Table Of ContentSymposiaReview
InternationalJournalofToxicology
32(6)405-414
Insights and Perspectives on Emerging ªTheAuthor(s)2013
Reprintsandpermission:
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DOI:10.1177/1091581813508436
Determinations for Food Safety: ijt.sagepub.com
Workshop Proceedings
Heidi Bialk1, Craig Llewellyn2, Alison Kretser3, Richard Canady4,
Richard Lane1, and Jeffrey Barach5
Abstract
This workshop aimed to elucidate the contribution of computational and emerging in vitro methods to theweight of evidence
used by risk assessors in food safety assessments. The following issues were discussed: using in silico and high-throughput
screening (HTS) data to confirm the safety of approved food ingredients, applying in silico and HTS data in the process of
assessingthesafetyofanewfoodingredient,andutilizinginsilicoandHTSdataincommunicatingthesafetyoffoodingredients
whileenhancingthepublic’strustinthefoodsupply.PerspectivesonintegratingcomputationalmodelingandHTSassaysaswellas
recommendations for optimizing predictive methods for risk assessment were also provided. Given the need to act quickly or
proceedcautiouslyasnewdataemerge,thisworkshopalsofocusedoneffectivelyidentifyingapathforwardincommunicatingin
silico andinvitro data.
Keywords
risk assessment,foodsupply, weightof evidence,high-throughput screening, insilico, computational modeling
Purpose of the Workshop exposures to chemicals; however, for food safety assessment,
there may be both a need to act quickly in the face of new
TheInternational Life Sciences Institute (ILSI) North Amer-
data and a need to proceed with caution. How do we begin to
ica Technical Committee on Food and Chemical Safety
sort through and prioritize the steps to integrate the new data
developed a workshop togain insight into how computational
types into food safety assessment?
and emerging in vitro methods could be used to contribute to
To begin to address these issues, the workshop (Table 1)
the weight of evidence that is used by risk assessors in the
broughttogetherover70expertsfromacademia, government,
food sector to determine safety. The practice of food safety
and industry as well as a public interest organization. The
assessment has continually evaluated and adopted new meth-
workshop began by posing the following questions to
ods; however, because of the pace and nature of change in
participants:
biological effects evaluation methods, it is not clear how
some newly developed in vitro data types or computational
1. Howcaninsilicoandhigh-throughputscreening(HTS)
approaches fit into current approaches to food safety assess-
data be used to confirm the safety of approved food
ment. For example, how would a ‘‘cancer gene’’ activation
ingredients?
shown in an in vitro assay at concentrations orders of mag-
nitude higher than could be achieved in foods contribute to
our understanding of the likelihood of harm of a commonly
used food additive with negative carcinogenicity data? How
1PepsiCoInc,Valhalla,NY,USA
should we communicate this information to the public while
2TheCoca-ColaCompany,Atlanta,GA,USA
we are trying to understand its significance? How in general
3InternationalLifeSciencesInstituteNorthAmerica,Washington,DC,USA
can we combine data from these new methods to improve 4InternationalLifeSciencesInstituteResearchFoundation,Washington,DC,
food safety? In particular, it is also not clear where and when USA
thesenewdatatypesmaysignal aneedfor promptevaluation 5BarachEnterprisesLLC,Oakton,VA,USA
using toxicity tests used in current practice to evaluate safety.
CorrespondingAuthor:
These kinds of questions have also been asked for environ-
HeidiBialk,PepsiCoInc,350ColumbusAve,Valhalla,NY10595,USA.
mental risk assessment for environmental and occupational Email:[email protected]
406 International Journal of Toxicology 32(6)
Table1.WeightofEvidenceWorkshopProgram.
WelcomeandPurposeoftheWorkshop—HeidiBialk,PhD,PepsiCoInc,Valhalla,NY
Session1—WeightofEvidenceInputs:InSilicoData
ComputationalorPredictiveToxicologyandMetabolism:ToolsfortheTrade?
EugeneAhlborn,PhD,GivaudanFlavorsCorporation,Cincinnati,OH
ComputationSafetyAnalysisforRegulatoryDecisionMaking
JamesRathman,PhD,OhioStateUniversity,Columbus,OH
Mid-DayPresentation—Values,TrustandScience—BuildingTrustinToday’sFoodSysteminanEraofRadical
Transparency,Mr.CharlieArnot,TheCenterforFoodIntegrity,Gladstone,MO
Session2—WeightofEvidenceInputs:InVitroData
TheNeedforaRoadmapforUsingTox21MethodsinRiskAssessment
ThomasHartung,MD,PhD,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,MD
RiskAssessmentRoadmapsandMethodsforUsing21stCenturyMethods
MichaelL.Dourson,PhD,DABT,ATS,ToxicologyExcellenceforRiskAssessment,Cincinnati,OH
EvaluationofFood-RelevantChemicalsinTox21
AgnesForgacs,ILSINorthAmerica2012SummerFellow/DoctoralCandidate,MichiganStateUniversity,EastLansing,MI
WeightofEvidenceforTargeting‘‘SecondTierTesting’’fromHTSandEDSPApproaches
MichaelDeVito,PhD,NIH/NIEHS,ResearchTrianglePark,NC
KeyEventsDoseResponseFramework(KEDRF)
SamuelCohen,MD,PhD,UniversityofNebraskaMedicalCenter,Omaha,NE
Session3—PerspectivesonApplyingInSilicoandInVitroDataintoaHolisticWeightofEvidenceApproach
FDAPerspectivesontheApplicationof21stCenturyToxicologyMethodsUsedinRiskAssessmentandRegulatoryDecisionMaking
DavidHattan,PhD,FDACFSAN,CollegePark,MD
PerspectivesfromtheHealthEnvironmentalScienceInstitute(HESI)Risk21Program
AlanBoobis,OBE,PhD,CBiol,FSB,FBTS,ImperialCollegeLondon,UK
DefiningtheElementsofaHolisticWeightofEvidenceApproach
JamesBus,PhD,DABT,ATS,TheDowChemicalCompany,Midland,MI
PanelSession—PracticalityofWeightofEvidence:WhereDoWeGoFromHere?
Moderator:RichardCanady,PhD,ILSIResearchFoundation,Washington,DC
Panelists:RoxiBeck,CenterforFoodIntegrity;JamesBus,TheDowChemicalCompany;KeithHouck,EPANCCT;MikeDeVito,
NIHNIEHSNTP;JamesRathman,OhioStateUniversity,Columbus,OH;andKenWallace,UniversityofMinnesotaMedicalSchool,
Minneapolis,MN
Abbreviations:EPA,EnvironmentalProtectionAgency;ILSI,InternationalLifeSciencesInstitute;NCCT,NationalCenterforComputationalToxicology;
NIEHS,NationalInstituteofEnvironmentalHealthSciences;NIH,NationalInstitutesofHealth;NTP,NationalToxicologyPlatform.
2. How can in silico and HTS data be applied in the pro- Introduction to Emerging Methods in
cess ofassessing the safety ofanew foodingredient? Predicting Toxicity
3. HowwouldinsilicoandHTSdatahelpincommunicat-
Speaker
ing the safety of food ingredients while enhancing the
public’strust inthe foodsupply?
The Need for a Roadmap for Using Tox21 Methods in Risk
Assessment.
The final session of the workshop culminated in a panel Thomas Hartung, MD, PhD, Johns Hopkins Bloomberg
sessionthatnotonlyhelpedtoaddressthesequestionsbutalso School of Public Health, Baltimore, MD, USA
focused on identifying a path forward in communicating in
silicoandinvitrodataeffectively.Thisworkshopisoneofthe In2005,theEnvironmentalProtectionAgency(EPA),with
projects by the ILSI North America Committee on Food and support from the National Institute of Environmental Health
Chemical Safety developed in an effort to help fulfill its mis- Sciences (NIEHS)/National Toxicology Platform (NTP),
sion and objectives of promoting a science-based determina- requested that the National Research Council (NRC) develop
tion of the chemical safety of foods in support of the avisionforthefutureoftoxicitytestingandastrategicplanto
advancement ofpublic health. accomplishit.1Theimpetusbehindthisrequestwasthestrong
Bialk et al 407
commitmentbytheEPAandNTPthatfuturetoxicitytesting enhance the confidence in these methodologies to support
meet evolving regulatory needs in that testing paradigms safety assessments.
should (1) readily accommodate the increasingly large num-
bers of substances in commercial use that are considered to
have incomplete toxicity data; (2) incorporate recent The Application of Computational Methods
advances in molecular toxicology, computational sciences, in Hazard Identification
and information technology; and (3) offer increased effi-
ciency in the design, costs, and extent of animal usage.2 Speakers
Two years after the requestfrom the EPA, the NRC released
Computational or Predictive Toxicology and Metabolism: Tools for
a report entitled Toxicity Testing in the 21st Century: A
the Trade?.
Vision and a Strategy.1 This strategy was aspirational, nev-
EugeneAhlborn,PhD,GivaudanFlavorsCorporation,Cincin-
ertheless it described a new approach to toxicity testing that
nati,OH,USA
would increase the use of in vitro high-throughput assays
and reduce the reliance on (and ultimately replace) animal Computation Safety Analysis for RegulatoryDecision Making.
studies using cells, cell lines, or cellular components to James Rathman, PhD, Ohio State University, Columbus, OH,
evaluate the effects that chemicals can have on biological USA
processes.3 The specific near- and long-term goals as
described in the NRC report are as follows: (1) to identify
Current State of the Science of Computational
patterns of compound-induced biological response in order
Methods in Hazard Identification
to characterize disease pathways, facilitate cross-species
extrapolation, and model low-dose extrapolation; (2) to Computationaltoxicologyisamultifacetedsciencethatincor-
prioritize compounds for more extensive toxicological eva- porates knowledge from a variety of disciplines including but
luation; and (3) to develop predictive models for biological not limited to toxicology, chemistry, computer science, and
response in humans.1 Numerous stakeholders are needed to statistics. Thetheorybehindtheuseofstructure–activity rela-
develop a roadmap for how to use Tox21 methods4 to sup- tionships in computational toxicology is that the chemical
port safety assessments. The Doerenkamp-Zbinden Founda- structure ofacompound determines itsphysical andchemical
tion and the Center for Alternatives to Animal Testing properties,whichinturndeterminesitsbiologicalactivity(i.e.,
(CAAT; http://caat.jhsph.edu) have established the Transatlan- toxicology,metabolism,andpharmacology).8Theinitialsteps
ticThinkTankofToxicology(t4;http://altweb.jhsph.edu/about_ in a safety assessment of a given compound may therefore
us/t4.html). The t4 was created in part to further the concept includepredictive computationalmethods(e.g.,insilicotech-
of evidence-based toxicology following the role of evidence- niques)suchasstructuralalerts,quantitativestructure–activity
based medicine (http://www.ebox.com) and to develop and relationships (QSAR), and read-across to chemical analogs.
assess the conceptual needs to enable the change approaches QSAR is currently used by the Flavor Extract Manufacturer’s
in predictive toxicology, integrated testing, and systems tox- Association (FEMA; http://www.femaflavor.org/) and Joint
icology.5 So far, 14 workshops have been held and 13 com- Expert Committee on Food Additives (JECFA; http://
missioned white papers have been published. In addition, the www.who.int/foodsafety/chem/jecfa/en/) to support the safety
HumanToxomeproject,aNationalInstitutesofHealth(NIH) assessments offlavor compounds.
Transformative Research grant of 6 partner organizations The call to refine and reduce animal testing in addition to
lead by CAAT, is working to identify the toxicity signatures the advances in computer technology have enhanced the
of substances with a focus on endocrine disruptors (ED). The opportunities to apply in silico techniques in informing
central goal of the Human Toxome project6 is to define, science-based health assessments. From an industry perspec-
experimentally verify, and systematically annotate Pathways tive, these tools can provide a faster and more accurate eva-
ofToxicity(PoT)pioneeringthis for ED (http://humantoxome. luation of molecules going through the discovery pipeline,
com). The area of ED is well suited for this work, as the provide additional insights into potential toxicological and
physiological pathways of the endocrine system are relatively metabolic activities, and provide a better understanding of
well understood, making PoT identification simpler for ED key end points and what testing may be necessary to ensure
than for other potential target organs. This project will also the safety of evaluated materials. In addition, regulatory
serve to develop a common, community-accessible frame- authorities around the world are either using or investigating
work and databases that will enable the toxicology commu- the potential use of computational toxicology in the safety
nity at large to comprehensively and cooperatively map the evaluation process.
Human Toxome combining ‘‘omics’’ data with computational In order to achieve these objectives, the strengths, weak-
models. On a shorter term, integrated testing strategies will nesses,andlimitationsofthesepredictivemethodsneedtobe
enable the combination of different methods to satisfy safety evaluated. In addition, extensive consideration should be
information needs.7 The following sections of this article giventohowtheoutputprovidedbycomputationalplatforms
describe the current state of the science of emerging, predic- should be properly applied when conducting a safety/risk
tive methods in general, and the next steps that are needed to assessment.Itiscriticalthattheseplatformsneedtoundergo
408 International Journal of Toxicology 32(6)
rigorousvalidationofendpointsinpartnershipwiththeplat- Evaluation of Food-Relevant Chemicals inTox21.
formproviderstocontinuallyupdateandimprovethepredict- Agnes Forgacs, ILSI North America 2012 Summer Fellow/
ability of toxicological and metabolic end points. Doctoral Candidate, Michigan State University, East Lansing,
MI, USA
Enhancing the Confidence in Computational Methods
Improving computational techniques with respect to the pre-
Background on Tox21 and HTS
dictabilityoftoxicologicalendpointsinadditiontometabolic
and pharmacokinetic performance are also key to advancing TheNationalCenterforComputationalToxicology(NCCT)at
thevalidationeffort.Identifyingoff-targetreceptorsandsub- the EPA initiated the ToxCast (http://www.epa.gov/ncct/tox-
structuresforcarcinogenicormutagenicsubstanceswouldbe cast/) program in 2005 with the initial screening of a phase I
helpfulinimprovingthepredictabilityoftoxicityendpoints. libraryof320chemicalsacross550assays.ToxCastisalarge-
Pharmacokinetic performance can also be strengthened by scale experiment using a battery of in vitro HTS assays to
understanding how these models can be developed to better develop activity signatures predicting the potential toxicity of
describedoseresponse,metabolism,andthepotentialforlocal environmental chemicals at a cost that is a fraction of that
versussystemictoxicity. required for full-scale animal testing.12 Phase I of the Tox21
Currently, combining evidence from multiple sources is (http://epa.gov/ncct/Tox21/) quantitative high-throughput
mainly a qualitative exercise in that multiple outputs are used screening (qHTS) initiative began in 2006 with 2800 com-
to determine whether there is a consensus on a given predic- pounds in >100 qHTS at 14 concentrations (5 nmol/L to
tion.Morequantitativeandrigorousstatisticalmethodsdevel- 92 mmol/L) tested at the NIH Chemical Genomics Center
oped for information theory applications may provide a path (NCGC), which has been renamed the National Center for
forwardforquantitativelyweighingandcombiningpredictions Advancing Translational Sciences (NCATS). The first Tox21
frommultiplesourcesinordertogenerateasinglepredictorfor Memorandum of Understanding (MOU) was signed in early
agiventoxicologicalendpoint(J.Rathman,personalcommu- 2008bytheEPA,NIEHS/NTP,andNationalHumanGenome
nication,2013).Asanillustrativeexamplerelevanttothefood ResearchInstitute.Arevised5-yearMOUwasreleasedinJuly
anddrugindustry,theDempster-ShaferTheoryhasbeenused 2010 and signed by the existing partners, and the FDA was
to combine the results from 3 different types of outputs (eg, addedtotheinteragencyproject.Tox21phaseIIwaslaunched
structural alerts, QSAR models, and read-across to defined shortly thereafter using a library of 10 000 chemicals (10K
chemical analogs) using Ames mutagenicity as the end point Library)screened byapproximately 30assays per year. Phase
ofinterest.9-11Estimatingtheuncertaintyforthefinalprediction II of the ToxCast program, which includes 800 compounds
dependsuponthereliabilitiesofthevariousevidencesources. across 550assays, isongoing.
ForQSARmodels,predictionaccuraciesprovideobviousmea- The goals of Tox21 are as follows: to identify patterns of
suresofreliability;however,forothersourcessuchasstructural compound-inducedbiologicalresponseinordertocharacterize
alerts or read-across, reliability may be best determined by toxicity/disease pathways, facilitate cross-species extrapola-
expertsintoxicology,includingpossiblyregulatoryexperts. tion, and model low-dose extrapolation; to explore the use of
Overall, predictive in silico models are based on a wide existing HTSassays for prioritization ofchemical compounds
variety of computational platforms that require ongoing vali- for in vivo toxicological testing; and to develop predictive
dation. The US Food and Drug Administration (FDA) is modelsforbiologicalresponseinhumans.Throughapplication
nearing the completion of the development of a tool referred of these assays to a test set of chemicals with well-
to as Chemical Evaluation and Risk Estimation (http:// characterized toxicity profiles, the aim is to identify ‘‘predic-
www.accessdata.fda.gov/FDATrack/track-proj?program¼cf- tive toxicity signatures or pathways’’ that can then be used to
san&id¼CFSAN-OFAS-Chemical-Evaluation-and-Risk-Esti- screen untested chemicals for potential biological activity of
mation-System). This tool will enable the FDA to fully concern and prioritization for further, in vivo, testing.13 In
leverage available datagenerated frommodern computational phase I of Tox21 and ToxCast, many of the assays used were
approaches with other data for the pre- and postmarket proprietary assays designed to screen for potent compounds
review of food ingredients and packaging materials. that exhibited targeted biological activity, similar to assays
used in the pharmaceutical industry. The pharmaceutical
approach necessitated that only 1 concentration of a commer-
The Application of High-Throughput
cially available compound be tested. By focusing on pharma-
Screening Assays in Hazard Identification ceutically active compounds, highly specific but relatively
insensitive assays were selected. The challenge with these
Speakers
assaysistheirlackofapplicabilitytochemicaltoxicityscreen-
Weight of Evidence for Targeting ‘‘Second Tier Testing’’ from HTS ingin which dose response ofa pure and stabile compound is
and EDSP Approaches. critical. Assay selection for Tox21 phase II is based on the
Michael DeVito, PhD, NIH /NIEHS, Research Triangle Park, following: information from in vivo toxicological investiga-
NC,USA tions; phase I experience, advice of basic researchers, and
Bialk et al 409
nominated assays; and maps of disease-associated cellular address this question, the ILSI North America Technical
pathways. A critical issue for Tox21 is the need to identify CommitteeonFoodandChemicalSafetysupportedasummer
humandisease-associatedpathwaysandusefulassaysforthose fellowship program in which a doctoral student worked with
pathways.AnothercriticalissueforTox21istheneedtoextra- the various regulatory agencies to identify the food-relevant
polate from acute to chronic exposure conditions and from in chemicals in scope under Tox21 and to understand how the
vitro concentrationtoinvivo dose. resulting data could be analyzed and interpreted (A. Forgacs,
personalcommunication,2013).Throughtwomaterialtransfer
agreements(MTA)betweentheEPAandILSINorthAmerica
Current State of the Science on HTS Assays
and the NIEHS and ILSI North America, the summer fellow
in Hazard Identification
was able to undertake the analysis of Tox21 data relevant to
High-throughput screening data currently provide information thefoodsector.UndertheMTAwithILSINorthAmerica,the
on the biological activity of chemicals that may aid in prior- doctoralstudentwastheonlyindividualwhohadaccesstothe
itizationandscreeningofchemicalsubstancesforhazards.The Tox21data.
HTSdatacollectionisinitsinitialstagesasistheidentification Todeterminetheextenttowhichfood-relevantcompounds
andselectionofappropriatescreeningassays.Astechnologies are included in the current phase of the Tox21 program, HTS
advance and the screening assays are developed, they will be chemical inventories were mined for food-relevant chemicals
incorporated into the Tox21 effort (M. DeVito, personal including food additives, Generally Recognized as Safe
communication, 2013). (GRAS) substances, and food contact substances (FCS). The
The qHTS program is currently evaluating approximately FDA publicly available databases were used as sources for
10000chemicalsacrossawidevarietyofbiochemicalassays. the list of indirect and direct FCS, including the Everything
Ifoneweretoassumethat5%ofthosechemicalsdemonstrate Added to Food in the United States (EAFUS) list and the
biologicalactivity,thiswouldleaveapproximately500chem- GRAS notification list. These lists were used as a reference
ical substances requiring further evaluation in order to priori- toidentifythosechemicalsubstancesintheHTSdatabasethat
tizeamoremanageablenumberofchemicals.Thisscenariohas are relevant to food. Over 5000 food-relevant chemicals were
called for the development of streamlined prioritization identified in which nearly 2000 were included in the qHTS
approaches. Streamlined approaches with mitochondrial dis- phase II of the Tox21 10K Library and nearly 1000 in phase
ruption assays have indicated that a single HTS assay alone II ofToxCast.
may not be sufficient for prioritization (M. DeVito, personal IncollaborationwithapostdoctoralfellowatNIEHS/NTP,
communication, 2013). Identification of false-positives and chemical and biological analysis involving the grouping of
specific mechanisms of mitochondrial disruption in addition chemicals based on the physical/structural properties using
totheevaluationoftissuesensitivityusingdifferentcelltypes Leadscope chemoinformatics software was conducted. This
are other factors that will play a key role in prioritization. groupingprovidesawaytoidentifychemicalclassesthathave
Prioritizationshouldbebasedonrisknotjusthazard.Informa- similar biological activity. Once the chemical clusters
tiononpotentialexposurelevelsinparticularwillbecrucialin were established, a modified Fisher’s test was used to deter-
incorporating riskintoprioritization efforts. mine which assays were statistically relevant. This approach
Tobetteridentifythosechemicalsthatareofhigherpriority allows for the identification of assays that exhibit significant
and to effectively develop targeted testing strategies for those activity for food-relevant chemicals while identifying those
chemicals, efforts are underway to evaluate the relationships structural clusters that elicit the greatest/least biochemical
(both qualitatively and quantitatively) between in vitro assays activity across the assays. It also provides valuable insight
and in vivo biological activity and toxicity (M. DeVito, per- as to how such complex data can be managed and analyzed
sonal communication, 2013). Validation work focused on tar- whichmaybeofinteresttoabroadaudiencebeyondthefood
geting testing in the liver, for example, is intended to test for industry.
thepresenceofinvivoactivityasseenininvitroassaysandto In summary, HTS phase II of Tox21 currently includes
confirmthatpreviouslyuntestedcompoundsshowpredictedin many food-relevant chemicals. Some of those food-relevant
vivo activity. Other approaches being researched to provide chemicals have demonstrated biological activity in certain
insight into the uncertainties in extrapolating HTS data to in assays.Futureworkwillseektoprovidecontextforthesefind-
vivobiologicalresponsesincludeinvitrotoinvivoextrapola- ings by further evaluating the food-relevant compounds with
tion via reverse toxicokinetics, short-term in vivo studies, respect to additional validation assays, published safety data,
orthogonal in vitro assays to help identify false-positives, and and biomonitoring datasets. This work will be discussed in
computational toxicology approaches (M. DeVito, personal moredetailinthefollowingsectionandiscriticaltoassessing
communication, 2013). thesignificanceofHTSdatageneratedonfood-relevantcom-
pounds.ThereisanopportunitytounderstandhowHTSassays
respond to food constituents, including beneficial nutrients,
Food-Relevant Compounds in HTS Assays
which by definition are biologically active substances. These
Of interest to the food industry is the extent to which food- compoundsmayplayacriticalroleininterpretingandanchor-
relevant compounds are being assessed by HTS assays. To ing‘‘toxicity signaturesor pathways’’ thatcould begenerated
410 International Journal of Toxicology 32(6)
byavarietyofchemicalsubstancesbyHTSassays(J.Busand doseandtoxicitywasexploredwith2,4-dichlorophenoxyacetic
A.Forgacs, personalcommunication, 2013). acid(2,4-D).Thenoobservableadverseeffectlevelfor2,4-D
basedonanimalstudiesis0.7mg/mLinplasma,whilehuman
biomonitoring data indicate that the real-world exposure to
Enhancing the Confidence in HTS Assays
2,4-Dis60ng/mLinplasma.Theconcentrationof2,4-Dtested
for Hazard Identification in the EDSP in vitro screening assay was significantly higher
(0.1mmol/Lequivalenttoanestimated22.1mg/mLinhuman
It is critical that the scientific community consider the exten-
plasma) than the real-world exposure level. Although in vitro
sive reservoir of knowledge gained from animal testing that
culturemediaandassociatedconditionsarenotdirectlyparal-
wouldbehelpfulinframingfuturetoxicologicaltestingstrate-
lel to blood serum or plasma (M. DeVito, personal communi-
gies(J.Bus,personalcommunication,2013).Effectiveimple-
cation, 2013),14 these data show that the top tested
mentation of the Tox21 HTS testing strategies calls for
concentrationof2,4-Dinvitrowasnotintherangeofphysio-
recognition and understanding of dose response, organ-
logicallyrelevantconcentrationsandthuswasoflimitedifany
specific responses, multiorgan interactions, and mechanisms
valuetoinforming the potentialfor humanhealth risks.
of action that drive the phenotypic expression of toxicity (J.
AsignificantlimitationtoHTSscreeningdataisthediscon-
Bus, personal communication, 2013). A significant concern
nect between in vitro test concentrations and exposure. By
with the reliance on in vitro assays is the identification of
applying dosimetric anchoring to HTS screening data, false-
biologicalactivitythatis(1)disconnectedfromnormalhomeo-
positives and false-negatives for chemical hazard assessment
static or compensatory mechanisms present in vivo and (2)
provoked due to high concentrations tested in vitro.13 This canbeavoided.Whilefalse-positivesleadtounnecessarybans
on valued compounds and misdirected resources away from
concern reinforces the need for extensive validation of HTS
issues ofgreater concern,false-negatives delayactiononche-
assays in order to gain confidence that the HTS output is rel-
micals that are of concern. If the goal of HTS is to identify
evanttopredicting hazardsthat mayoccur invivo.
adverse outcomes of real-world concern, then these assays
Based on the knowledge shared during the workshop, the
mustbegrounded bythe exposure-to-doseparadigm.
HTSapproachtoprioritizingchemicalsfortoxicitytestingisin
its initial stages of scientific verification and development.
Only through extensive rounds of HTS data collection, devel-
opment of prioritization approaches and targeted testing will Perspectives on Integrating Computational
the scientific community be in a position to fully evaluate the Modeling and HTS Assays into
utility of this approach (M. DeVito, personal communication,
Risk Assessment
2013).
Speakers
Linking High-Throughput Screening Assays Risk Assessment Roadmaps and Methods for Using 21st Century
Methods.
to Dosimetry
Michael L. Dourson, PhD, DABT, ATS, Toxicology Excel-
Speaker lence for Risk Assessment, Cincinnati,OH, USA
Defining theElements of aHolistic Weightof Evidence Approach.
PerspectivesfromtheHealthEnvironmentalScienceInstitute(HESI)
JamesBus,PhD,DABT,ATS,TheDowChemicalCompany,
Risk 21 Program.
Midland,MI, USA
AlanBoobis,OBE,PhD,CBiol,FSB,FBTS,ImperialCollege
High-throughputscreeningofteninvolvestheevaluationof London,UK
chemicalsatmultipletestconcentrationswithoutclearconsid-
eration on how these in vitro test concentrations compare to KeyEvents Dose ResponseFramework (KEDRF).
dosesusedinanimalstudiesandreal-worldhumanexposures. Samuel Cohen, MD, PhD, University of Nebraska Medical
Advances in analytical and modeling technologies in both Center, Omaha, NE, USA
animal and human exposure evaluations (ie, biomonitoring) Productiveintegrationofincreasinglycomplexdatasetsgen-
provide an opportunity to link in vitro test methods to whole erated by in silico and HTS technologies into chemical risk
animaltoxicitythroughdosimetry(J.Bus,personalcommuni- assessments that improve the confidence of health evaluations
cation, 2013). By translating in vitro test concentrations to willbechallenging(A.Boobis,personalcommunication,2013).
animal toxicology responses through‘‘dosimetric anchoring,’’ Tohelpaddressthischallenge,amultisector,internationalteam
hazards irrelevant to human risk and nonreflective of whole was assembled by the ILSI Health and Environmental
animal metabolismcan be identified. SciencesInstitute(HESI)toformtheHESIRisk21initiative.
The US EPA Endocrine Disruptor Screening Program ThegoalofHESIRisk21istodevelopascientificallybased
(EDSP) uses default top-dose concentrations of 100 mmol/L framework or roadmap that enables the incorporation of
or 1 mmol/L depending on the test system. The relationship advancesin1.)computationalandmoleculartoxicology,2.)con-
betweensuchinvitrodefaultconcentrationstoinvivoanimal ventionaltoxicitytestingand3.)exposureinordertoprovidethe
Bialk et al 411
basisofanintegratedevaluationstrategytochemicalriskassess- This approach has laid the foundation for a scientifically
ment(A.Boobis,personalcommunication,2013). based framework in which inputs from in silico, HTS, and
TheprinciplesofHESIRisk21havebeendesignedtobring conventional toxicity data can form the basis of an integrated
applicable, accurate, and resource-appropriate approaches to evaluation strategy for chemical risk assessment. It is impor-
the rapidly evolving world of human health risk assessment. tant to note that while HTS and in silico data may certainly
Thisisbestarticulatedbyashiftinriskassessmentphilosophy provide insight into MOA, the data need to be anchored to
away from conducting comprehensive toxicity testing to con- key events in animal studies in order to begin to assess
sideringtheproblemthatneedstobeaddressedandthenselect- whether the MOA can be extrapolated to humans (S. Cohen
ing sources of information that will have the most value (A. and M. L. Dourson, personal communication, 2013). The
Boobis and M. L. Dourson, personal communication, 2013). KEDRF framework also encourages transparency, provides
Wherever possible, prior knowledge on the physicochemical guidance for data presentation, identifies critical data defi-
characteristics of the chemical, its exposure profile, and its ciencies and needs, and promotes global harmonization
toxicity as well as that of related compounds is used to deter- (S. Cohen, personal communication, 2013).
mine the data needs for a new chemical that would be most
informative (A. Boobis and M. L. Dourson, personal commu-
nication,2013).Ratherthanassessingthehazarddatafirst,the Recommendations for Optimizing
HESI Risk 21 program emphasizes the use of exposure esti- Predictive Methods for Risk Assessment
matestoprovideinsightintothetoxicitydatathatmayormay and Regulatory Use
notbenecessary.
Speaker
In order to achieve this, HESI Risk 21 utilizes a tiered
approach to obtain ranges for estimates of exposure and of
FDA Perspectives on the Application of 21st Century Toxicology
toxicity in which the accuracy of those estimates increases
MethodsUsedinRiskAssessmentandRegulatoryDecisionMaking.
with higher tiers. Estimates of exposure, for example, begin
David Hattan, PhD, FDACFSAN, College Park,MD, USA
with tier 0 or worst-case approximations and then proceed to
higher tiers with deterministic, probabilistic, and biomonitor- The risk assessment process outlined by the FDA begins
ing data. In terms of toxicity, assessments begin at the lowest with assessing the toxicological hazards of a compound by
tier such as QSAR and continue to progress to more signif- qualitative identification of possible adverse health effects
icant data sets to in vitro, in vivo, and ultimately mode of (ie, hazard identification) followed by a quantitative assess-
action (MOA) data (A. Boobis, personal communication, ment to determine the dose at which toxicity may occur (ie,
2013). The importance of MOA data highlights the critical hazardcharacterization).Toputthesefindingsintocontext,the
role of the underlying biology in the expression of toxicity likely exposure or intake is then estimated. This process then
(M. L. Dourson, personal communication, 2013). The Key culminates inrisk characterization inwhich there is a qualita-
Events Dose Response Framework (KEDRF) is founded on tive and quantitative estimation of the probability of occur-
MOA analysis, which is defined as a sequence of key events rence and severity of known or potential adverse health
leading to the ultimate biological effect in an organism. The effectsinagivenpopulationbasedonthehazardidentification,
KEDRF focuses specifically on the dose response of each hazardcharacterization,andexposureassessment(Hattan,per-
key event as a way to evaluate the ultimate dose response sonalcommunication, 2013).
for the biologic effect being evaluated (S. Cohen and M. L. Inconductingareviewanddeterminationofthesafetyofa
Dourson, personal communication, 2013). foodadditive,theFDAweighsalloftheavailableandrelevant
The KEDRF is founded on first establishing a MOA by scientific evidence. The safety determination is not based
utilizing animal models. The Bradford Hill criteria are then on individual studies or isolated evidence. The mere presence
applied to assess the strength of the MOA data including an of a compound or its metabolite in sample tissues or fluids is
analysisofdose–responserelationships,temporalrelationship, not determinative in and of itself of a health risk. Important
biological plausibility, and the strength, consistency, and spe- criteria for assuring the validity and integrity of data used to
cificity of the association of the toxicological effect with the support thesafetyofachemicalsubstance have beenoutlined
key events identified (S. Cohen, personal communication, in the FDA’s Code of Federal Regulations (21 CFR) 58.185
2013).IftheanimalMOAmeetsthesecriteria,thekeyevents (Hattan,personalcommunication,2013).Ofsignificantimpor-
within the MOA are then evaluated both qualitatively and tanceisaclearanddetaileddescriptionofthemethodologyofa
quantitatively as to their relevance to humans. If any one of giventechnique,thestatisticalanalyses,circumstances,orcon-
the key events leading up to the development of the ultimate founders thatmay have affected thequality orintegrityofthe
biologicresponsewasconsiderednottoberelevanttohumans, data,alltabulatedrawdata,andanevidence-based interpreta-
the MOA was also considered not to be relevant in humans tionofthedatanottoexceedthelimitsofthefindings(Hattan,
(S.Cohen,personalcommunication,2013).Thislevelofanal- personal communication, 2013). One issue faced by many
ysisisconsistentwiththeknowledgethateachbiologiceffect scientists who seek to validate HTS assays is the lack of visi-
willbeevaluatedindividuallyevenifmultipleeffectsweredue bility to HTS protocols used in some of the assays that are
tothe same MOA. proprietary and cannot be shared. Without access to these
412 International Journal of Toxicology 32(6)
protocols, it is challenging to show that HTS assays meet the experimentalapproachestounderstandingbiology,andnoevi-
standardsofdatareliabilityandreproducibilityasdescribedby dence was presented that supports the use of individual assay
theFDA.Notonlydothetestingprotocolsthemselvesneedto resultsasabasisforsafetydecisions.Infact,mostparticipants
be validated but tests should also be conducted to determine agreed that there remains no clear consensus on the relevance
whethertheoutputcorrelateswithanimalstudies(possiblyvia of the output generated by these methods nor how the output
parallel study designs) to satisfy a representation of what shouldbeappliedinriskassessment.Ithasbeensuggestedthat
occurs invivo (Hattan,personal communication, 2013). insilicomodelingcanprovideguidanceinidentifyingpotential
chemicalhazards,andvalidationeffortsarebeingextendedto
newendpoints, andpanelistsgenerally supportedthiskindof
Panel Session: Practicality of Weight of approach.However,theuseofHTSassaysinidentifyingchem-
Evidence: Where Do We Go From Here? ical hazards is stillinitsinitial phasesofdevelopment.
Two high-level needs were often repeated by the partici-
Moderator
pants before input data could gain a position of proven utility
intheriskassessmentprocess.Thefirstistheneedtoidentify
RichardCanady,PhD,ILSIResearchFoundation,Washington,
therelationshipbetweeninsilicoandHTSdatatothepossible
DC,USA
exposure ranges of target chemicals through foods and the in
vivo effects considering ADME. The second is the call for
Panelists
simple validation of the methods. Validation would include
RoxiBeck, Center for FoodIntegrity showing that, for example, ‘‘off-the-shelf’’ cell line assays
JamesBus,PhD,TheDowChemicalCompany,Midland,MI, developed for one purpose (drug discovery) are relevant to
USA toxicity.Itwouldalsoincludemeasuresofreproducibilitywith
Keith Houck,PhD, EPANCCT respect to the outcomes of interest (eg, predicting toxicity
Mike DeVito,PhD, NIH NIEHS NTP across a range of chemicals known to produce or not produce
James Rathman, PhD, OhioState University, Columbus, OH, a particular effect). These are rough paraphrases of goals of
USA theToxCastandTox21programs,anditishopedthatovertime
Ken Wallace, PhD, University ofMinnesota Medical School, they can be achieved. As mentioned previously, showing the
Minneapolis, MN,USA relationshiptosafetyassessmentanddemonstratingvalidityof
these approaches begins with disclosure to the scientific
The workshop culminated in a panel discussion that revis-
communityonthemethodsthemselvesinordertoensurerepro-
itedthe followingquestions:
ducibility. All panelists expressed interest in advancing the
scientific verification of these methods so that they could
1. How would in silico and HTS data help in confirming
supportsafetyassessments.Meetingthischallengewillrequire
the safety ofapproved foodingredients?
2. Howdowebegintheprocesstoconsistentlyandeffec- an extensive and ongoing dialog between the toxicology, risk
tively use in silico and HTS data to help assess the assessment, and otherrelated scientific communities.
safety ofnew foodingredient?
3. HowwouldinsilicoandHTSdatahelpincommunicat- Communication
ing the safety of food ingredients while enhancing the
It is likely that the public release of data indicating alerts of
public’strust inthe foodsupply?
chemical substances (including food-relevant substances) will
In assessing these questions, the panelists agreed that the beinterpretedasriskstohumanhealthbythepublicandsome
immediate goal was to use these methods to help prioritize organizations.Giventheexperimentalnatureofthemethods,it
chemicalsforfuturetesting,especiallyforthosechemicalsthat is verylikelythatmanyif notmostfindingswillhave limited
havelittledata.Itwashighlightedthattherealvaluefromthese predictive value for actual health effects and will rather be
methodscouldbederivedbyanchoringtheoutputwithanimal informative of testing needs. However, at some point in the
and human data on exposure and health effects from development of the methods, it seems likely that ‘‘true’’ pre-
approaches widely accepted by risk assessment and risk man- dictorsofharmmayemerge.Forthisreason,thediscussionand
agement experts. For example, if a compound has an in silico expectationofpredictionofharmwillcontinueforthesemeth-
and HTS alert at estimated exposure levels relevant to a con- ods. Considering this developmental nature of the meaning of
cordant concern identified in animal testing, then all lines of the data, communication about the data requires careful con-
evidence could better inform future investigations. If concor- sideration. Neither undue harm nor undue delay should be
dance is not seen across the outputs from in silico, HTS, and conveyed in such communication, as each may cause unwar-
animaltesting,thenadditionalworkonvalidationandtheneed ranted effects to health through consumer response. For these
for moredataare warranted. reasons, consideration needs to be given to the key messages
Itwasclearthat,ashasalwaysbeenthecase,therearelimits thataccompanythedata.Inaddition,itisimportantthattrans-
totheconclusionsthatcanbebasedontheresultsofindividual parencybeconsideredbytheownersofdataandlaterusersof
assays. The HTS and in silico modeling are currently the released data in their communications with the media and
Bialk et al 413
thepublic.TheCenterforFoodIntegrityspokeabouttheneed (cid:2) Despite what the name ‘‘Tox21’’ implies, the assays
forcommunicationthatemphasizes3keyaspectsbasedonthe provide preliminary information on biological activity,
shared values, trust, and science in order to build trust in nottoxicity.
today’sfoodsysteminaneraofradicaltransparency withthe (cid:2) Tox21 may be an opportunity to reduce animal testing
emergence of social media. According to the Center for Food but more work is required to confirm that the methods
Integrity, confidence or shared values are 3 to 5 times more areaccurate.
important to consumers than scientific verification or compe- (cid:2) Publishingthedataandtheresultsshouldincludecareful
tence in determining who they will trust in the food system. considerationtowhatthefindingsmeanwithinthecon-
Thesefindingsshouldserveasacall-to-actionforthoseinthe textofacomprehensive foodsafety assessment.
governmentandthefoodsystem,andnolongeritissufficient (cid:2) The contribution of HTS data to a perception of public
torelysolelyonscienceortoattacktheattackersasameansof healthrisk willvary amongvarious stakeholders.
protecting self-interest. This new environment requires new (cid:2) Balancedmessagingconcerningtheresultsandthestate
ways of engaging and new methods of communication if the ofthescience is unlikelytooccur.
Tox21 community and industry want to build trust, earn and (cid:2) In light of the release of HTS data, developing a
maintain social license, and protect the freedom to operate communicationsstrategythatthepublicwillappreciate
whetherinaresearch laboratoryorinbusiness.Relatedtothe andunderstand is imperative.
themeoftheworkshop,theCenterforFoodIntegritysaidthat
weneedstakeholderswhocontrolsociallicensetounderstand
that while our use of technology has increased and our food Authors’ Note
systemshavechanged,ourcommitmenttodoingwhatisright InternationalLifeSciencesInstitute(ILSI)NorthAmericaisapublic,
hasneverbeenstronger.Theworkshopparticipantswereespe- nonprofitfoundationthatprovidesaforumtoadvanceunderstanding
cially interested in using these communication concepts and ofscientificissuesrelatedtothenutritionalqualityandsafetyofthe
approachestothesituationathandwhereriskassessmentmeth- food supply by sponsoring research programs, educational seminars
odsareevolvingintheabsenceofknowledgeabouttheirpos- and workshops, and publications. ILSI North America receives sup-
sible applications. Theconcern expressedwas thatthe lack of portprimarilyfromitsindustrymembership.Theviewsexpressedin
this research report are those of the authors and do not necessarily
understandingaboutthesenewinputsystemsandthemeaning
reflectthepositionorpolicyofPepsiCo,Inc.
of the data and results may be unfamiliar to many, and this
knowledgevoidcouldbefilledbyopponentsofthetechnology
Declaration ofConflicting Interests
withmisinformation,leadingtoasituationthatwouldbevery
difficult tomanage orreverse. The author(s) declared the following potential conflicts of interest
Based on this, it was recommended that industry and gov- with respect to the research, authorship, and/or publication of this
ernment stakeholders develop a communications strategy that article:HBisanemployeeofPepsiCoInc.
focuses on the data to be released, format of the data to be
released,and the shared commitment to the safety of the food Funding
supply. This strategy could begin with communicating how Theauthor(s)disclosedreceiptofthefollowingfinancialsupportfor
screening methods may contribute to food safety and the the research, authorship and/or publication of this article: Coauthor
additional work that is underway to build confidence in these JeffreyBarachsupportedthedevelopmentofthisarticleandreceived
methods. For example, stakeholders could generate talking fundsfromILSINorthAmericaforhisworkonthisarticle.Thework-
points, which state that these screening approaches are as shop was sponsored by the North American Branch of the Interna-
tional Life Sciences Institute Technical Committee on Food and
follows:(1) a part ofa scientific process tofind new methods
ChemistrySafety.
to analyze the safety of chemical substances, (2) represent a
potential opportunity to further reduce animal testing, (3)
remain under development and will require additional work References
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