PERSPECTIVE pubs.acs.org/NanoLett
Debunking Some Misconceptions about Nanotoxicology David B. Warheit* DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, Delaware 19714 ABSTRACT Nanotechnology is currently undergoing an impressive expansion in material science research and development of systems that have novel properties due to their small size. Most of the research efforts have been focused on applications, while the implications efforts (i.e., environmental health and safety) have lagged behind. As a consequence, the success of nanotechnology will require assurances that the products being developed are safe from an environmental, health, and safety standpoint. These concerns have led to a debate among governmental agencies and advocacy groups on whether implementation of special regulations should be required for commercialization of products containing nanomaterials. Therefore the assessments of nanomaterial-related health risks must be accurate and verifiable. A mechanism for conducting well-designed toxicology studies includes rigorous attention to nanoparticle physicochemical characterization, as well as consideration of potential routes of exposure, justification of nanoparticle doses, and inclusion of benchmark controls. Unfortunately, some results obtained from earlier studies have fostered general perceptions and fears about nanoparticle health hazardssbased mainly upon simple metrics such as particle size, surface area, and particle dose. In addition, there are currently held views that results of screening in silico or in vitro cell culture assays can serve as adequate screening substitutes for identifying health hazards. Some of these “misconceptions” should be challenged or confirmed by the implementation of thorough and accurately detailed nanotoxicology studies. In this article, the author briefly discusses some of the generalized “misconceptions” regarding nanomaterial toxicity and presents alternative views on these issues. KEYWORDS Nanoparticles, nanomaterials, ultrafine titanium dioxide particles, ultrafine particles, nanoquartz particles, pulmonary toxicity, physicochemical characterization, nanorisk framework, risk assessment, particle surface characteristics, surface reactivity
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anotechnology is an emerging, cross-disciplinary science that incorporates nanoscale particulates (i.e., 10-9 m) into the development of new products and applications. As this technology matures and thrives, governmental regulatory agencies throughout the world have now begun to focus on a perceptual “risk/benefit” equation as a societal value consideration. What exact does this mean? In this sense, products of nanotechnology have the potential to provide a wide range of beneficial consumer, industrial, and medical applications, but these attributes may be offset or compromised by some possible human health and/or environmental liabilities. Federal organizations such as the US National Nanotechnology Initiative have organized the focus of nanotechnology-based research to that of “applications” activity (i.e., constructing nanoparticles with novel properties), as well as “implications” components (i.e., EHS (environmental health and safety) effects), with the bulk of the research dollars going for applications research. Despite this funding disparity, it is now generally recognized that ongoing assessments of human health and ecological implications of exposure to nanoscale materials are necessary prerequisites before the commercial benefits of this technology can be fully realized.
Given the paucity of meaningful health effects data for nanomaterials that has emerged thus far, it has been rather easy to draw generalized (but possibly erroneous) conclusions regarding human health risks. This is because the hazard database for virtually all nanoparticle types is incomplete. Moreover, many of the published toxicity studies have limited relevance, due, in large part, to study design limitations, including inadequate justification for dose selection or route of exposure criteria. Accordingly, one objective of this short article is to stimulate a thoughtful consideration of pertinent testing issues for evaluating human health effects. It should be noted that implementation of fundamental risk assessment methodologies is predicated on two important components, namely, hazard potential and exposure. Most of these adverse effects have been attributed to the small particle size. However, some studies that are described below have demonstrated that factors other than particle size, such as particle surface reactivity may play important roles in defining nanomaterial toxicity. Perhaps the most important point to be made is that nanoparticle toxicological effects are complex and involve a variety of factors including physicochemical characteristics,1,2 particle-cellular interactions, routes and degrees of exposure, biokinetics, logistics, and other considerations. Unfortunately, these effects cannot yet be accurately modeled using simple systems. In the following discussion, some current misconceptions related to
* To whom correspondence should be addressed,
[email protected]. Published on Web: 10/29/2010
© 2010 American Chemical Society
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DOI: 10.1021/nl103432w | Nano Lett. 2010, 10, 4777–4782
PERSPECTIVE day inhalation studies that were conducted in three rodent species using pigment grade (particle size ∼380 nm; surface area ) 6 m2/g) or ultrafine/nano (particle size reported as 25 nm; 53 m2/g) titanium dioxide particle-types (Figure 1). In the first study, rats, mice, and hamsters were exposed to fine-sized TiO2 particles at aerosol concentrations of 10, 50, or 250 mg/m3.5 In a second study, the same rodent species were exposed to ultrafine (i.e., nano) TiO2 particulates at exposure concentrations of 0.5, 2, or 10 mg/m3 (ref6) and evaluated at several time points postexposure. The results of the two studies demonstrated that (1) rats were the most sensitive rodent species for lung effects and (2) the effects measured at 250 mg/m3 in the pigment-grade study were not dissimilar from those observed at 10 mg/m3 in the nanoscale TiO2 study. Accordingly, comparing the results of both studies would suggest that, on a mass basis, the effects measured following nanoscale TiO2 exposures were ∼25 times greater than those observed for the pigment-grade TiO2 particle types. However, a number of other factors require consideration before drawing this conclusion. These include the following issues: (1) Surface area indices of the nanoscale TiO2 particletypes were significantly greater compared to the finesized TiO2 particulates used in the study (53 m2/g vs 6 m2/g). (2) The crystal structure of the nano TiO2 particles was composed of 80% anatase and 20% rutile, whereas the fine TiO2 samples were 100% rutile. (3) The nano TiO2 particle surface was not passivated and contained no surface coatings, whereas the fine TiO2 particulate surfaces were both passivated and composed of alumina surface coatings.7 (4) Perhaps most importantly, the measured surface reactivity index (i.e., delta b* -using the vitamin C assay)
The relative dearth of substantive, hazard data on nanomaterials, concomitant with an abundance of high-dose, in vitro cellular findings has created a perception that the vast majority of nanoparticles are highly toxic.
nanotoxicology issues are presented and alternative viewpoints are offeredsergo the title “debunking some misconceptions regarding nanotoxicology”. Misconception 1. Nanoparticles are always more toxic than bulk particles of similar or identical composition. Misconception 2. Particle size and surface area are the critical indices that influence nanoparticle toxicity. Misconception 3. All forms of nano titanium dioxide particles have similar toxicity profilessor nano TiO2 is nano TiO2si.e., we can identify nanoparticle types by their “core identities” without specifying their compositional physicochemical characteristics. A number of reviews have concluded that pulmonary exposures in rats to ultrafine/nanoparticles (i.e., defined here as fine quartz > CI particles. It was concluded that the pulmonary toxicities of R-quartz particles appear to correlate better with surface reactivity indices when compared to particle size and surface area parameters.10 © 2010 American Chemical Society
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PERSPECTIVE TABLE 1. Base Set Hazard Tests nanomaterial physiochemical characterization
mammalian hazard tests
genotoxicity tests
aquatic screening battery
size & size distribution crystal structure chemical composition surface reactivity
pulmonary bioassay skin irritation skin sensitization acute oral toxicity eye irritation
bacterial reverse mutation chromosomal aberration
rainbow trout daphnia green algae
icity studies, acute dermal toxicity and sensitization studies, acute oral and ocular toxicity studies, along with screening type genotoxicity, and aquatic toxicity studies (see Table 1). Recently, the base set toxicity results on newly developed, well-characterized, ultrafine titanium dioxide (uf-TiO2) particle types were reported. In the aggregate, the results of base set studies demonstrated low hazard potential in mammals or aquatic species following acute exposures to the ultrafine TiO2 particle types tested in this program.12 Myth 5. Pulmonary hazard assessments for nanoparticles can be accurately evaluated using in vitro or in silico methodologies The development of reliable in silico or in vitro assays to function as predictive screening tools to assess hazard potency for nanoparticles would be a significant achievement, particularly during the incipient phases of product development. If the accuracy of these nonanimal tests can be properly validated, the potential advantages of screening tests would result in simpler, faster, and less expensive assessments than their in vivo counterparts. Additional important benefits would include a significant reduction or elimination of animal usage for hazard screening. Although implementation of in vitro lung toxicity screening systems for particulates would be a highly beneficial development, the accuracy and predictability of these assays have not been substantiated against currently accepted testing protocols. Seagrave and co-workers13,14 tested some forms of in vitro models as predictive screens for pulmonary toxicity to diesel extract particles but reported a lack of convergence in results when comparing in vivo lung toxicity effects and in vitro cell culture exposures. Subsequently, the capacity of in vitro screening studies to predict in vivo pulmonary toxicity of several fine or nanoparticle types was assessed in rats by Sayes et al.15 In the in vivo component of the study, rats were exposed by intratracheal instillation and evaluated using a pulmonary bioassay protocol with the following particle types: carbonyl iron, crystalline silica, amorphous silica, nanoscale zinc oxide, or fine zinc oxide particle types. Following exposures, lung inflammation and cytotoxicity biomarkers were measured at several postexposure time periods. For the in vitro studies, three different culture conditions were employed. Cultures of rat lung epithelial cells, primary alveolar macrophages from lavaged rats, and alveolar macrophage/L2 rat lung epithelial cell cocultures were incubated with the same particles as identified © 2010 American Chemical Society
above. The culture fluids were evaluated for cytotoxicity and inflammatory cytokines end points at several doses and four different time periods ranging from 1 to 48 h incubation. Data from the in vivo pulmonary toxicity studies demonstrated that instilled carbonyl iron particles were benign. Crystalline quartz silica particle exposures produced sustained inflammation and cytotoxic effects, while lung exposures to amorphous silica particles and fine or nanoscale zinc oxide produced transient inflammatory responses.
Evaluations of human health and ecological implications of nanoparticle exposures will be required to attain full commercialization potential.
In contrast to the in vivo results, cell culture pulmonary cytotoxicity studies demonstrated a variety of inconsistent patterns to the different particle types which did not reflect the in vivo results. Accordingly, it was concluded that in vitro cellular systems used in this study will need to be further developed, standardized, and validated (relative to in vivo effects) in order to provide useful predictive screening data on the relative pulmonary toxicities of inhaled particles.15 Similar disparate in vivo/in vitro findings have also been reported when the toxicities of underivatized and hydroxylated nano C60 fullerenes were investigated.16 A number of in vitro and in silico screening assays for nanoparticle toxicity have been proposed and currently are under development.17-21 It will be critical to validate the results of these screening tests relative to in vivo effects with the identical particle types in order to confirm the efficacy of these methodologies. Similar to the evaluation of most complex toxicological questions that face us today, the development of accurate in silico or in vitro methodologies would provide a significant advancement for facilitating early screening hazard evaluations of nanomaterials and is viewed as a significant “grand challenge 4781
DOI: 10.1021/nl103432w | Nano Lett. 2010, 10, 4777-–4782
PERSPECTIVE of safe nanotechnology”.22 However, current in vitro/in silico technology clearly requires further development to provide an acceptable level of human protection. Conclusions. Most of the federal research funding for nanomaterials has been devoted to applications investigations. Yet it seems clear that evaluations of human health and ecological implications of nanoparticle exposures will be required to attain full commercialization potential. The absence of meaningful toxicity data on nanoparticle types, has fostered a perception that many nanoparticulates are inherently hazardous. This is due, in part, to the fact that, for a given nanoscale material, as the particle size range is decreased below 100 nm, both the chemical and physical properties are known to change. Thus it is reasonable to postulate that the biological properties may also be altered. It seems clear that biological and toxicological effects of nanoparticle-cellular interactions are complex and influenced by a variety of factorssfirst and foremost, gauging nanoparticle physicochemical characteristics under conditions that best simulate the expected route of exposure. Unfortunately, hazard effects cannot yet be accurately modeled using simple in silico or in vitro systems. Because the hazard database for nanomaterials is very limited, it will be important to rigorously characterize the material of interest and generate substantive hazard data that is accurate and can be confirmed independently by other research investigators.
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