This NIOSH CIB, based on our assessment of the current available scientific in formation about this widely used material, (1) reviews the animal and human data relevant to assessing the carcinogenicity and other adverse health effects of TiO2, (2) provides a quantitative risk assessment using dose-response information from the rat and human lung dosimetry modeling and recommended occupational exposure limits for fine and ultrafine (including engineered nanoscale) TiO2, and (3) describes exposure monitoring techniques, exposure control strategies, and research needs. This report only addresses occupational exposures by inhalation, and conclusions derived here should not be inferred to pertain to nonoccupational exposures. NIOSH recommends exposure limits of 2.4 mg/m3 for fine TiO2 and 0.3 mg/m3 for ultrafine (including engineered nanoscale) TiO2, as time-weighted average (TWA) concentrations for up to 10 hours per day during a 40-hour work week. NIOSH has determined that ultrafine TiO2 is a potential occupational carcinogen but that there are insufficient data at this time to classify fine TiO2 as a potential occupational car cinogen. However, as a precautionary step, NIOSH used all of the animal tum or re sponse data when conducting dose-response modeling and determining separate iii RELs for ultrafine and fine TiO2. These recommendations represent levels that over a working lifetime are estimated to reduce risks of lung cancer to below 1 in 1,000. NIOSH realizes that knowledge about the health effects of nanomaterials is an evolving area of science. Therefore, NIOSH intends to continue dialogue with the scientific community and will consider any comments about nano-size titanium di oxide for future updates of this document.# Foreword
The purpose of the Occupational Safety and Health Act of 1970 (Public Law 91-596) is to assure safe and healthful working conditions for every working person and to preserve our human resources. In this Act, the National Institute for Occupational Safety and Health (NIOSH) is charged with recommending occupational safety and health standards and describing exposures that are safe for various periods of em ployment, including (but not limited to) the exposures at which no worker will suf fer diminished health, functional capacity, or life expectancy as a result of his or her work experience.
Current Intelligence Bulletins (CIBs) are issued by NIOSH to disseminate new sci entific information about occupational hazards. A CIB may draw attention to a for merly unrecognized hazard, report new data on a known hazard, or disseminate in formation about hazard control. CIBs are distributed to representatives of academia, industry, organized labor, public health agencies, and public interest groups as well as to federal agencies responsible for ensuring the safety and health of workers. Titanium dioxide (TiO2), an insoluble white powder, is used extensively in many commercial products, including paint, cosmetics, plastics, paper, and food, as an anticaking or whitening agent. It is produced and used in the workplace in varying particle-size fractions, including fine and ultrafine sizes. The number of U.S. work ers currently exposed to TiO2 dust is unknown.

# Executive Summary
In this Current Intelligence Bulletin, the National Institute for Occupational Safety and Health (NIOSH) reviews the animal and human data relevant to assessing the carcinogenicity of titanium dioxide (TiO2) (Chapters 2 and 3), presents a quantita tive risk assessment using dose-response data in rats for both cancer (lung tumors) and noncancer (pulmonary inflammation) responses and extrapolation to humans with lung dosimetry modeling (Chapter 4), provides recommended exposure limits (RELs) for fine and ultrafine (including engineered nanoscale) TiO2 (Chapter 5), describes exposure monitoring techniques and exposure control strategies (Chapter 6), and discusses avenues of future research (Chapter 7). This report only addresses occupational exposures by inhalation, and conclusions derived here should not be inferred to pertain to nonoccupational exposures. TiO2 (Chemical Abstract Service  Number 13463-67-7) is a noncombustible, white, crystalline, solid, odorless powder. TiO2 is used extensively in many commer cial products, including paints and varnishes, cosmetics, plastics, paper, and food as an anticaking or whitening agent. Production in the United States was an estimated 1.45 million metric tons per year in 2007 . The number of U.S. workers currently exposed to TiO2 dust is not available.
TiO2 is produced and used in the workplace in varying particle size fractions includ ing fine (which is defined in this document as all particle sizes collected by respirable particle sampling) and ultrafine (defined as the fraction of respirable particles with a primary particle diameter of <0.1 ^m ). Particles <100 nm are also defined as nanoparticles.
The Occupational Safety and Health Administration (OSHA) permissible exposure limit for TiO2 is 15 mg/m 3, based on the airborne mass fraction of total TiO2 dust (Chapter 1). In 1988, NIOSH recommended that TiO2 be classified as a potential oc cupational carcinogen and that exposures be controlled as low as feasible . This recommendation was based on the observation of lung tumors (nonmalignant) in a chronic inhalation study in rats at 250 mg/m3 of fine TiO2 [Lee et al. 1985[Lee et al. , 1986a (Chapter 3).
Later, a 2-year inhalation study showed a statistically significant increase in lung cancer in rats exposed to ultrafine TiO2 at an average concentration of 10 mg/m3 . Two recent epidemiologic studies have not found a relation ship between exposure to total or respirable TiO2 and lung cancer , although an elevation in lung cancer mortality was obv served among male TiO2 workers in the latter study when compared to the gen eral population (standardized mortality ratio  1.23; 95% confidence interval  = 1.10-1.38) (Chapter 2). However, there was no indication of an exposureresponse relationship in that study. Nonmalignant respiratory disease mortality was not increased significantly (P <0.05) in any of the epidemiologic studies.
In 2006, the International Agency for Research on Cancer (IARC) reviewed TiO2 and concluded that there was sufficient evidence of carcinogenicity in experimental animals and inadequate evidence of carcinogenicity in humans (Group 2B), "pos sibly carcinogenic to humans" . TiO2 and other poorly soluble, low-toxicity (PSLT) particles of fine and ultrafine sizes show a consistent dose-response relationship for adverse pulmonary responses in rats, including persistent pulmonary inflammation and lung tumors, when dose is expressed as particle surface area. The higher mass-based potency of ultrafine TiO2 compared to fine TiO2 is associated with the greater surface area of ultrafine particles for a given mass. The NIOSH RELs for fine and ultrafine TiO2 reflect this mass-based difference in potency (Chapter 5). NIOSH has reviewed and considered all of the relevant data related to respiratory effects of TiO2. This includes results from animal inhalation studies and epidemiologic studies. NIOSH has concluded that TiO2 is not a direct-acting carcinogen, but acts through a secondary genotoxicity mechanism that is not specific to TiO2 but primarily related to particle size and surface area. The most relevant data for assessing the health risk to workers are re sults from a chronic animal inhalation study with ultrafine (<100 nm) TiO2 in which a statistically significant increase in adenocarcinomas was observed . This is supported by a pattern of TiO2 induced responses that include persis tent pulmonary inflammation in rats and mice  and cancer responses for PSLT particles related to surface area. Therefore, on the basis of the study by Heinrich et al.  and the pattern of pulmonary inflam matory responses, NIOSH has determined that exposure to ultrafine TiO2 should be considered a potential occupational carcinogen.
For fine size (pigment grade) TiO2 (>100 nm), the data on which to assess carcino genicity are limited. Generally, the epidemiologic studies for fine TiO2 are incon clusive because of inadequate statistical power to determine whether they replicate or refute the animal dose-response data. This is consistent for carcinogens of low potency. The only chronic animal inhalation study , which demon strated the development of lung tumors (bronchioalveolar adenomas) in response to inhalation exposure of rats to fine sized TiO2 did so at a dose of 250 mg/m3 but not at 10 or 50 mg/m3. The absence of lung tum or development for fine TiO2 was also reported by Muhle et al.  in rats exposed at 5 mg/m 3. However, the re sponses observed in animal studies exposed to ultrafine and fine TiO2 are consistent with a continuum of biological response to TiO2 that is based on particle surface area. In other words, all the rat tum or response data on inhalation of TiO2 (ultrafine and fine) fit on the same dose-response curve when dose is expressed as total par ticle surface area in the lungs. However, exposure concentrations greater than 100 mg/m3 are generally not considered acceptable inhalation toxicology practice today. Consequently, in a weight-of-evidence analysis, NIOSH questions the relevance of the 250 mg/m3 dose for classifying exposure to TiO2 as a carcinogenic hazard to workers and therefore, concludes that there are insufficient data at this time to clas sify fine TiO2 as a potential occupational carcinogen. Although data are insufficient on the cancer hazard for fine TiO2, the tumor-response data are consistent with that observed for ultrafine TiO2 when converted to a particle surface area metric. Thus to be cautious, NIOSH used all of the animal tumor response data when conducting dose-response modeling and determining separate RELs for ultrafine and fine TiO2.
NIOSH also considered the crystal structure as a modifying factor in TiO2 carci nogenicity and inflammation. The evidence for crystal-dependent toxicity is from observed differences in reactive oxygen species (ROS) generated on the surface of TiO2 of different crystal structures (e.g., anatase, rutile, or mixtures) in cell-free systems, with differences in cytotoxicity in in vitro studies  and with greater inflammation and cell proliferation at early tim e points following intratracheal instillation in rats . However, when rats were exposed to TiO2 in subchronic inhalation studies, no difference in pulmonary inflammation response to fine and ultrafine TiO2 particles of different crystal structure (i.e., 99% rutile vs. 80% anatase/20% rutile) was observed once dose was adjusted for particle surface area . Therefore, NIOSH concludes that the scientific evidence supports surface area as the critical metric for occupational inhalation exposure to TiO2.
NIOSH also evaluated the potential for coatings to modify the toxicity of TiO2, as many industrial processes apply coatings to TiO2 particles. TiO2 toxicity has been shown to increase after coating with various substances . How ever, the toxicity of TiO2 has not been shown to be attenuated by application of coat ings. NIOSH concluded that the TiO2 risk assessment could be used as a reasonable floor for potential toxicity, with the notion that toxicity may be substantially in creased by particle treatment and process modification. These findings are based on the studies in the scientific literature and may not apply to other formulations, sur face coatings, or treatments of TiO2 for which data were not available. An extensive review of the risks of coated TiO2 particles is beyond the scope of this document.
NIOSH recommends airborne exposure limits of 2.4 m g/m3 for fine TiO2 and 0.3 mg/m3 for ultrafine (including engineered nanoscale) TiO2, as time-weighted aver age (TWA) concentrations for up to 10 hr/day during a 40-hour work week. These recommendations represent levels that over a working lifetime are estimated to re duce risks of lung cancer to below 1 in 1,000. The recommendations are based on using chronic inhalation studies in rats to predict lung tum or risks in humans.

# vii
In the hazard classification (Chapter 5), NIOSH concludes that the adverse effects of inhaling TiO2 may not be material-specific but appear to be due to a generic effect of PSLT particles in the lungs at sufficiently high exposure. While NIOSH concludes that there is insufficient evidence to classify fine TiO2 as a potential occupational carcinogen, NIOSH is concerned about the potential carcinogenicity of ultrafine and engineered nanoscale TiO2 if workers are exposed at the current mass-based exposure limits for respirable or total mass fractions of TiO2. NIOSH recommends controlling exposures as low as possible, below the RELs. Sampling recommenda tions based on current methodology are provided (Chapter 6).
Although sufficient data are available to assess the risks of occupational exposure to TiO2, additional research questions have arisen. There is a need for exposure assess ment for workplace exposure to ultrafine TiO2 in facilities producing or using TiO2. Other research needs include evaluation of the (1) exposure-response relationship of TiO2 and other PSLT particles and human health effects, (2) fate of ultrafine particles in the lungs and the associated pulm onary responses, and (3) effective ness of engineering controls for controlling exposures to fine and ultrafine TiO2. (Research needs are discussed further in Chapter 7).  . TiO2 is insoluble in water, hydrochloric acid, ni tric acid, or alcohol, and it is soluble in hot con centrated sulfuric acid, hydrogen fluoride, or alkali . TiO2 has several natu rally occurring mineral forms, or polymorphs, which have the same chemical formula and different crystalline structure. Common TiO2 polymorphs include rutile (CAS Number 1317 80-2) and anatase (CAS Number 1317-70-0). While both rutile and anatase belong to the te tragonal crystal system, rutile has a denser ar rangement of atoms (Figure 1).

# Abbreviations
At temperatures greater than 915oC, anatase reverts to the rutile structure . The luster and hardness of anatase and rutile are also similar, but the cleavage dif fers. The density (specific gravity) of rutile is 4.25 g/ml , and that of anatase is 3.9 g/ml . Com mon im purities in rutile include iron, tan talum , niobium , chrom ium , vanadium, and tin , while those in anatase include iron, tin, vana dium, and niobium .
The sulfate process and the chloride process are two main industrial processes that produce TiO2
Rutile Anatase pigment . In the sulfate process, anatase or rutile TiO2 is pro duced by digesting ilmenite (iron titanate) or titanium slag with sulfuric acid. In the chloride process, natural or synthetic rutile is chlori nated at temperatures of 850 to 1,000oC , and the titanium tetrachloride (TiCl4) is converted to the rutile form by vapor-phase oxidation . Both anatase and ru tile are used as white pigment. Rutile TiO2 is the most commonly used white pigment be cause of its high refractive index and relatively low absorption of light . Anatase is used for specialized applications (e.g., in pa per and fibers). TiO2 does not absorb visible light, but it strongly absorbs ultraviolet (UV) radiation. Commercial rutile TiO2 is prepared with an average particle size of 0.22 ^m to 0.25 ^m . Pigment-grade TiO2 refers to anatase and rutile pigments with a median particle size that usually ranges from 0.2 ^m to 0.3 ^m . Particle size is an important determinant of the prop erties of pigm ents and other final products .

# Uses
TiO2 is used mainly in paints, varnishes, lac quer, paper, plastic, ceramics, rubber, and printing ink. TiO2 is also used in welding rod coatings, floor coverings, catalysts, coated fab rics and textiles, cosmetics, food colorants, glassware, pharmaceuticals, roofing granules, rubber tire manufacturing, and in the produc tion of electronic components and dental im pressions . Both the anatase and rutile forms of TiO2 are semiconductors . TiO2 white pigment is widely used due to its high refractive index. Since the 1960s, TiO2 has been coated with other materials (e.g., silica, alumina) for commercial applications .

# Production and Number of Workers Potentially Exposed
An estimate of the num ber of U.S. workers currently exposed to TiO2 dust is not avail able. The only current information is an un referenced estimate submitted by industry to NIOSH in response to request for public comment on the draft document. Industry estimates that the num ber of U.S. workers in the "so-called 'white' end of TiO2 production plants" is "approximately 1,100 workers na tionwide" and that there is no reliable estimate of the num ber of workers involved in the "ini tial compounding of downstream products" .
In 2007, an estimated 1.45 million metric tons of TiO2 pigment were produced by four U.S. companies at eight facilities in seven states that employed an estimated 4,300 workers (jobs not described) . The paint (includes varnishes and lacquers), plastic and rubber, and paper industries accounted for an estimated 95% of TiO2 pigment used in the United States in 2004 . In 2006, the U.S. Bureau of Labor Statistics estimated that there were about 68,000 U.S. workers in all oc cupations (excluding self-employed workers) in paint, coating, and adhesive manufacturing (North American Industry Classification Sys tem  code 325500), 803,000 in plastics and rubber products manufacturing (NAICS code 326000), and about 138,000 employed in pulp, paper, and paperboard mills (NAICS code 322100) . In 1991, TiO2 was the 43rd highest-volume chemical produced in the United States .

# Current Exposure Limits and Particle Size Definitions
Occupational exposure to TiO2 is regulated by the Occupational Safety and Health Adminis tration (OSHA) under the permissible expo sure limit (PEL) of 15 mg/m3 for TiO2 as total dust (8-hr time-weighted average  con centration) .
The OSHA PEL for particles not otherwise reg ulated (PNOR) is 5 mg/m3 as respirable dust . These and oth er exposure limits for TiO2 and PNOR or par ticles not otherwise specified (PNOS) are listed in Table 1. PNOR/S are defined as all inert or nuisance dusts, whether mineral, inorganic or organic, not regulated specifically by substance name by OSHA (PNOR) or classified by the American Conference of Governmental Indus trial Hygienists (ACGIH)(PNOS). The same exposure limits are often given for TiO2 and PNOR/PNOS (Table 1). OSHA definitions for the total and respirable particle size fractions refer to specific sampling methods and devices , while the maximum concentra tion value in the workplace (MAK) and the ACGIH definitions for respirable and inhalable particle sizes are based on the internation ally developed definitions of particle size selec tion sampling .
Aerodynamic diameter affects how a particle behaves in air and determines the probability of deposition at locations within the respira tory tract. Aerodynamic diameter is defined as the diameter of a spherical particle that has the same settling velocity as a particle with a - See CFR in references.
density of 1 g/cm3 (the density of a water drop let) .
"Respirable" is defined as the aerodynamic size of particles that, when inhaled, are capable of depositing in the gas-exchange (alveolar) region of the lungs . Sampling methods have been developed to estimate the airborne mass concentration of respirable particles .
"Fine" is defined in this document as all parti cle sizes that are collected by respirable particle sampling (i.e., 50% collection efficiency for par ticles of 4 ^m, with some collection of particles up to 10 ^m). Fine is sometimes used to refer to the particle fraction between 0.1 ^m and ap proximately 3 ^m , and to pigment-grade TiO2 . The term "fine" has been replaced by "respirable" by some organizations, e.g., MAK , which is consistent with international sampling conventions .
"Ultrafine" is defined as the fraction of respirable particles with prim ary particle diame ter <0.1 ^m (<100 nm), which is a widely used definition. Particles <100 nm are also defined as nanoparticles. A primary particle is defined as the smallest identifiable subdivision of a par ticulate system . Additional methods are needed to determine if an airborne respirable particle sample includes ultrafine TiO2 (Chapter 6). In this document, the terms fine and respirable are used interchangeably to re tain both the common term inology and the international sampling convention.
In 1988, NIOSH classified TiO2 as a potential occupational carcinogen and did not establish a recommended exposure limit (REL) for TiO2 . This classification was based on the observation that TiO2 caused lung tumors   classifiable as a human carcinogen); TiO2 is under study by ACGIH .

#  Respirable
MAK1' 1' Inhalable fraction 4 Inhalable [DFG 2000[DFG , 2008 except for ultrafine particles; suspected carcinogen (MAK Category 3A)

# Respirable
Abbreviations: ACGIH = American Conference of Governmental Industrial Hygienists; MAK = Federal Republic of Germany Maximum Concentration Values in the Workplace; NIOSH = National Institute for Occupational Safety and Health; OSHA = Occupational Safety and Health Administration; PNOR/S = particles not otherwise regulated or specified; TiO2 = titanium dioxide; TWA = time-weighted average; TLV = threshold limit value Recommendations in effect before publication of this document. *Total, inhalable, and respirable refer to the particulate size fraction, as defined by the respective agencies. PNOS guideline (too little evidence to assign TLV). Applies to particles without applicable TLV, insoluble or poorly soluble, low toxicity (PSLT) . 11MAK values are long-term averages. Single shift excursions are perm itted within a factor of 2 of the MAK value.
The TiO2 MAK value has been withdrawn and pregnancy risk group C is not applicable .
in rats in a long-term, high-dose bioassay 

# Human Studies

# Case Reports
Case reports can provide information about the potential health effects of exposure to titanium dioxide (TiO2) that may lead to formal epide miologic studies of a relationship between oc cupational exposure and observed cases.
A few case reports described adverse health ef fects in workers with potential TiO2 exposure. These effects included adenocarcinoma of the lung and TiO2-associated pneumoconiosis in a male TiO2 packer with 13 years of potential dust exposure and a 40-year history of smok ing . Pulmonary fibrosis or fibrotic changes and alveolar macrophage responses were identified by thoracotomy or autopsy tissue sampling in three workers with 6 to 9 years of dusty work in a TiO2 factory. No workplace exposure data were reported. Two workers were "moderate" or "heavy" smokers (pack-years not reported) and smoking habits were not reported for the other worker . Small amounts of silica were present in all three lung samples, and significant nickel was present in the lung tissue of the autopsied case. Exposure was confirmed using sputum samples that contained macrophages with high concentrations of titanium 2 to 3 years after their last exposure . Ti tanium particles were identified in the lymph nodes of the autopsied case. The lung con centrations of titanium were higher than the lung concentration range of control autopsy specimens from patients not exposed to TiO2 (statistical testing and number of controls not reported). Moran et al.  presented cases of TiO2 ex posure in four males and two females. Howev er, occupation was unknown for one male and one female, and the lung tissue of one worker (artist/painter) was not examined (skin biopsy of arm lesions was performed). Smoking hab its were not reported. Diffuse fibrosing inter stitial pneumonia, bronchopneumonia, and al veolar metaplasia were reported in three male patients (a TiO2 worker, a painter, and a paper mill worker) with lung-deposited TiO2 (rutile) and smaller amounts of tissue-deposited silica . Titanium was also identi fied in the liver, spleen, and one peribronchial lymph node of the TiO2 worker, and talc was identified in the lungs of that patient and the paper mill worker.
A case of pulmonary alveolar proteinosis (i.e., deposition of proteinaceous and lipid material within the airspaces of the lung) was reported in a worker employed for more than 25 years as a painter, with 8 years of spray painting expe rience. He smoked two packs of cigarettes per day until he was hospitalized. Titanium was the major type of metallic particle found in his lung tissues .
According to a four-sentence abstract from the Toxic Exposure Surveillance System (TESS) of the American Association of Poison Control Centers, death occurred suddenly in a 26-year-old male worker while pressure-cleaning inside a tank containing TiO2; death was "felt to be due to inhalation of this particulate chemical" . There was no other information about the cause of death or indication that an autopsy was conducted.
TESS data are used for hazard identification, education, and training .
In pathology studies of TiO2 workers, tissuedeposited titanium was often used to confirm exposure. In many cases, titanium, rather than TiO2, was identified in lung tissues; the pres ence of TiO2 was inferred when a TiO2-exposed worker had pulmonary deposition of titanium (e.g., Ophus et al. ; Rode et al. ; Maatta and Arstila ; Elo et al. ; Humble et al. ). In other case reports, X-ray crystallography identified TiO2 (i.e., anatase) in tissue digests , and X-ray diffraction distinguished rutile from anatase . Similarly, with the excep tion of one individual in whom talc was identi fied , pathology studies (i.e., Elo et al. ; Moran et al. ) identified the silica as "SiO2" (silicon dioxide) or "silica" in tissue and did not indicate whether it was crys talline or amorphous.
In summary, few TiO2-related health effects were identified in case reports. None of the case reports provided quantitative industrial hygiene information about workers' TiO2 dust exposure. Lung particle analyses indicated that workers exposed to respirable TiO2 had par ticle retention in their lungs that included ti tanium, silica (form not specified), and other minerals sometimes years after cessation of exposure. The chronic tissue reaction to lungdeposited titanium is distinct from chronic sili cosis. Most cases of tissue-deposited titanium presented with a local macrophage response with associated fibrosis that was generally mild, but of variable severity, at the site of deposition. More severe reactions were observed in a few cases. The prevalence of similar histopathologic responses in other TiO2-exposed populations is not known. The effects of concurrent or sequential exposure to carcinogenic particles, such as crystalline silica, nickel, and tobacco smoke, were not determined.

# Epidemiologic Studies
A few epidemiologic studies have evaluated the carcinogenicity of TiO2 in humans; they are described here and in Table 2-1. Epidemiolog ic studies of workers exposed to related com pounds, such as TiCl4 or titanium metal dust (i.e., Fayerweather et al.  and Garabrant et al. ) were not included because those compounds may have properties and effects that differ from those of TiO2 and discussion of those differences is beyond the scope of this document.

# Chen and Fayerweather 
Chen and Fayerweather  conducted a mortality, morbidity, and nested case-control study of 2,477 male wage-grade workers em ployed for more than 1 year before January 1, 1984 in two TiO2 production plants in the United States. The objectives of the study were to determine if workers potentially exposed to TiO2 had higher risks of lung cancer, chronic respiratory disease, pleural thickening/plaques, or pulmonary fibrosis than referent groups.
Of the 2,477 male workers, 1,576 were poten tially exposed to TiO2. Other exposures includ ed TiCl4, pigmentary potassium titinate (PKT), and asbestos. (The TiCl4-exposed workers were evaluated in Fayerweather et al.   Small num ber of cases ever exposed to T i0 2 (n = 33).
Limitations include self-or proxy-reporting of occupa tional exposures.
Most T i0 2 fum e-exposed cases (n = 5) and controls (n = 1) were also exposed to chrom ium and nickel.

# See footnotes at end of table. (Continued)
NIOSH CIB 63 - Titanium Dioxide
No SMR = 1.0 SMR = 1.0 SMR = 0.8 SMR = 0.4 SMR = 0.8 SMR = 0.7 0 . 8 -1. 3 0.5-1.7 0.6-1.2 0.1-1.3 0.8-0.9 0.6-0.9
Lung cancer and nonm a lignant respiratory disease SMRs not elevated sig nificantly with exception of lung cancer death in the subgroup of shortest-term workers (0-9 years worked) with >20 years since first hire (SMR for cancer of trachea, bronchus, lung = 1.5; 95% Cl 1.0-2.3; P < 0.05; num ber of deaths in subgroup not reported). Internal analyses with models found no significant exposure-response trends for those diseases or total Study limitations:
( 1) short follow-up period (avg. 21 years) and about half the cohort born after 1940;
(2) m ore than half worked fewer than 10 years;
(3) lim ited data on nonoccupational factors (e.g., smoking).

# See footnotes at end of table.
(Continued)    Boffetta et al.  subgroups: 43 ever exposed, 9 substantial exposure; 29 low exposure; 9 medium exposure; 5 high exposure; 22 worked 1-21 years; 21 worked > 22 years. *90% acceptance range for the expected num ber of deaths or cases ^Reported as "not statistically significantly elevated." 90% Cl.
A cumulative exposure index, duration of ex posure, and TWA exposure were derived and used in the analyses (details not provided).
Chest radiographic examination was used to detect fibrosis and pleural abnormalities, and the most recent chest X-ray of active employ ees (on 1/1/1984) was read blindly by two Breaders. Chest X-ray films were not available for retired and terminated workers.
Observed numbers of cancer morbidity cases (i.e., incident cases) were compared to ex pected numbers based on company rates. Ob served numbers of deaths were compared to expected numbers from company rates and national rates. Ninety percent (90%) "accep tance ranges" were calculated for the expected numbers of cases or deaths. The nested casecontrol study investigated decedent lung can cer and chronic respiratory disease, incident lung cancer and chronic respiratory disease (not described), and radiographic chest abnor malities. Incidence data from the company' s insurance registry were available from 1956 to 1985 for cancer and chronic respiratory dis ease. Mortality data from 1957 to 1983 were obtained from the company mortality registry. The study reported the number of observed deaths for the period 1935-1983; the source for deaths prior to 1957 is not clear. Vital status was determined for "about 94%" of the study cohort and death certificates were obtained for "about 94%" of workers known to be deceased.
The observed number of deaths from all can cers was lower than the expected number based on U.S. mortality rates; however, the observed number of deaths from all causes was greater than the expected number when based on com pany mortality rates (194 deaths observed; 175.5 expected; 90% acceptance range for the expect ed number of deaths = 154-198). Lung cancer deaths were lower than the expected number based on national rates (9 deaths observed/17.3 expected = 0.52; 90% acceptance range for the expected number of deaths = 11-24) and com pany rates (9 deaths observed/15.3 deaths ex pected = 0.59; 90% acceptance range for the expected number of deaths = 9-22). Lung can cer morbidity was not greater than expected (company rates; 8 cases observed; 7.7 expected; 90% acceptance range for the expected num ber of cases = 3-13).
Nested case-control analyses found no associa tion between TiO2 exposure and lung cancer morbidity after adjusting for age and exposure to TiCl4, PKT, and asbestos (16 lung cancer cases; 898 controls; TiO2 odds ratio  = 0.6). The OR did not increase with increasing aver age exposure, duration of exposure, or cumu lative exposure index. No statistically signifi cant positive relationships were found between TiO2 exposure and cases of chronic respiratory disease (88 cases; 898 noncancer, nonrespiratory disease controls; TiO2 OR = 0.8). Chest X-ray findings from 398 films showed few abnormalities-there were four subjects with "questionable nodules" but none with fibrosis. Pleural thickening or plaques were present in 5.6% (n = 19) of the workers potentially ex posed to TiO2 compared with 4.8% (n = 3) in the unexposed group. Case-control analyses of 22 cases and 372 controls with pleural ab normalities found a nonstatistically significant OR of 1.4 for those potentially exposed and no consistent exposure-response relationship. This study did not report statistically significant increased mortality from lung cancer, chronic respiratory disease, or fibrosis associated with titanium exposure. However, it has limitations: (note: the study component or information af fected by the limitation is mentioned, when possible) (1) Existence of quantitative exposure data for respirable TiO2 after 1975 is implied; the type of measurement (e.g., total, respirable, or submicrometer), type of sample (e.g., area or personal), number of samples, sampling lo cation and times, nature of samples (e.g., epi demiologic study or compliance survey), and breathing zone particle sizes were not reported. (Exposure data were used in the nested casecontrol analyses of morbidity and mortality.) (2) The report did not describe the number of workers, cases, or deaths in each exposure dura tion quartile which could contribute to under standing of all component results. (3) The pres ence of other chemicals and asbestos could have acted as confounders. (4) Incidence and mortal ity data were not described in detail and could have been affected by the healthy worker effect.
(5) Company registries were the only apparent source for some incidence and mortality infor mation (e.g., company records may have been based on those workers eligible for pensions and thus not typical of the general workforce).

# Fryzek et al. 
Fryzek et al.  conducted a retrospective cohort mortality study of 4,241 workers with potential exposure to TiO2 employed on or af ter 1/1/1960 for at least 6 months at four TiO2 production plants in the United States.
The plants used either a sulfate process or a chlo ride process to produce TiO2 from the original ore. Nearly 2,400 records of air sampling mea surements of sulfuric acid mist, sulfur dioxide, hydrogen sulfide, hydrogen chloride, chlorine, TiCl4, and TiO2 were obtained from the four plants. Most were area samples and many were of short duration. Full-shift or near full-shift personal samples (n = 914; time-weighted av eraging not reported) for total TiO2 dust were used to estimate relative exposure concen trations between jobs over time. Total mean TiO2 dust levels declined from 13.7 mg/m 3 in 197(3-1980 to 3.1 mg/m3 during 1996-2000. Packers, micronizers, and addbacks had about 3 to 6 times higher exposure concentrations than other jobs. Exposure categories, defined by plant, job title, and calendar years in the job, were created to examine mortality patterns in those jobs where the potential for TiO2 expo sure was greatest.
M ortality of 409 female workers and 3,832 male workers was followed until 12/31/2000 (average follow-up time = 21 years; standard deviation = 11 years). The number of expected deaths was based on mortality rates by sex, age, race, time period, and the state where the plant was located, and standardized mortality ratios (SMRs) and confidence intervals (CIs) were calculated. Cox proportional hazards (PH) models, which adjusted for effects of age, sex, geographic area, and date of hire, were used to estimate relative risks (RR) of TiO2 exposure (i.e., average intensity, duration, and cumula tive exposure) in medium or high exposure groups versus the lowest exposure group.
Of the 4,241 workers (58% white, 22% non white, 20% unknown race, 90% male), 958 did not have adequate work history information and were omitted from some plant analyses. Some company records from the early period may have been lost or destroyed; however, the study authors "found no evidence to sup port such an assumption" . Wage status (i.e., hourly or salaried) was not described. Thirty-five percent of workers had been employed in jobs with the highest poten tial for TiO2 exposure. Workers experienced a significantly low overall mortality (533 deaths, SMR = 0.8, 95% CI = 0.8-0.9; P < 0.05). No significantly increased SMRs were found for any specific cause of death and there were no trends with exposure. (Results were not reported by category of race). The number of deaths from trachea, bronchus, or lung cancer was not greater than expected (i.e., 61 deaths; SMR = 1.0; 95% CI = 0.8-1.3). However, there was a significant 50% elevated SMR for workers employed 9 years or less with at least 20 years since hire (SMR = 1.5 ; 95% CI = 1.0-2.3; number of deaths not reported). SMRs for this cancer did not increase with increasing TiO2 concentra tions (i.e., as evidenced by job category in Ta ble 6 of the study). Workers in jobs with great est TiO2 exposure had significantly fewer than expected total deaths (112 deaths; SMR = 0.7; 95% CI = 0.6-0.9) and mortality from cancers of trachea, bronchus, or lung was not greater than expected (11 deaths; SMR = 1.0; 95% CI = 0.5-1.7). Internal analyses (i.e., Cox PH m od els) revealed no significant trends or exposureresponse associations for total cancers, lung cancer, or other causes of death. No associa tion between TiO2 exposure and increased risk of cancer death was observed in this study (i.e., Fryzek et al. ).
Limitations of this study include (1) about half the cohort was born after 1940, lung can cer in these younger people would be less fre quent and the latency from first exposure to TiO2 would be short, (2) duration of employ ment was often quite short, (3) no informa tion about ultrafine exposures (probably be cause collection methods were not available over the course of the study), and (4) limited data on nonoccupational factors (e.g., smok ing). Smoking information abstracted from medical records from 1960 forward of 2,503 workers from the four plants showed no im balance across job groups. In all job groups, the prevalence of smoking was about 55% and it declined over time by decade of hire. However, the information was inadequate for individual adjustments for smoking .
Fryzek et al.  performed additional analyses in response to a suggestion that the RRs may have been artifi cially low, especially in the highest category of cumulative exposure, because of the statistical methods used . These analyses yielded hazard ratios similar to those in the original analysis and found no signifi cant exposure-response relationships for lung cancer mortality and cumulative TiO2 expo sure (i.e., "low," "medium," "high") with either a time-independent exposure variable or a timedependent exposure variable and a 15-year exposure lag (adjusted for age, sex, geographic area, and date of hire) . The hazard ratio for trachea, bron chus, and lung cancer from "medium" cumu lative TiO2 exposure (15-year lag) was greater than 1.0 (hazard ratio for "medium" cumula tive exposure, time-dependent exposure vari able and 15-year lag = 1.3; 95% CI = 0.6-2.8) and less than 1.0 for "high": (hazard ratio = 0.7; 95% CI = 0.2-1.8; "low" was the referent group) . Boffetta et al.  reevaluated lung can cer risk from exposure to TiO2 in a subset of a population-based case-control study of 293 substances including TiO2 (i.e., Siemiatycki et al. ; see Table 2-1 for description of Si emiatycki et al. ).

# Boffetta et al. 
Histologically confirm ed lung cancer cases (n = 857) from hospitals and noncancer ref erents were randomly selected from the popu lation of Montreal, Canada. Cases were male, aged 35 to 70, diagnosed from 1979 to 1985, and controls were 533 randomly selected healthy residents and 533 persons with cancer in other organs.
Job information was translated into a list of po tential exposures, including all Ti compounds and TiO2 as dust, mist, or fumes. Using profes sional judgment, industrial hygienists assigned qualitative exposure estimates to industry and job combinations worked by study subjects, based on information provided in interviews with subjects, proxies, and trained interview ers and recorded on a detailed questionnaire. The exposure assessment was conducted blindly (i.e., case or referent status not known). Dura tion, likelihood (possible, probable, definite), frequency (30%), and extent (low, medium, high) of exposure were assessed. Those with probable or definite exposure for at least 5 years before the interview were classified as "exposed." Boffetta et al.  classified ex posure as "substantial" if it occurred for more than 5 years at a medium or high frequency and level. (Siemiatycki et al.  used a different definition and included five workers exposed to titanium slag who were excluded by Boffetta et al. ; see Table 2-1). Only 33 cases and 43 controls were classified as ever exposed to TiO2 (OR = 0.9; 95% CI = 0.5-1.5). Results of unconditional logistic models were adjusted for age, socioeconomic status, ethnicity, re spondent status (i.e., self or proxy), tobacco smoking, asbestos, and benzo(a)pyrene (BAP) exposure. No trend was apparent for estimated frequency, level, or duration of exposure. The OR was 1.0 (95% CI = 0.3-2.7) for medium or high exposure for at least 5 years. Results did not depend on choice of referent group, and no significant associations were found with TiO2 exposure and histologic type of lung cancer. The likelihood of finding a small increase in lung cancer risk was limited by the small number of cases assessed. However, the study did find an excess risk for lung cancer associated with both asbestos and BAP, indicating that the study was able to detect risks associated with potent carcinogens. The study had a power of 86% to detect an OR of 2 at the 5% level, and 65% pow er for an OR of 1.5.
Limitations of this study include (1) self reporting or proxy reporting of exposure in formation; (2) use of surrogate indices for exposure; (3) absence of particle size character ization; and (4) the nonstatistically significant lung cancer OR for exposure to TiO2 fumes, which was based on a small group of subjects and most were also exposed to nickel and chro mium (5 cases; 1 referent; OR = 9.1; 95% CI = 0.7-118). In addition, exposures were limited mainly to those processes, jobs, and industries in the Montreal area. For example, the study probably included few, if any, workers who manufactured TiO2. Most workers classified as TiO2-exposed were painters and motor vehicle mechanics and repairers with painting experi ence; the highly exposed cases mixed raw m a terials for the manufacture of TiO2-containing paints and plastics. Boffetta et al.  conducted a retrospective cohort mortality study of lung cancer in 15,017 workers (14,331 men, 686 women) employed at least 1 month in 11 TiO2 production facili ties in six European countries. The factories produced mainly pigment-grade TiO2. Esti mated cumulative occupational exposure to respirable TiO2 dust was derived from job title and work history. Observed numbers of deaths were compared with expected numbers based on national rates; exposure-response relation ships within the cohort were evaluated using the Cox PH model. Few deaths occurred in fe male workers (n = 33); therefore, most analyses did not include female deaths. The follow-up period ranged from 1950-1972 until 1997 2001; 2,619 male and 33 female workers were reported as deceased. (The follow-up periods probably have a range of years because the follow-up procedures varied with the partici pating countries.) The cause of death was not known for 5.9% of deceased cohort members. Male lung cancer was the only cause of death with a statistically significant SMR (SMR = 1.23; 95% CI = 1.10-1.38; 306.5 deaths ). However, the Cox regression analysis of male lung cancer mortality found no evidence of increased risk with increas ing cumulative respirable TiO2 dust exposure (P-value for test of linear trend = 0.5). There was no consistent and monotonic increase in SMRs with duration of employment, although workers with more than 15 years of employ ment had slightly higher SMRs than workers with 5 to 15 years of employment and an effect of time since first employment was suggested for workers employed more than 10 years. (The authors indicated that the increase in lung can cer mortality with increasing time since first employment could be "explained by the large contribution of person-years to the categories with longest time since first employment from countries such as Germany, with increased overall lung cancer mortality" .) For male nonm alignant respiratory disease mortality, the num ber of observed deaths was lower than the expected number (SMR = 0.88; 95% CI = 0.77-1.02; 201.9 deaths observed; 228.4 expected), and there was no evidence of an exposure-response relationship.

# Boffetta et al. 
The authors suggested that lack of exposureresponse relationships may have been related to a lack of (1) statistical power or (2) inclu sion of workers who were employed before the beginning of the follow-up period when exposure concentrations tended to be high. (Regarding the latter point, the authors stated that this phenom enon could have occurred and resulted in a loss of power, but "the results of the analysis on the inception cohort, com posed of workers whose employment is entirely covered by the follow-up, are remarkably simi lar to the results of the whole cohort, arguing against survival bias" ). The authors also suggested that the statistically sig nificant SMR for male lung cancer could rep resent (1) heterogeneity by country, which the authors thought should be explained by chance and differences in effects of confounders (see next item), rather than factors of TiO2 dust ex posure; (2) differences in the effects of potential confounders, such as smoking or occupational exposure to lung carcinogens; or (3) use of na tional reference rates instead of local rates. Ramanakumar et al.  analyzed data from two large, population-based case-control stud ies conducted in Montreal, Canada, and fo cused on lung cancer risk from occupational exposure to four agents selected from a large set of agents and mixtures: carbon black, TiO2, industrial talc, and cosmetic talc. Results from the first study (i.e., Study I by Boffetta et al. ) involving TiO2 were published and are described above (see Section 2.2.3). Subject interviews for Study I were conducted from 1982-1986 and included 857 lung cancer cases in men aged 35-70 years. Study II' s interviews were conducted from 1995-2001 and includ ed men and women aged 35-75 years (765 male lung cancer cases, 471 female lung can cer cases). In both studies, lung cancer cases were obtained from 18 of the largest hospitals in the metropolitan Montreal area and con firmed histologically. Controls were randomly sampled from the population. Study I had an additional control group of persons with non lung cancers (Study I: 533 population controls and 1,349 cancer controls; Study II: 899 male controls and 613 female controls). Exposure assessment methods were similar to those de scribed in Boffetta et al.  and Siemiatycki et al.  and included estimates by indus trial hygienists based on job histories (see Sec tion 2.2.3 and Table 2-1). Major occupations of cases and controls with TiO2 exposure (n = 206) were painting, paper hanging, and relat ed occupations (37%); construction laborer, grinder chipper and related occupations; motor-body repairmen; and paint plant laborer. Unconditional multivariate logistic regression models estimated the association between the exposure and lung cancer and included po tential confounders of age, ethnicity, educa tion, income, type of respondent (i.e., self or surrogate), smoking history, and exposure to at least one other known occupational hazard (i.e., cadmium compounds, silica, or asbestos). Association of substance with cell type (i.e., squamous cell, adenocarcinoma, small cell) was also assessed. ORs for lung cancer and ex posure to TiO2 (i.e., "any," "nonsubstantial," or "substantial") were not statistically significant ly increased, exposure-response trends were not apparent, and there was no evidence of a confounding effect of smoking or other confounder or an association with histologic type (results by histologic type were not shown). In the pooled analysis of cases, controls, and sexes from both studies, the ORs were 1.0 (95% CI = 0.8-1.5), 1.0 (95% CI = 0.6-1.7), and 1.2 (95% CI = 0.4-3.6) for TiO2 exposure categories of "any," "nonsubstantial," and "substantial," re spectively. ORs were adjusted for the possible confounders mentioned above .

# Ramanakumar et al. 
Limitations of this study are similar to those for the Boffetta et al.  study and include (1) self-reporting or proxy reporting of expo sure information, (2) use of surrogate indices for exposure, (3) absence of particle size char acterization, (4) few lung cancer cases in the "substantial exposure" to TiO2 category (n = 8, both studies combined) and no female cases in that category, and (5) exposures limited mainly to those processes, jobs, and industries in the Montreal area.

# Summary of Epidemiologic Studies
In general, the five epidemiologic studies of TiO2-exposed workers represent a range of environments, from industry to populationbased, and appear to be reasonably representa tive of worker exposures over several decades. One major deficiency is the absence of any co hort studies of workers who handle or use TiO2 (rather than production workers).
Overall, these studies provide no clear evi dence of elevated risks of lung cancer mortality or morbidity among those workers exposed to TiO2 dust.
Nonmalignant respiratory disease mortality was not increased significantly (i.e., P < 0.05) in any of the three epidemiologic studies that investigated it. Two of the three retrospective cohort mortality studies found small numbers of deaths from respiratory diseases other than lung cancer, and the number of pneumoco niosis deaths within that category was not re ported, indicating that these studies may have lacked the statistical power to detect an in creased risk of mortality from TiO2-associated pneumoconiosis (i.e., Chen and Fayerweather : 11 deaths from nonmalignant diseases of the respiratory system; Fryzek et al. : 31 nonmalignant respiratory disease deaths).
The third study had a larger number of male deaths from nonmalignant respiratory disease and found no excess mortality (SMR = 0.88; 201.9 deaths observed; 95% CI = 0.77-1.02) . None of the studies re ported an SMR for pneumoconiosis mortality; Boffetta et al.  did discuss four pleural cancer deaths, although the number of ob served deaths was lower than expected num ber, based on national rates. Boffetta et al.  suggested that "mortality data might not be very sensitive to assess risks of chronic respiratory diseases."
In addition to the methodologic and epide miologic limitations of the studies, they were not designed to investigate the relationship be tween TiO2 particle size and lung cancer risk, an important question for assessing the poten tial occupational carcinogenicity of TiO2. Fur ther research is needed to determine whether such epidemiologic studies of TiO2-exposed workers can be designed and conducted and to also study workers who manufacture or use products that contain TiO2 (see Chapter 7 Re search Needs).

# Experimental Studies in Animals and
Comparison to Humans

# In Vitro Studies

# Genotoxicity and M utagenicity
Titanium dioxide (TiO2) did not show genotoxic activity in several standard assays: cellkilling in deoxyribonucleic acid (DNA)-repair deficient Bacillus subtilis, mutagenesis in Salmo nella typhimurium or E. coli, or transformation of Syrian hamster embryo cells (particle size and crystal form not specified) . TiO2 was not genotoxic in a Drosphila wing spot test  or mutagenic in mouse lymphoma cells  (par ticle size and crystal structure not provided in either study). More recent genotoxicity stud ies have shown that TiO2 induced chrom o somal changes, including micronuclei in Chi nese hamster ovary cells (particularly when a cytokinesis-block technique was employed)  and sister chromatid exchanges and micronuclei in lymphocytes  (particle size and crystal form not provided in either study). Photo-illumination of TiO2 (anatase/rutile samples of various ra tios; particle size not known) catalyzed oxida tive DNA damage in cultured human fibroblast cells, which the assay indicated was due to hy droxyl radicals . Ultrafine TiO2 (particle diameter < 100 nm; crystal struc ture not provided) induced apoptosis in Syrian hamster embryo cells  and in cultured human lymphoblastoid cells . Sanderson et al.  provided additional physical-chemical data on the ul trafine TiO2 material studied in Wang et al.
 (anatase > 99%; mean particle diam eter = 6.57; surface area = 147.9 m 2/g). DNA damage (micronuclei) was produced in human lymphoblastoid cells at a 65 ig/m l dose with out excessive cell killing (20%) .
Ultrafine TiO2 (80% anatase, 20% rutile) was not genotoxic without UV/vis light irradiation in treated cells but did show dose-dependent increase in chromosome aberrations in a Chi nese hamster cell line with photoactivation . Greater photocatalytic activity by mass was observed for anatase than for rutile TiO2 (specific surface areas: 14.6 and 7.8 m 2/g, respectively)  and for anatase-rutile mixtures (e.g., 80% ana tase, 20% rutile ) com pared to either particle type alone (0.5% wt anatase due to photoinduced interfacial elec tron transfer from anatase to rutile ). Particle size influenced oxidative DNA damage in cultured human bronchial epithelial cells, which was detected for 10-and 20-nm but not 200-nm diameter anatase TiO2 . In the absence of photoac tivation, an anatase-rutile mixture (50%, 50%; diameter = 200 nm) induced higher oxidative DNA damage than did the pure anatase or ru tile (200 nm each) . Overall, these studies indicate that TiO2 exhibits geno toxicity (DNA damage) under certain condi tions but not mutagenicity (genetic alteration) in the assays used.

# Oxidant Generation and Cytotoxicity
TiO2 is considered to be of relatively low in herent toxicity, although the crystal phase can influence the particle surface properties and cytotoxicity in vitro. Sayes et al.  re ported that nano-anatase produced more ROS and was more cytotoxic than nano-rutile, but only after UV irradiation. ROS generation by cells in vitro (mouse BV2 microglia) treated with P25 ultrafine TiO2 (70% anatase and 30% rutile) was suggested as the mechanism for damaging neurons in complex grain cell cul tures . In contrast, Xia et al.  reported that TiO2 (80% anatase, 20% rutile; ~25 nm diameter) did not induce oxi dative stress or increase the heme oxygenase 1 (HO-1) expression in phagocytic cells (RAW 264.7), which the authors suggested may be due to passivation of the particle surfaces by culture medium components or neutralization by available antioxidants. In a study comparing in vitro cellular responses to P25 ultrafine TiO2 (21 nm particle size) and fine TiO2 (1 ^m parti cle size) at exposure concentrations of 0.5-200 Hg/ml, the generation of ROS was significantly elevated relative to controls after 4-hr expo sure to either fine or ultrafine TiO2, although the ROS induced by ultrafine TiO2 was greater than that of fine TiO2 at each exposure concen tration; no cytotoxicity was observed 24 hours after treatment at any of these doses . Thus, photoactivation appears to be an important mechanism for increasing cytotoxic ity of TiO2, especially for formulations contain ing anatase. However, TiO2 cytotoxicity is low relative to more inherently cytotoxic particles such as crystalline silica .

# Effects on Phagocytosis
Renwick et al.  reported that both fine and ultrafine TiO2 particles (250 and 29 nm mean diameter; 6.6 and 50 m2/g surface area, re spectively) reduced the ability of J774.2 mouse alveolar macrophages to phagocytose 2 ^m latex beads at doses of 0.39 and 0.78 ^g/mm2. Ultrafine particles (TiO2 and carbon black) im paired macrophage phagocytosis at lower mass doses than did their fine particle counterparts, although this effect was primarily seen with ul trafine carbon black (254 m2/g surface area).
Moller et al.  found that ultrafine TiO2 (20 nm diameter), but not fine TiO2 (220 nm2 diameter), significantly retarded relaxation and increased cytoskeletal stiffness in mouse alveolar macrophages (J774A.1 cell line) at a dose of 320 ^g/ml. Ultrafine TiO2 inhibited proliferation in J774A.1 cells to a greater ex tent than did fine TiO2 (100 |ig/ml dose; 50% and 90% of control proliferation, respectively). In primary alveolar macrophages (BD-AM, isolated from beagle dogs by bronchoalveolar lavage ), either fine or ultrafine TiO2 (100 ^g/ml dose) caused moderate retardation of relaxation. Neither fine nor ultrafine TiO2 caused impaired phagocytosis of latex micropheres in BD-AM cells, but both particle sizes significantly impaired phagocytosis in J774A.1 cells (~65% of control level by fine or ultrafine TiO2). Ultrafine TiO2 reduced the fraction of viable cells (either J774A.1 or BD-AM) to a greater extent than did fine TiO2 (at 100 ^g/ml dose).
These in vitro studies provide mechanistic in formation about how particle-macrophage interactions may influence cell function and disease processes in vivo. Overall, ultrafine TiO2 impairs alveolar macrophage function to a greater extent than does fine TiO2, which *Bronchoalveolar lavage (BAL) is a procedure for washing the lungs to obtain BAL fluid (BALF), which contains cellular and biochemical indicators of lung health and disease status.
may also relate to the greater inflammatory re sponse to ultrafine TiO2 at a given mass dose.

# In Vivo Studies in Rodent Lungs

# Intratracheal Instillation

# Short-term follow-up
Studies with male Fischer 344 rats instilled with 500 |ig of TiO2 of four different particle sizes and two crystal structures (rutile: 12 and 230 nm; anatase: 21 and 250 nm) indicate that the ultrafine TiO2 particles (~20 nm) are translocated to the lung interstitium (interstitialized) to a greater extent and cleared from the lungs more slowly than fine TiO2 particles (~250 nm) . Other-intratra cheal instillation (IT) studies conducted by the same laboratory showed that ultrafine TiO2 particles produced a greater pulmonary in flammation response than an equal mass dose of fine TiO2 particles. The greater toxicity of the ultrafine particles was related to their larger particle surface area dose and their increased interstitialization .
In a study of four types of ultrafine particles (TiO2, nickel, carbon black, and cobalt; particle sizes 14-20 nm), male Wistar rats (aged 10-12 weeks) were administered IT doses of 125 ^g of particles, and BAL was performed after 4 or 18 hours. Ultrafine TiO2 was the least toxic and inflammogenic, although it was still signifi cantly greater than the saline-treated controls . The adverse pulm onary responses were associated with the free radical activity of the particles, which was very low for ultrafine TiO2. Hohr et al.  observed that, for the same surface area, the inflam m atory response (as measured by BAL fluid (BALF) markers of inflammation) in female Wistar rats to un coated TiO2 particles covered with surface hydroxyl groups (hydrophilic surface) was similar to that of TiO2 particles with surface OCH3-groups (hydrophobic surface) replacing OH-groups. The IT doses in this study were 1 or 6 mg fine (180 nm particle size; 10 m 2/g specific surface area) or ultrafine (20-30 nm; 40 m 2/g specific surface area) TiO2, and BAL was performed 16 hours after the IT.
Ultrafine TiO2 was more damaging than fine TiO2 in the lungs of male Wistar rats . The particle mean diameters of the ultrafine and fine TiO2 were 29 and 250 nm, re spectively, and the specific surface areas were 50 and 6.6 m 2/g; the crystal structure was not specified . Twenty-four hours after instillation of ultrafine or fine TiO2, rats treated with ultrafine TiO2, but not fine TiO2, had BALF elevations in the percent age of neutrophils (indicating inflammation), y-glutamyl transpeptidase concentration (a measure of cell damage), protein concentra tion (a measure of epithelium permeability), and lactate dehydrogenase (LDH) (an indica tor of cytotoxicity and cell death) . The 125 |ig IT dose of either ultra fine or fine TiO2 did not cause any significant adverse lung response in 24 hours. At 500 |ig, the phagocytic ability of the alveolar macro phages was significantly reduced by exposure to particles of either size. The 500 ^g IT dose of ultrafine TiO2, but not fine TiO2, was asso ciated with an increased sensitivity of alveolar macrophages to a chemotactic stimulus, an effect which can reduce the macrophage m o bility and clearance of particles from the lungs.
In a study that included both inhalation (see Section 3.2.3) and IT exposures to six different formulations of fine rutile TiO2 (including un coated or alumina-or amorphous silica-coated; particle size 290-440 nm; specific surface area 6-28 m 2/g), male 8-week old Sprague Dawley rats were dosed with 2 or 10 mg/kg by IT. Pul monary inflammation (measured by polymor phonuclear leukocytes  in BALF) was statistically significantly elevated at 24 hours in the rats administered 10 mg/kg for each of the coated or uncoated formulations. The coated TiO2 formulations produced higher inflam mation than the uncoated TiO2 .
In a study of rutile TiO2 nanorods, inflamma tion responses were examined in BALF and whole blood in Wistar rats 24 hours after an IT dose of 1 or 5 mg/kg . At both doses, the neutrophilic inflammation in BALF was significantly greater than the ve hicle controls. The number of monocytes and granulocytes in blood was dose-dependently elevated, while the platelets were significantly reduced at the higher dose, indicating platelet aggregation.
Mice instilled with 1 mg fine TiO2 (250 nm mean diameter) showed no evidence of inflammation at 4, 24, or 72 hrs after instillation as assessed by inflammatory cells in BALF and expression of a variety of inflammatory cytokines in lung tissue .
Adult male ICR mice (2 months old, 30 g) were exposed to ultrafine (nanoscale) TiO2 (rutile, 21 nm average particle size; specific surface area of 50 m 2/g) or fine (microscale) TiO2 (180-250 nm diameter; specific surface area of 6.5 m2/g) by a single IT dose of either 0.1 or 0.5 mg per mouse . One week later, the lungs showed "significant changes in morphology and histology" in the mice receiv ing the 0.1-mg dose of nanoscale TiO2, includ ing disruption of the alveolar septa and alveolar enlargement (indicating emphysema), type II pneumocyte proliferation, increased alveolar epithelial thickness, and accumulation of par ticle-laden alveolar macrophages. Nanoscale TiO2 elicited a significantly greater increase in chemokines associated with pulmonary emphy sema and alveolar epithelial cell apoptosis than did the microscale TiO2.
A dose-response relationship was not seen, as the adverse effects of the 0.1-mg dose of na noscale TiO2 exceeded those of the 0.5-mg dose. "No significant pathological changes" were observed at either dose of the microscale TiO2.
In summary, these short-term studies show that while TiO2 was less toxic than several other particle types tested, TiO2 did elicit pulmonary inflammation and cell damage at sufficiently high surface area doses (i.e., greater response to ultrafine TiO2 at a given-mass dose). Rehn et al.  observed an acute (3-day) in flammatory response to instillation of ultrafine TiO2 and found that the response from a single instillation decreased over time, returning to control levels by 90 days after the instillation. The reversibility of the inflammatory response to ultrafine TiO2 contrasted with the progres sive increase in inflammation over 90 days that was seen with crystalline silica (quartz) in the same study. This study also compared a silanized hydrophobic preparation of ultra fine TiO2 to an untreated hydrophilic form and concluded that alteration of surface properties by silanization does not greatly alter the bio logical response of the lung to ultrafine TiO2.

# Interm ediate-term follow-up
Three recent studies of various types of na noscale or microscale TiO2 used a similar ex perimental design, which involved IT dosing of male Crl:CD(SD):IGS BR rats (approximate ly 8 weeks of age; 240-255 g body weight). In stilled particle doses were either 1 or 5 mg/kg, and BAL was perform ed at 24 hours, 1 week, 1 month, and 3 months after instillation [Warheit et al. 2006a,b;. Cell proliferation as says and histopathological examination were also performed. Min-U-Sil quartz was used as a positive control, and phosphate buffered saline (PBS) was the instillation vehicle in controls.
In the first study of two hydrophilic types of TiO2 ("R-100" or "Pigment A"), rats were ad ministered IT doses of either 1 or 5 mg/kg of either type of TiO2, carbonyl iron, or Min-U-Sil quartz. Primary average particle sizes were 300 nm, 290 nm, ~1.2 ^m, or ~1.5 |^m, re spectively . Significantly elevated PMNs in BALF was observed for the two types of TiO2 or carbonyl iron at 24 hours postexposure, but not at the later time points.
The second study compared two types of na noscale TiO2 rods (anatase, 92-233 nm length, 20-35 nm width; 26.5 m2/g specific surface area), nanoscale TiO2 dots (anatase, 5.8-6.1 nm spheres; 169 m 2/g specific surface area), and microscale rutile TiO2 (300 nm primary particle diameter; 6 m 2/g specific surface area) . A statistically significant increase in the percentage of PMNs in BALF was seen at the 5 mg/kg dose for all three TiO2 materials tested (which was higher in the rats administered the nanoscale TiO2) but returned to control levels at the 1-week time point. There were no statistically significant lung responses (inflammation or histopathology) to either the fine or the ultrafine TiO2 at either dose (1 or 5 mg/kg) compared to controls at the 1-week to 3-month time points.
In the third study, comparisons were made of the lung inflammation, cytotoxicity, cell pro liferation, and histopathological responses of two types of ultrafine rutile TiO2, a fine ru tile TiO2, an ultrafine anatase-rutile mixture (80%, 20%) TiO2, and Min-U-Sil quartz par ticles . Although the sur face area of these particles varied from 5.8 to 53 m 2/g, the median particle sizes in the PBS instillation vehicle were similar (2.1-2.7 (om), perhaps due to agglomeration. The pulmonary inflammation (% PMNs) of the 5 mg/kg-dose group of anatase/rutile TiO2 was statistically significantly greater than the PBS controls 24 hrs and 1 wk after IT (but not at 1 or 3 months), while the ultrafine and fine rutile TiO2 groups did not differ significantly from controls at any time point. The tracheobronchial epithelial cell proliferation (% proliferating cells) at the 5 mg/ kg-dose group of anatase/rutile TiO2 was also statistically significantly greater than controls 24 hrs after IT (but not at the later time points), while the ultrafine and fine rutile TiO2 groups did not differ significantly from controls at any time point. The two ultrafine rutile TiO2 prepa rations were passivated with amorphous silica and alumina coatings to reduce their chemi cal and photoreactivity to a low level similar to that of the fine rutile TiO2, while the ultra fine anatase/rutile TiO2 was not passivated and was also more acidic. The ultrafine anatase/ rutile TiO2 was more chemically reactive in a Vitamin C assay measuring oxidation poten tial. These results suggest that the crystal phase and surface properties can influence the lung responses to TiO2. In both studies, the Min-U-Sil quartz-instilled rats showed the expected persistent inflammation.

# Long-term follow-up
In a study of the role of lung phagocytic cells on oxidant-derived mutations, rats were dosed by IT with either 10 or 100 mg/kg of fine TiO2 (anatase; median diameter: 180 nm; surface area: 8.8 m 2/g) and held for 15 months . Type II cells isolated from the rats dosed with 100 mg/kg fine TiO2 exhibited an in creased hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation frequency, but type II cells isolated from rats treated with 10 mg/kg fine TiO2 did not. Neutrophil counts were sig nificantly elevated in the BALF isolated from rats instilled 15 months earlier with 100 mg/ kg fine TiO2, as well as by 10 or 100 mg/kg of a-quartz or carbon black. Hprt mutations could be induced in RLE-6TN cells in vitro by cells from the BALF isolated from the 100 mg/kg fine TiO2-treated rats. The authors concluded that the results supported a role for particle-elicited macrophages and neutrophils in the in vivo m u tagenic effects of particle exposure, possibly me diated by cell-derived oxidants.
An IT study in hamsters suggested that fine TiO2 (97% < 5 om, including 51% < 0.5 ^m) may act as a co-carcinogen . When BAP and fine TiO2 (<0.5 ^m par ticle size) were administered by IT to 48 ham sters (male and female Syrian golden hamsters, 6-7 weeks of age), 16 laryngeal, 18 tracheal, and 18 lung tumors developed, compared to only 2 laryngeal tumors found in the BAPtreated controls. In hamsters receiving an IT dose of 3 mg fine TiO2 only in 0.2 ml saline once a week for 15 weeks, no respiratory tract tumors were found. The animals were kept un til death, which occurred by 80 weeks in treat ed hamsters and by 120 weeks in controls.
TiO2 was included in an IT study of 19 differ ent dusts in female SPF Wistar rats . The types of TiO2 tested were ultrafine hydrophilic (P25; "majority anatase"; ~0.025 ^m mean particle size; 52 m 2/g specific surface area), ultrafine hy drophobic (coated) (P805; 21 nm particle size; 32.5 m 2/g specific surface area), and small-fine anatase (hydrophilic) (200 nm particle size;
9.9 m 2/g specific surface area). Groups of 48 rats were administered two or three IT doses, and then maintained for 26 weeks before ter minal sacrifice. The IT doses (number of doses x mass per dose) and the corresponding lung tum or response (percentages of rats with be nign or malignant tumors) included ultrafine hydrophilic TiO2 (5 doses of 3 mg, 5 doses of 6 mg, and 10 doses of 6 mg-52%, 67%, 69% tumors, respectively), ultrafine hydrophobic (coated) TiO2 (15 doses of 0.5 mg, and 30 doses of 0.5 mg-0% and 6.7% tumors, respectively); and small-fine anatase TiO2 (10 doses of 6 mg, and 20 doses of 6 mg-30% and 64% tumors, respectively) . The original 6-mg dose for hydrophobic coated TiO2 was reduced to 0.5 mg because of acute mortality at the higher dose. The TiO2 was analyzed with the tum or data for the other poorly soluble particles (1,002 rats surviving 26 weeks), and they found that the dose metric that provided the best fit to the tum or data was particle vol ume and particle size . Borm et al.  and Morfeld et al.  analyzed a subset of these data (709 rats) for five different poorly soluble particles of different sizes (TiO2 of low and high surface area, carbon black, diesel exhaust particles, and amorphous silica). Morfeld et al.  fit a multivariate Cox model to these pooled data (excluding silica) and reported a threshold dose of 10 mg and a saturation dose of 20 mg for lung tumors. Although ultrafine particles were more tumorigenic than fine par ticles, in their multivariate model no difference was seen between particle mass, volume, or surface area after accounting for particle type and rat survival time; they suggested a high de gree of agglomeration in these IT preparations may have reduced the effective particle surface area relative to that estimated from Brunauer, Emmett, and Teller (BET) analysis . Roller  considered the thresh old findings to be inconsistent with the sta tistically significant lung tum or incidences in three dose groups that were within the 95% confidence interval of the estimated thresh old in Morfeld et al. . All of the analyses of these pooled data including TiO2 showed a greater tum or potency of the ultrafine versus fine particles, whether the dose was expressed as particle volume and size or as particle sur face area . This was considered to be due to the greater translocation of ultrafine particles to the lung interstitium . Although these IT studies used relative ly high-mass doses, increasing dose-response relationships were observed for particles of a given size; greater tum or responses were also observed for the ultrafines compared to fine particles at a given mass dose. One study sug gests a genotoxic mechanism involving DNA damage from oxidants produced by phagocytic and inflammatory cells in response to the TiO2 particles in the lungs.

# Acute or Subacute Inhalation
Nurkiewicz et al.  investigated the role of particle size in systemic microvascular function in male SPF Sprague Dawley rats inhaling either fine TiO2 (1 om; 2.34 m2/g) or P25 ultrafine TiO2 (21 nm; 48.08 m2/g) at airborne exposures aimed at achieving similar particle mass deposition in the lungs (ultrafine: 1.5-12 mg/m3, 240-720 min; fine: 3-15 mg/m3, 240-480 min). No evidence of pulmonary inflammation or lung damage (based on BALF markers) was observed at these exposures. However, 24 hours after ex posure, the arteriolar vasodilation response (to Ca2+ intraluminal infusion of the spinotrapezius muscle) was found to be significant ly impaired in rats exposed to ultrafine TiO2 compared to either the control rats or the rats exposed to fine TiO2 with the same retained mass dose in the lungs. On an equivalent mass basis, ultrafine TiO2 was approximately an or der of magnitude more potent than fine TiO2 in causing systemic microvascular dysfunc tion. When converted to surface area dose, the potency of the fine TiO2 was greater, which the authors suggested was due to overestimation of ultrafine particle surface area delivered to the lungs due to agglomeration. Either fine or ul trafine TiO2 caused systemic microvessel dys function at inhalation doses that did not cause marked lung inflammation. This effect was related to the adherence of PMNs to the m i crovessel walls and production of ROS in the microvessels. This study indicates that cardio vascular effects may occur at particle exposure concentrations below those causing adverse pulmonary effects.
Rats (male WKY/Kyo@Rj, 246-316 g body weight) were exposed by inhalation (endo tracheal intubation) to ultrafine TiO2 (20 nm count median diameter) at an airborne mass concentration of approximately 0.1 m g/m 3 (7.2 x 106 mean num ber concentration) for 1 hour . BAL was performed either 1 or 24 hours after the inhalation expo sure. Elemental microanalysis of the particles provided evidence that the alveolar macro phages did not efficiently phagocytize the par ticles; rather particle uptake was "sporadic" and "unspecific."
Mice (C57Bl/6 male, 6 weeks of age) were exposed by whole-body inhalation to TiO2 nanoparticles (2-5 nm primary particle size; 210 m2/g specific surface area) for either 4 hours (acute) or 4 hr/day for 10 days (subacute) . Airborne TiO2 concentrations were 0.77 or 7.22 mg/m3 for the acute exposure and 8.88 mg/m3 for the subacute exposures. In the subacute study, groups of mice were necropsied at the end of the exposure period and at 1, 2, and 3 weeks postexposure. No adverse effects were observed after the 4-hour expo sure. A "significant but modest" inflammatory response was observed in the mice at 0, 1, or 2 weeks after the subacute exposures, with re covery at the 3rd week postexposure (the num ber of alveolar macrophages in BALF was sta tistically significantly greater than controls in the 1-and 2-week postexposure groups).

# Short-Term Inhalation
Short-term exposure to respirable fine TiO2 has been shown to result in particle accumulation in the lungs of rodents inhaling relatively high par ticle concentrations. The pulmonary retention of these particles increased as exposure concen trations increased. In one study, after 4 weeks of exposure to 5 mg/m3, 50 mg/m3, and 250 mg/ m 3, the fine TiO2 retention half-life in the lung increased (~68 days, ~110 days, and ~330 days, respectively) , which indi cates overloading of alveolar macrophage-me diated clearance of particles from the lungs.
In multiple studies, the most frequently noted change after 1 to 4 weeks of fine TiO2 inhala tion was the appearance of macrophages laden with particles, which were principally local ized to the alveoli, bronchus-associated lym phoid tissue, and lung-associated lymph nodes . Particle-laden macrophages in creased in number with increasing intensity of exposure and decreased in number after cessa tion of exposure . Alveolar macrophages from rats inhaling 250 mg/m3 fine TiO2 for 4 weeks also appeared to be func tionally impaired as demonstrated by persis tently diminished chemotactic and phagocytic capacity .
Inflammation in the lungs of fine rutile TiO2exposed rats was dependent upon exposure concentration and duration. Rats exposed to 250 mg/m3 fine TiO2 6 hr/day, 5 days/week for 4 weeks had markedly increased numbers of granulocytes in BALF .
The granulocytic response was muted after re covery, but numbers did not approach control values until 6 months after exposures ceased. Rats exposed to 50 mg/m 3 fine TiO2 6 hr/day, 5 days/wk for 4 weeks had a small but signifi cantly increased number of granulocytes in the BALF that returned to control levels at 3 months after exposures ceased . This study showed that high concen trations of poorly soluble, low toxicity (PSLT) dust (e.g., fine TiO2 and carbonyl iron) caused impaired pulmonary clearance and persistent inflammation in rats.
In a study of male Fischer 344 rats, inhaling 22.3 mg/m3 of ultrafine (20 nm particle size, anatase) TiO2 6 hr/day, 5 days/wk for up to 12 weeks, the antioxidant enzyme manganesecontaining superoxide dismutase (MnSOD) expression in the lungs increased dramatically and was correlated with pulmonary inflamma tion indicators . Fine TiO2 (23.5 m g/m3 of 250 nm particle size, anatase) did not produce this response at the exposure conditions in this study. Follow-up observa tion of the rats in this study showed that the in flammatory lesions "regressed" during a 1-year period following cessation of exposure . This observation suggests that the inflammatory response from short-term expo sures to TiO2 may be reversible to some degree, if there is a cessation of exposure.
In a separate study, rats exposed to airborne concentrations of 50 mg/m3 fine TiO2 7 hr/day, 5 days/week for 75 days had significantly ele vated neutrophil numbers, LDH (a measure of cell injury) concentration, and n-acetylglucosaminidase (a measure of inflammation) con centration in BALF . However, in this study the BALF of rats inhal ing 10 m g/m 3 or 50 m g/m 3 fine TiO2 7 hr/day, 5 days/week for 2 to 32 days had PMN num bers, macrophage numbers, and LDH concen trations that were indistinguishable from con trol values .
Rats exposed to airborne concentrations of 51 m g/m 3 fine TiO2 (1.0 ^m mass median aerodynamic diameter ) 6 hr/day for 5 days (whole body exposures) had no sig nificant changes in BALF neutrophil number, macrophage number, lymphocyte number, LDH concentration, n-acetylglucosaminidase concentration, or measures of macrophage ac tivation 1 to 9 weeks after exposure . The TiO2 lung burden at the end of exposure was 1.8 mg/lung, and the reten tion was 39% 28 days after the end of exposure. Similarly, rats exposed to 0.1, 1, or 10 mg/m3 6 h/ day, 5 days/week for 4 weeks had no evidence of lung injury as assessed by BAL 1 week to 6 months after exposure or by histopathology 6 months after exposure .
Pulmonary responses to six different formula tions of fine rutile TiO2 (including uncoated or alumina-or amorphous silica-coated; par ticle size 290-440 nm; specific surface area 6-28 m 2/g) were investigated in male, 8-week old Sprague Dawley rats, with both IT (see Section 3.2.1.1) and inhalation exposures. Rats were exposed to very high airborne concen trations (1130-1310 m g/m 3) of the different formulations of fine TiO2 for 30 days (6 hr/ day, 5 days/week). The pulmonary inflamma tion response (assessed by histopathology) remained significantly elevated 1 month after exposure to the TiO2 coated with alumina (7%)
and amorphous silica (8%). The coated TiO2 formulations produced higher inflammation than the uncoated TiO2 in the inhalation study (as in the IT study) .

# Subchronic Inhalation
In a study of two fine-sized, PSLT particles-TiO2 and barium sulfate (BaSO4)-no-observed -adverse-effect levels (NOAELs) were esti mated based on the relationship between the particle surface area dose, overloading of lung clearance, and neutrophilic inflammation in rats . These two PSLT parti cles had similar densities (4.25 and 4.50 g/cm3, respectively) but different particle sizes (2.1 and 4.3 ^m MMAD); since both of these fac tors influence particle deposition in the lungs, the exposure concentrations were adjusted to provide similar particle mass deposition in the lungs. Male Wistar rats (age 12 weeks, specificpathogen free) were exposed by whole body inhalation (7 hr/day, 5 days/wk) to either 25 m g/m 3 for 7.5 months (209 calendar days) or to 50 mg/m3 for 4 months (118 calendar days). The findings showed that the retardation of alveolar macrophage-mediated clearance, particle transfer to the lung-associated lymph nodes, and influx of PMNs were related to the lung burden as particle surface area dose. A mean airborne concentration of 3 mg/m3 fine sized TiO2 was estimated as the NOAEL, which was defined as a 95% probability that the lung responses would be below those predicted using the "no overload level" for the average animal.
The relationship between subchronic inhala tion of TiO2 and neutrophilic inflammation was also investigated in a study of Montserrat vol canic ash . Male Wistar rats (225 g, specific-pathogen free) were exposed by nose-only inhalation (6 hr/day, 5 days/wk) to 140 mg/m 3 of TiO2 (1.2 ^m MMAD) for up to 2 months. The concentration of ash (5.3 MMAD) was 253 mg/m3, which was predicted to provide the same retained lung burden as the TiO2. After the 8-week exposure, the histopathological examination showed relatively minor pathology changes in the lungs of rats treated with TiO2, although the pathological changes in the lungs of the ash-exposed rats were generally "more marked." The pulmonary inflammation response was also greater in the ash-exposed rats, based on the percentage of PMNs in the BALF, which ranged from 19%-53% PMNs for the ash and from 0.2%-16% for TiO2 at 14-56 days of exposure.
Fine TiO2 (pigmentary; 1.4 ^m MMAD) was studied in female rats (CDF/Crl-BR), mice (B3C3F1/CrlBR), and hamsters , 6 wk old, after a 13-week inhalation exposure to 10, 50, or 250 mg/m3, followed by up to 52 weeks without TiO2 expo sure .
Retained particle burdens in the lungs and lung-associated lymph nodes were measured at the end of exposure and at 4, 13, 26, and 52 weeks postexposure. The lung particle reten tion patterns indicated that after the 13 weeks of exposure to 50 or 250 mg/m3, clearance overload had occurred in rats and mice but not in hamsters. In mice and rats, the numbers of macrophages and the percentage of neutro phils were significantly increased in BALF after 13 weeks of exposure to 50 or 250 mg/m3 fine TiO2 and remained elevated through the 52week postexposure. These BALF cell responses were significantly elevated in hamsters after exposure to 250 mg/m3 but were no longer sig nificantly elevated by 26 weeks postexposure.
Alveolar cell proliferation was significantly el evated only at 52-week postexposure in the rats . Histopathology showed alveolar hypertrophy and hyperplasia of type II epithelial cells in rats after the 13-week exposure to 50 or 250 mg/m3 fine TiO2. In mice, alveo lar type II cell hypertrophy was observed (dose not given), while in hamsters minimal type II epithelial cell hypertropy and hyperplasia were observed at 50 or 250 mg/m 3. Foci of alveolar epithelial cell hypertrophy and hyperplasia were seen to be often associated with aggre gates of particle-laden alveolar macrophages in all three species. In rats, but not mice and hamsters, these foci of alveolar epithelial hy pertrophy became increasingly more prom i nent with time, even after cessation of expo sure, and at the high dose, rats progressed to bronchiolization of alveoli (metaplasia) and to fibrotic changes with focal interstitialization of TiO2 particles. Alveolar lipoproteinosis and cholesterol clefts were observed in rats 52 weeks after the 13-week exposure to 250 mg/m3. Al though "high particle burdens" were associ ated with "proliferative epithelial changes as sociated with particle-induced inflammation" in all three species, only rats developed meta plastic and fibrotic lesions .
P25 ultrafine TiO2 (21 nm prim ary particle size; 1.37 MMAD) was studied in female rats, mice, and hamsters after a 13-week inhalation exposure to 0.5, 2, or 10 m g/m 3, followed by a recovery period (without TiO2 exposure) for up to 52 weeks  (same rodent strains as Bermudez et al. 2002). Pul monary responses and retained particle bur dens in the lungs and lung-associated lymph nodes were measured at the end of exposure and at 4, 13, 26, and 52 weeks postexposure. Retardation of pulmonary clearance follow ing exposure to 10 m g/m 3 was observed in rats and mice but not in hamsters. Pulmonary in flammation was also observed at the 10 m g/m 3 dose in rats and mice but not in hamsters. The total number of cells in BALF was significantly elevated in rats and mice after 13 weeks of ex posure to 10 mg/m3, and the percentages of neutrophils and macrophages remained statis tically significantly elevated through 52 weeks postexposure in both species. The BALF cell responses were not statistically significant fol lowing exposure to 0.5 or 2 mg/m3 in mice or rats (except for significantly elevated neutro phils in rats immediately after the 13-wk expo sure to 2 mg/m3) (Tables S1-S3 in Bermudez et al. 2004). BALF cell responses were not signifi cantly elevated in hamsters at any exposure or observation time.
The alveolar cell replication index was statis tically significantly increased at 0, 4, and 13 weeks after the 13-wk exposure to 10 m g/m 3 ultrafine TiO2 in rats, while in mice it was sig nificantly increased at 13 and 26 weeks after exposure cessation . In rats inhaling 10 m g/m 3, the histopathologic responses included epithelial and fibroproliferative changes, interstitial particle accumu lation, and alveolar septal fibrosis. Although most of the epithelial proliferative lesions had regressed postexposure, some remaining le sions were believed to have progressed. At 52 weeks postexposure, minimal to mild m eta plastic changes and minimal to mild particleinduced alveolar septal fibroplasia were seen in rats. In mice inhaling 10 m g/m 3, lesions were described as aggregations of heavily par ticle laden macrophages in the centriacinar sites. During postexposure, these cell aggre gates were observed to move to the interstitial areas over time. No epithelial, metaplastic, and fibroproliferative changes were observed by histopathology in the mice or hamsters (although the mice had significantly elevated alveolar cell replication at 13 and 26 weeks postexposure in the 10 mg/m3 dose group, this response apparently did not result in histopathologically visible changes). The absence of adverse pulmonary responses in hamsters was considered to reflect their rapid clearance of particles from the lungs.

# Chronic Inhalation
TiO2 has been investigated in three chronic in halation studies in rats, including fine TiO2 in Lee et al.  and Muhle et al.  and ultrafine TiO2 in Heinrich et al. . These studies were also reported in other publica tions, including Lee et al. , Muhle et al. . In an other 2-year rat inhalation study, an increase in lung tumors was found in rats exposed to TiCl4 . TiCl4 is an intermediate in the production of pigment TiO2, including by hydrolysis of TiCl4 to produce TiO2 and HCl. However, TiCl4 is a highly volatile compound with different properties than TiO2, and thus it is not addressed further in this document.
In Lee et al. , groups of 100 male and 100 female rats (CD, Sprague-Dawley derived; strain not specified) were exposed by whole body inhalation to fine rutile TiO2 (1.5-1.7 MMAD) for 6 hr/day, 5 days/week, for up to two years, to 10, 50, or 250 mg/m3 (84% respirable; < 13 ^m MMAD). A fourth group (con trol) was exposed to air. In each group, twenty rats were killed at 3, 6, or 12 months; 80 rats were exposed for 2 years, and all surviving rats were killed at the end of exposure. No increase in lung tumors was observed at 10 or 50 mg/m3. At 250 mg/m3, bronchioalveolar adenomas were observed in 12 out of 77 male rats and 13 out of 74 female rats. In addition, squamous cell carcinomas were reported in 1 male and 13 females at 250 mg/m3. The squamous cell carci nomas were noted as being dermoid, cyst-like squamous cell carcinomas , lat er reclassified as proliferative keratin cysts , and later still as a continuum ranging from pulmonary keratinizing cysts through pulmonary keratinizing eptheliomas to frank pulmonary squamous carcinomas . A recent reanalysis of the 16 tu mors originally classified as cystic keratinizing squamous cell carcinomas in Lee et al.  had a similar interpretation: two were reclas sified as squamous metaplasia, one as a poorly keratinizing squamous cell carcinoma, and 13 as nonneoplastic pulmonary keratin cysts .
In both the Muhle et al.  and Heinrich et al.  studies, TiO2 was used as a nega tive control in 2-year chronic inhalation stud ies of toner and diesel exhaust, respectively. In Muhle et al. , the airborne concentration of TiO2 (rutile) was 5 mg/m3 (78% respirable, according to 1984 ACGIH criterion). Male and female Fischer 344 rats were exposed for up to 24 months by whole body inhalation and sac rificed beginning at 25.5 months. No increase in lung tumors was observed in TiO2-exposed animals; the lung tum or incidence was 2/100 in TiO2-exposed animals versus 3/100 in non exposed controls.
In the Heinrich et al.  study, 100 female Wistar rats were exposed to ultrafine TiO2 (80% anatase, 20% rutile; 15-40 nm primary particle size; 0.8 mm MMAD; 48 ( 2.0) m 2/g specific surface area) at an average of approxi mately 10 m g/m 3, 18 h/day 5d/wk, for up to 24 months (actual concentrations were 7.2 mg/m3 for 4 months, followed by 14.8 m g/m 3 for 4 months, and 9.4 m g/m 3 for 16 months).
Following the 2-year exposure, the rats were held without TiO2 exposure for 6 months . At 6 months of expo sure, 99/100 of the rats had developed bronchioloalveolar hyperplasia, and by 2 years all rats had developed slight to moderate in terstitial fibrosis . After 24 months of exposure, four of the nine rats examined had developed tum ors (including a total of two squamous cell carcinomas, one adenocarcinoma, and two benign squamous cell tumors). At 30 months (6 months after the end of exposure), a statistically significant increase in adenocarcinomas was observed (13 adenocarcinomas, in addition to 3 squa mous cell carcinomas and 4 adenomas, in 100 rats). In addition, 20 rats had benign keratin izing cystic squamous-cell tumors. Only 1 ad enocarcinoma, and no other lung tumors, was observed in 217 nonexposed control rats.
NMRI mice were also exposed to ultrafine TiO2 in Heinrich et al. . The lifespan of N M r2 mice was significantly decreased by inhaling approximately 10 mg/m3 ultrafine TiO2, 18 hr/ day for 13.5 months . This exposure did not produce an elevated tu mor response in the NMRI mice, but the 30% lung tumor prevalence in controls may have decreased the sensitivity for detecting carcino genic effects in this assay. In a study of several types of particles, 100 female Wistar (Crl: Br) rats were exposed to 10.4 m g/m 3 TiO2, 18 hr/day 5 days/wk, for 24 months (followed by 6 months in clean air). No information was provided on the particle size or crystal struc ture of TiO2 used in this study. Cystic keratizing epitheliomas were observed in 16% of the rats. In addition, 3.0% cystic keratinizing squamous-cell carcinomas and 1% nonkera tinizing squamous-cell carcinoma were ob served .
The primary data used in the dose-response model in the TiO2 risk assessment (Chapter 4) include the ultrafine TiO2 data of Heinrich et al.  and the fine TiO2 data of Lee et al. . Differences in postexposure follow-up (24 and 30 months, respectively, in Lee et al.  and Heinrich et al. ) may have increased the likelihood of detecting lung tumors in the ultrafine TiO2-exposed rats. However, the dif ferences in the hours of exposure per day (6 and 18 hrs, respectively, in Lee et al.  and Heinrich et al. )) were accounted for in the risk assessment models since the retained particle lung burden (at the end of 2-year in halation exposure) was the dose metric used in those models.
In summary, the chronic inhalation studies in rodents show dose-related pulmonary re sponses to fine or ultrafine TiO2. At sufficiently high particle mass or surface area dose, the re sponses in rats include reduced lung clearance and increased particle retention ("overload"), pulmonary inflammation, oxidative stress, tis sue damage, fibrosis, and lung cancer. Studies in mice showed impaired lung clearance and inflammation, but not fibrosis or lung cancer. Studies in hamsters found little adverse effect of TiO2 on either lung clearance or response.

# In Vivo Studies: Other
Routes of Exposure

# Acute Oral Administration
The acute toxicity of nanometer (25 and 80 nm) and submicron (155 nm) TiO2 was investigated after a large single dose of TiO2 (5 g/kg body weight) in male and female CD-I mice . The TiO2 was retained in liver, spleen, kidney, and lung tissues, indicating up take by the gastrointestinal tract and systemic transport to the other tissues. No acute toxicity was observed. However, statistically significant changes were seen in several serum biochemical parameters, including LDH and alpha-hydroxybutyrate (suggesting cardiovascular damage), which were higher in mice treated with either 25 and 80 nm nanoscale TiO2 compared to fine TiO2. Pathological evidence of hepatic injury and kidney damage was also observed. In fe male mice treated with the 80 nm nanoscale and the fine particles, the liver injury included hydropic degeneration around the central vein and spotty necrosis of hepatocytes; renal dam age included protein-filled liquid in the renal tubule and swelling of the renal glomerulus.
The tissue injury markers did not always re late to particle size; damage was often but not always greater in the mice treated with the nanoscale particles compared to the fine par ticles, and the 80-nm TiO2 was more damag ing than the 25-nm TiO2 by some indicators.
Given the single large dose in this study, it was not possible to evaluate the dose-response re lationship or to determine whether the effects were specific to the high dose.

# Chronic Oral Administration
The National Cancer Institute conducted a bio assay of TiO2 for possible carcinogenicity by the oral route. TiO2 was administered in feed to Fischer 344 rats and B6C3F1 mice. Groups of 50 rats and 50 mice of each sex were fed either 25,000 or 50,000 parts per million TiO2 (2.5% or 5%) for 103 weeks and then observed for an additional week. In the female rats, C-cell ade nomas or carcinomas of the thyroid occurred at an incidence of 1 out of 48 in the control group, 0 out of 47 in the low-dose group, and 6 out of 44 in the high-dose group. It should also be noted that a similar incidence of C-cell adeno mas or carcinomas of the thyroid, as observed in the high-dose group of the TiO2 feeding study, has been seen in control female Fischer 344 rats used in other studies. No significant excess tumors occurred in male or female mice or in male rats. It was concluded that, under the conditions of this bioassay, TiO2 is not car cinogenic by the oral route for Fischer 344 rats or B6C3F1 mice .
In a study of male and female Fischer 344 rats fed diets containing up to 5% TiO2-coated mica for up to 130 weeks, no treatment-related toxi cologic or carcinogenic effects were reported .

# Intraperitoneal Injection
Female Wistar rats received intraperitoneal in jections of P25 ultrafine anatase TiO2 (2 ml 0.9% NaCl solution) of either (1) a total dose of 90 mg per animal (once per week for five weeks); (2) a single injection of 5 mg; or (3) a series of injections of 2, 4, and 4 mg at weekly intervals . Controls received a single injection of saline alone. The average lifespan of rats in the three treatment groups were 120, 102, and 130, respectively, and 120 weeks for controls. In the first treatment group, 6 out of 113 rats developed sarcomas, mesothe liomas, or abdominal cavity carcinomas, com pared to 2 carcinomas in controls. No intra abdominal tumors were found in the other two treatment groups. This study may not be rele vant to inhalation exposure to TiO2 since stud ies have not shown that either fine or ultrafine TiO2 would be likely to reach the peritoneum after depositing in the lungs.

# Particle-Associated Lung Disease Mechanisms

# Role of Pulmonary

# Inflamm ation
Chronic pulmonary inflammation is charac terized by persistent elevation of the number of PMNs (measured in BALF) or by an increased number of inflammatory cells in interstitial lung tissue (observed by histopathology). Pul monary inflammation is a defense mechanism against foreign material in the lungs. PMNs are recruited from the pulmonary vasculature in response to chemotactic stimuli (cytokines) generated by lung cells including alveolar mac rophages, which patrol the lungs as part of the normal lung defense to phagocytose and clear foreign material . Additional alveolar macrophages are recruited from blood monocytes into the lung alveoli in response to particle deposition. Typically, the PMN response is short-lived but may become chronic in response to persistent stimuli, e.g., prolonged particle exposure .
Particle-induced pulmonary inflammation, oxidative stress, lung tissue damage, and epi thelial cell proliferation are considered to be the key steps leading to lung tum or develop ment in the rat acting through a secondary genotoxic mechanism . Oxidative stress is considered the underlying mechanism of the prolifera tive and genotoxic responses to poorly solu ble particles including TiO2 and other PSLT . Reactive oxygen or nitrogen species (ROS/RNS) are released by inflammatory cells (macrophages and PMNs) and/or by particle reactive surfaces. Oxida tive stress results from an imbalance of the damaging oxidants and the protective antiox idants. Oxidants can damage the lung epithe lial tissue and may also induce genetic dam age in proliferating epithelial cells, increasing the probability of neoplastic transformation . The mechanisms linking pulm onary inflammation with lung cancer may involve (1) induction of hprt mutations in DNA of lung epithelial cells by inflamma tory cell-derived oxidants and increased cell proliferation , and (2) inhibition of the nucleotide excision repair of DNA (with adducts from other exposures such as polycyclic aromatic hydrocarbons) in the lung epithelial cells . Both of these mechanisms involve induction of cell-derived inflammatory mediators, without requiring particle surface-generated oxidants.
A secondary genotoxic mechanism would, in theory, involve a threshold dose that triggers inflammation and overwhelms the body' s an tioxidant and DNA repair mechanisms . Antioxidant de fense responses vary across species, and inter individual variability is generally greater in hu mans than in laboratory animals , and the quantitative aspects of the inflammatory response (level and duration) that are sufficient to cause a high probability of lung tum or development are not known.
While there is a clear association between in flammation and genotoxicity, the specific link ages between key cellular processes such as cell cycle arrest, DNA repair, proliferation, and apoptosis are not well understood .
Thus, chronic pulmonary inflammation ap pears to be required in the development of lung tumors in rats following chronic inhala tion exposure to TiO2; i.e., acting through a secondary genotoxic mechanism involving oxidative DNA damage. An implication of this mechanism is that maintaining exposures be low those causing inflammation would also prevent tum or development, although the dis tribution of inflammatory responses in human populations is not known and a direct genotoxic mechanism cannot be ruled out for dis crete nanoscale TiO2 (see Section 3.5.2.1).

# Dose Metric and Surface

# Properties
High mass or volume dose of PSLT fine par ticles in the lungs has been associated with overloading, while ultrafine particles impair lung clearance at lower mass or volume dos es .
The increased lung retention and inflamma tory response of ultrafine PSLT particles com pared to fine PSLT particles correlates better to the particle surface area dose ]. Some evidence suggests that reduced lung clearance of ultrafine particles may involve mechanisms other than high-dose overload ing, such as altered alveolar macrophage func tion (phagocytosis or chemotaxis) .
The quantitative relationships between the particle dose (expressed as mass or surface area) and the pulmonary responses of inflam mation or lung tumors can be determined from the results of subchronic or chronic inha lation studies in rats. When the rat lung dose is expressed as particle mass, several different dose-response relationships are observed for pulmonary inflammation following subchron ic inhalation of various types of poorly soluble particles (Figure 3-1). However, when dose is converted to particle surface area, the different types (TiO2 and BaSO4) and sizes (ultrafine and fine TiO2) of PSLT particles can be described by the same dose-response curve (Figure 3-2), while crystalline silica, SiO2 (a high-toxicity particle) demonstrates a more inflammogenic response when compared to PSLT particles of a given mass or surface area dose. Similarly, the rat lung tum or response to vari ous types and sizes of respirable particles (in cluding fine and ultrafine TiO2, toner, coal dust, diesel exhaust particulate, carbon black, and talc) has been associated with the total particle surface area dose in the lungs (Figure 3-3). This relationship, shown by Oberdorster and Yu , was extended by Driscoll  to include results from subsequent chronic inha lation studies in rats exposed to PSLT particles and by Miller  who refit these data using a logistic regression model. The lung tumor response in these analyses is based on all lung tumors, since some of the studies did not dis tinguish the keratinizing squamous cell cysts from the squamous cell carcinomas . Keratinizing squamous cell cysts have been observed primarily in the lungs of female rats exposed to PSLT, including TiO2 (discussed further in Sections 3.2.5, 3.5.2.4, and 3.6).
Figure 3 -3 . Relationship between particle surface area dose in the lungs of rats after chronic inha lation to various types of poorly soluble, low toxicity (PSLT) particles and tum or proportion (all tumors including keratinizing squamous cell cysts)
Data source: toner , coal dust , diesel exhaust particulate , TiO2 , talc  Figures 3-4 and 3-5 show the particle mass and surface area dose-response relationships for fine and ultrafine TiO2 for chronic inhala tion exposure and lung tumor response in rats.
In these figures, the lung tumor response data are shown separately for male and female rats at 24 months in Lee et al.  and for fe male rats at 24 or 30 months, including either all tumors or tumors without keratinizing cystic tumors  since this study distinguished these tum or types. The data are plotted per gram of lung to adjust for differences in the lung mass in the two strains of rats (Sprague-Dawley and Wistar). Figure 3-4 shows that when TiO2 is expressed as mass dose, the lung tumor response to ultrafine TiO2 is much greater at a given dose than that for fine TiO2; yet when TiO2 is expressed as par ticle surface area dose, both fine and ultrafine TiO2 data fit the same dose-response curve (Figure 3-5). These findings indicate that, like other PSLT particles, TiO2 is a rat lung car cinogen, and for a given surface area dose, the equivalent mass dose associated with elevated tum or response would be much higher for fine particles than for ultrafine particles.
T i0 2 mass concentration (mg/g lung)
Figure 3 -4 . TiO2 mass dose in the lungs of rats exposed by inhalation for 2 years and tum or pro portion (either all tumors or tumors excluding keratinizing squamous cell cysts)
Note: Spline model fits to Lee data. (Heinrich dose data are jittered, i.e., staggered) Data source: Heinrich et al. , Lee et al.  Figure 3 -5 . TiO2 surface area dose in the lungs of rats exposed by inhalation for 2 years and tumor proportion (either all tumors or tumors excluding keratinizing squamous cell cysts)
Note: Spline model fits to Lee data. (Heinrich dose data are jittered, i.e., staggered) Data source: Heinrich et al. , Lee et al.  The difference in T^ crystal structure in these subchronic and chronic studies did not influence the dose-response relationships for pulmonary inflammation and lung tumors . That is, the particle sur face area dose and response relationships were consistent for the ultrafine (80% anatase, 20% rutile) and fine (99% rutile) T^ despite the differences in crystal structure. In contrast, differences in ROS generation and toxicity have been observed for different T^ crystal structures in cell-free, in vitro, and short-term in vivo studies. Cell-free assays have reported crystal structure (anatase, rutile, or mixtures) influences particle surface ROS generation . In a cellfree study designed to investigate the role of sur face area and crystal structure on particle ROS generation, Jiang et al.  observed that size, surface area, and crystal structure all contribute to ROS generation. The ROS generation was associated with the number of defective sites per surface area, which was constant for many of the particle sizes but varied for some of the smaller particle sizes (10-30 nm) due to dif ferences in particle generation methods. In an in vitro cell assay, cytotoxicity was associated with greater ROS generation from photoacti vated TiO2 . In a pulmonary toxicity study in rats, the surface activity of the TiO2 particle (related to crystal structure, pas sivation, and acidity) was associated with the inflammation and cell proliferation responses at early time points, but not at the later time points .
Although these studies (cited in preceding paragraph) indicate that the particle surface properties pertaining to the crystal structure of TiO2, including photoactivation, can influ ence the ROS generation, cytotoxicity, and acute lung responses, these studies also show that crystal structure does not influence the pulmonary inflammation or tumor responses following subchronic or chronic exposures.
The reason for these differences in the acute and longer-term responses with respect to TiO2 crystal structure are not known but could relate to immediate effects of exposure to pho toactivated TiO2 and to quenching of ROS on the TiO2 surfaces by lung surfactant. Unlike crystalline silica, which elicits a pronounced pulmonary inflammation response, the in flammatory response to TiO2 of either the ru tile or anatase/rutile crystal form was much less pronounced after subchronic exposure in rats [Bermudez et al. 2002[Bermudez et al. , 2004 (Figure 3-2).
Reactive species (ROS/RNS) are also produced by alveolar macrophages and inflammatory cells during lung clearance and immunologi cal responses to inhaled particles, and the oxidative stress that can occur when antioxi dant defenses are overwhelmed is considered an underlying mechanism of the proliferative and genotoxic responses to inhaled particles . PSLT particles, including TiO2, have relatively low surface reactivity compared to the more inher ently toxic particles with higher surface activ ity such as crystalline silica . These findings are based on the studies in the scientific literature and may not apply to other formulations, surface coatings, or treatments of TiO2 for which data were not available.

# Particle-Associated Lung Responses

# Rodent Lung Responses to Fine and Ultrafine TiO2
Like other PSLT, fine and ultrafine TiO2 can elicit persistent pulmonary inflammation at sufficiently high dose and/or duration of ex posure. This occurs at doses that impair the normal clearance of particles from the alveolar (gas exchange) region of the lungs, i.e., "over loading" of alveolar macrophage-mediated particle clearance from the lungs . As the lung burden increases, alveolar macrophages become activated and release ROS/RNS and cellular factors that stimu late pathological events . Lung overload is characterized in rats, and to some extent in mice and ham sters, as increased accumulation of particle laden macrophages, increased lung weight, infiltration of neutrophils, increased epithelial permeability, increased transfer of particles to lymph nodes, persistent inflammation, lipoproteinosis, fibrosis, alveolar epithelial cell hyperplasia, and (eventually in rats) metapla sia and nonneoplastic and neoplastic tumors .
Lung inflammation and histopathological re sponses to TiO2 and other PSLT particles were more severe in the rat than in the mouse or hamster strains studied . Qualitatively similar early lung responses have been observed in rodents, especially rats and mice, although differences in disease progression and severity were also seen . Hamsters continued to ef fectively clear particles from the lungs, while the mice and rats developed overloading of lung clearance and retained higher lung par ticle burdens postexposure . Mice and rats developed persistent pulm onary inflam m a tion through 52 weeks after a 13-week expo sure to 50 or 250 mg/m3 fine TiO2  or to 10 mg/m3 ultrafine TiO2 . The rat lung response to another PSLT (carbon black) was more inflammatory (e.g., greater and more sustained generation of ROS in lung cells and higher levels of some inflammatory cytokines in BALF) compared to mice or hamsters . All three rodent species developed proliferative epithelial changes at the higher doses; however, only the rat developed metapla sia and fibrosis , but only rats had a significantly elevated lung tumor response .
Different strains of mice were used in the sub chronic and chronic studies (B3C3F1/CrlBR in Bermudez et al.  and NMRI in Hein rich et al. ). The NMRI mice had a high background tumor response (in the unexposed control mice), which might have limited the ability to detect any particle-related increase in that study . The B3C3F1/ CrlBR mouse strain, which showed a prolif erative alveolar cell response to ultrafine TiO2 , has not been evaluated for tumor response to TiO2. Thus, the mouse data are limited as to their utility in evaluating a TiO2 lung tum or response. In addition, the rapid particle lung clearance in hamsters and their relative insensitivity to inhaled particles also limits the usefulness of that species in eval uating lung responses to TiO2. In the rat stud ies, three different strains were used (Sprague-Dawley , Wistar , and Fischer-344 ), although no heterogeneity was observed in the dose-response relationship when these data were pooled and the dose was expressed as particle surface area per gram of lung (Ap pendix A). Based on the available data, the rat is the most sensitive rodent species to the pul monary effects (inflammation and tumors) of inhaling PSLT particles such as TiO2.

# Comparison of Rodent and Human Lung Responses to PSLT Including TiO2

# Particle retention and lung response
The similarities and differences between hu man and rat lung structures, particle deposi tion, and responses to inhaled poorly soluble particles including TiO2 have been described . For example, there are structur al differences in the small airways in the lungs of rats and humans (e.g., lack of well-defined respiratory bronchioles in rats). However, this region of the lungs is a primary site of particle deposition in both species, and particles that deposit in this region can translocate into the interstitium where they can elicit inflamma tory and fibrotic responses .
Both similarities and differences in rat and hu man nonneoplastic responses to inhaled par ticles were observed in a comparative pathol ogy study (based on lung tissues from autopsy studies in workers and in chronic inhalation studies in rats, with chronic exposure to ei ther coal dust, silica, or talc in both species) . Humans and rats showed some consistency in response by type of dust; i.e., granulomatous nodular inflammation was more severe among workers and rats exposed to silica or talc compared to coal dust. Simi larly, humans and rats showed some graded response by dose; e.g., more severe centriacinar fibrosis at high versus low coal dust expo sure in both humans and rats. In humans, the centriacinar fibrotic response was more severe among individuals exposed to either silica or coal dust compared to the rat response to these dusts. In rats, intra-alveolar acute inflamma tion, lipoproteinosis, and alveolar epithelial hyperplasia responses were more severe fol lowing chronic exposure to silica, talc, or coal dust compared to those responses in humans.
The greater inflammatory and cell proliferation responses in rats suggest they may be more sus ceptible than humans to lung tumor responses to inhaled particles via a secondary genotoxic mechanism involving chronic pulmonary in flammation, oxidative stress, and cell damage and proliferation. However, it is also important to note that alveolar epithelial hyperplasia was clearly observed in workers exposed to silica, talc, or coal dust (and this response was more severe in humans compared to rats at "low" coal dust exposure). An important consid eration in interpreting these study results is that the quantitative dust exposures were well known in rats and poorly known in humans . The rat exposures to these dusts ranged from 2 to 18 mg/m3 for 2 years. In humans, the exposure concentrations and durations were not reported for silica and talc; for coal dust, the durations were not reported, and "high" versus "low" exposure was defined as having worked before or after the 2 mg/m3 standard. Since it is not possible from these data to determine how well the dust exposures and lung burdens compare between the rats and humans, the qualitative comparisons are probably more reliable than the quantitative comparisons, which could be due in part to unknown differences in the lung dose in rats and humans.
Different patterns of particle retention have been observed in rats, monkeys, and humans . Although no data are available on TiO2 particle retention patterns, coal dust (fine size) or diesel exhaust particles (ultrafine) were retained at a higher volume percentage in the alveolar lumen in rats and in the interstitium in monkeys . The animals had been exposed by inha lation to 2 mg/m3 of coal dust and/or diesel ex haust particulate for 2 years .
A greater proportion of particles was also retained in the interstitium in humans com pared to rats . In humans, the proportion of particles in the interstitium increased as the duration of exposure and es timated coal dust concentration increased. In rats, the particle retention pattern did not vary with increasing concentration of diesel exhaust particulate from 0.35 to 7.0 mg/m3. The authors . The increased interstitialization of particles in rats at high doses has also been inferred by the in creasing particle mass measured in the lungassociated lymph nodes  since the movement of particles from the al veolar region to the pulmonary lymphatics requires transport through the alveolar epithe lium and its basement membrane . Thus, the particle retention observed in rats at overloading doses may bet ter represent the particle retention in the lungs of workers in dusty jobs, such as coal miners, where little or no particle clearance was ob served among retired miners . In addition, similar particle retention patterns (i.e., location in lungs) have been observed in rats, mice, and hamsters , yet the rat lung response is more severe. The retained dose is clearly a main factor in the lung response, and the low response in the hamster has been attributed to its fast clearance and low retention of particles . These findings suggest that the particle retention pattern is not the only, or necessarily the most important, factor influencing lung response between species in cluding rat and human.
Particle size and ability to translocate from the lung alveolar region into the interstitium may also influence lung tissue responses. Borm et al.  noted that the rat lung tumor response increased linearly with chronic pulmonary inflammation (following IT of various types of fine-sized particles), but that the rat lung tum or response to ultrafine TiO2 was much greater relative to the inflammation response. They suggested that tumor development from ultrafine particles may be due to high interstitialization rather than to overload and its sequella as seen for the fine-sized particles. Al though this hypothesis remains unproven, it is clear that the rat lung tum or response to ul trafine TiO2 was greater than that of fine TiO2 on a mass basis . The rat lung tum or response was con sistent across particle size on a particle surface area basis, suggesting that the particle surface is key to eliciting the response.
The extent of particle disaggregation in the lungs could influence the available particle sur face as well as the ability of particles to trans locate from the alveolar lumen into the interstitium, each of which could also influence pulmonary responses, as shown with various sizes of ultrafine and fine particles such as TiO2 . In a study using simulated lung surfactant, disaggregation of P25 ultrafine TiO2 was not observed . An other nanoscale TiO2 particle (4 nm primary particle diameter; 22 nm count median diam eter; 330 m 2/g specific surface area) was ob served inside cells and cell organelles including the nucleus . Based on this observation, it has been suggested that an al ternative genotoxic mechanism for nanoscale particles might involve direct interaction with DNA [Schins and . NIOSH is not aware of any studies of the carcinogenic ity of discrete nanoscale TiO2 particles that would provide information to test a hypothesis of direct genotoxicity. However, this possible mechanism has been considered in the risk as sessment (Chapters 4 and 5).
In rats that have already developed particleassociated lung tumors, the percentage of PMNs in the lungs is relatively high (e.g., 40%-60%) . However, it is not known what sustained level of PMNs is required to trigger epithelial proliferation and tumorigenic responses. In a chronic inhalation study, an average level of approximately 4% PMNs in BALF was measured in rats at the first dose and time point that was associated with a sta tistically significantly increased lung clear ance half-time (i.e., a relatively early stage of overloading) . Similarly, in a subchronic inhalation study, 4% PMNs was predicted in rats at an average lung dose of TiO2 or BaSO4 that was not yet overloaded (based on measured particle lung burden) . The relatively slight differences in the rat responses in these studies  may be due in part to differences in rat strains (Fisher-344; Wistar) and measures to assess overloading (clearance half-time and lung burden retention).
In humans, chronic inflammation has been as sociated with nonneoplastic lung diseases in workers with dusty jobs. Rom  found a statistically significant increase in the percent age of PMNs in BALF of workers with respi ratory impairment who had been exposed to asbestos, coal, or silica (4.5% PMN in cases vs. 1.5% PMNs in controls). Elevated levels of PMNs have been observed in the BALF of miners with simple coal workers' pneumoco niosis (31% of total BALF cells vs. 3% in con trols)  and in patients with acute silicosis (also a ten-fold increase over controls) . Humans with lung diseases that are characterized by chronic in flammation and epithelial cell proliferation

# Inflammation in rats and humans
(e.g., idiopathic pulmonary fibrosis, diffuse interstitial fibrosis associated with pneumoco niosis) have an increased risk of lung cancer . Dose-related increases in lung cancer have been observed in workers exposed to respirable crystalline silica , which can cause inflammation and oxidative tissue dam age . Chronic inflammation appears to be important in the etiology of dustrelated lung disease, not only in rats, but also in humans with dusty jobs  and interstitial fibrosis .
The percentage of PMNs in BALF of normal con trols "rarely exceeded" 4% . In other human studies, "normal" PMN percent ages ranged from 2%-17% . An average of 3% PMNs (range 0%-10%) was observed in the BALF of nonsmoker controls (without lung disease), while 0.8% (range 0.2%-3%) was seen in smoker controls (yet the PMN count in smokers was higher; this was due to ele vation in other lung cell populations in smokers) . An elevation of 10% PMNs in the BALF of an individual has also been cited as being clinically abnormal .

# Noncancer responses to TiO2 in rats and humans
Case studies of workers exposed to TiO2 pro vide some limited information to compare the human lung responses to those observed in the rat. In both human and animal studies, TiO2 has been shown to persist in the lungs. In some workers, extensive pulmonary deposition was observed years after workplace exposure to TiO2 had ceased . This suggests that human lung retention of TiO2 may be more similar to that in rats and mice compared to hamsters, which continue to clear the particles effectively at exposure concentrations that caused over loading in rats and mice [Everitt et al. 2000;Bermudez et al. 2002Bermudez et al. , 2004.
Inflammation, observed in pathologic ex amination of lung tissue, was associated with titanium (by X-ray or elemental analysis) in the majority of human cases with heavy TiO2 deposition in the lung . Pulmonary inflammation has also been observed in studies in rats, mice, and hamsters exposed to TiO2 . Contin ued pulmonary inflammation in the lungs of some exposed workers after exposure cessation  also appears to be more consistent with the findings in rats and mice than in hamsters, where in flammation gradually resolved with cessation of exposure.
In one case study, pulmonary alveolar pro teinosis (lipoproteinosis) was reported in a painter whose lung concentrations of titanium (60-129 x 106 particles/cm3 lung tissue) were "among the highest recorded" in a database of similar analyses . The titani um-containing particles were "consistent with titanium dioxide" and were the major type of particle found in the workers' lungs. In this case, the lipoproteinosis appeared more extensive than the lipoproteinosis seen in TiO2-exposed rats . Thus, although the rat lipoproteinosis response was more severe than that in work ers exposed to various fine-sized dusts , this case study illustrates that hu mans can also experience severe lipoproteinosis response which is associated with particle retention in the lungs.
Mild fibrosis has been observed in the lungs of workers exposed to TiO2  and in rats with chronic inhalation exposure to TiO2 .
In laboratory animals, alveolar metaplasia has been observed in rats, but not mice or ham sters, with subchronic inhalation to fine and ultrafine TiO2 [Lee et al. 1985;Everitt et al. 2000;Bermudez et al. 2002Bermudez et al. , 2004.
Although these studies suggest some similari ties in the lung responses reported in case stud ies of workers exposed to respirable TiO2 and in experimental studies in mice and rats, the hu man studies are limited by being observational in nature and by lacking quantitative exposure data. Information was typically not available on various factors including other exposures that could have contributed to these lung responses in the workers. Also, systematic histopathological comparisons were not performed, for exam ple, on the specific alveolar metaplastic changes of the rat and human lungs.

# Lung tum or types in rats and humans
Lung tumors observed in rats following chronic inhalation of TiO2 include squamous cell keratin izing cysts, bronchioloalveolar adenomas, squa mous cell carcinomas, and adenocarcinomas  (see Sec tion 3.2.5). The significance of the squamous cell keratinizing cystic tum or (a.k.a. prolifera tive keratin cyst) for human risk assessment has been evaluated . Squamous cell keratinizing cystic tu mors are most prevalent in female rats exposed to high mass or surface area concentrations of PSLT. In a recent reanalysis of the lung tumors in the Lee et al.  chronic inhalation study of fine (pigment-grade) TiO2, the 15 lesions originally recorded as "squamous cell carci noma" were reclassified as 16 tumors including two squamous metaplasia, 13 pulmonary kera tin cysts, and one squamous cell carcinoma . This reevaluation is consistent with the earlier evaluation of the squamous cell keratinizing cystic tumors by Boorman et al. . The classification of the 29 bronchioloalveolar adenomas in Warheit and Frame  remained unchanged from that reported in Lee et al. .
Human and rat lung cancer cell types have simi larities and differences in their histopathologies . The respira tory tracts of humans and rats are qualitatively similar in their major structures and functions, although there are also specific differences such as the absence of respiratory bronchioles in rats . In humans, the major cell types of lung cancer worldwide are adenocarcinoma and squamous-cell carcinoma (also observed in rats) and small-and large-cell anaplastic carcinoma (not seen in rats) . In rats exposed to PSLT, most cancers are adenocarcinomas or squamous-cell carcinomas of the alveolar ducts . In recent years, there has been a shift in the worldwide prevalence of human lung tumors toward adenocarcinomas in the bronchoalveolar region . Maronpot et al.  suggest that some of the apparent difference in the bronchioloalveolar carcinoma incidence in rodents and humans can be explained by differences in terminology, and that a more accurate comparison would be to combine the adenocarcinomas and bronchioloalveolar carcinomas in humans, which would significantly reduce the apparent dif ference. Cigarette smoking in humans is also likely to contribute to the difference between the incidences of human and rodent tumor types. Maronpot et al.  suggest that if the smoking-related tum or types were eliminated from the comparison, then the major lung tum or types in humans would be adenocar cinomas and bronchioloalveolar carcinomas, which would correspond closely to the types of lung tumors occurring in rodents.

# Rat Model in Risk Assessment of Inhaled Particles
The extent to which the rat model is relevant to predicting human lung doses and responses to inhaled particles including TiO2 has been the subject of debate . Lung clearance of particles is slower in humans than in rats, by approximate ly an order of magnitude , and some humans (e.g., coal miners) may be exposed to concen trations resulting in doses that would overload particle clearance from rat lungs . Thus, the doses that cause overloading in rats appear to be relevant to estimating disease risk in work ers given these similarities in particle retention in the lungs of rats at overloading doses and in workers with high dust exposures.
Some have stated that the inhalation doseresponse data from rats exposed to PSLT particles should not be used in extrapolating cancer risks to humans because the rat lung tum or response has been attributed as a ratspecific response to the overloading of particle clearance from the lungs . While some of the tumors (kera tinizing cystic tumors) may not be relevant to humans, other types of tumors (adenomas, ad enocarcinoma, squamous cell carcinoma) do occur in humans.
Mice and hamsters are known to give false neg atives to a greater extent than rats in bioassays for some particulates that have been classified by the IARC as human carcinogens (limited or sufficient evidence), including crystalline silica and nickel subsulfide; and the mouse lung tu mor response to other known human particu late carcinogens-including beryllium, cadmi um, nickel oxide, tobacco smoke, asbestos, and diesel exhaust-is substantially less in mice than that in rats . These particulates may act by various plausible mechanisms, which are not fully understood . The risks of several known human particulate carcinogens would thus be underestimated by using dose-response data from rodent models other than the rat. Al though the mechanism of particle-elicited lung tumors remains to be fully elucidated, the rat and human lung responses to poorly soluble particles of low or high toxicity (e.g., coal dust and silica) are qualitatively similar in many of the key steps for which there are data, including pulmonary inflammation, oxidative stress, and alveolar epithelial cell hyperplasia . Case studies of lung responses in humans exposed to TiO2 are consistent in showing some similarities with rat responses (see Section 3.5.2.3). Semiquantitative com parisons of the lungs of rats and humans ex posed to diesel exhaust, coal dust, silica, or talc indicate both similarities and differences, in cluding similar regions of particle retention in the lungs but at different proportions , and greater fibrotic response in humans but greater inflammation and epithe lial hyperplasia responses in rats . These sensitive inflammatory and pro liferative responses to particles in the lungs are considered key to the rat lung tum or response . Although the data are limited for quantitative comparison of rat and hum an dose-response relationships to inhaled par ticles, the available data (e.g., crystalline silica and diesel exhaust particles) indicate that the rat-based estimates are not substantially great er, and some are lower, than the human-based risk estimates  and that, in the absence of mechanistic data to the contrary, it is reasonable to assume that the rat model can identify potential carcinogenic hazards of poorly soluble particles to humans , including PSLT such as TiO2.
4 Quantitative Risk Assessment

# Data and Approach
Dose-response data are needed to quantify risks of workers exposed to TiO2. Such data may be obtained either from human studies or extrapolated to humans from animal stud ies. The epidemiologic studies on lung cancer have not shown a dose-response relationship in TiO2 workers . However, dose-response data are avail able in rats, for both cancer (lung tumors) and early, noncancer (pulmonary inflammation) endpoints. The lung tumor data (see Table 4-4) are from chronic inhalation studies and in clude three dose groups for fine TiO2 and one dose group for ultrafine TiO2 (in addition to controls). The pulmonary inflammation data are from subchronic inhalation studies of fine and ultrafine particles . Various modeling approaches are used to fit these data and to estimate the risk of disease in workers exposed to TiO2 for up to a 45-year working lifetime.
The modeling results from the rat dose-response data provide the quantitative basis for develop ing the RELs for TiO2, while the mechanistic data from rodent and human studies (Chapter 3) provide scientific information on selecting the risk assessment models and methods. The prac tical aspects of mass-based aerosol sampling and analysis were also considered in the overall approach (i.e., the conversion between particle surface area for the rat dose-response relation ships and mass for the human dose estimates and RELs). Figure 4-1 illustrates the risk as sessment approach.

# Methods
Dose-response modeling was used to estimate the retained particle burden in the lungs asso ciated with lung tumors or pulmonary inflam mation. Both maximum likelihood (MLE) and 95% lower CI estimates of the internal lung doses in rats were computed. Particle surface area was the dose metric used in these models because it has been shown to be a better pre dictor than particle mass of both cancer and noncancer responses in rats (Chapter 3). In the absence of quantitative data comparing rat and human lung responses to TiO2, rat and human lung tissue are assumed to have equal sensitiv ity to an equal internal dose in units of particle surface area per unit of lung surface area .

# Particle Characteristics
Study-specific values of particle MMAD, geo metric standard deviation (GSD), and spe cific surface area were used in the dosimetric modeling when available (see Tables 4-1 and 4-4). The Heinrich et al.  study reported a specific surface area (48  2 m 2/g ultrafine TiO2) for the airborne particulate, as mea sured by the BET N2 adsorption method. For Risk assessment approach using rat dose-response data to derive recommended exposure limits for TiO2.
the Lee et al.  study, the specific surface area (4.99 m 2/g fine TiO2) reported by Driscoll  was used; that value was based on mea surement of the specific surface area of a ru tile TiO2 sample similar to that used in the Lee study . This specific surface area was also assumed for the fine TiO2 in the M uh le et al.  study. Otherwise, fine TiO2 was assumed to have the particle characteristics re ported by Tran et al.  and a specific sur face area of 6.68 m2/g, and ultrafine TiO2 was assumed to have the particle characteristics re ported by Heinrich et al.  and a specific surface area of 48 m 2/g.

# Critical Dose
The term "critical dose" is defined as the re tained particle dose in the rat lung (maximum likelihood estimate  or 95% lower confi dence limit ) associated with a specified response, including either initiation of inflam mation or a given excess risk of lung cancer. One measure of critical dose is the benchmark dose (BMD), which has been defined as ". . . a statistical lower confidence limit on the dose corresponding to a small increase in effect over the background level" . In cur rent practice, and as used in this document, the BMD refers to the MLE from the model; and the benchmark dose lower bound (BMDL) is the 95% LCL of the BMD , which is equivalent to the BMD as originally defined by Crump . For dichotomous noncancer responses the benchmark response level is typically set at a 5% or 10% excess risk; however, there is less agreement on benchmark response levels for continuous responses. The benchmark response level used in this analysis for pulmonary inflammation, as assessed by el evated levels of PMNs, was 4% PMNs in BALF  (see Section 3.5.2.2). Another measure of critical dose, used in an earlier draft of this current intelligence bulletin, was the esti mated threshold dose derived from a piecewise linear model fit to the pulmonary inflamma tion data (Appendix B). As discussed in Section 4.3.1.2, not all of the current data sets are com patible with a threshold model for pulmonary inflammation; however, the threshold estimates described in Appendix B have been retained in this document for comparative purposes. For lung tumors the approach to estimating critical lung doses was to determine the doses associ ated with a specified level of excess risk (e.g., 1 excess case per 1,000 workers exposed over a 45-year working lifetime), either estimated di rectly from a selected model or by model av eraging using a suite of models . The 1 per 1,000 lifetime excess risk level was considered to be a significant risk based on the "benzene" decision .

# Estimating Human Equivalent Exposure
The critical doses were derived using particle surface area per gram of rat lung, which was estimated from the mass lung burden data, rat lung weights, and measurements or estimates of specific surface area (i.e., particle surface area per mass). The use of a particle surface area per gram of lung dose for the rat doseresponse analyses was necessary in order to normalize the rat lung weights to a common dose measure, since several strains of rats with varying lung weights were used in the analyses (see The critical doses were then multiplied by 1.5 in order to normalize them to rats of the size used as a reference for lung surface area, as these were estimated to have lung weights of approximately 1.5 grams, based on the animal' s body weights. The critical doses were then ex trapolated to humans based on the ratio of rat lung to human lung surface areas, which were assumed to be 0.41 m 2 for Fischer 344 rats, 0.4 m2 for Sprague-Dawley rats, and 102.2 m2 for humans . These critical particle surface area doses were then converted back to particle mass dose for humans because the current hum an lung dosim etry models (used to estimate airborne concentration lead ing to the critical lung doses) are all mass-based and because the current occupational exposure limits for most airborne particulates including TiO2 are also mass-based.

# Particle Dosimetry Modeling
The multiple-path particle dosimetry model, version 2, (MPPD2) human lung dosimetry model  was used to es timate the working lifetime airborne mass con centrations associated with the critical doses in human lungs, as extrapolated from the rat dose-response data. The specific MPPD2 m od ule used was the Yeh/Schum Symmetric model, assuming 17.5 breaths per minute and a tidal volume of 1,143 ml, with exposures of 8 hours per day, 5 days per week for a working lifetime of 45 years. The total of alveolar TiO2 plus TiO2 in the lung-associated lymph nodes was con sidered to be the critical human lung dose.
The respiratory frequency and tidal volume were chosen to be consistent with the Inter national Commission on Radiological Protec tion (ICRP) parameter values for occupational exposure, which equate an occupational ex posure to 5.5 hours/day of light exercise and 2.5 hours/day of sedentary sitting, with a total inhalation volume of 9.6 m 3 in an 8-hour day . The ICRP assumes 20 breaths per minute and a tidal volume of 1,250 ml for light exercise and 12 breaths per minute and a tidal volume of 625 ml for sedentary sitting. The values assumed in this analysis for modeling TiO2 exposures-i.e., 17.5 breaths per minute and a tidal volume of 1,143 m l-are a weighted average of the respiratory values for the light exercise and sedentary sitting conditions and are designed to match the ICRP  value for total daily occupational inhalation volume.
In summary, the dose-response data in rats were used to determine the critical dose asso ciated with pulmonary inflammation or lung tumors, as particle surface area per surface area of lung tissue. The working lifetime air borne mass concentrations associated with the human-equivalent critical lung burdens were estimated using human lung dosimetry m od els. The results of these quantitative analyses and the derivation of the RELs for fine and ul trafine TiO2 are provided in the remainder of this chapter.

# Dose-Response Modeling of Rat Data and Extrapolation to Humans

# Pulmonary Inflamm ation 4.3.1.1 Rat data
Data from four different subchronic inhalation studies in rats were used to investigate the rela tionship between particle surface area dose and pulmonary inflammation response: (1) TiO2 used as a control in a study of the toxicity of volcanic ash , (2) fine TiO2 and BaSO4 in a study of the particle surface area as dose metric , (3) fine TiO2 in a multidose study , and (4) ultrafine TiO2 in a multidose study . Details of these studies are provided in

# Critical dose estimates in rats
The TiO2 pulmonary inflammation data from the Tran et al.  and Cullen et al.  studies could be fitted with a piecewise linear model which included a threshold parameter (described in Appendix B), and the threshold parameter estimate was significantly different from zero at a 95% confidence level. The MLE of the threshold dose was 0.0134 m 2 (particle surface area for either fine or ultrafine TiO2) for TiO2 alone (90% CI = 0.0109-0.0145) based on data from Tran et al.  and 0.0409 m2 (90% CI = 0.0395-0.0484) based on data from Cullen et al. . However, the fine and ul trafine TiO2 pulmonary inflammation data from the Bermudez et al.  and Bermudez et al.  data sets provided no indication of a nonzero response threshold and were not consistent with a threshold model. Therefore, critical dose estimation for the pulmonary in flammation data was carried out via a bench mark dose approach, since benchmark doses could be fit to all three of the data sets (Tran et al. , Cullen et al. , and the combined data from Bermudez et al.  and Bermudez et al. ).
Continuous models in the benchmark dose software (BMDS) suite  were fit ted to the pulmonary inflammation data us ing percent neutrophils as the response and TiO2 surface area (m2) per gram of lung as the predictor. The model that fit best for all three sets of TiO2 data was the Hill model, which converged and provided an adequate fit. Since there appears to be an upper limit to the degree of physiological response, the Hill model is able to capture this behavior better than the linear, quadratic, or power models. Models were fit ted using constant variance for the Cullen et al.  data and a nonconstant variance for the Tran et al.  data and the combined data from Bermudez et al.  and Bermu dez et al. . The model fits for these data sets are illustrated in Figures 4-2, 4-3, and 4-4, respectively. In all models the critical dose or BMD was defined as the particle surface area per gram of lung tissue associated with a 4% inflammatory response of neutrophils, which has been equated to a low level inflammatory response ; see Section 3.5.2.2. The benchmark dose estimates for pulmonary inflammation in rats are shown in Table 4-2.

# Estimated human equivalent exposure
The critical dose estimates from

# Lung Tumors

# Rat data
Dose-response data from chronic inhalation studies in rats exposed to TiO2 were used to estimate working lifetime exposures and lung cancer risks in humans. These studies are de scribed in more detail in Table 4-4 and include fine (pigment-grade) rutile TiO2  and ultrafine anatase TiO2 . The doses for fine TiO2 were 5 mg/m3  and 10, 50, and 250 mg/m3 . For ultra fine TiO2 there was a single dose of approxi mately 10 mg/m3 . Each of these studies reported the retained particle mass lung burdens in the rats. The internal dose measure of particle burden at 24 months of exposure was used in the dose-response models, either as particle mass or particle sur face area (calculated from the reported or esti mated particle surface area per gram).
The relationship between particle surface area dose of either fine or ultrafine TiO2 and lung tumor response (including all tumors or tu mors excluding the squamous cell keratinizing cysts) in male and female rats was shown in Chapter 3. Statistically significant increases in lung tumors were observed at the highest dose of fine TiO2 (250 mg/m3) or ultrafine TiO2 (ap proximately 10 mg/m3), whether or not the squamous cell keratinizing cysts were included in the tum or counts.
Different strains and sexes of rats were used in each of these three TiO2 studies. The Lee et al.  study used male and female Sprague-Dawley rats (crl:CD strain). The Heinrich study used female Wistar rats . The Muhle et al.  study used male and female Fischer-344 rats but reported only the average of the male and female lung tumor proportions.
The body weights and lung weights differed by rat strain and sex (Table 4-4). These lung mass differences were taken into account when cal culating the internal doses, either as mass (mg TiO2/g lung tissue) or surface area (m2 TiO2/m 2 lung tissue).

# Critical dose estimates in rats
Statistical models for quantal response were fitted to the rat tumor data, including the suite of models in the BMDS . The re sponse variable used was either all lung tumors or tumors excluding squamous cell keratiniz ing cystic tumors. Figure 4-5 shows the fit of the various BMD models  to the lung tum or response data (without squamous cell keratinizing cysts) in male and female rats chronically exposed to fine or ultrafine TiO2 .
The lung tum or response in male and female rats was significantly different for "all tum ors" but not when squamous cell keratinizing cys tic tumors were removed from the analysis (Appendix A, Table A-2). In other words, the male and female rat lung tum or responses were equivalent except for the squamous cell  Lee et al.  Muhle et al. ; Bellman et al.  Sprague-Dawley 557 780 (cri: CD)  At 24 months: 0/10 (controls) 4/9 (all tumors) At 30 months: 1/217 (controls) 19/100 (no keratinizing cysts) 32/100 (all tumors) GSD = geometric standard deviation; MMAD = mass median aerodynamic diameter; SA = surface area (mean or assumed mean); SD = arithmetic standard deviation; T i0 2 = titanium diox ide; crl:CD and crl:(WI)BR are the rat strain names from Charles River Laboratories, Inc. *Lung particle burdens in controls not reported; assumed to be zero.
Turn or types: controls, male: 2 bronchioloalveolar adenomas. At 10 mg/m3, males: 1 large cell anaplastic carcinoma and 1 bronchioloalveolar adenoma. At 50 mg/m3, male: 1 bronchioloalveolar adenoma. At 250 mg/m3, females: 13 bronchioloalveolar adenomas and 1 squamous cell carcinoma; males: 12 bronchioloalveolar adenomas. In addition to the tumors listed above, 13 keratin cysts were observed: 1 in a 10 mg/m3 male, 1 in a 250 mg/m3 male, and 11 in 250 mg/m3 females. Dose was averaged for male and female rats because the tumor rates were reported only for male and female rats combined. Tumor types: controls, 2 adenocarcinomas and 1 adenoma. At 5 mg/m3: 1 adenocarcinoma and 1 adenoma. ' " Tumor types: controls, at 30 months: 1 adenocarcinoma. At -10 mg/m3: 20 benign squamous-cell tumors, 3 squamous-cell carcinomas, 4 adenomas, and 13 adenocarcinomas (includes 8 rats with 2 tumors each). BMD models and three-model average fit to the lung tum or data (without squamous cell keratinizing cysts) in male and female rats chronically exposed to fine or ultrafine TiO2
Note: The specific models used in constructing the model average were the multistage, Weibull, and log-probit models. The confidence intervals represent 95% binomial confidence limits for the individual data points.
Data source: BMD models and three-model average , male and female rats exposed to TiO2  keratinizing cystic tumor response, which was elevated only in the female rats. To account for the heterogeneity in the "all tumor" response among male and female rats , a modified logistic regres sion model was developed (Appendix A); this model also adjusted for the combined mean tu mor response for male and female rats reported by Muhle et al. . As discussed in Chap ter 3, many pathologists consider the rat lung squamous cell keratinizing cystic tumor to be irrelevant to human lung pathology. Excess risk estimates of lung tumors were estimated both ways-either with or without the squamous cell keratinizing cystic tumor data. The full results of the analyses including squamous cell keratin izing cystic tumors can be found in Appendix A. Inclusion of the keratinizing cystic tumors in the analyses resulted in slightly higher excess risk es timates in females, but not males. Since the male and female rat tumor responses may be combined when the squamous cell keratinizing cystic tumors are excluded and exclusion does not have a major numeric impact on the risk esti mates, risk estimates for TiO2-induced lung tumors are based on the combined male and female rat lung tumors, excluding the squa mous cell keratinizing cystic tumors. All lung tumor-based risk estimates shown below have been derived on this basis. Classification of the squamous cell keratinizing cystic tumors in the Lee et al.  study was based on a reanalysis of these lesions by Warheit and Frame .
The estimated particle surface area dose associ ated with a 1/1000 excess risk of lung tumors is shown in Table 4-5 for lung tumors excluding squamous cell keratinizing cystic lesions. The estimated particle surface area doses-BMD and BMDL-associated with a 1/1000 excess risk of lung cancer vary considerably depend ing on the shape of the model in the low-dose region. The model-based estimates were then summarized using a model averaging technique , which weights the various models based on the model fit. Model averaging provides an approach for summariz ing the risk estimates from the various models, which differ in the low-dose region which is of interest for human health risk estimation, and also provides an approach for addressing the uncertainty in choice of model in the BMD ap proach. The specific model averaging method used was the three-model average procedure as described by Wheeler and Bailer , using the multistage, Weibull, and log-probit models. These received weights of 0.14, 0.382 and 0.478, respectively, in the averaging procedure. This approach was considered to be appropriate for the TiO2 data set because the dose-response re lationship is nonlinear, and the specific models used in the three-model average procedure do not impose low-dose linearity on the model av erage if linearity is not indicated by the data. In this case the best fitting models are all strongly sub-linear; the multistage model is cubic in form, the Weibull model is similar with a power of 2.94, and the log-probit model has a slope of 1.45. Therefore the dose-response relationship is quite steep, and the estimated risk drops sharply as the dose is reduced, as shown in Table 4-7.

# Estimated human equivalent exposure
The critical dose estimates from Table 4-5 were converted to mass dose and extrapolated to humans by adjusting for species differences in lung surface area, as described in Section 4.2.3. Pulmonary dosimetry modeling was used to estimate the occupational exposure concentra tions corresponding to the benchmark dose estimates, as described in Section 4.2.4. Life time occupational exposure concentrations estimated to produce human lung burdens equivalent to the rat critical dose levels for lung tumors are presented in Table 4-6 for fine and ultrafine TiO2. Estimated occupational ex posure levels corresponding to various levels of lifetime excess risk are shown in Table 4-7.
This table shows the risk estimates for both fine and ultrafine TiO2. The 95% LCL estimates of the occupational exposure concentrations ex pected to produce a given level of lifetime ex cess risk are shown in the right-hand column. The concentrations shown in bold for fine and ultrafine TiO2 represent 1 per 1000 risk levels, which NIOSH has used as the basis for estab lishing RELs. The REL for ultrafine TiO2 was rounded from 0.29 mg/m3 to 0.3.

# Alternate Models and Assumptions
The choice of dosimetry models influences the estimates of the mean airborne concentration.
A possible alternative to the MPPD model of MA = model average; BMD = maximum-likelihood estimate of benchmark dose; BMDL = benchmark dose lower bound (95% lower confidence limit for the benchmark dose); BMDS = benchmark dose software; TiO2 = titanium dioxide.
Response modeled: lung tum ors excluding cystic keratinizing squam ous lesions-from two studies of fine TiO2  and one study of ultrafine TiO 2 . Acceptable m odel fit determ ined by P > 0.05. BMDS did not converge. Model fitting and BMD and BMDL estim ation were carried out as described in W heeler and Bailer . Average model, as described by W heeler and Bailer , based on the multistage, Weibull, and log-probit models. P-values are not defined in m odel averaging because the degrees of freedom are unknown. Abbreviations: MA = m odel average; BMD = benchm ark dose; GSD = geometric standard deviation, LCL = lower confidence limit; MLE = m axim um likelihood estimate; TiO 2 = titanium dioxide, MPPD = m ultiple-path particle dosim etry  model. *MLE and 95% LCL were determ ined in rats (Table 4-5) and extrapolated to hum ans based on species differences in lung surface area, as described in Section 4.2.3. Mean concentration estimates were derived from the CIIT and RIVM  lung model. W ithout keratinizing cystic lesions. ?Model averaging combined estimates from the multistage, Weibull, and log-probit models .
Mass m edian aerodynamic diam eter (MMAD). Agglomerated particle size for ultrafine TiO 2 was used in the deposition model . Specific surface area was used to convert from particle surface area dose to mass dose; thus airborne particles with different specific surface areas w ould result in different mass concentration estimates from those shown here. "Abbreviations: MA = model average; BMD = benchm ark dose; GSD = geometric standard deviation, LCL = lower confidence limit; MLE = m axim um likelihood estimate; TiO 2 = titanium dioxide, M PPD = m ultiple-path particle dosim etry  model. fM odel averaging com bined estimates from the multistage, Weibull, and log-probit models . *MLE and 95% LCL were determ ined in rats (Table 4-5) and extrapolated to hum ans based on species differences in lung surface area, as described in Section 4.2.3. Mean concentration estimates were derived from the CIIT and RIVM  lung model. Without keratinizing cystic lesions. Mass m edian aerodynamic diam eter (MMAD). Agglomerated particle size for ultrafine TiO 2 was used in the deposition model . Specific surface area was used to convert from particle surface area dose to mass dose; thus airborne particles with different specific surface areas would result in different mass concentration estimates from those shown here. n The exposure levels shown in boldface are the 95% LCL estimates of the concentrations of fine and ultrafine TiO2 considered ap propriate for establishment of a REL. The ultrafine exposure level of 0.29 m g/m 3 was rounded to 0.3 for the REL.
particle deposition of CIIT and RIVM , which was used for particle dosimetry model ing in this analysis, would be an interstitialization/sequestration model that was developed and calibrated using data of U.S. coal miners  and later validated using data of U.K. coal miners . The MPPD model uses the ICRP alveolar clearance model, which was developed using data on the clearance of radiolabeled tracer particles in humans, and has been in use for many years . More data are needed to evaluate the model structures and determine how well each model would de scribe the retained doses associated with low particle exposures in humans, particularly for ultrafine particles. The MPPD model was se lected for use in this analysis on the grounds that it is widely used and well accepted for par ticle dosimetry modeling in general, while use of the interstitialization/sequestration model has to date been limited primarily to modeling expo sures to coal dust. Nevertheless, it should be not ed that the interstitialization/sequestration mod el predicts lung burdens of fine and ultrafine TiO2 which are approximately double those predicted by the MPPD model. Thus, the use of the interstitialization/sequestration model for particle do simetry would approximately halve the estimates of occupational exposure levels equivalent to the rat critical dose levels, compared to estimates de veloped using the MPPD model.
The method selected for extrapolating between rats and humans also influences the estimates of occupational exposure levels equivalent to the rat critical dose levels. To extrapolate the critical particle surface area dose in the lungs of rats to the lungs of humans, either the relative mass or surface area of the lungs in each species could be used. The results presented in this analysis are based on the relative alveolar surface area, as suming 0.41 m 2 for Fischer 344 rats, 0.4 m2 for Sprague-Dawley rats, and 102.2 m2 for humans . Alternatively, extrapolation could be based on the relative lung weights of rat and human lungs, using a dose metric of particle surface area per gram of lung tissue. In that case, the estimates of the working lifetime occupational exposure levels equivalent to the rat critical dose levels would be higher by a factor of approximately four. The lung surface area-based approach was selected for this anal ysis because insoluble particles deposit and clear along the surface of the respiratory tract, so that dose per unit surface area is often used as a normalizing factor for comparing particle doses across species .
The critical dose estimates in Table 4-5 also vary depending on the dose-response model used and on whether the MLE or the 95% LCL is used as the basis for estimation. The MLE dose estimates associated with a 1/1000 excess risk of lung tumors vary by a factor of 38, while the 95% LCL estimates vary by a factor of 14. All of the models provided statistically adequate fits to the data, and there is little basis to select one model over another. This uncertainty regarding model form has been addressed by the use of MA, as described by Wheeler and Bailer , which weights the models based on the model fit as as sessed by the Akaike information criterion. How ever, any of the models shown could conceivably be selected as the basis for human risk estimation. Use of the model-averaged 95% LCL value, as op posed to the model-averaged MLE, is intended to address both model uncertainty and variability in the rat data; however, it is possible that the 95% LCL may underestimate the true variability of the human population.

# Mechanistic Considerations
The mechanism of action of TiO2 is relevant to a consideration of the associated risks because, as discussed earlier, the weight of evidence sug gests that the tumor response observed in rats exposed to fine and ultrafine TiO2 results from a secondary genotoxic mechanism involving chronic inflammation and cell proliferation, rather than via genotoxicity of TiO2 itself. This effect appears related to the physical form of the inhaled particle (i.e., particle surface area) rather than the chemical compound itself. Other PSLT particles-such as BaSO4, carbon black, toner, and coal dust-also produce inflammation and lung tumors in proportion to particle surface area (Figures 3-2 and 3-3) and therefore appear to act via a similar mechanism.
Studies supporting this mechanism include em pirical studies of the pulmonary inflammatory response of rats exposed to TiO2 and other PSLT (see Sections 3.2.4 and 3.4.1); the tumor re sponse of TiO2 and other PSLT, which have con sistent dose-response relationships (see Section 3.4.2); and in vitro studies, which show that in flammatory cells isolated from BALF from rats exposed to TiO2 released ROS that could induce mutations in naive cells (see Section 3.2.1.3).
There is some evidence, though limited, that in flammation may be a factor in the initiation of human lung cancer as well (see Section 3.5.2.2).
In considering all the data, NIOSH has deter mined that a plausible mechanism of action for TiO2 in rats can be described as the accumula tion of TiO2 in the lungs, overloading of lung clearance mechanisms, followed by increased pulmonary inflammation and oxidative stress, cellular proliferation, and, at higher doses, tumorigenesis. These effects are better described by particle surface area than mass dose (see Section 3.4.2). The best-fitting dose-response curves for the tumorigenicity of TiO2 are non linear; e.g., the multistage model in Table 4-5 is cubic with no linear term , the quantalquadratic model is quadratic with no linear term , and the gamma and Weibull models have power terms of approximately 4 and 3, respectively. This nonlinearity is consistent with a secondary genotoxic mechanism and suggests that the carcinogenic potency of TiO2 would decrease more than proportionately with decreasing surface area dose as described in the best-fitting risk assessment models.

# Quantitative Comparison of Risk Estimates From Human and Animal Data
A quantitative comparison was performed (Appendix C) of the rat-based excess risk esti mates for human lung cancer due to exposure to fine TiO2 to the 95% upper confidence limit (UCL) of excess risk from the epidemiologic studies (Appendix D), in order to compare the rat-and human-based excess risks of lung cancer. If the sensitivity of the rat response to inhaled particulates differs from that of hu mans, then the excess risks derived from the rat data would be expected to differ from the excess risks estimated from the human studies.
The results of the comparison of the rat-and human-based excess risk estimates were used to assess whether or not there was adequate precision in the data to reasonably exclude the rat model as a basis for predicting the excess risk of lung cancer in humans exposed to TiO2.
The results of these comparisons showed that the MLE and 95% UCL excess risk estimates from the rat studies were lower than the 95% UCL from the human studies for an estimated working lifetime (Appendix C, Table C-1). These results indicate that, given the variability in the human studies , the rat-based excess risk es timates cannot reasonably be dismissed from use in predicting the excess risk of lung cancer in humans exposed to TiO2. Thus, NIOSH determined that it is prudent to use these rat dose-response data for risk assessment in workers exposed to TiO2.

# Possible Bases for an REL

# Pulmonary Inflamm ation
As discussed above, the evidence in rats sug gests that the lung tum or mechanism asso ciated with PSLT particles such as TiO2 is a secondary genotoxic mechanism involving chronic inflammation and cell proliferation.
One plausible approach to developing risk esti mates for TiO2 is to estimate exposure concen trations that would not be expected to produce an inflammatory response, thus preventing the development of responses that are secondary to inflammation, including cancer. A bench mark dose analysis for pulmonary inflamma tion in the rat was described in Section 4.3.1, and the results of extrapolating the rat BMDs to humans are presented in Table 4-3. Since two of the three studies available yielded 95% BMDLs of 0.78 and 1.03 mg/m3, a concentra tion of approximately 0.9 mg/m3 is reasonable as the starting point for development of rec ommendations for human exposures for fine TiO2. Similarly, a concentration of approxi mately 0.11 mg/m3 is appropriate as the start ing point for developing recommended expo sures to ultrafine TiO2.
As noted in Section 4.3.1.3, the human pulmo nary inflammation BMDs in Table 4-3 are es timates of frank-effect levels and should be ad justed by the application of uncertainty factors to allow for uncertainty in animal-to-human ex trapolation and interindividual variability. These uncertainty factors are commonly assumed to be ten-fold for animal-to-human extrapola tion and another ten-fold for interindividual variability; the animal-to-human uncertainty may be subdivided into a factor of 4 for toxi cokinetics and 2.5 for toxicodynamics (WHO 1994). Since the rat BMDs were extrapo lated to humans using a deposition/clear ance model, it is reasonable to assume that the animal-to-human toxicokinetic subfactor of 4 has already been accounted for; therefore, a total uncertainty factor of 25 (2.5 for animal to-human toxicodynamics times 10 for inter individual variability) should be applied. This results in estimated exposure concentrations designed to prevent pulmonary inflammation of 0.04 m g/m 3 for fine TiO2 and 0.004 mg/m3 for ultrafine TiO2.

# Lung Tumors
Rather than estimating an exposure concentra tion designed to avoid secondary toxicity by preventing pulmonary inflammation, another possible basis for developing a REL is to model the risk of lung tumors directly. In the absence of mechanistic data in humans, the tumorigenic mechanism operative in rats cannot be ruled out. Therefore, one approach for estimation of recommended levels of occupational exposure to TiO2 is to estimate the pulmonary particle surface area dose associated with a 1/1000 in crease in rat lung tumors and to extrapolate that dose to humans on the basis of particle surface area per unit of lung surface area. This approach was used to assess the excess risk of lung cancer at various working lifetime expo sure concentrations of fine or ultrafine TiO2 (Table 4 -6). Selection of the model for esti mating risks has a significant impact on the risk estimates. As shown in Table 4-6, the 95% LCL working lifetime mean concentration of fine TiO2 associated with a 1/1000 excess risk of lung cancer is 0.3 to 4.4 mg/m3, depending on the model used to fit the rat lung tum or data. For ultrafine TiO2, the 95% LCL working lifetime mean concentration associated with a 1/1000 excess risk of lung cancer is 0.04 to 0.54 mg/m 3, depending on the model.
Although any of the models evaluated in Table 4-6 could conceivably be used to develop rec ommendations for occupational exposures to TiO2, the model averaging procedure is attrac tive since it incorporates both statistical vari ability and model uncertainty into confidence limit estimation. However, an argument could also be made for basing recommendations on the multistage model, due to its long his tory of use for carcinogen risk assessment or the quantal-linear model, on the grounds that it generates the lowest BMD and BMDL and is thus arguably the most health-protective. The BMD and BMDL derived via each of these models are shown in Table 4-6 for both fine and ultrafine TiO2.
Since the various models produce different risk estimates and there is no clear mechanistically based preference for one model over another, it is appropriate to summarize the results by using a MA technique. MA, as implemented here, uses all the information from the various dose-response models, weighting each model by the Akaike information criterion for model fit and constructing an average dose-response model with lower bounds computed by boot strapping. This method was described by Wheeler and Bailer , who demonstrated via simulation studies that the MA method has superior statistical properties to a strategy of simply picking the best-fitting model from the BMDS suite. As shown in Table 4-6, the model average estimate of the working lifetime mean concentration of fine TiO2 associated with a 1/1000 excess risk of lung cancer is 13.2 mg/m3, with a 95% LCL of 2.4 mg/m3. The correspond ing estimates for ultrafine TiO2 are 1.6 mg/m3, with a 95% LCL of 0.3 mg/m3. NIOSH believes that it is reasonable and prudent to use the 95% LCL model-averaged estimates as the ba sis for RELs, as opposed to the MLEs, in order to allow for model uncertainty and statistical variability in the estimates.

# Comparison of Possible Bases for an REL
As discussed above, occupational exposure concentrations designed to prevent pulmo nary inflammation, and thus prevent the de velopment of secondary toxicity (including lung tumors), are 0.04 m g/m 3 for fine TiO2 and 0.004 mg/m3 for ultrafine TiO2. In comparison, modeling of the dose-response relationship for lung tumors indicates that occupational expo sure concentrations of 2.4 mg/m3 for fine TiO2 and 0.3 m g/m 3 for ultrafine TiO2 would be suf ficient to reduce the risk of lung tumors to a 1/1000 lifetime excess risk level. The discrep ancy between the occupational exposure con centrations estimated from modeling either pulmonary inflammation or lung tumors raises serious questions concerning the optimal basis for a TiO2 REL. However, it must be acknowl edged that the two sets of possible RELs are not based on entirely comparable endpoints. The pulmonary inflammation-based exposure concentrations are expected to entirely prevent the development of toxicity secondary to pul monary inflammation, resulting in zero excess risk of lung tumors due to exposure to TiO2. In contrast, the lung tumor-based exposure con centrations are designed to allow a small, but nonzero, excess risk of lung tumors due to oc cupational exposure to TiO2.
As discussed in Section 3.4.1, particle-induced pulmonary inflammation may act as a precur sor for lung tum or development; however, pulmonary inflammation itself is not a specific biomarker for lung cancer. As noted in Sec tion 3.5.2.2, the precise level of sustained inflammation necessary to initiate a tumorigenic response is currently unknown. It is pos sible that the 4% PMN response used in this analysis as the benchmark response level for pulmonary inflammation is overly protective and that a somewhat greater inflammatory re sponse is required for tum or initiation.
It is also possible that the 25-fold uncer tainty factor applied to the critical dose es tim ate for pulm onary inflamm ation may be overly conservative, since pulm onary in flammation is an early event in the sequence of events leading to lung tum ors. However, NIOSH has not previously used early events or secondary toxicity as a rationale for apply ing smaller than normal uncertainty factors. Given that in this case the primary objective of preventing pulmonary inflammation is to prevent the development of lung tumors, and given that lung tumors can be adequately con trolled by exposures many-fold higher than the inflammation-based exposure concentrations, NIOSH has concluded that it is appropriate to base RELs for TiO2 on lung tumors rather than pulmonary inflammation. However, NIOSH notes that extremely low-level exposures to TiO2-i.e., at concentrations less than the pul monary inflammation-based RELs-may pose no excess risk of lung tumors.

# Hazard Classification and Recommended Exposure Limits
NIOSH initiated the evaluation of titanium dioxide by considering it as a single substance with no distinction regarding particle size. However, a review of all the relevant scien tific literature indicated that there could be a greater occupational health risk with smaller size (ultrafine) particles and therefore NIOSH provides separate recommendations for the ul trafine and fine categories.
NIOSH has reviewed the relevant animal and hum an data to assess the carcinogenicity of titanium dioxide (TiO2) and has reached the following conclusions. First, the weight of evi dence suggests that the tum or response ob served in rats exposed to ultrafine TiO2 resulted from a secondary genotoxic mechanism involv ing chronic inflammation and cell proliferation, rather than via direct genotoxicity of TiO2. This effect appears to be related to the physical form of the inhaled particle (i.e., particle surface area) rather than to the chemical compound itself.
Second, based on the weight of the scientific data (including increase in adenocarcinoma tu mor incidence in a chronic inhalation study in rats of 10 mg/m3), NIOSH determined that in haled ultrafine TiO2 is a potential occupational carcinogen and is recommending exposure lim its to minimize the cancer risk from exposure to ultrafine TiO2. Finally, because the tumorigenic dose of fine TiO2 (250 mg/m3) in the Lee et al. studies [1985Lee et al. studies [ , 1986a was substantially higher than current inhalation toxicology practiceand because there was no significant increase in tumors at 10 or 50 mg/m3-NIOSH did not use the highest dose in its hazard identification and concluded that there is insufficient evidence to classify fine TiO2 as a potential occupational carcinogen. Although NIOSH has determined that the data are insufficient for cancer hazard classification of fine TiO2, the particle surface area dose and tum or response relationship is consistent with that observed for ultrafine TiO2 and warrants that precautionary mea sures be taken to protect the health of workers exposed to fine TiO2. Therefore, NIOSH used all of the animal tum or response data to con duct the dose-response modeling, and devel oped separate mass-based RELs for ultrafine and fine TiO2.

# Hazard Classification
NIOSH reviewed the current scientific data on TiO2 to evaluate the weight of the evidence for the NIOSH designation of TiO2 as a "potential occupational carcinogen." Two factors were considered in this evaluation: (1) the evidence in humans and animals for an increased risk of lung cancer from inhalation of TiO2, including exposure up to a full working lifetime, and (2) the evidence on the biologic mechanism of the dose-response relationship observed in rats, including evaluation of the particle character istics and dose metrics that are related to the pulmonary effects.
No exposure-related increase in carcinogenic ity was observed in the epidemiologic studies conducted on workers exposed to TiO2 dust in the workplace . In contrast, chronic inhalation exposures to ultrafine TiO2 at approximately 10 mg/m 3 re sulted in a statistically significant increase in malignant lung tumors in rats, although lung tumors in mice were not elevated . The lung tumors observed in rats after exposure to 250 mg/m3 of fine TiO2 were the basis for the original NIOSH designation of TiO2 as a "potential occupational carcinogen." However, because this dose is considered to be significantly higher than currently accepted in halation toxicology practice , NIOSH concluded that the response at such a high dose should not be used in making its haz ard identification. Therefore, NIOSH has come to different conclusions regarding the potential occupational carcinogenicity for fine versus ultrafine TiO2. NIOSH evaluated the doseresponse data in humans and animals, along with the mechanistic factors described below, in assessing the scientific basis for the current NIOSH designation of ultrafine but not fine TiO2 as a "potential occupational carcinogen."
In addition, NIOSH used the rat dose-response data in a quantitative risk assessment to develop estimates of excess risk of nonmalignant and malignant lung responses in workers over a 45-year working lifetime. These risk estimates were used in the development of RELs for fine and ultrafine TiO2.

# Mechanistic Considerations
As described in detail in Chapter 3, the mech anistic data considered by NIOSH were ob tained from published subchronic and chronic studies in rodents exposed by inhalation to TiO2 or other PSLT particles. These studies in clude findings on the kinetics of particle clear ance from the lungs and on the nature of the relationship between particle surface area and pulmonary inflammation or lung tum or re sponse. The mechanistic issues considered by NIOSH include the influence of particle size or surface area (vs. specific chemical reactivity) on the carcinogenicity of TiO2 in rat lungs, the relationship between particle surface area dose and pulmonary inflammation or lung tumor response in rats, and the mechanistic evidence on the development of particle-elicited lung tumors in rats. These considerations are dis cussed in detail in Chapter 3.
NIOSH also considered the crystal structure as a modifying factor in TiO2 carcinogenic ity and inflammation. As described in Chap ter 3, some short-term studies indicate that the particle surface properties pertaining to the crystal structure of TiO2, including pho toactivation, can influence the ROS genera tion, cytotoxicity, and acute lung responses. These studies also show that crystal structure does not influence the pulmonary inflamma tion or tum or responses following subchronic or chronic exposures . These findings are based on the studies in the scientific literature and may not apply to other formulations, surface coatings, or treatments of TiO2 for which data were not available.
After analysis of the issues, NIOSH concluded that the most plausible mechanism for TiO2 carcinogenesis is a nonchemical specific inter action of the particle with the cells in the lung, characterized by persistent inflammation and mediated by secondary genotoxic processes. The dose-response relationships for both the in flammation and tumorigenicity associated with TiO2 exposure are consistent with those for other PSLT particles. Based on this evidence, NIOSH concluded that the adverse effects produced by TiO2 exposure in the lungs are likely not sub stance-specific, but may be due to a nonchem ical-specific effect of PSLT particles in the lungs at sufficiently high particle surface area expo sures. However, because the tumorigenic dose for fine TiO2  of 250 mg/m3 was significantly higher than currently accepted in halation toxicology practice , NIOSH did not use the 250 mg/m3 dose in its hazard identification. Therefore, NIOSH con cluded that there are insufficient data to classify fine TiO2 as a potential occupational carcinogen but there are sufficient data indicating that ul trafine TiO2 has the potential to cause cancer after adequate occupational exposure.

# Limitations of the Rat Tumor Data
NIOSH evaluated all publicly available epide miology studies and laboratory animal inhala tion studies and determined that the best data to support a quantitative risk assessment for TiO2 were from rat inhalation studies [Lee et al. 1985;Muhle et al. 1991;Heinrich et al. 1995;Cullen et al. 2002;Tran et al. 1999;Bermudez et al. 2002Bermudez et al. , 2004. These studies provided exposureresponse data for both inflammation and tumorigenicity and were used as the basis of the quantitative risk assessment.
NIOSH considered the scientific literature that supported and disputed on the rat as an ap propriate model for human lung cancer after exposure to inhaled particles and reached the conclusion that the rat is an appropriate spe cies on which to base its quantitative risk as sessment for TiO2. Although there is not ex tensive evidence that the overloading of lung clearance, as observed in rats (Chapter 3), occurs in humans, lung burdens consistent with overloading doses in rats have been ob served in some humans with dusty jobs (e.g., coal miners) . Rather than ex cluding the rat as the appropriate model, the lung overload process may cause the rat to at tain lung burdens comparable to those that can occur in workers with dusty jobs. In addition, evidence suggests that, as in the rat, inhalation of particles increases the human inflammatory response, and increases in the inflammatory response may increase the risk of cancer (see Section 3.5.2.2). This information provides ad ditional support for the determination that the rat is a reasonable animal model with which to predict human tum or response for particles such as TiO2.
After evaluating all ofthe available data, NIOSH concluded that the appropriate dose metric in its risk assessment was particle surface area. Both tumorigenicity and inflammation were more strongly associated with particle surface area than particle mass dose. The separate risk estimates for fine and ultrafine TiO2 are sup ported by the higher lung cancer potency in rats of ultrafine TiO2 compared to fine TiO2, which was associated with the greater surface area of ultrafine particles for a given mass. In rats chronically exposed to airborne fine TiO2, statistically significant excess lung tumors were observed only in the 250 m g/m3 dose group. Al though exposure concentrations greater than 100 mg/m3 are not currently standard method ology in inhalation toxicity studies , and NIOSH questions the relevance of the 250 mg/m 3 dose for classifying exposure to TiO2 as a carcinogenic hazard to workers, the tumor-response data are consistent with that observed for ultrafine TiO2 when converted to a particle surface area metric. Thus to be cau tious, NIOSH used all of the animal tum or re sponse data when conducting dose-response modeling and determining separate RELs for ultrafine and fine TiO2. With chronic exposure to airborne ultrafine TiO2, lung tumors were seen in rats exposed to an average of approxi mately 10 mg/m3 (ranged from 7.2 mg/m 3 to 14.8 mg/m3) over the exposure period.
It would be a better reflection of the entire body of available data to set RELs as the inhaled sur face area of the particles rather than the mass of the particles. This would be consistent with the scientific evidence showing an increase in potency with increase in particle surface area (or decrease in particle size) of TiO2 and other PSLT particles. For this reason, the basis of the RELs for fine and ultrafine TiO2 is the rat doseresponse data for particle surface area dose and pulmonary response. However, current tech nology does not permit the routine measure ment of the surface area of airborne particles; therefore, NIOSH recommends sampling the mass airborne concentration of TiO2 as two broad prim ary particle size categories: fine (< 10 ^m) and ultrafine (< 0.1 ^m). These cate gories reflect current aerosol size conventions, although it is recognized that actual particle size distributions in the workplace will vary.

# Cancer Classification in Humans
Since the public comment and peer review draft of this document was made available, NIOSH has learned that the IARC has reassessed TiO2. IARC now classifies TiO2 as an IARC Group 2B carcinogen, "possibly carcinogenic to humans" . NIOSH supports this decision and the underlying analysis lead ing to this conclusion.
Based on the animal studies described in Chapter 3 and the quantitative risk assessment in Chapter 4, NIOSH has concluded that ultra fine but not fine TiO 2 particulate m atter is a potential occupational carcinogen. However, as a precautionary step, NIOSH conducted a quantitative risk assessment based on the tumor data for both fine and ultrafine TiO2.
The potency of ultrafine TiO2, which has a much higher surface area per unit mass than fine TiO2, was many times greater than fine TiO2, with malignant tumors observed at the lowest dose level of ultrafine TiO2 tested (10 mg/m3).
The lack of an exposure-response relation ship in the epidemiologic studies of workers exposed to TiO2 dust in the workplace should not be interpreted as evidence of discordance between the mechanism presumed to operate in rats and the human potential for carcino genicity. As demonstrated by the quantitative comparison between the animal and human studies (see Section 4.4 and Appendix C), the responses were not statistically inconsistent: the epidemiologic studies were not sufficiently precise to determine whether they replicated or refuted the animal dose-response. This is not a surprising finding for carcinogens of lower po tency, such as fine-sized TiO2.
The mechanistic data reviewed above, how ever, leave open the possibility of species dif ferences beyond what would be anticipated for a genotoxic carcinogen. Although it is plau sible that the secondary genotoxic mechanism proposed here operates in humans exposed to TiO2 dust, there is insufficient evidence to corroborate this. In addition, there is limited information on the kinetics or specific physi ological response to TiO2 particles in humans.
The evidence suggests that inhalation of lower surface area TiO2 is not likely to result in car cinogenicity in any test species. This concept is reflected in the quantitative risk assessment, in which the curvilinear dose response predicts that lower exposures have disproportionally less risk than higher exposures. For workers, this suggests that exposure to concentrations lower than the RELs will be less hazardous and may pose a negligible risk.
Although the analysis in this document is lim ited to consideration of the workplace hazard posed by TiO2, these findings suggest that other PSLT particles inhaled in the workplace may pose similar hazards, particularly nano-sized particles with high surface areas. NIOSH is concerned that other nano-sized PSLT particles may have similar health effects to those ob served for TiO2, in which ultrafine TiO2 parti cles were observed to be more carcinogenic and inflammogenic on a mass basis than fine TiO2 [Heinrich et al. 1995;Lee et al. 1985Lee et al. , 1986a.

# Recommended Exposure Limits
NIOSH recommends airborne exposure limits of 2.4 mg/m3 for fine TiO2 and 0.3 mg/m 3 for ultrafine (including engineered nanoscale) TiO2 as TWA concentrations for up to 10 hr/day during a 40-hour work week, using the inter national definitions of respirable dust  and the NIOSH Method 0600 for sampling airborne respirable particles . NIOSH selected these expo sure limits for recommendation because they would reduce working lifetime risks for lung cancer to below 1/1000. Cancer risks greater than 1/1000 are considered significant and wor thy of intervention by OSHA. NIOSH has used this risk level in a variety of circumstances, in cluding citing this level as appropriate for devel oping authoritative recommendations in Crite ria Documents and risk assessments published in scientific journal articles . It is noted that the true risk of lung cancer due to exposure to TiO2 at these concentrations could be much lower than 1/1000. To account for the risk that exists in work environments where airborne exposures to fine and ultrafine TiO2 occur, exposure measurements to each size fraction should be combined using the addi tive formula and compared to the additive REL of 1 (unitless) (see Figure 6-1 Exposure assess ment protocol for TiO2).
Because agglomerated ultrafine particles are frequently measured as fine-sized but behave biologically as ultrafine particles due to the sur face area of the constituent particles, exposures to agglomerated ultrafine particles should be controlled to the ultrafine REL.
"Respirable" is defined as particles of aero dynamic size that, when inhaled, are capable of depositing in the gas-exchange (alveolar) region of the lungs . Sampling methods have been developed to estimate the airborne mass concentration of respirable par ticles . "Fine" is defined in this document as all par ticle sizes that are collected by respirable parti cle sampling (i.e., 50% collection efficiency for particles of 4 ^m, with some collection of par ticles up to10 ^m). "Ultrafine" is defined as the fraction of respirable particles with primary particle diameter < 0.1 ^m (< 100 nm), which is a widely used definition. Additional methods are needed to determ ine if an airborne re spirable particle sample includes ultrafine TiO2 (Chapter 6).
The NIOSH RELs for fine TiO2 of 2.4 mg/m3 and ultrafine TiO2 of 0.3 mg/m3 were derived using the model averaging procedure (described in Sections 4. Based on the observed relationship between particle surface area dose and toxicity (Chap ters 3 and 4), the measurement of aerosol surface area would be the preferred method for evaluating workplace exposures to TiO2. However, personal sampling devices that can be routinely used in the workplace for measur ing particle surface area are not currently avail able. As an alternative, if the airborne particle size distribution of the aerosol is known in the workplace and the size distribution remains relatively constant with time, mass concentra tion measurements may be useful as a surro gate for surface area measurements. NIOSH is recommending that a mass-based airborne concentration measurement be used for m oni toring workplace exposures to fine and ultra fine (including engineered nanoscale) TiO2 un til more appropriate measurement techniques can be developed. NIOSH is currently evaluat ing the efficacy of various sampling techniques for measuring fine and ultrafine TiO2 and may make specific recommendations at a later date.
In the interim, personal exposure concentrations to fine (pigment-grade) and ultrafine (including engineered nanoscale) TiO2 should be determined with NIOSH Method 0(300 using a standard 10-mm nylon cyclone or equivalent particle sizeselective sampler . Measurement results from NIOSH Method 0600 should provide a reasonable estimate of the exposure concentra tion to fine and ultrafine (including engineered nanoscale) TiO2 at the NIOSH RELs of 2.4 and 0.3 mg/m3, respectively, when the predominant exposure to workers is TiO2. No personal sam pling devices are available at this time to spe cifically measure the mass concentrations of ultrafine aerosols; however, the use of NIOSH Method 0600 will permit the collection of most airborne ultrafine (including engineered nanoscale) particles and agglomerates.
In work environments where exposure to other types of aerosols occur or when the size distri bution of TiO2 (fine vs. ultrafine) is unknown, other analytical techniques may be needed to characterize exposures. NIOSH Method 7300  can be used to assist in differ entiating TiO2 from other aerosols collected on the filter while electron microscopy, equipped with X-ray energy dispersive spectroscopy (EDS), may be needed to measure and iden tify particles. In workplaces where TiO2 is pur chased as a single type of bulk powder, the pri mary particle size of the bulk powder can be used to determine whether the REL for fine or ultrafine should be applied if adequate airborne exposure data exist to confirm that the airborne particle size has not substantially been altered during the handling and/or material process ing of TiO2. Although NIOSH Methods 0600 and 7300 have not been validated in the field for airborne exposure to TiO2, they have been validated for other substances and, therefore, should provide results for TiO2 within the ex pected accuracy of the Methods.

# Exposure Assessment
A multitiered workplace exposure assessment might be warranted in work environments where the airborne particle size distribution of TiO2 is unknown (fine vs. ultrafine) and/ or where other airborne aerosols may inter fere with the interpretation of sample results. Figure 6-1 illustrates an exposure assessment strategy that can be used to measure and iden tify particles so that exposure concentrations can be determined for fine and ultrafine (in cluding engineered nanoscale) TiO2. An initial exposure assessment should include the simul taneous collection of respirable dust samples with one sample using a hydrophobic filter (as described in NIOSH Method 0600) and the other sample using a mixed cellulose ester filter (MCEF).- If the respirable exposure concentra tion for TiO2 (as determined by Method 0600) is less than 0.3 mg/m3, then no further action is required. If the exposure concentration ex ceeds 0.3 mg/m3, then additional characteriza tion of the sample is needed to determine the particle size and percentage of TiO2 and other extraneous material on the sample. To assist in this assessment, the duplicate respirable sample collected on a MCEF should be evaluated us ing transmission electron microscopy (TEM) to size particles and determine the percentage of fine (> 0.1 ^m) and ultrafine (< 0.1 ^m) TiO2. When feasible, the percent of fine and ultrafine TiO2 should be determined based on the mea surement of "primary" particles (includes ag glomerates comprised of primary particles). The identification of particles (e.g., TiO2) by TEM can be accomplished using EDS or *Note: The collection time for samples using a MCEF may need to be shorter than the duplicate samples col lected and analyzed by M ethod 0600 to ensure that particle loading on the filter doesn't become excessive and hinder particle sizing and identification by TEM.
energy loss spectroscopy. Once the percent of TiO2 (by particle size) has been determined, adjustments can be made to the mass concen tration (as determined by Method 0600) to assess whether exposure to the NIOSH RELs for fine, ultrafine (including engineered na noscale), or combined fine and ultrafine TiO2 had been exceeded.
To minimize the future need to collect sam ples for TEM analysis, samples collected us ing M ethod 0600 can also be analyzed us ing NIOSH M ethod 7300 or other equivalent methods to determine the amount of TiO2 on the sample. When using NIOSH Method 7300, it is important that steps be taken (i.e., pre treatment with sulfuric or hydrofluoric acid) to insure the complete dissolution and recovery of TiO2 from the sample. The results obtained using Method 7300, or other equivalent m eth od, should be compared with the respirable mass concentration to determine the relative percentage of TiO2 in the concentration mea surement. Future assessments of workplace ex posures can then be limited to the collection and analysis of samples using Method 0600 and Method 7300 (or equivalent) to ensure that airborne TiO2 concentrations have not changed over time.

# Control of Workplace Exposures to TiO2
Given the extensive commercial use of fine (pigment grade) TiO2, the potential for occu pational exposure exists in many workplaces. However, few data exist on airborne concen trations and information on workplaces where exposure might occur. Most of the available data for fine TiO2 are reported as total dust and not as the respirable fraction. Historical total dust exposure measurements collected in TiO2 production plants in the United States often exceeded 10 mg/m3 , while more contemporary exposure concentrations in these plants indicate that mean total inhalable dust measurements may be below 3 mg/m3 (1.1 mg/m3 median) for most job tasks . Given the particle size dimensions of fine TiO2 (~0.1 ^m to 4 ^m, avg. of 0.5 ^m) , it is reasonable to conclude that respirable TiO2 particles com prised a significant fraction of the total dust measurement. Estimates of worker exposures to respirable TiO2 determined in 1999 from 8 European plants producing fine TiO2 ranged from 0.1 to 5.99 mg/m3 (plant with highest measured concentrations) to 0.1 to 0.67 mg/m3 (plant with lowest measured concentrations) . The highest reported worker exposure concentrations to TiO2 in both U.S. and European production plants were among packers and micronizers and during maintenance .
Results of a NIOSH health hazard evaluation conducted at a facility using powder paint con taining TiO2 found airborne concentrations of respirable TiO2 ranging from 0.01 to 1.0 mg/m3 (9 samples) . TEM analysis con ducted on one airborne sample found 42.4% of the TiO2 particles less than 0.1 ^m in diameter. NIOSH is not aware of any extensive commer cial production of ultrafine anatase TiO2 in the United States although it may be imported for use. Ultrafine rutile TiO2 is being commercially produced as an additive for plastics to absorb and scatter ultraviolet light; 10%-20% of the ultrafine TiO2 is reported to be < 100 nm in size . Engineered TiO2 nanoparticles are also being manufactured, and like ultrafine TiO2, they are finding commercial application as a photocatalyst for the destruction of chemi cal and microbial contaminants in air and wa ter, in light-emitting diodes and solar cells, in plastics, as a UV blocker, and as a "self-cleaning" surface coating. While a paucity of data exist on worker exposure to engineered TiO2, expo sure measurements taken at a facility manu facturing engineered TiO2 found respirable exposure concentrations as high as 0.14 mg/m3 . The primary particle size determined by TEM analysis ranged from 25 to 100 nm.  Liu 1981, 1982]. Ventila tion systems must be properly designed, tested, and routinely maintained to provide maximum efficiency. fit-tested, half-facepiece particulate respirator will provide protection up to 10 times the re spective REL. When selecting the appropriate filter and determining filter change schedules, the respirator program manager should con sider that particle overloading of the filters may occur because of the size and character istics of TiO2 particles. Studies on the filtration performance of N95 filtering-facepiece respi rators found that the mean penetration levels for 40 nm particles ranged from 1.4% to 5.2%, indicating that N95 and higher performing respirator filters would be effective at captur ing airborne ultrafine TiO2 particles [Balazy et al. 2006;Rengasamy et al. 2007Rengasamy et al. , 2008.
In workplaces where there is potential worker exposure to TiO2, employers should establish an occupational health surveillance program. Occupational health surveillance, which in cludes hazard surveillance (hazard and expo sure assessment) and medical surveillance, is an essential component of an effective occupa tional safety and health program. An impor tant objective of the program is the systematic collection and analysis of exposure and health data for the purpose of preventing illness. In workplaces where exposure to ultrafine or en gineered TiO2 occurs, NIOSH has published interim guidance on steps that can be taken to minimize the risk of exposure  and recommendations for medical surveil lance and screening  that could be used in establishing an occupational health surveillance program.

# Research Needs
Additional data and information are needed to assist NIOSH in evaluating the occupational safety and health issues of working with fine and ultrafine TiO2. Data are particularly need ed on the airborne particle size distributions and exposures to ultrafines in specific opera tions or tasks. These data may be merged with existing epidemiologic data to determine if exposure to ultrafine TiO2 is associated with adverse health effects. Information is needed about whether respiratory health (e.g., lung function) is affected in workers exposed to TiO2. Experimental studies on the mechanism of toxicity and tumorigenicity of ultrafine TiO2 would increase understanding of whether fac tors in addition to surface area may be impor tant. Although sampling devices for all particle sizes are available for research purposes, prac tical devices for routine sampling in the work place are needed.

# Workplace Exposures and Human Health
- Quantify the airborne particle size dis tribution of TiO2 by job or process and obtain quantitative estimates of workers' exposures to fine and ultrafine TiO2. - Conduct epidemiologic studies of work ers manufacturing or using TiO2-containing products using quantitative estimates of exposure by particle size, including fine and ultrafine fractions (see bullet above).
- Evaluate the extent to which the specific surface area in bulk TiO2 is representative of the specific surface area of the airborne TiO2 particles that workers inhale and that are retained in the lungs. - Investigate the adequacy of current massbased human lung dosimetry models for predicting the clearance and retention of inhaled ultrafine particles.

# Experimental Studies
- Investigate the fate of ultrafine particles (e.g., TiO2) in the lungs and the associ ated pulmonary responses. - Investigate the ability of ultrafine parti cles (e.g., TiO2) to enter cells and interact with organelle structures and DNA in m i tochondria or the nucleus.

# Measurement, Controls, and Respirators
- Develop accurate, practical sampling de vices for ultrafine particles (e.g., surface area sampling devices). - Evaluate effectiveness of engineering con trols for controlling exposures to fine and ultrafine TiO2. - Determine effectiveness of respirators for ultrafine TiO2.

# References
ACGIH . Particle size-selective sam pling in the workplace. Report of the ACGIH Technical Committee on air sampling proce dures. Ann Am Conf Gov Ind Hyg 11:23-100.
ACGIH . 1994-1995  As seen in Figures 3-4 and 3-5, particle surface area is a much better dose metric than particle mass for predicting the lung tum or response in rats after chronic exposure to fine and ultra fine TiO2. The statistical fit of these models is shown in Table A-1, using either mass or par ticle surface area dose. These goodness-of-fit tests show that particle surface area dose pro vides an adequate fit to models using either the all tum or response or tumors excluding squa mous cell keratinizing cysts and that particle mass dose provides an inadequate fit to these data. The P-values are for statistical tests of the lack of fit; thus, P < 0.05 indicates lack of fit. Because of the observed differences in tum or response in males and females, when squa mous cell keratinizing cystic tum ors were in cluded in the analysis (Table 4-4), it was im portant to test for heterogeneity in response by rat sex. Since the data were from different studies and rat strains, these factors were also investigated for heterogeneity (the influence of study and strain could not be evaluated separately because a different strain was used in each study). Finally, the possibility of het erogeneity in response to fine and ultrafine TiO2 after adjustment for particle surface area was investigated to determine whether other factors may be associated with particle size that influence lung tum or response and that may not have been accounted for by particle sur face area dose. Table A-2 shows that there was statistically significant heterogeneity between male and female rats for the all lung tumors re sponse but not for the tumors excluding squa mous cell keratinizing cysts. No heterogeneity in tum or response was observed across study/ strain or for fine versus ultrafine when dose was expressed as particle surface area per gram of lung. These analyses showed that all of the data from the different studies, rat strains, and both sexes  could be pooled and the model fit was adequate when the dose metric used is particle surface area per gram of lung and the tum or response is neoplastic tumors (i.e., without squamous cell keratinizing cystic tumors).
In addition, a modified dose-response model was developed to examine the all-tumor re sponse (by adjusting for rat sex and to include the averaged male/female lung tumor response data in the Muhle et al.  study) (see Sec tion A-3). Data are from two studies of fine TiO2  and one study of ultrafine TiO2 . Estimated directly from model. Estimated from linear extrapolation of BMD and BMDL at 1/10 excess risk level.
Table A -4. All tumors: Benchmark dose (BMD) and lower 95% confidence limit (BMDL) estimates-expressed TiO2 particle surface area in the lungs (m2/g )-by model fit separately to male and female rat data FEMALE rats MALE rats   BMD (BMDL) by excess risk level BMD (BMDL) by excess risk level P-value___________________________ P-value ___________________________ Model (BMDS 2003) (for lack of fit) 1/10- 1/1000- 1/1000f (for lack of fit) 1/10- 1/1000- 1/1000f

# APPENDIX B: Threshold Model for Pulmonary Inflammation in Rats
A threshold model (i.e., piecewise linear or "hockeystick") was examined for its ability to adequately represent TiO2-induced pulmonary inflammation in rat lungs . As described in Section 4.3.1.2, the TiO2 pulmonary inflammation data from the Tran et al.  and Cullen et al.  studies could be fitted with a piecewise linear model which included a threshold pa rameter, and the threshold parameter estimate was significantly different from zero at a 95% confidence level. However, the fine and ultra fine TiO2 pulmonary inflammation data from the Bermudez et al.  and Bermudez et al.  data sets provided no indication of a nonzero response threshold and were not consistent with a threshold model. The piece wise linear modeling methodology is detailed below.
In modeling pulmonary inflammation (as neu trophilic cell count in BAL fluid) in rat lungs, the response was assumed to be normally distributed with the mean response being a function of the dose and the variance propor tional to a power of the mean. Thus for the ith rat given the dose d., the mean neutrophilic cell count would be jupmn (di ) with variance a (Hpmn (di ))P, where MP"m is any continuous function of dose, a is a proportionality con stant, and p represents a constant power. The mean response was modeled using a variety of functions of dose; these functions were then used to estimate the critical dose at which the mean neutrophil levels went above the back ground. For the continuous functions that did not include a threshold parameter, this criti cal level was found using the BMD method  and software . For purposes of calculation, the BMD was defined as the particle surface area dose in the lungs associated with /upmn (dt) corresponding to the upper 5th percentile of the distribution of PMN counts in control rat lungs.
For the piecewise linear model, which is a thresh old model, we assumed no dose-response, and thus no additional risk, above background prior to some critical threshold y . For points beyond the threshold, the dose-response was modeled using a linear function of dose. For example, As the parameter y is an unknown term, the above function is nonlinear and is fit using maximum likelihood (ML) estimation. Very approximate (1-a)% CIs can be found using profile likelihoods . As the con fidence limits are only rough approximations, the limits and significance of the threshold can be cross validated using parametric bootstrap methods .

# APPENDIX C: Comparison of Rat-and Human-Based Excess Risk Estimates for Lung Cancer Following Chronic Inhalation of TiO2
As described in Chapter 2, the epidemiologic studies of workers exposed to TiO2 did not find a statistically significant relationship between the estimated exposure to total or respirable TiO2 and lung cancer mortality , suggesting that the precision of these studies for estimating ex cess risks of concern for worker health (e.g., <1/1000) may be limited. The exposure data in Fryzek et al.  were based on the to tal dust fraction, whereas respirable dust data were used to estimate exposures in Boffetta et al. . Neither study had exposure data for ultrafine particles. Chronic inhalation stud ies in rats exposed to fine  and ultrafine TiO2  showed statistically significant dose-response relation ships for lung tumors (Chapter 3). However, the rat lung tum or response at high particle doses which overload the lung clearance has been questioned as to its relevance to humans . Recent studies have shown that rats inhaling TiO2 are more sensitive than mice and hamsters to pulmonary effects in cluding inflammation [Bermudez et al. 2002[Bermudez et al. , 2004, although the hamsters had much faster clearance and lower retained lung burdens of TiO2 compared to rats and mice. Because of the observed dose-response data for TiO2 and lung cancer in rats, it is important to quanti tatively compare the rat-based excess risk esti mates with excess risk estimates derived from results of the epidemiologic studies.
The purpose of these analyses is to quantita tively compare the rat-based excess risks of lung cancer with results from the human stud ies. If the sensitivity of the rat response to in haled particulates differs from that of humans, then the excess risks derived from the rat data would be expected to differ from the excess risks estimated from the human studies. The results of the comparison will be used to as sess whether or not the observed differences of excess risks have adequate precision for rea sonably excluding the rat model as a basis for predicting the excess risk of lung cancer in hu mans exposed to TiO2.

# Methods
Excess risk estimates for lung cancer in work ers were derived from the epidemiologic stud ies (Appendix D) and from the chronic inhala tion studies in rats . These excess risk estimates and associated standard errors were computed for a mean exposure concentration of 2.4 m g/m 3 over a 45-year working lifetime. This exposure concentration was selected to correspond to a low value relative to the rat data (which is also the NIOSH REL, see Chapter 5).
Excess risks were derived from the rat data based on the three-model average procedure described in Chapter 4. The Model Averaging for Dichotomous Response (MADR) software used for the model average risk estimation was modified to output the model coefficients and model weights associated with each model fit in a 2,000-sample bootstrap, based on the rat data. The model coefficients and weights were then used to construct the distribution of risk estimates from rats exposed to the equivalent of a lifetime occupational exposure to 2.4 mg/m3 fine TiO2. The rat-equivalent exposures were estimated by using the MPPD2 model to esti mate the human lung burden associated with a 45-year occupational exposure to 2.4 mg/m3 fine TiO2. This lung burden (2545 mg TiO2) was then extrapolated to rats following the steps described in Chapter 4 for extrapolat ing from rats to humans, but in reverse order. This procedure yielded an estimated rat lung burden of 6.64 mg TiO2. Since the rat-based, dose-response modeling was based on a par ticle surface area dose metric, the rat lung bur den was then converted to 0.0444 m 2 particle surface area per gram of lung tissue, assuming a specific surface area of 6.68 m 2/g TiO2. The 2,000 sets of model equations from the model average were then solved for a concentration of 0.0444 m2/g lung tissue, yielding a bootstrap estimate of the distribution of the excess risk associated with exposure to TiO2. Excess risks were estimated from each of the two worker cohort studies, using two different methods for each. For the cohort studied by Boffetta et al. , two different values for representing the highest cumulative exposure group were separately assumed; and for the cohort studied by Fryzek et al. , two dif ferent exposure lags (no lag, 15-year lag) were separately used.

# Results
Table C shows the rat-based maximum likeli hood estimate (MLE) and 95% upper confi dence limit (UCL) of excess risks for lung can cer and the human-based 95% UCL on excess risk from exposure to TiO2. There is consisten cy in the estimates of the 95% UCL from these two independent epidemiologic studies at the exposure concentration evaluated for both studies, 2.4 mg/m3 (Boffetta: 0.038 and 0.053; Fryzek: 0.046 and 0.056). The rat-based MLE and 95% UCL excess risk estimates for fine TiO2 exposures are lower than the 95% UCL risk estimates based on the human studies in Table C. This result suggests that the rat-based risk estimates for fine TiO2 are not inconsistent with the human risk estimates derived from the epidemiological studies.

# Discussion
These two epidemiologic studies are subject to substantially larger variability than are the rat studies. The results of the epidemiologic stud ies of TiO2 workers by Fryzek et al.  and Boffetta et al. [2003Boffetta et al. [ , 2004 are consistent with a range of excess risks at given exposures, in cluding the null exposure-response relation ship (i.e., no association between the risk of lung cancer and TiO2 exposure) and an exposure-response relationship consistent with the low-dose extrapolations from the rat stud ies (based on the method used, a three-model average as described in Chapter 4). Both the MLE and 95% UCL excess risk estimates from the rat studies were lower than the 95% UCL from the human studies for fine TiO2.
to differ from the referent population under unexposed conditions.
The estimators of Alpha and Beta are based on iteratively reweighted least squares with weights proportional to the reciprocal of the mean. Although these estimates are equiva lent to Poisson regression MLEs, the observed counts are not strictly Poisson. This is due to the adjustments made by Boffetta et al.  for missing cause of death arising from the lim ited time that German death certificates were maintained. The reported observed counts are 53+0.9, 53+2.3, 52+2.7, 53+2.4 where 0.9, 2.3, 2.7, and 2.4 have been added by Boffetta et al.  for missing cause of death that are esti mated to have been lung cancer deaths. Invok ing a Poisson regression model should work well given such small adjustments having been added to Poisson counts of 53, 53, 52, and 53. Hence, Alpha and Beta are estimated accord ingly but their standard errors and CIs do not rely on the Poisson assumption; instead, stan dard errors were estimated from the data and Cls were based on the t distribution with 2 de grees of freedom.
A similar approach using the results of Table 4-2 was not attempted since these categorical RR estimates are correlated and information on the correlations was not reported by Bof fetta et al. .  Boffetta et al.  with a linear effect of cumulative exposure are presented in Table D-1. These results are sensitive to the value used to represent the highest cumulative expo sure category, particularly the estimate of the effect of exposure. However, zero is contained in both of the 95% CIs for Beta indicating that the slope of the exposure-response is not sig nificant for these data.

# Results

# Results based on modeling the SMRs in
Estimates of excess risk based on application of the results given in Table D-1 to U.S. popula tion rates using the method given by BEIR IV  appear in Table D-2.

# Discussion
The exposure assessment conducted by Boffet ta et al.  relies heavily on tours of the fac tories by two occupational hygienists who first reconstructed historical exposures without using any measurements (as described in Bof fetta et al. ; Cherrie et al. ; Cherrie ; Cherrie and Schneider ). The sole use of exposure measurements by Boffetta et al.  was to calculate a single adjustment factor to apply to the previously constructed exposure estimates so that the average of the measurements coincided with the correspond ing reconstructed estimates. However, Boffetta et al.  offer no analyses of their data to support this approach. Also, the best value to use to represent the highest exposure interval (i.e., 13.20 + m g/m3^yr) is not known and the results for the two values examined suggest that there is some sensitivity to this value. Hence, these upper limits that reflect only statistical variability are likely to be increased if the ef fects of other sources of uncertainty could be quantified.   . Value representing the highest exposure category is 56.5 m g/m 3 yr based on the conditional mean given exposures greater than 13.20 using the conditional distribution derived from the lognormal distribution having median and 75th percentiles equal to 1.98 and 6.88 m g/m 3 yr, respectively.

# f i k t S H

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