GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

A

CXXXX American Chemical Society

Nanomaterial Categorization forAssessing Risk Potential To FacilitateRegulatory Decision-MakingHilary Godwin,*,†,‡,§, ) Catherine Nameth,§ David Avery,§ Lynn L. Bergeson,^ Daniel Bernard,#

Elizabeth Beryt,§,z William Boyes,0 Scott Brown,9 Amy J. Clippinger,O Yoram Cohen,§, ),b Maria Doa,4

Christine Olgilvie Hendren,2 Patricia Holden,§,3 Keith Houck,0 Agnes B. Kane,1 Frederick Klaessig,§,"

Toivo Kodas,` Robert Landsiedel,†† Iseult Lynch,‡‡ Timothy Malloy,‡,§,§§ Mary Beth Miller,^^ Julie Muller,##

Gunter Oberdorster,zz Elijah J. Petersen,00 Richard C. Pleus,99 Philip Sayre,4,`` Vicki Stone,OO Kristie

M. Sullivan,bb Jutta Tentschert,44 Philip Wallis,22 and Andre E. Nel*,§, ),33,11

†Department of Environmental Health Sciences, Fielding School of Public Health, ‡Institute of the Environment and Sustainability, §University of California Center forEnvironmental Implications of Nanotechnology, and )California NanoSystems Institute, University of California, Los Angeles, California 90095, United States,^Bergeson & Campbell, P.C., Washington, D.C. 20037, United States, #CEA Nanosafety Platform, Grenoble, France, zLuskin School of Public Affairs, University ofCalifornia, Los Angeles, California 90095, United States, 0Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NorthCarolina 27711, United States, 9DuPont Central Research & Development, DuPont Titanium Technologies, E.I. du Pont de Nemours and Company, Wilmington,Delaware 19803, United States, OPETA International Science Consortium Ltd., London, United Kingdom, bDepartment of Chemical and Biomolecular Engineering,University of California, Los Angeles, California 90095, United States, 4Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency, Washington,D.C. 20460, United States, 2Center for the Environmental Implications of Nanotechnology, Duke University, Durham, North Carolina 27708, United States, 3BrenSchool of Environmental Science and Management, University of California, Santa Barbara, California 93106, United States, 1Brown University, Providence, RhodeIsland 02912, United States, "Pennsylvania Bio Nano Systems, Doylestown, Pennsylvania 18901, United States, `Cabot Corporation, Boston, Massachusetts 02210,United States, ††Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany, ‡‡School of Geography, Earth & Environmental Science,University of Birmingham, Edgbaston B15 2TT, United Kingdom, §§University of California School of Law, Los Angeles, California 90095, United States, ^^AppliedNanoStructured Solutions, L.L.C., Lockheed Martin Company, Baltimore, Maryland 21220, United States, ##Nanocyl, Sambreville, Belgium, zzUniversity of Rochester,Rochester, New York 14627, United States, 00Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899,United States, 99Intertox, Inc., Seattle, Washington 98101, United States, OOSchool of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS,United Kingdom, bbPhysicians Committee for Responsible Medicine, Washington, D.C. 20016, United States, 44Federal Institute for Risk Assessment,Berlin, Germany, 22SouthWest NanoTechnologies, Norman, Oklahoma 73071, United States, and 33Center for Nanobiology and Predictive Toxicology and11Department of Medicine, Division of NanoMedicine, University of California, Los Angeles, California 90095, United States

Due to the tremendous potentialfor nanotechnology to revolutionizefields as diverse as electronics and

medicine, the global community has in-vested considerable time and resourcesin the research and development of engi-neered nanomaterials (ENMs) and nano-enabled products. In the past decade, in-creasing emphasis has been placed on de-veloping the science and tools neededto ensure that ENMs and nanoenabled

products are produced and used as safelyas possible. In many countries, the timewhen new materials and/or products arefirst brought to market is a critical check-point for regulatory agencies to evaluatethe potential risks of those materials andproducts and to put into place controls toensure that the health and well-being ofworkers, the public, and the environmentare well-protected. Although regulatorystrategies for new chemicals and materials

* Address correspondence tohgodwin@ucla.edu,anel@mednet.ucla.edu.

Received for review February 9, 2015

Published online10.1021/acsnano.5b00941

ABSTRACT

For nanotechnology to meet its potential as a game-changing and sustainable technology, it is important to ensure that the engineered nanomaterials and

nanoenabled products that gain entry to the marketplace are safe and effective. Tools and methods are needed for regulatory purposes to allow rapid

material categorization according to human health and environmental risk potential, so that materials of high concern can be targeted for additional

scrutiny, while material categories that pose the least risk can receive expedited review. Using carbon nanotubes as an example, we discuss how data from

alternative testing strategies can be used to facilitate engineered nanomaterial categorization according to risk potential and how such an approach could

facilitate regulatory decision-making in the future.

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

B

differ from country to country, almostall regulatory bodies grapple with thechallenges associated with assessingthe risks of ENMs in a timely fashion fordecision-making. Categorization stra-tegies are needed to enable regulatorsand industry either to predict ENMrisk better or to allow prioritizationof the testing (hazard, exposure,physicochemical) needed to estimatetheir potential risk while minimizingtime-consuming and costly in vivo

studiesor traditional risk assessments.1

Developing scientifically basedcategorization strategies for regula-tory purposes requires considera-tion of the needs, capacities, andinput of regulators, affected busi-nesses, and other stakeholders. To-ward this end, the University ofCalifornia Center for EnvironmentalImplications of Nanotechnology(UC-CEIN) and the UCLA Center forNanobiology and Predictive Toxicol-ogy convened aworkshop for repre-sentatives from industry, govern-ment agencies, non-governmentalorganizations (NGOs), and academiaat the Woodrow Wilson Center inWashington, DC, to discuss how cat-egorization of ENMs coupled withalternative testing strategies (ATS)might be used to expedite hazardcharacterization and risk analysis,allowing for integrated environ-mental and occupational health andsafety (EHS) decision-making forENMs. Below, we present a synthesis(but not necessarily a consensus)of the perspectives of this diversegroup of stakeholder representa-tives on this topic. To simplify thediscussion, we have chosen to focuson carbon nanotubes (CNTs) as anillustrative example. We focus onCNTs both because of their impor-tance in the world market andbecause grouping of CNTs currentlyposes a significant challenge to reg-ulators. We discuss how CNTs arecurrently handled under the U.S.Environmental Protection Agency's(EPA) New Chemicals program andthe challenges that have arisenwhen addressing these materials.Next, we discuss how new andemerging tools and approaches for

testing the safety of chemicals andmaterials (referred to herein as“alternative testing strategies” orATS; see Figure 1) can facilitategrouping, ranking, and read-acrossfor ENMs. In that section, we willintroduce the concepts of adverseoutcome pathways (AOPs) andstructure�activity relationships thatare based on ENM composition andphysicochemical properties. Finally,we provide an example of how datafromATS could be incorporated intoa revised decision analysis frameworkfor ENMs. Although we have chosento focus on CNTs and the EPA's NewChemicals program for the examplediscussed here, we feel that many ofthe principles and insights identifiedin this example are broadly applicableto other classes of ENMs and otherregulatory paradigms. Thus, discus-sion of the CNT example will help tomove the entire field forward.

Handling of CNTs under the U.S. EPA'sNew Chemicals program: Challenges andOpportunities. In the United States,when companies are planning tomanufacture a new chemical (e.g.,a new nanomaterial), a premanu-facturing notice (PMN) must be sub-mitted to the U.S. EPA. After review-ing the PMN, the EPA can take avariety of actions, which often in-volves placing the chemical on theToxic Substances Control Act (TSCA)Chemical Inventory without restric-tion or placing the chemical on theInventory with restrictions (mostcommonly through a section 5(e)Consent Order and/or a SignificantNew Use Regulation or SNUR). Sig-nificant New Use Regulations areissued because submissions underthe TSCA New Chemicals programare specific to the manufacturing,processing, and use that the submit-ter and downstream clients are un-dertaking; SNURs do not take intoaccount different manufacturing,processing, and use scenarios.

The EPA's treatment of CNTs un-der its New Chemicals program isillustrative of the actions that aretaken under this program (Figure 2).Under this program, the EPA uses56 chemical categories to streamline

the review of new chemicals. TheEPA has consistently consideredCNT risks in the context of its “Re-spirable, Poorly Soluble Particulates”category. If a CNT is determined tofall into this category and sufficientrisk exists as defined under section 5of TSCA, a 90 day inhalation study istypically requested (once sufficientprofits have accrued from commer-cialization of the subject CNT). TheEPA has also promulgated SNURs forboth generic single-walled CNTsand multiwalled carbon nanotubes(MWCNTs).2�10 These SNURs requirethat, before certain new uses ofCNTs (which are different from theuses provided in the initial submis-sions and with exposure potential)can begin, the company wishing touse the CNT in a new manner mustprovide notice to the EPA 90 daysbefore commencing that new use.This regulation provides the EPA anopportunity to review the use anddetermine if it may present an un-reasonable risk and take appropriateaction. The review is similar to thatconducted for the initial submissionand builds upon the EPA's review ofthe initial submission (see Figure 2).For instance, if a 90 day inhalationstudy has not been submitted by theinitial submitter, it may be requiredof the submitter of the notice of thenew use. The primary challengeposed by the current approach isthat it does not explicitly provide amethodology by which manufac-turers or regulators can determinewhether a “new” CNT is sufficientlysimilar to prior submissions suchthat the risk potential of the “new”CNT can be predicted based on datafor existing CNTs. (This process isknownas “readacross”; see Figure1.)Integration of methods for predict-ing the risk potential for CNTs basedon data other than a 90 day inhala-tion study into the decision-tree ap-proach would greatly facilitateimproved decision making by bothmanufacturers and regulators.

Furthermore, the developmentof categories for nanomaterialswould be helpful in the regulationof chemicals in countries other than

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

C

Figure 1. Key definitions.

Figure 2. Decision-tree approach currently used by the EPA to characterize risk potential of carbon nanotubes (CNTs). Thedecision tree above provides an example of a regulatory program where CNTs are currently characterized by their risk andexposure potential under the Toxic Substances Control Act section 5 program. Currently, inhalation testing is requestedmostfrequently as a result of concerns for worker exposure. Testing required as a result of risks to the general population orconsumers, rarely if ever, has occurred to date for CNTs.

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

D

the United States. In Canada, forinstance, under the New Substancesprogram, information on particlesize and distribution will be re-quested for those nanomaterialsand classes of nanomaterials thatare likely to behave differently thantheir non-nanoscale forms. Carbonnanotubes are one such class ofnanomaterials.11 Due to recent de-velopments by the U.S.�CanadaRegulatory Cooperation Council,the two countries will work to aligntheir nanomaterials regulatory workbetter so as to have consistent ap-proaches to risk assessment and theidentification of categories of nano-materials. In the European Union,under the Registration, Evaluation,Authorisation and Restriction ofChemicals (REACH) program, regis-trants can use read-across methodsand categorization methods to filldata gaps for chemical safety assess-ment and for alternatives analysis.3,12

Typically, these categories are basedon structural similarities and physico-chemical properties, as well as simi-larities in effects on human healthand the environment. At present, theEuropean Chemicals Agency (ECHA)has not issued formal guidanceon categorization specific to nano-materials, although ECHA and otherregulators have developed reportsand other materials addressing cate-gorization of nanomaterials.13

What Is Categorization and Why Is ItCritical? Preliminary grouping ofENMs could be helpful during theearly stages of qualitative risk anal-ysis either by manufacturers or reg-ulators to identify “nanomaterials ofconcern”, which could then be tar-geted for more detailed testing,analysis, and verification. Several ap-proaches involving different levelsof stringency and completenessof data collection have been pro-posed1,14,15 to group and to rankENMs. These are briefly summarizedin Table 1. Categorization methodsshould be premised on the bestavailable evidence and take intoconsideration uncertainties con-cerning the hazard potential ofspecific ENMs at sites of potential

occupational or environmental ex-posures. The nature of the attributesused for categorization varies de-pending upon the type and purposeof the grouping as well as otherfactors. The relevant attributes maybe human health outcomes (e.g.,carcinogenicity), environmental endpoints (e.g., aquatic toxicity), physi-cochemical features of the sub-stance, and/or the production vol-ume of the material. Prudent riskmanagement measures would in-clude greater precaution in thehandling and use of ENMs that lacksufficient data to estimate thehealth risks adequately.

The “holy grail” of this field is tobe able to categorize the risk poten-tial of ENMs based on their physico-chemical properties because suchan approach would allow manufac-turers and regulators to make rapiddecisions without requiring costlyand time-consuming in vivo and/orin vitro data. A decision-tree ap-proach for assessing nanomaterialhealth hazards has already beendeveloped jointly by entities suchas the U.S. EPA's New Chemicalsprogram, Health Canada, and Envi-ronment Canada under the Regula-tory Cooperation Council.13 How-ever, practical implementation ofsuch a decision tree for CNTs is notyet feasible. Carbon nanotubes havecomplex physical and chemicalstructures for which the contribu-tion of individual properties to thecausation of injury in relation to thecomplex, integrated structure hasnot yet been developed to the ex-tent that risk can be predicted basedon a selection of specific physico-chemical properties alone. In vitro

and in vivo toxicological testing hasindicated that CNT wall number,synthesis method (which deter-mines metal impurities and metallicor semiconductor status), length/aspect ratio, state of agglomeration,surface functionalization, hydropho-bicity, suspension stability in aqu-eous environments, embedded-ness in a matrix, and surface coatingcould all play roles in CNT toxicity. Itis often impossible when looking at

the safety data sheet for CNTs toidentify a single dominant featurethat can be used to describe thepotential hazard according to whichthe material can be classified. More-over, how exactly these propertiesdetermine fate and transport, bioa-vailability, persistence, and trigger-ing of biological injury responsesis not well understood. As a result,some direct measures of biologicalactivity and exposure potential arecurrently needed to categorize CNTsaccording to their risk potential. Apossible exception is a new materialthat is very similar to some priorexamples for which extensivehazard and exposure data are avail-able. In most other cases, however,information about hazard and/orexposure potential is required at thisstage for meaningful risk evaluation.

For decision makers to be able toidentify whether a new material issimilar to prior examples and tomake informed decisions about therisk potential of ENMs, the broadercommunity needs access to ashared data repository that inte-grates ENM physicochemical, haz-ard, and exposure data that havebeen curated for reliability and ac-curacy. In addition, we need more

Preliminary grouping of

engineerednanomaterials

could be helpful during

the early stages of

qualitative risk analysis

either by manufacturers

or regulators to identify

“nanomaterials of

concern”, which could

then be targeted for

more detailed testing,

analysis, and

verification.

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

E

sophisticated tools for interrogatingthese data. Although resources suchas NanoHUB and the NanomaterialRegistry provide a good start, com-prehensive tool and data sets forproviding these linkages and pre-dicting behavior do not yet exist.We advise that, until such toolsare developed, categorization ap-proaches that consider the bestavailable evidence are the only al-ternative for nano-EHS and regula-tory decision-making by govern-ment agencies.

How Alternative Testing StrategiesCould Facilitate Categorization of CNTs.While it is not currently possible todevelop risk categories based onENM physicochemical propertiesalone, the data obtained fromATS;including both in vitro assays as wellas linked, predictive short-term ani-mal studies;could provide data onwhich categorization can be based.Alternative testing strategy ap-proaches include the use of mecha-nistic and predictive toxicological

assays to assign the materials tocategories that are expected to havesimilar toxicological profiles amongmembers in the group. Illustrativeexamples include the use of high-content screening (HCS) (Figure 1)16,17

to develop hazard rankings forlibraries of ENMs that have beenassembled to represent systematicvariation of physicochemical com-position as well as accentuationof specific properties (e.g., size, crys-tallinity, solubility, aspect ratio,electronic properties, surface func-tionalization, surface coating) with-in compositions such as CNTs,graphene, metals, metal oxides (in-cluding silica), and rare earth oxides.Where possible, this screening ispremised on adverse outcome path-ways (AOPs; e.g., lysosome damagethat triggers chronic inflamma-tion and fibrosis; oxidative stressresponses that trigger acute pul-monary inflammation) that also de-termine the pathophysiology ofdisease and are of potential use for

hazard ranking based on modeof action (MOA) and, in someinstances, quantitative structure�activity relationships (QSARs). Differ-ent approaches for nanomaterialgrouping that are based on ATShazard assessment, exposure po-tential, risk assessments, and riskmanagement have been pub-lished or are currently being devel-oped.1,13�15

The assessment of mechanis-tic biological responses by high-content and rapid-throughput as-says can speed up data generationto establish category-specific hazardprofiling that can be used for initialregulatory decision analysis, includ-ing whether more costly and labor-intensive animal studies are recom-mended. Application of ATS data toENM risk categorization also confersthe advantage that these assays canbe used to test pristine particles aswell as nanoenabled products andenvironmentally aged particles. Inaddition, ATS approaches can be

TABLE 1. Summary of Approaches To Grouping and Ranking Engineered Nanomaterials

categorization according to physicochemical characteristics

•Similarities in chemical composition (e.g., carbon nanotubes, inorganic carbon, metal oxides, metalloid oxides, metals and metalloids, semiconductor quantum dots, organics,etc.)•Aggregation or agglomeration state (e.g., readily dispersed particles that could be easily respired into the lung vs heavily agglomerated particles that are not respirable)•The media or matrix (e.g., pure nanomaterials, liquid suspensions, and solid matrices; particles in diffuse coatings, durable coatings and composites, nanostructured products)•Classification according to physical state to improve safe handling and reduced occupational exposure (e.g., bound or fixed nanostructures, liquid suspensions or liquiddispersions, dry dispersible nanomaterials, and nanoaerosols and gas phase synthesis)•Characteristics affecting particle toxicology (e.g., size, shape, aspect ratio, surface area, surface reactivity or functionalization, surface coating, chemical composition and crystalstructure, agglomeration and aggregation)•Categorization according to modes of action that predict biological risk (e.g., higher solubility particles, poorly soluble/low toxicity particles, poorly soluble/high toxicityparticles, fibrous particles)•Categorization according to the effects of the ENMs based on overall composition (e.g., particles themselves, coatings, chemical effects such as ions or released molecules,molecules formed by the catalytic surface, molecules bound to the ENM)

categorization according to exposure and use scenarios

•Classification to predict likelihood of consumer exposure based on nanomaterial location in the product (e.g., part of a bulk substance, such as nanoelectronics; surface location,such as on films; particles in liquid suspensions) or use of the product•Likelihood of inhalation based on dustiness or dispersibility•Exposure potential based on volume of production and life cycle analysis•Exposure based on environmental release from a manufacturing/processing site or a product matrix•Exposure of workers during production

Categorization linking selected physicochemical properties to specific biological outcomes

•Categorization based on the potential to induce acute oxidative stress and inflammation (including consideration of the dissolution, electronic properties, and surface catalyticactivity of metal, metal oxide, semiconductor nanoparticles)•Categorization based on material aspect ratio and the capacity to induce lysosomal injury, inflammasome activation and initiation of chronic inflammatory responses andfibrosis (e.g., carbon nanotubes (CNTs); metal and metal oxide nanowires, nanobelts, and nanorods; rare earth oxides)•Categorization based on surface functionalization of material groups that catalyze oxidative stress or lysosomal injury (e.g., anionic, cationic, or neutral surface functionalizationon CNTs; silica ring structure and surface silanol display)

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

F

used to facilitate the use of groupingaccording to exposure potential andproduct-use scenarios. Alternativetesting strategy approaches that in-corporate HTS/HCS assays are alsopotentially useful for rapid compara-tive analysis of material collectionsor ENM libraries to provide hazardranking according to a tiered ap-proach that may be used for limitinganimal testing. Rapid-throughputscreening can also be used to estab-lish QSARs and to determine thehierarchical ranking of new materi-als that are assayed by HTS or HCStogether with libraries of well-char-acterized materials. Thus, ATS couldbe used to establish low-risk cate-gories that could receive rapid deci-sions, while materials of high-riskconcern could receive more exten-sive evaluation. However, for suchHTS/HCS assays to be broadly ac-cepted in a regulatory context, theyshould also be evaluated for theirrelevance and reliability.18

To illustrate how ATS could facil-itate grouping, ranking, and read-across of CNTs, a tiered approachto animal testing has beenproposedto assess the risk of lung-based oc-cupational exposures.19 Under thisparadigm, predictive toxicologicalparadigms based on AOPs couldbe used to prioritizematerials withina category for further testing. The

results could then contribute to de-termining when a 90 day rodentinhalation study (currently consid-ered by regulatory bodies as themost reliable indicator of adversepulmonary effects in mammals andas a surrogate for worker adversepulmonary effects) would be recom-mended. This approach enablesstepwise investigation of large num-bers and categories of materials,which can be compared, grouped,and prioritized for cellular, short-term in vivo, and, ultimately, long-term inhalation exposures ifrequired tomeet regulatory require-ments. Key to the interpretation ofthese studies is the appropriate useof a wide dose range in vitro, toensure that extrapolation (e.g., doseper unit surface area in the culturedish vs the lung) to the in vivo situa-tion represents a dose�responserelationship on the steep part ofthe dose�response curve, whichcan also be compared with occupa-tional exposure measurements(available for CNTs). Used appropri-ately and in an iterative fashion togain confidence during the intro-duction of new materials, the visionis to use the ATS-based tiered sys-tem to reduce animal testing. More-over, the rapid knowledge gath-ering through ATS use can expeditenew material testing as well asprovide rankings for early decisionanalysis.

We have specifically chosen tofocus on CNT risk categorizationby using ATS that reflects injury topulmonary cell types that can alsooccur in the intact lung during in-halation exposure in rodents and,by extrapolation, is possible for oc-cupational exposures in humans.Based on predictive toxicologicalstudies for lung injury, experimentaldata suggest that CNT propertiessuch as hydrophobicity, state of dis-persion, surface coating, length/as-pect ratio, and surface charge playkey roles in cellular uptake and bio-availability by lung cells. Other prop-erties such as metal impurities,surface catalytic groups that cangenerate oxygen radicals, aspect

ratio, stability of the surface coating,and state of suspension in a low pHenvironment (e.g., lysosomes) areimportant for cellular injury (e.g., tothe lysosome).19�23 These findingsstand in contrast with the popularimpression that the fiber-like dimen-sions of rigid MWCNTs are the defin-ing property ofmesothelial and lunginjury based on a frustrated phago-cytosis paradigm. Although AOPshave not yet been developed forall pathways leading to pulmonarytoxicity by CNTs (work is ongoing inthis area), at least some AOPs areamenable to ATS. This strategy in-cludes use of HTS/HCS approachesthat assess profibrogenic effects at acellular level as a result of lysosomeinjury, which predicts CNT fibrosis inthe lung.20�23 Based on these stud-ies, it follows that ATS and groupingcould be used to develop a tieredapproach to assess CNT risk potentialin the lung. Below, we discuss howsuch an approach potentially couldbe applied in a regulatory setting.

A Proposed New Approach to Categor-ization of Engineered Nanomaterials forRegulatory Purposes. Although no sin-gle categorization strategy is likelyto work for all classes of ENMs in allregulatory situations, it may be pos-sible to develop a general frame-work that can be adapted andcustomized for specific ENM com-positions and specific regulatorycontexts. For illustrative purposes,we will discuss a decision-tree ap-proach for CNTs based on a predic-tive toxicological approach usingATS to prioritize material categoriesfor further testing. We will also dis-cuss potential barriers to incorporat-ing ATS approaches into regulatorydecision-making and how indus-try�academia�government�NGOcooperation could work to reducethese barriers.

A decision tree for assessing na-nomaterial health hazards has al-ready been developed jointly bythe U.S. EPA's New Chemicals pro-gram, Health Canada, and Environ-ment Canada under the Regula-tory Cooperation Council (RCC).13

However, the existing RCC decision

Alternative testing

strategy approaches

include the use of

mechanistic and

predictive toxicological

assays to assign the

materials to categories

that are expected to

have similar

toxicological profiles

among members in the

group.

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

G

tree relies on physicochemical prop-erties alone tomake decisions about

risk potential, which is not feasibleas yet given gaps in our knowledgeabout the exact relationship of theseproperties to hazard and risk out-come. As a result, when regulators(e.g., in the EPA New Chemicalsprogram) determine that there ispotential for inhalation exposurefor a new CNT, they typically recom-mend that the manufacturer per-form a 90 day inhalation study (seeFigure 2). By contrast, the new deci-sion tree proposed below explicitlyarticulates how ATS data potentiallycould be incorporated into decisionpoints andminimize time-consumingand costly animal studies.

An example of such a decisiontree for CNTs is depicted in Figure 3.This tree is structured around atiered approach once a potentialfor inhalation exposure is identified.In the first tier, one or more in vitro

assays that reliably recapitulate thepotential to cause pulmonary in-flammation could be used as an

initial hazard screen (Tier 1 Test-ing).20�23 If (and only if) the resultsof the Tier 1 Testing suggest po-tential risk, then Tier 2 Testing isundertaken based on the rankingprovided in Tier 1. Tier 2 Testing in-volves short-term bolus administra-tion to the lung for determininginjury potential (e.g., lung fibrosis24).This testing also serves to confirmthe predictive hazard potentialbased on the Tier 1 Test. If (andonly if) the results of the Tier 2Testing confirm and suggest in vivo

risk potential, then performing Tier 3Testing should be given considera-tion. Tier 3 Testing involves aerosoladministration in rodents (e.g., a 90day study). This approach is prefer-able to asking for a 90 day inhalationstudy for every new material expo-sure and inhalation potential be-cause 90 day inhalation studies aretime-consuming and expensive: theEPA estimates that the cost is morethan $500,000 to perform such a

Figure 3. Proposal for how alternative testing strategy (ATS) data could be incorporated into a decision-tree approach forcategorizing carbon nanotubes (CNTs) according to a tiered testing approach to rule out the need for 90 day inhalation studieswhen possible. We propose the use of ATS as a Tier 1 approach to determine whether short-term animal studies (Tier 2) areneeded. A 90 day inhalation study would only be required if Tier 2 testing comes back positive or if validation studies suggestTier 1 and/or Tests are not predictive for this class ofmaterials. Prior to implementation, the tests used in Tier 1 and Tier 2mustbe determined to be “fit for purpose” (i.e., to yield results that are predictive of results obtained by Tier 3 tests and henceprovide accurate predictions of risk potential for the class of materials being investigated).

Although no single

categorization strategy

is likely to work for all

classes of engineered

nanomaterials (ENMs)

in all regulatory

situations, it may be

possible to develop a

general framework that

can be adapted and

customized for specific

ENM compositions and

specific regulatory

contexts.

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

H

study on a single respirable, poorlysoluble particulate material.25

The coordinated development ofa decision-tree approach based onrelevant and reliable ATS datawouldcontribute to better-informed regu-latory decisions about the potentialrisks of ENMswith data that could becollected in days to weeks and atrelatively low cost. A decision-treeapproach would also enable indus-try to anticipate the types of infor-mation that should be provided tothe EPA for an expeditious EPAreview. Similar approaches for as-sessing risk of ENMs postmanufac-turing could be considered withinthe European regulatory system,REACH.26

Critically, for the type of decision-tree approach described above tobe effective, both manufacturersand regulators will need to be inagreement that the assays used forTier 1 and Tier 2 Testing accuratelypredict the risk potential for thespecific class of materials beingqueried. Fortunately, significant pro-gress has been made in the pastdecade in developing both in vitro

assays and short-term in vivo assaysto provide predictions that correlatewell with data from conventionalstudies.20�24 Nonetheless, employ-ing ATS data in decision-making forregulatory purposes would begreatly facilitated by the establish-ment of government�academia�industry�NGO consortiums thatcould systematically investigatehow results from different ATS ap-proaches for a collection of well-characterized ENMs compare tothose in historical in vivo outcomestudies (e.g., from 90 day inhalationstudies). Carbon nanotubes provideone possible example. The cross-comparison of ATS with standardassays would be an important stepin a validation process for ap-proaches such as those proposedhere. Because the term “validation”has complex and variable meaningsdepending on the situation, for thepurposes of this Nano Focus, weassume that the tools and methodsemployed would be sufficiently

validated to support the intendedrisk-assessment purpose. This con-cept is referred to as “fit for purpose”and recognizes that certain applica-tions require different levels of strin-gency for validation than others.27

Cross-comparison of results fromtests in different tiers would alsobe greatly facilitated by the creationof a shared data repository similar tothe one proposed earlier, whichintegrates ENM physicochemical,hazard, and exposure data that havebeen curated for reliability and ac-curacy together with effective toolsfor querying the data therein.

CONCLUSIONS AND PROSPECTS

Categorization strategies areneeded to allow regulators and in-dustry to predict ENM risk and toprioritize the level of testing (hazard,exposure, physicochemical) neededto estimate potential risk whilemini-mizing time-consuming and costlyin vivo studies that characterize tra-ditional risk assessment. To date,progress toward categorizing ENMsaccording to risk potential has beenstymied by gaps in data and knowl-edge of how nanomaterial proper-ties correlate with risk. Using inputfrom a multistakeholder group ofrepresentatives from industry, gov-ernment agencies, NGOs, and aca-demia, we have synthesized thefollowing insights that we feel pro-vide constructive guidance on howto improve and to expedite categor-ization of ENMs according to riskpotential:

• Physicochemical propertiesare not currently sufficient forENM categorization for regula-tory purposes.

• Categorization methods forregulatory purposes should in-clude indicators of both hazardand exposure potential.

• Alternative testing strategies(ATS) may provide a usefulmeans for expedited hazardscreening for ENMs.

• Decision-tree approaches forcategorizing CNTs accordingto their risk potential post-manufacturing could facilitate

decision-making in the EPA'sNew Chemicals program andin other frameworks.

• Targeted cross-comparison ofATS with standard assays maybe needed for ATS to be in-corporated as an acceptedcomponent of categorizationstrategies in some regulatorycontexts.

Building upon these insights, wehave developed an example of atiered decision-tree approach forcategorizing the risk of ENMs thatincorporates data from ATS assays,which would minimize the need forcostly and time-consuming long-term animal studies and greatly ex-pedite effective decision-making inregulatory contexts. We have illu-strated how this approach wouldwork using available assays that linkto adverse outcome pathways toassess the risk of CNTs in the contextof the US EPA New Chemicals pro-gram. Many of the principles andinsights identified through this ex-ample are broadly applicable toother classes of ENMs and otherregulatory paradigms, includingthose for small molecular weighttoxicants, where the use of ATS datafor material characterization canalso play a prominent role in guid-ing decisions by a broad range ofstakeholders in different regulatorycontexts.

Disclosure: This article has been sub-jected to review by the EPA and NIST andapproved for publication. The viewsexpressed are those of the authors anddo not necessarily reflect EPA/NIST policy,nor does mention of trade names or com-mercial products constitute endorsementor recommendation for use.

Conflict of Interest: The authors declarethe following competing financial interest(s):Several of the authors (S. Brown, T. Ko-das, R. Landsiedel, M. B. Miller, J. Muller,and P. Wallis) are employed by compa-nies which manufacture or utilize nano-objects to create materials or products.The authors alone are responsiblefor the content of the paper. The otherauthors declare no competing financialinterest. ``Formerly with U.S. EPA inWashington, D.C.

Acknowledgment. The UC Center forEnvironmental Implications of Nano-technology is funded by a cooperativeagreement from the National Science

NANO

FOCUS

GODWIN ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX

www.acsnano.org

I

Foundation and the EnvironmentalProtection Agency (NSF-DBI-0830117and NSF-DBI-1266377). The Center forNanoBiology and Predictive Toxicology issupported by the National Instituteof Environmental Health Sciences(U19ES019528).

REFERENCES AND NOTES1. Stone, V.; Pozzi-Mucelli, S.; Tran, L.;

Aschberger, K.; Sabella, S.; Vogel, U.;Poland, C.; Balharry, D.; Fernandes,T.; Gottardo, S.; et al. ITS-NANO-Prioritising Nanosafety Research ToDevelop a Stakeholder Driven Intel-ligent Testing Strategy. Part. FibreToxicol. 2014, 11, 1–11.

2. Multi-walled Carbon Nanotubes andSingle-Walled Carbon Nanotubes;Significant New Use Rules, 75 FR56880; Office of the Federal Register,National Archives and RecordsAdministration, 2010; Vol. 75, pp56880�56889.

3. ECHA Guidance on the Preparationof an Application for Authorisation.http://echa.europa.eu/documents/10162/13637/authorisation_appli-cation_en.pdf (accessed January25, 2015).

4. Multi-walled CarbonNanotubes; Sig-nificant New Use Rule, 76 FR 26186;Office of the Federal Register,National Archives and RecordsAdministration, 2011; Vol. 76, pp26186�26192.

5. Significant NewUse Rules on CertainChemical Substances, 78 FR 27048;Office of the Federal Register, Na-tional Archives and Records Admin-istration, 2013; Vol. 78, pp27048�27057.

6. Significant NewUse Rules on CertainChemical Substances, 78 FR 38210;Office of the Federal Register,National Archives and Records Ad-ministration, 2013; Vol. 78, pp38210�38223.

7. Significant NewUse Rules on CertainChemical Substances, 78 FR 48051;Office of the Federal Register,National Archives and Records Ad-ministration, 2013; Vol. 78, pp48051�48068.

8. Significant NewUse Rules on CertainChemical Substances, 79 FR 51899;Office of the Federal Register,National Archives and Records Ad-ministration, 2014; Vol. 79, pp51899�51913.

9. Significant NewUse Rules on CertainChemical Substances, 79 FR 8273;Office of the Federal Register, Na-tional Archives and Records Admin-istration, 2014; Vol. 79, pp8273�8293.

10. Significant NewUse Rules on CertainChemical Substances, 79 FR 38464;Office of the Federal Register,National Archives and Records Ad-ministration, 2014; Vol. 79, pp38464�38475.

11. Environment Canada, Advisory Note2014-02 Program, N.S., 2014.

12. ECHA Guidance on InformationRequirements and Chemical SafetyAssessment. http://echa.europa.eu/guidance-documents/guidance-on-information-requirements-and-chemical-safety-assessment(accessed January 25, 2015).

13. Arts, J. H. E.; Hadi, M.; Keene, A. M.;Kreiling, R.; Lyon, D.; Maier, M.;Michel, K.; Petry, T.; Sauer, U. G.;Warheit, D.; et al. A Critical Appraisalof Existing Concepts for the Group-ing of Nanomaterials. Regul. Toxicol.Pharmacol. 2014, 70, 492–506.

14. Hansen, S. F.; Larsen, B. H.; Olsen, S. I.;Baun, A. Categorization FrameworkTo Aid Hazard Identification of Nano-materials. Nanotoxicology 2007, 1,243–250.

15. Som, C.; Nowack, B.; Krug, H. F.; Wick,P. Toward the Development of Deci-sion Supporting Tools That Can BeUsed for Safe Production and Use ofNanomaterials. Acc. Chem. Res.2013, 46, 863–872.

16. Xia, T.; Malasarn, D.; Lin, S.; Ji, Z.;Zhang, H.; Miller, R. J.; Keller, A. A.;Nisbet, R. M.; Harthorn, B. H.; Godwin,H. A.; et al. Implementation of aMultidisciplinary Approach To SolveComplex Nano EHS Problems by theUC Center for the EnvironmentalImplications of Nanotechnology.Small 2013, 9, 1428–1443.

17. Nel, A.; Xia, T.; Meng, H.; Wang, X.; Lin,S.; Ji, Z.; Zhang, H. NanomaterialToxicity Testing in the 21st Century:Use of a Predictive ToxicologicalApproach and High-ThroughputScreening. Acc. Chem. Res. 2012, 46,607–621.

18. OECD, Guidance Document on theValidation and International Accep-tance of New or Updated Test Meth-ods for Hazard Assessment. As-sessment, O. S. o. T. a., Ed. ENV/JM/MONO: Paris, France, 2005.

19. Nel, A. E.; Nasser, E.; Godwin, H.;Avery, D.; Bahadori, T.; Bergeson, L.;Beryt, E.; Bonner, J. C.; Boverhof, D.;Carter, J.; et al. A Multi-stakeholderPerspective on theUse of AlternativeTest Strategies for NanomaterialSafety Assessment. ACS Nano 2013,7, 6422–6433.

20. Wang, X.; Xia, T.; Duch, M. C.; Ji, Z.;Zhang, H.; Li, R.; Sun, B.; Lin, S.; Meng,H.; Liao, Y.-P.; et al. Pluronic F108Coating Decreases the Lung FibrosisPotential of Multiwall Carbon Nano-tubes by Reducing Lysosomal Injury.Nano Lett. 2012, 12, 3050–3061.

21. Wang, X.; Xia, T.; Ntim, S. A.; Ji, Z.; Lin,S.; Meng, H.; Chung, C.-H.; George, S.;Zhang, H.; Wang, M.; et al. DispersalState of Multiwalled Carbon Nano-tubes Elicits Profibrogenic CellularResponses That Correlate with Fibro-genesis Biomarkers and Fibrosis inthe Murine Lung. ACS Nano 2011, 5,9772–9787.

22. Li, R.; Wang, X.; Ji, Z.; Sun, B.; Zhang,H.; Chang, H. C.; Lin, S.; Meng, H.; Liao,Y.; Wang, M.; et al. The SurfaceCharge and Cellular Processing of

Covalently Functionalized MultiwallCarbon Nanotubes Determine Pul-monary Toxicity. ACS Nano 2013, 7,2352–68.

23. Wang, X.; Duch, M. C.; Mansukhani,N.; Ji, Z.; Liao, Y.-P.; Wang, M.; Zhang,H.; Sun, B.; Chang, C. H.; Li, R.; et al.Use of a Pro-Fibrogenic Mechanisms-Based Predictive Toxicological Ap-proach for Tiered Testing and De-cision Analysis of CarbonaceousNanomaterials. ACS Nano 2015,10.1021/nn507243w.

24. Shvedova, A. A.; Kisin, E.; Murray,A. R.; Johnson, V. J.; Gorelik, O.;Arepalli, S.; Hubbs, A. F.; Mercer,R. R.; Keohavong, P.; Sussman, N.Inhalation vs. Aspiration of Single-Walled Carbon Nanotubes inC57BL/6 Mice: Inflammation, Fibro-sis, Oxidative Stress, and Mutagen-esis. Am. J. Physiol. Lung Cell Mol.Physiol. 2008, 295, L552–L565.

25. Office of Pollution Prevention andToxics, TSCA New Chemicals Pro-gram. Agency, Environmental Pro-tection Agency, 2010.

26. European Union Environment Direc-torate-General and the EuropeanUnion Enterprise and Industry Direc-torate-General, Fourth Meeting ofthe Competent Authorities forREACH and CLP (CARACAL). Brussels,Belgium, 2010.

27. Judson, R.; Kavlock, R.; Martin, M.;Reif, D.; Houck, K.; Knudsen, T.;Richard, A.; Tice, R. R.; Whelan, M.;Xia, M.; et al. Perspectives on Valida-tion of High-Throughput AssaysSupporting 21st Century ToxicityTesting. ALTEX 2013, 30, 51–66.

NANO

FOCUS