ABSTRACTS - NanoValid · Safe use of nanomaterials has been subject to intense research activities...

ABSTRACTS8th International Symposium on Nanotechnology,Occupational and Environmental Health29 May – 1 June 2017, Elsinore, Denmark

8th International Symposium on Nanotechnology, Occupational and Environmental Health29 May 1 June 2017, Elsinore, Denmark

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Main sponsors


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WELCOMEAwarm welcome to the 8th International Symposium on Nanotechnology,Occupational and Environmental Health, NanOEH2017, in Elsinore, Denmark!

On behalf of the organizing committee, the scientific committee and the international advisorycommittee, we are very pleased to welcome you to this 8th International Symposium onNanotechnology, Occupational and Environmental Health. We hope that you will spend someenjoyable and scientifically rewarding days here at Konventum in Elsinore.

We have made a great effort to present an interesting and exciting program – and we believethat we have succeeded. We are also very pleased to welcome five prominent key notespeakers and 32 invited speakers representing many different fields of nanosafety.

Safe use of nanomaterials has been subject to intense research activities during the last 15years, and we hope that this conference will help providing an overview of theaccomplishments. Participants from many countries from both the scientific community andfrom a variety of companies and institutions are attending the conference. We hope that thisbroad range of participants will promote the dialogue across professional boundaries for thebenefit of nanosafety.

In addition to the scientific program, we hope that you will take the time to visit the exhibitionsand join us for the sightseeing tour at Kronborg Castle as well as the conference dinner.

Once again, welcome to Elsinore and have a great conference.

Ulla Vogel, Professor and head of The Danish Nano Safety Centre at the National ResearchCentre for the Working Environment, Denmark

Paul Anker Lund, Senior Advisor at the National Research Centre for the WorkingEnvironment, Denmark

Inger Schaumburg, Director General at the National Research Centre for the WorkingEnvironment, Denmark


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INTERNATIONAL ADVISORY COMMITTEEAndrew Maynard, Director, Risk Science Center, University of Michigan, USACandace Tsai, Assistant Professor, Occupational and Environmental Health, Department ofEnvironmental & Radiological Health Sciences, Colorado State University, USACharles L Geraci, Senior Scientist and Coordinator of the Nanotechnology Research Center at theNational Institute for Occupational Safety and Health (NIOSH), USAChuen Jinn Tsai, Professor, Institute of Environmental Engineering, National Chiao Tung University,TaiwanDavid YH Pui, Professor, Mechanical Engineering, University of Minnesota, USAPaul Schulte, PhD, Director, Education and Information Division and Co Manager of the NanotechnologyResearch Center, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USAGaku Ichihara, Professor, Tokyo University of Science, Faculty of Pharmaceutical Sciences, Departmentof Pharmacy, JapanKai Savolainen, Research professor, Finnish Institute of Occupational Health (FIOH), Helsinki, FinlandMary Gulumian, Professor, Head of the Toxicology Section, National Institute for Occupational Health(NIOH), South AfricaMichael Riediker, DSc, Postdoc, Adj. Asst. Professor NTU, Research Director, Institute of OccupationalMedicine (IOM), SingaporeRob Aitken, Director of Strategic Consulting, Institute of Occupational Medicine (IOM), Singapore.

SCIENTIFIC COMMITTEEAlison Elder, Associate Professor, Department of Environmental Medicine, University of RochesterMedical Center, New York, USAAnders Baun, Professor, Department of Environmental Engineering, Technical University of Denmark(DTU), DenmarkAnne T Saber, Senior Researcher, National Research Centre for the Working Environment, DenmarkChristof Asbach, Dr. Ing. & Head of unit, Institute of Energy and Environmental Technology (IUTA),GermanyDana Kühnel, Dr., the Helmholtz Centre for Environmental Research GmbH (UFZ), GermanyEnrico Bergamaschi, Associate Professor of Occupational Medicine and Industrial Hygiene, University ofParma, ItalyFlemming Cassee, Professor F.R., Dutch National Institute for Public Health and the Environment(RIVM), NetherlandsHanna Karlsson, Senior Researcher, Institute of Environmental Medicine (IMM), Karolinska Instituttet,SwedenHåkan Wallin, Group leader, Department of Chemical and Biological Work Environment, the NationalInstitute of Occupational Health (STAMI), NorwayIseult Lynch, Professor, School of Geography, Earth and Environmental Sciences, University ofBirmingham, United KingdomIsmo Koponen, Senior Researcher, National Research Centre for the Working Environment, DenmarkJoonas Koivisto, Postdoc, National Research Centre for the Working Environment, DenmarkJun Kanno, Director, Japan Bioassay Research Center (JBRC), Japan Organization of Occupational Healthand Safety and Visiting Researcher, National Institute of Health Sciences (NIHS), JapanKaarle J Hämeri, Professor, University of Helsinki, FinlandKarin S Hougaard, Senior Researcher, National Research Centre for the Working Environment, Denmark


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Keld A Jensen, Senior Researcher, National Research Centre for the Working Environment, DenmarkKirsten Kling, Postdoc, National Research Centre for the Working Environment, DenmarkKristina B Knudsen, Postdoc, National Research Centre for the Working Environment, DenmarkMartin Roursgaard, Associate Professor, Department of Public Health, University of Copenhagen,DenmarkMartin Seipenbusch, Managing Partner, ParteQ GmbH, University of Stuttgart, GermanyMiikka Dal Maso, Professor, Tampere University of Technology, University of Helsinki, FinlandNicklas R Jacobsen, Senior Researcher, National Research Centre for the Working Environment,DenmarkPenny Nymark, PhD, Postdoc, Institute of Environmental Medicine, Karolinska Instituttet, SwedenPeter Møller, Professor, Department of Public Health, University of Copenhagen, DenmarkRambabu Atluri, Postdoc, National Research Centre for the Working Environment, DenmarkRoel Schins, PhD, Head of Research Group, Leibniz Research Institute for Environmental Medicine (IUF),GermanySabina Halappanavar, Adjunct Professor, Department of Biology, University of Ottawa, CanadaSarah Søs Poulsen, Postdoc, National Research Centre for the Working Environment, DenmarkTobias Stöger, Researcher, Helmholtz Zentrum München, German Research Center for EnvironmentalHealth, GermanyTrine Berthing, Postdoc, National Research Centre for the Working Environment, DenmarkUlla Vogel, Professor, National Research Centre for the Working Environment, DenmarkWendel Wohlleben, Senior Scientist, BASF SE, Ludwigshafen am Rhein, GermanyWouter Fransman, Senior Scientist, TNO, the Netherlands.

NATIONAL ORGANISING COMMITTEEAnne Thoustrup Saber, Senior ResearcherJohannes Pries Pries, Web CoordinatorKirsten Jürgensen, Head of ServicesKirsten Rydahl, Communication ConsultantPaul Anker Lund, Senior AdviserSarah Søs Poulsen, PostdocUlla Vogel, Professor

SPONSOR LIST

TSI/Danalytic tsi.com/VitroCell vitrocell.com/Testo/Buhl&Bønsøe buhl bonsoe.dk/da/Eurotox eurotox.com/StatPeel statpeel.com/Calibrate EU project nanocalibrate.eu/homeSmartNanoTox EU project smartnanotox.eu/index.php


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PROGRAMME

Day 1 Monday 29 May09:00 11:45 Registration

10:00 11:30 Free guided art tour at Konventum

11:45 12:45 Lunch

12:45 13:15 Welcome to the conferenceRoom 1 (Conference room)

13:15 14:00 Keynote ICarbon nanotube toxicityJun KannoSession Chairs: Rachel Smith & Ulla VogelRoom 1 (Conference room)

14:00 16:00 Session 1ACarbon nanotube toxicitySession Chairs: Jun Kanno & Nicklas R JacobsenRoom 1 (Conference room)

14:00 14:30 Invited presentationPulmonary toxicity of multi walled carbon nanotubes in rats, a subacuteinhalation studyLaurent Gaté

14:30 15:00 Invited presentationDemonstration of carcinogenicity of multi walled carbon nanotubes by transtracheal intrapulmonary spray (TIPS) method in the rat A simple carcinogenicityscreening test of fibrous carbon nanomaterialsHiroyuki Tsuda

15:00 15:30 Invited presentationThe recent industrial applications of CNTs considering toxicological evaluationsand Redox Potential estimating ROS toxicityShuji Tsuruoka

15:30 15:45 Dose dependent lung tumor promotion after multi walled carbon nanotubeinhalationDale Wayne Porter

15:45 16:00 Fragmented centromeres, translocations, aneuploidy, aberrant mitotic spindles,and fragmented centrosomes in human lung epithelial cells exposed to Mitsui 7,heat treated, and nitrogen doped multi walled carbon nanotubeLinda Marie Sargent


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14:00 16:00 Session 1BPhysic chemical characterization requirements and methods for grouping and riskassessmentSession Chairs: Wendel Wohlleben & Rambabu AtluriRoom 2

14:00 14:30 Invited presentationChallenges and lessons learnt during the implementation into the GUIDEnanoTool of a systematic evaluation of similarity between nanomaterialsGemma Janer

14:30 15:00 Invited presentationMulti assay implementation of the ECETOC grouping framework: Reactivity &dissolution results on families of pigments & silicatesWendel Wohlleben

15:00 15:20 Characterizing nano sized particles using scanning electron microscopyAnders B Bluhme

15:20 15:40 First order risk assessment for nanoparticle inhalation exposure based uponpulmonary inflammation and surface area doseAntti Joonas Koivisto

15:40 16:00 Grouping of manufactured nanomaterials: Strategies within NANoREG andNanoReg2Rambabu Atluri

16:00 16:30 Coffee break

16:30 18:00 Session 2AInhalation toxicity – drivers of toxicitySession Chairs: Tobias Stöger & Håkan WallinRoom 1 (Conference room)

16:30 17:00 Invited presentationThe cellular events governing inflammasome activation and IL 1 processing inresponse to inhaled particlesFrançois Huaux

17:00 17:30 Invited presentationMacrophage responses to particlesTobias Stöger

17:30 17:45 The role of interleukin 1 cytokine in the fibrotic responses of multi walled carbonnanotubes injected into the pleural cavityShan Zienolddiny

17:45 18:00 Inhalation toxicity of 5–10 nm cerium dioxide nanoparticlesRachel Smith


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16:30 18:00 Session 2BNanomaterial characterization in toxicology – Reactivity, dissolution, andbiomolecule interactionsSession Chairs: Iseult Lynch & Trine BerthingRoom 2

16:30 17:00 Invited presentationNanoparticle behavior variability: sources, characterization needs, and bestpracticesDonald R Baer

17:00 17:30 Invited presentationThe importance of characterisation of bionanointeractions in NM (eco)toxicityassessment: new approaches and insightsIseult Lynch

17:30 17:45 Dissolution and transformation of Zn containing nanomaterials in artificial lungfluidsAleksandr Stefaniak

17:45 18:00 Evaluation of intrinsic versus cell based oxidative properties of nanomaterialsand associated oxidative stress responses using multiple assaysBryan Hellack

18:00 19:30 Poster Session 1

19:30 Dinner (Konventum)


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Day 2 Tuesday 30 May09:00 10:00 Keynote II

Developmental toxicity of nanomaterialsKarin S HougaardSession Chairs: Tim Nurkiewicz & Gaku IchiharaRoom 1 (Conference room)


10:30 12:30 Session 3AReprotoxicitySession Chairs: Flemming Cassee & Karin S HougaardRoom 1 (Conference room)

10:30 11:00 Invited presentationEngineered nanomaterial inhalation during gestation: Ramifications on uterinemicrovascular function, the fetal genome and cardiac transcriptomeTimothy Robert Nurkiewicz

11:00 11:30 Invited presentationMaternal exposure to diluted diesel engine exhaust alters placental function ofthe first and second generation in rabbit modelAnne Couturier Tarrade

11:30 11:45 Induction of behavioral changes and astrogliosis in the brain of offspring micefollowing maternal inhalation of carbon black nanoparticleAtsuto Onoda

11:45 12:00 Pulmonary exposure to metallic nanoparticles during pregnancy irreversiblyimpairs lung development of the offspringSophie Lanone

12:00 12:15 In utero exposure to ultrafine particles induces 11 hydroxysteroid dehydrogenasetype 2 related placenta stress in miceAndrea De Vizcaya Ruiz

12:15 12:30 Effects of pulmonary exposure to nano particles on male reproductive functionAstrid Skovmand

10:30 11:30 Session 3BIToxicity of soluble nanomaterialsSession Chairs: Hanna Karlsson & Andrea HartwigRoom 2

10:30 11:00 Invited presentationSoluble nanoparticles – toxicity, dissolution testing and risk assessmentHanna Karlsson

11:00 11:30 Invited presentationUse of gene expression profiling to assess the impact of partly soluble metalbased nanoparticles on genomic stabilityAndrea Hartwig


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11:30 12:30 Session 3BIIEcotoxicity – closing gapsSession Chairs: Dana Kühnel & Anders BaunRoom 2

11:30 12:00 Invited presentationMetal uptake and distribution in the zebrafish (Danio rerio) embryo: differencesbetween nanoparticles and metal ionsDana Kühnel

12:00 12:30 Invited presentationEcotoxicity testing of nanoparticles – The quest for disclosing the nano effectAnders Baun

12:30 13:30 Lunch

13:30 15:30 Session 4ANew hazard endpoints of nanomaterial exposureSession Chairs: Roel Schinz & Peter MøllerRoom 1 (Conference room)

13:30 14:00 Invited presentationTelomere length as a new hazard measure in nanoparticle toxicologyPeter Møller

14:00 14:15 Effect of age, diet, rat strain, and particle exposure on lung and peripheral bloodmononuclear cell (PBMC) telomere regulation and lengthAaron Erdely

14:15 14:30 Cellular senescence induced by combustion derived nanoparticles in endothelialcells and lung epithelial cellsKlaus Unfried

14:30 14:45 Invited presentationEffects of nanoparticles in the 5xFAD mouse model of Alzheimer’s DiseaseRoel Schins

14:45 15:00 Integrated in vitro neurotoxicity testing: an approach to improve hazardassessment of inhaled substancesHarm J Heusinkveld

15:00 15:15 Thrombotic potential of intravenous engineered nanoparticlesJennifer Bridget Raftis

15:15 15:30 Acute fine particulate matter exposure reduces heart rate variability in roadmaintenance workersMichael Riediker


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13:30 15:30 Session 4BYoung researcher grantsSession Chairs: Michael Riediker & Sarah Søs PoulsenRoom 2

13:30 13:40 Introduction

13:40 13:55 NanoStreeM, the European Semiconductor Industry Consortium as a startingpoint for developing a medical surveillance consensus for nano workersEline Vandebroek

13:55 14:10 Induction of behavioral changes and astrogliosis in the brain of offspring micefollowing maternal inhalation of carbon black nanoparticleAtsuto Onoda

14:10 14:25 A comparison of the biokinetics of nanoceria after intravenous, inhalation,instillation and oral exposure using PBPK modelingTshepo Moto

14:25 14:40 Inhaled multi walled carbon nanotubes induced gene expression profile in ratlungCarole Seidel

14:40 14:55 Dispersion and aerosolization of engineered nanoparticles in liquid suspensionfor in vitro toxicity testingYaobo Ding

14:55 15:25 Global health impacts of nanotechnology law: The legend of asbestos that hauntsfuture innovation in nanotechnologyIlise Charoy


16:00 18:00 Session 5AIn vivo models of hazardSession Chairs: Alison Elder & Kristina B KnudsenRoom 1 (Conference room)

16:00 16:30 Invited presentationPhysicochemical predictors of MWCNT induced pulmonary histopathology andtoxicity 1 year after pulmonary deposition of 11 different MWCNT in C57BL/6NmiceKristina B Knudsen

16:30 17:00 Invited presentationEffects in the lungs following inhalation exposures to nanoparticle containingslurries used in semiconductor manufacturingAlison Elder

17:00 17:15 Characterization of pulmonary toxicity following acute exposure to a boronnitride nanotube suspensionAaron Erdely


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17:15 17:30 The influence of the redox activity of inhaled nano sized cerium dioxide onrespiratory, immune, cardiovascular and neurological effects in various mousemodelsFlemming R Cassee

17:30 17:45 Lung remodeling after pulmonary exposure of mice to cerium oxide nanoparticlesrole of autophagy

Sophie Lanone

17:45 18:00 Carbon nanoparticle and lung interaction is crucial for systemic effects: Lessonslearnt from inhalation versus intra arterial infusion exposure studies in miceKoustav Ganguly

16:00 18:00 Session 5BRelease characterization: from concentrations to quantitative releaseSession Chairs: Kaarle Hämeri & Joonas KoivistoRoom 2

16:00 16:30 Invited presentationOccupational exposure assessment to NOAA during production, formulation andassembly of nanoenabled products in several European platforms and pilot plantsSimon Clavaguera

16:30 17:00 Invited presentationRelease of nanomaterials from consumer products during use and end of lifeAlessio Boldrin

17:00 17:15 Engineered nanoparticle containing consumer products in the Singapore retailmarket and likelihood of release into the aquatic environmentMichael Riediker

17:15 17:30 Comparing the Venturi and rotating drum dustiness testing methodsDouglas E Evans

17:30 17:45 Bayesian Belief Network to forecast nanomaterial release during waste shreddingNeeraj Shandilya

17:45 18:00 Estimation of aerodynamic and diffusion diameters of carbon nanotube aerosolagglomerates using electron microscopyBon Ki Ku

18:00 19:30 Poster Session 2

18:00 19:00 Parallel workshopSafe production and use of nanomaterials in the cera mic industry: outcomes andprogress of CERASAFE projectSession Chair: Mar VianaRoom 2

19:30 Dinner (Konventum)


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Day 3 Wednesday 31 May09:00 10:00 Keynote III

Nanomaterial dosimetry in inhalation toxicology: Bridging the gaps between invitro and in vivo models as well as real world exposureOtmar SchmidSession Chair: Alison Elder & Ulla VogelRoom 1 (Conference room)


10:30 12:30 Session 6AIn vitro models of toxicity testingSession Chairs: Penny Nymark & Martin RoursgaardRoom 1 (Conference room)

10:30 11:00 Invited presentationIn vitro based high throughput/high content screening and omics driveninformatics support rapid and effective safety evaluation of engineerednanomaterialsPenny Nymark

11:00 11:30 Invited presentationHow to make in vitro models more realistic, and compare the data to in vivooutcomesMartin Roursgaard

11:30 11:45 Genotoxicity assessment of 31 nanomaterials in human bronchial epithelial cellsHannu Norppa

11:45 12:00 Assessment of adverse effects of silver nanoparticles using an air liquid interfacesmall airway epithelial exposure modelChang Guo

12:00 12:15 Adverse effects of summer and winter ambient air in human lung cell cultures, analternative model to assess occupational and environmental healthChristoph Bisig

12:15 12:30 Dispersion and aerosolization of engineered nanoparticles in liquid suspensionfor in vitro toxicity testingYaobo Ding

10:30 12:30 Session 6BWork place measurement & modellingSession Chairs: Mikka Dal Maso & Ismo K KoponenRoom 2

10:30 11:00 Invited presentationSimultaneous modelling of dispersion and aerosol dynamics of workplace aerosolMiikka Dal Maso

11:00 11:30 Invited presentationAirborne particle exposure modelings in indoor settingsAntti Joonas Koivisto


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11:30 11:45 Testing geometrical layouts for a multi box aerosol dispersion model in chamberstudiesAlexander CØ Jensen

11:45 12:00 Workplace exposure assessment in the recycling of nano compositesMartin Seipenbusch

12:00 12:15 Workplace carbon nanotube exposure: Real time measurements using anaethalometerKarin Elin Lovén

12:15 12:30 Sensing solution for airborne carbon nanotube exposure in workplaces based onsurface enhanced Raman SpectroscopyRudolf Bieri

12:30 13:30 Lunch

13:30 15:30 Session 7AToxicogenomics and adverse outcome pathways (AOP) as tools for nano riskassessmentSession Chairs: Sabina Halappanavar & Sarah Søs PoulsenRoom 1 (Conference room)

13:30 14:00 Invited presentationDeveloping an adverse outcome pathway based on toxicogenomic data for ENMinduced risk of developing atherosclerotic plaquesSarah Søs Poulsen

14:00 14:30 Invited presentationUtilizing an adverse outcome pathway framework to investigate the essentialityof Interleukin and STAT signaling in the pathology of a carbon nanotube in miceSabina Halappanavar

14:30 14:50 TiO2 induced gene expression and protein profiles in rat lung: a subacuteinhalation studyLaëtitia Chezeau

14:50 15:10 Nanomaterial impact on models of asthma and allergic airway disease:mechanistic insight as an aid for hazard assessmentMartin Leonard

15:10 15:30 Proteomic analysis of nasal lavage samples collected from weldersexperimentally exposed to welding fume nanoparticlesNeserin Ali

13:30 15:30 Session 7BExposure managementSession Chairs: Martin Seipenbusch & Christof AsbachRoom 2

13:30 14:00 Invited presentationAssessment strategy for nanoparticles in workplace airDirk Dahmann


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14:00 14:30 Invited presentationOccupational exposure to nanoparticles: monitoring and management inindustrial settingsMar Viana

14:30 14:45 Exposure assessment to noaa at workplace – an opportunity towards a safer andmore responsible development of nanocomposites: NANOLEAP projectCécile Ducros

14:45 15:00 Evaluation of and risk management measures for the handling of carbonnanotubes in a laboratoryJohannes Pelzer

15:00 15:15 Assessment of personal exposure to airborne nanomaterials A review ofmeasurement equipmentChristof Asbach

15:15 15:30 Explosion hazards of nanoparticlesDouglas E Evans

16:30 Transport (busses leaves at 16:30)

17:00 19:00 Social programme (Kronborg Castle)

19:00 Transport (walk to restaurant)

20:00 23:00 Conference dinner (Marienlyst)

23:00 Transport (busses leaves at 23:00)

23:00 Social programme, discoteque at Konventum


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Day 4 Thursday 1 June09:00 10:00 Keynote IV

Occupational risk assessment of nanomaterial exposurePaul A SchulteSession Chair: Sally Tinkle & Hannu NorppaRoom 1 (Conference room)


10:30 12:00 Session 8ANanomaterial epidemiology & biomonitoringSession Chairs: Enrico Bergamaschi & Anne T SaberRoom 3

10:30 11:00 Invited presentationHealth surveillance and epidemiology in nanomaterial workers worldwide –where are we today?Michael Riediker

11:00 11:30 Invited presentationEpidemiological surveillance of nanotechnology workers: Past and newchallenges based on the French exampleIrina Guseva Canu

11:30 11:45 Occupational exposure and health effects of multi walled carbon nanotubesLiliya Fatkhutdinova

10:30 12:00 Session 8BRisk assessment & managementSession Chairs: Wouter Fransman & Keld A JensenRoom 1 (Conference room)

10:30 11:00 Invited presentationCurrent status of nanomaterial risk assessment models and strategies to riskmanagementWouter Fransman

11:00 11:15 Invited presentationSelection of proven risk management measures (RMMs) to control the exposureto ENMsCarlos Fito

11:15 11:30 An analysis of the OECD WPMN dossier regarding the availability of data for riskassessmentMichael Riediker

11:30 11:45 A critical and in depth analysis of the environmental aspect of the OECD SPdossiersSteffen F Hansen

11:45 12:00 Development of a Nano Exposure and Contextual Information Database (NECID)Johannes Pelzer


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12:00 13:00 Keynote VEnvironmental risk assessment of nanomaterials

Bernd NowackSession Chairs: Michael Riediker & Anders BaunRoom 1 (Conference room)

13:00 13:15 Thank you and welcome in 2019

13:15 14:15 Lunch/grab a sandwich


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ABSTRACTS


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Keynote session I

Monday 29 May, 13:15 – 14:00Location: Room 1 (Conference room)Session chairs: Rachel Smith & Ulla Vogel

Carbon nanotube toxicity Jun Kanno

Japan Bioassay Research Center, Japan Organization of Occupational Health and Safety, Japan;[email protected]

One of the reasons that the chronic toxicity study on nanomaterials (NMs) has not been well developed,compared to general chemicals or “soluble” compounds, is because they are one of the particulatematter (PM), and PM toxicology is a very difficult subfield of Toxicology to engage. Perhaps the mostimportant route of exposure of PMs is inhalation. This inhalation is another big hurdle for toxicologists;inhalation facilities are usually expensive to build and run, and requires skillful operators. Methods togenerate well dispersed aerosol and quantitatively measure the aerosol density often need case bycase innovation for each NM specimen.

And yet, fortunately, we find a few hints from the PM related human diseases that are reported in thepast. One example is asbestos. Studies ranges from foreign body inflammation, granulomatousresponses, responses towards fibrosis, to the most important “frustrated phagocytosis” for a specificsize and shape of a fiber related to the oxidative stress mechanisms to initiate and promotemesotheliomagenesis and lung adenocarcinomagenesis.

Here, biological responses against multi wall carbon nanotubes (MWCNT) monitored in intraperitonealstudies and whole body inhalation studies are presented. Histologically, non granulomatous persistentchronic inflammatory micro lesions with single fibers were considered to be important for thedevelopment of mesothelioma, whereas granulomas formed against aggregates/agglomerates were notdirectly involved in the process.

The most challenging part of the inhalation study is to generate well dispersed aerosol. We would liketo report some data on the “Taquann” dispersion method and direct injection system using a MWCNT(Mitsui MWNT 7) at NIHS. In addition, the summary of the result of two year rat whole body inhalationcarcinogenesis study of MWNT 7 performed at JBRC will be presented.

In vitro and in vivo acute toxicity data are accumulating fast. The extrapolation of such data to in vivochronic toxicity awaits further accumulation of bridging data. For the sound growth of nanomaterialindustry and protection of workers and users, usage of both acute and chronic toxicity data in areasonable balance is practical and essential.


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Session 1A: Carbon nanotube toxicity

Monday 29 May, 14:00 – 16:00Location: Room 1 (conference room)Session chairs: Jun Kanno & Nicklas R Jacobsen

Pulmonary toxicity of multi walled carbon nanotubes in rats, a subacuteinhalation study

Laurent Gaté, Sylvie Sébillaud, Mylène Lorcin, Laetitia Chézeau, Carole Seidel, Christian Darne,Sébastien Bau, Stéphane Grossmann, Stéphane Viton, Hervé Nunge, Laurine Douteau, SylvieMichaux, Frédéric CosnierInstitut National de Recherche et de Sécurité, France; [email protected]

Due to their physical and chemical properties, multi walled carbon nanotubes (MWCNTs) are amongthe most promising nanomaterials in terms of industrial use. In order to assess their toxicologicalproperties, inhalation experiments performed in laboratory rodents remain the most suitable andreliable approach. We have performed sub acute inhalation experiments in female Sprague Dawleywith two CNTs: the “long and thick” NM 401 and the “short and thin” NM 403. Animals were exposedin nose only chambers to these aerosols 6 hours/day, 5 days/week for 4 weeks. CNT aerosols weregenerated at a concentration of 0.5 and 1.5 mg/m3 using an acoustic generator. Aerosols were fullycharacterized in terms of mass and number concentration; number and mass size distribution as wellas morphology.

Tissues were collected 3, 30, 90 and 180 days after the end of the exposure period. Only at the highestdose, NM 401 induced a pulmonary inflammatory response 3 days after the end of exposure; this wasdemonstrated by an important neutrophilia in the broncho alveolar lavage fluid (BALF) which howeverdecreased overtime. On the other hand, NM 403 at both concentrations induced a lung inflammationwhich also decreased overtime. Despite the presence of CNTs within the lung, no significanthistopathological changes were found in the lungs of exposed animals. According to the Multiple PathParticle Dosimetry model (MPPD v3.04), the deposited mass of NM 401 was about 2 times higher thanthat of NM 403 (377 μg vs 192 μg per lung respectively following an exposure to 1.5 mg/m3ofMWCNTs), however the specific surface area of the latter was about 10 fold higher than that of NM 401(189 vs 18 m2/g respectively). Then in term of specific surface area, the pulmonary deposited dose ofNM 403 was much higher than that of NM 401 and this can in part explain the differential toxicitybetween the two MWCNTs.

This work is supported by the European Commission through the 7th Framework Project (ProjectNanoReg, Grant agreement n° 310584).


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Demonstration of carcinogenicity of multi walled carbon nanotubes by transtracheal intrapulmonary spray (TIPS) method in the rat A simplecarcinogenicity screening test of fibrous carbon nanomaterials

Hiroyuki Tsuda1, Mohamed Abdelgied1, Ahmed M El Gazzar1, David B Alexander1, William T.Alexander1, Takamasa Numano1, Aya Naiki2, Satoru Takahashi2, Hirotsugu Takase3, Masumi Suzui4,Akihiko Hirose5, Yuhji Taquahashi6, Jun Kanno7

1Nanotoxicology Project, Nagoya City University, Nagoya, Japan; 2Department of Experimental Pathology and Tumor Biology,Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 3Core Laboratory, Nagoya City University GraduateSchool of Medical Sciences, Nagoya, Japan; 4Department of Molecular Toxicology, Graduate School of Medical Sciences,Nagoya City University, Japan; 5Division of Risk Assessment, National Institute of Health Sciences, Tokyo, Japan; 6Division ofCellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan; 7Japan Industrial Safety and HealthAssociation, Japan Bioassay Research Center, Kanagawa, Japanrch Center, Kanagawa, Japan; [email protected] cu.ac.jp

Multi walled carbon nanotubes (MWCNT) have a fibrous structure similar to asbestos, raising concernthat MWCNT exposure may lead to fatal asbestos like pleural lesion development. We showed thatMWCNT translocates from the lung to the pleural cavity and causes pleuritis and mesothelialhyperplastic proliferation in the rat. Conditioned cell culture medium of alveolar macrophagesphagocytosing MWCNT induced an obvious increase in the proliferation of human mesothelial (MESO1) and lung cancer (A549) cells. Furthermore, we showed sustained alveolar and pleural inflammationand mesothelial proliferation after exposure to MWCNTs. Based on these findings, we have developed asimple assay system to screen the carcinogenicity of MWCNTs that can be used as a potentialalternative method to inhalation testing.

Method:MWCNT N was fractionated by passing it through a sieve with a pore size of 25 um. Theaverage lengths of the MWCNT fractions were: whole, 4.2 um; flow through, 2.6um; retained, over2.6um. Male F344 rats were dosed with MWCNT N (Nikkiso) suspended in 125ug/0.5 ml salinecontaining PF68 co polymer, 8 times (total 1mg/rat) over a 2 week period by our tran trachealintrapulmonary spraying (TIPS) method and observed without any further treatment until sacrifice at107 weeks after the last dose.

Results:MWCNT N (Nikkiso) induced lung bronchiolo alveolar tumors (adenoma and carcinomacombined, 36.8%) and malignant pleural mesothelioma (15.8%), both being significant compared to thevehicle control. MWCNTs were found mostly in the alveolar wall, macrophages in the lung, andmediastinal lymph nodes. No differences of the incidence was found among the different sievefractions. The amount of MWCNT N in the whole, filtrated, and retained fractions was 25.4, 48.0, and26.3%, respectively, of the week 2 values. We also found MWCNT 7 and other types were carcinogenicto the lung using this same protocol.

Conclusions:We demonstrated carcinogenicity of MWCNT N in the lung and mesothelial cells after 2week TIPS administration followed by two years observation. Collection of pleural cavity lavage cellpellets is a useful method to evaluate inflammatory events in the pleural cavity. These protocols are apotential screening method of MWCNT carcinogenicity replacing costly inhalation tests.

Financial Support: Supported by the Health and Labor Sciences Research Grants of the Ministry ofHealth, Labour and Welfare, Japan and by the Princess Takamatsu Cancer Research Fund and the LongRange Research Initiative Fund.


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The recent industrial applications of CNTs considering toxicological evaluationsand Redox Potential estimating ROS toxicity

Shuji Tsuruoka1, Hidetoshi Matsumoto2, Vincent Castranova31Shinshu University, Japan; 2Tokyo institute of Technology; 3West Verginia University; [email protected]

The recent CNT applications demonstrate the remarkable progress and will enter the major marketplaces. CNTs have been applied to secondary battery and additives into resins for these two decades,where CNTs are regarded as the auxiliary materials. On the other hand, the new approaches show thatCNTs are the key materials to cause the paradigm shift in industries. The primary fields with the CNTapplications are ink, yarn, and cloth, and then these materials are applied to paint heater, strain sensor,artificial muscle, armor, robot, artificial body parts, non metal mechanical parts including auto andaero space industries, and so on. CNTs also materialize the next generation semi conductors, but ittakes a lot of time. These products and applications are realized with thinner CNTs such as SWCNTs andDWCNTs; particularly the latter is important industrially due to the physical characteristics. Toxicologicalstudies on those CNTs have not been studied well because there exists few samples practically. In thispresentation, those recent industrial efforts and approaches by a group of small and mediumcompanies are presented, which also contributes to toxicological study to investigate the thin CNTtoxicity. In addition, the relationship between Redox Potential of CNTs and ROS is discussed, as it is animportant physicochemical property to estimate toxicity of nano materials. The kinetics of a hydroxylradical scavenging reaction for CNTs is examined regarding to redox potential. The reaction kinetics forCNTs agreed well with the theoretical model and will be used to estimate ROS using the physicalproperties.

ST was partially supported by the research program "Center of Innovation" by Japan Science andTechnology Agency.

Dose dependent lung tumor promotion after multi walled carbon nanotubeinhalation

Dale W Porter1, David Lowry1, Lori Battelli1, Katelyn Siegrist1, Michael Kashon1, Bean Chen1, LaurenStaska2, Ann Hubbs1, Walter McKinney1, Michael Andrew1, Shuji Tsuruoka3, Morinobu Endo3, VincentCastranova4, Steven Reynolds1, Linda Sargent11National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA; 2Charles River Laboratories, Durham, NC,USA 27703; 3Shinshu University, Nagano, Japan 380 8533; 4West Virginia University, Morgantown, WV 26506, USA;[email protected]

In previous work, we demonstrated that exposure to a DNA damaging agent, 3 methylcholanthrene(MCA), followed by multi walled carbon nanotube (MWCNT) inhalation, caused potent promotion oflung tumors in B6C3F1 mice. In the present study, to investigate the dose response for MWCNTinduced carcinogenesis, we exposed B6C3F1 mice to a single dose of either MCA (10 μg/g BW, i.p.) orvehicle (corn oil). One week after i.p. injection, mice were exposed by inhalation to MWCNT (5 mg/m3, 5hours/day) or filtered air (controls) for a total of 2, 5 or 10 days. At 17 months post exposure, micewere euthanized and examined for lung tumor formation. For air exposed mice the tumor incidencerate (percent of mice with tumors) was 30% and tumor multiplicity (number of tumors/mouse) was 0.4,whereas in air exposed mice which also received MCA the tumor incidence rate was slightly higher at45% with tumor multiplicity at 0.6. For mice exposed to MWCNT only, the tumor incidence rate andtumor multiplicity were not related to MWCNT dose and averaged 27% and 0.30. In contrast, forMWCNT exposed mice which received MCA, the tumor incidence rate and tumor multiplicity were


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related to dose and ranged from 75 87% and 1.5 2.5, respectively. Thus, the data suggest a doserelationship between tumor promotion and MWCNT exposure, but only in mice treated with MCA. Thedata presented here suggests that MWCNT are lung tumor promoters and this response is related toMWCNT dose. The data also suggest that MWCNT did not initiate lung tumor formation in the absenceof a tumor initiator, and thus we found no evidence that they are a complete carcinogen in this model.

Fragmented centromeres, translocations, aneuploidy, aberrant mitotic spindles,and fragmented centrosomes in human lung epithelial cells exposed to Mitsui7, heat treated, and nitrogen doped multi walled carbon nanotube

Katelyn Jayne Siegrist1, Steven H Reynolds1, Robert R Mercer1, Dale W Porter1, Lorenzo Cena2,Michael L. Kashon1, John Edward Wiley3, John Mastovich4, Jeffrey L Salisbury5, Kristin Bunker4, Jason SLupoi4, Shuji Tsuruoka7, Mark Sparrow4, Moribu Endo7, Terrones Mauricio6, Kimberly McKinstry1,Linda Marie Sargent11CDC/NIOSH, Morgantown WV 26505 United States of America; 2West Chester University, West Chester PA United States ofAmerica; 3East Carolina Medical School, Greenville NC United States of America; 4RJ LeeGroup, Monroeville, PA, 15146 UnitedStates of America; 5Mayo Clinic, Rochester MN 55902 United States of America; 6Pennsylvania State University, State College,PA, 16801 United States of America; 7Shinshu University, Nagano, Japan; [email protected]

Multi walled carbon nanotubes (MWCNT) have been used and studied extensively due to their uniquephysicochemical properties. Due to their low density and small size MWCNT are easily aerosolized inthe workplace making respiratory exposures possible in workers. The International Agency for Researchon Cancer (IARC) designated the pristine Mitsui 7 MWCNT (MWCNT 7) as a Group 2B carcinogen or“possibly carcinogenic to humans”, but there was insufficient data to classify all other MWCNT. MWCNTexposed to very high temperatures (MWCNT HT) or synthesized with nitrogen (MWCNT ND) havealtered physicochemical properties that have been shown to induce attenuated toxic effects. Therefore,the objective of this study was to determine the differences in genotoxicity of MWCNT HT and NDcompared to MWCNT 7 by using immortalized human lung epithelial cell BEAS 2B and primary lungepithelial cell SAEC. Each MWCNT material was observed within the nucleus and associated with theDNA, mitotic spindle and centrosomes. Quantification of nuclear uptake found that MWCNT 7 materialwas taken up at a higher rate than MWCNT HT and ND, respectively. Twenty four hours of exposure tothe MWCNT 7 and HT material induced a significant arrest in the G1/S phase in BEAS 2B cell cyclewhereas the MWCNT ND induced a G2 arrest. Each MWCNT material produced a significant arrest inthe G1 and G2 phases of the SAEC cell cycle 24 hours after exposure. However, 72 hours of exposureresulted in a significant G1/S phase arrest in MWCNT exposed SAEC. The rate of mitotic spindleaberrations and centrosome fragmentation was significantly increased with exposure to each MWCNTmaterial. Mono and multipolar mitotic aberrations were observed, however the monopolar morphologypredominated. Significant aneuploidy was foundin a dose dependent manner from exposure to eachMWCNT material with 24 μg/mL, the highest dose, of MWCNT 7, HT, and ND producing a rate of 66.5,61.1, and 55.3%, respectively. Chromosome analysis demonstrated translocations, as well asfragmentation of the centromere. Clonal growth was increased following exposure to 0.024 and 0.24μg/mL MWCNT 7, 2.4 μg/mL MWCNT HT, and 0.024 μg/mL MWCNT ND material. These data indicatethat although each MWCNT material investigated in this study produced significant genotoxicity, thephysicochemical alterations of the MWCNT HT and ND reduced nuclear uptake and, therefore,magnitude of genotoxic effect.


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Session 1B: Physic chemical characterization requirementsand methods for grouping and risk assessment

Monday 29 May, 14:00 – 16:00Location: Room 2Session chairs: Wendel Wohlleben & Rambabu Atluri

Challenges and lessons learnt during the implementation into the GUIDEnanoTool of a systematic evaluation of similarity between nanomaterials

Gemma Janer1, Margriet Park2, Julia Catalán3, Natalia Ferraz4, Joan Cabellos1, Ralph Vanhauten5,Socorro Vázquez Campos11LEITAT Technological Center, C/ de la Innovació 2, 08225 Terrassa (Barcelona), Spain; 2National Institute for Public Health andthe Environment, Centre for Health Protection PO box 1 3720BA Bilthoven, The Netherlands; 3Finnish Institute of OccupationalHealth, P.O. Box 40, FI 00251 Helsinki, Finland; 4Uppsala University, Dept Engineering Sciences Regementsvägen 1 75237Uppsala, Sweden; 5ThinkWorks, B.V. Rotterdamseweg 183c 2629 HD Delft, The Netherlands; [email protected]

Due to the high diversity of synthesized nanomaterials (NM) and transformations that can occur aftertheir synthesis, practical approaches to conclude on the similarity between different materials areneeded. During the development of the GUIDEnano tool, we designed and implemented a systematicassessment of similarity between two NMs.

The main steps of the process involved:1. the identification of physical and chemical properties that would be used in the assessment for a

given purpose;2. the definition of algorithms to generate a similarity value as a function of descriptors for each

property; and3. the definition of an algorithm to generate an overall similarity score (i.e., integrating all the

properties evaluated).

Within the human hazard assessment module, we aimed at comparing an exposure relevant NM with aNM for which hazard data is available. The resulting similarity score can be used to select hazard dataobtained with sufficiently similar NMs and to inform on the uncertainty related to dissimilarity. Thesimilarity assessment differed among exposure routes. In the case of inhalation toxicity, the selectedproperties were: chemical composition (including crystallinity and purity), dissolution, shape, primarysize distribution, and aggregated size distributions. In the case of oral or dermal toxicity, the limiteddata available for local toxicity and systemic absorption hinders the selection of the properties to beconsidered beyond chemical composition. Even when the relevant properties are identified, selectingthe descriptors, the algorithm to generate a similarity score, and the thresholds to accept or reject datais a complex and arbitrary process.

Coated or functionalized particles could not be included in this assessment for now, due to the limitedcharacterization data commonly available for such surface modifications. Standardizing characterizationmethods for NM surface modifications so that their effect on NM toxicity can be established is anurgent need because surface modifications are one of the most relevant engineered as well asunintended modifications of NMs.


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A systematic assessment of similarity is necessary for a transparent risk assessment of NMs. The majorchallenges are the lack of scientific evidence to back up several of the choices that need to be takenduring the similarity assessment process, and the limited characterization data in hands of productdevelopers for the NM used in real applications. (Funded by Grant Agreement No. 604387).

Multi assay implementation of the ECETOC grouping framework: Reactivity &dissolution results on families of pigments & silicates

Wendel Wohlleben1, Johannes Keller1, Kai Werle1, Bryan Hellack2, Carmen Nickel2, JohannaKoltermann Juelly1, Martin Wiemann3

1BASF SE, Germany; 2IUTA, Institute of Energy and Environmental Technology, Germany; 3IBE R&D gGmbH, Germany;[email protected]

The ECETOC grouping framework proceeds in three tiers: Tier 1 is consistent with the ECHA definition ofa „nanoform“ (composition, size, shape, descriptive surface chemistry). Compositions with knownsolubility are grouped with their bulk analogues; otherwise the ECETOC framework proceed to tier 2,where composition, shape and exposure/release are used to define testing needs and „relevant media“of system dependent properties. Intrinsic surface chemistry is often considered as proxy for mobilityand reactivity, but tier 2 uses the directly measured system dependent properties instead of the proxy.Methods and benchmark materials for the direct measurement of mobility, reactivity and dissolutionhave been proposed, but need refinement and calibration against in vivo studies, which remain the lastresort in tier 3.

The talk presents extensive results on surface reactivity and dissolution kinetics, developed in the frameof the nanoGRAVUR project (BMBF, 2015 2018). Calibration of the new multi dose FRAS and prestandardized ESR against in vitro macrophage NR8383 reactivity and in vivo rat inhalation effects onfamilies of pigments & silicates highlights the need for multi assay strategies to reduce false positivesand false negatives from tier 2 grouping. Calibration of re discovered methods for dynamic dissolutionin relevant medium against NR8383 macrophage processing and in vivo STIS inhalationprocessing/clearance can much reduce tier 3 testing based on similarity of dissolution.

Characterizing nano sized particles using scanning electron microscopyAnders B Bluhme1,2, Kirsten Kling2, Ismo Kooponen2, Kristian Mølhave11DTU, Denmark; 2NRCWE, Denmark; [email protected]

The use of nano materials have grown rapidly in recent years, bringing new exposure scenarios forworkers in industries such as cleaning and construction, as well as in various productions e.g. paint andcosmetics. The public is furthermore exposed to nano materials when using or disposing of theseproducts, as well as from both natural and anthropogenic emissions, where especially the transportsector is a major contributor. This has brought a need for exposure and risk assessments associatedwith the fabrication, use, and disposal of nano containing products, as well as in rural areas and ingeneral ambient conditions. The assessments are usually based on models and online measurements,providing integrated number and size information with a high time and spatial resolution. However themost commonly used instruments bring no knowledge of the composition or shape of particles, and it istherefore not possible to determine their source. This proves an issue, since some particles are moreharmful than others, and since it is diffcult to develop preventive strategies when it is not certain how


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many of the measured particles are process generated, transported inside through ventilation, ororiginate from alternative sources.

Here we present the current development of an automated software based analysis of nano particlesusing Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)coupled with Energy Dispersive X ray Spectroscopy (EDS). The goal of the analysis is to complementonline measurements and modelling, providing both detailed physical and chemical single particleinformation not provided by the current standard methods. Physical parameters such as area, diameter,aspect ratio, and aggregation state can be obtained for each individual particle based on the acquiredimages, while automated EDS analysis yields single particle elemental composition data. This will allowsize resolved chemical classication of each individual particle, which can be used to determine thefraction of particles originating from in house processes, ambient background, or other sources. Theautomated analysis can furthermore systematically map large areas of a sample without userintervention, enabling a fast and repeatable measurement, while obtaining suffcient data for statisticalanalysis.

First order risk assessment for nanoparticle inhalation exposure based uponpulmonary inflammation and surface area dose

Antti Joonas Koivisto1, Kirsten Inga Kling1, Marcus Levin1, Wouter Fransman2, Ilse Gosens3, FlemmingRalph Cassee3,4, Keld A Jensen1

1National Research Centre for the Working Environment, Copenhagen, Denmark; 2TNO, Zeist, The Netherlands; 3NationalInstitute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; 4Utrecht University, Utrecht, TheNetherlands; [email protected]

There is a need to develop risk assessment techniques where (nano)particle exposure and doseassessment is closely related to the biological response. Inhalation exposure to low toxicity granularbiodurable particles (GBPs) causes polymorphonuclear neutrophilia (PMN) in the lungs of experimentalanimals. PMN is a strong indicator for lung inflammation. The total particle surface area (cm2) that isdeposited in the lung (lung deposited surface area; LDSA, μm2 cm 3) is recognized to correlate well withthe PMN influx in the lungs. The relation between particles dry powder Brunner Emmett Teller (BET)surface area dose and PMN influx for granular GBPs (e.g. TiO2, carbon black) and transition metal oxides(e.g. ZnO, Ni, Co3O4) was recently assessed by Schmid and Stoeger [1]. This was used to derive theNOEL1/100 , the one hundredth of no observed effect levels [2]. The NOEL1/100 levels were compared withmeasured LDSA concentration levels in seven occupational settings: injection molding of polypropylenecar bumpers [2], tungsten carbide cobalt (WCCo) powder production [2], nanodiamond handling [3],handcraft workshop [4], F 16 jet engine emissions, liquid TiO2 coating, and thermal treatment ofceramics coated with TiO2. The LDSA exposure levels varied from 1.8 to >7900 μm2 cm 3, lowest being inhandcraft workshop and highest in F 16 jet engine emissions. This corresponds to the 8 hour surfacearea dose normalized with human lung weight from 3×10 4 to >1.1 cm2 g 1 while the NOEL1/100 assignedfor GBPs was 0.11 cm2g 1 and for transition metal oxide particles was 9×10 3 cm2 g 1. The comparison ofsurface area doses with NOEL1/100 values provides a first order estimate to predict workers risk sufferingpulmonary inflammation during an 8 hour exposure. Currently, this prediction has large degree ofuncertainties in e.g. how dry powder BET surface area equals to LDSA dose, which should be studied indetail [2]. However, even in its current state the approach might be useful for a first step in riskassessment.

1. Schmid O., Stoeger T., 2016. J. Aerosol. Sci. 99:133 143.2. Koivisto A.J., et al., 2016. Nanoimpact, http://dx.doi.org/10.1016/j.impact.2016.11.002.3. Koivisto A.J., et al., 2014. Int. J. Environ. Res. Public. Health. 11:5382 5402.4. Mølgaard B., et al., 2015. Int. J. Environ. Res. Public. Health. 12:3756 3773.


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Grouping of manufactured nanomaterials: Strategies within NANoREG andNanoReg2

Rambabu Atluri1, Christain Micheletti2, Blanca Suarez Merino3, Andrea Haase4, Nicklas R Jacobsen1,Keld A Jensen1

1NRCWE, Denmark; 2ECAMRICERT SRL, Italy; 3GAIKER IK4 Technological Center, 48.170 Zamudio – Bizkaia Spain;4Bundesinstitut für Risikobewertung, Max Dohrn Strasse 8 10, 10589 Berlin; [email protected]

The establishment of a grouping paradigm for manufactured nanomaterials (MNM) is to develop astructured scheme for regulatory agencies and industries to identify substances with similar propertiesand/or biological effects and thereby allow read across.

Within NANoREG (2013 2017), a revised substance identification and grouping scheme was proposedbased on the principle structure in for REACH registration of substances while introducing relevantphysicochemical properties. This physicochemical scheme grouped MNM according to their1. Structure/Chemical composition,2. Shape/Porosity, and3. Specific Physicochemical properties.

This grouping system was demonstrated by several nanostructured silicas with variations in theircomposition and structural complexity. ECHA recently published Draft Appendix R.6 1 (Jan. 2017) onguidance on end points suitable for QSAR, grouping and read across for MNM, which considersgrouping according to: 1) What they are, 2) Where they go, and 3) What they do, described in Sellers etal. (2015).

In NanoReg2 (2015 2018), it is suggested to widen this regulatory grouping scheme taking into accountboth intrinsic and system dependent properties of the MNMs along their life cycle. This is achieved bycombining the NANoREG approach with the approach in Sellers et al. (2015) and further refined in theEU FP7 MARINA project. Here we present a preliminary set of grouping criteria defined in the NanoReg2

project, which is an important step towards a full fledged grouping strategy for MNNs within theregulatory frameworks.

Reference:K. Sellers, N. Deleebeeck, M. Messiaen, M. Jackson, E. Bleeker, D. Sijm and F. van Broekhuizen, RIVM Report 2015 0061. 2015.http://www.rivm.nl/bibliotheek/rapporten/2015 0061.html.


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Session 2A: Inhalation toxicity – drivers of toxicity Monday 29 May, 16:30 – 18:00Location: Room 1 (Conference room)Session chairs: Tobias Stöger & Håkan Wallin

The cellular events governing inflammasome activation and IL 1 processing inresponse to inhaled particles

François HuauxUniversité catholique de Louvain, Belgium; [email protected]

The innate immune system is the first line of defense against inhaled particles. Macrophages serveimportant roles in particle clearance and inflammatory reactions. Following recognition andinternalization by phagocytes, particles are taken up in vesicular phagolysosomes. Intracellularphagosomal leakage, redox unbalance and ionic movements induced by toxic particles result in pro IL1 expression, inflammasome complex engagement, caspase 1 activation, pro IL 1 cleavage,biologically active IL 1 release and finally inflammatory cell death termed pyroptosis. Researchershave recently identified the emerging signals and pathways involved in the expression, maturation andsecretion of IL 1 during these responses to particles. Physicochemical characteristics of particles (size,surface and shape) determine their capacity to induce inflammasome activation and IL 1 processing.From these findings, in vitro IL 1 (and IL 1 ) release from macrophages has been proposed to predictthe acute inflammogenic activity of particles.

Macrophage responses to particles

Tobias StögerHelmholtz Zentrum München, Germany; tobias.stoeger@helmholtz muenchen.de

Depending on the aerodynamic size, inhaled particles penetrate into different areas of the lung.Nanoparticles are known to deposit in the entire respiratory tract, and particularly efficiently in themost vulnerable, alveolar region, which is not protected by a thick mucus layer and mucociliaryclearance. Therefore phagocytic clearance by alveolar macrophages, is the major route of particleremoval from the alveolar surface. These also dust cells called tissue phagocytes, develop from fetalmonocytes that differentiate into long lived cells in the first week of life, they leave their alveolarresidence only sporadically to get themselves cleared by the mucociliary escalator. Accordingly thephysiological cleansing function works well for biodegradable particles, like organic dust, cell debris oreven many pathogens, but reaches its limits for inorganic and biopersistent nanoparticles. Alveolarmacrophages are in particular among the first and primary cell types that process nanoparticles andmediate host inflammatory and immunological biological responses in a material dependent mannerand length of time.

In this context, special surface receptors, expressed by macrophages, will be discussed, which can beresponsible for nanoparticle recognition, and may also induce specific internalization mechanisms, withand without downstream inflammatory effects. The acute, pulmonary inflammatory response one ofthe most used short term effects applied to comparatively rate the toxicity of nanomaterials may thusbe triggered with or without a direct involvement of alveolar macrophages. Accordingly it has been


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suggested that materials such as inhaled carbon black like nanoparticles are rapidly internalized bymacrophages, but do not cause any cytotoxic or pro inflammatory stimulation of these phagocytesitself, while other particles, like particular carbon nanotubes, cause cellular inflammasome activationand subsequent macrophage specific cytokine release.

We will discuss macrophage mediated differences between the acute inflammatory responses toparticles, and chronic, long term effects. While the first, acute pathway of inflammation may beresolved after sable isolation of inert nanomaterials, inside the phagocytes (clearance), the secondpathway of toxicity may involve a persistent vicious circle of nanotube phagocytosis mediatedmacrophage cytotoxicity and inflammatory macrophage recruitment.

The role of interleukin 1 cytokine in the fibrotic responses of multi walledcarbon nanotubes injected into the pleural cavity

Yke Jildouw Arnoldussen1, Vidar Skaug1, Mona Aleksandersen2, Erik Ropstad3, Kristine HaugenAnmarkrud1, Elin Einarsdottir1, Fang Chin Lin1, Cesilie Granum3, Mayes Kasem1, Einar Eilertsen1, Ron NApte4, Shan Zienolddiny11National Institute of Occupational Health, Norway; 22Department of Basic Sciences and Aquatic Medicine, NorwegianUniversity of Life Sciences, Faculty of Veterinary Medicine, P.O.Box 8146 Dep, 0033 Oslo,Norway; 33Department of ProductionAnimal Clinical Sciences, Norwegian University of Life Sciences, Faculty of Veterinary Medicine, P.O. Box 8146 Dep, 0033 Oslo,Norway; 44The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben Gurion University of the Negev, BeerSheva 84105, Israel; [email protected]

Despite numerous studies on the effects of exposure to multi walled carbon nanotubes (CNTs), there isinsufficient data on in vivo intra pleural effects. Here, fibroplasia in the pleura and the adjacent lungtissue of mice, and a role for interleukin 1 (IL 1) cytokines in mice following intra pleural injection ofthree CNTs and crocidolite asbestos were investigated. IL1 WT and IL1 / KO mice were injected withtwo doses of each fiber type in the pleural cavity and sacrificed after 4 or 24 weeks. Histopathologicalexaminations showed that CNTs had the highest effect on inducing fibroplasia, mesothelial cellproliferation and increasing leukocyte infiltration both in the pleura and lungs compared to asbestos.IL1 KO mice were significantly less prone to development of fibrosis and leukocyte infiltration than IL1WT mice. Genetic and epigenetic analysis of a panel of 84 fibrosis specific genes and miRNAs wasinvestigated in the pleura and lung tissue. CNTs had again the greatest impact on mRNA levels in thepleura. Furthermore, the effects were more significant in the pleura than in lung tissue. Specifically, upregulation of the genes Ccl12, Ccl3,Mmp9 and Timp1 and downregulation of Serpina1a, Timp4 and Bcl2were prominent, but between IL1 WT and IL1 KO mice there were only very few significant differences.Epigenetic analyses showed that the levels of miRNA were differentially regulated in the pleuracompared to the lung in the exposed animals, and was affected differently by the types of fibers. Inconclusion, CNTs induce fibrotic and inflammatory responses depending on the physico chemicalproperties of the CNTs, and the responses may not entirely depend on IL 1.

Inhalation toxicity of 5 – 10 nm cerium dioxide nanoparticles

Chang Guo1, Alison Buckley1, Martin Leonard1, Ralf Weber2, Sarah Robertson1, Tim Marczylo1, KirstyMeldrum1, Alan Hodgson1, Mark Viant2, Rachel Smith1

1Public Health England, United Kingdom; 2University of Birmingham, United Kingdom; [email protected]

Cerium dioxide nanoparticles (CeO2NPs) are used in a number of diesel fuel additives to improve fuelcombustion efficiency and exhaust filter operation, and they have been detected in ambient air in


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locations where such additives are in use. Concerns have been raised about the potential impacts onhuman health of inhaling CeO2NPs and an in vivo inhalation study has been undertaken to investigate.Male Sprague Dawley rats were exposed in a nose only inhalation exposure system to aerosolisedCeO2NPs (mass concentration 1.8 mg/m3, primary particle size 5 – 10 nm, aerosol count mediandiameter 40 nm) or water (controls) for 3 hours per day for 4 days per week for one or two weeks.Animals were sacrificed at 3 and 7 days post exposure. Bronchoalveolar lavage was undertaken andsamples of lung and other tissues generated. Analysis of total and differential cell type counts, LDH, TP,AP and cytokine release in bronchoalveolar lavage fluid, and lung histopathology indicates aninflammatory response greater than that seen in other studies using primarily micron sized CeO2NPagglomerates, thus emphasising the importance of particle size. Gene expression analysis of lung tissuealso confirms an inflammatory response. ICP MS analysis shows the presence of cerium in kidney andliver sections indicating translocation from the lungs with the potential for interference in biochemicalprocesses.


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Session 2B: Nanomaterial characterization in toxicology – Reactivity, dissolution, and biomolecule interactionsMonday 29 May, 16:30 – 18:00Location: Room 2Session chairs: Iseult Lynch & Trine Berthing

Nanoparticle behavior variability: sources, characterization needs, and bestpractices

Donald R Baer, Brian D ThrallPacific Northwest National Laboratory, United States of America; [email protected]

Nanoparticles of various types are of increasing research and technological importance in biological andother applications. In addition to fundamental research on properties and relevance to newtechnologies, questions of environmental effects and toxicity are receiving increased attention.Difficulties in the production and delivery of nanoparticles with consistent and well defined propertiesimpact both toxicity studies and technology development. These delivery problems appear in manyforms and have a variety of causes. Consistent with those of other researchers from around the world,we have found that the detailed history of particles, including the synthesis method, treatment aftersynthesis including time and nature of storage and the detailed nature of any sample processing ormodification can all influence particle behaviors. Some factors that influence particle behavior aredeliberate or intended while others occur as particles respond to time and their environment. Thetendency of particles to change with time or environmental condition highlights the importance of bothtimely characterization and the need for careful records[1]. Our studies involving ceria and silvernanoparticles will be used to highlight inherent characteristics of nanomaterials that explain some ofthe variations in toxicity studies as well as inconsistent results in other types of studies. We summarizemany of these effects with three simple statements: nanoparticles are not created equal; surfaces areimportant and often unknown; and particles are dynamic, not static [1, 2]. During the presentationimpacts of synthesis on the behaviors of ceria[3] and silver nanoparticles[4], the role of deliberate andenvironmentally induced surface coatings on particle behavior, and how particles vary with time indifferent environments will be demonstrated.

1. D. R. Baer, P. Munusamy, and B. D. Thrall, "Provenance information as a tool for addressing engineered nanoparticlereproducibility challenges," Biointerphases, vol. 11, p. 04B401, 2016.

2. D. R. Baer, et al., "Surface Characterization of Nanomaterials and Nanoparticles: important needs and challengingopportunities," Journal of Vacuum Science & Technology A, vol. 31, p. 050820 2013.

3. A. S. Karakoti, et al., "Preparation and characterization challenges to understanding environmental and biological impactsof ceria nanoparticles," Surface and Interface Analysis, vol. 44, pp. 882 889, 2012.

4. P. Munusamy, et al., "Comparison of 20 nm silver nanoparticles synthesized with and without a gold core: structure,dissolution in cell culture media, and biological impact on macrophages," Biointerphases, vol. 10, p. 031003, 2015.


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The importance of characterisation of bionanointeractions in NM (eco)toxicityassessment: new approaches and insights

Iseult LynchUniversity of Birmingham, United Kingdom; [email protected]

Nanomaterials (NMs) have a high surface energy that they seek to lower by binding to availablebiomolecules from their surroundings such as components of product formulations, proteins or lipids inliving systems, natural organic matter (NOM) components of water or soil or potentially exuded andsecreted biomolecules in complex ecosystems.1 3 Formation of a protein corona around NMs is aubiquitous phenomenon that occurs instantaneously upon contact with available macromolecules.Most research to date has focussed on the interactions of NPs with blood proteins (human or animalsera) or lung surfactant proteins, to correlate corona composition with NM uptake and impacts on livingsystems.2 However, the focus has typically been on relatively static systems, rather than on addressingthe interplay between the living system and the NMs and the effect of cellular secretions on theevolution of the NM corona.4 Even less work has investigated the potential for NMs to bind the secretedbiomolecules central to much of the plant and microorganism world, where secretion of biomoleculescan be a defensive response to repel insect attack, or an offensive habit to repel other incompatible orcompetitive plants.2

The impacts of biomolecule binding on NM transformation, including release of ions, dispersionstability, uptake and toxicity will be discussed. Data from a range of NMs, including polystyrene (modelparticles) with positive and negative surface charges, gold NMs and nanorods of different length andsurface charge, and organic inorganic perovskite particles widely used in solar panels will be presented,and from cellular systems (A549 cells) and whole organisms (Daphnia magna) as exemplars of thechallenges for in vitro toxicity and ecotoxicity assessment, respectively.

Finally, some recommendations for redesign of NMs characterisation and toxicity testing protocols toaccount for NMs interactions with their surroundings, and the dynamic responses of the cells ororganisms to the presence of NM, are provided.

Dissolution and transformation of Zn containing nanomaterials in artificial lungfluids

Aleksandr Stefaniak1, Diane Schwegler Berry1, Kelly Pisane21National Institute for Occupational Safety and Health, United States of America; 2West Virginia University, United States ofAmerica; [email protected]

Background: Zinc oxide (ZnO) is among the most widely exploited nanomaterials and has numerouscommercial applications. Studies indicate that the respiratory toxicity of ZnO nanomaterials isinfluenced by particle size, shape, and rapid release of ions. Despite the importance of dissolution inrelease of zinc ions, there is little understanding of the kinetics of ion release in the lung and interactionof dissolved material with lung fluid constituents.

Methods: Dissolution kinetics of well characterized ZnO (nanowires, nanoparticles, micronscalebenchmark particles) and Zn metal (nanoparticles) materials were evaluated in artificial extracellularairway lining fluid (ALF, pH 7.4) and artificial alveolar macrophage phagolysosomal fluid (PSF, pH 4.5).Additionally, the interaction of zinc ions with constituents of these lung fluid models were evaluated tounderstand the potential to form transformation products.


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Results: Average diameters of the ZnO nanowires, ZnO nanoparticles, and Zn nanoparticles were below100 nm. The benchmark ZnO material was a mixture of spheroidal (diameter 274±336 nm) and rodshaped particles (diameter 146±68 nm, length 587±259 nm). Values of specific surface area ranged from3.9 to 23.8 m2/g among materials. X ray diffraction analysis indicated that all ZnO materials werecrystalline zincite. Upon addition of zinc ions to ALF, solid transformation products formed immediately.Cysteine was the main reactant in ALF that yielded crystalline zinc/sulfur precipitates, though we couldnot identify the specific phases from a search of x ray diffraction reference spectra. When zinc ionswere added to PSF there was no evidence that any transformation products were formed. Hence,dissolution was only measured in PSF. On a mass basis, 14 to 55% of the zinc dissolved in 72 hours.Dissolution was biphasic for all materials. In the initial rapid phase, dissolution half times (t1/2) were 0.5to 1.8 days and dissolution rate constants (k) were about 2 x 10 5 g/(cm2 d) and in the latter slowerphase t1/2 ranged from 18 to 36 days and k from 1 to 7 x 10 7 g/(cm2 d). Values of t1/2 and k were shorterand faster, respectively for the ZnO benchmark material compared to all other materials.

Conclusions: These data support the hypothesis that the solubility of ZnO in the acidic environment ofphagolysosomes is an important component of the process underlying toxicity in tissues, cells andorganelles. A better understanding of nanomaterial characteristics that influence dissolution and toxicinteractions with the lung can be used to improve existing health protection efforts and in the design ofnewer safer nanomaterials.

Evaluation of intrinsic versus cell based oxidative properties of nanomaterialsand associated oxidative stress responses using multiple assays

Bryan Hellack1, Thomas AJ Kuhlbusch2,3, Sonja Boland4, Armelle Baeza Squiban4, Catrin Albrecht5, RoelPF Schins51Institute of Energy and Environmental Technology (IUTA) e.V., Duisburg, Germany; 2Federal Office for Occupational Safety andOccupational Medicine, Dortmund, Germany; 3CENIDE, University Duisburg Essen, Duisburg, Germany; 4University ParisDiderot (UPD) (Sorbonne Paris Cité), Unit of Functional and Adaptive Biology, UMR CNRS 8251, Paris, France; 5IUF LeibnizResearch Institute for Environmental Medicine, Düsseldorf, Germany; [email protected]

The surface of nanomaterials (NMs) and their ability to generate oxidants/reactive oxygen species(ROS), often referred also as Oxidative Potential (OP), are promising metrics to predict theirtoxicological potency. The evaluation and testing of this hypothesis formed the central component ofthe project nanOxiMet.

We investigated a representative panel of 16 NMs in suspensions (deionized water, cell culture media)regarding their OP by spin trap/probe based Electron Paramagnetic Resonance spectroscopy (EPR),dithiothreitol (DTT) and 2’,7’ Dichlorofluorescein acetate (DCF DA) assays as well as by measurement ofantioxidant depletion (i.e. glutathione (GSH) and ascorbic and uric acid) in synthetic lung lining fluid.

Cell based OP was analysed using similar approaches, i.e. fluorescence spectroscopy, EPR andmeasurement of GSH depletion in macrophage and epithelial cell lines. Oxidative stress responses wereaddressed by analysis of (oxidative) DNA damage, lipid peroxidation and protein oxidation in differentcell lines.

When comparing the main different intrinsic OP assays complementary results were found wherebytwo principle groups of ROS detection approaches could be specified: (1) assays being sensitive to(transition) metals (e.g. showing reactivity for Cu, Ni) and (2) assays sensitive to materials triggering


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electron transfer (e.g. reactive for Ag and Carbon Black). The antioxidant depletion assay could notclearly be assigned to one of these two groups.

The most suitable method to determine (intra)cellular ROS formation was fluorescence using DCFH DA.Comparison of the intrinsic OP and the cellular OP revealed the best correlation between thefluorescence based assays. Among the oxidative damage endpoints evaluated the Fpg modified cometassay was most sensitive. GSH depletion appeared particularly relevant since it allowed for a clearcategorisation of NMs into those that did not cause depletion, those that caused transient GSH loss andthose that triggered progressive GSH depletion.

NMs may generate oxidative stress via two principal pathways, i.e. due to their intrinsic OP and upontheir interaction with cells. In our hands, several NMs caused marked oxidative stress responses whilehaving low/negligible OP, whereas other NMs with a marked OP only triggered minimal oxidative stressin cells. In conclusion, intrinsic OP assays are useful tools to assess for the potential toxicity of NMs.However, cell based OP/ROS analyses should be included at least to minimal extent, to reducemisclassification.

Acknowledgement. The nanOxiMet project was supported by the Federal Ministry of Education andResearch (BMBF) and the French National Research Agency (ANR), under the ERA NET “SafeImplementation of Innovative Nanoscience and Nanotechnology (SIINN)” initiative.


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Keynote session II Tuesday 30 May, 9:00 10:00Location: Room 1 (Conference room)Session chairs: Tim Nurkiewicz & Gaku Ichihara

Developmental toxicity of nanomaterialsKarin Sørig Hougaard

National Research Centre for the Working Environment

Karin S Hougaard1,2, Morana Vitezic2, Mette Boyd2, Astrid Skovmand2, Petra Jackson1, Alexander CØJensen1, Sabina Halappanavar3, Keld A Jensen1, Ismo Koponen1, Håkan Wallin4, Albin Sandelin2, UllaVogel1,51National Research Centre for the Working Environment, Denmark; 2University of Copenhagen, Denmark; 3Health Canada,Canada; 4National Institute of Occupational Health, Norway; 5Tecnical University of Denmark; [email protected]

Testing of nanomaterials (NM) for effects on pregnancy and fetal development has been a latecomerwithin nanotoxicology. However, as more and more studies emerge so do indications that NM mayinterfere with developmental events. Thus several recent studies in rodents support the existence of anassociation between exposure to particles during pregnancy and induction of a broad range ofphysiological changes in the offspring. In studies of exposure applying the airway route of exposure,effects manifest mainly as functional alterations in, e.g., the central nervous, male reproductive,immune and cardiovascular systems. Several plausible toxicological mechanisms have been described.From the maternal lungs NM may translocate to the placenta and fetus and here exert their effectsdirectly. Alternatively, maternal airway inflammation may translate into low grade systemicinflammation, a potent modulator of pregnancy and fetal development. We have previously reportedthat maternal inhalation of particles change gene expression in liver from two day old mouse offspring.New data show that the placenta may indeed be a central organ in developmental toxicity – thus geneexpression in the placenta changed massively 24 hours after intratracheal instillation of TiO2 particles –and so did expression in fetal liver. While existing data suffice for generation of hypotheses, thetremendous variation in study designs and tested particles hampers generalization and risk assessment.There is a need for confirmatory studies using standardized study protocols and NM – do maternalairway exposure to NM in fact pose a risk to the central nervous, male reproductive, immune andcardiovascular systems? This would also allow for initial grouping of particle toxicity relative todevelopmental effects – does such a thing as a “particle pattern” exist? Interestingly, in contrast tomost NM, studies of diesel exhaust and particles often report effects on offspring weight, indicatingthat the latter does not owe to particulate fractions but rather to associated chemicals such as PAHs.Here it is an issue that developmental toxicity guideline protocols are often not well developed forassessment of functional deficits, such as cardiovascular reactivity and immune response to allergens.For developmental neurotoxicity, the guideline is established but may be difficult to apply in full.


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Session 3A: Reprotoxicity

Tuesday 30 May, 10:30 12:30Location: Room 1 (conference room)Session chairs: Flemming R Cassee & Karin S Hougaard

Engineered nanomaterial inhalation during gestation: Ramifications on uterinemicrovascular function, the fetal genome and cardiac transcriptome

Timothy Robert NurkiewiczWest Virginia University / School of Medicine, United States of America; [email protected]

Engineered nanomaterials (ENM) are firmly established in diverse human activities from surfacecoatings and ingredients in common consumer products, to additives in industrial processes, andcomponents of clinical applications. The risk for inadvertent ENM related health effects resulting fromintentional or unintentional introduction to the body is a complex function of ENM state and theexposure conditions. Notwithstanding this dynamic risk, the most common ENM exposure route acrosstheir life cycle is via inhalation. Despite their ubiquitous inclusion and projected proliferation in humanendeavors; the consequences of maternal ENM inhalation on the developing fetus and their impacts onfuture health are poorly understood. We and others have consistently reported that such exposures(typically via the lung) negatively impact cardiovascular function through a variety of direct and indirectmechanisms. Over the past three years of rat based inhalation studies, we have reported that maternalENM inhalation (nano titanium dioxide, Evonik P25, primary particle diameter = 21 nm, count modeaerodynamic diameter ~150 nm, 10 12 mg/m3, 4 5 hrs/day) during pregnancy (days 6 20) not onlyimpairs uterine microvascular function, but also creates a hostile gestational environment (1) thatmanifests as fetal mitochondrial and cardiovascular dysfunction (2) that persists into adulthood (3).Data and pathway analyses will be presented that identify specific dysregulation in the fetal epigenomeand transcriptome. These data will be supported over a continuum of cellular, tissue and whole animalexperiments. The impact of these apoptotic, hypertrophic, and developmental outcomes on futurecardiovascular health will be discussed.Support: NIH R01 ES015022

References1. Stapleton PA, Minarchick VC, Yi J, Engels K, McBride CR, Nurkiewicz TR. Maternal Engineered Nanomaterial Exposure and

Fetal Microvascular Function: Does the Barker Hypothesis Apply? Am.J Obstet.Gynecol. 209(3):227. e1 11. 2013.(PMC3757100)

2. Hathaway QA, Nichols CE, Shepherd DL, Stapleton PA, McLaughlin SL, Stricker JC, Rellick SL, Pinti MV, Abukabda AB,McBride CR, et al. Maternal Engineered Nanomaterial Exposure Disrupts Progeny Cardiac Function and Bioenergetics.Am.J.Physiol Heart Circ.Physiol. Dec 23. 2016. [Epub ahead of print] (PM28011589)

3. Stapleton PA, Nichols CE, Yi J, McBride CR, Minarchick VC, Shepherd DL, Hollander JM, Nurkiewicz TR. Microvascular andmitochondrial dysfunction in the female F1 generation after gestational TiO2 nanoparticle exposure. Nanotoxicology.9(8):941 51. 2015. (PMC4736545)


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Maternal exposure to diluted diesel engine exhaust alters placental function ofthe first and second generation in rabbit model

Anne Couturier Tarrade1,2, Sarah Valentino1,2, Delphine Rousseau Ralliard1,2, Marie ChristineAubrière1,2, Michèle Dahirel1,2, Marie Sylcie Lallemand1,2, Catherine Archilla1, Luc Jouneau1, EveMourier1, Christophe Richard1, Natalie Fournier3,4, Marine Guinot1,2, Josiane Aïoun1,2, SylvaineCamous1,2, Rémy Slama5, Véronique Duranthon1, Flemming Cassee6,7, Pascale Chavatte Palmer1,21UMR BDR, INRA, ENVA, Université Paris Saclay, 78350, Jouy en Josas, France; 2PremUp Foundation, Paris, France; 3Univ ParisSud, EA 4041/4529 Lip (Sys)2, UFR de Pharmacie, Châtenay Malabry, France; 4Hôpital Européen Georges Pompidou (AP HP),Laboratoire de Biochimie, UF Cardio Vasculaire, Paris, France; 5Inserm and Univ. Grenoble Alpes, U823, IAB Research Center,Team of Environmental Epidemiology Applied to Reproduction and Respiratory Health, Grenoble, France; 6Centre forSustainability, Environment and Health, National Institute for Public Health and the Environment, Bilthoven, Netherlands;7Institute of Risk Assessment Sciences, Utrecht University, Utrecht, Netherlands.; [email protected]

Atmospheric pollution is a rising concern in urban areas and a major contributor to prematuremortality. So far, little is known about the intergenerational effects of in utero exposure. Our aim was toevaluate the effects of exposure during pregnancy to diluted diesel exhaust (DE) on feto placentaldevelopment in the first (F1) and the second generation (F2).

Pregnant female rabbits (F0) were exposed to diluted and filtered DE (1mg/m3, nanoparticle (NP)diameter 69nm) (Exposed group) or clean air (Control group) for 2h/day, 5days/week by nose onlyexposure from 3 to 27 days postconception (dpc).

DE exposure induced early signs of growth retardation at mid gestation with decreased head length andumbilical pulse. Near term, fetal head length and plasma insulin and IGF1 concentrations were reduced,maternal and fetal biochemistry being otherwise undisturbed. Placental function was also affected, withreduced placental efficiency, decreased placental blood flow, fetal vessel and trophoblastic volume, thelatter being confirmed by the reduced concentrations of fatty acids in phospholipids, reflectingdecreased cellular membrane density. NP like structures were observed in maternal blood space, introphoblastic cells and in the fetal blood, demonstrating transplacental transfer.

F1 adult female offspring were bred with control males. Maternal F1 biochemistry was similar betweengroups although total hepatic neutral lipid (NL) contents were significantly increased in Exposed F1females, indicating hepatic steatosis. F2 fetoplacental biometry was not affected at 28dpc but fetalmetabolism was modified by grand dam exposure with decreased plasma cholesterol and increasedtriglyceride concentrations. In F2 placentas, total NL concentration was increased in the Exposed groupmostly in monounsaturated fatty acids while arachidonic acid (AA, pro inflammatory precursor) wasproportionally reduced. In F2 fetal plasma, the proportion of n 3 polyunsaturated fatty acids (precursorsof anti inflammatory molecules) was increased whereas AA was decreased in Exposed compared toControl fetal plasma.

Gene Set Enrichment Analysis (GSEA) analyses of placental transcriptomic data showed thatproteasome complex and ubiquitin pathways, involved in protein degradation, were enriched inExposed F2 placentas. On the opposite, ion channel and inflammation regulation pathways wereenriched in Control compared to Exposed F2 placentas.

In conclusion, repeated daily gestational exposure to DE can affect feto placental development in thefirst, but also affect placental function and offspring lipid metabolism in the second generation.


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Induction of behavioral changes and astrogliosis in the brain of offspring micefollowing maternal inhalation of carbon black nanoparticle

Atsuto Onoda1,2,3, Masakazu Umezawa1,4, Ken Takeda1, Alexander CØ Jensen5, Ismo K Koponen5, KeldA Jensen5, Ulla Vogel5,6, Karin S Hougaard5,7

1Research Institute for Science and Technology, Organization for Research Advancement, Tokyo University of Science, Noda,Chiba, Japan.; 2Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Tokyo University of Science,Noda, Chiba, Japan.; 3Research Fellow of Japan Society for the Promotion of Science, Chiyoda, Tokyo, Japan.; 4Department ofMaterials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Katsushika,Tokyo, Japan.; 5National Research Centre for the Working Environment, Copenhagen Ø., Denmark.; 6National Food Institute,Technical University of Denmark, Søborg, Denmark.; 7Institute of Public Health, University of Copenhagen, Copenhagen K,Denmark.; [email protected]

There is increasing concern that exposure to nanoparticle (NPs) during the sensitive stages ofdevelopment may predispose the developing brain to diseases later in life. However, available data onthe effects of maternal NP exposure in developmental neurotoxicity studies are still limited. Therefore,we examined outcomes related to the function of the central nervous system in the offspring followingmaternal inhalation exposure to carbon black nanoparticle (CB NP).

Time mated mice were exposed by inhalation 45 min/day to 0, 4.6 or 37 mg/m3 aerosolized carbonblack (Printex 90; 3.61×105 or 2.43×106 n/cm3; peak size: 70 120 nm) on gestation days 4 18. The brainswere collected from 6 week old mice offspring and subjected histopathological analysis. The behavioraltest was performed 12 weeks after birth.

In the open field test, maternal exposure to CB NP affected behavior in both male and female offspringcompared to controls. Exposed female offspring moved a longer total distance during the 3 min testperiod, and especially males spent longer time significantly in the central zone of the maze. Spendingmore time in the central part may be a sign of reduced anxiety or awareness. This type of effects maybe caused by a change in the developmental balance between excitatory (glutamatergic) and inhibitory(GABAergic) input in the basal brain areas, but also the noradrenergic system or neurosteroid releasemay be affected. Evaluation of neurochemical change by maternal inhalation of CB NP requires furtherstudies. Frozen sections of mouse brain were subjected into immunohistochemical GFAP visualizationwith PAS staining. Inhalation exposure of pregnant mice to CB NP dose dependently increased GFAP inthe astrocytes around blood vessels. In particular, GFAP expression was significantly up regulated in thecerebral cortex and hippocampus of the high compared to low dose and control groups. Enlargedlysosomal granules were also observed in perivascular macrophages of the maternal CB NP exposuregroups compared to air controls. These results indicate that maternal inhalation exposure to CB NPinduced perivascular abnormalities including histopathological changes in perivascular macrophagesand reactive astrogliosis in the cerebral cortex and hippocampus of their offspring.

Our results demonstrate the need to find means of preventing and controlling the developmental effectof maternal NP exposure on the developing brain.


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Pulmonary exposure to metallic nanoparticles during pregnancy irreversiblyimpairs lung development of the offspring

Emmanuel Paul1, Marie Laure Franco Montoya1, Erwan Paineau2, Jérôme Rose3, Jorge Boczkowski1,Christophe Delacourt1, Jean Claude Pairon1, Sophie Lanone11IMRB Inserm U955, France; 2Laboratoire de Physique des Solides, UMR CNRS, France; 3CEREGE, France;[email protected]

Due to the growing commercial applications of manufactured nanoparticles (NP), knowledge on thetoxicity of those NP is of great importance. It has been shown that pulmonary exposure of adult mice toNP can lead to an inflammatory response, together with the development of a granuloma and/orpulmonary fibrosis. However less is known on the effect of such exposure to NP during the criticalwindow of lung development, although it could represent a major public health issue.

Therefore, the aim of this study is to assess the impact of respiratory exposure to various NP duringpregnancy, on the lung development of the offspring, and to determine the key parameters involved inthe observed lung alterations. Pregnant mice were weekly exposed to 100 μg (total dose 300 μg) oftitanium dioxide (TiO2), cerium oxide (CeO2), silver (Ag) NP or saline solution by nonsurgicalintratracheal instillation. The offspring lungs were analyzed at different stages of lung development:fetal stage (gestational day 17.5), pulmonary alveolarization (post delivery day 14.5), and lung maturity(post delivery day 49.5) with the following end points: fetotoxicity, histological, morphological,functional analysis of the lungs, inflammatory mediators production (q PCR, Luminex). The presence ofNP in placenta and fetal lungs were detected by ICP MS, micro X Ray, and micro XANES fluorescence.

Results showed that, regardless of the type of NP, maternal exposure during gestation induced a longlasting impairment of lung development in the offspring. This effect was accompanied by: i) decreasedplacental efficiency together with the presence of NP in the placenta, ii) no increase of inflammatorymediators present in amniotic fluid, placenta or offspring lungs, and iii) decreased pulmonaryexpression of vascular endothelial growth factor (VEGF ) and matrix metalloproteinase 9 (MMP 9) atthe fetal stage, and fibroblast growth factor 18 (FGF 18) at the alveolarization stage.

In conclusion, our study demonstrates that respiratory exposure to metallic NP during pregnancyinduces stereotyped impairment of lung development with a lasting effect in adult mice, independentlyof the chemical nature of the NP.

In utero exposure to ultrafine particles induces 11 hydroxysteroiddehydrogenase type 2 related placenta stress in mice

Russell Morales Rubio1, Marisela Uribe Ramirez1, Jessica Monroy Cruz2, Daniela Gomez Colin1,Andrea De Vizcaya Ruiz11Departamento de Toxicologia, Cinvestav, Mexico; 2Escuela Superior de Ingenieria Química e Industrias Extractivas, IPN,Mexico; [email protected]

Exposure to incidental nanoparticles or ultrafine particles (UFP) with an aerodynamic diameter of 100nm (from anthropogenic sources: fossil combustion activity and vehicular traffic) during sensitivedevelopmental stages may predispose to disease later in life. In addition, epidemiological studiessuggest that exposure to airborne particulate matter is associated with adverse gestational outcomes,such as premature birth, reduced birth number, and small size for gestational age. Moreover, placentainsufficiency or stress may influence these adverse outcomes. The placental 11 hydroxysteroid


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dehydrogenase type 2 (11 HSD2) enzyme protects the fetus from excess maternal cortisol maintaininga normal pregnancy. Maternal stress conditions induced from UFP exposure either by directtranslocation to placenta or indirect toxic mechanisms affects the levels of placental 11 HSD2, and canlead to increased fetal cortisol concentrations and contribute to adverse gestational outcomes. Thisstudy aimed to investigate if in utero exposure to UFP affects gestation, through the evaluation ofplacental 11 HSD2 enzyme, fetal cortisol concentration and gestational outcomes in mice, as an UFPexposure hazard endpoint. First, we collected and characterized UFP from ambient air using an aerosolconcentrator. Afterwards, pregnant female C57Bl/6J mice were exposed to sterile H2O (sham) or UFP (insterile H2O) by intratracheal instillation during gestation. Dams received UFP in suspension (2 μg/40μl/dose, or 400 μg/kg accumulated dose) at day 6.5, 8.5, 10.5, 12.5, 14.5 y 16.5 of pregnancy orembryonic day (E). At embryonic day 17.5, uterus, placenta and fetuses were collected to measureweight and evaluate gestation parameters. Cortisol was measured in maternal serum and whole proteinfetus by ELISA, and levels of the placental 11 HSD2 were measured by Western blot. Our results showthat the collected UFP exhibited nanometric diameters (60%: <100 d nm) and spherical morphology,measured by DLS and SEM. We observed an increase in embryo reabsorption, and decreased weight inuterus, placenta, and fetuses, that was statistical significant compared to the sham group. Importantly,serum cortisol levels in dams and fetus were increased as well as a decrease in placental 11 HSD2levels. Fetal cortisol increase together with the reduction in placental 11 HSD2 levels suggest that inutero exposure to UFP induces placenta stress leading to placental dysfunction, which could contributeto alterations during developmental stages and also predispose to diseases in adulthood.

Effects of pulmonary exposure to nano particles on male reproductive function

Astrid Skovmand1,2, Anna Lauvås2, Preben Christensen3, Ulla Vogel1, Karin S Hougaard1, SandraGoericke Pesch2

1National Research Center for the Working Environment, Denmark; 2University of Copenhagen, Department of VeterinaryClinical Sciences, Section of Veterinary Reproduction and Obstetrics; 3SPZ Lab A/S; [email protected]

Recent findings indicate that exposure to nanosized particles adversely affect male reproductivefunction. Particles potentially affect reproductive parameters in several ways: When inhaled, particlesare potent inducers of pulmonary inflammation, which leads to the release of inflammatory mediators.Small amounts of particles may translocate from the lungs into the lung capillaries, entering thesystemic circulation and ultimately reaching the testes. Both the inflammatory response and theparticles may induce increased production of reactive oxygen species and the deriving oxidative stresscan directly impair spermatogenesis and increase sperm DNA damage. Furthermore, spermatogenesismay be indirectly affected by a change in the hormonal milieu, as systemic inflammation is a potentialmodulator of endocrine function. The aim of this study is to investigate the effects of a pulmonaryexposure of nanoparticles on male reproduction function, focusing mainly on semen quality, in anexperimental mouse model. NMRI mice were exposed once weekly to either graphene oxide, 2different carbon black nanoparticles Flammruss 101 and Printex 90, or diesel exhaust particleSRM1650b via intratacheal instillation for 7 consecutives weeks (~one spermatogenic cycle in mice).Three control groups were added to the experimental design: a vehicle control, a unhandled and apositive control on high fat diet. Pulmonary inflammation levels were determined by differential cellcount in bronchoalveolar lavage fluid. Epididymal sperm concentration, motility and viability wereassessed using a computer assisted sperm analysis system. Sperm morphology was assessed manually.The Sperm DNA Integrity test was performed to determine the percentage of sperm with damaged DNA(DFI). Daily sperm production was measured in the testis and testosterone levels were measured inplasma. Macrophage and neutrophil numbers in the bronchoalveolar lavage fluid indicate aninflammatory response in the particle exposed group, but no response in the controls as expected.Preeliminary statistical analysis suggest that there was no significant changes in the epididymal sperm


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concentration, motility and viability. Morphological abnormalities were unchanged amongst all groups.In addition, no significant changes were found in the daily sperm production, DNA integrity, or plasmatestosterone levels. In conclusion, a 7 week exposure to graphene oxide, flammruss 101, printex 90 andthe diesel SRM1650b does not appear to affect semen quality in the NMRI mouse.


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Session 3BI: Toxicity of soluble nanomaterials Tuesday 30 May, 10:30 11:30Location: Room 2Session chairs: Hanna Karlsson & Andrea Hartwig

Soluble nanoparticles – toxicity, dissolution testing and risk assessment

Hanna KarlssonKarolinska Institutet, Sweden; [email protected]

The bio solubility (dissolution) of nanoparticles has emerged as a critical parameter to determine in riskassessment, not least due to the fact that poorly soluble particles are more likely to accumulate in thelung following inhalation. Although being less prone to accumulate, several partly soluble nanoparticleshave shown to be more acute toxic. By using reporter cell lines, we have compared the (geno)toxicity of24 different nanomaterials and demonstrated reporter cell activation and toxicity mainly followingexposure to the partly soluble materials. In this presentation, results related to toxicity of various partlysoluble nanoparticles including CuO, NiO and silver nanoparticles will be discussed. For suchnanoparticles the so called “Trojan horse” effect is believed to be of importance. This means that thecellular uptake is facilitated by the particle structure but once inside the cells, the intracellularenvironment, such as acidic lysosomes, cause nanoparticle dissolution and release of toxic metal ions.There may, on the other hand, also be mechanisms involved not related to a high cell uptake. In a studyusing BEAS 2B cells, in which the genotoxicity of Ni and NiO nanoparticles as well as ionic nickel (NiCl2)was compared, we showed chromosomal damage for all exposures despite a lack of cell uptake of theNiCl2. Our results indicate involvement of calcium dependent mechanisms.

For assessing nanoparticle dissolution, many studies to date have analyzed the release of ions in cellmedium or in other fluids mimicking the extra or intracellular environment. Fewer studies haveattempted to study dissolution quantitatively in the presence of cells. In a recent study we investigatedsize dependent dissolution of gold nanoparticles in the presence of macrophages. We coulddemonstrate that in particular if the macrophages were triggered using LPS (lipopolysaccharide), aremarkable dissolution of the smallest gold nanoparticles took place. Such information is likely usefulfor understanding the biological fate of gold nanoparticles as well as other “poorly soluble” particles.Indeed, assays involving cells as well as assays simulating dissolution in the air blood barrier may beimportant to better assess bio solubility of (nano)particles in the lung.

Use of gene expression profiling to assess the impact of partly soluble metalbased nanoparticles on genomic stability

Andrea Hartwig, Bettina M. Strauch, Mathias HufnagelKarlsruhe Institute of Technology (KIT), Germany; [email protected]

Metal based nanoparticles are increasingly applied as catalysts or antimicrobial additives. This increasesthe risk of adverse health effects, since toxic metals may be released, and even essential trace elementsmay be toxic under overload conditions. Within our study, we investigated copper oxide nanoparticles(CuO NP) with regard to solubility and intracellular bioavailability of copper ions, cytotoxicity, direct andindirect genotoxicity as well as the modulation of signaling pathways in different cell lines and


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compared the effects to those evoked by the corresponding microparticles (CuO MP) and copperchloride (CuCl2). CuO NP and CuCl2 caused a pronounced and dose dependent cytotoxicity in differentcell lines, whereas CuO MP exerted no cytotoxicity based on the same copper content; cell deathinduced by CuO NP was at least in part due to apoptosis. Only CuO NP induced significant amounts ofDNA strand breaks in HeLa S3 cells, whereas all three compounds elevated the level of H2O2 inducedDNA strand breaks. All copper compounds diminished the H2O2 induced poly(ADP ribosyl)ation, butagain, CuO NP exerted the strongest effect. With respect to the intracellular bioavailability anddistribution, copper derived from CuO NP, CuO MP and CuCl2 accumulated in cytoplasmic extracts andin case of CuO NP and CuO MP also in the nucleus; however, highest copper concentrations in bothcompartments were observed after incubation with CuO NP. Gene expression analysis via highthroughput RT qPCR were conducted in the immortalized human bronchial epithelial cell line BEAS 2Band the human lung adenocarcinoma cell line A549. CuO NP affected multiple signaling pathways likemetallothionein induction, cell cycle regulation, apoptosis, inflammation, oxidative stress response aswell as DNA damage response more pronounced than their microscale counterparts and CuCl2. Similarreactions were also observed after incubation at the air liquid interface. The modulation of cellularsignaling by the nanoparticles seems to be evoked both by elevated levels of ROS and particlemembrane receptor interactions. With respect to direct and indirect genotoxicity, especially the highcopper content in the cell nucleus derived after cell treatment with CuO NP appears to be decisive formost pronounced effects.

ReferenceSemisch A, Ohle J, Witt B and Hartwig A. Cytotoxicity and genotoxicity of nano and microparticulate copper oxide: role ofsolubility and intracellular bioavailability. Part Fibre Toxicol, 11, 10 (2014).


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Session 3BII: Ecotoxicity – closing gaps Tuesday 30 May, 11:30 12:30Location: Room 2Session chairs: Dana Kühnel & Anders Baun

Metal uptake and distribution in the zebrafish (Danio rerio) embryo: differencesbetween nanoparticles and metal ions

Steffi Böhme1, Marta Baccaro1, Matthias Schmidt1, Annegret Potthoff2, Hans Joachim Stärk1, ThorstenReemtsma1, Dana Kühnel11Helmholtz Centre for Environmental Research UFZ, Germany; 2Fraunhofer IKTS, Dresden, Germany; [email protected]

There is in general little knowledge on the internalization of nanomaterials (NM) in environmentalorganisms, and how internal concentrations of NM relate to toxic effects. Hence, this contributionpresents recent results on metal internalization and distribution in zebrafish (Danio rerio) embryo, aswell as on the underlying kinetic processes. To shed light on metal ZFE interaction, the uptake ofdifferent metal composed nanoparticles (Ag , Au , CuO , ZnO NP) and their dissolved cations wasstudied in terms of temporal and spatial distribution as well as internal concentrations consideringdifferent zebrafish embryo compartments (whole egg, embryo, chorion, perivitelline space).

The quantification of uptake of metal containing nanoparticles by embryos was performed by digestionof the organic material to dissolve the particles, followed by Inductively Coupled Plasma MassSpectrometry (ICP MS) as a robust element detection method 1. By applying laser ablation ICP MS andelectron microscopy, element specific differences in metal distribution at the surface and within thetest organisms were observed. As determined by nebulization ICP MS, all metals showed abioconcentration in the eggs. Gold showed the highest accumulation, followed by silver, zinc andcopper, with distinct differences when comparing the uptake of nanoparticles and metal ions. Uponexposure to nanoparticles, more silver and zinc was detected in the organisms, whereas more copperaccumulated upon exposure to the cations. For silver, gold, and copper, the major share was found toaccumulate at the chorion structures, irrespective of exposure to NPs or cations. In contrast, for zinc thehighest portion reached the inner embryo.

In summary, our results show that the type of metal seems to be crucial for the amount of metalassociated to the zebrafish eggs, but also the metal form the organism is exposed to, NM or cation,plays a role. We demonstrate an element specific distribution of metal over the different zebrafishembryo compartments. As demonstrated by the internal concentrations, a bioconcentration of allmetals inside the embryo was observed, irrespective of exposure to NM or cations2.

1. Bohme, S.; Stark, H. J.; Kuhnel, D.; Reemtsma, T., Exploring LA ICP MS as a quantitative imaging technique to studynanoparticle uptake in Daphnia magna and zebrafish (Danio rerio) embryos. Analytical and Bioanalytical Chemistry 2015,407, (18), 5477 5485.

2. Bohme, S.; Baccaro, M.; Schmidt, M.; Potthoff, A.; Staerk, H. J.; Reemtsma, T.; Kuehnel, D., Metal uptake and distributionin the zebrafish (Danio rerio) embryo: differences between nanoparticles and metal ions. Environmental Science: Nano2017. DOI 10.1039/C6EN00440G


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Ecotoxicity testing of nanoparticles – The quest for disclosing the nano effect

Anders Baun, Lars M Skjolding, Sara N Sørensen, Rune Hjorth, Steffen F Hansen, Nanna B HartmannTechnical University of Denmark, Denmark; [email protected]

While the literature on ecotoxicological effects and uptake of ENPs is rapidly expanding, theapplicability of reported data of ENPs for hazard assessment purposes is questionable. A majorknowledge gap is whether nanoparticle effects occur when test organisms are exposed to ENPs inaquatic test systems. This knowledge gap is not straightforward to fill, due to the high variability in ENPtypes, and the different behavior of ENPs compared to "ordinary" (dissolved) chemicals in theecotoxicity test systems. The risk of generating false negative, as well as false positive, results in thecurrently used tests is high, but in most cases difficult to assess. Based on a literature review, thispresentation outlines some of the pitfalls in aquatic toxicity testing of ENPs which may lead tomisinterpretation of test results. In this paper, we are describing the effect of nanoparticles towardsaquatic organisms. The presentation revolves around three response types that are not directly relatedto the nanoparticle per se, but which can mask that nanoparticle effects can be quantified. Theseresponse types are1. Physical effects caused by attachment of nanoparticles,2. Dissolution of nanoparticles leading to toxic effects corresponding to that of ions, and3. Uptake, internalization and translocation of nanoparticles.

We further propose that already known toxic modes of action should be carefully characterized andquantified before claiming that novel nanoparticle effects have been discovered.


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Session 4A: New hazard endpoints of nanomaterial exposure

Tuesday 30 May, 13:30 15:30Location: Room 1 (Conference room)Session chairs: Roel Schinz & Peter Møller

Telomere length as a new hazard measure in nanoparticle toxicology

Peter Møller1, Martin Roursgaard1, Ditte M Jensen1, Ana CD Gouveia1, Ismo K Koponen2, Steffen Loft11University of Copenhagen, Denmark; 2National Research Centre for the Working Environment; [email protected]

Telomere shortening is an important aspect in cellular senescence and aging. Interestingly, oxidativestress is implicated in the aging process as well as being an important mechanism of action ofparticulate matter (PM) toxicity. Certain molecular epidemiological studies have shown associationsbetween air pollution exposure and short telomere length in leukocytes from humans. Wehypothesized that exposure to PM is associated with telomere shortening in animal organs and culturedcells. Telomere length was assessed in two different animal studies after exposure to PM. In the firststudy, ApoE knockout mice were exposed to combustion particles from burning candles by i.t.instillation (0.5 or 5 mg/kg once a week for 5 weeks). Both of the doses were associated with anapproximately 50% shorter telomere length in the spleen of mice (p<0.05), which also had slightlyincreased level of atherosclerosis. In the second study, lean Zucker rats were exposed to food gradetitanium dioxide (E171) or vegetable carbon (E153) once a week for 10 weeks. The exposure tovegetable carbon was associated with a 25% shorter telomere length in lung tissue (p<0.01), whereasthe levels were not statistically significantly different in the liver (18% decrease) and spleen (2%decrease). A cell culture experiment demonstrated that lung epithelial A549 cells had approximately50% shorter telomeres after 72 h exposure to NM400 MWCNTs (Borghini et al., Mutagenesis 32: 173180, 2017). The results demonstrate an association between exposure to different types of particle andtelomere length. The mechanisms of action of particle induced telomere shortening have not yet beenresolved. Nevertheless, telomere shortening might be an important new hazard and mechanism ofaction of particle induced toxicity in cells and tissues.

Effect of age, diet, rat strain, and particle exposure on lung and peripheralblood mononuclear cell (PBMC) telomere regulation and length

Mohammad Shoeb, Aaron Erdely, Jenny Roberts, Vasmsi Kodali, Pius Joseph, Gul Mustafa, BreanneFarris, Christina Umbright, Patti Zeidler Erdely, James AntoniniNIOSH, Morgantown, WV, United States of America; [email protected]

The exposome measures all exposures to an individual in a lifetime and their impact on health.Successful ‘mapping’ of the exposome could substantially improve the understanding of diseasemechanisms and facilitate the development of prevention strategies. Lifestyle (e.g., diet) and relevantoccupational and environmental exposures are linked with specific internal biological endpoints. In a ratmodel, we investigated the influence of genetic background, diet, age and particle exposure onregulation and length of telomeres, the protective ends of each chromosome. To assess geneticcontributions and diet, male rats from three different strains [Sprague Dawley (SD), Fischer 344 (F344),and Brown Norway (BN)] were maintained on a high fat Western (HF) or standard diet for 24 wk. Toassess particle effects, SD rats were exposed to stainless or mild steel welding fumes (2 mg/rat by


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intratracheal instillation) and F344 rats were exposed to silica (15 mg/m3 x 6 h/d x 5 d). Lung tissue andPBMCs were collected at baseline, and at multiple times up to 44 wk after particle exposure and up to24 wk after initiating the HF diet for analysis of telomere regulatory protein expression and relativetelomere length. Interestingly, relative telomere length in PBMCs decreased in the HF diet groupcompared to the standard diet group at 12 wk for SD; 4, 12, and 24 wk for the F344; and 12 and 24 wkfor the BN strain. Relative telomere length decreased in PBMCs at 12 and 24 wk compared to baselinein all strains, indicating an age related effect on telomere shortening. Stainless steel, but not mild steel,welding fume significantly increased telomere length in PBMCs up to 30 d after a single lung exposurecompared to control. Similarly, significant increases in telomere length and altered expression ofmultiple telomere regulatory proteins were observed in the lung of silica exposed animals at 3, 36, and44 wk compared to air controls. Thus diet, age, and lung particle exposure, important components ofthe exposome, caused telomere length changes in PBMCs and lung from rats with diverse geneticbackgrounds. Importantly, this study indicated that proteins involved in telomere regulation and lengthare potential biomarkers of occupational and environmental exposures and their alterations may offerinsight into the molecular mechanisms of particle induced pulmonary pathology.

Cellular senescence induced by combustion derived nanoparticles inendothelial cells and lung epithelial cells

Klaus Unfried1, Niloofar Ale Agha1, Tim Spannbrucker1, Christine Goy1, Nadine Dyballa Rukes1,Joachim Altschmied1, Judith Haendeler1,21IUF Leibniz Research Institute for Environmental Medicine, Germany; 2Clinical Chemistry, University of Duesseldorf, Germany;klaus.unfried@uni duesseldorf.de

Particulate air pollution in humans causes adverse health effects primarily in the airways and thecardiovascular system. In these organs, typical age associated diseases like chronic obstructivepulmonary disease, idiopathic fibrosis, and atherosclerosis are induced by chronic exposure toenvironmental particles. As a major feature of aging, cellular senescence might be responsible for thedevelopment of these degenerative diseases after exposure to particulate air pollution. Our earlierstudies demonstrated that carbon nanoparticles as model particles for combustion derivedenvironmental particles trigger intracellular oxidative stress in exposed cells. Besides eliciting particlespecific signalling pathways, oxidative stress might be responsible for the induction of cellularsenescence.

We therefore investigated whether combustion derived carbon nanoparticles trigger senescencerelated molecular changes in lung epithelial cells, in primary human endothelial cells, and in vivo inexposed animals. For this purpose cultured cells and mice or rats were subjected to short term as wellas repetitive exposure with non cytotoxic doses of carbon nanoparticles. Cell type specific molecularchanges occurring during senescence and responsible for the development of diseases were monitored.Short term exposure to carbon nanoparticles increased the formation of intracellular reactive oxygenspecies and decreased mitochondrial telomerase activity in lung epithelial cells and in endothelial cells.Under theses conditions in endothelial cells, activation of the endothelial NO synthase (eNOS) andbioavailability of nitric oxide were reduced. Repetitive exposure to carbon nanoparticles for two weeksinduced senescence in both cell types measured by increased p21 protein levels and senescenceassociated beta galactosidase. Moreover, the protein levels of eNOS were decreased by more than 50 %in EC. In lung epithelial cells a reduction of the regenerative capacity due to this treatment wasobserved. Thus, carbon nanoparticles induce a senescent phenotype ex vivo. To investigate whetherthese particle effects also occur in vivo, we applied carbon nanoparticles in non inflammatory dosesinto mice. Indeed, eNOS expression was reduced in the abdominal aorta of particle treated animals. As


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a further feature of the degenerative effects, the adverse influence of carbon nanoparticles on cell cellcommunication was observed in both cell types ex vivo and in vivo.

The data demonstrate that carbon nanoparticles reduce cellular functions of the pulmonary andvascular systems and, thus, could serve as an environmental factor, which precludes healthy aging.

Acknowledgment: The work leading to these results has received funding from the European UnionSeventh Framework Programme (FP7/2007 2013) under grant agreement n° NMP4 LA 2013 310451and the German Federal Ministry of Education and Research (BMBF/InnoSysTox Verbund 031L0020A).

Effects of nanoparticles in the 5xFAD mouse model of Alzheimer’s Disease

Roel PF Schins1, Tina Wahle1, Adriana Sofranko1, Harm J Heusinkveld2, Flemming R Cassee2,3, CatrinAlbrecht11IUF Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany; 2Institute for Risk Assessment Sciences(IRAS), Utrecht, The Netherlands; 3National Institute for Public Health and the Environment (RIVM), Bilthoven, TheNetherlands; roel.schins@uni duesseldorf.de

There is growing concern that long term exposure to ambient ultrafine particles may contribute to thedevelopment and progression of age related neurodegenerative diseases like Alzheimer`s Disease (AD).To address this hypothesis, we have employed the transgenic 5X Familial Alzheimer's Disease (5XFAD)mouse model, with evaluation of effects on working memory and motor function as well as onhippocampal and cortical amyloid plaque load. We previously showed that inhalation exposure todiesel engine exhaust (DEE) causes accelerated plaque formation and motor function impairment inthese mice. DEE represents a major source of ultrafine particles in urban environments, thus suggestingthat exposure to manufactured nanoparticles may also contribute to AD pathogenesis. However, inlong term exposure studies with CeO2 and SiO2 nanoparticles (by inhalation or oral exposure), we didnot observe significant aggravation of AD like features in the 5xFAD mouse model. The observeddifferences can be explained by contrasting physicochemical properties of the nanosize particles as wellas the dose and route of exposure. Moreover, the findings from the DEE inhalation study warrantfurther investigation on the specific contributions of the particulate versus non particulate componentsof this pollutant mixture.

Integrated in vitro neurotoxicity testing: an approach to improve hazardassessment of inhaled substances

Harm J Heusinkveld1, Flemming R Cassee2, Roel PF Schins3, Remco HS Westerink11Institute for Risk Assessment Sciences (IRAS), Utrecht, The Netherlands; 2National Institute for Public Health and theEnvironment (RIVM), Bilthoven, The Netherlands; 3Leibniz Research Institute for Environmental Medicine (IUF), Düsseldorf,Germany; [email protected]

To comply with international regulations such as REACH, a vast amount of toxicity testing is required.Dedicated mode of action specific endpoints are valuable tools to reveal the neurotoxic potential ofcompounds. Current neurotoxicity testing still relies heavily on expensive, time consuming and ethicallydebated in vivo animal experiments that are unsuitable for high throughput testing. A more advanced invitro approach should be developed for which the data are highly appreciated in risk assessment. Tocapture the complexity of (disturbance of) neuronal function in a highly sensitive, predictive andreproducible manner, it is important that the in vitro system relies on a set of functional endpoints such


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as neurotransmission and disturbance of neuronal network function, rather than crude measures likecytotoxicity.

Micro electrode array (MEA) recordings provide a novel approach to measure neuronal networkfunctionality with high throughput. Using this technique, the effects of both acute and chronic (>21days) exposure to (inhalable) compounds on spontaneously active neuronal networks can be assessed.Rat primary cortical cultures grown on MEAs consist of excitatory and inhibitory neurons as well assupporting cell types (e.g. astrocytes and glia) that cover a wide range of targets, including ion channelsand neurotransmitter receptors. The MEA approach has been used as an in vitro alternative forscreening of marine neurotoxins, insecticides and drugs of abuse and demonstrated high sensitivity(>90% of the neuroactive compounds were correctly identified). As exposure via dissolution ordispersion of compounds in medium not necessarily resembles a realistic exposure to inhaled chemicalsincluding nanomaterials, we combined an air liquid interface (ALI) exposure system with measurementsin the MEA to assess neurotoxicity following inhalation exposure. This tandem was used to test theneurotoxicity of pyrolysis products of (aviation) oil. The results demonstrate that it is possible tocombine these two novel techniques to assess neurotoxicity of (small) inhalable compounds.

Considering the observation that neurotoxicity of nanoparticles likely involves the interplay betweendifferent cell types, the MEA provides a particularly suitable platform. Therefore, the projectN3rvousSystem aims at comparing the effects of acute and (sub)chronic exposure to nano TiO2 andnano Ag2O to dedicated in vivo neurotoxicity studies to assess the applicability of the MEA forneurotoxicity testing of nanomaterials.

This work was funded by the National Centre for the Replacement, Refinement and Reduction ofAnimals in Research (NC3Rs; project number 50308 372160) and the Netherlands Organisation forHealth Research and Development (ZonMW; InnoSysTox project number 114027001).

Thrombotic potential of intravenous engineered nanoparticles

Jennifer Bridget RaftisUniversity of Edinburgh, United Kingdom; [email protected]

Aims: Engineered nanoparticles, developed to facilitate imaging of atherosclerosis and as a drugdelivery tool, share properties with combustion derived particles in air pollution that have beenassociated with thrombosis and acute myocardial infarction. We hypothesize that engineerednanoparticles will exert similar pro thrombotic effects in man.

Methods:We assessed the effects of ultra small superparamagnetic iron oxide nanoparticles onplatelet activation, aggregation, and thrombus formation using flow cytometric techniques and theBadimon chamber (an ex vivomodel of thrombosis). In a randomised, double blind, cross over study 12healthy volunteers received saline control or ultra small superparamagnetic iron oxide nanoparticles(150μg/mL & 300μg/mL) added to whole blood in the extra corporeal circuit of the Badimon chamber.In 8 patients with abdominal aortic aneurysms platelet activation and thrombus formation weremeasured both before and 1 hour after intravenous delivery of ultra small superparamagnetic ironoxide nanoparticles (4 mg/kg) bolus injection.

Results: In healthy volunteers extra corporeal infusion of ultra small superparamagnetic iron oxidenanoparticles at 300μg/mL increased thrombus area compared to saline control (16,916 ± 1,766 μm2,versus 11,678 ± 1,045 μm2; P=0.001), but not at 150 μg/mL. In patients, intravenous bolus injection of


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ultra small superparamagnetic iron oxide nanoparticles increased thrombus formation compared tobaseline (27,670 ± 709 μm2versus 12,156 ± 1156 μm2; P=0.006), and increased platelet p selectin andCD36 expression (P<0.05 for both) at 1 hour.

Conclusions:We demonstrate that engineered nanoparticles cause platelet activation and haveprothrombotic effects in healthy persons and patients with vascular disease. The clinical consequencesof these findings are unknown but warrant further investigation.

Acute fine particulate matter exposure reduces heart rate variability in roadmaintenance workers

Michael Riediker1,2,3, Yannick Franc4, Valentin Rousson4, Reto Meier5, Murielle Bochud4, WayneCascio6

1IOM Singapore, Singapore; 2Nanyang Technological University, Singapore; 3Institut de Santé au Travail, University of Lausanne,Switzerland; 4Institut universitaire de médecine sociale et préventive, Université de Lausanne, Switzerland; 5Federal Office forthe Environment, Bern, Switzerland; 6Environmental Public Health Division, NHEER, ORD, U.S. EPA, Chapel Hill, NC, UnitedStates; michael.riediker@iom world.sg

Heart Rate Variability (HRV) reflects the adaptability of the heart to internal and external stimuli.Reduced HRV is a predictor of post infarction mortality. We previously found in road maintenanceworkers HRV increases several hours after exposure to fine particulate matter (PM2.5), interpreted as arecovery response.

Here we assessed the within an hour effects of PM2.5 exposure onto HRV in the same population. Onfive non consecutive days, workers carried nephelometers providing 1 minute interval PM2.5 exposure.Five minute HRV intervals (SDNN and PNN50) were extracted from 24 hour electrocardiograms.Analyses were done for time at work, at home and during the night. Following 60 minutes PM2.5

exposure intervals, the changes in HRV parameters were assessed during 120 minute intervals visuallyand by moving average method accounting for autocorrelation.

Correlation analysis showed a significant association of decreasing SDNN and PNN50 with increasingPM2.5 exposure. When stratifying for daytime, most associations remained significant. Short term PM2.5

exposure was followed by reduced HRV. This may pose a health risk in particular to people having preexisting cardiovascular risk factors such as previous infarction or arrhythmic disorders. The role thatultrafine particles play in this response will need to be assessed in future analyses.


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Session 4B: Young researcher grants

Tuesday 30 May, 13:30 15:30Location: Room 2Session chairs: Michael Riediker & Sarah Søs Poulsen

Due to the generosity of our sponsors, we have been able to provide 5 young researchers with aconference grant. The five grant awardees are presenting their projects in this session. Finally thesession includes an oral presentation on the legal perspectives of nanotechnology.

The presentations will be repetitions of contributions in other sessions where they thematically belong:

P54 ElineVandebroek

NanoStreeM, the European Semiconductor Industry Consortium as a startingpoint for developing a medical surveillance consensus for nano workers.

3A AtsutoOnoda

Induction of behavioral changes and astrogliosis in the brain of offspring micefollowing maternal inhalation of carbon black nanoparticle

P61 TshepoMoto

A comparison of the biokinetics of nanoceria after intravenous, inhalation,instillation and oral exposure using PBPK modeling

P49 CaroleSeidel

Inhaled multi walled carbon nanotubes induced gene expression profile in ratlung

6A Yaobo Ding Dispersion and aerosolization of engineered nanoparticles in liquid suspensionfor in vitro toxicity testing

P58 Ilise Charoy Global health impacts of nanotechnology law: The legend of asbestos thathaunts future innovation in nanotechnology


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Session 5A: In vivo models of hazard Tuesday 30 May, 16:00 18:00Location: Room 1 (Conference room)Session chairs: Alison Elder & Kristina B Knudsen

Physicochemical predictors of MWCNT induced pulmonary histopathology andtoxicity 1 year after pulmonary deposition of 11 different MWCNT in C57BL/6Nmice

Kristina B Knudsen1, Trine Berthing1, Petra Jackson1, Sarah Søs Poulsen1, Alicja Mortensen1, Nicklas RJacobsen1, Vidar Skaug2, Józef Szarek3, Karin S Hougaard1, Henrik Wolff4, Håkan Wallin2, Ulla Vogel1,51NRCWE, Denmark; 2Stami, Norway; 3University of Warmia and Mazury, Poland; 4FIOH, Finland; 5DTU, Denmark;[email protected]

Multi walled carbon nanotubes (MWCNT) are promising nanomaterials used in a wide range ofindustrial applications. Long term inhalation studies in rodents indicate that long, thick MWCNT arecarcinogenic, but as MWCNT vary considerably in physicochemical properties, this may not beapplicable to all MWCNT. Few studies have addressed the effects of different physicochemicalproperties on the long term toxicity of MWCNT by comparison of many different MWCNT.

Here, we have evaluated histological changes one year after a single intratracheal instillation of 11 wellcharacterized MWCNT and carbon black in female C57BL/6N BomTac mice. The dose of 54 μg MWCNTcorresponds to three times the estimated dose accumulated during a work life at a NIOSHrecommended exposure limit (1 μg/m3). Short and thin MWCNT were observed as agglomerates in lungtissue even one year after exposure, whereas thicker and longer MWCNT were detected as single fibers,suggesting biopersistence of both types of MWCNT. There was no MWCNT related fibrosis or tumors inthe lungs or pleura compared to vehicle controls. Most MWCNT induced varying degree of pulmonaryinflammation, in terms of lymphocytic and macrophage infiltration and granuloma formation. Bymultiple regression analysis, we identified that a larger diameter and greater content of Fe predictedless histopathological changes, whereas greater cobalt content significantly predicted morehistopathological changes. No effect of total oxygen content was observed. We conclude that thephysicochemical properties of MWCNTs were predictive of the histopathological changes. The resultsmay direct safe by design of MWCNT to reduce human health effects following pulmonary exposure.

Effects in the lungs following inhalation exposures to nanoparticle containingslurries used in semiconductor manufacturing

Alison Elder1, Sara Brenner2, Uschi Graham3, Andrea Kennell1, Robert Gelein1, Günter Oberdörster11University of Rochester, USA; 2SUNY Polytechnic Institute, USA; 3University of Kentucky, Lexington, USA;[email protected]

Chemical mechanical planarization of semiconductor surfaces with nanoparticle (NP) containing slurriescould lead to NP aerosolization and subsequent workplace exposures. Beginning with a panel of 8slurries containing amorphous silicon dioxide (31±7 nm), aluminum oxide (~60 nm), or cerium oxide(53±19 nm) NPs, we conducted a series of in vitro cytotoxicity, bolus in vivo, and short term andrepeated inhalation exposure studies in rats (male F 344, 200 300 g) to determine the inflammatory


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potency of the mixtures in the lungs. Intratracheal instillation exposures (200 μg per rat) revealed arange of potencies based on slurry type and that responses persisted through 7 days post exposure forsome slurry types. A subset of the slurries (one per NP type) was chosen for further study and werediluted in water, ultrasonically nebulized, and delivered to rats as dry aerosols via whole bodyinhalation exposures that achieved a range of lung deposited doses. Single exposures (4 6 hrs) showedthat the silica NP containing slurry had higher inflammatory potency than the alumina or ceria NPcontaining slurries. Surprisingly, the response to the latter two slurry types persisted through 7 dayspost exposure, albeit at low levels. The silica slurry was used for repeated inhalation exposures (4hrs/day, 5 days/week for 4 weeks; 0, 0.2, 1.0, or 4.7 mg/m3; MMADs, 0.38 0.5 μm; GSDs, 1.8 2.4).Controls were exposed to filtered air. Lung, bronchoalveolar lavage fluid, and other tissues wereharvested at 1, 7, and 27 days post exposure. Retained lung burdens at 1 day post exposure were 14±3,47±9, and 196±7 μg of silicafor the low, mid, and high doses, respectively. These values are lower thanwhat was predicted from dosimetry modeling, suggesting that the particles are highly biosoluble.Immediately after exposure, the mid and high concentrations produced statistically significantelevations in lavage neutrophils (5.8%±1.7; 33.9%±1.8) and other inflammatory parameters and theseresponses persisted through 27 days post exposure. Evaluations via high resolution analytical electronmicroscopy of lung tissue from rats exposed at the highest aerosol concentration revealed that silicaNPs underwent in vivo dissolution, but remained in the tissue through 27 days post exposure. Thelowest concentration did not produce any statistically significant changes in inflammatory parametersand was, thus, the no observed adverse effect concentration. These data and dosimetric extrapolationswere used to calculate human equivalent exposure concentrations of 0.2 0.8 mg/m3 (depending on theparameters used in modeling) that should be protective for workplace exposures.

This research was funded by NIH P30 ES01247 and SUNY Research Foundation subaward 13 15.

Characterization of pulmonary toxicity following acute exposure to a boronnitride nanotube suspension

Jenny Roberts1,2, Aaron Erdely1,2, Dale Porter1,2, Aleksandr Stefaniak1,2, Mark Barger1, KatherineRoach2, Michael Wolfarth1, Mohammad Shoeb1, Diane Schwegler Berry1, Tracy Eye1, Vamsi Kodali11NIOSH, Morgantown, WV, United States of America; 2West Virginia University, Morgantown, WV, United States of America;[email protected]

Boron nitride nanotubes (BNNT), multi walled nanotubes composed of hexagonal B N bonds, is anemerging nanomaterial offering superior electrical, chemical, and thermal properties. Potential toxicityis largely unknown. The goal of the study was to characterize pulmonary toxicity of BNNT in vitro and invivo. The BNNT sample was manufactured to have nanotubes with a diameter of ~5 nm and range inlength up to 200 m. The sample also contained boron and hexagonal boron nitride particles. The BNNTsample was dispersed by sonication in ethanol, followed by drying then suspension in a physiologicaldispersion medium (DM). The dispersion process yielded a sample with surface area ~180 m2/g and sizedistribution on average of 13 23 nm in diameter x 0.6 1.6 m. For in vitro studies, human monocyte cellline THP 1 wild type and NACHT, LRR and PYD domains containing protein 3 (NLRP3) knockout cellswere exposed to 0 100 μg BNNT sample. The exposure caused a dose dependent increase incytotoxicity, as well as cathepsin B, caspase 1, and protein levels of IL 1 in THP 1 cells. Effects wereattenuated in NLRP3 knockout cells. Dose response time course studies were also conducted in vivo.Male C57BL/6J mice were exposed to 4 or 40 g of the dispersed BNNT sample or DM by oropharyngealaspiration. Bronchoalveolar lavage (BAL) was performed and lung lymph nodes (LN) were collected at 4hours, 1 day, 7 days, 1 month, and 2 months post exposure. Lung histopathology was evaluated in aseparate set of animals. No adverse effects were observed with the low dose exposure. One day post


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exposure, the high dose caused an increase in lung injury (elevated BAL lactate dehydrogenase) andcellular influx into the lungs marked by neutrophils, eosinophils and lymphocytes. In addition, the highdose caused a loss in mitochondrial membrane potential, increased 4 hydroxynonenal, and destabilizedthe macrophage lysosomal membrane. Lung injury remained significantly elevated in the high dosegroup up to 1 month with resolution by 2 months. Neutrophils and eosinophils remained elevated up to7 days. BAL lymphocytes were significantly elevated at 1 and 7 days post exposure primarilyattributable to increased CD4+ and CD8+ T cells. LN lymphocytes were also significantly increased in thehigh dose group at 7 days. Histopathology confirmed the acute inflammatory response with resolutionby 1 2 months post exposure. In summary, BNNT caused a dose dependent increase in cytotoxicity andinflammasome activation in vitro, and acutely increased inflammation and lung injury in vivo followinghigh dose exposure.

The influence of the redox activity of inhaled nano sized cerium dioxide onrespiratory, immune, cardiovascular and neurological effects in various mousemodels

Susan Dekkers1, Mark Miller2, Roel Schins3, Rob Vandebriel1, Isabella Römer4, Mike Russ5, CatrinAlbrecht3, Wim de Jong1, Flemming R Cassee11Dutch National Institute for Public Health and the Environment (RIVM), Netherlands, The; 2University of Edinburgh; 3LeibnizResearch Institute for Environmental Medicine (IUF); 4University of Birmingham; 5Promethean Particles Ltd;[email protected]

To be able to better assess the potential risk of nanomaterials, more knowledge is needed on theconsequences of their different (physicochemical) properties and their mechanisms of toxicity. Here weassess the influence of redox modification via zirconium (Zr) doping of cerium dioxide nanoparticles(Ce2O NPs) on respiratory, immune, cardiovascular and neurological effects in mice following subacuteinhalation.

Healthy (C57BL/6J) mice, mice prone to cardiovascular disease (Western diet fed ApoE / ), a mice modelof neurological susceptibility (5xFAD) and ovalbumin (OVA) sensitised BALB/C mice were used. All micewere exposed to CeO2 NPs with different amounts of Zr doping, or to clean air, by inhalation over a 4week period (4 mg/m3 for 3 h/day, 5 days/week). BALB/C mice were also intranasally exposed to OVAon the last 3 days of the 4 week CeO2 NPs exposure period and on the last 3 days of the 2 week postexposure period to evaluate the adjuvant activity of the CeO2 NPs. In the other mice models, effectswere assessed 4 weeks post exposure to coincide with the formation of complex atheroscleroticplaques as well as changes in the brain.

CeO2 NP exposure had no major effects on clinical parameters, body weight, organ weights, blood cellcounts (total and cell differentials) and the bronchoalveolar lavage fluid. CeO2 NP exposure causedmodest inflammatory histopathological findings in the lung, which were not different between theredox modifications. Zr doping related differences in cytokine production of spleen and lymph nodecells and a slight, non Zr doping related increase in serum OVA specific IgG1, but not IgE suggested amild adjuvant effect of CeO2 NPs. CeO2 NPs did not accelerate amyloid plaque formation in the brain ofthe 5xFAD mice and did not alter the size of atherosclerotic plaques in the brachiocephalic arteries ofthe ApoE / mice, although there was a trend toward an increased inflammatory cell content in plaqueswith increases Zr content of the NP.

These findings suggest that Ce2O NPs had minimal toxicological effects following subacute inhalationand that redox modification via Zr doping of CeO2 NPs has limited effects on these responses. Further


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studies with alternative nanomaterials of greater inherent toxicity or a wider range of redox activitiesare required to fully assess the influence of redox modification on the toxicity of nanomaterials.

Contribution from Marie France Belinga, Shea Connell, Ian Smith,Paul Fokkens, John Boere, DaanLeseman, Jolanda Vermeulen, Eric Gremmer, Liset de la Fonteyne, Harm Heusinkveld, Tina Wahle,Adriana Sofranko, Christine Soputan

Lung remodeling after pulmonary exposure of mice to cerium oxidenanoparticles role of autophagy

Balasubramanyam Annangi, Audrey Ridoux, Jorge Boczkowski, Sophie LanoneIMRB U955 Inserm, France; [email protected]

Cerium and cerium compounds are mainly used as diesel fuel catalysts resulting in the emission ofcerium oxide nanoparticles (CeO2NPs) in the exhaust, which could lead to the exposure of the generalpopulation to these NPs. Some data in the literature suggest that pulmonary exposure to CeO2NPscould induce the development of lung fibrosis, but the exact underlying mechanism remains to beelucidated. Autophagy, a physiological self renewal mechanism wherein dysfunctional or damagedorganelles, misfolded or aged proteins are degraded for cell survival, has been recently proposed as apotential underlying mechanism of NP toxicity. We therefore designed a study aimed to understand therole of autophagy in CeO2NPs induced pulmonary fibrosis in mice. We first characterized themodulation of autophagy by CeO2NPs (5, 10 and 20μg/mL) in isolated peritoneal macrophages fromGFP LC3 mice. CeO2NPs were able to accumulate autophagosomes in a dose dependent manner, asevaluated by fluorescence microscopy. Besides, the co localization of LC3 and LAMP1 markers wasobserved in macrophages treated with CeO2NPs studied using confocal microscopy, indicating an activeautophagy pathway. To delineate the role of autophagy in CeO2NPs induced lung fibrosis, we used micewith mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5fl/fl LysM Cre+ mice, (Atg5 / ) andtheir wild type (WT) counterparts. Mice were exposed to CeO2NPs (5 or 50μg) by a single intratrachealinstillation. After 28 days, histological analysis of WT mice revealed a dose dependent increase inbronchial and alveolar wall thickening characterized by inflammatory cells infiltration, total and type IIIcollagen deposition, elevated expression of fibrotic markers such as SMA, TGF 1 in peribronchiolarand alveolar spaces. Atg5 / mice were protected from the alveolar phenotype (less alveolar thickening,insignificant total and type III collagen deposition, no increased expression of SMA, TGF 1 in alveoli),whereas no effect of Atg5 deletion could be detected in the bronchi region. In conclusion, the lack ofAtg5 gene protects from CeO2NPs induced alveolar wall thickening but not from CeO2NPs inducedbronchial wall remodeling. From these studies, it could be inferred that macrophagic autophagy maypossibly play a dual role in CeO2NPs induced lung fibrosis. The exact underlying mechanisms arecurrently under investigation.


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Carbon nanoparticle and lung interaction is crucial for systemic effects: Lessonslearnt from inhalation versus intra arterial infusion exposure studies in mice

Koustav Ganguly1, Dariusch Ettehadieh2, Swapna Upadhyay3, Shinji Takenaka2, Thure Adler4, ErwinKarg5, Fritz Krombach6, Wolfgang Kreyling2, Holger Schulz7, Otmar Schmid2, Tobias Stoeger21Units of Lung and Airway Research and Work Environment Toxicology; Institute of Environmental Medicine (IMM), KarolinskaInstitutet, SE 171 77 Stockholm, Sweden; 2Institute of Lung Biology and Disease, Comprehensive Pneumology Center,Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany D85764; 3Unit ofLung and Airway Research; Institute of Environmental Medicine (IMM), Karolinska Institutet, SE 171 77 Stockholm, Sweden;4German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center forEnvironmental Health, Neuherberg, Germany D85764; 5Institute of Lung Biology and Disease, Comprehensive PneumologyCenter; and Cooperationgroup Comprehensive Molecular Analytics (CMA), Joint Mass Spectrometry Centre (JMSC), HelmholtzZentrum München, German Research Center for Environmental Health, Neuherberg, Germany D85764; 6Walter Brendel Centreof Experimental Medicine, Ludwig Maximilians Universität München Germany D81377; 7Institute of Epidemiology I, HelmholtzZentrum München, German Research Center for Environmental Health, Neuherberg, Germany D85764; and ComprehensivePneumology Center Munich (CPC M), Member of the German Center for Lung Research, Munich, Germany D85764;[email protected]

Rationale: Cardiovascular impairments represent a major cause of death and emergency room visitsdue to inhalation of ambient particulate matter (PM) during pollution episodes. However, it has been along standing question whether the PM related cardiovascular events are driven by particletranslocation from lung to circulation (direct effect) or by pulmonary particle cell interactions (indirecteffects). We attempted to address this issue experimentally by exposing healthy mice via inhalation andintra arterial infusion (IAI) routes to carbon nanoparticles (CNP). CNPs are considered as surrogate forsoot, a major constituent of (ultrafine) urban PM.

Methods:Mice were exposed to CNPs by inhalation (spark discharge generated particles) andequivalent surface area CNP doses in the blood (ca. 100 mm2 per animal) by IAI (Printex90). Assessmentfor changes in hematology and molecular markers of endothelial/epithelial dysfunction, proinflammatory reactions, oxidative stress, and blood coagulation in both lung and extra pulmonaryorgans (including aorta, heart and liver) after CNP inhalation (4h and 24h) and infusion (4h) wereperformed. Due to methodological constraints, we used two different types of CNPs (sparkdischarge/Printex 90) with very similar physicochemical properties [chemical composition: 98%/ 95%elemental carbon and no bioavailable organic compounds; primary particle diameter: 10±2nm/14±2nm;and Brunauer–Emmett–Teller (BET) surface area: 800 m2/g / 300 m2/g] for inhalation and IAI exposurerespectively.

Results:Mild pulmonary inflammatory reaction and significant systemic effect was detected following4h and 24h of CNP inhalation exposure.Increased retention of activated leukocytes, secondarythrombocytosis, and pro inflammatory responses in extra pulmonary organs were detected following4h and 24h of CNP inhalation only. Interestingly, among the investigated tissues other than lung (i.e.aorta, heart, and liver); aorta revealed as the most susceptible extra pulmonary target followinginhalation exposure. However, to our surprise, bypassing the lungs by IAI, did not induce anyappreciable extra pulmonary effects at 4h compared to inhalation. Thus, 24h time point was notpursued for IAI experiments.

Conclusions: Our findings indicate that extra pulmonary effects due to CNP inhalation are primarilydriven by indirect effects (particle cell interactions in the lung) rather than direct effects (translocatedCNPs) within the first 24 h after exposure. This led us to conclude that CNP translocation may not be thekey event inducing early cardiovascular impairment following air pollution episodes. The considerableresponse detected in the aorta after CNP inhalation warrants detailed mechanistic studies focused onthis target tissue.


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Session 5B: Release characterization: from concentrations toquantitative release

Tuesday 30 May, 16:00 18:00Location: Room 2Session chairs: Kaarle Hämeri & Antti Joonas Koivisto

Occupational exposure assessment to NOAA during production, formulationand assembly of nanoenabled products in several European platforms and pilotplants

Simon Clavaguera1, Sébastien Artous1, Cécile Ducros1, Cécile Philippot1, Manuel Carmona Franco2,Irene Izarra2, Jose Luis Valverde Palomino2, Juan Francisco Rodriguez Romero2, Briac Lanfant3, MarcLeparoux3, Antaryami Mohanta3, Ari Setyan4, Jing Wang4, Bryony Ross5, Martie van Tongeren5, MaidaDomat6, Carlos Fito6

1NanoSafety Platform, CEA, Univ. Grenoble Alpes, F 38054 Grenoble, France; 2UCLM, Ciudad Real 13071, Spain; 3EMPA, 3603Thun, Switzerland; 4EMPA, 8600 Dübendorf, Switzerland; 5IOM, Research Avenue North Riccarton, Currie EH14 4AP / UK;6ITENE, 46980 Paterna, Valencia, Spain; [email protected]

Nanotechnology has rapidly promoted the development of smart and innovative processes and nanoenabled products that have created a tremendous growth potential for a large number of industrysector. In particular, nanotechnology offers substantial possibilities for improving the competitiveposition of the EU and for responding to key societal challenges. Ensuring the safe, sustainable andresponsible development of nanotechnologies through the implementation of the ‘safer by design’concept in the development of new European pilot plants is a key objective of the Horizon 2020 WorkProgramme and the EU NanoSafety Cluster. Exposure plays an important role in the area of riskassessment, as ‘safer by design’ refers not just to reducing the hazard potential of the engineerednanomaterials (ENMs) but also to reducing the release, emission and exposure potential of productsand processes.

This work presents the results of comprehensive experimental campaigns focused on the assessment ofoccupational exposure to next generation nanomaterials covering several case studies throughout thelife cycle of nano enabled products for energy harvesting, energy storage and building applications. Thefield measurements were performed in several European platforms and pilot plants for which differentexposure scenarios were investigated. The boundaries of the system relevant for the present study arethe indoor air and indoor surfaces which are considered potential occupational endpoints for inhalationand dermal exposure. The methodology used for the exposure evaluation followed the OECD Tieredapproach. Thanks to several granulometers, particle counters and personal devices, measurementsincluded concentration of particles, size distribution, state of agglomeration / aggregation, specificsurface area, morphology and chemical composition.

The key findings from the undertaken actions are:Increased understanding of some of the tasks and activities undertaken along the life cycle ofnano enabled products and the potential for exposure to airborne ENMs. The generated data onrelease, emission and exposure throughout the life cycle of ENMs is used to provide inputs forpractical and cost effective risk management, common safety guidelines, and safer by designstrategies.


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The identification of the remaining experimental challenges to address the speciation of thereleased particles (i.e. pristine vs aged and transformed) and to discriminate high and fluctuatingbackground in workplaces.Harmonization of the measurement strategy adapted to pilot plants and small businesses in orderto measure potential airborne emissions of ENMs at workplace.

The research leading to these results has received funding from the European Union's FP7 GrantAgreement n.310584 (NanoReg project), n.604602 (FutureNanoNeeds project) and H2020 GrantAgreement n.646397 (NanoLeap project).

Release of nanomaterials from consumer products during use and end of life

Alessio Boldrin, Laura Heggelund, Aiga Mackevica, Steffen Foss HansenTechnical University of Denmark, Denmark; [email protected]

A significant number of products containing engineered nanomaterials (ENMs) is currently alreadyavailable and easily accessible on the market. The presence of ENMs in commercial products could beassociated with human and environmental exposure during both the use and the end of life phases ofthe product. The release during the use phase will depend on several aspects, among which the productmatrix and the location of the ENMs in the product. Our tests on a range of products – food packagingmaterials (Ag), toothbrushes (Ag), textiles (TiO2), surface coatings (Ag, CuO) – showed that ENMs aregenerally released in small amounts. While the release could occur in long term, this may indicate thatmost of the ENMs initially present in products may end up in waste flows. With respect to the end oflife phase, the type of material (e.g. plastic, paper, glass, metals) and the waste treatment process (e.g.mechanical pre treatment, recycling, incineration, landfilling) will significantly influence the release ofENMs. For example, our experimental results show that during mechanical size reduction significantrelease could occur during crushing of hard materials (e.g. tiles, glass) because of ducting, whereasshredding/cutting of malleable materials (e.g. plastic, paper) seems to be associated with almostinsignificant exposure levels. During recycling operations, the temperature of the process and theaffinity of ENMs towards the air, solid, or liquid phase are key aspects. We identified that ENMS with aboiling/melting point below the process temperature and affinity for the air phase may be release tosome extent, whereas ENMs which are persistent and have affinity for the solid phase may accumulatein the recycled material thereby potentially being an issue again for consumers. With respect toincineration, a key role is played by boiling/melting point of the ENMs, which determine whether theyenter the flue gas stream or end up in the bottom ash residues. In both cases, the subsequenttreatment/handling of these flows is crucial to limit the release. In a landfill scenario, release of ENMsmay almost solely occur via the liquid phase. The amount of ENMs release and their mobility willdepend, among other things, on the degradability of the waste matrix, on the specific ENMs in question,and the composition of the surrounding waste and the variety of physical and chemical processesoccurring in the landfill body. For example, we reported limited release from tiles coated with TiO2, nomatter the chemical conditions of the leachate.


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Engineered nanoparticle containing consumer products in the Singapore retailmarket and likelihood of release into the aquatic environment

Michael Riediker1,2,3, Robert J Aitken1, Yuanyuan Zhang1,41IOM Singapore, Singapore; 2Nanyang Technological University, Singapore; 3Institut de Santé au Travail, University of Lausanne,Switzerland; 4NTU Hyundai Urban System Centre, Hyundai Engineering & Construction Co. Ltd, Singapore;michael.riediker@iom world.sg

We recently inventoried consumer products in the Singapore retail market with regard to their likelycontents of engineered nanoparticles (ENP). These products were further assessed regarding theirpotential for releasing ENP into the aquatic environment. For this we attributed to each product typeusage patterns. By estimating release factors, we obtained approximate quantities of ENP that arereleased into the aquatic environment in Singapore. In total 1,432 products were investigated. Of these138 were “confirmed” and 293 were “likely” to contain ENP. Product types in these categories includedsunscreens, cosmetics, health and fitness, automotive, food, home and garden, clothing and footwear,and eyeglass/lens coatings. The ENP confirmed or likely to be contained in these products included SiO2(predominant), followed by TiO2, ZnO, Carbon Black, Ag, and Au. The amounts released into the aquaticenvironment were in the range of several hundred tons per year. We are currently modelling theaquatic environment in Singapore to describe in more detail the fate of these ENP after their release.

Comparing the Venturi and rotating drum dustiness testing methods

Douglas E Evans, Leonid A Turkevich, Gregory J DeyeCDC/NIOSH, United States of America; [email protected]

During production and downstream use of nanomaterials there is inherent potential for workers to beexposed to nanoscale particulate, with inhalation exposure of primary concern. Open dry powderhandling, product harvesting and clean up operations, significantly contribute to worker exposures.Several factors may influence the degree to which a worker may be exposed, one of which is thepowder dustiness. Dustiness may be defined as the propensity for a powder to aerosolize under aprescribed stimulus. Dustiness testing should replicate the dispersal mechanism that results in fugitiveairborne dust. One of the most widely recognized dustiness methods is the EN15051 rotating drum,which replicates many general powder handling and transfer operations. One limitation of the drum, isthat it requires ~100 g of powder for testing. A method such as the Venturi, which uses ~30 mg ofpowder, has been successfully applied to nanomaterials.

Numerous fine and nanoscale powders have been tested by both the Venturi and rotating drummethods and mean average dustiness data compared. Although there are significant differencesbetween the underlying dispersal mechanisms for the two testing methods, both of which are observedin the workplace, there appears to be good correlation between dustiness derived from the twomethods. Further powders are required for a more definitive comparison.


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Bayesian Belief Network to forecast nanomaterial release during wasteshredding

Neeraj Shandilya1, Tom Ligthart1, Imelda van Voorde1, Burkhard Stahlmecke2, Simon Clavaguera3,Thomas Kuhlbusch4, Richard Laucournet3, Bas Henzing1, Yaobo Ding5, Henk Goede11TNO, Netherlands, The; 2IUTA, Germany; 3CEA, France; 4BAuA, Germany; 5IST, Switwerland; [email protected]

For the nanomaterial containing Waste of Electronic and Electrical Equipment (WEEE), shredding is themain treatment before further recycling. A nanomaterial flow analysis showed that for several valuechains, the end of life of these products is a potential ‘hotspot’ of nanomaterial release. However, dataon the release of nanomaterials during shredding are very sparse. Since the nanomaterial containingWEEE is expected to grow significantly, this poses a forthcoming problem for recycling plants. A way toovercome this problem is to predict the release of nanomaterials during shredding by establishing aBayesian Belief Network (BBN). BBNs have multiple advantages: they provide a coherent framework formaking a priori assumptions about unknowns, present the formal rules to update that knowledge whendata later become available, and explicitly model the uncertainty in the BBN’s release forecasts.

We developed a BBN for three value chains: photovoltaics, lithium ion batteries and displaytechnologies. Shredder manufactures and recyclers were consulted to obtain process relatedinformation. To set up the BBN three steps were taken during an expert elicitation procedure:1. assigning appropriate variables,2. developing the network structure and conditional links between them, and3. quantification of the model by filling the conditional probability tables.

Measurement data from literature and field data obtained from shredding processes in the FNN projectwere considered as information sources.

The final BBN release model consists of fourteen material and process related variables, sevenintermediate variables and four goal variables. The goal variables for the initial release of nanoparticlesare the number of particles, their total mass, their size classes and their composition. The network wasimplemented in Genie software©. The model output will be presented using a number of availablerelease studies on shredding processes.

Acknowledgment: The research leading to the results has received funding from the European ResearchCouncil under the European Union's FP7 (FP/2007 2013)/ERC Grant Agreement n.604602 (FNN project).

Estimation of aerodynamic and diffusion diameters of carbon nanotube aerosolagglomerates using electron microscopy

Bon Ki Ku, Pramod KulkarniCDC/NIOSH, United States of America; [email protected]

Transport characteristics of aerosol particles are important in assessing their fate in respiratory systemsas well as in engineered systems such as particulate filters. Diffusion and aerodynamic diameters ofnear spherical particles, with density close to standard density and dynamic shape factors close to 1,are nearly equal. For nonspherical particles with high aspect ratios, such as aerosolized carbonnanotubes in the workplace, these diameters can be very different, requiring their independentmeasurement. The objective of this study was to develop an approximate approach for estimatingaerodynamic and diffusion equivalent diameters of airborne carbon nanotube (CNT)agglomerates using


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their electron microscopy images. The as received CNT nanomaterials were aerosolized using differentdry dispersion and pneumatic atomization techniques. The aerosolized particles were classified by adifferential mobility analyzer and their mass was measured using tandem mobility massinstrumentation. This tandem measurement yielded test particles with known mobility and mass tocharge ratio. Subsequently, these particles with known mass to charge ratio and electrical mobilitywere collected on a microscopy grid and analyzed by transmission electron microscopy (TEM). The TEMimages were processed using image processing tool to obtain projected area, maximum projectedlength, and 2 D radius of gyration. These morphological measurements were then used to obtaindiffusion and aerodynamic equivalent diameters of the CNT particles using fractal theory. Afteraccounting for the dynamic shape factor, most of the estimated aerodynamic diameters reasonablyagreed with the direct measurements within 30 40% while the diffusion diameters are comparable withthe reference values within 40 50% in the size range of 100 800 nm except some particles smaller than200 nm. Also, the aerodynamic diameter was found to be estimated within 35% and 91% for CNTaerosols generated from the workplace (Birch et al., 2011) and CNT particles aerosolized in thelaboratory (Chen et al., 2012), respectively. It was observed that as the agglomerate size increases, itsestimated aerodynamic diameter is underestimated, because of artifacts introduced by overlappingstructures in the micrographs. This study indicates that this approach could be useful in calculatingapproximate property equivalent diameters from micrographs of carbon nanotube agglomerates withrelatively open structures, when other more accurate measurements are not readily available.


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Keynote IIIWednesday 31 May, 9:00 10:00Location: Room 1 (Conference room)Session chairs: Alison Elder & Ulla Vogel

Nanomaterial dosimetry in inhalation toxicology: Bridging the gaps between invitro and in vivo models as well as real world exposureOtmar Schmid

Helmholtz ZentrumMünchen, Germany; otmar.schmid@helmholtz muenchen.de

Aerosolized delivery of engineered nanomaterials (ENMs) to animal (in vivo) or cell based (in vitro)models of the lung is a technological challenge, which typically requires highly skilled aerosol expertsand advanced aerosol equipment for controlled ENM exposure. For translation of preclinical ENMtoxicity into risk assessment it is expected that not only exposure levels, but tissue delivered dose playan important role. Hence, it is essential to understand the relationship between exposure and tissuedelivered dose, which depends on various parameters including aerosol size, density and shape as wellas lung morphology, respiratory conditions and exposure time.

In this talk, various methods for in vitro and in vivo testing of aerosolized ENMs will be discussed withparticular emphasis on the tissue delivered dose. For in vitro experiments air liquid interface (ALI)rather than submerged cell cultures of the lung are more physiologic and offer the possibility for directmeasurement of the tissue delivered dose using dosimetric methods such as Inductively CoupledPlasma Mass Spectrometry (ICP MS) and Quartz Crystal Microbalance (QCM). An overview of currentlyavailable ALI cell exposure systems will be discussed with emphasis on aerosol cell deposition efficiencyand delivery rate. Similarly, animal inhalation systems will be presented in the context of lung delivereddose (rates) and put into perspective by comparison with physiological doses encountered in real worldexposure settings.

A dose based comparison of in vitro and in vivo toxicity of ENMs is likely to provide insights into gapsbetween currently applied in vitro and in vivo testing strategies. The potential for bridging these gaps isrelated to the use of biomimetic and clinically relevant models of the lung and to our understanding ofphysiological doses encountered during realistic exposure scenarios.


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Session 6A: In vitro models of toxicity testing Wednesday 31 May, 10:30 12:30Location: Room 1 (Conference room)Session chairs: Penny Nymark & Martin Roursgaard

In vitro based high throughput/high content screening and omics driveninformatics support rapid and effective safety evaluation of engineerednanomaterials

Penny Nymark1,2, Vesa Hongisto2, Pekka Kohonen1,2, Roland Grafström1,2

1Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden; 2Division of Toxicology, Misvik Biology, Turku,Finland; [email protected]

The rapidly developing “Big Data era” in toxicology involves potential for generation of large amounts ofsafety testing data, including the associated need to interpret the results relative steadily increasingamounts of related information in databases. Cell culture models serve excellently for high throughputscreening (HTS) analysis, allowing entire libraries of engineered nanomaterials (ENMs), to be rapidlyassessed and ranked for cytotoxicological properties. In parallel, microscopy driven high contentanalyses of morphology and immunochemical markers, and genomic profiling data opens formechanistic cytopathological interpretation. A steadily increasing diversity of bioinformatics tools, e.g.Chipster, additionally permits extensive overview and visualization of the generated results relativeexisting relevant data. Ideally, the combined use of the above tools allow for coupling of the results toadverse outcome pathway descriptions, e.g., for pulmonary fibrosis or other pathological statesconsidered in ENM safety evaluations. Overall, data driven workflows combined into tiered approachesallow for systems toxicology driven means to safety evaluation parallel to the innovation of novel ENMvariants. The applicability and challenges associated with the above technologies will be discussed inrelation to the conference theme.

How to make in vitro models more realistic, and compare the data to in vivooutcomes

Martin Roursgaard1, Yi Cao1, Henrik Klingberg1, Ali Kermanizadeh1, Kim Jantzen1, Kristina B Knudsen2,Peter Møller11University of Copenhagen, Denmark; 2National Research Centre for the Working Environment, Denmark; [email protected]

When setting up new in vitro systems to make better alternatives to animal experimental models, it isimportant to test the system thoroughly. An in vitromodel is often supposed to mimic a whole organ ororganism, which is a complex multi cellular compartment. To close the gap between in vivo and in vitromodels, the inclusion of co cultures or more realistic growth conditions can be used.

The presentation will focus on mechanistic investigations of disease development using in vitromodels.Examples will be on atherosclerosis in simple models, as well as on other endpoints in more complexorgano typical models.


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Another approach is the implementation of High Content Screening that combined with standardcytotoxicity and genotoxicity measurements as initial screening can be performed for predictivetoxicological screening. For the in vitromodels to be relevant they need to be validated relative to thefinal endpoint, e.g. the adverse outcome in humans or animals, why often initial screening needs to beperformed in both cell cultures and animals.

In a study of drug delivery particles the general effect level showed to be higher for an in vitro studythan a previous animal study, possibly making cell studies relevant as a sensitive initial evaluation toolwith the High Content Screening as a good method for predictive toxicological investigations.

A VITROCELL CLOUD system was used to aerosolize and deliver suspended nanoparticles to cells via airliquid interface exposure. It was shown that high ENP dose rate can be achieved (1000 ng/cm2/min,<12% insert insert variability). The cell delivered dose can be quantified with an equipped QCM (QuartzCrystal Microbalance).

Next, we will further test different nanomaterials of toxicological concerns (e.g., CNTs), consideringabove mentioned parameters. How salt to particle ratio (mass and surface area) influences dispersionquality will be explored. Dependence of necessary sonication time (or total power) on particleconcentration in suspensions will be experimented.

Genotoxicity assessment of 31 nanomaterials in human bronchialepithelial cells

Gerard Vales1, Julia Catalán1, Manuel Correia2, Zahraa Al ahmady3, Julie Muller4, Juri Fedutik5, JaimeRuiz6, Alexei Antipov5, Kostas Kostarelos3, Didier Astruc6, Sergio Moya7, Erik Huusfeldt Larsen2, KaiSavolainen1, Hannu Norppa11Finnish Institute of Occupational Health, Helsinki, Finland; 2Technical University of Denmark, National Food Institute, Søborg,Denmark; 3University of Manchester, United Kingdom; 4Nanocyl SA, Sambreville, Belgium; 5PlasmaChem GmbH, Berlin,Germany; 6Université de Bordeaux, Talence, France; 7CIC biomaGUNE, Donostia, Spain; [email protected]

The possible carcinogenicity of engineered nanomaterials is one of the key questions in evaluating theirhazard to human health. In vitro assays for genotoxicity are used to detect carcinogens that act througha primary genotoxic mechanism. We have assessed the genotoxicity of 31 nanomaterials in humanbronchial epithelial BEAS 2B cells using the alkaline comet assay for the detection of primary DNAdamage (24 h treatment) and the cytokinesis block micronucleus assay (48 h treatment) forchromosome damage. The nanomaterials tested included CdTe quantum dots (QDs), CuO nanoparticles(NPs; primary particles 10 20 nm), Ag NPs (10 20 nm), two sizes (3 5 nm and 10 20 nm) of Au NPs, TiO2

NPs (anatase; 10 20 nm) and nanorods (10 x 50 nm), nanodiamonds (3 5 nm), and multiwalled carbonnanotubes (MWCNTs; 10 15 nm x 1 1.5 μm). All materials were studied with three different surfacemodifications – ammonium (positive charge), carboxylate (negative) and polyethylene glycol (PEG;neutral) functionalization. A non functionalized form (core) was additionally available for CuO, TiO2, andMWCNTs.

Dose selection for the genotoxicity assays was based on cytotoxicity assessment. QDs were highlycytotoxic regardless of functionalization, indicating that the effect was driven by the chemicalcomposition of the nanomaterial. All forms of MWCNTs showed low cytotoxicity. For the othernanomaterials, except nanodiamonds, ammonium functionalization strongly increased cytotoxicity.Lower cytotoxicity was associated with PEGylation for QDs, CuO, and both sizes of Au NPs and withcarboxylate for Ag and TiO2 NPs.


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DNA damage was increased by ammonium functionalized 10 20 nm Au NPs, QDs and TiO2 NPs,PEGylated Ag NPs and 3 5 nm Au NPs, and core TiO2 nanorods, with an equivocal result (significantincrease only at one non top dose) for core TiO2 NPs and ammonium functionalized MWCNTs. No DNAdamage induction was seen for CuO NPs, nanodiamonds, or carboxylate functionalized nanomaterials.

Micronuclei were induced by all functionalized QDs, core and ammonium functionalized CuO NPs,PEGylated and carboxylate Ag NPs, ammonium functionalized 3 5 nm Au NPs, PEGylated 10 20 nm AuNPs, and PEGylated and core TiO2 NPs, with an equivocal effect by the carboxylate forms of 10 20 nmAu NPs and MWCNTs. TiO2 nanorods or nanodiamonds did not produce micronuclei.

In conclusion, the chemical composition of the nanomaterials determined their cytotoxicity andgenotoxicity, but ammonium functionalisation tended to be associated with high cytotoxicity andPEGylation with low cytotoxicity. Genotoxicity was seen more often with the ammonium and PEGylatedforms, but general rules could not be derived for the effect of functionalisation on genotoxicity. Thehigh cytotoxicity of the ammonium functionalized nanomaterials may have partly masked their possiblegenotoxic effects. [Supported by NMP4 LA 2013 309329 NANOSOLUTIONS]

Assessment of adverse effects of silver nanoparticles using an air liquidinterface small airway epithelial exposure model

Chang Guo, Alison Buckley, Tim Marczylo, Rachel Smith, Martin LeonardCentre for Radiation, Chemical and Environmental Hazards, Public Health England, UK; [email protected]

The increased use of silver nanoparticles (AgNPs) in applications ranging from biomedical therapeuticsto consumer products has led to concern over inadvertent exposure and the potential for hazardouseffects in humans. Experimental modelling to identify specific inhalation hazards for nanomaterials hasmainly focussed on in vivo approaches such as those utilising rats or mice. Attempts at hazardidentification using these systems have suffered from uncertainties surrounding species specificdifferences. Developments in human in vitromodelling have the potential to address these concerns. Interms of pulmonary exposure, approaches which combine ‘inhalation like’ nanoparticulate aerosoldeposition with relevant human cell and tissue air liquid interface cultures are considered at theforefront of model development. In this study we utilized such a model system to compare and build onin vivo exposures aimed at characterizing mechanisms underlying the adverse effects of inhaled AgNPsin Sprague Dawley rats. Pulmonary exposure resulted in alterations in mRNA and miRNA within the lungconsistent with inflammatory processes and metal response effects. We also identified the small airwayepithelium as an important site for AgNPs deposition within the lung using laser ablation ICP/MS. Withthis information, we selected organotypic reconstituted 3D human primary small airway epithelial(HPSAE) cell cultures in an air liquid interface aerosol exposure (ALI AE) system and exposed them tothe same spark generated AgNPs using an integrated electrostatic deposition device. Adverse effectswere characterised using lactate levels, LDH release and alterations in mRNA and miRNA expression.Modest toxicological effects were paralleled by significant regulation in gene expression reflectivemainly of specific inflammatory events. Importantly, there was a high correlation between geneexpression changes observed in vitro and in vivo. Moreover, this correlation also extended toobservations found upon exposure of HPSAE ALI AE cultures to mass equivalent silver ion solutions(Ag+). In addition to key mechanistic information identified as important for our understanding of thepotential health risks associated with AgNP inhalation exposure, this work also highlights the potentialfor specialized in vitro systems to capture in vivo effects relevant for human exposure.


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Adverse effects of summer and winter ambient air in human lung cell cultures,an alternative model to assess occupational and environmental health

Christoph Bisig1, Pierre Comte2, Jan Czerwinski2, Andreas Mayer3, Alke Fink1, Barbara RothenRutishauser11Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 2Bern University of Applied Sciences, Nidau,Switzerland; 3Technik Thermische Maschinen, Niederrohrdorf, Switzerland; [email protected]

Background: Ambient air consists of nitrogen and oxygen, additionally it is polluted by particulatematter (PM), nitrogen oxides (NOx), and ozone (O3). Epidemiological studies have associated these airpollutants with cardiovascular and pulmonary diseases, as well as premature deaths and asthma.However, epidemiological studies are time consuming and confounders (smoking) are an issue,therefore complementing this research area with in vitro or in vivomodels are important.Our aim was to evaluate a portable in vitro exposure system with ambient air, where a multi cellularhuman lung model was directly exposed at the air liquid interface to ambient air in summer or inwinter.

Methods: A portable system containing two exposure chambers was placed on a balcony in Fribourg,Switzerland. Ambient air was pumped through one chamber with a constant flow of 2 L/min. In parallel,filtered medicinal air was applied to the other chamber. The multi cellular lung model (epithelial16HBE14o cells combined with macrophages, and dendritic cells) was placed in the chambers andexposed on up to three subsequent days each for 12 h (7am to 7pm) to ambient air. Biologicalendpoints included cell morphology assessment, gene expression analysis, and cytotoxicity. Particlenumber (PN) concentrations were measured with a DiSCmini. Additionally, the official air pollutionobservation station of the canton of Fribourg is within 300m of the exposure site providing PM10, NOx,and O3 levels.

Results: PN and PM10 concentrations varied around 10’000 and 20’000 particles/cm3 and 15 and25μg/m3 for the summer and winter ambient air, respectively. Ambient air did not change cellmorphology nor increase cytotoxicity. The summer ambient air did not lead to increased proinflammatory response or induction of oxidative stress compared to the filtered medicinal air at thegene expression level. On the other hand, winter ambient air showed an increase in the expression ofpro inflammatory genes already after one day.

Conclusion: The novelty of our approach is to directly expose human lung cells in vitro in a very realisticmanner to ambient air. Results revealed that the summer air (low PN) did not induce oxidative stress orpro inflammation over the three days repeated exposures, while the winter exposure (higher PN)increased pro inflammation.With this work we have demonstrated that our simple, fast, and cost effective approach can be used toassess (adverse) effects of ambient air, and the system could be placed at specific locations (e.g. workplace, production facilities), where epidemiological long term studies are not possible.


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Dispersion and aerosolization of engineered nanoparticles in liquid suspensionfor in vitro toxicity testing

Yaobo Ding1,2, Patrick Weindl1,2, Clara Wimmer1,2, Andreas Schröppel1,2, Tobias Krebs3, OtmarSchmid1,2

1Institute of Lung Biology and Disease, Helmholtz Zentrum München, Germany; 2Comprehensive Pneumology Center Memberof the German Center for Lung Research (DZL), Munich, Germany; 3VITROCELL Systems GmbH, Waldkirch, Germany;yaobo.ding@helmholtz muenchen.de

In vitro cell exposure studies using particle suspensions are often utilized as a high throughput toxicityscreening method for hazardous engineered nanoparticles (ENPs) (DeLoid 2017). Certain air liquidinterface (ALI) cell exposure systems also require preparing ENP suspensions for aerosolized delivery vianebulization (Lenz 2014). In order to compare the outcomes of in vitro and in vivo toxicity studies, thetissue delivered dose must be determined. In liquid suspensions this is governed by particle kinetics,which is governed by dispersion properties (Teeguarden 2007). Electrolyte rich dispersion medium (e.g.cell culture mediums) may change particle properties dramatically (Jiang 2008). Therefore, wellcharacterized suspensions are imperative for accurate dose response determination in cell exposurestudies.

We explored several important factors affecting dispersion quality including salt concentration andsonication parameters. Different types of nanoparticles were tested in suspensions of varied particleconcentrations. The powders were suspended in distilled water containing small amounts ofphysiological salt, followed by vortex shaking and probe sonication. The suspensions were thencharacterized using dynamic light scattering (DLS) for hydrodynamic particle size.

Our preliminary results showed that sonication decreased the particle size over time reaching itsasymptotic limit after reaching the critical delivered sonication energy of 161 J/ml (DeLoid 2017).However, re agglomeration indicated by increasing particle size was observed, if the sonicationenergy transfer rate (sonicator power setting) was too high (ca. 1 J/ml/s). Interestingly, dispersionquality improved with higher suspension concentrations (shown by decreased particle sizes) and reagglomeration at 1 J/ml/s only occurred for ENP concentrations less than 1 mg/ml. Adding saline in thesuspension increased the particle size (e.g., from 200 nm to >1 μm for TiO2). However, this well known“salting” effect was noticeably reduced for increasing particle concentrations. Moreover, materials withsimilar primary particle size but different chemical composition showed very different dispersionbehaviour (e.g., significantly higher agglomeration for NiFe2O4 than for TiO2). This may be attributed tothe magnetic properties of NiFe2O4.


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Session 6B: Work place measurement & modelling

Wednesday 31 May, 10:30 12:30Location: Room 2Session chairs: Miikka Dal Maso & Ismo K Koponen

Simultaneous modelling of dispersion and aerosol dynamics of workplaceaerosol

Miikka Dal Maso1, Mikko Poikkimäki1, Niko Leskinen1, Ismo K Koponen2, Antti Joonas Koivisto2, Keld AJensen2

1Tampere University of Technology, Finland; 2National Research Centre for the Working Environment, Denmark;[email protected]

Occupational and environmental exposure to aerosol particles presents a complex field of study, as theparticulate matter sources are often strongly localized and both the aerosol concentration and sizedistribution vary temporally and spatially with the workplace activities; in addition, the presence ofbackground aerosol complicates the picture[1,2]. In addition to heterogeneous aerosol sources, severalprocesses such as coagulation, agglomeration, diffusional deposition, and gravitational settling affectthe evolution of the aerosol number size distribution in the workplace. Moreover, it is possible that gasphase vapour compounds condense or partition to the particle phase, changing its composition andtoxicity.

Understanding the dispersion and dynamic evolution of the aerosol once it has been released requiressimultaneous solving of the aerosol general dynamic equation (GDE)[3] and dispersion equations. Thisusually requires discretization or making some assumptions and simplifications, for example in the caseof particle coagulation and aggregation [4].

In this presentation, we will present comparisons of three different modelling approaches (Gaussiandispersion[5,6], the near field/far field model, and multicompartment modelling) for workplace sources,and discuss their applicability for detailed aerosol dynamics modelling and for estimating bothworkplace concentrations and assessment of personal exposure.

1. Brouwer, D. et al., Toxicology 2010, 269, 120–127.2. Hämeri, K. et al., Inhalation Toxicol. 2009, 21, 17–24.3. Seinfeld & Pandis, Atmospheric Chemistry and Physics, Wiley, 19984. Lehtinen, K. E. J., et al., J. Coll. Int. Sci. 1996, 182 (2) , 606–608.5. Drivas, P.J. et al., Indoor Air 1996, 6 (4): 271–277,6. Cheng, K. C. et al Environmental Sci & Tech 2011, 45 (9): 4016–4022.


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Airborne particle exposure modelings in indoor settings

Antti Joonas Koivisto1, Ismo K Koponen1, Miikka Dal Maso2, Kirsten Inga Kling1, Marcus Levin1, AnaSofia Fonseca1, Alexander CØ Jensen1, Tareq Hussein3, Kaarle Hämeri4, Mingzhou Yu5,6, WendelWohlleben7, Wouter Fransman8, Keld A Jensen1

1National Research Centre for the Working Environment, Copenhagen, Denmark; 2Tampere University of Technology, Tampere,Finland; 3University of Helsinki, Helsinki, Finland; 4The University of Jordan, Amman, Jordan; 5China Jiliang University,Hangzhou, China; 6Chinese Academy of Sciences,Xi'an, China; 7BASF SE, Ludwigshafen, Germany; 8TNO, Zeist, The Netherlands;[email protected]

Chemical safety assessment (CSA) consists of a substance hazard and exposure assessment. In REACHregulation CSA is required when a substance is manufactured or imported >10 tons per year and hasharmful properties or are classified as (very) Persistent, (very) Bioaccumulative and/or Toxic chemicals(PBT and/or vPvB) [1]. CSA may be required by national authorities (e.g. in Denmark AVP).

Exposure modelings consist of three parts: emission sources, dispersion model, and exposure andemission controls. Here we present emission library [2], dispersion models [3], and emission controlefficiency library [4]. Emission source characterization is one critical part considering exposuremodelings. Thus, we present how to characterize particle emission source strengths usingmeasurements and mass balance modelings [5] or with the concept of powder dustiness [6].Exposure scenarios are used to estimate substance emissions from a product during its different lifecycle stages. Here, we give examples how to create exposure scenarios involving emission sources (e.g.laser printer [7], sanding, and shredding) and exposure and emission controls (e.g. respirator,encapsulation, local exhaust ventilation, and air circulation through a filter [8]). We show how well themodels can predict concentration levels in laboratory conditions [9], during nanodiamond handling, in apaint factory [10], and during electrostatic spray coating. Finally, we present concept of inhalation doserate [11] and how it can be used to estimate regional deposited doses in respiratory tract in differentexposure scenarios.

1. ECHA, 2016. DOI: 10.2823/288625.2. Koivisto AJ et al., 2017. Nanoimpact, in press.3. Nazaroff WW, 1989. http://thesis.library.caltech.edu/576/4. Fransman W, et al., 2008. Ann. Occup. Hyg. 52:567 575.5. Hussein T and Kulmala M, 2008. Water Air Soil Poll. 8:23 34.6. Schneider T and Jensen KA, 2008. Ann. Occup. Hyg. 52:23–34.7. Koivisto et al., 2010. Atmos. Environ. 44:2140 2146.8. Mølgaard et al., 2014. Aerosol Sci. Technol. 48:409 417.9. Koivisto et al., 2012. J. Aerosol Sci. 47:58 69.10. Koivisto et al., 2015. Environ. Sci.: Processes Impacts. 17:62.11. Koivisto et al., 2012. ACS Nano. 1195 1203.

Testing geometrical layouts for a multi box aerosol dispersion model inchamber studies

Alexander CØ Jensen1, M Dal Maso2, E Belut3, AJ Koivisto1, A Meyer Plath4, M Van Tongeren5, ASánchez Jiménez5, I Tuinman6, M Domat7, J Toftum8, IK Koponen1

1NRCWE, Denmark; 2TUT, Finland; 3INRS, France; 4BAuA, Germany; 5IOM, UK; 6TNO, The Netherlands; 7ITENE, Spain; 8DTU,Denmark; [email protected]

The European REACH chemical legislation requires nanomaterials produced or imported in more than 1metric ton to be registered, and the potential exposure of workers across the full life cycle of thenanomaterial to be estimated (ECHA, 2016). In this framework it is also possible to use modelling data


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as a basis for estimation of exposure. Typical aerosol dispersion models use a two box based near field(NF)/far field (FF) approach, where it is assumed that there is instant and constant mixing throughoutboth boxes (Ganser & Hewett, 2017). However, in large rooms such as industrial facilities there is a riskof underestimating the aerosol concentrations and therefore exposure in the FF. Additionally theventilation might not be equally efficient in all parts of the room, thus further increasing theunderestimation (Jensen et al., 2015). Here we investigate if a two box model is the best way torepresent aerosol dispersions in large chambers by combining the information from detailed chambermeasurements with the output from the model when using different geometric inputs and using one ,two , or three box systems. In the present study we dispersed a 0.1 %w/w NaCl solution using anebulizer into a well ventilated 2.65 x 4.72 x 6.45 m3 chamber and monitored the development of theaerosol concentrations at four main/seven different measurement locations in the chamber. We usedinstruments measuring both size distributions and number concentrations in each location. The modelused includes source generation, ventilation and transport, coagulation, and deposition. Usingmeasured particle number concentration and size distribution data from the source measurementposition, positioned in one corner of the room, as source input in the model we were able to modelconcentrations in each of the boxes. A two box geometry resulted in good estimates for theconcentrations in the NF and the measurement position in the centre of the room. However, measuredconcentrations in the far end of the room were overestimated in this setup. To improve the estimateand increase granularity three box models were tested with different geometrical layouts and weinvestigated if alternative geometries would provide better estimates for measurement positionsfurther from the source while maintaining a good estimate of concentrations in the NF and at thecentral measurement point. While differences in concentration estimates between the differentgeometrical layouts were found, in general, the three box model provided a better fit for the measureddata than either the one box or two box model systems.

Workplace exposure assessment in the recycling of nano composites

Martin Seipenbusch1, Frederik Weis2, Christoph Hübner3, Dirk Dietrich4

1University of Stuttgart, Germany; 2Palas GmbH; 3Fraunhofer Institut für Chemische Technologie ICT; 4PALLMANNMaschinenfabrik GmbH & Co. KG; [email protected] stuttgart.de

Nanoparticle filled polymers, so called nano composites, are widely in use e.g. for their positive UVprotection and oxygen barrier properties in the packaging industry.

The question whether these materials pose a potential risk in the workplace when they are recycled isthe basis for the federally funded german project ProCycle in which two recycling pathways arefollowed. The first is typical for the production process but also a representative end of life scenario,where material is milled for reuse. The second is thermal recycling, where the thermal energy stored inthe polymeric part of the composite is recovered. The first pathway and the particle emissions in aworkplace setting are the focus of this contribution.

Three different polymers, PMMA, PS and LDPE, were compounded with nanoscale titania and copperoxide at 2 and 10% filling ratio. The particle emissions were evaluated using a Naneos Partector, a USMPS and a U Range system (Palas) to cover the size range from 4 nm to 40 microns. A strong peak inthe nanometer regime was found for all materials, regardless if nanomaterial fillers were used or not.Experiments showed that the most likely origin for this peak is the recondensation of organics broughtinto the gas phase by the milling process. The presence of fillers however reproducibly influences theposition of the nano peak.


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Workplace carbon nanotube exposure: Real time measurements using anaethalometer

Karin Elin Lovén1, Maria Hedmer2, Linus Ludvigsson3, Maria E Messing3, Anders Gudmundsson1,Joakim Pagels11Ergonomics and Aerosol Technology, Lund University, Sweden; 2Occupational and Environmental Medicine, Lund University,Sweden; 3Solid State Physics, Lund University, Sweden; [email protected]

The field of nanotechnology have generated innovations in many different areas, among these materialand electronic developments. Carbon nanotubes (CNTs) are one type of nanomaterial that has manypotential applications, but these fiber shaped nanoparticles are biopersistent and therefore considereda high toxicity nanomaterial. The potential risk of exposure for workers increases as these materialsbecome more and more used in the development of new products. Worker exposure measurements ofCNTs are still limited and the exposure is often characterized with filter based methods. However, filtermethods give limited information of the specific work place activities that generates CNT emissions.

This study aimed to characterize the possible exposures of CNTs in real time during production,processing and testing of multi walled CNT composites. Measurements were performed in the personalbreathing zone (PBZ) and the background zone (BG). Two μ aethalometers (AE51, Aethlabs. SanFrancisco, USA) were used to measure the equivalent Black Carbon (BC) concentrations in real time,interpreted from the measured changes in attenuation of infrared radiation (880 nm) through a filter.The method is sensitive to elemental carbon (EC) and CNTs. Some of the work tasks that weremonitored included direct mixing of CNT epoxy resin, electrophoretic deposition, cutting and shortbeam shear testing of CNT nanocomposites.

During “direct mixing of CNT epoxy resin”, clear BC peaks above background was observed in thepersonal breathing zone. The specific activities that generated the BC peaks within this work task wasrefilling of dry CNT powder and stirring the epoxy mixture with CNTs. The highest peak (18 μg m 3)comes from the first refilling of CNT powder, during which the worker openly transferred CNT powderfrom the bulk container into a small plastic container. The National Institute for Occupational Safety andHealth (NIOSH, USA) have suggested an 8 h exposure limit for CNTs based on EC of 1 μg C m 3. Some ofthe observed BC peaks are temporarily well above this proposed limit and they may impose risks for theworkers. The EC filter samples collected alongside the aethalometers were below the limit of detection,while Electron Microscopy analysis of filter samples did show CNT material for a few work tasks. As theaethalometers are easy to use and relatively cheap, they are a particularly promising tool to use in afirst screening stage at work places where CNTs are handled. They also provide complementaryinformation to time integrated filter collection by linking exposures to specific work tasks.

Sensing solution for airborne carbon nanotube exposure in workplaces basedon surface enhanced Raman Spectroscopy

Rudolf Bieri1, Burcu Celikkol Zijlstra1, Joanna Borek Donten1, Stefano Cattaneo2, Thomas Bürgi31Stat Peel AG, Switzerland; 2CSEM Swiss Center for Electronics and Microtechnology, Switzerland; 3University of Geneva,Switzerland; [email protected]

Today’s advances in man made nanomaterials pose new and unprecedented health risks, arisingespecially from airborne, inhalable fiber shaped nanomaterials, like carbon nanotubes (CNTs). In vivostudies indicate that inhalation of CNTs can cause adverse pulmonary effects including inflammation,granulomas and pulmonary fibrosis [1, 2]. As a result, the National Institute of Occupational Health and


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Safety (NIOSH) in USA recommends an exposure limit of 1μg/m3 of CNTs as a respirable mass 8 hourtime weighted average concentration [3]. However, detecting this amount is extremely challenging withthe current sensing solutions.

Here, we would like to present a wearable, cost effective badge sampler with an air filtration system[4,5]. The sampler is capable of collecting airborne carbon nanotubes from the surrounding atmosphereon a disposable membrane filter, which acts as a SERS substrate. The badge system is integrated with abench top sized optical reader for fast and automated inspection of collected samples.

Our system enables detection of sub nanogram quantities of collected CNTs and, by utilizing theadvantages of Raman spectroscopy, is a solution able to uniquely distinguish carbon nanotubes frombackground aerosols present in air.

[1] Nat Nanotechnol. 4(11): 747–751 (2009)[2] Am J Physiol Lung Cell Mol Physiol 289: 698 708 (2005)[3] NIOSH CIB 65: Carbon Nanotubes and Nanofibers[4] Patent pending[5] Swiss Technology and Innovation Project 17623


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Session7A: Toxicogenomics and adverse outcome pathways(AOP) as tools for nano risk assessment

Wednesday 31 May, 13:30 15:30Location: Room 1 (Conference room)Session chairs: Sabina Halappanavar & Sarah Søs Poulsen

Developing an adverse outcome pathway based on toxicogenomic data forENM induced risk of developing atherosclerotic plaques

Sarah S Poulsen1, Anne T Saber1, Nicklas R Jacobsen1, Petra Jackson1, AndrewWilliams2, JulieBourdon Lacombe2, Mainul Husain2, Stefan Bengtson1, Carole Yauk2, Håkan Wallin3, SabinaHalappanavar2, Ulla Vogel11The National Research Centre for the Working Environment, Denmark; 2Environmental Health Science and Research Bureau,Health Canada, Ottawa, Ontario, K1A 0K9, Canada; 3Statens Arbeidsmiljøinstitutt, Gydas vei 8, Majorstuen, 0363 Oslo, Norway;[email protected]

Adverse lung effects following pulmonary exposure to engineered nanomaterials (ENMs) are welldocumented in rodents. With the aim of understanding the molecular changes accountable for theseeffects, we have the in recent years investigated the global transcriptional changes in the lung and liversof mice pulmonary exposed to multi walled carbon nanotubes, carbon black, TiO2 or graphene byintratracheal instillation. Besides aiding the identification of nanomaterial specific changes, this alsoenabled a cross evaluation of processes and changes common for all types of ENM exposures.

Despite the large variances in physicochemical properties of the ENMs tested, they all commonlyinduced a strong and persistent pulmonary acute phase response. The most differentially regulatedgene across all exposure types was the acute phase protein serum amyloid A isoform 3 (Saa3), with theother isoforms (Saa1 and Saa2) also being strongly induced. The SAA proteins are traditionally thoughtto originate from the liver, however, in our further analyses on plasma protein levels, we established astrong correlation between pulmonary Saa3 mRNA levels and plasma SAA3 protein levels afterexposure. This indicates a significant impact of the pulmonary derived Saa expression on plasma proteinlevels.

Increased plasma levels of SAA protein are a predictor of cardiovascular disease in epidemiologicalstudies. A proposed mechanism is through a replacement of ApoA 1 with SAA on the HDL lipoproteins,which impairs HDL's ability to mediate cholesterol efflux from macrophages.This results in reducedreverse cholesterol transport, sequestering of peripheral cholesterol, and transport of cholesterol fromHDL to macrophages, thereby facilitating the transformation of macrophages into foam cells.Based on our current data, we hypothesize that pulmonary exposure to a stressor, in this case ENMs,initiates a potent and persistent Saa induction in the lungs. The pulmonary derived SAA proteintranslocates to the bloodstream and replaces ApoA 1 in HDL. This leads to reverse cholesteroltransport, foam cell transformation, and increased atherosclerotic plaque areas. We have constructed aputative AOP illustrating this hypothesis. Besides providing a pathway for this poorly understoodsystemic adverse effect of particle and ENMs exposure, this AOP can also be used as a scaffold forhighlighting key event relationships needing additional investigation.


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Utilizing an adverse outcome pathway framework to investigate the essentialityof Interleukin and STAT signaling in the pathology of a carbon nanotube in mice

Sabina Halappanavar1, Jake Nikota1, Allyson Banville1, Laura Rose Goodwin1, Dongmei Wu1, AndrewWilliams1, Håkan Wallin2, Ulla Vogel21Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9, Canada; 2National ResearchCentre for the Working Environment, Lerso Parkallé 105, DK 2100 Copenhagen, Denmark; Department of Public Health,University of Copenhagen, DK 1353 Copenhagen K, Denmark; sabina.halappanavar@hc sc.gc.ca

Background: The accumulation of MWCNTs in the lung environment leads to inflammation and thedevelopment of disease similar to pulmonary fibrosis in rodents. Adverse Outcome Pathways (AOPs) area framework for defining and organizing the key events that comprise the biological changes leading toundesirable events. A putative AOP has been developed describing MWCNT induced pulmonaryfibrosis; inflammation and the subsequent healing response induced by inflammatory mechanisms havebeen implicated in disease progression.

Objectives: The objective of the present study was to address a key data gap in this AOP: empirical datasupporting the essentiality of pulmonary inflammation as a key event prior to fibrosis. Specifically,Interleukin 1 Receptor1 (IL 1R1) and Signal Transducer and Activator of Transcription 6 (STAT6) knockout (KO) mice were employed to target inflammation and the subsequent healing response usingMWCNTs as a model pro fibrotic stressor to determine whether this altered the development offibrosis.

Results:Wild type (WT) C57BL/6, IL 1R1 (KO) or STAT6 KO mice were exposed to a high dose of Mitsui 7MWCNT by intratracheal administration. Inflammation was assessed 24 hours and 28 days post MWCNTadministration, and fibrotic lesion development was assessed 28 days post MWCNT administration.MWCNT induced acute inflammation was suppressed in IL 1R1 KO mice at the 24 hour time pointrelative to WT mice, but this suppression was not observed 28 days post exposure, and IL 1R1 KO didnot alter fibrotic disease development. In contrast, STAT6 KO mice exhibited suppressed acuteinflammation and attenuated fibrotic disease in response to MWCNT administration compared toSTAT6 WT mice. Whole genome analysis of all post exposure time points identified a subset ofdifferentially expressed genes associated with fibrosis in both KO mice compared to WT mice.

Conclusion: The findings support the essentiality of STAT6 mediated signaling in the development ofMWCNT induced fibrotic disease. The IL 1R1 KO results also highlight the robust nature of theinflammatory response associated with MWCNT exposure, and indicate a system with multipleredundancies. These data add to the evidence supporting an existing AOP, and will be useful indesigning screening strategies that could be used by regulatory agencies to distinguish betweenMWCNTs of varying toxicity.

TiO2 induced gene expression and protein profiles in rat lung: a subacuteinhalation study

Laëtitia Chézeau1, Ramia Safar2, Olivier Joubert2, Frédéric Cosnier1, Bertrand Rihn2, Laurent Gaté11Institut National de Recherche et de Sécurité, Vandoeuvre lès Nancy, France; 2Université de Lorraine, Faculté dePharmacie,EA 3452 CITHEFOR, Nancy, France; [email protected]

Due to the growing use of nanoparticles in industrial processes, the number of workers potentiallyexposed is increasing while the toxicological properties of these compounds are not fully known.


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Inhalation represents the main route of occupational exposure. In this respect, the experimentaltoxicology studies conducted by inhalation in animals appear to be the most relevant for the earlyevaluation of the hazard associated with exposure to nanoaerosols. In this work we study thepulmonary toxicological properties of titanium dioxide using conventional and molecular toxicologicalapproaches. For this, we exposed Fischer 344 rats by nose only inhalation 6 hours/day, 5 days/week for4 weeks to a nanoaerosol of TiO2 (10 mg/m3). Lung samples have been collected up to 180 days afterthe end of exposure. Biochemical and cytological analyses of the broncho alveolar lavage fluid (BALF)showed a strong inflammatory response up to 3 days which decreased overtime but persisted 180 daysafter exposure. In addition, a gene expression profiling experiment showed an overexpression of genesinvolved in inflammation that persisted in the chronic phase. These observations are consistent withphysiopathological changes. Moreover, this experimental approach revealed also an overexpression ofgenes involved in vasodilatation and oxidative stress. This transcriptomics approach has beencompleted with proteomics analysis in the BALF. The preliminary data also showed an overexpression ofproteins involved in inflammation and oxidative stress. To confirm these data, additional experimentswill be performed at the protein level using western blot or ELISA. Finally, analysis of long termepigenetic modifications (DNA methylation, acetylation) will be performed to observe possible chronicgene expression modification, involved in physio pathological processes.

Nanomaterial impact on models of asthma and allergic airway disease:mechanistic insight as an aid for hazard assessment

Kirsty Meldrum, Sarah Robertson, Rachel Smith, Martin LeonardPublic Health England, United Kingdom; [email protected]

Asthma is a chronic respiratory disease considered for its susceptibility to environmental exposure. Theimpact of nanomaterials on this condition is a major challenge for modelling approaches attempting toassign hazard potential to nanoscale properties. We have carried out a comprehensive review of thosestudies focussed to safety assessment of different nanomaterials and their unique characteristics inasthma and allergic airway disease relevant after respiratory exposure. These mainly include in vivoapproaches, with the weight of evidence suggesting a modifying role for nanomaterial exposure. Thereis however concern for how one may assign relative hazard potential to individual nanoscale propertiesacross these studies, primarily due to the variability in modelling approaches and endpoints of diseaseused to attribute dysfunction. It has been suggested that key to refinement of appropriate testingsystems is a more complete understanding of how these materials may influence disease at the cellularand molecular level. Such detailed mechanistic information has the potential not only to extract themost relevant information across different model systems but also has enhanced potential to linknanoscale properties to disease outcome appropriate for human exposure. In order to further ourunderstanding of the range of mechanistic information that can be extracted from such model systemswe used RNA SEQ analysis to map transcriptional alterations in the lung, which occur in a murine modelof allergen induced airway disease after exposure to cerium dioxide nanoparticles. House dust miteexposure resulted in eosinophilic lung inflammation, mast cell accumulation, mucin production andincreased airway hyperresponsiveness (AHR). Co exposure with cerium dioxide nanoparticles resultedin a further enhancement of these inflammatory disease parameters, including increased IgE levels,consistent with a type 2 immune response within the lung. RNA SEQ was carried out using 101PEsequencing after 1 day and 3 week repeat exposures and alterations in gene expression werecategorised according to cellular and molecular pathways. Characterisation of cerium dioxidenanoparticles was achieved using electron microscopy, dynamic light scattering and nanoparticletracking analysis allowing for alterations in biological response to be associated with the properties ofthis particular material. Adoption of this type of approach to nanomaterial testing for disease


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susceptibility, including standardised models and focussed in vitro and in silico approaches togetherwith integrating mechanistic information from high content analysis has the potential to more reliablyidentify properties of concern relevant for human safety assessment.

Proteomic analysis of nasal lavage samples collected from weldersexperimentally exposed to welding fume nanoparticles

Neserin Ali1, Stefan Ljunggren2, Helen M Karlsson2, Katrin Dierschke1, Jörn Nielsen1, Mats Bohgard3,Aneta Wierzbicka3, Joakim Pagels3, Christina Isaxon3, Anders Gudmundsson3, Jenny Rissler3, ChristianH Lindh1, Monica Kåredal11Occupational and environmental medicine, Lund University, Sweden; 2Occupational medicine, Linköping university, Sweden;3Ergonomics and aerosol technology, Lund university, Sweden; [email protected]

IntroductionExposure to welding fume is associated with symptoms from the respiratory tract and alsocardiovascular health effects. During welding a mixture of gases and particles, including nanosizedparticles ranging between 2 70 nm, are produced at high numbers. The primary particels agglomeratefast to form larger complexes but may still be inhalable and able to reach the lungs. There aresuggestions to explain the pathogensis but much knowledge is still lacking.

AimThe aim of the study was to study physiological mechanisms associated with airway symptoms inducedby welding fume particle exposure by measuring protein changes in nasal lavage from subjectsexperimentally exposed to welding fume particles.

MethodNasal lavage samples were obtained from symptomatic welders experimentally exposed to weldingfume (1000 μg /m3). Subjects were also exposed to filtered air. Nasal lavage samples were collectedbefore exposure and then immediately at the end of and the day after exposure.

Mechanistic pathways were first investigated using a shotgun proteomic hypothesis generating methodby analyses of pooled samples. Samples from all subjects were pooled at each time point. Proteindigests were analyzed with liquid chromatography coupled to a high resolution mass spectrometry (LCMS/MS, Orbitrap Velos Pro). Identification and relative quantification was performed with MAXQuantsoftware against a human database downloaded from uniprot.

Findings from shotgun analysis were further investigated by analyses of individual samples using atargeted label free LC/MS method (SRM LC/MS, 5500 QTRAP, AB Sciex, UFLCXR; ShimadzuCorporation).

Data was statistically evaluated using a linear mixed model and Wilcoxon signed rank test. Proteins withsignificantly changed levels were included in the mechanistic analysis performed using ingenuitypathway analysis (IPA).

Results and discussionIn this study, pathway analysis of pooled samples suggested that there were three major pathwaysassociated with the exposure. These were LXR/RXR activation of monocytes, the inflammatory defensemechanism by macrophages and anti inflammatory mechanisms. Data also showed that thesemechanisms influenced tha status of the extracellular matrix (ECM).


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Analyses of indvidual samples identified 46 proteins with significantly changed levels. Pathway analysisshowed that these proteins had two pathways in common; activation of acute phase response signalingand suppression of LXR/RXR. Main findings were decreased levels of MMP9 and increased levels ofMMP8 following welding exposure. Both MMP8 and MMP9 are proteases that degrade extracellularmatrix proteins and therefore play a central role in normal tissue structure to maintain the balancebetween formation and degradation of extracellular matrix proteins.


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Session 7B: Exposure management

Wednesday 31 May, 13:30 15:30Location: Room 2Session chairs: Martin Seipenbusch & Christof Asbach

Assessment strategy for nanoparticles in workplace air

Dirk DahmannInstitut für Gefahrstoff Forschung, Bochum, BGRCI, Germany; [email protected]

AbstractIn order to perform a proper risk analysis and an educated management of technical or organizationalmeasures with the purpose of generating safe conditions in workplaces with a possible inhalationexposure to nanoparticles and their agglomerates and aggregates (NOAA), it is necessary to quantify orat least asses this exposure with a sufficient degree of validity.

One part of the assessment process is the selection of proper measurement techniques and procedures.This presentation will concentrate on the proper planning of measurements or the assessment strategy.

Workplaces with a possible inhalation exposure to NOAA do not only pose specific problems forinstrumentation but also create new challenges for the selection of a proper measurement strategy.Some examples of these problems are:

The necessity of distinguishing NOAA exposure from a high and fluctuating background ofincidentally created ultrafine particlesThe fact that currently not even a uniform metric to be determined, i.e. particle numberconcentration, particle surface concentration, or mass concentration, is clearly defined orinternationally agreed uponThe availability of measurement equipment in a wide range of “capability” and cost (in terms ofmoney and operating effort), which makes cost effectiveness a primary issue in selectingappropriate equipment. That selection, however, has then significant consequences for the validityand the use of the result obtainable from the assessmentsThe possibly high differences in hazard level of the investigated NOAA could be reflected in themeasurement procedures applied or they could be disregarded. In other words: would a “highhazard” NOAA (example: CNT) be assessed using identical procedures as the ones for “low hazard”NOAA (example: BaSO4), and how is the hazard level defined if the answer would be “no”.

Recently there have been some suggestions to structure the measurement strategy according to a socalled tiered approach, which groups the assessment procedure into three consecutive steps (tiers) ofgrowing complexity, cost, and also “depth” of results. The idea is that one just applies “what isnecessary” to get enough information to make the necessary decisions within the risk assessmentprocedure. The European Standardization Organization (CEN) has recently started work within themandate M 461 (“Nanotechnologies and Nanomaterials”). The responsible working group is currentlyworking on a standard describing general procedure within an appropriate assessment strategy. In thispresentation an overview on the current state of discussions is tried, which take into account the newstandard prEN 17058 being developed within that group.


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Occupational exposure to nanoparticles: monitoring and management inindustrial settings

Mar Viana1, Apostolos Salmatonidis1, Eliseo Monfort2, Vicenta Sanfélix2, Ana López Lilao2, Germán FDe la Fuente3, Luis A. Angurel3, Carlos Estepa3, Jordi Díaz41IDAEA CSIC, Spain; 2ITC UJI, Spain; 3ICMA CSIC, Zaragoza University, Spain; 4CCiTUB, Barcelona University, Spain;[email protected]

Inhalation exposure to particles is a well known health hazard, with nanoparticles (<100 nm) being anespecially harmful component due to their ability to penetrate deep into the respiratory tract (Strak etal., 2012; Meng et al., 2013). Recent research in the aerosol field has developed a strong focus on thecharacterisation of nanoparticle release and exposure scenarios, with the aim to minimise and/ormanage potential health risks. Industrial settings are an especially interesting source of potentialexposure scenarios, due to the large variability of processes (mechanical, thermal, etc.) and input andoutput materials (engineered nanoparticles, micron sized powders, etc.) involved, as well as the varietyof mitigation measures implemented. This work aims to present an overview of exposure scenarios, andtheir related nanoparticle emissions, characterised under real world operating conditions in industrialplants and pilot plants. Examples of the industrial processes evaluated are ceramic tile sintering inconventional and laser furnaces, ceramic tile ablation, atmospheric plasma spraying, inkjet printing withengineered nanoparticles, engineered nanoparticle handling as additives, spray drying, ceramic tileshaping, and mechanical processing of raw materials. Results evidenced a wide range of particleemission concentrations between 104/cm3 and 106/cm3 in terms of particle number concentration, aswell as high concentrations in terms of mass (102 104μgPM2.5/m3) and lung deposited surface area (upto 5000 μm2/cm3). The in depth characterisation in each setting evidenced the engineered and nonengineered nature of the nanoparticles, thus highlighting the relevance of the processes themselves assources of nanoparticle emissions and occupational exposures. Nanoparticle emission mechanismsdiffered for each process under study, including particle formation by nucleation and direct emission ofprimary particles, as well as particle growth. When implemented, the effectiveness of mitigationstrategies was assessed. Where unavailable, risk prevention protocols were proposed andimplemented. Over all, our work evidences the relevance of occupational exposure to nanoparticles inindustrial settings and the need for specific real world studies on this topic, given the highly specificnature of each process under study. A link with risk assessment modelling is also necessary, in view ofmodel validation and extrapolation of the experimental results obtained.

References:Meng et al. (2013) Environ Health Perspect 121.Strak et al. (2012) Environ Health Perspect 120.

Exposure assessment to noaa at workplace – an opportunity towards a saferand more responsible development of nanocomposites: NANOLEAP project

Cécile Ducros1, Sébastien Artous1, Jose Luis Valverde Palomino2, Juan Francisco Rodriguez2, IsabelRodriguez3, Simon Clavaguera11CEA, France; 2UCLM, Ciudad Real 13071, Spain; 3IMDEA Nano, Madrid 28049, Spain; [email protected]

Nanotechnology has rapidly promoted the development of a new generation of smart and innovativeproducts and processes that are nano enabled, and have created a tremendous growth potential for alarge number of industry sector. In particular, nanotechnology offers substantial possibilities forimproving the competitive position of the EU and for responding to key societal challenges. Ensuring


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the safe, sustainable and responsible development of nanotechnologies is a key objective of theHorizon 2020 Work Programme.

NANOLEAP project aims at the development of a coordinated network of specialized pilot lines for theproduction of nanocomposite based products for different civil infrastructure and building applications.The goal of this infrastructure is to support the research activities of European SMEs in the Constructionsector in nanocomposite products enabling the progress of the product to next steps of technologydeployment such as installation of industrial lines and enter in the commercialization stage. NANOLEAPproject will also help to road map nanosafety issues related to the production, use and handling ofnanocomposite based products ahead of any realized risk to society.

The aim of this paper is to present our approach to assess potential occupational exposure toengineered nanomaterials (ENMs) in the 10 different pilot lines (nanoparticle production,nanocomposite manufacturing, aerogel preparation etc…). The followed exposure assessment strategyis the OECD harmonized tiered approach:

tier one evaluates the available information through questionnaires sent to the pilot plan partnersor scoping visits.tier two identify sources of potential release of airborne ENMs.tier three was reached when exposure was likely, and consists on measurements of personalexposure during tasks using personal samplers, size resolved direct reading instruments andoffline methods.

The 10 different pilot lines and the identified exposure scenarios to ENMs were classified according tothe volume of ENMs manufactured or handled, frequency and duration of use and the type of processas well as risk management measures. Several scoping visits and field measurements were performedto gather information on emissions and occupational exposure. The aim consists in determiningwhether a process, or part of a process, could potentially release nanoparticles that can be emitted toindoor air and that could eventually lead to potential occupational exposure. After the implementationof risk minimization procedures by the pilot plant partners, risk assessment will be updated. Specifictraining and organizational procedures will be implemented on each pilot plan facilities that will help totackle the main nanosafety issues.

Evaluation of and risk management measures for the handling of carbonnanotubes in a laboratory

Christian Schumacher, Bianca Oeffling, Johannes Pelzer, Carsten MöhlmannInstitut für Arbeitsschutz der Deutschen Gesetzlichen Unfallversicherung, Germany; [email protected]

The Institute for occupational safety and health of the German Social Accident Insurance (IFA) wasasked by a technical laboratory how they could handle aluminium or silicon wafers that were coatedwith different kinds of carbon nanotubes (CNT) in a safe way.

As a first step the material safety data sheets (MSDS) from the manufacturers were checked. Then theIFA examined some material samples of the coated wafers under a scanning electron microscope (SEM)and simulated the planned handling procedure by adopting parameters like air stream and physicalimpact in a glove box. A possible release of CNT was sampled. The air samples were evaluated underSEM analysis and showed that in case of careless handling with impact onto the coating an emission ispossible and some CNT agglomerates fitted to the so called WHO criteria for hazardous fibres (aspectratio 3:1, length > 5μm). Consequently some improvements of the construction of the coated wafers


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were suggested, like sealing the edge of the wafer. Additional advice was given how to handle the CNTcoated wafers, especially during mounting and transportation.

To evaluate the suggested risk management measures (RMM), the IFA examined the possible emissionand exposure by workplace measurements. For this purpose a measurement strategy by stationarysampling in the near field, far field and background as well as sampling at the person were applied. Anew analysing strategy for SEM with optimized technical parameters as well as counting routines wasdeveloped. The aim was to compare the results with a suggested occupational exposure limit value of10.000 fibres per cubic meter.

The results show that during this application no emission and thus no exposure took place at theworkplace. Nevertheless recommendations were given to improve technical measures and how toimplement a protocol for the case of unintentional release, including the use of personal protectiveequipment.

Assessment of personal exposure to airborne nanomaterials A review ofmeasurement equipment

Christof Asbach1, Simon Clavaguera2, Dirk Dahmann3, Martin Fierz4, Luca Fontana5, Ivo Iavicoli5,6,Heinz Kaminski1, Laura MacCalman7, Asmus Meyer Plath8, Barbara Simonow8, Martie Van Tongeren7,Ana Maria Todea11Institut für Energie und Umwelttechnik e. V. (IUTA), Germany; 2NanoSafety Platform, Commissariat à l’Energie Atomique etaux Energies Alternatives (CEA), France; 3Institute for the Research on Hazardous Substances (IGF), Germany; 4University ofApplied Sciences Northwestern Switzerland (FHNW), Switzerland; 5Catholic University of the Sacred Heart (UCSC), Italy;6University of Naples, Federico II (UNINA), Italy; 7Institute of Occupational Medicine (IOM), UK; 8Federal Institute ofOccupational Safety and Health (BAuA), Germany; [email protected]

Exposure to airborne granular or fibrous dusts needs to be assessed in the personal breathing zone, i.e.within a 30 cm hemisphere around mouth and nose, by the use of personal measurement equipment.Specific measurement devices for assessing personal exposure to airborne nanomaterials have onlybecome available in the recent years. They can be differentiated into direct reading personal monitorsand personal samplers that collect the airborne nanomaterials for subsequent analyses. Most personalmonitors are based on unipolar diffusion charging of the incoming aerosol particles and subsequentmeasurement of the particle induced current to derive the lung deposited surface area (LDSA)concentration. Some of them contain additional means for also determining the particle numberconcentration and the mean particle size. As of now, only one type of personal condensation particlecounter (CPC) exists. Personal samplers mostly remove particles above a certain size from the airflowprior to collecting the remaining fraction onto filters. However, the cut off aerodynamic diameter oftypically used pre separators is mostly above the nanometer size range.

A detailed study on their accuracy, comparability and field applicability had been lacking and thus oneaim of the recently finished SIINN Eranet project nanoIndEx was to scrutinize the possibilities andlimitations of this new measurement equipment. The data show that the currently commerciallyavailable samplers and monitors are robust and ready for field use with sufficient accuracy andcomparability. However, several limitations were identified, e.g. regarding the applicable particle sizerange of the personal monitors and their in general lower accuracy and comparability with regard totheir stationary counterparts. For example, diffusion charger based personal monitors were found to beaccurate within ±30%, provided the main fraction of the particles is within a size range from 20 nm to400 nm. The personal CPC was shown to have a higher accuracy of about ±10%, but is limited in therange of detectable particle concentrations to a maximum of 200,000 1/cm³, whereas the diffusion


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charger instruments are still applicable at concentrations exceeding 1,000,000 1/cm³. The accuracy ofthe personal samplers is strongly related to the downstream analysis techniques used.

A review of the available personal monitors and samplers will be presented along with data fromlaboratory and field measurements from the project as well as from the literature. The results will bediscussed in view of the use of these instruments in personal exposure assessment, e.g. in theframework of a tiered approach.

Explosion hazards of nanoparticles

Leonid A. Turkevich, Douglas E. EvansCDC/NIOSH, United States of America; [email protected]

Many hydrocarbon (e.g. coal) and carbohydrate (e.g. grain) dusts present an explosion hazard. There isa concern that engineered carbon nanoparticles, when manufactured on an industrial scale, will pose asimilar explosion hazard. Nanocellulose, currently at the cusp of commercialization, potentially presentsa similar problem. Some finely divided metals exhibit pyrophoric behavior, and their potentialexplosivity at the nanoscale is also a cause for concern. With collaborators, NIOSH has conductedsystematic explosion and flammability testing of a variety of these materials in order to evaluate theextent and severity of these hazards. This presentation summarizes our current understanding derivedfrom that testing.

A fairly coherent picture emerges for the carbonaceous nanomaterials. Explosion testing (ASTM E1226protocol) has been performed on SWCNTs (single walled carbon nanotubes), MWCNTs (multi walledcarbon nanotubes) and CNFs (carbon nanofibers), graphene, diamond, fullerene, as well as severaldifferent control carbon blacks and graphites. Samples typically exhibit overpressures of 5 7 bar, anddeflagration index KSt ~ 10 80 bar m/s, which places these materials in European Dust Explosion ClassSt 1 (similar to cotton and wood dust). There is minimal variation between these different materials.The explosive characteristics are uncorrelated with primary particle size (BET specific surface area) andagglomeration state.

We have measured minimum explosive concentration (MEC), minimum ignition energy (MIE), andminimum ignition temperature (MITcloud) for selected materials. The nanocarbons exhibit MEC ~ 101 102

g/m3, an order of magnitude lower than that for coals and for fine particle carbon blacks and graphites.We estimate MIE ~ 102 – 103 J, an order of magnitude higher than that for coals but an order ofmagnitude lower than that for fine particle graphites. Thus the explosion severity of the nanocarbons iscomparable to that of the coals, but their explosion susceptibility (ease of ignition) is lower. MITcloud >550oC, comparable to that of the coals and carbon blacks.

Testing has also been conducted on nanocellulose. The explosion mechanism differs from that of thenanocarbons but appears to correspond to that of macroscopic wood dust, with comparable explosionparameters. More limited testing has been performed on several of the nanometals. These powdersinvariably have oxidized surfaces, the thickness of which inhibits explosive reaction. Thus nominallyidentical materials may exhibit disparate explosion characteristics due to differences in surfaceoxidation.


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Keynote IV Thursday 1 June, 9:00 10:00Location: Room 1 (Conference room)Session chairs: Sally Tinkle & Hannu Norppa

Occupational risk assessment of nanomaterial exposurePaul Alexis Schulte

National Institute for Occupational Safety and Health, United States of America; [email protected]

Research interestsFor the last 40 years the focus of my research has been the protection of workers. This is still myinterest. I have done this in diverse areas of standards development, hazard evaluation, epidemiology,risk assessment, communication, and education. For the last 13 years in leading the NanotechnologyResearch Center I have promoted the utilization and development of quantitative risk assessment.

Keynote – Occupational Risk Assessment of Nanomaterial ExposureResponsible development of a technology, such as nanotechnology requires the development ofguidance to protect people and the environment from adverse effects and unintended consequences.With regard to workers — the first people exposed to nanotechnology — occupational exposure limitsand attendant risk management guidelines are the critical components of prevention and protection.The driving activity for development of these preventive components is risk assessment, and moreparticularly, quantitative risk assessment (QRA). Quantitative and semi quantitative risk assessmentapproaches have been applied to nanomaterials and resultant Occupational Exposure Limits have beenissued.

In this presentation the history of QRA with regard to nanomaterials will be reviewed and the issuesthat have arisen will be identified and discussed. For a few single nanoparticle substance riskassessments there have generally been limited, but adequate data, on which to conduct theassessment. However, for the myriad of nanomaterials in commerce or anticipated to be in commercethere are practically no data. Consequently various categorical approaches have been developed. Thesewill be compared and contrasted. Finally, new generations of nanomaterials are in development.Questions of how risk assessment might be approached for these new nanomaterials will be addressed.


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Session 8A: Nanomaterial epidemiology & biomonitoring

Thursday 1 June, 10:30 12:00Location: Room 3Session chairs: Enrico Bergamaschi & Anne T Saber

Health surveillance and epidemiology in nanomaterial workers worldwidewhere are we today?

Michael Riediker1,2,31IOM Singapore, Singapore; 2Nanyang Technological University, Singapore; 3Institut de Santé au Travail, University of Lausanne,Switzerland; michael.riediker@iom world.sg

Epidemiological studies and health surveillance are essential to understand the risk to workers exposedto potentially harmful substances, and to evaluate the effectiveness of “safe exposure levels” that werederived from animal and cell experiment data. Globally, a few epidemiological studies already started toassess the health of workers potentially exposed to manufactured nanomaterials. However, asanticipated in early discussions, most companies have still relatively small numbers of workers. To allowpooling international cohorts, five years ago, several leading groups defined together a road map for aglobally harmonized framework for exposure characterization, identi cation of study populations,de nition of health endpoints, evaluation of appropriateness of study designs, data collection andanalysis, and interpretation of the results. These studies have progressed but it is not clear how stronglyfunding limitations impacted their ability for a coordinated approach. This presentation will reviewstudies ongoing to data and compare the progress made to the initial roadmap.

Epidemiological surveillance of nanotechnology workers: Past and newchallenges based on the French example

Irina Guseva CanuInstitut universitaire romand de santé ou travail (IST), France; irina.guseva [email protected]

France was a pioneer country developing the first registry of engineered nanomaterials (ENM) in Europeand launching a program of prospective epidemiological surveillance of nanotechnology workersfocused on potential long term health effects of ENM. While the former was ordered by the FrenchMinistry of Ecology, the latter was a joint demand of the Ministries of Health and of Labor. A challengewas both, political and scientific: the definition of ENM was imprecise and ENM physicochemicalproperties influencing their exposure potential, biological and clinical effects following the exposure,and related risks were unknown.

In 2012, the French Institute for Public Health Surveillance proposed a step by step project where thefirst step was to identify companies dealing with ENM, and to register potentially exposed workers. Anexploratory study showed that the ENM producing companies are more willing to cooperate ascompared to ENM using companies. A strong involvement of the ministries was required to establishcollaboration with them; however, it also appeared challenging. Moreover, lacking standardizedmethodology of ENM exposure assessment made challenging identification of eligible workers even incollaborating companies.


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Thanks to the “Quintet ExpoNano” partnership, involving national experts in ENM metrology,occupational hygiene and epidemiology, and collaboration with the Occupational Health Inspectorate,academic researchers, and the national registry “R nano”, the program has been moved forward, and ageneral protocol with standardized methodological tools and questionnaires for workers’ exposure andhealth assessment, and two communication strategies to recruit companies into the program and tomotivate eligible workers to participate and to retain their participation constant over time weredeveloped. Implementation of this program resulted in recruitment of about 25 companies and of morethan 150 workers between January 2014 and June 2016. However the program was deemed inefficient.The recruitment of companies remained the biggest challenge given the lack of interest to cooperateamong large industrial companies.

In a situation of financial crisis, political and economic reforms, the program could be discontinued, thatis politically challenging, or substantially modified by simplifying its protocol, the procedure of exposureassessment and the content of questionnaires. If the second option is proceeded, it would bring achallenge to solve essentially a non scientific problem by scientific means.

Occupational exposure and health effects of multi walled carbon nanotubes

Liliya Fatkhutdinova1, Timur Khaliullin2, Elena Kisin2, Anna Shvedova21Kazan State Medical University, Russian Federation; 2National Institute for Occupational Safety and Health, WV 26505,Morgantown, USA; [email protected]

Studying the behavior of nanoparticles (NPs) in living systems and safety aspects of new nanomaterialsis one of the strategic objectives of nanotechnology network development. The urgency of the problemis due to lack of sufficient information regarding behavior of engineered nanoparticles in humans. Thecarbonaceous NPs occupy a leading position on the market of nanomaterials. The number ofenterprises, which are produced or used carbon nanotubes (CNT) and other types of carbonaceousnanomaterials, is growing from year to year. Investigation of the cell and molecular mechanisms ofbiological and toxic effects in interaction with the human body is necessary for risk assessment andcontrol. In 2009 2015, in the frame of the joint Russian American CNT ERA project (Carbon NanoTubesExposure and Risk Assessment), exposure and human health risks associated with industrial exposure toinhaled MWCNTs had been investigated. The study included a hygienic, toxicological andepidemiological stages. Respirable fraction concentration of aerosol in the employee's breathing zone,averaged over the 8 hour period, were in the range of 0.54 to 6.11 mg / m3 (calculated as elementalcarbon). Our data suggest that exposure to MWCNTs at the workplace could change the content ofsome markers of fibrosis in serum and induced sputum samples. In particular, the KL 6 levels in inducedsputum were significantly related to exposure to MWCNTs. Moreover, we could detect exposurerelated increase in serum TGF 1, but only for young workers. Osteopontin in this study did not showitself as informative human biomarker, but it is still recommended to be included in the battery tests forfuture research. The significant changes in several miRNAs and mRNAs expression as well as theirregulatory networks identified in this study revealed important insights into the molecular details ofMWCNT induced toxicity and pathogenesis in humans.


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Session 8B: Risk assessment & management

Thursday 1 June, 10:30 12:00Location: Room 1 (Conference room)Session chairs: Wouter Fransman & Keld A Jensen

Current status of nanomaterial risk assessment models and strategies to riskmanagement

Wouter FransmanTNO, The Netherlands; [email protected]

Environmental release and human exposure to manufactured nanomaterials may be associated with arange of industries and use of nano enabled products. The nanomaterials will have highly differentlevels of potential hazard, which will require different control strategies for precautionary riskmanagement. The uncertainties on the risks urgently needs to be carefully addressed to prevent thatexcessive stakeholder risk perception blocks the benefits of nano enabled products. Sound scientificinformation and predictive models needs to be developed to identify potential risks of nanomaterialexposure scenarios and nano enabled products and, when considered unacceptable, efficiently mitigatesuch human health risks. This has to be done in a holistic manner, taking into consideration all stages ofthe life cycle of these products. Various risk assessment models have been developed in recent years,with different levels of quantitative details and input requirements. Some Higher Tier models exist thatrequire a lot of input information and expert knowledge. On the other hand, control bandingapproaches have been launched to assist SME in assessing and managing the risks of exposure tonanomaterials. The relevance of different engineering and PPE controls will be dependent on the lifecycle stage where the nanomaterial is used, incl. the industry and scenario of concern. Availableinformation on the efficiency of Risk Management Measures (RMM) have been reviewed focusing onworkplace control measures that are relevant for activities or processes associated with nanomaterials.Where required, evidence on the effectiveness of control measures relevant for ‘conventional’substances has also been considered and integrated with nano specific data. This information was usedto determine whether effectiveness of existing RMM for conventional substances require updating fornano specific scenarios. This effectiveness classification can be used for exposure and risk modellingpurposes. This ensures that the risks associated with a nano enabled product, throughout its wholevalue chain, will be appropriately evaluated and mitigated to an acceptable level.

Selection of proven risk management measures (RMMs) to control theexposure to ENMs

Carlos FitoITENE, Spain; [email protected]

For a comprehensive risk assessment of ENMs, information is needed with respect to the intrinsicharmfulness of the particle, likelihood of exposure, and efficacy of workplace controls. Majorinvestments have been done so far on the characterization of the toxicological profile. However,research aiming to improve our understanding of the possible exposure, as well as on the effectivenessof common risk management measures is far less advanced. This work presents experimental data on


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the effectiveness of respiratory and dermal protection equipment, and local exhaustive ventilation(LEV) systems to control the exposure to ENMs in occupational settings. New experimental data on theprotection factors achieved under representative exposure scenarios, as well as recommendations forthe design of PPE and ECs will be presented.

The testing activities were conducted after the validation of a set of standardized procedures, includingthe evaluation of the permeation to ENMs for dermal protective equipment, total inward leakage (TIL)inward leakage for respirators and filters, and capture efficiency for ventilation systems. Theexperimental work was conducted in a dedicated nano aerosol exposure chamber where severalexposure scenarios can be reproduced. The results from the test suggest that the control of exposurevia inhalation is a key priority. Respirators provided medium performance levels of filtration efficiencyagainst NMs. The performance levels determined suggest that face seal leakage, and not filterpenetration, is a key parameter to be considered when working with NMs. The evaluation of dermalprotective equipment showed very low permeation levels, meaning that common measures areeffective.

The capture efficiency of the LEV systems was demonstrated to be adequate. The data are compiled in alibrary of nano specific RMMs developed using Microsoft Excel®. The library helps stakeholders to selectproper measures depending of the type of ENM and process, guiding the user in the selection of provenrisk management measures. The research leading to these results has received funding from theEuropean Union's FP7 Grant Agreement n. 310584 (NanoReg project), and the LIFE project NanoRISK(LIFE ENV/ES/000178).

An analysis of the OECD WPMN dossier regarding the availability of data for riskassessment

Michael Riediker1,2,3, Ting YU1, Robert J Aitken1

1IOM (Institute of Occupational Medicine) Singapore; 2Nanyang Technological University, Singapore; 3Institut de Santé auTravail, University of Lausanne, Switzerland; michael.riediker@iom world.sg

The OECD Working Party on Manufactured Nanomaterials (WPMN) recently published dossiers of theirSponsorship Programme for testing 11 important Manufactured Nanomaterials (MNM). Our studyaimed to assess its usefulness for regulatory risk management of MNM.

We first identified information available in the dossiers on Test guidelines used; Environmental fate andpathways; Ecotoxicological/Toxicological information; Manufacture, use and exposure ofnanomaterials; and Human exposure scenarios. In a refined analysis we assessed the Endpoint StudyRecords (ESR) of three types of nanomaterials (fullerenes, SWCNT and zinc oxide) regarding availabilityof characteristics recommended by nanosafety experts: chemistry, nanoscale descriptors andcircumstances of the (eco)tox experiments.

All 6075 dossier pages were thoroughly reviewed and 5279 annex pages were referred to whennecessary. The testing programme adopted 131 test guidelines, 65 from OECD. Good overall coverageof endpoints was seen for aquatic, acute and geno toxicity; Moderate coverage for terrestrial systems;and poor coverage for long term effects and human toxicity. No ESR were identified for carcinogenicityand almost no data was available about the manufacture, use and exposure of nanomaterials. Most ESR(> 96%) provided only chemical description and circumstances of the experiments, while only very fewstudy records provide nanospecific characteristics of actually delivered preparations.


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The dossiers represent an enormous chemical testing effort. However, the data provided is mostlyinsufficient to make statements regarding the nanospecificity of observed hazard data, and to whatnano scale properties they may be linked. The dossiers also do not allow conclusions on theappropriateness of current Testing Guidelines for assessing ENM hazards. Finally, the possibility ofartefacts and thus the potential for false negative toxicity results cannot be assessed on basis ofreported data.

The first version of the software was realised and has been tested by PEROSH members and threeexternal partners. It is planned to offer the software to organisations, which perform exposuremeasurements for nanomaterials, and companies that produce or use nanomaterials. Aim is to enlargethe number of users and datasets and thus enhance the benefit for the users. Participating companiese.g. can benchmark themselves amongst their peer group. Scientific users can enlarge data pools tostrengthen statistical conclusions or validate their measurement results with reference data. Finallypublic access shall be enabled to publish consolidated and anonymised information.

Conclusion:Due to the limited amount of data in the field of exposure to nanomaterials, a harmonised exposuredatabase linked to other databases, e. g. on material or toxicological properties, is able to acceleratethe improvement in occupational safety.

A critical and in depth analysis of the environmental aspect of the OECD SPdossiers

Steffen F Hansen1, Rune Hjorth1, Lars M Skjolding1, Diana M Bowman2, Andrew Maynard2, AndersBaun1

1Technical University of Denmark, Denmark; 2Arizona State University, USA; [email protected]

In 2015, the OECD finally published the findings of its seven year testing programme for manufacturednanomaterials. Here, we present the first in depth analysis of the published OECD dossiers with regardsto data on physical and chemical properties, environmental fate and ecotoxicology. Each individualstudy in the dossiers was reviewed with regard to, among other, which OECD Test Guidelines (TG) wereused, and the reliability assigned to the study. We furthermore analyzed in detail the suspensionmethods used, how media quality was quantified and physical and chemical characterization performedprior, during and/or at the end of the study. We find that the information in the dossiers present anincomplete portfolio of nanomaterial ecotoxicological evaluations that are difficult to draw substantiveconclusions from and that most of the studies were not designed to investigate the validity of the OECDtest guidelines. We acknowledge the effort of the OECD WPMN and recommend that a follow onprogram is established with well defined goals, end points and direct funding to qualified researchlaboratories to ensure valid, rigorous, reproducible and efficient research.


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Development of a Nano Exposure and Contextual Information Database (NECID)

Johannes Pelzer1, Wouter Fransman2, Wiho Stoeppelmann1, Arto Saamanen3, Delphine Bard4, IsmoKoponen5, Oliver Witschger6, Elzbieta Jankowska8, Miren Agurtzane Zugasti Makazaga7, ChristianSchumacher11DGUV German Social Accident Insurance, Germany; 2TNO Inovation for life; 3FIOH Finnish Institute of Occupational Health;4HSL Health and Safety Laboratory; 5NRCWE National Research Centre for the Working Environment; 6INRS Santé etsécurité au travail; 7INSHT Instituto Nacional de Seguridad e Higiene en el Trabajo; 8CIOP PIB Centralny Instytut OchronyPracy Pa stwowy Instytut Badawczy; [email protected]

Research in exposure to nanoparticles requires sufficient and detailed data on occupational exposure.In addition to a uniform and harmonised way to collect and store information, data sharing and theconnection to other sources of information is a key factor to increase the quality and amount of data.Only with a sufficient number of datasets of high quality the next steps in risk assessment and also thededuction of future limit values will be feasible.

Under the leadership of IFA and TNO a working group of PEROSH institutes developed and tested adatabase software called NECID (Nano Exposure and Contextual Information Database). In addition tomeasurement data of individual instruments the collection and documentation of work conditions is afocus of this project.

The NECID software includes a nanomaterial specific exposure database, as well as features for datasharing and data assessment. The software runs locally on a computer but also offers a web based,central database for the exchange of information. As far as possible NECID uses a harmonised ontologyto enable a link to other databases. A direct link to the database of the EU project eNanoMapper isprojected.


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Keynote session V

Thursday 1 June, 12:00 13:00Location: Roome 1 (Conference room)Session chairs: Michael Riediker & Anders Baun

Environmental risk assessment of nanomaterialsBernd Nowack

Empa, Switzerland; [email protected]

Research interestsThe current research of Bernd Nowack deals with the chances and risks of engineered nanomaterialsand microplastics, comprising a wide spectrum of different approaches: development and application ofmethods for material flow modeling, exposure modeling, environmental risk assessment and life cycleassessment; experimental studies about release of nanomaterials from products and investigationsabout their behavior and effects in the environment. Bernd Nowack has published more than 140 peerreviewed publications and has an h factor of 49. He is founding co Editor in Chief of the journalNanoImpact and is Associate Editor of Environmental Pollution. He is listed in “The World’s mostinfluential scientific minds 2015” from Thomson Reuters in the category “EnvironmentalSciences/Ecology”.

Key note Environmental risk assessment of nanomaterialsThe environmental risks of engineered nanomaterials (ENM) can be quantified by comparing modelledPEC values (predicted environmental concentrations) with PNEC values (predicted no effectconcentrations), obtained from an analysis of the ecotoxicological literature. As a starting point for theenvironmental exposure assessment, exploring sources and pathways of release helps to identifyrelevant applications and situations where the environment may face exposure to ENM. By tracking thelife cycle of products, it is possible to explore whether and in which situations a release of ENM fromapplications may occur. Using material flow modeling as a basis, we can quantitatively identify thedetermining steps in the life cycle of nano products that result in release to the environment. Usingprobabilistic species sensitivity distributions, we are able to obtain PNEC values ENM that can then becompared to probability distributions of PEC values. Environmental risks were quantified so far for thefollowing nanoparticles in water and soils: TiO2, ZnO, Ag, CNT, fullerenes, Au, SiO2 and Fe oxides. Thispresentation gives an overview of the problems we are facing when performing this risk assessmentand critically discusses the results for different materials and environmental compartments.


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POSTER SESSIONSMonday 29 May, 18:00–19:30Tuesday 30 May, 18:00–19:30

Poster awards are sponsored by Mutagenesis and Environmental Science: Nano

Session 1A Carbon nanotube toxicity

P1Modulation of the latent infection specific gene expression signature ingamaherpesvirus infected macrophages by nanoparticles

Youjia Yu, Martin Irmler, Christine Sattler, Johannes Beckers, Heiko Adler, Tobias StögerHelmholtz Zentrum München, Germany; youjia.yu@helmholtz muenchen.de

BackgroundNanotoxicity is of great interest because environmental nanoparticles (NPs) have been described to beof cardio pulmonary health concern and various engineered NPs may even provoke the risk of chronicand debilitating lung diseases. Herpesviruses are among the most prevalent human pathogensworldwide and cause latent, recurring infections. By evading the immune system their infection laststhe whole life of the host and particularly high burden of gammaherpesvirus infection has beenassociated with pulmonary fibrosis. In our previous study, we revealed that carbon nanoparticles (CNP)and double wall carbon nanotubes (DWCNT) could reactivate latent murine gammaherpesvirus 68(MHV 68) in vitro and in vivo (mice). However, the mechanisms underlying nanoparticles induced virusreactivation remained elusive.

MethodThe murine monocyte/macrophage like cell line ANA 1, with or without persistently infected with MHV68 was exposed to 50ug/ml CNP (Printex 90; Degussa) or DWCNT (NC2100; Nanocyl) for 24h and totalRNA was extracted and applied for gene expression analysis with Affymetrix Mouse Gene 2.0 ST arrays.Pathway analysis was carried out by Genomatix and Ingenuity pathway analysis. Selected differentiallyexpressed genes of interest were verified by qRT PCR and western blot.

ResultCNP or DWCNT treatment had no noticeable effect on the expression signature in ANA 1 macrophages,with fold changes between 2.8 and 0.4 for CNP, and 2.2 and 0.4 for DWCNT treatment versus control


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cells. In contrast, persistent MHV 68 infection caused changes from 11.9 to 0.1 fold in ANA 1/MHV 68versus ANA 1 cells. This induction of several genes such as Hp, Trim13, Saa3, Cxcl2 and C3 was howeversignificantly attenuated 24h after treatment with 50ug/ml NPs. Pathway analysis further indicated thatthe by MHV 68 latent infection upregulated inflammatory response pathway was downregulated by theNPs treatment, as both CNP and DWCNT suppressed part of virus induced genes involved in hostresponse to oxidative stress.

ConclusionOur data showed that while NPs exposure had only a very moderate effect on the expression profile ofuninfected macrophages, the treatment could reverse a specific signature of the latentgammaherpesvirus in macrophages in vitro. Future investigations shall elucidate how far the abrogationof this signature is related to the reactivation of dormant virus.

P2Physicochemical related differences in pulmonary distribution and lymphatictranslocation of MWCNT

Trine Berthing1, Kristina B Knudsen1, Petra Jackson1, Sarah Søs Poulsen1, Nicklas R Jacobsen1, VidarSkaug2, Alicja Mortensen1, Józef Szarek3, Karin S Hougaard1, Henrik Wolff4, Håkan Wallin2, UllaVogel1,51National Research Centre for the Working Environment, Denmark; 2Department of Biological and Chemical WorkEnvironment, National Institute of Occupational Health, Oslo, Norway; 3Department of Forensic and Administration VeterinaryMedicine, University of Warmia and Mazury in Olsztyn, 10 717 Olsztyn, Poland; 4Finnish Institute of Occupational Health,Helsinki, Finland; 5Department of Micro and Nanotechnology, DTU, DK 2800 Kgs. Lyngby, Denmark; [email protected]

The fate in the organism is an important aspect of carbon nanotube toxicity. Here, standard transmittedbright field and enhanced darkfield microscopy are used to compare the pulmonary distribution andlymphatic translocation of 11 different MWCNT after intratracheal instillation in mice. The imagesillustrate rapid phagocytosis and mucociliary clearance, and with time granuloma encapsulation,perivascular accumulation and translocation to mediastinal lymph nodes. One year after exposure,aggregates of thin and short MWCNT are still present in the lung and associated with low levelinflammation whereas thick and straight MWCNT are retained as single fibers in the interstitium,showing no tissue reaction. Visualizing MWCNT distribution in tissues reveals interactions with cells andbiological barriers, and thereby helps to uncover mechanisms of toxicity.

P3Carcinogenic potential of multi walled carbon nanotubes in a human lung celltransformation assay in vitro

Santosh Phuyal1, Mayes Kasem1, Laura Rubio Lorente2, Hanna L Karlsson3, Ricardo Marcos Dauder2,4,Vidar Skaug1, Shan Zienolddiny11National Institute of Occupational Health, Norway; 22Grup de Mutagènesi, Departament de Genètica i de Microbiologia,Facultat de Biociències , Universitat Autònoma de Barcelona , Bellaterra , Spain; 33Unit of Biochemical Toxicology, Institute ofEnvironmental Medicine, Karolinska Institutet, SE 171 77, Stockholm, Sweden; 44CIBER Epidemiología y Salud Pública, Institutode Salud Carlos III, Madrid, Spain.; [email protected]

The toxicity of manufactured nanomaterials (MNM) has been extensively studied using in vitro assaysduring recent years. The most plausible route of exposure to MNM is through pulmonary inhalation.


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Yet, only very limited number of studies have attempted to assess carcinogenic properties in vitrofollowing long term exposure of human pulmonary cells to low and occupationally relevant doses. Themost advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs), most of themrelying on rodent cells and short term exposure. We hypothesized that long term exposure of humanlung cells with a normal phenotype could be a valuable assay for testing carcinogenicity ofnanomaterials in vitro. This study was designed to assess the carcinogenic potential of two multi walledcarbon nanotubes (MWCNT, NM400 and NM401) and were compared to two TiO2 materials (NM62002a and KC7000) by using chronic exposure (up to 6 month) of normal human bronchial epithelialcells to low doses of MNMs. The study was performed within the framework of the EU FP7 NANoREGproject. In order to harmonize and standardize experiments, standard operating protocols of MNMdispersion (NANOGENOTOX) were used by three different NANoREG project partners. All nanomaterialsshowed low cytotoxicity in short term tests for the tested doses (0.08 to 1.92 mg/cm2). NM 400 andTiO2 (NM62002a) stimulated colony formation after 3 months. However, the NM 401 clearly affectedcell proliferation but no cell transformation was observed for this CNT. We conclude that differentnanomaterials may have different potential for cell transformation in the CTA assay used here.

P4Carbon nanotubes: Biodistribution in animal models

Nicklas R Jacobsen1, Peter Møller2, Per Axel Clausen1, Anne T Saber1, Christian Micheletti3, Keld AJensen1, Håkan Wallin4, Ulla Vogel1,51NRCWE, Denmark; 2University of Copenhagen, Denmark; 3ECAMRICERT, Italy; 4STAMI, Norway; 5Technical University ofDenmark, Denmark; [email protected]

The many interesting physical and chemical properties of carbon nanotubes (CNT) make it one of themost commercially attractive materials in the era of nanotechnology. Here, we review the recentpublications on in vivo biodistribution of pristine and functionalized forms of single walled and multiwalled CNT.

Pristine CNT remain in the lung for months or even years after pulmonary deposition. If cleared, themajority of CNT move to the gastrointestinal (GI) tract via the mucociliary escalator. However, thereappears to be no uptake of CNT from the GI tract, with a possible exception of the smallestfunctionalized SWCNT. Importantly, a significant fraction of CNT translocate from the alveolar space tothe near pulmonary region including lymph nodes, sub pleura and pleura (<7% of the pulmonarydeposited dose) and to distal organs including liver, spleen and bone marrow (~ 1%). These resultsclearly demonstrate the main sites of long term CNT accumulation, which also includes pleura, a majorsite for fibre induced pulmonary diseases.

Studies on intravenous injection show that CNT in blood circulation are cleared relatively fast with ahalf life of minutes or hours. The major target organs were the same as identified after pulmonaryexposure with the exception of urine excretion of especially functionalized SWCNT and accumulation inlung tissue. Overall, there is evidence that CNT will primarily be distributed to the liver where theyappear to be present at least one year after exposure.

Reference:Biodistribution of Carbon Nanotubes in Animal Models (Review). Jacobsen NR, Møller P, Clausen PA, Saber AT, Micheletti C,Jensen KA, Wallin H, Vogel U. Basic Clin Pharmacol Toxicol. 2016 [In Press]


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P5Morphological changes in mouse liver one year after intratracheal instillation tocarbon nanotubes

Alicja Mortensen1, Józef Szarek2, Kristina B Knudsen1, Trine Berthing1, Petra Jackson1, Sarah SøsPoulsen1, Niklas R Jacobsen1, Vidar Skaug3, Karin S Hougaard1, Henrik Wolff4, Håkan Wallin1

1National Research Centre for Working Environment, Denmark; 2University of Warmia and Mazury in Olsztyn, Olsztyn, Poland;3Department of Biological and Chemical Work Environment, National Institute of Occupational Health, Oslo, Norway; 4FinnishInstitute of Occupational Health, Helsinki, Finland; [email protected]

Introduction: Engineered nanoparticles are smaller than 100 nm in at least one direction and designedto improve or achieve new physicochemical properties. Consequently, toxicological properties may alsochange. Carbon nanotubes (CNT) have attracted industrial interest due to their unique properties.

Materials and Methods: Samples of livers (9 10/group) from female mice (C57BL/6BomTac, TaconicEurope, Denmark) exposed by intratracheal instillation to a single dose of 54 g of one of 11 differentmulti walled carbon nanotubes (MWCNT) were taken for histopathological examination (haematoxylinand eosin staining) 1 year after treatment. Concurrent vehicle control livers (29/group) from femalemice exposed to a vehicle (Nanopure water with 2% mouse serum) and from mice (9/group) exposed toCarbon Black (CB) were also included.

Results:Microscopical changes of inflammatory, proliferative/hypertrophic, necrotic, degenerative,regenerative or other types were recorded in all livers. Two samples were diagnosed with lymphaticleucaemia (from NRCWE 040 and NRCWE 0420 groups). Qualitative differences were observed forcertain types of changes in the livers from the exposed mice when compared to vehicle controls. Thisconcerned the degree of intensity of microscopic changes such as inflammatory changes, extend ofnecrosis and vacuolar (hydropic) degeneration.

Conclusions: Our results suggest that a single intratracheal exposure to MWCNT was associated withdifferent intensity and extend of microscopical changes in the liver one year after the exposure.

Session 1B Physic chemical characterization requirements andmethods for grouping and risk assessment

P6Guidance for substance identification and categorization of nanomaterials inREACH and CLP and supporting characterization methods: Proposal for revisionsfrom the EU FP7 NANoREG project

Keld Alstrup Jensen, Rambabu AtluriThe National Research Centre for the Working Environment, Denmark; [email protected]

Introduction: The European Chemicals Agency (ECHA) is revising the guidance for substanceidentification and categorization of manufactured nanomaterials (MNM) to enable registration andassessment MNM in REACH. Several documents were released between November 2016 and January2017; e.g., Appendix 4: Recommendations for nanomaterials applicable to the Guidance on Registration


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Draft (Public) v. 1.0 January 2017 and Appendix R.6 1: Recommendations for nanomaterials applicableto Guidance on QSARs and Grouping of Chemicals (Public) Draft v. 2.0 December 2016 on).

Methods: The schemes in these new documents for MNM were first reviewed for applicability andconsistency with the parent Guidance for bulk materials. Second, the applicability of the recommendedphysico chemical characterization methods was assessed considering the developments in the EU FP7NANoREG project.

Analysis: ECHA still adhere to the conventional substance identification scheme dividing substancesinto: 1) well defined monoconstituent substances considering the minimum 80 wt.% purity rule, 2) welldefined multiconstituent substances when consisting 10 to 80 wt.% of a single substance, and finally 3)UVCB (Unspecified or Variable compositions, Complex reaction products or Biological materials)substances when the chemistry is poorly defined or variable. New nanoforms are defined within theconventional Substance Identity Profiles (SIPs) considering as a minimum the size, shape and surfacechemistry. To define shape, ECHA has developed four new shape categories (near spheroidal, platy,high aspect ratio, and other), which differ from the shape definitions for bulk materials. It is mandatoryto report the surface chemistry and describe any surface chemical treatments of the nanoforms definethe nanosubstances. Similar scheme and definitions do not exist for bulk materials. It is evident that thenanospecific ECHA guidance is not applicable to distinguish between all types of principle forms ofMNM (solid, capsules/hollow, porous, doped) or the more advanced higher generation nanomaterials(pore filled, matrix embedded, physically coated, and chemically functionalized NM and combinationsthereof).

Recommendations:1. The guidance and recommendations for bulk materials and nanomaterials are harmonized to avoid

confusion and inability to perform read across and grouping;2. The ECHA substance identification and naming scheme is further revised to prepare for structured

identification and registration of higher generation chemically and structurally complex materials;3. The purity for identification a well defined monoconstituent substances must be considerably

increased to avoid grouping with materials that compositionally clearly must have been chemicallymodified;

4. An update of the MNM characterization methods needs an update and establishment ofinternationally approved technical guidelines is urgently needed.

P7An electrochemical toxicity biosensor based on carbon nanotubes/polymers forassessment of air pollutants

Wen Lian William LeeChung Shan Medical University, Taiwan, Republic of China; [email protected]

The study will present a series of sensitive and miniaturized cell based electrochemical sensors to assessthe sensitivities of a variety of pollutant gases. A varieties of sensing array wafers were fabricated withthe assistance by carbon nanotubes composited with eight different polymers in a facile and greenprocess including, polycarbonate(PC), polystyrene, polyisoprene(PIP), poly (epichlorhydrin)(PECH),poly[alpha methylstysrene] (PMS), poly[vinyl benzyl chloride] (PVBC), poly[4 vinyl phenpl co methylmethacrylate](PVCMM), and polymethyltrifluoropropylsiloxane(OV 210) and s. Based on the differenthybrid composites modified electrode, the data of the sensitization of the adsorbent gases withdifferent resistance change were collected. These sensors were used to evaluate the sensitivities of six


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different chemical gases or air pollutants at room temperature, including chloroform, methanol,methyl ethyl ketone, tetrahyrofuran(THF), toluene, and m xylene. Through the process of usingdifferent prepared concentrations of carbon nanotubes to the electrode adjusted below 1k ohms, theperformance of sixteen different sensors shows that the sensitivity could reach to ppm level, and withfive times reproducible. The results demonstrate that these types sensors worth to development a newtype of chemical gas detector system.

P8Ultrafine particles size distribution and their metallic elements emitted from ahigh tech industrial park

Yen Ping Peng, Ting Yu ChenTunghai University, Taiwan, Republic of China; [email protected]

This study used a 8 stage micro orifice uniform deposit impactor (MOUDI) sampler to collect the sizedistribution of various heavy metals (Al, Fe, Na, Mg, Ca, K, In, Mo, Zn, Cr, Cu, Pb, Mn, Ni, Sn, V, Cd, As,Ga and Ge) in airborne particles from January to September, 2013.

Comparing with other researches, results indicated that concentration of Ni (2.18 ng/m3), As (0.16ng/m3), Ge (0.01 ng/m3), Ga (0.03 ng/m3) and Cd (0.12 ng/m3) were both higher other researches. Thecharacteristic size peak distributions was identified by accumulation mode curve and could be classifiedinto three categories: (i) main peak at 5.6 m, (Al, Fe, Na, Mg and Ca), (ii) peak spread around fine,intermediate and coarse modes (K, Zn, In, Mo, Cr, Cu, Pb, Mn, Ni and Sn), and (iii) metals mass peakmainly less than 1 m (V, Cd, As, Ga and Ge). The correlation analysis could both tell which metal hadthe similar sources and its characteristic of pollution. Both in upwind and in downwind sites, theelements: Ga Ge As Cd observed having the high correlation in two tailed significant level analysis. Themobile sources elements Mn Ni, Pb Zn In Cr in upwind had high correlation, while in downwind Mn Ni,Zn Cu and Pb As Cd Zn K Cr had high correlation while the crustal metals had high correlation in bothupwind and in downwind samples. We used hierarchical cluster analysis (HCA) to classify the metals, itcould divide three different clusters: (i) crustal cluster, (ii) mobile cluster, (iii) station cluster. In bothupwind and downwind samples had Al, Fe, Na. Mg and Ca within the same cluster, those elements bothwere emit from crustal, so the cluster was the crustal sources while the cluster of station sources had V,As, Ni, Pb, Ga and Ge in upwind, but the cluster of station sources had Ga, Ge, V, As, Cd, Pb, Mn and Niin downwind. The cluster of mobile sources had Mn, Cd, Zn, K, Sn, Cr, In, Cu and Mo in upwind while thecluster of mobile sources had Cr, Sn, In, K, Cu, Mo and Zn in downwind.5. Finally, combining all themethods, it could realize the metals most frequent were As, Ga and Cd. Using expected value theory toidentify the possible sources from high tech industries.


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Session 2A Inhalation toxicity – drivers of toxicity

P9Alteration in metabolism by pulmonary exposure of carbon black in mice

Hsiao Chi Chuang1, Ta Chih Hsiao2, Chii Hong Lee3, Chun Te Lin4, Kai Jen Chuang5, Tsun Jen Cheng61School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan; 2Graduate Institute ofEnvironmental Engineering, National Central University, Taoyuan, Taiwan; 3Department of Pathology, Shuang Ho Hospital,Taipei Medical University, New Taipei City, Taiwan; 4Department of Environmental Engineering and Science, Feng ChiaUniversity, Taichung, Taiwan; 5School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei,Taiwan; 6Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University,Taipei, Taiwan; [email protected]

The bioreactivity of carbon black (CB) has been studied due to its unique physicochemical parametersand relatively low toxic potency. However, CB is still recognized as a carcinogen in rodents, and theburden of CB in the respiratory system is likely to play a critical role in these effects. To evaluate howphysical parameters of CB influence metabolism, we investigated CB and diesel exhaust particles (DEPs)and attempted to relate various physical parameters, including the hydrodynamic diameter, zetapotential, and particle number concentrations, to lung energy metabolism in BALB/c mice. A bodyweight increase was arrested by 3 months of exposure to CB of smaller size fractions, which wasnegatively correlated with pyruvate in plasma. There were no significant differences in cytotoxic lactatedehydrogenase or total protein in bronchoalveolar lavage fluid after 3 months of CB exposure.However, we observed alterations in the acetyl CoA and NADP/NADPH ratio in lung tissues with CBexposure. Additionally, the NADP/NADPH ratio was associated with the zeta potential of CB. Mildperibronchiovascular and interstitial inflammation and multinucleated giant cells (macrophages) with atransparent and rhomboid appearance and containing foreign bodies in the lungs were observed in lungsections. These observations may be related to disruption of energy metabolism caused by CB. Wefurther observed that inhaled CB remained in the lungs after 3 months of exposure withperibronchiovascular and interstitial inflammation. The results underscore the need for more thoroughand continued investigation of the effects of CB exposure on energy metabolism.

P10Genotoxic effects of different types of nanofibrillated cellulose materials

Kukka Aimonen1, Hanna Lindberg1, Julia Catalán1, Henrik Wolff1, Irene Wedin2, Markus Nuopponen3,Kai Savolainen1, Hannu Norppa11Finnish Institute of Occupational Health, Finland; 2Stora Enso Oyj, Finland; 3UPM Kymmene Oyj, Finland; [email protected]

Nanofibrillar cellulose (NFC) is among the most promising innovations in the forest industry with a widevariety of possible applications. As NFC consists of thin and long fibres with a high aspect ratio, it isimportant to investigate the safety of NFC at an early stage of product development. Our objective wasto examine the genotoxicity of four NFC materials (in gel form; fibril diameter 2 15 nm, length severalμm) in comparison with a bulk sized cellulose material. Multiwalled carbon nanotubes (MWCNTs)served as a comparative control. In vitro genotoxicity was assessed in human bronchial epithelial (BEAS2B) cells by the comet assay (24 h exposure, 9.5 950 μg/ml) to detect DNA strand breakage and by thecytokinesis block micronucleus (MN) assay (48 h exposure, 25 1250 μg/ml) to show possiblechromosomal damage. For genotoxicity assessment in vivo, the comet assay in lung andbronchoalveolar lavage (BAL) cells and the bone marrow erythrocyte MN assay were performed after


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single pharyngeal aspiration of female C57BL/6 mice (24 h and 28 d follow up; 10, 40, 80 and 200μg/mouse). The NFCs did not induce DNA damage or micronuclei in vitro. However, all NFC materials,except one, induced DNA damage in mouse lung or BAL cells. For one NFC, the effect was dosedependent in lungs both 24 h and 28 days post exposure. The comparative materials, bulk sized pulpand MWCNTs, also induced DNA damage after 24 h and 28 days. None of the NFCs or comparativematerials showed systemic genotoxicity, as measured by the micronucleus assay in bone marrow. Theoutcome of the in vivo studies was not predicted by the in vitro tests, as none of the NFCs or the bulksized pulp were genotoxic in vitro. This suggested that the effects in vivo were not present in the in vitrocell system used. All materials studied were biopersistent in the lungs for 28 days, which raises someconcern, particularly as an increase in DNA damage was still seen at this time point. A longer follow upwould be required to better define the fate of the NFCs in the lungs and the duration of the increasedlevel of DNA damage. The exposure route used in this study was chosen to mimic a tentative worstcase scenario in NFC manufacture, where nanocellulose in liquid may be aerosolized in the atmosphereand inhaled by workers. [Supported by UPM Kymmene Oyj, Stora Enso Oyj, the Finnish Safety andChemicals Agency, and the European Commission, NANoREG 310584].

P12Effect of the agglomeration state of TiO2 aerosols on their pulmonary toxicity inrat

Laurent Gaté, Stéphane Grossmann, Sylvie Sébillaud, Stéphane Viton, Mylène Lorcin, Sébastien Bau,Carole Seidel, Christian Darne, Hervé Nunge, Laurine Douteau, Sylvie Michaux, Frédéric CosnierINRS, France; [email protected]

Due to the growing use of nanoparticles in industrial processes, the number of workers potentiallyexposed by inhalation in the workplaces is increasing, while the toxicological properties of thesecompounds are not fully known. In this respect, inhalation experiments performed in laboratoryrodents remain the most suitable and reliable approach to assess their toxicological properties.

In addition to the size of the primary particles composing the material, the state of agglomeration ofairborne particles emitted from/released by this material may impact its toxicity, in particular thetoxicological profiles in terms of inflammatory response and pulmonary biopersistence ortoxicokinetics. To assess this point, this work investigates the effect of the agglomeration state oftitanium dioxide (TiO2) aerosols produced from the same TiO2 powder (TiO2 P25 from Evonik). Subacute nose only inhalation experiments were performed in Fischer 344 rats with two aerosols: (1) anagglomerated aerosol (AA) and (2) a desagglomerated aerosol (DA). Tissues were collected 0, 3, 30, 90and 180 days after the end of the exposure period. Biological effects were investigated byhistopathology, cytology and biochemistry of the bronchoalveolar lavage fluid (BALF). Rats wereexposed to 10 mg/m3 AA and 5 mg/m3 DA. Since it is a crucial aspect for the interpretation of thebiological effects, aerosols were first deeply characterized in terms of mass and number concentrationsand size distributions, as well as particle morphology. Furthermore, aerosols were continuouslymonitored directly within the animals’ breathing zone during exposure periods. Number concentrationswere found to be 24,000 and 265,000 particles/cm3 for AA and DA, respectively. Count median mobilitydiameters determined for both aerosols were 347 (AA) and 218 nm (DA) with geometric standarddeviation of 2.29 and 1.80. The mass modal aerodynamic diameters were 1.70 (AA) and 0.40 μm (DA).

In addition, the main respiratory parameters of rats were recorded during exposure. This allows theestimation of deposited fractions and mass within the respiratory tract, as well as clearance, using theMultiple Path Particle Dosimetry model (MPPD v3.04). These values were further compared to the


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retained mass dose during the 180 days follow up period measured by inductively coupled plasma massspectrometry.

Biochemical and cytological analyses of the BALF of rats exposed to AA showed a strong inflammatoryresponse up to 3 days which decreased overtime but persisted 180 days after the end of exposure.Analysis of results obtained with rats exposed to DA is ongoing.

P13Prediction of acute lung toxicity of impregnation products using an in vitromethod based on lung surfactant inhibition

Jorid B Sørli, Yishi Huang, Emilie Da Silva, Jitka S Hansen, Søren T Larsen, Karin S HougaardThe National Research Centre for the Working Environment, Denmark; [email protected]

Private consumers and professionals may be affected by acute inhalation toxicity after use ofimpregnation products. The distinction between toxic and non toxic products is difficult to make bothfor manufacturers and product users, as there is no clear link between the chemical composition of theproducts and the toxicity. The current available method for determining acute inhalation toxicity isbased on experiments involving laboratory animals. This is time consuming, expensive and causes stressfor the animals. The toxic products furthermore show an exceptionally steep dose response curve,resembling an all or nothing type response. This implies that pinpointing the exact toxic dose is oftenvery difficult. We have used an in vitromethod, the constrained drop surfactometer (CDS), whichmimics the functional lung alveoli and is based on lung surfactant inhibition. We aimed to determine ifthe acute airway toxicity could be predicted by lung surfactant inhibition. We tested 22 impregnationproducts in vitro in the CDS, and the same products were tested in a bioassay where the breathingpattern of mice was monitored during product exposure to determine the acute inhalation toxicity. Thein vitro method could predict the acute inhalation toxicity of the thirteen products that caused toxicityin vivo. An additional three products inhibited surfactant function in the CDS, but without overt toxicityobserved in vivo in mice. Thus the accuracy of the method was 86%, whereas the sensitivity was 100%.Seven of the tested products have been involved in incidences of acute inhalation toxicity in humansafter accidental inhalation by users. Of these, six were both toxic to mice and inhibited lung surfactantfunction in vitro. The last product was one of several that workers were exposed to, this was the onlyexposure product tested, and it could be excluded as the culprit based on the experiments. The CDS invitromethod can be used as a fast, cheap and predictive screening tool to assess the acute inhalationtoxicity of impregnation products.

P14Potential exposure to air toxics in the indoor environment from the use ofmosquito coils

Jonathan N Hogarh, Thomas P Agyekum, Kwasi Obiri DansoKwame Nkrumah University of Science and Technology, Kumasi, Ghana; [email protected]

Mosquito coils are commonly applied in many developing countries across Africa, Asia and SouthAmerica as a cheap approach to repelling the mosquito at the household level. In applying these coils,users have mainly been interested in the environmental health gains derived from repelling themosquito, oblivious of the environmental health implications from potential exposure to gaseous


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emissions from the mosquito coil smoke. The objective of this study was to investigate gaseousemissions from experimental application of mosquito coils under varying indoor conditions of roomspace and ventilation. The target pollutants were carbon monoxide (CO), total volatile organiccompounds (TVOCs), sulfur dioxide (SO2) and nitrogen dioxide (NO2). Eight hour concentrations of thesepollutants were derived from continuous burning of mosquito coils in the indoor environment, applyingAeroqual Series 500 (S500) gas monitors. Among the various pollutants, CO concentration exceededhealth based air quality guideline of 10 mg/m3 when the indoor environment lacked ventilation. CO,TVOCs and SO2 were consistently highest in the 19 m3 room compared to those with room volumes 34and 8.5 m3. Pollutant concentrations in the indoor environment expectedly decreased with ventilation.Burning one strand of mosquito coil in a closed indoor environment yielded CO emissions of an order ofmagnitude quite comparable to the rate emanated from environmental tobacco smoke. Exposure tosmoke from these coils should be avoided, especially in a closed indoor environment.

P15In vivo toxicological effects of a self cleaning agent containing silver doped TiO2

nanoparticles

Kukka Johanna Aimonen1, Julia Catalán1,2, Satu Suhonen1, Mira Hartikainen1, Joan Cabellos3,Alejandro Vílchez3, Camilla Delpivo3, Delphine Boutry4, Henrik Wolff1, Kai Savolainen1, Hannu Norppa11Finnish Institute of Occupational Health, Helsinki, Finland; 2Department of Anatomy, Embryology and Genetics, University ofZaragoza, Zaragoza, Spain; 3Leitat Technological Center, Terrassa (Barcelona), Spain; 4CEA Tech, Grenoble, France;[email protected]

Due to their photocatalytic and oxidative properties, titanium dioxide (TiO2) nanoparticles (NPs) can beused to create dirt repelling and antimicrobial coatings. Previous studies suggest that pulmonaryexposure to TiO2 NPs can cause acute inflammation, however, the genotoxicity of TiO2 NPs remainscontroversial. In the present study, a self cleaning agent containing TiO2 NPs was tested in vivo forpulmonary and systemic genotoxicity and inflammatory effects in mice.

A water based self cleaning agent containing ~0.5 wt% nanoTiO2 (particle size <8 nm, doped with silver(Ag) NPs) was administrated to C57Bl/6 mice by repeated (3x) pharyngeal aspiration using threedifferent doses (corresponding to 10, 30 and 80 μg of TiO2/mouse/aspiration). One and 28 days afterthe last administration, DNA damage was assessed by the comet assay locally in bronchoalveolar lavage(BAL) and lung cells, and systemically in liver cells. Micronuclei, a biomarker of chromosome damage,were analysed in bone marrow (24 h) and peripheral blood erythrocytes (28 d). Immunotoxicity wasevaluated by BAL cell counting. Furthermore, histopathological effects on the lungs and biodistributionof TiO2 were assessed. To differentiate the possible effect of the Ag doping, exposure of the highesttested dose (3 x 80 μg of TiO2/mouse/aspiration) was repeated with a corresponding dispersion ofundoped TiO2.

At 24 h, a mild, statistically significant increase in DNA damage was seen at the lowest tested dose inBAL and liver cells. 28 days after the last administration, a slight, statistically significant increase in DNAdamage was measured in the BAL cells. The undoped material was genotoxic in BAL and lung cells at 24h, and in liver cells at both tested time points. No chromosomal damage was observed by theerythrocyte micronucleus assay.

The self cleaning agent tested induced an acute dose dependent inflammatory response. Recruitmentof neutrophils, accompanied with mild influx of eosinophils and lymphocytes was detected by BAL cellcounting 24 h after the last administration, but 28 days later, the inflammation had mostly resolved.


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Histological findings support these results, although, residues of the material were still visible in thelung tissue 28 days after the last exposure. In addition, biodistribution analyses indicate accumulation ofthe undoped material in the liver tissue.

Our findings show that short term exposure of mice to nanoTiO2 containing self cleaning agent inducespulmonary inflammation and slight DNA damage. TiO2 is biopersistent in the body for at least a month.(Funded by GUIDEnano, Grant Agreement No.604387)

P16Visualizing nanoparticles in histological tissue samples using enhanced darkfieldhyperspectral microscopy

Trine Berthing1, Kristina B Knudsen1, Justyna Swietek1, Petra Jackson1, Sarah Søs Poulsen1, Nicklas RJacobsen1, Vidar Skaug2, Alicja Mortensen1, Karin S Hougaard1, Henrik Wolff3, Håkan Wallin2, Anne TSaber1, Katrin Löschner4, Gitte Ravn Haren4, Ulla Vogel1,51National Research Centre for the Working Environment, Denmark; 2Department of Biological and Chemical WorkEnvironment, National Institute of Occupational Health, Oslo, Norway; 3Finnish Institute of Occupational Health, Helsinki,Finland; 4DTU Food, DK 2800 Kgs. Lyngby, Denmark; 5Department of Micro and Nanotechnology, DTU, DK 2800 Kgs. Lyngby,Denmark; [email protected]

The fate of nanomaterials in the organism is an important aspect of nanotoxicology.

Enhanced darkfield hyperspectral microscopy is a new approach for visualizing the distribution ofnanomaterials in histological tissue samples. This technique can detect and spectrally characterizeunlabeled nanoparticles with a diameter down to 10 nm. Here, we show the applicability of enhanceddarkfield hyperspectral imaging of carbon based and metal oxide nanomaterials in lung and liver tissue.Visualizing nanoparticle distribution in tissues can provide information on cell types, biological barriersand fluid flows involved in accumulation and translocation of nanomaterials. As a result, the techniquehelps to uncover mechanisms of nanoparticle toxicity.

P17Phospholipid corona formed on functionalized nanoparticles after incubationwith lung surfactant

Marie Frederiksen, Thomasz Berezniak, Kirsten Kling, Rambabu Atluri, Asger W Nørgaard, Per AxelClausen, Håkan Wallin, Jorid B SørliNational Research Centre for the Working Environment, Denmark; [email protected]

Nanoparticles may affect lung surfactant composition and consequently impair lung function. Lungsurfactant consists of a complex mixture of phospholipids (PLs) and proteins. Its main function is todecrease the surface tension in the lungs in order to keep the alveoli open during respiration. Ifsurfactant composition is altered lung function can be affected. Therefore, we examined the PL coronaformed on nanoparticles following incubation with lung surfactant. In this study functionalizednanosized maghemite (SPIONs) were used as model particles, since they can be held back with amagnet when washing off excess surfactant. The SPIONs were modified with five types of surfacecoatings, incubated with lung surfactant (Curosurf®) and the formed phospholipid corona was studied.Surface modifications of different polarity were produced including hydroxyl (hydrophilic),aminopropylsilane (hydrophilic), octylsilane (lipophilic) and perfluorooctylsilane (very lipophilic). The


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SPIONs were incubated at 37°C for 2 hrs with Curosurf®, a commercially available lung surfactant. Thenthe excess surfactant was washed off using saline in three consecutive wash cycles while retaining theSPIONs with a strong magnet. The PL corona was extracted using methanol:chloroform (1:1) andinternal deuterated standard was added. The extracts were analysed by LC qTOF MS for nine differentPLs including 1,2 dipalmitoyl sn glycero 3 phosphocholine (DPPC). The lipid coronas were generallydominated by DPPC, which is the major constituent of Curosurf®. Currently, the PL patterns on thedifferent surface modifications are being investigated.

Session 2B Nanomaterial characterization in toxicology – Reactivity,dissolution, and biomolecule interactions

P18Considerations in the characterization and analysis of engineerednanomaterials in exposure matrices

Petra Krystek1,2, Wouter Fransman1, Peter Tromp1

1 TNO, Zeist & Utrecht, The Netherlands2 VU University, Dep. Environment & Health, Amsterdam, The Netherlands

As nanotechnology is still an emerging field with an increasing variety of engineered nanomaterials(ENMs), products and applications, there is a great relevance in assessing health and environmentalhazards. This presentation focuses on analytical approaches for the identification of ENMs in productsand in various biological matrices after different routes of exposure.

A project dedicated selection of exposure matrices and analytical techniques must be made. Theanalytical challenges of a few cases will be presented in details.

• For exposed tissues, the matrix dissolution prior to measurement by asymmetric flow field flowfraction hyphenated to inductively coupled plasma mass spectrometry (AF4 ICPMS) is challengingbecause the ENMs must stay stable regarding to particle size and composition. A new enzymaticapproach is explored in comparison to the classical dissolution approaches in speciation analysis.

• Next to in vivo studies, in vitro cell exposure testing has been put forward as a faster approach toscreen ENMs on their toxic potential but also to study the possible cellular uptake which dependson the characteristics of the ENMs. The analysis delivers new insights into the understanding ofENMs in mainly complex media. Additional aspects, like e.g. the composition of the cell culturemedium that affects aggregation of ENMs and this in turn may affect exposure levels, are of greatrelevance.

• Other examples by imaging techniques likes scanning electron microscopy (SEM) will beillustrated. In this case, we focussed on nanoparticles made of silver which are bioavailable.


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P19Surface modifications of silica nanoparticles affect their uptake by the cells andsubsequent pulmonary toxicity

Sandra Vranic1,2, Eri Watanabe1, Yurika Osada1, Sahoko Ichihara3, Wenting Wu4, Toshihiro Sakurai1,Toshihiro Suzuki1, Ryo Abe1, Sonia Boland5, Lang Tran6, Gaku Ichihara11Tokyo University of Science, Japan; 2University of Manchester, UK; 3Jichi Medical University, Japan; 4Toray Chemical, China;5University of Paris VII Diderot, France; 6Institute of Occupational Medicine, UK; [email protected]

It is necessary to clarify the relationship between specific properties of nanomaterial (NM) and inducedbiological effect, as this could lead to the development of NM “safe by design”. Silica nanoparticles(SiO2NPs) are one of NPs most widely used in the cosmetic, medicine or food. The present studyinvestigated effect of surface modification of SiO2NPs on their uptake into macrophages and inductionof pulmonary inflammation. Rhodamine labeled SiO2NPs of 25 nm (Italian NPs) or 30 nm (German NPs)in diameter were surface modified with amino groups, carboxyl groups or hydroxyl groups renderingthem either positively (+q NPs), negatively ( q NPs) or neutrally charged (N NPs), respectively. First thehydrodynamic diameter and surface charge density of each surface group in Endotoxin free water andin tissue culture medium were measured. Next Male C57BL/6J mice were exposed to 2 mg/kg or 10mg/kg SiO2NPs by pharyngeal aspiration. After 24 hours, the bronchoalveolar lavage fluid (BALF) wascollected to determine the total and differential cell count. Cells taking up SiO2NPs were observed usingconfocal microscope. Then RAW264.7 was exposed to at 0 100 μg/mL of Italian and German SiO2NPsfor 4 and 24 hours and their cell viability was measured by MTS assay. The cells taking up German NPswere observed using confocal microscope and internalized SiO2NPs with Rhodamine were quantified byflow cytometer. Exposure to +q NPs and q NPs increased the number of total cells, macrophages andneutrophils dose dependently and exposure to q NPs increased neutrophils to a greater extent thanexposure to +q NPs. On the other hand, exposure to N NPs increased the number of total cells at 2mg/kg bw. Observation using confocal microscope revealed that the ratio of macrophage with q NPwas higher than ratio of macrophage with +q NPs or N NPs. In MTS assay, +q NPs and q NPs exhibitedlittle cytotoxicity at even highest dose (100 μg/mL), although exposure to N NPs significantly decreasedthe cell viability at 10 25 μg/mL. RAW264.7 internalized q NPs the most among three types of NPs andthis result was consistant with the result of flow cytometry. We concluded that induction of theinflammatory response in the murine lung was dependent on the surface modifications of NPs. Whenwe exposed mice to positively and negatively charged SiO2NPs, there was a positive associationbetween uptake and inflammation. The cytotoxicity of N NPs to RAW264.7 was the highest amongthree types of NPs but not associated with the degree of NPs’ uptake.

P20An innovative in vitro model for the study of acute inhalation toxicity followingexposure to nanomaterials

Emilie Da Silva, Ismo K Koponen, Søren T Larsen, Jorid B SørliThe National Research Center for the Working Environment, Denmark; [email protected]

Health effects induced by inhalation of nanoparticles is of great concern. Toxicological assessment ofsuch particles is necessary and the only OECD test guideline currently available is the test guideline 436where rats or mice are exposed to up to 20 g/m3 for 4 hours. The test causes the animals distress, istime consuming and expensive to perform. Development of an alternative method to assess acuteinhalation toxicity is therefore warranted. The objective of this study was to investigate the toxicological


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profile of a range of metal oxide nanoparticles using an in vivomodel and an innovative in vitro systemand to compare the results. In the in vivomodel, mice were restrained in plethysmographs to monitortheir breathing pattern during 60 min of exposure to a particle aerosol. A modified version of theconstrained drop surfactometer was used as the in vitromodel. The modifications allow modeling ofthe air liquid interface of a drop of lung surfactant (LS). LS is the first barrier that inhaled nanoparticlesencounter. This thin layer of liquid in the alveolar region of the lungs has the vital function of reducingsurface tension at the end of expiration, making breathing effortless. Damage of LS function, seen as anincrease in minimum surface tension, manifests as alveolar collapse in vivo.Minimum surface tensionwas used as a marker of acute inhalation toxicity in vitro in this study. Particle size distribution anddeposited doses were determined. We show that all particles induced a decrease in tidal volume in vivo,marker of lung damage. The response was dependent on nanoparticle physicochemical properties,deposited dose and duration of exposure. These results were in agreement with the observation in vitroof a dose dependent inhibition of the surface activity of LS after exposure to the same particles.

P21Radicals generated by nanoparticles in contact with liquid water using spintrapping and mass spectrometry

Per Axel Clausen, Ingrid Elise K Weydahl, Vivi Kofoed Sørensen, Nicklas R JacobsenNational Research Centre for the Working Environment, Denmark; [email protected]

Several studies have shown associations between inhalation of particles and airway inflammatoryeffects which have been suggested, at least partly, to be induced by reactive oxygen species (ROS)generated by the particles. ROS are often measured by non specific methods and the most wellestablished method is using 2’,7’ dichlorodihydrofluorescein (DCFH2). ROS comprises in addition toradicals (hydroxyl, hydroperoxyl, and superoxide), also the non radicals singlet oxygen and hydrogenperoxide. Both radicals and non radicals contribute to the response of DCFH2. The aim of this study wasto develop a method which can measure the specific radicals that are formed when nanoparticles (NP)get into contact with liquid water.

The method is based on spin trapping which uses compounds that after reaction with a radical form aso called spin trap radical adduct which retain the radical function. Traditionally, this spin trap adducthas been measured by electron spin resonance spectroscopy (ESR). One problem for ESR is that the spintrap radical adduct can lose the radical function and become ESR silent. The advantage of using liquidchromatography mass spectrometry (LC MS) coupled with spin trapping is that it detects not only theESR active spin trap radical adducts, but also the oxidized and reduced forms, which are ESR silent.Liquid chromatography mass spectrometry (LC MS) with electrospray ionization was used foridentification and quantification of spin trapped radicals in solution. However, if only radicals in solutionwere measured the formation potential appeared to be very low. We then got the idea that the spintrap radical adducts might be adsorbed to the surface of the NP. Therefore we used matrix assistedlaser desorption ionization time of flight mass spectrometry (MALDI TOF MS) for direct analysis of spintrap radical adducts on the NP surfaces. In short, NPs were dispersed in pure water added the spin trap,incubated over night, and centrifuged. The supernatant was analysed with LC MS and the “pellet”consisting of the NPs was analysed with MALDI TOF MS.

The preliminary results show that the carbon black, Printex 90, produced both the hydroxyl radical ( OH)and the hydroperoxyl radical ( OOH), but mainly OH, that the carbon nanotube, NM 400, produced OHonly and that the silica NP, NM 203, produced no radicals. The radical formation potential of Printex 90


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was 13 46 nmol/mg NP and that of NM 400 was 8 20 nmol/mg NP depending on the amount of NPdispersed in the water.

P22Fluorescence enhancement of dyes by nanoparticles

I Chia ChenNational Tsing Hua University, Taiwan, Republic of China; [email protected]

The interaction between gold nanoparticles (GNPs) with silica shell as spacer, Au@SiO2 NPs, anduorophores like dye rose bengal (RB) and others are studied using time resolved spectroscopy. Rose

bengal (4,5,6,7 tetrachloro 2',4',5',7' tetraiodofluorescein) is a stain commonly used in eye drops tostain damaged cells. Varied sizes of GNPs with controlled thickness of silica shell were synthesized toinvestigate the e ects on metal enhanced uorescence. Fluorophore RB covalently connected toprefunctionlized silica surface has spectral overlap with the plasmon resonance of the goldnanoparticle. The enhancement factor for uorescence displaying a maximum at spacer separation 10nm is 2.4, 3.8, 4.6, and 5.5 for diameters 45, 65, 80, and 100 nm Au@SiO2 NPs, respectively.Biexponential decay of emission is observed for small thicknesses of spacer, indicating multiplepathways for relaxation of the excited states. Both time constants 1 and 2 are consistently increasedwith increased separation of the silica spacer. For 100 nm GNPs, we nd that the rate constant forenergy transfer from RB to GNP is 9 × 106 to 2.0 × 1010 s 1 (bright + dark modes) for separation 5 45 nm,displaying a dependence on the separation of the silica shell d n with n 2.5. The interaction of otherdye with silver nanoparticles and the time resolved fluorescence spectroscopy will be presented as well.

P23Analysis of the redox and acid base reactivity during transformation ofmanufactured nanomaterials in hydrous mediums

Keld A Jensen, Yahia KemboucheThe National Research Centre for the Working Environment, Denmark; [email protected]

In a regulatory context, manufactured nanomaterials (MNM) are defined to consist of nano objectswith primary minimum diameters between 1 and 100 nm, however, still including fullerene, graphene,multi walled carbon nanotubes. They may be single compounds (first generation MNM) ormulticonstient compounds (2nd, 3rd, or higher generation MNM). Due to the small sizes, which oftenresult in quantum effects, and chemical complexity, the transport and reactivities (including redoxactivity, dissolution, phase transformation, and overall reaction rates) of MNMmay differ from that ofcomparable μm size phases. Consequently, uncertainty has been raised about the potential risksassociated with MNM and the validity of existing data on their reactivity, fate and behaviour in theenvironment and in biological systems. To assess the potential reactivity of MNM, we have developedtwo methods for assessment of MNM reactivity in biological fluids. The 1st method is a Sensor DishReader (SDR) System, which can be used to screen the effect on the O2 and pH balance in liquids underexternal atmospheric conditions. The 2nd method is an Atmosphere Temperature pH controlled StirredBatch Reactor (AtempH SBR) with online monitoring of the redox potential. The results generated bythese systems are intended for use in predictive reaction chemical modelling using e.g., GeochemistWorkbench.


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Pocedures and examples of results are given to illustrate the reactivity of MNM in cell media andbiological simulant fluids. The results are applied in modelling of MNM reaction rates and phasetransformaton using Geochemist Workbench.

Session 3A Reprotoxicity

P24Effects of maternal exposure in pregnancy on male reproductive parameters infuture generations

Astrid Skovmand1,2, Anna Lauvås2, Ulla Vogel1, Sandra Goericke Pesch2, Karin S Hougaard1

1Danish National Research Center for the Working Environment, Denmark; 2University of Copenhagen, Department ofVeterinary Clinical Sciences, Section of Veterinary Reproduction and Obstetrics; [email protected]

Evidence suggests that nanoparticles may affect fetal development of the male reproductive system.Maternal pulmonary exposure of certain nanoparticles during gestation have been associated withdecreased sperm counts and testicular structural changes in the offspring. In a previous study of malereproduction from our laboratory, effects on the male offspring did not appear until the secondgeneration. This indicates that the effect is potentially heritable and possibly due to epigeneticmodifications of the genome. The aim of this study was to investigate the effects of carbon blackPrintex90 inhalation during maternal gestation on male reproductive function in NMRI mice. Themothers were exposed in an inhalation chamber to a control of clean air and to two doses of Printex90:a low dose of 4.8 mg/m3 or a high dose of 33.5 mg/m3 . Effects on male reproductive parameters wereinvestigated in four generations: the male offpring were raised with the mothers until weaning andreared until sexually mature, then mated with naïve females (non exposed). Once they had successfullymated, the males were euthanized and the following male reproductive parameters were assessed:Epididymal sperm concentration, motility and viability. These were examined using computer assistedsperm analysis (CASA). Daily sperm production was investigated in the testis and testosterone levelswere measured in plasma. Results on the epididymal sperm concentration, motility and viability showedno difference amongst the three groups. Results on the daily sperm production, plasma testosteroneconcentrations and the statistical analysis are pending. The pending analysis and a final conlusion willbe presented at the conference.

P25ZnO nanoparticles: Pulmonary toxicity and mortality in mice after intratrachealinstillation in three laboratories

Nicklas R Jacobsen1, Tobias Stoeger2, Sybille van den Brûle3, Anne T Saber1, Andrea Beyerle2, GiuliaVietti3, Alicja Mortensen1,4, Józef Szarek5, Hans Christian Budtz1, Ali Kermanizadeh6, AtrayeeBanerjee7, Nuran Ercal7, Ulla Vogel1,4, Håkan Wallin6,8, Peter Møller61NRCWE, Denmark; 2Helmholtz Zentrum München, Germany; 3Université catholique de Louvain, Belgium; 4Technical Universityof Denmark, Denmark; 5University of Warmia and Mazury in Olsztyn, Poland; 6University of Copenhagen, Denmark; 7MissouriUniversity of Science and Technology, USA; 8STAMI, Norway; [email protected]

Inhalation is the main pathway of ZnO exposure in the occupational environment but few studies haveaddressed toxic effects of pulmonary exposure to ZnO nanoparticles (NPs). Here we present results


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from studies in three laboratories of pulmonary intratracheal instillation exposure and toxicity of ZnONP in mice.

Three different ZnO NPs were used; the particle size and BET surface area ranged between 12–94 nmand 13–60 m2/g for the three materials. Two of the three studies were prematurely terminatedbecause interim results unexpectedly showed severe toxicity. In the third study a high bolus doses ofZnO NPs (25–100 g (1.4 5.4 mg/kg) were clearly associated with a dose dependent mortality in themice. Lower doses (from 6 g (0.3 mg/kg) elicited acute toxicity in terms of reduced weight gain,desquamation of epithelial cells with concomitantly increased barrier permeability of the alveolar/blood as well as DNA damage. Oxidative stress was shown in terms of a strongly increased lipidperoxidation and reduced glutathione in the pulmonary tissue. Blood parameters showed increasedneutrophils, red blood cells, hematocrit, platelets and more. Two months post exposure revealed noobvious toxicity for 12.5 and 25 g/mouse using a range of parameters. However, mice that survived ahigh dose (50 g; 2.7 mg/kg) had an increased pulmonary collagen accumulation (fibrosis) at a similarlevel as a high bolus dose of crystalline silica. The recovery from these end points appeared dosedependent.

The results indicate that alveolar deposition of ZnO NP may cause significant adverse health effects.Some effects could be hypothesized transient due to the suluble nature of ZnO in some biological fluids.

P26Optimization of wet digestion methods for the determination of silvernanoparticles on rice (Oryza sativa L.cv. KDML105), sticky rice (Oryza sativa var.glutinosa cv. RD 6) and Chinese water convolvulus (Ipomoea aquatic Forsk. Var.reptan)

Sujitra Srisung, Jakkrisn Kunthup, Nootcharin WasukanSrinakharinwirot University, Thailand; [email protected]

Currently, agriculture is widely which the properties of silver nanoparticles (AgNPs) can be used as antibacterial and fungi. AgNPs may be released into the environment and impact on agricultural crops havebeen exposed. In this work, to investigate the effects of AgNPs to accumulate translocation and impacton three plants including rice (Oryza sativa L. cv. KDML 105), sticky rice (Oryza sativa var. glutinosa cv.RD 6) and Chinese water convolvulus (Ipomoea aquatic Forsk. Var. reptan). In the experiment, theAgNPs were synthesized by pure natural honey as a reducing agent. The characterization and particlesize of the silver nanoparticles were evaluated with UV Vis spectrophotometry and transmissionelectron microscope (TEM). The results of AgNPs showed the maximum absorption at 423 nm and theparticle size in the range of 20 50 nm. In addition, to study on the wet digestion of D1, D2 and D3methods. The methods of D1 and D2 using nitric acid as a solvent. While the time and temperature aredifferent for digestion on the roots and the shoots of the plants. Then, a D3 method was performed bymixtures of concentrated nitric acid and hydrogen perchloric acid. Moreover, the three plants wereexposed with AgNPs to various concentrations of 0.02, 0.05, 0.1 and 1 mg/L. After wet digestion theAgNPs were determined by Graphite furnace atomic absorption spectroscopy (GFAAS). Therefore, theD2 method was the optimum wet digestion process for the determination of AgNPs in plants, resultingshow the percent recovery of between 81.67 to 94.00. While the effect of AgNPs to aggregation andaccumulation within the roots and shoots of three plants. The result showed that the roots of sticky riceat a concentration of 0.05 mg/L was accumulated and penetrated to the cell wall and cell in root lead to


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effect on structural features and transporting nutrients to the plants, including the development on thegrowth of plant cells.

P27Oxidative potential of nanomaterials in the eluate of quartz sand and naturalsoils?

Bryan Hellack1, Carmen Nickel1, Karsten Schlich2, Kerstin Hund Rinke2, Tim Huelser11Institute of Energy and Environmental Technology (IUTA) e.V., Bliersheimerstrasse 58 60, Duisburg, Germany; 2FraunhoferInstitute for Molecular Biology and Applied Ecology (IME), Auf dem Aberg 1, 57392 Schmallenberg, Germany; [email protected]

Nanomaterials (NM) are commonly used in many everyday life products and in the end NM will enterthe environmental compartments. Soils were identified as one important sink for NM with the potentialof accumulation of biopersistent NM and/or passage into the groundwater. During this processes theywill undergo certain environmental transformation processes which potentially affect the hazardpotential of the NM. As one promising metric describing the potential hazard of NM the reactivity oroxidative potential (OP) of NM is discussed.

Within this study we primarily investigated, if it is somehow feasible to apply the spin probe basedelectron paramagnetic resonance spectroscopy for OP detection in quartz sand eluate after NMtreatment and, second, if NM (when passing the soil column) change their OP.

Therefore, within the DENANA project in a first approach we performed soil column experiments with asilver and a copper NM in unsaturated quartz sand and detected the OP in the eluate at three differenttimepoints. At a first time we additionally analysed six types of highly diluted natural reference soils totheir OP by EPR to investigate whether a soil induced OP background can be differentiated to a NMelicit OP.

First results indicate that the spin probe based OP analysis by EPR for eluate is feasible. However, achange of the reactivity of the NM are indicated after passing quartz sand filled soil columns but theresults are not evident so far. Furthermore, the OP backgrounds of the highly diluted reference soilsdiffer but seem to be too high for a distinction of NM and/or soil elicit OP. The results have to beconfirmed in further experiments and will be presented at the conference.

Session 3BII Ecotoxicity – closing gaps

P28Revising REACH technical guidance on information requirements and chemicalsafety assessment for engineered nanomaterials for aquatic ecotoxicityendpoints – recommendations from the EnvNano project

Steffen F Hansen, Sara N Sørensen, Lars Michael Skjolding, Nanna B Hartmann, Anders BaunDepartment of Environmental Engineering Technical University of Denmark; [email protected]

The European Chemical Agency (ECHA) is in the process of revising its guidance documents on how toaddress the challenges of ecotoxicological testing of nanomaterials. In these revisions, outset is taken in


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the hypothesis that ecotoxicological test methods, developed for soluble chemicals, can be madeapplicable to nanomaterials. European Research Council project EnvNano Environmental Effects andRisk Evaluation of Engineered, which ran from 2011 2016, took another outset by assuming that: “Thebehaviour of nanoparticles in suspension is fundamentally different from that of chemicals in solution.The aim of this paper is to present the findings of the EnvNano project and through these provide thescientific background for specific recommendations on how ECHA guidance could be further improved.Key EnvNano findings such as the need to characterize dispersion and dissolution rates in stock and testmedia have partially been addressed in the updated guidance. However, it has to be made clear thatmultiple characterization methods have to be applied to describe state of dispersion and dissolutionover time and for various test concentration. More detailed information is called for on the specificcharacterization methods and techniques available and their pros and cons. Based on findings inEnvNano, we recommend that existing algal tests are supplemented with tests where suspensions ofnanomaterials are aged for 1 3 days for nanomaterials that dissolve in testing media. Likewise, fordaphnia tests we suggest to supplement with tests where a) exposure is shortened to a 3h pulseexposure in daphnia toxicity tests with environmentally hazardous metal and metal oxide nanomaterialsprone to dissolution; and b) food abundance is three to five times higher than normal, respectively. Wefurther suggest that the importance of considering the impact of shading in algal tests is made moredetailed in the guidance and that it is specified that determination of uptake, depuration and trophictransfer of nanomaterials for each commercialized functionalization of the nanomaterials is required.Finally, as an outcome of the project a method for assessing the regulatory adequacy of ecotoxicologicalstudies of nanomaterials is proposed.

Session 5A In vivo models of hazard

P29Long fibre carbon nanotubes induce pleural mesothelioma via silencing and/orloss of key tumour suppressor genes

Tatyana Chernova1, Fiona A Murphy2, Sara Galavotti1, Xiao Ming Sun1, Ian R Powley1, Stefano Grosso1,Anja Schinwald2, David Dinsdale1, John Le Quesne1, Jonathon Bennett3, Apostolos Nakas3, PeterGreaves4, Craig A Poland2, Ken Donaldson2, Martin Bushell1, Anne E Willis1, Marion MacFarlane11MRC Toxicology Unit, United Kingdom; 2MRC/University of Edinburgh, Centre for Inflammation Research, QMRI, UK; 3UHL NHSTrust, Glenfield Hospital, Leicester, UK; 4Department of Cancer Studies, University of Leicester, UK; [email protected]

Exposure to asbestos fibres causes pathological changes in the pleural cavity including malignantmesothelioma. Length dependent retention of asbestos fibres in the pleural cavity is crucial for diseasedevelopment. Chronic inflammation induced by pathogenic asbestos fibres plays a key role incarcinogenesis and epigenetic events, rather than driver mutations, are considered to be majorcausative factors. Manufactured carbon nanotubes (CNT) are similar to asbestos in terms of their highaspect ratio and thus may pose an asbestos like inhalation hazard, however the molecular mechanismsunderlying their carcinogenic potential have not been sufficiently explored. Using a model of directinstilation into the pleural cavity, we compared the molecular changes which occur at the mesotheliumafter exposure to short and long asbestos fibres and short and long CNT over 20 months followinginjection.

We show a common pro oncogenic activity of long CNT and long asbestos throughout diseaseprogression. The common key molecular events encompass changes in gene expression and signalingpathway activation, oxidative DNA damage, increased mitosis and proliferation. Instillation of long CNT


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into the pleural cavity of mice induces chronic inflammation and pro oncogenic changes leading todevelopment of mesothelioma with deletion of p19/Arf and silencing of p16/Ink4a and NF2. Epigeneticchanges induced by pathogenic fibres occur at the pre neoplastic stage of disease and may play a keyrole in progression of pleural inflammatory lesions to malignant mesothelioma.

Together these data demonstrate that exposure to long CNT induces development of pleuralmesothelioma replicating the pathogenesis of human disease and highlights commonality in the hazardmechanism of long pathogenic fibres at the molecular level. Epigenetic changes precede malignanttransformation of both asbestos and CNT induced inflammatory lesions. Crucially, our findingsreinforce concerns that long CNT may pose an asbestos like hazard, leading to malignant mesothelioma.

P30Real time and non invasive imaging of biodistribution of nanoparticles by usingOTN NIR fluorophore in mice

Masakazu Umezawa1,2, Masao Kamimura1,3, Moe Yoshida1, Kohei Soga1,31Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science,Japan; 2Research Institute for Science and Technology (RIST), Tokyo University of Science, Japan; 3Imaging Frontier Center,RIST, Tokyo University of Science, Japan; masa [email protected]

Easy screening techniques to assay the biodistribution and its kinetics of nanoparticles inside the bodiesare needed to better utilize the data on nanoparticle safety and toxicity. Here we show thatnanoparticle fluorescent probes in over thousand nanometer near infrared (OTN NIR) region, which isapplicable to the deep bioimaging in the second biological window, are useful for non invasiveinvestigation of the kinetics of nanoparticles without any radiation facility. Rare earth doped ceramicsnanoparticles, oxygen doped single walled carbon nanotubes, and near infrared dye micelles arepreparable as OTN NIR fluorescent nanoparticles at high fluorescence with surface modification bypoly(ethylene glycol) (PEG) for improving biocompatibility. To investigate the distribution of thesenanoparticles following pulmonary exposure of mice, the particles were administered by intranasalinstillation to mice under anesthesia. Diffusion of the fluorescent particles to deeper region of the nasalcavity by spontaneous breathing was visualized by real time monitoring using a portable in vivo NIRfluorescence imaging system (SAI 1000; Shimadzu Co., Japan) within 10 min post instillation. Thedistribution of OTN NIR fluorescent nanoparticles from respiratory tract to the gastrointestinal tract(especially the stomach) via the mucociliary escalator in the bronchi was also detected non invasivelywithin 24 h post instillation in mice. The use of chemically modified PEG for surface modification of thenanoparticles provides us OTN NIR fluorescent nanoparticles with different surface charge and withdifferent modification by functional peptides for further comparative analysis of the kinetics ofnanoparticles. This emerging and non invasive deep bioimaging technique will facilitate the easyscreening of kinetics of nanoparticles and effective designing safer and useful nanomaterials.


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P31Influence of vehicle on nanomaterial induced inflammation and DNA strandbreaks

Niels Hadrup1, Stefan Bengtson1, Nicklas R Jacobsen1, Petra Jackson1, Anne T Saber1, Karin SHougaard1, Keld A Jensen1, Håkan Wallin2, Ulla Vogel11National Research Centre for the Working Environment, Denmark; 2Department of Biological and Chemical WorkEnvironment, National Institute of Occupational Health, Oslo, Norway; [email protected]

Background: Intratracheal instillation serves as a model for inhalation exposure to e.g. nanomaterials inrodents. However, to allow exposure by instillation of materials, they need to be dispersed in a liquidvehicle. The question is whether the selected vehicle influences the toxicological effects.

Aim: To investigate the influence of different vehicles on pulmonary inflammation and DNA strandbreaks of 14 nm Printex90 carbon black (CB), NM 400 carbon nanotubes (CNT) and two titaniumdioxide (TiO2) nanoparticles, one with endogenous negative surface charge (NRCWE 001) and onewhich was modified to be positively charged (NRCWE 002).

Methods:Mice were intratracheally instilled with 162 μg/mouse (CB, TiO2), or 54 μg/mouse (CNT). Theinvestigated vehicles were: Nanopure water (CB, TiO2); 2% serum in nanopure water (CB, CNT, TiO2);0.05% serum albumin in nanopure water and powder pre wetting with 0.5% (v/v) ethanol (CB, CNT,TiO2); 10% bronchoalveolar lavage fluid in 0.9% NaCl (CB), 10% bronchoalveolar lavage fluid in nanopurewater (CB) or 0.1% Tween80 in nanopure water (CB). Inflammation and DNA strand breaks weremeasured in bronchoalveolar lavage fluid; inflammation as influx of neutrophils and DNA strand breaksas the DNA tail length in bronchoalveolar lavage fluid cells using the Comet assay. Time points were 1, 3,5, 28 and 90 days after exposure.

Results: Intratracheal instillation of nanomaterials increased neutrophil numbers in bronchoalveolarlavage fluid for nanomaterials in all vehicles compared to vehicle controls. For CB, the inflammatoryresponse at 1 day was somewhat vehicle dependent. However overall, concordance was observed inbetween the different vehicles. This is in contrast to observations for DNA damage. An increase in DNAtail length was only observed for CB in water, CB in 2% serum, and CB in 10% bronchoalveolar lavagefluid in 0.9% NaCl; But not for 0.05% serum albumin in water with ethanol pre wetting nor for 10%bronchoalveolar lavage fluid in nanopure water. CNT had no effect on DNA tail length for any of the twotested vehicles (2% serum in water and 0.05% serum albumin in water with ethanol pre wetting). ForTiO2, the tested vehicles (water, 2% serum in water, 0.05% serum albumin in water with ethanol prewetting) did not influence that genotoxicity was lower than in controls.

Conclusion: The choice of vehicle had a small effect on nanomaterial induced inflammation, whereasvehicle was an important determinant of nanomaterial induced genotoxicity. Pure water was thepreferred vehicle, but can only be used for a limited number of nanomaterials.


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P32An occupational life cycle approach to engineered nanomaterial toxicity toprovide context to potential health effects

Aaron Erdely1,2, Lindsey Bishop1,2, Marlene Orandle1, Naveena Yanamala1, Matthew Dahm3, EileenBirch3, Douglas Evans3, Vamsi Kodali1, Tracy Eye1, Lori Battelli1, Patti Zeidler Erdely1,2, Gary Casuccio4,Kristen Bunker4, Aleksandr Stefaniak1,2, Tina Sager1, Aliakbar Afshari1, Diane Schwegler Berry1, DavidLowry1, Machael Kashon1, Robert Mercer1, Charles Geraci3, Mary Schubauer Berigan3, Linda Sargent1,Lorenzo Cena1,51NIOSH, Morgantown, WV, USA; 2West Virginia University, Morgantown, WV, USA; 3NIOSH, Cincinnati, OH, USA; 4RJ Lee Group,Monroeville, PA, USA; 5West Chester University, West Chester, PA, USA; [email protected]

Pulmonary toxicity studies primarily focus on as produced engineered nanomaterials and rarely areguided by a life cycle perspective or integration with exposure assessment. Understanding toxicitybeyond the as produced (AP), or pure native material is critical, due to modifications needed toovercome barriers to commercialization of applications. We evaluated the toxicity of extensivelycharacterized AP multi walled carbon nanotubes (MWCNT: 14 nm diameter and 1 2 μm length), theirpolymer coated (PC) counterparts [polyurethane or a proprietary poly(arylene ethynylene)], and therespirable aerosols generated from sanding (fine grit, P320) the PC nanotube embedded or neatcomposites from two separate companies. Polymer coating, an increasing trend in distribution andapplications of MWCNT, reduces the percent MWCNT loading required for incorporation into thecomposite, simplifies handling, and can decrease dustiness thereby reducing potential downstreaminhalation exposures. Male C57BL/6J mice exposed by oropharyngeal aspiration to 4 or 40 μg wereharvested 1, 7, 28, and 84 d post exposure. Using workplace exposure assessment to guide in vivo studydesign (e.g. deposited dose and material preparations to emulate personal breathing zone collections),dose and time dependent measures of pulmonary cytotoxicity, inflammation, and histopathology wereobserved as expected for AP MWCNT. Polymer coating the MWCNT did not enhance the pulmonarytoxicity of AP nanotubes and toxicity was significantly attenuated in some instances. One specificpolymer coating resulted in auto agglomeration of PC MWCNT. The agglomerates did not stimulate alocalized inflammatory response. As an initial screen for genotoxicity, micronuclei were evaluated incultured primary small airway epithelial cells exposed to 2.4 g/ml MWCNT. Micronuclei significantlyincreased in all AP and PC material exposed primary cells. For analysis of PC MWCNT containingcomposites, an adaptable system was developed to generate and characterize particles using acombination of direct read instruments and automated electron microscopy. No free nanotubes weredetected in aerosols generated from sanding composites containing embedded PC nanotubes (0.15 3%by weight). Sanding produced primarily micron sized particles with some MWCNT protrusions. Thecollected respirable particulate fraction affected acute in vivo pulmonarytoxicity if the incorporation ofPC nanotubes and composite matrix altered the particulate size distribution; when size distribution wasunaltered, toxicity was unchanged. Our study provides insights into MWCNT toxicity during transitionsfrom dry powder to an end product: although the number of workers and potential consumers mayincrease along the life cycle, the toxicity and/or exposure potential of the MWCNT may be greatlymodified.


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P33Long fibre carbon nanotubes and asbestos induce pleural pathology with acommon molecular signature

Tatyana Chernova1, Fiona A Murphy1, Sara Galavotti1, Xiao Ming Sun1, Ian R Powley1, Stefano Grosso1,Anja Schinwald2, David Dinsdale1, John Le Quesne1,3, Jonathan Bennett3, Apostolos Nakas3, PeterGreaves4, Craig A Poland2, Ken Donaldson2, Martin Bushell1, Anne E Willis1, Marion MacFarlane11MRC, United Kingdom; 2MRC/University of Edinburgh; 3UHL NHS Trust, Glenfield Hospital; 4Department of Cancer Studies,University of Leicester; [email protected]

Recent studies have highlighted the potential pathogenicity of carbon nanotubes (CNT) in the pleura,including mesothelioma. Although the histological changes observed are similar to those induced byasbestos exposure and replicate human mesothelioma, the molecular mechanisms involved and howthese compare with asbestos induced disease are yet to be investigated. Using a model of directinjection into the pleural cavity, we compared the molecular changes in the pleura after exposure toshort and long asbestos fibres and short and long CNT over 20 months. Exposure to long, but not short,asbestos and CNT led to activation of pro oncogenic signalling pathways, including Akt, mTOR, ERK1/2and Src family kinases, which was detected at 1 week post exposure and sustained for the entireduration of the study. The differential contribution of mesothelial cells and stroma to aberrant cellularsignalling was evident throughout lesion development and, importantly, was similar to that detected inend stage mesothelioma in human patients. Oxidative DNA damage, increased mitosis and proliferationbecame evident as long asbestos and long CNT induced lesions progressed. Proliferation of themesothelial cell layer and the occurrence of ‘reactive’ mesothelial cells suggests cross talk betweenstromal cells, with activated pro oncogenic pathways, and target mesothelial cells. Together, these datademonstrate that exposure to long CNT induces development of pleural pathology which replicates thepathogenesis of human disease and highlights a common molecular signature of long asbestos and longCNT induced lesions. Crucially, our findings reinforce concerns that long CNT may pose an asbestos likehazard, leading to malignant mesothelioma.

P34Toxic effects of a photocatalytic mixture containing TiO2 nanoparticles in mice

Julia Catalán1,2, Kukka Aimonen1, Satu Suhonen1, Mira Hartikainen1, Joan Cabellos3, AlejandroVílchez3, Gemma Janer3, Socorro Vázquez Campos3, Christian Cortés de la Fuente4, Henrik Wolff1, KaiSavolainen1, Hannu Norppa11Finnish Institute of Occupational Health, P.O. Box 40, FI 00251 Helsinki, Finland; 2University of Zaragoza, Miguel Servet 177,50013 Zaragoza, Spain; 3Leitat Technological Center, Innovació 2, 08225 Terrassa, Spain; 4Servià Cantó SA, Balmes, 36, 08007Barcelona, Spain; [email protected]

Due to their photocatalytic and oxidative properties, titanium dioxide (TiO2) nanoparticles (NPs) can beused in asphalt pavements as a means to decrease urban pollution, especially to reduce theconcentration of nitrogen oxides. The material is applied on road surfaces by spraying, which raisesconcerns about occupational exposure, as workers may inhale the aerosols during the applicationprocess.

In the present study, a water based photocatalytic mixture containing 3% (w/v) nanosized TiO2

(anatase, particle size 5 10 nm) and 2% (w/v) synthetic resins was administrated to C57Bl/6 mice bypharyngeal aspiration at three different doses (corresponding to 10, 100 and 200 μg of TiO2/mouse).One and 28 days post administration, DNA damage was assessed by the comet assay locally in


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bronchoalveolar lavage (BAL) cells and lung cells, and systematically in liver cells. Micronuclei, abiomarker of chromosome damage, were analysed in bone marrow (at 24 h) and peripheral blooderythrocytes (at 28 d). Inflammation was evaluated by BAL cell counting. Furthermore, histopathologyof the lungs and biodistribution of the NPs (ICP MS analysis of Ti in several organs) were assessed. Todifferentiate the possible effect of TiO2 and the resins, groups of mice were exposed to TiO2 NPs aloneat the same three concentrations and to the resins at a dose corresponding to the highestconcentration in the mixture.

At 24 h, the only statistically significant genotoxic effect induced by the mixture was a 2 fold increase inthe level of DNA damage in BAL cells at the intermediate dose (100 μg of TiO2/mouse). No local orsystemic genotoxic effects were observed at 28 d. TiO2 NPs or the resins produced no genotoxic effectswhen tested separately. A dose dependent recruitment of neutrophils, accompanied by a minor influxof eosinophils, was detected in both BAL fluid and lung tissue 24 h post administration of thephotocatalytic mixture. TiO2 NPs and the resins showed a similar inflammatory response, although witha lower degree of neutrophilia. At 28 d, the inflammation had almost completely resolved.

Results from the biodistribution analyses showed a dose dependent accumulation of Ti in lung tissue.By 28 d, Ti had partly been eliminated more efficiently when administrated in the mixture than alone.No accumulation of Ti was observed in other organs.

Our findings show that short term exposure of mice to the photocatalytic mixture containing nanosizedTiO2 induces transient pulmonary inflammation but no clear genotoxic effects. (Funded by GUIDEnano,grant agreement no.604387).

Session 5B Release characterization: from concentrations toquantitative release

P35Evidence of carbon nanotubes in 3 dimensional printer emissions

Aleksandr Stefaniak1, Lauren Bowers1, Alycia Knepp1, Chalong Qi2, Diane Schwegler Berry1, SherriFriend1, Alyson Johnson1, Yong Qian1, Duane Hammond2

1National Institute for Occupational Safety and Health, Morgantown, WV, United States of America; 2National Institute forOccupational Safety and Health, Cincinnati, OH, United States of America; [email protected]

Fused deposition modeling (FDM®) is a type of 3 dimensional printing which uses a heated nozzle tomelt a thermoplastic filament and deposit material layer by layer to build an object. In order to improvethe electrical properties of printed objects, some manufacturers have begun to use carbon nanotubes(CNTs) as filament additives. CNTs are known respiratory toxicants; however, it is currently unknownwhether these materials are emitted during 3 D printing.

Two different filaments that were labeled as containing CNTs were purchased on the open market: 3DXESD (acrylonitrile butadiene styrene [ABS]) polymer and F electric (poly lactic acid [PLA]) polymer. Eachas received filament was inspected using field emission scanning electron microscopy (FE SEM). Next,we evaluated whether CNTs were emitted during printing using a commercially available 3 D printer ina 13 m3 chamber. The chamber atmosphere was monitored using a real time fast mobility particle sizer(FMPS) and a condensation particle counter (CPC) to determine submicrometer particle size distributionand number concentration, respectively. Airborne particles were collected onto track etched


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polycarbonate filters using 37 mm open faced cassette samplers and inspected using FE SEM. Finally,we inspected the surfaces of printed objects using FE SEM.

Mean diameters of CNTs in the as received filaments were 16±3 nm (3DX ESD) and 19±4 nm (F electric).During printing, particle generation rates from the CPC data were 5.6 x 107 particles/min (3DX ESD) and7.3 x 106 particles/min (F electric). The particle size distribution from the FMPS exhibited a maximumconcentration of 1.8 x 105 particles/cm3 for the 10.8 to 12.4 nm size channel (3DX ESD) and 1.9 x 105

particles/cm3 for the 52.3 to 60.4 nm size channel (F electric). Emitted particles had morphologyconsisting of aggregates of nanoscale spherical particle or discrete solid compact micronscale particles.CNTs visibly protruded from some particles emitted during printing with both filament types. No free(unbound) CNTs were observed on filter samples. CNTs were visible on and protruding from surfaces ofprinted objects.

CNTs were emitted during 3 D printing thereby presenting a potential respiratory hazard. Additionalresearch is needed to understand factors influencing these emissions, toxicity of ENM/thermoplasticparticles and to develop appropriate engineering controls to mitigate exposures.

P36Nanoparticle release mechanisms during laser ablation of ceramic tiles

Apostolos Salmatonidis1, Mar Viana1, Noemi Perez1, Andres Alastuey1, Eliseo Monfort2, VicentaSanfelix2, Luis Alberto Angurel Lambán3, German F De la Fuente31IDAEA CSIC, Barcelona, Spain; 2ITC UJI, Castellon, Spain; 3ICMA CSIC, Zaragoza, Spain; [email protected]

The ceramic industry is one of the most ancient on the planet. Even though the manipulation ofmaterials to obtain new properties sounds like a modern concept, there is evidence that Co metalnanoparticles (NPs) were unintentionally used in glass matrix since the Neolithic era as pigment [1].Hence, NPs have been present for centuries in ceramic production processes.

In addition to engineered NPs, other types of NPs (referred to as process generated, or non engineeredNPs) may be detected in workplaces during the manufacturing or processing of ceramic products (e.g.tiles). The reason behind this is that high energy processes such as laser sintering or ablation andplasma spraying have a high potential for NP emission [2].

Laser ablation is widely used in the ceramic industry for surface structuring and decoration of tiles. Thiswork aims to understand the mechanisms controlling NP formation and release into workplace airduring ablation of different types of ceramic tiles (conventional and advanced), using different lasersetups (n IR and mid IR). The measurements took place at laboratory as well as at pilot plant scale, andthe process parameters evaluated were laser energy potential, frequency, velocity, and pulse duration.NP characteristics assessed were particle number concentration and size distribution (using SMPS withnano DMA, DiscMini, and butanol CPC), particle mass concentration (PM2.5; DustTrak DRX), and NPmorphology and chemical characterization (NPs were collected on TEM grids). Similar measurementstook place at the worker’s breathing zone, and in indoor background. In total, the combination of 4types of ceramic tiles and 2 lasers was assessed. We report high NP emissions (from 3.5*104/cm3 to2.5*106/cm3 on average; TSI 3775 CPC) from all of the materials tested and under all the laser setups,with mean diameters between 35 135 nm (with DiscMini). A strong dependence was observed betweenNP emissions (as number concentration) and ceramic tile properties (body and glaze composition). Interms of particle mass, emissions depended on a combination of the laser conditions and materialcharacteristics. Emissions in terms of particle mass were markedly lower than in terms of particle


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number. The developed methodology has allowed the effectiveness of NP mitigation strategies to beevaluated.

References[1] P. Colomban, Arts, 2, (2013), 77 110[2] A.S. Fonseca, M. Viana, X. Querol, N. Moreno, I. de Francisco, C. Estepa, G.F. de la Fuente, Journal ofAerosol Science, 88, (2015), 48 57

P37A simple tool for massflow analysis of nanomaterials along their lifecycle

Burkhard Stahlmecke1, Uta Sager1, Simon Clavaguera2, Tom Ligthart3, Henk Goede4, Christof Asbach1

1IUTA e.V., Germany; 2CEA, France; 3TNO Utrecht, The Netherlands; 4TNO Zeist, The Netherlands; [email protected]

Release of nanomaterials can occur at all stages of their lifecycle. Due to the often unknown hazards ofa given nanomaterial it is essential to identify the main release points as well as the compartment intowhich the materials may be released. Thus, to better understand possible impacts of specificnanomaterials during their product life a simplified “life cycle analysis” with regard to release/emissionof a nanomaterial was conducted for three characteristic applications of selected end products. As faras available, the analysis tool is based on measurements, literature data and read across fromcomparable materials and processes. Expert judgement may be required to fill remaining knowledgegaps. The scope of the analysis is to sum up the nanomaterial emissions into the environmentalcompartments air, water, soil and the technosphere compartments landfill and recycling. Asintermediaries, also waste water treatment plants and municipal solid waste incineration plants aretaken into account. The calculations are based on simple spreadsheet software and use relative valuesof mass released into a certain compartment per lifecycle step.

We will present the results of this simple massflow analysis for three test cases: a) nanomaterials forlithium ion batteries, b) silicon based nanomaterials for thermoelectrics and c) inorganic fullerenes fornano based lubricants. It is expected that for cases a) and b) the main nanomaterial mass can eventuallybe found in recycling and landfill with minor or even negligible amounts emitted into the environmentalcompartments. For case c) it is also expected that the major amount is recycled or landfilled but theamounts ending up in environmental compartments account for about 20% of the total nanomaterialmass of which 50% are emitted into air. It has to be noted that possible transformations are not takeninto account during the analysis.

In part, the mass flow analysis presented is based on cross reading and assumptions and, thus, has tobe interpreted carefully due to the many unknowns. All case studies showed that generally the majorityof the nanomaterials will not be emitted until the end of life stage or are at least emitted undercontrolled settings within a specific lifecycle stage. Nevertheless, the analysis shows where emissionsare likely and indicate further research needs.

The research leading to these results has received funding from the European Research Council underthe European Union's FP7 (FP/2007 2013)/ERC Grant Agreement n.280765 (Buonapart E project) andGrant Agreement n.604602 (FNN project).


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P38Improving the survival rate of impinger for nano bioaerosols by using differentsampling liquid

Chih Ching ChengNational Yang Ming University, Taiwan, Republic of China; [email protected]

The fact that disease causing airborne viruses, such as SARS, MERS coV, and influenza virus, spread fastamong human beings is partially due to their nanoscale particle size. The liquid impingers are widelyused to sampling air bioaerosols, which have advantages for the preservation of bioaerosols. However,the lowest cutoff diameter of commercially available samplers for bioaerosols is about 0.3 m and theirphysical collection efficiency for nanoparticle is only about 10 20%. Therefore, this study aims toimprove the collection efficiency and survival rate of the liquid impinge for the collection of nanobioaerosols with different sampling solution.

Our previous research has shown that the excellent collection efficiency (~99%) of AGI 30 with packedglass beads for nanoparticles occurred in the condition of 4 lpm, 6 lpm sampling flow rate, grain size of3 mm, 10 cm depth of packed bed with 30 ml of collection liquid.

In this study, the above conditions and MS2 aerosols mixed with a physical tracer (uranine) was used.After periods of 10, 20 and 30 min, sampling liquids were titred using the plaque assay to test biologicalcollection efficiency and survival rate. The experimental system was combined with zero gas supplysystem, nano bioaerosols particle production system, bioaersols sampling system. We will try to obtainthe optimal efficiency by changing following conditions, including type of sampling liquid (deionizedwater, peptone, LB broth) and sampling flow rates (4 lpm, 6 lpm, 12.5 lpm).

The results showed the highest collection efficiency for airborne nano bioaerosols occurred in thecondition of AGI 30 sampler packing with glass beads, 4 lpm sampling flow rate, peptone and LB brothas sampling liquid.

The study provides a fundamental basis for sampling nano airborne virus.

Session 6A In vitro models of toxicity testing

P39Determining the permeation parameters of chemicals in metalworking fluidsthrough skin absorption

Yu Jou Lin1, Shih Wei Tsai1, Po Chen Hung21Department of Public Health & Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei,TAIWAN; 2Institute of Labor, Occupational Safety and Health, Ministry of Labor, Executive Yuan, Taipei, TAIWAN;[email protected]

Objective:The main task in a metalworking factory is to process the metal products, which involves the use ofmetalworking fluids (MWFs) to lubricate and cool the tools and metal pieces. However, due to itscomplex ingredients, there are many potential health hazards, which might be occurred with the use of


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MWFs. For example, it has been reported from various literatures that the exposures of MWFs arehighly associated with the occurrences of contact dermatitis. In order to protect people from theoccurring of occupational skin diseases in the MWFs operating environment, this study determined thepermeation parameters of skin absorption for the contact of MWFs. An in vitro system, i.e., the Hansonvertical diffusion cell (VDC), was performed.

Methods:This study focused on chemicals (e.g., formaldehyde, amines and so on.) in the metalworking fluids thatmight cause skin exposures. Parameters, including chemical compositions and contact conditions (e.g.,whether gloves and barrier cream were in use or not), were examined to elucidate their effects on skinabsorption. In addition, the Hanson vertical diffusion cell (VDC) was performed to simulate the commonsituation of workers’ skin exposures. Finally, all the samples were pre treated and analyzed by the solidphase microextraction (SPME) and triple quadruple gas chromatography tandem mass spectrometry(GC/MS/MS). Besides, questionnaires were administrated to the metalworkers to collect theinformation regarding the disease history of contact dermatitis, work conditions and related symptoms.

Results:From questionnaires, nearly half of the metalworkers had symptoms of skin abnormality. Nearly all themetalworkers used gloves for protection and none of them had the experience of using barrier creams.Nevertheless, wrong types of gloves using were also observed (e.g., cotton, where chemicals might bepermeated easily). On the other hand, the SPME procedure coupled with GC/MS/MS analysis for thedetermination of chemicals in the PBS solution was established in this study. It was observed thatchemicals can permeate through skin via dermal exposure. The permeability, including flux and Kp(permeation coefficient), for chemicals through skin were different with various protective measures.

Conclusions:VDC system was applied in this study to determine the permeation parameters of chemicals in MWFsthrough skin absorption. The results indicated that dermal exposure of MWFs is a problem worthy ofattention in the metalworking factories. With the findings of this study, suggestions were made to theworkers on how to protect from possible skin exposures.

Session 6B Work place measurement & modelling

P40Sensitivity analysis of a model characterizing nanoparticle agglomeration,dispersion and deposition processes in the atmosphere

Mikko Poikkimäki, Paxton Juuti, Joni Kalliokoski, Miikka Dal MasoAerosol Physics, Faculty of Natural Sciences, Tampere University of Technology, Tampere, Finland; [email protected]

An increasing amount of nanoscale materials such as engineered nanoparticles (ENPs) are produced forvarious industrial applications and everyday products. Recent studies have shown that some ENPs maycause adverse effects on human health (Card et al, 2008) and the environment (Hegde et al, 2016).Therefore, a need to assess and govern the risks resulting from their production, use and disposal hasarisen. However, present risk assessment models include dynamic microphysical processes affectingENPs after their release into the atmosphere only superficially. For example, conventional modellingtools generally assume chemically inert compounds, and therefore they neglect many nanoparticlespeci c processes such as coagulation, deposition (both wet and dry) and gas to particle conversions,


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including condensation, evaporation and hygroscopic growth. In addition, current models still lackproper testing and calibration, thus can result in uncertain risk estimates (Hristozov et al, 2016).

In this study, the nanoparticle Agglomeration, Dispersion and Deposition model (ADD, Anttila and DalMaso, 2015) is further developed to be used in risk assessment of an atmospheric release of ENPs. Ithas been especially improved to compute predicted environmental concentrations (PECs) in soil and thenanoparticle dose in the human respiratory system.

A sensitivity analysis, using the one at a time method (Saltelli et al, 2008), is performed on the ADDmodel. The main goal of the analysis is to map changes in model output, i.e., PECs, the lung depositednumber and mass of particles, as a function of input parameters. The analysis reveals, e.g., howaccurate input data should be in order to achieve reliable results.

Information acquired in sensitivity analysis can be then used as a guide to generating new exposure andhazard data. Hence, it increases accuracy and reliability of the risk assessment models. Themethodology used here can be applied to all currently available nano specific environmental andhuman risk assessment models.

We thank Dr. T. Anttila for developing the ADD model. M.P. was supported by the European Union’sHorizon 2020 research and innovation programme (caLIBRAte project Grant Agreement No. 686239).M.P. also acknowledges TUT Grad school for financial support.

Anttila, T. and Dal Maso, M. (2015) Aerosol Technology 2015 –conference, Tampere, Finland.Card, J. W. et al (2008) Am. J. Physiol. Lung Cell Mol. Physiol. 295(3), L400 L411.Hegde, K. et al (2016) Nanotechnol. Environ. Eng. 1(1), 5.Hristozov, D. et al (2016) Env. International 95, 36 53.Saltelli, A. et al (2008) Global sensitivity analysis: the primer, John Wiley & Sons.

P41Human risk assessment models input requirements and applicability duringproduct innovation stage gates: Results from EU H2020 ‘CaLIBRAte’

Wouter Fransman1, Thies Oosterwijk1, Remy Franken1, Miikka Dalmaso2, Miikko Poikkimaki2, ArtoSäämänen3, Helene Stockmann Juvala3, Ulla B Vogel4, Rambabu Atluri4, Keld A Jensen4, Rob Stierum1

1TNO, The Netherlands; 2Tampere University of Technology, Tampere, Finland; 3Tyoeterveyslaitos, FIOH, Helsinki, Finland;4NCRWE, Copenhagen, Denmark; [email protected]

The EU H2020 caLIBRAte project (http://www.nanocalibrate.eu/) aims to design, calibrate andimplement a next generation systems of systems risk governance framework for manufacturednanomaterials (MNM). Aim of this framework is to allow for screening of existing and emerging risks,quantitative risk assessment for humans and the environment and implementation of safe by designdecision support systems and risk management. A central part of the project is to support theassessment of MN along the “Cooper Stage Gate®” product innovation model. This idea to launchinnovation model allows for the screening and scoping of ideas, proceeding towards productdevelopment, testing, launch and post launch review. It is also applicable to MNM, either during thedevelopment of MNMs themselves, or for development of end products involving the application ofMN. An important part of this framework is concerned with human risk assessment models (WP2), inwhich integrated hazard, exposure and risk assessment models (HRA models) are being adopted andfurther developed for stage gate specific requirements.


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This talk describes the initial results of the identification of HRA models and proposal for refinement ofthese models based on stakeholder requirements at the different stage gates of innovation. First, inconsultation with external stakeholders from SMEs, industry, government, stage gate specific criteriahave been defined to which (existing) HRA models need to comply with. These involve general criteria,such as transparency of the model, operational costs, duration, capacity to deal with data gaps,compatibility of the model to accept new data etc.. More specific criteria for HRA models were alsodefined concerning both the model output (e.g. type of risk indicators, type of uncertaintycharacterization provided, accuracy of resulting risk estimate) and model input criteria (e.g. variousphysical chemical characteristics, the inclusion of kinetic endpoints, in vitro or in vivo hazard endpointincluded, exposure duration, aggregated exposures, inclusion of non intentional use). Secondly, existinghuman hazard, exposure (a.o. Consexpo, dART, FIOH Indoor Air Quality Model) and risk assessment(a.o. Guidenano, SUN, Stoffenmanager Nano, Licara) models have been evaluated for their applicabilitytowards the different stage gates of innovation bearing these predefined criteria in mind. Steps towardsfurther refinement of the existing HRA models for application along the stage gate model as well aspotential inclusion of novel approaches (e.g. physicochemical modelling, internal dose assessment andPBPK modelling systems toxicology, high throughput screening, adverse outcome pathway basedapproaches, bioinformatics) will be discussed.

P42Dermal model for nanomaterials

Eelco Kuijpers1,2, Stijn Schmeink1, Henk Goede1, Bernice Schaddelee Scholten1, Wouter Fransman1

1TNO, Netherlands, The; 2IRAS Institute for Risk Assessment Sciences, Molecular Epidemiology and Risk Assessment Utrecht,the Netherlands; [email protected]

Recent health related nanoparticle research has focused on biological activity, toxicity and(occupational) exposure through inhalation. However, it has been estimated that dermal exposure canbe a major route of exposure in occupational settings. For bulk compounds models are available toestimate dermal exposure, both in occupational settings and for consumer products. However, currentdermal exposure models need to be examined for their reliability and usability in the assessment ofnanoparticles, nanospecific weaknesses will need to be identified and, when necessary, adaptations willhave to be made to eliminate these weaknesses. Within the Sustainable Nanotechnology (SUN) projectwe have continued the work on the pre normative research under CEN Mandate/461Nanotechnologies. We answered the following questions: 1) What are the unique properties ofnanoparticles and which variables are necessary to model them? 2) How do the current dermal modelswork, are they suitable for nanoparticles and what are their nanospecific weaknesses? 3) What would ananoparticle specific model look like and where does it deviate from non NP models?

In this SUN study, first a systemic literature review was performed, exploring the state of the art in thefield of nanoparticles in relation to dermal exposure. Subsequently, a short review was performed onrecent studies illuminating the nanospecific properties that affect dermal penetration. Next, a selectionof dermal exposure models and their underlying framework were examined. This was followed by anevaluation of their nanospecific weaknesses and strengths using the insights into the unique propertiesof nanoparticles and which of these properties are relevant for dermal exposure. The results werediscussed with an expert panel in order to develop a concrete (theoretical and not data driven)framework for a nanospecific dermal exposure model.

The physiochemical properties of nanoparticles which are expected to be important based on thereview are primary particle size, exposed size distribution, particle number concentrations, particle


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mobility, specific surface area, surface properties & coating, coagulation, adhesion, cohesion andremoval.

The dART exposure model was selected as it is a quantitative based model (based on non nano data)with a broad applicability domain. Theoretical adaptations were made for the dART model to becomenanospecific. In addition, adaptations are general applicable and can also be made for other dermalexposure models. Future research should focus on the calibration and validation of the model.The authors would like to acknowledge the EU Commission FP7 program for funding this SUN project(Grant agreement No: 604305).

P43Measurements of the hygroscopicity of fresh and aged nanoparticles

Spyridon Bezantakos1, George Biskos21Université du Littoral Côte d' Opale, France; 2The Cyprus Institute; [email protected]

The ability of ultrafine aerosol particles (UFPs) to take up water and increase in size (i.e., theirhygroscopicity) can severely change their deposition characteristics in the human respiratory tract(Löndahl et al., 2007). The hygrosocpicity of particles depends mainly on their size and chemicalcomposition, which may differ between freshly emitted and aged nanoparticles. Hygroscopicitymeasurements conducted with the hygroscopic tandem differential mobility analyzer (HTDMA; Raderand McMurry, 1986) are widely used for modeling the deposition characteristics of UFPs in the humanrespiratory tract (e.g. Löndahl et al., 2008).

In this work we present laboratory measurements using an HTDMA system to determine thehygroscopicity of sampling antimony oxide (SbO3,SbO5) nanoparticles. In brief, an emulsion containingSbO3,SbO5 particles was aerosolized using an atomizer and their hygroscopicity was measured using theHTDMA system under two distinct conditions: i) without any additional treatment, and ii) when ozonewas inserted in the system for simulating conditions of atmospheric aging. Since these particles werecontained in an emulsion, surfactant agents were used to prevent their coagulation and therefore athermal denuder (TD) able of reaching temperatures of 380 °C was used upstream for evaporating mostof these surfactants. Initially hygroscopicity measurements were conducted when operating the TD atthree different temperatures (i.e., 380, 200 and 30 °C) without ozone. The hygroscopicity of freshSbO3,SbO5was moderate in all cases, but reduced when the TD was operated at the maximumtemperature, indicating that the presence of surfactants affected their hygroscopicity. When ozone wasintroduced into the system, however, and even when the TD was operated at its maximumtemperature, a chemical reaction, most probably caused by ozone interacting with the surfactantagents, leading to particle growth even at low relative humidity, was observed. These findings underlinethe importance of the purity of the sampled nanoparticles, which should be as close as possible to theirpristine chemical composition, as well as on the suitability of methods used for introducing particlesinto the gas phase. For instance, the spark ablation method (Tabrizi et al., 2009) can be used forgenerating metal oxide particles, like SbO3,SbO5, into the gas phase for further evaluating theirphysicochemical properties without the need of additional chemical agents.

ReferencesLöndahl J. et al., (2007), Inhalation Toxicology, 19:2, 109 116.Löndahl, J. et al., (2008), Inhalation Toxicology,20:10, 923 933.Rader, D. J. and McMurry P. H (1986).: J. Aerosol Sci. 17, 771–787Tabrizi, N. et al., (2009), J. Nanopart. Res., 11:315 332.


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P44Exposure and emission measurements of semiconductor nanowires duringproduction at a small scale company

Maria Hedmer1, Christina Isaxon2, Karin Lovén2, Linus Ludvigsson3, Jenny Rissler2, AndersGudmundsson2, Maria E Messing3, Håkan Tinnerberg1, Joakim H Pagels21Occupational and Environmental Medicine, Lund University, Sweden; 2Ergonomics and Aerosol Technology, Lund University,Sweden; 3Solid State Physics, Lund University, Sweden; [email protected]

Objective: The industrial use of novel manufactured nanomaterials with enhanced or completely newproperties is increasing globally. During the last decade there have been growing concerns of adversehealth effects of nanofibers e.g. carbon nanotubes. One type of nanofibers is the semiconductornanowires (NW), which is used in applications such as solar cells, light emitting diodes and batteries.Semiconductor NWs are fibre shaped manufactured nano objects with a length of 1 2 μm, and theindustrial use is growing – along with the risk for worker exposure. Aerotaxy is a new method with largepotential for up scaling the production of NWs. Production takes place in closed reactor systems andNWs of GaAs are grown in the gas phase on catalytic seed nanoparticles of Au by addition of gaseousprecursor molecules. The aerotaxy reactor system is opened up during maintenance e.g. manualcleaning operation. The aim of the study was to quantify the personal breathing zone (PBZ) exposureand emissions of NWs to air during production work at a small scale company. The potential forsecondary inhalation exposures and dermal exposure through resuspension of semiconductor materialdeposited on workplace surfaces was also assessed.

Methods: PBZ and emission filter samples were collected on 25 mm filters for 8 production stages fordetermination of particle number concentration, mass concentration, and metal content. Also directreading instruments were used in the emission zone and background to measure particle numberconcentrations and number size distributions with high time resolution. Tape samples (N=14) werecollected from workplace surfaces in the facility. The samples were analysed gravimetrically, with SEM,and ICP MS.

Results: Concentrations up to 98 NWs/cm3 were detected in the emission zone during the 8 productionstages. In the PBZ a concentration of 0.025 NWs/cm3 was measured. The emitted NWs had a meanlength of 4 μm (range 0.5 24 μm). One surface inside the tool enclosure was found to be contaminatedwith NWs, Au and semiconductor material. The cleaning operation was identified as the productionstage with highest emission.

Conclusion: During maintenance NWs and particles of Au and semiconductor material were emitted tothe workplace air and surface. The emitted NWs were typically longer than the produced ones. Thepersonal protective equipment used for this production stage was essential to avoid worker exposure.Based on the sampled surfaces the potential for secondary inhalation and dermal exposures was low.


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P45Particle emissions during lab scale metal oxide synthesis and handling

Ana Sofia Fonseca, Marcus Levin, Antti Joonas Koivisto, Kirsten Kling, Keld A Jensen, Signe H Nielsen,Ismo K KoponenNational Research Centre for the Working Environment (NRCWE), Copenhagen, Denmark; [email protected]

The production and use of engineered nanoparticles (ENPs; particle with at least one dimension 100nm) is one of the key technologies in Horizon 2020 (Savolainen et al. 2013). The earliest stage of ENPsexposure will likely occur in laboratories or pilot plants where researchers and/or technicians develop,produce and handling ENPs in a dusty form (O’Shaughnessy 2013; Jensen et al. 2009; Gomez et al. 2014;Tsai et al. 2009). Given the absence of reliable information about the toxicity of ENPs, studies focusingon occupational exposure assessment are particularly important for a precautionary principle.In this study, the potential exposure to airborne emissions during lab scale synthesis and handling CuO(mean primary Dp=40 nm), TiO2 (mean primary Dp=13.2 nm), and ZnO (mean primary Dp=1 10 nm) wasassessed. Near field (NF) and far field (FF; 4 m away from the fume hood) real time particlemeasurements were performed and personal samples were collected for gravimetric and microscopyanalysis. Additionally, assessment of the potential nanoparticle escape from fume hood and exposurerisk was conducted during a simulated worst case scenario which consisted in spillage of ultrafine TiO2

with very high dustiness index from 40 cm drop height. Overall, this study showed that there is a risk ofENPs emission and exposure associated with synthesis and handling different nanopowders only if thefume hood is not working properly. Activities monitored inside the fume hood such as milling,transferring and cooling of CuO nanoparticles, generated particles with significantly low particlediameters (< 50 nm) in terms of particle number concentration (up to 1 x 105 cm 3). However, theseconcentrations were markedly lower than the FF (constant order of magnitude of 103 cm 3). Thepersonal and stationary respirable samples collected were below the detection limit and only sootparticles could be detected during milling of the solid phase inorganic precursor under the hood.Additional study showed no detectable emission of ENPs into the workplace air from processes such asspilling of a high dustiness powder. Hence, this study confirms that the use of appropriate strategiesmay prevent worker exposure to nanoparticles in laboratory setups.

ReferencesGomez, V. et al., 2014. Journal of Hazardous Materials, 279, pp.75–84.Jensen, K.A. et al., 2009. Journal of Nanoparticle Research, 11(1), pp.133–146.O’Shaughnessy, P.T., 2013. Environmental Science: Processes & Impacts, 15(1), p.49.Savolainen, K. et al., 2013. Available at: www.ttl.fi/en/publications/electronic_publications/pages/default.aspx%5CnThis.Tsai, S.J. et al., 2009. Journal of Nanoparticle Research, 11(1), pp.147–161.

P46Characteristics of ultrafine and submicron metal containing paint particlesreleased from a spray painting workplace

Chia Hsiang Lai, Pei Chen Wu, Ting Yu YanCentral Taiwan University of Science and Technology, Taiwan, Republic of China; [email protected]

This study estimates the concentrations of metal containing paint particles at various particle sizedistributions in a spray painting process at an industrial ventilation equipment manufacturing plant on30 days in September, October and December of 2014. A total of 360 integrated air samples werecollected using a micro orific uniform deposition impactor (MOUDI). Metallic particles were analyzedusing an inductively coupled plasma with atomic emission spectroscop (ICP AES). The mass ratios of


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thoracic PM to total PM were in the range 0.36–0.56. The most abundant metal elements in particles ofall sizes on all sampling days were Pb (55.10±19.50 mg/ m3) and Fe (42.10±4.12 mg/m3). Significantly,the MMD of Cd, Ni, As and Cr of carcinogenic metals were in the range 0.19–0.88 mm (below respirableparticles diameter), indicating that 50% carcinogenic metals could possibly be deposited in the nonciliated gas exchange regions of the lung. In particular, most metal content contributed approximately57.48% 98.09% to the total PM content in submicron particles (PM1), except Pb. However, since metalcontent values may vary, further studies on health effects of solvent based paint emissions in differentpaint types, dilution proportion of diluting solvent and paints, types of pneumatic paint brush and arerequired in different industrial applications.

Session 7A Toxicogenomics and adverse outcome pathways (AOP) astools for nano risk assessment

P47Association of alveolar macrophage polarization genes in carbon nanoparticletoxicity and chronic obstructive pulmonary disease susceptibility

Tania A Thimraj1, Leema George1, Sangeetha Vishweswaraiah1, Swapna Upadhyay2, Holger Schulz3,Tobias Stoeger4, Koustav Ganguly51SRM Research Institute, SRM University, Chennai 603203 India; 2Lung and Airway Research, Institute of EnvironmentalMedicine, Karolinska Institutet, Stockholm, SE 171 77 Sweden; 3Institute of Epidemiology I, Helmholtz ZentrumMuenchen,German Research Center for Environmental Health, Neuherberg, Munich, 85764 Germany and Comprehensive PneumologyCenter Munich (CPC M), Member of the German Center for Lung Research, Munich, Germany D85764; 4Institute of LungBiology and Disease, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Munich,85764 Germany; 5Units of Lung and Airway Research and Work Environment Toxicology, Institute of Environmental Medicine,Karolinska Institutet, Stockholm, SE 171 77 Sweden; [email protected]

Rationale: Ambient particulate matter (PM) exposure and alveolar macrophage (AM) polarization hasbeen implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Previously, wereported a 2 fold macrophage influx in JF1/MsJmice (smaller lung volume) compared to C3H/HeJ(greater lung volume) following carbon nanoparticle (CNP) challenge. Therefore, in this study weinvestigated the transcriptomic signature ofM1/inflammatory andM2/healingmacrophagepolarization genes following CNP exposure comparing the two mouse strains. We also performedassociation studies of M1/M2 genes in a COPD cohort consisting of 98 patients and 91 control subjects.

Methods: PCR array consisting of 42 M1/M2 polarization genes was used to assess the transcriptomicsignature in lung tissue (n=3/strain/group) and total BAL cells (pooled n=3/strain/group, duplicates) offemale C3H/HeJ and JF1/MsJ mice intratracheally instilled with 20 g CNP (Printex 90). Samples werecollected after 1, 3 and 7 days of exposure. Sham instilled strain, age and sex matched mice served ascontrol. Cut off was set at 2 fold increase/decrease at any one investigated time point (p<0.05).Publicly available lung RNAseq data (GSE51748) from a COPD cohort was used to perform associationstudies of M1/M2 gene panel.

Results: Our data shows that M1/M2 transcriptomic signature in the mouse lung tissue and BAL cells(>80% macrophages) are independent of each other with most effects being detected in thebronchoalveolar lavage cells. C3H/HeJ BAL cells showed increased M1 markers CCl3 (on days 1,3),Cxcl15 (days 1,3,7) and increased M2 marker (Chil3) at all three times points investigated.Msr1 andTgfb1 (M2 markers) were decreased on days 3 and 7.


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In case of JF1/MsJ BAL cells, M1 markers Cxcl1 and Il6 decreased at all 3 investigated times.Interestingly, many genes exhibited altered regulation only on day 3. M1 markers (Cd86, Nos2) and M2markers (Ctsc, Mrc2, Cxcl13, Ms4a6d) were decreased on the third day. Fas, which is involved in bothM1 and M2 polarization and Clec7a (M2 marker) was increased on days 3 and 7.

Significant association of missense and/ or splice junction polymorphisms located within the M1/M2genes (APOL6, CD163, CD200R1, CIITA, CTSC, FN1, HEXB, IL7R, PSME2 and TGM2) imparting highersusceptibility to COPD was detected.

Conclusions: Altered AM polarization in mice with lower lung function, may, at least in part, explaintheir impaired pulmonary inflammation resolution efficiency to CNP challenge. Moreover, significantassociation of functionally relevant variations located within M1/M2 genes to higher COPDsusceptibility indicate genetic predisposition to the phenomenon.

P48Exploration of the modifying effects of cerium dioxide nanoparticles on dieselexhaust particles in a murine model of allergen exposure

Kirsty Meldrum3, Sarah Robertson1, Rachel Smith1, Teresa Tetley2, Martin Leonard1

1Public Health England, United Kingdom; 2Imperial College London; 3Public Health England and Imperial College London;[email protected]

In recent years, concerns have been raised regarding possible health effects due to constituents of airpollution, including those that may occur with increasing use of diesel engines and addition of fuelcatalysts such as cerium dioxide nanoparticles (CeO2NPs). There is limited information on the hazardpotential of this nanomaterial as part of diesel exhaust on respiratory conditions, including asthma andallergic airway disease.

We therefore examined the effects diesel exhaust particles (DEP) alone and in combination withCeO2NPs in an in vivo house dust mite (HDM) model of allergen exposure. Female Balb/c mice wereintranasally exposed to HDM alone or with DEP (50μg) alone or in combination with either low (50ng) orhigh (1.5μg) concentrations of CeO2NPs in single and repeat dose protocols over three weeks.

HDM exposure alone resulted in increased inflammatory cell counts within bronchoalveolar lavage fluid(BAL), increased airway epithelial mucin staining as well as increased inflammatory gene expressionwithin the lung tissue. When compared to control treatments, repeat DEP (over 3 weeks) resulted inincreased inflammatory cell counts (specifically lymphocytes, macrophages and neutrophils). Theaddition of DEP to HDM caused an increase in the inflammatory cell counts within BAL fluid, specificallyeosinophils and total cell counts. There was also an increase in the protein levels of IL 13 within the lungtissue. Inflammatory gene expression within lung tissue was synergistically altered on exposure to bothHDM and DEP when compared to each treatment alone. Addition of CeO2NPs to this combinationresulted in a further increase in BAL cell counts, specifically lymphocytes and eosinophils. This was alsoparalleled by CeO2NPs dependent alterations in inflammatory gene expression.

Preliminary investigations therefore suggest that cerium dioxide nanoparticles have the potential tomodify DEP effects in a mouse model of airway allergen exposure. Further investigation to characterisethis response will provide important information regarding the inhalation hazard potential of thisnanomaterial in conditions such as asthma.


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P49Inhaled multi walled carbon nanotubes induced gene expression profile in ratlung

Carole Seidel, Sylvie Sébillaud, Mylène Lorcin, Laetitia Chézeau, Christian Darne, Sébastien Bau,Stéphane Grossmann, Stéphane Viton, Hervé Nunge, Laurine Douteau, Sylvie Michaux, FrédéricCosnier, Laurent GatéInstitut National de Recherche et de Sécurité, France; [email protected]

Due to the attractive physicochemical properties of nanomaterials, in particular those of carbonnanotubes (CNTs), their industrial use is constantly increasing. Because of the insufficient knowledgeabout the CNTs toxicity the assessment of their potential hazard for workers health is a necessity. Sincethe main route of occupational exposure is inhalation, evaluation of the toxicological properties wasassessed by inhalation in laboratory rodents with two multi walled CNTs: NM 401 and NM 403, whichdiffer in length, diameter and specific surface area. These CNTs were aerosolized at two concentrationsof 0.5 and 1.5 mg/m3 and the resulting aerosols were fully characterized. Female Sprague Dawley ratswere nose only exposed 6 hours/day, 5 days/week for 4 weeks to these aerosols and tissues werecollected 3, 30, 90 and 180 days after the end of the exposure period. Results obtained fromconventional approaches, including biochemical and cytological analyses of broncho alveolar lavagefluid, histopathology and genotoxicity studies, demonstrated an induction of pulmonary inflammatoryresponse mainly at the shortest post exposure time point, which decreased overtime. On the otherhand, molecular approach allows identification of changes in lung gene expression overtime after theend of exposure. In accordance with the observed induction of inflammatory response 3 days after theend of exposure by CNTs, an up regulation of several chemokine genes as well as other genes involvedin inflammation was observed. Further analyses could result in establishing a link between carbonnanotube properties and key events associated with the observed toxicity. Then, transcriptomicexperiments could participate in the identification of molecular initiating events that lead to CNTpulmonary toxicity.

Session 7B Exposure management

P50Exposure assessment of fragrance allergens in personal care products in Taiwan

Chia Hsuan Wang, Shih Wei TsaiInstitute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan, Republic of China;[email protected]

Objective:Fragrances are widely used in most of personal care products, cosmetics and household productsnowadays. However, there are more and more health concerns related to the exposures of fragrancesthat have been observed elsewhere, including, dermatitis and asthma attacks, etc. In 1999, theEuropean Scientific Committee on Cosmetic Products and Non Food Products identified a set of 26fragrance allergens. The European Union also regulates the labels of cosmetics. Though these allergenshave been announced, there are still a lot of fragrance contained products on the market. Hence, to


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assess the possible health risks in Taiwan, there is a need to develop a convenient and sensitive methodfor the determinations of these fragrance allergens in PCPs and assess the exposure risk.

Methods:24 of the 26 fragrance allergens, except tree moss and oak moss, were dissolved in methanol as thestandard solution. For the preparation of calibration curve, deionized water was used to dilute thestandard solution. The PCPs samples were first diluted by water and equilibrated for 4 minutes beforethe extraction. The extraction was performed at 50°C for 20 minutes with 500 rpm. The 65 mPDMS/DVB fiber was exposed to the headspace over the samples. After the adsorption equilibrium, thesolid phase microextraction (SPME) fiber was inserted into the injector of the gas chromatography withtandem mass spectrometry (GC/MS/MS) for thermal desorption and further analysis. Exposureparameters, such as the usage amount and frequency in Taiwan, were collected by the designedquestionnaires.

Results:The headspace SPME procedure coupled with GC/MS/MS analysis for the determinations of 24fragrance allergens in PCPs was established in this study. The SPME fiber provided good extractionefficiency of the fragrance allergens in several kinds of PCPs. No carry over effect was observed fromthe thermal desorption of the sample. The linear ranges of these substances ranged from 0.1 to 500ng/ml, depending on their properties. Good linearity and precision were presented. More than 100PCPs samples were determined for the concentrations of the fragrance allergens. Exposure risk of theseallergens in PCPs was also calculated.

Conclusions:The SPME procedure coupled with GC/MS/MS analysis for the determinations of the fragrance allergensin PCPs was established in this study. With SPME procedure, advantages, such as solvent free and timesaving, were reached. The analytical method for different compounds was sensitive enough todetermine the concentrations from PCPs. Health risk associated with the possible exposures in Taiwanwere also assessed.

P51Nanoparticles exposure mitigation in plasma spraying

Eliseo Monfort1, Ana López Lilao1, Mar Viana2, Xavier Querol Querol2, Apostolos Salmatonidis2, AnaSofia Fonseca3, Pablo Carpio4

1Instituto de Tecnología Cerámica, Universitat Jaume I, Spain; 2Institute of Environmental Assessment and Water Research(IDAEA CSIC). Barcelona, Spain; 3National Research Centre for the Working Environment (NRCWE). Copenhagen, Denmark;4Institute of Materials Technology (ITM), Universitat Politècnica de València. Valencia, Spain.; [email protected]

In the present work, a case study of implementation of mitigation measures in an atmospheric plasmaspraying system used for applying ceramic coating is presented.

Firstly, a preliminary phase was developed to identify and quantify particle emissions to indoor air fromplasma spraying with special interest in ultrafine (UFPs <100 nm in diameter) and nanoparticles (NPs<50 nm).

For this purpose, particle number, mass concentrations, alveolar lung deposited surface areaconcentration and size distributions in the 10 20000 nm size range were simultaneously monitored atthree sites: the potential breathing area (worker’s area), inside the plasma spraying chamber and in


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outdoor air. The experimental protocol has included the use of different particle monitoringinstrumentation (NanoScan SMPS, Grimm optical particle counter 1108, DiscMini and CPC).

During plasma projection of different ceramic materials (containing micro and nano sized feedstocks asraw materials) significant increases in UFPs and NPs number concentrations were observed. For thisreason, a complete risk prevention protocol to plasma spraying scenario was implemented, based onapplying corrective measures in the emission zone, in the extraction system of the spraying chamberand in the ventilation of the worker’s area. It should be highlighted that the application of this protocolled to a very significant reduction in the potential breathing zone (worker’s area) concentration from>2.5 106 to 0.05 106 p.cm 3 (reduction >95%).

In summary, these outcomes evidence, on the one hand the potential risk of occupational exposure toUFPs and NPs during plasma spraying process, but on the other hand the relevance of implementingeffective mitigation protocols, above all in high energy processes. Therefore, this study raises the needto further investigate UFPs and NPs formation mechanisms in high energy processes and the realeffectiveness of mitigation measures to minimise occupational exposure.

P52Determining the permeation parameters of synthetic musks through skinabsorption

Shih Wei Tsai, Ya Ling Hsu, Chia Hsuan Wang, Yu Jou LinNational Taiwan University, Taiwan, Republic of China; [email protected]

Aims:Synthetic musks, which have been widely added in various PCPs, are suspected to cause different healtheffects. The purpose of this study was hence to determine the permeation parameters of skinabsorption for the contact of synthetic musks. An in vitro system, i.e., the Hanson vertical diffusion cell(VDC), was performed.

Methods:Ten commonly used synthetic musk were chosen. To simulate skin exposures, the vertical diffusion cell(VDC) was applied in this study. Porcine skin was selected as the substituted skin for human exposure,while phosphate buffered saline (PBS) solution was used as the receptor media. Samples from the VDCsystem were taken periodically to determine the permeation profiles. The 65 m PDMS/DVB solid phasemicroextraction (SPME) fiber was exposed to the headspace over the samples. After adsorptionequilibrium has been reached, the SPME fiber was inserted into the injector of the gas chromatographywith tandem mass spectrometry (GC/MS/MS) for thermal desorption and further analysis.

Results:The SPME procedure coupled with GC/MS/MS analysis for the determination of synthetic musks in PBSsolution was established in this study. It was observed that synthetic musks can permeate through skinvia dermal exposure. The permeabilities, including flux and Kp, for synthetic musks (e.g., AHTN andHHCB) through skin were different for PCPs with various sample matrixes. Compared with lotion, theflux and Kp for shower bath gel was higher, which means the synthetic musks in shower bathpermeated through skin easier. In addition, the permeation coefficients through skin were 10^ 10 10^8 cm/hr and 10^ 7 10^ 6 cm/hr for synthetic musks contained in lotion and shower bath gel,respectively. It was also observed that around 0.001~0.4% and 3~12% of the synthetic musks in lotionand shower bath gel, respectively will permeate through skin. Moreover, the concentrations of


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synthetic musks in PCPs affected the permeability as well. As the concentration increased, the flux washigher no matter which kind of sample matrix was tested. Besides, the Kp (permeation coefficient) alsodepended on the sample matrix and the concentrations of synthetic musks. It was observed that the lagtime for AHTN, HHCB, and AHMI were all below 0.5 hour, which meant the synthetic musks permeatedshortly once the PCPs were applied on the skin.

Conclusions:This research demonstrated that the hypothetical parameters for skin absorption were not consistentwith what were found the in vitro testing system.

P53Non intentional nanoparticles exposure, the unexpected guest intoexperimental process: who can we manage it?

Cecile Philippot, Cecile Ducros, Dominique Locatelli, Jean François DamlencourtUniv. Grenoble Alpes, CEA Tech, PNS, F 38000 Grenoble, France; [email protected]

In this paper, we will discuss about non intentional nanoparticles (NPs) released during the recyclephase at high temperature of photovoltaic panel and who we can manage it to protect workers and theenvironment. This case study is quite specific of experimental processes where researches try todevelop new process working with prototypes. All the conventional risk such as electrical, chemical orlaser, are taken in consideration but in this case the experimental condition induce non intentional NPsreleased.

The starting point of this measurement campaign comes from the formation of a quite volatile dustduring the process inducing a silicon contamination of the nearest environment (other equipments andthe platform). The silicon fume leakage seems to hail from a non waterproof connection between theprototype (a high temperature furnace) and an additional equipment.

The CEA NanoSafety Plateform (PNS), have been solicited to analyze and characterize theses fumesand also to provide EHS recommendations and some workstation improvement axes. The methodologyused by PNS team for the exposure evaluation is described in the French methodological guide. Tostudy the NPs liable to be released in air at workplace, we use several real time techniques tocharacterize particles aerosol in terms of concentration in particles and size distribution, coupled withthe SEM/EDS observations.

The measurement campaign allows to confirm that the silicon contamination is due to a nonwaterproof connection which induces some silicon fumes leakage at high temperature. These fumescondensate on coldest part of the furnace in the form of crystallized silicon nanowires with a diameterof 30 nm. We have also identified all the trick steps where the workers have to manage the nano risk inaddition of all the other during the process such as open and close furnace operations or maintenancephases. Moreover, the cleaning procedure and the waste have been also investigated and validated toavoid any additional NPs dispersion. In parallel, some chemical air samples have been done to confirmthe good cleaning practices.


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Session 8A Nanomaterial epidemiology & biomonitoring

P54NanoStreeM, the European Semiconductor Industry Consortium as a startingpoint for developing a medical surveillance consensus for nano workers

Eline Vandebroek, Luc HonsOccupational Health Service Premed VZW, Leuven; [email protected]

Nano electronics has a huge transformative potential for the society. The pace of innovation is very fastbringing a variety of new materials functionalized at the nanoscale into everyday life. This is recognizedas a shared concern by the industry; therefore a number targeted process safety and occupationalhealth monitoring campaigns have been conducted.

Timelines of obtaining better scientific understanding greatly mismatch those of product innovation andmarket distribution. Such a situation presents a challenge for both mitigation of the occupational andenvironmental risks and the overall risk governance and policymaking. As a step in the direction ofaddressing this challenge, 14 industrial and academic institutions from 6 EU member states, initiated acollaborative project named NanoStreeM, funded under Horizon 2020 EU Framework Program forResearch and Innovation. The project is providing the European Semiconductor Industry theopportunity to grow its knowledge regarding its utilisation of nanomaterials. The timeline of the projectis 2016 – 2018. The overall objectives are:

1. to build inventories of materials, research topics and directions relevant for nanomaterial use andexposure in nano electronics manufacturing;

2. to identify gaps in knowledge and methodologies to assess the risk of engineered nanomaterialsused in semiconductor manufacturing or incidentally released as by products;

3. to apply obtained results for better training, management and governance of the risks related toengineered nanomaterials and industrial processes.

An important focus of the consortium activities will be the comparison of current practices of medicalsupervision on occupational exposure to engineered nanomaterials. Indications exist that nanoparticlescan affect health (cardiovascular effects, possible carcinogenicity of multi walled carbon nanotubes,etc.). However a consensus concerning occupational screening is lacking.

Therefore, Premed, a Belgian Occupational Health Service participating in NanoStreeM, will collectinformation from all (industrial) partners in the project about the current practices of medicalsupervision of persons handling nanomaterials.

Based on the received information and a literature study, a recommendation regarding medical datacollection and screening will be formulated.

In our presentation for NanOEH 2017 we would like to focus on:A general view on the different working packages of the consortium. This may be inspiring for otherconsortia in other industrial sectors working on the health and safety aspects of nanomaterials.A summary of the results collected through the questionnaire on current practices.Approach and preliminary results of the literature study concerning the medical surveillance foremployees exposed to nanomaterials.


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P55A biomonitering study of firefighters and human volunteers during fireextinction exercises

Anne T Saber1, Maria Helena Andersen2, Per Axel Clausen1, Niels Ebbehøj3, Julie Elbæk3, Anne HeleneGarde1,2, Åse Marie Hansen1,2, Ismo Koponen1, Steffen Loft2, Peter Møller2, Eva Carina Nørskov3, PeterB Pedersen3, Ulla Vogel1,41NFA, Denmark; 2University of Copenhagen, Denmark; 3Danish Technological Institute, Denmark; 4Department of Micro andNanotechnology Technical University of Denmark, Denmark; [email protected]

Fire smoke contains many different carcinogens and firefighting is classified as possibly carcinogenic tohumans (2B) by the International Agency for Research on Cancer [1]. So far only a limited number ofDanish studies on cancer risk among firefighters have been published. In the BIOBRAND project we aimat obtaining more knowledge on the adverse health effects among Danish firefighters after fireextinguishing by the current use of protection equipment.

The potential health risk for firefighters using protective equipment is studied 1) in a controlledbiomonitoring study where we measure biomarkers in samples from young conscripts training to smokedivers at the Danish Emergency Management Agency, and 2) in firefighters before and after fireextinguishing. Particle exposure levels are measured during fire development as well as inside the maskand on the skin (PAH). Moreover biomarkers of cancer (DNA strand breaks in peripheral lymphocytes)and cardiovascular disease (e.g. C reactive protein, serum amyloid A) in blood samples are measured.As a marker of exposure to tars, 1 hydroxypyrene is measured in urine. Furthermore lung function andvascular function (EndoPat) is assessed.

The project runs from January 2015 to October 2017. The project involves scientists from the NationalResearch Centre for the Working Environment, Department of Occupational and EnvironmentalMedicine, Bispebjerg Hospital, Copenhagen University and the Danish Technological Institute. Theproject is implemented in close collaboration with the Danish Emergency Management Agency andCopenhagen Fire Brigade. A National Advisory Board including stakeholders from eg. fire brigades, tradeunions and The Danish Emergency Management Agency has been established. The National AdvisoryBoard has been involved in the overall study design and will be involved in the dissemination of theresults to stakeholders. The National Advisory Board is also reference group for our sister projectEPIBRAND which is an epidemiological registry study assessing possible health risks for Danishfirefighters. The project is supported by the Working Environment Research Fund. More informationcan be found on our homepage [2].

[1] IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Painting, Firefighting, andShiftwork, Volume 98 (2010)[2] http://www.arbejdsmiljoforskning.dk/da/projekter/biobrand


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P56Exhaled breath condensate nanoparticulate content and comparison with deeplung nanoparticulate burden

Véronique Chamel Mossuz1, Caroline Marie Desvergne1, Muriel Dubosson1, lara Leclerc2,3,4, JeremiePourchez2,3,4, Michele Cottier2,3,5, Jean Michel Vergnon2,3,5

11CEA Grenoble, NanoSecurity Platform (PNS), Medical Biology Laboratory (LBM), 17 rue des martyrs, 38054 Grenoble Cedex 9,France; 2INSERM, U1059, SAINBIOSE, Saint Etienne, F 42023, France; 3Université de Lyon, Saint Etienne, F 42023, France;4Ecole Nationale Supérieure des Mines de Saint Etienne, CIS EMSE, SAINBIOSE, F 42023 Saint Etienne, France; 5CHU SaintEtienne, Saint Etienne, F 42055, France; [email protected]

The use of engineered nanoparticles (NP) is expanding very fast worldwide, but there is still a lack ofclear answer on their toxicity. There is a special concern for the inhalation route of exposure based onultrafine particles health effects and the asbestos drama. In this context, it is of main importance to beable to evaluate NP exposure of individuals, especially at work places where exposure might beelevated. Exhaled breath condensate (EBC) is sampled in a non invasive way, and contains non volatilescompounds that emanate from the respiratory tract. In that respect, EBC is particularly attractive forthe biomonitoring of inhalation exposures and their potential respiratory impact. There is emergingliterature reinforcing the hypothesis that NP might be found in EBC following inhalation and might beused as biomarkers of exposure to NP.

Prior to the use of EBC for inhalation exposure monitoring in occupational health, better knowledge onEBC particulate content is required and the question whether EBC is representative of the alveolarregion of the lung in terms of NP burden needs to be elucidated. The goal of this study is therefore,first, to describe the NP content of EBC, and second, to compare broncho alveolar lavages (BAL) andEBC from the same subjects in terms of nanoparticle composition.

This has been investigated in the project EXPO NANO supported by the Canceropole Lyon AuvergneRhone Alpes (CLARA, France). BAL and EBC were collected from 100 patients who had a medicalprescription for BAL in the pneumology department of St Etienne hospital over one year. EBC werecollected with the RTube device after pre treatment to avoid nanoparticle contamination. Thecomposition of EBC for 21 elements was determined by inductively coupled mass spectrometry, and theparticulate content was evaluated by dynamic light scattering. EBC were characterized by their volume,their content in sodium and total proteins, in order to evaluate different modes of standardization.

First results indicated that EBC were mainly composed of Si, Na, and Zn. Also, based on the DLS results,a particulate content was brought out in EBC from patients in comparison with blank RTubes.

This study brings new information on the feasibility to measure NP in EBC and their characterization.This is a first step in the development of potentially new biomarkers of exposure to NP. The next stepwill focus on the comparison between EBC data and BAL data so as to evaluate EBC representativenessof deep lung.


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P57Analytical developments for oxidative stress biomonitoring in exhaled breathapplied to the field of nanoparticle human exposure

Caroline Marie Desvergne1, Muriel Dubosson1, Jean Luc Ravanat2, Melissa Revol1, Véronique ChamelMossuz11University of Grenoble Alpes, CEA Grenoble, NanoSecurity Platform (PNS), Medical Biology Laboratory (LBM), 17 rue desmartyrs, 38054 Grenoble Cedex 9, France; 2University of Grenoble Alpes, CEA Grenoble, INAC/SyMMES/CIBEST, UMR 5819CEA/CNRS/UGA, 17 rue des martyrs, 38054 Grenoble Cedex 9, France; [email protected]

Although it is increasingly widely accepted that nanoparticle (NP) exposure and health monitoring forworkers is needed, there is currently no consensual proposal for adequate biomarkers of exposure andeffect. The development of biomarkers of exposure is limited by the difficulties to find and characterizeNP in biological media, and for biomarkers of effect, the main issue lies in the non specificity ofavailable biomarkers. The inhalation route of exposure is of main concern for NP potential toxicity. Thusexhaled breath presents the advantage of being specific of that route of exposure and is more and morestudied in the field of nanoparticle exposure monitoring. This abstract is dedicated to the analyticaldevelopments of a biomarker of oxidative stress (8 isoprostane) in exhaled breath as part of a projectselected by the French Envitéra platform (funding from the region, ARS and DREAL Auvergne RhôneAlpes).

High performance liquid chromatography coupled to tandem mass spectrometry (HPLC MS/MS), andenzyme immuno assay (EIA) were compared in terms of sensitivity and specificity for 8 isoprostanedetection. Moreover, two devices of collection of exhaled breath were compared based on the analysisof 8 isoprostane: the RTube device from Respiratory Research and the SensAbues device. Exhaledbreath samples were obtained from volunteers after informed consent.

An original HPLC MS/MS method was developed based on the quantification of the ammonium adductof 8 isoprostane in the positive ionization mode. Preliminary results indicate that the main advantage ofHPLC MS/MS lies in the specificity of the measurement, while EIA might be more sensitive. Jointly, thetwo collection devices for 8 isoprostane in exhaled breath have their own advantages and limitations;the collection efficiency of the SensAbues device might be higher, meanwhile the versatility of theRTube device might be more appropriated for the simultaneous monitoring of several biomarkers.

The main issue about biomarker detection in exhaled air is that the analytical methods need to behighly sensitive since the biomarker levels are very low. The collection efficiency is also of mainimportance to obtain sufficient biological matter and has to be optimized. The next step will consist indeveloping other biomarkers of oxidative stress to be measured simultaneously with 8 isoprostane, inorder to obtain oxidative stress fingerprints. Such analytical developments are intended to be applied infuture field studies for the monitoring of respiratory early effects due to occupational exposure to NP.


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Session 8B Risk assessment & management

P58Global health impacts of nanotechnology law: The legend of asbestos thathaunts future innovation in nanotechnology

Ilise CharoyLegal Advisor the Greek National Platform on Nanomedicine; [email protected]

Experts quoted in popular articles on the web and in serious law articles have asked whether“nanotechnology is the next asbestos” and some have claimed that “nanotechnology torts” are asleeping giant. This presentation examines the recent vogue of assuming that asbestos litigation is avalid paradigm that foreshadows litigation about Nanotechnology. Although anyone can be sued at anytime for any reason, far more dangerous risks than asbestos exposure have passed through commercewithout the notorious litigation of asbestos fame. Why? This presentation suggests that risk or danger isnot the relevant criterion. Litigation surrounding asbestos exposure is an outgrowth of failure to warnabout known dangers of potential harm; many judges and juries found a willful plan to hide informationabout dangers. Therefore, people who were exposed in the workplace or as consumers could not takeprecautions to mitigate risk. This presentation outlines key cases that broke open the pandora's box ofmedical evidence regarding asbestos and regulations that were an outgrowth of the public demand forprotection in response to those harms. International laws, USA laws and the global harmonization ofchemical safety agreements are examples of modern law that encourages use of toxic and hazardoussubstances, so long as safeguards are applied and known dangers have been disclosed. Thispresentation concludes that candid discussion of known risks, absent in the early history of asbestos useis the best prevention against potential litigation. Disclosing that problems are unquantifed is not thesame as hiding known dangers; disclosure of the known but unquantifiable risks can avoid potentialliability so long as the disclosure is accurate. Candid disclosure of accurate statements about risk is thecriterion that will distinguish any effort to shackle nanotechnology in toxic torts compared to the ghostsof asbestos litigation that haunt us today.

P59Estimation of the recommended occupational exposure limits for multi walledcarbon nanotubes (MWCNT) and graphene

Young Sub Lee1, Jae Hyuck Sung2, Kyung Seuk Song2, Byung Sun Choi1, Il Je Yu3, Jung Duck Park11Chung Ang University, Korea, Republic of (South Korea); 2Korea Conformity Laboratory, Korea, Republic of (South Korea);3Hoseo University, Korea, Republic of (South Korea); [email protected]

This study was performed to provide the recommended occupational exposure limits (OELs) for multiwalled carbon nanotubes (MWCNT) and graphene of nano materials at workplace from animal datausing lung dosimetry model. In this study, we used rat NOAELs of 0.98 mg/ and 3.02 mg/ inMWCNT and graphene, respectively, which was suggested in the previous 13 weeks inhalation study ofrats in each (KCL, 2014, 2016). The deposition fractions of nano materials in the lungs of rat and humanwere calculated by using the multi path particle dosimetry model (MPPD model, v3.04). The depositionfraction of MWCNT in the lungs was calculated at 0.0577 and 0.0893 in rats and human, respectively,and 0.1529 and 0.1499 in rats and human, respectively, for graphene. Then, the human equivalentexposure concentrations (HECs) of MWCNT and graphene were calculated using the NIOSH method. As


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a result, the HEC was estimated at 0.17 mg/ for MWCNT and at 0.82 mg/ for graphene which wasrelevant to the rat NOAEL of 0.98 mg/ and 3.02 mg/ for MWCNT and graphene, respectively.Finally, we estimated the recommended OELs by applying the uncertainty factor to the HEC asfollowings, 3 for rat to human, 2 for subchronic to chronic, 5 for interindividual variation in workers, and1 for severity of endpoint. As a result, OEL was estimated to be 6 / for MWCNT and 27 / forgraphene.

P60A promising approach towards responsible and safe nano innovations

Susanne Resch, Christa Schimpel, Andreas FalkBioNanoNet Forschungsgesellschaft mbH, Austria; [email protected]

With large amounts of nanotoxicology studies delivering contradicting results and an unstable, movingregulatory framework, potential risks surrounding nanotechnology appear complex and confusing. TheEuropean chemical regulation, REACH, is trying to adapt its annexes to fit nanomaterials [1], however,this is not a process achieved swiftly. In order to develop safe and legally compliant products, wefocussed on the development of a safety concept implementable in real life innovation processes [2],which may help to plot a sensible path through the nano risk landscape, without stifling innovation.Presently, the model is used in ongoing H2020 pilot line projects synergistically (e.g., Hi RESPONSE,INSPIRED, R2R Biofluidics), and encompasses 6 main pillars:

1. Information Gathering2. Hazard Assessment3. Exposure Assessment (Occupational & Environmental Exposure)4. Risk Characterisation5. Refined Risk Characterisation and Exposure Monitoring6. Risk Mitigation

The approach provides a lifecycle hazard , exposure and risk profile for a given nanomaterial (e.g.,classification of which materials/process operation pose greater risk, where these risks occur in thelifecycle, and the impact of these risks on society), while following the general REACH (CSA) approach[3] applied to chemicals. The risk profile for a given nanomaterial (e.g., categorisation of whichmaterials and process operations pose greater risk, where these risks occur in the lifecycle, and theimpact of these risks on society) is created using state of the art safety assessment approaches/tools(ECETOC TRA, Stoffenmanager Nano and ISO 12901 2) [4 6]. If critical hotspots are identified, riskmitigation actions are taken that focus on hazard/risk avoidance (e.g., “design out” physico chemicalparameters that are identified as being drivers of toxicity whilst retaining the functional aspects, “redesign” process and operational conditions, develop solutions for exposure reduction). Thus, concreteand practical guidance to industry on how to deal with environmental health and safety (EHS) issues ofmanufactured nanomaterials and nano enabled products is provided, including, as appropriate,legislation/sector specific issues.

The proposed approach aims to be universally applicable for various nano related innovations and thusbringing them closer to the market.

References[1] Kathrin Schwirn et al., 2014.[2] European Commission, 2013.


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[3] Christa Schimpel et al., 2017.[4] Targeted Risk Assessment Ecetoc, 2004.[5] Birgit Van Duuren Stuurman et al., 2012.[6] ISO, ISO/TS 12901 2:2014, 2014.

P61A comparison of the biokinetics of nanoceria after intravenous, inhalation,instillation and oral exposure using physiologically based pharmacokineticmodeling

Tshepo Paulsen Moto1,2, Ulrika Carlander2, Gunnar Johanson21University of Pretotia, Faculty of Health Sciences, South Africa; 2Karolinska Institutet, Institute of Environmental Medicine,Unit of Work Environment Toxicology; [email protected]

BackgroundNanoceria can act as an antioxidant due to its ability to change between oxidation state, and thus istargeted for use in scavenging free radicals. Uptake of nanoceria from inhalation and oral exposure islimited though bioaccumulation may occur as dissolution and excretion is slow. Understanding adverseeffects of nanoceria requires understating of nanoceria biokinetics. Physiologically basedpharmacokinetic (PBPK) models can be employed to investigate biokinetics. PBPK models can bedeveloped, calibrated, refined and validated with the use of biodistribution experimental data. A PBPKmodel was developed to study nanoceria biokinetics.

AimUse PBPK modelling to compare the systemic biodistribution after inhalation, instillation and oralexposure to that of intravenously (iv) administered nanoceria.

Materials and MethodsExperimental biokinetic data in rats were collected from the literature and investigated with ourpreviously developed PBPK model. The model was first calibrated and validated for 5 nm and 30 nmnanoceria given iv, and then used to predict the biokinetics of various sizes. In a third step, the modelwas modified to allow biokinetic predictions for inhalation, instillation and oral exposure. These threeroutes were also compared to the iv route by calculating tissue: liver concentration ratios. Ratios ratherthan absolute concentrations were used to account for differences in dose and absorbed fractionbetween studies and routes.

ResultsThe PBPK model adequately described the biokinetics of 5 nm nanoceria administered by iv. Thebiokinetics of the other sizes and exposure routes were less well described by our model. A limitationwhen modeling the inhalation, instillation and oral exposure data was that, due to the low uptake,tissue levels were very low, variable and unreliable. Yet, the PBPK model and the comparison ofconcentration ratios both suggest route dependent biokinetics of nanoceria.

ConclusionPrediction of the tissue/organ timecourses of 5 nm administered by iv was adequately described by thePBPK model but other sizes and routes were not so well described and predicted. Furthermore, thebiokinetics of nanoceria seem to depend on nanoparticle properties, dose and route of exposure.


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P62European standardization in nanotechnologies and relation with Internationalwork. How standardization can help industry and regulators in developing safeproducts?

Patrice ConnerAFNOR, France; [email protected]

Nanotechnologies have enormous potential to contribute to human flourishing in responsible andsustainable ways. They are rapidly developing field of science, technology and innovation. As enablingtechnologies, their full scope of applications is potentially very wide. Major implications are expected inmany areas, e.g. healthcare, information and communication technologies, energy production andstorage, materials science/chemical engineering, manufacturing, environmental protection, consumerproducts, etc. However, nanotechnologies are unlikely to realize their full potential unless theirassociated societal and ethical issues are adequately attended. Namely nanotechnologies andnanoparticles may expose humans and the environment to new health risks, possibly involving quitedifferent mechanisms of interference with the physiology of human and environmental species.

One of the building blocks of the “safe, integrated and responsible” approach is standardization. Boththe Economic and Social Committee and the European Parliament have highlighted the importance tobe attached to standardization as a means to accompany the introduction on the market ofnanotechnologies and nanomaterials, and a means to facilitate the implementation of regulation. ISOand CEN have respectively started in 2005 and 2006 to deal with selected topics related to thisemerging and enabling technology.

In the beginning of 2010, EC DG "Enterprise and Industry" addressed the mandate M/461 to CEN,CENELEC and ETSI for standardization activities regarding nanotechnologies and nanomaterials. ThusCEN/TC 352 "Nanotechnologies" has been asked to take the leadership for the coordination in theexecution of M/461 (46 topics to be standardized) and to contact relevant European and InternationalTechnical committees and interested stakeholders as appropriate

(56 structures have been identified). Prior requests from M/461 deal with characterization andexposure of nanomaterials and any matters related to Health, Safety and Environment. Answers will begiven to: What are the structures and how they work? Where are we right now and how work is goingfrom now onwards? How CEN’s work and targets deal with and interact with global matters in thisfield?

P63EC4SafeNano: European Centre for Risk Management and Safe Innovation inNanomaterials and Nanotechnologies

Tomasz Berezniak1, Keld A Jensen1, Ulla Vogel1, Emeric Frejafon2, Benoit Hazebrouck31National Research Centre for the Working Environment, Denmark; 2Institut National de l’Environnement Industriel et desRisques, France; 3European Virtual Institute for Integrated Risk Management, Germany; [email protected]

A significant challenge to ensuring sustainable production and use of nanotechnologies is to understandsafety and health risks of the technology and its end products, and to implement practical strategies tomanage these risks. Knowledge is growing rapidly, but effective use of this knowledge for risk


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management is lagging behind. We therefore need to bridge the gap between knowledge on hazardand risk, and ‘fit for purpose’ risk management tools and strategies supported by measurement andcontrol methods.

EC4SafeNano will bridge this gap in an efficient and sustainable way by setting up an independent,science based, managed Centre (hub) linked with several networks (spokes) to act at the interfacebetween research organisations, industry, regulatory bodies, and civil society.

The objectives are to:1. understand the needs of all stakeholders along the innovation value chain for nanotechnologies,

ensuring safer, marketable, regulated and accepted long lived products;2. identify the resources and capabilities available to address these needs, and evaluate the capacity

to provide technical solutions and actions;3. build, test and benchmark a range of services, based on selected resources that answer stakeholder

needs across the innovation value chain;4. develop mechanisms and operating procedures to facilitate periodic updating of the “needs and

resources” mapping and of the service provision;5. develop networking activities aiming to share, benchmark and promote the EC4SafeNano services

thereby enhancing and harmonizing the overall expertise, at EU level and beyond; and6. develop governance rules and a strategic plan to prepare for self sufficient operation beyond the

project lifetime.

P64Readiness of control banding tools for safe innovation and regulatoryoccupational exposure assessment of nanomaterials

Biase Liguori1, Flor Angela Quintero2, Mehmood Ahmad2, Aleksandar Jovanovic2, Blanca SuarezMerino3, Isabel Rodriguez Llopis3, Christian Micheletti4, Rambabu Atluri1, Nicklas R Jacobsen1, AndersBaun5, Steffen F Hansen5, Keld A Jensen1

1National Research Centre for the Working Environment, Copenhagen, Denmark; 2Steinbeis Advanced Risk TechnologiesGmbH, Stuttgart, Germany; 3GAIKER Technology Center, Parque Tecnológico de Bizkaia ed. 202, 48170 Zamudio, Bizkaia, Spain;4ECSIN ECAMRICERT, Viale Porta Adige 45, Rovigo, I 45100, Italy; 5DTU Environment, Technical University of Denmark, Building113, Kgs. Lyngby DK 2800; [email protected]

The nanotechnology industry has been referred to as the “new industrial revolution” because of thenovel material properties of nanomaterials. Nanotechnology applications occur to diverse sectors suchas electronics, clean energy, information and communication, chemistry, biotechnology, health, and theconstruction industry. It is estimated that by 2020, approximately 20% of all goods manufacturedworldwide will involve nanotechnology, which will lead to an increased development, production andapplication of engineered nanomaterials. Consequently, there will be further increased risk ofnanomaterial exposures in the working environment. Because adequate exposure limits have not beenestablished for nanomaterials, there is a growing risk that an increasing number of workers may beexposed to concentrations at which hazardous effects can occur.

In order to protect workers from potential risks related to nanomaterials a number of methods havebeen developed including the Control Banding Nanotool, IVAM Technical Guidance, StoffenmanagerNano, ANSES CB Tool, NanoSafer, the Precautionary Matrix and the ISO/TS 12901 control bandingscheme.


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Most of these models were assessed in Liguori et al. (2016). Here we further evaluate the methods inregards to their use domains; types, extent, use and availability of input parameters; their outputformat; and finally their potential use and maturity in regard tomeeting the minimum requirements foroccupational exposure assessment under REACH. We also assess the ability of the models to supportSbD (Safe by Design) in a NANOREG2 industry case study on risk innovation governance.

The analyses showed that the models were developed for different purposes and also have differentapplication domains and inclusion criteria. Moreover, the exposure assessment methods and derivedrisk levels are based on different assumptions and give outputs in different formats. The use ofrequested input parameters for exposure assessment differs greatly among the tools. Despite, stillgiving qualitative risk management results, the regulatory readiness is high for Stoffenmanager Nanoand NanoSafer, where the information requirements exceed requirements for exposure assessmentunder REACH and CLP. Only the US Control Banding Nanotool was specifically developed to supportsafety assessment during innovation. Further harmonization and technical development of the modelsare required to support SbD approaches (NANOREG2) as well as risk innovation governance frameworkas proposed in the EU H2020 caLIBRAte project (www.nanocalibrate.eu).ReferencesLiguori B., Hansen S.F., Baun A., and Jensen K.A., 2016. Control banding tools for occupational exposure assessment ofnanomaterials — Ready for use in a regulatory context? NanoImpact, 2, 1 17. http://dx.doi.org/10.1016/j.impact.2016.04.002

P65Regulatory adequacy of ecotoxicity data for risk assessment of nanomaterials –the NanoCRED framework

Nanna B. Hartmann1, Marlene Ågerstrand2, Holten Lützhøft H.C.1, Anders Baun1

1Technical University of Denmark, Denmark; 2Stockholm University, Sweden; [email protected]

Environmental hazard and risk assessment is the foundation for regulatory decisions to protect theenvironment from unintended adverse effects caused by chemical substances including nanomaterials.The risk assessment process requires relevant and reliable environmental hazard data upon whichPredicted No Effect Concentration (PNEC) values can be estimated and upon which classification andlabelling can be based. In a regulatory context ecotoxicological data is often considered more valid forregulatory use if obtained according to accepted and validated test guidelines, preferably also followingGood Laboratory Practice (GLP). It is known, however, that engineered nanomaterials behave verydifferently in ecotoxicity tests when compared to the ‘conventional’ soluble chemicals, for which mosttest guidelines were developed. Therefore non standard tests, or tests following modified testguidelines, can provide valuable information on nanomaterial hazards and should not per se beconsidered less reliable. To support expert judgement and facilitate a transparent evaluation ofavailable ecotoxicity data for nanomaterials, we propose twenty criteria for evaluation of ecotoxicitydata reliability. These criteria take into account the testing challenges and characterisationrequirements associated with nanomaterial ecotoxicity testing. The criteria were developed to be usedin combination with the method developed through the ‘Criteria for Reporting and EvaluatingEcotoxicity Data (CRED)’ project. Combining criteria for evaluation data relevance with criteria for datareliability an overall evaluation of data adequacy can be made. This approach was developed toaccommodate all types of nanomaterials, all types of aquatic ecotoxicity studies and support qualitativeas well as quantitative data evaluation requirements. Furthermore, it is intended to be practicallyfeasible to implement and directly applicable in European as well as international regulatoryframeworks.


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P66Understanding occupational safety needs concerning handling of nanomaterials and the development of good practice guidelines

Marie Louise Kirkegaard, Pete Kines, Keld A JensenNational Research Centre for the Working Environment, Denmark; [email protected]

A wide range of manufacturing industries, marketing , research and educational institutions use nanomaterials and products. The emerging knowledge of nano hazards combined with the large range ofoccupational ‘users’ has led to different risk perceptions, risk control strategies and risk assessmenttools. As a part of the EU H2020 project caLIBRAte, this sub study will investigate the identification andperception of nano risks and the formal and informal introduction and instruction practices forhandling nano materials. The results will be used to develop a ‘good practice’ guideline for effectiveintroduction, instruction and follow up on the safe handling of nanomaterials.

The principle of focusing on culture to understand and influence safety conditions and behaviour isbased on safety culture maturity models, leadership communication training, and the Vision Zerostrategy for occupational health, safety and well being. For the analysis, the industries will be dividedinto three groups: Research, manufacturing and marketing to create meaningful analytical entities andto create source specific out put. Key informants will be interviewed, including OSH coordinators,representatives, sales personnel, managers and key employees. The interviews will exploreunderstandings of risk, risk attitudes, risk management (including policies and procedures), emergingrisks, and risk communication.

Nano OSH policies, good practice tools, brochures, information and instruction materials to newemployees etc. will be collected along with the interviews to form the basis for the ‘Good practice’guidelines. These guidelines can be used in the research community, manufacturing and marketing, tostrengthen their policies, procedures and practices in regards to the safe handling of nano materials. Inaddition, they will be used in risk communication within the organizations and to downstream users aswell as administrative and regulatory stakeholders. The guidelines will consist of generalrecommendations that are similar across the different groups, as well as a section withrecommendations directed at the specific needs and challenges of the group.

P67Risk assessment analysis on candidate nanomaterials at different stages of theinnovation change prior to Safe by Design implementation. First results fromNanoReg2 H2020 project (WP2)

Isabel Rodríguez Llopis1, Christian Micheletti2, Nicklas R Jacobsen3, Rambabu Atluri3, Raquel Puelles4,Alegria Caballero4, Erica García Heras5, Mariada Malvindi6, Stefania Sabella7, Eldara Rodríguez Cobo8,Yohan Oudart9, Federico Benetti2, Jean François Perrin9, Cesar Merino5, Blanca Suarez Merino1

1Gaiker Technology Centre, Spain; 2ECAMRICERT, Italy; 3NRCWE, Denmark; 4Avanzare, Spain; 5Grupo Antolin, Spain; 6HiQ Nano,Italy; 7IIT, Italy; 8NanoGap, Spain; 9Nanomakers, France; [email protected]

The NanoReg2 project builds from the challenge to couple Safe by design (SbD) to regulation taking intoaccount three main pillars: safe product, safe production and safe use, all addressed in the innovationchain. The aims of this exercise are two fold; on one hand to provide regulators with a set of tools which


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are flexible enough to deal with a rapidly diversifying market, and secondly to provide industry with asystem which could be rapidly implemented in a cost effective manner.

To put to the test this approach, the NanoReg2 project has designed seven case studies where SbD isbeing implemented. An initial step was the identification of hot spots for SbD and gaps in knowledgethrough the application of risk assessment tools to the selected case studies. The work presented hereintroduces 1) the description of the case studies in terms of production processes and materials and theformulation of the potential exposure and hazard profiles for workers, professional users, consumers,and the environment, 2) the comparison of the different tools for the risk assessment, 3) the strategybehind selection of risk assessment tools adapted to the particular cases 4) the results of the exerciseand lessons learnt and 5) the plan to cover knowledge gaps prior to the implementation of SbD. Ingeneral, not all the data required by the tools were available and, in these instances, data derived fromthe literature or the bulk material were used to feed the tools. Knowledge gaps identified in all casestudies included biopersistance, exposure assessment information, dustiness and toxicological reactions(generation of ROS species and inflammation). The main hotspots identified were, in general, theuse/handling of nanomaterials in powder form, and 2) chemical composition including toxic substances(eg. Cadmium, Silver). Environmental release was also a potential issue during disposal of wastes and, insome cases, due to accidental dispersion inside the working environment. Hot spots during theinnovation chain susceptible for SbD implementation have been identified.

P68Identifying criteria for environmental risk assessment models at different stagegates of nano material/product innovation considering requirements of variousstakeholders

Sara N Sørensen1, Steffen F Hansen1, Anders Baun1, Dave Spurgeon2, Marianne Matzke2, KristinSchirmer3, Michael Burkard3, Miikka Dal Maso4, Mikko Poikkimäki4, Anja Verschoor5, Joris Quik5,Willie Peijnenburg5, Henning Wigger6, Bernd Nowack61Department of Environmental Engineering Technical University of Denmark; 2Centre for Ecology and Hydrology; 3SwissFederal Institute of Aquatic Science and Technology; 4Tampere University of Technology; 5Netherlands National Institute forPublic Health and the Environment; 6Swiss Federal Laboratories for Materials Science and Technology; [email protected]

The EU H2020 project “calibrate” overall aims to establish a state of the art versatile risk governanceframework for assessment and management of human and environmental risks of manufacturednanoparticles (MN) and MN enabled products applicable throughout the innovation process (stagegates) for these materials and products.

Initial efforts have focused on identifying criteria for environmental risk assessment (ERA) models andtools for such governance framework. It was recognized that some criteria are applicable to bothenvironmental and human risk assessment (HRA), and these so called “overall” criteria were identifiedthrough joint efforts of the ERA and HRA working group experts in caLIBRAte. The identified “overall”criteria relate to RA model features and resources needed to use the tools, whereas the criteria specificto ERA models relate to model outcome on hazard, exposure and risks

The identified criteria were listed against the Cooper stage gates®, thus forming a table in which theimportance or relevance of each criterion could be assessed for each of the stage gates. This wasformed into questionnaires with defined response options for each criterion and stage gatecombination, such as picklists and ratings of importance of the criterion. These questionnaires weresent to stakeholders representing regulators, consultants, researchers and industries, who provided


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their feedback, either by filling the questionnaires or by listing general input on their current RAapproaches or needs. Efforts to obtain input from NGOs and insurers remain ongoing.

The feedback clearly illustrated different requirements between stakeholder groups. For example, notall use the (same) stage gate approach or have the same level of expertise for RA. Other criteria weresimilar or similarly important to most stakeholders. For example, the middle stage gates are reported asessential for RA issues with regulatory compliance being the main driver. Criteria suggested useful forusers by caLIBRAte partners included the use of modeling/estimations and safety by designconsiderations as low cost options to identify “red flags” for hazard and/or exposure at early stagegates of MN innovation.

The criteria and stakeholder feedback generated will be applied to evaluate existing models/toolsagainst, but also to enable the creation of a “System of systems” for RA along stage gates whendeveloping MN and MN enabled products, incorporating the needs of different specific user groups.


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ABSTRACTS - NanoValid · Safe use of nanomaterials has been subject to intense research activities...

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