CURRENT INTELLIGENCE BULLETIN 65

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

National Institute for Occupational Safety and Health

Occupational Exposure

to Carbon Nanotubes

and Nanofibers

2 μm
On the cover: High-resolution electron microscope image of a single multi-walled carbon 

nanotube (MWCNT) penetrating out of the lung surface into the pleural space. Figure 7D 

from Mercer et al. Particle and Fibre Toxicology 2010, 7:28 

Article can be found at: http://www.particleandfibretoxicology.com/content/7/1/28

Image courtesy of Robert Mercer and Diane Schwegler-Berry, NIOSH.
Occupational Exposure to

Carbon Nanotubes and Nanofibers

Current Intelligence Bulletin 65
ii NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

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DHHS (NIOSH) Publication No. 2013–145

April 2013

Safer • Healthier • PeopleTM
NIOSH CIB 65 • iii Carbon Nanotubes and Nanofibers

Foreword

The Occupational Safety and Health Act of 1970 (Public Law 91-596) was passed to assure 

safe and healthful working conditions for every working person and to preserve our human resources. This Act charges the National Institute for Occupational Safety and Health 

(NIOSH) with recommending occupational safety and health standards and describing 

exposures that are safe for various periods of employment, including (but not limited to) 

the exposures at which no worker will suffer diminished health, functional capacity, or life 

expectancy because of his or her work experience. 

NIOSH issues Current Intelligence Bulletins (CIBs) to disseminate new scientific information about occupational hazards. A CIB may draw attention to a formerly unrecognized 

hazard, report new data on a known hazard, or disseminate information about hazard control. CIBs are distributed to representatives of academia, industry, organized labor, public 

health agencies, and public interest groups, as well as to federal agencies responsible for 

ensuring the safety and health of workers. 

NIOSH is the leading federal agency conducting research and providing guidance on the 

occupational safety and health implications and applications of nanotechnology. As nanotechnology continues to expand into every industrial sector, workers will be at an increased 

risk of exposure to new nanomaterials. Today, nanomaterials are found in hundreds of 

products, ranging from cosmetics, to clothing, to industrial and biomedical applications. 

These nanoscale-based products are typically called “first generation” products of nanotechnology. Many of these nanoscale-based products are composed of engineered nanoparticles, such as metal oxides, nanotubes, nanowires, quantum dots, and carbon fullerenes 

(buckyballs), among others. Early scientific studies have indicated that some of these nanoscale particles may pose a greater health risk than the larger bulk form of these materials. 

Results from recent animal studies indicate that carbon nanotubes (CNT) and carbon nanofibers (CNF) may pose a respiratory hazard. CNTs and CNFs are tiny, cylindrical, large aspect 

ratio, manufactured forms of carbon. There is no single type of carbon nanotube or nanofiber; one type can differ from another in shape, size, chemical composition (from residual 

metal catalysts or functionalization of the CNT and CNF) and other physical and chemical 

characteristics. Such variations in composition and size have added to the complexity of 

understanding their hazard potential. Occupational exposure to CNTs and CNFs can occur 

not only in the process of manufacturing them, but also at the point of incorporating these 

materials into other products and applications. A number of research studies with rodents 

have shown adverse lung effects at relatively low-mass doses of CNT and CNF, including pulmonary inflammation and rapidly developing, persistent fibrosis. Although it is not known 

whether similar adverse health effects occur in humans after exposure to CNT and CNF, the 

results from animal research studies indicate the need to minimize worker exposure. 

This NIOSH CIB, (1) reviews the animal and other toxicological data relevant to assessing 

the potential non-malignant adverse respiratory effects of CNT and CNF, (2) provides a 
iv NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

quantitative risk assessment based on animal dose-response data, (3) proposes a recommended exposure limit (REL) of 1 μg/m3

 elemental carbon as a respirable mass 8-hour 

time-weighted average (TWA) concentration, and (4) describes strategies for controlling 

workplace exposures and implementing a medical surveillance program. The NIOSH REL 

is expected to reduce the risk for pulmonary inflammation and fibrosis. However, because 

of some residual risk at the REL and uncertainty concerning chronic health effects, including whether some types of CNTs may be carcinogenic, continued efforts should be made to 

reduce exposures as much as possible. 

Just prior to the release of this CIB NIOSH reported at the annual meeting of the Society 

of Toxicology [03/11/2013] preliminary findings from a new laboratory study in which 

mice were exposed by inhalation to multi-walled carbon nanotubes (MWCNT) [see http://

blogs.cdc.gov/niosh-science-blog/2013/03/mwcnt/]. The study was designed to investigate 

whether MWCNT have the potential to initiate or promote cancer. Mice receiving both an 

initiator chemical plus inhalation exposure to MWCNT were significantly more likely to 

develop tumors (90% incidence) and have more tumors than mice receiving the initiator 

chemical alone. These results indicate that MWCNT can increase the risk of cancer in mice 

exposed to a known carcinogen. The study did not indicate that MWCNTs alone cause cancer in mice. This research is an important step in our understanding of the hazards associated with MWCNT, but before we can determine whether MWCNT pose an occupational 

cancer risk, we need more information about workplace exposures, the types and nature 

of MWCNT being used in the workplace, and how that compares to the material used in 

this study. Research is underway at NIOSH to learn more about worker exposures and the 

potential occupational health risks associated with exposure to MWCNT and other types of 

CNTs and CNFs. As results from ongoing research become available, NIOSH will reassess 

its recommendations for CNT and CNF and make appropriate revisions as needed. 

NIOSH urges employers to share this information with workers and customers. NIOSH 

also requests that professional and trade associations and labor organizations inform their 

members about the potential hazards of CNT and CNF. 

John Howard, M.D.

Director, National Institute for Occupational 

 Safety and Health

Centers for Disease Control and Prevention 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers v

Executive Summary

Overview

Carbon nanotubes (CNTs) and nanofibers (CNFs) are some of the most promising materials to result from nanotechnology. The introduction of these materials and products 

using them into commerce has increased greatly in the last decade [Thostenson et al. 2001; 

Invernizzi 2011]. The development of CNT-based applications in a wide range of products 

is expected to provide great societal benefit and it is important that they be developed responsibly to achieve that benefit [Sanchez et al. 2009; Schulte et al 2012]. Worker safety 

and health is a cornerstone of responsible development of an emergent technology because 

workers are the first people in society to be exposed to the products of the technology and 

the workplace is the first opportunity to develop and implement responsible practices.

In this Current Intelligence Bulletin, NIOSH continues its long-standing history of using 

the best available scientific information to assess potential hazards and risks and to provide 

guidance for protecting workers. Since it is early in the development of these materials and 

their applications, there is limited information on which to make protective recommendations. To date, NIOSH is not aware of any reports of adverse health effects in workers using 

or producing CNT or CNF. However, there are studies of animals exposed to CNT and 

CNF that are informative in predicting potential human health effects consistent with ways 

in which scientists traditionally have used such data in recommending risk management 

strategies. NIOSH systematically reviewed 54 laboratory animal studies, many of which 

indicated that CNT/CNF could cause adverse pulmonary effects including inflammation 

(44/54), granulomas (27/54), and pulmonary fibrosis (25/54) (Tables 3–1 through 3–8). 

NIOSH considers these animal study findings to be relevant to human health risks because 

similar lung effects have been observed in workers exposed to respirable particulates of 

other materials in dusty jobs [Rom and Markowitz 2006; Hubbs et al. 2011]. There are well 

established correlations between results of animal studies and adverse effects in workers exposed to particulates and other air contaminants [NIOSH 2002, 2006, 2011a, b]. Moreover, 

in animal studies where CNTs were compared with other known fibrogenic materials (e.g., 

silica, asbestos, ultrafine carbon black), the CNTs were of similar or greater potency [Lam et 

al. 2004; Muller et al. 2005; Shvedova et al. 2005; Murray et al. 2012], and the effects, including fibrosis, developed soon after exposure and persisted [Shvedova et al. 2005, 2008; Porter 

et al. 2010; Mercer et al. 2011]. These are significant findings that warrant protective action. 

NIOSH conducted a quantitative assessment of risk using the animal studies with sufficient 

dose-response data, which included two subchronic (90-day) inhalation studies [Ma-Hock 

et al. 2009; Pauluhn 2010a] and five additional studies [Lam et al. 2004; Muller et al. 2005; 

Shvedova et al. 2005,2008; Mercer et al. 2011] conducted by other routes or durations. The 

estimated risk of developing early-stage (slight or mild) lung effects over a working lifetime 

if exposed to CNT at the analytical limit of quantification (NIOSH Method 5040) of 1 μg/m3

(8-hr time-weighted average [TWA] as respirable elemental carbon) is approximately 0.5% 
vi NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

to 16% (upper confidence limit estimates) (Table A–8). In addition, the working lifetime 

equivalent estimates of the animal no observed adverse effect level (NOAEL) of CNT or 

CNF were also near 1 μg/m3 (8-hr TWA) (Sections A.6.3.3 and A.7.6). Therefore, NIOSH 

recommends that exposures to CNT and CNF be kept below the recommended exposure 

limit (REL) of 1 μg/m3 of respirable elemental carbon as an 8-hr TWA. Because there may 

be other sources of elemental carbon in the workplace that could interfere in the determination of CNT and CNF exposures, other analytical techniques such as transmission 

electron microscopy are described that could assist in characterizing exposures. Studies 

have shown that airborne background (environmental and in non-process areas in the 

workplace) concentrations to elemental carbon are typically less than 1 μg/m3 and that an 

elevated exposure to elemental carbon in the workplace is a reasonable indicator of CNT 

or CNF exposure [Evans et al. 2010; Birch 2011a, b; Dahm et al. 2011]. Studies have also 

shown in some manufacturing operations that exposures can be controlled below the REL 

when engineering controls are used [Dahm et al. 2011]. However, NIOSH has not assessed 

the extent to which exposures can be controlled during the life cycle of CNT/CNF product 

use, but since airborne CNT/CNF behave as classical aerosols, the control of worker exposures appears feasible with standard exposure control techniques (e.g., source enclosure, 

local-exhaust ventilation) [NIOSH 2009a]. Previously in a 2010 draft of this CIB for public 

comment, NIOSH indicated that the risks could occur with exposures less than 1 μg/m3 but 

that the analytic limit of quantification was 7 μg/m3. Based on subsequent improvements 

in sampling and analytic methods, NIOSH is now recommending an exposure limit at the 

current analytical limit of quantification of 1 μg/m3.

More research is needed to fully characterize the health risks of CNT/CNF. Long-term animal studies and epidemiologic studies in workers would be especially informative. However, the toxicity seen in the short-term animal studies indicates that protective action is 

warranted. The recommended exposure limit is in units of mass/unit volume of air, which 

is how the exposures in the animal studies were quantified and it is the exposure metric that 

generally is used in the practice of industrial hygiene. In the future, as more data are obtained, a recommended exposure limit might be based on a different exposure metric better 

correlated with toxicological effects, such as CNT/CNF number concentration [Schulte et 

al. 2012]. 

There are many uncertainties in assessing risks to workers exposed to CNT/CNF. These 

uncertainties, as described and evaluated in this document, do not lessen the concern or diminish the recommendations. Other investigators and organizations have been concerned 

about the same effects and have recommended occupational exposure limits (OELs) for 

CNT within the range of 1–50 μg/m3 [Nanocyl 2009; Aschberger et al. 2010; Pauluhn 2010b; 

Nakanishi (ed) 2011a,b]. The relative consistency in these proposed OELs demonstrates the 

need to manage CNT/CNF as a new and more active form of carbon. To put this in perspective, since there is no Occupational Safety and Health Administration (OSHA) permissible 

exposure limit (PEL) for CNT/CNF, the PEL for graphite (5,000 μg/m3) or carbon black 

(3,500 μg/m3) [NIOSH 2007] might inappropriately be applied as a guide to control worker 

exposures to CNT/CNF. Based on the information presented in this document, the PELs 

for graphite or carbon black would not protect workers exposed to CNT/CNF. 

The analysis conducted by NIOSH was focused on the types of CNT and CNF included in 

published research studies. Pulmonary responses were qualitatively similar across the various types of CNT and CNF, purified or unpurified with various metal content, and different 

dimensions [Lam et al. 2004; Shvedova et al. 2005, 2008; Muller et al. 2005; Ma-Hock et al. 
NIOSH CIB 65 • vii Carbon Nanotubes and Nanofibers

2009; Pauluhn 2010a; Porter et al. 2010; Mercer et al. 2011; Murray et al. 2012; DeLorme 

et al. 2012]. The fibrotic lung effects in the animal studies developed early (within a few 

weeks) after exposure to CNT or CNF, at relatively low-mass lung doses, and persisted or 

progressed during the post-exposure follow-up (~1–6 months) [Shvedova et al. 2005, 2008; 

Mercer et al. 2008; Porter et al. 2010; Pauluhn 2010a; Murray et al. 2012]. However, the 

studied CNT and CNF only represent a fraction of the types of CNT and CNF that are, or 

will be, in commerce and it is anticipated that materials with different physical and chemical parameters could have different toxicities. At this time, however, given the findings in 

the published literature, NIOSH recommends that exposures to all CNT and CNF be controlled to less than 1 μg/m3

 of respirable elemental carbon as an 8-hr TWA, and that the risk 

management guidance described in this document be followed. Until results from research 

can fully explain the physical-chemical properties of CNT and CNF that define their inhalation toxicity, all types of CNT and CNF should be considered a respiratory hazard and 

exposure should be controlled below the REL.

In addition to controlling exposures below the REL, it is prudent for employers to institute medical surveillance and screening programs for workers who are exposed to CNT 

and CNF for the purpose of possibly detecting early signs of adverse pulmonary effects 

including fibrosis. Such an assessment can provide a secondary level of prevention should 

there be inadequacies in controlling workplace exposures. In 2009, NIOSH concluded that 

there was insufficient evidence to recommend specific medical tests for workers exposed to 

the broad category of engineered nanoparticles but when relevant toxicological information became available, specific medical screening recommendations would be forthcoming 

[NIOSH 2009b]. As described in this document, the toxicologic evidence on CNT/CNF has 

advanced to make specific recommendations for the medical surveillance and screening of 

exposed workers. That is, the strong evidence for pulmonary fibrosis from animal studies 

and the fact that this effect can be detected by medical tests is the basis for NIOSH specific 

medical screening recommendations. NIOSH also recommends other risk management 

practices in addition to controlling exposure and medical surveillance. These include education and training of workers and the use of personal protective equipment (e.g., respirators, clothing, and gloves). 

In summary, the findings and recommendations in this Current Intelligence Bulletin are 

intended to minimize the potential health risks associated with occupational exposure to 

CNT and CNF by recommending a working lifetime exposure limit (1 μg/m3, 8-hr TWA, 45 

years), a sampling and analytical method to detect CNT and CNF, medical surveillance and 

screening and other guidelines. The expanding use of CNT/CNF products in commerce 

and research warrants these protective actions. 

Background 

The goal of this occupational safety and health guidance for carbon nanotubes (CNT) and 

carbon nanofibers (CNT) is to prevent the development of adverse respiratory health effects in workers. To date, NIOSH is not aware of any reports of adverse health effects in 

workers producing or using CNT or CNF. The concern about worker exposure to CNT or 

CNF arises from the results of recent laboratory animal studies with CNT and CNF. Shortterm and subchronic studies in rats and mice have shown qualitatively consistent noncancerous adverse lung effects including pulmonary inflammation, granulomas, and fibrosis 

with inhalation, intratracheal instillation, or pharyngeal aspiration of several types of CNT 
viii NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

(single or multiwall; purified or unpurified). These early-stage, noncancerous adverse lung 

effects in animals include: (1) the early onset and persistence of pulmonary fibrosis in 

CNT-exposed mice [Shvedova et al. 2005, 2008; Porter et al. 2010; Mercer et al. 2011], (2) 

an equal or greater potency of CNT compared with other inhaled particles known to be 

hazardous (e.g., crystalline silica, asbestos) in causing pulmonary inflammation and fibrosis 

[Lam et al. 2004; Shvedova et al. 2005; Muller et al. 2005], and (3) reduced lung clearance 

in mice or rats exposed to relatively low-mass concentrations of CNT [Mercer et al. 2009; 

Pauluhn 2010a]. Findings of acute pulmonary inflammation and interstitial fibrosis have 

also been observed in mice exposed to CNF [Murray et al. 2012]. The extent to which these 

animal data may predict clinically significant lung effects in workers is not known. However, NIOSH considers these animal study findings of pulmonary inflammation, granulomas, 

and fibrosis associated with exposure to CNT and CNF to be relevant to human health risk 

assessment because similar lung effects have been observed in workers in dusty jobs [Rom 

and Markowitz 2006; Hubbs et al. 2011]. 

Some studies also indicate that CNT containing certain metals (nickel, 26%) [Lam et al. 

2004] or higher metal content (17.7% vs. 0.2% iron) are more cytotoxic in vitro and in vivo 

[Shvedova et al. 2003, 2008]. However, in experimental animal studies, both unpurified and 

purified (low metal content) CNT are associated with early onset and persistent pulmonary 

fibrosis and other adverse lung effects [Lam et al. 2004; Shvedova et al. 2005; 2008]. Other 

studies indicate that differences in physical-chemical properties, including functionalization or bio-modification, may alter the lung retention and biological responses [Kagan et al. 

2010; Osmond-McLeod et al. 2011; Pauluhn 2010a; Oyabu et al. 2011]. Although a number 

of different types of CNT and CNF have been evaluated, uncertainty exists on the generalizability of the current animal findings to new CNT and CNF. 

In addition to the early-stage non-cancer lung effects in animals, some studies in cells or 

animals have shown genotoxic or carcinogenic effects. In vitro studies with human lung 

cells have shown that single-walled carbon nanotubes (SWCNT) can cause genotoxicity 

and abnormal chromosome number by interfering with mitosis (cell division) [Muller et 

al. 2008b; Sargent et al. 2009, 2011; Kisin et al. 2011]. Other in vitro studies did not show 

evidence of genotoxicity of some MWCNT [Wirnitzer et al. 2009; Kim et al. 2011]. 

Studies in mice exposed to multi-walled carbon nanotubes (MWCNT) have shown the 

migration of MWCNT from the pulmonary alveoli to the intrapleural space [Hubbs et al. 

2009; Porter et al. 2010; Mercer et al. 2010]. The intrapleural space is the same site in which 

malignant mesothelioma can develop due to asbestos exposure. Intraperitoneal injection of 

CNT in mice has resulted in inflammation from long MWCNT (> 5 μm in length), but not 

short MWCNT (< 1 μm in length) or tangled CNT [Poland et al. 2008; Takagi et al. 2008; 

Muller et al. 2009; Murphy et al. 2011]. In rats administered CNT by peritoneal injection, 

the pleural inflammation and mesothelioma were related to the thin diameter and rigid 

structure of MWCNT [Nagai et al. 2011]. In a study of rats administered MWCNT or crocidolite by intrapulmonary spraying, exposure to either material produced inflammation in 

the lungs and pleural cavity in addition to mesothelial proliferative lesions [Xu et al. 2012]. 

Pulmonary exposure to CNT has also produced systemic responses including an increase 

in inflammatory mediators in the blood, as well as oxidant stress in aortic tissue and increase plaque formation in an atherosclerotic mouse model [Li et al. 2007; Erdely et al. 

2009]. Pulmonary exposure to MWCNT also depresses the ability of coronary arterioles 

to respond to dilators [Stapleton et al. 2011]. These cardiovascular effects may be due to 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers ix

neurogenic signals from sensory irritant receptors in the lung. Mechanisms, such as inflammatory signals or neurogenic pathways causing these systemic responses, are under 

investigation. 

Additional research is needed to fully explain the mechanisms of biological responses to 

CNT and CNF, and the influence of physical-chemical properties. The findings of adverse 

respiratory effects and systemic effects reported in several animal studies indicate the need 

for protective measures to limit worker exposure to CNT and CNF. 

CNT and CNF are currently used in many industrial and biomedical applications, including electronics, lithium-ion batteries, solar cells, super capacitors, thermoplastics, polymer composites, coatings, adhesives, biosensors, enhanced electron-scanning microscopy 

imaging techniques, inks, and in pharmaceutical/biomedical devices. CNT and CNF can 

be encountered in facilities ranging from research laboratories and production plants to 

operations where CNT and CNF are processed, used, disposed, or recycled. The data on 

worker personal exposures to CNT and CNF are extremely limited, but reported workplace 

airborne concentrations for CNT [Maynard et al. 2004; Han et al. 2008a; Bello et al. 2009, 

2010; Tsai et al. 2009; Lee et al. 2010; Cena and Peters 2011; Dahm et al. 2011] and CNF 

[Methner et al. 2007; Evans et al. 2010; Birch 2011a; Birch et al. 2011b] indicate the potential 

for worker exposures in many tasks or processes and the reduction or elimination of exposures when measures to control exposure are used.

Assessment of the Health Risk and Recommended 

Exposure Limit

NIOSH has determined that the best data to use for a quantitative risk assessment and 

as basis for a recommended exposure limit (REL) are the nonmalignant pulmonary data 

from the CNT animal studies. At present, data on cancer and cardiovascular effects are not 

adequate for a quantitative risk assessment of inhalation exposure. NIOSH considers the 

pulmonary responses of inflammation and fibrosis observed in short-term and subchronic 

studies in animals to be relevant to humans, as inflammatory and fibrotic effects are also 

observed in occupational lung diseases associated with workplace exposures to other inhaled particles and fibers. Uncertainties include the extent to which these lung effects in 

animals are associated with functional deficits and whether similar effects would be clinically significant among workers. However, these fibrotic lung effects observed in some of 

the animal studies developed early (e.g., 28 days after exposure) in response to relatively 

low-mass lung doses, and also persisted or progressed after the end of exposure [Shvedova 

et al. 2005, 2008; Ma-Hock et al. 2009; Pauluhn 2010a; Porter et al. 2010; Mercer et al. 2011; 

DeLorme et al. 2012; Murray et al. 2012]. Given the relevance of these types of lung effects 

to humans, the REL was derived using the published subchronic and short-term animal 

studies with dose-response data of early stage fibrotic and inflammatory lung responses to 

CNT exposure (Section 5 and Appendix A). 

Critical effect levels for the noncancerous lung effects estimated from the animal doseresponse data (e.g., BMD, benchmark dose and BMDL, the 95% lower confidence limit estimates of the BMD) have been extrapolated to humans by accounting for the factors influencing the lung dose in each animal species. The no observed adverse effect level (NOAEL) 

and lowest observed adverse effect level (LOAEL) estimates reported in the subchronic 

inhalation studies were also evaluated as the critical effect levels. Working-lifetime exposure 
x NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

concentrations were calculated based on estimates of either the deposited or retained alveolar lung dose of CNT assuming an 8-hour time-weighted average (TWA) exposure during 

a 40-hour workweek, 50 weeks per year, for 45 years. Based on BMD modeling of the subchronic animal inhalation studies with MWCNT [Ma-Hock et al. 2009; Pauluhn 2010a], a 

working lifetime exposure of 0.2–2 μg/m3 (8-hour TWA concentration) was estimated to be 

associated with a 10% excess risk of early-stage adverse lung effects (95% lower confidence 

limit estimates) (Tables 5–1 and A–5). Risk estimates derived from short-term animal studies (Tables A–3 and A–4) were consistent with these estimates. 

In addition to the BMD-based risk estimates, NOAEL or LOAEL values were used as the 

critical effect level in animals. As with the BMD(L) estimates, the human-equivalent working 

lifetime concentrations were estimated, although using dosimetric adjustment and uncertainty factors (Section A.6.3). The estimated human-equivalent working lifetime concentrations based on this approach were approximately 4–18 μg/m3

 (8-hr TWA), depending on the 

subchronic study and the interspecies dose retention and normalization factors used. Dividing these estimates by data-suitable uncertainty factors (e.g., UFs of 20–60), and assuming a 

threshold model, the estimated zero risk levels were <1 μg/m3 as working lifetime 8-hr TWA 

concentrations. A recent subchronic inhalation (13-wk exposure plus 3 months follow-up) 

study of CNF in rats [DeLorme et al. 2012] showed qualitatively similar lung response as 

in a shorter-term (28-day) study of CNF administered by pharyngeal aspiration in mice 

[Murray et al. 2012] (Sections 3.5 and A.7). Using the NOAEL-based approach, the humanequivalent working lifetime concentration estimates were 1–4 μg/m3

 (8-hr TWA), depending on the data and assumptions used to estimate the human-equivalent dose (Section A.7).

In the 2010 draft Current Intelligence Bulletin (CIB) Occupational Exposure to Carbon 

Nanotubes and Nanofibers, NIOSH proposed a REL of 7 μg/m3 elemental carbon (EC) 8-hr 

TWA, which was set at the upper limit of quantitation (LOQ) for NIOSH Method 5040 

[NIOSH 2010a]. In the draft CIB, NIOSH acknowledged that workers may still have an 

excess risk of developing early-stage pulmonary effects including fibrosis if exposed over 

a full working lifetime at the proposed REL. In view of these health risks, and ongoing 

improvements in sampling and analytical methodologies, NIOSH is recommending a REL 

of 1 μg/m3 EC as an 8-hr TWA respirable mass concentration using NIOSH Method 5040 

(Section 6.1, Appendix C). The 45-yr working lifetime excess risk estimates of minimal level 

(grade 1 or greater) lung effects in rats observed by histopathology at 1 μg/m3 (8-hr TWA 

concentration) range from 2.4% to 33% (maximum likelihood estimates, MLE) and 5.3% to 

54% (95% upper confidence limit, UCL) estimates (Table A–7). The 45-yr working lifetime 

excess risk estimates of slight/mild (grade 2) lung effects at 1 μg/m3 (8-hr TWA) range from 

0.23% to 10% MLE and 0.53% to 16% (95% UCL) (Tables 5–2 and A–8). These estimates 

are based on a risk assessment using dose-response data from the rat subchronic inhalation 

studies of two types of MWCNT. The range in these risk estimates reflects differences across 

studies and/or types of MWCNT and the uncertainty in the estimation of working lifetime 

CNT lung burden. The lung burden estimates are based on either the retained lung dose 

(normal clearance) or deposited lung dose (no clearance). Although data from animal studies with CNF are more limited [Murray et al. 2012; DeLorme et al. 2012], physical-chemical 

similarities between CNT and CNF and findings of acute pulmonary inflammation and 

interstitial fibrosis in animals exposed to CNF [Murray et al. 2012] indicate the need to also 

control occupational exposure to CNF at the REL of 1 μg/m3 EC. Because of uncertainties 

in the risk estimates some residual risk for adverse lung effects may exist at the REL; therefore, efforts should be made to reduce airborne concentrations to CNT and CNF as low as 
NIOSH CIB 65 • xi Carbon Nanotubes and Nanofibers

possible. Until the results from animal research studies can fully explain the mechanisms 

(e.g., shape, size, chemistry, functionalized) that potentially increase or decrease their toxicity all types of CNT and CNF should be considered a respiratory hazard and occupational 

exposures controlled at the REL of 1 μg/m3.

Exposure Measurement and Controls

Occupational exposure to all types of CNT and CNF can be quantified using NIOSH Method 5040. A multi-tiered exposure measurement strategy is recommended for determining worker exposure to CNT and CNF [Section 6.1]. When exposure to other types of 

EC (e.g., diesel soot, carbon black) are absent or negligible, environmental background 

EC concentrations are typically < 1 μg/m3 including in facilities where CNT and CNF are 

produced and used [Evans et al. 2010; Birch 2011a, b; Dahm et al. 2011]. Thus, an elevated 

airborne EC concentration relative to background (environmental and in non-process areas in the workplace) is a reasonable indicator of CNT or CNF exposure. When exposure 

to other types of EC is possible, additional analytical techniques may be required to better 

characterize exposures. For example, analysis of airborne samples by transmission electron 

microscopy (TEM) equipped with energy dispersive x-ray spectroscopy (EDS) can help to 

verify the presence of CNT and CNF (Section 6.1.2).

Published reports of worker exposure to CNT and CNF using NIOSH Method 5040 (EC 

determination) are limited but in the study by Dahm et al. [2011] worker personal breathing zone (PBZ) samples collected at CNT manufacturers frequently found low to nondetectable mass concentrations of EC when engineering controls were present. In a study 

by Birch et al. [2011a], the outdoor air concentrations over four survey days, two months 

apart, were nearly identical, averaging about 0.5 μg/m3. Respirable EC area concentrations 

inside the facility were about 6–68 times higher than outdoors, while personal breathing 

zone samples were up to 170 times higher. In studies where airborne particle concentrations 

were used as a surrogate for measuring the potential release of CNT and CNF, the use of 

engineering controls (e.g., local exhaust ventilation, wet cutting of composites, fume hood/

enclosures) appeared to be effective in reducing worker exposure [Han et al. 2008; Bello et 

al. 2009; Tsai et al. 2009; Methner et al. 2010a; Cena and Peters 2011] (Section 2.1). However, direct reading instruments used in these studies are non-selective tools and often subject 

to interferences due to other particle sources, especially at low concentrations [Evans et al. 

2010; Birch et al. 2011]. Control strategies and technologies developed by several industrial trade associations have proven successful in managing micrometer-sized fine powder 

processes, and should have direct application to controlling worker exposures from CNT 

and CNF processes. Examples include guidance issued for containing dry powder during 

manufacturing of detergents by the Association Internationale de la Savonnerie, de la Détergence et des Produits d’Entretien (AISE) [AISE 2001]. Following these guidelines makes 

it possible, at a minimum, to control enzyme-containing dust exposures below 60 ng/m3

for enzymes. Additional guidance on a broader process and facility approach is available 

from the International Society for Pharmaceutical Engineering (ISPE). This organization 

offers guidance on the design, containment, and testing of various processes that handle 

finely divided dry powder formulations. One guide in particular, Baseline Guide Volume 1, 

2nd Edition: Active Pharmaceutical Ingredients Revision to Bulk Pharmaceutical Chemicals, has broad applicability to CNT and CNF processes and is available from ISPE [ISPE 

2007]. Finally, the Institute for Polyacrylate Absorbents (IPA) has developed guidelines for 
xii NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

its member companies to assist them in controlling worker exposures to fine polyacrylate 

polymer dust in the micrometer-size range through a combination of engineering controls 

and work practices [IPA 2013]. The extent to which worker exposure to CNT and CNF can 

be controlled below 1 μg/m3 respirable mass concentration as an 8-hr TWA is unknown, 

but should be achievable in most manufacturing and end-use job tasks if engineering controls are used and workers are instructed in the safe handling of CNT/CNF materials. 

Until results from research studies can fully explain the physical-chemical properties of 

CNT and CNF that define their inhalation toxicity, all types of CNT and CNF should be 

considered a respiratory hazard, and exposures should be controlled as low as possible 

below the REL. The REL is based on the respirable airborne mass concentration of CNT 

and CNF because the adverse lung effects in animals were observed in the alveolar (gasexchange) region. “Respirable” is defined as the aerodynamic size of particles that, when 

inhaled, are capable of depositing in the alveolar region of the lungs [ICRP 1994]. Sampling 

methods have been developed to estimate the airborne mass concentration of respirable 

particles [ACGIH 1984; CEN 1993; ISO 1995; NIOSH 1998]. Reliance on a respirable EC 

mass-based REL will provide a means to identify job tasks with potential exposures to CNT 

and CNF so that appropriate measures can be taken to limit worker exposure. 

Recommendations

In light of current scientific evidence from experimental animal studies concerning the hazard potential of CNT and CNF, steps should be taken to implement an occupational health 

surveillance program that includes elements of hazard and medical surveillance. NIOSH 

recommends that employers and workers take the following steps to minimize potential 

health risks associated with exposure to CNT and CNF.

1. Recommendations for Employers

• Use available information to continually assess current hazard potential related to CNT 

and CNF exposures in the workplace and make appropriate changes (e.g., sampling and 

analysis, exposure control) to protect worker health. At a minimum, follow requirements of the OSHA Hazard Communication Standard [CFR 1910.1200(h)] and the 

Hazardous Waste Operation and Emergency Response Standard [29 CFR 1910.120]. 

• Identify and characterize processes and job tasks where workers encounter bulk 

(“free-form”) CNT or CNF and materials that contain CNT/CNF (e.g., composites).

• Substitute, when possible, a nonhazardous or less hazardous material for CNT and 

CNF. When substitution is not possible, use engineering controls as the primary 

method for minimizing worker exposure to CNT and CNF.

• Establish criteria and procedures for selecting, installing, and evaluating the performance 

of engineering controls to ensure proper operating conditions. Make sure workers are 

trained in how to check and use exposure controls (e.g., exhaust ventilation systems).

• Routinely evaluate airborne exposures to ensure that control measures are working 

properly and that worker exposures are being maintained below the NIOSH REL of 

1 μg/m3 using NIOSH Method 5040 (Section 6 and Appendix C).
NIOSH CIB 65 • xiii Carbon Nanotubes and Nanofibers

• Follow exposure and hazard assessment procedures for determining the need for and 

selection of proper personal protective equipment, such as clothing, gloves, and respirators (Section 6). 

• Educate workers on the sources and job tasks that may expose them to CNT and CNF, 

and train them about how to use appropriate controls, work practices, and personal 

protective equipment to minimize exposure (Section 6.3).

• Provide facilities for hand washing and encourage workers to make use of these facilities before eating, smoking, or leaving the worksite. 

• Provide facilities for showering and changing clothes, with separate facilities for storage of nonwork clothing, to prevent the inadvertent cross-contamination of nonwork 

areas (including take-home contamination). 

• Use light-colored gloves, lab coats, and workbench surfaces to make contamination 

by dark CNT and CNF easier to see. 

• Develop and implement procedures to deal with cleanup of CNT and CNF spills and 

decontamination of surfaces.

• When respirators are provided for worker protection, the OSHA respiratory protection standard [29 CFR 1910.134] requires that a respiratory protection program be 

established that includes the following elements: 

ȣ A medical evaluation of the worker’s ability to perform the work while wearing 

a respirator.

ȣ Regular training of personnel.

ȣ Periodic workplace exposure monitoring.

ȣ Procedures for selecting respirators. 

ȣ Respirator fit testing.

ȣ Respirator maintenance, inspection, cleaning, and storage.

• The voluntary use of respirators are permitted, but must comply with the provisions 

set forth in CFR 1910.134(c)(2)(i) and CFR 1910.134(c)(2)(ii).

• Information on the potential health risks and recommended risk management practices contained in this CIB should, at a minimum, be used when developing labels and 

Safety Data Sheets (SDS), as required [http://www.osha.gov/dsg/hazcom]. 

1.1 Medical Screening and Surveillance

The evidence summarized in this document leads to the conclusion that workers occupationally exposed to CNT and CNF may be at risk of adverse respiratory effects. These workers may 

benefit from inclusion in a medical screening program to help protect their health (Section 6.7). 

1.1.1 Worker Participation

Workers who could receive the greatest benefit from medical screening include the following: 

• Workers exposed to concentrations of CNT or CNF in excess of the REL (i.e., all 

workers exposed to airborne CNT or CNF at concentrations above 1 μg/m3 EC as an 

8-hr TWA). 
xiv NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

• Workers in areas or jobs that have been qualitatively determined (by the person 

charged with program oversight) to have the potential for intermittent elevated airborne concentrations to CNT or CNF (i.e., workers are at risk of being exposed when 

they are involved in the transfer, weighing, blending, or mixing of bulk CNT or CNF, 

or the cutting or grinding of composite materials containing CNT or CNF, or workers 

in areas where such activities are carried out by others).

1.1.2 Program Oversight

Oversight of the medical surveillance program should be assigned to a qualified healthcare professional who is informed and knowledgeable about potential workplace exposures, routes of exposure, and potential health effects related to CNT and CNF.

1.1.3 Screening Elements

Initial Evaluation

• An initial (baseline) evaluation should be conducted by a qualified health-care professional and should consist of the following:

ȣ An occupational and medical history, with respiratory symptoms assessed by 

use of a standardized questionnaire, such as the American Thoracic Society 

Respiratory Questionnaire [Ferris 1978] or the most recent.

ȣ A physical examination with an emphasis on the respiratory system. 

ȣ A spirometry test (Anyone administering spirometry testing as part of the medical 

screening program should have completed a NIOSH-approved training course 

in spirometry or other equivalent training; additionally, the health professional 

overseeing the screening and surveillance program should be expert in interpreting 

spirometry testing results, enabling follow-up evaluation as needed.). 

ȣ A baseline chest X-ray (digital or film-screen radiograph). All baseline chest 

images should be clinically interpreted by a board eligible/certified radiologist 

or other physician with appropriate expertise, such as a board eligible/certified 

pulmonologist. Periodic follow up chest X-rays may be considered, but there is 

currently insufficient evidence to evaluate effectiveness. However, if periodic 

follow up is obtained, clinical interpretation and classification of the images by 

a NIOSH-certified B reader using the standard International Classification of 

Radiographs of Pneumoconioses (ILO 2011 or the most recent equivalent) are 

recommended. 

ȣ Other examinations or medical tests deemed appropriate by the responsible 

health-care professional (The need for specific medical tests may be based on 

factors such as abnormal findings on initial examination—for example, the 

findings of an unexplained abnormality on a chest X-ray should prompt further 

evaluation that might include the use of high-resolution computed tomography 

scan of the thorax.). 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers xv

Periodic Evaluations

• Evaluations should be conducted at regular intervals and at other times (e.g., postincident) as deemed appropriate by the responsible health-care professional based 

on data gathered in the initial evaluation, ongoing work history, changes in symptoms such as new, worsening, or persistent respiratory symptoms, and when process 

changes occur in the workplace (e.g., a change in how CNT or CNF are manufactured 

or used or an unintentional “spill”). Evaluations should include the following:

ȣ An occupational and medical history update, including a respiratory symptom 

update, and focused physical examination—performed annually.

ȣ Spirometry—testing less frequently than every 3 years is not recommended 

[OSHA NIOSH 2011]; and

ȣ Consideration of specific medical tests (e.g., chest X-ray). 

Written reports of medical findings

• The health-care professional should give each worker a written report containing 

the following:

ȣ The individual worker’s medical examination results.

ȣ Medical opinions and/or recommendations concerning any relationships 

between the individual worker’s medical conditions and occupational exposures, 

any special instructions on the individual’s exposures and/or use of personal 

protective equipment, and any further evaluation or treatment. 

• For each examined employee, the health-care professional should give the employer a 

written report specifying the following:

ȣ Any work or exposure restrictions based on the results of medical evaluations.

ȣ Any recommendations concerning use of personal protective equipment.

ȣ A medical opinion about whether any of the worker’s medical conditions is likely 

to have been caused or aggravated by occupational exposures.

• Findings from the medical evaluations having no bearing on the worker’s ability 

to work with CNT or CNF should not be included in any reports to employers. 

Confidentiality of the worker’s medical records should be enforced in accordance 

with all applicable regulations and guidelines.

1.1.4 Worker Education 

Workers should be provided information sufficient to allow them to understand the nature 

of potential workplace exposures, potential health risks, routes of exposure, and instructions for reporting health symptoms. Workers should also be provided with information 

about the purposes of medical screening, the health benefits of the program, and the procedures involved. 

1.1.5 Periodic Evaluation of Data and Screening Program 

• Standardized medical screening data should be periodically aggregated and evaluated 

to identify worker health patterns that may be linked to work activities and practices 
xvi NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

that require additional primary prevention efforts. This analysis should be performed 

by a qualified health professional or other knowledgeable person to identify worker 

health patterns that may be linked to work activities or exposures. Confidentiality of 

workers’ medical records should be enforced in accordance with all applicable regulations and guidelines. 

• Employers should periodically evaluate the elements of the medical screening program to ensure that the program is consistent with current knowledge related to exposures and health effects associated with occupational exposure to CNT and CNF.

Other important components related to occupational health surveillance programs, including medical surveillance and screening, are discussed in Appendix B.

2. Recommendations for Workers

• Ask your supervisor for training in how to protect yourself from the potential hazards 

associated with your job, including exposure to CNT and CNF.

• Know and use the exposure control devices and work practices that keep CNT and 

CNF out of the air and off your skin.

• Understand when and how to wear a respirator and other personal protective 

equipment (such as gloves, clothing, eyewear) that your employer might provide.

• Avoid handling CNT and CNF in a ‘free particle’ state (e.g., powder form).

• Store CNT and CNF, whether suspended in liquids or in a powder form, in closed 

(tightly sealed) containers whenever possible. 

• Clean work areas at the end of each work shift (at a minimum) using a HEPA-filtered 

vacuum cleaner or wet wiping methods. Dry sweeping or air hoses should not be used 

to clean work areas. 

• Do not store or consume food or beverages in workplaces where bulk CNT or CNF, 

or where CNT- or CNF-containing materials, are handled. 

• Prevent the inadvertent contamination of nonwork areas (including take-home contamination) by showering and changing into clean clothes at the end of each workday.
NIOSH CIB 65 • xvii Carbon Nanotubes and Nanofibers

Contents

Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Executive Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Assessment of the Health Risk and Recommended Exposure Limit. . . . . . . . . . . . ix

Exposure Measurement and Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

1. Recommendations for Employers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

1.1 Medical Screening and Surveillance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1.1.1 Worker Participation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1.1.2 Program Oversight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1.1.3 Screening Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

1.1.4 Worker Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

1.1.5 Periodic Evaluation of Data and Screening Program . . . . . . . . . . . . . . . xv

2. Recommendations for Workers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii

1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Potential for Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 Exposure to Carbon Nanotubes during Research and Development,

Small-scale Manufacturing, and Use Applications. . . . . . . . . . . . . . . . . . . . . . . . 3

2.2 Exposure to Carbon Nanotubes (other sources). . . . . . . . . . . . . . . . . . . . . . . . . 9

2.3 Exposure to Carbon Nanofibers during Research and Small-scale

Manufacturing Operations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3 Evidence for Potential Adverse Health Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.1 Single-Walled Carbon Nanotubes (SWCNT). . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.1.1 IT Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.1.2 Pharyngeal Aspiration Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.1.3 Inhalation Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.2 Multi-Walled Carbon Nanotubes (MWCNT). . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.2.1 Pharyngeal Aspiration Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.2.2 IT Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.2.3 Inhalation Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.3 SWCNT and MWCNT Intraperitoneal Studies. . . . . . . . . . . . . . . . . . . . . . . . . . 19
xviii NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

3.4 Systemic Responses to Pulmonary Exposure to SWCNT and MWCNT. . . . . 20

3.4.1 Translocation of CNT to Systemic Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.4.2 Systemic Inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3.5 Carbon Nanofibers (CNF). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4 Conclusions—Hazard and Exposure Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

5 CNT Risk Assessment and Recommended Exposure Limit. . . . . . . . . . . . . . . . . . . . . . 37

5.1 Risk Assessment and Recommended Exposure Limit (REL). . . . . . . . . . . . . . 37

5.2 Other Derived Occupational Exposure Limits for CNT . . . . . . . . . . . . . . . . . . 43

5.3 Evaluation of Uncertainties in CNT Risk Assessment and REL. . . . . . . . . . . . 46

5.3.1 Strength of Evidence for Estimating a Health-Based REL

for CNT and CNF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.3.2 Major Areas of Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.3.3 Minor Areas of Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

6 Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.1 Exposure Assessment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.1.1 Exposure Monitoring Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.1.2 CNT and CNF Measurement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.1.3 Method 5040 Limit of Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.2 Engineering Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

6.3 Worker Education and Training. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

6.4 Cleanup and Disposal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.5 Personal Protective Clothing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.6 Respirators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

6.7 Medical Screening and Surveillance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.7.1 Worker Participation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.7.2 Program Oversight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

6.7.3 Screening Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

6.7.4 Worker Education. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6.7.5 Periodic Evaluation of Data and Surveillance Program . . . . . . . . . . . . . 69

7 Research Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

7.1 Workplace Exposures, Measurement, and Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . 71

7.2 Experimental and Human Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Appendix A—Quantitative Risk Assessment of CNT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

A.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

A.2 Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

A.2.1 Rodent Dose-response Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

A.2.2 Estimated Lung Dose in Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

A.2.3 Animal Dose-response Modeling and BMD Estimation. . . . . . . . . . . . . . . 103

A.3 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

A.3.1 Benchmark Dose and Working Lifetime Exposure Estimates. . . . . . . . 110
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers xix

A.3.2 Comparison of Short-term and Subchronic Dose-response Data. . . . . 115

A.3.3 Estimated Excess Risks of Working Lifetime Exposures to

Low-mass Concentrations of MWCNT. . . . . . . . . . . . . . . . . . . . . . . . . . . 115

A.4 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

A.4.1 The Use of Short-term Data to Predict Longer-term Response. . . . . . . 119

A.4.2 Physical-Chemical Properties and Metal Content. . . . . . . . . . . . . . . . . . 120

A.4.3 Lung Dose Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A.4.4 Critical Effect Level Estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

A.4.5 Animal Dose-response Data Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

A.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

A.6 Sensitivity Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

A.6.1 Lung Dose Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

A.6.2 Critical Effect Level Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

A.6.3 Alternative OEL Estimation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

A.6.4 Summary of Sensitivity Analyses Findings . . . . . . . . . . . . . . . . . . . . . . . . 137

A.7 Evaluation of Carbon Nanofiber Studies in Mice and Rats. . . . . . . . . . . . . . . . 137

A.7.1 Particle Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.2 Experimental Design and Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.3 Lung Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.4 Effects in Other Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

A.7.5 Equivalent Lung Dose Estimation Methods. . . . . . . . . . . . . . . . . . . . . . . 140

A.7.6 Equivalent Lung Dose Estimation Results. . . . . . . . . . . . . . . . . . . . . . . . . 141

Appendix B—Occupational Health Surveillance: Informing Decisions Concerning. . 145

B.1 Key Terms Related to Medical Surveillance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

B.2 Medical Surveillance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

B.2.1 Planning and Conducting Medical Surveillance. . . . . . . . . . . . . . . . . . . 146

Appendix C—NIOSH Method 5040. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

C.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

C.2 Method Evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

C.3 Inter-laboratory Comparisons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

C.4 Carbonates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

C.5 Organic Carbon Sampling Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

C.6 Carbon Nanotubes and Nanofibers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
xx NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Abbreviations

AF adjustment factor

ALvSA Alveolar lung surface area

APS aerodynamic particle sizer

APF assigned protection factor

BEL benchmark exposure limit

BET Brunauer-Emmett-Teller

BMC benchmark concentration (maximum likelihood estimate)

BMCL 95% lower confidence limit estimate of BMC

BMC(L) Refers to both the BMC and BMCL estimates 

BMD benchmark dose (maximum likelihood estimate)

BMDL 95% lower confidence limit estimate of BMD

BMD(L) Refers to both the BMD and BMDL estimates

BMDS benchmark dose software

BMR benchmark response

BW body weight

C carbon

CIB Current Intelligence Bulletin

cm2 centimeter squared

CNF carbon nanofibers

CNM carbon-based nanomaterial

CNT carbon nanotubes

Co cobalt

CPC condensation particle counter

CVD chemical vapor deposition

d day

DAF dosimetric adjustment factor

DF deposition fraction

DPM diesel particulate matter

EC elemental carbon

EDS energy dispersive spectroscopy

ELPI electrical low pressure impactor

EPA Environmental Protection Agency
NIOSH CIB 65 • xxi Carbon Nanotubes and Nanofibers

ESP electrostatic precipitator

Fe iron

FMPS fast mobility particle sizer

FPSS fast particulate size spectrometer

g grams

GM geometric mean

GSD geometric standard deviation

HCL hydrogen chloride

HEC human equivalent concentration

HEPA high efficiency particulate air

hr hour(s)

ISO International Organization for Standardization

IT intratracheal instillation

kg kilogram

L liters

LCL lower confidence limit

LEV local exhaust ventilation

LOAEL lowest observed adverse effect level

LOD limit of detection

LOQ limit of quantitation

lpm liters per minute

m2 meter squared

mg milligram(s)

mg/kg milligram per kilogram body weight

mg/m3 milligrams per cubic meter

m/s meters per second

min minute

ml milliliters

MLE maximum likelihood estimate

MMAD mass median aerodynamic diameter

MPPD multiple-path particle dosimetry

MWCNT multi-walled carbon nanotubes

NEDO New Energy and Industrial Technology Development Organization

NF normalizing factor

Ni nickel

NIOSH National Institute for Occupational Safety and Health

nm nanometer(s)

NOAEL no observed adverse effect level
xxii NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

NR not reported

NTRC Nanotechnology Research Center

OC organic carbon

OEL occupational exposure limit

OSHA Occupational Safety and Health Administration

PA pharyngeal aspiration

PBZ personal breathing zone

PMN polymorphonuclear neutrophils

POD point of departure

PPE personal protective equipment

REL recommended exposure limit

ROS reactive oxygen species

RPD relative percent difference 

RSD relative standard deviation

RT retention half-time

SD standard deviation

SDS Safety Data Sheets

SEM scanning electron microscopy

SMPS scanning mobility particle sizer

SWCNT single-walled carbon nanotubes

TC total carbon

TD toxicodynamic

TEM transmission electron microscopy

TK toxicokinetic

TP thermal precipitator

TWA time-weighted average

UF uncertainty factor

μg microgram(s)

μg/m3 micrograms per cubic meter

μm micrometer(s)

UCL upper confidence limit

U.S. United States

VE ventilation rate

wk week

Y yttrium

yr year

% percent
NIOSH CIB 65 • xxiii Carbon Nanotubes and Nanofibers

Acknowledgements

This Current Intelligence Bulletin (CIB) was developed by the scientists and staff of the National Institute for Occupational Safety and Health (NIOSH) who participate in the NIOSH 

Nanotechnology Research Center (NTRC). Paul Schulte is the manager, and Charles Geraci 

is the coordinator of the NIOSH National Occupational Research Agenda (NORA) nanotechnology cross-sector program. The document manager was Ralph Zumwalde. The document writing team was Ralph Zumwalde, Eileen Kuempel, Eileen Birch, Douglas Trout, and 

Vincent Castranova. The data analyses were performed by Eileen Kuempel, Randall Smith, 

Ralph Zumwalde, and Eileen Birch. Document internal review was conducted by Thomas 

Lentz, Faye Rice, John Bailer, David Dankovic, Christine Sofge, Leonid Turkevich, Douglas 

Evans, Ronald Shaffer, Mary Schubauer-Berigan, Aleksandr Stefaniak, Charles Geraci, and 

Paul Schulte. 

Special thanks goes to NIOSH researchers who provided rodent dose-response data used 

in the risk assessment and contributed to the interpretation of the pulmonary response and 

aerosol data: Ann Hubbs, Robert Mercer, Elena Kisin, Anna Shvedova, Dale Porter, Bean 

Chen, and Pramod Kulkarni. 

Special acknowledgement also goes to the NTRC scientists whose research cited in this CIB 

has significantly contributed to the scientific data used to support recommendations for 

minimizing worker exposure to carbon nanotubes and nanofibers. 

The NIOSH NTRC acknowledges the contributions of Vanessa Williams and Rachel Thieman for graphic design and document production and John Lechliter for editing the CIB.

Acknowledgment is also due to Julie Muller, Ph.D., Dominique Lison, Ph.D., Lan Ma-Hock, 

Ph.D., Robert Landseidel, Ph.D., and Jürgen Pauluhn, Ph.D. for providing additional data 

and information from their published studies that were used in the risk assessment.

Peer Reviewers

The NIOSH NTRC greatly appreciates the time and effort of the expert peer reviewers who 

provided comments and suggestions on the December 2010 public and stakeholder draft 

of the CIB. 

Robert Aitken, Ph.D

Director of Strategic Consulting, Institute of Occupational Medicine (IOM)

Nanotechnology Programme Director, IOM

Director of SAFENANO

Institute of Occupational Medicine

Riccarton, Edinburgh

Andrew D. Maynard, Ph.D

Charles and Rita Gelman Professor of Risk Science

Director, University of Michigan Risk Science Center
xxiv NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Philip Sayre, Ph.D

Associate Director, Risk Assessment Division

Office of Pollution Prevention & Toxics, U.S. EPA

Ronald H. White, M.S.T.

Deputy Director, Risk Sciences and Public Policy Institute

Johns Hopkins University

Bloomberg School of Public Health

Public Review

NIOSH greatly appreciates the public comments on the December 2010 draft document 

that were submitted to the NIOSH docket. The comments and responses to them can be 

seen at: www.cdc.gov/niosh/docket/archive/docket161A.html 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 1

1 Introduction

Many nanomaterial-based products are now commercially available. These include nanoscale powders, solutions, and suspensions of nanoscale materials, as well as composite materials and devices 

incorporating nanomaterials. The International Organization for Standardization (ISO) has developed 

nomenclature and terminology for nanomaterials 

[ISO/TS 2008]. According to ISO 27687:2008, a 

nano-object is material with one, two, or three external dimensions in the size range from approximately 1–100 nanometers (nm). Sub-categories of 

a nano-object are (1) nanoplate, a nano-object with 

one external dimension at the nanoscale (i.e., 1–100 

nm); (2) nanofiber, a nano-object with two external dimensions at the nanoscale, with a nanotube 

defined as a hollow nanofiber and a nanorod as a 

solid nanofiber; and (3) nanoparticle, a nano-object 

with all three external dimensions at the nanoscale. 

Nano-objects are commonly incorporated in a larger matrix or substrate called a nanomaterial. This 

taxonomy differs slightly from that suggested by the 

title of this CIB “Occupational Exposure to Carbon 

Nanotubes and Nanofibers.” By this title, NIOSH is 

not suggesting an alternative taxonomy, but rather 

identifying the nano-objects (nanoscale carbon fiber and tube structures) that have been evaluated 

to date in toxicology and workplace exposure measurement studies.

Carbon nanotubes (CNT) are nanoscale cylinders of carbon (essentially consisting of seamlessly 

“rolled” sheets of graphene) that can be produced 

with very large aspect ratios. There is no single type 

of carbon nanotube. They may differ in shape, dimension, physical characteristics, surface coatings, 

chemical composition, or surface functionalization. This includes “raw” CNT, which contain residual metal catalysts vs. “purified” CNT, from which 

most of the metal catalysts have been removed. 

Single-walled carbon nanotubes (SWCNT) consist 

of a single rolled graphene sheet and have a typical 

diameter of approximately 1–2 nm. Multi-walled 

carbon nanotubes (MWCNT) consist of many 

single-walled tubes stacked one inside the other 

with diameters in the range of 2–100 nm, depending on the number of encapsulated tubes forming 

the CNT structure. SWCNT and MWCNT can 

vary in length, with some being up to many tens of 

micrometers long [Thostenson et al. 2001]. Carbon 

nanofibers (CNF), which are structurally similar to 

MWCNT, have typical diameters approximately 40 

to 200 nm [Ku et al. [2006]. CNF have lengths ranging from tens of micrometers to several centimeters, average aspect ratios (length to diameter ratio) 

of >  100, and they display various morphologies, 

including cupped or stacked graphene structures. 

The primary characteristic that distinguishes CNF 

from CNT resides in graphene plane alignment. If 

the graphene plane and fiber axis do not align, the 

structure is defined as CNF, but when parallel, the 

structure is considered a CNT [ISO/TS 2008]. 

The synthesis of CNT and CNF requires a carbon 

source and an energy source [Sanchez et al. 2009]. 

CNT and CNF are synthesized by several distinct 

methods, including chemical vapor deposition 

(CVD), arc discharge, laser ablation, and highpressure CO conversion (HiPco). Depending on 

material and method of synthesis, a metal catalyst 

maybe used to increase yield and sample homogeneity, and to reduce the synthesis temperature. 

The diameter of the fibers depends on the dimensions of the metal nanoparticle used as a catalyst; 

the shape, symmetry, dimensions, growth rate, and 

crystallinity of the materials are influenced by the 

selection of the catalyst, carbon source, temperature, and time of the reaction. Different amounts 

of residual catalyst often exist following synthesis; 

consequently, post-synthesis treatments are used 

to increase the purity of the product. The most 
2 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

common purification technique involves selective oxidation of the amorphous carbon and/or 

carbon shells at a controlled temperature followed 

by washing or sonicating the material in an acid 

(HCL, HNO3, H2SO4) or base (NaOH) to remove 

the catalyst. As there are many types of purification processes, purified CNT and CNF will exhibit 

differences in the content of trace elements and residual materials [Liu et al. 2008; Hou et al. 2008]. 

A growing body of literature indicates a potential 

health hazard to workers from exposure to various 

types of carbon nanotubes and nanofibers. A number of research studies with rodents have shown 

adverse lung effects at relatively low-mass doses of 

CNT (Tables 3–2 and 3–7), including pulmonary 

inflammation and rapidly developing, persistent fibrosis. Similar effects have been recently observed 

with exposure to CNF (Table 3–6). It is not known 

how universal these adverse effects are, that is, 

whether they occur in animals exposed to all types 

of CNT and CNF, and whether they occur in additional animal models. Most importantly, it is not 

yet known whether similar adverse health effects 

occur in humans following exposure to CNT or 

CNF, or how airborne CNT in the workplace may 

compare in size and structure to the CNT aerosols 

generated in the animal studies. 

Because of their small size, structure, and low surface charge, CNT and CNF can be difficult to separate in the bulk form and tend to be agglomerated 

or to agglomerate quickly when released in the air, 

which can affect their potential to be inhaled and 

deposited in the lungs. The extent to which workers are exposed to CNT and CNF in the form of 

agglomerates or as single tubes or structures is 

unclear because of limited exposure measurement 

data, but airborne samples analyzed by electron microscopy have shown both individual and agglomerated structures [Johnson et al. 2010; Methner et 

al. 2010b; Birch et al. 2011b; Dahm et al. 2011]. 

This Current Intelligence Bulletin (CIB) summarizes 

the adverse respiratory health effects that have been 

observed in laboratory animal studies with SWCNT, 

MWCNT, and CNF. A recommended exposure limit (REL) for CNT and CNF is given to help minimize 

the risk of occupational respiratory disease in workers as well as guidance for the measurement and 

control of exposures to CNT and CNF. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 3

2 Potential for Exposure 

The novel application of CNT and CNF has been 

extensively researched because of their unique 

physical and chemical properties. CNT and CNF 

are mechanically strong, flexible, lightweight, heat 

resistant, and they have high electrical conductivity [Walters et al. 1999; Yu et al. 2000]. The commercial market for CNT and CNF is expected to 

grow substantially over the next decade [Lux Research 2007] with global capacity in 2013 estimated 

at 2,000 tons/year for MWCNT and 6 tons/year for 

SWCNT [Nanotech 2013]. Carbon nanotubes and 

nanofibers are commercially used in a variety of applications. These include electronics, lithium-ion 

batteries, solar cells, super capacitors, thermoplastics, polymer composites, coatings, adhesives, biosensors, enhanced electron/scanning microscopy 

imaging techniques, and inks. They are also used in 

pharmaceutical/biomedical devices for bone grafting, tissue repair, drug delivery, and medical diagnostics [WTEC 2007; Milne et al. 2008]. 

The potential for worker exposure to CNT and CNF 

can occur throughout the life cycle of CNT- and 

CNF-product use (processing, use, disposal, recycling) [Maynard and Kuempel 2005] (Figure 2–1), 

but the extent to which workers are exposed has 

not been completely characterized. Available data 

indicate that airborne exposures to CNT and CNF 

can occur during the transfer, weighing, blending, 

and mixing of the bulk powders, and during the 

cutting and drilling of CNT- and CNF-composite 

materials. A recent study of U.S. companies manufacturing carbonaceous nanomaterials identified 

43 companies manufacturing CNT (14 primary, 18 

secondary, and 11 primary and secondary users) 

[Schubauer-Berigan et al. 2011]. The purpose of 

the study was to enumerate the companies directly 

manufacturing (or using in other manufacturing 

processes) engineered carbonaceous nanomaterials 

in the United States, and to estimate the workforce 

size and characteristics of nanomaterials produced. 

The number of workers engaged in the manufacturing of CNT was estimated at 375, with a projected 

growth rate in employment of 15% to 17% annually. The quantity of CNT (SWCNT and MWCNT) 

produced annually by each company was estimated 

to range from 0.2 to 2500 kg. The size of the workforce involved in the fabrication or handling of 

CNT/CNF-enabled materials and composites is unknown, but it is expected to increase as the market 

expands from research and development to industrial high-volume production [Invernizzi 2011]. 

2.1 Exposure to Carbon

Nanotubes during

Research and Development,

Small-scale Manufacturing, 

and Use Applications

Recent assessments of airborne exposure to 

MWCNT in a research laboratory that manufactures and handles MWCNT found totalparticulate concentrations ranging from 37 μg/m3

(weighing operation) to 430 μg/m3 (blending 

process) in the absence of exposure controls 

[Han et al. 2008a]. The implementation of engineering controls (e.g., ventilated enclosure 

of MWCNT blending process) significantly reduced airborne particulate concentrations, often 

to non-detectable results. Transmission electron 

microscopy (TEM) analysis (NIOSH Method 

7402) of personal breathing zone (PBZ) and 

area samples collected during the blending of 

MWCNT found airborne concentrations ranging from 172.9 tubes/cm3

 (area sample) to 193.6 

tubes/cm3 (PBZ sample) before the installation 

of exposure controls. The subsequent introduction of exposure controls significantly reduced 
4 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

airborne MWCNT concentrations to 0.018–0.05 

tubes/cm3. Aerosolized MWCNT structures were 

reported to have 52–56 nm diameters and 1473–

1760 nm (avg. 1.5 μm) lengths. 

Maynard et al. [2004] also assessed the propensity 

for SWCNT to be released during the agitation of 

unprocessed SWCNT material in a laboratorybased study and during the handling (e.g., furnace 

removal, powder transfer, cleaning) of unrefined 

material at four small-scale SWCNT manufacturing facilities in which laser ablation and highpressure carbon monoxide techniques were used 

to produce SWCNT. Particle measurements taken 

during the agitation of unprocessed material in the 

laboratory indicated the initial airborne release of 

material (some visually apparent) with the particle 

concentration of the aerosol (particles < 0.5 μm in 

diameter) observed to decrease rapidly over time. 

With no agitation, particles around 0.1 μm in diameter appeared to be released from the SWCNT 

material, probably because of the airflow across the 

powder. At the four manufacturing facilities, shortterm SWCNT mass concentrations were estimated 

(using a catalyst metal as the surrogate measurement) to range from 0.7 to 53 μg/m3

 (area samples) 

in the absence of exposure controls. When samples 

were evaluated by scanning electron microscopy 

(SEM), most of the aerosolized SWCNT were agglomerated, with agglomerated sizes typically larger than 1 μm. 

Airborne particle and MWCNT concentrations were 

determined by Bello et al. [2008] during chemical 

vapor deposition (CVD), growth, and handling of 

vertically aligned CNT films. Continuous airborne 

particle measurements were made using a real-time 

fast mobility particle sizer (FMPS) and a condensation particle counter (CPC) throughout the furnace 

operation. No increase in total airborne particle 

concentration (compared with background) was 

observed during the removal of MWCNT from the 

reactor furnace or during the detachment of MWCNT from the growth substrate (a process whereby 

MWCNT are removed from the substrate with a razor blade). Electron microscopic analysis of a PBZ 

sample collected on the furnace operator found no 

detectable quantity of MWCNT, either as individual 

tubes or as agglomerates. No mention was made 

about the use of engineering controls (e.g., local 

exhaust ventilation, fume hood) to prevent exposure to MWCNT.

The potential for airborne particle and SWCNT 

and MWCNT release was determined in a laboratory setting in which both types of CNT were produced using CVD [Tsai et al. 2009]. A qualitative 

assessment of the exposure (i.e., particle morphology, aerosol size) was made during the synthesis 

of SWCNT and MWCNT in which modifications 

of the manufacturing methods were made to ascertain how changes in the production of CNT influenced airborne particle size and concentration 

(e.g., SWCNT synthesis with and without a catalyst, 

and growth of MWCNT on a substrate and with no 

substrate). An FMPS and an aerodynamic particle 

sizer (APS) were used to monitor particle size and 

concentrations. Background particle concentrations were determined to assist in quantifying the 

release of SWCNT and MWCNT during their synthesis and handling. Samples were also collected for 

analysis by TEM to determine particle morphology 

and elemental composition. Particle measurements 

made inside a fume hood during the synthesis of 

SWCNT were found to be as high as 107 particles/

cm3 with an average particle diameter of 50  nm; 

PBZ samples collected on workers near the fume 

hood were considerably lower (<  2,000 particles/

cm3). The difference between particle concentrations obtained during SWCNT growth using a 

catalyst and the control data (no catalyst) was small 

and was postulated to be a result of particles being 

released from the reactor walls of the furnace even 

when no SWCNT were being manufactured. Particle measurements made during the synthesis of 

MWCNT were found to peak at 4 × 106 particles/

cm3 when measured inside the fume hood. Particle 

size ranged from 25 to 100 nm when a substrate 

was used for MWCNT growth and from 20 to 200 

nm when no substrate was present. Airborne particle concentrations and particle size were found to 

vary because of the temperature of the reactor, with 

higher particle concentrations and smaller particle 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 5

sizes observed at higher temperatures. PBZ samples 

collected on workers near the fume hood during 

MWCNT synthesis had particle concentrations 

similar to background particle concentrations. 

TEM analysis of MWCNT samples indicated the 

presence of individual particles as small as 20 nm 

with particle agglomerates as large as 300  nm. 

Some individual MWCNT were observed, but were 

often accompanied by clusters of carbon and iron 

particles. The diameters of the tubes were reported 

to be about 50  nm. The use of a fume hood that 

was extra wide and high and operated at a constant 

velocity of 0.7 m/s face velocity, appeared to be effective in minimizing the generation of turbulent 

airflow at the hood face, which contributed to the 

good performance of the fume hood in capturing 

the airborne release of SWCNT and MWCNT during their synthesis. 

Lee et al. [2010] investigated the potential airborne release of MWCNT at seven facilities (e.g., 

research laboratories, small-scale manufacturing) 

where MWCNT was either being synthesized by 

CVD or handled (e.g., ultrasonic dispersion, spraying). Real-time aerosol monitoring was conducted 

using a scanning mobility particle sizer (SMPS) and 

a CPC to determine particle size and concentration. 

PBZ and area samples were collected for determining airborne mass concentrations (total suspended 

particulate matter) and for TEM (NIOSH Method 

7402) and SEM analysis for particle identification 

and characterization. Background measurements 

of airborne nanoparticle exposures were determined at two of the seven worksites before starting 

work to assist in establishing a baseline for airborne 

nanoparticle concentrations. Most of the handling 

of MWCNT during synthesis and application was 

performed inside a laboratory fume hood, where 

most of the measurements were made. Exposure 

concentrations of total suspended particulate matter ranged from 0.0078 to 0.3208 mg/m3

 for PBZ 

samples and 0.0126 to 0.1873 mg/m3 for area 

samples. TEM and SEM analysis of filter samples 

found no detectable amounts of MWCNT but only 

aggregates of metal particles (e.g., iron and aluminum) which were used as catalysts in the synthesis 

of MWCNT. The highest airborne particle releases 

were observed in area samples collected during 

catalyst preparation (18,600–75,000 particles/cm3

for 20–30  nm diameter particles) and during 

the opening of the CVD reactor (6,974–16,857 

particles/cm3 for 20–50 nm diameter particles). 

Other handling processes such as CNT preparation, 

ultrasonic dispersion, and opening the CNT spray 

cover also generated the release of nanoparticles. 

The ultrasonic dispersion of CNT generated particles in the range of 120 to 300 nm, which were larger 

in size than those released from other processes.

The release of airborne carbon-based nanomaterials (CNMs) was investigated during the transfer 

and ultrasonic dispersion of MWCNT (10–20 nm 

diameters), fullerenes, and carbon black (15 nm 

diameter) inside a laboratory fume hood with the 

airflow turned off and the sash halfway open [Johnson et al. 2010]. Airborne exposure measurements 

were made during the weighing and transferring 

of dry CNMs to beakers filled with reconstituted 

freshwater with and without natural organic matter 

and then sonicated. The study was designed to determine the relative magnitude of airborne nanomaterial emissions associated with tasks and materials used to evaluate environmentally relevant 

matrices (e.g., rivers, ponds, reservoirs). Direct 

reading real-time instruments (i.e., CPC, OPC) 

were used to determine airborne particle number 

concentrations, with the results compared with 

particle number concentrations determined from 

general air samples collected in the laboratory before and after the laboratory process. Samples were 

also collected for TEM analysis to verify the presence of CNMs. Airborne particle number concentrations for all tasks exceeded background particle 

concentrations, which were inversely related to 

particle size, with the size distribution of particles 

skewed toward those CNMs with an aerodynamic 

diameter < 1 μm. Airborne particle number concentrations for MWCNT and carbon black, during 

the sonication of water samples, were significantly 

greater than those found during the weighing and 

transferring of dry CNMs. TEM analysis of airborne area samples revealed agglomerates of all 
6 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

CNMs, with MWCNT agglomerates observed to 

be 500 to 1,000 nm in diameter.

The National Institute for Occupational Safety and 

Health (NIOSH) conducted emission and exposure 

assessment studies at 12 sites where engineered 

nanomaterials were produced or used [Methner et 

al. 2010a]. Studies were conducted in research and 

development laboratories, pilot plants, and smallscale manufacturing facilities handling SWCNT, 

MWCNT, CNF, fullerenes, carbon nanopearls, 

metal oxides, electrospun nylon, and quantum 

dots. Airborne exposures were characterized using 

a variety of measurement techniques (e.g., CPC, 

OPC, TEM) [Methner et al. 2010b]. The purpose 

of the studies was to determine whether airborne 

exposures to these engineered nanomaterials occur and to assess the capabilities of various measurement techniques in quantifying exposures. In 

a research and development laboratory handling 

CNF, airborne particle number concentrations (determined by CPC) were reported as 4000 particles/

cm3 during weighing/mixing and 5000 particles/

cm3 during wet sawing. These concentrations were 

substantially less than the reported background 

particle concentration of 19,500 particles/cm3. 

Samples collected for TEM particle characterization indicated the aerosol release of some CNF. All 

handling of CNF was in a laboratory hood (with 

HEPA filtered vacuum) for the weighing/mixing 

and wet saw cutting of CNF composite materials. 

In a facility making CNF in a chemical vapor phase 

reactor, OPC particle count concentrations ranged 

from 5,400 particles/cm3 (300–500 nm particle 

size) to a high of 139,500 particles/cm3 (500–100 

nm particle size). Higher airborne particle concentrations were found during the manual scooping of 

CNF in the absence of exposure control measures. 

Samples collected for TEM particle characterization indicated the aerosol release of some CNF. In 

another research and development laboratory, the 

potential for airborne exposure to MWCNT was 

evaluated during weighing, mixing, and sonication. All handling of MWCNT was performed in 

a laboratory hood (without HEPA filtered vacuum). Particle concentrations were determined by 

CPC (particle size 10–1000nm) and OPC (particle 

size 300–500nm, 500–100nm). CPC particle concentrations ranged from 1480–1580 particles/cm3 

(weighing MWCNT in hood) to 2200–2800 particles/cm3

 (sonication of MWCNT). The background 

particle concentration determined by CPC was 700 

particles/cm3. Airborne particle concentrations 

determined by OPC ranged from 3,900–123,400 

particles/cm3 (weighing) to 6,500–42,800 particles/

cm3 (sonication). Background particle concentrations determined by OPC ranged from 700 

particles/cm3 (1–10 μm particle size) to 13,700

particles/cm3 (300–500 nm particle size). The higher 

particle concentrations determined with the OPC 

indicated the presence of larger, possibly agglomerated particles. Samples collected for TEM particle 

characterization indicated the aerosol release of agglomerated MWCNT. 

Subsequent studies conducted by NIOSH at six 

primary and secondary pilot or small-scale manufacturing facilities (SWCNT, MWCNT, CNF) 

employed a combination of filter-based samples 

to evaluate PBZ and area respirable and inhalable 

mass concentrations of EC as well as concentrations of CNT and CNF structures determined by 

TEM analysis [Dahm et al. 2011]. A total of 83 

filter-based samples (30 samples at primary and 

22 at secondary manufacturers) were collected for 

EC determination (NIOSH Method 5040) and 31 

samples for TEM analysis (NIOSH Method 7402). 

Similar processes and tasks were reported in the 

three primary and three secondary manufacturers of CNT. These processes and tasks consisted of: 

(1) similar production and harvesting methods for 

CNT, and common cleaning/housekeeping procedures in primary manufacturers, and (2) common 

CNT handling practices such as weighing, mixing, 

sonication, manual transfer, cleaning, and spray 

coating operations in secondary manufacturers. 

Worker PBZ inhalable mass concentrations found 

at primary manufacturers ranged from 0.68 to 

5.25 μg/m3 EC with an average concentration of 

2.42 μg/m3. Area samples for EC determination 

from these samples ranged from non-detectable 

to 4.62 μg/m3 while outdoor background samples 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 7

ranged from non-detectable to 0.89 μg/m3. [Note: 

these are inhalable mass fractions and may not be 

equivalent to the unmeasured respirable mass fractions. Thus, these concentrations cannot be compared directly to the NIOSH REL]. The highest 

airborne exposures were found during harvesting 

of CNT when no exposure control measures were 

used. In secondary manufacturers, PBZ inhalable 

mass concentrations for EC ranged from non-detectable to 7.86 μg/m3

 with area sample concentrations ranging from non-detectable to 2.76 μg/m3

. 

No EC was found in an outdoor background air 

sample. The highest airborne exposures were found 

during extrusion and weighing of CNT when using a fume hood that was reported as not always in 

operation or being utilized properly during material handling. A majority of the reported EC mass 

concentrations in primary and secondary facilities 

were determined from airborne samples found 

to contain detectable amounts of EC between the 

LOD and LOQ of Method 5040. 

Samples for TEM analysis (collected side-by-side 

with PBZ and area mass samples) reported CNT 

concentrations for PBZ samples ranging from nondetectable to 1.613 structures/cm3

 with the highest concentration for an area sample reported as 

0.295 structures/cm3. A statistical correlation between side-by-side mass concentrations and TEM 

structure counts was reported (p-0.01) with a corresponding Pearson correlation coefficient of 0.44. 

Various types of exposure prevention measures 

were reported for most workplaces including the 

use of PPE (e.g., respirators, gloves, safety glasses) 

and implementation of different exposure control 

techniques (e.g., glove box, chemical fume hood, 

clean rooms). A limitation of the study was that 

most workers were not handling CNT/CNF full 

shift, thus many samples were collected over a relatively short sample time due to the short duration 

of processes and tasks. 

In a study designed to investigate the release of 

CNT during the dry and wet cutting of composite 

materials containing CNT, airborne samples were 

collected to determine particle number, respirable 

mass, and nanotube concentrations [Bello et al. 2009]. 

Two different composites containing MWCNT 

(10–20 nm diameters) were cut using a band saw 

or rotary cutting wheel. The laboratory study was 

designed to simulate the industrial cutting of CNTbased composites. PBZ and area samples (close 

to the emission source) were collected during dry 

cutting (without emission controls) and during 

wet cutting (equipped with a protective guard surrounding the rotary cutting wheel). The cutting of 

composite materials ranged from 1 to 3 minutes. 

The dry cutting of composite materials generated 

statistically significant (P < 0.05) quantities of airborne nanoscale and fine particles when compared 

with background airborne particle concentrations. 

Although the particle number concentration was 

dominated by the nanoscale and fine fractions, 71% 

to 89% of the total particle surface area was dominated by the respirable (1–10 μm) aerosol fraction. 

During the dry cutting of composites, reported 

mean PM10 mass concentrations for area samples 

were 2.11 and 8.38 mg/m3, and 0.8 and 2.4 mg/m3

for PBZ samples. Submicron and respirable fibers 

were generated from dry cutting of all composites. 

TEM analysis of area samples found concentrations 

that ranged from1.6 fibers/cm3 (during the cutting 

of CNT-alumina) to 3.8 fibers/cm3 (during the cutting of carbon-base composite materials). A PBZ 

fiber concentration of 0.2 fibers/cm3 was observed 

during the dry cutting of base-alumina composite 

materials. No fiber measurement data were reported for the wet cutting of composite materials. No 

increase in mean PM10 mass concentrations were 

observed in 2 of 3 area samples collected during the 

wet cutting of composites. In the third sample, the 

observed high particle concentration was attributed to extensive damage of the protective guard 

around the rotary cutting wheel.

Bello et al. [2010] also investigated the airborne 

release of CNT and other nanosized fibers during solid core drilling of two types of advanced 

CNT-hybrid composites: (1) reinforced plastic 

hybrid laminates (alumina fibers and CNT), and 

(2) graphite-epoxy composites (carbon fibers and 

CNT). Worker PBZ and area samples were collected to determine exposures during the drilling of 
8 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

composite materials with local exhaust ventilation 

turned off. Four potential exposure-modifying factors were assessed: (1) by composite type, (2) drilling rpm (low and high), (3) thickness of the composite, and (4) dry versus wet drilling. Replicate test 

measurements (10–30 measurements) lasting <  5 

minutes were performed on each composite material. A combination of real-time and integrated 

samples were collected at the source and PBZ using 

an FMPS, aerodynamic particle sizer (APS), CPC, 

diffusion charger, and cascade impactor to measure 

aerosol particle size, concentration, and chemical 

identification. An electrostatic precipitator (ESP) 

and thermal precipitator (TP) were used to collect 

particles directly on TEM grids for electron microscopy analysis. Aerosol concentrations during 

high rpm drilling generally were higher than for 

low rpm drilling for all composite materials with 

aerosol concentrations found to be higher from 

alumina composites. Wet drilling was observed to 

suppress the release of particles > 10 nm in diameter. High aspect-ratio fiber concentrations were 

determined using the sizing and counting criteria 

in NIOSH Method 7400 (> 5 μm long, aspect ratio 

> 3). Airborne exposure to both alumina fiber and 

CNT structures were found ranging in concentration from 1.0  fibers/cm3

 (alumina composite) to 

1.9  fibers/cm3 (carbon and CNT composite) for 

PBZ samples; similar concentrations were observed 

in area samples. Because sampling volume and fiber surface density on the samples were below the 

optimal specification range of Method 7400, fiber 

concentration values were determined to be first 

order approximations. The authors concluded that 

higher input energies (e.g., higher drilling rpms, 

larger drill bits) and longer drill times associated 

with thicker composites generally produced higher 

exposures, and that the drilling of CNT-based composites generated a higher frequency of nanofibers 

than had been previously observed during the cutting of CNT-based composites [Bello et al. 2009]. 

Cena and Peters [2011] evaluated the airborne 

release of CNT during the weighing of bulk CNT 

and the sanding of epoxy nanocomposite sticks 

measuring 12.5 × 1.3 × 0.5 cm. Epoxy reinforced 

test samples were produced using MWCNT (Baytubes®) with 10–50 nm outer diameters and 1–20 

μm lengths. The purpose of the study was to (1) 

characterize airborne particles during handling of 

bulk CNT and the mechanical processing of CNT 

composites, and (2) evaluate the effectiveness of 

local exhaust ventilation (LEV) hoods to capture 

airborne particles generated by sanding CNT composites. Airborne particle number and respirable 

mass concentrations were measured using a CPC 

(particle diameters 0.01 to 1 μm) and OPC (particle diameters 0.3 to 20 μm). Respirable mass concentrations were estimated using the OPC data. 

Samples for TEM analysis were also collected for 

particle and CNT characterization. PBZ and source 

airborne concentrations were determined during 

two processes: weighing bulk CNT and sanding 

epoxy nanocomposite test sticks. Exposure measurements were taken under three LEV conditions 

(no LEV, a custom fume hood, and a biological 

safety cabinet). CPC and OPC particle concentrations were measured inside a glove box in which 

bulk CNT (600 mg) was transferred between two 

50-ml beakers; background particle concentrations were measured inside the glove box before 

the process began. To study the sanding process, a 

worker manually sanded test sticks that contained 

2% by weight CNT. Aerosol concentrations were 

measured for 15–20 min in the worker’s breathing 

zone and at a site adjacent to the sanding process. 

The sanding process with no LEV was conducted 

on a 1.2 m by 2.2 m worktable. The sanding was 

also conducted inside a custom fume hood that 

consisted of a simple vented enclosure that allowed 

airflow along all sides of the back panel but had no 

front sash or rear baffles. The average face velocity 

of the fume hood was 76 ft/min. Exposures from 

the sanding process were also assessed while using 

a biological safety cabinet (class II type A2). 

Particle number concentrations determined during the weighing process contributed little to that 

observed in background samples (process to background ratio [P/B] = 1.06), however it did influence the mass concentration (P/B = 1.79). The 

GM respirable mass concentration inside the glove 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 9

box was reported as 0.03 μg/m3 (background GM was 

0.02 μg/m3). During the sanding process (including 

no LEV, in a fume hood, and in a biological safety 

cabinet) the PBZ nanoparticle number concentrations were negligible compared with background 

concentrations (P/B ratio = avg. 1.04). Particles generated during sanding were reported to be predominantly micron sized with protruding CNT and very 

different from bulk CNT that tended to remain in 

large (>1 μm) tangled agglomerates. Respirable mass 

concentrations in the worker’s breathing zone were 

elevated. However, the concentrations were lower 

when sanding was performed in the biological safety 

cabinet (GM = 0.2 μg/m3) compared with those with 

no LEV (GM was 2.68 μg/m3) or those when sanding was performed inside the fume hood (GM = 

21.4 μg/m3; p value <0.0001). The poor performance 

of the fume hood was attributed to the lack of a front 

sash and rear baffles and its low face velocity. 

2.2 Exposure to Carbon

Nanotubes (other sources)

Exposure to CNT and other carbon nanocrystalline structures (e.g., spheres, shells) can occur during the burning of natural gas, propane, and other 

methane-series gases. CNT exposures have been 

observed in indoors areas where gas stoves are in 

use, and in outdoor air near gas-burning industrial 

sites [Murr et al. 2004 a, b]. Samples collected near 

a methane (CH4)/air flame exhaust revealed carbon nanocrystal aggregates that ranged from 0.4 

to 2 μm, and contained several thousand individual MWCNT and other nanocrystal (polyhedral) 

structures that averaged 20 nm in diameter. 

2.3 Exposure to Carbon

Nanofibers during

Research and Small-scale

Manufacturing Operations

Some research has been conducted to date on 

workplace exposure to carbon nanofibers (CNF) 

[Methner et al. 2007; Evans et al. 2010; Birch 2011a; 

Birch et al. 2011b]. In a NIOSH health hazard evaluation conducted at a university-based research 

laboratory, the potential release of airborne CNF 

was observed at various processes using real-time 

aerosol instruments (e.g., CPC, ELPI, aerosol photometer) [Methner et al. 2007]. General area exposure measurements indicated slight increases in 

airborne particle number and mass concentrations 

relative to background measurements (outdoors 

and offices) during the transfer of CNF prior to 

weighing and mixing, and during the chopping 

and wet saw cutting of a polymer composite material. Airborne total carbon mass concentrations 

(per NIOSH Method 5040, with correction for 

adsorbed vapor) within the laboratory processing area were 2 to 64 times higher than those of a 

nearby office area, with the highest peak exposure 

concentration (1094 μg/m3) found during the wet 

saw cutting of the CNF composite material. No indoor particle concentrations exceeded the outdoor 

background concentrations. Particles having a diameter of about 400 nm or greater were found in 

greater number during wet-saw cutting, while the 

number of particles having a diameter of about 

500 nm or greater were elevated during the weighing and mixing of CNF. Airborne samples collected 

directly on TEM grids were analyzed for the presence of CNF. Some fibers observed by TEM had 

diameters larger than the 100 nm criterion used to 

define a nanofiber, which was consistent with results reported by Ku et al. [2006], in which the mobility diameter of aerosolized CNF was observed to 

be larger than 60 nm, with a modal aerodynamic 

diameter of about 700  nm. The majority of CNF 

observed by TEM were loosely agglomerated, rather than single fibers, which was in general agreement with the particle size measurements made by 

real-time instruments. 

Detailed investigations of exposures at different job 

tasks were conducted at a facility manufacturing 

and processing CNF [Evans et al. 2010; Birch 2011a; 

Birch et al. 2011b] in which CNF production totaled 14,000 kg a year. Filter, sorbent, cascade impactor, bulk, and microscopy samples, combined with 

direct-reading instruments, provided complementary 
10 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

information on air contaminants. Samples were 

analyzed for organic, elemental, and total carbon 

(OC + EC = TC), metals, and polycyclic aromatic 

hydrocarbons (PAHs), with EC as a measure of 

CNF. TEM with energy-dispersive X-ray spectroscopy (TEM-EDS) also was applied. Study results for 

the time-integrated (filter/sorbent) samples were 

reported in companion papers, with OC-EC, metals, and microscopy results in part I [Birch et al. 

2011b] and PAH results in part II [Birch 2011a]. 

Fine/ultrafine, iron-rich soot, PAHs, and carbon 

monoxide were production byproducts. Area concentrations of respirable EC inside the facility were 

about 6 to 68 times higher than outdoors, while 

PBZ samples were up to 170 times higher. 

As part of the same study, multiple direct-reading 

instruments (e.g., CPC, ELPI, photometer/with 

cyclone, diffusion charger, fast particulate size 

spectrometer [FPSS]) and respirable particle mass 

concentrations were used to assess CNF exposures 

on site and to evaluate instrument performance. A 

transient increase in respirable mass concentration 

was observed during manual bagging of the final 

product and was attributed to aerosolized CNF. 

The tamping of the bag to settle contents and the 

subsequent closing dispersed CNF through the bag 

opening into the workplace. High particle number 

and active surface area concentrations were found 

during the opening of the dryer and during the 

manual redistribution of the CNF product. This 

was attributed to the presence of ultrafine particles 

emitted from the dryer and as by-products formed 

through the high-temperature thermal processing 

of CNF. No elevations in respirable mass concentrations were observed during these operations, 

suggesting that significant quantities of CNF were 

not released into the workplace. However, the 

transfer or dumping of dried CNF from a dryer to 

a drum, and subsequent bag change-out of final 

product, contributed the largest transient increases 

in respirable mass concentrations, with concentrations exceeding 1.1 mg/m3

 for transfer or dumping and 0.5 mg/m3

 for bag change-out. The authors 

concluded that integrated particle number and active surface area concentrations (i.e., using CPC 

and diffusion charger) were not useful in assessing the contribution of emissions from CNF in the 

workplace, because measurements were dominated 

by ultrafine particle emissions [Evans et al. 2010]. 

Respirable particle mass concentrations estimated 

by the photometer appeared to be the most useful 

and practical metric for measuring CNF when using direct-reading instruments. Results obtained 

for filter samples support the direct-reading instrument findings. The TEM analyses of size-selective 

area samples indicated that large fiber bundles were 

present [Birch et al. 2011b]. In addition, sizeclassified samples (collected with impactors) analyzed for EC (by NIOSH 5040) indicated CNF particles in the micrometer range [Birch et al. 2011b].
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 11

Figure 2–1. Workplaces and job tasks with potential for occupational exposure to carbon nanotubes and 

nanofibers. Adapted from Schulte et al. 2008.

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 13

3 Evidence for Potential Adverse Health Effects 

Various types of laboratory animal studies have 

been conducted with CNT and CNF using different routes of exposure to evaluate potential toxicity (Tables 3–1 through 3–8). These studies have 

shown a consistent toxicological response (e.g., 

pulmonary inflammation, fibrosis) independent 

of the study design (i.e., intratracheal, aspiration, 

and inhalation). Exposure to SWCNT, MWCNT, 

and CNF are of special concern because of their 

small size and fibrous structure. The nanometer 

diameters and micrometer lengths of some materials closely resemble the dimensions of some mineral fibers (e.g., asbestos). Results from laboratory 

animal studies with SWCNT, MWCNT, and CNF 

also show pulmonary responses similar to those 

reported for some respirable particles and durable 

fibers. In some studies, CNT-induced lung fibrosis 

developed more rapidly and at a lower mass burden 

than either ultrafine carbon black or quartz [Lam et 

al. 2004; Shvedova et al. 2005; Ryman-Rasmussen 

et al. 2009b]. Pulmonary exposure to CNT has also 

produced systemic responses including an increase 

in inflammatory mediators in the blood, as well as 

oxidant stress in aortic tissue and increase plaque 

formation in an atherosclerotic mouse model [Li et 

al. 2007; Erdely et al. 2009; Stapleton et al. 2011]. 

CNT and CNF are widely considered durable 

in biological systems because of the process they 

undergo during synthesis in which contaminating catalytic metals are frequently removed by 

high temperature vaporization or acid/base treatment [Sanchez et al. 2009]. Researchers [OsmondMcLeod et al. 2011] have measured the durability 

(in vitro) of four types of CNT, one type of glass 

wool fiber, and two asbestos fiber types in simulated biological fluid (Gambles solution) followed 

by an assessment of their ability to induce an inflammatory response when injected into the abdominal cavities of mice. Three of the four types 

of CNT tested for durability showed no signs, or 

minimal loss of mass, or change in fiber length, or 

morphology. When the four CNTs were injected 

into the peritoneal cavity of mice, an inflammatory 

and fibrotic response was induced for three of the 

CNTs that had retained their long discrete structures, whereas the other CNT, which was less durable and had shorter structures and/or formed tight 

bundles, caused minimal inflammation. The findings were consistent with those found for asbestos 

and glass wool fibers after intraperitoneal injection 

into mice, in which an inflammatory and fibrotic 

response was elicited by the two asbestos samples 

that were both durable and contained a high percentage of long fibers, and the glass wool fiber that 

was not very durable and caused only minimal inflammation.

The physical-chemical properties (e.g., dimension, composition, surface characteristics) of CNT 

and CNF can often be modified to accommodate 

their intended commercial use. CNT and CNF 

can also be coated or functionalized, thus changing their surface chemistry. Toxicological effects 

of such changes remain largely unexplored [Yan 

et al. 2011] except for some limited evidence indicating that structural defects [Muller et al. 2008a; 

Fenoglio et al. 2008], surface [Sayes et al. 2006] and 

oxidative modification [Allen et al. 2008], nitrogen 

doping [Carrero-Sanchez et al. 2006], surface functionalization [Liu et al. 2010], and polymer (acidand polystyrene-based) surface coating [Tabet et 

al. 2011] of CNT can influence their toxicity potential. Recent studies indicate that functionalization of MWCNT with –COOH groups significantly 

decreases the inflammatory and fibrotic response 

after aspiration in a mouse model [Sager et al. 

2011; Wang et al. 2012] and when SWCNT were 

functionalized by carboxylation and subjected 

to phagolysosomal fluid, longitudinal splitting 
14 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

and oxidative degradation of the tubes occurred 

[Liu et al. 2010]. Kagan et al. [2010] reported that 

in vitro myeloperoxidase, which is found in high 

concentrations in polymorphonuclear neutrophils 

(PMN), degraded SWCNT. However, it is uncertain as to whether PMN-derived myeloperoxidase 

would degrade SWCNT in vivo (e.g., in the lung) 

because of the following: (1) PMN recruitment after SWCNT exposure is a transient rather than persistent response, (2) there is no strong evidence for 

SWCNT phagocytosis by PMN, and (3) SWCNT 

and MWCNT are found in the lungs of mice 

months after pharyngeal aspiration [Shvedova et 

al. 2005; Mercer et al. 2008; Porter et al. 2010]. 

Several animal studies have shown that the size 

(e.g., length) of MWCNT and SWCNT may have an 

effect on their biological activity [Takagi et al. 2008; 

Poland et al. 2008; Muller et al. 2009]. Intraperitoneal 

injection of mice with long MWCNT (20 μm length), 

but not short MWCNT (< 5 μm length), caused granulomatous lesions on the diaphragm in a 2-week 

post-exposure study [Poland et al. 2008]. Fibrotic 

peritoneal adhesions and mesothelioma were also 

observed after exposure to MWCNT in which approximately 28% of the tubes were > 5 μm in length 

[Takagi et al. 2008]. However, when rats were exposed to short MWCNT (< 1 μm length) by intraperitoneal injection, only acute inflammation was 

observed, with no evidence of mesothelioma over 

the 2 year post-exposure period [Muller et al. 2009].

Nagai et al. [2011] provided evidence that the carcinogenic potential of MWCNT may be related to 

the fiber-like properties and dimensions. Fischer 

344/Brown Norway (male and female, 6 wk old) 

were injected with doses of 1 or 10 mg of one of 

five types of MWCNT with different dimensions 

and rigidity. The thin diameter MWCNT (~50 

nm) with high crystallinity caused inflammation 

and mesothelioma, whereas thick (~150 nm) or 

tangled structures (~2–20 nm) were less cytotoxic, 

inflammogenic, or carcinogenic. A specific mutation to tumor suppressor genes (Cdkn2a/2b) was 

observed in the mesotheliomas, which is similar 

to that observed in asbestos-associated mesotheliomas induced by asbestos. In vitro studies with 

mesothelial cells showed that the thin MWCNT 

pierced cell membranes and caused cytotoxicity. 

Numerous studies have investigated the genotoxic 

properties of CNT with results from in vitro assays 

indicating that exposure to SWCNT and MWCNT 

can induce DNA damage, micronuclei formation, 

disruption of the mitotic spindle, and induction of 

polyploidy [Li et al. 2005; Kisin et al. 2007; Muller et 

al. 2008a; Pacurari et al. 2008; Lindberg et al. 2009; 

Sargent et al. 2009; Asakura et al. 2010]. Other in 

vitro studies of some MWCNT did not show evidence of genotoxicity [Wirnitzer et al. 2009; Kim et 

al. 2011]. The presence of residual metal catalysts 

was also found to promote the generation of reactive oxygen species (ROS), thereby enhancing the 

potential for DNA damage [Pulskamp et al. 2007; 

Barillet et al. 2010]. The results from in vitro studies 

with CNF have also shown that exposure can cause 

genotoxicity [Magrez et al. 2006; Lindberg et al. 

2009; Kisin et al. 2011] including aneugenic as well 

as clastogenic events. In addition, low-dose, longterm exposure of bronchial epithelial cells to SWCNT or MWCNT has been reported to transform 

these cells to exhibit unregulated proliferation, loss 

of contact inhibition of division, enhanced migration and invasion, and growth in solf agar [Stueckle 

et al. 20011]. When SWCNT-transformed epithelial cells were subcutaneously injected into the 

hind flanks of immunodeficient nude mice, small 

tumors were observed at one week post-injection. 

Histological evaluation of tumors showed classic 

cancer cell morphology, including the presence of 

multinucleated cells, an indicator of mitotic dysfunction [Wang et al. 2011]. 

When CNT and CNF are suspended in test media, 

agglomerates of various sizes frequently occur. This 

is particularly evident in test media used in recent 

studies where animals have been exposed to CNT 

suspensions by intratracheal instillation, intraperitoneal injection, or by pharyngeal aspiration (a technique where particle deposition closely resembles 

inhalation). The agglomerate size for CNT and CNF 

is normally smaller in a dry aerosol than when suspended in physiological media. Evidence from toxicity studies in laboratory animals indicates that 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 15

decreasing agglomerate size increases the pulmonary response to exposure [Shvedova et al. 2007, 

2008; Mercer et al. 2008]. The extent to which agglomerates of CNT and CNF de-agglomerate in 

biological systems (e.g., in the lung) is unknown. 

However, a diluted alveolar lining fluid has been 

shown to substantially improve dispersion of CNT 

in physiological saline [Porter et al. 2008; Wang et 

al. 2010a]. 

3.1 Single-Walled Carbon 

Nanotubes (SWCNT) 

Mice or rats exposed to SWCNT by IT or pharyngeal aspiration have developed granulomatous lesions 

at sites in the lung where agglomerates of SWCNT 

deposited [Lam et al. 2004; Warheit et al. 2004]. In 

addition, interstitial fibrosis has also been reported 

[Shvedova et al. 2005; Mangum et al. 2006]. This 

fibrotic response was associated with the migration 

of smaller SWCNT structures into the interstitium 

of alveolar septa [Mercer et al. 2008].

3.1.1 IT Studies

Lam et al. [2004] investigated the toxicity of SWCNT obtained from three different sources, each 

with different amounts of residual catalytic metals 

being present. Mice were exposed by IT to three 

different types of SWCNT (containing either 27% 

Fe, 2% Fe, or 26% Ni and 5% Y) at concentrations 

of 0.1 or 0.5 mg and to carbon black (0.5 mg) or 

to quartz (0.5 mg). The mice were toxicologically 

assessed 7 or 90 days post exposure. All types of 

SWCNT studied produced persistent epithelioid 

granulomas (which were associated with particle 

agglomerates) and interstitial inflammation that 

were dose-related. No granulomas were observed 

in mice exposed to carbon black, and only mild to 

moderate inflammation of the lungs was observed 

in the quartz exposure group. High mortality (5/9 

mice) occurred within 4 to 7 days in mice instilled 

with the 0.5 mg dose of SWCNT containing nickel 

and yttrium. 

Warheit et al. [2004] exposed rats via IT to concentrations of 1 or 5 mg/kg SWCNT, quartz, carbonyl 

iron, or graphite particles, and evaluated effects 

at 24-hr, 1-week, 1-month, and 3-months post exposure. The SWCNT were reported to have nominal diameters of 1.4 nm and lengths > 1 μm, which 

tended to agglomerate into micrometer size structures. In this study, ~15% of the SWCNT-instilled 

rats died within 24 hours of SWCNT exposure, apparently due to SWCNT blockage of the upper airways. In the remaining rats, a transient inflammatory response of the lung (observed up to 1-month 

post exposure) and non-dose dependent multifocal 

granulomas that were non-uniform in distribution 

were observed. Only rats exposed to quartz developed a dose-dependent lung inflammatory response that persisted through 3 months. Exposures 

to carbonyl iron or graphite particles produced no 

significant adverse effects. 

3.1.2 Pharyngeal Aspiration Studies

Progressive interstitial fibrosis of alveolar walls 

has also been reported in mice when exposed via 

pharyngeal aspiration to purified SWCNT at doses 

of 10, 20, 40 μg/mouse [Shvedova et al. 2005]. As 

with studies by Lam et al. [2004] and Warheit et 

al. [2004], epithelioid granulomas were associated 

with the deposition of SWCNT agglomerates in 

the terminal bronchioles and proximal alveoli. This 

granuloma formation was rapid (within 7 days), 

dose-dependent, and it persisted over the 60-day 

post exposure period. A rapid, dose-dependent, 

and progressive development of interstitial fibrosis in pulmonary regions distant from deposition 

sites of SWCNT agglomerates was observed, and it 

appeared to be associated with deposition of more 

dispersed SWCNT structures. At equivalent mass 

lung burdens, nano-sized carbon black failed to 

cause any significant pulmonary responses. These 

findings were consistent with those reported by 

Mangum et al. [2006], in which rats exposed to 

2 mg/kg via pharyngeal aspiration developed granulomas at sites of SWCNT agglomerates and diffuse interstitial fibrosis at 21 days post exposure. 

Also noted was the formation of CNT structures 
16 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

bridging alveolar macrophages, which may affect 

normal cell division and/or function. When a more 

dispersed delivery of SWCNT was given by aspiration to mice (10 μg) [Mercer et al. 2008], an accelerated increase in collagen production in the alveolar 

interstitium occurred that progressed in the absence 

of persistent inflammation, with the development of 

few granulomatous lesions. A significant submicrometer fraction of the dispersed SWCNT was observed 

to rapidly migrate into alveolar interstitial spaces 

with relatively little of the material being a target 

for macrophage engulfment and phagocytosis. 

3.1.3 Inhalation Studies

Shvedova et al. [2008] compared the responses resulting from exposure via pharyngeal aspiration 

[Shvedova et al. 2005] with exposure via inhalation 

of more-dispersed SWCNT [Baron et al. 2008]. One 

set of mice were exposed by inhalation to 5 mg/m3, 

5 hr/day for 4 days, while mice exposed by aspiration were given a single dose of 10 or 20 μg. The 

SWCNT for both studies had dimensions of 0.8–

1.2 nm diameters and 100–1000 nm lengths with 

a measured surface area (Brunauer-Emmett-Teller 

method [BET]) of 508 m2/g. Both studies reported 

acute lung inflammation followed by the development of granulomatous pneumonia and persistent 

interstitial fibrosis; these effects were observed for 

both purified (0.2% Fe) [Shvedova et al. 2005] 

and unpurified (17.7% Fe) [Shvedova et al. 2008] 

SWCNT. The finding that the acute lung inflammation resolved after the end of exposure while 

the pulmonary fibrotic response persisted or progressed is unusual compared with lung responses 

observed from other inhaled particles. The findings 

indicate that the mechanism may involve the direct 

stimulation of fibroblasts by dispersed SWCNT 

that translocate to the lung interstitium [Wang et 

al. 2010a,  b]. Quantitatively, mice exposed by inhalation (dispersed SWCNT) were 4-fold more 

prone to developing an inflammatory response, 

interstitial collagen deposition, and fibrosis, when 

compared (at an estimated equivalent lung dose) 

with mice exposed by aspiration to a less dispersed 

suspension of SWCNT. The exposure of mice 

by inhalation of 5  mg/m3 SWCNT [Shvedova et 

al. 2008] is relevant, because the Occupational 

Safety and Health Administration (OSHA) permissible exposure limit (PEL) for respirable synthetic 

graphite of 5 mg/m3 is sometimes used for controlling workplace exposures to CNT. 

3.2 Multi-Walled Carbon 

Nanotubes (MWCNT)

3.2.1 Pharyngeal Aspiration Studies

Exposures to well-dispersed MWCNT in mice via 

pharyngeal aspiration have resulted in dose- and 

time-dependent pulmonary inflammation [Han et 

al. 2008b; Wolfarth et al. 2009; Hubbs et al. 2009; 

Han et al. 2010; Porter et al. 2010; Mercer et al. 

2011], as well as central nervous system effects [Sriram et al. 2007; Sriram et al. 2009], at doses ranging from 10 to 80 μg/mouse. Exposure of mice to 

dispersed suspension of purified MWCNT at doses 

of 10, 20, 40, or 80  μg resulted in pulmonary inflammation and damage, granulomas, and a rapid 

and persistent fibrotic response [Porter et al. 2010]. 

Morphometric analyses indicated that the interstitial fibrotic response was dose-dependent and progressed through 56 days post-exposure [Mercer et 

al. 2011]. There was also evidence that MWCNT can 

reach the pleura [Porter et al. 2010] and that alveolar macrophages containing MWCNT can migrate 

to the lymphatics and cause lymphatic inflammation [Hubbs et al. 2009]. Some of the MWCNT 

(mean diameter of 49 nm and mean length of 4.2 

μm) were observed penetrating the outer lung wall 

and entered the intrapleural space [Hubbs et al. 

2009; Mercer et al. 2010]. Morphometric analyses 

indicated that 12,000 MWCNT entered the intrapleural space at 56 days post-exposure to 80 μg of 

MWCNT [Mercer et al. 2010].

3.2.2 IT Studies

Lung inflammation and fibrosis have also been 

observed in rats exposed by IT to long (5.9  μm) 

or short (0.7  μm) MWCNT at doses of 0.5, 2, or 

5 mg of either ground or unground MWCNT and 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 17

examined up to 60 days post-exposure [Muller 

et al. 2005]. Rats that received ground MWCNT 

(0.7  μm) showed greater dispersion in the lungs, 

and fibrotic lesions were observed in the deep lungs 

(alveolar region). In rats treated with unground 

MWCNT (5.9  μm), fibrosis appeared mainly in 

the airways rather than in the lung parenchyma. 

The biopersistence of the unground MWCNT was 

greater than that of the ground MWCNT (81% vs. 

36 %). At an equal mass dose, ground MWCNT 

produced a similar inflammatory and fibrogenic response as chrysotile asbestos and a greater response 

than ultrafine carbon black [Muller et al. 2005]. 

Similar findings have been reported by Aiso et 

al. [2010], in which rats exposed to IT doses of 

0.04 and 0.16 mg of dispersed MWCNT (mean 

length-5  μm, diameter-88  nm) caused transient 

inflammation, and persistent granulomas and alveolar wall fibrosis. These acute effects have also 

been reported in guinea pigs at IT doses of 12.5 mg 

[Grubek-Jaworska et al. 2005] and 15 mg [Huczko 

et al. 2005]; in mice at doses of 0.05 mg (average diameter of 50 nm, average length of 10 μm) [Li et al. 

2007], and at 5, 20, and 50 mg/kg [Park et al. 2009]; 

and in rats [Liu et al. 2008] dosed at 1, 3, 5, or 7 mg 

(diameters of 40 to 60 nm, lengths of 0.5 to 5 μm). In 

contrast, Elgrabli et al. [2008a] reported cell death 

but no histopathological lesions or fibrosis in rats exposed at doses of 1, 10, or 100 μg MWCNT (diameters of 20 to 50 nm, lengths of 0.5 to 2 μm). Likewise, 

Kobayashi et al. [2010] observed only transient lung 

inflammation and a granulomatous response in 

rats exposed to a dispersed suspension of MWCNT 

(0.04–1 mg/kg). No fibrosis was reported, but the 

authors did not use a collagen stain for histopathology, which would have compromised the sensitivity 

and specificity of their lung tissue analysis.

In a study of rats administered MWCNT or crocidolite asbestos by intrapulmonary spraying (IPS), 

exposure to either material produced inflammation 

in the lungs and pleural cavity in addition to mesothelial proliferative lesions [Xu et al. 2012]. Four 

groups of six rats each were given 0.5 ml of 500 μg 

suspensions, once every other day, five times over 

a 9-day period and then evaluated. MWCNT and 

crocidolite were found to translocate from the lung 

to the pleural cavity after administration. MWCNT 

and crocidolite were also observed in the mediastinal lymph nodes suggesting that a probable route 

of translocation of the fibers is lymphatic flow. 

Analysis of tissue sections found MWCNT and 

crocidolite in focal granulomatous lesions in the 

alveoli and in alveolar macrophages. 

3.2.3 Inhalation Studies

Several short-term inhalation studies using mice or 

rats have been conducted to assess the pulmonary 

[Mitchell et al. 2007; Arkema 2008; Ma-Hock et al. 

2009; Porter et al. 2009; Ryman-Rasmussen et al. 

2009b; Pauluhn 2010a; Wolfarth et al. 2011] and 

systemic immune effects [Mitchell et al. 2007] from 

exposure to MWCNT. Mitchell et al. [2007] reported the results of a whole-body short-term inhalation 

study with mice exposed to MWCNT (diameters of 

10 to 20 nm, lengths of 5 to 15 μm) at concentrations of 0.3, 1, or 5 mg/m3

 for 7 or 14 days (6 hr/

day) (although there was some question regarding 

whether these structures were actually MWCNT 

[Lison and Muller 2008]). Histopathology of lungs 

of exposed animals showed alveolar macrophages 

containing black particles; however, there was no 

observed inflammation or tissue damage. Systemic 

immunosuppression was observed after 14 days, although without a clear concentration-response relationship. Mitchell et al. [2009] reported that the immunosuppression mechanism of MWCNT appears 

to involve a signal originating in the lungs that activates cyclooxygenase enzymes in the spleen. Porter 

et al. [2009] reported significant pulmonary inflammation and damage in mice 1 day after inhalation of 

well-dispersed MWCNT (10 mg/m3, 5 hr/day, 2–12 

days; mass aerodynamic diameter of 1.3 μm, count 

aerodynamic diameter of 0.4 μm). In addition, granulomas were also observed encapsulating MWCNT 

in the terminal bronchial/proximal alveolar region 

of the lung. In an inhalation (nose-only) study with 

mice exposed to 30 mg/m3 MWCNT (lengths of 0.5 

to 50 μm) for 6 hours, a high incidence (9 of 10 mice) 

of fibrotic lesions occurred [Ryman-Rasmussen et 

al. 2009b]. MWCNT were found in the subpleural 
18 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

region of the lung 1  day post exposure, with subpleural fibrosis occurring at 2 weeks post exposure 

that progressed through 6  weeks of follow-up. No 

fibrosis was observed in mice exposed to 1 mg/m3 of

MWCNT or in mice exposed to 30  mg/m3 of nanoscale carbon black. 

Subchronic inhalation studies with MWCNT have 

also been conducted in laboratory studies with 

rats to assess the potential dose-response and time 

course for developing pulmonary effects [Arkema 

2008; Ma-Hock et al. 2009; Pauluhn 2010a]. MaHock et al. [2009] reported on the results of a 90-

day inhalation (head-nose) study with rats exposed 

at concentrations of 0.1, 0.5, or 2.5 mg/m3 MWCNT 

(BASF Nanocyl NC 7000) for 6 hr/day, 5 days/week 

for 13 weeks with a resultant lung burden of 47–

1170 μg/rat. No systemic toxicity was observed, 

but the exposure caused hyperplastic responses 

in the nasal cavity and upper airways (larynx and 

trachea), and granulomatous inflammation in the 

lung and in lung-associated lymph nodes at all exposure concentrations. The incidence and severity 

of the effects were concentration-related. No lung 

fibrosis was observed but pronounced alveolar lipoproteinosis did occur. 

Ellinger-Ziegelbauer and Pauluhn [2009] conducted a short-term inhalation bioassay (before 

the Pauluhn 2010a subchronic study) to investigate the dependence of pulmonary inflammation 

resulting from exposure to one type of MWCNT 

(Bayer Baytubes®), which was highly agglomerated 

and contained a small amount of cobalt (residual 

catalyst). Groups of rats were exposed to 11 mg/m3 

MWCNT containing either 0.53% or 0.12% cobalt 

to assess differences in pulmonary toxicity because 

of metal contamination. Another group of rats was 

exposed to 241  mg/m3 MWCNT (0.53% cobalt) 

to serve the purpose of hazard identification. All 

animals were exposed to a single nose-only inhalation exposure of 6  hr followed by a post-exposure period of 3 months. Time course of MWCNTrelated pulmonary toxicity was compared with rats 

exposed to quartz in post-exposure weeks 1, 4, and 

13 to distinguish early, possibly surface area/activityrelated effects from retention-related poorly soluble 

particle effects. Rats exposed to either quartz or 

MWCNT resulted in somewhat similar patterns 

of concentration-dependent pulmonary inflammation during the early phase of the study. The 

pulmonary inflammation induced by quartz increased during the 3  months post-exposure period, whereas that induced by MWCNT regressed in 

a concentration-dependent manner. The time course 

of pulmonary inflammation associated with retained 

MWCNT was independent on the concentration 

of residual cobalt. Pauluhn [2010a], using the same 

MWCNT (0.53% cobalt) used in the study by 

Ellinger-Ziegelbauer and Pauluhn [2009] exposed 

rats (nose-only) at concentrations 0.1, 0.4, 1.5, and 

6 mg/m3 for 6 hr/day, 5 days/week for 13 weeks. The 

aerosolized MWCNT were described as being highly agglomerated (mean diameter of 3  μm). Lung 

clearance of MWCNT at the low doses was slow, 

with a marked inhibition of clearance at 1.5 and 

6 mg/m3. Histopathology analysis at 6 months post 

exposure revealed exposure-related lesions in the 

upper respiratory (e.g., goblet cell hypermetaplasia 

and/or metaplasia) and lower respiratory (e.g., inflammation in the bronchiole-alveolar region) tract 

in animals exposed at concentrations of 0.4, 1.5, and 

6  mg/m3, as well as inflammatory changes in the 

distal nasal cavities that were similar to those found 

by Ma-Hock et al. [2009]. In rats exposed at 6 mg/

m3, a time-dependent increase of bronchioloalveolar hyperplasia was observed, as well as changes in 

granulomas and an increase in collagen deposition 

that persisted through the 39-week post-exposure 

observation period. No treatment-related effects 

were reported for rats exposed at 0.1 mg/m3. 

In a report submitted by Arkema [2008] to EPA, rats 

exposed (nose only) to agglomerates of MWCNT 

(Arkema) at concentrations of 0.1, 0.5, and 2.5 mg/

m3 for 6 hr/day for 5 days exhibited histopathological effects that were consistent with those reported 

by Ma-Hock et al. [2009], Ellinger-Ziegelbauer and 

Pauluhn [2009] and Pauluhn [2010a]. An increase of 

various cytokines and chemokines in the lung, along 

with the development of granulomas were found in 

the 0.5 and 2.5 mg/m3 exposure groups, while no 

treatment-related effects were reported at 0.1 mg/m3. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 19

3.3 SWCNT and MWCNT 

Intraperitoneal Studies

Intraperitoneal injection studies in rodents have 

been frequently used as screening assays for potential mesotheliogenic activity in humans. To date, 

exposures to only a few fiber types are known to 

produce mesotheliomas in humans; these include 

the asbestos minerals and erionite fibers. Several 

animal studies [Takagi et al. 2008; Poland et al. 

2008; Muller et al. 2009; Varga and Szendi 2010; 

Murphy et al. 2011] have been conducted to investigate the hazard potential of various sizes and doses 

of MWCNT and SWCNT to cause a carcinogenic 

response. Takagi et al. [2008] reported on the intraperitoneal injection of 3 mg of MWCNT in p53 

+/- mice (a tumor-sensitive, genetically engineered 

mouse model), in which approximately 28% of the 

structures were > 5 μm in length with an average 

diameter of 100 nm. After 25 weeks, 88% of mice 

treated with MWCNT revealed moderate to severe 

fibrotic peritoneal adhesions, fibrotic peritoneal 

thickening, and a high incidence of macroscopic 

peritoneal tumors. Histological examination found 

mesothelial lesions near fibrosis and granulomas. 

Similar findings were also seen in the crocidolite 

asbestos-treated positive control mice. Minimal 

mesothelial reactions and no mesotheliomas were 

produced by the same dose of (nonfibrous) C60 

fullerene. Poland et al. [2008] reported that the 

peritoneal (abdominal) injection of long MWCNT—but not short MWCNT—induced inflammation and granulomatous lesions on the abdominal side of the diaphragm at 1 week post-exposure. 

This study, in contrast to the Takagi et al. [2008] 

study, used wild type mice exposed to a much lower dose (50 μg) of MWCNT. Although this study 

documented acute inflammation, it did not evaluate whether this inflammation would persist and 

progress to mesothelioma. Murphy et al. [2011] 

found similar findings in C57BI/6 mice that were injected with different types of MWCNT composed of 

different tube dimensions and characteristics (e.g., 

tangled) or injected with mixed-length amosite asbestos. Mice were injected with a 5 μg dose directly 

into the pleural space and evaluated after 24 hours, 1, 

4, 12, and 24 weeks. Mice injected with long (> 15 

μm) MWCNT or asbestos showed significantly 

increased granulocytes in the pleural lavage, compared with the vehicle control at 24 hours post exposure. Long MWCNT caused rapid inflammation 

and persistent inflammation, fibrotic lesions, and 

mesothelial cell proliferation at the parietal pleural 

surface at 24 weeks post exposure. Short (< 4 μm) 

and tangled MWCNT did not cause a persistent inflammatory response and were mostly cleared from 

the intrapleural space within 24 hours. 

A lack of a carcinogenic response was reported by 

Muller et al. [2009] and Varga and Szendi [2010] 

in rats, and by Liang et al. [2010] in mice, following intraperitoneal injection or implantation of 

MWCNT or SWCNT. No mesotheliomas were 

noted 2  years after intraperitoneal injection of

MWCNT in rats at a single dose of 2 or 20  mg 

[Muller et al. 2009] or MWCNT (phosphorylcholine-grafted) in mice when given daily doses of 

either 10, 50, or 250  mg/kg and evaluated at day 

28 [Liang et al. 2010]. However, the MWCNT samples used in the Muller et al. [2009] and Liang et 

al. [2010] studies were very short (avg. < 1 μm in 

length observed by Muller et al. [2009] and < 2 μm 

in length observed by Liang et al. [2010]), and the 

findings were consistent with the low biological 

activity observed in the Poland et al. [2008] study 

when mice were exposed to short MWCNT. Varga 

and Szendi [2010] reported on the implantation of 

either MWCNT or SWCNT in F-344 rats (six per 

group) at a dose of 10 mg (25 mg/kg bw). Gelatin 

capsules containing either SWCNT (< 2 nm diameters × 4–15 μm lengths), MWCNT (10–30 nm diameters × 1–2 μm lengths), or crystalline zinc oxide 

(negative control) were implanted into the peritoneal cavity. Histological examination at 12 months 

revealed only a granulomatous reaction of foreign 

body type with epithelioid and multinucleated giant 

cells in CNT-exposed animals. No information was 

reported on what effect the delivery of SWCNT and 

MWCNT in gelatin capsules had on their dispersion in the peritoneal given the tendency of CNT to 

agglomerate. If SWCNT and MWCNT remained 

agglomerated following delivery, this may have 
20 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

resulted in the lack of a mesothelioma-inducing effect. The low biological activity observed for the 

short MWCNT sample (≤ 2 μm) used in the study 

was consistent with the findings from Poland et al. 

[2008], Muller et al. [2009], and Liang et al. [2010], 

in which short MWCNT were also used. 

3.4 Systemic Responses to 

Pulmonary Exposure to 

SWCNT and MWCNT

Li et al. [2007] reported that multiple aspirations of 

SWCNT (20 μg/mouse, every 2 weeks, for 2 months) 

in Apo E -/- mice caused a 71% increase in aortic 

plaques. Inhalation of MWCNT by rats (26 mg/m3

for 5 hr; lung burden of 22 μg) resulted in a 92% depression of the responsiveness of coronary arterioles to dilators 24 hr post-exposure [Stapleton 

et al. 2011], while aspiration of MWCNT has been 

shown to increase baroreflex activity in rats [Legramante et al. 2009; Coppeta et al. 2007]. Furthermore, pharyngeal aspiration of MWCNT (80 μg/

mouse) results in induction of mRNA for certain 

inflammatory mediators and markers of blood/

brain barrier damage in the olfactory bulb, frontal 

cortex, midbrain and hippocampus brain regions 

24 hr post-exposure [Sriram et al. 2009]. Several 

mechanisms have been suggested to explain these 

systemic responses:

3.4.1 Translocation of CNT to 

Systemic Sites

Translocation of intraperitoneal instilled MWCNT 

from the abdominal cavity to the lung has been reported [Liang et al. 2010]; however, there is no evidence that the reported systemic effects are associated with translocation of CNT from the lung to 

the affected tissue. Aspirated gold-labeled SWCNT 

were not found in any organ 2 weeks post-exposure 

[Mercer et al. 2009].

3.4.2 Systemic Inflammation

Pulmonary exposure to particles causes localized 

inflammation at the sites of particle deposition in 

the alveoli. Erdely et al. [2009] reported that aspiration of SWCNT or MWCNT (40 μg/m) induced a 

small but significant increase in blood neutrophils, 

mRNA expression, and protein levels for certain 

inflammatory markers in the blood at 4 hr postexposure, but not at later times. Pulmonary CNT 

exposure also significantly elevated gene expression for mediators, such as Hif - 3α and S100α, in 

the heart and aorta at 4 hr post-exposure. Evidence 

also exists that pulmonary exposure to particles alters systemic micro-vascular function by potentiating PMN as they flow through pulmonary capillaries in the close proximity to affected alveoli. These 

potentiated blood PMN adhere to micro-vessel 

walls and release reactive species that scavenge 

NO produced by endothelial cells [Nurkiewicz et 

al. 2006; Nurkiewicz et al. 2009]. Therefore, less 

dilator-induced NO diffuses to vascular smooth 

muscle resulting in less dilation. 

3.5 Carbon Nanofibers (CNF) 

Recent observations indicate that exposure to CNF 

can cause respiratory effects similar to those observed in animals exposed to CNT [Murray et al. 

2012]. In this study, female mice were exposed by 

pharyngeal aspiration to SWCNT (40 μg), CNF 

(120 μg) or crocidolite (120 μg) and evaluated post 

exposure at 1, 7, and 28 days. Delivered structure 

number or particle surface area at the highest doses 

were 1.89 × 106 and 0.042 m2 for SWCNT, 4.14 × 106

and 0.05 m2 for CNF, and 660 × 106 and 0.001 m2 for 

asbestos. SWCNT and CNF were purified and contained 0.23 and 1.4% iron, respectively compared to 

the 18% iron of the asbestos sample. SWCNT had 

diameters of 1 to 4 nm and lengths ranging from 

1 to 3 μm whereas the diameters of CNF ranged 

from 60 to 150 nm and lengths approximately 5 to 

30 μm. The fiber lengths of asbestos ranged from 

2 to 30 μm. On a mass dose bases, inflammation 

and lung damage at 1 day post-exposure followed 

the potency sequence of SWCNT>CNF>asbestos. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 21

The same potency sequence was observed for TNF 

and IL-6 production at 1 day post-exposure. SWCNT agglomerates were associated with the rapid 

(7 days) development of granulomas, while neither 

CNF nor asbestos (being more dispersed) caused 

granulomatous lesions. Interstitial fibrosis (noted 

as TGF production, lung collagen, and Sirius red 

staining of the alveolar septa) was observed at 28 

days post-exposure with a mass-based potency sequence of SWCNT>CNF=asbestos. The potency 

sequence for fibrosis was not found to be related 

to structure number or particle surface area (determined by BET gas absorption method) delivered 

to the lung. However, it is likely that gas absorption overestimates the surface area of agglomerated 

SWCNT structures delivered to the lung. Estimates 

of effective surface area, based on geometrical analysis of structures including agglomeration, provided an improved dose metric that was correlated to 

the toxicological responses to CNT and CNF.

Respiratory effects after a subchronic inhalation 

exposure of rats to CNF (purity > 99.7%) were 

recently reported by DeLorme et al. [2012]. Both 

male and female Sprague Dawley rats were exposed 

nose-only inhalation to CNF (VGCF-H Showa 

Denko), for 6 hrs/day, 5 days/week at concentrations of 0, 0.54, 2.5, or 25 mg/m3

 over a 90-day 

period and evaluated 1 day post exposure. Histopathological assessment included bronchoalveolar 

lavage fluid (BALF) analysis and cell proliferation 

studies of the terminal bronchiole, alveolar duct, 

and subpleural regions of the respiratory tract. 

The 25 mg/m3 exposed rats and the non-exposed 

control group were also evaluated after a 3-month 

recovery period. The aerosol exposure to rats was 

characterized using SEM and TEM to determine 

the size distribution and fiber concentrations using NIOSH Method 7400. At an aerosol concentration of 0.54 mg/m3

 the fiber concentration was 

4.9 fibers/cc with a MMAD of 1.9 μm (GSD 3.1), at 

2.5 mg/m3 the concentration was 56 fibers/cc with 

a MMAD of 3.2 μm (GSD 2.1), and at 25 mg/m3 the 

concentration was 252 fibers/cc with a MMAD of 

3.3 μm (GSD 2.0). The mean lengths and diameters 

of fibers were 5.8 μm and 158 nm, respectively with 

surface area measurements (by BET) of 13.8 m2/g. 

At 1-day post exposure wet lung weights were significantly elevated compared to controls in male 

rats at 25 mg/m3 and in female rats at 2.5 and 25 

mg/m3. Small increases in inflammation of the terminal bronchiole and alveolar duct regions were 

also observed in rats exposed to 2.5 mg/m3 while 

histopathological assessments of rats exposed at 

25 mg/m3 found subacute to chronic inflammation of the terminal bronchiole and alveolar duct 

regions of the lungs along with thickening of the 

interstitial walls and hypertrophy/hyperplasia of 

type II pneumocytes. No adverse histopathological findings were reported for the 0.54 mg/m3

 exposure group. After the 3-month recovery period, 

lung weights remained elevated in each sex in the 

25 mg/m3 exposure group. Inflammation and the 

numbers (> 70%) of fiber-laden alveolar macrophages still persisted in the lung of rats exposed to 

25 mg/m3 with the inflammatory response reported to be relatively minor but significantly increased 

when compared to the non-exposed control group. 

Fibers were also observed to persist in the nasal 

turbinate’s at 3-months post-exposure in all rats exposed at 25 mg/m3

 causing a nonspecific inflammatory response. In contrast to Murray et al. [2012], 

no fibrosis was noted in this inhalation study. The 

most likely reason for this discrepancy is a difference in alveolar lung burden between the Murray 

et al. [2012] and the DeLorme et al. [2012] study. 

In the former, the lung burden was 120 μg/mouse 

lung. In contrast, lung burden was not reported or 

estimated in the DeLorme et al. [2012] rat study. 

However, with a MMAD as large as 3.3 μm, nasal 

filtering would be expected to be high and alveolar 

deposition relatively low.
22 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 3–1. Findings from an uncharacterized carbon nanotube short-term intratracheal instillation 

(IT) toxicology study

Study design and exposure/dose Observed pulmonary effects

Author/year Species Exposure route

Exposure

or dose Granuloma Inflammation Fibrosis

Huczko et al. 

[2001]

G. pigs IT of soot 

containing CNT 

(uncharacterized)

25 mg

(eval: 28 days 

post exposure)

NR – NR

NR: Not Reported

+ = effect observed

– = no effect observed 

Table 3–2. Findings from published SWCNT short-term intratracheal instillation 

(IT) toxicology studies

Study design and exposure/dose Observed pulmonary effects

Author/year Species Exposure route

Exposure

or dose Granuloma Inflammation Fibrosis

Warheit et al. 

[2004]

Rats IT 1, 5 mg

(eval: 24-hr, 

1-wk, 1 and 3 mo. 

post exposure)

+ non-dose 

dependent

transient –

Lam et al. 

[2004]

Mice IT 0.1, 0.5 mg

(eval: 7 or 

90 days post 

exposure]

+ + NR

Inoue et al. 

[2008]

Mice IT 4 mg

(eval: 24-hr post 

exposure)

NR + NR

NR: Not Reported

+ = effect observed

– = no effect observed 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 23

Table 3–3. Findings from published SWCNT short-term aspiration toxicology studies

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure 

or dose Granuloma Inflammation Fibrosis

Shvedova

et al. [2005]

Mice Pharyngeal 

aspiration

10, 20, 40 μg

(eval: 1, 3, 7, 28, 

and 60 days post 

exposure)

+ + +

Mangum

et al. [2006]

Rats Pharyngeal 

aspiration

2 mg/kg

(eval: 1 or 

21 days post 

exposure)

+ – + 

(interstitial 

lesions)

Shvedova

et al. [2007]

Mice 

(vitamin E 

deficient)

Pharyngeal 

aspiration

40 μg

(eval: 1, 7, and 

28 days post 

exposure)

+ + +

Mercer

et al. [2008]

 Mice Pharyngeal 

aspiration

10 μg

(eval: 1-hr, 1 and 

7 days and 

1 mo. post 

exposure)

+ (undispersed)

– (dispersed)

+ +

Shvedova

et al. [2008]

Mice Pharyngeal 

aspiration

5,10, 20 μg

(eval: 1, 7, and 

28 days post 

exposure)

+ + +

NR=Not Reported

+ = effect observed

– = no effect observed
24 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 3–4. Findings from published SWCNT and CNF short-term inhalation toxicology studies 

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure

or dose Granuloma Inflammation Fibrosis

Shvedova

et al. [2008]

Mice Inhalation SWNCT—5 mg/m3 

5 hr/day for

4 days (eval: 1, 7, 

and 28 days post 

exposure)

+ + +

DeLorme et 

al. [2012]

Rats Nose-only 

inhalation

CNF—0.54, 2.5 or 

25 mg/m3 6 hr/day 

for 90 days. (eval: 

1 and 90 days post 

exposure)

– –(0.54 mg/m3)

+(2.5 and

25 mg/m3)

–

NR = Not Reported

+ = effect observed

– = no effect observed
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 25

Table 3–5. Findings from published MWCNT short-term intratracheal (IT)

instillation and intrapulmonary spraying toxicology studies

Study design and exposure/dose Observed pulmonary effects

Author/year Species Exposure route

Exposure

or dose Granuloma Inflammation Fibrosis

Muller et al. [2005] Rats IT 0.5, 2, 5 mg

(eval: 1 hr, 3, 15, 28, 

and 60 days post exp)

+ + +

Huczko et al. 

[2005]

G. 

pigs

IT 15 mg

(eval: 90 days

post exp)

NT

(pneumonialike reaction)

+ (Increased 

lung 

resistance) +/–

Grubek-Jaworska 

et al. [2005]

G. 

Pigs

IT 12.5 mg

(eval: 90 days

post exp)

+ + +

Carrero-Sanchez

et al. [2006]

Mice IT 1, 2.5,

5 mg/kg

(eval: 1, 2, 3, 7 and 30 

days post exp)

+ + +

Deng et al. [2007] Mice IT 600 μg

(eval: 1 day

post exp)

NR – NR

Li et al. [2007] Mice IT 0.05 mg

(eval: 8, 16, and 24 

days post exp)

NR + NR

Liu et al. [2008] Rats IT 1, 3, 5, and 7 mg/kg

(eval: 1 and 7 days, 1 

and 3 mo. post exp)

+ + NR

Muller et al. 

[2008a]

Rats IT 2 mg

(eval: 3 and 60 days 

post exp)

+ + NR

Muller et al. 

[2008b]

Rats IT 0.5 or 2 mg

(eval: 3 days post exp)

NR + NR

Inoue et al. [2008] Mice IT 4 mg/kg

(eval: 1 day post exp)

NR + NR

Elgrabli et al. 

[2008a]

Rats IT 1, 10, 100 μg

(eval: 1, 7, 30, 90 and 

180 d post exp)

– – –

See footnotes at end of table. (Continued)
26 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Study design and exposure/dose Observed pulmonary effects

Author/year Species Exposure route

Exposure

or dose Granuloma Inflammation Fibrosis

Park et al. [2009] Mice IT 0.04, 0.2 or 1 mg/kg

(eval: 3 days, 1 week, 

1-mo, 3-mo, 6-mo 

post exp)

+ + NR

Aiso et al. [2010] Rats IT 5, 20, or 50 mg/kg

(eval: 1, 3, 7 or 14 

days post exp)

+ transient +

Kobayashi et al. 

[2010]

Rats IT 0.04 or 0.16 mg

(eval: 1, 7, 28 or 91 

days post exp)

transient transient –

Xu et al. [2012] Rats Intrapulmonary 

spray

MWCNT—0.5 ml of 

500 ug suspensions, 

5 times over 9-days 

and then evaluated

+ + NA

NR = Not Reported 

+ = effect observed

– = no effect observed

Table 3–5 (Continued). Findings from published MWCNT short-term intratracheal (IT)

instillation and intrapulmonary spraying toxicology studies
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 27

Table 3–6. Findings from published MWCNT or CNF short-term aspiration toxicology studies 

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route Exposure or dose Granuloma Inflammation Fibrosis

Sriram et al. 

[2007]

Mice Pharyngeal 

aspiration

MWCNT—

10, 20, or 40 μg

+ +

Including 

neuroinflammation 

of the brain

NR

Han et al. 

[2008b]

Mice Pharyngeal 

aspiration 

with ozone 

exposure

MWCNT—20 μg

(eval: 5 and 24-hr

post exp)

NR + NR

Hubbs et al. 

[2009]

Mice Pharyngeal 

aspiration

MWCNT—20 or 80 μg 

(eval: 7 and 56 days 

post exp)

+ + +

Sriram et al. 

[2009]

Mice Pharyngeal 

aspiration

MWCNT—10 or 80 μg 

(eval: 1, 7, 28 days

post exp)

NR neuroinflammation NR

Wolfarth et al. 

[2009]

Mice Pharyngeal 

aspiration

MWCNT—40 μg

(eval: 1, 7, 28, and 56 

days post exp)

+ + +

Porter et al. 

[2010]

Mice Pharyngeal 

aspiration

MWCNT—10, 20, 40, 

or 80 μg (eval: 1, 7, and 

28 days post exp)

+ + +

Han et al. 

[2010]

Mice Pharyngeal 

aspiration

MWCNT—20 or 40 μg 

(eval: 1 and 7 days

post exp)

NR + NR

Mercer et al. 

[2011]

Mice Pharyngeal 

aspiration

MWCNT—10, 20,40, 

or 80 μg (eval: 1, 7, 28, 

and 56 days)

+ + +

Murray et al. 

[2012]

Mice Pharyngeal 

aspiration

CNF—120 μg

(eval: 1, 7, and 28 days 

post exp)

+ + +

NR = Not Reported

+ = effect observed

– = no effect observed
28 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 3–7. Findings from published MWCNT short-term inhalation toxicology studies 

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure

or dose Granuloma Inflammation Fibrosis

Li et al. [2007] Mice Inhalation Est. lung 

deposition 

dose: 0.07, 0.14, 

.21 mg. (eval: at 

days 8, 16, and 

24)

NR – NR

Mitchell et al. 

[2007]

Mice Inhalation 0.3, 1, 

5 mg/m3

6 hr/day for 7 or 

14 days. (eval: at 

days 7 and 14)

– – –

Arkema 

[2008]

Rats Head-nose 

inhalation

0.1, 0.5,

2.5 mg/m3

6 hr/day for 5 

days. (eval: at 

days 7 and 28)

– (0.1 mg/m3)

+ (0.5, 2.5 

mg/m3)

– (0.1 mg/m3)

+ (0.5, 2.5 mg/

m3)

–

RymanRasmussen

et al. [2009a]

Mice w/

preexisting 

allergic 

inflammation

Nose-only 

inhalation

100 mg/m3 for 

6 hr (~10 mg/kg 

alveolar dose). 

(eval: at days 1 

and 14)

Lung injury + + when 

preexisting 

allergic 

inflammation 

exists

Ma-Hock

et al. [2009]

Rats Head-nose 

inhalation

0.1, 0.5, 

2.5 mg/m3 6 hr/

day–5 days/wk. 

for 13 weeks. 

(eval: at week 13) 

+ + –

Porter et al. 

[2009]

Mice Whole 

body 

inhalation

10 mg/m3

5 hr/day for 2, 4, 

and 8 days, then 

evaluated

+ + +

Sriram et al. 

[2009]

Mice Whole 

body 

inhalation

10 mg/m3

5 hr/day for 

2, 4, and 8 days, 

then evaluated

NR NeuroinflammationNR

See footnotes at end of table. (Continued)
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 29

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure

or dose Granuloma Inflammation Fibrosis

EllingerZiegelbauer 

[2009]

Rats Nose-only 

inhalation

11 and 241

mg/m3 for 6 hr 

(eval: at days 7, 

28, and 90)

NR + – (11 mg/m3)

+ (241 mg/m3)

RymanRasmussen et 

al. [2009b]

Mice Nose-only 

inhalation

1 or 30 mg/

m3 for 6 hr 

(~0.2 mg/kg and 

4 mg/kg alveolar 

dose), (eval: at 

1 day, and 2, 6, 

and 14)

– – (1 mg/m3)

+ (30 mg/m3)

– (1 mg/m3)

+ (30 mg/m3)

Pauluhn 

[2010a]

Rats Nose-only 

inhalation

0.1, 0.4, 1.5 and 

6 mg/m3 for 6 hr, 

5 days/week for 

13 weeks

+(6 mg/m3) – (0.1 mg/m3)

+ (0.4,1.5,

 6 mg/m3)

– (0.1 mg/m3)

(0.4 mg/m3, 

focal septal 

thickening)

+(1.5, 6 mg/m3)

NR = not reported

+ = effect observed

– = no effect observed

Table 3–7 (Continued). Findings from published MWCNT short-term inhalation toxicology studies
30 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 3–8. Findings from published MWCNT or SWCNT short-term injection/implantation 

toxicology studies 

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure

or dose Mesothelioma Inflammation Fibrosis

Deng et al. 

[2007]

Mice Intravenous 

injection

(also gavage)

1–600 μg 

MWCNT 

depending on 

exp. route

NR – NR

Takagi et al. 

[2008]

Mice Intraperitoneal 

injection

27.5 % longer 

than 5 μm; 

1 × 109

MWCNT/1 mL 

(corresponds 

to 3 mg) (eval: 

week 25)

mesothelioma + +

Poland et al. 

[2008]

Mice Intraperitoneal 

injection

Long and short 

MWCNT

50 μg (eval: 1 

and 7 days 

post exp)

Increase in 

response with 

increasing fiber 

length

Increase in 

response with 

increasing 

fiber length

NR

Muller et al. 

[2009]

Rats Intraperitoneal 

injection

MWCNT 

< 1 μm on avg. 

length;

2 or 20 mg w/

defects, 

20 mg wo/

defects. (eval: 

month 24) 

No mesotheliomas

Sakamoto et al. 

[2009]

Rats Intrascrotal 

injection

MWCNT > 5 

μm in length; 

0.24 mg (1 mg/

kg body weight) 

27.5 % 

mesothelioma NR NR

Varga and 

Szendi [2010]

Rats Peritoneal 

implantation 

(in gelatin 

capsule)

10 mg of 

MWCNT (1–2 

μm length) or 

SWCNT (4–

15 μm length)

No mesotheliomas – –

See footnotes at end of table. (Continued)
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 31

Study design and exposure/dose Observed pulmonary effects

Author/year Species

Exposure 

route

Exposure

or dose Mesothelioma Inflammation Fibrosis

Liang et al. 

[2010]

Mice Intraperitoneal 

injection

MWCNT 

(200 nm–2 μm 

length); 10, 50, 

or 250 mg/kg 

(eval: 28 days)

No mesotheliomas - (10 and 50 

mg/kg)

+ (250 mg/kg)

- (10 and 50 

mg/kg) 

+ (250 mg/

kg)

Murphy et al. 

[2011]

Mice Intrapleural 

injection

MWCNT 

different 

lengths; 5 μg; 

(eval: 1 day, 

1,4, 12 and 24 

weeks)

No mesotheliomas + long 

MWCNT

+ long 

MWCNT

Nagai et al. 

[2011]

Rats Intrapleural 

injection

MWCNT 

(mean lengths 

~4-5 μm); 1 or 

10 mg; (eval: 

up to 1 yr.) 

agglomerated 

and nonagglomerated 

+ 1mg (nonagglomerated) 

mesothelioma 

at higher 

frequency than 

agglomerated 

+ (1 and

10 mg)

+ (1 and

10 mg)

NR = not reported 

+ = effect observed 

– = no effect observed 

Table 3–8. Findings from published MWCNT or SWCNT short-term injection/implantation 

toxicology studies 

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 33

4 Conclusions—Hazard and Exposure Assessment 

Results of laboratory animal studies with both 

SWCNT and MWCNT report qualitatively similar 

pulmonary responses including acute lung inflammation, epithelioid granulomas (microscopic nodules), and rapidly developing fibrotic responses at 

relatively low-mass doses (Section 3). Animal studies with CNT and CNF have shown the following: 

1. Early onset and persistent pulmonary fibrosis 

in SWCNT-, MWCNT-, and CNF- exposed 

animals in short-term and subchronic studies 

[Shvedova et al. 2005, 2008; Mercer et al. 2008; 

Ma-Hock et al. 2009; Porter et al. 2010; Pauluhn 

2010a; Mercer et al. 2011; Murray et al. 2012]. 

2. Similar pulmonary responses in animals (e.g., 

acute lung inflammation, interstitial fibrosis) 

when exposed to purified and unpurified SWCNT 

[Lam et al. 2004; Shvedova et al. 2005, 2008]. 

3. Equal or greater potency of SWCNT, MWCNT, 

and CNF compared with other inhaled particles (ultrafine carbon black, crystalline silica, 

and asbestos) in causing adverse lung effects 

including pulmonary inflammation and fibrosis [Shvedova et al. 2005; Muller et al. 2005; 

Murray et al. 2012]. 

4. CNT agglomeration affects the site of lung deposition and response; large agglomerates tend 

to deposit at the terminal bronchioles and 

proximal alveoli and induce a granulomatous 

response, while more dispersed structures deposit in the distal alveoli and cause interstitial 

fibrosis [Mercer et al. 2008; Porter et al. 2010]. 

Agglomerated SWCNT tend to induce granulomas, while more dispersed CNF and asbestos 

did not [Murray et al. 2012].

5. Intraperitoneal injection of long (>  5 μm) 

MWCNT in mice causes fibrotic lesions and 

mesothelial cell proliferation [Takagi et al. 2008; 

Murphy et al. 2011].

Although pulmonary responses to SWCNT and 

MWCNT are qualitatively similar, quantitative 

differences in pulmonary responses have been reported. In mice exposed to CNT by pharyngeal aspiration (10 μg/mouse), SWCNT caused a greater 

inflammatory response than MWCNT at 1  day 

post exposure [Shvedova et al. 2005, 2008; Porter 

et al. 2010]. Morphometric analyses indicate that 

well-dispersed purified SWCNT (< 0.23% iron) are 

not well recognized by alveolar macrophages (only 

10% of the alveolar burden being within alveolar 

macrophages) [Shvedova et al. 2005], and that 90% 

of the dispersed SWCNT structures have been observed to cross alveolar epithelial cells and enter 

the interstitium [Mercer et al. 2008]. In contrast, 

approximately 70% of MWCNT in the respiratory 

airways are taken up by alveolar macrophages, 8% 

migrate into the alveolar septa, and 22% are found 

in granulomatous lesions [Mercer et al. 2010, 

2011]. These findings suggest that well-dispersed 

SWCNT may be more potent in causing interstitial 

fibrosis on an equal mass lung burden basis than 

MWCNT [Mercer et al. 2008], possibly due to the 

greater tube count per mass of SWCNT [Mercer 

2011; Wang et al. 2010a,b; Mercer et al. 2011]. In 

addition, although both SWCNT and MWCNT 

have been reported in the subpleural tissue of the 

lung [Mercer et al. 2008; Ryman-Rasmussen et al. 

2009], penetration of the visceral pleura and translocation to the intrapleural space has been reported 

only for MWCNT [Hubbs et al. 2009; Mercer et al. 

2010]. Despite these differences, CNTs of various 

types, both purified and unpurified, dispersed or 

agglomerated, all cause adverse lung effects in rats 

or mice at relatively low mass doses that are relevant to potential worker exposures.

Animal studies have also shown asbestos-type pathology associated with the longer, straighter CNT 

structures [Poland et al. 2008; Takagi et al. 2008; 

Murphy et al. 2011]. Mesothelial tumors have been 

reported in mice receiving intraperitoneal injection 

of long MWCNT (5–20 μm in length) [Takagi et al. 

2008; Murphy et al. 2011]; whereas chronic bioassays of short MWCNT (avg. < 1 μm and < 2 μm in 
34 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

length, respectively) [Muller et al. 2009; Liang et al. 

2010] did not produce mesothelioma. These findings are consistent with those reported by Yamashita et al. [2010] and Nagai et al. [2011] who found 

that MWCNT injected into the peritoneal cavity of 

mice or rats generated inflammation/genetic damage and mesothelioma that were related to the dimension of the CNT. Results from these peritoneal 

assay studies indicate that CNT of specific dimensions and durability can cause inflammation, fibrosis, and mesothelial tumors in mice and in rats; 

however, additional experimental animal research 

is needed to: (1) provide quantitative data on the 

biopersistence of different types of CNT in the lung 

and, (2) address the key question as to the precise 

dimensions (and possibly other physical-chemical 

characteristics) of CNT that pose a potential pathogenic risk for cancer including mesothelioma. 

As synthesized, raw (unpurified) CNT, contain as 

much as 30% catalytic metals. Catalytic metals, 

such as iron-rich SWCNT, can generate hydroxyl 

radicals in the presence of hydrogen peroxide and 

organic (lipid) peroxides [Kagan et al. 2006], and 

when human epidermal keratinocytes cells are exposed to unpurified SWCNT (in vitro cellular studies), oxidant injury occurs [Shvedova et al. 2003]. 

These catalytic metals can be removed from raw 

CNT by acid treatment or by high temperature to 

yield purified CNT with low metal content. Removal of catalytic metals abolishes the ability of 

SWCNT or MWCNT to generate hydroxyl radicals. However, in laboratory animal studies the pulmonary bioactivity of SWCNT does not appear to 

be affected by the presence or absence of catalytic 

metals. Lam et al. [2004] compared the pulmonary 

response of mice to intratracheal instillation of raw 

(containing 25% metal catalyst) with purified (~2% 

iron) SWCNT and found that the granulomatous 

reaction was not dependent on metal contamination. Likewise, the acute inflammatory reaction of 

mice after aspiration of raw (30% iron) versus purified (< 1% iron) SWCNT was not affected by metal 

content [Shvedova et al. 2005, 2008]. 

Pulmonary exposure to CNT have shown systemic 

responses including an increase in inflammatory 

mediators in the blood, as well as oxidant stress in 

aortic tissue and increase plaque formation in an 

atherosclerotic mouse model [Li et al. 2007; Erdely et al. 2009]. Pulmonary exposure to MWCNT 

also depresses the ability of coronary arterioles to 

respond to dilators [Stapleton et al. 2011]. These 

cardiovascular effects may be due to neurogenic 

signals from sensory irritant receptors in the lung. 

Mechanisms, such as inflammatory signals or neurogenic pathways causing these systemic responses, 

are under investigation. 

Results from in vitro cellular studies have shown 

that SWCNT can cause genotoxicity and abnormal 

chromosome number, because of interference with 

mitosis (cell division), by disrupting the mitotic 

spindles in dividing cells and inducing the formation of anaphase bridges among the nuclei [Sargent 

et al. 2009]. In vitro studies also indicate that exposure to CNF can cause genotoxicity (micronuclei) 

as a result of reactive oxygen species (ROS) production, which in turn reacts with DNA, and by 

interfering physically with the DNA/chromosomes 

and/or mitotic apparatus [Kisin et al. 2011]. Lowdose, long-term exposure of bronchial epithelial 

cells to MWCNT has been shown to induce cell 

transformation, and these transformed cells induce 

tumors after injection into nude mice [Stueckle et 

al. 2011; Wang et al. 2011].

Currently, there are no studies reported in the literature on the adverse health effects in workers producing or using CNT or CNF. However, because 

humans can also develop lung inflammation and 

fibrosis in response to inhaled particles and fibers, 

it is reasonable to assume that at equivalent exposures (e.g., lung burden/alveolar epithelial cell surface) to CNT and CNF, workers may also be at risk 

of developing these adverse lung effects. 

Although data on workplace exposures to CNT and 

CNF are limited, aerosolization of CNT and CNF 

has been shown to occur at a number of operations 

during research, production, and use of CNT and 

CNF, including such work tasks as transferring, 

weighing, blending, and mixing. Worker exposure 

to airborne CNT and CNF has frequently been 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 35

observed to be task-specific and short-term in duration, with exposure concentrations (frequently 

reported as particle number or mass concentrations) found to exceed background exposure measurements when appropriate engineering controls 

are not used to reduce exposures [Maynard et al. 

2004; Methner et al. 2007; Han et al. 2008a; Bello et 

al. 2009; Tsai et al. 2009; Bello et al. 2010; Evans et al. 

2010; Johnson et al. 2010; Lee et al. 2010; Cena and 

Peters 2011; Dahm et al. 2011]. Results from studies also suggest that the airborne concentration and 

the physical-chemical characteristics of particles 

(e.g., discrete versus agglomerated CNT) released 

while handling CNT may vary significantly with 

production batch and work process. Comprehensive workplace exposure evaluations are needed to 

characterize and quantify worker exposure to CNT 

and CNF at various job tasks and operations, and 

to determine what control measures are the most 

effective in reducing worker exposures. 

The findings of adverse respiratory effects (i.e., pulmonary fibrosis, granulomatous inflammation) and 

systemic responses in animals indicate the need 

for protective measures to reduce the health risk to 

workers exposed to CNT and CNF. Available 

evidence also indicates that the migration of MWCNT into the intrapleural space could potentially 

initiate mesothelial injury and inflammation that 

over time cause pleural pathology, including mesothelioma. Long-term inhalation studies are needed 

to determine whether CNT and CNF of specific 

dimension and chemistry can cause cancer in 

laboratory animals at doses equivalent to potential 

workplace exposures. In addition, the potential for 

migration of CNT through the lungs and for accumulation in the intrapleural space with time after 

inhalation requires further investigation. Until results from animal research studies can fully explain 

the mechanisms in which inhalation exposure to 

CNT and CNF cause adverse lung effects and possible systemic effects, all types of CNT and CNF 

should be considered an occupational respiratory 

hazard, and the following actions should be taken 

to minimize health concerns: 

1. Minimize workplace exposures. 

2. Establish an occupational health surveillance 

program for workers exposed to CNT and CNF 

(Section 6, Appendix B).

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 37

5 CNT Risk Assessment and Recommended

Exposure Limit

5.1 Risk Assessment and

Recommended Exposure

Limit (REL)

NIOSH bases its recommended exposure limits 

(RELs) on quantitative risk assessments when possible. Quantitative risk assessment provides estimates of the severity and likelihood of an adverse 

response associated with exposure to a hazardous 

substance. The hazard and quantitative risk assessments (Section 4 and Appendix A) provide the 

health basis for developing a recommended exposure limit (REL) for CNT and CNF. Establishing 

health-based exposure limits is the first consideration by NIOSH in setting a REL. The analytical 

feasibility of measuring worker exposures to airborne CNT and CNF is also taken into account in 

the establishment of the REL (Section 6.1).

In general, quantitative risk assessment involves 

the following steps: first a data set is selected that 

best depicts a dose-response relationship, in this 

case, the relationship between exposure to CNT 

and pulmonary effects in animals. Then, a critical 

dose in the animal lungs is calculated. A frequently 

used indicator of critical dose is the benchmark 

dose (BMD) which is defined as the dose corresponding to a small increase in response (e.g. 10%) 

over the background level of response [Crump 

1984]. Next, the dose in humans, that is equivalent 

to the critical dose in the animals, is estimated. This 

requires adjusting for species differences between 

animals and humans. It is assumed in the absence 

of specific data that an equivalent dose in animals 

and humans will result in the same risk of disease, 

based on the assumption that the same mechanism 

of action is operating in both animals and humans. 

After the critical average dose in human lungs 

is estimated from the animal data, an equivalent 

workplace concentration over a full working lifetime is derived. This is accomplished by using 

mathematical and physiological models to estimate 

the fraction of the dose that reaches various parts 

of the respiratory tract and is deposited and cleared 

[Kuempel et al. 2006; Schulte et al. 2010; NIOSH 

2011a]. Variability in human dose and response, 

including sensitive subpopulations, and uncertainty in the extrapolating animal data to humans are 

typically addressed with uncertainty factors in the 

absence of specific data.

NIOSH determined that the best data to use for a 

quantitative risk assessment and as the basis for a 

REL were the nonmalignant pulmonary data from 

short-term and subchonic animal studies. In these 

studies, lung exposures to CNT (i.e., various types 

of MWCNT and SWCNT, purified and unpurified, 

dispersed or agglomerated, and with different metal 

content) were observed to cause early-stage adverse 

lung effects including, pulmonary inflammation, 

granuloma, alveolar septal thickening, and pulmonary fibrosis (Section 3 and Appendix A). NIOSH 

considers these animal lung effects to be relevant 

to workers because similar lung effects have also 

been observed in workers with occupational lung 

disease associated with exposure to various types of 

inhaled particles and fibers [Rom and Markowitz 

2006; Hubbs et al. 2011]. Human-equivalent risk 

estimates were derived from animal dose-response 

data (in rats and mice). Human-equivalent exposures over a 45-year working lifetime were estimated to be associated with either a specified risk level 

(e.g., 10%) of early-stage lung effects or with a no 

observed adverse effect level based on the animal 

studies. In the absence of validated lung dosimetry 

models for CNT, lung doses were estimated using 
38 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

spherical particle-based models and CNT airborne 

size data, assuming either deposited or retained 

lung dose in animals or humans. 

The findings from this analysis indicate that workers are potentially at risk of developing adverse lung 

effects if exposed to airborne CNT during a 45-year 

working lifetime. Table 5–1 provides a summary 

of the estimated exposure concentrations (8-hour 

TWA) associated with 10% excess risk based on the 

animal data. Table 5–2 provides a summary of the 

risk estimates at the REL of 1 μg/m3 (8-hour TWA). 

Working lifetime exposures to 0.2–2 μg/m3 (8-hour 

TWA concentration) were estimated to be associated with 10% excess risk of early stage lung effects 

(minimal granulomatous inflammation or alveolar 

septal thickening, grade 1 or higher) (95% lower 

confidence limit, LCL estimates) based on results 

from the subchronic animal inhalation studies with 

MWCNT [Ma-Hock et al. 2009; Pauluhn 2010a] 

(Tables 5–1 and A–5). For slight/mild (grade 2 or 

higher) lung effects, the working lifetime exposure 

estimates are 0.7–19 μg/m3 (8-hour TWA concentration; 95% LCL estimates) (Tables 5–1 and 

Table 5–1. Estimated exposure concentration associated with a 10% risk of adverse lung effects 

above background.*

Lung disease indicator†

Estimated working lifetime exposure concentration (8-hr TWA)‡

Maximum likelihood estimate

95% Lower confidence

limit estimate

Minimal lung effects (grade 1 or higher) 0.5 to 4 μg/m3 0.2 to 2 μg/m3

Slight or mild lung effects (grade 2

or higher) 1 to 44 μg/m3 0.7 to 19 μg/m3

Abbreviation: TWA=Time-weighted average.

*

Excess (exposure-attributable) risk during a 45-year working lifetime.

†

Histopathology findings of granulomatous inflammation [Ma-Hock et al. 2009] or alveolar septal thickening [Pauluhn 2010] in 

rat subchronic inhalation studies of multiwall carbon nanotubes. ‡

Estimates vary by rat study and lung burden estimation method (Appendix A, Tables A–7 and A–8).

Table 5–2. Estimated risk of adverse lung effects at recommended exposure limit of 1 μg/m3

(8-hour TWA) during a 45-year working lifetime.

Lung disease indicator*

Excess risk†

Maximum likelihood estimate

95% Upper confidence

limit estimate

Minimal lung effects (grade 1 or higher) 2.4% to 33% 5.3% to 54%

Slight or mild lung effects (grade 2

or higher) 0.23% to 10% 0.53% to 16%

Abbreviation: TWA=Time-weighted average.

*

Histopathology findings of granulomatous inflammation [Ma-Hock et al. 2009] or alveolar septal thickening [Pauluhn 2010] in 

rat subchronic inhalation studies of multiwall carbon nanotubes. †

Exposure-attributable risk (added risk above background). Estimates vary by rat study and lung burden estimation method 

(Appendix A, Tables A–7 and A–8).
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 39

A–6). Risk estimates derived from other animal 

studies (e.g., single dose with up to 90-day followup) using SWCNT and other types of MWCNT 

(Tables A–3 and A–4) are consistent with these 

estimates, i.e., 0.08–12 μg/m3 (8-hour TWA) (95% 

LCL estimates). These working lifetime exposure 

concentration estimates vary by approximately two 

orders of magnitude (across the different types of 

CNT, study design, animal species/strain and gender, route of exposure, and response endpoints); 

yet all of these estimates are relatively low airborne 

mass concentrations, most within ~1–10 μg/m3

(8-hour TWA). NIOSH does not consider a 10% 

estimated excess risk over a working lifetime to be 

acceptable for these early-stage lung effects, and 

the REL is set at the optimal limit of quantification 

(LOQ) of the analytical method carbon (NIOSH 

method 5040) (Appendix C). 

Additional estimates were derived from the no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL) in the rat subchronic inhalation studies of MWCNT [Pauluhn 

2010a; Ma-Hock et al. 2009] (Section A.6.2 and 

A.6.3). The human-equivalent working lifetime 

concentrations of ~4-18 μg/m3 (8-hour TWA) were 

estimated to be equivalent to the rat subchronic 

NOAEL or LOAEL of 0.1 mg/m3 (Table A–13). Applying data-appropriate uncertainty factors (e.g., 

Table A–14) to estimate safe working lifetime exposure (essentially zero risk assuming a threshold 

model) results in concentrations of less than 1 μg/m3

(8-hour TWA) over a 45-yr working lifetime. 

A more detailed summary of the working lifetime 

exposure estimates—shown by animal study, lung response, and effect level estimate—is provided in Table 

5–3. Table 5–4 summarizes the factors, assumptions, 

and options involved in the CNT risk assessment 

(see Appendix A for complete information). Evaluations of the variability and uncertainty in these risk 

estimates are provided in Sections 5.3, A.4, and A.6. 

Among the uncertainties in this risk assessment 

using animal data, there is uncertainty in extrapolating the respiratory effects observed in shortterm or subchronic animal studies to estimate 

the probability of chronic respiratory effects in 

humans. In the absence of chronic data, these animal studies provide the best available information 

to derive initial estimates of health risk for use in 

REL development. Subchronic (13  wk.) exposure 

studies are a standard toxicity assay used in human 

health risk assessment, although the studies with 

shorter exposure and post-exposure durations also 

provide useful information about the relationship 

between CNT lung dose and response. To the extent that the precursor effects to chronic disease are 

observed in these shorter-term studies, the hazard 

and risk estimates would be expected to provide 

useful information for chronic disease prediction 

and prevention. Although there is uncertainty in 

the benchmark dose estimates from the subchronic 

studies because of the dose-spacing and high response proportions, these estimates are similar to 

the NOAEL and LOAEL values reported in these 

studies [Ma-Hock et al. 2009; Pauluhn 2010a] 

(Table A–12).

One of the measures of pulmonary fibrosis used 

in the shorter-term studies [Shvedova et al. 2005, 

2008; Mercer et al. 2008, 2011]—alveolar epithelial 

cell thickness (due to collagen deposition)—was 

previously used in the U.S. EPA ozone standard. 

This biological response was selected by EPA as 

the adverse lung response for cross-species doseresponse extrapolation because it indicates “fundamental structural remodeling” [US EPA 1996; 

Stockstill et al. 1995]. 

Some of these studies provide data comparing the 

potency of CNT with that of other particles or fibers 

for which animal and human data are available on 

the long-term adverse health effects. These studies 

show that on a mass basis, CNT had equal or greater 

potency (pulmonary inflammation or fibrosis response at a given mass dose) to that of ultrafine carbon black, crystalline silica, or chrysotile asbestos 

[Lam et al. 2004; Muller et al. 2005; Shvedova et al. 

2005]. These comparative toxicity findings between 

CNT and other well-studied particles or fibers help 

to reduce the uncertainty about whether the lung 

effects in these short-term studies are relevant to 

evaluating the chronic respiratory hazard of CNT. 
40 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 5–3. Summary of human-equivalent working lifetime exposure concentration estimates 

associated with animal effect levels

Animal 

effect level Animal lung response

Working lifetime 8-hr TWA (μg/m3)

Animal study 

Table

in CIB

Assuming 

estimated 

deposited

lung dose* 

Assuming 

estimated 

retained

lung dose*

Subchronic studies

NOAEL No statistically significant 

lung response

nd 3.5 Pauluhn 2010a A–13

LOAEL Minimal granulomatous 

inflammation (grade 1+)

nd 4.0 Ma-Hock et al. 

2009

A–13

BMD10 Minimal granulomatous 

inflammation or alveolar 

septal thickening (grade 1+) 

0.51, 0.77 2.7, 4.2 Ma-Hock et al. 

2009; Pauluhn 

2010a, respectively

A–5

BMDL10 0.19, 0.38 1.0, 1.9

BMD10 Slight/mild granulomatous 

inflammation or alveolar 

septal thickening (grade 2+)

1.0, 6.4 6.2, 44 Ma-Hock et al. 

2009; Pauluhn 

2010a, respectively

A–6

BMDL10 0.69, 3.3 4, 19

Short-term studies

BMD10 Alveolar connective tissue 

thickness

0.11, 1.8, 4.7 nd Shvedova et al. 

2005; Mercer et 

al. 2011; Shvedova 

et al. 2008, 

respectively 

A–3

BMDL10 0.075, 1.0, 2.5

BMD10 Hydroxyproline amount 18 nd Muller et al. 2005 A–3

BMDL10 12

BMD10 

BMDL10

Granuloma 10

 1.7

nd Lam et al. 2004 A–4

Abbreviations: nd=not determined; BMD10=benchmark dose (maximum likelihood estimate) associated with 10% additional risk 

of the adverse response; BMDL10=95% lower confidence limit estimate of the BMD10; LOAEL= lowest observed adverse effect 

level; NOAEL=No observed adverse effect level *

Interspecies dose normalized by alveolar surface area in animal and humans (Section A.2.3.4).
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 41

Table 5–4. Factors, assumptions, and options evaluated in the CNT risk assessment. 

Factor Assumption Options evaluated

CNT type Risk estimates for various types of 

CNT are relevant to worker exposures, 

based on animal dose-response data of 

administered or estimated lung dose and 

early-stage, persistent lung responses, using 

standardized methods

SWCNT (Fe 2%)

SWCNT (Fe 0.2–0.3%)

SWCNT (Fe 18%)

MWCNT (Co 2%, Fe 0.5%)

MWCNT (Al2O3 9.6%)

MWCNT (Co 0.5%)

Route of exposure Lung dose is associated with animal 

response regardless of route of exposure 

and is relevant to human inhaled dose

Inhalation

Pharyngeal aspiration

Intratracheal instillation

Duration of exposure Subchronic or short-term exposure is 

associated with observed lung responses 

and relevant to humans 

13-week inhalation, 1d–26wk PE

Short-term inhalation, 56d PE

Single dose, 28–91d PE

Species/strain Animal model is relevant for humans Rat

• Sprague-Dawley

• Wistar

• Mouse

• B6C3F1

• C57BL/6

Sex No sex-specific effect Male

Female

Critical effect Animal response is relevant to humans Dichotomous response:

Granuloma

Granulomatous inflammation*

• grade 1+

• grade 2+

Lipoproteinosis

• grade 1+

Alveolar septal thickening 

• grade 1+

• grade 2

Continuous response:

Alveolar connective tissue (septal) 

thickness 

Hydroxyproline amount

Critical effect level Dose to target tissue (as administered, 

estimated deposited, or estimated retained 

mass in alveolar region of lungs) is 

associated with lung response 

BMDL

BMD

NOAEL

LOAEL

See footnotes at end of table. (Continued)
42 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Factor Assumption Options evaluated

Human equivalent dose

• Interspecies 

normalization

Humans would have an equal lung 

response (on average) to an estimated 

equivalent alveolar dose

Alveolar surface area

Alveolar macrophage cell volume

• Duration of exposure Working lifetime average cumulative 

exposure would be associated with the 

human-equivalent lung response

45-years (8-hr TWA, 40-hr workweek, 

50 wk/yr)

• Clearance kinetics True average working lifetime exposure 

lies between deposited and retained CNT 

dose estimates based on spherical particle 

dosimetry model

Deposited dose (no clearance)

Retained dose (normal clearance)

• Ventilation rate Workers on average breath the same 

amount of air in a day; normal oronasal 

augmenter breathing pattern

Reference worker (9.6 m3/d)

• Alveolar deposition 

fraction

Airborne particle size distribution predicts 

deposition fraction 

Spherical particle-based model 

(MPPD), incl. two versions

• Density of 1

• Density of <1 

*

Grade 1: minimal; grade 2: slight/mild.

Table 5–4 (Continued). Factors, assumptions, and options evaluated in the CNT risk assessment. 

Based on currently available data, it is difficult to assess the relative toxicity of the various types of CNT 

and CNF because there has been limited systematic 

study of various CNT and CNF using the same study 

design. These available studies differ in factors that 

include the rodent species and strain, the techniques 

and assays for measuring lung effects, and the exposure and post-exposure durations. Despite differences 

in the type and composition of SWCNT and MWCNT 

used in the animal studies, the risk estimates across 

the different types of CNT and studies are associated 

with relatively low mass exposure concentrations. 

Although data from laboratory animal studies with 

CNF are limited, the similarities in physical-chemical 

properties and adverse lung effects between CNF 

and CNT support the need to control exposures to 

CNF at the REL derived for CNT. 

NIOSH is recommending an occupational exposure limit for CNT and CNF to minimize the risk 

of developing adverse lung effects over a working 

lifetime. A mass-based airborne exposure limit is 

being recommended because this exposure metric 

is the same as that used in determining the dose-response relationship in animal studies and deriving 

the risk estimates, as well as being the most common exposure metric currently used in monitoring 

workplace exposures to CNT and CNF. The REL 

is based on the respirable particle-size fraction because the adverse lung effects in the animal studies were observed in the alveolar (gas-exchange) 

region. “Respirable” is defined as the aerodynamic 

size of particles that, when inhaled, are capable 

of depositing in the alveolar region of the lungs 

[ICRP 1994]. Sampling methods have been developed to estimate the airborne mass concentration 

of respirable particles [ACGIH 1984; CEN 1993; 

ISO 1995; NIOSH 1998]. Although the goal is to 

establish a REL that would eliminate any potential 

risk for developing respiratory disease, limitations 

exist in reliably measuring airborne CNT and CNF 

using currently available mass-based sampling 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 43

and analytical methods. NIOSH is recommending that NIOSH Method 5040 [NIOSH 1994; Birch 

2004a, b] be used to measure workplace airborne 

exposure to respirable CNT and CNF. 

An upper estimate of the LOQ of Method 5040 

(7 μg/m3) was proposed as the draft REL (8-hr TWA) 

for CNT and CNF [NIOSH 2010]. This upper limit 

was based on total carbon (TC) results found for 

filter media from different vendors and lots, by different laboratories, and during a 6-month period. 

In practice, when elemental carbon (EC) results are 

used from media blanks submitted with the samples 

to estimate the LOQ, a much lower value can be 

achieved (Section 6.1). It is important to note that 

the LOQ for NIOSH Method 5040 depends on the 

media blank variability, filter area sampled, portion 

of filter analyzed, and the collected sample air volume. As discussed in Section 6.1, under optimum 

conditions an LOQ of 1 μg/m3 can be obtained for 

an 8-hr respirable sample collected on a 25-mm 

filter at a flow rate of 4 liters per minute (lpm). 

Considering the potential uncertainties in estimating the health risks and current limitations of analytical methodologies, NIOSH is recommending a 

REL of 1 μg/m3 EC as an 8-hr TWA airborne respirable mass concentration for up to a 40-hr work week. 

For working lifetime exposures at 1 μg/m3, the MLE 

risk estimates for slight/mild level of lung effects 

(based on the rat subchronic inhalation studies of 

MWCNT) range from 0.23% to 10%, and the 95% 

UCL estimates range from 0.53% to 16% depending 

on the rat study and the assumptions used in estimating the CNT lung dose (Tables 5–2 and A–8). 

The more sensitive endpoint of minimal level of 

lung effects in the rat subchronic inhalation studies 

were associated with MLE risk estimates of 2.4% to 

33% and 95% UCL estimates of 5.3% to 54% (Tables 

5–2 and A–7). Estimates of a 45-yr working lifetime no-effect concentration (8-hr TWA) based on 

the NOAEL or LOAEL estimates from the rat subchronic studies are < 1 μg/m3

 (8-hr TWA) (Section 

A.6.3). The estimates based on the short-term 

studies of SWCNT and MWCNT in rats and mice 

are consistent with those from the subchronic studies of MWCNT in rats (Section A.3). 

NIOSH recognizes that the REL may not be completely health protective and that there is uncertainty 

in these risk estimates, but maintaining exposures 

below the REL should help to lower workers’ risk 

of developing occupational lung disease over a 

working lifetime. The REL and other recommendations in this CIB should also assist employers in 

establishing an occupational health surveillance 

program that includes elements of hazard and 

medical surveillance. Until improved methods to 

measure airborne exposures to CNT and CNF are 

established, continued efforts should be made to 

reduce airborne concentrations to as low as possible below the REL. Approaches to optimize the 

sampling and analysis of exposures are discussed in 

Section 6.1 and Appendix C. Examples of engineering controls to reduce or eliminate workers’ exposures to CNT and CNF are provided in Section 6.2. 

Additional guidance, including personal protective 

equipment, respirators, training, and medical surveillance is provided in Sections 6.3 through 6.7. 

Based on available workplace exposure data, it is 

not possible for NIOSH to determine whether the 

NIOSH REL can be achieved in all workplaces 

where exposure to CNT and CNF occur; however, exposure data that have been reported indicate 

that implementing appropriate engineering control 

measures (e.g., local exhaust ventilation, enclosures) can eliminate or greatly reduce worker exposures [Han et al. 2008a; Methner et al. 2008; Tsai 

et al. 2009; Johnson et al. 2010; Lee et al. 2010; Cena 

and Peters 2011; Dahm et al. 2011].

5.2 Other Derived

Occupational Exposure

Limits for CNT 

One of the earliest OELs for CNT was proposed 

by the British Standards Institute [BSI 2007]—the 

benchmark exposure limit (BEL) of 0.01 fiber/cm3, 

or one-tenth of their asbestos exposure limit (Table 

5–5). Nanocyl [2009] derived an estimated OEL 

of 2.5 μg/m3 for an 8-hr TWA exposure based on 

applying an overall assessment (a.k.a. uncertainty) 

factor of 40 to the LOAEL of 0.1 mg/m3 in the 
44 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Ma-Hock et al. [2009] subchronic rat inhalation 

study of MWCNT. Aschberger et al. [2010] proposed OELs of 1 μg/m3

 for MWCNT studied by 

Ma-Hock et al. [2009] and 2 μg/m3 for MWCNT 

from Pauluhn [2010a], by adjusting 0.1 mg/m3 (the 

LOAEL in Ma-Hock et al. [2009] and the NOAEL 

in Pauluhn [2010a]) for rat-to-human daily exposure and respiratory volume, and applying an overall assessment factor of 50 and 25, respectively. 

Pauluhn [2010b] derived an OEL using subchronic 

data in rats inhaling MWCNTs (Baytubes®) [Pauluhn 2010a]. This approach was based on the biological mechanism of volumetric overloading of alveolar macrophage-mediated clearance of particles 

from the lungs of rats [Morrow 1988]. Increased 

particle retention half-time (an indication of lung 

clearance overload) was reported in rats exposed 

by subchronic inhalation to MWCNT (Baytubes®) 

at 0.1, 0.4, 2.5, or 6 mg/m3 The overloading of rat 

lung clearance was observed at lower-mass doses of 

MWCNT (Baytubes®) compared with other poorly 

soluble particles; and the particle volume dose was 

better correlated with retention half-time among 

poorly soluble particles including CNT [Pauluhn 

2010a, b]. Pauluhn [2010b] reported benchmark 

concentration (BMC) estimates of 0.16 to 0.78 mg/

m3 for rat lung responses of pulmonary inflammation and increased collagen, but selected the lower 

NOAEL of 0.1 mg/m3 to derive a human-equivalent 

concentration. The NOAEL was adjusted for human and rat differences in factors affecting the 

estimated particle lung dose (i.e., ventilation rate, 

alveolar deposition fraction, retention kinetics, and 

total alveolar macrophage cell volume in each species). The product of these ratios resulted in a final 

factor of 2, by which the rat NOAEL was divided, 

to arrive at a human-equivalent concentration of 

0.05 mg/m3 (8-hr TWA) as the OEL for MWCNT 

(Baytubes®). No uncertainty factors were used in 

deriving that estimate. 

The Japanese National Institute of Advance Industrial Science and Technology (AIST) derived an 

OEL for CNT of 30 μg/m3 [Nakanishi 2011a,b], 

based on studies supported by the New Energy and 

Industrial Technology Development Organization 

(NEDO) of Japan. Rat NOAELs for pulmonary 

inflammation were identified in 4-week inhalation studies of SWCNT and MWCNT [Morimoto 

et al. 2011a,b]. Human-equivalent NOAELs were 

estimated by accounting for rat and human differences in exposure duration, ventilation rate, particle 

deposition fraction, and body weight [Nakanishi 

2011b]. The rat NOAELs of 0.13 and 0.37 mg/m3 for 

SWCNT and MWCNT, respectively, were estimated 

to be equivalent to 0.03 and 0.08 mg/m3 in humans 

including adjustment by an uncertainty factor 

of 6. This total uncertainty factor included a factor of 2 for uncertainty in subchronic-to-chronic 

extrapolation and a factor of 3 for uncertainty in 

rat to human toxicokinetic differences (factors of 1 

were assumed for toxicodynamic differences in rats 

and humans and for worker inter-individual variability). A relationship was reported between the 

BET specific surface area of various types of CNT 

and pulmonary inflammation (percent neutrophils 

in bronchoalveolar lavage fluid) (Figure V.2 in Nakanishi [2011b]). Thus, the OEL of 0.03 mg/m3

 was 

proposed for all types of CNT, based on the data for 

the SWCNT with the relatively high specific surface area of ~1,000 m2

/g (which was noted would 

be more protective for other CNTs with lower specific surface area). A period-limited (15-yr) OEL 

was proposed due to uncertainty in chronic effects 

and based on the premise that the results will be reviewed again within that timeframe with further 

data [Nakanishi 2011a].

In summary, these currently proposed OELs for 

CNT range from 1 to 50 μg/m3 (8-hr TWA concentration) [Aschberger et al. 2010; Nanocyl 2009; 

Pauluhn 2010b; Nakanishi (ed) 2009a], including 

the NIOSH REL of 1 μg/m3. Despite the differences 

in risk assessment methods and assumptions, all of 

the derived OELs for CNT are low airborne mass 

concentrations relative to OELs for larger respirable carbon-based particles. For example, the current U.S. OELs for graphite or carbon black are approximately 2.5 to 5 mg/m3

. Each of these CNT risk 

assessments supports the need to control exposures 

to CNT in the workplace to low airborne mass concentrations (μg/m3

) to protect workers’ health.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 45

Table 5–5. Recommended occupational exposure limits for CNT

Reference Occupational exposure limit (OEL) Comments

Pauluhn [2010b] 0.05 mg/m3 (8-hr TWA) for MWCNT 

(Baytubes®)

Based on rat subchronic (13-wk) inhalation 

study of MWCNT (Baytubes®) and 

prevention of lung clearance overload and 

associated pulmonary effects. Rat NOAEL 

of 0.1 mg/m3 adjusted by a factor of 2 for 

worker exposure day, air intake, deposition, 

and clearance kinetics. No uncertainty 

factors were applied. 

Nakanishi (ed) [2011a,b] 30 μg/m3 (8-hr TWA) for CNT Based on 4-wk inhalation studies of 

SWCNT and MWCNT in rats. Lowest 

NOAEL of 0.13 mg/m3 (for high surface 

area SWCNT) used as basis for CNT 

OEL). Adjusted for worker exposure day, 

air intake, deposition fraction, and body 

weight; uncertainty factor of 6. OEL is 

period-limited (15-yr).

Nanocyl [2009] 2.5 μg/m3 (8-hr TWA) for MWCNT Adjusted rat LOAEL of 0.1 mg/m3

(subchronic inhalation) [Ma-Hock et al. 

2009] to workers and applied assessment 

factor of 40.

Aschberger et al. [2010] 2 μg/m3 (8-hr TWA) for MWCNT

1 μg/m3 (8-hr TWA) for SWCNT

Adjusted rat NOAEL of 0.1 mg/m3

(subchronic inhalation) [Pauluhn 2010a] 

for worker exposure day and air intake; 

assessment factor of 25. 

Adjusted rat LOAEL of 0.1 mg/m3

(subchronic inhalation) [Ma-Hock et al. 

2009] for worker exposure day and air 

intake; assessment factor of 50. 

BSI [2007] 0.01 fibers/ml for fibrous nanomaterials 

with high aspect ratios (> 3:1 and length 

> 5000 nm)

Benchmark exposure level (BEL) based on 

one tenth of the asbestos exposure limit
46 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

5.3 Evaluation of Uncertainties

in CNT Risk Assessment

and REL

Animal data have been used to evaluate the health 

hazard and risk of occupational exposure to CNT 

and CNF. Limited human exposure data are available, and NIOSH is not aware of any studies or reports at this time of any adverse health effects in 

workers producing or using CNT or CNF. The best 

available scientific information to develop recommended exposure limits is from the subchronic 

(13-wk) animal inhalation studies of two types of 

MWCNT and the shorter-term animal studies of 

SWCNT and other types of MWCNT. 

The analysis of animal data in this risk assessment 

includes: (1) identifying the adverse health effects 

that are associated with exposure to CNT or CNF 

in laboratory animals; (2) evaluating the severity of 

the response and the relevance to humans; and (3) 

estimating the human-equivalent dose and likelihood (risk) of adverse effects in workers. Ideally, 

sufficient evidence is desired to derive exposure 

limits that are estimated to be associated with essentially a zero risk of an adverse health effect even 

if exposed 8 hr/d, 40 hr/wk. over a 45-yr working 

lifetime. However, limitations in the scientific data 

result in uncertainties about those hazards and risk 

estimates. Characterizing the degree of that uncertainty, and the extent to which use of those data are 

useful for occupational health risk management 

decision-making, is an important step in risk assessment. Alternative models and methods contribute to the differences in the risk estimates, and 

there is uncertainty about which biological endpoints, animal models, and interspecies and dose 

rate extrapolation methods may be most predictive 

of possible human health outcomes. 

5.3.1 Strength of Evidence for

Estimating a Health-Based REL

for CNT and CNF 

NIOSH and others have used the published animal 

dose-response data to develop OELs for various 

types of CNT (see Section 5.2). The methods and 

assumptions differ across these studies and have 

resulted in mass-based OELs ranging from 1 μg/m3

to 50 μg/m3. A proposed benchmark exposure limit 

(BEL) of 0.01 fiber/cm3 for CNT has also been proposed [BSI 2007]. 

In the NIOSH risk assessment (Appendix A), the 

animal effect level estimates (and also the equivalent working lifetime estimates) for early-stage 

noncancer lung effects (granulomatous inflammation, interstitial thickening, or fibrosis) differ by 

approximately two orders of magnitude depending 

on the animal study, CNT type, and lung dose estimation methods (Tables A–3 through A–6; Table 

A–13). However, these estimated human-equivalent 45-yr working lifetime exposure concentrations (8-hr TWA) are all relatively low airborne 

mass concentrations (approximately 0.1–19 μg/m3

before adjusting for uncertainty in these estimates). 

The major areas of uncertainty in the CNT risk 

assessment include: (1) the critical effect or lung 

response measure including level of severity; (2) 

dose rate and retention assumptions for extrapolation from subchronic or short-term animal studies 

to chronic exposure in humans; and (3) low dose 

extrapolation using the benchmark dose models to 

estimate risks below the 10% benchmark dose. The 

relatively minor areas of uncertainty include: (1) 

benchmark dose estimation; (2) impact of route of 

exposure; and (3) rat lung dose estimation. 

These uncertainties can result in either under-estimation or over-estimation of the true health risk to 

workers at a given exposure scenario. Each of these 

areas is discussed further below.

5.3.2 Major Areas of Uncertainty 

(1) Lung response and severity level

The REL is based on estimates of excess risk of earlystage noncancer lung effects, which NIOSH has determined are relevant to human health risk assessment (Section A.2.1.3). The extent to which these 

lung responses would be associated with functional 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 47

deficits in animals or clinically significant effects in 

humans is uncertain. However, these lung responses 

include early onset fibrosis which persisted or progressed after the end of exposure [Shvedova et al. 

2005. 2008; Porter et al. 2010; Mercer et al. 2011]. 

Limited evidence in animals suggests that these 

effects may be associated with some lung function 

decrement (reduced breathing rate in mice) [Shvedova et al. 2008]. A quantitative measure of pulmonary fibrosis—alveolar interstitial (septal, or 

connective tissue) thickening [Shvedova et al. 2005, 

2008; Mercer et al. 2011]—was previously used in 

developing the health basis for the U.S. air contaminant standards for another lung toxicant, ozone [US 

EPA 1994]. EPA selected this health endpoint as 

the critical lung effect in animals and extrapolated 

to humans the lung dose associated with this effect 

[US EPA 1996; Stockstill et al. 1995]. Additional 

measures of early stage lung effects in rats and mice 

that were used in this CNT risk assessment include 

minimal or greater alveolar septal thickening, granulomas, or granulomatous inflammation [Pauluhn 

2010a; Ma-Hock et al. 2009; Lam et al. 2004].

The choice of health endpoint and severity level 

from the subchronic studies resulted in different 

REL estimates by factors of several-fold to an order 

of magnitude (Table A–5 and A–6). In addition to 

quantitative differences in risk estimates for noncancer effects, there is also qualitative uncertainty 

about the risk of other disease endpoints, including cancer. Possible cancer risk (e.g., associated 

with fiber-like structures) is an area of considerable 

uncertainty for CNT and CNF which warrants targeted research and a high level of exposure control 

until the risk is understood [Schulte et al. 2012]. 

(2) Dose rate and retention

Appendix A–6 provides some analyses to show the 

quantitative influence of dose rate and lung retention assumptions on the risk estimates and REL 

derivation (Tables A–5 and A–6; Section A.6.3.2.2). 

The lung effects were assumed to be associated with 

the total lung dose, regardless of the dose rate. If 

the average daily deposited lung dose is assumed 

(i.e., no difference in rat or human clearance rates), 

then the human-equivalent concentration would 

be ~30 times higher than that based on the ICRP 

[1994] clearance model, and ~10 times higher than 

that assuming simple first-order kinetics [Snipes et 

al. 1989; Pauluhn 2010b]. The human-equivalent 

working lifetime (8-hr TWA) estimates based on 

deposited lung dose (assuming no clearance) are 

lower by a factor of ~5–7 than those estimates 

based on retained lung burden (assuming normal 

clearance) (Tables A–5 and A–6). 

(3) Inter-species dose normalization

Alternative assumptions about the biologicallyrelevant measure of equivalent dose can result in 

considerable differences in the human-equivalent 

dose. NIOSH normalized the inter-species lung dose 

based on the ratio of the human-to-animal average 

alveolar surface area. Alternatively, Pauluhn [2010b] 

normalized the dose from rat to humans based on 

the average total alveolar macrophage cell volume. 

This difference resulted in a factor of ~4 in the humanequivalent lung burdens and working lifetime 8-hr 

TWA concentration estimates (Table A–13).

(4) Low dose extrapolation

All of these animal data and methods result in low 

equivalent working lifetime exposure estimates. 

The animal NOAEL, LOAEL, and BMD(L)* estimates, and the equivalent human working lifetime 

exposure estimates, indicate low mass concentrations (8-hr TWA) over a 45-year working lifetime 

(<0.1—19 μg/m3) (95% LCL, Tables A–3 through 

A–6). As discussed in Section A.2.1, the animal 

dose-response data from the CNT subchronic inhalation studies were limited (and minimally acceptable) for benchmark dose estimation. Only 

the multistage model provided unique MLE estimates that adequately fit the data. Yet, the rat 10% 

BMD(L) estimates are similar to the NOAEL and 

LOAEL estimates, indicating that the BMD(L) estimates are in reasonable agreement (Table A–12). 

In standard risk assessment procedure [US EPA 

1994, 2012; Kuempel et al. 2006; Schulte et al. 2010], 

*

Abbreviation for both BMD and BMDL estimates.
48 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

the BMDL, NOAEL, or LOAEL is the estimated 

point of departure (POD) for extrapolation below 

the range of the data. This low dose extrapolation 

may be risk-based (e.g., from the 10% BMDL) by 

linear or nonlinear modeling (depending on the 

mode of action information); or in noncancer risk 

assessment, the POD may be adjusted downward 

to account for uncertainty in the extrapolation 

from animals to humans and other factors (Section 

A.6.3.3). 

The risk estimates based on the subchronic and 

short-term animal studies (Section A.3) generally indicate that risks less than 10% would be associated with working lifetime exposures below 

1 μg/m3 (8-hr TWA). This is estimated by either 

linear or model-based low-dose extrapolation of 

the rat BMDL estimates (Section A.3.3). Although 

an acceptably low level of risk for early-stage pulmonary effects has not been established, some of 

the working lifetime excess risk estimates at 1 μg/m3

(8-hr TWA) are less than 10% (e.g., approximately 

0.5% to 16% for the slight/mild, grade 2) lung effects in the rat subchronic inhalation studies; 95% 

UCL estimates) (Table A–8). Given the uncertainty 

about the early-stage lung effects and the shape 

of the dose-response relationship, the actual risk 

could be much lower, even zero (if exposures are 

below an effect threshold). Alternatively, adjusting 

the POD downward using standard uncertainty 

factors, also generally results in estimates of <1 μg/

m3 (8-hr TWA concentration) as the working lifetime exposure likely to be without appreciable risk 

of adverse effects (Section A.6.3.3). 

5.3.3 Minor Areas of Uncertainty 

(1) Effect level estimation

An effect level is a dose associated with a specified effect (or lack of observed effect). A BMD is a 

statistical estimate of an effect level [Crump 1984, 

1995; US EPA 2012]. BMD estimates have several 

advantages over NOAEL or LOAEL estimates, including use of all the dose-response data, statistical accounting of sample size and variability, and a 

standard, risk-based definition of effect level (e.g., 

10%) in the low region of the dose-response data. 

The data to estimate BMD(L)s were limited (see 

Appendix A), but NIOSH considered these data to 

be minimally acceptable, and adequate statistical fit 

was obtained for each data set included in the risk 

assessment (Appendix A). 

NOAELs and LOAELs can also be uncertain, as 

observation of effects can depend on the dose spacing and the number of animals. For example, a low 

number of animals in a study can result in an apparent NOAEL due to chance, whereas a larger sample 

size would have more power to detect an effect. A 

statistical comparison of the NOAEL and BMD estimates showed that the NOAEL was statistically 

consistent with the 10% BMD estimate (Section 

A.6.2). In practice, the effect level estimate had 

little influence on the risk estimates or REL derivation because the BMD(L) estimates were similar to 

the LOAEL or NOAEL values (Table A–12). 

(2) Impact of route of exposure

Different routes of exposure were used in the CNT 

animal studies, including intratracheal instillation 

(IT), pharyngeal aspiration (PA), and inhalation. 

IT and PA are each single administered doses, followed by approximately 1 to 2 months post-exposure time to determine whether the observed effects were reversible, persistent, or progressive. PA 

permits the delivery of the substance to the lungs of 

animals that results in a deposition pattern that is 

more dispersed than that of IT and therefore more 

similar to inhalation. Inhalation is the most physiologically relevant route of exposure to workers. A 

study that compared mouse lung responses to SWCNT by PA or inhalation exposure found qualitatively similar responses, although the inhalation 

exposure was four times more potent at an estimated equivalent lung dose [Shvedova et al. 2008]. 

Among the various studies and routes of exposure 

(Tables A–3 through A–5), no clear differences in 

exposure and risk estimates are seen for route of 

exposure (versus variability due to other differences among studies).
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 49

(3) Rat lung dose estimation

In the absence of CNT-specific lung models, standard rat and human lung dosimetry models were 

used to estimate either the deposited or the retained 

lung dose. These two estimates are considered to 

represent the upper or lower bounds on the possible lung burden estimates. The effect of assuming deposited dose (no clearance) versus retained 

dose (normal clearance) resulted in a difference of 

approximately five-fold. However, the actual estimates are expected to lie within this range. The 

working lifetime exposure estimates (associated 

with 10% excess risk of early stage lung effects) 

were lower based on deposited dose estimates than 

those based on retained dose estimates (Appendix 

A, Table A–5).

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 51

6 Recommendations

In light of current scientific evidence on the hazard potential of CNT and CNF, appropriate steps 

should be taken to minimize worker exposure 

through the development of a risk management 

program and implementation of an exposure control strategy. Elements of that program should include the following: 

1. Control worker exposure to CNT and CNF 

below 1 μg/m3 8-hr TWA, respirable fraction 

(elemental carbon) during a 40-hr work week.

2. Conduct comprehensive exposure assessments 

(including exposures to other potential hazards) as part of an overall hazard surveillance 

program.

3. Develop guidelines for selecting, installing, and 

evaluating engineering controls (e.g., local exhaust ventilation, dust collection systems).

4. Educate and train workers on the recognition of 

potential exposures and in the use of good work 

practices in the handling of bulk CNT and CNF, 

as well as CNT- and CNF-containing materials.

5. Develop procedures for the selection and use 

of personal protective equipment (i.e., clothing, 

gloves, respirators).

6. Implement a medical surveillance program for 

workers potentially exposed to CNT or CNF 

with conduct of specific medical screening tests 

when warranted (Section 6.7).

7. Conduct routine (e.g., annual) and systematic 

evaluation of worker exposure to CNT or CNF 

when there is a process change in how CNT or 

CNF are manufactured or handled.

8. Encourage workers to wash hands before eating, 

smoking, or leaving the worksite.

9. Establish facilities for showering and changing 

clothes, with separate facilities for storage of 

non-work clothing, to prevent the inadvertent 

cross contamination of other areas (including 

take-home).

6.1 Exposure Assessment 

NIOSH is recommending that a respirable massbased airborne concentration measurement be 

used to monitor worker exposure to all types of 

CNT and CNF until additional data are available 

to determine whether other measurement metrics 

or techniques would be more effective in protecting 

workers’ health. NIOSH is currently evaluating the 

efficacy of various sampling techniques for measuring CNT and CNF and may make additional 

recommendations at a later date. 

Personal exposure concentrations to CNT and CNF 

can be determined as elemental carbon (EC) by 

NIOSH Method 5040 [NIOSH 1994; Birch 2004a, 

b]. Whenever possible, a bulk sample of the CNT/

CNF material should be analyzed to establish the 

thermal profile for the material(s) (Appendix C). 

Measurement results from NIOSH Method 5040 

should provide a reasonable estimate of a worker’s 

respirable exposure to CNT and CNF at the NIOSH 

REL of 1 μg/m3 8-hr TWA when the predominant 

workplace exposure to EC material is CNT or CNF. 

6.1.1 Exposure Monitoring Program

An exposure-monitoring program should be established to ensure that worker exposures to CNT and 

CNF are maintained below the REL. The program 

should consist of a plan designed to do the following: (1) characterize exposures of all exposed workers; (2) identify sources of potential EC exposures 

(e.g., diesel soot, carbon black) that may interfere 

with the interpretation of worker CNT and CNF 

exposures; (3) identify specific work areas or job 
52 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

tasks where worker exposures exceed or may exceed the REL; and (4) assess the effectiveness of engineering controls, work practices, PPE, training, 

and other factors used in reducing airborne exposures. To implement the plan an exposure assessment strategy should be developed. The details of 

the strategy will depend on a number of factors, including the number of workers potentially exposed 

to CNT or CNF and the day-to-day and worker-toworker variability in airborne concentrations. 

An important first step in applying any strategy is 

to develop an inventory of the processes and job 

activities (e.g., handling of dry powders, use of 

composite materials) that place workers at risk of 

exposure. This inventory can be used to determine 

the number of workers potentially exposed and a 

qualitative assessment as to the workers and processes with the highest potential for exposure. 

The strategy should also incorporate provisions to 

quantify the airborne release of CNT and CNF occurring at specific processes or job activities to provide “activity pattern data” [Duan and Mage 1997]. 

Activity pattern data are useful for identifying 

possible causes of high exposure for remediation; 

however, these data are vulnerable to spatial variation in exposure concentrations and should not be 

used in predicting worker exposures. For example, 

in the study by Birch et al. [2011b], personal exposure to CNF was much higher than area samples, 

depending on location. Respirable EC exposure for 

two employees working mainly in a thermal treatment area was approximately 45 μg/m3

, and a CNF 

reactor area was about 80 μg/m3, while the corresponding area samples were about 32 μg/m3

 in the 

thermal treatment area and 13 μg/m3 in the CNF 

reactor area. The EC concentration in the reactor 

area was less than half that in the thermal treatment area, but the personal sample collected in 

the reactor area was nearly twice as high. Because 

area samples are often not predictive of personal 

exposure, extrapolating personal exposure from 

area concentrations should not be done without 

a thorough assessment of the workplace to establish whether a valid extrapolation is possible [Birch 

et al. 2011b]. NIOSH [NIOSH 2009a] and others 

[Brouwer et al. 2009; Methner et al. 2010a; Ramachandran et al. 2011] have developed exposure assessment guidance for determining the release of 

engineered nanoparticles that can be adapted for 

determining sources of exposure to CNT and CNF. 

To ensure that worker exposure to CNT or CNF is 

being maintained below the REL, several exposure 

measurement strategies are available [NIOSH 1977; 

Corn and Esmen 1979; Leidel and Busch 1994; 

Rappaport et al. 1995; Lyles et al. 1997; Bullock and 

Ignacio 2006]. These strategies can be tailored to 

the specific workplace depending on the number 

of workers, complexity of the work environment 

(e.g., process type and rate of operation, exposure 

control methods, physical state and properties of 

material) and available resources. One approach 

for determining worker exposure would be to initially target similarly exposed groups of workers 

[Corn and Esmen 1979; Leidel and Busch 1994]. 

This initial sampling effort may be more time efficient and require fewer resources for identifying 

workers with exposures to CNT or CNF above the 

REL. However, this measurement strategy may 

produce incomplete and upwardly biased exposure estimates if the exposures are highly variable 

[Kromhout 2009]. Therefore, repeated measurements on randomly selected workers may be required to account for between- and within-worker 

variation in exposure concentrations [Rappaport et 

al. 1995; Lyles et al. 1997]. Because there is no ‘best’ 

exposure measurement strategy that can be applied 

to all workplaces, multi-day random sampling of 

workers (all workers, if the exposed workforce is 

small) may be required to have an accurate assessment of worker airborne exposure concentrations 

to CNT and CNF.

6.1.2 CNT and CNF Measurement

A multi-tiered exposure measurement strategy is 

recommended for determining worker exposure 

to CNT and CNF (see Figure 6–1). The selection 

of workers and the frequency in which they should 

be sampled should follow guidelines established for 

the exposure monitoring program (Section 6.1.1). 
NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers 53Figure 6–1. Exposure measurement strategy for CNT and CNF
54 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

As part of the evaluation of worker exposures to 

CNT and CNF, ‘background’ samples for EC determination should be collected outdoors (or at 

air intakes of the facility) and at indoor locations 

where exposure to CNT or CNF is unlikely. The EC 

concentrations (using Method 5040) determined 

from ‘background’ samples should be subtracted 

from the EC personal sample results to determine 

whether worker exposures exceeded the REL. Initially, more samples may be required to characterize 

the workplace thoroughly. This initial assessment 

will help refine the sampling approach and determine whether EC interference is an issue. Careful 

consideration of environmental background is essential. For example, outdoor EC may sometimes 

be higher than indoor background depending on 

the facility’s air handling system. If so, the indoor 

EC background may be more representative of area 

and worker samples.

In workplaces where exposure to other types of EC 

(e.g., diesel soot, carbon black) may occur, the initial evaluation of a worker’s exposure should include 

the simultaneous collection of a personal respirable 

EC sample and a personal sample for electron microscopy analysis (e.g., TEM, SEM). Electron microscopy analysis, in conjunction with energy dispersive x-ray spectroscopy (EDS), can be used for 

CNT and CNF identification. In addition, consideration should be given to the sizing and counting of 

CNT and CNF structures during electron microscopy analysis should future efforts to control occupational exposures be based on a different exposure 

metric (e.g., number concentrations of airborne 

CNT and CNF structures in a given size bin). While 

no specific electron microscopy (e.g., TEM, SEM) 

method exists for the sizing and counting of CNT 

and CNF structures, methods used in the analysis 

of other ‘fibrous’ materials are available [NIOSH 

1994a; ISO 1999, 2002] and could be adapted in the 

characterization of exposures. 

NIOSH investigators have conducted a number 

of surveys at CNT and CNF producers and/or 

secondary users [Evans et al. 2010; Birch 2011a; 

Birch et al. 2011b; Dahm et al. 2011]. In many 

cases ‘background’ EC concentrations were <1 μg/

m3. In the study reported by Dahm et al. [2011], 

results for PBZ samples were often “non-detect” 

or between the LOD and LOQ (i.e., semi quantitative results) for Method 5040. In these facilities 

[Dahm et al. 2011], only small amounts of material 

were being handled and the tasks were over a short 

duration. Thus, time-weighted air concentrations 

determined over extended periods were low. These 

data indicate that in workplaces where airborne exposures to CNT and CNF are expected to be low, or 

where worker exposure may be sporadic or shortterm, a higher flow rate (e.g., 4 lpm) respirable dust 

sampler (cyclone) and a 25-mm filter should be 

used to increase the amount of sample collected for 

quantification. 

At job tasks where dusts are generated during cutting, sanding, or grinding of CNT/CNF polymer 

composites, the accuracy of determining the EC 

fraction of the dust can vary depending on the 

polymer and sample loading (Appendix C). Depending on the dust concentration, the sample can 

be easily overloaded with organic carbon (OC) because of the high relative OC content of the polymer. Due to OC overload and/or polymer pyrolysis, it may not be possible to quantify exposures to 

CNT and CNF by Method 5040 if they are incorporated as powders in composite operations. Further, even if the EC fraction of a composite dust 

can be determined with reasonable accuracy, both 

unbound and polymer-bound CNT and CNF are 

determined by Method 5040, if present. Also, in 

a polymer composite particle, the CNT/CNF is 

usually bound within the polymer (or resin) matrix, dissimilar to a particle of unbound material. 

Analysis of samples by electron microscopy may be 

helpful in identifying the presence of matrix bound 

and unbound CNT or CNF and for determining a 

course of action in controlling exposures. An effort 

to improve the analysis of samples containing dusts 

of polymer composites is ongoing. 

Metals employed as catalysts in the synthesis of 

CNT/CNF also were considered by NIOSH as potential markers of CNT/CNF airborne exposure, 

but purified CNT and CNF have low metal contents (1% or less by weight). Detection limits for 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 55

inductively coupled plasma atomic emission spectroscopy (ICP-AES) may not be adequate at low 

CNT/CNF concentrations [Birch et al. 2011b]. Inductively coupled plasma mass spectrometry (ICPMS) offers detection limits superior to ICP-AES 

and may be useful, depending on the amount of 

CNT/CNF collected, metal and percent metal content, and whether other aerosol sources are present that would interfere with analysis. However, if a 

metal is employed as a surrogate measure of CNT/

CNF, minimal background interference and correlation with CNT/CNF mass (or other relevant metric) would be required [Birch et al. 2011b]. Iron was 

not a useful indicator of CNF exposure in a study 

reported by Birch et al. [2011b]. There was no correlation between the iron and CNF concentrations 

found in a CNF manufacturing facility, because the 

major iron source was not CNF-derived. In addition, the LOD for ICP-AES was not adequate for its 

determination at low EC concentrations (e.g., near 

the EC LOQ). 

6.1.3 Method 5040 Limit

of Detection

As with all analytical methods, the LOD is a varying number. However, the airborne EC LOD originally reported for NIOSH Method 5040 (i.e., about 

2 μg/m3), or an LOQ of 7 μg/m3 was a high estimate 

[NIOSH 2010]. The LOD was based on analysis of 

pre-cleaned media blanks from different filter lots, 

during a 6-month period, and by different analysts 

at two different laboratories. Further, variability for 

the total carbon (TC) results was used to estimate 

the LOD rather than EC results. These combined 

factors gave a conservative (high) estimate of the 

EC LOD. 

In practice, a much lower EC LOD is obtained by 

NIOSH Method 5040 than was originally reported, 

because the variability for EC results for a set of 

media blanks submitted (with the sample set) for 

the LOD (LOQ) determination is much lower than 

reported for the total carbon (TC) results. Thus, 

if EC is of primary interest, as with CNT/CNF 

measurement, and the level of organic carbon 

(OC) contamination is acceptable (with respect to 

the OC and TC LOD), EC results for as-received 

filters should be used to determine the EC LOD 

(Appendix C). 

Estimates of the EC LODs and LOQs (in units μg 

EC/cm2 of air) determined with 25-mm and 37-mm 

quartz filter media from a given lot, and with 

manual splits assigned are reported in Table 6–1. 

OC-EC splits for the media blanks were assigned at 

the point when oxygen is introduced so the baseline signal is integrated over the region in which 

EC is removed (oxidized) from the filter.

Because there are many possible OC sources, and 

bulk CNT/CNF contain little OC, EC is a better 

indicator of exposure than TC. Nevertheless, high 

particulate OC concentrations indicate air contamination, and these data can be useful for general industrial hygiene purposes if care is taken to correct 

for OC media contamination (Appendix C). Unlike EC, OC contamination (e.g., through contact 

with a contaminated surface and/or vapor adsorption) of the quartz filter media is common. Consequently, the OC (and TC) LOD is higher than for 

EC, and the OC (and TC) results may have significant positive bias. Bias is especially apparent when 

the particulate OC air concentrations and sampled 

air volumes are low. To obtain a more accurate 

estimate of the particulate OC air concentration, 

an OC blank correction should be applied. Blank 

correction can be accomplished by subtracting the 

OC media blank or (preferably) by a tandem filter 

correction (organic carbon sampling artifacts section in Appendix C), with the latter generally being 

more accurate. Mean OC blanks, LODs, and LOQs 

for 25-mm and 37-mm quartz filter media are reported in Table 6–2 (units are μg OC/cm2

). 

Two additional sets (n  =  10 for each set of five 

filters) of 37-mm filters were analyzed several 

months apart in 2010. The pooled EC results are 

comparable to those obtained previously. Results 

(μg C/cm2), including OC results for both sets and 

TC results for one are given in Table 6–3. The results in Table 6–3 represent duplicates on two sets 

of 5 filters.
56 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

As stated on previous page, NIOSH Method 5040 

LOD depends on the air volume, filter size, sample 

portion analyzed (usually 1.5 cm2), and the media 

blank variability. The latter is used to determine the 

LOD in unit’s μg/cm2. Expressed as an air concentration, the EC LOD (μg/m3

) corresponding to the 

EC LOD (μg/cm2) determined with media blanks 

(i.e., LOD = 3 times the standard deviation for the 

blanks) can be calculated by the following equation:

This equation explains why a lower LOD (μg/m3) 

can be obtained by reducing the filter size (deposit 

area), increasing the air volume, and minimizing 

the variability for the media blanks (i.e., the EC 

LOD in μg/cm2). The LOD is improved using a 

smaller filter size since the deposit density is higher 

for an equivalent mass deposited. The same applies to the LOQ, commonly defined as 10 times 

the standard deviation (SD) for the blanks, or 3.3 

times the LOD. 

If 0.02 μg  EC/cm2 is taken as the SD for media 

blanks (with manual OC-EC split adjustment), 

the LODs and LOQs (in μg EC/m3) for different 

Table 6–1. EC LODs and LOQs for 25- and 37-mm filters (ug EC/cm2)

25-mm filter EC (n = 10) 37-mm filter EC (n = 6)

Mean 0.063 Mean 0.033

Standard deviation 0.030 Standard deviation 0.028

LOD 0.09 LOD 0.08

LOQ 0.30 LOQ 0.28

Table 6–2. OC LODs and LOQs for 25- and 37-mm filters (ug OC/cm2)

25-mm filter OC (n = 10) 37-mm filter OC (n = 6)

Mean 1.41 Mean 1.94

Standard deviation 0.413 Standard deviation 0.281

LOD 1.24 LOD 0.84

LOQ 4.13 LOQ 2.81

Table 6–3. OC, EC, and TC LODs and LOQs for 37-mm filters (ug C/cm2)

37-mm filter OC EC OC EC TC

Mean 1.31 0.03 1.44 0.03 1.52

Standard deviation 0.164 0.016 0.304 0.024 0.318

LOD 0.49 0.05 0.91 0.07 0.95

LOQ 1.64 0.16 3.04 0.24 3.18
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 57

air volumes, 25-mm and 37-mm filters, and a 

1.5 cm2 filter portion analyzed would be as listed 

in Table 6–4. Results for a SD double this value 

(i.e., SD  =  0.04  μg EC/cm2) also are reported as 

worst-case estimates, but are seldom this high. If 

SDs for media blanks are frequently above 0.02 

ug EC/cm2) the cause of the high blank variability 

should be identified and corrected.

Based on EC results for media blanks (Tables 6–1 

and 6–3), a filter loading of about 0.3 μg/cm2 (i.e., 

at or above the EC LOQs reported in Tables 6–1 

and 6–3) will provide quantitative results. 

As described above (6.1.2), higher flow rate respirable samplers (cyclones) with a 25 mm cassette 

can improve the sample collection to permit 

measurement of CNT and CNF above the LOQ 

(i.e., 1 μg/m3) for samples collected for less than 

full work shift. Examples of sampling periods and 

flow rates that provide air volumes of about a halfcubic meter or higher (shaded area in table below) 

are listed in Table 6–5. A larger filter portion also 

can be used to further lower the LOD, but the instrument’s small (quartz tube) oven and need for 

proper sample alignment limit the amount of sample that can be analyzed. 

Table 6–4. EC LODs and LOQs estimated with media blanks

SD blank 

(μg EC/cm2) Limit

EC limit 

(μg/cm2)

EC LOD and LOQ (μg EC/m2) 

3 m3 air 1 m3 air 0.5 m3 air

37-mm 

filter

25-mm 

filter

37-mm 

filter

25-mm 

filter

 37-mm 

filter

25-mm 

filter

0.02 LOD 0.06 0.17 0.07 0.51 0.21 1.02 0.42

LOQ 0.20 0.57 0.23 1.70 0.69 3.40 1.38

0.04 LOD 0.12 0.34 0.14 1.02 0.42 2.04 0.83

LOQ 0.40 1.13 0.46 3.40 1.38 6.80 2.77

Table 6–5. Examples of sampling periods and flow rates

Air volumes (m3) for indicated sampling periods (hours) and flow rates

Flow rate (lpm)† 1 h‡ 2 h 4 h 8 h

2 120 240 480 960

4 240 480 960 1920

6 360 720 1440 2880

7 420 840 1680 3360

†

Liters per minute (lpm).

‡

Sampling period, hours, and highest flow rate tested at NIOSH laboratory. Tested with a Leland Legacy pump and 25-mm 

quartz-fiber filter.
58 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

6.2 Engineering Controls

One of the best ways to prevent adverse health effects from exposure to CNT and CNF is to eliminate 

exposure and minimize risks early in the design or 

re-design of manufacturing and down-stream user 

processes (see NIOSH prevention through design 

(PtD) at: www.cdc.gov/niosh/topics/PtD/). This 

can be accomplished through the establishment 

of a process safety management (PSM) program. 

PSM entails the development and implementation 

of programs or systems to ensure that the practices 

and equipment used in potentially hazardous processes are adequate and appropriately maintained. 

An integral part of the PSM program is the conduct 

of a process hazard analysis prior to the initiation 

of work to identify where sources of exposure to 

CNT or CNF may occur so that process equipment 

can be designed or re-designed to minimize the 

risk of exposure. At a minimum, the elements of 

the PSM program should be consistent with those 

required in the OSHA Process Safety Management 

Standard [29 CFR 1910.119]. 

In workplaces where CNT or CNF can’t be substituted with a less hazardous or nonhazardous material then all process equipment and other equipment involved with the handling of CNT and CNF 

should incorporate the necessary engineering control measures to prevent worker exposure to CNT 

and CNF. Because of limited published workplace 

exposure data for CNT and CNF, it is unknown 

whether worker respirable mass exposures to CNT 

and CNF can be maintained at all workplaces below the NIOSH REL of 1 μg/m3

 EC as an 8-hour 

TWA. However, exposure control techniques such 

as source enclosure (i.e., isolating the generation 

source from the worker) and well-designed local 

exhaust ventilation (LEV) systems equipped with 

high efficiency particulate air (HEPA) filters have 

been shown to be effective for capturing airborne 

nanoparticles including CNT and CNF [Old and 

Methner 2008; NIOSH 2009a; Evans et al. 2010]. A 

general description of exposure control techniques 

and their advantages and disadvantages is given in 

Table 6–6. The selection of the exposure control 

technique should take into account the quantity 

and physical form of the nanomaterial (e.g., dispersible powder, liquid slurry, contained in a matrix) and the task duration and frequency in which 

workers come into contact with the material (Table 

6–7). For instance, working with materials containing CNT or CNF (e.g., encapsulated in a solid) may 

require a different type of an exposure control system than would be required for large quantities of 

CNT and CNF in a highly dispersed free form. Processes involved in the cutting, grinding, or drilling 

of solid materials containing CNT or CNF should 

incorporate appropriate engineering controls (e.g., 

local exhaust ventilation) to prevent aerosol release, whereas the manufacturing (i.e., product collection at reactor) and handling of dry bulk CNT or 

CNF should be performed in enclosed, and when 

warranted, HEPA-ventilated systems. HEPA filtration has been shown to be effective in capturing 

nanoscale particles and should be considered in 

situations where emissions may be regular, where 

processes are repeated, and where higher quantities 

are used in a way that may lead to emissions. The 

handling of research quantities of CNT and CNF 

in laboratories is best performed using a laboratory 

fume hood, such as a low-flow or air-curtain hood 

[Tsai et al. 2010], or use of a glove box to minimize 

worker exposure [NIOSH 2012]. All exposure control systems should be properly designed, tested, 

and routinely maintained to ensure maximum 

efficiency [ACGIH 2007]. 

6.3 Worker Education

and Training 

Establishing a program that includes the education 

and training of workers on the potential hazards of 

CNT and CNF and their safe handling is critical 

to preventing adverse health effects from exposure. 

Research has shown that training can attain immediate and long-term objectives when (1) workers 

are educated about the potential hazards of their 

job, (2) there are improvements in knowledge and 

work practices, (3) workers are provided the necessary skills to perform their job safely, and (4) there 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 59

Table 6–6. Examples of engineering controls

Containment category

and description Advantages Disadvantages

A. Dilution ventilation and no 

engineering controls

Supply and exhaust large 

volumes of air (typically > 10 

air changes/hr.) throughout the 

work area to dilute airborne 

emissions.

For general facility HVAC 

needs. Not recommended for 

controlling worker exposure to 

CNT and CNF.

No local exhaust ventilation (LEV) or 

equipment enclosures required.

Disperses/dilutes airborne emissions 

throughout work area. 

Does not control exposure at the source, 

spreads emissions throughout work area 

potentially exposing other workers.

Often requires large airflow exhausts 

to dilute contaminants to below OEL 

increasing operating costs.

Should only be considered when 

contaminant generation is reasonably 

uniform and toxicity of material is low.

B. Local exhaust Ventilation 

(LEV)

Hoods or enclosures on process 

equipment that exhaust air 

at the emission source to 

collection equipment and away 

from the worker’s breathing zone. 

Includes: 

B.1. Laboratory fume hoods 

(typically 80–120 ft/min 

face velocity) with HEPA 

filter 

B.2. Biological safety cabinet 

Class II 

B.3. LEV incorporated at 

source of exposure and 

can be built into handheld tools 

Capture emissions at their source with 

well-designed hoods.

Hoods can be tailored to the process 

or work task to optimize the capture of 

emissions.

Usually requires less overall exhaust 

airflow rates than dilution ventilation 

systems.

Air volumes and face velocity of LEV 

must be maintained to ensure the 

capture of emissions.

Workers must be trained in the

correct use.

Fume hood sash opening needs to

be adjusted to ensure proper hood

face velocity.

System exhaust flow rate may need 

careful evaluation to ensure adequate 

capture while minimizing loss of 

product.

C. Down flow booths

Small room or enclosure with 

low velocity (100 ft/min) 

downward airflow to push/pull 

contaminants away from the 

worker’s breathing zone. 

Emissions pushed away from the 

worker’s breathing zone.

Flexible control that can be used for 

several tasks/operations.

Useful for manual operations for 

which a more contained enclosure is 

not feasible (e.g., larger amounts of 

materials or equipment).

Air volumes and control velocities of 

booth must be monitored/maintained 

to ensure proper performance.

Worker technique and interface with 

the work process can interfere with the 

capture of emissions.

Workers must be trained in the

correct use.

(Continued)
60 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Containment category

and description Advantages Disadvantages

D. Closed process design 

(isolation)

All steps of the process or job 

task are sealed with little chance 

of worker exposure.

Examples:

D.1. Glove box isolators (with 

HEPA filtered exhaust)

D.2. Biological safety cabinet 

class III 

Emission source confined. 

Minimizes external contamination. 

Need for worker PPE (e.g., respirator) 

reduced.

More time required moving materials 

and equipment in and out of enclosure.

Difficulty in manipulating materials 

when wearing gloves.

Limitations on size of material that can 

be placed inside of an isolator.

Need to periodically clean the 

enclosure.

Adapted from Industrial Ventilation [ACGIH 2007]

Table 6–6 (Continued). Examples of engineering controls
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 61

Table 6–7. Engineering controls to reduce CNT and CNF exposures

Process/activity

Potential exposure source and recommended

containment of exposure*

A. Pilot and research development 

operations 

Exposure Source: Synthesis of CNT and CNF by fluidized-bed, chemical 

vapor deposition, etc.: a) collection/harvesting after synthesis, b) powder 

transfer, c) cleaning reactor, d) removal of CNT and CNF from a substrate, 

e) purification and/or functionalization of CNT or CNF [note: potential 

exposures are generally to small quantities of CNT and CNF (i.e., μg, 

mg) compared to exposure to larger amounts (e.g., kg) during full-scale 

manufacturing/synthesis (see C below)].

Exposure Controls: a) laboratory fume hood (with HEPA filtered exhaust 

when warranted), b) HEPA-filtered exhausted enclosure (glove box), or c) 

biological safety cabinet. Local exhaust ventilation (LEV) may be required 

when opening reactor and during harvesting. 

B. Research laboratories Exposure Source: Handling (e.g., mixing, weighing, blending, transferring) 

small quantities (e.g., μg, mg) of CNT or CNF powder or during sonication 

of a CNT or CNF liquid suspension.

Exposure Controls: a) laboratory fume hood (with HEPA filtered exhaust 

when warranted), b) HEPA-filtered exhausted enclosure (glove box 

isolator), or c) biological safety cabinet. 

C. CNT and CNF manufacturing 

and synthesis

Exposure Source: Synthesis of CNT and CNF by fluidized-bed, chemical 

vapor deposition, etc., including: a) collection/harvesting after synthesis, 

b) drum and bag filling, c) powder transfer, d) cleaning reactor, e) removal 

of CNT and CNF from a substrate, f) purification and/or functionalization 

of CNT or CNF [Note: potential exposures are generally to large quantities 

(e.g., kg) of CNT and CNF].

Exposure Controls: Dedicated ventilated room with HEPA filtered exhaust, 

and/or LEV at source of exposure with HEPA filtered exhaust. Examples: 

ventilated bagging/weighing station and/or laminar down-flow booth or 

non-ventilation options such as continuous liner off-loading systems for 

bagging operations. Ventilated bag dumping stations for product transfer. 

See footnotes at end of table. (Continued)
62 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Process/activity

Potential exposure source and recommended

containment of exposure*

Production and use of CNT and CNF 

enabled materials and composites

Exposure Source: Mixing, weighing, and transferring of small quantities of 

CNT or CNF powder or liquid suspension, including the: a) incorporation 

of CNT or CNF into matrices (e.g., polymer composites) and into coatings 

(e.g., inks) and, b) spraying CNT or CNF on surfaces.

Exposure Controls: a) Laboratory fume hood (with HEPA filtered exhaust 

when warranted), b) HEPA-filtered exhausted enclosure (glove box 

isolator), or c) biological safety cabinet.

Exposure Source: Handling large quantities of CNT or CNF powder that 

involves pouring and blending into other matrices. In addition, spinning, 

twisting, weaving of CNT into making rope, cloth, etc.; spray coating of 

surfaces.

Exposure Controls: Isolation techniques such as a dedicated ventilated 

room or process enclosure with HEPA filtered exhaust. Process-based 

controls such as ventilated bagging/weighing station, laminar down-flow 

booth or non-ventilation options such as continuous liner off-loading 

systems for bagging operations. Ventilated bag dumping stations for 

product transfer.

Exposure Source: Grinding, sanding, cutting, drilling or other mechanical 

energy applied to enabled-materials/composites containing CNT or CNF.

Exposure Controls: For the handling of small pieces of CNT or CNF 

enabled materials/composites: a) laboratory fume hood (with HEPA 

filtered exhaust when warranted), b) HEPA filtered exhausted enclosure 

(glove box isolator), or c) biological safety cabinet.

Exposure Controls: For handling large CNT or CNF enabled materials/

composites and where use of isolation techniques such as large ventilated 

enclosures are not feasible: a) use LEV at exposure source with HEPA 

filtered exhaust (may include LEV built into a hand-held tool), b) ventilated 

down-flow booths with HEPA filtered exhaust, c) laboratory fume hood 

(with HEPA filtered exhaust) and/or d) wet dust suppression machining 

techniques such as wet saws (if applicable).

*Note: Factors that influence selection of appropriate engineering controls and other exposure control strategies include the 

physical form (e.g., dry dispersible powder, liquid slurry, in a matrix/composite), task duration, frequency, and quantity of CNT 

or CNF handled. Measurement of airborne exposure at the potential source of emission should be performed to confirm the 

effectiveness of the control measure.

Table 6–7 (Continued). Engineering controls to reduce CNT and CNF exposures
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 63

is management commitment and support for workplace safety [NIOSH 2010b]. The requirements for 

the education and training of workers as specified 

in the OSHA Hazard Communication Standard

(29 CFR 1910.1200), the Hazardous Waste Operation and Emergency Response Standard (29 CFR 

1910.120), and as described by Kulinowski and 

Lippy [2011] for workers exposed to nanomaterials, provide a minimum set of guidelines that can 

be used for establishing an education and training 

program. The establishment of a program should 

have written procedures (e.g., standard operating 

procedures [SOPs]) for: (a) ensuring management 

commitment to control exposures, (b) identifying 

and communicating potential hazards to workers, (c) assessing workplace exposures to CNT and 

CNF, (d) identifying and implementing engineering and work practice controls, (e) establishing 

documentation of risk management actions taken, 

and (f) periodically reviewing the adequacy of controls and other preventive practices. Management 

should systematically review and update these procedures and convey to workers actions taken to resolve and/or improve workplace conditions. 

A program for educating workers should also include both instruction and “hands-on” training 

that addresses the following:

• The potential health risks associated with exposure to CNT and CNF. 

• The safe handling of CNT, CNF, and CNTand CNF-containing materials to minimize 

the likelihood of inhalation exposure and 

skin contact, including the proper use of 

engineering controls, PPE (e.g., respirators, 

gloves), and good work practices. 

6.4 Cleanup and Disposal

Procedures should be developed to protect workers 

from exposure to CNT and CNF during the cleanup 

of CNT and CNF spills and CNT- or CNF-contaminated surfaces. Inhalation and dermal exposures will 

likely present the greatest risks. The potential for inhalation exposure during cleanup will be influenced 

by the likelihood of CNT and CNF becoming airborne, with bulk CNT and CNF (powder form) 

presenting a greater inhalation potential than CNT 

and CNF in solution (liquid form), and liquids in 

turn presenting a greater potential risk than CNTand CNF-encapsulated materials. 

It would be prudent to base strategies for dealing 

with spills and contaminated surfaces on the use of 

current good practices, together with available information on exposure risks. Standard approaches 

for cleaning powder spills can be used for cleaning 

surfaces contaminated with CNT or CNF. These 

include using HEPA-filtered vacuum cleaners, wiping up CNT and CNF (powder form) using damp 

cloths, or wetting the powder before wiping. Liquid spills containing CNT or CNF can typically 

be cleaned by applying absorbent materials/liquid 

traps. If vacuum cleaning is employed, care should 

be taken that HEPA filters are installed properly 

and bags and filters changed according to manufacturer’s recommendations. Dry sweeping or air 

hoses should not be used to clean work areas. 

The handling and disposal of waste (including all 

cleaning materials) and other contaminated materials (e.g., gloves) should comply with all applicable 

regulations (e.g., federal, state, local). 

6.5 Personal Protective 

Clothing

There are no regulations or guidelines for the selection of protective clothing or other apparel 

against exposure to CNT and CNF; however, the 

Occupational Safety and Health Administration 

(OSHA) requires employers to provide employees 

with hand protection when exposed to hazards 

[OSHA 1910.138(a)]. Currently, limited information is available to assess the exposure and health 

hazards of skin exposure to CNT and CNF. In a 

study to determine potential airborne and dermal 

exposures to SWCNT during manufacturing and 

handling, workers’ dermal exposure was estimated 

by placing cotton gloves over the rubber gloves 

used by workers [Maynard et al. 2004]. Dermal 
64 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

exposure estimates for SWCNT on individual gloves 

(total hand area) ranged from 217  μg to 6020  μg, 

with most of the SWCNT material appearing on the 

parts of the gloves in direct contact with surfaces. Results from experimental studies with various types 

of nanoparticles found that dermal penetration of 

nanoparticles may occur under certain conditions of 

exposure (e.g., flexing of skin) [Ryman-Rasmussen 

et al. 2006; Rouse et al. 2007] and that factors such 

as size, shape, water solubility, and surface coating 

directly affect a nanoparticle’s potential to penetrate 

the skin [Sayes et al. 2004; Ryman-Rasmussen et al. 

2006]. The results from in vitro studies, using primary 

or cultured human skin cells and engineered human 

skin, show that SWCNT and MWCNT are able to 

enter cells and cause the release of pro-inflammatory 

cytokines, induce free radical generation and oxidative stress, and decrease cell viability [Shvedova et 

al. 2003; Monteiro-Riviere et al. 2005; Murray et al. 

2009; Vankoningsloo et al. 2010]. Vankoningsloo 

et al. [2010] also found the surface properties of

MWCNT played a determinant role in their interaction with cells, and when nanotube agglomeration was decreased, there was an increase in cytotoxicity on keratinocytes. The topical application of 

SWCNT (160 μg) to SKH-1 mice caused inflammation that was localized around or within the hair follicles; however, no significant changes were observed 

at the lowest dose (40 μg) tested [Murray et al. 2009]. 

It was concluded that topical exposure to unpurified 

SWCNT at doses > 80 μg/mouse are capable of inducing free radical generation, oxidative stress, and 

inflammation. However, the results of dermal toxicity 

testing with one type of MWCNT (Baytubes®) found 

no evidence of acute skin irritation or sensitization 

and only mild eye irritation in rabbits when tested according to OECD test guidelines [Pauluhn 2010b]. 

Given the limited amount of data on dermal exposure to CNT and CNF, it would be prudent to wear 

protective clothing and gloves when 

• all technical measures to eliminate or control the release of exposure to CNT and CNF 

have not been successful, or 

• in emergencies. 

If protective clothing and/or gloves are worn, 

particular attention should be given to preventing CNT and CNF exposure to abraded or lacerated skin. Based on limited experimental evidence, 

airtight fabrics made of nonwoven textile seem 

to be more efficient in protecting workers against 

nanoparticles than fabrics made of woven cotton 

or polyester [Golanski et al. 2009; Golanski et al. 

2010]. The results of a study designed to evaluate 

the penetration of nano- and submicron particle 

penetration through various nonwoven fabrics 

found minimal penetration (<  5%) of iron oxide 

particles (<  100  nm) through nonwoven fabrics 

typically used for hospital frocks, hoodless coveralls, and firefighter ensemble insulation [Gao et al. 

2011]. The challenge when selecting appropriate 

protective apparel is to strike a balance between 

comfort and protection. Garments that provide the 

highest level of protection (e.g., an impermeable 

Level A suit) are also the least comfortable to wear 

for long periods of time, while garments that are 

probably the least protective (e.g., thin cotton lab 

coat) are the most breathable and comfortable to 

wear. The efficiency of commercial gloves to prevent dermal exposure to nanoparticles varies depending on the glove material, its thickness, and 

the manner in which it is used (e.g., long exposure 

times, other chemical exposures) [NanoSafe 2008; 

Golanski et al. 2009, 2010]. The proper selection of 

gloves should take into account the resistance of 

the glove to the chemical attack by both the nanomaterial and, if suspended in liquids, the liquid 

[USDOE 2007]. If protective gloves (e.g., nitrile, 

neoprene, latex) are used then “double gloving” 

may be needed when the worker requires physical 

protection (e.g., working with sharp instruments) 

in addition to chemical protection. Special attention should also be given to the proper removal and 

disposal of contaminated gloves to prevent skin 

contamination. Gloves should also be visually inspected for tears and routinely replaced. 

6.6 Respirators

When engineering controls and work practices 

cannot reduce worker CNT and CNF exposures 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 65

to below the REL, then workers should be provided respiratory protection. The use of respirators may also be advisable for certain work tasks 

that place workers at risk of potentially high 

peak concentrations of CNT and CNF (e.g., the 

cleanup of CNT and CNF spills or debris, maintenance of equipment used to process CNT- and 

CNF-materials, the cleaning or disposal of filtration 

systems used to capture CNT and CNF aerosols). 

The OSHA respiratory protection standard (29 

CFR 1910.134) sets out the elements of a respirator 

program for both voluntary and required respirator use. When respirators are provided for worker 

protection, the OSHA respiratory protection standard requires that a respiratory protection program 

be established 29 CFR 1910 (c)(1). Elements of the 

program include (1) a medical evaluation of the 

worker’s ability to perform the work while wearing 

a respirator; (2) regular training of personnel; (3) 

periodic workplace exposure monitoring; (4) procedures for selecting respirators; (5) respirator fittesting; and (6) respirator maintenance, inspection, 

cleaning, and storage. The effectiveness of the program should be evaluated regularly and respirators 

should be selected by the person who is in charge 

of the program and knowledgeable about the workplace and the limitations associated with each type of 

respirator. The voluntary use of respirators are permitted, but must comply with the provisions set forth 

in CFR 1910.134(c)(2)(i) and 1910.134(c)(2)(ii). 

Based on published workplace monitoring data 

for CNT [Maynard et al. 2004; Han et al. 2008a; 

Bello et al. 2008, 2009, 2010; Dahm et al. 2011] 

and CNF [Methner et al. 2007; Evans et al. 2010], 

a NIOSH-approved filtering facepiece respirator, 

or elastomeric half-facepiece particulate respirator 

equipped with a 95 or 100 series filter, should provide adequate protection when properly fit-tested 

on the worker [Shaffer and Rengasamy 2009], and 

where engineered controls have been installed to 

reduce exposures. A properly fit-tested, half-facepiece 

particulate respirator or a filtering facepiece respirator will provide protection at exposure concentrations up to 10 times the REL. Other classes of 

respirators are available that provide a higher level 

of protection (Table 6–8). The publication NIOSH 

Respirator Selection Logic 2004 provides guidance 

for selecting an appropriate respirator [NIOSH 2005]. 

When selecting the appropriate respirator, the 

respirator program manager should consider the 

particle size in which workers will be potentially 

exposed [Rengasamy and Eimer 2011], and the 

presence of other workplace aerosols. Based on this 

information, the respirator program manager may 

decide to choose a respirator with a higher assigned 

protection factor (APF) or choose a respirator 

with a higher level of filtration performance (e.g., 

changing from an N95 to a P100). Studies on the 

filtration performance of N-95 filtering facepiece 

respirators have found that the mean penetration 

levels for 40 nm particles range from 1.4% to 5.2%, 

indicating that 95 and higher performing respirator filters would be effective at capturing airborne 

CNT and CNF [Bałazy et al. 2006; Rengasamy 

et al. 2007, 2008]. Recent studies also show that 

nanoparticles <20 nm are also effectively captured 

by NIOSH-approved filtering facepiece respirators 

as predicted by the single fiber theory [Rengasamy 

et al. 2008, 2009]. 

6.7 Medical Screening

and Surveillance

The toxicological evidence summarized in this 

document leads to the conclusion that workers occupationally exposed to CNT and CNF may be at 

risk of adverse respiratory effects. These workers 

may benefit from inclusion in a medical screening 

and surveillance program recommended to help 

protect their health (Figure 6–2) [NIOSH 2009b]. 

6.7.1 Worker Participation

Workers who could receive the greatest benefit 

from medical screening include the following: 

• Workers exposed to concentrations of CNT 

or CNF in excess of the REL (i.e., workers exposed to airborne CNT or CNF at concentrations above 1 μg/m3

 EC as an 8-hr TWA). 
66 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table 6–8. Respiratory protection for exposure to CNT and CNF

Airborne concentrations of CNT and CNF or 

conditions of use* options Minimum respiratory protection

1–10 μg/m3 (10 × REL) Any filtering facepiece respirator or air-purifying, elastomeric 

half-facepiece respirator equipped with appropriate type of 

particulate filter†

Any negative pressure (demand), supplied-air respirator 

equipped with a half-mask

≤ 25 μg/m3 (25 × REL) Any powered, air-purifying respirator equipped with a hood or 

helmet and a high-efficiency particulate air filter (HEPA filter)‡

Any continuous flow supplied air respirator equipped with a 

hood or helmet

≤ 50 μg/m3 (50 × REL) Any air-purifying full-facepiece respirator equipped with 

N-100, R-100, or P-100 filter

Any powered air-purifying respirator equipped with a tightfitting half-facepiece and a high-efficiency particulate air filter.

Any negative pressure (demand) supplied-air respirator 

equipped with a full-facepiece

Any continuous flow supplied-air respirator with a tight-fitting 

half-facepiece

Any negative pressure (demand) self- contained respirator 

equipped with a full-facepiece

≤ 1000 μg/m3 (1,000 × REL) Any pressure-demand supplied-air respirator equipped with a 

full-facepiece

*

The protection offered by a given respirator is contingent upon (1) the respirator user adhering to complete program 

requirements (such as those required by OSHA in 29 CFR 1910.134), (2) the use of NIOSH-certified respirators in their approved 

configuration, and (3) individual fit testing to rule out those respirators that cannot achieve a good fit on individual workers. 

†

The appropriate type of particulate filter means: Any 95 or 100 series (N, R, or P) filter. Note: N-95 or N-100 series filters should 

not be used in environments where there is potential for exposure to oil mists. 

‡

Some powered air purifying respirators with a hood/helmet are considered to have an APF of 1000 and thus could be used in 

situations involving higher airborne concentrations of CNT and CNF (< 1000 μg/m3). Contact the respirator manufacturer to 

determine whether this would apply. Absent such a determination, powered air purifying respirators with helmets/hoods are to 

be treated as loose-fitting facepiece respirators, and receive an APF of 25.

Note: complete information on the selection of respirators can be found at (1) OSHA 3352-02 2009, Assigned Protection Factors for 

the Revised Respiratory Protection Standard at http://www.osha.gov/Publications/3352-APF-respirators.html, and (2) NIOSH at 

[http://www.cdc.gov/niosh/docs/2005-100/default.html].
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 67

Figure 6–2. Medical screening and surveillance recommendations
68 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

• Workers in areas or in jobs who are qualitatively determined (by the person charged 

with program oversight) to have the potential 

for exposure to intermittent elevated airborne 

concentrations of CNT or CNF (i.e., workers are at risk of being exposed if they are involved in the transfer, weighing, blending, or 

mixing of bulk CNT or CNF; or in the cutting, 

grinding, or drilling of composite materials 

containing CNT or CNF; or in areas where 

such activities are carried out by others).

6.7.2 Program Oversight

Oversight of the medical surveillance program 

should be assigned to a qualified health-care professional who is informed and knowledgeable about 

potential workplace exposures, routes of exposure, 

and potential health effects related to CNT and CNF.

6.7.3 Screening Elements 

Initial evaluation

• An initial (baseline) evaluation should be 

conducted by a qualified health professional 

and should consist of the following:

ȣ An occupational and medical history 

with respiratory symptoms assessed by 

use of a standardized questionnaire such 

as the American Thoracic Society Respiratory Questionnaire [Ferris 1978], or 

the most recent equivalent. 

ȣ A physical examination with an emphasis on the respiratory system.

ȣ A spirometry test. (Anyone administering 

spirometry testing as part of the medical 

screening program should have completed 

a NIOSH-approved training course in spirometry or other equivalent training; additionally, the health professional overseeing the screening and surveillance program should be expert in the interpretation of spirometry testing results, enabling 

follow-up evaluation as needed.)

ȣ A baseline chest X-ray (digital or filmscreen radiograph). All baseline chest images should be clinically interpreted by a 

board eligible/certified radiologist or other physician with appropriate expertise, 

such as a board eligible/certified pulmonologist. Periodic follow up chest X-rays 

may be considered, but there is currently 

insufficient evidence to evaluate effectiveness. However, if periodic follow up is obtained, clinical interpretation and classification of the images by a NIOSH-certified 

B reader using the standard International 

Classification of Radiographs of Pneumoconioses (ILO 2011 or the most recent equivalent) are recommended.

ȣ Other examinations or medical tests 

deemed appropriate by the responsible 

health-care professional. (The need for 

specific medical tests may be based on 

factors such as abnormal findings on 

initial examination—for example, the 

findings of an unexplained abnormality 

on a chest X-ray should prompt further 

evaluation that might include the use of 

high-resolution computed tomography 

scan of the thorax.)

Periodic evaluations

• Evaluations should be conducted at regular 

intervals and at other times (e.g., post-incident) 

as deemed appropriate for the individual worker by the responsible health-care professional. 

Evaluations should be based on data gathered in the initial evaluation, ongoing work 

history, changes in symptoms such as new or 

worsening respiratory symptoms, and when 

process changes occur in the workplace (e.g., 

a change in how CNT or CNF are manufactured or used or an unintentional spill). Evaluations should include the following:

ȣ An occupational and medical history update, including a respiratory symptom update, and focused physical examination—

performed annually.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 69

ȣ Spirometry testing less frequently than 

every 3 years is not recommended [OSHA 

NIOSH 2011]

ȣ Consideration of specific medical tests 

(e.g., chest X-ray). 

Written reports of medical findings

• The health-care professional should give 

each worker a written report containing the 

following:

ȣ The individual worker’s medical examination results.

ȣ Medical opinions and/or recommendations concerning any relationships between the individual worker’s medical 

conditions and occupational exposures, 

any special instructions on the individual’s exposures and/or use of personal 

protective equipment, and any further 

evaluation or treatment. 

• For each examined employee, the health-care 

professional should give the employer a written report specifying the following:

ȣ Any work or exposure restrictions based 

on the results of medical evaluations.

ȣ Any recommendations concerning use 

of personal protective equipment.

ȣ A medical opinion as to whether any of 

the worker’s medical conditions is likely 

to have been caused or aggravated by occupational exposures.

• Findings from the medical evaluations having no bearing on the worker’s ability to work 

with CNT and CNF should not be included 

in any reports to employers. Confidentiality 

of the worker’s medical records should be enforced in accordance with all applicable regulations and guidelines.

6.7.4 Worker Education

Workers should be provided information sufficient to allow them to understand the nature of 

potential workplace exposures, routes of exposure, 

and instructions for reporting health symptoms. 

Workers should also be given information about 

the purposes of medical screening, the health benefits of the program, and the procedures involved. 

6.7.5 Periodic Evaluation of Data 

and Surveillance Program 

Standardized medical screening data should be periodically aggregated and evaluated to identify patterns of worker health that may be linked to work 

activities and practices that require additional primary prevention efforts [i.e., medical surveillance]. 

This analysis should be performed by a qualified 

health-care professional or other knowledgeable 

person to identify patterns of worker health that 

may be linked to work activities or exposures. Confidentiality of worker’s medical records should be 

enforced in accordance with all applicable regulations and guidelines. 

Employers should periodically evaluate the elements of the medical screening program to ensure 

that the program is consistent with current knowledge related to exposures and health effects associated with occupational exposure to CNT and CNF.

Other important components related to occupational health surveillance programs, including 

medical surveillance and screening, are discussed 

in Appendix B.

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 71

7 Research Needs

Additional data and information are needed to assist NIOSH in evaluating the occupational safety 

and health concerns of working with CNT and 

CNF. Data are particularly needed on workplace 

exposures to CNT and CNF, as well as information 

on whether in-place exposure control measures 

(e.g., engineering controls) and work practices are 

effective in reducing worker exposures. Additional 

assessment of NIOSH Method 5040 is needed to 

better understand potential interferences or other 

method limitations, improve the sensitivity and 

precision of the analytical method, and establish 

validity through the use of reference materials. 

The conduct of experimental animal studies with 

various types of CNT and CNF would help to explain potential mechanisms of toxicity and would 

provide a better understanding of the exposure parameters (e.g., mass, fiber/structure number, and 

particle size) that best describe the toxicological responses. Chronic studies in animals are needed to 

better estimate the long-term risks of lung disease 

in workers. 

The following types of information and research 

are needed:

7.1 Workplace Exposures,

 Measurement, and Controls

• Quantify worker airborne exposures to CNT 

and CNF.

• Evaluate NIOSH Method 5040 and other appropriate sampling and analytical methods in 

CNT and CNF workplaces. For example, validate 

Method 5040 against EC reference material and 

ruggedize against several CNT and CNF types. 

• Improve the sensitivity and precision of NIOSH 

Method 5040 and other appropriate methods for 

measuring airborne concentrations of CNT and 

CNF, including those based on metrics that may 

be more closely associated with the potential 

adverse effects (e.g., electron microscopy- based 

CNT or CNF structure counts).

• Develop improved sampling and analytical 

methods for measuring airborne exposures to 

CNT and CNF. Apply these different methods in 

toxicological studies to determine which exposure metric best predicts the health endpoints in 

laboratory animal studies. 

• Determine the effectiveness of engineering controls to control airborne exposures to CNT and 

CNF below the NIOSH REL of 1 μg/m3. 

• Confirm the effectiveness of using HEPA filters 

in an exhaust ventilation system for removing exposures to CNT and CNF. 

• Determine the effectiveness of gloves and other 

PPE barrier materials in preventing dermal exposure to CNT and CNF. 

• Identify, quantify, and develop CNT and CNF 

reference materials for toxicology studies and for 

measurement quality control. 

• Conduct workplace studies to measure total inward leakage (TIL) of respirators for workers exposed to nanoparticles (e.g., CNT/CNF). 

7.2 Experimental and

Human Studies

• Conduct chronic animal inhalation studies to assess respiratory and other organ (e.g., heart and 

other circulatory system) effects. Special emphasis should be placed on assessing the risk for developing lung fibrosis and cancer. Studies should 

evaluate different types of CNT and CNF and use 

various exposure metrics (e.g., mass, tube, and 

structure counts, surface area) for assessing toxicological responses. 

• Determine the mechanisms and other causative 

factors (e.g., tube, fiber and agglomerate size, 

surface area, and surface reactivity) by which 
72 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

CNT and CNF induce adverse effects (e.g., lung 

fibrosis) in animals. 

• Develop early markers of exposure and pulmonary response to CNT and CNF, given evidence 

from animal studies that CNT and CNF persist 

in the lungs and result in the development and 

progression of pulmonary fibrosis and/or cancer 

at relatively low-mass doses. 

• Quantitatively and qualitatively compare the 

CNT and CNF materials used in the animal 

studies with the CNT and CNF materials found 

in workplace air.

• Determine the potential for CNT and CNF to 

penetrate the skin and cause toxicity.

• Evaluate the predictive value of using in vitro

screening tests for assessing the hazard (e.g., 

fibrogenic potential) of various types of CNT 

and CNF. 

• Assess the feasibility of establishing exposure 

registries for workers potentially exposed to 

CNT and CNF for conducting future epidemiologic studies and surveillance activities. 

• Conduct cross-sectional and prospective studies 

of workers exposed to CNT and CNF.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 73

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APPENDIX A

Quantitative Risk Assessment of CNT

NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 93

Contents

A.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

A.2 Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

A.2.1 Rodent Dose-Response Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

A.2.1.1 Data selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

A.2.1.2 Dose rate evaluation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

A.2.1.3 Lung responses evaluated. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

A.2.1.4 Summary of dose-response data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

A.2.2 Estimated Lung Dose in Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

A.2.3 Animal Dose-Response Modeling and BMD Estimation. . . . . . . . . . . . . . . . . . . 101

A.2.3.1 Dichotomous Response Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

A.2.3.2 Continuous response data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

A.2.3.3 BMD model fitting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

A.2.3.4 Human-equivalent dose and working lifetime exposure. . . . . . . . . 103

A.3 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

A.3.1 Benchmark Dose and Working Lifetime Exposure Estimates. . . . . . . . . . . . 108

A.3.2 Comparison of Short-Term and Subchronic Dose-Response Data. . . . . . . . 113

A.3.3 Estimated Excess Risks of Working Lifetime Exposures

to Low-Mass Concentrations of MWCNT. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

A.4 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

A.4.1 The Use of Short-Term Data to Predict Longer-Term Response. . . . . . . . . . . . . . 117

A.4.2 Physical-Chemical Properties and Metal Content. . . . . . . . . . . . . . . . . . . . . . 118

A.4.3 Lung Dose Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

A.4.4 Critical Effect Level Estimates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

A.4.5 Animal Dose-Response Data Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A.6 Sensitivity Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A.6.1 Lung Dose Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A.6.1.1 Lung dosimetry model-based deposition fraction

and dose estimates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

A.6.1.2 Cobalt tracer vs. dosimetry model estimates of

MWCNT lung dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

A.6.1.3 Pulmonary Ventilation Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

A.6.2 Critical Effect Level Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

A.6.3 Alternative OEL Estimation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

A.6.3.1 Illustration of human-equivalent concentration estimation. . . . . . 129

A.6.3.2 Evaluation of dosimetric adjustment Factors . . . . . . . . . . . . . . . . . . 130

A.6.3.3 Selection of uncertainty factors and OEL derivation. . . . . . . . . . . . 133

A.6.4 Summary of Sensitivity Analyses Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
94 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

A.7 Evaluation of Carbon Nanofiber Studies in Mice and Rats. . . . . . . . . . . . . . . . . . . . 139

A.7.1 Particle Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.2 Experimental Design and Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.3 Lung Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

A.7.4 Effects in Other Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

A.7.5 Equivalent Lung Dose Estimation Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . 140

A.7.6 Equivalent Lung Dose Estimation Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 95

A.1 Introduction

The increasing production and use of CNT and the 

preliminary significant toxicology findings necessitate an assessment of the potential adverse health 

effects in workers who produce or use these materials. Risk assessment is a process that uses standardized tools and procedures to characterize the 

health risk of exposure to a hazardous substance, as 

well as the uncertainties associated with those risk 

estimates. Research studies in toxicology, epidemiology, exposure measurement, and other areas 

provide the data needed to perform the risk assessment. The standard risk assessment paradigm in 

the United States includes four basic steps: hazard 

assessment, exposure assessment, dose-response 

analysis, and risk characterization [NRC 1983, 

2009]. Risk assessment also involves the initial 

steps in problem formulation and evaluation of the 

various risk management options [NRC 2009]. The 

most recent guidance [NRC 2009] recommends 

asking these questions: ”What are the options available to reduce the hazards or exposures that have 

been identified, and how can risk assessment be 

used to evaluate the merits of the various options?” 

Risk assessment is intended to provide information 

needed to determine risk management options.

Risk assessment practice seeks to use the best available data and scientific methods as the basis for 

public and occupational health decision-making 

[NRC 2009]. When sufficient dose-response data 

are available (e.g., from animal studies), quantitative risk assessment can be performed. Quantitative risk assessment provides estimates of the severity and likelihood of an adverse response associated 

with exposure to a hazardous agent [Piegorsch and 

Bailer 2005; NRC 2009]. Risk assessments are used 

in developing occupational exposure limits and in 

selecting and evaluating the effectiveness of exposure controls and other risk management strategies 

to protect workers’ health. 

The best data available for risk assessment, in the 

absence of epidemiological studies of workers producing or using CNT, are from animal studies 

with CNT. These studies include two subchronic 

inhalation studies of MWCNT in rats and several 

short-term studies of SWCNT, MWCNT, or CNF 

in rats or mice. These studies provide the data and 

information on the dose-response relationships 

and the biological mechanisms of early-stage inflammatory and fibrotic lung effects from exposure to CNT. No chronic animal studies of CNT 

were available for this risk assessment.

The biological mode of action for CNT and CNF, as 

for inhaled particles and fibers, generally relates to 

their physical and chemical properties. These properties include: (1) nano-structure which increases 

the surface area and associated inflammogenic and 

fibrogenic response; (2) fiber shape which may 

decrease clearance of long structures, resulting in 

translocation to the interstitial and pleural tissues 

of the lungs; and (3) the graphitic structure of CNT 

and CNF which influences their durability and 

biopersistence [Donaldson et al. 2006; Shvedova et 

al. 2009; Castranova 2011]. CNT and CNF are heterogeneous structures, and differences can include 

size (length and diameter), metal contaminants 

(type and amount), surface chemistry, and tendency to aggregate/agglomerate. CNT and CNF typically form agglomerates in air but may also exist as 

individual structures Johnson et al. 2010, Methner 

et al. 2010, Dahm et al. 2011]. Evidence from shortterm and subchronic studies in animals indicates 

that CNT may be biopersistent in the lungs [Muller 

et al. 2005; Deng et al. 2007; Elgrabli et al. 2008b; 

Mercer et al. 2009; Pauluhn 2010a,b] However, 

some evidence suggests that functionalization may 

increase biodegradation of CNT [Kagan et al. 2010; 

Osmond-McLeod et al. 2011]. 

Dose metrics that have been associated with lung 

responses to CNT or CNF in animal studies include 

mass, volume, number, and surface area. The CNT 

volume dose was associated with the overloading of 

CNT clearance from rat lungs and to the lung responses [Pauluhn 2010a]. The specific surface area 

(m2/g) dose of various types of CNTs was associated with the pulmonary inflammation response in 

rats [Nakanishi 2011a]. Mercer et al. [2011] showed 

that on a mass basis, SWCNT is more fibrogenic 

than MWCNT, but this difference disappeared after 

accounting for the greater specific surface area of 

the SWCNT than MWCNT. Murray et al. [2012] 
96 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

found that the effective surface area (estimated from 

the geometry of the structures observed by electron 

microscopy) was more closely associated with the 

pulmonary inflammation and fibrosis in mice. 

In addition to exhibiting some of the same physical-chemical properties of other poorly-soluble 

particles and/or fibers, the nanoscale structure of 

CNT and CNF may relate to more specific biological modes (or mechanisms) of action. For example, 

evidence in vitro suggests that disperse CNT may 

act as a basement membrane, which enhances fibroblast proliferation and collagen production 

[Wang et al. 2010]. This mechanism is consistent 

with the observation in mice of the rapid onset of 

diffuse interstitial fibrosis, which progressed in the 

absence of persistent inflammation, following exposure to SWCNT or MWCNT by pharyngeal aspiration [Shvedova et al. 2005, 2008; Porter et al. 

2010]. As fibrosis progresses, it causes thickening 

of the alveolar septal air/blood barrier, which can 

result in a decrease of gas-exchange between lung 

and blood [Hubbs et al. 2011]. 

The focus of this quantitative risk assessment is on the 

early-stage noncancer lung responses (fibrotic and inflammatory) from studies in rats and mice, for which 

dose-response data are available. These responses are 

relevant to humans as observed in workers in dusty 

jobs [Rom and Markowitz 2006; Hubbs et al. 2011]. 

Dose-response relationships are based on mass dose, 

because the mass of CNT and CNF was associated 

with lung responses in all of the animal studies and 

because it is the metric typically used to measure airborne exposure in the workplace (Section 6 and Appendix C). The evidence for cancer effects from CNT 

and CNF (Section 3 and 4) is insufficient for quantitative risk assessment, and may also depend on specific 

types of CNT or CNF structures [Donaldson et al. 

2011; Nagai et al. 2011; Schulte et al. 2012]. 

A.2 Methods

NIOSH used dose-response data from subchronic 

and short-term studies in rats and mice exposed to 

SWCNT or MWCNT to estimate the lung doses associated with early-stage inflammatory and fibrotic 

lung responses. Benchmark dose (BMD) modeling 

[Crump 1984; 1995; US EPA 2010] of rodent doseresponse data was used to estimate an adverse effect 

level (10% excess risk of early-stage lung effects). 

The animal BMD was extrapolated to humans to 

estimate the risk of working lifetime exposures 

and provided the scientific basis for developing 

a NIOSH recommended exposure limit (REL) 

for CNT. Dose-response data from subchronic 

and short-term studies in rats and mice exposed 

to SWCNT or MWCNT were used to estimate 

the BMDs associated with benchmark responses 

(BMRs) of early-stage inflammatory and fibrotic 

lung responses. The rodent-based BMD estimates 

were extrapolated to humans by accounting for 

species differences in factors influencing lung dose 

in order to estimate the working lifetime risk of airborne exposure to CNT. Sensitivity analyses were 

performed to evaluate the influence of the various 

methods and assumptions on the risk estimates. 

When feasible, NIOSH utilizes BMD estimates in 

risk assessment rather than a lowest observed adverse effect level (LOAEL) or a no observed adverse 

effect level (NOAEL) for the following reasons: (1) 

BMD methods provide a standardized approach 

for risk estimation; (2) BMD methods provide 

both maximum likelihood (BMD) and 95% lower 

confidence limit, or BMDL, estimates, which explicitly account for the sample size and variability in the 

data; and (3) BMD models efficiently use all of the 

dose-response data in estimating the BMD(L)s. In 

contrast, NOAEL and LOAEL estimates are: (1) not 

risk-based; (2) generally interpreted as estimates 

of a threshold; and (3) sensitive to the study size 

and dose group spacing. However, BMD estimation may require more dose-response data than 

does a NOAEL or LOAEL. Sparse data provide 

limited information for BMD estimation and can 

result in model uncertainty. In addition, the NOAEL estimate may depend on the size of the study, 

such that larger studies (e.g., greater number of animals) could detect effects at smaller exposures. This 

would not occur with the BMDL estimate, which 

would “simply approach the (true) BMD” with increasing study size [Crump 2002]. Comparisons of 

the BMD(L) estimates to the LOAELs or NOAELs 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 97

provide an informal check on the estimated and observed responses in the low-dose region of the data; 

and sensitivity analyses of the methods and assumptions in these analyses provides information on the 

qualitative and quantitative uncertainty in these risk 

estimates and derived OELs (Section A.6). 

A.2.1 Rodent Dose-response 

Data

A.2.1.1 Data Selection 

The published rodent studies on pulmonary responses to CNT (Section 3, Tables 1–3c) were examined for possible inclusion in this risk assessment. Pulmonary effects were examined because of 

their relevance to workers who may be exposed to 

CNT in workplace air. The studies with adequate 

quantitative dose-response data to estimate BMDs 

were included in these analyses. These studies reported on the size and characterization of the CNT 

(Table A–1) as well as the route of exposure, doses, 

duration of exposure or post-exposure, number of 

animals per group, and lung responses. In general, 

the CNT animal studies have limited data, with 

few (4–20) animals per dose group and sparse dose 

group spacing, especially in the low range of the 

dose-response curve. Some of these studies just 

meet the minimum data criteria for BMD estimation, that is, a significant dose-related trend in the 

selected endpoint [US EPA 2012]. It is preferable to 

have data with one or more doses near the benchmark response (e.g., 10%) [US EPA 2012]; however, in some studies the response proportions were 

quite high at each dose (e.g., 30–100%) [Lam et al. 

2004; Ma-Hock et al. 2009; Pauluhn 2010a]. In addition, one study [Shvedova et al. 2008] had only 

one dose group in addition to the control, but the 

study was included because it is the only animal inhalation study for SWCNT currently available and it 

provides a useful comparison by route of exposure. 

No other deficiencies were noted in the selected 

studies that would have resulted in their omission.

Either the individual animal dose-response data 

or the mean and standard deviation of the group 

response are required for BMD model fitting. The 

dose was either the intratracheal instillation (IT) 

or pharyngeal aspiration (PA) administered mass 

dose (mg/lung) or the inhaled mass concentration 

(mg/m3). Datasets with treatment-related mortality of animals were not used. Data on special preparations of CNT (e.g., ground CNT) or studies using 

sensitive animal models (e.g., vitamin E deficient) 

were not included (although these data may be of 

interest for subsequent analyses using animal models to investigate biological mechanisms, including 

in sensitive human populations, or to evaluate the 

effect of specific alterations in CNT properties on 

hazard potential). 

Study details of the data selected for this risk assessment are provided in Table A–1. These studies include the two recently published subchronic 

inhalation studies of MWCNT in rats [Ma-Hock 

et al. 2009; Pauluhn 2010a] and several IT, PA, or 

short-term inhalation studies in rats or mice exposed to SWCNT [Lam et al. 2004; Shvedova et al. 

2005, 2008] or MWCNT [Muller et al. 2005; Mercer et al. 2011] with post-exposure durations and 

examination from 4 to 26 weeks after exposure. In 

the subchronic inhalation studies, rats were headnose exposed [Ma-Hock et al. 2009] or nose-only 

exposed [Pauluhn 2010a] to three or four different 

airborne mass concentrations (6  hr/d, 5  d/week) 

for 13 weeks. Lung responses were examined at the 

end of the 13-week exposure in both studies; postexposure follow-up was extended to 6 months in 

the Pauluhn [2010a] study. 

The IT, PA, and short-term inhalation studies provide additional dose-response data for comparison 

to other MWCNT or SWCNT with different types 

and amounts of metal contaminants. Although 

both IT and PA routes bypass the head region and 

deliver the CNT material directly to the trachea and 

lung airways, PA is to be considered more similar 

to inhalation than IT because PA provides greater 

dispersion of deposited material in the lungs [Shvedova et al. 2005, 2008]. Following the administered 

dose (on day 1), the lung responses were evaluated 

after a post-exposure period (e.g., 1, 7, 28, 60, and/

or 90 days). For studies with more than one postexposure duration, the longest post-exposure duration data are used in these risk analyses. Some 
98 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

of these studies also provide dose-response data on 

other particles or fibers (e.g., ultrafine carbon black, 

crystalline silica, and asbestos) for comparison of 

dose and response to that observed from exposure 

to MWCNT or SWCNT [Lam et al. 2004; Muller et 

al. 2005; Shvedova et al. 2005]. Two other short-term 

exposure studies (Porter et al. [2010] and EllingerZiegelbauer-Pauluhn [2009]), which were included 

in the external review draft of the CIB [NIOSH 

2010], have been omitted in this analysis This is because the dose-response data were of equivocal fit to 

the minimum data criteria for BMD analysis and 

because updates of these studies are available for the 

same CNT material and from the same laboratory 

(i.e., Mercer et al. [2011] and Pauluhn [2010a]). 

Those studies are included in these risk analyses. 

A.2.1.2 Dose Rate Evaluation

A study of 1-day inhalation exposure to MWCNT 

(Baytubes) in rats and examined 13 weeks after the 

end of exposure [Ellinger-Ziegelbauer and Pauluhn 2009] provided an opportunity to compare 

the dose-response relationships of the 1-day inhalation exposure study with that of the 13-week 

(subchronic) inhalation study [Pauluhn 2010a] in 

order to examine the influence of dose rate on the 

rat lung responses (Section A.3.2). These findings 

are relevant to interpreting and using the results 

from the short-term exposure studies of the SWCNT and other MWCNT.

A.2.1.3 Lung Responses Evaluated

The lungs are the target organ for adverse effects as 

shown in animal studies of CNT (Sections 3 and 4). 

Granulomatous inflammation, alveolar interstitial 

thickening, and pulmonary fibrosis are among the 

benchmark responses evaluated in this risk assessment (Table A–1). These responses are considered 

to be relevant to workers since inflammatory and 

fibrotic lung diseases have been associated with 

occupational exposure to various types of inhaled 

particles and fibers [Rom and Markowitz 2006]. 

These pulmonary inflammation and fibrotic effects 

in animals were observed at relatively early stages, 

although they developed earlier in mice exposed to 

SWCNT than from exposure to crystalline silica, 

which is a known fibrogenic particle [Shvedova et 

al. 2005; Lam et al. 2004]. 

The most quantitative measure of fibrosis was reported by the studies that measured the thickening 

of the gas-exchange region of the lungs (alveolar 

interstitial or septal connective tissue) due to increased collagen (as observed by lung tissue staining in histopathology examination) [Mercer et al. 

2010, 2011; Shvedova et al. 2005, 2008; Hubbs et 

al. 2011]. This alveolar thickening was observed to 

progress with time after administration of a single 

dose in mice administered by PA [Shvedova et al. 

2005; Mercer et al. 2008; Porter et al. 2010; Mercer et al. 2011]. Alveolar thickening was also observed in a subchronic study, which persisted up 

to 6 months after the end of exposure in a 13-wk 

inhalation study in rats [Pauluhn 2010a]. Alveolar 

interstitial (epithelial cell) thickness has been used 

as the adverse response in other risk assessment (of 

ozone) because it indicates “fundamental structural 

remodeling” [US EPA 1996; Stockstill et al. 1995].

Alveolar interstitial fibrosis can be detected by 

Sirius red staining of septal collagen [Hubbs et al. 

2011]. Interstitial thickening with fibrosis has been 

demonstrated by Sirius red staining of lungs from 

mice exposed to SWCNT or MWCNT [Shvedova 

et al. 2005, 2008; Mercer et al. 2011]. In SWCNT 

exposed mice, the septal fibrosis has been further 

confirmed by transmission electron microscopy 

[Mercer et al. 2008]. Pauluhn [2010a] also reported 

alveolar interstitial thickening in rats exposed to 

MWCNT, but distinguished the focal effects observed at 0.4 mg/m3 from those at higher exposures. 

That is, Pauluhn [2010a] reported: “Increased interstitial collagen staining (Sirius red) occurred at 

1.5 and 6 mg/m3. Focal areas of increased collagen 

staining were adjacent to sites of increased particle 

deposition and inflammatory infiltrates (onset at 

0.4 mg/m3, see Table 3). Increased septal collagen 

staining was depicted as equal to interstitial fibrosis (for details, see Fig 12).” In that study, a severity 

level of minimal (category 1) or greater persisted 

or progressed up to 26 weeks after the end of the 

13-week inhalation exposure to either 0.4, 1.5, or 6 

mg/m3 [Pauluhn 2010a, Table 3]. Hypercellularity 

in the bronchial alveolar junctions was observed in 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 99

these same dose groups; this effect persisted after 

the end of exposure, but resolved by the 39th week 

in the 0.4 mg/m3 group. The 0.4 mg/m3 dose group 

was considered the LOAEL for inflammatory lung 

effects, while 0.1 mg/m3 was considered the NOAEL [Pauluhn 2010a]. Concerning the focal septal 

thickening observed at 0.4 mg/m3, pathologists’ interpretations may differ as to whether these earlystage responses would be considered adverse or 

to have the potential to become adverse. NIOSH 

interpreted the alveolar septal thickening (and associated effects) in the 0.4 mg/m3 and higher dose 

groups as being adverse changes of relevance to human health risk assessment due to their persistence 

and consistency with the early-stage changes in the 

development of pulmonary fibrosis. For these reasons, the alveolar septal thickening of minimal or 

higher grade (i.e., proportion of rats with this response, which included rats exposed at 0.4 mg/m3

and higher doses) was selected as the benchmark 

response in the Pauluhn [2010a] study. Although 

these data were reported as the average histopathology score in each dose group [Pauluhn 2010a, 

Table 3], NIOSH requested the response proportion data as these were needed for the dichotomous 

BMD modeling. These data were provided by Dr. 

Pauluhn in response to this request [personal communication, J. Pauluhn and E. Kuempel, 1/27/10].

Pulmonary inflammation has been associated with 

exposure to airborne particles and fibers, and it is a 

hallmark of occupational lung disease in humans. It 

is also a precursor to particle-associated lung cancer in rats [IARC 2010; NIOSH 2011a]. Pulmonary 

inflammation can be measured by the increase in 

polymorphonuclear leukocytes (PMNs) in bronchoalveolar lavage (BAL) fluid following exposure 

to various particles including CNT. However, for 

some CNT, the inflammation resolves, while the 

fibrosis continues to develop [Shvedova et al. 2005, 

2008; Mercer et al. 2010; Pauluhn 2010a]. This indicates that neutrophilic inflammation in BAL fluid 

may not be a good predictor of adverse lung effects 

from some CNT, which appear to cause fibrosis 

by a different mechanism than for other types of 

particles and fibers (by resembling the lung basement membrane and serving as a framework for 

fibroblast cell growth, without eliciting a persistent 

inflammatory response) [Wang et al. 2010b]. In 

other studies, the inflammatory effects of MWCNT 

were associated with granuloma development [MaHock et al. 2009] and with alveolar lipoproteinosis, 

a more severe inflammatory lung response, observed at higher doses of MWCNT [Ma-Hock et 

al. 2009]. 

Minimal or higher levels of severity of these lung responses were selected as the benchmark responses. 

This included minimal level (grade 1 or higher) of 

pulmonary inflammation [Ma-Hock et al. 2009] 

or alveolar septal thickening [Pauluhn 2010a] as 

observed by histopathology. The incidence data on 

the minimal level of effect that is persistent provides 

a sensitive measure of a critical effect, which is of 

interest for health risk assessment. It is not known 

whether the human-equivalent effects to those observed in the animal studies would be associated 

with abnormal lung function or clinical disease, or 

if progression to more severe levels could occur if 

these effects developed as a result of chronic exposure. To evaluate sensitivity of risk estimates to the 

selection of a minimal level of disease, risk estimates 

were also derived for the next level of response 

(grade 2 or higher) in the subchronic animal studies.

The lung response measures in this risk assessment are either dichotomous (proportion of animals observed with the response endpoint) or continuous (amount or level of response in individual 

animals) (Table A–1). The dichotomous responses 

include the incidence of lung granulomas [Lam et 

al. 2004]; granulomatous inflammation [Ma-Hock 

et al. 2009]; and histopathology grade of alveolar 

interstitial (septal) thickening [Pauluhn 2010a]. 

The continuous responses include the amount of 

hydroxyproline (as mass) [Muller et al. 2005] and 

alveolar interstitial connective tissue thickness 

[Shvedova et al. 2005, 2008; Mercer et al. 2011]. 

A.2.1.4 Summary of

Dose-response Data

Collectively, the data available for CNT risk assessment include dose-response data from several 

rodent species and strains, both males and females, 
100 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

and three routes of exposure to several types of 

SWCNT and MWCNT with varying types and 

amounts of metal contaminants (Table A–1). The 

dose metric used in this risk assessment is the mass 

dose of CNT in the lungs, either the administered 

dose (IT or PA studies) or the lung burden (deposited or retained) estimated from the airborne particle 

size distribution and exposure concentration data 

(inhalation studies). Mass dose was used because all 

of the studies reported this dose metric and because 

mass dose was associated with the inflammatory 

and fibrotic lung responses in the animal studies. 

A.2.2 Estimated Lung Dose

in Animals

For the IT and PA studies [Lam et al. 2004; Shvedova et al. 2005; Muller et al. 2005], the administered CNT mass dose was assumed to be equivalent to the deposited lung dose. In the inhalation 

studies [Shvedova et al. 2008; Ma-Hock et al. 2009; 

Ellinger-Ziegelbauer and Pauluhn 2009; Pauluhn 

2010a], the deposited lung dose was estimated 

from the exposure concentration and duration, the 

species-specific ventilation rate, and the alveolar 

deposition fraction (estimated from the CNT aerodynamic particle size data), as follows:

Equation A–1:

Deposited lung dose (μg) = 

Exposure Concentration (μg/m3) × Duration (hr/d × d/wk × wk) 

× Minute Ventilation (L/min) × 0.001 m3/L × 60 min/hr 

× Alveolar Deposition Fraction 

The exposure concentration and duration, as reported in the animal studies, are shown in Table 

A–1. The values used for respiratory minute ventilation were based on the species and body weight: 

0.037 L/min for mice [US EPA 1988; 2006]; 0.25 L/

min for male rats in Pauluhn [2010] (369 g body 

weight); and 0.21 L/min for male and female rats 

in MaHock et al. [2009], assuming average body 

weight of 300 g [US EPA 1994, 2006]. The alveolar 

lung deposition fraction in rats was estimated from 

the MPPD 2.0 model for inhaled poorly soluble 

spherical particles [CIIT and RIVM 2006] using 

the mass-median aerodynamic diameter (MMAD) 

and geometric standard deviation (GSD) data reported for SWCNT and MWCNT (Table A–2). 

In the mouse inhalation study [Shvedova et al. 

2008], an alveolar deposition fraction of 0.01 was 

estimated based on the MMAD (Table A–2) and 

interpolating from the deposition fractions for 

monodisperse spherical particles reported in Table 

2 of Raabe et al. [1988]. For the two subchronic 

inhalation studies of MWCNT [Ma-Hock et al. 

2009; Pauluhn 2010a], in addition to the deposited 

dose, the retained lung dose was also estimated. 

The MPPD 2.0 model [CIIT and RIVM 2006] was 

used to estimate the lung burden at the end of the 

13-week exposure based on the particle MMAD 

and GSD (Table A–2) reported in those studies, assuming unit density (the lowest density accepted 

by MPPD 2.0). However, Ma-Hock et al. [2009] 

reported the MWCNT particle density of 0.043 g/

ml, and Pauluhn [2010a] reported the MWCNT 

particle density of 0.1–0.3 g/ml. The sensitivity of 

the lung dose estimates to the assumption of density 1 or lower is evaluated in Section A.6.1.1. Also 

evaluated is the effect of MPPD model version 2.0 

or 2.1 on the lung dose estimates. A recent update 

of MPPD 2.0 [CIIT and RIVM 2007] to MPPD 2.1 

[ARA 2011] included revised estimates of the rat 

head/extra thoracic deposition efficiency based on 

the equations in Raabe et al. [1988], which resulted 

in lower predicted deposition fractions in the rat 

pulmonary region [personal communication, O. 

Price and E. Kuempel, 9/24/10] (Section A.6.1.1). 

These lung dosimetry models have not been evaluated for CNT. However, the measured aerodynamic 

diameter is considered to provide a reasonable 

estimate of the deposition efficiency in the respiratory tract for CNT which have MMAD in the 

micrometer size range (Table A–2) (see Section 

A.6.1.1 for further discussion). The estimates of 

CNT clearance and retention in the lungs may be 

more uncertain than those for deposition fraction, 

given the slower clearance reported for CNT in 

some animal studies [Mercer et al. 2009; Pauluhn 

2010a,b]. Reasonable bounds on the uncertainty of 

the CNT lung dose estimates are considered to be 
NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers 101

Table A–1. Rodent study information

Rodent study

CNT type and 

main metal 

component 

Species, 

strain, gender

Route of 

exposure*

Number of 

animals per 

dose group

Dose

(& exposure 

duration, if 

inhalation)

Postexposure 

days Lung response

Lam et al. [2004] SWCNT

Fe 2.0%

Mouse, B6C3F1, male

IT 5 0, 0.1, or 0.5 mg 90 Granuloma†

Shvedova et al. [2005] SWCNT

Fe 0.2%

Mouse, C57BL/6, female

PA 6 (28 d)

3 (60 d)

0, 10, 20, 40 μg 1, 3, 7, 28, 

60

Alveolar connective tissue thickness‡

Muller et al. [2005] MWCNT Al 2%, 

Co 0.5%, Fe 0.5%

Rat, SpragueDawley, female IT 5 0, 0.5, 2, 5 mg 28 and 60 Hydroxyproline amount‡

Shvedova et al. [2008] SWCNT

Fe 18%

Mouse, C57BL/6, female

Inhal 5 5 mg/m3

(5 hr/d, 4 d)

1, 7, 28 (after 4 d exposure)

Alveolar connective tissue thickness‡

Ma-Hock et al. [2009] MWCNT Al O2 3

9.6%

Rat, Wistar (Crl:WI), male and female

Inhal 20

(10 each gender)

0, 0.1, 0.5,2.5 mg/m3(6 hr/d, 5 d/wk, 13 wk)

1 Granulomatous 

inflammation (minimal or greater)†

Pauluhn [2010] MWCNT Co 

0.5%

Rat, Wistar (HsdCpb: WU), male

Inhal 10 0, 0.10, 0.45, 

1.62, 5.98 mg/m3(6 hr/d, 5 d/wk, 13 wk)

1, 28, 91, 182 

Alveolar septal thickening (minimal or greater)†

Mercer et al. [2011] MWCNT Fe 

0.3%

Mouse, C57BL/6J, male

PA 6 0, 10, 20, 40, 

80 μg

1, 7, 28, 56 Alveolar connective 

tissue thickness‡

*Intratracheal instillation (IT); pharyngeal aspiration (PA); inhalation (inhal).†Dichotomous response.‡Continuous response.
102 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table A–2. CNT particle size and alveolar deposition fraction in rodent and human

Study Particle size information

Human DFalv*

and MMAD 

(GSD) used

Rodent DFalv 

(same MMAD 

and GSD)

Lam et al. [2004] Not reported; same SWCNT source as Shvedova 

et al. [2005]. (assume same MMAD [GSD] as 

Shvedova et al. [2008])

0.076

3.5 (2.14)

ad¶

Shvedova et al. [2005] 1–4 nm width (primary particles) (assume same 

MMAD [GSD] as Shvedova et al. [2008])

0.076

3.5 (2.14)

ad

Muller et al. [2005] 9.7 nm width; 5.9 μm length (primary particles) 

(assume same MMAD [GSD] as Ma-Hock et al. 

[2009])

0.099

1.2 (2.7)

ad

Shvedova et al. [2008] 0.8–1.2 nm width; 100–1000 nm length (primary 

particles); 4.2 μm mass mode diameter; 240 nm 

count mode diameter; 3.5 μm MMAD (2.14 GSD)§

0.076

3.5 (2.14)

0.01‡

Ma-Hock et al. [2009] 1.5 (3.6); 1.2 (2.7); 0.8 (2.8) μm MMAD (GSD) at 

0.1, 0.5, and 2.5 mg/m3, respectively; (median of 3 

values at each concentration). Primary particles: 

5–15 nm width; 0.1–10 μm length

0.099

1.2 (2.7)

0.072§

Pauluhn [2010a] 3.05 (1.98); 2.74 (2.11); 3.42 (2.14) μm MMAD 

(GSD) at 0.4, 1.5, and 6 mg/m3, respectively. Primary 

particles: ~10 nm width; 200–1,000 nm length

0.086

2.74 (2.11)

0.046§

Mercer et al. 2011 1.5 μm MMAD from Porter et al. [2010] (GSD not 

reported; assume 2); count mean width (49 nm; 13.4 

SD); median length 3.86 μm (1.94 GSD) 

0.10

1.5 (2)

ad¶

*

MPPD 2.0 human; Yeh and Schum deposition model; 9.6 m3/8 hr d (20 L/min, or 1143 ml tidal volume at 17.5 breaths/min); inhalability adjustment; assumed unit density.

†

MMAD and GSD in Shvedova et al. [2008] were estimated from data reported in Baron et al. [2008] [personal communication 

from B. Chen to E. Kuempel, August 4, 2009].

‡

Raabe et al. [1988]: mouse DFalv interpolated from values in Table 2 of Raabe et al. [1988]. §

MPPD 2.0 rat; 0.21 L/min or 2.45 ml tidal volume (assuming 300 g male and female rats) [Ma-Hock et al. 2009]; and 0.25 L/min 

or 2.45 ml tidal volume (369 g male rats) [Pauluhn 2010; US EPA 1994; 2006]; inhalability adjustment; assumed unit density.

¶

ad—administered dose by intratracheal instillation or pharyngeal aspiration.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 103

the deposited (no clearance) and the retained (normal clearance) doses predicted from the sphericalparticle based models [MPPD, CIIT and RIVM 

2007, ARA 2011]. This is because the CNT deposited in the lungs may undergo some clearance, although evidence from animal studies suggests the 

clearance rate may be slower than for other poorly 

soluble particles at relatively low-mass doses in the 

rats and mice [Deng et al. 2007; Mercer et al. 2009; 

Pauluhn 2010a; 2011; Elgrabli et al. 2008b]. 

A.2.3 Animal Dose-response 

Modeling and BMD 

Estimation

The dose-response data in rats and mice exposed 

to SWCNT or MWCNT were modeled using 

benchmark dose methods [Crump 1984; US EPA 

2010]. A benchmark dose has been defined as “. . . 

a statistical lower confidence limit for the dose 

corresponding to a specified increase in level of 

[adverse] health effect over the background level” 

[Crump 1984]. The increased level of adverse effect 

(called a benchmark response, or BMR) associated 

with a BMD is typically in the low region of the 

dose-response data (e.g., a 10% excess risk). In this 

document, the term BMD is used to describe the 

point estimate based on maximum likelihood estimation, and the term BMDL is used to describe the 

lower 95% confidence limit (i.e., as originally defined by Crump [1984]). A 10% excess risk, based 

on dichotomous or quantal data, is used because 

it is at or near the limit of sensitivity in the animal 

bioassay [US EPA 2012; Crump 2002]. The BMDL 

associated with a 10% BMR is used as a point of 

departure (POD) for low-dose extrapolation using linear or nonlinear methods (depending on 

the mode of action evidence) [US EPA 2012]. The 

low-dose extrapolation may include estimation of 

the probability of effects at low doses or below a reference value (not risk-based) by accounting for uncertainties in the dose estimation (e.g., extrapolation 

from animal to human, inter-individual variability, 

limitations in the animal data [US EPA 2012]. 

A.2.3.1 Dichotomous Response Data

For dichotomous data (yes/no response), a BMD 

is defined as the dose associated with a specified 

increase in the probability of a given response, either as an excess risk (i.e., additional probability 

above background) or as a relative risk (i.e., relative 

to the background probability of having a normal 

response) [Crump 2002].

In this analysis, the BMD (using dichotomous data) is 

the dose d corresponding to a specified excess (added) risk (e.g., 10%) in the proportion of animals with 

a given adverse lung response (BMR), as follows: 

Equation A–2:

BMR = P(d) – P(0)

where P(d) is the probability of an adverse response at the BMD, and P(0) is the probability of 

that adverse response in an unexposed population 

[Crump 2002; US EPA 2010]. 

The dichotomous BMR lung responses include the 

presence or absence of granulomatous inflammation [Ma-Hock et al. 2009] or alveolar septal thickening [Pauluhn 2010a] (Table A–1). The proportion of animals responding with the minimal or 

higher severity was selected as the benchmark response. The BMD(L) estimates are expressed as the 

mass dose of SWCNT or MWCNT in rodent lungs 

associated with the specified BMR. These animalbased BMD(L)s are extrapolated to humans based 

on species-specific differences in the estimated deposition and retention of CNTs in the lungs (Section A.2.4).

A.2.3.2 Continuous Response Data

BMD estimation using continuous data requires 

specifying a BMR level along the continuum of responses. Continuous response data provide information on the amount or degree of a biological 

response. Continuous response measures may include nonzero levels that are associated with the 

normal structure or function (e.g., a certain number immune cells or amount of protein in healthy 

lungs). These levels can become elevated in response 
104 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

to a toxicant, and at some point, they may result 

in irreversible, functional impairment of the lungs 

[NIOSH 1986]. If data are available, the BMRs can 

be based on a biologically significant response that 

is associated with, or expected to result in, a material impairment of health. However, there may be 

insufficient data to determine a specific level that 

is associated with a measurable adverse response. 

In that case, a statistical criterion may be used as a 

BMR for continuous data [Crump 1995].

A statistical method (originally referred to as a 

“hybrid” method) is described by Crump [1995] to 

provide BMD(L) estimates from continuous data 

that are equivalent to a 10% excess risk based on 

dichotomous data, assuming that an abnormal or 

biologically significant response is defined as the 

upper 99th percentile of the unexposed (control) 

distribution. According to this method, “for a normal distribution with constant variance, setting 

BMR = 0.1 and PO = 0.01 is equivalent to choosing the BMD to be the dose that results in an increase in the mean equal to 1.1 times the standard 

deviation,” assuming a normal distribution with 

constant variance [Crump 1995]. That is, if one assumes that the probability of the specified adverse 

response in the unexposed population is the upper 

1% of a normal distribution of responses, then selecting a BMR of 1.1 standard deviations above the 

control mean response is equivalent to a 10% BMD 

as estimated in dichotomous data. 

In evaluating possible BMRs for the continuous 

data of CNT in mice, earlier studies of chronic 

ozone exposure in rats were examined to determine if a biologically-based BMR could be identified for pulmonary fibrosis (measured as alveolar 

connective tissue thickening) associated with abnormal pulmonary function [Chang et al. 1992; 

Costa et al. 1995; Stockstill et al. 1995]. However, 

those rat findings did not appear to extrapolate well 

to the mice in Shvedova et al. [2005, 2008]. That 

is, the observed abnormal response in rats (associated with a persistent lung function deficit) was a 

36% increase in the control mean alveolar connective tissue thickness [Chang et al. 1992; Costa et al. 

1995]; however, this amount of response occurred 

in up to 30% of the control (unexposed) mice in 

Shvedova et al. [2005, 2008] (vs. 2.5% of controls 

in Chang et al. [1992], in part due to the greater 

variability in the alveolar tissue thickness in the 

unexposed mice. In addition, no data were found 

of a biologically relevant BMR for the amount of 

hydroxyproline in the lungs of rats or mice. In the 

absence of an identified biological basis for a BMR 

for the continuous response measures of alveolar 

connective tissue thickening or the amount of hydroxyproline, NIOSH used the statistical criterion 

described by Crump [1995], in which a BMR of 

1.1 standard deviations above the control mean 

response is equivalent to a 10% excess risk in the 

dichotomous data, assuming the 99th percentile of 

the distribution of control responses is abnormal or 

biologically significant.

That is, the BMR for the continuous data (alveolar connective tissue thickness and hydroxyproline 

amount) is defined as follows:

Equation A–3:

BMR = μ(d) – μ(0) 

where μ(d) is the mean response at the BMD (d); 

μ(0) is the control mean response; and BMR is the 

specified number of standard deviations (SDs) (i.e., 

1.1 in these analyses). Thus, the continuous databased BMD is the dose associated with a 10% increase in the proportion of animals exposed at dose 

d with response greater than the 99th percentile of 

the control mean response. The estimates of μ(d)

and μ(0) are derived from the fitted dose-response 

models (polynomial) (Section A.2.3.3). 

A.2.3.3 BMD Model Fitting

The animal dose-response data were fit using the 

benchmark modeling software (BMDS 2.1.2) [US 

EPA 2010]. The dichotomous data were fit with a 

multistage (polynomial degree 2) model. This is the 

only model that provided an adequate fit to the subchronic inhalation data, each of which [Ma-Hock et 

al. 2009; Pauluhn 2010] had only one dose between 

zero and 100% response for the endpoints evaluated (granulomatous inflammation or alveolar septal thickening, histopathology grade 1 or higher). 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 105

The other BMDS models failed to converge or, in 

further statistical evaluation, showed non-unique 

parameter solutions. The continuous dose-response 

data were fit with a polynomial model of degree 2 

for all data with three or more dose groups, and degree 1 (linear) for data with two groups (see Table 

A–1 for dose groups). 

P values for goodness of fit were computed for the 

individual BMDS models (based on likelihood 

methods) [US EPA 2007]. Model fit was considered adequate at P > 0.05 (i.e., testing for lack of 

fit), although the P values based on likelihood ratio tests may not be a reliable indicator of model 

fit in the studies with few animals per group. The 

number of animals per dose group in each study 

is given in Table A–1. EPA typically uses a P > 0.1 

criteria for BMD model fit [US EPA 2012]. Either 

criteria is considered reasonable and represents 

a trade-off in the type I or type II error. That is, 

P  >  0.1 provides more power to reject an incorrect model, while P > 0.05 provides less chance of 

rejecting a correct model. The BMD model fits to 

each data set are shown in Figure A–1 (subchronic 

studies), Figure A–2 (short-term studies, dichotomous response), and Figure A–3 (short-term studies, continuous response). 

A.2.3.4 Human-equivalent Dose and 

Working Lifetime Exposure

The rodent BMD(L)s were extrapolated to humans 

based on species-specific differences in the alveolar 

epithelial surface area of the lungs (i.e., by normalizing the dose per unit of cell surface area). It is assumed that humans and animals would have equal 

response to an equivalent dose (i.e., mass of CNT 

per unit surface area of lungs). The human-equivalent 

BMD and BMDL estimates were the target lung 

doses used to estimate, respectively, the maximum 

likelihood estimate (MLE) and 95% lower confidence limit (95% LCL) estimate of the MLE, as 

an 8-hr TWA exposure concentration during a 45-

year working lifetime. 

The human-equivalent BMD and BMDL estimates 

were calculated as follows: 

Equation A–4:

Human-equivalent BMD(L) =

Rodent BMD(L) × [AlvSA human / AlvSA rodent] 

where the values used for alveolar lung surface area 

(AlvSA) were 102 m2 (human) [Stone et al. 1992]; 

0.4 m2 (rat) and 0.055 m2 (mouse) [Mercer et al. 

2008]. In Tables A–3 through A–5, the humanequivalent BMD(L)s were multiplied by 0.001 mg/

μg to obtain the units of mg per lung.

The human-equivalent BMD(L)s are expressed as 

the mass (mg) of CNT in the lungs. The working 

lifetime airborne mass concentration that would 

result in the BMD(L) human-equivalent lung mass 

dose was calculated based on either deposition 

only (no lung clearance) or retention (lung deposition and clearance), as described below. 

(a) Deposited lung dose

Equation A–5:

Estimated 8-hr TWA (μg/m3) = 

Human-equivalent BMD(L) (μg) /

[8-hr worker air inhaled (m3/day) × Alveolar Deposition

Fraction × Work Days]

The values assumed include 9.6 m3 8-hr air intake 

(reference worker [ICRP 1994]); alveolar deposition fraction based on aerodynamic particle size 

(Table A–2); and working lifetime days (250 days/

yr × 45 yr). 

(b) Retained lung dose

The MPPD 2.0 human model [CIIT and RIVM 

2006] for inhaled poorly soluble spherical particles 

was used to estimate the working lifetime exposure 

concentration that would result in the humanequivalent BMD(L) lung burden estimates. This was 

done by a systematic search to identify the 8-hr time 

weighted average (TWA) airborne concentration 

over a 45-year working lifetime that predicted the 

target lung burden. The input parameters used in 

the MPPD human model (Yeh and Schum human 

deposition model option) include CNT aerodynamic particle size (MMAD, GSD) (Table A–2); inhalability adjustment; oronasal-normal augmenter; 
106 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Figure A–1. Benchmark dose model (multistage, polynomial degree 2) fit to rodent dose-response data 

from the two subchronic inhalation studies of MWCNT in rats: Ma-Hock et al. [2009], response: granulomatous inflammation, Pauluhn [2010a], response: alveolar septal thickening, minimal or greater. P values 

are 0.99 for Ma-Hock et al. [2009] and 0.88 for Pauluhn [2010a].
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 107

Figure A–2. Benchmark dose model (multistage, polynomial degree 2) fit to rodent dose-response data 

from short-term studies with dichotomous response: Lam et al. [2004] (SWCNT, mouse, intratracheal instillation; response: lung granulomas) (P value is 0.35).
108 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Figure A–3. Benchmark dose model fit to rodent dose-response data from short-term studies with continuous response: Shvedova et al. [2005] (SWCNT, mouse, pharyngeal aspiration; response: alveolar connective 

tissue thickening) (P value is 0.23) (polynomial model, degree 2, coefficients restricted to non-negative—fit 

to all data except top dose group due to nonhomogeneous variance); and Shvedova et al. [2008] (SWCNT, 

mouse, inhalation; response: alveolar connective tissue thickening) (P value is not applicable, linear). Benchmark response level: 1.1 standard deviations about the control mean response. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 109

Figure A–3 (continued). Benchmark dose model fit to rodent dose-response data from short-term studies 

with continuous response: Muller et al. [2005] (MWCNT, rat, intratracheal instillation; response: hydroxyproline amount) (P value is 0.67); Mercer et al. [2011] (MWCNT, mouse, pharyngeal aspiration response: 

alveolar connective tissue thickening) (P value is 0.35). Benchmark response level: 1.1 standard deviations 

about the control mean response. 
110 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

and reference worker conditions, including 9.6 m3

of air inhaled per 8-hr day (corresponding to 17.5 

breaths/min and tidal volume of 1143 ml), and 

work for 8 hr/d, 5 d/wk, 50 wk/yr, for 45 years. 

In the two subchronic inhalation studies for MWCNT, excess risk estimates were derived based on 

either the estimated deposited lung dose or the estimated retained lung dose [Ma-Hock et al. 2009; 

Pauluhn 2010a]. 

A.3 Results

A.3.1 Benchmark Dose and 

Working Lifetime 

Exposure Estimates

The estimates of the rodent BMD(L)s, the humanequivalent BMD(L)s, and the associated working 

lifetime 8-hr TWA exposure concentrations (MLE 

and 95% LCL)—called the benchmark concentration (BMC) and the BMCL (95% LCL of the 

BMC)—are shown in Tables A–3 through A–5. 

All dose-response models used in this risk assessment provided adequate fit (P > 0.05) to the rodent data for BMD(L) estimation (P values for the 

Pearson X2 goodness of fit test shown in Tables 

A–3 through A–5). 

In Table A–3, the BMD(L) and BMC(L)* estimates 

are based on the IT, PA, or short-term inhalation 

exposure studies of SWCNT or MWCNT with 

continuous response measures. Lung responses in 

rodents were evaluated at 32 to 60 days after first 

exposure. Rodent dose is the administered (IT or 

PA) or estimated deposited dose (inhalation). The 

BMR is the specified adverse lung response at 1.1 

standard deviations above the estimated rodent 

control mean response (i.e., alveolar connective 

tissue thickness or amount of hydroxyproline) (as 

explained in Section A.2.3.2). Considerably higher 8-hr TWA concentrations are estimated based 

on the endpoint of lung hydroyxproline amount 

[Muller et al. 2005] compared with those based on 

*Abbreviation for both BMC and BMCL estimates.

the alveolar connective tissue thickness endpoint, 

which is a more sensitive (earlier) indicator of fibrosis [Mercer et al. 2008].

In Table A–4, the BMD(L) and BMC(L) estimates 

are based on the IT exposure study of SWCNT with 

dichotomous response measures. Lung responses 

were evaluated 90 days after the first exposure. The 

BMR is the 10% excess risk of the specified adverse 

lung response (proportion of rats with lung granulomas). Although Lam et al. [2005] report doseresponse data for three different preparations of 

SWCNT (containing either 2% Fe, 27% Fe, or 26% 

Ni), the BMD(L) and BMC(L) estimates are provided only for the SWCNT with 2% Fe, which was 

the only dataset of the three reported by Lam et al. 

[2005] that was adequately fit by the BMD model 

(Table A–4). 

Table A–5 provides the BMD(L) and BMC(L) estimates based on the two subchronic inhalation studies of MWCNTs, which also report dichotomous 

response measures. Lung responses were evaluated 

at the end of the 13-week (91 d) exposure period. 

Rodent dose is either the total deposited lung dose 

or the retained lung dose at the end of exposure. 

The BMR is estimated as the 10% excess risk of the 

specified adverse lung response (granulomatous 

inflammation or alveolar septal thickening of histopathology grade 1 or higher). As expected, the estimates based on deposited lung dose are lower than 

those based on the retained lung dose, because the 

assumption of no clearance in the deposited lung 

dose results in a lower estimated 8-hr TWA concentration to attain the human-equivalent BMD(L) 

lung burdens. The estimates for MWCNT (with 

9.6% Al2O3) based on the rat granulomatous inflammation response are lower than those for MWCNT (Baytubes) (with 0.53% Co) based on the rat 

alveolar septal thickening response. 

Table A–6 shows the animal and human BMD(L) 

estimates and equivalent working lifetime 8-hr 

TWA concentration estimates, BMC(L), associated 

with grade 2 (slight/mild) or higher lung responses 

in the subchronic inhalation studies, based on the 

estimated deposited lung dose. As expected, higher 

BMD(L)s and BMC(L)s are estimated from the 
NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers 111

Table A–3. Benchmark dose estimates* and associated human working lifetime airborne concentrations—continuous response data in rats or mice exposed to SWCNT or MWCNT by IT, PA,† or short-term inhalation (dose metric: administered or estimated deposited lung dose)

Rodent study, CNT type, and response

Rodent Human

Human working 

lifetime airborne 

concentration‡

(μg/m3)

BMD§, ¶

(μg/lung)

BMDL

(μg/lung)

BMD

(mg/lung)

BMDL 

(mg/lung) BMC BMCL

IT or PA 

Muller et al. [2005]—MWCNT (2% Al)Hydroxyproline amount (at 60 d) in rats

760 486 194 124 18 12

Shvedova et al. [2005]—SWCNT (0.2% Fe)Alveolar connective tissue thickness (at 60 d) in mice

7.8 6.5 14 12 1.8 1.5

Mercer et al. [2011]—MWCNT (0.3% Fe)Alveolar connective tissue thickness (at 60 d) in mice

27.1 14.1 50 27 4.7 2.5

Inhalation (5 hr/d, 4 d)

Shvedova et al. [2008]—SWCNT (18% Fe)Alveolar connective tissue thickness (at 32 d) in mice

0.48 0.33 0.89 0.62 0.11 0.075

*Benchmark response level: 1.1 standard deviations above estimated control mean response [Crump 1995; US EPA [2010]; associated with a 10% increase in abnormal 

response (assumed greater than the 99th percentile of the distribution of control responses).

†IT—intratracheal instillation [Muller et al. 2005]; PA—pharyngeal aspiration [Shvedova et al. 2005] and [Mercer et al. 2011].

‡8-hr time weighted average (TWA) concentration associated with the human-equivalent BMD(L)s; BMC: maximum likelihood estimate of the benchmark concentration. 

BMCL: 95% lower confidence limit of the BMC.

§BMD: estimated benchmark dose (maximum likelihood estimate). BMDL: estimated 95% lower confidence limit of the BMD; polynomial (degree 2) model [US EPA 2010].¶P values for the rodent dose response models: 0.67 for Muller et al. [2005], 0.23 for Shvedova et al. [2005], 0.41 for Mercer et al. [2011], and not applicable (linear) for Shvedova et al. [2008].
112 NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers

Table A–4. Benchmark dose estimates* and associated human working lifetime airborne concentrations—dichotomous response data in rats or mice exposed to SWCNT by intratracheal instillation (dose metric: administered lung dose)

Rodent study, CNT type, and response

Rodent Human

Human working lifetime 

airborne concentration

(μg/m3) †

BMD‡, §

(μg/lung)

BMDL

(μg/lung)

BMD

(mg/lung)

BMDL

(mg/lung) BMC BMCL

Lam et al. [2004]— SWCNT (2% Fe)Granuloma (at 90 d) in mice

45 7.6 84 14 10 1.7

*Benchmark response level—10% excess (added) risk in exposed animal [US EPA 2010]. †8-hr time weighted average (TWA) concentration associated with the human-equivalent BMD(L)s. BMC: maximum likelihood estimate of the benchmark concentration. 

BMCL: 95% lower confidence limit of BMC. 

‡BMD: estimated benchmark dose (maximum likelihood estimate). BMDL: estimated 95% lower confidence limit of the BMD; multistage (polynomial degree 2) model 

[US EPA 2010].

§P value: 1.0 
NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers 113

Table A–5. Benchmark dose estimates and associated working lifetime airborne concentrations—grade 1 or higher severity of lung responses in rats after subchronic inhalation of MWCNT (dose metric: estimated deposited or retained dose in lungs)

Rodent study and response*

Rodent Human

Human working lifetime 

airborne concentration‡

(μg/m3)

BMD†

(μg/lung)

BMDL

(μg/lung)

BMD

(mg/lung)

BMDL

(mg/lung) BMC BMCL

Deposited lung dose (assuming no clearance)

Ma-Hock et al. [2009]Granulomatous inflammation 

21 8.1 5.4 2.1 0.51 0.19

Pauluhn [2010a]Alveolar septal thickening 

28 14 7.2 3.5 0.77 0.38

Retained lung dose(assuming normal clearance)

Ma-Hock et al. [2009]Granulomatous inflammation 

11 3.8 2.7 0.97 2.7 1.0

Pauluhn [2010a]Alveolar septal thickening 

14 6.5 3.6 1.7 4.2 1.9

*Histopathology grade 1 (minimal) or higher severity. Benchmark response level—10% excess (added) risk in exposed animals.†BMD: estimated benchmark dose (maximum likelihood estimate). BMDL: estimated 95% lower confidence limit of the BMD; multistage (polynomial degree 2) [US EPA 

2010]. P values for the rodent dose-response models: 0.99 for Ma-Hock et al. [2009] and 0.88 for Pauluhn et al. [2010a] (deposited dose); 1.0 for Ma-Hock et al. [2009] and 0.94 for Pauluhn [2010a] (retained dose).

‡8-hr time weighted average (TWA) concentration associated with the human-equivalent BMD(L)s. BMC: maximum likelihood estimate of the benchmark concentration; 

BMCL: 95% lower confidence limit of the BMC. 
114 NIOSH CIB 65 

• Carbon Nanotubes and Nanofibers

Table A–6. Benchmark dose estimates and associated working lifetime airborne concentrations—grade 2 or higher severity of lung responses in rats after subchronic inhalation of MWCNT (dose metric: estimated deposited or retained dose in lungs)

Rodent study and response*

 Rodent Human 

Human working lifetime 

airborne concentration‡

(μg/m3)

BMD†

(μg/lung)

BMDL

(μg/lung)

BMD

(mg/lung)

BMDL

(mg/lung) BMC BMCL

Deposited lung dose

Ma-Hock et al. [2009]Granulomatous inflammation

44 29 11 7.4 1.0 0.69

Pauluhn [2010a]Alveolar septal thickening

235 120 60 31 6.4 3.3

Retained lung dose

Ma-Hock et al. [2009]Granulomatous inflammation

24 16 6.3 4.0 6.2 4.0

Pauluhn [2010a]Alveolar septal thickening

150 66 38 17 44 19

*Histopathology grade 2 is slight [Ma-Hock et al. 2009] or slight/mild [Pauluhn 2010a] severity (proportion of animals with that response). Benchmark response 

level—10% excess (added) risk in exposed animals. 

†BMD: estimated benchmark dose (maximum likelihood estimate). BMDL: estimated 95% lower confidence limit of the BMD; multistage (polynomial degree 2) [US EPA 

2010]. P values for the rodent dose-response models: 0.67 for Ma-Hock et al. [2009] and 0.98 for Pauluhn et al. [2010a] (deposited dose); 0.76 for Ma-Hock et al. [2009] and 0.99 for Pauluhn [2010a] (retained dose).

‡8-hr time weighted average (TWA) concentration associated with the human-equivalent BMD(L)s. BMC: maximum likelihood estimate of the benchmark concentration; 

BMCL: 95% lower confidence limit of the BMC.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 115

histopathology grade 2 or higher lung responses 

(Table A–6) compared with those estimated from 

histopathology grade 1 (minimal) or higher responses (Table A–5) because more animals developed the grade 1 or higher response at a given dose 

(i.e., histopathology grade 1 or higher is a more 

sensitive response).

A.3.2 Comparison of Shortterm and Subchronic

Dose-response Data

Two studies of MWCNT (Baytubes) provided an 

opportunity to examine the effect of dose-rate on 

the same lung response measured at the same time 

point. Wistar rats were exposed by inhalation for 

either one 6-hr day [Ellinger-Ziegelbauer and Pauluhn 2009] or 13 weeks (6 hr/d, 5 d/wk) [Pauluhn 

2010a]. Lung responses were examined in both 

studies at 13  weeks after the first exposure day. 

Histopathology severity scores for alveolar septal 

thickening were available for each study. The number of male rats with alveolar septal thickening (of 

minimal or higher grade) and the respective exposure concentrations are as follows: 

• Ellinger-Ziegelbauer and Pauluhn [2009]: 1, 

0, and 6 rats (6 total per group) at 0, 11.0, and 

241.3 mg/m3.

• Pauluhn [2010a, extended by personal communication]: 0, 0, 9, 10, 10 rats (10 total per 

group) at 0, 0.1, 0.45, 1.62, and 5.98 mg/m3.

The dose metric used for this comparison was the 

deposited lung dose, estimated from MPPD 2.0 

[CIIT and RIVM 2006] based on the particle size 

data (MMAD and GSD) and the rat exposure conditions reported in each study. 

To evaluate whether these data [Pauluhn 2010a; 

Ellinger-Ziegelbauer and Pauluhn 2009] can be 

described by the same dose-response relationship, 

a multistage (polynomial degree 2) model [US 

EPA 2010] was fit to the combined data (Figure 

A–4). This model provided adequate fit to the data 

(P = 0.37), suggesting that these data can be described by the same dose-response model using the 

estimated total deposited lung dose, regardless of 

the dose rate differences (i.e., dose administered in 

1 day vs. during 91 days). This finding is consistent 

with the impaired clearance and biopersistence of 

the deposited MWCNT in the rat lungs at these 

doses as shown in Pauluhn [2010a]. 

A.3.3 Estimated Excess Risks 

of Working Lifetime 

Exposures to Low-mass 

Concentrations of MWCNT

Standard risk assessment approaches for non-cancer endpoints have typically assumed a threshold 

model, with extrapolation beyond the point of departure based on uncertainty factors, e.g., US EPA 

[1994]. NRC [2009] and others have recommended 

using risk-based low-dose extrapolation for noncancer endpoints. NIOSH practice has also included risk-based low-dose extrapolation for noncancer endpoints. In the absence of information on 

the shape of the dose-response relationship in the 

low-dose region, assumptions can include linear 

and nonlinear model-based extrapolation. Linear 

extrapolation is the most protective (i.e., unlikely 

to underestimate the risk). However, the actual risk 

could be much lower, including zero.

Low-dose linear extrapolation of the working lifetime-equivalent 10% excess risk estimates in Table 

A–5 (deposited dose assumption) results in BMC 

(BMCL) estimates of 0.051 (0.019) μg/m3 or 0.077 

(0.038) μg/m3 associated with 1% excess risk (MaHock et al. [2009] or Pauluhn [2010a], respectively). The corresponding BMC (BMCL) estimates associated with 0.1% excess risk are 0.0051 (0.0019) 

μg/m3 or 0.0077 (0.0038) μg/m3. Multistage modelbased estimates are higher for the BMCs, but nearly 

identical for the BMCLs: 0.16 (0.019) μg/m3 or 0.24 

(0.042) μg/m3 associated with 1% excess risk; and 

0.050 (0.0020) μg/m3 or 0.075 (0.0042) μg/m3 associated with 0.1% excess risk (Ma-Hock et al. [2009] 

or Pauluhn [2010a], respectively). 
116 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table A–7. Working lifetime percent, excess risk estimates of low-mass concentrations of CNT

associated with minimal (grade 1) lung effects

Subchronic inhalation 

study in rats

Working lifetime 

8-hr TWA airborne 

concentration (μg/m3)

Working lifetime excess risk (%)*

Maximum likelihood 

estimate (MLE)

95% Upper 

confidence limit of 

MLE

Deposited lung burden (assumes no clearance)

Ma-Hock et al. [2009] 1 33 54

2 80 96

7 >99 >99

Pauluhn [2010] 1 16 30

2 50 72

7 >99 >99

Retained lung burden (assumes normal clearance)

Ma-Hock et al. [2009 1 3.7 10

2 7.4 20

7 49 73

Pauluhn [2010] 1 2.4 5.3

2 4.8 10

7 25 42

*

45-year working lifetime; estimated from multistage model (degree 2) [US EPA 2010] for exposures greater than 10% BMC(L) 

and by linear extrapolation from the 10% BMC(L) in Table A–5 for lower exposures.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 117

Table A–8. Working lifetime percent, excess risk estimates of low-mass concentrations of CNT 

associated with slight/mild (grade 2) lung effects

Subchronic inhalation 

study in rats

Working lifetime 

8-hr TWA airborne 

concentration (μg/m3)

Working lifetime excess risk (%)*

Maximum likelihood 

estimate (MLE)

95% Upper 

confidence limit of 

MLE

Deposited lung burden (assumes no clearance)

Ma-Hock et al. [2009] 1 10 16

2 31 44

7 99 >99

Pauluhn [2010] 1 1.6 3.0

2 3.1 6.1

7 12 24

Retained lung burden (assumes normal clearance)

Ma-Hock et al. [2009 1 1.6 2.5

2 3.2 5.0

7 12 21

Pauluhn [2010] 1 0.23 0.53

2 4.5 1.0

7 1.6 3.7

*

45-year working lifetime; estimated from multistage model (degree 2) [US EPA 2010] for exposures greater than 10% BMC(L) 

and by linear extrapolation from the 10% BMC(L) in Table A–6 for lower exposures.
118 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Tables A–7 and A–8 provide working lifetime excess risk estimates of early stage-lung effects (minimal or higher histopathology grade of granulomatous inflammation or alveolar septal thickening) 

associated with 1, 2, or 7  μg/m3 as an 8-hr TWA 

concentration. These concentrations were selected as 

possible limits of quantification (LOQs) that were 

under evaluation for the analytical method to measure airborne CNT in the workplace (NIOSH method 5040). These estimates are based on lung dose 

estimates assuming either total deposited lung dose 

(no clearance) or retained dose (normal, spherical 

particle-based clearance). Risk estimates are higher 

for the no clearance assumption than those assuming normal clearance, within either the minimal 

(grade 1) (Table A–7) or slight/mild (grade 2) (Table A–8) lung responses. These excess (exposureattributable) risk estimates were derived from the 

multistage (degree 2) model fit to the rat subchronic dose-response data, or by linear extrapolation 

below the 10% BMC(L) estimates shown in Tables 

A–5 and A–6. 

A.4 Discussion

NIOSH conducted a quantitative risk assessment of 

CNTs by evaluating dose-response data of early-stage 

adverse lung effects in rats and mice exposed to several types of SWCNT or MWCNT (with different metal 

contaminants), by several routes of exposure (inhalation, PA, or IT), and duration of exposure (single 

day or subchronic) and post-exposure period (up to 

26 weeks). Because of the different study designs and 

response endpoints used in the rodent studies, limited information was available to evaluate the extent 

to which the differences in the risk estimates across 

studies are due to differences in the CNT material or 

are attributable to other study differences. Some evidence indicates that CNT with certain metals (nickel, 

26%) [Lam et al. 2004] or with higher metal content 

(18% vs. 0.2% Fe) [Shvedova et al. 2008] are more 

toxic and fibrogenic. However, some studies have 

shown that both unpurified and purified (low metal 

content) CNT were associated with early-onset and 

persistent pulmonary fibrosis at relatively low-mass 

doses [Shvedova et al. 2005, 2008]. The LOAELs for 

MWCNT (containing either 9.6% Al2O2 or 0.5% Co) 

were 0.1 mg/m3 [Ma-Hock et al. 2009] and 0.4 mg/

m3 [Pauluhn 2010a], which are more than an order 

of magnitude lower than the LOAEL of 7 mg/m3 for 

ultrafine carbon black [Elder et al. 2005] in the same 

animal species and study design (13-week inhalation 

studies in rats, although with different strains, Wistar 

(male and female) [Pauluhn 2010a] and F-344 

(female) [Elder et al. 2005]). 

Because no chronic animal studies or epidemiological studies of workers producing or using CNT 

have been published to date, the best available data 

for risk assessment were the subchronic inhalation 

studies of MWCNT in rats [Ma-Hock et al. 2009; 

Pauluhn 2010a]. For SWCNT, no subchronic studies were available, and several short-term studies 

(IT, PA, or inhalation exposure) in rats or mice 

provide the only available dose-response data for 

either SWCNT [Lam et al. 2004; Shvedova et al. 

2005, 2008] or for other types of MWCNT (with 

different metal content) [Muller et al. 2005; Mercer 

et al. 2011] (Table A–1). 

All of these studies reported inflammatory, granulomatous, and/or fibrotic lung effects of relevance 

to human health risk assessment. These lung effects 

in the animal studies were relatively early-stage 

and were not reversible after exposure ended (up to 

approximately 6  months post-exposure [Pauluhn 

2010a]). In the studies with multiple post-exposure 

follow-up times, the amount of pulmonary fibrosis 

persisted or progressed with longer follow-up [Shvedova et al. 2005, 2008; Mercer et al. 2008; Porter 

et al. 2010; Pauluhn 2010a]. One of the measures 

of pulmonary fibrosis used in the short-term studies [Shvedova et al. 2005, 2008; Mercer et al. 2008, 

2011]—alveolar epithelial cell thickness (due to increased collagen deposition associated with CNT 

mass lung dose)—was also used to develop the 

EPA ozone standard. This response endpoint was 

selected by EPA as the adverse lung response for 

cross-species dose-response extrapolation, because 

it indicates “fundamental structural remodeling” 

[US EPA 1996; Stockstill et al. 1995].

The excess risk estimates based on the subchronic 

and short-term studies of MWCNT and SWCNT 

suggest that workers are at >10% excess risk of 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 119

developing early-stage adverse lung effects (pulmonary inflammation, granulomas, alveolar septal 

thickening, and/or fibrosis) if exposed for a working lifetime at the estimated upper LOQ of 7 μg/m3

based on NIOSH Method 5040 for measuring the 

airborne concentration of CNT [NIOSH 2010a] 

(Appendix C; Tables A–3 through A–8). Working 

lifetime airborne concentration (8-hr TWA) estimates of 0.51–4.2 μg/m3 MLE and 0.19–1.9 μg/m3

95% LCL were associated with a 10% excess risk 

of early-stage lung effects (histopathology grade 1 

minimal or higher) based on the subchronic inhalation studies (Table A–5). For histopathology 

grade 2 (slight [Ma-Hock et al. 2009] or slight/mild 

[Pauluhn 2010a]), the working lifetime 8-hr TWA 

concentrations associated with an estimated 10% 

excess risk are 1.0 to 44 μg/m3 MLE and 0.69 to 19 

μg/m3 95% LCL (Table A–6). 

As discussed in Section A.2.3, the 10% BMDL estimates are a typical POD for extrapolation to lower 

risk. NIOSH does not consider 10% or greater excess risk levels of these early-stage lung effects to 

be acceptable if equivalent effects were to occur in 

workers as a result of working lifetime exposures 

to CNT. Linear extrapolation by application of uncertainty factors (e.g., Table A–14) would result 

in lower 8-hr TWA concentrations. However, the 

lowest LOQ of NIOSH Method 5040 (1 μg/m3) is 

the best that can be achieved at this time in most 

workplaces and is similar to or greater than the 

8-hr TWA concentrations estimated to be associated with 10% excess risk of minimal (grade 1) 

effects (Table A–7). Some of the risk estimates are 

less than 10% at the LOQ of 1 μg/m3 (8-hr TWA), 

in particular those based on the slight/mild (grade 

2) rat lung effects and assumed normal clearance 

(Table A–8). 

Although uncertainties and limitations exist in 

these animal studies, the evidence supports the 

health-based need to reduce exposures below 1 μg/

m3. These risk estimates indicate the need for research to develop more sensitive measurement 

methods for airborne CNT in the workplace, to 

demonstrate effective exposure control, and to 

evaluate the need for additional risk management 

measures such as the use of respirators and other 

personal protective equipment and medical screening (Section 6, Appendix B). Chronic bioassay data 

are also needed to reduce the uncertainty concerning the potential for chronic adverse health effects 

from long-term exposure to CNT. Evaluation of the 

factors that influence the risk estimates and the areas of uncertainty are discussed below.

A.4.1 The Use of Short-term 

Data to Predict Longerterm Response

Several factors suggest that in the absence of 

chronic data these short-term and subchronic animal data may be reasonable for obtaining initial estimates of the risk of human noncancer lung effects 

from exposure to CNT. First, some fraction of CNT 

that deposit in the lungs are likely to be biopersistent based on studies in animals [Muller et al. 2005; 

Deng et al. 2007; Elgrabli et al. 2008b; Mercer et al. 

2009; Pauluhn 2010a, b] and studies of other poorly soluble particles in human lungs [ICRP 1994; 

Kuempel et al. 2001; Gregoratto et al. 2010]. Second, the pulmonary fibrosis developed earlier and 

was of equal or greater severity than that observed 

from exposure to the same mass dose of other inhaled particles or fibers (silica, carbon black, asbestos) examined in the same study [Shvedova et al. 

2005; Muller et al. 2005]. Third, the adverse lung 

responses persisted or progressed after the end of 

exposure up to 90 days after a single- or multipleday exposure to SWCNT or MWCNT [Lam et al. 

2004; Muller et al. 2005; Shvedova et al. 2005, 2008; 

Ellinger-Ziegelbauer and Pauluhn 2009; Porter et 

al. 2010] or 26 weeks after a 13-week inhalation exposure to MWCNTs (Baytubes) [Pauluhn 2010a]. 

There is uncertainty in estimating working-lifetime 

health risk from either subchronic or short-term 

animal studies, and perhaps from the shorter-term 

studies. The strength of the subchronic inhalation studies is that they provide exposure conditions that are more similar to those that may 

be encountered by workers exposed to airborne 

CNT. However, there is some uncertainty about 

the deposited and retained dose in the rat lungs 
120 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

(see Section A.6.1 for sensitivity analysis of the 

lung dose estimates). In the PA or IT studies, the 

administered lung dose is known, although the 

pattern of lung deposition (especially for IT administration) may differ from that of inhalation. 

The subchronic inhalation studies and some of 

the PA studies include multiple doses, which can 

provide better information about the shape of the 

dose-response relationship. However, in the subchronic studies, steep dose-response relationships 

were observed for lung response proportions based 

on histopathology score, reaching 100% response 

for minimal or higher severity (grade 1) (Figure 

A–1). Although the data are sparse in the low dose 

region (near a 10% response level), the BMD(L) 

estimates are generally similar to the LOAEL and 

NOAEL values reported in those studies (Section 

A.6.2 and Table A–12). 

A comparison of data from 1-day and 13-week 

inhalation exposures in rats [Ellinger-Ziegelbauer 

and Pauluhn 2009; Pauluhn 2010a], indicates that 

the dose-response relationship was consistent despite the differences in dose-rate in those two studies (Figure A–4). This finding indicates that it may 

be reasonable to assume that the dose-response 

relationships for the IT, PA, and short-term inhalation exposure studies would be consistent with 

the subchronic study results if the same response 

is examined at the same time point, although additional study is needed to confirm this finding. 

The BMC(L) estimates among the subchronic and 

short-term studies (Tables A–3 through A–5) are 

reasonably consistent.

A.4.2 Physical-Chemical 

Properties and Metal 

Content

There are limited data to evaluate the role of 

physical-chemical properties of CNT on the lung 

responses. Although the dose estimates vary for 

the early-stage lung effects in rats and mice (and 

in the human-equivalent concentrations (Tables 

A–3 through A–6), all estimates are relatively low 

mass concentrations. It is difficult to tease out the 

CNT-specific factors affecting these estimates from 

those due to the other study differences (e.g., exposure route, duration, animal species, lung response 

measures). 

The two subchronic inhalation studies of MWCNT 

[Ma-Hock et al. 2009; Pauluhn 2010a], based on 

the same study design (13 week inhalation) and 

animal species/strain (Wistar rats), facilitates comparison. Different types of MWCNT and different 

generation methods for aerosolizing exposures 

were used in each study, although the primary particle sizes reported were similar—approximately 10 

nm in width and 0.1–10 μm in length, with specific surface area of approximately 250–300 m2/g 

[Ma-Hock et al. 2009; Pauluhn 2010a]. The aerodynamic diameter (and resulting alveolar deposition 

fraction) estimates were also fairly similar (Table 

A–2); yet the bulk densities differed (approximately 

0.04 and 0.2 g/ml, respectively, in Ma-Hock et al. 

[2009] and Pauluhn [2010a]). The metal content 

also differed, with 9.6% Al2O3 in the MWCNT in 

the Ma-Hock et al. [2009] study vs. 0.5% Co in 

the MWCNT (Baytubes) in the Pauluhn [2010a] 

study. The lung responses differed both qualitatively and quantitatively, including “pronounced 

granulomatous inflammation, diffuse histiocytic 

and neutrophilic inflammation, and intra-alveolar 

lipoproteinosis” with a LOAEL of 0.1 mg/m3 in MaHock et al. [2009], vs. “inflammatory changes in the 

bronchioloalveolar region and increased interstitial 

collagen staining” with a LOAEL of 0.45 mg/m3

[Pauluhn 2010a]. Yet, both MWCNT studies reported LOAELs that are lower by more than an order 

of magnitude compared to the LOAEL (7 mg/m3) 

reported in a 13-week inhalation study of ultrafine 

carbon black [Elder et al. 2005]. 

A recent study provides a quantitative comparison 

of the effects of SWCNT and MWCNT on pulmonary interstitial fibrosis [Mercer et al. 2011]. In 

this study, MWCNTs were administered to mice 

by pharyngeal aspiration at several different doses 

(0 [control], 10, 20, 40, or 80 μg); the lung tissues 

(stained for collagen using Sirius red) were examined at 56 days post-exposure. At the 80-μg dose 

of MWCNT, the average thickness of the alveolar interstitial connective tissue was significantly 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 121

Figure A–4. Dose-response relationship between estimated retained lung doses of MWCNT (Baytubes) 

based on cobalt-tracer measurements and early-stage pulmonary fibrosis (proportion of rats with minimal 

or greater alveolar interstitial thickening) examined at 13 weeks, following either a 1-day [Ellinger-Ziegelbauer and Pauluhn 2009] or 13-week inhalation exposure [Pauluhn 2010a]. Dose groups include n=10 [Pauluhn 2010a] or n = 6 [Ellinger-Ziegelbauer and Pauluhn 2009]. Data were fit with a multistage (polynomial 

degree 2) model in BMDS 2.2 [US EPA 2010]. Error bars are the 95% confidence limits.

increased at 28 days, and a progressive increase in 

thickness was observed at 56 days. The 40-μg MWCNT dose group also showed a significant increase 

in the interstitial connective tissue thickness at 56 

days. These data were compared with those of an 

earlier study of SWCNT [Mercer et al. 2008] using 

the same study design. The individual MWCNTs 

had a mean diameter of 49 nm and a mean length 

of 3.9 μm. The individual SWCNTs were 1–4 nm 

in diameter and several hundred nanometers in 

length. Both SWCNT and MWCNT were rapidly 

incorporated into the alveolar interstitial spaces 

(within 1 hour individual CNT or small clumps of 

CNT were observed), although the percentage of 

the administered SWCNT observed in the alveolar 

interstitium (~90%) was much higher than that for 

MWCNT (~8%). After accounting for the differences in the target tissue dose, SWCNTs were still 

~8.5–fold more fibrogenic than MWCNTs. However, the surface area of SWCNT was ~20-fold greater 

per unit mass than that of MWCNTs (508 m2/g for 

SWCNT vs. 26 m2/g for MWCNT), suggesting that 

the greater fibrogenic potency of SWCNT may be 

due to its greater surface area. When the lung response was evaluated per unit CNT surface area 

dose, SWCNT was no longer more potent, and the 

MWCNT were 2.5-fold more potent on a surface 

area basis. There is uncertainty about the degree of 

dispersion (and hence available surface) of these 

materials in vivo, which precludes assigning exact 

potency factors [Mercer et al. 2011]. However, these 

findings suggest that the greater fibrotic potency of 
122 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

SWCNT on a mass basis is likely due to its greater 

surface area available to react with lung tissue. 

Comparison of other CNT types and metal content 

is generally impeded by differences in study design. 

In one of the few studies to investigate CNT with 

different metal content, Lam et al. [2004] reported 

lung granuloma and inflammation responses in 

mice administered IT doses of SWCNT containing 

either 2% Fe, 27% Fe, or 26% Ni. The number of 

mice developing granulomas by group (each containing 5 mice) were the following:

• 0.1 mg dose: 2 (2% Fe); 5 (27% Fe); and 0 

(26% Ni)

• 0.5 mg dose: 5 (2% Fe); 5 (27% Fe); and 5 

(26% Ni)

In addition, three mice died in the first week in the 

0.5 mg dose of the 26% Ni group.

Because of the sparse data and the steep doseresponse relationship, only the SWCNT containing 

2% Fe were adequately fit by the BMDS model. The 

high mortality rate in mice exposed to the SWCNT 

containing Ni suggests this material is highly toxic. 

The greater response proportion in the mice exposed 

to 0.1 mg SWCNT with 27% Fe (5/5) compared with 

rats exposed to the same dose of SWCNT with 2% 

Fe (2/5) suggests that the CNT with higher Fe content are more toxic than CNT with lower Fe content. 

In Shvedova et al. [2005, 2008], higher iron content was also associated with greater lung response 

and thus lower BMD(L) estimates. The BMD(L) estimates for SWCNT with 18% Fe were lower than 

those for SWCNT with 0.2% Fe (Table A–3), even 

though the post-exposure time was longer (60 vs. 

28 days) for the 0.2% Fe SWCNT [Shvedova et al. 

2005, 2008]. All types of CNT (including SWCNT 

and MWCNT, purified or unpurified, and with various types and percentages of metals) were of similar or greater potency (i.e., similar or greater lung 

responses at the same mass dose) in these animal 

studies compared to the other types of particles 

or fibers tested (asbestos, silica, ultrafine carbon 

black) [Lam et al. 2004; Muller et al. 2005; Shvedova et al. 2005, 2008]. 

A.4.3 Lung Dose Estimation 

In any CNT risk assessment, there may be greater 

uncertainty in the estimated lung dose of respirable 

CNT than there is for spherical airborne particles 

for which lung dosimetry models have been developed and validated. Evaluations have not been 

made on the influence of particle characteristics 

(e.g., shape and density) on the inhalability and 

deposition of CNT in the human respiratory tract, 

and on the clearance or biopersistence of CNT. 

However, the available data on the aerodynamic 

size of CNT provides an initial estimate (based 

on validated models for spherical particles) of the 

deposited mass fraction of airborne CNT in the 

human respiratory tract, and specifically in the alveolar (gas exchange) region. The clearance rate of 

CNT from the lungs may be more uncertain than 

the deposition efficiency, as animal studies indicate 

that CNT clearance becomes impaired in rat lungs 

at lower mass doses than for larger particles of 

greater density [Pauluhn 2010a, b]. The NIOSH risk 

assessment helps to characterize this uncertainty 

by providing bounds on the range of possible lung 

dose estimates, from assuming normal clearance to 

assuming no clearance of the deposited CNT. This 

approach also provides a framework for introducing improved dose estimates when validated lung 

dosimetry models for CNT become available.

The assumptions used in the lung dose estimation have a large influence on the animal and 

human-equivalent BMD(L) or BMC(L) estimates 

(Tables A–5 and A–6), as well as on the estimated 

human-equivalent NOAEL (Section A.6.3). The rat 

BMD(L) estimates based on the estimated retained 

lung dose after subchronic inhalation exposure in 

rats are lower than those based on the estimated 

deposited lung dose (Table A–5). This is because 

the retained dose estimates allow for some lung 

clearance to occur during the 13-week exposure in 

rats, and a lower dose estimate is therefore associated with a given fixed response proportion. The 

human-equivalent BMD(L) estimates based on 

retained dose are also lower because they are proportional to the rat BMD(L)s (i.e., calculated based 

on the ratio of the human to rat alveolar epithelial 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 123

cell surface area). However, the working lifetime 

8-hr TWA concentrations, BMC(L)s, based on the 

estimated retained lung doses are higher than those 

based on the estimated deposited lung dose. This is 

because the retained dose estimates (which assume 

some particle clearance from workers’ lungs during 

the 45 years of exposure), require a higher inhaled 

airborne concentration to reach the estimated human-equivalent BMD(L) lung doses.

The estimated deposited lung dose of CNT (assuming no clearance) may overestimate the actual CNT 

lung dose, given that the short-term kinetic data 

have shown some CNT clearance in rats and mice 

[Muller et al. 2005; Deng et al. 2007; Elgrabli et al. 

2008b; Mercer et al. 2009; Pauluhn 2010a, b]. On 

the other hand, the estimated retained lung dose of 

CNT, based on models for poorly soluble spherical particles, may underestimate the retained CNT 

lung burden, given that overloading of rat lung 

clearance has been observed at lower mass doses of 

MWCNT (Baytubes) than for other poorly soluble 

particles [Pauluhn 2010a,b]. Thus, although there 

is uncertainty in the deposition and retention of 

CNT in the animal and human lungs, the deposited 

and retained lung dose estimates reported in this 

risk assessment may represent reasonable upper 

and lower bounds of the actual lung doses. 

A.4.4 Critical Effect

Level Estimates 

The response endpoints in these animal studies of 

CNT are all relatively early-stage effects. Although 

these effects were persistent or progressive after 

the end of exposure in some studies, there was no 

information on whether these responses were associated with adverse functional effects. More advanced-stage responses (grade 2 or higher severity 

on histopathology examination) were also evaluated, and as expected, these responses resulted in 

lower risk estimates (Table A–6). It is expected that 

exposure limits derived from these early response 

data would be more protective than those based on 

frank adverse effects. On the other hand, because 

of the lack of chronic studies, there is considerable 

uncertainty about the potential chronic adverse 

health endpoints. 

The excess risk estimates at the lower LOQ (1 μg/

m3) are considerably lower than those at the upper 

LOQ (7 μg/m3) of NIOSH Method 5040, for either 

minimal (TableA–7) or slight/mild (Table A–8) lung 

effects based on the rat subchronic inhalation data. 

The range in the estimates in Table A–7 and A–8 

reflects the low precision in the animal data and the 

uncertainty about CNT retention in the lungs. There 

is also uncertainty about the relationship between 

the lung dose and response, including whether there 

is a threshold. For example, for slight/mild lung 

effects (Table A–8), the actual risk could be as low as 

zero or as high as 16% at the REL of 1 μg/m3.

NIOSH utilized BMD modeling methods to estimate the critical effect level (i.e., the dose associated 

with the critical effect or benchmark response) in 

order to provide a standardized method for risk 

estimation across studies. In contrast, the NOAELbased approaches do not estimate risk, but may assume safe exposure or zero risk below the derived 

OEL. BMD modeling also uses all of the doseresponse data, rather than only a single dose for a 

NOAEL or LOAEL, and takes appropriate statistical account of sample size, unlike NOAEL-based 

approaches. However, the BMD modeling options 

for some of these CNT data were limited because 

of sparse data, and the dose groups with 100% 

response (observed in the subchronic inhalation 

studies) contribute little information to the BMD 

estimation. A common challenge in risk assessment is defining a biologically relevant response for 

continuous endpoints, which was also encountered 

in this risk assessment. A standard practice of using 

a statistical definition of the benchmark response 

was used for the continuous BMD estimation in 

the absence of data on the functional significance 

of the early-stage pulmonary inflammation and fibrotic responses (Section A.2.3.2). 

For CNT, as with other chemicals, there is uncertainty in whether a NOAEL or a BMDL from a 

short-term or subchronic study in animals would 

also be observed in a chronic study. For example, 

in the Pauluhn [2010a] study, 0.1 mg/m3 was the 
124 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

NOAEL based on subchronic inhalation exposure 

in rats, but there was some indication that lung 

clearance overloading may have already begun 

(i.e., retention half-time about two-fold higher 

than normal, although imprecision in the low dose 

measurement was noted) [Pauluhn 2010a,  b]. A 

comparison of the BMD and the NOAEL estimates 

shows that these estimates are statistically consistent (Section A.6.2). Thus, there is uncertainty as 

to whether chronic exposure at 0.1  mg/m3 might 

result in adverse lung effects that were not observed during subchronic exposure. It is also uncertain whether these subchronic effects (without 

additional exposure) would resolve with longer 

post-exposure duration (beyond the 26-week 

post-exposure period in the Pauluhn [2010a] 

study). Yet, workers may be exposed to CNT daily 

for many years, e.g., up to a working lifetime. The 

NIOSH REL is intended to reduce the risk of lung 

disease from exposures to CNT and CNF up to a 

45-year working lifetime.

A.4.5 Animal Dose-response

Data Quality

In the absence of epidemiological data for CNT, the 

two subchronic inhalation studies of two types of 

MWCNT, in addition to the short-term studies of 

SWCNT and MWCNT, provide the best available 

dose-response data to develop initial estimates of 

the risk of early-stage adverse lung responses associated with exposure to CNT. The availability 

of animal dose-response data for different types 

of CNT—and the consistent low mass concentration BMC(L) estimates—suggests these risk estimates are relatively robust across a range of CNT 

types, including SWCNT or MWCNT, either purified or unpurified (containing different types and 

amounts of metal), dispersed or agglomerated. Although a formal comparison of the potency of the 

different CNT is not feasible because of differences 

in study design, these studies consistently show 

that relatively low-mass doses of CNT are associated with early-stage adverse lung effects in rats and 

mice. Consequently, the human-equivalent benchmark dose and working lifetime exposure estimates 

derived from these studies are also relatively low on 

a mass basis. The excess risk estimates of early-stage 

adverse lung responses to CNT generally indicate 

> 10% excess risk (lower 95% confidence limit estimates) at the upper LOQ (7 μg/m3) of the measurement method (NIOSH Method 5040) regardless of 

the CNT type or purification (Tables A–3 through 

A–5). Lower risks are estimated at the optimal LOQ 

(1  μg/m3), depending on lung dose assumptions 

(Tables A–7 through A–8).

A more in-depth analysis of specific areas of uncertainty in this CNT risk assessment is provided in 

Section A.6. This includes quantitative evaluation 

of the methods and assumptions used in the CNT 

risk assessment for the derivation of a REL.

A.5 Conclusions

Risk estimates were developed using benchmark dose 

methods applied to rodent dose-response data of adverse lung effects following subchronic or short-term 

exposure to various types of SWCNT and MWCNT. 

In the absence of validated lung dosimetry models for 

CNT, lung doses were estimated assuming either deposited or retained lung dose in animals and humans. 

These findings suggest that workers are at risk of developing adverse lung effects, including pulmonary 

inflammation and fibrosis, if exposed to CNT over 

a working lifetime. Based on the two rat subchronic 

inhalation studies for two types of MWCNT (with 

different metal content), working lifetime exposures 

of 0.2–2 μg/m3 (8-hr TWA; 95% LCL estimates) are 

estimated to be associated with a 10% excess risk of 

early-stage lung effects (minimal severity grade 1) 

(Table A–5). For a severity level of slight/mild (grade 

2), the 45-year working lifetime excess risk estimates 

are approximately 0.7–19 μg/m3 (8-hr TWA; 95% 

LCL estimates) (Table A–6). 

These working liftetime 8-hr TWA concentrations 

are below the estimated upper LOQ (7  μg/m3) of 

NIOSH Method 5040 for measuring the respirable 

mass concentration of CNT in air as an 8-hr TWA. 

Similar risk estimates relative to the LOQ were also 

derived for SWCNT and MWCNT from the shortterm studies, regardless of whether the CNT were 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 125

purified or unpurified (with different types and 

amounts of metals), i.e., 0.08–12 μg/m3 (Tables A–3 

and A–4). Lower risks are estimated at the lower 

LOQ of 1 μg/m3, which are approximately 0.5% to 

16% based on the rat subchronic dose-response 

data for the slight/mild lung effects and different 

lung dose estimation (95% UCL estimates) (Table 

A–8). Higher risks are estimated for the more sensitive endpoint of minimal grade 1 lung effects 

(Table A–7). Additional analyses and risk estimates 

based on other methods and assumptions are provided in Section A.6. 

A.6 Sensitivity Analyses

Specific areas of uncertainty in this CNT risk assessment are evaluated in this section, including: (1) the 

rat lung dose estimation; (2) the critical effect level 

selection in animals and relevance to humans; and 

(3) alternative assumptions used in the OEL estimation methods. Sensitivity analyses in these areas 

were performed to qualitatively and quantitatively 

evaluate the influence of the different options and 

assumptions on the draft REL [NIOSH 2010]. 

A.6.1 Lung Dose Estimation 

Key factors that influence the estimates of CNT 

lung burden in rats and humans include: (a) the 

lung geometry and airway dimensions; (b) lung and 

breathing parameters (including, functional residual capacity, total lung capacity, breathing frequency, 

and tidal volumes; (c) lung retention kinetics; and 

(d) interspecies dose normalization. The deposition 

fraction is based on the airborne particle size (and 

to some extent shape for nonspherical particles), on 

the breathing pattern (nasal, oral, or combination) 

and minute ventilation, and on the lung airway geometry. The ventilation rate depends on the species 

and on the activity level. Reference values are available for the average ventilation rates in rats and humans [EPA 1988, 1994; ICRP 1994]. The airborne 

particle size data (as reported in the animal studies) (Table A–2) were used to estimate the deposited 

lung dose of CNT in rats and humans, using spherical particle based models. The long-term clearance 

kinetics have been well studied and validated for 

inhaled poorly soluble spherical particles in rats 

[Anjilvel and Asgharian 1995; Asgharian et al. 2001, 

2003] and in humans [ICRP 1994; Kuempel et al. 

2001a, b; Gregoratto 2010, 2011], but models specifically for CNT are not yet available. 

This section examines some of the key parameter values used in the lung dose estimation, and 

also characterizes the quantitative influence of 

alternative models and assumptions. Two studies were available to evaluate the lung dose estimates in rats. Pauluhn [2010a] and EllingerZiegelbauer and Pauluhn [2009] provided cobalt 

tracer-based measurements of the CNT lung 

burden based on cobalt-tracer measurements. 

These data were used to compare MPPD modelbased estimates. Because of prediction equation 

changes in the MPPD model from version 2.0 to 

2.1, which affect the model-predicted rat alveolar deposition fraction predictions (discussed 

further in Section A.2.2), the cobalt tracer-based 

estimates are compared to each model version 

(Section A.6.1.2). The influence of assumed 

density on the CNT lung deposition fraction is 

quantified in addition to the evaluation of the 

MPPD model version 2.0 vs. 2.1 predictions 

(Section A.6.1.1). The derivation of allometricbased (body weight scaled) lung ventilation rate 

estimates is also discussed (Section A.6.1.3). 

A.6.1.1 Lung Dosimetry Modelbased Deposition Fraction 

and Dose Estimates

The fraction of inhaled CNT that is deposited in 

the respiratory tract is predicted from the aerosol 

characteristics. The deposition mechanisms include 

impaction, sedimentation, interception, and diffusion. The aerodynamic diameter, by definition, 

represents the gravitational settling (sedimentation) 

behavior of particles [Hinds 1999]. The definition 

of aerodynamic diameter standardizes the shape 

(to spherical) and density (to that of water, 1 g/ml). 

The aerodynamic diameter of a particle, regardless 

of its shape and density, is the diameter of a sphere 

with the same gravitational settling velocity as the 
126 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

particle in question. Conventionally, aerodynamic 

diameter has been used as a reference diameter to 

represent total particle deposition in the respiratory 

system over a wide particle size range. Models such 

as MPPD [CIIT and RIVM 2006; ARA 2011] use 

particle density (specified by the user), to convert 

aerodynamic to physical diameter and vice versa, 

and in this manner capture the key particle deposition mechanisms for spherical particles. 

However, for high-aspect ratio particles and particles less than 500 nm diameter, including some 

individual or airborne agglomerates of CNT, the 

aerodynamic diameters are much smaller than 

their diffusion-equivalent diameter (i.e., the measure of diameter that captures the diffusional deposition mechanism) [Baron et al. 2006; Kulkarni et 

al. 2009]. When the different equivalent diameters 

could significantly differ, it is recommended to experimentally measure these property-equivalent 

diameters, and subsequently use the measured diameters in the lung deposition models to provide a 

reliable representation of each relevant deposition 

mechanism [Kulkarni et al. 2011]. 

In the animal inhalation studies of CNT [Shvedova 

et al. 2008; Ma-Hock et al. 2009; Pauluhn 2010a], 

the airborne particle sizes (MMAD) were in the 

micrometer size range (~1–3 μm) (Table A–2) and 

the airborne CNT structures in those studies were 

roughly spherical agglomerates—suggesting that 

deposition from diffusional mechanisms may be 

negligible and aerodynamic diameter may provide 

a reasonable estimate of the deposition efficiency of 

CNT in the respiratory tract. However, the density 

of the airborne structures can affect the deposition 

efficiency predictions in MPPD [ARA 2011]. An 

evaluation of the effect of the CNT density assumptions on the rat alveolar deposition fraction is provided in this section.

In the rat model, MPPD version 2.1 (but not 2.0) 

accepts density values less than one. The MMAD 

(GSD) values reported in the subchronic rat inhalation studies varied slightly with particle concentration and sampling device [Ma-Hock et al. 2009; 

Pauluhn 2010a]. The central MMAD (GSD) values 

were used for the deposition fraction and lung 

burden estimates. The influence of the alternative 

particle size estimates was not fully evaluated but appeared to be minimal compared with other factors 

(MPPD rat model version and assumed density). 

In addition, the MPPD model estimates of CNT lung 

burden in rats are compared to the measured CNT 

lung burdens from two rat inhalation studies. Pauluhn [2010a] reported the amount of cobalt tracer 

in the rat lungs as well as the amount of Co that was 

matrix-bound to the CNT. The Ellinger-Ziegelbauer 

and Pauluhn [2009] 1-day inhalation study with 91-

day post-exposure follow-up also reported Co data. 

These data provided a basis for comparison to lung 

burden estimates from the MPPD models. 

Results in Table A–9 show that the rat deposition 

estimates (at the same density) vary by a factor of 

approximately two depending on the version of 

the MPPD model (2.0 or 2.1). As discussed in Section A.2.2, this is apparently because of a change in 

MPPD 2.1 in the deposition efficiency equations 

for the head region of the rat model, which reduces 

the deposition efficiency of the alveolar region. The 

lower density further reduces the alveolar deposition efficiency estimates. These findings suggest that 

rat alveolar lung dose estimates based on MPPD 

2.1 (regardless of density assumption) would result 

in greater estimated potency of the CNT (because 

the response proportions do not change) and thus 

lower BMD(L) estimates in rats and lower OEL estimates (by approximately a factor of two) than those 

shown in the main analyses. Table A–9 also shows 

the human alveolar deposition fraction estimates 

from MPPD 2.0 and 2.1 (Yeh and Schum deposition 

model). MPPD 2.0 and 2.1 provide similar deposition fraction estimates for particle density of 1 g/ml. 

Different density assumptions (within MPPD 2.1) 

also had less effect (up to approximately 20%).

A.6.1.2 Cobalt Tracer vs. Dosimetry 

Model Estimates of MWCNT 

Lung Dose 

Table A–10 provides a comparison of the dose estimates from either the MPPD 2.0 or 2.1 rat lung 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 127

Table A–9. Comparison of rat or human alveolar deposition fraction of inhaled particles, by MPPD 

version and density assumption*

Rat subchronic 

inhalation study

 MPPD 2.0 MPPD 2.1

Density = 1 (g/ml) Density = 1 (g/ml) Density < 1 (g/ml)*

 Rat estimates

Ma-Hock et al. [2009] 0.072 0.044 0.024

Pauluhn [2010a] 0.046 0.027 0.023

 Human estimates

Ma-Hock et al. [2009] 0.099 0.10 0.080

Pauluhn [2010a] 0.086 0.090 0.084

*

Density: 0.043 g/ml [Ma-Hock et al. 2009]; 0.2 g/ml [Pauluhn 2010a]. Particle MMAD (GSD): 1.2 (2.7) [Ma-Hock et al. 2009]; 

2.74 (2.11) [Pauluhn 2010]; tidal volume 2.1 ml [Ma-Hock et al. 2009]; 2.45 ml [Pauluhn 2010a]; inhalability adjustment (all).

Table A–10. Comparison of MPPD model and cobalt-tracer based estimates of MWCNT lung 

burden—subchronic inhalation exposure in rats [Pauluhn 2010a]

Exposure 

concentration 

(mg/m3)

 MPPD 2.0* MPPD 2.1†

MWCNT Retained 

lung dose (μg) 

estimated from 

cobalt-tracer‡

Deposited 

lung dose 

(μg) 

Retained 

lung dose 

(μg)

Deposited 

lung dose 

(μg) 

Retained 

lung dose 

(μg)

0.1 27 12 14 5.6 8.7

0.45 121 63 50 23 109

1.62 436 271 222 125 391

5.98 1,610 1,230 594 392 1,433

*

MMAD (GSD)—2.74 (2.11); assumed density of 1 g/ml; tidal volume—2.45 ml; alveolar deposition fraction estimate was 0.046.

†

Assumed density of 0.2 g/ml; tidal volume—2.45 ml; alveolar deposition fraction estimate was 0.023 for MMAD (GSD) of 

2.74 (2.11).

‡

Mass of cobalt was estimated from Figure 6 in Pauluhn [2010a] to be approximately 10, 125, 450, and 1,650 ng, respectively, by 

increasing exposure concentration. The CNT amount in the lungs was estimated from the reported 0.115% Co that was matrixbound to the CNT [Pauluhn 2010a]; the remaining mass (99.885% was assumed to be CNT). The CNT mass was thus calculated 

as CNT (ng) = [0.99885 × Co mass (ng)] / 0.00115. CNT (ng) × 0.001 μg/ng equals CNT (μg).
128 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table A–11. Comparison of MPPD model and cobalt-tracer based estimates of MWCNT lung

burden—single day (6-hr) inhalation exposure in rats [Ellinger-Ziegelbauer and Pauluhn 2009]

Exposure 

concentration 

(mg/m3)

 MPPD 2.0* MPPD 2.11†

MWCNT retained 

lung dose (μg) 

estimated from 

cobalt-tracer‡

Deposited 

lung dose 

(μg) 

Retained 

lung dose 

(μg)

Deposited 

lung dose 

(μg) 

Retained 

lung dose 

(μg)

11 50 12 18 3.4 ≤26

241 933 568 557 285 339

*

MMAD (GSD)—2.9 (1.8) and 2.2 (2.6), respectively, for 11 and 241 mg/m3, from Ellinger-Ziegelbauer and Pauluhn [2009]; alveolar deposition fraction—0.050 and 0.043, respectively, for 11 and 241 mg/m3

; assumed density of 1 g/ml; tidal volume—2.45 ml. †

Assumed density of 0.2 g/ml; tidal volume—2.45 ml; alveolar deposition fraction—0.019 and 0.026, respectively for 11 and 

241 mg/m3. ‡

Mass of cobalt at 91 d post-exposure was estimated from Figure 2 in Ellinger-Ziegelbauer and Pauluhn [2009] to be approximately 

0.03 μg (11 mg/m3) and 0.39 μg (241 mg/m3). The CNT amount in the lungs was estimated from the reported 0.115% Co that 

was matrix-bound to the CNT [Pauluhn 2010a]; the remaining mass (99.885% was assumed to be CNT). The CNT mass was 

thus calculated as CNT (μg) = [0.99885 × Co mass (μg)] / 0.00115. 

model to those from the cobalt tracer measurements reported in two studies [Pauluhn 2010a; 

Ellinger-Ziegelbauer and Pauluhn 2009].

Table A–10 shows that the cobalt-based estimate 

of CNT in the rat lungs is numerically between the 

deposited and retained dose estimated by MPPD 

2.0 (density of 1). The MPPD 2.11 model (density of 0.2) [Pauluhn 2010b] underestimated the 

Co-based lung burden, even the deposited dose 

estimate (assuming no clearance). These findings 

suggest that the model-based estimates of the deposited and retained rat lung doses in the main 

analyses (MPPD 2.0, density 1) provided reasonable estimates of the bounds on the estimated 

lung burden. Moreover, these findings are consistent with the animal toxicokinetic data that show 

CNT overloads alveolar clearance at lower mass 

doses than for particles with lower total surface 

area or volume lung dose, resulting in increased 

retention of CNT in the lungs of rats and mice 

than expected for other poorly soluble respirable 

particles [Pauluhn 2010a; Mercer et al. 2009]. The 

finding that the cobalt-tracer estimates were between the deposited and retained lung doses is 

consistent with CNT reduced clearance compared 

with spherical particles. 

Similar comparisons were made of the cobalt-tracer 

or lung model estimated lung dose of MWCNT in a 

study of rats exposed for 1 day (Table A–11). Results 

show that the MPPD 2.0 model overestimated the 

retained lung dose of CNT by nearly a factor of two 

(at the higher dose) compared with the estimates 

based on the cobalt tracer in the Ellinger-Ziegelbauer and Pauluhn [2009] study (Table A–11). This 

suggests greater clearance than would be predicted 

at this high dose (241 mg/m3) based on overloading of lung clearance in the rat model (MPPD 

2.0). If the retained lung dose estimated by cobalt 

tracer is the best estimate (closest to actual), this 

suggests that the BMD estimates using the modelestimated lung burdens may be overestimates (i.e., 

they underestimate potency because the response 

proportion is constant while the actual lung burden causing the effect may be lower). Some error 

may also exist in the cobalt-tracer measurements 

of the MWCNT mass (estimated from Figure 2 in 

Ellinger-Ziegelbauer and Pauluhn [2009]).
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 129

A.6.1.3 Pulmonary Ventilation Rate 

The pulmonary ventilation rate influences the deposited and retained lung dose estimates. Rat inhalation rate estimates vary slightly among different sources [US EPA 1994; Pauluhn 2010a, citing 

Snipes 1989]. Species-specific ventilation rates can 

be calculated based on the following allometric 

scaling equation [US EPA 1994]:

Equation A.6:

ln(VE) = b0 + b1 ln(BW) 

where VE is the minute ventilation (L/min); BW is 

body weight; and b0 + b1 are species-specific parameters.

For the rat, b0 + b1 are -0.578 and 0.821, respectively (Table 4–6 of US EPA [1994]). For a 300 g 

rat, the ventilation rate can be calculated as follows:

Equation A–7:

0.21 L/min = Exp [-0.578 + 0.821 × Ln(0.3)] 

This is also the default minute ventilation in MPPD 

[CIIT and RIVM 2007; ARA 2011]. 

Rat mean body weights in Pauluhn [2010a] were 

reported as 369 g (male) and 245 g (female) in the 

control (unexposed) group at 13 weeks. Because 

the alveolar septal thickening response data in Pauluhn [2010a] were based on male rats only, a male 

rat minute ventilation of 0.25 L/min (calculated 

from equation A–6) was used to estimate lung dose 

in that study. 

Ma-Hock et al. [2009] did not report the rat body 

weight, although the rat strain (Wistar) and study 

duration (13 weeks) were the same as in Pauluhn 

[2010a]. Because the granulomatous inflammation 

response data in Ma-Hock et al. [2009] were combined for the 10 male and 10 female rats in each 

dose group (since response proportions were statistically consistent), an average rat body weight in 

male and female rats of 300 g was assumed, which 

is similar to the male and female average body 

weight of 307 g reported in Pauluhn [2010a] and 

the default value of 300 g in MPPD. Subsequently, body weights were obtained for the Ma-Hock 

et al. [2009] study [personal communication, L. 

Ma-Hock and E. Kuempel, 10/14/10]. The average 

male and female rat body weight at 13 weeks was 

nearly identical (305 g) to that reported in Pauluhn [2010a]. Other rat minute ventilation rates of 

0.8–1 L/min per kg [Pauluhn 2010a, citing Snipes 

1989] would result in somewhat higher lung dose 

estimates. 

Based on equation A–6, a minute ventilation of 

0.21 L/min is calculated for female and male rats in 

Ma-Hock et al. [2009], and 0.25 L/min for male rats 

in Pauluhn [2010a]. Minute ventilation is the product of tidal volume and breathing frequency. Assuming the same breathing frequency (102 min-1), a 

tidal volume of 2.45 ml is calculated (equation A–6) 

and used instead of the default value in MPPD 2.0 

[CIIT and RIVM 2006] in estimating the rat lung 

dose in the Pauluhn [2010a] data. 

In humans, based on the MPPD 2.0 model [CIIT 

and RIVM 2006], the default pulmonary ventilation rate is 7.5 L/min, based on default values of 12 

min-1 breathing frequency and 625 ml tidal volume. 

The “reference worker” ventilation rate is 20 L/min 

[ICRP 1994] or 9.6 m3/8 hr (given 0.001m3/L, and 

480 min/8-hr). In these estimates, 17.5 min-1 breathing frequency and 1143 ml tidal volume [NIOSH 

2011a] were used in MPPD 2.0 to correspond to a 

20 L/min reference-worker ventilation rate.

A.6.2 Critical Effect 

Level Selection 

A key step in the dose-response analyses of any risk 

assessment is estimating the critical effect level. 

A critical effect level from an animal study is extrapolated to humans to derive a POD for low dose 

extrapolation (Section A.2.3). A critical effect is 

typically the most sensitive effect associated with 

exposure to the toxicant (i.e., the effect observed 

at the lowest dose) which is adverse or is causally 

linked to an adverse effect. The early-stage lung 

effects discussed in Section A.2.1.3 are the critical effects used in both the main risk assessment 

and in these sensitivity analyses. The primary 
130 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table A–12. Effect level estimates in rats after subchronic (13-wk) inhalation exposure to 

multiwall carbon nanotubes (MWCNT)

Study

Effect level in rats

BMR

LOAEL

(mg/m3)

NOAEL

(mg/m3)

BMC

(mg/m3)

BMCL

(mg/m3)

Ma-Hock et al. [2009] 0.1 nd 0.060 0.023 Granulomatous 

inflammation 

(≥ minimum, severity 

grade 1+)

 0.5 0.1 0.12 0.082 Granulomatous 

inflammation 

(≥ mild, severity 

grade 2+)*

Pauluhn et al. [2010] 0.45 0.1 0.10 0.051 Alveolar septal 

thickening 

(≥ minimal, severity 

grade 1+)

 1.5 0.45 0.87 0.45 Alveolar septal 

thickening (≥ mild, 

severity grade 2+)

Abbreviations: NOAEL: No observed adverse effect level; LOAEL: Lowest observed adverse effect level; BMC: Benchmark concentration (maximum likelihood estimate) associated with 10% excess risk of specified BMR. BMCL: 95% lower confidence limit of 

the BMC; based on a multistage model, polynomial degree 2, P = 0.88); BMR: Benchmark response; nd: not determined

*

Same response proportion per dose, and therefore the same BMD(L) estimates, for alveolar lipoproteinosis.

critical effect selected is the proportion of rats 

with minimal (grade 1) or higher severity of pulmonary inflammation or alveolar septal thickening 

(as reported by Ma-Hock et al. [2009] and Pauluhn [2010a]). In addition, grade 2 (slight/mild) 

or greater effects (as reported in the same studies) 

were also evaluated as a response endpoint since 

the interpretation of the histopathology, for a slight 

or mild response, may be less variable than that for 

a minimal response, and may also be more relevant 

to a potential adverse health effect in humans. 

The critical effect levels in the main analysis are the 

BMD(L) estimates from the dose-response modeling of the rat estimated deposited or retained 

lung dose and to the human-equivalent lung dose 

estimates. The working lifetime exposure concentration that would result in that equivalent lung 

dose was then calculated, assuming either particlesize specific lung deposition only (assuming no 

clearance) or the estimated retained lung dose (assuming normal spherical particle clearance). 

In the main risk analysis, BMD methods were selected over NOAELs or LOAELs because of several statistical advantages (Section A.2). However, 

BMD(L) estimates may also be uncertain, for example, when the dose spacing is not optimal, as 

occurred in the CNT subchronic studies (Figures 

A–1 and A–4). In this sensitivity analysis, NOAELs 

and LOAELs reported in the subchronic inhalation 

studies [Ma-Hock et al. 2009; Pauluhn 2010a] are 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 131

used as the effect levels in evaluations of alternative methods to derive OEL estimates. A quantitative comparison of possible critical effect levels 

is shown in Table A–12. The BMDL estimates are 

generally similar to the NOAEL estimates (within a 

factor of approximately 1 to 4), which suggests that 

the BMDL estimates may be reasonable despite the 

sparse data in the low dose region of the subchronic inhalation studies (Figure A–1). 

A statistical analysis was performed to compare the 

NOAEL and BMD estimates (in this example, the 

BMD is an exposure concentration, or BMC). The 

maximum likelihood estimate of the excess risk (of 

a minimal or higher grade of alveolar septal thickening) at 0.1 mg/m3 is 0.10 (i.e., 10%), based on the 

BMD model fitted to the dose-response data in the 

Pauluhn [2010a] study (Table A–12). Yet, 0.1 mg/m3

was identified as a NOAEL based on zero adverse 

response being observed [Pauluhn 2010a]. In order 

to assess the precision of the estimate of the excess 

risk associated with this NOAEL, the likelihood of 

the data in the NOAEL and control groups was reparameterized in terms of the respective sum and 

difference of the expected response proportions; 

and an upper confidence limit for the difference 

was assessed by inverting its likelihood ratio test 

statistic. When a nominal confidence coefficient of 

95% for a two-sided interval was applied, a value 

of 0.17 (i.e., 17%) was obtained for the UCL of the 

difference. Hence, the results supporting the use of 

0.1 mg/m3 as a NOAEL are also statistically consistent with the results from the BMD model since 

the MLE of excess risk based on the model is less 

than the UCL.

In a standard risk assessment approach, BMDL 

estimates may be considered equivalent to a NOAEL for use as a POD in risk assessment [US EPA 

1994]. Once an effect level is selected in a given animal study, it is extrapolated to a human-equivalent 

effect level (e.g., as 8-hr TWA concentration), 

or human-equivalent concentration (HEC). This 

HEC_POD (human-equivalent point-of-departure) 

is the POD for either extrapolating to a lower (acceptable) risk level or applying uncertainty factors in 

the derivation of an OEL. These steps are discussed 

further in Section A.6.3. 

A.6.3 Alternative OEL 

Estimation Methods 

As mentioned in the previous section, a standard 

risk assessment method using animal data typically involves first identifying a critical effect level 

in animals (e.g., NOAEL or BMDL), which is the 

PODanimal. A HEC_POD is estimated by extrapolating 

the animal dose to humans by accounting for the 

biological and physical factors that influence the 

lung dose across species†. Lung dosimetry models can account for these interspecies differences 

and provide equivalent dose estimates in animals 

and humans given the exposure concentration and 

duration, the breathing rates and patterns, and 

the physical properties of the aerosol. A simplified standard approach in lieu of a lung dosimetry 

model to apply a total dosimetric adjustment factor to the animal effect level (Section A.6.3.1). It is 

useful to evaluate both approaches given that the 

lung dosimetry models have not been specifically 

validated for respirable CNT. 

A.6.3.1 Illustration of HumanEquivalent Concentration 

Estimation

The human equivalent concentration HEC) to a 

PODanimal (e.g., NOAEL) in an animal study can be 

calculated as: 

Equation A–8:

HEC_POD = PODanimal / DAF

where DAF is the dosimetric adjustment factor, 

and 

Equation A–9:

DAF = (VEH/VER) × (DFH/DFA) × (RTH/RTA) × (NFA/NFH) 

 

where VE is the ventilation rate (e.g., as total volume of air inhaled per exposure day, m3/d) in 

†

HEC_POD is then divided by appropriate uncertainty 

factors (UFs) to account for variability and uncertainty in its estimation (Section A.6.3.3).
132 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

humans (H) or animals (A); DF is the deposition 

fraction, in this case, in the alveolar region of the 

respiratory tract; RT is retention half-time of particles in the lungs, and NF is the interspecies dose 

normalization factor. 

The basic method shown in equation A–9 is consistent with the reference concentration (RfC) method 

[US EPA 1994] and the method used by Pauluhn 

[2010b].‡ The NOAEL of 0.1 mg/m3 and the specific 

adjustment factors reported by Pauluhn [2010b] are 

used below as an example of this standard risk assessment approach to estimating a HEC_POD: 

Equation A–10:

AF‡ = (0.14/0.29) × (0.118/0.057) × (10/1) × 

(8.66 × 1010/4.99 × 1011) = 1.73 

VE (Equations A–9 and A–10) is expressed as volume per body weight (m3/kg) Pauluhn [2010b]. 

The rat value of 0.29 is calculated from a ventilation rate of 0.8 L/min/kg BW × 360 min (i.e., rat 

6-hr exposure day) × 0.001 m3/L. The human value 

of 0.14 is based on 9.6 m3 (per 8-hr workday)/70 

kg. The alveolar DF is for a MMAD of 3 μm and estimated from the MPPD2.0 rat and human models 

[CIIT and RIVM 2006]. RT is based on an assumed 

average retention half-time of approximately 1–2 yr 

in humans and 60 days in rats [Snipes et al. 1989] 

(the factor was rounded to 10 in Pauluhn 2010b). 

NF is expressed as the total alveolar macrophage 

cell volume (μm3) per kg BW in each species, calculated from the average AM cell volume (1166 μm3

rat, 4990 μm3 human), the total average number of 

AMs per lung (2.6 × 107 rat, 7.0 × 109 human), and 

‡

There are some differences in the terminology and 

presentation of standard methods to estimate a 

HEC_POD. For example, the placement of the 

animal or human factors in the numerator or denominator of the ratios determines whether the 

effect level is multiplied or divided by the DAF). 

The term AF (adjustment factor [Pauluhn 2010b]) 

is equivalent in concept to the term DAF [US EPA 

1994]. AF is used here to clarify that the adjustment factor illustrated here is specific to the estimation of the human-equivalent dose from the 

Pauluhn [2010b] study.

the average BW in rats and humans (0.35 and 70 

kg, respectively) [Pauluhn 2010b] (Table A–2).

The AF of 1.7 was rounded up to 2 in Pauluhn 

[2010b]. Thus, 

Equation A–11: 

HEC_POD = 0.1mg/m3 / 2 = 0.05 mg/m3

The method shown in equations A–8 to A–11 is 

seen to be identical to the derivation of the humanequivalent NOAEL in Pauluhn [2010b]. In that 

study, the HEC_NOAEL was not divided by UFs, 

but was proposed as an OEL [Pauluhn 2010b]. 

To simplify further, the BW factors in equation 

A–10 can be dropped. The BW terms cancel out 

such that the estimate is the same whether or not 

BW is used in these calculations. Thus, substituting the terms expressed per kg BW in equation 

A–10 with the animal or human values of VEH/VER

(9.6/0.1015) and NFA/NFH (3.03 × 1010 /3.49 × 1013) 

results in the same AF of 1.7.

A.6.3.2 Evaluation of Dosimetric 

Adjustment Factors 

As illustrated in Section A.6.3.1 and equation A–9, 

the four adjustment factors that make up the total 

DAF include:

1. Air intake (ventilation rate)

2. Deposition fraction 

3. Dose retention

4. Normalizing factor

To evaluate the quantitative effect of the different assumptions in extrapolating the animal dose 

to humans, it is of interest to examine alternative assumptions in these four adjustment factors 

(Equation A–9). The first two factors—ventilation 

rate (VE) and deposition fraction (DF)—do not 

vary much among the various sources because 

they are based on well-known physiological and 

physical measurements and models. There is some 

uncertainty in the DF estimates for CNT as they 

are based on models for spherical particle aerosols, and also the density assumption can influence 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 133

the DF estimate, although a larger difference (approximately a factor of two) is due to differences in 

the spherical particle model-based estimates (e.g., 

MPPD 2.0 vs. 2.1) (Sections A.2.2; A.6.1.1; and 

A.6.1.2). 

The values used by NIOSH [2010] and Pauluhn 

[2010b] are similar for the VE and DF, i.e., for human and rat, respectively:

• VE (m3/d): 10 and 0.015 [Pauluhn 2010b],

9.6 and 0.09 [NIOSH 2010]; and

• DF: 0.118 and 0.057 [Pauluhn 2010b],

0.086 and 0.046 [NIOSH 2010]. 

The other two factors—retention half-time (RT) 

and interspecies normalization factor (NF)—can 

differ largely depending on the assumed mode of 

action concerning how the deposited CNT interacts with the lung tissue over time. These factors 

are discussed below.

A.6.3.2.1 Interspecies dose 

normalization factor

The interspecies NF adjusts for the size difference 

in the lung (surface area or volume) into which the 

CNT dose deposits. Studies of other inhaled particles or fibers are relevant to evaluating mechanisms 

that may also apply to CNT in the lungs. Possible 

dose metrics related to the modes of action for 

pulmonary inflammation and fibrosis include the 

CNT mass, surface area, or volume dose per alveolar epithelial cell surface area or alveolar macrophage cell volume in each species. Normalizing 

the dose (e.g., NOAEL) across species to the total 

average alveolar macrophage cell volume in rat or 

human lungs is based on the experimental observation of overloading of alveolar clearance in rats and 

mice exposed to respirable poorly soluble particles 

or fibers [Bolton et al. 1983; Morrow 1988; Bellmann et al. 1991; Elder et al. 2005; Pauluhn 2010b]. 

(a) Alveolar macrophage cell volume

At a sufficiently high particle dose, pulmonary 

clearance can become impaired due to overloading 

of alveolar macrophage-mediated clearance. In rats, 

the overloading dose has been observed as particle 

mass (~1 mg/g lung), volume (~1 μl/g lung for 

unit density particles) [Morrow 1988; Muhle et al. 

1990], or surface area (200–300 cm2 particles per 

rat lung) [Tran et al. 2000]. On a volume basis, an 

overloading particle dose corresponds to approximately 6%–60% of total alveolar cell volume, when 

overloading begins and is complete, respectively 

[Morrow 1988]. The 60% value has been observed 

experimentally [Oberdörster et al. 1992], although 

particle clearance impairment may start at lower 

particle volume lung dose [Bellmann et al. 1991; 

Kuempel et al. 2001a]. Biological responses to overloading include: accumulation of particle-filled 

macrophages in the alveoli, increased permeability 

of the epithelial cell barrier, persistent inflammation, increased particle translocation to the alveolar interstitium and lung-associated lymph nodes, 

as well as increasing alveolar septal thickening, lipoproteinosis, impaired lung function, and fibrosis 

[Muhle et al. 1990, 1991]. 

Although the overload mode of action in the rat has 

been well-studied, the extent to which overloading 

is involved in human lung responses to inhaled 

particles is not as clear due to observed differences 

in both the kinetics and the pattern of particle retention in the lungs of rats and humans. Whereas 

particle clearance in rats is first-order at doses below overloading, studies in workers have shown 

that human lung clearance of respirable particles is 

not first-order even at relatively low retained particle mass lung low doses [Kuempel 2000; Kuempel 

et al. 2001; Tran and Buchanan 2000; Gregoratto 

et al. 2010, 2011]. That is, some portion of the particle dose that deposits in the pulmonary region is 

retained for a very long time (retention half-time 

of several years) [ICRP 1994; Kuempel et al. 2001; 

Gregaratto et al. 2010]. Humans also apparently retain a greater portion of the particles in the alveolar 

interstitium, whereas rats retain more particles in 

the alveolar space [Nikula et al. 1997, 2001]. The 

greater interstitial particle retention may increase 

the dose to the target tissue for pulmonary fibrosis in humans relative to that for the same deposited dose in rats lungs. Given the differences in the 

particle clearance kinetics and retention patterns 

in rats and humans, normalizing the dose across 
134 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

species based on the total alveolar macrophage volume may not be the best dose metric for predicting 

adverse lung responses in humans. 

(b) Alveolar epithelial cell surface area

Another dose metric that may be relevant to the inflammatory and fibrotic lung responses is the particle or CNT dose per surface area of alveolar epithelial cells [US EPA 1994; Donaldson et al. 2008]. 

It is the epithelial cell surface with which particles 

interact when they migrate through the epithelial 

cell layer into the interstitium, and epithelial cells are 

also involved in recruitment of inflammatory and fibrotic cells [Bohning and Lippmann 1992; Driscoll 

et al. 1996; Tran et al. 2000]. For this reason, normalizing the dose based on the total alveolar epithelial 

cell surface area may be more predictive of the human lung response. However, since both the alveolar 

macrophages and epithelial cells are involved in the 

lung responses to inhaled particles, some combination of dose metrics may ultimately be most predictive in this dynamic biological system. 

In the absence of a more complete biologicallybased model, an evaluation of the quantitative influence of each assumed dosimetric mode of action 

(e.g., based on either the alveolar macrophage cell 

volume or the epithelial cell surface area) provides 

information on the sensitivity of the risk assessment and OEL derivation to the interspecies dose 

normalization factor. Thus, replacing the alveolar 

macrophage volume ratio in equation A–10 with a 

NFA/NFR of 0.4m2/102m2 [Stone et al. 1992] results 

in a total AF that is 4.5 × greater. That is,

Equation A–12: 

AF = (9.6m3/0.102m3) × (0.118/0.057) × (10/1)

 × (0.4m2/102m2) = 7.7 

Equation A–13:

HEC_NOAEL = 0.1mg/m3 / 7.7 = 0.013 mg/m3

The larger AF results in a corresponding smaller 

human-equivalent concentration. This illustrates 

that the risk estimates for CNT—as for other inhaled 

particles—is sensitive to the assumed mode of action concerning the interspecies normalizing factor.

A.6.3.2.2 Interspecies Dose Retention Factor 

The retained dose to the target tissue is influenced 

by the clearance mechanism in the lung region in 

which the particles deposit. RT in equation 2 (as 

the kinetic factor in Pauluhn [2010b]) is intended 

to account for the differences in the rat and human 

particle retention half-time. This factor is also dependent on the assumptions concerning the biological mode of action. In the rat, evidence suggests 

that doses of poorly soluble low toxicity particles 

below those causing overloading of lung clearance 

(i.e., at steady-state) would not be associated with 

adverse lung effects. A steady-state lung burden 

means that the rate of particle deposition equals 

the rate of clearance such that once the steady-state 

burden had been achieved, the lung burden would 

be the same over time if exposure conditions did 

not change. For example, if steady-state lung burden was reached after subchronic (13 week) exposure to a given exposure concentration, then the 

chronic (2 yr) lung burden would be the same given 

the same rates of exposure and clearance. However, 

the steady-state lung burden may not been entirely 

reached by 13 weeks in the rat or in an equivalent 

time in humans. Based on the rat overload mode 

of action, Pauluhn [2010b] assumed that humans 

would achieve a steady-state lung burden if exposed at an equivalent total particle volume dose in 

the alveolar macrophages (over a roughly equivalent human exposure duration of 10 years to a rat 

3 month exposure). A ratio of 10/1 for human/rat 

retention half-time rate was used [Pauluhn 2010b], 

based on a simple first-order clearance rate model 

of particle clearance from the lungs in both rats and 

humans [Snipes et al. 1989]. The volumetric dose 

of CNT associated with overloading in the rat was 

equivalent to a relatively low mass dose compared 

to other poorly soluble particles [Pauluhn 2010b. 

2011]. Moreover, human lung-particle retention 

data have shown that a simple first-order clearance 

model would underpredict the human long-term 

lung dose at similar low mass doses [ICRP 1994; 

Kuempel et al. 2001; Gregoratto et al. 2010]. That is, 

the human long-term retained lung burden would 

be expected to exceed a steady-state lung burden 

predicted from the rat model (i.e., low-dose firstorder clearance with dose-dependent impairment, 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 135

or overloading, of particle clearance after reaching 

a critical lung dose). 

An alternative approach evaluated was to use the 

MPPD 2.0 [CIIT and RIVM 2006] human lung 

dosimetry model to estimate directly the retained 

lung burden in humans over a working lifetime. 

This approach assumes a mode of action in which 

the cumulative retained particle dose is related to 

the adverse lung responses, regardless of the dose 

rate (i.e., the time required to reach that dose). The 

cumulative exposure concept (concentration × 

time), known as “Haber’s Law,” is a typical default 

assumption in risk assessment for long-term exposures in the absence of other data [US EPA 1994]. 

Some studies in workers (coal miners) have shown 

that the working lifetime cumulative exposure and 

the retained lung dose are better predictors of pulmonary fibrosis than the average exposure concentration without consideration of duration [NIOSH 

2011b]. Yet, there remains uncertainty about how 

well a cumulative dose received over a short duration may predict the response to the same cumulative dose received over a longer duration (i.e., at a 

lower dose rate). The direction of error could go 

either way, depending on the biological mechanisms of response. For example, a lower dose rate 

may allow the lung defense mechanisms to adapt 

to the exposure (e.g., by increasing clearance or repair mechanisms), which could reduce the adverse 

response at a later time point. On the other hand, a 

longer time in which a substance is in contact with 

the tissue may exacerbate the response, resulting 

in a more severe effect at the later time point. The 

actual lung response may be some combination of 

these effects. 

To evaluate the assumptions used to estimate the 

human and rat retention kinetics, estimates were 

compared from the MPPD2.0 lung dosimetry 

model [CIIT and RIVM 2006] to the ratio of 10/1 

for RTH/RTA used by Pauluhn [2010a]. The rat and 

human lung dosimetry models take into account 

the ventilation rates, the deposition fraction by respiratory tract region (predicted from particle size 

and breathing rate and pattern, nasal vs. oronasal), 

and the normal average clearance rates. Using 

the particle size and breathing rate values for the 

Pauluhn 2010a study (Table A–2), the rat retained 

lung burden at the end of the 13 week exposure 

to 0.1 mg/m3 was estimated to be ~12 μg (Table 

A–10). This is similar to the 8.7 μg lung burden estimated from the cobalt-tracer based measurement 

(Table A–10).

Assuming that the rat has achieved steady-state 

lung burden after 13 wk exposure to 0.1 mg/m3, the 

chronic lung burden should also be approximately 

12 μg.§ Extrapolating the rat lung dose of 0.012 mg 

to the human-equivalent lung burden would result 

in either: 

• 13.5 mg—estimated by dividing the rat lung 

dose by an interspecies NF for the average total alveolar macrophage cell volume (i.e., 3.03 

× 1010 μm3/3.49 × 1013 μm3) (rat/human); or 

• 3.0 mg based on the average total alveolar 

epithelial cell surface area (0.4 m2/102 m2) 

(rat/human). 

The associated 8-hr TWA concentration for 45-yrs 

would result in human-equivalent lung burdens (estimated from MPPD 2.0 human model [CIIT and 

RIVM 2006]) of 16 μg/m3 and 3.5 μg/m3, respectively, for the normalized lung burdens based on 

the alveolar macrophage cell volume or the alveolar 

epithelial cell surface area (Table A–13). The value of 

16 μg/m3 is approximately 3-fold lower than the ~50 

μg/m3 as the human equivalent concentration to the 

rat NOAEL reported in Pauluhn [2010b] (or 3.5 × 

lower than the 58 μg/m3 HEC_LOAEL by applying 

an AF of 1.7 without rounding to 2). This difference 

is due to the approximately 3x higher retained lung 

dose estimates after a 45-year working lifetime (Table A–14) to that estimated as a 10-year steady-state 

lung burden [Pauluhn 2010a]. This suggests that the 

RT of 10 may underestimate the rat to human lung 

retention kinetics, and that a factor of 35 (i.e., 10 × 

3.5) may be more realistic. Since the MPPD model 

already takes into account the ventilation rate and 

deposition fraction, the difference in the human 

retained lung dose estimates is due to greater 

§

At 2 years, the MPPD model predicted a lung burden 

of 13 μg and a lung burden plus lung-associated 

lymph node burden of 23 μg.
136 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

particle retention predicted by the MPPD model 

(which includes the ICRP [1994] clearance model) 

[CIIT and RIVM 2006] compared to that of the 

first-order kinetic model used to estimate the factor 

of 10/1 [Snipes et al. 1989; Pauluhn 2010b]. 

When this same method was applied to the rat 

LOAEL of 0.1 mg/m3 in the Ma-Hock et al. [2009] 

subchronic inhalation study, but using the particle 

size data and rat minute ventilation specific for 

that study (Table A–2 and Section A.2.2), similar 

human-equivalent estimates were obtained. The 

slightly higher doses are due to the greater DF for 

the MWCNT in the Ma-Hock et al. [2009] study. In 

addition, the POD from the Ma-Hock et al. [2009] 

study is based on a LOAEL (vs. NOAEL in Pauluhn 

[2010a], so an additional uncertainty factor would 

be applied (as discussed in the next section). In 

each case, the estimates using an interspecies normalizing factor based on the alveolar epithelial cell 

surface area are lower by a factor of approximately 

four. The estimates in Table A–13 are working lifetime human-equivalent concentrations to the rat 

NOAEL or LOAEL with no uncertainty factors 

applied to these values. Lower estimated rat lung 

doses and human-equivalent lung doses and associated working lifetime concentrations would be 

expected if using MPPD 2.1 and density <1 g/ml 

(Section A.6.1.1 and Table A–9). 

A.6.3.3 Selection of uncertainty 

factors and OEL derivation

Standard noncancer risk assessment often assumes 

a threshold model, such that exposures below the 

OEL are assumed to be associated with essentially 

zero risk. Uncertainty factors (UFs) are applied to 

the POD estimates (e.g., HEC_NOAEL) to derive 

health-based OELs. Uncertainty factors include not 

only factors to account for the uncertainty in the 

models and estimates used in the risk assessment, 

but also to account for variability in the distribution of responses in the human population. Possible 

UFs are shown in Table A–14. Various criteria and 

systems for UF selection have been developed (e.g., 

US EPA [1994]; WHO [2005]). NIOSH has used 

these standard systems to select uncertainty factors 

in the derivation of RELs on a case by case basis. 

The selection of UFs for a given study may depend 

in part on the criteria or system used and the interpretation of the available dose and response data 

within that system. Other risk assessments of CNT 

that have used uncertainty factors include Aschberger et al. [2010] and Nakanishi [2011a]. 

To obtain an OEL estimate based on the rat NOAEL 

or LOAEL data, the human-equivalent NOAEL or 

LOAEL (e.g., Table A–13) would be divided by 

study or data-based UFs (e.g., Table A–14). Using 

this approach, the human-equivalent NOAEL or 

LOAEL estimates of 3.5 to 18 μg/m3 (Table A–13) 

divided by example uncertainty factors of 20 or 60 

(Table A–14) results in estimates of approximately 

<1 μg/m3 as the working lifetime exposures likely to 

be without appreciable risk of adverse effects. Such 

OEL estimates are below the upper LOQ (7 μg/m3) 

of the analytical method to measure airborne CNT 

[NIOSH method 50540] and approximately equal 

to or less than the lower LOQ of 1 μg/m3 [NIOSH 

method 5040]. These findings are consistent with 

the BMD-based estimates, which generally indicate >10% excess risk at the LOQ, depending on 

the effect level (BMR of grade 1+ or grade 2+ lung 

effects) and lung dose (deposited or retained) assumptions (Tables A–3 through A–6). That is, the 

working lifetime 10% BMC(L) estimates are generally less than 1 or 7 μg/m3. Other estimates indicate <10% excess risk at the lower LOQ of 1 μg/m3, 

based on an effect level (BMR) of histopathology 

grade 2 or higher. At the REL of 1 μg/m3, the 45-

yr working lifetime risk estimates of slight or mild 

(grade 2) lung effects, based on the rat subchronic 

inhalation studies to MWCNT, were approximately 

0.5 to 16% (8-hr TWA; 95% UCL estimates) (Table 

A–8), depending on the type of MWCNT and the 

estimated lung dose in animals and humans. 

Excess risk estimates based on the short-term 

studies of SWCNT and MWCNT in rats and mice 

(Tables A–3 and A–4) are consistent with those 

from the rat subchronic inhalation studies (Tables 

A–5 and A–6). However, the uncertainty factors applied to the short-term studies would be expected 

to be higher (e.g., by factor of 2) [Nakanishi 2011a] 

than those for the subchronic studies. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 137

Table A–13. Human-equivalent retained lung burden and working lifetime 8-hr TWA concentrations to rat subchronic NOAEL or LOAEL of 0.1 mg/m3

Subchronic rat study and 

normalizing factor

Rat lung burden 

(μg)†

Human-equivalent 

lung burden (mg)‡

Working lifetime 

8-hr TWA (μg/m3)

§

Pauluhn [2010a]

Alveolar macrophage volume 11.7 13.5 16 

Alveolar epithelial cell surface area 3.0 3.5 

Ma-Hock et al. [2009]

Alveolar macrophage volume 16.0 18 18 

Alveolar epithelial cell surface area 4.1 4.0

†

Estimated retained lung burdens in rats from MPPD 2.0 [CIIT and RIVM 2006], assuming particle size (MMAD and GSD) in 

Table A–2 and unit density. MPPD adjusts for the rat ventilation rate, deposition fraction by respiratory tract region, and uses a 

first-order clearance model (with rat overload at higher doses).

‡

Human-equivalent retained lung burden estimated by dividing the rat lung burden by a normalizing factor for interspecies differences—either the total alveolar macrophage cell volume (3.03 x 1010 μm3

/3.49 × 1013 μm3 ) (rat/human) or the total alveolar 

epithelial cell surface area (0.4 m2/102 m2) (rat/human). §

Human-equivalent concentration and point of departure (HEC_POD) based on the rat NOAEL [Pauluhn 2010a] or LOAEL 

[Ma-Hock et al. 2009], as 8-hr TWA over a 45-year working lifetime, estimated using MPPD 2.0 [CIIT and RIVM 2006] (Yeh 

and Schum human deposition model), reference worker ventilation rate and patterns [ICRP 1994], and the same particle size in 

Table A–2. 

A.6.4 Summary of Sensitivity 

Analyses Findings 

Many of the areas of uncertainty in these risk estimates for CNT also occur in other standard risk 

assessments based on subchronic or short-term 

animal study data. Potential chronic effects of CNT 

are an important area of uncertainty because no 

chronic study results were available. Uncertainty 

exists about the estimated lung doses for the inhalation studies because of lack of experimental 

evaluation or validation of lung dosimetry models 

to predict deposition and retention of CNT. Information is also limited on the relative potency of different types of CNT to cause specific lung effects in 

animals because of study differences. Despite the 

variability in the risk estimates across the various 

types of CNT, all of the risk estimates were associated with low-mass concentrations (below the upper and lower LOQ or 7 or 1 μg/m3, respectively). 

In conclusion, these sensitivity analyses show that 

the estimates of a health-based OEL are not strongly dependent on the BMD-based risk assessment 

methods, and the use of an alternative (POD/UF) 

method provides supporting evidence indicating 

the need for a high level of exposure containment 

and control for all types of CNT.

A.7 Evaluation of Carbon 

Nanofiber Studies in 

Mice and Rats

Two in vivo studies of carbon nanofibers (CNF) 

have been recently published in mice and rats [Murray et al. 2012 and DeLorme et al. 2012]. In order 

to compare the lung responses to CNF observed in 

these studies, estimates of the lung doses normalized 

across species are provided in this section. 
138 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Table A–14. Example of uncertainty factor (UF) selection for human-equivalent concentrations to 

rat effect level in MWCNT subchronic inhalation studies†

Type of UF

Possible 

UF value

NOAEL 

[Pauluhn 

2010a] 

LOAEL

[Ma-Hock et 

al. 2009] Rationale

1. Animal to human extrapolation Up to 10 (4 for 

TK; 2.5 for TD) 

[WHO 2005] 

2 2 TK: Dosimetry based on estimated 

retained lung burden (in Table A–13); 

additional uncertainty about slower 

clearance of CNT (ad hoc TK factor of 

2). 

TD: Assume equal average subchronic 

response at equivalent dose in each 

species (TD factor of 1). 

2. Subchronic 

animal doseresponse data 

used in absence 

of chronic data

Up to a factor 

of 10 [US EPA 

1994]

2 2 Subchronic data were used, assuming 

steady-state lung burden in rats 

uncertainty about chronic effects (ad hoc 

UF of 2).

3. Human interindividual 

variability for 

sensitive subpopulation 

10 (3.16 each 

for TK and TD) 

[WHO 2005]

5 5 Variability in workers (TK and 

TD components); factor of 5 from 

Aschberger et al. [2010]. 

4. LOAEL used instead of NOAELUp to a factor 

of 10 [US EPA 

1994]

1 3 NOAEL: factor of 1.

LOAEL: Responses at LOAEL were 

“minimal” severity by histopathology 

[Ma-hock et al. 2009].

5. Modifying factor 

(e.g., poor data 

quality; severe 

effect)

Up to a factor 

of 10 [US EPA 

1994]

1 1 Subchronic studies were standard quality 

assays, and lung effects were early-stage.

Total UF 3,000‡ 20 60 OEL is derived as HEC_POD/total UF. 

Abbreviations: TK—toxicokinetic. TD—toxicodynamic. NOAEL—no observed adverse effect level. LOAEL—lowest observed 

adverse effect level. POD—point of departure. UF—uncertainty factor.

†

These UF examples refer to estimates in Table A–13. 

‡

Total uncertainty factor is typically capped at 3,000 [US EPA 1994].
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 139

A.7.1 Particle Characteristics

Both types of CNF were vapor grown, but obtained 

from different sources. In Murray et al. [2012], the 

CNF was supplied by Pyrograf Products, Inc. The 

chemical composition was 98.6% wt. elemental carbon and 1.4% wt. iron. CNF structures were 80 to 

160 nm in diameter, and 5 to 30 μm in length. The 

specific surface area (SSA) measured by BET was 35-

45 m2/g; the effective SSA was estimated as ~21 m2/g 

[Murray et al. 2012]. In DeLorme et al. [2012], the 

CNF was supplied by Showa Denko KK, Tokyo, 

Japan. The chemical composition was >99.5% carbon, 0.03% oxygen and < 0.003% iron. CNF structures were 40-350 nm (158 nm average) in diameter 

and 1-14 μm in length (5.8 μm average). The BET 

SSA was 13.8 m2/g [DeLorme et al. 2012]. 

A.7.2 Experimental Design 

and Animals

The species and route of exposure also differed in 

the two studies. In Murray et al. [2012], six female 

C57BL/6 mice (8-10 wk of age, 20.0 + 1.9 g body 

weight) were administered a single dose (120 μg) of 

CNF by pharyngeal aspiration [Murray et al. 2012]; 

mice were examined at 1, 7, and 28 days post-exposure. In DeLorme et al. [2012], female and male 

Crl:CD Sprague Dawley rats (5 wk of age) were exposed to CNF by nose-only inhalation at exposure 

concentrations of 0, 0.54, 2.5, or 25 mg/m3 (6 hr/d, 

5 d/wk, 13 wk). The rats were examined 1-d after 

the end of the 13-wk exposure and 3 months postexposure. Body weights were reported as: 252 g +

21.2 female; 520 g + 63.6 male (unexposed controls, 1-d post-exposure); 329 g + 42.2 female; 684 

g + 45.8 male (unexposed controls, 3 mo. post-exposure) [DeLorme et al. 2012]. 

A.7.3 Lung Responses 

In mice, the lung responses to CNF included pulmonary inflammation (polymorphonuclear lymphocytes, PMNs, measured in bronchioalveolar 

lavage fluid, BALF); PMN accumulation in CNFexposed mice was 150-fold vs. controls on day 1. 

By day 28 post-exposure, PMNs in BALF of CNFexposed mice had decreased to 25-fold vs. controls. 

Additional lung effects included increased lung 

permeability (elevated total protein in BALF), cytotoxicity (elevated lactate dehydrogenase, LDH), 

which remained significantly elevated compared to 

controls at day 28 post-exposure. Oxidative damage (elevated 4-hydroxynonenol, 4-HNE, and oxidatively modified proteins, i.e., protein carbonyls) 

was significantly elevated at days 1 and 7, but not 

at day 28. Collagen accumulation at day 28 postexposure was 3-fold higher in CNF-exposed mice 

vs. controls by biochemical measurements. Consistent with the biochemical changes, morphometric 

measurement of Sirius red-positive type I and III 

collagen in alveolar walls (septa) was significantly greater than controls at day 28 post-exposure 

[Murray et al. 2012]. 

In rats, the respiratory effects observed in the DeLorme et al. study [2012] were qualitatively similar 

to those found in the Murray et al study [2012]. The 

wet lung weights were significantly elevated compared to controls in male rats at 25 mg/m3

 CNF and 

in female rats at 2.5 and 25 mg/m3 CNF at 1-day 

post-exposure; lung weights remained elevated in 

each sex in the 25 mg/m3 exposure group at 3 mo. 

post-exposure. Histopathologic changes at 1 day 

post-exposure included inflammation in the terminal bronchiolar and alveolar duct region in the 

2.5 and 25 mg/m3 exposure groups, and interstitial 

thickening with type II pneumocyte proliferation 

in the 25 mg/m3 exposure group. Cell proliferation 

assays confirmed increased cell proliferation in that 

highest dose group in the subpleural, parenchymal 

and terminal bronchiolar region; the subpleural 

proliferation in this dose group did not resolve in 

the females by the end of the 3 month recovery period. Cell proliferation appeared to resolve in males 

after a 3 month recovery period but numerically remained higher in the parenchyma and subpleural 

regions. Histopathologic evidence of inflammation and the presence of fiber-laden macrophages 

were reported to be reduced but still present in the 

high dose group after a 3 month recovery period. 

Inflammation within the alveolar space (as measured by PMN levels in BALF) was statistically sig-
140 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

nificant only in the rats exposed to 25 mg/m3 CNF. 

However, the percent PMNs increased in a doseresponsive manner: 1.2 (± 0.81), 1.4 (± 0.79), 2.7 (± 

0.67), and 11 (± 2.0), respectively, in the 0, 0.54, 2.5, 

and 25 mg/m3 exposure groups. LDH and other 

BALF markers were elevated at the end of the 13-

wk exposure only in the 25 mg/m3 exposure group, 

and LDH remained elevated at 3-mo. post-exposure 

in that group. The observed no adverse effect level 

(NOAEL) in rats was reported to be 0.54 mg/m3. The 

lowest observed adverse effect level (LOAEL) was 

reported to be 2.5 mg/m3 “...based on the minimal 

inflammation observed in terminal bronchioles 

and alveolar ducts of male and female rats...” [DeLorme et al. 2012].

The sample size and sensitivity of the markers or 

assays are factors that could influence the statistical power and likelihood of observing exposurerelated effects in these animal studies. In Murray 

et al. [2012], six animals per group were used for 

the BAL analysis, histopathology evaluation, oxidative stress markers, and lung collagen measurements. Five animals per group were used for the 

BAL and cell proliferation assays in the DeLorme 

et al. [2012] study (male and female data were analyzed separately). The Murray et al. [2012] study 

used a more sensitive marker of interstitial fibrosis 

in measuring the average thickness of the alveolar 

connective tissue, while the DeLorme et al. [2012] 

study did not report using that assay. 

A.7.4 Effects in Other Tissues

In rats, CNF were observed in the nasal turbinates of the high-dose group (25 mg/m3

) at 1 day 

post-exposure, which was accompanied by hyaline 

droplet formation in the epithelium; CNF persisted in the nasal turbinates at 3-mo. post-exposure 

in the high dose group [DeLorme et al. 2012]. In 

all exposure groups, CNF translocated to the tracheobronchial lymph nodes and CNF fibers were 

seen in brain, heart, liver, kidney, spleen, intestinal 

tract, kidneys, and mediastinal lymph nodes, but 

no associated histopathologic abnormalities were 

detected [DeLorme et al. 2012]. In CNF-exposed 

mice, T cell mitogen (concavalin A) responsiveness 

indicated decrease T cell responses in the spleen at 

28 days post-exposure [Murray et al. 2012].

A.7.5 Equivalent Lung Dose 

Estimation Methods

In order to quantitatively compare the results of the 

two CNF studies in mice and rats, equivalent lung 

doses were estimated by accounting for differences 

in route of exposure and particle size characteristics and by normalizing to either the mass or alveolar surface area of the lungs in each species. The 

respiratory tract region where the adverse effects 

were observed is the pulmonary (a.k.a. alveolar) region, which is where gas exchange occurs between 

the lungs and blood circulatory system across the 

alveolar septal walls. In mice, the lung dose estimate is simply the proportion of the administered 

dose (by pharyngeal aspiration) [Murray et al. 

2012] that is estimated to deposit in the alveolar region. Mercer et al. [2010] reported that 81% of the 

aspirated MWCNT by pharyngeal aspiration deposited in the alveolar region of the mouse. If this 

figure applies to the CNF reported in Murray et al. 

[2012], then approximately 97 μg of the 120 μg administered dose would be deposited in the alveolar 

region. In the absence of CNF-specific data, 100% 

alveolar deposition of the administered dose was 

also assumed. 

In rats, the airborne particle size data are used to 

estimate the inhalable, deposited, and retained 

lung doses of CNT, based on the exposure concentrations and particle size characteristics reported 

[DeLorme et al. 2012]. The multipath particle deposition model (MPPD), version 2.90 [ARA 2009], 

was used to estimate these lung doses. MPPD version 2.11 was originally used to obtain some particle deposition estimates, but some output indicated 

errors in estimating the tracheobronchial regional 

deposited dose, which appeared to lower the alveolar deposition estimates. This issue was apparently 

resolved in the updated version (2.90). 

Particle characteristic input values used in MPPD 

include the mass median aerodynamic diameter 

(MMAD), geometric standard deviation (GSD), 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 141

and density. The following MMAD and GSD values were reported by airborne exposure concentrations: 0.54 mg/m3

 (MMAD 1.9 μm; GSD 3.1); 

2.5 mg/m3 (MMAD 3.2 μm; GSD 2.1); and 25 mg/

m3 (MMAD 3.3 μm; GSD 2.0). The density assumed 

for this CNF is 0.08 g/ml. Density was not reported 

in DeLorme et al. [2012] and was obtained from 

the manufacturer’s data analysis sheet, which indicates it is the same material as that reported in 

DeLorme et al. [2012]. 

The default breathing rates and parameters were 

assumed, and inhalability adjustment was selected. 

In MPPD 2.90, nonspherical particle shape can be 

taken into account in the respiratory tract deposition estimates, but some of the required input parameters (GSD of structure diameter and length 

and correlation) were not reported in DeLorme 

et al. [2012]. So, the spherical particle assumption (aspect ratio of 1.0) was assumed, which may 

not be unreasonable given that the fiber interception mechanism may be less for CNF structures of 

length 5.8 μm than for longer fibers. The default 

breathing parameters (including 0.21 ml tidal volume and 102 breaths/min) may be reasonable for 

the female Sprague Dawley rats in the DeLorme et 

al. [2012] study based on similar body weight (300 

g) associated with the default values [Kuempel and 

Castranova 2011], but may be too low for the male 

Sprague Dawley rats. The average body weights in 

control rats (air-only exposed) at the end of 13-wk 

exposure period and the 90-d post-exposure period, respectively, were: 252 and 329 g (females); 

520 and 684 g (males) [DeLorme et al. 2012]. The 

retained lung burden at the end of the 13-wk exposure was also estimated in MPPD 2.90 using the 

particle size data for each exposure concentration 

(using MMAD and GSD values reported above). 

The lung dose estimates in rats and mice were normalized by the lung weight or alveolar surface area to 

estimate the equivalent dose across species. The average lung weights of rats were those reported in DeLorme et al. [2012] 1-d post-exposure in the control rats 

(1.9 g and 1.3 g in males and females, respectively). 

The average mouse lung weight was 0.15 g [personal 

communication, A. Shvedova to E. Kuempel, Aug. 

2012]. The average alveolar surface area assumed for 

the rat lungs was 0.4 m2 [Stone et al. 1992], and that of 

mice was 0.055 m2 [Mercer et al. 2010].

The total deposited CNF dose in the alveolar region 

was estimated in rats in the DeLorme et al. [2012] 

study in the following equation:

Deposited lung dose (mg) =

Exposure Concentration (mg/m3) × Duration (hr/d × d/wk × wk) 

× Minute Ventilation (L/min) × 0.001 m3/L × 60 min/hr 

× Alveolar Deposition Fraction

where the exposure concentrations are 0.54, 2.5, 

or 25 mg/m3; the duration of exposure is 6 hr/d, 5 

d/wk, 13 wk; the minute ventilation is 0.21 L/min; 

and the alveolar deposition fractions are reported 

in Section A.7.5.

A.7.6 Equivalent Lung Dose 

Estimation Results

The inhalable fraction estimates of CNF in rats were 

0.79, 0.73, and 0.72, respectively, in rats at the reported particles sizes for concentrations of 0.54 mg/

m3 (MMAD 1.9 μm; GSD 3.1); 2.5 mg/m3 (MMAD 

3.2 μm; GSD 2.1); and 25 mg/m3 (MMAD 3.3 μm; 

GSD 2.0) in DeLorme et al. [2012] (based on MPPD 

v. 2.90 [ARA 2009] as described in Section A.7.4). 

The alveolar deposition fraction estimates were 

0.0715, 0.0608, and 0.054, respectively, for the 0.54, 

2.5, and 25 mg/m3 exposure concentrations. 

The normalized dose estimates in mice and rats 

(as CNF mass per alveolar surface area or mass of 

lungs) and associated lung responses are shown in 

Tables A–15 and A–16. In mice, these lung dose estimates are similar to or higher than the deposited 

lung dose estimate in the rat at the LOAEL (2.5 mg/

m3), but less than the deposited lung doses estimated in rats at the highest concentration (25 mg/m3

) 

(Tables A–15 and A–16). The mouse deposited lung 

burden estimates are higher than the rat retained 

lung burden estimates at all doses, assuming spherical-particle model clearance in MPPD 2.90 [ARA 

2009]. If CNF is cleared in a similar manner as that 

reported for MWCNT in Pauluhn [2010b], the actual retained lung dose in rats may be intermediate 
142 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

between the estimated deposited and retained lung 

burdens. Thus, the mouse fibrotic lung response 

was observed at an administered lung dose that 

was similar to, or higher than, the rat lung doses 

estimated at the LOAEL. This suggests a roughly 

similar dose-response relationship in the rat and 

mouse lungs to CNT, based on the limited data in 

these two studies.

As discussed above (Section A.7.3), the mouse lung 

responses to CNF (at a 120 μg dose) included alveolar septal thickening identified as pulmonary 

fibrosis based on collagen deposition observed by 

Sirius Red staining and the measured thickness 

of the alveolar connective (septal) tissue [Murray 

et al. 2012]. In the DeLorme et al. [2012] study, 

similar qualitative lung responses were observed at 

the 25 mg/m3 (as discussed in Section A.7.3). The 

DeLorme et al. [2012] did not report fibrosis at 25 

mg/m3 although the description of the responses is 

consistent with early stage fibrosis reported in the 

Murray et al. [2012].

NOAELs were reported for one type of CNF in DeLorme et al. [2012] and for one type of MWCNT in 

Pauluhn [2010a], which were 0.1 and 0.54 mg/m3, 

respectively. It follows that the human-equivalent 

working lifetime exposure estimates to the NOAEL would be roughly 5-fold higher for the CNF 

than that for the MWCNT (although not exactly, 

due to particle size differences and lung deposition 

estimates). Table A–13 shows estimates of humanequivalent concentrations to effect levels in the Pauluhn and Ma-Hock subchronic inhalation studies, 

based on different assumptions in extrapolating the 

rat lung dose to humans. The application of uncertainty factors (e.g., Table A–14) with the CNF used 

in the DeLorme et al. [2012] study would result in 

estimated working lifetime no-effect levels in humans of roughly 1–4 μg/m3

. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 143

Table A–15. CNT lung dose normalized by alveolar surface area in rats and mice.

Species and dose*

Deposited lung 

dose†(mg/m2 lung) 

Retained lung 

dose†(mg/m2 lung) Lung response

Rat: exposure concentration (mg/m3)

0.54 0.47 0.084 NOAEL

2.5 1.9 0.25 LOAEL

25 16 1.1 Septal thickening (slight, 

grade 2) & hypertrophy/ 

hyperplasia of type II 

pneumocytes

Mouse: administered dose (μg)

120 2.2‡ nd Septal thickening and 

pulmonary fibrosis

Abbreviations: NOAEL=no observed adverse effect level; LOAEL= lowest observed adverse effect level; nd=not determined

*

Study references: rat [DeLorme et al. 2012]; mouse [Murray et al. 2012].

†

In rats, the pulmonary deposition fraction and 13-wk retained lung burdens were estimated from MPPD 2.9 [ARA 2009].

‡

In mice, this estimate assumes 100% alveolar deposition of the administered by pharyngeal aspiration. If 81% alveolar deposition 

is assumed as for MWCNT [Mercer et al. 2010], this estimate would be 1.8 mg/m2 lung.

Table A–16. CNT dose normalized by lung weight in rats and mice. 

Species and dose* Deposited dose (mg/g lung)† Retained dose (mg/g)†

Rat (male): exposure concentration (mg/m3)

0.54 0.10 0.018

2.5 0.40 0.054

25 3.4 0.24

Rat (female): exposure concentration (mg/m3)

0.54 0.14 0.025

2.5 0.55 0.074

25 4.7 0.33

Mouse (female): administered dose (μg)

120 0.80‡ nd

Abbreviation: nd=not determined

*

Study references: rat [DeLorme et al. 2012]; mouse [Murray et al. 2012].

†

In rats, the pulmonary deposition fraction and 13-wk retained lung burdens were estimated from MPPD 2.9 [ARA 2009].

‡

In mice, this estimate assumes 100% alveolar deposition of the administered by pharyngeal aspiration. If 81% alveolar deposition 

is assumed as for MWCNT [Mercer et al. 2010], this estimate would be 0.65 mg/g lung. 

APPENDIX B

Occupational Health Surveillance: 

Informing Decisions Concerning Medical 

Surveillance in Workplaces with Potential 

Exposure to CNT and CNF
146 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

B.1 Key Terms Related to

Medical Surveillance

Occupational health surveillance involves the 

ongoing systematic collection, analysis, and dissemination of exposure and health data on groups 

of workers for the purpose of preventing illness and 

injury. Occupational health surveillance, which includes hazard and medical surveillance, is an essential component of an effective occupational safety 

and health program [Harber et al. 2003; Baker and 

Matte 2005; NIOSH 2006; Wagner and Fine 2008; 

Trout and Schulte 2009], and NIOSH continues to 

recommend occupational health surveillance as 

an important part of an effective risk management 

program.

Hazard surveillance includes elements of hazard 

and exposure assessment 

• The hazard assessment involves reviewing 

the best available information concerning 

toxicity of materials. Such an assessment may 

come from databases, texts, and published 

literature or available regulations or guidelines (e.g., from NIOSH or the Occupational 

Safety and Health Administration [OSHA]). 

Human studies, such as epidemiologic investigations and case series or reports, and 

animal studies may also provide valuable information. In most instances involving CNT 

there are limited toxicological data and a lack 

of epidemiologic data with which to make a 

complete hazard assessment. 

• The exposure assessment involves evaluating 

relevant exposure routes (inhalation, ingestion, 

dermal, and/or injection), amount, duration, 

and frequency (i.e., dose), as well as whether 

exposure controls are in place and how protective they are. When data are not available, 

this will be a qualitative process. 

B.2 Medical Surveillance

Medical surveillance targets actual health events 

or a change in a biologic function of an exposed 

person or persons. Medical surveillance involves 

the ongoing evaluation of the health status of a 

group of workers through the collection and aggregate analysis of health data for the purpose of 

preventing disease and evaluating the effectiveness 

of intervention programs (primary prevention). 

NIOSH recommends the medical surveillance of 

workers when they are exposed to hazardous materials, and therefore are at risk of adverse health 

effects from such exposures. Medical screening is 

one form of medical surveillance that is designed 

to detect early signs of work-related illness in individual workers by administering tests to apparently 

healthy persons to detect those with early stages of 

disease or risk of disease. Medical screening generally represents secondary prevention. 

Medical surveillance is a second line of defense 

behind the implementation of engineering, administrative, and work practice controls (including personal protective equipment). Integration 

of hazard and medical surveillance is important to 

an effective occupational health surveillance program, and surveillance of disease or illness should 

not proceed without having a hazard surveillance 

program in place.

B.2.1 Planning and Conducting

Medical Surveillance

Important factors when considering medical surveillance include the following:

1. A clearly defined purpose or objective.

2. A clearly defined target population.

3. The availability of testing modalities to accomplish the defined objective. Testing modalities 

may include such tools as questionnaires, physical examinations, and medical testing.

A clear plan should be established before beginning 

a medical surveillance program. The plan should 

include the following:

1. A rationale for the type of medical surveillance.

2. Provisions for interpreting the results.
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 147

3. More frequent and detailed medical examinations as indicated, based on findings from these 

examinations.

4. Post-incident examinations and medical screening following uncontrolled or nonroutine increases in exposures such as spills.

5. Worker training to recognize symptoms of exposure to a given hazard.

6. A written report of medical findings.

7. Employer actions in response to identification of 

potential hazards.

3. Presentation of the findings to workers and 

management of the affected workplace. 

4. Implementation of all the other steps of a complete 

medical surveillance program [Harber et al. 2003].

The elements for conducting a medical surveillance 

program generally include the following: 

1. An initial medical examination and collection of 

medical and occupational histories.

2. Periodic medical examinations at regularly scheduled intervals, including specific medical screening tests when warranted.

APPENDIX C

NIOSH Method 5040
150 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

C.1 Background 

NIOSH Method 5040 is based on a thermal-optical analysis technique [Birch and Cary 1996] for 

organic and elemental carbon (OC and EC). The 

analysis quantifies total carbon (TC) in a sample as 

the sum of OC and EC. The method was developed 

to measure diesel particulate matter (DPM) in occupational settings, but it can be applied to other 

types of carbonaceous aerosols. It is widely used for 

environmental and occupational monitoring. 

For the thermal-optical analysis, a portion (typically a 1.5-cm2 rectangular punch) of a quartz-fiber filter sample is removed and placed on a small 

quartz spatula. The spatula is inserted in the instrument’s sample oven, and the oven is tightly sealed. 

Quartz-fiber filters are required for sample collection because temperatures of 850 °C and higher are 

employed during the analysis. The thermal-optical 

analyzer is equipped with a pulsed diode laser and 

photo detector that permit continuous monitoring 

of the filter transmittance. This optical feature corrects for the “char” that forms during the analysis 

because of carbonization of some materials. 

Thermal-optical analysis proceeds in inert and oxidizing atmospheres. In both, the evolved carbon is 

catalytically oxidized to carbon dioxide (CO2). The 

CO2 is then reduced to methane (CH4), and CH4 is 

quantified with a flame ionization detector (FID). 

The OC (and carbonate, if present) is first removed 

in helium, as the temperature is increased to a preset maximum. If sample charring occurs, the filter transmittance decreases as the temperature is 

stepped to the maximum. After the OC is removed 

in helium, an oxygen-helium mix is introduced, 

and the temperature is again stepped to a maximum (850 oC or higher, depending on the sample) 

to effect combustion of the remaining material. As 

the light-absorbing carbon (mainly EC and char) 

is oxidized from the filter, the filter transmittance increases. The split between the OC and EC 

is assigned when the initial (baseline) value of the 

filter transmittance is reached. All carbon removed 

before the OC-EC split is considered organic, and 

that removed after the split is considered elemental. 

If no charring occurs, the split is assigned before 

removal of EC. If the sample chars, the split is not 

assigned until enough light-absorbing carbon is 

removed to increase the transmittance to its initial value. 

OC and EC results are reported as micrograms 

per square centimeter (μg/cm2) of sample deposit. 

The total OC and EC on the filter are calculated by 

multiplying the reported values by the deposit area. 

Because only a portion of the sample is analyzed, it 

must be representative of the entire deposit. Thus, a 

homogeneous deposit is assumed. The entire filter 

must be analyzed (in portions if a 37-mm filter is 

used) if the filter deposit is uneven. 

C.2 Method Evaluation

The reported accuracy of NIOSH 5040 is based on 

analysis of TC in different sample types. Accuracy 

was based on TC, because there is no analytical 

standard for determining the OC-EC content of a 

complex carbonaceous aerosol. In the method evaluation, five different organic compounds were analyzed to examine whether the instrument response 

is compound dependent. Linear regression of the 

data (43 analyses total) for all five compounds gave 

a slope and correlation coefficient (r) near unity 

[slope = 0.99 (± 0.01), r2 = 0.999, n = 43], indicating 

a compound-independent response. Eight different 

carbonaceous materials also were analyzed by three 

methods, in-house by thermal-optical analysis and 

by two other methods used by two external laboratories. Sample materials included, DPM, coals, urban dust, and humic acid. Thermal-optical results 

agreed well with those reported by the two other 

laboratories. The variability of the TC results for 

the three laboratories ranged from about 1%–7%. 

These findings [Birch and Cary 1996] demonstrate 

that carbon can be accurately quantified irrespective of the compound or sample type. 

In sampling DPM, different samplers gave comparable EC results because particles from combustion 

sources are generally less than one μm (diameter). 

As such, the particles are collected with high efficiency (near 100%) and evenly deposited on the 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 151

filter. In the method evaluation, different sampler 

types (open-face 25-mm and 37-mm cassettes, 

298 personal cascade impactors, and four prototype impactors) were used to collect diesel exhaust 

aerosol at an express mail facility. The relative standard deviation (RSD) for the mean EC concentration was 5.6% [Birch and Cary 1996]. Based on the 

95% confidence limit (19%; 13 degrees of freedom, 

n = 14) on the accuracy, the NIOSH accuracy criterion [Kennedy et al. 1995] was fulfilled. Variability for the OC results was higher (RSD = 12.3%), 

which is to be expected when different samplers are 

used to collect aerosols that contain semi-volatile 

(and volatile) components, because these may have 

a filter face velocity dependence. The method precision (RSD) for triplicate analyses (1.5  cm2

 filter 

portions) of a 37-mm quartz-fiber filter sample of 

DPM was normally better than 5%, and often 2% 

or less [NIOSH 1994a]. 

In the method evaluation, the limit of detection 

(LOD) was estimated two ways: (1) through analysis of low-level calibration standards [Birch and 

Cary 1996; NIOSH 1994], and (2) through analysis 

of pre-cleaned media blanks. In the first approach, 

OC standard solutions (sucrose and ethylene diaminetetraacetic acid [EDTA]) covering a range from 

0.23 to 2.82 μg C (or from 0.15 to 1.83 μg C per cm2

of filter) were analyzed. An aliquot (usually 10 μL) 

of the standard was applied to one end of a 1.5-cm2

rectangular filter portion that was pre-cleaned in the 

sample oven just before application of the aliquot. 

The filter portion was pre-cleaned to remove any 

OC contamination, which can greatly increase the 

EC LOD when TC results are used for its estimation. 

After cleaning the filter portion, metal tweezers are 

used to remove from the sample oven the quartz 

spatula that holds the portion. External to the oven, 

the spatula is held in place by a metal bracket such 

that the standard can be applied without removing 

the filter portion from the spatula. This avoids potential contamination from handling. 

Results of linear regression of the low-level calibration data were used to calculate the LOD as 3 σy/m, 

where σy is the standard error of the regression and 

m is the slope of the regression line. TC results were 

used rather than OC because the pyrolysis correction may not account for all of the char formed 

during analysis of the standard (because of low 

sample loading and/or the position of the aliquot 

in the laser). If not, a small amount of the OC will 

be reported as EC, introducing variability in the 

OC results and increasing the LOD. The LOD estimated through the linear regression results was 

0.24 μg C per filter portion, or 0.15 μg/cm2. 

A simpler approach for LOD determination is 

through analysis of media blanks. In the method 

evaluation, TC results for pre-cleaned, 1.5-cm2

portions of the filter media were used to calculate 

the LOD estimate. The mean (n =  40) TC blank 

was 0.03 ±0.1 μg TC. Thus, the LOD estimated as 

three times the standard deviation for pre-cleaned 

media blanks (3 σ blank) was about 0.3 μg C. This 

result agrees well with the value (0.24 μg C) estimated through analysis of the standard solutions. 

Considering a 960-L air sample collected on a 37-

mm filter and a 1.5-cm2 sample portion, this LOD 

translates to an air concentration of about 2 μg/m3

([0.3 μg TC/1.5 cm2] [8.5 cm2]/0.960 m3 = 1.78 μg/

m3), corresponding to the reported upper LOQ of 

about 7 μg/m3 (LOQ = 3.3 × LOD). 

As with all analytical methods, the LOD is a varying number. However, the EC LOD (about 2 μg/m3

, 

or an LOQ of 7 μg/m3) reported for NIOSH Method 5040 is a high estimate. As discussed in Section 6 of the CIB, it was based on analysis of precleaned media blanks from different filter lots, over 

a 6-month period, and by different analysts at two 

different laboratories. Further, variability for the 

TC results, rather than the EC results, was used to 

estimate the LOD. These combined factors gave a 

conservative (high) estimate of the EC LOD. More 

typical values, under different sampling conditions, 

are discussed in Section 6.1 of the CIB. 

C.3 Inter-Laboratory

Comparisons

When results of the initial method evaluation 

were published [Birch and Cary 1996], an interlaboratory comparison was not possible because 
152 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

the thermal-optical instrument was available in 

only one laboratory. After additional laboratories 

acquired thermal-optical instruments, a round 

robin comparison [Birch 1998] was conducted. 

Matched sets of filter samples containing different 

types of complex carbonaceous aerosols were distributed to 11 laboratories. Six of the eleven analyzed the samples according to NIOSH 5040, while 

five used purely thermal (i.e., no char correction) 

methods. Good interlaboratory agreement was obtained among the six laboratories that used NIOSH 

5040. In the analysis of samples containing DPM, 

the variability (RSD) for the EC results ranged 

from 6% to 9%. Only low EC fractions were found 

in wood and cigarette smoke. Thus, these materials 

pose minimal interference in the analysis of EC. In 

addition, only minor amounts of EC were found in 

two OC standards that char: about 1% for sucrose 

and 0.1% for the disodium salt of ethylene diaminetetraacetic acid (EDTA). Two aqueous solutions 

of OC standards were included in the comparison 

as a check on the validity of the char correction and 

accuracy of the TC results. Variability (RSD) of the 

TC results for the two standard solutions and five 

filter samples ranged from 3% to 6%. 

A second interlaboratory comparison study using 

NIOSH 5040 was also conducted [Schauer et al. 

2003]. Seven environmental aerosol samples were 

analyzed in duplicate by eight laboratories. Four 

samples were collected in U.S. cities, and three were 

collected in Asia. Interlaboratory variability for the 

EC results ranged from 6% to 21% for six samples 

having EC loadings from 0.7 to 8.4 μg/cm2. Four of 

the six had low EC loadings (0.7 μg/cm2 to 1.4 μg/

cm2). The variability for the OC results ranged from 

4% to 13% (OC loadings ranged from about 1 to 

25 μg/cm2). Results for TC were not reported, but 

the variability reported for the OC results should 

be representative of that for TC, because the samples were mostly OC (75% to 92%). Similar findings were also reported by Chai et al. [2012] from 

seven laboratories in which analysis was performed 

using Method 5040 on four sample filter sets containing OC and EC. The summary RSDs for EC results were <12% for all four sample sets. 

C.4 Carbonates

Carbonate in a sample is indicated by a relatively 

narrow peak during the fourth temperature step 

in helium [Birch 2004a]. Its presence is verified by 

exposing a second portion of the filter to hydrogen chloride (HCL) vapor before analysis. When 

the acidified portion is analyzed, a diminished (or 

absent) peak during the fourth temperature step 

is indicative of carbonate in the original sample 

[Birch 2004a]. Environmental samples typically 

contain little (if any) carbonate, but carbonate (e.g., 

in limestone, trona, concrete) levels in some occupational samples can be quite high. In such cases, 

it is important to ensure that all of the carbonate is 

removed during the first stage of the analysis. If it is 

not completely removed (because of high loading), 

the sample should be acidified. 

C.5 Organic Carbon

Sampling Artifacts 

Problems commonly referred to as ”sampling artifacts“ have been reported when collecting particulate OC on quartz fiber filters. These artifacts do 

not affect the EC results, but they cause positive or 

negative bias in the measurement of particulate OC 

(and TC). Eatough et al. [1995, 1996] observed loss 

of semi-volatile OC from particles during sampling, 

referred to as the “negative” or evaporation artifact. 

This artifact causes a negative bias in the particulate OC (and TC) concentration, because OC initially collected as condensed matter is subsequently 

lost through evaporation from the filter during 

sampling. Conversely, several studies have demonstrated a “positive” or adsorption artifact because 

of filter adsorption of gas phase OC. A quartz-fiber 

filter collects airborne particulate matter and allows 

gases and vapors to pass through, but some adsorption of gas phase (and vapor) OC occurs, resulting 

in overestimation of the true airborne particulate 

OC concentration [Turpin et al. 2000; McDow and 

Huntzicker 1990; Turpin et al. 1994; Olson and Norris 2005; Kirchstetter et al. 2001; Mader et al. 2003; 

Subramanian et al. 2004; Mader et al. 2001; Noll and 

Birch 2008; Schauer et al. 1999]. 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 153

Most of the studies on sampling artifacts apply to 

environmental air sampling. Occupational sampling 

methods and conditions are generally much different than environmental. Environmental samples 

are usually collected at much higher face velocities: 

20–80 cm/s as opposed to 3–4 cm/s for occupational 

samples. In addition, the concentrations of carbon 

are much lower in environmental air than in most 

occupational settings [Fruin et al. 2004; Sheesley 

et al. 2008], and the types of aerosols sampled are 

different (e.g., aged aerosol from multiple environmental sources, as opposed to aerosols close to 

source). These differences are important because 

OC sampling artifacts depend upon conditions 

such as filter face velocity, air contaminants present, sampling time, and filter media. Given the 

much lower filter face velocities typical of occupational sampling, adsorption (i.e., positive artifact) 

is expected over evaporation for occupational samples. Turpin et al. [1994], Kirchstetter et al. [2001], 

Noll and Birch [2008], and Schauer et al. [1999] 

have reported adsorption as the dominant artifact. 

To correct for the positive adsorption artifact, tandem quartz filters have been applied. When sampling with tandem filters, particulate matter is 

collected by the first filter, while both the first and 

second filters are exposed to and adsorb gaseous 

and vaporous OC. For the correction to be effective, both filters must be in equilibrium with the 

sampled airstream, adsorb the same amount of gas/

vapor OC, and not have a significant amount of OC 

loss through evaporation. The OC on the second 

filter can then be subtracted from the OC on the 

first filter to account for the adsorbed OC. Several 

studies have found the tandem filter correction to 

underestimate the adsorption artifact [Turpin et al. 

2000; McDow and Huntzicker 1990; Turpin et al. 

1994; Olson and Norris 2005], while others have 

shown effective correction [Kirchstetter et al. 2001; 

Mader et al. 2003; Subramanian et al. 2004; Mader 

et al. 2001; Noll and Birch 2008].

Air samplers containing a Teflon® and quartz filter 

also have been used for correction of the positive 

OC artifact. In theory, the Teflon top filter collects 

particulate matter with negligible OC gas/vapor 

adsorption, so only the quartz filter beneath it adsorbs gas and vapor OC. Studies on tandem filter 

corrections have shown the quartz filter beneath 

Teflon to have a greater OC value than quartz beneath quartz [Turpin et al. 2000; Olson and Norris 2005]. This finding was attributed to the quartz 

beneath quartz not reaching equilibrium with the 

sampling stream and underestimating the adsorption artifact. Others have attributed it to the evaporation artifact being more prevalent when using a 

Teflon filter instead of a quartz filter, and they reported the quartz behind Teflon to overestimate 

the adsorption artifact [Subramanian et al. 2004]. 

Several studies have shown no difference when using either type of correction [Mader et al. 2003; 

Mader et al. 2001]. 

Noll and Birch [2008] conducted studies on OC 

sampling artifacts for occupational samples to test 

the accuracy of the tandem quartz-filter correction. 

In practice, using two quartz filters for air sampling 

is preferable to the Teflon-quartz combination because both the collection and blank filters are in 

the same sampler. The tandem quartz correction 

effectively reduced positive bias for both laboratory and field samples. Laboratory samples were 

collected under conditions that simulated DPM 

sampling in underground mines. Without correction, TC on the sample filter was 30% higher than 

the actual particulate TC for 50% of the samples, 

but it was within 11% of the particulate TC after the 

tandem quartz-fiber correction. For field samples, 

this correction significantly reduced positive bias 

due to OC adsorption artifact. Little artifact effect 

was found after the correction was made.

C.6 Carbon Nanotubes

and Nanofibers

Method 5040 was developed to measure DPM in 

occupational environments, but it can be applied 

to other types of carbonaceous aerosols. When applied to materials such as carbon black or CNT/

CNF, particle deposition on a filter may be more 

variable because particles in these materials are 

much larger than DPM. Variability depends on the 
154 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

sampler type, and as expected, different samplers 

(e.g., cyclones, open- and closed-face cassettes) will 

give different air concentration results, depending 

on the particle size distribution [Birch et al. 2011]. 

Diesel emissions, and combustion aerosols generally, are composed of ultrafine (< 100 nm diameters) particles. Because of the small size, DPM normally deposits evenly across the quartz-fiber filter 

used for sample collection. As already discussed, 

even deposition is required because only a portion 

of the filter is normally analyzed. Thus, it must be 

representative of the entire sample deposit. 

When applying NIOSH 5040 to CNT/CNF, it is 

important to verify an even filter deposit so that 

an accurate air concentration (based on results for 

the filter portion) can be calculated. Alternatively, 

the entire filter can be analyzed if the deposit is uneven, but this requires analysis of multiple portions 

of a 37-mm filter because of the relatively small diameter (about 1 cm) of the carbon analyzer’s quartz 

sample oven. Quality assurance procedures should 

include duplicate analyses of the 37-mm filter to 

check precision, especially if the deposit appears 

uneven. If a 25-mm filter is used, the entire filter 

can be analyzed, which improves the LOD and 

obviates the need for an even deposit, but a repeat 

analysis (or other chemical analysis) of the sample 

is not possible if the entire filter is analyzed. In addition, the filter must be cut into portions, and the 

portions must be properly loaded in the analyzer 

so the sample transmittance can be monitored. 

Additional details on the evaluation and use of 

NIOSH 5040 are provided elsewhere [Birch and 

Cary 1996; Birch 1998; Birch et al. 1999; Birch 

2002; Birch 2003; Birch 2004a].

As discussed in the CIB, NIOSH 5040 has been applied to several field studies on CNT/CNF [Methner et al. 2007; Birch et al. 2011b; Dahm et al. 2011]. 

In one study, it was employed for area monitoring 

at a laboratory facility that processes CNF in the 

production of polymer composites [Methner et al. 

2007]. Carbon nanofibers and CNT have negligible 

(if any) OC content, making EC a good indicator 

of these materials. Survey results were reported in 

terms of TC, which is subject to OC interferences, 

but the OC results were blank corrected by the tandem filter method described in the preceding section (organic carbon adsorption artifacts) to minimize the positive sampling artifact. Further, based 

on the thermal profiles for the air samples and the 

bulk materials (CNF and composite product), the 

blank-corrected TC was a good measure of the 

CNF air concentration, except in an area where a 

wet saw was operating. In that area, TC was a measure of the composite aerosol released during the 

sawing operation, which contained a high OC fraction due to the composite matrix. 

There are several issues and limitations when analyzing dusts generated during cutting, sanding, or 

grinding CNT/CNF composites. First, the accuracy of determining the EC fraction of a polymer 

composite is questionable and expected to vary, depending on polymer type and sample loading. Further, EC in both the polymer and bulk CNT/CNF 

materials will be measured (i.e., not speciated) if 

both are present. In addition, the EC loading in a 

polymer composite is usually a low percentage (e.g., 

1%). Therefore, if the composite dust is the only EC 

source, and if its EC content is determined accurately, 

an EC concentration of 2  μg/m3 would correspond 

to a dust concentration (at 1% EC) of 200 μg/m3, 

considerably higher than the EC concentration. As 

such, the sample can be easily overloaded with OC 

because of the high relative OC content, which can 

both overload the analyzer and cause positive bias 

in the EC result. Further, in a composite particle, 

the CNT/CNF is bound within a polymer (or resin) 

matrix, which is dissimilar to a particle of unbound 

material. An effort to improve the analysis of samples containing dusts of polymer composites is ongoing; however, in the context of the NIOSH REL, 

the intended application is CNT/CNF in powder 

form, purified or unpurified. Whenever possible, a 

bulk sample of the material (and, if available, other 

materials that may be aerosolized) should be analyzed as the thermal properties of CNT/CNF are 

material dependent (e.g., CNF, SWCNT, MWCNT, 

functionalized or not functionalized). The OC-EC 

split for a bulk material is not reliable because it 

depends on how the powder is applied to the filter 

punch, but a small amount of the CNT/CNF should 
NIOSH CIB 65 • Carbon Nanotubes and Nanofibers 155

be analyzed to determine the onset of oxidation of 

the material and confirm its complete oxidation.

NIOSH investigators also conducted extensive air 

monitoring at a facility that manufactures and processes CNFs [Evans et al. 2010; Birch 2011a; Birch 

et al. 2011b]. Both personal breathing zone and area 

samples were collected. To evaluate the method 

precision, paired samples were collected and repeat 

analyses of the filters were performed. The relative 

percent difference (RPD) and RSD (%) for repeat 

analyses of 12 samples collected in different areas 

of the facility are listed in Table 1. Total, thoracic, 

and respirable dust samples are included. Total (inhalable equivalent) dust was collected with 37-mm 

cassettes, while cyclones were used to collect thoracic and respirable dust. The RPD was determined 

by analyzing either two punches from the same 

filter (duplicates) or one punch from two different 

filters (paired samplers); the RSD was determined 

by analyzing one filter in triplicate. The precision 

for the EC results ranged from about 3% to 14% 

except for one respirable sample, where the RPD 

was about 22%. Higher variability for the latter 

may relate to spatial variation, because the two filter punches analyzed were from different samplers. 

Spatial variation, rather than sampler variability, is 

a likely explanation for this particular result as two 

other sets of paired samplers do not show higher 

variability. The RPDs for these are about 8% and 

13%, comparable to results for multiple punches 

from the same filter. 
156 NIOSH CIB 65 • Carbon Nanotubes and Nanofibers

Sample OC*

RPD or RSD†

(%) ECc‡

RPD or RSD 

(%) TC

RPD or 

RSD (%) Comments

Respirable 16.42 0.97 [1.87]§ 13.37 18.28 2.19 paired¶

Respirable 22.19 8.25 3.41 22.29 25.66 10.60 paired

Total 27.17 13.40 21.52 12.04 48.69 12.80 duplicate**

Respirable 60.87 0.74 79.59 12.14 140.31 6.36 duplicate

Respirable 25.47 4.46 20.72 8.48 46.09 6.28 duplicate

Total 12.42 6.84 4.14 4.59 16.60 4.88 triplicate††

Respirable 19.89 3.22 3.05 4.59 22.93 2.22 triplicate 

Total 15.11 1.29 9.89 9.37 25.01 3.63 triplicate 

Total 17.80 9.72 11.07 7.97 28.88 9.15 paired

Thoracic 27.16 10.80 11.23 6.79 38.46 6.68 triplicate 

Respirable 22.81 2.50 23.67 13.86 46.48 8.26 duplicate 

Respirable 18.64 6.77 8.44 3.15 27.14 5.63 duplicate

Table 1. NIOSH 5040 precision for air samples collected in different areas of a CNF manufacturing 

facility with 37-mm cassettes (total dust) and cyclone samplers (thoracic and respirable dust). OC, 

EC, TC are reported as air concentrations (μg C/m3).

*OC = organic carbon.

†

RPD is relative percent difference. RSD is relative standard deviation. 

‡

EC = elemental carbon.

§

Result in brackets lies between method LOD and LOQ.

¶

Results for two identical, paired samplers. 

**Duplicate analysis of same filter. 

††Triplicate analysis of same filter.

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