health effects of nanomaterials: a critical appraisal approach and research to practice

Human Factors and Ergonomics in Manufacturing, Vol. 18 (3) 293–341 (2008)C© 2008 Wiley Periodicals, Inc.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hfm.20113

Health Effects of Nanomaterials: A Critical AppraisalApproach and Research to Practice

Reynold Sequeira and Ash GenaidyIndustrial and Manufacturing Engineering Program, University of Cincinnati,Cincinnati, Ohio 45221-0116, USA

Gary WeckmanDepartment of Industrial and Systems Engineering, Ohio University,Athens, Ohio 45701, USA

Richard ShellIndustrial and Manufacturing Engineering Program, University of Cincinnati,Cincinnati, Ohio 45221-0116, USA

Waldemar KarwowskiIndustrial Engineering and Management Systems, University of Central Florida,Orlando, FL 32816-2993, USA

Adriana Acosta-LeonIndustrial and Manufacturing Engineering Program, University of Cincinnati,Cincinnati, Ohio 45221-0116, USA

ABSTRACT

Background Researchers across the globe are studying health effects of various nanomaterials andnanostructures. Most of these studies are qualitative in nature and lack parallel research to compareand validate the reliability of the findings.Objective This research targets a critical appraisal of such clinical studies by first developing a suitabletool and then evaluating the quality of reporting of these trials.Methods The published articles were collected from a search of electronic databases and bibliogra-phies of identified articles. A critical appraisal (ExpAI) suitable for assessment of such experimentalstudies was developed and the articles were appraised using this instrument. The main findings of thestudy were evaluated for their use in the development of new techniques for practice using a linguisticscale.Results Twenty-eight articles were found to satisfy the inclusion criteria adopted in this research. Themethodological qualities of the published studies ranged from marginal to average.Conclusions Though the studies evaluated in this research have proven that nanoparticles do haveadverse health effects, nanoparticles should not be discounted without exploring the possibility ofusing them to benefit companies and the general population. Reengineering should be consideredwhile creating these new systems to overcome the health effects found by various studies. C© 2008Wiley Periodicals, Inc.

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1. INTRODUCTION

Nanotechnology refers to technology that exploits phenomena and structures occurring at thenano or 10−9 scale, that is, the scale of several atoms and small molecules. Nanotechnologyis based on the fact that the properties of materials become markedly different when theirsize approaches that of a few hundredths or tenths of an atom. Nanomaterials have foundtheir use in many sectors, such as electronics, health, personal care, and automotive. Theapplication of nanotechnology is anticipated to increase greatly in the coming decades.Nanomaterials like nanotubes, filaments, and other nanoelements play a fundamental rolein nanotechnology and have been considered for a variety of applications (Kulinowski,2004). These nanodevices will have a tremendous impact on the development of newmaterials and devices, and their application is expected to facilitate progress in almost everyfield, aiding and improving the quality of life. Although the world is preparing itself for theadvent of widespread commercial use of nanotechnology, awareness of the risks involved,if any, is still in a nascent stage.

Processing of nanomaterials manipulates matter at a near-atomic level to produce newmaterials or structures, resulting in products and by-products that may possess characteristicsthat are different from those possessed by the original bulk-sized particles, in spite of the factthat the chemical composition has remained unchanged (Sequeira et al., 2006). Interestingly,an overwhelming majority of material safety data sheets document the properties of the bulk-sized particles; but no special requirements pertaining to safety, exposure, and usage havebeen mandated for people exposed at the workplace and beyond to the nano “cousins” ofthese very same substances.

With the observed and anticipated profusion of nanotechnology within different industriesand research communities, it is imperative that relevant, validated research be reanalyzedand compiled to formulate or benchmark a set of guidelines that can serve as a roadmap forconceptualizing and developing safety procedures, precautionary measures, and codes forcreation, use, and disposal of nano and nano-related products (Genaidy et al., 2006).

The objective of the research is twofold: (1) to investigate whether nanomaterials posea health hazard as concluded by various researchers and (2) to transform these researchfindings into preliminary work practices, if possible, using a linguistic scale. To achievethe first goal, an assessment tool was developed by Shell and Genaidy (2008) to criticallyappraise the research articles published in various journals across the globe to determinetheir quality and conclusively validate the results that were presented. To transform theseresearch findings into work practices, a linguistic scale is used based on the knowledge ofthe authors and the published literature.

2. BACKGROUND

Nanomaterials and nanostructures have found their way into everyday life. From beinga fuzzy, futuristic technology in the early half of the 20th century, they have now beenestablished as a beachhead in the economy. Stain-free clothing, cosmetics, sunscreens,vitamins and additives, and scratch-free parts and paints for the automotive industry aresome of the current uses. These modest, fairly low-tech applications are just a tip of the vasticeberg—an iceberg that will influence our lives both directly and indirectly. Today, withcutting-edge technologies and corporate and government investments running into billionsof dollars, researchers are attempting to evolve this technology to a higher level and makeuse of it in almost every conceivable walk of life. From outer space research to medicine,

Human Factors and Ergonomics in Manufacturing DOI: 10.1002/hfm

HEALTH EFFECTS OF NANOMATERIALS 295

agriculture to military warfare, nuclear science to computing systems, DNA research andbiological cloning to intrabody devices, nanotechnology is being studied vigorously todiscover if it can contribute to existing products and systems, to make things smaller, faster,more energy efficient and durable (Donaldson, Stone, Tran, Kreyling, & Borm, 2004).

Like any other technology, nanotechnology also cuts both ways. In the past, technologieshave proliferated into the consumer market first, only to discover a darker side later whenmuch of the damage already has been done. But these dreadful experiences have madegovernments and societies wary, and scientists worldwide have taken the initiative to lookinto the negative side of this technology before it can cause irreversible damage. But theseworks are isolated and have not conclusively established the detrimental properties ofnanotechnology. In fact, some of these works have given birth to the newer applications,which were unnoticed until now.

An evidence-based treatment approach in medical care has been popular for quite sometime now. Researchers have tried to adopt this technique in other fields in order to investigatetheir interests from previous works. This approach depends on the published works of otherresearchers, critically appraising their work for the aims, the designs of experiments, thepopulation on whom the experiment was conducted along with their findings, and thenusing these findings to draw conclusions about the current problem. Genaidy et al. havesuccessfully tried this approach by adopting the principles of critical appraisal in developingthe Epidemiological Appraisal Instrument (EAI) to investigate the issues in epidemiologyand ergonomics and substantiated their interests based on the numbers. Inspired by this,Shell and Genaidy (2008) has expanded the instrument to investigate the detrimental effectsof nanomaterials and transformed the results concluded by various researchers to the benefitof product-producing companies and the general population.

3. METHODS

The benchmark assessment methodology for experimental studies, the Experimental Ap-praisal Instrument (ExpAI), was developed and tested, followed by the description andtranslation of this critical appraisal tool with respect to its ratings. The articles were ap-praised using this methodology to evaluate their quality and compared with other studieson this basis.

3.1. Application of the ExpAI

Research and use of nanotechnology and nano-based materials has increased exponentiallyin the last few years. The estimated value associated with various nano-based activities isanticipated to be approximately $50 billion in 2006. Despite all this profusion, research rel-evant to the subsequent health, safety, and ecological effects of such use, though numerous,has not been able to substantiate the negative impacts in this process. In order to validatethe results obtained by worldwide research, it was necessary to compare the experimentaldesign procedures to a benchmarked assessment method. Twenty-eight such works wereselected from an observed sample of approximately 2,500 peer-reviewed published worksfor this purpose. Fifteen of these focused on carbon-based nanomaterials, primarily becauseof the significantly higher number of works that have been done in that area. The other 13included various types of nanomaterials like oxides of titanium, beryllium, and silicon.


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3.1.1. Search strategy and inclusion criteria. Publications were retrieved througha search of electronic databases including PubMed, Medline, Academic Search Premier,COMPENDEX, and Elsevier Journals, as well as search engines like Google and Yahoousing the following search words: (1) carbon nano materials and health effects, (2) carbonnanotubes and health effects, (3) nanomaterials and health effects, (4) nanotechnology andadverse effects, and (5) nanoparticles and occupational health. To make this search as broadas possible, no time limitation on the publication date of the articles was used. In addition tothe electronic search, bibliographies of selected papers were reviewed for additional studies.

The search was restricted to reports in English. Abstracts, reviews, and surveys were notincluded. All studies were scanned for input, output, study objectives, and main results, andthe ones that used particle sizes less than 100 nm and reported the adverse health effectswere included. The search was concluded in April 2005, and the critical appraisal wascompleted on the selected articles.

3.1.2. Data extraction . The selected articles were appraised carefully for the input,output, study objective, study design, study population, and main outcomes, and the find-ings were documented as the description of evidence. Each article was evaluated by fiveresearchers independently using the ExpAI and results were compared during subsequentmeetings. Disagreements were discussed and mutual consensus was reached.

3.1.3. Quality assessment . The methodological quality of the studies that enteredthe review was assessed by means of the Experimental Appraisal Instrument, or ExpAI,developed by Shell and Genaidy (2008). The ExpAI consists of 34 questions groupedinto five scales: (1) Study Description (15 items); (2) Subject Selection (4 items); (3)Measurement Quality (9 items); (4) Data Analysis (4 items); and (5) Generalization ofResults (2 items). Every item in the ExpAI is rated for a given study using any one ofthe following options: (1) Not Applicable (not included in score calculation); (2) Yes (2points); (3) Partial (1 point); (4) No (0 points); and (5) Unable to Determine (0 points). Thefinal score for each scale was taken as the average of the values recorded for each item.The five graduate student members of the research team conducted the critical appraisalsindependently. The results of the evaluations were compared, and areas of disagreementwere discussed and resolved in follow-up sessions.

It should be noted that each article was critically appraised for the research questionformulated in this research. The article was not evaluated on the basis of the aims citedin the article. This practice should be the basis of systematic reviews dealing with criticalappraisals in order to compare the included studies on a uniform basis of reference. As such,the quality of each study is compared with the research question in this paper.

3.2. Development of Linguistic Scale

Prior to the critical appraisal tool, a linguistic scale (Appendix F) was developed to explorethe usefulness of the main findings of each study into building the new systems for practice,such as medical applications. The findings of each study were evaluated carefully using theexisting literature and research articles. The purpose of this is to demonstrate how thesefindings can be used constructively rather than putting off the nanomaterials as a bane basedon these toxicological studies and is limited to the current knowledge of the authors.

The ratings of the linguistic scale are based on the plausibility of the main results of thestudy and are as follows:



Level 0: Absolutely not useful—the applications of the findinga of the study forbuilding a new system are not possible and are not consistent with the lines on whichthe current research is taking place.Level 1: A little useful—the use of the study results is very limited, but it is possiblethat at least a few of the observations can be used in future research.Level 2: Moderately useful—the findings can be used in research of new systems toa moderate extent and a few of the observations are considered by the researcherswho are working on developing the systems.Level 3: Very useful—the main findings of the study can be used to develop newsystems and some research is being conducted to investigate their usefulness.Level 4: Entirely useful—the results of the study are being used entirely to developthe new system or the results are consistent with the research aimed at developingthe new system.

4. RESULTS

4.1. Testing the ExpAI Pilot Version

Each member of the research team’s evaluation in the two studies using the ExpAI(Step 5 in Appendix B) was benchmarked against that of the primary author (Genaidy). The

TABLE 1. Description of Evidence of Carbon Articles

Study Description

Barrett et al. (2003)Input Ultrafine carbon particles in dogsOutput Airway resistance and lung inflammationStudy Design Between groupStudy Population -6 beagle dogs 18 months of age

-6 beagle dogs 18 months of ageStudy Objectives Investigate if acute inhalation of ultrafine carbon particles will alter lung

function in an animal model of allergic asthmaMain Results The allergic dogs developed increased pulmonary resistance to ragweed,

whereas nonallergic dogs did not• The IgE level in allergic dogs is 100% more than nonallergic dogs.• The IgG level in allergic and nonallergic dogs are nearly equivalent.

Chen et al. (1998)Input C17 fullerenes in miceOutput Immune system response to fullereneStudy Design Unable to determineStudy Population Group of BALB/c miceStudy Objectives Whether the immune system can process fullerene as-protein conjugateMain Results -Immune response to 1-TG: no antibody or nonspecific binding to pre-immune

serum-IgG response directed to the fullerenes, not at the functional groups linkingthe fullerenes to the carriers

(Continued)


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TABLE 1. Continued

Study Description

Cui et al. (2005)Input SWCNT on human HEK293 cellsOutput Inhibition of HEK293 cells growth by inducing cell apoptosis and

decreasing cellular adhesion abilityStudy Design Inferred between groupStudy Population -24-well plates of HEK293 cells for cellular viability

-6-well plates of HEK293 cells for cellular adhesion-Group of cell for control

Study Objectives Explore the effect of SWCNT on human HEK293 cells and its potentialbiochemical mechanism and exploring potential application for moleculartherapy

Main Results -Compare to the controls, the cell viability of cells exposed to SWCNTdecreased with a time and dose dependent relationship up to 90%

-The same behavior was noted for cell addition, where it went down to 100%-Apoptosis was present on HEK293 cells on a dose and time dependent

manner

Harder et al. (2005)Input Ultrafine carbon particles in ratsOutput Cardiovascular performance and pulmonary inflammationStudy Design Inferred between groupStudy Population -22 male Wistar Kyoto rats; 12–15 weeks old for the exposure

-22 male Wistar Kyoto rats; 12–15 weeks old for the controlStudy Objectives Effects of UFCP on cardiac autonomic function in ratsMain Results -UFCP increases the transient heart rate and activates a neural pathway in

response to particle inhalation-Cardiac rate was increase in 4.8 ± 3.1%-BALF glutathione concentration increase in 0.22 μg/ml-Increase in polymorphonuclear cells was double in the first 4 hr. After24 hr the increase was fivefold-There was no significant change in blood coagulation and clot lysis

Kim et al. (2005)Input Ultrafine carbon particles in normal human bronchial epithelial (NHBE) cellsOutput Activation of MAPKStudy Design Unable to determineStudy Population NHBE cells

Study Objectives Determine if exposition to UFCP will induce IL-8 expression in NHBE cellsMain Results -UFCP appear to be potent inducer of proinflammatory response

in NHBE cells-IL-8 protein increase around 4.5-fold over control with 33 μg/ml after 24 hr

of exposure, and has a fourfold increase over control with 56 μg/ml after24 hr of exposure

-There is no difference in cell viability after exposure

(Continued)



TABLE 1. Continued

Study Description

Lam et al. (2004)Input Raw nanotubes (RNT), purified nanotubes (PNT) and carbon nanotubes

(CNT). For reference carbon black (CB) and quartz in miceOutput Inflammation and granulomas in the lungStudy Design Inferred between groupStudy Population 9 B6C3F1, 2-month-old male mice for each of the following groups:

• RNT at 01 and 05 mg/mouse• PNT at 01 and 05 mg/mouse• CNT at 01 and 05 mg/mouse• CB at 01 and 05 mg/mouse• Quartz at 01 and 05 mg/mouse• Control

Study Objectives Investigate pulmonary toxicity of SWCNT in intratracheally instilled miceMain Results -All NT products produce dose-dependent lung lesions and granulomas

-NT that reach lungs are much more toxic that CB and can be more toxicthan quartz

Lundborg et al.(2001)

Input Ultrafine carbon particles in human alveolar macrophages (AM)Output Phagocytic function and oxidative metabolismStudy Design Inferred mixed groupStudy Population 500 human alveolar macrophages cells 6 rats, weight 250–300 gStudy Objectives Study the effect of ingested UFCP on phagocytosis by human AMMain Results -High concentration (10 μg/ml) of ingested particles impaired the

phagocytic capacity of AM cells-With ingested particles phagocytic activity decreases by 70% with a dose of

3 μg/ml-Accumulated attachment and ingested fraction decreases by 50%

respectively compared to the control

Monteiro-Riviereet al. (2005)

Input MWCNT on human epidermal keratinocytes (HEK)Output Presence of MWCNT in HEKStudy Design Inferred as between groupStudy Population 96-well culture plates of HEK cellsStudy Objectives Interaction between MWCNT and HEK using proinflammatory cytokine in-

terleukin (IL) 8 as an early marker of biological response.Main Results -Chemically unmodified MWCNT are capable of entering the HEK as well

as causing irritation to it-Amount of MWCNT in the cell increased when treatment concentration

and exposure time increased:• From 59.1% at 24 hr to 84% at 48 hr at 0.4 mg/ml

(Continued)


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TABLE 1. Continued

Study Description

-Cell viability after 24 hr:• For control group is 100%• For 0.1 mg/ml dose is 87%• For 0.2 mg/ml dose is 75%• For 0.4 mg/ml dose is 72%

Oberdorster et al.(2002)

Input Ultrafine 13C particles as aerosols in ratsOutput δ-13C measurements in lung and liverStudy Design Between groupStudy Population 9 male Fisher-344 rats, weight 250 to 260 g for the exposure 6 male

Fisher-344 rats, weight 250 to 260 g for the controlStudy Objectives Determine if inhaled UFCP translocate to the liver and other extrapulmonary

organsMain Results -Large amount of particles reach the blood circulation and subsequently the

liver. They were also found in the lower respiratory tract-Maximum deposition of 13C in lungs occurs during the first half hour-13C concentration in liver is fivefold higher after 18 and 24 hr than

in the lung-There is no significant decrease in 13C in lung from 0.5 to 24 hr of post

exposure


Input Ultrafine 13C particles in ratsOutput Measurement of the δ-13C particles in different sections of the brainStudy Design Between groupStudy Population -12 male Fisher-344 rats, 14 weeks old, weight 284 ± 9 g for the exposure

-3 male Fisher-344 rats, 14 weeks old, weight 284 ± 9 g for the controlStudy Objectives Determine if there is translocation of UFCP across blood–brain barrier to

different regions of the brainMain Results -There is translocation of inhaled UFCP into the CNS via the olfactory nerve

-13C particle concentration in lungs changes from 1.39 ± 0.6 μg on the firstday to 0.59 ± 0.15 μg on the seventh day

-In olfactory bulb there is an increase from 0.35 ± 0.17 μg/g tissue on dayone to 0.43 ± 0.08 μg/g tissue on day seven

-The concentrations in cerebellum and cerebrum were not consistent andvary between 0.11 to 0.44 μg/g per tissues

Shvedova et al.(2003)

Input SWCNT on cell culture of immortalized human epidermal keratinocytes(HaCaT)

Output Oxidative stress and cellular toxicityStudy Design Inferred as between groupStudy Population 96-well plates of HaCaT cellsStudy Objectives Adverse effects of SWCNT exposed to HaCaT

(Continued)



TABLE 1. Continued

Study Description

Main Results -SWCNT produced oxidative stress and cellular toxicity on HaCaT-Indicators, after 18 hr, for these harmful effects are:

• Formation of free radicals• Accumulation of peroxidative products• Antioxidant depletion• Loss of cell viability

-Cell viability decreased with increase in dosage:• Control 0%• 0.06 mg/ml 11.3%• 0.12 mg/ml 24.5%• 0.24 mg/ml 37.6%

-SWCNT produced a significant concentration dependent decrease inantioxidant reserve of 35.2% for 0.66 mg/ml, 53.8% for 0.12 mg/ml, and76.8% for 0.24 mg/ml after 18 hr of incubation

Yamago et al. (1995)Input Water-soluble C60 fullerene in ratsOutput Chronic and acute toxicity of water-soluble fullereneStudy Design Unable to determineStudy Population Group of ddY female, 18–20 g mice

Group of male Fisher rats, 85–95 gStudy Objectives Oral absorption, distribution and excretion of water-soluble fullereneMain Results -Oral absorption of fullerenes was found to be very poor in rats; however,

traces of them are found in liver and other tissues after 6 hr. 97% wasexcreted after 48 hr

-Intravenously administrated fullerene:• Total amount of fullerene retain in the body when administered

intravenously = 90%• Total fullerene retain in the liver after 30 hr = 80%• Approximately a dose of 500 mg/kg fullerene is found to be lethal

-C60 Fullerenes may be regarded as a compound with weak acute toxicityWarheit et al. (2004)

Input SWCNT in ratsOutput Lung toxicity: Transient inflammation and cell injury effectsStudy Design Between groupStudy Population Group of male rats 8 weeks old, Mean weight 240–255 g

Control group: CD(SD)IGS BR. 8 weeks oldFrom Charles River Lab, Raleigh, NC

Study Objectives Evaluate the acute lung toxicity of intratracheally instilled SWCNT, quartzand carbonyl iron particles.

Main Results -Overall mortality at 5 mg/kg of CNT = 15% after 24 hr-Pulmonary inflammation in cells at 5 mg/kg of quartz = 61%.-Pulmonary inflammation in cells at 1 mg/kg of quartz = 31%-Pulmonary exposure to SWCNT produced a nondose dependent series of

multifocal granulomas


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TABLE 2. Description of Evidence of Noncarbon Articles

Study Description

Jani et al. (1989)Input Fluorescent polystyrene nanoparticles and microparticles in ratsOutput Uptake and translocation after oral administrationStudy Design Between groupStudy Population Groups of at least 3 female Sprague-Dawley adult rats 15–20 weeks old, mean

weight 200 g for• 2 groups for oral administration of (100 nm and 1 m dia) ionized

carboxylated polystyrene microspheres• 4 groups for oral administration of (100 nm, 500 nm, 1 m, and 3 m)

non-ionized polystyrene microspheres• 2 groups of 2 rats for intravenous administration of (100 nm and 1 m

dia) microspheresControl Group: A group of at least 3 rats

Study Objectives To determine the uptake and subsequent translocation from the GI tract oforally administered polystyrene micro particles with varied surfacecharacteristics

Main Results -Uptake of 100 nm and 500 nm microspheres is higher than 1 mm dia particles-Absorption of non-ionized particles is more evident than the ionized particles-Uptake by Peyer’s patches and Mesentery node is highest in comparison

with other parts of the GI tract-There is no evidence of particles in kidney, heart, and lungs

Nemmar et al.(2002)

Input Unmodified, amine-modified and carboxylate-modified 60 nm diapolystyrene particles.

Output Impact of ultrafine particles on thrombus formationStudy Design Within-group studyStudy Population • Groups of 4 hamsters (100–150 g) for intravascular administration of

unmodified particles at 50, 500, and 5000 g/kg of dosage each• Groups of 4–6 hamsters (100–150 g) for intravascular administration of

both amine-modified and carboxylate-modified particles at 50,100, and500 g/kg of dosage each

• A group of 13 hamsters as control group• Groups of 6 hamsters each for intratracheal instillation of 5 mg/kg of

unmodified, amine modified, carboxylate modified and as a controlgroup

Study Objectives To investigate whether the Ultrafine particles have effect on thrombusformation

Main Results For intravascular instillation:-Unmodified particles do not interfere with the thrombus formation.-Negatively charged particles significantly (approx. 52–55%) inhibit the

thrombus formation at higher doses and do not have effect at a dosage of50 mg/kg

-Positively charged particles enhance the thrombus formation by 219% for50 mg/kg and 307% for the 500 mg/kg dosage

(Continued)



TABLE 2. Continued

Study Description

-For intratracheal instillation of 5 mg/kg of particles, there is no significanteffect on thrombus formation by unmodified and carboxylated particles,while amine modified particles induce 368% increase in thrombusformation

Nemmar et al.(2003)

Input Unmodified, amine modified, and carboxylate modified 60 nm polystyreneparticles and 400 nm amine-modified polystyrene particles

Output Effect on bronchoalveolar lavage and peripheral thrombus formationStudy Design Inferred within group designStudy Population Groups of male and female hamsters (100–110 g) (Pfd gold, University of

Leuven, Leuven, Belgium)• A group of 6 animals for 500 mg/animal dose of 60 nm unmodified

polystyrene particles• Group of 6 animals for 500 mg/animal dose of 60 nm carboxylate

modified polystyrene particles• 3 groups of 6 animals each for 5, 50, 500 mg/animal dosages of 60 nm

amine modified polystyrene particles• A group of 6 animals for 500 mg/animal dose of 400 nm amine modified

polystyrene particles• 15 animals as control group

Study Objectives To determine the acute effects and the size effect of ultrafine particles onlung inflammation and peripheral thrombosis

Main Results Both 60 nm and 400 nm particles caused pulmonary inflammation, but onlypositively charged 60 nm particles enhanced thrombus formation asopposed to negatively unmodified or negatively charged 400 nm particles

Jani et al. (1990)Input Fluorescein linked mono dispersed non-ionized polystyrene microspheres

(50, 100, 300, 500 nm, 1 and 3 mm)Output Uptake and translocation of UFP in systemic organs after oral administration.Study Design Between-groupStudy Population -Groups of 3 female Sprague-Dawley rats (200 g, 15–20 weeks old) for

doses of each of 50, 100, 300, 500 nm, 1 and 3 mm polystyrene particles.-Groups of 3 female Sprague-Dawley rats (200 g, 15–20 weeks old) for

doses of each of 50, 100, 300, 500 nm, 1 and 3 mmI polystyrene beads-3 female Sprague-Dawley rats as control group

Study Objectives Quantitative analysis of uptake following oral administration and toestablish the extent of uptake of nano and microspheres as a percentage ofadministered dose

Main Results -Total uptake of 50 nm particles is about 33% of the administered dose-Uptake of 1 mm particles is about 7% of the administered dosage-3 mm particles were found in GI tract but do translocate to other organs-100 nm particles seem to represent the upper limit for transport into the

bone marrow and 300 nm represents the upper limit for the particles toenter into the blood circulation

(Continued)


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TABLE 2. Continued

Study Description

Takenaka et al.(2001)

Input Ultrafine elemental silver (EAg) particles in female Fischer ratsOutput Pulmonary and systematic distribution of EAg particlesStudy Design Inferred within-groupStudy Population Female Fischer 344 rats of body weight 150–200 g

-16 animals as whole body inhalation study group-A group of 12 animals for intratracheal instillation of 4.4 mg Ag-A group of 7 animals for intratracheal instillation of 50 mg of EAg-8 animals as control

Study Objectives To determine the pulmonary and systematic distribution of EAg afterinhalation at relatively low concentrations.

Main Results In inhalation study:-No accumulation of particles in alveolar macrophages in morphological

analysis-Within 7 days the lung burden reduced to 4% of the initial burdenIn instillation study:-Particles were observed in macrophages even till day 7.-The EAg content in lungs remains unchanged from day 1 to 7-With prolonged inhalation of particles, particles enter the alveolar walls

Takenaka et al.(2004)

Input Ultrafine CdO particles on female Fischer ratsOutput Concentration in different organs and pathological response of the lungsStudy Design Between-groupStudy Population 68 female Fischer 344 rats (150–200 g)

-24 animals for inhalation exposure of 70 mg Cd/m3 mass concentration ofCdO particles

-16 animals for inhalation exposure of 550 mg Cd/m3 mass concentration ofCdO particles

-24 animals as control groupStudy Objectives To determine the distribution and concentration of cadmium in different

organs after the exposure at different doses and to assess the pathologicalresponse of the lung

Main Results • For lower concentration of CdO:-The cadmium content in lung was 0.53 mg/lung immediately after

exposure and was constant till the seventh day-No significant increase of cadmium content in liver as well as kidneys

during the study period• For higher concentration of CdO:

-Liver shows a significant elevation of cadmium content from day 0 today 1

-Cadmium content in the kidneys shows a significant elevation• Cadmium can be detected in blood at higher dosages of exposure

(Continued)



TABLE 2. Continued

Study Description

• The lung weight increases with the higher concentrations but at lowerconcentrations it does not change

• Lungs show multifocal alveolar inflammation characterized bypolymorphonuclear and mononuclear cell infiltration in alveolar lumen

• Neutrophils count increase significantly with higher concentration

Churg et al. (1999)Input Fine and ultrafine titanium diaoxide (TiO2) in rat tracheal explantsOutput Effects on growth factor, fibrogenic mediators and procollagenStudy Design Inferred as between groupStudy Population 2 × 2 mm tracheal Explants from 250 g male Sprague-Dawley ratsStudy Objectives To find the effects of ultrafine and fine particles on the expression of growth

factor, firogenic mediators, and procollagenMain Results -There is an effect on size of the particles on mediator and matrix gene

expression-There is no increase in the gene expression for the fine and ultrafine dust

exposures till day 7-At the highest exposure (5 mg) of ultrafine particles there is a 20% increase

in procollagen expression. But there is no effect for fine dusts-On day 7 the gene expression increases, but it is not consistent for all the

genesFernandez-Urrusunoet al. (1997)

Input Poly(isobutyl Cyanoacrylate)(PIBCA) and polystyrene(PS) particles in ratsOutput Oxidative stress in hepatocytesStudy Design Unable to determineStudy Population Groups of male Sprague-Dawley Rats, 300–350 g in weightStudy Objectives Investigate the endogeneous defense of hepatocytes against the oxidative

stress due to injected polymeric nanoparticles.Main Results -Uptake of polymeric particles by Kupffer cells in the liver induce the release

of oxidative species altering the antioxidant systems of hepatocytes-PIBCA and PS both reduced (GHS) and oxidized (GSSG) glutathione content-PIBCA and PS nanoparticles significantly reduce the superoxide dismutase

(SOD) activity and catalase (CT) activity

Beck-Speier et al.(2001)

Input Ultrafine particles of elemental carbon (EC) and titanium dioxide (TiO2) oncanine alveolar macrophages

Output Release of lipid mediators by alveolar macrophagesStudy Design Unable to determineStudy Population Alveolar macrophages and polymorphonuclear neutrophils (PMN) from

healthy beaglesStudy Objectives Explore the effects of agglomerates of EC and TiO2 on alveolar

macrophages inducing the release of lipid mediators

(Continued)


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TABLE 2. Continued

Study Description

Main Results -The surface area rather than mass concentration determines the effects ofagglomerates of ultrafine particles (AUFP).

-Both low and high concentrations of UFPs cause the inhibitory effects onPMNs.

-There is an inflammatory response of the lung to the AUFPs

Lademann et al.(1999)

Input Titanium dioxide (TiO2) microparticles on hair folliclesOutput Penetration into horny layer and hair folliclesStudy Design Unable to determineStudy Population Tape stripping of a marked area on left volar forearm of a volunteerStudy Objectives Investigate the penetration of TiO2 microparticles inside and outside of the

hair folliclesMain Results • Largest amount of TiO2 localizes to the upper part of the stratum

corneum• TiO2 microparticles can enter the orifices of hair follicles• In the deeper part of horny layer, TiO2 particles exclusively localize

in the follicle channelsDriscoll et al. (1997)

Input Saline suspensions of α-quartz, carbon black, and titanium dioxide in ratsOutput Lung inflammation and mutation in alveolar cellsStudy Design Inferred between-groupStudy Population Groups of 9 female F344 Fischer rats (180–200 g)

• 3 groups for a dosage of 5 mg/kg of α-quartz, carbon black, and titaniumdioxide each

• 3 groups for a dosage of 50 mg/kg of α-quartz, carbon black, andtitanium dioxide each

• 12 rats as control groupStudy Objectives To investigate the role of inflammation in the lung tumor response to the

particles through their mutagenic effects on alveolar epithelial cellsMain Results -In vivo exposures to poorly soluble particles produce a neutrophilic

inflammatory response resulting in increased dose related to mutageniceffect in alveolar type-II cells

-Carbon black and α-quartz produce mutagenic effects in epithelial cells

Renwick et al.(2005)

Input Fine and Ultrafine carbon black and titanium particles in ratsOutput Inflammation and epithelial injury in lungsStudy Design Unable to determineStudy Population Male Wister rats body weight between 370–470 gStudy Objectives To compare the ability of fine and ultrafine particles of the same material to

induce inflammation, epithelial cell damage, and the affect onphagocytosis and chemotoxis

(Continued)



TABLE 2. Continued

Study Description

Main Results -Ultrafine and nanoparticles cause more inflammation than the same mass ofthe fine particles

-Ultrafine carbon black particles have greater activity than ultrafine TiO2particles as well as fine carbon black and TiO2

-Both fine and ultrafine particles impair the alveolar macrophagephagocytosis only at the higher doses except for fine TiO2

-The increased sensitivity of macrophages, inflamed by ultrafine particlesmigrates toward C5a chemotactic stimulus, which hinders the normalmucociliary clearance


Input Titanium oxide particles (TiO2-F and TiO2-D)Output Lung injury and particle translocationStudy Design Between-groupStudy Population -8 male Fisher 344 rats, weight approx. 220 g for the intratracheal instillation

-27 male Fisher 344 rats for reinstillation study-12 male Fisher 344 rats for dose-response study-5 male Fisher 344 rats as control group

Study Objectives To determine the ability of the UFPs to enter interstitium faster than thelarger-sized particles of the same material, the prevention ofinterstitialization by the alveolar macrophages and how increased lungoverload increase the lung injury and macrophages effects

Main Results -Ultrafine particles deposited in the alveoli of the lung enter the interstitiummore readily than larger-sized particles

-Ultrafine particles cause greater inflammatory response in alveolar regionthan larger sized particles

-Once the particles are phagocytized, the inflammatory events caused byalveolar macrophages are inhibited

average overall degree of agreement was 0.67%. The average degrees of agreement for thefive scales were 0.65% (study description), 0.80% (subject selection), 0.62% (measurementquality), 0.65% (data analysis), and 0.90% (generalization of results), respectively.

4.2. Identification of Studies

The search of computerized databases identified a total of 2,185 peer-reviewed articles untilApril 2005. The databases used in the search were located in PubMed, Medline, AcademicSearch Premier, COMPENDEX, and Elsevier Journals. Figure 1 provides a summary of thesearch process consisting of four steps. Upon review of published articles, additional studieswere excluded because of, among other things, the size of the nanoparticle used (more than100 nm) and type of outcome (drug delivery, sensing). After applying all exclusion criteria, atotal of 28 studies were identified from an electronic search of databases and bibliographiesof published articles. Fifteen of the studies dealt solely with carbon nanoparticles, whereas


308 SEQUEIRA ET AL.

Figure 1 Summary of steps involved in the electronic search of databases.

13 investigated the effects of noncarbon particles. Two of the randomly chosen carbon-basedarticles were used to calibrate the ExpAI tool.

4.3. Description of Evidence

Tables 1 and 2 display the description of evidence for the 26 studies in terms of exposure,outcome, study design, study population, and main results. The highlights of this evidenceare:

1. The numbers of studies conducted on carbon-based nanomaterials are relativelyhigher than the studies conducted on any other nanomaterials.

2. All of the studies conducted so far are either in vitro or on animals. Eighteen of thestudies have been conducted on animals and the remainder of the studies (n= 8) wereon extracted human cell specimens.

3. All but seven of the studies were either between- or within-group studies. One studyhad a mixed design (Lundborg, Johard, Lastbom, Gerde, & Cammer, 2001), and forsix of the studies, the study design was unclear.

After evaluating the description of evidence of the 13 articles based on carbon-basednanostructures (Table 1) for their quality in terms of exposure, outcome, study population,and main results, the following points were evident.

1. Almost an equal number of studies have been conducted on animals (n = 7) and invitro human tissue cells (n= 6). One study (Lundborg et al., 2001) investigated thetoxic effects on both human cells and animals.



2. Six of the studies (Barrett, Rudolph, Bowen, Muggenburg, & Bice, 2003; Harderet al., 2005; Kim et al., 2005; Lam, James, McCluster, & Hunter, 2004; Lundborget al., 2001; Warheit et al., 2004) were conducted on the response of lungs or thelung tissues; three of the studies (Chen, Wilson, Das, Coughlin, & Erlanger, 1998;Cui, Tian, Ozkan, Wang, & Gao, 2005; Monteiro-Riviere, Nemanich, Inman, Wang,& Riviere, 2005) investigated the cell response to carbon nanomaterials; and onestudy (Shvedova et al., 2003) investigated skin cell response to exposure to carbonnanomaterials. Two of the studies (Oberdorster et al., 2002, 2004) investigated thetranslocation of the nanomaterials following exposure among the various organs,and one study (Yamago et al., 1995) investigated both translocation and retention ofnanomaterials in the body.

3. All of the studies established a relationship between the exposure to carbon nanopar-ticles and health effects

4. The study conducted by Cui et al. (2005) demonstrates that the toxicity of carbonnanotubes is dose and time dependent. Barrett et al. (2003) tested airway resistanceto carbon nanotubes.

Based on evaluations of the descriptions of evidence (Table 2) of the 13 noncarbonarticles, the following are observed:

1. All studies but one was conducted on animal subjects. Lademann et al. (1999) is theonly study that was performed on human skin samples.

2. Six of the studies were between-group designs, three of the studies were within-groupdesign, and the dosing in four of the studies could not be determined.

3. Two of the studies (Jani, Halbert, Langridge, & Florence, 1989, 1990) investigatedthe uptake of nanomaterials and translocation thereafter. Nemmar et al. (2002) andNemmar, Hoylaerts, Hoet, Vermylen, and Nemery (2003) investigated the impact ofUFP on thrombus formation. Three of the studies (Driscoll et al., 1997; Oberdorster,Ferin, Gelein, Soderholm, & Finkelstein, 1992; Renwick, Brown, Clouter, &Donaldson, 2005) investigated the response of the lung and the lung tissues to ul-trafine particles. Takenaka et al. (2001) investigated the pulmonary response andsystematic translocation of the UFP after inhalation, whereas another study reportedthe pathological response of organs to UFP (Takenaka et al., 2004).

4. Churg, Gilks, and Dai (1999) investigated the effects of ultrafine TiO2 on geneexpression, and Lademann et al. (1999) investigated the presence of UFP in skincells and hair follicles.

5. All of the studies established the relationship between exposure to UFP and healtheffects.

4.4. Critical Appraisal of Study Quality

The studies identified from the literature search were subjected to a rigorous critical appraisalusing the Experimental Appraisal Instrument. Each study was evaluated separately byfive assessors. Afterward, a consensus was established through follow-up meetings. Thedifferences among the assessors stemmed directly from the research question outlined inthe introduction. The research question was based on expert knowledge and informationextracted from the published literature. As such, each study was not evaluated based onits own objectives. Indeed, critical appraisal reflects its methodological quality and theknowledge gap with respect to the research question established in this study.


310 SEQUEIRA ET AL.

Figure 2 Overall quality of all the articles.

An overall appraisal of the studies (Tables 3 and 5) suggests that the quality of individualstudies range from marginal (0.5 on 0–2 scale) to average (1.0 on 0–2 scale; Figure 2).Figures 2 through 7 provide a summary of the different methodological qualities for eachstudy. The following can be deduced:



Figure 3 Critical appraisal of evidence reporting of all the articles.

1. Studies scored average in reporting the different elements of methodological qualities.2. There was a variance in the subject selection criteria among the studies. About one

third of the studies did an average job, with the remaining two thirds roughly splitbetween marginal and poor scores.


312 SEQUEIRA ET AL.

Figure 4 Critical appraisal of subject selection of all the articles.

3. The majority of the studies were uniformly marginal in terms of measurement qualitywith the remainder scoring somewhat poor.

4. The data analysis methods were widely scattered between poor and average scores.This was greatly impacted by the lack of consideration for some important individualand environmental covariates/confounders.



Figure 5 Critical appraisal of measurement quality of all the articles.

5. The generalization of the studies is greatly in question. This may be attributed in partto the lack of information about the participation rate. In addition, the criteria forsample size calculations were not reported.


314 SEQUEIRA ET AL.

Figure 6 Critical appraisal of data analysis of all the articles.

Figure 5 provides a closer look at the measurement quality of individual studies. Investi-gators paid more attention to the exposure and outcome methods, and here again, the qualityof methods used centered on marginal values.



Figure 7 Generalization of results.

The overall quality was marginal. The reporting attribute was the highest, scoring aver-age values. The subject selection and data analysis methods were, on average, marginal.Measurement quality was somewhat poor and study generalization was in question. Therewas a wide variance among the studies in terms of quality attributes.


316 SEQUEIRA ET AL.

TABLE 3. Appraisal Scores for 13 Articles on Carbon-Based Nanomaterials

Overall Evidence Reporting Subject Selection(Q1–Q34) (Q1–Q15) (Q16–Q19)

Article R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 R1 R2 R3 R4 R5

1 0.71 0.71 0.76 0.82 0.88 0.73 0.67 0.60 0.73 0.87 1.00 1.00 1.00 1.00 1.002 0.88 0.68 0.91 0.97 0.91 0.87 0.73 0.80 0.93 1.00 1.00 0.50 1.00 1.00 1.003 0.71 0.76 0.82 0.85 0.82 0.60 0.80 0.73 0.87 0.87 1.00 0.75 1.00 1.00 1.004 0.88 0.85 0.44 0.47 0.79 0.87 0.67 0.60 0.27 0.73 1.00 1.00 0.00 0.25 0.505 0.79 0.94 0.82 0.79 0.85 0.80 1.00 0.80 0.73 0.80 1.00 1.00 0.75 0.50 1.006 0.74 0.91 0.79 0.68 0.88 0.73 0.87 0.73 0.73 0.80 0.75 1.00 0.75 0.00 1.007 0.88 0.85 0.91 0.53 0.91 0.87 0.93 0.87 0.53 0.87 1.00 1.00 1.00 0.00 1.008 0.74 0.79 0.85 0.59 0.76 0.73 0.87 0.87 0.73 0.93 0.75 1.00 0.75 0.00 0.759 0.79 1.00 0.71 0.88 0.94 0.73 1.00 0.73 0.87 0.93 1.00 1.00 0.75 0.75 1.0010 0.88 0.91 0.85 0.97 0.85 0.93 0.87 0.67 1.00 0.93 1.00 0.75 1.00 0.75 0.7511 0.85 0.71 0.76 0.76 0.88 0.80 0.67 0.73 0.87 0.87 0.75 0.25 1.00 0.50 0.7512 0.94 0.91 0.82 0.88 0.88 1.00 0.93 0.87 0.93 0.87 1.00 1.00 0.75 0.75 0.7513 0.88 0.71 0.88 0.82 0.91 0.93 0.53 0.87 0.80 0.87 0.75 1.00 1.00 0.75 1.00

Measurement Quality Data Analysis Generalization of(Q20–Q28) (Q29–Q32) Results (Q32–Q34)


1 0.67 0.67 0.78 0.78 0.78 0.25 0.50 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.002 0.78 0.67 1.00 1.00 0.78 1.00 0.50 1.00 1.00 0.75 1.00 1.00 1.00 1.00 1.003 0.89 0.78 1.00 0.89 0.67 0.25 0.50 0.50 0.50 0.75 1.00 1.00 1.00 1.00 1.004 0.78 1.00 0.22 0.78 0.89 1.00 1.00 0.50 0.50 1.00 1.00 1.00 1.00 1.00 1.005 0.78 0.89 0.78 0.89 0.78 0.50 0.75 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.006 0.78 0.89 0.89 0.78 0.89 0.50 1.00 0.75 0.75 1.00 1.00 1.00 1.00 1.00 1.007 0.89 0.67 1.00 0.67 1.00 0.75 0.75 0.75 0.50 0.75 1.00 1.00 1.00 1.00 1.008 0.78 0.56 0.78 0.56 0.44 0.50 0.75 1.00 0.50 0.75 1.00 1.00 1.00 1.00 1.009 0.89 1.00 0.67 1.00 0.89 0.50 1.00 0.50 0.75 1.00 1.00 1.00 1.00 1.00 1.0010 0.89 1.00 1.00 1.00 0.89 0.50 1.00 1.00 1.00 0.50 1.00 1.00 1.00 1.00 1.0011 1.00 0.89 0.78 0.89 0.89 0.75 0.75 0.50 0.25 1.00 1.00 1.00 1.00 1.00 1.0012 1.00 0.89 0.89 0.89 0.89 0.50 0.75 0.50 0.75 1.00 1.00 1.00 1.00 1.00 1.0013 0.78 0.78 0.89 0.78 1.00 1.00 0.75 0.75 1.00 0.75 1.00 1.00 1.00 1.00 1.00

4.5. Research to Practice

Critical evaluation of the selected studies using a linguistic scale for developing new nano-based technologies, with in vivo drug delivery systems in specific, suggests that the charac-teristics of these nanoparticles can be used in the design of new systems for practice. Theevaluation based on the type of nanoparticles tested, the study population, and the mainfindings suggest the following.



TAB

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8267

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9000

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6500

0.90

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0.65

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8000

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9111

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8444

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7588

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8000

0.74

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8294

0.82

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8353

0.74

710.

8706

0.90

590.

7882

0.88

820.

8235


318 SEQUEIRA ET AL.

TABLE 5. Appraisal Scores for 13 Articles on Noncarbon Nanomaterials

Evidence Reporting Subject SelectionOverall (Q1–Q34) (Q1–Q15) (Q16–Q19)


1 0.68 0.74 0.91 0.71 0.76 0.60 0.67 0.93 0.73 0.67 1.00 1.00 1.00 1.00 1.002 0.82 0.91 0.74 0.62 0.85 0.93 0.93 0.73 0.80 0.67 1.00 1.00 1.00 0.25 1.003 0.85 0.82 0.76 0.53 0.88 0.93 0.87 0.73 0.73 0.80 0.75 1.00 1.00 0.50 1.004 0.82 0.71 0.85 0.71 0.79 0.80 0.60 0.80 0.73 0.93 1.00 1.00 1.00 1.00 1.005 0.85 0.76 0.85 0.82 0.71 0.80 0.73 0.73 0.67 0.67 1.00 0.75 1.00 1.00 0.506 0.91 0.82 0.82 0.85 0.71 0.93 0.87 0.87 0.93 0.67 1.00 0.75 1.00 0.75 0.757 0.88 1.00 0.91 0.88 0.56 0.87 1.00 0.80 0.93 0.53 1.00 1.00 1.00 0.75 0.508 0.82 0.94 0.76 0.85 0.79 0.93 1.00 0.87 1.00 0.73 0.75 1.00 1.00 0.75 0.759 0.88 0.97 0.88 0.88 0.68 1.00 1.00 0.93 0.93 0.53 1.00 1.00 1.00 0.75 0.7510 0.88 0.88 0.76 0.85 0.74 0.93 0.80 0.73 0.93 0.67 1.00 1.00 1.00 1.00 1.0011 0.88 1.00 0.74 0.94 0.85 0.87 1.00 0.60 0.93 0.73 1.00 1.00 1.00 1.00 1.0012 0.88 1.00 0.88 0.97 0.85 0.93 1.00 0.80 1.00 0.87 0.75 1.00 1.00 1.00 1.0013 0.97 0.91 0.82 0.76 0.76 1.00 0.87 0.60 0.67 0.67 1.00 1.00 1.00 1.00 1.00



1 0.78 0.78 0.89 0.67 0.89 0.25 0.50 0.75 0.75 0.50 1.00 1.00 1.00 0.00 1.002 0.67 0.89 0.78 0.56 1.00 0.50 0.75 0.25 0.25 1.00 1.00 1.00 1.00 1.00 1.003 1.00 0.67 0.67 0.22 1.00 0.25 0.75 0.75 0.25 0.75 1.00 1.00 1.00 1.00 1.004 0.78 0.89 0.89 0.67 0.44 0.75 0.25 0.75 0.25 0.75 1.00 1.00 1.00 1.00 1.005 0.78 0.89 0.89 1.00 0.89 1.00 0.50 1.00 0.75 0.50 1.00 1.00 1.00 1.00 1.006 0.89 0.89 0.78 0.78 0.78 0.75 0.50 0.50 0.75 0.50 1.00 1.00 1.00 1.00 1.007 0.89 1.00 1.00 0.78 0.67 0.75 1.00 1.00 1.00 0.25 1.00 1.00 1.00 1.00 1.008 0.78 0.89 0.67 0.67 0.89 0.50 0.75 0.25 0.75 0.75 1.00 1.00 1.00 1.00 1.009 0.78 1.00 0.78 0.78 0.78 0.50 0.75 0.75 1.00 0.75 1.00 1.00 1.00 1.00 1.0010 0.89 1.00 0.78 0.56 0.89 0.50 0.75 0.50 1.00 0.25 1.00 1.00 1.00 1.00 1.0011 0.89 1.00 1.00 1.00 1.00 0.75 1.00 0.25 0.75 0.75 1.00 1.00 1.00 1.00 1.0012 0.89 1.00 1.00 1.00 0.78 0.75 1.00 0.75 0.75 0.75 1.00 1.00 1.00 1.00 1.0013 1.00 0.89 1.00 0.78 0.78 0.75 1.00 1.00 0.75 0.75 1.00 1.00 1.00 1.00 1.00

The main finding of nine of the studies (Beck-Speir et al., 2001; Cui et al., 2005; Harderet al., 2005; Lam et al., 2004; Lundborg et al., 2001; Nemmar et al., 2002, 2003; Renwicket al., 2005; Shvedova et al., 2003) cannot be used to directly design any new systems. Thesestudies are aimed at observing the detrimental effects of nanomaterials on the tissues andcells associated with them that were directly exposed to the materials, but the mechanismsand dynamics behind such effects are not reported. These observations could be used toimprove the existing systems rather than developing a completely new system. The findingsof Lademann et al. (1999) can be used in developing new systems for a larger extent.



In their study they observed that the TiO2 particles can penetrate the skin surface. Thisproperty of TiO2 can be used in devising the drug delivery through skin systems. Findingsof 12 of the studies (Chen et al., 1998; Driscoll et al., 1997; Fernandez-Urrusuno, Fattal,Feger, Couvreur, & Therond, 1997; Jani et al., 1989; Kim et al., 2005; Oberdorster, 2004;Oberdorster et al., 1992, 2002, 2004; Takenaka et al., 2001, 2004; Yamago et al., 1995) couldbe used to a moderate extent. In these studies the researchers observed the proliferation of thenanomaterials in the cells and found the elevated concentrations within organs suggestingthat these observations could be used in the development of site-specific drug deliverysystems. Observations made in six of the studies (Barrett et al., 2003; Churg et al., 1999;Jani et al., 1990; Monteiro-Riviere et al., 2005; Tsuchiya, Oguri, Yamakoshi, & Miyata,1996; Warheit et al., 2004) have minimal use. In these studies, the observations made weremainly for the negative effects of the nanoparticles on organs based on the introductionand translocation through different pathways. This could be translated as though there aredetrimental effects that the course taken by the nanomaterials to reach the different organsand cells could be used to our advantage to develop the new system.

5. DISCUSSION

Nanotechnology has been predicted to be the technology of the future because of the ex-traordinary properties exhibited by the materials at this scale. Although newer materials andproperties are being researched for their possible use in the commercial and technologicalfield, few studies demonstrated the adverse health effects of nanomaterials. Numerous pub-lished reviews have raised various safety and health concerns resulting from nanomaterials.The studies conducted to date are mainly on animal or in vitro human tissues. Though therecould be a relationship between the actual exposure of humans to nanomaterials and healtheffects, it is very difficult to substantiate this statement. This is mainly because of the limitednumber of toxicology studies on any particular area/organ. This research was conductedto bridge this gap by critically appraising the selected articles using ExpAI as a primarytool.

5.1. Critical Appraisal With ExpAI

The purpose of the ExpAI tool is to quantify the quality of the articles in terms of the studydescription, subject selection, data analysis, measurement quality, and the generalization ofresults. Study description, which evaluates if the different aspects of the study are clearlyreported, is between marginal and average for most of the studies. This is mainly because offailure of these studies to explain various aspects such as reason for selecting the particularsubjects, from where the study subjects were selected, and identifying whether any selectioncriterion was used and, if so, identifying what type was used. Also lacking was the mention ofwhether all the confounders and covariates considered in the study, the design of the study,and so forth. For most of the studies, the subject selection criterion was not clear. Thiscriterion looks at the duration of the experiment and the randomization of subjects, as wellas the observers, all of which would minimize the bias. This was not explained in any of thearticles. Quality was measured as fairly good for few articles and ranged between marginaland average. This is because the authors failed to explain the reliability and validity of theinputs considered as well as the results obtained. Though the measurement methods areclear and comparable in every study for all different groups and inputs, the randomizationwas lacking, resulting in a biased study. Data analysis has been reported by 14 of the


320 SEQUEIRA ET AL.

TAB

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8000

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8222

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7706

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8471

0.83

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0.82

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8824

0.91

760.

8471



TABLE 7. Evaluation of Study Results Based on Linguistic Scale

Source Main Findings Rating Comments

Barrett et al.(2003)Main Results The allergic dogs developed in-

creased pulmonary resistance to rag-weed whereas nonallergic dogs didnot

• The IgE level in allergic dogsis 100% more than nonallergicdogs

• The IgG levels in allergic andnonallergic dogs are nearlyequivalent

Level 1 The level of ultrafine particlesin the environment is alreadyon the higher side. Therefore,exposure of persons with al-lergies to these particles arevery likely

Chen et al. (1998)Main Results -Immune response to 1-TG: no anti-

body or nonspecific binding to pre-immune serum-IgG response directed to thefullerenes, not at the functionalgroups, linking the fullerenes to thecarriers

Level 2 No binding of antibodies spe-cific to the fullerene wereobserved and the hygro-scopic nature of the particlescould make it useful in drugdelivery

Cui et al. (2005)Main Results -Compared to the controls, the cell

viability of cells exposed to SWCNTdecreased with a time and dose de-pendent relationship up to 90%-The same behavior was noted forcell addition, where it went down to100%-Apoptosis was present on HEK293cells on a dose and time-dependentmanner

Level 0

Harder et al.(2005)Main Results -UFCP increases the transient heart

rate and activates a neural pathwayin response to particle inhalation-Cardiac rate was increased in4.8 ± 3.1%-BALF glutathione concentration in-crease in 0.22 μg/ml-Increase in polymorphonuclearcells was double in the first 4 hr. Af-ter 24 hr the increase was fivefold-There was no significant change inblood coagulation and clot lysis

Level 0 -The dose concentration of180 mg/m3 is quite high inany normal conditions-The mechanism of nerve ac-tivation and the cause of HRincrease is not clear

(Continued)


322 SEQUEIRA ET AL.

TABLE 7. continued


Kim et al. (2005)Main Results -UFCP appears to be a potent in-

ducer of proinflammatory responsein NHBE cells-IL-8 protein increased around4.5-fold over control with 33 μg/mlafter 24 hr of exposure, and has afourfold increase over control with56 μg/ml after 24 hr of exposure-There is no difference in cell viabil-ity after exposure

Level 2 The concentration of theUFCP used in the study ishigher than normal thoughthe condition in the ambi-ent conditions is possible atlower dosages

Lam et al. (2004)Main Results -All NT products produce dose-

dependent lung lesions and granu-lomas-NT that reach lungs are much moretoxic than CB and can be more toxicthan quartz

Level 0 Dosage in the study is toohigh, which is highly unlikelyto occur in any environment

Lundborg et al.(2001)Main Results -High concentration (10 μg/ml)

of ingested particles impaired thephagocytic capacity of AM cells-With ingested particles, phagocyticactivity decreases by 70% with adose of 3 μg/ml-Accumulated attachment and in-gested fraction decreases by 50%compared to the control

Level 0 The UHF are likely to effectthe people exposed to them,that is, the concentration inthe environment is sufficientenough to cause these effects

Monteiro-Riviereet al. (2004)Main Results -Chemically unmodified MWCNT

are capable of entering the HEK aswell as causing irritation to it.-Amount of MWCNT in the cell in-creased when treatment concentra-tion and exposure time increased:

• From 59.1% at 24 hr to 84%at 48 hr at 0.4 mg/ml

-Cell viability after 24 hr:• For control group is 100%• For 0.1 mg/ml dose is 87%• For 0.2 mg/ml dose is 75%• For 0.4 mg/ml dose is 72%

Level 1 Dose dependent and chancesof such exposure (24 hrcontinuously) is minimal innormal conditionsThe capability of theMWCNT to enter the cellscould be used in the de-velopment of drug deliverysystems

(Continued)



TABLE 7. Continued


Oberdorster et al.(2002)Main Results -Large amount of particles reach the

blood circulation and subsequentlythe liver. They were also found inthe lower respiratory tract-Maximum deposition of 13C inlungs occurs during the first halfhour-13C concentration in liver is 5-foldhigher after 18 and 24 hr than in thelung-There is no significant decrease in13C in lung from 0.5 to 24 hr of postexposure

Level 2 In case of chronic exposure,the buildup of carbon parti-cles in the organs is likely,due to slow removal of par-ticles from the organsThis property of the nanopar-ticles can be used in site-specific drug delivery and insensing

Oberdorster et al.(2004)Main Results -There is translocation of inhaled

UFCP into the CNS via the olfac-tory nerve-13C particle concentration in lungschanges from 1.39 ± 0.6 μg on thefirst day to 0.59 ± 0.15 μg on theseventh day-In the olfactory bulb there is an in-crease from 0.35 ± 0.17 μg/g tissueon day 1 to 0.43 ± 0.08 μg/g tissueon day 7-The concentrations in cerebellumand cerebrum were not consistentand vary between 0.11 and 0.44 μg/gtissues

Level 2 170 mg/kg3 is a very highconcentration of particles.which is very unlikely tohappenThe capability of the parti-cles to cross the BBB couldbe used in drug delivery

Warheit et al.(2004)Main Results -Overall mortality at 5 mg/kg of

CNT = 15% after 24 hr-Pulmonary inflammation in cells at5 mg/kg of quartz = 61%-Pulmonary inflammation in cells at1 mg/kg of quartz = 31%-Pulmonary exposure to SWCNTproduced a nondose dependent se-ries of multifocal granulomas

Level 1 The mortality in study sub-jects is due to mechani-cal blockage of upper air-ways. At dosage of 5 mg/kg,granulomas were found inthe lungs. But the levelof SWCNT in manufactur-ing facilities is undetectableand is <0.1 mg/kg (Litera-ture search for the level ofSWCNT, Baron et al., 2002;Joseph et al., 2002; Maynardet al., 2003)

(Continued)


324 SEQUEIRA ET AL.

TABLE 7. Continued


Shvedova et al.(2003)Main Results -SWCNT produced oxidative stress

and cellular toxicity on HaCaT-Indicators after 18 hr for theseharmful effects are:

• Formation of free radicals• Accumulation of peroxidative

products• Antioxidant depletion• Loss of cell viability

-Cell viability decreased with in-crease in dosage:

• Control 0%• 0.06 mg/ml 11.3%• 0.12 mg/ml 24.5%• 0.24 mg/ml 37.6%

-SWCNT produced a significantconcentration dependent decrease inantioxidant reserve of 35.2% for0.66 mg/ml, 53.8% for 0.12 mg/ml,and 76.8% for 0.24 mg/ml after 18 hrof incubation

Level 0 The onset of effects is ob-served within 4 hrSignificant effects were ob-served at 18 hr of exposure

Yamago et al.(1995)Main Results -Oral absorption of fullerenes was

found to be very poor in rats, how-ever traces of them are found in liverand other tissues after 6 hr. 97% wasexcreted after 48 hr-Intravenously administratedfullerene:

• Total amount of fullereneretained in the body whenadministered intravenously= 90%

• Total fullerene retained in theliver after 30 hr = 80%

• Approximately a dose of500 mg/kg fullerene is foundto be lethal

-C60 fullerenes may be regarded as acompound with weak acute toxicity

Level 2 Even though the fullerenesare regarded compound withweak toxicity, the retention offullerenes in the vital organsfor a longer time raises theconcern about chronic toxic-ity. The dosage of 500 mg/kgis comparatively a very highdosageThis condition is unlikely tohappen in any normalconditions except in case ofaccidental releases

(Continued)



TABLE 7. Continued


Tsuchiya et al.(1996)Main Results -An intraperitoneal dose of 137

mg/kg of C60 proves to be deadlyto pregnant mice as well as em-bryos. Also the yolk sac function wasseverely disrupted-50% of the embryos had abnormalshape after a dose of 50 mg/kg to thepregnant mouse-At 25 mg/kg there was only oneabnormal embryo

Level 1 The dosage to cause the em-bryo damage is higher, whichis not like to happen throughinhalationThe transport to embryothrough the placenta andproducing active oxygen,with modifications, makesfullerenes useful in the treat-ment during pregnancy

Oberdorster(2004)Main Results -nC60 seems to be able to improve

water quality, possibly by interferingwith bacterial growth.After 48 hr of exposure:-There was significant lipid peroxi-dation in the brain as well as othertissues due to nC60. No protein oxi-dation was found-In the brain, 0.5 ppm nC60 causedseventeenfold increase in lipid per-oxidation-In the brain, at 1 ppm there is a slightreduction (around 4 mM/mg) of pro-tein oxidation-In the gill, at both 5 ppm and 1 ppmthere is a slight reduction of pro-tein oxidation, and there is almostno change for the liver-GSH depletion on the gill was onlystatistically significant in the indi-vidual fish analysis, not to aquariumaverages-Fullerenes absorption andtransportation by olfactory cells =2.90 ± 0.21 mm/hr

Level 2 Emphasis is needed onthe proper handling andwaste disposal of fullerenes.Though the oral absorption offullerenes is very poor, the in-take through potable water orthe use of fullerenes in wa-ter treatment can have an ad-verse effect. Since they af-fect the wildlife and can bein the organs for a longertime, fullerenes get in thefood chain and cause higheradverse effects to humansThe ability to cross theblood–brain barrier could beused in the treatment of ill-nesses pertaining to the brain

Jani et al. (1989)Main Results -Uptake of 100 nm and 500 nm mi-

crospheres is higher than 1 mm diaparticles-Absorption of non-ionized particlesis more evident than the ionized par-ticles

Level 2 Could be used in drug deliv-ery and detection

(Continued)


326 SEQUEIRA ET AL.

TABLE 7. Continued


-Uptake by Peyer’s patches andMesentery node is highest in com-parison with other parts of the GItract-There is no evidence of particles inkidney, heart, and lungs

Nemmar et al.(2002)Main Results For intravascular instillation:

-Unmodified particles do not inter-fere with the thrombus formation-Negatively charged particles signif-icantly (approx. 52–55%) inhibit thethrombus formation at higher doseswhile they do not have effect at adosage of 50 mg/kg-Positively changed particles en-hance the thrombus formation by219% for 50 mg/kg and 307% forthe 500 mg/kg dosage-For intratracheal instillation of5 mg/kg of particles, there is nosignificant effect on thrombus for-mation by unmodified and carboxy-lated particles, whereas amine mod-ified particles induce 368% increasein thrombus formation

Level 0

Nemmar et al.(2003)Main Results -Both 60 nm and 400 nm particles

caused pulmonary inflammation, butonly positively charged 60 nm par-ticles enhanced thrombus formationas opposed to negatively unmodifiedor negatively charged 400 nmparticles

Level 0

Jani et al (1990)Main Results -Total uptake of 50 nm particles is

about 33% of the administered dose-Uptake of 1 mm particles is about7% of the administered dosage-3 mm particles were found in GItract but do translocate to otherorgans

Level 1 100 nm could be useful in thedrug delivery

(Continued)



TABLE 7. Continued


-100 nm particles seem to representthe upper limit for transport into thebone marrow and 300 nm representsthe upper limit for the particles toenter into the blood circulation

Takenakaet al. (2001)Main Results In inhalation study:

-No accumulation of particles inalveolar macrophages in morpho-logical analysis-Within 7 days the lung burdenreduced to 4% of the initial burdenIn instillation study:-Particles were observed inmacrophages even till day 7-The EAg content in lungs remainsunchanged from day 1 to 7-With prolonged inhalation ofparticles, particles enter the alveolarwalls

Level 2 The EAg particles could beused in the drug delivery.The rapid clearance of theseparticles minimizes any ad-verse effects

Churg et al.(1999)Main Results -There is an effect of size of the par-

ticles on mediator and matrix geneexpression-There is no increase in the geneexpression for the fine and ultrafinedust exposures till day 7-At the highest exposure (5 mg) ofultrafine particles, there is a 20%increase in procollagen expression.But there is no effect for fine dusts-On day 7 the gene expression in-creases, but it is not consistent forall the genes

Level 1 Could be used in the genetherapy

Takenaka et al.(2004)Main Results • For lower concentration of

CdO-The cadmium content in lung was0.53 mg/lung immediately after ex-posure and was constant till theseventh day

Level 2 The modified particles couldbe used as sensors in the de-tection of functioning of thevital organs

(Continued)


328 SEQUEIRA ET AL.

TABLE 7. Continued


-No significant increase of cadmiumcontent in the liver as well as kidneysduring the study period• For higher concentration of CdO- Liver shows a significant

elevation of cadmium contentfrom day 0 to day 1

- Cadmium content in the kidneysshows a significant elevation

• Cadmium can be detected inblood at higher dosages ofexposure

• The lung weight increases withthe higher concentrations but atlower concentrations it does notchange

• Lungs show multifocal alveolarinflammation characterized bypolymorphonuclear andmononuclear cell infiltration inalveolar lumen

• Neutrophils count increasesignificantly with higherconcentration

Fernandez-Urrusunoet al. (1997)Main Results -Uptake of polymeric particles by

Kupffer cells in the liver induce therelease of oxidative species alteringthe antioxidant systems of hepato-cytes.-PIBCA and PS both reduced (GHS)and oxidized (GSSG) glutathionecontent-PIBCA and PS nanoparticles sig-nificantly reduce the superoxide dis-mutase (SOD) activity and catalase(CT) activity

Level 2 The particles to reach the vitalorgans could be used in thedrug delivery and detection

Beck-Speier et al.(2001)Main Results -The surface area rather than mass

concentration determines the effectsof agglomerates of ultrafine particles(AUFP)

Level 0

(Continued)



TABLE 7. Continued


-Both low and high concentrationsof UFPs cause the inhibitory effectson PMNs-There is an inflammatory responseof the lung to the AUFPs

Lademann et al.(1999)Main Results • Largest amount of TiO2

localizes to the upper part of thestratum corneum

• TiO2 microparticles can enter theorifices of hair follicles

• In the deeper part of horny layer,TiO2 particles exclusivelylocalize in the follicle channels

Level 3 Results could be used in thedevelopment of drug deliverythrough skin as well as skintreatment

Driscoll et al.(1997)Main Results -In vivo exposures to poorly solu-

ble particles produce a neutrophilicinflammatory response resulting inincreased dose-related mutagenic ef-fect in alveolar type-II cells-Carbon black and α-quartz producemutagenic effects in epithelial cells

Level 2 The results could be used inthe development of drugs forgene therapy

Oberdorster et al.(1992)Main Results -Ultrafine particles deposited in the

alveoli of the lung enter the intersti-tium more readily than larger-sizedparticles-Ultrafine particles cause greater in-flammatory response in alveolar re-gion than larger-sized particles-Once the particles are phagocytized,the inflammatory events caused byalveolar macrophages are inhibited

Level 2 These particles could be usedin drug delivery through in-halation as a carrier

Renwick et al.(2005)Main Results -Ultrafine and nanoparticles cause

more inflammation than the samemass of the fine particles-Ultrafine carbon black particleshave greater activity than ultrafineTiO2 particles as well as fine carbonblack and TiO2

Level 0

(Continued)


330 SEQUEIRA ET AL.

TABLE 7. Continued


-Both fine and ultrafine particlesimpair the alveolar macrophagephagocytosis only at the higherdoses except for fine TiO2-The increased sensitivity ofmacrophages, inflamed by ultra-fine particles migrates toward C5achemotactic stimulus, which hindersthe normal mucociliary clearance

28 studies. The remaining 14 studies are limited qualitative studies, and the raw data oranalysis was not reported by these articles. Lack of any other literature on the experimentson the same subjects and the same materials makes it difficult to generalize the study’sfindings. This could be attributed to the nascency of the research in the field of health effectsarising from nanotechnology.

The evidence reported across all of the studies was between average and better thanaverage, which indicates that nanomaterials do in fact have adverse effects. However, dueto the lack of information about the sample size calculation and the participation rate,it cannot be generalized. Another reason for not being able to generalize the evidencereported is the lack of any other parallel studies or statistics to check the reliability ofthe outcome measures. The poor data analysis in some of the studies is attributed to thelack of individual and environmental covariates/confounders. Due to the lack of reportedraw data in these studies, the relationship between the exposure and the adverse out-come cannot be established quantitatively. The qualitative evaluation of the articles fromthe evidence description suggests that the hazard posed by these materials cannot be dis-counted. The similar results in several studies conducted on lungs and lung tissues implythat there is, in fact, a co-relation between exposure and outcome. Apart from inhalation,the studies have demonstrated that the nanomaterials can cause similar effects throughingestion as well as from diffusion through the skin. Several of the investigators foundthe traces of nanomaterials in different vital organs substantiating their translocation afterabsorption.

Lam et al. (2004) have observed that carbon nanostructures can infiltrate deeper into thelung tissue and can cause granuloma, which is dose dependent, whereas the carbon black aswell as carbon and quartz dust seem to be less toxic in comparison. Similar observation hasbeen described by Warheit et al. (2004) in their study using carbon nanotubes. Kim et al.(2005) and Monteiro-Riviera et al. (2005), in their investigations of the effects of carbonparticles on lungs, have demonstrated proinflammatory response by the bronchial cells,and Lam et al. (2004) have found granulomas in the lungs due to exposure. Oberdorsteret al. (2004), in their experiment with 13C carbon particles, have found that apart fromlung alveoli, nanomaterials can enter the body through the olfactory bulb. The same studyalso found elevated levels of nanomaterials in the brain and the central nervous system.Oberdorster et al. (2002), in their study on translocation of nanomaterials after inhalation,found nanomaterials in the inner vital organs.



Since carbon-based and non-carbon-based studies have reported similar outcomes, itcould be safely deduced that the size and shape of the particle does have an effect onlung function, even though it is not material specific. The studies conducted using carbonnanotubes on lungs and lung tissues have reported an elevated response in contrast withplain carbon dust particles of similar size.

5.2. Limitations

Overall, the studies appraised in this research indicate that there are health risks due tonanoparticles. However, due to the lack of a sufficient number of studies on any particularorgan or a group of cells, the risks cannot be quantified. Moreover, these studies are either invitro or are conducted on animals in lab conditions. The results may not be the same for invivo experiments because metabolism and the defense mechanism of the body may influencethe results significantly. Also, the concentrations of the input applied in these studies aremuch higher than the ones that exist in the normal environment because materials at thisscale are lighter and can spread and traverse in the surrounding air. On the contrary, the sameproperties of the materials cause them to suspend in the air for a much longer time beforesettling, resulting in a sustained dosage over a longer period of time. Due to these factors,it is not possible to draw solid conclusions based on these studies emphasizing the needfor further research targeting the subjects who are exposed to such nanoparticles in theenvironment.

5.3. Research to Practice

Although these studies were conducted to determine the extent of adverse effects caused bynanomaterials, the same findings can be used to benefit mankind by building new systems andpractices, which were not possible until the advent of nanotechnology. Many of the studiesevaluated in this research have identified the mechanisms and pathways through whichthese nanomaterials infiltrate different organs and cells. The same mechanisms and pathwayscould be used to build new imaging systems, treatment systems, or even monitoring systemsfor vital functions. Oberdorster et al. (2004) have reported the presence of nanoparticles inthe brain and central nervous system, which were absorbed through inhalation. This impliesthat nanoparticles could cross the blood–brain barrier, which could be used to design a newdrug delivery carrier for the treatment of psychological problems as well as the treatmentof diseases pertaining to the brain. Another study (Lademann et al., 1999) found traces oftitanium dioxide nanospears in skin tissue, which could make it possible to deliver treatmentthrough the skin as an ointment. Several studies have found the presence of nanoparticles invital organs, such as the liver and kidneys, which were absorbed and translocated from theprimary exposure area, which was either the lung or the GI tract. This opens a new horizonfor detection and site-specific drug delivery systems. Other aspects of this phenomenonare time delayed and sustained drug delivery. The retention of nanoparticles in the body bythese organs could be used in formulating time delayed and sustained drug delivery systems,which could either be triggered through external impulses or be completely autonomous.

In conclusion, though the studies evaluated in this research have proven that nanoparticlesdo have adverse health effects, nanoparticles should not be discounted without exploring thepossibility of using them to benefit companies and the general population. Reengineeringshould be considered when creating these new systems to overcome the health effects foundby various studies.


332 SEQUEIRA ET AL.

APPENDIX A: DEVELOPMENT OF ExpAI TOOL

Appendix B illustrates a road map for the development and testing of the ExpAI pilot andsubsequent revised versions. The ten steps of the road map are detailed in the followingsections.

Development of the EAI Pilot Version

A first draft of the ExpAI pilot version was developed by an epidemiologist/biostatisticianand safety and health engineering professional ergonomist (Ash Genaidy) after reviewingpublished literature on experimental designs (see Genaidy & Shell, 2008). Apart from re-ferring to experimental design and analysis principles (Keppel, 2004), the first draft chieflyinvolved customizing the EAI (Genaidy et al., 2007) to suit the evaluation of experimen-tal studies (Step 1). The ExpAI is organized around two main components: (a) StudyDescription—an evaluation of the extent to which the different aspects of the study areclearly reported; and (b) Study Execution—an evaluation of how well the study is designedand analyzed, and how well the results can be generalized. The ExpAI consists of 34questions, grouped into five categories:

1. Study Description (15 items)—Evaluates whether the different aspects of the study areclearly reported and if it allows the reader to make an accurate evaluation (includesthe main hypothesis/aim/objective, study design, exposure/intervention, outcome,covariates and confounders, statistical tests, and main findings).

2. Subject/Record Selection (4 items)—Evaluates how the subject selection and re-cruitment have been conducted and whether the methods of selection minimizedbias.

3. Measurement Quality (9 items)—Evaluates whether the methods of measurementand recording of information have been uniformly followed and applied with thesame precision to all groups.

4. Data Analysis (4 items)—Evaluates how the study has been analyzed, whether ap-propriate statistics have been used, and if confounders and covariates have beenaccounted for.

5. Generalization of Results (2 items)—An evaluation of the study in terms of whetherthe results are applicable to the eligible population and if the results can be extendedto other groups.

To respond to each item in the ExpAI, the assessor is provided with two or more of thefollowing levels to choose from:

1. Yes—Designates that the information provided is complete.2. Partial—Designates that the information provided is partially complete.3. No—Designates that the information provided is not described but should have been

provided.4. Unable to Determine—Applicable whenever the information provided is unclear or

insufficient to answer the question.5. Not Applicable—A means for skipping an item. For example, an item that targets a

study design different from the one evaluated.



The ExpAI provides detailed explanations for each answer choice. These explanationswere drawn from statistical sources and based on the experiences of the primary author(please see Appendix B for an example; an electronic file is available upon request fromthe authors). As shown in Appendix C, if demographically similar, internal controls areconsidered the top choice for a comparison group, and are assigned a Yes versus a Noassigned to a very dissimilar population.

Testing the ExpAI Pilot Version

The content validity of the ExpAI was discussed in several meetings over a period of 2months by members of the research team, which consisted of an epidemiologist/healthengineering professional (Genaidy) and five researchers (Step 2). These meetings resultedin modifying the content and/or the corresponding descriptions of some of the items in theinstrument so as to eliminate ambiguity in responses and to make the items more effectiveat analyzing experimental studies.

Following the content validity testing (Step 2) and criterion validity testing (Step 3),a refined pilot version of the instrument was developed that incorporated the suggestedimprovements (Step 4). The six-member research team used this refined pilot version toevaluate two articles (Step 5). The pilot testing by the research team ended with additionalmeetings to discuss the evaluation results. Further modifications were made, such as theneed to incorporate additional types of responses (e.g., not applicable) in some questions, theneed for further explanations and examples to be provided in the detailed description of theresponses, and the need to include new items (such as accounting for possible adverse effectsto the experimental intervention; Step 6). The items in this refined version are illustrated inthe next section.

Development and Testing of the ExpAI Revised Version

Following the testing of the refined pilot version, discussions among the research teammembers led to additional improvements in the description of the responses and in the in-structions, thus resulting in the development of the first draft of the refined ExpAI instrument(Step 6). Some of these refinements were:

1. Reporting—To respond to the items for the reporting scale, one may select one ofthree levels: No, Partial, and Yes (the exceptions are Items 5, 7, 9, and 10, which havethe additional level of Not Applicable; please see Genaidy and Shell (2008) for itemexplanation). Thus, the decision for selecting a response from among No/Partial/Yesshould be based on a two-step process. First, one needs to decide if the answer is No(i.e., a certain aspect of the study is not reported or described) or Partial/Yes (i.e.,the evidence is described). Second, if the answer is Partial/Yes, one needs to decideif it is a Partial or Yes. If the article is read several times to make the decision, thereis a good chance that the answer is Partial. Another way to decide if a response is aPartial or Yes is by reviewing the scale below. The end points of the scale are labeledas No and Yes, with Partial occupying the entire space between the two end points.

|−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−|NO�−−−−−−−−−−−−−−−−−−−−−− Partial −−−−−−−−−−−−−−−−−−−−− �Yes


334 SEQUEIRA ET AL.

2. Subject Selection/Measurement Quality/Data Analysis/Generalization of Results—Responding to these items, which include Unable to Determine as one of the levels,should be based on a two step-process. First, one needs to decide if the answer isUnable to Determine (that is, there is insufficient information to answer the question)or No/Partial/Yes (that is, there is sufficient information and the question can beanswered using one of these levels). Second, if the answer is No/Partial/Yes, oneneeds to decide if it is one of these levels by reading closely the description attachedwith each response.

In addition to the above, items related to basic subject characteristics, confounders, andcovariates (Items 8, 9, 10, 28, and 29, respectively, in Genaidy and Shell (2008)) requireknowledge of the subject matter. Therefore, one needs to determine, a priori, the informationrequested for each of the above-mentioned items.

The revised version of the ExpAI was subjected to further scrutiny (Step 7 and Step 8).The same group of five graduate students participated in this phase of testing the revisedExpAI version. Once the suggested modifications (mainly attempts at making the itemsmore lucid) were incorporated to create the final refined version (Step 9), the team of fivegraduate students employed the instrument to evaluate 15 observational studies (Step 10).The goals of this phase were: (1) to determine the interrater degree of agreement for eachof the five levels of responses utilized in the ExpAI (i.e., No, Partial, Yes, Unable toDetermine, Not Applicable); (2) to investigate the interrater reliability for each of the fivescales across 13 carbon-based and 13 noncarbon-based articles; and (3) to examine theinterrater degree of agreement for each of these articles. With reference to the third goal,the five raters were asked to initially appraise each study individually, followed by a teammeeting where the team members verified their individual responses and reached consensusover the differentially classified responses, without the intervention of the primary author(Genaidy).

Prior to this application of the ExpAI instrument (Step 10), the primary author (Genaidy)informed the raters about the research question involving the systematic review and the topic-specific knowledge required to answer Questions 7, 8, 9, 10, 29, and 30 in the instrument.Though the team worked using the refined version (Step 9), the process of improvement wasongoing, with an iterative process that continuously improved the efficacy of the instrument(Step 6, Step 7, Step 8, and Step 9).



APPENDIX B: ROAD MAP FOR THE DEVELOPMENT AND TESTING OFTHE EXPAI PILOT AND REVISED VERSIONS


336 SEQUEIRA ET AL.

APPENDIX C: AN EXAMPLE OF DETAILED CRITERIA FOR LEVELS OFANSWERS FOR A GIVEN QUESTION IN THE ExpAI

16. Is the comparison/reference group comparable to the primary group (s)?

Not Applicable NA • Within-subject designsYes – Comparable Y • All groups are drawn from the same eligible

population (between-subject and mixed designs)Partial – Somewhat

comparableP • Comparison groups are not drawn from the same

eligible population, but recruited from similarpopulations elsewhere (between-subject andmixed designs)

No – low comparability N • Dissimilar comparative groupComparative groups are not used

Unable to determine UTD • Insufficient details

APPENDIX D

Description of Evidence for Articles Used in Testing the Pilot

Tsuchiya (1996)Input C60 fullerenes on pregnant miceOutput Cell damage and cell growth inhibitionStudy Design Unable to determineStudy Population -2 pregnant SLC-mice

-Mice embryosStudy Objectives Examine harmful effects of C60 on mice embryos in vitro and

in vivo.Main Results -An intraperitoneal dose of 137 mg/kg of C60 proves to be deadly

to pregnant mice as well as embryos. Also the yolk sacfunction was severely disrupted

-50% of the embryos had abnormal shape after a dose of 50mg/kg to the pregnant mouse

-At 25 mg/kg there was only one abnormal embryoOberdorster(2004a)

Input C60 fullerenes in juvenile largemouth bass (JLB)Output Lipid peroxidation of the brain and glutathione depletion of the

gill



Study Design Between groupStudy Population 28 JLB (Micropterus salmoides) in a 10l aquarium:

-9 JLB exposed to C16 fullerenes at 0.5 ppm-4 JLB exposed to C16 fullerenes at 0.1 ppm-6 JLB exposed to 100 μM H2O2-9 JLB as control group

Study Objectives Evaluate the toxicity of uncoated fullerenes on a relevantenvironmental species

Main Results -nC60 seems to be able to improve water quality, possibly byinterfering with bacterial growth

After 48 hr of exposure:-There was significant lipid peroxidation in the brain as well as

other tissues due to nC60. No protein oxidation was found-In the brain, 0.5 ppm nC60 caused seventeenfold increase in

lipid peroxidation-In the brain, at 1 ppm there is a slight reduction (around 4

mM/mg) of protein oxidation-In the gill, at both 5 ppm and 1 ppm there is a slight reduction of

protein oxidation, and there is almost no change for the liver-GSH depletion on the gill was only statistically significant in the

individual fish analysis, not to aquarium averages-Fullerenes absorption and transportation by olfactory cells

= 2.90 ± 0.21 mm/hr

APPENDIX E

Interrater Degree of Agreement for Pilot Version of ExpAI

Overall Evidence Reporting Subject Selection(Q1–Q34) (Q1–Q15) (Q16–Q19)


1 0.65 0.62 0.74 0.71 0.71 0.60 0.67 0.67 0.67 0.73 0.50 0.50 0.75 0.75 0.502 0.71 0.44 0.79 0.74 0.74 0.67 0.27 0.87 0.73 0.73 1.00 1.00 1.00 1.00 1.00



1 0.67 0.56 0.78 0.67 0.78 0.75 0.50 0.75 1.00 0.50 1.00 1.00 1.00 0.00 1.002 0.78 0.56 0.56 0.44 0.44 0.25 0.00 0.75 1.00 1.00 1.00 1.00 1.00 1.00 1.00


338 SEQUEIRA ET AL.

APPENDIX F: LINGUISTIC SCALE

In order to evaluate studies for their use in practice, specifically in the development of newdrug delivery systems, a linguistic scale was used. The linguistic scale consists of five levelsfrom Level 0 to Level 4 based on the probability and the percentage of the use of the majorfindings in developing the systems, which could benefit mankind and other life on earth andthe environment. The reasons behind using these two criterions are:

1. The percentage determines to what extent a particular delivery system can be devel-oped for the betterment of mankind and the environment.

2. The probability gives an explanation as to the feasibility and efficiency of a drugdelivery system, for example, a particular delivery system may already exist butusing a different material can make the system more efficient.

The distinction between the various levels is as follows:

Level 0—Less than 10% chance that the study findings are directly useful in devel-oping the system or less than 10% of the study findings could be used.Level 1—Less than 25% and greater than 10% chance that the study’s findings areuseful or around 25% of the findings could be used.Level 2—Less than 50% and greater than 25% chance that the study’s findings areuseful or around 50% of the findings could be used.Level 3—More than 50% and less than 100% chance the study’s findings are usefulor more than 50% of the findings could be used.Level 4—100% chance that the study’s findings are useful or the study is aimed atdeveloping a new system successfully.

The above criterion is demonstrated in the following examples:

Level 0—One of the studies evaluated for this research (Lundborg et al., 2001)had reported that ultrafine particles impair the phagocytic activity of the alveolarmacrophages. This is a very crucial observation and is very useful when defining theemission norms or specifying the control measures. But this observation has hardlyany input when developing a new engineering system directly. Meanwhile, theseobservations might lead to the concept of developing a new emission control system,such as filters and condensers that contain UHF, but would need further research inorder to identify the mechanism of such a system. Such studies are categorized atLevel 0.Level 1—Jani et al. (1990) observed uptake of polystyrene particles in the GI tract,among others. Other studies have observed the translocation of the particles at thenanometer size. Though major observations made by Jani et al. have health effects, theabove-mentioned observation can be used in developing a noninvasive drug deliverysystem because the uptake of the particles is at a lower percentage in comparison withthe total dosage. Though this observation can be used, there could be other materialsthat have a better absorption level leading to an effective system. Therefore this andother such studies are categorized at Level 1.Level 2—Fernandez-Urrusuno et al. (1997), in their experiment with polystyreneparticles, observed a higher concentration in the liver. This observation could be



used to develop an organ-specific drug delivery system using the same particles ascarriers. Another use of such particles would be as carriers in detection when usedin conjunction with proper fluoresce or other detection dyes. The rapid clearance ofthe materials from the organs would reduce the risk of any damage whatsoever. Suchstudies have been categorized as Level 2.Level 3—Lademann et al. (1999) observed in their study that the TiO2 particlescan penetrate the horny layer of the skin and reach the inner tissues beside the hairfollicles and localize in follicular channels. These observations could be used in thedevelopment of a new skin-based delivery system. Though all of these observationscould be used in the development of a new system, many skin surface treatmentsystems have been proven to be effective with no or negligible side effects. In suchcases, the new system developed for the treatment would be an improvement overthe older system rather than an entirely new system. The property of these particlesto reach the inner tissues, and thus circulation, could be used to develop a system thatis not already in use. Hence, such studies are categorized as Level 3.Level 4—Research conducted by Cheng et al. (2006) on using magnetic nanoparticles(iron chloride) to deliver human insulin reported very promising results. Moreoverthey were successful in releasing the insulin in the desired area using external modu-lation. This study was aimed specifically at developing a drug delivery transport thatis efficient with minimal side effects. These findings can be directly used in massproducing the system using the same nanomaterials. Such studies are categorized asLevel 4.

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health effects of nanomaterials: a critical appraisal approach and research to practice

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