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# F o r e w o r d
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 hu man 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 informa tion 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 con trol. 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 nano technology 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 nano technology. Many of these nanoscale-based products are composed of engineered nanopar ticles, such as metal oxides, nanotubes, nanowires, quantum dots, and carbon fullerenes (buckyballs), among others. Early scientific studies have indicated that some of these na noscale 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 nano fibers (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 nano fiber; 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 pul monary 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 quantitative risk assessment based on animal dose-response data, (3) proposes a recom mended exposure limit (REL) of 1 ^g/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, includ ing 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  preliminary findings from a new laboratory study in which mice were exposed by inhalation to multi-walled carbon nanotubes (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 can cer in mice. This research is an important step in our understanding of the hazards associ ated 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. Carbon nanotubes (CNTs) and nanofibers (CNFs) are some of the most promising ma terials to result from nanotechnology. The introduction of these materials and products using them into commerce has increased greatly in the last decade . 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 re sponsibly to achieve that benefit . 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 recommenda tions. 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 . There are well established correlations between results of animal studies and adverse effects in workers ex posed to particulates and other air contaminants , and the effects, includ ing fibrosis, developed soon after exposure and persisted . 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  and five additional studies  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 ^g/m3 (8-hr time-weighted average  as respirable elemental carbon) is approximately 0.5% 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 ^g/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 ^g/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 deter mination 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 ^g/m3 and that an elevated exposure to elemental carbon in the workplace is a reasonable indicator of CNT or CNF exposure . Studies have also shown in some manufacturing operations that exposures can be controlled below the REL when engineering controls are used . 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 expo sures appears feasible with standard exposure control techniques (e.g., source enclosure, local-exhaust ventilation) . Previously in a 2010 draft of this CIB for public comment, NIOSH indicated that the risks could occur with exposures less than 1 ^g/m3 but that the analytic limit of quantification was 7 ^g/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 ^g/m3. More research is needed to fully characterize the health risks of CNT/CNF. Long-term ani mal studies and epidemiologic studies in workers would be especially informative. How ever, 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 ob tained, a recommended exposure limit might be based on a different exposure metric better correlated with toxicological effects, such as CNT/CNF number concentration .
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 di minish 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 ^g/m3 . 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 perspec tive, since there is no Occupational Safety and Health Administration (OSHA) permissible exposure limit (PEL) for CNT/CNF, the PEL for graphite (5,000 ^g/m3) or carbon black (3,500 ^g/m3)  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 vari ous types of CNT and CNF, purified or unpurified with various metal content, and different dimensions . 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) . 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 chemi cal 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 con trolled to less than 1 ^g/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 inha lation 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 insti tute 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 informa tion became available, specific medical screening recommendations would be forthcoming . 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 edu cation and training of workers and the use of personal protective equipment (e.g., respira tors, 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 ^g/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.

# B a c k g r o u n d
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 ef fects 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. Short term 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 (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 , (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 , and (3) reduced lung clearance in mice or rats exposed to relatively low-mass concentrations of CNT . Findings of acute pulmonary inflammation and interstitial fibrosis have also been observed in mice exposed to CNF . The extent to which these animal data may predict clinically significant lung effects in workers is not known. Howev er, 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 .
Some studies also indicate that CNT containing certain metals (nickel, 26%)  or higher metal content (17.7% vs. 0.2% iron) are more cytotoxic in vitro and in vivo . 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) . Other in vitro studies did not show evidence of genotoxicity of some MWCNT .
Studies in mice exposed to multi-walled carbon nanotubes (MWCNT) have shown the migration of MWCNT from the pulmonary alveoli to the intrapleural space 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 ^m in length), but not short MWCNT (< 1 ^m in length) or tangled CNT . In rats administered CNT by peritoneal injection, the pleural inflammation and mesothelioma were related to the thin diameter and rigid structure of MWCNT . 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 .
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 in crease plaque formation in an atherosclerotic mouse model Erdely et al. 2009]. Pulmonary exposure to MWCNT also depresses the ability of coronary arterioles to respond to dilators . These cardiovascular effects may be due to neurogenic signals from sensory irritant receptors in the lung. Mechanisms, such as in flammatory 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, includ ing electronics, lithium-ion batteries, solar cells, super capacitors, thermoplastics, poly mer 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  and CNF  indicate the potential for worker exposures in many tasks or processes and the reduction or elimination of expo sures when measures to control exposure are used. 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 in haled 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 clini cally 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 . 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 es timates of the BMD) have been extrapolated to humans by accounting for the factors influ encing 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 concentrations were calculated based on estimates of either the deposited or retained alveo lar 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 sub chronic animal inhalation studies with MWCNT , a working lifetime exposure of 0.2-2 ^g/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 stud ies (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 uncer tainty factors (Section A.6.3). The estimated human-equivalent working lifetime concentra tions based on this approach were approximately 4-18 ^g/m3 (8-hr TWA), depending on the subchronic study and the interspecies dose retention and normalization factors used. Divid ing 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 ^g/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  showed qualitatively similar lung response as in a shorter-term (28-day) study of CNF administered by pharyngeal aspiration in mice  (Sections 3.5 and A.7). Using the NOAEL-based approach, the humanequivalent working lifetime concentration estimates were 1-4 ^g/m3 (8-hr TWA), depend ing 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 ^g/m3 elemental carbon (EC) 8-hr TWA, which was set at the upper limit of quantitation (LOQ) for NIOSH Method 5040 . 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 ^g/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 ^g/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 ^g/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 stud ies with CNF are more limited , physical-chemical similarities between CNT and CNF and findings of acute pulmonary inflammation and interstitial fibrosis in animals exposed to CNF  indicate the need to also control occupational exposure to CNF at the REL of 1 ^g/m3 EC. Because of uncertainties in the risk estimates some residual risk for adverse lung effects may exist at the REL; there fore, efforts should be made to reduce airborne concentrations to CNT and CNF as low as 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 toxic ity all types of CNT and CNF should be considered a respiratory hazard and occupational exposures controlled at the REL of 1 ^g/m3. E x p o s u r e M e a s u r e m e n t a n d C o n t r o l s Occupational exposure to all types of CNT and CNF can be quantified using NIOSH Meth od 5040. A multi-tiered exposure measurement strategy is recommended for determin ing worker exposure to CNT and CNF . When exposure to other types of EC (e.g., diesel soot, carbon black) are absent or negligible, environmental background EC concentrations are typically < 1 ^g/m3 including in facilities where CNT and CNF are produced and used . Thus, an elevated airborne EC concentration relative to background (environmental and in non-process ar eas 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.  worker personal breath ing 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. , the outdoor air concentrations over four survey days, two months apart, were nearly identical, averaging about 0.5 ^g/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  (Section 2.1). Howev er, 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 . Control strategies and technologies developed by several indus trial 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 De tergence et des Produits d'Entretien (AISE) . 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 Chemi cals, has broad applicability to CNT and CNF processes and is available from ISPE . Finally, the Institute for Polyacrylate Absorbents (IPA) has developed guidelines for 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 . The extent to which worker exposure to CNT and CNF can be controlled below 1 ^g/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 con trols 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 (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 . Sampling methods have been developed to estimate the airborne mass concentration of respirable particles . 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.

# R e c o m m e n d a t i o n s
In light of current scientific evidence from experimental animal studies concerning the haz ard 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.

# . R e c o m m e n d a t i o n s f o r E m p l o y e r s
- 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 require ments of the OSHA Hazard Communication Standard  and the Hazardous Waste Operation and Emergency Response Standard .
- 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 ^g/m3 using NIOSH Method 5040 (Section 6 and Appendix C).
- Follow exposure and hazard assessment procedures for determining the need for and selection of proper personal protective equipment, such as clothing, gloves, and res pirators (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 facili ties before eating, smoking, or leaving the worksite.
- Provide facilities for showering and changing clothes, with separate facilities for stor age 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 protec tion standard  requires that a respiratory protection program be established that includes the following elements:
-A medical evaluation of the worker's ability to perform the work while wearing a respirator.
-Regular training of personnel.
-Periodic workplace exposure monitoring.
-Procedures for selecting respirators.
-Respirator fit testing.
-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 prac tices contained in this CIB should, at a minimum, be used when developing labels and Safety Data Sheets (SDS), as required .
1 .1 M e d i c a l S c r e e n i n g a n d S u r v e i l l a n c e
The evidence summarized in this document leads to the conclusion that workers occupation ally 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).

# W orker 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 ^g/m3 EC as an 8-hr TWA).
- Workers in areas or jobs that have been qualitatively determined (by the person charged with program oversight) to have the potential for intermittent elevated air borne 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).

# 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 expo sures, routes of exposure, and potential health effects related to CNT and CNF.

# Screening Elements
In i t ia l E v a lu a t io n
- An initial (baseline) evaluation should be conducted by a qualified health-care profes sional and should consist of the following:
-An occupational and medical history, with respiratory symptoms assessed by use of a standardized questionnaire, such as the American Thoracic Society Respiratory Questionnaire  or the most recent.
-A physical examination with an emphasis on the respiratory system.
-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.).
-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.
-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.).
- Evaluations should be conducted at regular intervals and at other times (e.g., post incident) as deemed appropriate by the responsible health-care professional based on data gathered in the initial evaluation, ongoing work history, changes in symp toms 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:
-An occupational and medical history update, including a respiratory symptom update, and focused physical examination-performed annually.
-Spirometry-testing less frequently than every 3 years is not recommended ; and -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:
-The individual worker's medical examination results.
-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:
-Any work or exposure restrictions based on the results of medical evaluations.
-Any recommendations concerning use of personal protective equipment.
-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.

# W orker 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 instruc tions 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 pro cedures involved.

# 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 P eriodic E valuations 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 regula tions and guidelines.
- Employers should periodically evaluate the elements of the medical screening pro gram to ensure that the program is consistent with current knowledge related to ex posures and health effects associated with occupational exposure to CNT and CNF.
Other important components related to occupational health surveillance programs, includ ing medical surveillance and screening, are discussed in Appendix B.

# . R e c o m m e n d a t i o n s f o r W o r k e r s
- 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 con tamination) by showering and changing into clean clothes at the end of each workday.  . CNF have lengths rang ing from tens of micrometers to several centime ters, 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 .
The synthesis of CNT and CNF requires a carbon source and an energy source . 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 homo geneity, and to reduce the synthesis temperature. The diameter of the fibers depends on the dimen sions 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, tempera ture, 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 common purification technique involves selec tive 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 purifica tion processes, purified CNT and CNF will exhibit differences in the content of trace elements and re sidual materials .
A growing body of literature indicates a potential health hazard to workers from exposure to various types of carbon nanotubes and nanofibers. A num ber 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 fi brosis. 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 ad ditional 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 sur face charge, CNT and CNF can be difficult to sepa rate 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 work ers 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 mi croscopy have shown both individual and agglom erated structures .
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 lim it (REL) for CNT and CNF is given to help minimize the risk of occupational respiratory disease in work ers as well as guidance for the measurement and control of exposures to CNT and CNF.

# P o t e n t i a l f o r E x p o s u r e
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 conductiv ity . 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 manufactur ing of CNT was estimated at 375, with a projected growth rate in employment of 15% to 17% annu ally. 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 work force involved in the fabrication or handling of CNT/CNF-enabled materials and composites is un known, but it is expected to increase as the market expands from research and development to indus trial high-volume production .   Maynard et al.  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 manufac turing 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 ^m in diameter) observed to decrease rapidly over time.
With no agitation, particles around 0.1 ^m in di ameter appeared to be released from the SWCNT material, probably because of the airflow across the powder. At the four manufacturing facilities, short term SWCNT mass concentrations were estimated (using a catalyst metal as the surrogate measure ment) to range from 0.7 to 53 ^g/m3 (area samples) in the absence of exposure controls. When samples were evaluated by scanning electron microscopy (SEM), most of the aerosolized SWCNT were ag glomerated, with agglomerated sizes typically larg er than 1 ^m.
Airborne particle and MWCNT concentrations were determined by Bello et al.  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 condensa tion 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 MW-CNT from the growth substrate (a process whereby MWCNT are removed from the substrate with a ra zor 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 expo sure to MWCNT.
The potential for airborne particle and SWCNT and MWCNT release was determined in a labora tory setting in which both types of CNT were pro duced using CVD . A qualitative assessment of the exposure (i.e., particle morphol ogy, aerosol size) was made during the synthesis of SWCNT and MWCNT in which modifications of the manufacturing methods were made to as certain how changes in the production of CNT in fluenced 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 concentra tions were determined to assist in quantifying the release of SWCNT and MWCNT during their syn thesis 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 concentra tions 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. Par ticle measurements made during the synthesis of MWCNT were found to peak at 4 x 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 par ticle concentrations and particle size were found to vary because of the temperature of the reactor, with higher particle concentrations and smaller particle 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 ef fective 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 dur ing their synthesis. Lee et al.  investigated the potential air borne 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, spray ing). 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 determin ing 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 deter mined 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 mat ter 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 alumi num) 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 (6,857 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 parti cles 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 nanomate rials (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 . 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 de termine the relative magnitude of airborne nano material emissions associated with tasks and ma terials 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 be fore and after the laboratory process. Samples were also collected for TEM analysis to verify the pres ence of CNMs. Airborne particle number concen trations 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 ^m. Airborne particle number con centrations 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 air borne area samples revealed agglomerates of all 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 . 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) . The purpose of the studies was to determine whether airborne exposures to these engineered nanomaterials oc cur and to assess the capabilities of various mea surement techniques in quantifying exposures. In a research and development laboratory handling CNF, airborne particle number concentrations (de termined 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 characteriza tion 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 concen trations were found during the manual scooping of CNF in the absence of exposure control measures. Samples collected for TEM particle characteriza tion 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 vacu um). Particle concentrations were determined by CPC (particle size 10-1000nm) and OPC (particle size 300-500nm, 500-100nm). CPC particle con centrations ranged from 1480-1580 particles/cm3 (weighing MWCNT in hood) to 2200-2800 parti cles/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 concen trations determined by OPC ranged from 700 particles/cm 3 (1-10 ^m 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 agglomer ated particles. Samples collected for TEM particle characterization indicated the aerosol release of ag glomerated MWCNT.
Subsequent studies conducted by NIOSH at six primary and secondary pilot or small-scale man ufacturing 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 concentra tions of CNT and CNF structures determined by TEM analysis . 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 manufactur ers of CNT. These processes and tasks consisted of:
(1) similar production and harvesting methods for CNT, and common cleaning/housekeeping proce dures in primary manufacturers, and ( 2 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 . 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 sur rounding 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 air borne 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 domi nated by the respirable (1-10 ^m) 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 cut ting 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 report ed 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 attrib uted to extensive damage of the protective guard around the rotary cutting wheel. Bello et al.  also investigated the airborne release of CNT and other nanosized fibers dur ing 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 col lected to determine exposures during the drilling of composite materials with local exhaust ventilation turned off. Four potential exposure-modifying fac tors were assessed: (1) by composite type, (2) drill ing rpm (low and high), (3) thickness of the com posite, and (4) dry versus wet drilling. Replicate test measurements (10-30 measurements) lasting  10 nm in diam eter. High aspect-ratio fiber concentrations were determined using the sizing and counting criteria in NIOSH Method 7400 (> 5 ^m long, aspect ratio > 3). Airborne exposure to both alumina fiber and CNT structures were found ranging in concentra tion 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 fi ber 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 com posites generated a higher frequency of nanofibers than had been previously observed during the cut ting of CNT-based composites . Cena and Peters  evaluated the airborne release of CNT during the weighing of bulk CNT and the sanding of epoxy nanocomposite sticks measuring 12.5 x 1.3 x 0.5 cm. Epoxy reinforced test samples were produced using MWCNT (Bay-tubes) with 10-50 nm outer diameters and 1-20 ^m 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 com posites. Airborne particle number and respirable mass concentrations were measured using a CPC (particle diameters 0.01 to 1 ^m) and OPC (par ticle diameters 0.3 to 20 ^m). Respirable mass con centrations 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 mea surements were taken under three LEV conditions (no LEV, a custom fume hood, and a biological safety cabinet). CPC and OPC particle concentra tions were measured inside a glove box in which bulk CNT (600 mg) was transferred between two 50-ml beakers; background particle concentra tions 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 dur ing the weighing process contributed little to that observed in background samples (process to back ground ratio  = 1.06), however it did influ ence the mass concentration (P/B = 1.79). The GM respirable mass concentration inside the glove box was reported as 0.03 ^g/m3 (background GM was 0.02 ^g/m3). During the sanding process (including no LEV, in a fume hood, and in a biological safety cabinet) the PBZ nanoparticle number concentra tions were negligible compared with background concentrations (P/B ratio = avg. 1.04). Particles gen erated during sanding were reported to be predomi nantly micron sized with protruding CNT and very different from bulk CNT that tended to remain in large (>1 ^m) 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 ^g/m3) compared with those with no LEV (GM was 2.68 ^g/m3) or those when sand ing was performed inside the fume hood (GM = 21.4 ^g/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.   Some research has been conducted to date on workplace exposure to carbon nanofibers (CNF) . In a NIOSH health hazard eval uation 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 pho tometer) . General area ex posure 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 ma terial. Airborne total carbon mass concentrations (per NIOSH Method 5040, with correction for adsorbed vapor) within the laboratory process ing area were 2 to 64 times higher than those of a nearby office area, with the highest peak exposure concentration (1094 ^g/m3) found during the wet saw cutting of the CNF composite material. No in door particle concentrations exceeded the outdoor background concentrations. Particles having a di ameter 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 weigh ing and mixing of CNF. Airborne samples collected directly on TEM grids were analyzed for the pres ence of CNF. Some fibers observed by TEM had diameters larger than the 100 nm criterion used to define a nanofiber, which was consistent with re sults reported by Ku et al. , in which the mo bility 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, rath er than single fibers, which was in general agree ment 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   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 concen trations 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 concentra tions exceeding 1.1 mg/m3 for transfer or dump ing and 0.5 mg/m3 for bag change-out. The authors concluded that integrated particle number and ac tive surface area concentrations (i.e., using CPC and diffusion charger) were not useful in assess ing the contribution of emissions from CNF in the workplace, because measurements were dominated by ultrafine particle emissions .
Respirable particle mass concentrations estimated by the photometer appeared to be the most useful and practical metric for measuring CNF when us ing direct-reading instruments. Results obtained for filter samples support the direct-reading instru ment findings. The TEM analyses of size-selective area samples indicated that large fiber bundles were present . In addition, sizeclassified samples (collected with impactors) ana lyzed for EC (by NIOSH 5040) indicated CNF par ticles in the micrometer range .
F ig u re 2 -1 . Workplaces and job tasks with potential for occupational exposure to carbon nanotubes and nanofibers. Adapted from Schulte et al. 2008. Various types of laboratory animal studies have been conducted with CNT and CNF using differ ent routes of exposure to evaluate potential toxic ity (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 materi als closely resemble the dimensions of some min eral 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 . 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 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 contaminat ing catalytic metals are frequently removed by high temperature vaporization or acid/base treat ment . Researchers  have measured the durability (in vitro) of four types of CNT, one type of glass wool fiber, and two asbestos fiber types in simu lated biological fluid (Gambles solution) followed by an assessment of their ability to induce an in flammatory response when injected into the ab dominal 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 struc tures, whereas the other CNT, which was less dura ble and had shorter structures and/or formed tight bundles, caused minimal inflammation. The find ings 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 per centage of long fibers, and the glass wool fiber that was not very durable and caused only minimal in flammation.
The physical-chemical properties (e.g., dimen sion, 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 chang ing their surface chemistry. Toxicological effects of such changes remain largely unexplored  except for some limited evidence indi cating that structural defects , surface  and oxidative modification , nitrogen doping , surface functionalization , and polymer (acidand polystyrene-based) surface coating  of CNT can influence their toxicity po tential. Recent studies indicate that functionalization of MWCNT with -COOH groups significantly decreases the inflammatory and fibrotic response after aspiration in a mouse model  and when SWCNT were functionalized by carboxylation and subjected to phagolysosomal fluid, longitudinal splitting and oxidative degradation of the tubes occurred . Kagan et al.  reported that in vitro myeloperoxidase, which is found in high concentrations in polymorphonuclear neutrophils (PMN), degraded SWCNT. However, it is uncer tain as to whether PMN-derived myeloperoxidase would degrade SWCNT in vivo (e.g., in the lung) because of the following: (1) PMN recruitment af ter SWCNT exposure is a transient rather than per sistent 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 .
Several animal studies have shown that the size (e.g., length) of MWCNT and SWCNT may have an effect on their biological activity . Intraperitoneal injection of mice with long MWCNT (20 ^m length), but not short MWCNT ( 5 ^m in length . However, when rats were ex posed to short MWCNT (< 1 ^m length) by intra peritoneal injection, only acute inflammation was observed, with no evidence of mesothelioma over the 2 year post-exposure period . Nagai et al.  provided evidence that the car cinogenic 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 muta tion to tumor suppressor genes (Cdkn2a/2b) was observed in the mesotheliomas, which is similar to that observed in asbestos-associated mesothe liomas 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 . Other in vitro studies of some MWCNT did not show evi dence of genotoxicity . The presence of residual metal catalysts was also found to promote the generation of reac tive oxygen species (ROS), thereby enhancing the potential for DNA damage . The results from in vitro studies with CNF have also shown that exposure can cause genotoxicity  including aneugenic as well as clastogenic events. In addition, low-dose, long term exposure of bronchial epithelial cells to SW-CNT or MWCNT has been reported to transform these cells to exhibit unregulated proliferation, loss of contact inhibition of division, enhanced migra tion and invasion, and growth in solf agar . When SWCNT-transformed epithe lial 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 dys function .
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, intraperito neal injection, or by pharyngeal aspiration (a tech nique where particle deposition closely resembles inhalation). The agglomerate size for CNT and CNF is normally smaller in a dry aerosol than when sus pended in physiological media. Evidence from tox icity studies in laboratory animals indicates that decreasing agglomerate size increases the pulmo nary response to exposure . The extent to which ag glomerates 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 . Mice or rats exposed to SWCNT by IT or pharynge al aspiration have developed granulomatous lesions at sites in the lung where agglomerates of SWCNT deposited . In addition, interstitial fibrosis has also been reported . This fibrotic response was associated with the migration of smaller SWCNT structures into the interstitium of alveolar septa .
3.1.1 IT Studies Lam et al.  investigated the toxicity of SW CNT 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.  exposed rats via IT to concen trations 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 ex posure. The SWCNT were reported to have nomi nal diameters of 1.4 nm and lengths > 1 ^m, which tended to agglomerate into micrometer size struc tures. In this study, ~15% of the SWCNT-instilled rats died within 24 hours of SWCNT exposure, ap parently due to SWCNT blockage of the upper air ways. In the remaining rats, a transient inflamma tory 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 de veloped a dose-dependent lung inflammatory re sponse that persisted through 3 months. Exposures to carbonyl iron or graphite particles produced no significant adverse effects.

# 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 ^g/mouse . As with studies by Lam et al.  and Warheit et al. , 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 fibro sis 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. , in which rats exposed to 2 mg/kg via pharyngeal aspiration developed gran ulomas at sites of SWCNT agglomerates and dif fuse interstitial fibrosis at 21 days post exposure. Also noted was the formation of CNT structures bridging alveolar macrophages, which may affect normal cell division and/or function. When a more dispersed delivery of SWCNT was given by aspira tion to mice (10 ^g) , an acceler ated 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 submicrom eter 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. Shvedova et al.  compared the responses re sulting from exposure via pharyngeal aspiration  with exposure via inhalation of more-dispersed SWCNT . One set of mice were exposed by inhalation to 5 mg/m3, 5 hr/day for 4 days, while mice exposed by aspira tion were given a single dose of 10 or 20 ^g. 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 ) of 508 m2/g. Both studies reported acute lung inflammation followed by the develop ment of granulomatous pneumonia and persistent interstitial fibrosis; these effects were observed for both purified (0.2% Fe)  and unpurified (17.7% Fe)  SWCNT. The finding that the acute lung inflam mation resolved after the end of exposure while the pulmonary fibrotic response persisted or pro gressed 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 . Quantitatively, mice exposed by in halation (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  is relevant, because the Occupational Safety and Health Administration (OSHA) permis sible exposure limit (PEL) for respirable synthetic graphite of 5 mg/m3 is sometimes used for control ling workplace exposures to CNT.

# Inhalation Studies

# Pharyngeal Aspiration Studies
Exposures to well-dispersed MWCNT in mice via pharyngeal aspiration have resulted in dose-and time-dependent pulmonary inflammation , as well as central nervous system effects , at doses rang ing from 10 to 80 ^g/mouse. Exposure of mice to dispersed suspension of purified MWCNT at doses of 10, 20, 40, or 80 ^g resulted in pulmonary in flammation and damage, granulomas, and a rapid and persistent fibrotic response . Morphometric analyses indicated that the intersti tial fibrotic response was dose-dependent and pro gressed through 56 days post-exposure . There was also evidence that MWCNT can reach the pleura  and that alveo lar macrophages containing MWCNT can migrate to the lymphatics and cause lymphatic inflamma tion . Some of the MWCNT (mean diameter of 49 nm and mean length of 4.2 ^m) were observed penetrating the outer lung wall and entered the intrapleural space Mercer et al. 2010]. Morphometric analyses indicated that 12,000 MWCNT entered the intra pleural space at 56 days post-exposure to 80 ^g of MWCNT .

# IT Studies
Lung inflammation and fibrosis have also been observed in rats exposed by IT to long (5.9 ^m) or short (0.7 ^m) MWCNT at doses of 0.5, 2, or 5 mg of either ground or unground MWCNT and examined up to 60 days post-exposure . Rats that received ground MWCNT (0.7 ^m) 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 ^m), 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 re sponse as chrysotile asbestos and a greater response than ultrafine carbon black .
Similar findings have been reported by Aiso et al. , in which rats exposed to IT doses of 0.04 and 0.16 mg of dispersed MWCNT (mean length-5 ^m, diameter-88 nm) caused transient inflammation, and persistent granulomas and al veolar wall fibrosis. These acute effects have also been reported in guinea pigs at IT doses of 12.5 mg  and 15 mg ; in mice at doses of 0.05 mg (average di ameter of 50 nm, average length of 10 ^m) ], and at 5, 20, and 50 mg/kg ; and in rats  dosed at 1, 3, 5, or 7 mg (diameters of 40 to 60 nm, lengths of 0.5 to 5 |im). In contrast, Elgrabli et al.  reported cell death but no histopathological lesions or fibrosis in rats ex posed at doses of 1, 10, or 100 ^g MWCNT (diame ters of 20 to 50 nm, lengths of 0.5 to 2 ^m). Likewise, Kobayashi et al.  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 . Four groups of six rats each were given 0.5 ml of 500 ^g 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 medias tinal 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.

# Inhalation Studies
Several short-term inhalation studies using mice or rats have been conducted to assess the pulmonary  and systemic immune effects  from exposure to MWCNT. Mitchell et al.  report ed 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 ^m) at concentra tions 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 ). 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, al though without a clear concentration-response rela tionship. Mitchell et al.  reported that the im munosuppression mechanism of MWCNT appears to involve a signal originating in the lungs that acti vates cyclooxygenase enzymes in the spleen. Porter et al.  reported significant pulmonary inflam mation 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 ^m, count aerodynamic diameter of 0.4 |im). In addition, gran ulomas 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 |im) for 6 hours, a high incidence (9 of10 mice) of fibrotic lesions occurred . MWCNT were found in the subpleural region of the lung 1 day post exposure, with sub pleural 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 na noscale 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 . Ma-Hock et al.  reported on the results of a 90day 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 ^g/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 ex posure 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  con ducted a short-term inhalation bioassay (before the Pauluhn 2010a subchronic study) to investi gate 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 inha lation exposure of 6 hr followed by a post-expo sure 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 inflam mation during the early phase of the study. The pulmonary inflammation induced by quartz in creased during the 3 months post-exposure peri od, 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 , using the same MWCNT (0.53% cobalt) used in the study by Ellinger-Ziegelbauer and Pauluhn  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 high ly agglomerated (mean diameter of 3 ^m). 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., in flammation 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. . 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  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. , Ellinger-Ziegelbauer and Pauluhn  and Pauluhn . 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.

# . S W C N T a n d M W C N T
I n t r a p e r i t o n e a l S t u d i e s Intraperitoneal injection studies in rodents have been frequently used as screening assays for poten tial 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  have been conducted to investi gate the hazard potential of various sizes and doses of MWCNT and SWCNT to cause a carcinogenic response. Takagi et al.  reported on the in traperitoneal 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 ^m 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.  reported that the peritoneal (abdominal) injection of long MW-CNT-but not short MWCNT-induced inflam mation and granulomatous lesions on the abdomi nal side of the diaphragm at 1 week post-exposure. This study, in contrast to the Takagi et al.  study, used wild type mice exposed to a much low er dose (50 ^g) of MWCNT. Although this study documented acute inflammation, it did not evalu ate whether this inflammation would persist and progress to mesothelioma. Murphy et al.  found similar findings in C57BI/6 mice that were in jected with different types of MWCNT composed of different tube dimensions and characteristics (e.g., tangled) or injected with mixed-length amosite as bestos. Mice were injected with a 5 ^g dose directly into the pleural space and evaluated after 24 hours, 1, 4, 12, and 24 weeks. Mice injected with long (> 15 ^m) MWCNT or asbestos showed significantly increased granulocytes in the pleural lavage, com pared with the vehicle control at 24 hours post ex posure. 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 ^m) and tangled MWCNT did not cause a persistent in flammatory response and were mostly cleared from the intrapleural space within 24 hours.
A lack of a carcinogenic response was reported by Muller et al.  and Varga and Szendi  in rats, and by Liang et al.  in mice, follow ing 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  or MWCNT (phosphorylcholine-grafted) in mice when given daily doses of either 10, 50, or 250 mg/kg and evaluated at day 28 . However, the MWCNT sam ples used in the Muller et al.  and Liang et al.  studies were very short (avg. < 1 ^m in length observed by Muller et al.  and < 2 ^m in length observed by Liang et al. ), and the findings were consistent with the low biological activity observed in the Poland et al.  study when mice were exposed to short MWCNT. Varga and Szendi  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 diam eters x 4-15 ^m lengths), MWCNT (10-30 nm di ameters x 1-2 ^m lengths), or crystalline zinc oxide (negative control) were implanted into the perito neal 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 disper sion in the peritoneal given the tendency of CNT to agglomerate. If SWCNT and MWCNT remained agglomerated following delivery, this may have resulted in the lack of a mesothelioma-inducing ef fect. The low biological activity observed for the short MWCNT sample (< 2 ^m) used in the study was consistent with the findings from Poland et al.
, Muller et al. , and Liang et al. , in which short MWCNT were also used.   reported that multiple aspirations of SWCNT (20 ^g/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 ^g) resulted in a 92% de pression of the responsiveness of coronary arte rioles to dilators 24 hr post-exposure , while aspiration of MWCNT has been shown to increase baroreflex activity in rats . Further more, pharyngeal aspiration of MWCNT (80 ^g/ 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 . Several mechanisms have been suggested to explain these systemic responses:

# Translocation of CNT to Systemic Sites
Translocation of intraperitoneal instilled MWCNT from the abdominal cavity to the lung has been re ported ; however, there is no evi dence that the reported systemic effects are associ ated 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 . Pulmonary exposure to particles causes localized inflammation at the sites of particle deposition in the alveoli. Erdely et al.  reported that aspira tion of SWCNT or MWCNT (40 ^g/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 post exposure, but not at later times. Pulmonary CNT exposure also significantly elevated gene expres sion for mediators, such as Hif -3a and S100a, in the heart and aorta at 4 hr post-exposure. Evidence also exists that pulmonary exposure to particles al ters systemic micro-vascular function by potentiat ing PMN as they flow through pulmonary capillar ies 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 . Therefore, less dilator-induced NO diffuses to vascular smooth muscle resulting in less dilation.

# . 5 C a r b o n N a n o f i b e r s ( C N F )
Recent observations indicate that exposure to CNF can cause respiratory effects similar to those ob served in animals exposed to CNT . In this study, female mice were exposed by pharyngeal aspiration to SWCNT (40 ^g), CNF (120 ^g) or crocidolite (120 ^g) and evaluated post exposure at 1, 7, and 28 days. Delivered structure number or particle surface area at the highest doses were 1.89 x 106 and 0.042 m2 for SWCNT, 4.14 x 106 and 0.05 m2 for CNF, and 660 x 106 and 0.001 m2 for asbestos. SWCNT and CNF were purified and con tained 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 ^m whereas the diameters of CNF ranged from 60 to 150 nm and lengths approximately 5 to 30 ^m. The fiber lengths of asbestos ranged from 2 to 30 ^m. On a mass dose bases, inflammation and lung damage at 1 day post-exposure followed the potency sequence of SWCNT>CNF>asbestos.

# System ic Inflam m ation
The same potency sequence was observed for TNF and IL-6 production at 1 day post-exposure. SW CNT 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 se quence of SWCNT>CNF=asbestos. The potency sequence for fibrosis was not found to be related to structure number or particle surface area (deter mined by BET gas absorption method) delivered to the lung. However, it is likely that gas absorp tion overestimates the surface area of agglomerated SWCNT structures delivered to the lung. Estimates of effective surface area, based on geometrical anal ysis of structures including agglomeration, provid ed 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. . 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 concentra tions 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 us ing NIOSH Method 7400. At an aerosol concen tration of 0.54 mg/m3 the fiber concentration was 4.9 fibers/cc with a MMAD of 1.9 ^m (GSD 3.1), at 2.5 mg/m3 the concentration was 56 fibers/cc with a MMAD of 3.2 ^m (GSD 2.1), and at 25 mg/m3 the concentration was 252 fibers/cc with a MMAD of 3.3 ^m (GSD 2.0). The mean lengths and diameters of fibers were 5.8 ^m and 158 nm, respectively with surface area measurements (by BET) of 13.8 m2/g. At 1-day post exposure wet lung weights were sig nificantly 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 ter minal 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 inflamma tion 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 ex posure 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 macro phages still persisted in the lung of rats exposed to 25 mg/m3 with the inflammatory response report ed 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 ex posed at 25 mg/m3 causing a nonspecific inflamma tory response. In contrast to Murray et al. , no fibrosis was noted in this inhalation study. The most likely reason for this discrepancy is a differ ence in alveolar lung burden between the Murray et al.  and the DeLorme et al.  study.
In the former, the lung burden was 120 ^g/mouse lung. In contrast, lung burden was not reported or estimated in the DeLorme et al.  rat study. However, with a MMAD as large as 3.3 ^m, nasal filtering would be expected to be high and alveolar deposition relatively low.         Ma-Hock et al.  Porter et al.  Sriram et al.  Mice Inhalation  Ellinger-Ziegelbauer  Rats Nose-only inhalation 11 and 241 mg/m3 for 6 hr (eval: at days 7, 28, and 90)

# C o n c l u s i o n s -H a z a r d a n d E x p o s u r e A s s e s s m e n t
Results of laboratory animal studies with both SWCNT and MWCNT report qualitatively similar pulmonary responses including acute lung inflam mation, epithelioid granulomas (microscopic nod ules), and rapidly developing fibrotic responses at relatively low-mass doses (Section 3). Animal stud ies 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 .
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. 2005Shvedova et al. , 2008.
3. Equal or greater potency of SWCNT, MWCNT, and CNF compared with other inhaled par ticles (ultrafine carbon black, crystalline silica, and asbestos) in causing adverse lung effects including pulmonary inflammation and fibro sis .
4. CNT agglomeration affects the site of lung de position and response; large agglomerates tend to deposit at the terminal bronchioles and proximal alveoli and induce a granulomatous response, while more dispersed structures de posit in the distal alveoli and cause interstitial fibrosis . Agglomerated SWCNT tend to induce granu lomas, while more dispersed CNF and asbestos did not .

# Intraperitoneal injection of long (> 5 ^m)
MWCNT in mice causes fibrotic lesions and mesothelial cell proliferation .
Although pulmonary responses to SWCNT and MWCNT are qualitatively similar, quantitative differences in pulmonary responses have been re ported. In mice exposed to CNT by pharyngeal as piration (10 ^g/mouse), SWCNT caused a greater inflammatory response than MWCNT at 1 day post exposure . 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) , and that 90% of the dispersed SWCNT structures have been ob served to cross alveolar epithelial cells and enter the interstitium . 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 , possibly due to the greater tube count per mass of SWCNT . In addition, although both SWCNT and MWCNT have been reported in the subpleural tissue of the lung , penetration of the visceral pleura and trans location to the intrapleural space has been reported only for MWCNT . 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 rele vant to potential worker exposures.
Animal studies have also shown asbestos-type pa thology associated with the longer, straighter CNT structures . Mesothelial tumors have been reported in mice receiving intraperitoneal injection of long MWCNT (5-20 ^m in length) ; whereas chronic bioas says of short MWCNT (avg. < 1 ^m and < 2 ^m in length, respectively)  did not produce mesothelioma. These find ings are consistent with those reported by Yamashita et al.  and Nagai et al.  who found that MWCNT injected into the peritoneal cavity of mice or rats generated inflammation/genetic dam age and mesothelioma that were related to the di mension of the CNT. Results from these peritoneal assay studies indicate that CNT of specific dimen sions and durability can cause inflammation, fi brosis, 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 patho genic 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 , and when human epidermal keratinocytes cells are ex posed to unpurified SWCNT (in vitro cellular stud ies), oxidant injury occurs . These catalytic metals can be removed from raw CNT by acid treatment or by high temperature to yield purified CNT with low metal content. Re moval of catalytic metals abolishes the ability of SWCNT or MWCNT to generate hydroxyl radi cals. However, in laboratory animal studies the pul monary bioactivity of SWCNT does not appear to be affected by the presence or absence of catalytic metals. Lam et al.  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 contamina tion. Likewise, the acute inflammatory reaction of mice after aspiration of raw (30% iron) versus puri fied (< 1% iron) SWCNT was not affected by metal content [Shvedova et al. 2005[Shvedova et al. , 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 Erdely et al. 2009]. Pulmonary exposure to MWCNT also depresses the ability of coronary arterioles to respond to dilators . These cardiovascular effects may be due to neurogenic signals from sensory irritant receptors in the lung. Mechanisms, such as inflammatory signals or neu rogenic 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 forma tion of anaphase bridges among the nuclei . In vitro studies also indicate that expo sure to CNF can cause genotoxicity (micronuclei) as a result of reactive oxygen species (ROS) pro duction, which in turn reacts with DNA, and by interfering physically with the DNA/chromosomes and/or mitotic apparatus . 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 .
Currently, there are no studies reported in the liter ature on the adverse health effects in workers pro ducing 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 expo sures (e.g., lung burden/alveolar epithelial cell sur face) 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 observed to be task-specific and short-term in du ration, with exposure concentrations (frequently reported as particle number or mass concentra tions) found to exceed background exposure mea surements when appropriate engineering controls are not used to reduce exposures . Results from stud ies 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. Comprehen sive 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., pul monary 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 MW CNT into the intrapleural space could potentially initiate mesothelial injury and inflammation that over time cause pleural pathology, including meso thelioma. 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 accu mulation in the intrapleural space with time after inhalation requires further investigation. Until re sults from animal research studies can fully explain the mechanisms in which inhalation exposure to CNT and CNF cause adverse lung effects and pos sible 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 bases its recommended exposure limits (RELs) on quantitative risk assessments when pos sible. Quantitative risk assessment provides esti mates of the severity and likelihood of an adverse response associated with exposure to a hazardous substance. The hazard and quantitative risk assess ments (Section 4 and Appendix A) provide the health basis for developing a recommended expo sure limit (REL) for CNT and CNF. Establishing health-based exposure limits is the first consider ation by NIOSH in setting a REL. The analytical feasibility of measuring worker exposures to air borne 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 corre sponding to a small increase in response (e.g. 10%) over the background level of response . 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 life time 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 . Variability in human dose and response, including sensitive subpopulations, and uncertain ty 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 pulmo nary 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 . Human-equivalent risk estimates were derived from animal dose-response data (in rats and mice). Human-equivalent expo sures over a 45-year working lifetime were estimat ed 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 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 work ers are potentially at risk of developing adverse lung effects if exposed to airborne CNT during a 45-year working lifetime.  Minimal lung effects (grade 1 or higher) 0.5 to 4 ^g/m3 0.2 to 2 ^g/m3
Slight or mild lung effects (grade 2 or higher) 1 to 44 ^g/m3 0.7 to 19 ^g/m3 Abbreviation: TWA=Time-weighted average. *Excess (exposure-attributable) risk during a 45-year working lifetime. tHistopathology findings of granulomatous inflammation  or alveolar septal thickening  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). 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  or alveolar septal thickening  in rat subchronic inhalation studies of multiwall carbon nanotubes. fExposure-attributable risk (added risk above background). Estimates vary by rat study and lung burden estimation method (Appendix A, Tables A-7 and A-8).
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 ^g/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 gen der, route of exposure, and response endpoints); yet all of these estimates are relatively low airborne mass concentrations, most within ~1 -10 ^g/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). Among the uncertainties in this risk assessment using animal data, there is uncertainty in extrapo lating the respiratory effects observed in short term or subchronic animal studies to estimate the probability of chronic respiratory effects in humans. In the absence of chronic data, these ani mal 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 ex tent 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 re sponse proportions, these estimates are similar to the NOAEL and LOAEL values reported in these studies  (Table A -12).
One of the measures of pulmonary fibrosis used in the shorter-term studies -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 "fun damental structural remodeling" .
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 re sponse at a given mass dose) to that of ultrafine car bon black, crystalline silica, or chrysotile asbestos . 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.    Based on currently available data, it is difficult to as sess 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 expo sure 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 expo sure 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 com mon exposure metric currently used in monitoring workplace exposures to CNT and CNF. The REL is based on the respirable particle-size fraction be cause the adverse lung effects in the animal stud ies 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 . Sampling methods have been devel oped to estimate the airborne mass concentration of respirable particles   One of the earliest OELs for CNT was proposed by the British Standards Institute -the benchmark exposure limit (BEL) of 0.01 fiber/cm3, or one-tenth of their asbestos exposure limit (Table 5-5). Nanocyl  derived an estimated OEL of 2.5 Mg/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 Ma-Hock et al.  subchronic rat inhalation study of MWCNT. Aschberger et al.  pro posed OELs of 1 Mg/m3 for MWCNT studied by Ma-Hock et al.  and 2 Mg/m3 for MWCNT from Pauluhn , by adjusting 0.1 mg/m3 (the LOAEL in Ma-Hock et al.  and the NOAEL in Pauluhn ) for rat-to-human daily expo sure and respiratory volume, and applying an over all assessment factor of 50 and 25, respectively.
Pauluhn  derived an OEL using subchronic data in rats inhaling MWCNTs (Baytubes) . This approach was based on the bio logical mechanism of volumetric overloading of al veolar macrophage-mediated clearance of particles from the lungs of rats . 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  reported benchmark concentration (BMC) estimates of 0.16 to 0.78 mg/ m3 for rat lung responses of pulmonary inflamma tion and increased collagen, but selected the lower NOAEL of 0.1 mg/m3 to derive a human-equivalent concentration. The NOAEL was adjusted for hu man 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 spe cies). 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 Indus trial Science and Technology (AIST) derived an OEL for CNT of 30 Mg/m3 , 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 inhala tion studies of SWCNT and MWCNT . Human-equivalent NOAELs were estimated by accounting for rat and human differ ences in exposure duration, ventilation rate, particle deposition fraction, and body weight . 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 fac tor 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 vari ability). 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 Na kanishi 
). 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 sur face area of ~1,000 m2/g (which was noted would be more protective for other CNTs with lower spe cific 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 re viewed again within that timeframe with further data .
In summary, these currently proposed OELs for CNT range from 1 to 50 Mg/m3 (8-hr TWA con centration) , including the NIOSH REL of 1 Mg/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 cur rent U.S. OELs for graphite or carbon black are ap proximately 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 con centrations (Mg/m3) to protect workers' health. Nanocyl  2.5 ^g/m3 (8-hr TWA) for MWCNT Adjusted rat LOAEL of 0.1 mg/m3 (subchronic inhalation)  to workers and applied assessment factor of 40.  Animal data have been used to evaluate the health hazard and risk of occupational exposure to CNT and CNF. Limited human exposure data are avail able, and NIOSH is not aware of any studies or re ports at this time of any adverse health effects in workers producing or using CNT or CNF. The best available scientific information to develop recom mended 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 likeli hood (risk) of adverse effects in workers. Ideally, sufficient evidence is desired to derive exposure limits that are estimated to be associated with es sentially 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 uncer tainty, and the extent to which use of those data are useful for occupational health risk management decision-making, is an important step in risk as sessment. Alternative models and methods con tribute to the differences in the risk estimates, and there is uncertainty about which biological end points, animal models, and interspecies and dose rate extrapolation methods may be most predictive of possible human health outcomes. These uncertainties can result in either under-esti mation or over-estimation of the true health risk to workers at a given exposure scenario. Each of these areas is discussed further below.

# 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 de termined are relevant to human health risk assess ment (Section A.2.1.3). The extent to which these lung responses would be associated with functional deficits in animals or clinically significant effects in humans is uncertain. However, these lung responses include early onset fibrosis which persisted or pro gressed after the end of exposure .
Limited evidence in animals suggests that these effects may be associated with some lung function decrement (reduced breathing rate in mice) . A quantitative measure of pul monary fibrosis-alveolar interstitial (septal, or connective tissue) thickening [Shvedova et al. 2005[Shvedova et al. , 2008 (

# 2) Dose rate and retention
Appendix A-6 provides some analyses to show the quantitative influence of dose rate and lung reten tion 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  clearance model, and ~10 times higher than that assuming simple first-order kinetics . 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  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

# Minor Areas of Uncertainty
(1) Effect level estimation
An effect level is a dose associated with a speci fied effect (or lack of observed effect). A BMD is a statistical estimate of an effect level . 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 differenc es among studies).
In the absence of CNT-specific lung models, stan dard 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 pos sible lung burden estimates. The effect of assum ing deposited dose (no clearance) versus retained
(3 ) R a t lu n g dose e s tim a tio n dose (normal clearance) resulted in a difference of approximately five-fold. However, the actual es timates 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).  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 pro cesses with the highest potential for exposure.
The strategy should also incorporate provisions to quantify the airborne release of CNT and CNF oc curring at specific processes or job activities to pro vide "activity pattern data" .
Activity pattern data are useful for identifying possible causes of high exposure for remediation; however, these data are vulnerable to spatial varia tion in exposure concentrations and should not be used in predicting worker exposures. For example, in the study by Birch et al. , personal expo sure to CNF was much higher than area samples, depending on location. Respirable EC exposure for two employees working mainly in a thermal treat ment area was approximately 45 ^g/m3, and a CNF reactor area was about 80 ^g/m3, while the corre sponding area samples were about 32 ^g/m3 in the thermal treatment area and 13 ^g/m3 in the CNF reactor area. The EC concentration in the reactor area was less than half that in the thermal treat ment 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 estab lish whether a valid extrapolation is possible . NIOSH  and others  have developed exposure as sessment 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 . 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 ini tially target similarly exposed groups of workers . This initial sampling effort may be more time ef ficient 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 expo sure estimates if the exposures are highly variable . Therefore, repeated measure ments on randomly selected workers may be re quired to account for between-and within-worker variation in exposure concentrations . 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 assess ment of worker airborne exposure concentrations to CNT and CNF.

# CNT and CNF M easurem ent
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). Ini tially, more samples may be required to characterize the workplace thoroughly. This initial assessment will help refine the sampling approach and deter mine whether EC interference is an issue. Careful consideration of environmental background is es sential. 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 ini tial evaluation of a worker's exposure should include the simultaneous collection of a personal respirable EC sample and a personal sample for electron mi croscopy analysis (e.g., TEM, SEM). Electron mi croscopy analysis, in conjunction with energy dis persive x-ray spectroscopy (EDS), can be used for CNT and CNF identification. In addition, consider ation should be given to the sizing and counting of CNT and CNF structures during electron micros copy analysis should future efforts to control occu pational 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 1999ISO , 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

# M ethod 5040 Limit of Detection
As with all analytical methods, the LOD is a vary ing number. However, the airborne EC LOD origi nally reported for NIOSH Method 5040 (i.e., about 2 ^g/m3), or an LOQ of 7 ^g/m3 was a high estimate . 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 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      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 ^g/cm2. Expressed as an air concen tration, the EC LOD (^g/m3) corresponding to the EC LOD (^g/cm2) determined with media blanks (i.e., LOD = 3 times the standard deviation for the blanks) can be calculated by the following equation:
EC LOD (figfm ) = EC LOD (ptgfcm ^ ) x deposit area fa n ^ J
This equation explains why a lower LOD (^g/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 ^g/cm2). The LOD is improved using a smaller filter size since the deposit density is higher for an equivalent mass deposited. The same ap plies to the LOQ, commonly defined as 10 times the standard deviation (SD) for the blanks, or 3.3 times the LOD.
If 0.02 ^g EC/cm2 is taken as the SD for media blanks (with manual OC-EC split adjustment), the LODs and LOQs (in ^g EC/m3) for different 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 ^g 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 ^g/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 ^g/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 in strument's small (quartz tube) oven and need for proper sample alignment limit the amount of sam ple that can be analyzed.

# . 3 W o r k e r E d u c a t i o n a n d T r a i n i n g
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 imme diate 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 neces sary skills to perform their job safely, and (4) there 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)  A program for educating workers should also in clude both instruction and "hands-on" training that addresses the following:
- The potential health risks associated with ex posure 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.

# . 4 C l e a n u p a n d D i s p o s a l
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 in halation exposure during cleanup will be influenced by the likelihood of CNT and CNF becoming air borne, 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 in formation on exposure risks. Given the limited amount of data on dermal expo sure to CNT and CNF, it would be prudent to wear protective clothing and gloves when - all technical measures to eliminate or con trol 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 prevent ing CNT and CNF exposure to abraded or lacer ated 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 . 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 cover alls, and firefighter ensemble insulation . 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 pre vent dermal exposure to nanoparticles varies de pending on the glove material, its thickness, and the manner in which it is used (e.g., long exposure times, other chemical exposures) , 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 respira tor filters would be effective at capturing airborne CNT and CNF [Balazy et al. 2006;Rengasamy et al. 2007Rengasamy et al. , 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[Rengasamy et al. , 2009.

# . 7 M e d i c a l S c r e e n i n g a n d S u r v e i l l a n c e
The toxicological evidence summarized in this document leads to the conclusion that workers oc cupationally 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) .

# 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 ex posed to airborne CNT or CNF at concentra tions above 1 Mg/m3 EC as an 8-hr TWA).

# Program Oversight
Oversight of the medical surveillance program should be assigned to a qualified health-care profes sional who is informed and knowledgeable about potential workplace exposures, routes of exposure, and potential health effects related to CNT and CNF.

# Screening Elements

# I n i t ia l e v a lu a t i o n
- An initial (baseline) evaluation should be conducted by a qualified health professional and should consist of the following:
-An occupational and medical history with respiratory symptoms assessed by use of a standardized questionnaire such as the American Thoracic Society Respi ratory Questionnaire , or the most recent equivalent.
-A physical examination with an empha sis on the respiratory system. -An occupational and medical history up date, including a respiratory symptom up date, and focused physical examinationperformed annually.
-Spirometry testing less frequently than every 3 years is not recommended  -Consideration of specific medical tests (e.g., chest X-ray).

# W r i t t e n r e p o r t s o f m e d ic a l f in d in g s
- The health-care professional should give each worker a written report containing the following:
-The individual worker's medical exami nation results.
-Medical opinions and/or recommenda tions concerning any relationships be tween the individual worker's medical conditions and occupational exposures, any special instructions on the individ ual'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 writ ten report specifying the following:
-Any work or exposure restrictions based on the results of medical evaluations.
-Any recommendations concerning use of personal protective equipment.
-A medical opinion as to whether any of the worker's medical conditions is likely to have been caused or aggravated by oc cupational exposures.
- Findings from the medical evaluations hav ing 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 en forced in accordance with all applicable regu lations and guidelines.

# Worker Education
Workers should be provided information suffi cient 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 ben efits of the program, and the procedures involved.

# Periodic Evaluation of Data and Surveillance Program
Standardized medical screening data should be pe riodically aggregated and evaluated to identify pat terns of worker health that may be linked to work activities and practices that require additional pri mary prevention efforts . 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. Con fidentiality of worker's medical records should be enforced in accordance with all applicable regula tions and guidelines.
Employers should periodically evaluate the ele ments of the medical screening program to ensure that the program is consistent with current knowl edge related to exposures and health effects associ ated with occupational exposure to CNT and CNF.
Other im portant components related to occupa tional health surveillance programs, including medical surveillance and screening, are discussed in Appendix B.

# R e s e a r c h N e e d s
Additional data and information are needed to as sist 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 ex plain potential mechanisms of toxicity and would provide a better understanding of the exposure pa rameters (e.g., mass, fiber/structure number, and particle size) that best describe the toxicological re sponses. 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: - Evaluate NIOSH Method 5040 and other ap propriate 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 expo sure metric best predicts the health endpoints in laboratory animal studies.
- Determine the effectiveness of engineering con trols to control airborne exposures to CNT and CNF below the NIOSH REL of 1 |ig/m3.
- Confirm the effectiveness of using HEPA filters in an exhaust ventilation system for removing ex posures to CNT and CNF.
- Determine the effectiveness of gloves and other PPE barrier materials in preventing dermal ex posure 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 in ward leakage (TIL) of respirators for workers ex posed to nanoparticles (e.g., CNT/CNF).

# . 2 E x p e r i m e n t a l a n d H u m a n S t u d i e s
- Conduct chronic animal inhalation studies to as sess respiratory and other organ (e.g., heart and other circulatory system) effects. Special empha sis should be placed on assessing the risk for de veloping 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 CNT and CNF induce adverse effects (e.g., lung fibrosis) in animals.
- Develop early markers of exposure and pulmo nary 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 epidemio logic studies and surveillance activities.
- Conduct cross-sectional and prospective studies of workers exposed to CNT and CNF.  Ellinger-Ziegelbauer H, Pauluhn J . Pulmo nary toxicity of multi-walled carbon nanotubes (Baytubes) relative to quartz following a single 6h inhalation exposure of rats and a 3 months post exposure period. Toxicology 266(1-3):16-29.
Erdely A, Hulderman T, Salmen R, Liston A, Zeidler-Erdely PC, Schwegler-Berry D, Castranova V, Koyama S, Kim YA, Endo M, Simeonova PP . Cross-talk between lung and systemic cir culation during carbon nanotube respiratory expo sure. Potential biomarkers. Nano Lett 1( 9 Golanski L, Guiot A, Rouillon F, Pocachard J, Tar dif F . Experimental evaluation of personal protection devices against graphite nanoaerosols: fibrous filter media, masks, protective clothing, and gloves. Hum Exp Toxicol 28(6-7):353-359. Grubek-Jaworska H, Nejman P, Czuminska K, Przybylowski T, Huczko A, Lange H, Bystrzejewski M, Baranowski P, Chazan R . Preliminary re sults on the pathogenic effects of intratracheal ex posure to one-dimensional nanocarbons.  . Cytotoxicity of multi-walled carbon nano tubes in three skin cellular models: effects of sonication, dispersive agents and corneous layer of re constructed epidermis. Nanotoxicol 4(1):84-97.

# Golanski L, Guiot

# A P P E N D I X A Q u a n t i t a t i v e R is k A s s e s s m e n t o f C N T Contents

# A.1 Introduction
The increasing production and use of CNT and the preliminary significant toxicology findings neces sitate an assessment of the potential adverse health effects in workers who produce or use these mate rials. Risk assessment is a process that uses stan dardized 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, epide miology, exposure measurement, and other areas provide the data needed to perform the risk assess ment. The standard risk assessment paradigm in the United States includes four basic steps: hazard assessment, exposure assessment, dose-response analysis, and risk characterization . The most recent guidance  recommends asking these questions: "What are the options avail able 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 avail able data and scientific methods as the basis for public and occupational health decision-making . When sufficient dose-response data are available (e.g., from animal studies), quantita tive risk assessment can be performed. Quantita tive risk assessment provides estimates of the sever ity and likelihood of an adverse response associated with exposure to a hazardous agent . Risk assessments are used in developing occupational exposure limits and in selecting and evaluating the effectiveness of expo sure 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 pro ducing 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 in flammatory and fibrotic lung effects from expo sure 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 prop erties 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  However, some evidence suggests that functionalization may increase biodegradation of CNT .
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 re sponses . The specific surface area (m2/g) dose of various types of CNTs was associ ated with the pulmonary inflammation response in rats . Mercer et al.  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 phys ical-chemical properties of other poorly-soluble particles and/or fibers, the nanoscale structure of CNT and CNF may relate to more specific biologi cal modes (or mechanisms) of action. For example, evidence in vitro suggests that disperse CNT may act as a basement membrane, which enhances fi broblast proliferation and collagen production . 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 ex posure to SWCNT or MWCNT by pharyngeal as piration . 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 .
The focus of this quantitative risk assessment is on the early-stage noncancer lung responses (fibrotic and in flammatory) 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 . 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 air borne exposure in the workplace (Section 6 and Ap pendix C). The evidence for cancer effects from CNT and CNF (Section 3 and 4) is insufficient for quantita tive risk assessment, and may also depend on specific types of CNT or CNF structures .

# 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 as sociated with early-stage inflammatory and fibrotic lung responses. Benchmark dose (BMD) modeling . It is preferable to have data with one or more doses near the bench mark response (e.g., 10%) ; howev er, in some studies the response proportions were quite high at each dose (e.g., 30-100%) . In ad dition, one study  had only one dose group in addition to the control, but the study was included because it is the only animal in halation 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 mortal ity of animals were not used. Data on special prepa rations 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 m od els 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 as sessment are provided in Table A-1. These stud ies include the two recently published subchronic inhalation studies of MWCNT in rats  and several IT, PA, or short-term inhalation studies in rats or mice ex posed to SWCNT  with post-exposure durations and examination from 4 to 26 weeks after exposure. In the subchronic inhalation studies, rats were headnose exposed  or nose-only exposed  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; post exposure follow-up was extended to 6 months in the Pauluhn  study.
The IT, PA, and short-term inhalation studies pro vide 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 . Two other short-term exposure studies (Porter et al.  and Ellinger-Ziegelbauer-Pauluhn ), which were included in the external review draft of the CIB , have been omitted in this analysis This is be cause 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.  andPauluhn ).
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  provided an opportunity to compare the dose-response relationships of the 1-day in halation exposure study with that of the 13-week (subchronic) inhalation study  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 SW-CNT and other MWCNT.

# A.2.1.3 Lung R esponses 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 assess ment (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 . 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 .
The most quantitative measure of fibrosis was re ported by the studies that measured the thickening of the gas-exchange region of the lungs (alveolar interstitial or septal connective tissue) due to in creased collagen (as observed by lung tissue stain ing in histopathology examination) . This alveolar thickening was observed to progress with time after administration of a single dose in mice administered by PA . Alveolar thickening was also ob served in a subchronic study, which persisted up to 6 months after the end of exposure in a 13-wk inhalation study in rats . Alveolar interstitial (epithelial cell) thickness has been used as the adverse response in other risk assessment (of ozone) because it indicates "fundamental structural remodeling" .
Alveolar interstitial fibrosis can be detected by Sirius red staining of septal collagen . Interstitial thickening with fibrosis has been demonstrated by Sirius red staining of lungs from mice exposed to SWCNT or MWCNT . In SWCNT exposed mice, the septal fibrosis has been further confirmed by transmission electron microscopy . Pauluhn  reported: "Increased in terstitial 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 fibro sis (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 . Hypercellularity in the bronchial alveolar junctions was observed in 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 NO-AEL . Concerning the focal septal thickening observed at 0.4 mg/m3, pathologists' in terpretations 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 as sociated effects) in the 0.4 mg/m3 and higher dose groups as being adverse changes of relevance to hu man health risk assessment due to their persistence and consistency with the early-stage changes in the development of pulmonary fibrosis. For these rea sons, the alveolar septal thickening of minimal or higher grade (i.e., proportion of rats with this re sponse, which included rats exposed at 0.4 mg/m3 and higher doses) was selected as the benchmark response in the Pauluhn  study. Although these data were reported as the average histopathology score in each dose group , NIOSH requested the response propor tion data as these were needed for the dichotomous BMD modeling. These data were provided by Dr. Pauluhn in response to this request .
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 can cer in rats . 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 . This in dicates 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 base ment membrane and serving as a framework for fibroblast cell growth, without eliciting a persistent inflammatory response) . In other studies, the inflammatory effects of MWCNT were associated with granuloma development  and with alveolar lipoproteinosis, a more severe inflammatory lung response, ob served at higher doses of MWCNT .
Minimal or higher levels of severity of these lung re sponses were selected as the benchmark responses. This included minimal level (grade 1 or higher) of pulmonary inflammation  or alveolar septal thickening  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 ob served 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 expo sure. 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 assess ment are either dichotomous (proportion of ani mals observed with the response endpoint) or con tinuous (amount or level of response in individual animals) (Table A-1). The dichotomous responses include the incidence of lung granulomas ; granulomatous inflammation ; and histopathology grade of alveolar interstitial (septal) thickening .
The continuous responses include the amount of hydroxyproline (as mass)  and alveolar interstitial connective tissue thickness .

# A.2.1.4 Sum m ary of D ose-response Data
Collectively, the data available for CNT risk as sessment include dose-response data from several rodent species and strains, both males and females, 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 (deposit ed 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 E s t i m a t e d L u n g D o s e i n A n i m a l s
For the IT and PA studies , the adminis tered CNT mass dose was assumed to be equiva lent to the deposited lung dose. In the inhalation studies , 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 aero dynamic particle size data), as follows: x Minute Ventilation (L/min) x 0.001 m3/L x 60 min/hr
x Alveolar Deposition Fraction
The exposure concentration and duration, as re ported in the animal studies, are shown in Table A-1. The values used for respiratory minute venti lation were based on the species and body weight: 0.037 L/min for mice  (369 g body weight); and 0.21 L/min for male and female rats in MaHock et al. , assuming average body weight of 300 g  using the mass-median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) data re ported for SWCNT and MWCNT (Table A-2).
In the mouse inhalation study , 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. . For the two subchronic inhalation studies of MWCNT , in addition to the deposited dose, the retained lung dose was also estimated.
The MPPD 2.0 model  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, as suming unit density (the lowest density accepted by MPPD 2.0). However, Ma-Hock et al.  reported the MWCNT particle density of 0.043 g/ ml, and Pauluhn  reported the MWCNT particle density of 0.1-0.3 g/ml. The sensitivity of the lung dose estimates to the assumption of den sity 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  to MPPD 2.1  included revised estimates of the rat head/extra thoracic deposition efficiency based on the equations in Raabe et al. , 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 evalu ated for CNT. However, the measured aerodynamic diameter is considered to provide a reasonable estimate of the deposition efficiency in the respi ratory 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 . Reasonable bounds on the uncertainty of the CNT lung dose estimates are considered to be    *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. fMMAD and GSD in Shvedova et al.  were estimated from data reported in Baron et al.  . *Raabe et al. : mouse DFalv interpolated from values in Table 2 of Raabe et al. .
MPPD 2.0 rat; 0.21 L/min or 2.45 ml tidal volume (assuming 300 g male and female rats) ; and 0.25 L/min or 2.45 ml tidal volume (369 g male rats) . This is because the CNT depos ited in the lungs may undergo some clearance, al though 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 .

# A . 2 . 3 A n i m a l D o s e -r e s p o n s e M o d e l i n g a n d B M D E s t i m a t i o n
The dose-response data in rats and mice exposed to SWCNT or MWCNT were modeled using benchmark dose methods . A benchmark dose has been defined as ". . . a statistical lower confidence limit for the dose corresponding to a specified increase in level of  health effect over the background level" . 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 esti mation, and the term BMDL is used to describe the lower 95% confidence limit (i.e., as originally de fined by Crump ). 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 . The BMDL associated with a 10% BMR is used as a point of departure (POD) for low-dose extrapolation us ing linear or nonlinear methods (depending on the mode of action evidence) . The low-dose extrapolation may include estimation of the probability of effects at low doses or below a ref erence value (not risk-based) by accounting for un certainties in the dose estimation (e.g., extrapolation from animal to human, inter-individual variability, limitations in the animal data .

# A.2.3.1 Dichotom ous 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, ei ther 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) .
In this analysis, the BMD (using dichotomous data) is the dose d corresponding to a specified excess (add ed) risk (e.g., 10%) in the proportion of animals with a given adverse lung response (BMR), as follows:
Equation A-2: BM R = P(d) -P(0)
where P(d) is the probability of an adverse re sponse at the BMD, and P(0) is the probability of that adverse response in an unexposed population .
The dichotomous BMR lung responses include the presence or absence of granulomatous inflamma tion  or alveolar septal thick ening  (Table A-1). The propor tion of animals responding with the minimal or higher severity was selected as the benchmark re sponse. The BMD(L) estimates are expressed as the mass dose of SWCNT or MWCNT in rodent lungs associated with the specified BMR. These animal based BMD(L)s are extrapolated to humans based on species-specific differences in the estimated de position and retention of CNTs in the lungs (Sec tion A.2.4).

# A.2.3.2 C ontinuous Response Data
BMD estimation using continuous data requires specifying a BMR level along the continuum of re sponses. Continuous response data provide in formation on the amount or degree of a biological response. Continuous response measures may in clude nonzero levels that are associated with the normal structure or function (e.g., a certain num ber immune cells or amount of protein in healthy lungs). These levels can become elevated in response to a toxicant, and at some point, they may result in irreversible, functional impairment of the lungs . If data are available, the BMRs can be based on a biologically significant response that is associated with, or expected to result in, a mate rial 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 .
A statistical method (originally referred to as a "hybrid" method) is described by Crump  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 nor mal distribution with constant variance, setting BMR = 0.1 and PO = 0.01 is equivalent to choos ing the BMD to be the dose that results in an in crease in the mean equal to 1.1 times the standard deviation," assuming a normal distribution with constant variance . That is, if one as sumes that the probability of the specified adverse response in the unexposed population is the upper 1% of a normal distribution of responses, then se lecting 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 deter mine if a biologically-based BMR could be identi fied for pulmonary fibrosis (measured as alveolar connective tissue thickening) associated with ab normal pulmonary function . However, those rat findings did not appear to extrapolate well to the mice in Shvedova et al. ; however, this amount of response occurred in up to 30% of the control (unexposed) mice in Shvedova et al. , 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 , 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 (alveo lar connective tissue thickness and hydroxyproline amount) is defined as follows:
Equation A-3: BM R = |j(d) -|j(0)
where ^(d) is the mean response at the BMD (d); ^(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% in crease in the proportion of animals exposed at dose d with response greater than the 99th percentile of the control mean response. The estimates of d) and fi(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) . The dichotomous data were fit with a multistage (polynomial degree 2) model. This is the only model that provided an adequate fit to the sub chronic inhalation data, each of which  had only one dose between zero and 100% response for the endpoints evalu ated (granulomatous inflammation or alveolar sep tal thickening, histopathology grade 1 or higher).
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 de gree 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) . Model fit was consid ered adequate at P > 0.05 (i.e., testing for lack of fit), although the P values based on likelihood ra tio 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 . 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 incor rect model, while P > 0.05 provides less chance of rejecting a correct model. The BMD model fits to each data set are shown in

# A.2.3.4 H um an-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 normal izing the dose per unit of cell surface area). It is as sumed 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 con fidence limit (95% LCL) estimate of the MLE, as an 8-hr TWA exposure concentration during a 45year working lifetime.
The human-equivalent BMD and BMDL estimates were calculated as follows: where the values used for alveolar lung surface area (AlvSA) were 102 m2 (human) ; 0.4 m2 (rat) and 0.055 m2 (mouse) . In Tables A-3 through A-5, the humanequivalent BMD(L)s were multiplied by 0.001 mg/ ^g 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 deposi tion and clearance), as described below.
(a) Deposited lung dose The values assumed include 9.6 m 3 8-hr air intake (reference worker ); alveolar deposi tion fraction based on aerodynamic particle size (Table A-2); and working lifetime days (250 days/ yr x 45 yr).

# (b) Retained lung dose
The MPPD 2.0 human model  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 aerody namic particle size (MMAD, GSD) (Table A-2); inhalability adjustment; oronasal-normal augmenter;
F ig u re 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. , response: granulo matous inflammation, Pauluhn , response: alveolar septal thickening, minimal or greater. P values are 0.99 for Ma-Hock et al.  and 0.88 for Pauluhn . F ig u re A -3 . Benchmark dose model fit to rodent dose-response data from short-term studies with continu ous response: Shvedova et al.  (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.  (SWCNT, mouse, inhalation; response: alveolar connective tissue thickening) (P value is not applicable, linear). Bench mark response level: 1.1 standard deviations about the control mean response. All dose-response models used in this risk assess ment provided adequate fit (P > 0.05) to the ro dent 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 high er 8-hr TWA concentrations are estimated based on the endpoint of lung hydroyxproline amount  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 fi brosis .
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 granu lomas). Although Lam et al.  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 pro vided only for the SWCNT with 2% Fe, which was the only dataset of the three reported by Lam et al.  that was adequately fit by the BMD model (Table A-4).
Table A-5 provides the BMD(L) and BMC(L) esti mates based on the two subchronic inhalation stud ies 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 esti mates 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 con centration to attain the human-equivalent BMD(L) lung burdens. The estimates for MWCNT (with 9.6% Al2O3) based on the rat granulomatous in flammation response are lower than those for MW-CNT (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 Table A-3. Benchmark dose estimates- and associated hum an working lifetime airborne concentrations-continuous response data in rats or mice exposed to SWCNT or MWCNT by IT, PA,f or short-term inhalation (dose metric: adm inistered or estim ated deposited lung dose)
Table A-5. Benchmark dose estim ates and associated working lifetime airborne concentrations-grade 1 or higher severity of lung responses in rats after subchronic inhalation of MWCNT (dose metric: estim ated deposited or retained dose in lungs) histopathology grade 2 or higher lung responses (Table A-6) compared with those estimated from histopathology grade 1 (minimal) or higher re sponses (Table A-5) because more animals devel oped the grade 1 or higher response at a given dose (i.e., histopathology grade 1 or higher is a more sensitive response).
A . Histopathology severity scores for alveolar septal thickening were available for each study. The num ber of male rats with alveolar septal thickening (of minimal or higher grade) and the respective expo sure concentrations are as follows:
- Ellinger-Ziegelbauer and Pauluhn : 1, 0, and 6 rats (6 total per group) at 0, 11.0, and 241.3 mg/m3.
- Pauluhn : 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  based on the particle size data (MMAD and GSD) and the rat exposure con ditions reported in each study.
To evaluate whether these data  can be described by the same dose-response relationship, a multistage (polynomial degree 2) model  was fit to the combined data . This model provided adequate fit to the data (P = 0.37), suggesting that these data can be de scribed 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 .
A .  . NRC  and others have recommended using risk-based low-dose extrapolation for non cancer endpoints. NIOSH practice has also in cluded risk-based low-dose extrapolation for non cancer 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 life time-equivalent 10% excess risk estimates in Table A-5 (deposited dose assumption) results in BMC (BMCL) estimates of 0.051 (0.019) ^g/m3 or 0.077 (0.038) ^g/m3 associated with 1% excess risk (Ma-Hock et al.  or Pauluhn , respective ly). The corresponding BMC (BMCL) estimates as sociated with 0.1% excess risk are 0.0051 (0.0019) ^g/m3 or 0.0077 (0.0038) ^g/m3. Multistage modelbased estimates are higher for the BMCs, but nearly identical for the BMCLs: 0.16 (0.019) ^g/m3 or 0.24 (0.042) ^g/m3 associated with 1% excess risk; and 0.050 (0.0020) ^g/m3 or 0.075 (0.0042) ^g/m3 asso ciated with 0.1% excess risk (Ma-Hock et al.  or Pauluhn , respectively).  Tables A-7 and A-8 provide working lifetime ex cess risk estimates of early stage-lung effects (mini mal or higher histopathology grade of granuloma tous inflammation or alveolar septal thickening) associated with 1, 2, or 7 ^g/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 mea sure airborne CNT in the workplace (NIOSH meth od 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 assum ing normal clearance, within either the minimal (grade 1) (Table A-7) or slight/mild (grade 2) (Ta ble A-8) lung responses. These excess (exposureattributable) risk estimates were derived from the multistage (degree 2) model fit to the rat subchron ic 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 sever al types of SWCNT or MWCNT (with different metal contaminants), by several routes of exposure (inha lation, 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, lim ited 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 evi dence indicates that CNT with certain metals (nickel, 26%)  or with higher metal content (18% vs. 0.2% Fe)  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  and 0.4 mg/ m3 , which are more than an order of magnitude lower than the LOAEL of 7 mg/m3 for ultrafine carbon black  in the same animal species and study design (13-week inhalation studies in rats, although with different strains, Wistar (male and female)  and F-344 (female) ).
Because no chronic animal studies or epidemio logical 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 . For SWCNT, no subchronic stud ies 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 
(Table A-1).
All of these studies reported inflammatory, granu lomatous, 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 ). In the studies with multiple post-exposure follow-up times, the amount of pulmonary fibrosis persisted or progressed with longer follow-up . One of the measures of pulmonary fibrosis used in the short-term stud ies -alveolar epithelial cell thickness (due to in creased 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" .
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 developing early-stage adverse lung effects (pul monary inflammation, granulomas, alveolar septal thickening, and/or fibrosis) if exposed for a work ing lifetime at the estimated upper LOQ of 7 ^g/m3 based on NIOSH Method 5040 for measuring the airborne concentration of CNT  (Appendix C; Tables A-3 through A-8). Working lifetime airborne concentration (8-hr TWA) esti mates of 0.51-4.2 ^g/m3 MLE and 0.19-1.9 ^g/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 in halation studies (Table A-5). For histopathology grade 2 (slight  or slight/mild ), the working lifetime 8-hr TWA concentrations associated with an estimated 10% excess risk are 1.0 to 44 ^g/m3 MLE and 0.69 to 19 ^g/m3 95% LCL (Table A-6).
As discussed in Section A.2.3, the 10% BMDL esti mates are a typical POD for extrapolation to lower risk. NIOSH does not consider 10% or greater ex cess 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 un certainty factors (e.g., Table A -14) would result in lower 8-hr TWA concentrations. However, the lowest LOQ of NIOSH Method 5040 (1 ^g/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 asso ciated 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 ^g/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 ^g/ m3. These risk estimates indicate the need for re search 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 screen ing (Section 6, Appendix B). Chronic bioassay data are also needed to reduce the uncertainty concern ing the potential for chronic adverse health effects from long-term exposure to CNT. Evaluation of the factors that influence the risk estimates and the ar eas of uncertainty are discussed below.
A . Several factors suggest that in the absence of chronic data these short-term and subchronic ani mal data may be reasonable for obtaining initial es timates 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 biopersis tent based on studies in animals  and studies of other poor ly soluble particles in human lungs . Sec ond, the pulmonary fibrosis developed earlier and was of equal or greater severity than that observed from exposure to the same mass dose of other in haled particles or fibers (silica, carbon black, asbes tos) examined in the same study . 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. 2005Shvedova et al. , 2008 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 inhala tion studies is that they provide exposure con ditions 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 (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 ad ministration) 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 sub chronic 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 , indicates that the dose-response relationship was consistent de spite the differences in dose-rate in those two stud ies . This finding indicates that it may be reasonable to assume that the dose-response relationships for the IT, PA, and short-term inha lation exposure studies would be consistent with the subchronic study results if the same response is examined at the same time point, although ad ditional study is needed to confirm this finding. The BMC(L) estimates among the subchronic and short-term studies (Tables A-3  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., expo sure route, duration, animal species, lung response measures).
The two subchronic inhalation studies of MWCNT , based on the same study design (13 week inhalation) and animal species/strain (Wistar rats), facilitates com parison. Different types of MWCNT and different generation methods for aerosolizing exposures were used in each study, although the primary par ticle sizes reported were similar-approximately 10 nm in width and 0.1-10 ^m in length, with spe cific surface area of approximately 250-300 m 2/g . The aerody namic 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.  and Pauluhn ). The metal content also differed, with 9.6% ALO3 in the MWCNT in the Ma-Hock et al.  study vs. 0.5% Co in the MWCNT (Baytubes) in the Pauluhn  study. The lung responses differed both qualita tively and quantitatively, including "pronounced granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis" with a LOAEL of 0.1 mg/m3 in Ma-Hock et al. , vs. "inflammatory changes in the bronchioloalveolar region and increased interstitial collagen staining" with a LOAEL of 0.45 mg/m3 . Yet, both MWCNT studies report ed 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 .
A recent study provides a quantitative comparison of the effects of SWCNT and MWCNT on pul monary interstitial fibrosis . In this study, MWCNTs were administered to mice by pharyngeal aspiration at several different doses (0 , 10, 20, 40, or 80 ^g); the lung tissues (stained for collagen using Sirius red) were exam ined at 56 days post-exposure. At the 80-^g dose of MWCNT, the average thickness of the alveo lar interstitial connective tissue was significantly increased at 28 days, and a progressive increase in thickness was observed at 56 days. The 40-^g MW CNT 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  using the same study design. The individual MWCNTs had a mean diameter of 49 nm and a mean length of 3.9 ^m. 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 differ ences in the target tissue dose, SWCNTs were still ~8.5-fold more fibrogenic than MWCNTs. Howev er, 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 re sponse 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 . However, these findings suggest that the greater fibrotic potency of 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.  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 con taining 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 con tent are more toxic than CNT with lower Fe content.
In Shvedova et al. [2005Shvedova et al. [ , 2008, higher iron con tent was also associated with greater lung response and thus lower BMD(L) estimates. The BMD(L) es timates 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[Shvedova et al. , 2008. All types of CNT (including SWCNT and MWCNT, purified or unpurified, and with var ious types and percentages of metals) were of simi lar 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;Shve dova et al. 2005Shve dova et al. , 2008.

# A . 4 . 3 L u n g D o s e E s t i m a t i o n
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 de veloped 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 al veolar (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 . 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 introduc ing improved dose estimates when validated lung dosimetry models for CNT become available.
The assumptions used in the lung dose estima tion 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 associ ated with a given fixed response proportion. The human-equivalent BMD(L) estimates based on retained dose are also lower because they are pro portional to the rat BMD(L)s (i.e., calculated based on the ratio of the human to rat alveolar epithelial 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 hu man-equivalent BMD(L) lung doses.
The estimated deposited lung dose of CNT (assum ing 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 . On the other hand, the estimated retained lung dose of CNT, based on models for poorly soluble spheri cal 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 . 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 C r i t i c a l E f f e c t L e v e l E s t i m a t e s
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 as sociated with adverse functional effects. More advanced-stage responses (grade 2 or higher severity on histopathology examination) were also evalu ated, 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 ^g/ m3) are considerably lower than those at the upper LOQ (7 ^g/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 ^g/m3.
NIOSH utilized BMD modeling methods to esti mate 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 as sume 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 statisti cal 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 assess ment 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 uncer tainty 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  study, 0.1 mg/m3 was the 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) . A comparison of the BMD and the NOAEL estimates shows that these estimates are statistically consis tent (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 ob served during subchronic exposure. It is also un certain whether these subchronic effects (without additional exposure) would resolve with longer post-exposure duration (beyond the 26-week post-exposure period in the Pauluhn  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 A n i m a l D o s e -r e s p o n s e D a t a Q u a l i t y
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 as sociated with exposure to CNT. The availability of animal dose-response data for different types of CNT-and the consistent low mass concentra tion BMC(L) estimates-suggests these risk esti mates are relatively robust across a range of CNT types, including SWCNT or MWCNT, either puri fied or unpurified (containing different types and amounts of metal), dispersed or agglomerated. Al though 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 associ ated with early-stage adverse lung effects in rats and mice. Consequently, the human-equivalent bench mark 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 esti mates) at the upper LOQ (7 ^g/m3) of the measure ment 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 ^g/m3), depending on lung dose assumptions (Tables A-7 through A-8).
A more in-depth analysis of specific areas of uncer tainty 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 ad verse 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 de posited or retained lung dose in animals and humans. These findings suggest that workers are at risk of de veloping 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 ^g/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 ^g/m3 (8-hr TWA; 95% LCL estimates) (Table A-6).
These working liftetime 8-hr TWA concentrations are below the estimated upper LOQ (7 ^g/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 short term studies, regardless of whether the CNT were purified or unpurified (with different types and amounts of metals), i.e., 0.08-12 ^g/m3 (Tables A-3 and A-4). Lower risks are estimated at the lower LOQ of 1 ^g/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 sen sitive endpoint of minimal grade 1 lung effects (Table A-7). Additional analyses and risk estimates based on other methods and assumptions are pro vided in Section A.6. (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 ge ometry. The ventilation rate depends on the species and on the activity level. Reference values are avail able for the average ventilation rates in rats and hu mans . The airborne particle size data (as reported in the animal stud ies) (Table A-2) were used to estimate the deposited lung dose of CNT in rats and humans, using spheri cal 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. 2001Asgharian et al. , 2003 and in humans [ICRP 1994;Kuempel et al. 2001a, b;Gregoratto 2010Gregoratto , 2011, but models spe cifically for CNT are not yet available.

# A.6 Sensitivity Analyses
This section examines some of the key param eter values used in the lung dose estimation, and also characterizes the quantitative influence of alternative models and assumptions. Two stud ies were available to evaluate the lung dose es timates in rats. Pauluhn  and Ellinger-Ziegelbauer and Pauluhn  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 alveo lar 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 Dosim etry 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 dif fusion. The aerodynamic diameter, by definition, represents the gravitational settling (sedimentation) behavior of particles . 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 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  use particle density (specified by the user), to convert aerodynamic to physical diameter and vice versa, and in this manner capture the key particle deposi tion mechanisms for spherical particles.
However, for high-aspect ratio particles and par ticles 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 mea sure of diameter that captures the diffusional depo sition mechanism) . When the different equivalent diameters could significantly differ, it is recommended to ex perimentally measure these property-equivalent diameters, and subsequently use the measured di ameters in the lung deposition models to provide a reliable representation of each relevant deposition mechanism .
In the animal inhalation studies of CNT , the airborne particle sizes (MMAD) were in the micrometer size range (~1-3 ^m) (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 . An evaluation of the effect of the CNT density assump tions on the rat alveolar deposition fraction is pro vided 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 inha lation studies varied slightly with particle concen tration and sampling device . 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 ap peared 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  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  1-day inhalation study with 91day 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 Sec tion 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 deposi tion 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 esti mates (by approximately a factor of two) than those shown in the main analyses.   --------------------------------------------------------------------------------------------------------------------------- 6 in Pauluhn  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 ; the remaining mass (99.885% was assumed to be CNT). The CNT mass was thus calculated as CNT (ng) =  / 0.00115. CNT (ng) x 0.001 |ig/ng equals CNT (|ig). *MMAD (GSD)-2.9 (1.8) and 2.2 (2.6), respectively, for 11 and 241 mg/m3, from Ellinger-Ziegelbauer and Pauluhn ; alveo lar 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. fAssumed 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  to be approximately 0.03 |ig (11 mg/m3) and 0.39 |ig (241 mg/m3). The CNT amount in the lungs was estimated from the reported 0. 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 (den sity of 0.2)  underestimated the Co-based lung burden, even the deposited dose estimate (assuming no clearance). These findings suggest that the model-based estimates of the de posited and retained rat lung doses in the main analyses (MPPD 2.0, density 1) provided reason able estimates of the bounds on the estimated lung burden. Moreover, these findings are consis tent 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 . The finding that the cobalt-tracer estimates were be tween 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  study (Table A -11). This suggests greater clearance than would be predicted at this high dose (241 mg/m3) based on overload ing 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 bur den 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 ). where VE is the minute ventilation (L/min); BW is body weight; and bo + bi are species-specific pa rameters.
For the rat, bo + bi are -0.578 and 0.821, respec tively (Table 4-6 of US EPA ). 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 x Ln(0.
This is also the default minute ventilation in MPPD .
Rat mean body weights in Pauluhn  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  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.  did not report the rat body weight, although the rat strain (Wistar) and study duration (13 weeks) were the same as in Pauluhn . Because the granulomatous inflammation response data in Ma-Hock et al.  were com bined for the 10 male and 10 female rats in each dose group (since response proportions were sta tistically 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  and the default value of 300 g in MPPD. Subsequent ly, body weights were obtained for the Ma-Hock et al.  study . The average male and female rat body weight at 13 weeks was nearly identical (305 g) to that reported in Pau luhn . 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. , and 0.25 L/min for male rats in Pauluhn . Minute ventilation is the prod uct of tidal volume and breathing frequency. As suming 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  in estimating the rat lung dose in the Pauluhn  data.
In humans, based on the MPPD 2.0 model , the default pulmonary ventila tion 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  or 9.6 m3/8 hr (given 0.001m3/L, and 480 min/8-hr). In these estimates, 17.5 min-1 breath ing frequency and 1143 ml tidal volume  were used in MPPD 2.0 to correspond to a 20 L/min reference-worker ventilation rate.
A . 6 . 2 C r i t i c a l E f f e c t L e v e l S e l e c t i o n
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 ex trapolated 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 criti cal effects used in both the main risk assessment and in these sensitivity analyses. The primary Ma-Hock et al.  0. Lowest observed adverse effect level; BMC: Benchmark concen tration (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 pul monary inflammation or alveolar septal thickening (as reported by Ma-Hock et al.  and Pau luhn ). 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 m od eling of the rat estimated deposited or retained lung dose and to the human-equivalent lung dose estimates. The working lifetime exposure concen tration 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 (as suming normal spherical particle clearance).
In the main risk analysis, BMD methods were se lected over NOAELs or LOAELs because of sev eral statistical advantages (Section A.2). However, BMD(L) estimates may also be uncertain, for ex ample, 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  are used as the effect levels in evaluations of alterna tive methods to derive OEL estimates. A quanti tative 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 subchron ic 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 thick ening) 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  study (Table A-12). Yet, 0.1 mg/m3 was identified as a NOAEL based on zero adverse response being observed . 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 re parameterized 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 con sistent 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 NO AEL for use as a POD in risk assessment . Once an effect level is selected in a given ani mal 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 (ac ceptable) 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 A l t e r n a t i v e O E L E s t i m a t i o n M e t h o d s
As mentioned in the previous section, a standard risk assessment method using animal data typi cally involves first identifying a critical effect level in animals (e.g., NOAEL or BMDL), which is the POD . ,. A HEC POD is estimated by extrapolating anim al j l o the animal dose to humans by accounting for the biological and physical factors that influence the lung dose across species*. Lung dosimetry m od els 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 simpli fied standard approach in lieu of a lung dosimetry model to apply a total dosimetric adjustment fac tor 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.  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 parti cles 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 al veolar epithelial cell surface area or alveolar mac rophage 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 observa tion of overloading of alveolar clearance in rats and mice exposed to respirable poorly soluble particles or fibers .

# (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 ^l/g lung for unit density particles) , or surface area (200-300 cm2 particles per rat lung) . On a volume basis, an overloading particle dose corresponds to approxi mately 6%-60% of total alveolar cell volume, when overloading begins and is complete, respectively . The 60% value has been observed experimentally , although particle clearance impairment may start at lower particle volume lung dose . Biological responses to over loading include: accumulation of particle-filled macrophages in the alveoli, increased permeability of the epithelial cell barrier, persistent inflamma tion, increased particle translocation to the alveo lar interstitium and lung-associated lymph nodes, as well as increasing alveolar septal thickening, lipoproteinosis, impaired lung function, and fibrosis [Muhle et al. 1990[Muhle et al. , 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 re tention in the lungs of rats and humans. Whereas particle clearance in rats is first-order at doses be low overloading, studies in workers have shown that human lung clearance of respirable particles is not first-order even at relatively low retained par ticle mass lung low doses . Humans also apparently re tain a greater portion of the particles in the alveolar interstitium, whereas rats retain more particles in the alveolar space . The greater interstitial particle retention may increase the dose to the target tissue for pulmonary fibro sis in humans relative to that for the same depos ited dose in rats lungs. Given the differences in the particle clearance kinetics and retention patterns in rats and humans, normalizing the dose across species based on the total alveolar macrophage vol ume 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 in flammatory and fibrotic lung responses is the par ticle or CNT dose per surface area of alveolar epi thelial cells .
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 fi brotic cells . For this reason, normal izing the dose based on the total alveolar epithelial cell surface area may be more predictive of the hu man lung response. However, since both the alveolar macrophages and epithelial cells are involved in the lung responses to inhaled particles, some combina tion of dose metrics may ultimately be most predic tive in this dynamic biological system.
In the absence of a more complete biologicallybased model, an evaluation of the quantitative in fluence 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 assess ment 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  results in a total AF that is 4.5 x greater. That is, Equation A-12: AF = (9.6m3/0.102m3) x (0.118/0.057) x (10/1) x (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 ac tion concerning the interspecies normalizing 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 ) is intended to account for the differences in the rat and human particle retention half-time. This factor is also de pendent on the assumptions concerning the bio logical 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 bur den was reached after subchronic (13 week) expo sure 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  assumed that humans would achieve a steady-state lung burden if ex posed at an equivalent total particle volume dose in the alveolar macrophages (over a roughly equiva lent 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 , based on a simple first-order clearance rate model of particle clearance from the lungs in both rats and humans . 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 . 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 first order clearance with dose-dependent impairment,

# A .6 .3 .2 .2 In te r s p e c ie s D o s e R e te n t io n F a c t o r
-r overloading, of particle clearance after reaching a critical lung dose).
An alternative approach evaluated was to use the MPPD 2.0  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 x time), known as "Haber's Law," is a typical default assumption in risk assessment for long-term expo sures in the absence of other data . Some studies in workers (coal miners) have shown that the working lifetime cumulative exposure and the retained lung dose are better predictors of pul monary fibrosis than the average exposure concen tration without consideration of duration . Yet, there remains uncertainty about how well a cumulative dose received over a short dura tion may predict the response to the same cumula tive 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 mecha nisms 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 re pair 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  to the ratio of 10/1 for RTh/RTa used by Pauluhn . The rat and human lung dosimetry models take into account the ventilation rates, the deposition fraction by re spiratory 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 ^g (Table A -10). This is similar to the 8.7 ^g lung burden es timated 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 ^g.  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 to tal alveolar macrophage cell volume (i.e., 3.03 x 1010 ^m3/3.49 x 1013 ^m3) (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 (es timated from MPPD 2.0 human model ) of 16 ^g/m3 and 3.5 ^g/m3, respec tively, 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 ^g/m3 is approximately 3-fold lower than the ~50 ^g/m3 as the human equivalent concentration to the rat NOAEL reported in Pauluhn  (or 3.5 x lower than the 58 ^g/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 (Ta ble A-14) to that estimated as a 10-year steady-state lung burden . 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 x 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 ^g and a lung burden plus lung-associated lymph node burden of 23 ^g.
particle retention predicted by the MPPD model (which includes the ICRP  clearance model)  compared to that of the first-order kinetic model used to estimate the factor of 10/1 .
When this same method was applied to the rat LOAEL of 0.1 mg/m3 in the Ma-Hock et al.  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.  study. In addition, the POD from the Ma-Hock et al.  study is based on a LOAEL (vs. NOAEL in Pauluhn , so an additional uncertainty factor would be applied (as discussed in the next section). In each case, the estimates using an interspecies nor malizing factor based on the alveolar epithelial cell surface area are lower by a factor of approximately four. The estimates in 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 ap plied to the short-term studies would be expected to be higher (e.g., by factor of 2)  than those for the subchronic studies. Alveolar macrophage volume 11.7 13.5 16
Alveolar epithelial cell surface area 3.0 3.5
Ma-Hock et al.  Alveolar macrophage volume 16.0 18 18 A . 6 . 4 S u m m a r y o f S e n s i t i v i t y
A n a l y s e s F i n d i n g s Many of the areas of uncertainty in these risk es timates 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 in halation studies because of lack of experimental evaluation or validation of lung dosimetry models to predict deposition and retention of CNT. Infor mation is also limited on the relative potency of dif ferent 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 associ ated with low-mass concentrations (below the up per and lower LOQ or 7 or 1 ^g/m3, respectively).
In conclusion, these sensitivity analyses show that the estimates of a health-based OEL are not strong ly 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 . 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.  A . 7 . 1 P a r t i c l e C h a r a c t e r i s t i c s Both types of CNF were vapor grown, but obtained from different sources. In Murray et al. , the CNF was supplied by Pyrograf Products, Inc. The chemical composition was 98.6% wt. elemental car bon and 1.4% wt. iron. CNF structures were 80 to 160 nm in diameter, and 5 to 30 ^m in length. The specific surface area (SSA) measured by BET was 35 45 m2/g; the effective SSA was estimated as ~21 m2/g . In DeLorme et al. , the CNF was supplied by Showa Denko KK, Tokyo, Japan. The chemical composition was >99.5% car bon, 0.03% oxygen and < 0.003% iron. CNF struc tures were 40-350 nm (158 nm average) in diameter and 1-14 ^m in length (5.8 ^m average). The BET SSA was 13.8 m2/g .

# A . 7 . 2 E x p e r i m e n t a l D e s i g n a n d A n i m a l s
The species and route of exposure also differed in the two studies. In Murray et al. , six female C57BL/6 mice (8-10 wk of age, 20.0 + 1.9 g body weight) were administered a single dose (120 ^g) of CNF by pharyngeal aspiration ; mice were examined at 1, 7, and 28 days post-ex posure. In DeLorme et al. , female and male Crl:CD Sprague Dawley rats (5 wk of age) were ex posed 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 post exposure. Body weights were reported as: 252 g + 21.2 female; 520 g + 63.6 male (unexposed con trols, 1-d post-exposure); 329 g + 42.2 female; 684 g + 45.8 male (unexposed controls, 3 mo. post-ex posure) .

# A . 7 . 3 L u n g R e s p o n s e s
In mice, the lung responses to CNF included pul monary inflammation (polymorphonuclear lym phocytes, 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), cy totoxicity (elevated lactate dehydrogenase, LDH), which remained significantly elevated compared to controls at day 28 post-exposure. Oxidative dam age (elevated 4-hydroxynonenol, 4-HNE, and oxi datively modified proteins, i.e., protein carbonyls) was significantly elevated at days 1 and 7, but not at day 28. Collagen accumulation at day 28 post exposure was 3-fold higher in CNF-exposed mice vs. controls by biochemical measurements. Consis tent with the biochemical changes, morphometric measurement of Sirius red-positive type I and III collagen in alveolar walls (septa) was significant ly greater than controls at day 28 post-exposure .
In rats, the respiratory effects observed in the De Lorme et al. study  were qualitatively similar to those found in the Murray et al study . The wet lung weights were significantly elevated com pared 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 ter minal 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 pe riod. Cell proliferation appeared to resolve in males after a 3 month recovery period but numerically re mained higher in the parenchyma and subpleural regions. Histopathologic evidence of inflamma tion 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 mea sured by PMN levels in BALF) was statistically sig nificant only in the rats exposed to 25 mg/m3 CNF. However, the percent PMNs increased in a dose responsive manner: 1.2 ( 0.81), 1.4 ( 0.79), 2.7 ( 0.67), and ( 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 13wk 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 r a t s . " .
The sample size and sensitivity of the markers or assays are factors that could influence the statisti cal power and likelihood of observing exposurerelated effects in these animal studies. In Murray et al. , six animals per group were used for the BAL analysis, histopathology evaluation, oxi dative stress markers, and lung collagen measure ments. Five animals per group were used for the BAL and cell proliferation assays in the DeLorme et al.  study (male and female data were ana lyzed separately). The Murray et al.  study used a more sensitive marker of interstitial fibrosis in measuring the average thickness of the alveolar connective tissue, while the DeLorme et al.  study did not report using that assay.

# A . 7 . 4 E f f e c t s i n O t h e r T i s s u e s
In rats, CNF were observed in the nasal turbi nates of the high-dose group (25 mg/m3) at 1 day post-exposure, which was accompanied by hyaline droplet formation in the epithelium; CNF persist ed in the nasal turbinates at 3-mo. post-exposure in the high dose group . In all exposure groups, CNF translocated to the tra cheobronchial 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 . In CNF-exposed mice, T cell mitogen (concavalin A) responsiveness 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 characteris tics and by normalizing to either the mass or al veolar 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) re gion, which is where gas exchange occurs between the lungs and blood circulatory system across the alveolar septal walls. In mice, the lung dose esti mate is simply the proportion of the administered dose (by pharyngeal aspiration)  that is estimated to deposit in the alveolar re gion. Mercer et al.  reported that 81% of the aspirated MWCNT by pharyngeal aspiration de posited in the alveolar region of the mouse. If this figure applies to the CNF reported in Murray et al. , then approximately 97 ^g of the 120 ^g ad ministered 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 concen trations and particle size characteristics reported . The multipath particle de position model (MPPD), version 2.90 , was used to estimate these lung doses. MPPD ver sion 2.11 was originally used to obtain some parti cle deposition estimates, but some output indicated errors in estimating the tracheobronchial regional deposited dose, which appeared to lower the alveo lar 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), and density. The following MMAD and GSD val ues were reported by airborne exposure concen trations: 0.54 mg/m3 (MMAD 1.9 ^m; GSD 3.1); 2.5 mg/m3 (MMAD 3.2 ^m; GSD 2.1); and mg/ m3 (MMAD 3.3 ^m; GSD 2.0). The density assumed for this CNF is 0.08 g/ml. Density was not reported in DeLorme et al.  and was obtained from the manufacturer's data analysis sheet, which in dicates it is the same material as that reported in DeLorme et al. .
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 deposi tion estimates, but some of the required input pa rameters (GSD of structure diameter and length and correlation) were not reported in DeLorme et al. . So, the spherical particle assump tion (aspect ratio of 1.0) was assumed, which may not be unreasonable given that the fiber intercep tion mechanism may be less for CNF structures of length 5.8 ^m than for longer fibers. The default breathing parameters (including 0.21 ml tidal vol ume and 102 breaths/min) may be reasonable for the female Sprague Dawley rats in the DeLorme et al.  study based on similar body weight (300 g) associated with the default values , 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 pe riod, respectively, were: 252 and 329 g (females); 520 and 684 g (males) . The retained lung burden at the end of the 13-wk ex posure 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 nor malized by the lung weight or alveolar surface area to estimate the equivalent dose across species. The aver age lung weights of rats were those reported in DeLo rme et al.  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 . The average alveolar surface area assumed for the rat lungs was 0.4 m2 , and that of mice was 0.055 m2 .
The total deposited CNF dose in the alveolar region was estimated in rats in the DeLorme et al.  study in the following equation: The inhalable fraction estimates of CNF in rats were 0.79, 0.73, and 0.72, respectively, in rats at the re ported particles sizes for concentrations of 0.54 mg/ m3 (MMAD 1.9 ^m; GSD 3.1); 2.5 mg/m3 (MMAD 3.2 ^m; GSD 2.1); and 25 mg/m3 (MMAD 3.3 ^m; GSD 2.0) in DeLorme et al. ] (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 es timates 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 estimat ed 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 . If CNF is cleared in a similar manner as that reported for MWCNT in Pauluhn , the ac tual retained lung dose in rats may be intermediate 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 ^g dose) included al veolar 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 . In the DeLorme et al.  study, similar qualitative lung responses were observed at the 25 mg/m3 (as discussed in Section A.7.3). The DeLorme et al.  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. .
NOAELs were reported for one type of CNF in De Lorme et al.  and for one type of MWCNT in Pauluhn , which were 0.1 and 0.54 mg/m3, respectively. It follows that the human-equivalent working lifetime exposure estimates to the NO-AEL 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 Pau luhn and Ma-Hock subchronic inhalation studies, based on different assumptions in extrapolating the rat lung dose to humans. The application of uncer tainty factors (e.g., Table A -14) with the CNF used in the DeLorme et al.  study would result in estimated working lifetime no-effect levels in hu mans of roughly 1-4 ^g/m3.  fIn rats, the pulmonary deposition fraction and 13-wk retained lung burdens were estimated from MPPD 2.9 . *In mice, this estimate assumes 100% alveolar deposition of the administered by pharyngeal aspiration. If 81% alveolar deposition is assumed as for MWCNT , this estimate would be 1.8 mg/m2 lung. fIn rats, the pulmonary deposition fraction and 13-wk retained lung burdens were estimated from MPPD 2.9 . *In mice, this estimate assumes 100% alveolar deposition of the administered by pharyngeal aspiration. If 81% alveolar deposition is assumed as for MWCNT , this estimate would be 0.65 mg/g lung. Trout and Schulte 2009], and NIOSH continues to recommend occupational health surveillance as an important part of an effective risk management program.
H a z a rd s u rv e illa n c e 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 guide lines (e.g., from NIOSH or the Occupational Safety and Health Administration ).
Human studies, such as epidemiologic in vestigations and case series or reports, and animal studies may also provide valuable in formation. 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 pro tective they are. When data are not available, this will be a qualitative process.
B . 2 M e d i c a l S u r v e i l l a n c e 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 ag gregate 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 ma terials, 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 indi vidual workers by administering tests to apparently healthy persons to detect those with early stages of disease or risk of disease. Medical screening gener ally represents secondary prevention.
Medical surveillance is a second line of defense behind the implementation of engineering, ad ministrative, and work practice controls (includ ing personal protective equipment). Integration of hazard and medical surveillance is important to an effective occupational health surveillance pro gram, and surveillance of disease or illness should not proceed without having a hazard surveillance program in place.

# B.2.1 Planning and C onducting Medical Surveillance
Important factors when considering medical sur veillance include the following:
1. A clearly defined purpose or objective.
2. A clearly defined target population.
3. The availability of testing modalities to accom plish the defined objective. Testing modalities may include such tools as questionnaires, physi cal 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.
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 .
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 sched uled intervals, including specific medical screen ing tests when warranted.
3. More frequent and detailed medical examina tions as indicated, based on findings from these examinations.
4. Post-incident examinations and medical screen ing following uncontrolled or nonroutine increas es in exposures such as spills.
Worker training to recognize symptoms of expo sure to a given hazard.
6. A written report of medical findings.
7. Employer actions in response to identification of potential hazards.  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 oc cupational 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 (typi cally 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 instru ment's sample oven, and the oven is tightly sealed. Quartz-fiber filters are required for sample collec tion 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 cor rects for the "char" that forms during the analysis because of carbonization of some materials.
Thermal-optical analysis proceeds in inert and oxi dizing 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 pre set maximum. If sample charring occurs, the fil ter 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 maxi mum (850 C 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 transm it tance 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 ini tial value.
OC and EC results are reported as micrograms per square centimeter (^g/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 M e t h o d E v a l u a t i o n
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 eval uation, five different organic compounds were ana lyzed 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 , 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 labora tories. Sample materials included, DPM, coals, ur ban 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  demonstrate that carbon can be accurately quantified irrespec tive of the compound or sample type.
In sampling DPM, different samplers gave compa rable EC results because particles from combustion sources are generally less than one ^m (diameter).
As such, the particles are collected with high ef ficiency (near 100%) and evenly deposited on the filter. In the method evaluation, different sampler types (open-face 25-mm and 37-mm cassettes, 298 personal cascade impactors, and four proto type impactors) were used to collect diesel exhaust aerosol at an express mail facility. The relative stan dard deviation (RSD) for the mean EC concentra tion was 5.6% . Based on the 95% confidence limit (19%; 13 degrees of freedom, n = 14) on the accuracy, the NIOSH accuracy cri terion  was fulfilled. Variabil ity 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 pre cision (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 .
In the method evaluation, the limit of detection (LOD) was estimated two ways: (1) through analy sis of low-level calibration standards , and (2) through analysis of pre-cleaned media blanks. In the first approach, OC standard solutions (sucrose and ethylene diaminetetraacetic acid ) covering a range from 0.23 to 2.82 ^g C (or from 0.15 to 1.83 ^g C per cm2 of filter) were analyzed. An aliquot (usually 10 ^L) 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 po tential contamination from handling.
Results of linear regression of the low-level calibra tion data were used to calculate the LOD as 3 oy/m, where oy 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 correc tion 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 es timated through the linear regression results was 0.24 ^g C per filter portion, or 0.15 ^g/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 ^g TC. Thus, the LOD estimated as three times the standard deviation for pre-cleaned media blanks (3 o blank) was about 0.3 ^g C. This result agrees well with the value (0.24 ^g C) esti mated 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 ^g/m3 ( /0.960 m3 = 1.78 ^g/ m3), corresponding to the reported upper LOQ of about 7 ^g/m3 (LOQ = 3.3 x LOD).
As with all analytical methods, the LOD is a vary ing number. However, the EC LOD (about 2 ^g/m3, or an LOQ of 7 ^g/m3) reported for NIOSH Meth od 5040 is a high estimate. As discussed in Sec tion 6 of the CIB, it was based on analysis of pre cleaned 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. When results of the initial m ethod evaluation were published , an inter laboratory comparison was not possible because the thermal-optical instrument was available in only one laboratory. After additional laboratories acquired thermal-optical instruments, a round robin comparison  was conducted. Matched sets of filter samples containing different types of complex carbonaceous aerosols were dis tributed to 11 laboratories. Six of the eleven ana lyzed the samples according to NIOSH 5040, while five used purely thermal (i.e., no char correction) methods. Good interlaboratory agreement was ob tained 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 . 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 ^g/cm2. Four of the six had low EC loadings (0.7 ^g/cm2 to 1.4 ^g/ cm2). The variability for the OC results ranged from 4% to 13% (OC loadings ranged from about 1 to 25 ^g/cm2). Results for TC were not reported, but the variability reported for the OC results should be representative of that for TC, because the sam ples were mostly OC (75% to 92%). Similar find ings were also reported by Chai et al.  from seven laboratories in which analysis was performed using Method 5040 on four sample filter sets con taining OC and EC. The summary RSDs for EC re sults were <12% for all four sample sets. 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 O r g a n i c C a r b o n S a m p l i n g A r t i f a c t s Problems commonly referred to as 'sampling artifacts" have been reported when collecting particu late 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.  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 particu late OC (and TC) concentration, because OC ini tially collected as condensed matter is subsequently lost through evaporation from the filter during sampling. Conversely, several studies have demon strated 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 adsorp tion of gas phase (and vapor) OC occurs, resulting in overestimation of the true airborne particulate OC concentration .
Most of the studies on sampling artifacts apply to environmental air sampling. Occupational sampling methods and conditions are generally much differ ent 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 , and the types of aerosols sampled are different (e.g., aged aerosol from multiple envi ronmental 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 pres ent, sampling time, and filter media. Given the much lower filter face velocities typical of occupa tional sampling, adsorption (i.e., positive artifact) is expected over evaporation for occupational sam ples. Turpin et al. , Kirchstetter et al. , Noll andBirch , and Schauer et al.  have reported adsorption as the dominant artifact.
To correct for the positive adsorption artifact, tan dem quartz filters have been applied. When sam pling 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 effec tive, 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 , while others have shown effective correction .
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 ad sorbs gas and vapor OC. Studies on tandem filter corrections have shown the quartz filter beneath Teflon to have a greater OC value than quartz be neath quartz . This finding was attributed to the quartz beneath quartz not reaching equilibrium with the sampling stream and underestimating the adsorp tion artifact. Others have attributed it to the evapo ration artifact being more prevalent when using a Teflon filter instead of a quartz filter, and they re ported the quartz behind Teflon to overestimate the adsorption artifact .
Several studies have shown no difference when us ing either type of correction .
Noll and Birch  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 be cause both the collection and blank filters are in the same sampler. The tandem quartz correction effectively reduced positive bias for both labora tory and field samples. Laboratory samples were collected under conditions that simulated DPM sampling in underground mines. Without correc tion, 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. Method 5040 was developed to measure DPM in occupational environments, but it can be applied to other types of carbonaceous aerosols. When ap plied 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 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 . Diesel emissions, and combustion aerosols gener ally, are composed of ultrafine (< 100 nm diame ters) particles. Because of the small size, DPM nor mally 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 un even, but this requires analysis of multiple portions of a 37-mm filter because of the relatively small di ameter (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 ad dition, 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 .
As discussed in the CIB, NIOSH 5040 has been ap plied to several field studies on CNT/CNF .
In one study, it was employed for area monitoring at a laboratory facility that processes CNF in the production of polymer composites . 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 tan dem filter method described in the preceding sec tion (organic carbon adsorption artifacts) to mini mize 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 mea sure of the composite aerosol released during the sawing operation, which contained a high OC frac tion due to the composite matrix.
There are several issues and limitations when ana lyzing dusts generated during cutting, sanding, or grinding CNT/CNF composites. First, the accu racy of determining the EC fraction of a polymer composite is questionable and expected to vary, de pending on polymer type and sample loading. Fur ther, 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 ^g/m3 would correspond to a dust concentration (at 1% EC) of 200 ^g/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 sam ples containing dusts of polymer composites is on going; 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 ana lyzed 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 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 pro cesses CNFs . 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 tho racic 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 fil ter 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.

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