaniumdioxide particles. The following expression wasproposed to calculate the inhalability:

IF(φ, θ, φ) = 11 + αdδ

ei(φ, θ, φ)Qγ(2)

where Q is the inhalation flow rate. By fit-ting Eq. (2) to inhalability measurements inrats, coefficients α, δ, and γ were found tobe 19.87, –1.5532, and –0.7466, respectively.In humans, coefficients α, δ, and γ werereported as 0.1361, 0.9906, and –0.8527 fororal breathing and 4.451 × 10−4, 2.168, and0.3778 for nasal breathing (Asgharian and Price,2006). A plot of CNT/CNF inhalability versusequivalent diameter for impaction is given in

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE A-1. CNT/CNF inhalability for humans and rats.

CNT/CNF inhalability for humans via oral and nasal, andrats nasal breathings. Equivalent impaction diameter in the fig-ure includes shape and orientation effects. dei is found fromEquation (1) for cylindrical shapes. Additional size measurementsare needed to develop more realistic models of dei for CNT/CNFwhich, while being elongated, generally are not cylindrical.

Figure A-1. Breathing flow rates of 15 LPMand 7.4 ml/s were used in Eq. (2) for humansand rats, respectively. CNT/CNF inhalability isgreater for humans than for rats and increasesfurther during by oral breathing than nasalbreathing. In humans, CNT/CNF of 10-μmimpaction diameter or smaller CNT/CNF are100% inhalable. Rats have a significantly lowerinhalability than humans, with about 90% of1-μm and 50% of 3-μm CNT/CNF beinginhalable.

The shape of CNT/CNF affects their aero-dynamic properties and thus transport anddeposition in lung airways. The shape has beenfound to be complex and to vary quite con-siderably based on filter sample observations;however, it has not been characterized so farand thus cannot be described mathematicallyat the present time. The current version ofMPPD assumes CNT/CNF to be shaped ascylinders. In addition, those CNT/CNF that arenot straight may be described by equivalentdiameters for impaction, sedimentation, anddiffusion. Deposition of these particles can be

estimated from the MPPD model for sphericalparticles. Thus, the fiber and spherical parti-cle models of MPPD may be jointly used topredict deposition as first order of approxi-mation. The degree of inaccuracy cannot beestimated until a mathematical model for dif-ferent shape CNT/CNF are developed. Thefiber version of the model will be made avail-able in the near future. CNT/CNF sizes (1 and3 μm) are for equivalent diameter for impactionin Eq. (2). This diameter can take on dif-ferent dimensions as described in Eq. (1) togive the desired impaction diameters of 1 and3 μm. Experimentally, impaction diameter canbe measured by an impactor.

Thoracic FractionA fraction of inhaled materials is deposited

in the extrathoracic airways, and the remain-ing inhaled materials that escape deposition areconsidered to be penetrable into the thoracicregion. The thoracic fraction of CNT/CNF isthe net effect of two filtering mechanisms:inhalability (IF) and removal efficiency in thehead airways (η). Therefore, the thoracic frac-tion (TF) of CNT can be described by

TF = IF × (1 − η) (3)

Losses of inhalable CNT/CNF in the headairways are by inertial impaction. Hence, thesame as for inhalability, CNT/CNF thoracic frac-tion will depend on equivalent diameter forimpaction and inhalation flow rate. Loss effi-ciency expressions for CNT/CNF are obtainedby replacing particle diameter with CNT/CNFimpaction diameters. For humans, nasal andoral breathing deposition efficiencies may begiven by (Rudolf, Kobrich, and Stahlhofen1990; Rudolf et al. 1986; and Stahlhofen,Rudolf, and James 1989)

ηH−n = 1 − 11 + 3 × 10−4d2

eiQ(4)

ηH−o = 1 − 1

1 + 10−4(d2eiQ0.6V−0.2

t)1.4 (5)

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


where Vt is the tidal volume. Similarly, forrats, the following relationship was proposed byZhang and Yu (1993):

ηR−n =[ (

d2eiQ

)2.553

105 + (d2

eiQ)2.553

]0.627

(6)

Respirable FractionAdditional losses of CNT/CNF occur while

passing through conducting airways of the lung,mainly by inertial impaction for micrometer andlarger size CNT/CNF and Brownian diffusionof smaller CNT/CNF sizes. Consequently, theamount of CNT/CNF that reaches the alveolarregion or the respirable fraction is lower thanthoracic fraction. A lung deposition model suchas MPPD is needed to calculate the totalconducting airway losses based on availabledeposition efficiency expressions for impactionand diffusion losses. The respirable fraction ofCNT/CNF is found by correcting TF for thetotal loss fraction of CNT/CNF in the conduct-ing airways. The respirable fraction dependson CNT/CNF size and lung and breathingparameters.

APPENDIX B: EXTRAPOLATION

Extrapolation Based on Inhaled DoseInhaled dose refers to the amount of

CNT/CNF that reaches different regions of therespiratory tract, and it should not serve as abasis for human risk estimates deduced fromlaboratory animal studies. Since the ability ofCNT to reach various locations of the lungdepends on the filtering efficiencies in thepassing airways, one needs to calculate theinhalable, thoracic, and respirable fractions touse as the basis for interspecies extrapolations.

Extrapolation across lab testing and humanexposure scenarios may involve two cases. Theintention in case 1 is to set up an exposureatmosphere in the lab setting (animal inhala-tion studies) based on the human exposurescenario.

Thus, an equivalent CNT/CNF size andconcentration will be sought for the exposuresystems to deliver the same dose to humansand laboratory animals. Size equivalency incase 1 will be based on an equal inhaled dosemetric of exposure (i.e., inhalable, thoracic,or respirable fractions). The opposite is to beachieved in case 2, where human equiva-lent concentration is sought for a given ani-mal inhalation exposure concentration. Again,extrapolation across exposure scenarios will bebased on the dose-metric of equal inhalable,thoracic, or respirable fraction. The same doseequivalent can also be considered as a goal forin vitro studies, either for conventional expo-sure via suspension in culture medium or viaaerosol in an Air Liquid Interface (ALI) sys-tem. The medium concentration could be inter-preted as the equivalent exposure atmospherein the lung regions of interest.

Figure B-1 describes scenario 1, in whichequivalent CNT/CNF sizes will be calculatedfor the design of an animal inhalation study.Given that the thoracic fraction depends onthe size of CNT/CNF, there will be an equiva-lent CNT/CNF size in animal studies for eachCNT/CNF size in the human exposure sce-nario. It is assumed in Figure B-1 that humansare exposed to 2.5-μm-diameter CNT/CNF.The equivalent CNT/CNF size in animals alsodepends on the breathing route in humans. Thecorresponding impaction diameters for animalstudies are 0.4 μm and 1.6 μm for humanoral breathing and nasal breathing, respectively.Minute ventilations of 15 LPM and 7.4 ml/swere assumed in the calculations. If the expo-sure environment is made of various size parti-cles, calculations must be repeated for each sizeinterval. Ideally, an equivalent exposure envi-ronment must be generated with equivalentCNT/CNF sizes and concentrations in each sizeinterval.

For case 2, a similar approach to the pre-ceding one may be applied if the airborne sizeof a rat inhalation study is to be used to deter-mine the human inhaled size of CNT/CNF.However, since the CNT/CNF thoracic frac-tion is greater in humans than rats (Figure B-1),CNT/CNF size correction is not needed in case

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE B-1. CNT/CNF thoracic fractions for humans and rats.

CNT/CNF Thoracic fraction for different sizes for humans and rats at minute ventilations of 15 LPM and 7.4 mL/s, respectively.

2, for which toxicity assessment is based onobserved laboratory outcomes. Human healthrisk is inferred based on in vivo results, whichrequire estimation of human equivalent expo-sure (HEC) concentration for a given laboratorysetting. For example, by assuming the samedelivered dose for human exposure scenariosand rats in an inhalation study, the followingrelationship is obtained for the calculations ofHEC:

HECCR

= TFR(dei)TFH(dei)

×(VE

)R(

VE)

H

× δtRδtH

(7)

where CR is the rat exposure concentration,reported from rodent inhalation studies, VE isthe minute ventilation, and δt is the expo-sure duration. Subscripts R and H refer to ratsand humans, respectively. Figure B-2 presents

the ratio of HEC/CRversus impaction diame-ter of CNT for equal exposure duration forhumans and animals. Minute ventilations of15 LPM and 7.4 ml/s were used in the calcu-lations of Eq. (7). Exposure concentration ratiodecreases with increasing impaction diameterof CNT/CNF. In addition, since the concentra-tions ratio is smaller than unity, HEC must besmaller than the exposure concentration in ratsto yield the same delivered dose.

Extrapolation Based on Deposited DoseWhile inhalable, thoracic, and respirable

fractions are good indicators of the delivereddose to the lung, they do not directly corre-late to the biological response because onlya fraction of inhaled CNT/CNF is depositedon lung airway surfaces. The deposited dose iswhat triggers the biological response and musttherefore be linked to it. Given that human

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE B-2. Human to rat ratios for equal thoracic fractions.

Ratio of human equivalent concentration to rat exposure con-centration based on equal thoracic fractions for minute ventila-tions of 15 LPM and 7.4 mL/s in humans and rats respectively.

exposure studies are not possible due to ethicalconcerns of possible adverse health effects andthat there is a limitation to conducting animalstudies from the standpoint of practicality andcost, lung dosimetry models have been devel-oped to assess deposition of airborne materialsin the lungs of humans and laboratory animals.These models allow direct prediction of bothdeposited and retained doses in the lungs ofhumans and animals and dosimetric extrapo-lation and comparison of such doses betweenanimals and humans for cases 1 and 2.

Predictive models of inhaled aerosols in thelungs of humans and laboratory animals havebeen around for a few decades. First-generationdeposition models were compartmental andlimited in predictive capability. More realis-tic deposition models assumed the lung tobe dynamic and to undergo expansion andcontract during a breathing cycle. The lunggeometry also resembled a trumpet in shapewhen symmetric lung geometry was assumed(Scherer, Shendalman, and Greene 1972; Yu1978). These models have been validated forregional deposition fractions. The human lunghas an asymmetric branching structure thataffects both airflow and particle deposition.

FIGURE B-3. CNT/CNF respiratory fractions for humans at differ-ent aspect ratios.

CNT/CNF deposition fraction in the human lung via nasalbreathing for different aspect ratios. A minute ventilation of7.5 LMP is used in the calculations.

Asgharian, Hofmann, and Bergmann (2001)developed a multiple-path particle dosimetrymodel (MPPD) and used it to predict site-specific and regional deposition of particlesin 10 stochastically generated lungs of humanadults. The stochastic lungs were based on mor-phometric measurements of Raabe et al. (1976)and developed by Koblinger and Hofmann(1985). Significant dose variations among thelung structures were found, which indicatedthe significance of intersubject variability in thepopulation.

Particle deposition models were extendedto CNT/CNF by adjusting for shape and sizeeffects of CNT/CNF (Asgharian and Price 2008for cylindrical shapes). The deposition modelconsisted of four components. First is theextrathoracic component for calculating headlosses of inhalable CNT/CNF so that the deliv-ered dose to the lung could be obtained asalready described . The next component wasthe lung geometry that enabled mechanisticcalculations of CNT/CNF deposition in thisgeometry. Due to complexity of airway struc-ture, multiscale dimensions of the lung, and the

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


FIGURE B-4. HEC/CR ratios for CNT/CNF (100 nm—2 μmdiameter) at aspect ratio of 10.

Ratio of HEC/CR for different diameter CNT/CNF and aspectratio of 10. Minute ventilations for humans and rats were7.5 LPM and 0.241 LPM respectively.

large population of airways, a complete mea-surement of the entire airway tree was infea-sible. Airway measurements typically includedselecting a number of pathways extending fromthe trachea down to the alveolar airways andmeasuring airway dimensions and angles alongthese paths (Raabe et al. 1976). By assuminglung airways to be shaped as a cylinder andperforming statistical analysis of airway param-eters, various lung geometries were devel-oped (Koblinger and Hofmann, 1985; Yeh andSchum 1980) and implemented in MPPD. Lunggeometries in MPPD extended from a typicalpath geometry such as that proposed by Weibel(1963) to a complete asymmetric geometry(Koblinger 1985).

The third component of the deposition wasthe lung ventilation, which in effect was themechanism of transport of CNT/CNF to variouslocations within the lung. The dose (and hencethe effect) at any site in the lung depends on theamount of inhaled material that reaches there.Hence, lung ventilation directly impacts biolog-ical response. Various ventilation models basedon lung compliance and airway resistance were

developed and implemented in the deposi-tion model. At normal low frequency breathing,lung lobar expansion is quasi-steady and lin-ear among lobes. Hence, MPPD implementeda uniform airway expansion and contractionduring a complete breathing cycle (Asgharian,Hofmann, and Bergmann 2001).

The last component of the depositionmodel entailed the application of the first prin-ciples to develop lung-specific conservation ofmass and momentum equations for inhaledCNT/CNF. It was assumed that CNT/CNF con-centration varied with lung depth and time andwas constant across an airway cross-section.The movement of CNT/CNF in an expanding–contracting airway was described by the simpli-fied, cross-sectional area averaged, convective-diffusion (general dynamic) equation:

∂ (AC)

∂t+ ∂ (QC)

∂x= ∂

∂x

(DA

∂C∂x

)− λAC. (8)

where A is the total airway cross-sectional areaand includes the contribution of the surround-ing the airways in the pulmonary region, C isthe CNT/CNF concentration, D is the apparentdiffusion coefficient, Q is the flow rate throughthe airway, λC is the number of CNT/CNF lostto the airways by diffusion per unit time, perunit volume, and x and t are the distance intothe lung and elapsed time, respectively. Theapparent diffusion coefficient D is the sum ofmolecular diffusion coefficient and convectivediffusion (dispersion) coefficient by dispersion.

Losses of uncharged particles in lung air-ways (λAC) are designated by the last termin Eq. (8). Losses occurred by major mecha-nisms of impaction, sedimentation, diffusion,and interception, which were time-invariantfor a given aerosol size and lung and breath-ing parameters. It should be noted that whilethe functional form of Eq. (8) is the samefor different shape particles, CNT/CNF specificshape and orientation effects were embed-ded in models for diffusion coefficient (D) andloss term (λAC) by different deposition mecha-nisms (Asgharian and Price 2008). Currently, theonly deposition model for CNT/CNF is basedon long, straight geometry. The model will

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


be extended to other shapes of CNT/CNF byaccounting for geometry-specific effects, whichalter orientation and hence transport and depo-sition in lung airways.

Prediction of CNT/CNF deposition in thelung provides the missing link between expo-sure and biological response. In humans,CNT/CNF losses are determined based ona given exposure scenario, which com-prises exposure concentration, duration, andCNT/CNF size characteristics. Losses in the lungare then directly linked to the response if thetarget site is within the respiratory tract. Forextrathoracic target sites, the deposition modelis linked to a pharmacokinetic or pharma-codynamic model to establish exposure-dose-response relationships. Deposition fraction ofelongated CNT/CNF in the lung is calculated atdifferent aspect ratios (β) for various CNT/CNFdiameters ranging from 0.01 to 10 μm for thecase of nasal breathing (Figure B-3). The minuteventilation is 7.5 LPM. For diameters below0.4 μm, lung deposition fraction increaseswith decreasing CNT/CNF diameter and aspectratio. Given that CNT/CNF equivalent diameterdepends on its diameter and aspect ratio, depo-sition of submicrometer-diameter CNT/CNF isinversely proportional to its size. For diametersgreater than 0.4 μm, lung deposition shouldincrease with size due to sedimentation andimpaction effects. However, the nasal filteringeffect reduces the amount entering the lung,and as a result, deposition first increases andthen drops sharply to zero. It is also interest-ing to note that the influence of aspect ratiois minimized near a CNT/CNF diameter of0.3 μm.

The predictions in Figure B-3 are basedon the assumption that CNT/CNF are cylin-drically shaped. However, collected CNT/CNFon the stages of nano-MOUDI showed highlyirregular structures and were often tangledtogether. In fact, very few, if any, were straight.Nonetheless, the shapes were clearly elongatedwith large aspect ratios. Therefore, modelingCNT/CNF behavior in the air and depositionon airway surfaces based on cylindrical geom-etry is a fair representation in the absence

of experimental data. The agglomerates takeon various shapes, including compact (spheri-cal), elongated (cylinders), and other (irregular)geometries. However, the majority is believedto be elongated based on observations and thuscan be described by MPPD models for fibers.

In lab animals, predicted deposition canbe extrapolated to human exposure scenar-ios to find the HEC for risk analysis. Labexposure concentrations at which biologicalresponses are observed may be used to esti-mate CNT/CNF deposition in the rat lung.A dose metric such as deposition per unitsurface area that is relevant to the observedeffect is selected and used to extrapolate ani-mal exposure settings to human exposure sce-narios. Under the assumption that the samedose (deposition per unit surface area) inhumans and rats gives the same response,HEC is obtained from the following relationship(Jarabek, Asgharian, and Miller 2005):

HECCR

= δtRδtH

×(VE

)R(

VE)

H

× (DF/SA)R

(DF/SA)H(9)

where DF is the CNT/CNF deposition frac-tion in the lung during one breathing cycleand SA is the surface area for the regionof interest in the lung. Alternatively, deposi-tion per unit lung weight can be selected asthe dose metric if the response of concerninvolves the pulmonary interstitium rather thanthe epithelium, for example a fibrotic response.However, because this is a long-term effectknowledge about pulmonary retention kineticsin rats and humans needs to be available so thatthe HEC can be based on the retained ratherthan the deposited dose (Figure B-3). A longerterm multidose inhalation study (subchronic)that includes a NOAEL should be the goal fordosimetric extrapolation to derive the HEC; theselection of a unit, surface area or mass of thelung as the basis depends on the effect seenat higher concentrations/doses. Preferably, sur-face area should primarily be selected unlessthere is strong evidence of mainly interstitialresponses.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


For human/rat and rat/human extrapola-tion modeling the requirements are:

Determining the CNT/CNF concentration bestin the breathing zone of a worker, sec-ondbest from area monitor.

Determining airborne particle size distribution.Determining what size distribution for rodent

exposure would mimic human exposureinterms of deposited doses in specific regionsof the respiratory tract.

Exposing rodents (preferably whole-body)to different concentrations to establishacute, subchronic, and chronic exposure-dose-response relationships, includingexposureconcentrations that are predictedto result in rodent retained lung burdens(per unit epithelial surface, or per unit lungweight) that correspond to those in exposedworkers.

Analyzing results to determine rodent NOAELfor extrapolating to humans.

The MPPD deposition model was used tocalculate CNT/CNF deposition fraction in ratsfor a given exposure atmosphere (concentrationand CNT/CNF size characteristics) and rat lungand breathing parameters (functional residualcapacity, tidal volume, breathing frequency, andbreathing interval times). A dose metric was cal-culated based on deposition fraction per unitsurface area or mass of the region of interestin the lung (i.e., tracheobronchial, pulmonary,lobar, etc.). The dose metric was used as thebasis to calculate HEC by iteration: Depositionfraction in the human lung was calculated fordifferent exposure concentrations of CNT/CNFuntil the predicted deposited mass per unit sur-face area (or mass) in humans matched thatin rats. Figure B-4 presents HEC/CR calcula-tions for different size CNT/CNF at an aspectratio of 10 with minute ventilations of 7.5 LPMand 0.241 LPM for humans and rats, respec-tively. Equal short exposure duration is usedin the calculations (δtR = δtH). The ratio wascalculated based on tracheobronchial (TB) andpulmonary (PUL) regions as well as for the

entire lung. The surface areas of all airways ineach region were added together to find thetotal surface areas for the TB and PUL regions.The surface area in the PUL region did notinclude the alveoli surface areas, because itis not yet included in the present version ofthe model. For losses by diffusion in the pul-monary region at diameters below 0.5 μm,HEC rose with increasing CNT/CNF diame-ter. Otherwise, HEC decreased with increasingCNT/CNF diameter where losses were by sed-imentation and impaction. Predicted HEC wassimilar for the PUL region and the entire lungbecause the majority of deposition occurred inthe PUL region. In addition, HEC was smallestif it was based on the TB region. When the ratiowas above 1, HEC was larger than rat exposureconcentration and vice versa.

Depending on the extent of availabledata—both from experimental animal inhala-tion and from human exposure—the HEC canbe estimated based on the deposited or also—even more useful—on the long-term retaineddose as explained in Figure B-3. While FigureB-4 was based on equal acute exposure dura-tion and deposition per surface area as the dosemetric, a more relevant dose metric should bebased on a lifelong exposure for both humansand rats. Since CNT/CNF that deposit on lungsurfaces are cleared over time, the dose met-ric should be based on retained dose ratherthan on deposited dose. Unlike the depositeddose, the retained dose of CNT/CNF in the lungdepends on the exposure duration taking clear-ance into account. Thus, an expression similarto Eq. (9) could not be obtained to predict HECfor a lifelong exposure. Instead, an iterativeprocedure needs to be undertaken in whichdifferent human exposure concentrations areselected to calculate the dose (Figure B-3). HECwill correspond to the human exposure con-centration for which the calculated retaineddose is the same as for the animal. Calculationsof HEC based on a dose related to the retaineddose have indicated that HEC will always belower than CR (Jarabek, Asgharian, and Miller2005).

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


GLOSSARY

ACGIH American Conference of Governmental Industrial HygienistsAPM aerosol particle mass analyzerAPS aerodynamic particle sizerBALF bronchoalveolar lavage fluidBAM beta attenuation mass monitorBET Brunauer, Emmet, and TellerBrDU bromodeoxyuridineBW body weightCIB Current Intelligence Bulletin (NIOSH)CMAD count median aerodynamic diameterCMD count median diameterCNF carbon nanofibersCNT carbon nanotubesCNC condensation nuclei counterCPC condensation particle counterCNS central nervous systemCRP C-reactive proteinDF deposition fractionDMA differential mobility analyzerDMA-APM, DMA particle mass analyzerDPBS Dulbecco’s phosphate-buffered salineDSPC disaturated phosphatidyl cholineDWCNT double-walled carbon nanotubeEM electron microscopeENM engineered nanomaterialEPA Environmental Protection AgencyFRC functional residual capacityGSD geometric standard deviationHEC human equivalent concentrationHPLC high-performance liquid chromatographyIARC International Agency for Research on CancerILSI International Life Sciences InstituteISO International Organization for StandardizationLOAEL lowest-observed-adverse-effect levelLDH lactate dehydrogenaseLPM liters per minuteMAC maximum aerosol concentrationMFTD maximum functionally tolerated doseMMAD mass median aerodynamic diameterMPPD multiple path particle dosimetrymRNA messenger ribonucleic acidMOUDI micro-orifice uniform deposition impactorMTD maximum tolerated doseNM nanomaterialNIOSH National Institute for Occupational Safety and HealthNOAEL no-observed-adverse-effect levelNIR near infrared

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015


OECD Organization for Economic Cooperation and DevelopmentOEL occupational exposure levelPBS phosphate-buffered salinePDGF platelet-derived growth factorPMN polymorphonuclear neutrophilsPPE personal protective equipmentPSP poorly soluble particle of low cytotoxicityPTA programmed thermal analysisPUL region, pulmonary regionSA surface areaSEM scanning electron microscopeSMPS scanning mobility particle sizerSTEM scanning transmission electron microscopeSWCNT single-walled carbon nanotubeTB tert-butylalcoholTEM transmission electron microscopeTEOM tapered-element oscillating microbalanceTF thoracic fractionTGF transforming growth factorTLC total lung capacityTV tidal volumeWHO World Health Organization

Dow

nloa

ded

by [

Uni

vers

ity o

f R

oche

ster

] at

07:

54 1

4 Se

ptem

ber

2015

Inhalation Exposure to Carbon Nanotubes (CNT) and Carbon ...

Documents

Transcript of Inhalation Exposure to Carbon Nanotubes (CNT) and Carbon ...