kemikalieinspektionen.se

Nanomaterials upptag och spridning i kroppen och miljön

Svenska myndigheter och nanomatrial

PM 3/15

Kemikalieinspektionen är en myndighet under regeringen. Vi arbetar i Sverige, inom EU och internationellt för att utveckla lagstiftning och andra styrmedel som främjar god hälsa och bättre miljö. Vi har tillsyn över reglerna för kemiska produkter, bekämpningsmedel och ämnen i varor och gör inspektioner. Vi granskar och godkänner bekämpningsmedel innan de får användas. Vårt miljökvalitetsmål är Giftfri miljö.

© Kemikalieinspektionen, Stockholm 2015.

Artikelnummer: 511 153.

Förord Kemikalieinspektionen har på uppdrag av regeringen tagit fram en handlingsplan för en giftfrivardag Handlingsplan för en giftfri vardag 2011– 2014 – Skydda barnen bättre. Insatser sker nu på flera områden både nationellt, inom EU och internationellt och ofta i samarbete med andra myndigheter. Att minska kemiska risker i vardagen är ett steg på vägen att nå riksdagens miljökvalitetsmål Giftfri miljö – det mål Kemikalieinspektionen ansvarar för. Inom ramen för handlingsplanen tar KemI fram sammanställningar som publiceras i Kemikalieinspektionens rapport respektive PM-serie. Publikationerna, som är kostnadsfria, finns på webbplatsen www.kemikalieinspektionen.se

I Kemikalieinspektionens handlingsplan för en Giftfri vardag 2011-2014 framhålls behovet av insatser för att nå en hög skyddsnivå för eventuella hälso- och miljörisker orsakade av nanomaterial. Som ett led i arbetetet sammankallades myndigheter/statliga aktörer vars verksamheter är berörda av nanoteknik och nanomaterial till ett fjärde möte för kunskaps- och erfarenhetsutbyte samt diskussion. Myndighetsmötet ordnades denna gång i samarbete med Nordiska Nanomaterial Gruppen sponsrat av Nordiska Kemikalie Gruppen (NKG). Mötet bestod av en gemensam workshop på förmiddagen och ett myndighetsmöte med nordiska deltagare på eftermiddagen.

Föreläsarna har själva bidragit med sammanfattningar från sina presentationer. I detta PM ingår bidrag från Kungliga Tekniska högskolan, Danska Miljöstyrelsen, Totalförsvarets forskningsinstitut, Lunds Universitet, representanter för OECD och EU kommissionen, Läkemedelsverket, Kemikalieinspektionen och Miljödepartementet.

Samordning av arbetet och sammanställning från mötet har gjorts av Lena Hellmér, Kemikalieinspektionen. Ansvarig för arbetet var Lisa Anfält, chef för enheten EU koordinering på Kemikalieinspektionen.

Innehåll Inledning ............................................................................................................ 5

Summary ........................................................................................................... 6

1 Sammanfattningar från föredragen ..................................................... 9 Nanomaterials in a Life Cycle Perspective .................................................................................... 9 Dermal absorption of nanomaterials ............................................................................................. 9 Interactions of nanoparticles with organs protected by internal biological barriers ..................... 10 Measurement Techniques for Airborne Nanoparticles ................................................................ 11 Effekter efter inandning av metalloxider i friska och känsliga individer ....................................... 12 Nanosäkerhet ur ett EU- och OECD perspektiv .......................................................................... 13 Update on NM projects for the method, guidance and testing strategy development in the OECD

Test Guidelines programme .......................................................................................... 14 Nytt om nano från Kemikalieinspektionen ................................................................................... 15 Nanomaterial i kosmetiska produkter – vad har hänt sedan de nya reglerna infördes? ............. 15 Danske aktiviteter vedr. nanomaterialer ...................................................................................... 16 Nanocellulose and NANoREG- project ....................................................................................... 17

2 Bilagor ................................................................................................. 19 Bilaga 1. Deltagarlista .................................................................................................................. 19 Bilaga 2. Föreläsarnas power-point presentationer .................................................................... 21

Inledning Detta PM är en sammanställning av det fjärde myndighetsmötet om nanoteknik och nano-material Syftet med mötet som redovisas i detta PM var att få kunskap om aktuell forskning och de frågor som svenska och statliga aktörer arbetar med inom nanoområdet samt att fortsätta utveckla myndighetsnätverket.

Denna gång deltog även forskare och myndighetsrepresentanter från övriga Norden. En inbjudan till myndighetsmötet skickades till samtliga departement inom Regeringskansliet och de statliga myndigheter eller offentliga finansiärer som bedömdes ha aktiviteter eller intressen med anknytning till nanomaterial och nanoteknik i Sverige. Totalt deltog 49 personer från 24 aktörer på seminariet den 27 november 2014.

Dessa var Bioforsk, Danmarks tekniska universitet, Europeiska kommissionen, Finska Arbetshälsoinstitutet, Totalförsvarets forskningsinstitut (FOI), Formas, Försvarets materiel-verk, Försvarsmakten, Generalläkaren, Kemikalieinspektionen, Kommerskollegium, Kungliga Tekniska högskolan (KTH), Livsmedelsverket, Lund universitet, Läkemedelsverket, Natur-vårdsverket, Norwegian institute for water research, norska Miljødirektoratet, OECD sekretariat, Regeringskansliet, SINTEF Materials and Chemistry, Socialstyrelsen, Danska miljöstyrelsen och Trafikverket.

Workshopen/seminariet hölls omväxlande på engelska och ”skandinaviska” och därför varierar språket även i rapporten. Som moderator för mötet fungerade Gregory Moore från Kemikalieinspektionen.

Introduction This PM is a compilation of speeches made at the fourth network meeting with authorities on nanotechnology and nanomaterials. The purpose of the meeting accounted for in this PM was to acquire knowledge about current research and topical questions at Swedish and government authorities within the nano area and to continue developing the authority network on nanotechnology and nanomaterials.

Participants at this meeting were researchers and authority representatives from the Nordic countries and the invitation was sent to all Swedish ministries and official financiers estimated to carry out activities or having interests in nanotechnology and nanomaterials in Sweden. 50 participants from 26 actors participated at the seminar on 27 November 2014, which was held alternately in English and a Scandinavian language and that is why the language varies in the report too. Moderator of the meeting was Dr. Gregory Moore, the Swedish Chemicals Agency.

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Summary David Lazarevic, from KTH gave an introductory lecture on Nanomaterials in a Life Cycle Perspective. Engineered nanomaterials are being used in a growing number of products. The assessment of the impacts, or benefits, upon human health and the environment should be conducted from a life cycle perspective. However, current assessments are inadequate due to the lack of data on the emissions from engineered nanomaterials and nanoparticles during the production, use and disposal phases of a product life cycle, and the lack of characterisation factors for nanomaterials in life cycle impact assessment models.

From the Danish EPA Anne Mette Zenner Boisen, presented two projects on dermal absorption of nanomaterials. The literary project from 2013 performed a scientific appraisal of the reliability and relevance of available studies on dermal absorption using Klimisch criteria and nanomaterial characterisation, database available from the Danish EPA. Recommendations include that study designs (in vivo and in vitro) should consist of a sufficient post-exposure duration to account for potential lag-times of dermal absorption. An experimental project on dermal absorption of TiO2 and ZnO in a realistic exposure scenario using in vitro and in vivo test methods is ongoing and will be published in 2015.

Anders Bucht introduced us to the area of interactions of nanoparticles with organs protected by internal biological barriers. Inhalation of nanoparticles leads to surface adsorption of biomolecules in the lung lining fluid that influence responses of cells in the lung epithelium and uptake in the body. When translocated into the blood circulation a corona of blood plasma proteins will be formed which can activate the contact dependent coagulation system. This activation may elicit tromboinflammation, blood clot formation, as well as activate innate and adaptive immune responses.

The important topic of Measurement Techniques for Airborne Nanoparticles was presented by Jakob Löndahl, Lund University. A major pathway for exposure to nanoparticles is through the air during breathing. Thus, it is necessary to have adequate monitoring techniques that ideally are able to separate engineered nanoparticles from the background. There is a range of instrumental options for measurement of airborne nanoparticles, but often it is necessary to choose between simplicity and accuracy (or high time resolution and relevant particle characteristics). It is still also difficult to measure relevant exposure metrics such as only the engineered nanoparticles. However, new methods are emerging that might overcome this problem at least partly. Åsa Gustafsson held a presentation about her thesis entitled ”Nanomaterials, respiratory and immunological effects following inhalation of engineered nanoparticles”. It shows that exposure to nano sized titanium dioxide (TiO2) induced long-lasting immune response in the airways. The long-term activation of the immune system could potentially trigger the development of diseases. The particles that were deposited in the alveolar region were also retained in the lung for a long time, up to three months after exposure. By comparing different inbred rat strains it was demonstrated that genetically determined factors influence the immune and respiratory responses to TiO2. An important issue of this thesis was to study the responses in sensitive individuals. These sensitive individuals were represented by groups of animals with an allergic airway inflammation or by rat strains that were genetically susceptible to inflammatory disorders. One study showed that when the mice were exposed to particles and an allergen during the same period, a decline in general health was observed. Altogether, this thesis emphasises the complexity of assessing health risks associated with nanoparticle exposure and the importance of including sensitive populations when evaluating adverse health effects of ENMs.

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Eva Hellsten, former Head of the Chemicals unit at the European Commission, vice president in the OECD WPMN, and member of the Swedish Nano Commission, informed about nanosafety from an EU and OECD perspective and her experiences in the area. Within the EU, work on health and environment safety aspects of nanomaterials started in 2004. Via the EU research programmes, 25 million Euros have been invested each year in this area. The EU legislation has been reviewed to analyse in which ways legislation needs to be adapted to better cover nanomaterials. For legislative purposes adaptation of existing test methods is needed, e.g. testing of (eco)toxicology, exposure and measurements. Work to ensure that test methods are standardised and internationally harmonised is on-going within the OECD WPMN since 2006. In Sweden, a Government Nano Commission was initiated in 2012. The conclusions from this investigation (SOU 2013:70) stresses the importance of additional Swedish efforts in participating in EU and OECD work, as well as improved co-ordination and communication between Swedish authorities, academia industry and civil society. A Nano Council, with a Nano Centre as secretariat, was proposed to be established for this purpose.

Jukka Ahtiainen, presently Senior researcher at the OECD secretariat continued with the topic and gave an update on NM projects for the method, guidance and testing strategy development in the OECD Test Guidelines programme. The OECD test guidelines for testing chemicals have been widely used for regulatory purposes all over the world since the establishment of the MAD principle in 1984. This Mutual Acceptance of Data ensures that if a chemical is tested under the GLP conditions according to an OECD Test Guideline, the data should be accepted in all OECD countries. The rationale behind this agreement is to save resources and avoid especially duplicate vertebrate testing. Eventually the OECD test methods are referred to or taken into the national chemicals legislation such as the test method regulation (440/2008/EU) in the European Union.

Sofia Tapper from the Swedish Ministry of the Environment and Energy informed among other things about the Nano Commission and an action plan for safe use and handling of nanomaterials. The new Swedish government has shown interest in the commission and its conclusions and intends to continue pressing the European Commission for the importance of adequate EU-rules on nano materials. Work on establishing a possible Nano Centre or Nano Council continues.

Elin Simonsson, the Swedish Chemicals Agency, briefly presented ongoing work at the agency on nano materials, i.e. amendments of REACH annexes, overview of the EU Commission recommended definition of nanomaterials and its survey of consequences with regard to measures for increased transparency of nanomaterials on the market.

Tomas Byström, the Swedish Medical Products Agency, informed about the rules on nanomaterials in cosmetic products, which came into force in 2013. Some regulatory support is, however, lacking with regard to approved substances in nano form. Within the EU; 25 000 cosmetic products and nanomaterials have been notified. The European Commission will within short publish a catalogue listing nanomaterials used in cosmetic products.

Flemming Ingerslev, gave an account of the activities of the Danish Environmental Protection Agency

- 6 million DKK have been allocated per year during 2012-2015 to ”Bedre styr på nano”, with the overall aim to get an overview of where in Denmark there may be a risk to consumers and the environment from the use of nano products.

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- One part of the effort included to set up a register of consumer products releasing nanomaterials. Danish manufacturers have to report their production and import of nano products aimed for consumers to this register.

- A number of other projects are also included in the work, from which reports are published continuously.

- Denmark also contributes to the OECD work on developing ecotoxicological methods for testing nano materials.

How can authorities make use of the results of nano research and how can authority work have an effect on research?

Jukka Ahtiainen opened the discussion by presenting the Finish participation in the NANoREG project.The safety of nanomaterials is investigated in the EU’s just-beginning extensive NANoREG project as a joint effort by the authorities and the industry. Finland’s goal in the project is to continue to study the safety of microfibrillar and nanofibrillar cellulose. In Finland, participation in the NANoREG project is coordinated by the Finnish Safety and Chemicals Agency (Tukes) that is also responsible for oversight and guidance concerning the REACH regulation. Also included in the project are the Finnish Institute of Occupational Health, responsible for the experimental in vitro and in vivo studies, and Stora Enso and UPM as a joint Nordic Cellulosa consortium.

Finland’s national research portion of the Nanoreg project focuses on microfibrillar and nanofibrillar cellulose materials that have several potential industrial applications in different products. Microcellulose and nanocellulose comprise wood fibre and fibre bundles originating from wood cellulose. The intention is to study the safety of biodegradable microcellulose and nanocellulose experimentally by means of biological testing. This is important in order for it to be possible to use nanocellulose, for example, as raw material for cosmetics, food additives or packaging.

The ensuing discussion raised the cooperation between the OECD and NANoREG, the design of a possible Nano centre in Sweden and ways in which Nordic colleagues treat different aspects of nanomaterials. In conclusion, it was agreed that a combination of a Nordic workshop and authority meeting was fruitful, particularly with respect to the discussions. The moderator extended his thanks to speakers and participants.

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1 Sammanfattningar från föredragen

Nanomaterials in a Life Cycle Perspective David Lazarevic, PhD, Researcher, KTH

Nanotechnology and nanomaterials have been promoted as having the potential to bring benefits to many areas of research, and to positively contribute to sustainable development. As such, this rapidly growing field is increasing attracting investments from governments and businesses worldwide. However, it is also recognised that engineered nanomaterials may pose a risk to human health and the environment.

There is a general consensus that the potential health and environmental risks of engineered nanomaterial should be evaluated over their entire life cycle. Life cycle assessment is one tool which has been promoted for such an evaluation. This work reviewed the literature on the application of life cycle assessment to engineered nanomaterials to identify current research and difficulties in applying life cycle assessment in this field.

Twenty five LCA studies of nanomaterials were identified, including nanomaterial such as cadmium telluride, calcium carbonate, carbon black, carbon nanofibres, carbon nanotubes, nanoclay, nanoscale platinum-group metals, silica, silver, silicon, titanium and titanium oxide. Product systems studied include: auto-body panels, biopolymers, coatings, electronic displays, electronic sensors, lithium-ion batteries, photo voltaic systems, packaging and agriculture polymer films, nanomaterial production processes, textiles and wind turbine blades. These studies only looked at parts of the life cycle, with no quantitative studies addressing the impact of nanomaterials to human health and the environment from the cradle to the grave. Results from these studies showed the potential for a significant cumulative energy demand in the production of nanomaterials such as carbon nanotubes and carbon nanofibres. However, this is reduced when taking into consideration the small amounts of nanomaterials in products and the potential benefits during the use phase, such as weight reduction.

Due to the different properties and functions of engineered nanomaterials when compared to conventional materials and products, special attention is required during the goal and scope definition phase in order to obtain meaningful results. The life cycle inventories of current LCA studies cannot be classified as comprehensive as they often lack nanomaterial specific data related to the outputs of processes. Hence, populating life cycle inventory databases with nanomaterial specific information, such as size and shape, is of critical importance. Although the UNEP/SETAC framework for toxic impacts can in principle be used for specific impacts causes by nanoparticles, life cycle impact assessment methods currently lack characterisation factors for the release of nanoparticles indoors and outdoors. Hence, no LCA studies to date have considered the human toxicity and eco-toxicity of nanomaterials from a life cycle perspective with consideration of the nano-specific properties.

Dermal absorption of nanomaterials Anne Mette Zenner Boisen,PhD Danish Ministry of the Environment

Under the Danish Nano Initiative ‘Better Control of Nano’ the Danish Environmental Protection Agency (EPA) has initiated a series of projects with the aim of further clarifying possible risks to consumers and the environment from nanomaterials. Two of these projects investigating dermal absorption of nanomaterials were presented at the workshop on 27th November 2014.

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The first, Danish EPA Environmental Project No. 1504, 2013 was a literary project conducted in 2013 by the Institute of Occupational Medicine IOM, UK. The project performed a scientific appraisal of the reliability and relevance of available studies on dermal absorption using Klimisch criteria and nanomaterial characterization (as described in Card and Magnuson 2010). The appraised studies are available in a database, which can be found in a link within the project report (original version). A link to an updated version of the database, which included more studies were also performed in 2013 and can be found on the Danish EPA website (mst.dk). This update did not change any of the conclusions found in the project report. Results from the project showed that a very low dermal absorption of nanomaterials is possible in some cases; Nano-specific characteristics that may influence dermal absorption include size, shape and surface chemistry; test method(s) which most closely simulate the transport of nanomaterials through human skin are evaluated in the report and research gaps concerning dermal absorption of nanomaterials are described. It has been noted in several studies that there can be a considerable lag time between application of a test substance and appearance in the circulation. Gulson et al. 2010 noted a lag time of ~30 h before the first detection of 68Zn in the blood and urine of human volunteers. Therefore one recommendation from the project is that study designs (in vivo and in vitro) should consist of a sufficient post-exposure duration to account for potential lag-times.

The second project, which is experimental is ongoing and will be concluded in the summer of 2015. This project is conducted by Aarhus University in Denmark. The main goal of this project is to investigate if TiO2 and ZnO nanoparticles are able to penetrate the skin in a realistic exposure scenario. Sun screen is used as the vehicle and 2.5% nanoparticles by weight are added to the sunscreen. The Main goal of the project is to investigate if TiO2 and ZnO nanoparticles are able to penetrate the skin in a realistic exposure scenario. Nano-specific characteristics investigated in the project are size and coating. Test methods include: The in vitro Epiderm model; An in vivo mouse inflammation model and an in vivo mouse xenograft transplantation model with human skin. The results from this project will be published in peer-reviewed scientific articles and in a Danish EPA project report in 2015.

Interactions of nanoparticles with organs protected by internal biological barriers Anders Bucht, Division of CBRN Defence and Security, Swedish Defence Research Agency, and Department of Respiratory Medicine, Umeå University.

The widespread exploitation of new types of nanoparticles in a variety of industrial applications and cosmetics has raised concerns about toxicity when dispersed in occupational and public environments, especially when humans are exposed by the inhalation route. We and others have demonstrated cellular uptake of titanium dioxide nanoparticles (TiO2 NP) when exposed to lung epithelial cells, and that the NP interactions with the cells results in inflammatory responses and oxidative stress (1 2). The ability of NPs to translocate intracellularly and activate cells is, however, highly dependent on particle properties (e.g. size, crystal structure surface chemistry and agglomeration), cell culture conditions and type of responder cells. For example, addition of serum proteins to cell cultures will greatly influence the responses to the NPs, most likely due to formation of a protein corona on the surface of the particles. The composition of biomolecules adsorbed on the surface will

1 Ekstrand-Hammarström B, Akfur CM, Andersson PO, Lejon C, Österlund L and Bucht A. Nanotoxicology 2012, 6:623-634 2 Andersson PO, Lejon C, Ekström Hammarström B, Akfur C, Bucht A and Österlund L. Small 2011,7:514-23

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gradually shift when particles translocate into the cells, i.e. when exposed through the airways the NP corona will initially consist of components of the lung lining fluid and after cellular uptake biomolecules from the cell interior will attach to the particles. When NPs are further translocated into the blood circulation, a corona of blood plasma proteins will be formed, including components of the complement and the coagulation systems.

Detailed analysis of adsorbed plasma proteins bound to TiO2 particles after incubation with human plasma has shown enrichment in proteins of the contact dependent coagulation system on the surface. Using a whole-blood model utilizing fresh non-anticoagulated human blood, it was shown that TiO2 NPs at very low concentrations (50 ng/mL) induce strong activation of the contact coagulation system, which in this model elicits thromboinflammation and blood clot formation (3). These data are in line with the finding of components of the contact system in the protein corona of the TiO2 NPs after exposure to blood. From that study it was concluded that TiO2 NPs, generally considered to be relatively harmless, are highly thrombogenic when they enter the body and cross epithelial and endothelial borders. Such activation may potentially induce immune activation, inflammation and tissues damage in vivo.

Measurement Techniques for Airborne Nanoparticles Jakob Löndahl, Div. of Ergonomics and Aerosol Technology, Lund University

The rapidly growing nanotechnology sector has resulted in an increasing need to understand health risks associated with exposure to the new materials emerging. A major pathway for exposure is through the air during breathing. To investigate, control and limit airborne exposure to nanoparticles it is necessary to have adequate measurement techniques. Such techniques should ideally be able to separate engineered nanoparticles from the background and to measure the most relevant particle properties. Relevant particle properties may for instance be size, shape, surface area, chemical composition, solubility and biological activity.

Among the easiest real time techniques to use are the instruments based on electrical charge. These measure particle number and/or surface area with reasonable accuracy, but are generally not able to sort out particles smaller than 100 nm. More advanced instruments include for instance the scanning mobility particle sizer (SMPS), the electrical low pressure impactor (ELPI), the aerosol particle mass analyser (APM) and the aerosol mass spectrometer (AMS). The latter being the most informative in terms of time resolved chemical composition with simultaneous particle size distribution. To measure certain particle properties it is necessary to perform sample collection with subsequent analysis by for instance electron microscopy, x-ray, PCR etc.

There is a range of possibilities to measure a wide range of properties of airborne (nano)particles. However, due to instrumental limitations and cost effectivity it is often necessary to choose between simplicity and accuracy (or high time resolution and relevant particle characteristics). It is still also difficult to measure relevant exposure metrics such as only the engineered nanoparticles, but new methods are emerging.

3 Ekstrand-Hammarström B, Hong J, Davoodpour P, Sandholm K, Ekdahl KN, Bucht A, Nilsson B. Manuscript.

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Effekter efter inandning av metalloxider i friska och känsliga individer Åsa Gustafsson, Forskningsingenjör PhD student, FOI

Syftet med denna avhandling har varit att få en mer detaljerad förståelse av lungornas och immunsystemets påverkan efter inandning av nanopartiklar. En viktig del av arbetet har varit att skapa en förståelse om hur känsliga grupper i samhället påverkas av nanopartiklar. I detta arbete representeras speciellt känsliga individer av försöksdjur med inducerad allergisk luft-vägsinflammation samt av djur med särskilt stor benägenhet att utveckla inflammatoriska sjukdomar. Dessa exponeringar är tänkta att efterlikna de som förekommer i arbetsmiljöer vid framställning av sådana nanomaterial. I denna avhandling har friska möss och råttor samt djur med en allergisk luftvägsinflammation andats in nanopartiklar av titandioxid eller järnoxid varefter de respiratoriska, inflammatoriska och immunologiska svaren studerats.

Studierna visade att kroppen har svårt att göra sig av med titandioxidpartiklar som hamnar i lungblåsorna. Partiklarna inducerade en tidig ökning av celler i lungan redan efter 1 dag, och fortfarande efter tre månader kunde en förhöjd ökning av inflammatoriska celler observeras i lungblåsorna. Histologisk analys visade att det även fanns partiklar kvar i lungvävnaden. De djur som har en genetisk benägenhet för autoimmun-liknande sjukdomar utvecklade ett kraftigare immunologiskt svar efter partikelexponering jämfört med de djur som har medfödd benägenhet för allergiska sjukdomar. Djur med en allergisk luftvägsinflammation fick inga förvärrade andningsbesvär eller förvärrade inflammationer i lungan efter partikelexponering. Däremot var cellsammansättningen i lungan annorlunda jämfört med de allergiska djuren som inte fick partiklar. Dessutom påverkade tidpunkten för partikelexponeringen det inflamma-toriska och immunologiska svaret i djuren beroende på om de ges vid allergensensibilisering eller senare då redan sensibiliserade djur utsätts för allergenet på nytt.

Studien av järnoxidexponeringar visade att allergiska och friska möss som fick partiklarna i lungorna fick helt olika inflammatoriska svar. De friska mössen utvecklade en inflammation i lungan och i de lymfkörtlar som dränerar lungorna. Däremot observerade vi färre inflamma-toriska celler hos möss med en pågående allergisk luftvägsinflammation en dag efter expo-nering för partiklar. Minskningen kunde även noteras i både luftvägar och lymfkörtlar. Cell-minskningen kan bero på att lungor har förhöjda nivåer av fria syreradikaler vid pågående inflammation samt att järnoxid kan generera ytterligare reaktiva syreradikaler. Detta till-sammans kan leda till ökad oxidativ stress som i sig kan leda till celldöd.

Studierna visade att titandioxidpartiklar ligger kvar i lungblåsorna under lång tid samt att en långvarig aktivering av immunsystemet kan uppstå vid lungexponering för nanopartiklar. En sådan immunaktivering skulle kunna leda till utveckling av immunmedierade sjukdomar. I råtta visades att nedärvda faktorer har betydelse för hur immunsystemet aktiveras efter inand-ning av titandioxidpartiklar. Allmäntillståndet hos allergiska möss försämrades efter titan-dioxidexponering men detta observerades inte i allergiska råttor. Däremot kunde en ökning av neutrofiler konstateras i möss och den råttstam som är benägen för autoimmuna sjukdomar. Den stora skillnaden mellan friska och allergiska djur vid lungexponering för nanopartiklar pekar på hur viktigt det är att inkludera känsliga individer vid hälsoriskbedömning av nanomaterial.

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Nanosäkerhet ur ett EU- och OECD perspektiv Eva Hellsten, tidigare avdelningschef vid EU-kommissionen, vice ordf i OECD WPMN samt medverkan i svenska Nanoutredningen

EU-kommissionen publicerade 2004 ”Towards an European Strategy for Nanotechnology” som framhöll nödvändigheten av att studera nanomaterialens miljö- och hälsoeffekter i samband med planerat utökat forsknings- och innovationsstöd till nanoteknologi via EU:s ramforskningsprogram. Året efter publicerades ”EU Action plan for Nanotechnology 2005-2009” som listar ett stort antal åtgärder inom forskning, etik, lagstiftning, internationellt samarbete etc. som EU-kommissionen och medlemsstaterna borde genomföra för att säkerställa en ”säker, integrerad och ansvarsfull” utveckling av nanoteknologi. Aktionsplanen slutredovisades av EU-kommissionen i en skrivelse 2009.

Forskningssatsningarna har ökat radikalt i EU:s ramprogram sedan år 2000. Genom sjunde ramprogrammet har totalt flera miljarder Euro satsats. Vad gäller miljö- och hälsoforskningen ligger budgeten på cirka 25 miljoner Euro per år. Detta utgör mellan 5 och 10 % av den totala nano-budgeten (uppgifter från kommissionen varierar beroende på hur den totala nano-forskningen definieras inom ramprogrammen).

Nano-området kräver långtgående samordning mellan olika politikområden. Inom EU-kommissionen skapades en horisontell grupp med representanter från forskning, industri, miljö, konsumenthälsa och arbetsmiljö tidigt i processen för att koordinera åtgärder av olika slag, främst samordna lagstiftning med satsningar på forskning och kunskapsuppbyggnad om nano-säkerhet. Ett flertal medlemsländer har skapat statliga tvärgående organisationer för att säkerställa samordning på nationell nivå, inte minst när det gäller landets ställningstagande i olika nano-relevanta EU-frågor.

EU-kommissionen har genomfört två översyner av lagstiftningen för nano-säkerhet (2008 respektive 2012). Kemikalielagstiftningen, Reach, är den viktigaste lagen för att se till att grundläggande information om potentiella risker tas fram. Dock krävs en anpassning av de metoder som används för ”vanliga” kemikalier till nanomaterial. Arbete med detta har därför pågått sedan ett antal år inom EU och internationellt. I arbetet har OECD en nyckelroll genom att länder världen över här enas om internationella standarder för testning inom kemikalie-området. År 2006 etablerades OECD Working Party on Manufactured Nanomaterial (OECD-WPMN) i vilket EU:s medlemsstater, EU-kommissionen, USA, Kanada, Australien, Japan, Korea m.fl. arbetar tillsammans för att se över hur testmetoder för kemikalier ska anpassas till att gälla även för nanomaterial. Även om lagstiftningen kan skilja sig åt mellan olika delar av världen bör de underliggande vetenskapliga testerna av farlighet inte ge olika resultat beroende på var i världen de är utförda.

Det svenska regeringsuppdraget att utveckla nationell handlingsplan för nanosäkerhet samt säkerställa en god samordning av det nationella arbetet med nanosäkerhet redovisades i oktober 2013 (SOU 2013:70, www.regeringen.se ). Rapporten ger en bred översikt av området. Huvudförslagen innebär en ökad satsning på forskning om säkerhetsaspekter, ökade insatser inom EU och OECD samt en samordning av svenska myndigheter, forskare och intressenter genom bildandet av ett Nanoråd med ett operativt sekretariat, Nanocentrum.

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Update on NM projects for the method, guidance and testing strategy development in the OECD Test Guidelines programme Jukka Ahtiainen, Senior researcher, OECD secretariat

Historically the test guidelines have been developed and validated to be used for hazard identification and risk assessment of various chemicals. But are these test guidelines applicable for the regulatory testing of nanoforms of a substances or chemicals? In principle, most of the existing "endpoints" or more precisely measurement variables are applicable. However, the dosage of the test material and the characterization of the exposure need specific guidance in order to gain regulatory relevant data. The Guidance on sample preparation and dosimetry has been developed in the OECD (OECD 2012). As testing has to be adapted, and in a worst case for testing of each type of nanomaterial, will this challenge also the principle of MAD?

The test conditions e.g. organic matter content during the test will affect the form and bioavailability of the nanomaterial, and detailed guidance is needed on the test conditions, Also, the conditions during the test should be documented carefully in order to achieve comparable and understandable results. The additional guidance for testing of nanomaterials with OECD test guidelines shall be seen as a refinement of the methods. This approach enables proper use of test guidelines and production of good quality data under the MAD principle for the regulatory purposes.

The way forward Currently the harmonized guidance on testing the fate and effects of nanomaterials is being developed at various stages. New harmonized and validated OECD test guidelines are needed especially for the physical-chemical characterization (e.g. size, shape, surface chemistry and charge, zeta-potential) as well as for physic-chemical interactions (dissolution of ions and dispersion stability) of nanomaterials. This basic knowledge of the nanomaterial under assessment is crucial to guide further (eco)toxicity and fate testing – which tests in which compartment are relevant, but also to better understand and interpret the results. The basic reactions and transformations of the nanomaterial for example in aquatic media should be tested at various conditions e.g. pH, ion strength and organic matter content.

Several OECD guidance documents are under development e.g. for aquatic toxicity testing (http://www.oecd.org/env/ehs/nanosafety). These comprise of decision frameworks based on the basic physical-chemical properties of the nanomaterial to be tested. One specific example in the environment fate testing is to develop technical guidance for dietary bioaccumulation of nanomaterials in fish using the OECD 305 test guideline.

There are regulatory needs for harmonized guidance for testing the effects and fate of nanomaterials, however at the same time OECD is also developing guidance for grouping and categorization of nanomaterials. The grouping of nanomaterials will be based on their chemical composition including coatings, size, shape, basic physical-chemical reactions and biological effects. This will hopefully help to understand some basic mechanisms of the effects and provide possibilities for read-across interpretation between some nanomaterials.

References

OECD 2012 Guidance on sample preparation and dosimetry for the safety testing of manufactured nanomaterials. Series on the Safety of Manufactured Nanomaterials No. 36, OECD Paris, France

14

Nytt om nano från Kemikalieinspektionen Elin Simonsson, Kemikalieinspektionen

Ändringar av bilagorna till EU:s kemikalieförordning Reach har planerats på att genomföras sedan 2013. Ändringarna är tänkta att anpassa informationskraven i bilagorna till nano-materials speciella egenskaper. Arbetet har blivit ytterligare försenat och tidigaste tidpunkten för ett förslag från Europeiska kommissionen är nu satt till sommaren 2015. Diskussioner förs i en av Europeiska kommissionens expertgrupper (CASG Nano).

En annan viktig fråga är Europeiska kommissionens rekommenderade definition som är avsedd att användas i lagstiftningssammanhang. Under 2014 har en översyn av definitionen pågått som förväntas bli färdig under 2015. Definitionen ska särskilt ses över med hänsyn till kravet på att minst 50 % av partiklarna ska vara inom nanoskalan (1-100 nanometer). Över-synen är uppdelad i en vetenskaplig del och en policy del. Den vetenskapliga delen består av tre rapporter från Europeiska kommissionens vetenskapliga gren (JRC). Policydelen kommer bl.a. bestå av ett offentligt samråd där det kommer att vara möjligt lämna synpunkter på hur definitionen bör se ut.

Den tredje frågan som är aktuell i EU just nu är Europeiska kommissionens konsekvensut-redning om åtgärder för ökad transparens på marknaden. Den åtgärd som främst diskuteras är att eventuellt införa ett EU-register för nanomaterial. Kemikalieinspektionen har svarat på ett offentligt samråd i frågan. Konsekvensutredningen färdigställs under 2015 varpå Europeiska kommissionen kommer att avgöra om och vilka åtgärder de tänker vidta.

Nanomaterial i kosmetiska produkter – vad har hänt sedan de nya reglerna infördes? Tomas Byström, Läkemedelsverket

Reglerna för kosmetiska produkter har ändrats från att vara EU-direktiv som implementerades nationellt till att vara en direkt gällande EU-förordning. Förordning (EG) nr 1223/2009 om kosmetiska produkter trädde i kraft i slutet av 2009, men den tillämpades fullt ut först den 11 juli 2013.

Bland de tydligaste nyheterna i EU-förordningen var särskilda regler för nanomaterial i kosmetiska produkter. Nanomaterial definieras som: ett olösligt eller biopersistent material som är avsiktligt tillverkat, med en eller fler yttre dimensioner, eller en inre struktur, med ett spann på mellan 1 och 100 nm.

De regler som gäller för nanomaterial i kosmetiska produkter är:

· Varje användning av nanomaterial måste anmälas till EU-kommissionen 6 månaderföre produkter släpps ut på marknaden

· EU-kommissionen avgör från fall till fall om den anmälda användningen behövergranskas av en vetenskaplig kommitté

Vissa ämneskategorier förhandsgranskade innan tillåtande (UV-filter, färgämnen, konserveringsmedel)

Övriga nanomaterial granskas specifikt för den anmälda användningen, anmälan ska innehålla uppgifter om nanomaterialets egenskaper

· Innehåll av nanomaterial måste framgå av innehållsförteckningen på förpackningen

Exempel: Titanium Dioxide (nano)

15

Sedan reglerna för nanomaterial började tillämpas fullt ut har det inom EU anmälts cirka 25000 kosmetiska produkter med nanomaterial. Vanligaste kategorierna är solskydds-produkter (7300 st.), sminkprodukter (7000 st.) och ansiktsprodukter (2700 st.). De anmälda ansiktsprodukterna är av allt att döma dagkrämer som innehåller UV-filter.

Från tillverkare/importörer Sverige är cirka 150 produkter anmälda, varav 80 st. sminkpro-dukter och 60 st. solskyddsprodukter. Läkemedelsverket kan dock konstatera att flera aktörer som sätter solskyddsprodukter på marknaden inte anmält dessa på rätt sätt.

Delar av det regulatoriska arbetet med nanomaterial har dock inte varit i tid. Rent formellt är de vanligaste nanomaterialen (Titanium dioxide, CI 77266) ännu inte tillåtna i nanoform. Detta delvis på grund av oklara definitioner av produkttyper som innebär inhalationsrisk (”spayprodukter”). EU-kommissionen skulle senast den 11 januari 2014 ha publicerat en katalog över de nanomaterial som används. Denna publicering har dock dröjt på grund av felaktiga anmälningar från företagen. Katalogen är sammanställd, och ska publiceras så snart den är översatt.

Danske aktiviteter vedr. nanomaterialer Flemming Ingerslev, The Danish Environmental Protection Agency

Nanomaterialer (NM) indgik i den danske kemikaliehandlingsplan fra 2010-2013, hvor fokus især var på at skabe overblik viden over de vigtigste nanomaterialer4 samt at bidrage til EU-arbejde med nanomaterialer 5. I den opfølgende kemikalieindsats (2014-2017) er der fokus på at sikre, at dansk viden om nanoteknologi bidrager til udviklingen af en fælles EU-løsning (http://kemikalieindsatsen.dk).

Den ny danske regering vedtog i 2011 initiativet ”bedre styr på nano”, som har til formål at skabe øget klarhed over eksponeringsveje og konsekvenserne for forbrugere og miljø ved anvendelse af nanomaterialer. Den styrkede indsats på nanoområdet omfatter blandt andet udvikling af et nanoproduktregister. Der er i årene 2012-2015 afsat 6 millioner danske kroner til indsatsen. Samlet skal initiativet skabe overblik over situationen med hensyn miljø- og forbrugersikkerhed i forhold til nanomaterialer i Danmark. Initiativet omfatter således projekter som skal 1) skabe overblik over eksisterende viden vedr. nanomaterialers sundhedsegenskaber og deres miljøegenskaber, 2) bidrage til ny viden om nanomaterialers hudgennemtrængelighed og nanomaterialers opløselighedshastighed i miljøet, 3) undersøge udbredelsen, anvendelse og risici af konkrete nanoprodukter, samt 4) skabe generelt overblik over anvendelsen af nanomaterialer i Danmark.

Et vigtigt element til at skabe overblik over nanomaterialer er det danske nanoproduktregister, som blev oprettet i juni 2014 i forbindelse med offentliggørelse af bekendtgørelsen om det danske nanoproduktregister (BEK nr 644 af 13/06/2014). Denne bekendtgørelse beskriver den registreringspligt som gælder alle virksomheder der sætter nanoprodukter på forbrugermarkedet i Danmark. Bekendtgørelsen omfatter således både producenter og importører af nanoprodukter. Nanoprodukter defineres som udgangspunkt som artikler eller kemiske blandinger, der indeholder nanomaterialer (jf. EU’s definition) og som frigiver disse nanomaterialer. Bekendtgørelsen undtager dog produkter, som reguleres under reglerne for fødevarer, fødevarekontaktmaterialer, medicinsk udstyr, kosmetik, pesticider og affald.

4 The Danish Environmental Protection Agency. Survey on basic knowledge about exposure and potential environmental and health risks for selected nanomaterials. Environmental Project 1370, 2011. 5 The Danish Environmental Protection Agency. Information Requirements for nanomaterials – IRNANO. Environmental Project 1469, 2013.

16

Endvidere undtager den produkter, hvor nanomaterialerne ikke bevidst er fremstillet i nanostørrelse og endelig undtager bekendtgørelsen visse konkrete produkttyper (bl.a. maling og gummiprodukter) med indhold af titandioksid, carbon black og silicium dioksid. Første indberetningsår slutter d. 30. august 2015.

I Nanoproduktregisteret er der dels nogle tvungne informationskrav og dels nogle frivillige krav. De tvungne krav omfatter information om den indberettende virksomhed, om produktet, om nanomaterialets kemiske sammensætning og om det er registreret under REACH. Endvidere er det frivilligt at indberette en række oplysninger om selve nanomaterialet (blandt andet partikel størrelse, størrelsesfordeling, oplysninger om aggregering m.m.). De frivillige oplysninger svarer til dem, som OECD har vedtaget harmoniserede standard formater for (OHTs). Som hjælp til de virksomheder der skal indberette, har Miljøstyrelsen udarbejdet vejledninger på dansk og engelsk 6 7. Der er endvidere oprettet en help-desk og en FAQ-side, hvor virksomheder kan få svar på spørgsmål (http://mst.dk/virksomhed-myndighed/kemikalier/miljoestyrelsens-nanoindsats/).

Nanocellulose and NANoREG- project Finnish Safety and Chemicals Agency

The safety of nanomaterials is investigated in the EU’s just-beginning extensive NANoREG project as a joint effort by the authorities and the industry. Finland’s goal in the project is to continue to study the safety of microfibrillar and nanofibrillar cellulose. To date, only a little is known about the health and environmental hazard impacts of nanoparticles, although the industrial use of nanomaterials has increased rapidly. For example, they are already common in sports equipment, sunscreens and other cosmetics products.

Nanotechnology is a technique for building nanometre-scale structures (one nanometre is one millionth of a millimetre). Interest in the use of nanoparticles is great, as they can be used to improve product characteristics; for example, nanoparticles help make a coat of paint more scratch-resistant. On the other hand, the safety of nanoparticles raises questions.

It is difficult to identify risks, as matter can have an unknown behaviour in nanoscale. The effect on cellular level can also vary according to what impurities have attached to the nanoparticle or which substance has been purposefully used to coat it.

The industrial use of nanomaterials has stirred up animated discussions in the EU. The REACH Regulation regulating the registration, evaluation, authorisation and restriction of chemicals plays a central role in legislation. The idea of safety-promoting dialogue between the industry and the authorities has come up to complement the idea of just using regulatory measures. This forms the basis of the NANoREG project to be implemented in the years 2013 to 2017.

Authorities, research institutes and companies and consortiums from fourteen European countries will participate in NANoREG. The project aims at developing guidelines for safe usage risk management and safety instructions, while assessing the need for new legislation.

6 Miljøstyrelsen. Vejledning om indberetning til det danske nanoproduktregister. Vejledning fra Miljøstyrelsen nr. 5, 2014. 7 The Danish Environmental Protection Agency. Guideline for the Danish Inventory of Nanoproducts. Guidance from the Danish Environmental Protection Agency No. 5, 2014

17

A stable and safe operating environment is a benefit that is shared by both the authorities and the industry. It is necessary for new applications and innovations, and the resulting investments, to be possible in the first place.

In Finland, participation in the NANoREG project is coordinated by the Finnish Safety and Chemicals Agency (Tukes) that is also responsible for oversight and guidance concerning the REACH regulation. Also included in the project are the Finnish Institute of Occupational Health, responsible for the experimental in vitro and in vivo studies, and Stora Enso and UPM as a joint Nordic Cellulosa consortium.

Finland’s national research portion of the Nanoreg project focuses on microfibrillar and nanofibrillar cellulose materials that have several potential industrial applications in different products. Microcellulose and nanocellulose comprise wood fibre and fibre bundles originating from wood cellulose. The intention is to study the safety of biodegradable microcellulose and nanocellulose experimentally by means of biological testing. This is important in order for it to be possible to use nanocellulose, for example, as raw material for cosmetics, food additives or packaging.

In the responsible development of products manufactured from micro and nanocellulose, research plays a key role. Research works aim to find and confirm potential benefits in microcellulose and assess the safety measures required for manufacturing and utilization of the materials. For this reason, the Nordic Cellulosa consortium acts in close cooperation with other fields and authorities, and promotes research in safe applications of microfibrillar and nanofibrillar cellulose.

18

2 Bilagor

Bilaga 1. Deltagarlista

Bioforsk Joner Erik Danmarks Tekniske Universitet Bloch Hartmann Nanna

Cupi Denisa Europeiska kommissionen Hellsten Eva Finnish Institute of Occupational Health Stockmann Juvala Helene

Hyytinen Eija-Riitta Finnish Safety and Chemicals Agency Einola Juha

Palomäki Jaana FOI Bucht Anders

Ekstrand-Hammarström Barbro Gustafsson Åsa

Formas Vikström Anna Försvarets Materielverk Henningsson Kenth

Ramfjord Birgit Westlund Robert

Försvarsmakten Jalalian Nazli Generalläkaren Duf Jessica

Kängström Marianne Kemikalieinspektionen Andersson Yvonne

Anfält Lisa Gellerstedt Therese Hellmér Lena Moore Gregory Simonsson Elin Wendt - Rasch Lina Virefjord Tania

Kommerskollegium Housset Cedric KTH Lazarevic David Livsmedelsverket Pihlström Tuija

Svensson Kettil Lund University Löndahl Jakob

Nilsson Annika Bohgard Mats

19

Läkemedelsverket Byström Tomas Hillgren Anna Salin Kia

Naturvårdsverket Hedlund Britta Mattsson Cecilia

Norwegian Institute for water research Macken Ailbhe Norwegian Environment Agency Andersen Sjur

Gudbrandsen Marius OECD Secretariate Ahtiainen Jukka Regeringskansliet Tapper Sofia SINTEF Materials and Chemistry Roman Netzer

Socialstyrelsen Domeij Helena The Danish Environmental Protection Agency Zenner Boisen Anne Mette

Ingerslev Flemming Trafikverket Bengtsson Malena

Reuithe Anna

20

Bilaga 2. Föreläsarnas power-point presentationer

21

Nanomaterials in a Life Cycle Perspective

David Lazarevic dalaz@kth.se

Division of Industrial Ecology

Department of Sustainable Development, Environmental Science &

Engineering

KTH – Royal Institute of Technology

• National action plan for the safe use and handling ofnanomaterials (2013)

• As part of this action plan:

• Lazarevic and Finnveden, 2013. Life cycle aspects ofnanomaterials. Environmental Strategies Research,KTH, Stockholm. (In English)

• Finnveden and Lazarevic, 2013. Livscykelaspekter ochnanomaterial. Avdelningen för miljöstrategisk analys,KTH, Stockholm. (In Swedish)

• Liljenström, C., Lazarevic, D., & Finnveden, G. 2013.Silicon-based nanomaterials in a life-cycle perspective:including a case study on self-cleaning coatings. KTH,Stockholm.

2

Benefits and Risks of Nanomaterials

Potential Benefits

• Expected significant impact on virtually all industrial

sectors including healthcare, agrifood, transport, energy,

materials, and ICT.

• Potential areas

• Monitoring

• Remediation and pollution

• Resource saving (energy)

3

“To some it represents the miracle cure for all that ails us. To others,

it could be the end of the world as we know it” (Maynard, 2010).

Benefits and Risks of Nanomaterials

Potential impacts

• Toxicological risks to humans and the environment

• Increase in the extraction of raw materials

• Increase energy use during production phases

• Higher material requirement (better specifications)

• Increased waste production during production (hazardous

waste)

• End-of-life: what happens to waste products?

4

Nanomaterials in a Life cycle perspective

5

The general consensus among scientists, researchers, and

regulatory agencies is that the potential health and

environmental risks of engineered nanomaterials (ENMs)

should be evaluated over their entire life-cycle (Grieger et al.

2012).

• Life cycle Assessment

• Risk Assessment

Life Cycle Assessment

• A Tool for assessing thepotential environmentalimpacts associated with aproduct/service system by:

o compiling an inventory ofrelevant inputs andoutputs

o evaluating potentialimpacts associated withinputs and outputs

o interpreting results ofinventory and impactassessment

• LCA follows a cradle-to-grave approach

6

(Lazarevic & Finnveden, 2013)

7

Components and semi - products Manufacture

Use and maintenance

Recycling and disposal

Resources and materials

Components and semi - products Manufacture

Use and maintenance

Recycling and disposal

Resources and materials

Process

Raw materials

Energy

Waste Emissions

Products

0 20 40 60 80 100 120

Global Warming

Acidification

Photochemical ozone formation

Eutrphoication

Human toxicity

Exotoxicity

Land use

Waste (volume)

Hazardous waste

B

A

LCA Phases

• Goal and Scope Definition

• Life Cycle Inventory (LCI)

• Life Cycle Impact

Assessment (LCIA)

• Interpretation

Life Cycle Assessment

• Enables the study and comparison of different options to

supply a given function

• Enables the identification of environmental ‘hotspots’

throughout the product/service life cycle

• LCA attempts to be comprehensive with respect to the

environmental interventions and environmental issues

considered.

8

9

(Grieger et al. 2012)

• LCA’s focus on the

product/service system

and RA’s focus on the

emissions of a single

substance

• The results of LCA are

comparative whereas

the results of RA are

absolute

• LCA covers a range of

environmental impacts

whereas RA primarily

cover toxicological and

(eco)toxicological

impacts.

Raw

Materials

Manufacturing Use E-O-L

Babaizadeh and Hassan (2013): Comparison of TiO2 coated class with float glass

Bauer et al. (2008)

Greijer et al. (2011) Nanocrystaline dye sensitized solar cell (from nano-TiO2 and carbon black)

Griffiths and O’Byrne (2013) MWCNT formation via catalytic chemical vapour deposition.

Grubb and Bakshi (2011a, 2011b) Life cycle energy use of nano TiO2 production and e.g. steel, aluminium, polysilicon production Isaacs et al. (2010) Production of single wall carbon nanotubes

Joshi (2008) Comparison of nanoclay composite biopolymer with biobased polymers

Khanna et al. (2008) & Khanna and Bakshi (2009) polymer nanocomposites compared to steel, aluminum and PP Kushnir and Sandén (2008) Energy requirements for fullerene and nanotube synthesis

Lloyd and Lave (2003) Clay polypropylene ENM's instead of steel or aluminum in vehicle body panels

Lloyd et al. (2005) Nanofabrication technique of platinum group metal (PGM) particles in automotive catalysts Merugula et al. (2010) Comparison of Glass fibre reinforced plastics an vapour grown CNts for wind turbine blades Meyer et al. (2011) Socks with and without Ag ENM's

Moign et al. (2010) Manufacturing of yttria-stabilized-zirconia ENM coating

Osterwalder et al. (2006) Energy comparison of wet and dry synthesis methods for oxide nanoparticle production Roes et al. (2007) Comparison of polypropylene nanocomposite with conventional polypropylene

Şengül and Theis (2011) LCA of quantum dot photovoltaic (QDPV) module compared to silicon and thin film PV’s Singh et al. (2008) SWCNT production

Walser et al. (2011) Comparison of nanosilver t-shirts and conventional t-shirts

Environmental impact categories

Energy use

Toxicological impact categories (non-nanoparticle release based)

Nanoparticle Toxicity impact (nanoparticle release based)

10

Nanomaterials in a Life cycle perspective Environmental Burden

• Potentially energy intensive raw

material extraction, processing, and

nanomanufacturing processes

• Potential depletion of non-renewable

resources

• Possible release of toxic emissions

& unconventional liquid streams

Missing Information

• Impacts of raw material use on

supply chains of other products

• Potential release, fate and transport

of ENMs

• Performance of ENM products

• Amount & characterisation of waste

streams

Uncertainties

• Waste & emissions from ENM

production

• ENM exposure & toxicity

• Preferred method of ENM disposal

11

Use

Extraction ofRaw Materials

Recycling

Distribution

Recovery

Release ofENMs to the Environment

Release of ENMs to the

Environment

End of life

Incineration &Landfill

Reuse

Engineered nanomaterial

Product life cycle

Production

(Lazarevic & Finnveden, 2013)

Current use of LCA: Cradle to Gate

(Reproduced from Khanna et al. (2008))

12

CED for CNFs compared to aluminium, steel and polypropylene

Current use of LCA: Cradle to Gate

(Reproduced from Khanna and Bakshi (2009))

13

CED of polymer nanocomposites that provide equal

stiffness to a steel component

Current use of LCA: Cradle to Use

(Reproduced from Hischier and Walser (2012))

14

as part of a

car, driving

280000 km

Current limitations of Applying LCA to nanomaterials

15

CNF emissions and impacts not included

Khanna et al. (2007)

Life Cycle Assessment of Nanomaterials

Goal and Scope Definition

• ENMs may have specific functions and material propertiesthat provide additional gains when used as a substitute fortraditional materials

Life Cycle Inventory

• Lack ENM specific data related to the outputs of theprocesses

o Data on Input side, no data on output side

o It is important to know if ENMs change their form duringtheir life cycle, due to aging and other influences such asweather, mechanical stress/pressure, etc.

• LCI databases do not distinguish between bulk materials andENMs

• Populating LCI databases with ENM specific information,such as size and shape, is of critical importance

16

Life Cycle Assessment of Nanomaterials

Life Cycle Impact Assessment

• ENMs may exhibit unconventional behaviour, leading to

unexpected fate, transport, and toxicity mechanisms in human

and ecological systems

• A complete lack of characterization factors for release of

nanoparticles indoors and outdoors

• LCIA methods such as CML 2001, Eco-Indicator 1999 or Impact

2002 do not cover toxicological effects of nanoparticles

• The current understanding of effect mechanisms, dose-response

relationships, as well as transport and transformations in the

environment may not be sufficient to ascertain a representative

characterization of ENMs.

17

Conclusions

• LCA of nanomaterials is still in the very early stages

• Consideration of the impacts of ENMs in LCA is currently

inadequate, due to:

– Lack of data on nanomaterial production

– Lack of data and uncertainties on what should be

included in the LCI

– Uncertainties on how to assess the impacts of ENMs

(LCIA)

• Need for cooperation between the LCA community and the

nanotoxicology community

• LCA needs to be complemented by RA to assess risk

related to specific life cycle stages

18

References

Greijer, H., Karlson, L., Lindquist, S.E., Hagfeldt, A., 2001. Environmental aspects of electricity generation from a nanocrystalline dye sensitized solar cell system. Renewable Energy 23, 27–39.

Hischier, R., Walser, T., 2012. Life cycle assessment of engineered nanomaterials: State of the art and strategies to overcome existing gaps. Science of The Total Environment 425, 271–282.

Khanna, V., Bakshi, B.R., 2009. Carbon nanofiber polymer composites: evaluation of life cycle energy use. Environmental Science & Technology 43, 2078–2084.

Khanna, V., Bakshi, B.R., Lee, L.J., 2007. Life cycle energy analysis and environmental life cycle assessment of carbon nanofibers production, in: Electronics & the Environment, Proceedings of the 2007 IEEE International Symposium On. IEEE, pp. 128–133.

Khanna, V., Bakshi, B.R., Lee, L.J., 2008. Carbon nanofiber production: Life Cycle Energy Consumption and Environmental Impact. Journal of Industrial Ecology 12, 394–410.

Lazarevic, D. and Finnveden, G., 2013. Life cycle aspects of nanomaterials. Environmental Strategies Research, KTH, Stockholm.

19

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Dermal absorption of nanomaterials

Projects from the Danish EPA

Anne Mette Boisen

Chemicals Unit,

Danish Environmental

Protection Agency (EPA)

Presentation outline

Introduction to the Danish Nano Initiative

Better Control of Nano

Dermal absorption of nanomaterials – Literary project

(conducted by IOM from UK)

Dermal absorption of nanomaterials – Eksperimental

project

(conducted by Aarhus University from DK)

Better control of Nano initiative (24 mio. DKK in 2012-15):

Dermal absorption –Literary project

Danish EPA Environmental Project No. 1504, 2013

Report and database

Scientific assessment of available studies on dermal absorption

Aims

• assessment of the extent of absorption of nanomaterials;

• identification of nano-specific characteristics that may influence the absorption of nanomaterials;

• evaluation of which test method(s) would most closely simulate the transport of nanomaterials through human skin

• candidates for testing and assessment of the specific research areas that require more knowledge.

Search strategy

Assessment of reliability and relevance of studies -Klimisch criteria and NM characterization (Card and Magnuson 2010)

Results and recommendations

Absorption of NM into systemic circulation:

Possible (small fraction) few robust, well performed studies, no

clear guidelines (hard to compare/interpret)

Parameters:Size, surface chemistry (coating), shape

Results and recommendations

Test methods: Harmonised testing approaches (specific technical guidance

given in the report). Human golden standard (but ethics and

cost)

Candidates for testing:

Systematic test of priority candidate properties (size,

surface chemistry)

Research gaps:

Effect of flexed skin, follicular penetration, children,

extending the study period

Dermal absorption –Experimental project

Project manager Christiane Beer, Aarhus University Started spring 2014 ends summer 2015

Overview

• Candidates for testing:

Nanomaterials used in sunscreen:

Titanium dioxide and Zinc Oxide

• Investigation of characteristics:

Size and coating

• Test methods:

In vitro Epiderm model

In vivo mouse inflammation model

In vivo mouse xenograft model

Candidates for testing

Titanium Dioxide

Size:

(TiO2, rutile, high purity 99.9%, 30 nm)

(TiO2, rutile, high purity 99.9%, 100 nm)

Coating:

(TiO2, rutile, 30 nm) coated: Silicone Oil (hydrophobe)

(TiO2, rutile, 30 nm) coated: Silicon and Aluminium (hydrophile)

UV-Titan M 161: TiO2 nanoparticles, rutile, 17 nm, coated: Alumina, stearic acid

UV-Titan M 262: TiO2 nanoparticles, rutile, ~20 nm, coated: Alumina, silicone

Zinc Oxide:

Coating: (ZnO, high purity 99.95%, 18 nm, uncoated)

(ZnO, 99+%, 20 nm, coated: 1 wt% Silane Coupling Agent)

(hydrophile)

Vehicle

Wt% Trade name Phase INCI

61.90

% Water 1 Aqua

0.20

% Keltrol AP 1 Xanthan Gum

0.10

% Dermofeel PA-3 1 Sodium Phytate, Aqua, Alcohol

1.00

% Verstatil PC 1 Phenoxyethanol, Caprylyl Glycol

2.00

% Propylen Glycol 1 Propylene Glycol

2.00

% Glycerin 1 Glycerin

10.00

%

Waglinol AB

1215 2 C12-C15 Alkyl Benzoate

2.80

% Parsol 340 2 Octocrylene

4.00

% Parsol MCX 2 Ethylhexyl Methoxycinnamate

3.00

% Parsol 1789 2 Butyl Methoxydibenzoylmethane

3.00

% Mulsifan RT 11 2 Ceteareth-22

10.00

% Soldoc EB 29 2 Isostearyle Isostearate

C12-C15 Alkyl Benzoate is used to pre-disperse nanoparticles before adding them to the sun cream.

2.5 wt% NP

In vitro model

3D in vitro skin model

in vitro EpiDerm™ system (MatTek)

Analyses

• TEER (trans epithelial electric resistance)

• electron microscopy (TEM)

• Histology

• skin corrosion/irritation tests (MTT assay)

• ICP-MS

• Cytokine analysis of medium (11 cytokines by multiplex flow cytometer assay)

Set-up

Day 1 – Arrival of the cells

Day 2 – TEER measurement

Exposure to sunscreen +/- nanoparticles and untreated control for 20 hours

Day 3 – Washing of the EpiDerm models (Medium samples for ICP-MS and

cytokine analysis)

Day 5 – Medium exchange (Medium samples for cytokine analysis)

Day 7 – TEER measurement

Sample preparation for EM and histology

Medium samples for cytokine analysis

MTT assay skin irritation/skin corrosion

Each sample will be treated with 30 μl of the test substances (All 8 NP are tested).

In vivo Inflammation model

Acute irritant contact dermatitis model

8 hours 24 hours

4 hours 0 hours

Induced: Erythema Oedema Scaling Epidermal hyperplasia Infiltrates of monocytes lymphocytes neutrophils IL-1b, TNFa, IL-6

TPA: 12-O-tetradecanoylphorbol-13 acetate

Set-up

A = Untreated (no sun spray, no nanoparticles) B = Control sun screen C = NP1 : (TiO2, rutile, high purity 99.9%, 30 nm) D = NP2 : (TiO2, rutile, high purity 99.9%, 100 nm) E = NP3 : (TiO2, rutile, 30 nm coated with Silicone Oil ) F = NP4 : (TiO2, rutile, 30 nm Coated with Silicon and Aluminium )

Set-up

Day TPA treatment

Sun screen application

1 X Ear thickness 2 X X Ear thickness 3 X X Ear thickness 4 X Ear thickness 8 Ear thickness

EM, histology and ICP-MS sample preparation

Storage blood and organs

2 x 3 mm biopsies are taken from each ear for TEM and histology; leftover from the ear is frozen and stored for ICP-MS

10 µl sun screen per cm2

In vivo xenograft transplantation model

Transplantation Engraftment

Mouse skin

Human skin

In vivo xenograft model

Vehicle and NP1-4 are applied on day 1, 2, and 3

(10 µl sun screen per cm2).

On day 8, mice are killed (4 days post last treatment with

NPs):

A = Untreated (no sun screen, no nanoparticles)

B = vehicle control sun screen no NPs

C = NP1 : (TiO2, rutile, high purity 99.9%, 30 nm)

D = NP2 : (TiO2, rutile, high purity 99.9%, 100 nm)

E = NP3 : (TiO2, rutile, 30 nm coated with Silicone Oil )

F = NP4 : (TiO2, rutile, 30 nm coated with Silicon and Aluminium )

Analyses • ICP-MS • TEM • histology

Expected Outcome Experimental project

• Project report (summer 2015)

• Articles in peer reviewed scientific journals (summer 2015)

Acknowledgements

Literary project

Dr. Craig Poland, Steve Hankin, Craig Poland, Sheona Read, Julia Varet,

Gillian Carse, Steven M. Hankin, IOM, United Kingdom,

Frans M. Christensen, COWI Denmark.

Reference group: Maxine McCall, CSIRO, Australia,

Jesper Bo Nielsen, Southern University of Denmark.

Experimental project

Christiane Beer, Herman Autrup , Karin Stenderup, Lars Iversen, Duncan

Sutherland, Jing Wang, Jens Randel Nyengaard, Torben Sigsgaard, Jakob

Bønløkke, Aarhus University, Denmark.

Reference group: Craig Poland, Steve Hankin, IOM, United Kingdom.

Interactions of nanoparticles with organs protected by internal biological barriers

Anders Bucht, Swedish Defence research AgencyDivision of CBRN Defence and Security

Nucleus

ER

TiO2-nanoparticles

Nucleus

Mitochondria

TiO2-nanoparticles30nm

80.000 x magnification 120.000 x magnfication

Uptake of TiO2-nanoparticles in lung epithelial cellsUptake of TiO2-nanoparticles in lung epithelial cells

Ekstrand-Hammarström B, Akfur CM, Andersson PO, Lejon C, Österlund L and Bucht A. Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B. Nanotoxicology 2011.

Raman imaging measures polymorph and size specific NP uptake and distribution in living lung epithelial cells

a) Optical micrograph of A549 cell exposed to 50/50 wt% mixture of P25/R9 TiO2 nanoparticles.

b) Raman mapping performed within the rectangular area visualized in (a) of P25 anatase characterized by the Eg vibrational mode at 145 cm-1.

c) Raman mapping of rutile characterized by the Eg vibrational mode at 450 cm-1.

x /m

b)

c)

y/

my

/m

2

-5

0

5

-5 0 5 100

50

100

150

200

2

4-5

0

5

-5 0 5 10

10

20

30

40

50

P25

R9

-10

10

0

a)

-10 100

P25/R9

wvib=448 cm-1

wvib=144 cm-1

P25 NP ontop of nucleus

Small, soft NP agglomerates (such as anatase ”P25”) penetrates membranes more easily than hard agglommerates (such as rutile)

Leakage into nucleus region

Andersson PO, Lejon C, Ekström Hammarström B, Akfur C, Bucht A and Österlund L. Polymorph- and size-dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells. Small 2011

TiO2 induce oxidative stress and pro-inflammatory response in lung epithelial cells

Exposure of A549 cells for 24 h exposure to 50 g TiO2 nanoparticles.

Sample IL-8[pg ml-1]

MCP-1[pg ml-1]

control 150 4 1254 19

A14 256 20* 1209 133

A60 411 59* 1076 48

R5 487 73* 1193 121

R9 424 25* 1414 383

P25 840 126** 2178 131**

Intracellular Superoxide production

Andersson PO, Lejon C, Ekström Hammarström B, Akfur C, Bucht A and Österlund L.Polymorph- and size-dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells. Small 2011.

Changed properties of TiO2 NPs in serum

Z-potencial -24mV

From studies of lung cells we know that:

• Nanoparticles can penetrate the cell membrane andnucleus, and the penetration depends on size andparticle structure.

• NPs can induce oxidative stress and inflammatory response in cells, and this response depends on type ofparticle, cell type and exposure conditions.

Tubing loops coated with heparin. Freshly drawn human blood is added to the loop together with the test nanoparticles. Rotated at 37°C in a heat cabinet. Aliquots of blood are removed at different time points during incubation for analysis

Analaysis of blood coagulation in vitro

Bo Nilsson and colleagues at Uppsala University

From studies of blood coagulation we know that:

• TiO2 NPs at very low concentrations (50 ng/mL)induce strong activation of the contact system, whichin this model elicits thromboinflammation.

• this is in line with the finding of components of thecontact system in the protein corona of the TiO2 NPsafter exposure to blood.

Overall conclusions

• A corona of biomolecules will be formed around the NPsbefore meeting the epithelial cell layer.

• The corona will influence translocation through theepithelial barrier and the subsequent systemic response.

• In the blood circulation, nanoparticles may have animpact on coagulation cascade, as well as induce innateand adaptive immune responses.

Remaining issues

• Do the adaptive immune system recognize biomoleculesbound to the NPs as foreign bodies, thereby providing arisk for autoimmune reaction?

• Do NPs in complex with biomolecules penetrate theblood-brain-barrier in concentrations that may harm thebrain?

Contributors and Collaborators

The Toxicology teamBarbro Ekstrand-HammarströmÅsa GustafssonSofia JonassonElisabeth WigenstamLinda ElfsmarkBo KochChristine AkfurMona KochLina ThorsLina ÅgrenUlrika Bergström

Raman spectroscopy at FOIPer-Ola AnderssonChristian Lejon Linnea Ahlinder

Dep. of Respiratory Medicine and Allergy, Umeå UniversityThomas Sandström and Anders Blomberg

Dep.of Engineering Sciences and solid phase physics, Uppsala UniversityLars Österlund and colleagues

Dep. of Immunology, Genetics and Pathology, Uppsala UniversityBo Nilsson and colleagues

Swedish Defence Research Agency Collaborators

anders.bucht@foi.se

Measurement Techniques for Airborne NanoparticlesJAKOB LÖNDAHL

Research on Nanosafety in Three CompetenceCentres at Lund University

METALUND, Medicine and Technology for Working Life and Society

nmC@LU, Nanometer StructureConsortium

CAST, Consortium for Aerosol Science and Technology CAST

The Lund Aerosol Group –One laboratory, two departments

Ergonomics and Aerosol TechnologyMats Bohgard, Ville Berg, Anders Gudmundsson, Christina Isaxon, Jonas Jakobsson, Jakob Löndahl, Patrik Nilsson, Erik Nordin, Joakim Pagels, Jenny Rissler, Christian Svensson, Aneta Wierzbicka, et al.

Nuclear PhysicsErik Swietlicki, Birgitta Svenningsson, Adam Kristensson, Göran Frank, Emelie Hermansson, Erik Ahlberg, Johan Martinsson, Pontus Roldin, Moa Sporre, Axel Eriksson, Cerina Wittbom et al.

Nanoparticles: what should wemeasure?Physical properties

SizeNumber / surface area / volumeSurface structureElectrical chargeRadioactivityShape

Chemical propertiesChemical composition (metals, toxins, …)Solubility

Biological activity(viruses)

Avoid measuringbackground particles

What we want to measure is not similarto what we are able to measure

Nanoparticles: how could we measure?

They are only a small fraction of the air (by mass)

Nanoparticles smaller than the wavelength of visible light

Particle surface area monitors

Examples: miniDiSC, NanoTracer, AeroTrak, NSAM, Partector

Particle surface area monitors

Advantages: small, cheap, easy to use, suitable for personal exposure, hith time resolution (seconds),lung deposited surface area (??)

Disadvantages: total concentration (10-300 nm), limited precision

Particle size distribution, SMPS (FMPS)

DMA CPC

0E+0

1E+5

2E+5

3E+5

10 100 1000

dN/dlogD

p (cm

‐3)

Dry mobility diameter (nm)

Idle engine

Transient driving

Advantages: precise, high time resolution (~1 min)

Disadvantages: complex, expensive

Particle size distribution, ELPI

Advantages: high time resolution (seconds), some size information, possibility to analyse particles afterwards

Disadvantages: complicated, not ideal for nanoparticles, lower precision than SMPS

Particle mass (and surface area) distribution

APM, aerosol particle mass analyzer

Particle mass (and surface area) distributionAPM, aerosol particle mass analyzer (10-18 grams)

DMA APM CPC

0.80

1.00

1.20

10 100 1000

Effective den

sity [gcm‐³]

Diameter [nm]

Cigarette smoke

Waterpipe smoke

Fitted density

Why? A way to assess surfacearea, mass and lung deposition

Aerosol Mass SpectrometerParticle composition with high time and size resolution (30-1000 nm), but requires a dedicated operator

Analysis of sampled nanoparticlesNo online methods available to obtain many important particlecharacteristics. Analysis of sampled particles needed:

• Particle shape: electron microscopy (TEM, SEM)

• Detailed chemical composition: e.g. x-ray, MS

• Toxicity: in vivo (animals, humans) or in vitro (cell culture)

• Biological properties: e.g. PCR

Lung deposition measurements

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.001 0.01 0.1 1 10 100

Deposition Fraction

Particle diameter [µm]

Total

Extrathoracic

Tracheobronchial

Alveolar

Lung deposition measurements Summary

Slow

FastCheapSimple(Unspecific)

ExpensiveComplex(Specific)

Particle shapeElectron microscopy, APM

Particle size distribution(SMPS, ELPI, FMPS…)

Chemical analysisx-ray (PIXE, XRF, MAX-lab…), MS…

Biological analysisPCR, proteomics, biomarkers…

Particle massTEOM, gravimetric…

Particle number and surface areaCPC, electrostatic… AMS, particle size and chemistry

Summary• Possibilities to measure a wide range of properties of

airborne (nano)particles

• Sometimes a choice between simplicity and accuracy (or high time resolution and relevant particle characteristics)

• Difficult to measure relevant exposure:- background particles vs produced particles- appropriate health metric unclear

• New methods are emerging…

Respiratory and immunological effects following

inhalation of engineered nanoparticles

Nanomaterial

Åsa Gustafsson

Background

Åsa Gustafsson

Particle properties

Åsa Gustafsson

Agglomeration

Material composition

Concentration

Shape

Size

Size distribution

Surface charge, ζ-potential

Surface functionality

NP

Studied effects

Åsa Gustafsson

Cells in lung lavage and in lymph nodes

Cytokines and chemokines in

Lung lavage and in blood

Lung function

How are susceptible individuals affected?

Åsa Gustafsson

Genetics

Study I

• In vivo, Rat

• Administration

• Dose

• Particle size

• Analysis

Dark Aguoti

Intratracheal

5 mg/kg body weight

200 nm and 2 µm

1, 2, 8, 16, 30 and 90 daysfollowing exposure

Åsa Gustafsson

Gustafsson et al (2011) J Immunotoxicol

Inflammatory and immunological responses in the airways following one exposure to titanium dioxide.

Nanoparticle

Pro-inflammatory cytokines IL-1β, IL-6, GM-CSF, TNF-α etc.

Pro-inflammatory cytokines IL-1β, IL-10, IFN-γ, TNF-α, IL-8

Lung epithelial cellsAlveolar macrophages

Dendritic cell

0 1 2 8 16 30 90 days after exp. Instillation

Åsa Gustafsson

Gustafsson et al (2011) J Immunotoxicol

Lymph nodes

Lymphocyte- activation

I. Innate and adaptive immune system

Åsa Gustafsson

Gustafsson et al (2011) J Immunotoxicol

0 1 2 8 16 30 90 90 (C)0

50

100

150

200

250 Neutrofiler

**

**

**

** **

Dagar efter instillering

Anta

l cel

ler (

x104 )

#

0 1 2 8 16 30 90 90 (C)0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5 Lymfocyter **

**

**

Dagar efter instillering

Anta

l cel

ler (

x104 )

# #

Num

ber

of c

ells

(x1

04 )

Num

ber

of c

ells

(x1

04 )

Days after exposure Days after exposure

Neutrophils Lymphocytes

I. Cell differentiation

Åsa Gustafsson

Neu: Neutrophil

TH1: Virus and bacteria

TH2: Parasites

TH9: Reglation of parasites?

TH17: Bacteria and fungal

TREG: T-regulatory lymphocyte

M1: Macrophage type 1 – Virus and bacteria

M2: Macrophage type 2 - Parasite

IL-1

IL-2

IL-4

IL-5

IL-6

IL-9

IL-7

IL-10

IL-12

IL-13

IFN-

TNF-α

GM-CSF

GCS-F

IL-18

IL-17

M1TH1

TH2

TH17

TH9TREG

M2

IL-4, IL

-5, IL-13

IL-17, IL-6

IL-4

, IL

-10

, IL

-13

TNF-α, IL-6, IL-1, GM-CSF, IL-8

Neu

Gustafsson et al (2011) J Immunotoxicol

I. Titanium dioxide particles in lungtissue

Åsa Gustafsson

20x10x 40x

64x100x100x

2 days 90 days30 days

Gustafsson et al (2011) J Immunotoxicol

Study II

• In vivo

• Nanomaterial

• Particle administration

• Number of exposures

• Deposition, each exposure

• Dose, each exposure

• Allergen

Mice Balb/c

TiO2

Aerosol

8 times á 2h

32±1 µg

1.5-1.8 mg/kg body weight

Ovalbumin

Åsa Gustafsson

Jonasson et al (2013) Inhal Toxicol

Responses on the immune system and lung function following exposure to titanium dioxide in mice at different time points

during the developement of allergic airway inflammation.

II. Allergy schedule

Åsa Gustafsson

Immunisation of allergen

OVA allergen OVA allergen

Allergen provocation

ImmunizedOVA group

OVA

2. Eosinophil inflammation

1. Allergen specificIgE antibodies

4. TH2 immune activation

IL-4, IL-5, IL-13

Jonasson et al (2013) Inhal Toxicol

3. Airway reactivity

2015-03-10

3

II. Allergy schedule-Particle exposure

Åsa Gustafsson

Immunisation of allergen

OVA allergen OVA allergen

Allergen provocation

ImmunizedOVA group

TiO2

OVA

ImmunizedTiO2/OVA (gr. 1)

ImmunizedTiO2/OVA (gr. 2)

ImmunizedTiO2/OVA (gr. 3)

Jonasson et al (2013) Inhal Toxicol

II. OVA-specific antibodiesAirway inflammation

Åsa Gustafsson

2. Airway inflammation1. OVA specificIgE antibodies

Jonasson et al (2013) Inhal Toxicol

O.D

kontroll

OVAGr.

1Gr.

2Gr.

30.0

0.5

1.0

1.5

2.0*

*** ***

Eosinofiler

An

tal c

elle

r i B

AL

(x1

05 )

kontroll

OVAGr.

1Gr.

2Gr.

30

20

40

60

*** ******

***

Neutrofiler

An

tal c

elle

r i B

AL

(x1

05 )

kontroll

OVAGr.

1Gr.

2Gr.

30

2

4

6

8

***

Eosinophils Neutrophils

Nu

mbe

r of

cel

ls in

BA

LF

(x1

05 )

Nu

mbe

r of

cel

ls in

BA

LF

(x1

05 )

II. Airway reactivity and TH2 immune activation

Åsa Gustafsson

3. Airway reactivity 4. TH2 immune activation (IL-4, IL-5, IL-13)

Jonasson et al (2013) Inhal Toxicol

RR

S(c

mH

2Os

/ml)

kontroll

OVAGr.

1Gr.

2Gr.

30

2

4

6 ***

pg/

ml i

n B

AL

IL-4

IL-5

IL-1

30

50010001500

20002500

500010000150002000025000

KontrollOVA

Gr. 1Gr. 2Gr. 3

** * *

*** ***

***

******

*** *********

Study III

• In vivo, Rat

• Nanomaterial

• Particle administration

• Number of exposures

• Deposition, each exposure

• Dose each exposure

• Allergen

Dark Aguoti (DA)

Brown Norwegian (BN)

TiO2

Aerosol

10 times á 2h

160-170 µg

0.64-0.8 mg/kg body weight

Ovalbumin

Åsa Gustafsson

Gustafsson et al (2014) Toxicol

Genetic influence on immunologic responses and lung physiology following exposure to titanium

dioxide.

DA Råtta

An

tal c

elle

r ( x

106)

1 2 3

0.00

0.25

0.50

0.75

1.00

1.25

1.50PBS OVA TiO2/OVA

Eosinofiler Neutrofiler Lymfocyter***

***

*

*

III. Allergen specific antibodiesAirway inflammation

Åsa Gustafsson

1. OVA specificIgE antibodies

2. Airway inflammation

Gustafsson et al (2014) Toxicol

Immunized TiO2/OVA

Immunisation of allergen Allergen provocation

DA Råtta

An

tal c

elle

r ( x

106)

1 2 3

0.00

0.25

0.50

0.75

1.00

1.25

1.50 Kontroll OVA TiO2/OVA

Eosinofiler Neutrofiler Lymfocyter*

*

**

O.D

Kontro

llOVA

/OVA

2TiO Kon

troll

OVA/O

VA

2TiO

0.0

0.1

0.2

0.3

0.4

*

DA ratBN rat

Nu

mbe

r of

cel

ls in

BA

LF

(x1

06)

Control

Eosinophils Neutrophils Lymphocytes

DA Rat

III. Airway reactivity

Åsa Gustafsson

DA. Central airways DA. Small airways

Gustafsson et al (2014) Toxicol

MCh (mg×mL-1)

RR

S(c

mH

2Os×

mL-1

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 Kontroll n=8OVA n=7TiO2+ OVA n=7

Saline 5

***

MCh (mg×mL-1)

G (c

mH

2Os×

mL-1

)

0.000.250.500.751.001.251.501.752.002.25 Kontroll n=8

OVA n=7OVA+TiO2 n=7

Saline 5

**

III. Mediators in healthy DA rats aftertitanium dioxid exposures

Åsa Gustafsson

Healthy (control)

Healthy (TiO2) Pro-inflammatoric

TH1

TH2

Cardiovascular

T- , B-cell mature

50

40

30

10

50

10

20

40

30

5040

3020

10

15 00012 000

9 0006 000

3 000

125

100

75

50

25

100

80

60

40

20

50

40

10

20

30

100 80

4020

60

125100

7550

25

25 000

20

5 000

10 000

15 000

IL-13

IL-6

GCS-F

GM-CSF

MIP-3α

IL-12 (p70)

IL-18

IFN-γ

IL-7

VEGF

20 000

Gustafsson et al (2014) Toxicol

Study IV

• In vivo, Mice

• Nanomaterial

• Particle administration

• Dose

• Analysis

• Allergen

• Balb/c

• Iron oxide (Hematite)

• Intratracheally

• 5 mg/kg body weight

• 1, 2, och 7 days after exposure

• Ovalbumin

Åsa Gustafsson

Gustafsson et al. Manuskript

Immunologic responses in airways and lymph nodes following inhalation to iron oxide (hematite) in healthy

and in mice with allergic airway inflammation.

IV. Inflammation in healthy mice

Åsa Gustafsson

Neutrophils

Num

ber o

f cel

ls (x

106 )

0.0

0.1

0.2

0.3

0.4

0.5

Day 1 Day 2 Day 7

*

***

Eosinophils

Num

ber o

f cel

ls (x

106 )

0.000

0.005

0.010

0.015

0.020

Day 1 Day 2 Day 7

p=0.09

**

**

Lymphocytes

Num

ber o

f cel

ls (x

106 )

0.00

0.01

0.02

0.03

0.04

0.05

Day 1 Day 2 Day 7

*****

Vehikel Hematit Macrophages

Num

ber o

f cel

ls (x

106 )

0.00

0.05

0.10

0.15

0.20

Day 1 Day 2 Day 7

p=0.06

Vehicle Hematite

IV. Cellular reduction i mice with anallergic airway inflammation

Åsa Gustafsson

Eosinophils

Macrophages

Num

ber o

f cel

ls (x

106 )

0.0

0.5

1.0

1.5

Day 1 Day 2 Day 7

*

Neutrophils

Num

ber o

f cel

ls (x

106 )

0.00

0.05

0.10

0.15

Day 1 Day 2 Day 7

Eosinophils

Num

ber o

f cel

ls (x

106 )

0

2

4

6

8

Day 1 Day 2 Day 7

p=0.08

Lymphocytes

Num

ber o

f cel

ls (x

106 )

0.0

0.5

1.0

1.5

2.0

Day 1 Day 2 Day 7

*

Vehikel HematitVehicle Hematite

IV. Production of oxygen species

Åsa Gustafsson

Conclusions

Åsa Gustafsson

TiO2-particles that are deposited in the alveolar region may remain over a long period of time (Study I and III).

TiO2-particles induce a long term activation of the innate- and adaptive immune system (Study I and III).

Inhalation of TiO2-particles before and during allergen provocation in immunized mice induced a neutrophil inflammation, body weight reduction and impaired general condition (Study II).

Inhalation of TiO2-particles does not exacerbate characteristics of alleric airway inflammation in rat, although there is a genetic variety regarding cellular composition (Study III).

Rats with inhereted predisposition towards developement of TH1 immune responses was more sensitive and expressed more mediators (Study III).

The cellular response in a lung with established allergic airway inflammation and associated draining lymph nodes does probably induce cell death following exposure to hematite NPs (StudyIV).

It is important to include sensitive individuals when evaluating risk assessments of nanomaterials.

Nanosäkerhet ur ett EU- och OECD perspektiv

Erfarenheter från EU-kommissionen, OECD och svenska regeringsuppdraget om nanosäkerhet

KemI myndighetsmöte, 27 November 2014Eva Hellsten

Förväntad tillväxt i världen år 2000-2020

2020 – marknadsvärde3 000 miljarder $

År 2000 – marknadsvärde 30 miljoner $

Årlig tillväxt 2000 – 2008 ekonomi, sysselsättning, patent, vetenskapliga publikationer, investering i R&D ~ 20-35 %

Adapted from M. Rocco et al 2011

EU Kommissionen år 2004…”….Overall spending on R&D should beincreased in Europe to balance the heavyinvestments that have been initiated by ourmain competitors”

“….Any negative impact on public health,safety or the environment must be addressedupfront and as an integral part oftechnological development”

“Towards a European Strategy for Nanotechnology, 2004”

R&D finansiering av Nanotech från EU

FP5 1998-2002 280 miljoner €

FP6 2002-2006 1,4 miljarder €

FP7 2007-2014 > flera miljarder € (?)

FP5 1998-2002 280 miljoner €

FP6 2002-2006 1,4 miljarder €

FP7 2007-2014 > flera miljarder € (?)

Satsningen på miljö och hälsa har under åren ökat till dagens nivå på cirka 25 miljoner € per år

EU:s Aktionsplan 2005-2009

50-tal åtgärder som Kommissionen ochmedlemsländerna skulle genomföra för attsäkerställa en “säker, integrerad och ansvarsfull”utveckling av nanoteknologi

Helhetsgrepp om forskning & innovation,industriell utveckling, etik, kunskapsuppbyggnadoch lagstiftning för skydd av miljö och hälsa,internationellt samarbete etc.

Antogs inom EU 2005

50-tal åtgärder som Kommissionen ochmedlemsländerna skulle genomföra för attsäkerställa en “säker, integrerad och ansvarsfull”utveckling av nanoteknologi

Helhetsgrepp om forskning & innovation,industriell utveckling, etik, kunskapsuppbyggnadoch lagstiftning för skydd av miljö och hälsa,internationellt samarbete etc.

Antogs inom EU 2005

“Nanosafety” som ett integrerat policy område

Lagstiftning för

nanomaterial

Forskning och

Innovation

Nanosafety

forskning

Koordinering Kommunikation

Inom EU kommissionen…..samordning mellan generaldirektoraten

DG ENV

DG ENTR

DG SANCO

DG EMPL

DG INFSO

DG RTD

Nano och REACH – en kapplöpning i tiden

2002 Nanotech R&D ökar i EU forskning

2003 REACH antas i EU Kommissionen (ton-gräns för registrering)

2004 ”Towards”-strategin

2005 ”Action Plan 2005-2009”

2006 REACH-lagstiftningen antas av EUParlamentet och Rådet

Flertalet lagstiftningar för nanomaterial

Kemikalier – REACH, Biocider, PesticiderArbetsmiljöKonsument produkter – Läkemedel, Livsmedel,Kosmetika m.m.Miljö– Luft, Vatten, Avfall m.m.

Kemikalier – REACH, Biocider, PesticiderArbetsmiljöKonsument produkter – Läkemedel, Livsmedel,Kosmetika m.m.Miljö– Luft, Vatten, Avfall m.m.

EU Kommissionens översyn av EU lagstifning 2008 resp 2012

EU:s Rekommendation av en definition för nanomaterial 2011

Vetenskapliga frågeställningar om nano och REACH

Nya och/eller modifierade testmetoder och guidelines

Validering av testmetoder.

Metoder för att mäta och bedöma exponering

Nya och/eller modifierade testmetoder och guidelines

Validering av testmetoder.

Metoder för att mäta och bedöma exponering

Risk-bedömning och nanomaterial

Risk assessment

(eco)toxicity testsfate, transport

exposureassessment

Risk management

Hazards Exposurescharacterization, standards, characterization, standards,

reference materials, metrics, dosimetry,

validation

Scientific uncertainty

Precautionaryprinciple

Proportionalityprinciple

Internationell samordning och harmonisering

OECD Working Party on ManufacturedNanomaterials (WPMN) – utveckla internationellt harmoniserade test metoder och test guidelines att användas i lagstiftning

ISO – skapa internationella standards,nomenklatur, mät- och analytiska metoder

Testmetoder

Test Guidelines

Risk assesment

Lagstiftning

Forskning

Databas

OECD WPMNstart 2006

In vitro test

lys

Exponeringsana

lys

Livscykelanalys

Central koordinering genom myndigheter &

departement:

TysklandUKNederländerna

Jfr USA där NNIkoordinerar

Central koordinering genom myndigheter &

departement:

TysklandUKNederländerna

Jfr USA där NNIkoordinerar

Samordning i medlemsländerna

Myndigheter

Forskare

Industri

NGO

Myndigheter

Forskare

Industri

NGO

Sverige 2012 –regeringsuppdrag om nanosäkerhet

Utveckla nationell handlingsplan.Säkerställa en god samordning avarbetet med nanosäkerhet

Redovisades I oktober 2013. SOU2013:70, www.regeringen.se

Remissrunda under våren 2014 medca 70 svar

Huvudförslagen – i linje med vad som görs inom EU och andra medlemsstater

Satsning på forskning för att öka kunskap om miljö ochhälsoeffekter I ett livscykelperspektiv.

Anpassning av lagstiftning och testmetoder genom attförstärka arbetet inom EU och OECD

Bygga upp ett Nanoråd och Nanocentrum för att bättrekoordinera svenska insatser

Satsning på forskning för att öka kunskap om miljö ochhälsoeffekter I ett livscykelperspektiv.

Anpassning av lagstiftning och testmetoder genom attförstärka arbetet inom EU och OECD

Bygga upp ett Nanoråd och Nanocentrum för att bättrekoordinera svenska insatser

Ahtiainen Jukka | 27.6.2013Myndighetsmöte  Nanomaterials upptag och spridning i kroppen och miljön 

OECD‐arbetet för nanomaterialsäkerhet idag

Jukka Ahtiainen, senior forskare, PhD

OECD Sekretariat ochSäkerhets‐ och kemikalieverket (Tukes)  

OECD‐arbetet för nanomaterialsäkerhet idag –

Innehåll:

27.11.2014, Kemi myndighetsmöte‐ Jukka Ahtiainen 

• OECD Working Party on Manufactured Nanomaterials (WPMN)• Säkerhetsbedömning och testning• Testmetodernas lämplighet• Riskhantering I arbetsmiljön

• OECD testmetoder (TG) och nanomaterialer• Test Guidelines (TG) utveckling och WNT• TG tillämplighet att testa nanomateraler or behov för nya metoder och

riktlinjer (OECD GD) t.ex. testningstrategi

• Idag viktiga OECD projekten• Fysikalisk‐ kemisk‐ egenskaper• Miljöspridning och akkumulering• Ekotoksisitet• Hälsoeffekter (med toksikokinetik)

• Slutsatser och sammanfatning

OECD Working Party on ManufacturedNanomaterials (WPMN)

27.11.2014 Kemi myndighetsmöte, Ahtiainen Jukka

Working Party on Manufactured Nanomaterials (WPMN) under OECD:s 

kemikaliekomitteen var inträttade i September 2006 med uppgiften att verka för 

internationell samverkan om hälso‐ och miljöriskrelaterade frågor när det gäller avsiktligt 

tillverkade nanomaterialer.

För detta ändamål har ett antal styrgrupper bildats under WPMN

I vilka man samlar information om:

befintlig forskning (SG1 och SG3), 

testar globalt och systematiskt givna representativa nanomaterialgrupper (SG3), 

bedömer och förbättrar kemikalieprovningsmetodernas tillämpbarhet (SG4 och SG7)

och 

uppgör anvisningar för riskbedömning och riskhanering (SG6 och SG8).

http://www.OECD.org/env/chemicalsafetyandbiosafety/safetyofmanufacturednanomaterials/

OECD Working Party on Manufactured

Nanomaterials (WPMN) ‐ Vad har gjort hittills?

27.9.2012 | Nordisk råds Miljö‐och naturesursutvalg, Ahtiainen Jukka

Publikationer och riktlinjer för testning:

Guidance on sample preparation and dosimetry for the safety testing of

manufactured nanomaterials (2012)

Systematisk analysering av resultat från “Sponsorship Programme” har börjat

redan 2012 och det andra projektet (phase 2) för verklig risk bedömning skall

börjas snart (linken till EU projekten NANoREG som startade 2013).

Några riktlinjer och metoder måste förbättras, men i allmänhet test metoder

och riktlinjer för kemikalier kan följas

Varför måste OECD testmetoder utnytjas?

OECD test guideline

Good Laboratory Practise

Mutual Acceptance of data

For testing intrinsic properties of chemicals (substances)

Binding OECD countries andselected non‐members

►Avoids duplication of testing

►Reduces use of animals

►160 million euros saved eachyear (2010)

►“easily” adopted to EU regulation (440/2008) for EU regulatory needs

27.11.2014, Kemi myndighetsmöte, Jukka Ahtiainen

National Coordinators of the test guideline programme

(WNT)

TG proposal (SPSF) by MC

Stakeholders (authority, NGO, industry, academia) initiative

Draft TGs

Validation package

Expert Group

Commenting rounds

OECD secretariat

Final approval by WNT at the meeting or written procedure

Joint Meeting , policy level and publication 

3/9/

2015

Aht

iain

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Göt

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g

OECD‐metoders tillämplighet att testa nanomaterialer (NM)

27.11.2014 Kemi myndihetsmöte, Jukka Ahtiainen 

Biologiska “endpoints” (vad man observerar) är relevanta och tillämpliga

för NM testing till ex:

Number of offspring in reproduction tests‐ hur många babysar

Bioaccumulation into tissues‐ akkumulering och spridning i kroppen

CO2 production in biodegradation test‐ biologisk nedbrytning

Many apical and other endpoints in mammal tests‐ inflammationer

Några nya “nanorelevant” Phys‐Chem‐metoder behövs

Dosing and dosimetry of the test material and NM detection and

characterization very important – Riktlinjer måste utveklas

Med harmoniserade riktlinjer oh metoder MAD‐principen torde hållas

MAD = Mutual Acceptance of Data

OECD Test Guideline Programme – Hur att handla nya

nanospesifika riklinjer och instrukrioner för NM testning

27.11.2014 Kemi myndighetsmöte, Jukka Ahtiainen 

TG development and guidance documents (GDs) and MAD

If the guidance given in the separate GDs can be seen as refinement of

the test guideline, should the result be still under MAD?

Or should the NM specific guidance be inserted as annex in the TG?

Guidance for similar matrices and test or for nanomaterial groups?Metals Metal

oxidesCNTs Fullerenes NFC NCC

Soil tests

Sediment tests

Aquatic tests

Bioaccumulation

Degradation

OECD Test Guideline Programme – Hur att handla

nya nanospesifika riklinjer och instrukrioner för NM 

testning

27.11.2014 Kemi myndighetsmöte, Jukka Ahtiainen 

Inhalation toxicity testing as an example

Minor changes in Test Guidelines

More extensive revisions in Guidance Documents (e.g. GD 29)

New Guidance Document for NM testing

Guidance for similar tests or for nanomaterial groups?Metals Metal

oxidesCNTs Fullerenes NFC NCC

Inhalation tox

Oral tox

Genotox

Immunotox

In vitro

Current  OECD guidance on  NM testing

Preliminary review of OECD test guidelines for their applicability to

manufactured nanomaterials (2009)

Guidance manual for the testing of manufactured nanomaterials: OECD

sponsorship programme – rev1(2009)

Guidance on sample preparation and dosimetry for the safety testing of

manufactured nanomaterials revision published 2012

Dispersion protocols for in vitro testing (on-going work)

Development of integrated testing strategy for NM testing (started)

OECD Horizontala mötena för detta (WNT & 

WPMN)

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

Aims: Review in terms of…the need to amend existing or develop new OECD Test guidelines,…the need to develop specific Guidance for the testing of NM… identification of knowledge gaps regarding regulatory need

Topic Venue Date

Inhalation Toxicology The Hague, NL 19‐20 October 2011

Environmental Fate and Ecotoxicology Berlin, DE 29‐31 January 2013

Physical‐Chemical Properties Querétaro, MEX 28 February – 01 March 2013

Genotoxicity Ottawa, CAN 19‐21 November 2013

Toxicokinetics Seoul, COR 26‐28 February 2014

Physical‐Chemical Parameters: Measurements and Methods

Washington DC, USA 18‐19 June 2014

Categorization Washington DC, USA 17‐19 September 2014

OECD metoder och instruktioner –

phys‐chem characterization methods

27.11.2014, Kemi myndighetsmöte‐ Jukka Ahtiainen 

• The phys‐chem data is crucial for substance ID, to guide furthertesting and understand the results• Overlaps partly to env.fate methods (e.g. dissolution)• Reliable methods (MAD?) will enable triggering or waiwing of some

testing‐ verkligen viktiga och nyttiga och behövs snart!

• Proposed methods• Size, form, fiber rigidity, surface charge, zeta‐potential, surface

chemistry…• Approved list of WPMN‐men skulle vara på WNT listan!

• Current status• Not any included in the WNT project list!• Cooperation with ISO TC 229 – samarbetet borde förbättras!

OECD‐metoder och instruktioner–

ecotoxicity and environmental fate methods

27.11.2014, Kemi myndighetsmöte‐ Jukka Ahtiainen 

TG: dissolution rate of nanomaterials in the aquatic environment

Lead: US (US Army Corp) in cooperation with DTU

TG: agglomeration behaviour of nanomaterials in different aquatic media

Lead: GER (UBA), contracted to University of Vienna

GD: agglomeration and dissolution of nanomaterials in aquatic media –

decision tree

Lead: GER (UBA), contracted to University of Vienna

GD Aquatic (and Sediment) Toxicology Testing of Nanomaterials

Lead: US (US Army Corp) and UK (Uni Plymoth & Heriot‐Watt

‐ Alt dessa är viktiga, men mera behövs till ex. för jordtestning

OECD metoder och instruktioner–

ecotoxicity and environmental fate methods

27.11.2014, Kemi myndighetsmöte‐ Jukka Ahtiainen 

GD: Assessing the Apparent Accumulation Potential of Nanomaterials

Lead: UK (Uni Plymouth) with ES, NL, DE, FI

TG: Nanomaterial Removal from Wastewater

Lead: US (EPA)

GD: Leaching in soil column 

Cooperation: CAN (ENV CAN), US (EPA), GER (UBA)

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD: agglomeration and dissolution of nanomaterials in aquatic media – decision tree

• Lead: Germany (UBA); contracted to University of Vienna with EC, DTU(DK)

• Should support decision making regarding environment fate& behaviour test performance/test strategy e.g. Identification whethernanospecific test is needed,

• First information on mobility or possible  target compartments• Same timeframe like TG on agglomeration behaviour

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD: agglomeration and dissolution of nanomaterials in aquatic media – decision tree

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD Aquatic (and Sediment) Toxicology Testing of Nanomaterials

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD Aquatic (and Sediment) Toxicology Testing of Nanomaterials

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD Aquatic (and Sediment) Toxicology Testing of Nanomaterials

Current OECD activities –

ecotoxicity and environmental fate methods

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD: Guidance on Fish Dietary Accumulation Studies for Engineered Nanomaterials (content)

• Introduction

• scope of the guidance, 

• limitations of TG 305, 

• Bioaccumulation/ digestability

• kinetics with nanomaterials…)

• Decision tree and triggers for needing to do the test

• Fate indications

• In vitro digestability

• Mixing into food

• Practical in vivo testing:

• Test designs, verification of exposure, detection and analytics

• Calculations and interpretations of the results

• Use in the risk assessment

• Not BCF or BMF as such but useful information

Current OECD activities –

human health

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

GD: Guidance on nanomaterial inhalation testing

Project approved into the WNT work programme:

“Amendments to the Inhalation TGs and GD toAccommodate Nanomaterial Safety Testing (lead: US andNL)

Expertise to be broadened to WNT experts

Current OECD activities –

human health

26.11.2014, Nordic Nano WS‐ Jukka Ahtiainen 

Guidances on nanomaterial genotoxicity testing

Not yet included in the WNT work programme

OECD Workshop in Canada 2013‐ report available late 2014

Modifications of 487 (In vitro Micronucleus Test toaccommodate NMs)

Comet assay

Not to use bacterial assays

OECD‐ arbetet för nano‐säkerhet idag‐

slutsatser och sammanfattning 

27.11.2014, Kemi myndighetsdag‐ Jukka Ahtiainen 

Mera phys‐chem‐metoder (OECD TGs) behövs

Många instruktioner (guidance documents, GDs) måste utvecklas för att testa NM med OECD metoder

Alt detta menar att ECHA och dess instruktioner (ECHA guidance for registration and fulfilling the informationrequirements) spelar mycket viktig roll

Tusen tack‐ några frågor?

Nanomaterial- en uppdatering om KemI:s arbete

Elin Simonsson, Stockholm 27 november 2014

Ändringar av bilagorna till EU:s kemikalieförordning Reach

• Kommissionens arbete med bilagorna blir ytterligareförsenade.

• Tidigare utlovades ändringar innan utgången av 2014senaste beskeden är nu våren 2015.

• Arbetet pågår i CASG Nano (Reach CompetentAuthorities subgroup on Nanomaterials )

Översyn av kommissionens rekommenderade definition

• Definitionen ses över särskilt med hänsyn till kravet påatt minst 50 % av partiklarna ska vara inom nanoskalan(1-100 nm).

• Översynen ska vara klar senast i december 2014 menkommer sannolikt att försenas till våren 2015.

• Översynen är uppdelad i en vetenskaplig del och enpolicy del.

Åtgärder för ökad transparens på marknaden

• Kommissionen arbetar med en konsekvensutredning omåtgärder för ökad transparens på marknaden bl.a. ettEU-register för nanomaterial

• Ett offentlig samråd har varit en del av arbete

• Konsekvensutredningen kommer att färdigställas under2015 varpå kommissionen kan välja att föreslå åtgärder

Frågor?

Nanomaterial i kosmetiska produkter –vad har hänt sedan de nya reglerna?

Tomas ByströmEnheten för Kosmetiska ProdukterLäkemedelsverket

Snabb regelrepetition

• Förordning (EG) nr 1223/2009 om KosmetiskaProdukter

• Egen definition‘nanomaterial: ett olösligt eller biopersistent material som är avsiktligt tillverkat, med en eller fler yttre dimensioner, eller

en inre struktur, med ett spann på mellan 1 och 100 nm

• Arbetsgrupp har diskuterat justering av definitionen– Ingen ändring aktuell– Avvaktar hur definitionen i livsmedelsreglerna fungerar

Snabb regelrepetition

• Varje användning av nanomaterial måste anmälas till EU-kommissionen 6 månader före produkter släpps ut på marknaden

• EU-kommissionen avgör från fall till fall om användningen behöver granskas av envetenskaplig kommitté

– Förhandsgranskade och tillåtna nanomaterial (UV-filter, Färgämnen, Konserveringsmedel)– Granskning av produktspecifik användning (information om nanomaterialets egenskaper ska

ingå i anmälan)– Vägledningsdokument om säkerhetsbedömning av nanomaterial

• Safety assessment of nanomaterials in Cosmetic Products http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_s_005.pdf

• Relevance, Adequacy and Quality of Data in Safety Dossiers on Nanomaterialshttp://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_142.pdf

• Innehåll av nanomaterial måste framgå av innehållsförteckningen på förpackningen– Exempel: Titanium Dioxide (nano)

• EU-kommisionen ska publicera en katalog över de nanomaterial som används i kosmetiska produkter

Vad har hänt?

• 25000 produkter anmälda i hela EU– Spritt över så gott som alla kategorier– 7300 solskyddsprodukter– 7000 sminkprodukter (Carbon Black/TiO2)– 2700 ansiktsprodukter (troligen stor del dagkräm med SPF)

• 150 produkter anmälda med hemvist i Sverige– 60 solskyddsprodukter– 80 sminkprodukter

• Dålig kunskap hos en del företag som anmäler produkter (t ex har vatten och etanol anmälts som nanomaterial) – dock inte noterat i Sverige

• Ett ämne (UV-filter) godkänt som nanomaterial – flera släpar

Utmaningar för regelutveckling

• Arbetet med säkerhetsvärderingar ligger aningensent

• Data saknas vilket gör att värderingarna inte kanfullföljas

• Oklara definitioner som berör restriktioner ianvändning av nanomaterial (t ex sprayprodukteroch inhalationsrisk)

• Trög process för regeländringar som resultat av ovanstående

Vilka nanomaterial har utvärderats

• Ett nanomaterial tillåtet– Tris-biphenyl triazine (UV-filter)

• Nanomaterial som har utvärderats av vetenskapliga kommittén, regeländring om tillåtande kvarstår– Titanium Dioxide (UV-filter)– Zinc Oxide (UV-filter)– Carbon black (CI 77266, Färgämne)– Methylene Bis-Benzotriazolyl Tetramethylbutylphenol (UV filter) –

utvärdering av kompletterande data kvarstår• Pågående utvärdering

– Silica med derivat• Annan utvärdering

– Säkerheten hos sprayprodukter (när finns inhalationsrisk?)

Kommande händelser

• Katalogen över nanomaterial är på översättning försnar publicering

• Fullfölja regeluppdateringarna rörandeförhandsgranskade och tillåtna nanomaterial

• EU-kommissionen har uppmanat medlemsstaternaatt utöva tillsyn för att säkerställa att företagen görrätt– Detta med anledning av de många tokiga anmälningarna

Tack för att ni lyssnade!

Definition av Nanomaterial i livsmedel

• Förordning (EU) nr 1169/2011 om tillhandahållande av livsmedels-information till konsumenterna

• Definitionkonstruerat nanomaterial: avsiktligt tillverkat material som har en eller fler dimensioner i storleksordningen 100 nm eller mindre eller som består av åtskilda funktionella delar, antingen i sitt inre eller på ytan, varav många har en eller flera dimensioner i storleksordningen 100 nm eller mindre, inbegripet strukturer, agglomerat eller aggregat, som kan vara i en storleksordning över 100 nm men behåller egenskaper som är utmärkande för nanonivån.Egenskaper som är utmärkande för nanonivå inbegriperi) de egenskaper som avser de stora särskilda ytorna hos materialet i fråga, och/ellerii) särskilda fysisk-kemiska egenskaper som skiljer sig från egenskaperna hos samma materials icke-nanoform.

2015‐03‐26

1

Danish initiatives on nanomaterials

Flemming Ingerslev, Section of ChemicalsThe Danish Environmental Protection Agency

The Danish Environmental Protection Agency PAGE 2

Overview

• Research initiatives, consumer concern and general collaboration

• Action plans for chemicals

• “Better control of nanomaterials”

• Overview of initiatives

• The nano product register

• Future

DK outside DK-EPA

• Research

• Environmental Chemistry, DTU and Aarhus University

• National Research Center for the Working Environment (Danish Nanosafety Center)

• University of Copenhagen and others

• Industry

• Consumers

• Politicians

• Danish EPA network group for nanomaterials!

The Danish Environmental Protection Agency PAGE 3

Danish Action Plans for chemicals (in total around 2 mio DKK for nano)

• 2010-2013:

• Overview of important nanomaterials

• Proposal for REACH-annexes (IR-nano!)

• 2014-16:

• Specific products

• International work

The Danish Environmental Protection Agency PAGE 4

Better control of nanomaterials

• New government i Denmark 3. October 2011 with a green Government Programme

• National Budget Agreement 2012 on “Better Control of Nanomaterials and their Safety” (~ 3.2 mio. € 2012-2015, )

• Subproject 1:Knowledge building with focus on exposure pathways and implications for consumers and the environment with regard tothe use of nanomaterials (approx. 3/4 of budget)

• Subproject 2:Development of a nano product register in cooperation withother countries (approx. 1/4 of budget)

The Danish Environmental Protection Agency PAGE 5

Potential Environmental and health risks due to

nanomaterials in DK

Projects on NMs under the DK nano-initiative

Use/occurrence of NM

Assessment of specific products

Processes and fate of NM

Effects of NM

NP - register

Supplementary surveys

1. NM’s in food, food contactmaterials, cosmetics, pesticides

2. NM’s in pigment

3. NM’s in waste

1. Products with photocatalytic titanium dioxide

2. Aerosol products withnanomaterials

3. Textiles with nanosilver

1. Oral and dermal uptake

2. Fate processes in the enviroment

3. Human exposure

4. OECD test methods

1. Ecotoxicology

2. Toxicology

2015‐03‐26

2

The Danish Environmental Protection Agency PAGE 7

Potential Environmental and health risks due to

nanomaterials in DK

Projects on NMs under the DK nano-initiative

Use/occurrence of NM

Assessment of specific products

Processes and fate of NM

Effects of NM

NP - register

Supplementary surveys

1. NM’s in food, food contactmaterials, cosmetics, pesticides

2. NM’s in pigment

3. NM’s in waste

1. Products with photocatalytic titanium dioxide

2. Aerosol products withnanomaterials

3. Textiles with nanosilver

1. Oral and dermal uptake

2. Fate processes in the enviroment

3. Human exposure

4. OECD test methods

1. Ecotoxicology

2. Toxicology

Potential Environmental and health risks due to

nanomaterials in DK

Projects on NMs under the DK nano-initiative

Use/occurrence of NM

Assessment of specific products

Processes and fate of NM

Effects of NM

NP - register

Supplementary surveys

1. NM’s in food, food contactmaterials, cosmetics, pesticides

2. NM’s in pigment

3. NM’s in waste

1. Products with photocatalytic titanium dioxide

2. Aerosol products withnanomaterials

3. Textiles with nanosilver

1. Oral and dermal uptake

2. Fate processes in the enviroment

3. Human exposure

4. OECD test methods

1. Ecotoxicology

2. Toxicology

Potential Environmental and health risks due to

nanomaterials in DK

Projects on NMs under the DK nano-initiative

Use/occurrence of NM

Assessment of specific products

Processes and fate of NM

Effects of NM

NP - register

Supplementary surveys

1. NM’s in food, food contactmaterials, cosmetics, pesticides

2. NM’s in pigment

3. NM’s in waste

1. Products with photocatalytic titanium dioxide

2. Aerosol products withnanomaterials

3. Textiles with nanosilver

1. Oral and dermal uptake

2. Fate processes in the enviroment

3. Human exposure

4. OECD test methods

1. Ecotoxicology

2. Toxicology

2015‐03‐26

3

The Danish Environmental Protection Agency PAGE 13

Nano Product Register

Objective:

• To provide an overview of the nanoproducts that are on the Danish market, the extent ofuse and the purposes they are used for.

• To provide information to sub-project 1 (knowledge building).

• To inspire to an EU-solution for registration of nano products

The Ministerial Order no. 644 of 13/06/2014/ Bekendtgørelse nr. 644 of 13/06/2014

The Danish Environmental Protection Agency PAGE 14

The Danish Environmental Protection Agency PAGE 15

Purpose and scope of the nanoproduct register

§ 1. The purpose of this Order is to establish a register of mixtures and articles that contain nanomaterials (nano products) and which are intended for sale to the general public, as well as require producers and importers of these mixtures and articles to report to the nano product register information on said mixtures and articles and the nano materials they contain.

§ 2. The reporting requirement to the nano product register includes mixtures and articles that are intended for sale to the general public and which contain nanomaterials, where the nanomaterial itself is released under normal or reasonably foreseeable use of the mixture or article or where the nanomaterial itself is not released but substances in soluble form that are classified as CMRs or environmentally dangerous substances are released from the nanomaterial; except as provided for in § 3.

§ 3. Excemptions1. Foodstuffs and food contact

materials.

2. Feed.

3. Medicinal products.

4. Medical devices.

5. Cosmetic products.

6. Pesticides.

7. Waste.

8. Nanomaterials listed Annex IV or V of REACH (Natural substances).

9. Not intentionally produced at the nanoscale.

10. Nanomaterial is part of a fixed matrix

11. Articles or their labels on which the nanomaterial is used directly as ink, in newspapers etc.

12. Textiles with nanomaterial used as ink or fordyeing.

13. Paint, wood preservative, glue and filler that contains nanopigment added solely for colouring the mixture.

14. Articles of rubber, or rubber parts of articles, that contain the nanomaterials carbon black or silicon dioxide

The Danish Environmental Protection Agency PAGE 16

Product information

Mandatory

Information on company: ID#, adress, contakt person etc.

Productinformation: name, amount, use, professional use (Y/N)

Information on the nanomaterial:Name, REACH-registration, occurrence in product

Chemical information: IUPAC, CAS no., EU-number, formula

Voluntary

REACH: Use descriptor categories (PC, PROC. ERC, AC)

Content of nanomaterial: in product or mixture (gram or %)

Fhysical information on nanomaterial: particle size, number size distribution, aggregation, agglomeratino, form, specific surface area, crystalline state, surface chemistry, surface charge

The Danish Environmental Protection Agency PAGE 17

Important features/characteristics

• Registration is mandatory - not all information

• Registrants are those introducing the nanoproduct to the Danish marked (i.e. no intention of traceability)

• Registration is not triggered by hazards or risk of nanomaterials

• Re-use of information from the Danish product register

• Data only available for authorities and registrant (own data)

The Danish Environmental Protection Agency PAGE 18

2015‐03‐26

4

Guidance and help for registrants

• Guidance in Danish and English

• Should I register my product (nano or not?)

• 18 illustrative examples of products

• How to use IT-system?

• Example of letter to supplier

• List of common nanomaterials

• List of product-groups that may be subject to registration

• FAQ on www

• Help-desk

The Danish Environmental Protection Agency PAGE 19

Ideas for future collaboration

Danish EPA network group for nanomaterials

OECD test methods

Shared knowledge (reports, advisory groups)

Research projects

EU-work (IR-nano)

The Danish Environmental Protection Agency PAGE 20

EU‐projekt NANoREG och nano‐cellulosa

27.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

NANoREG ‐ A common European approach to the 

regulatory testing of nanomaterials

27.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

Vad och varför?

Vetenskapliga svar för administrativa frågor

Snabbare och bättre

Tillförlitligt omgivning för industrin och myndigheterna

Allt med samma takt med innovationer och inte att förhindra dom

A common European approach to the regulatory testing of nanomaterials

NANoREG project‐ facts and figures:

The NANoREG project is funded by the EU Framework 7 Programme with € 10.000.000, total budget € 50.000.000

The project started on March 1st, 2013 and runs until August 31st, 2016 (42 months)

61 partners from 15 countries (Austria, Belgium, Denmark, Finland, France, Germany, Ireland, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom)

Coordination:Ministry of Infrastructure and the Environment, The Netherlands, Tom van Teunenbroek (coordinator)

3

NANoREG ‐målsättning

27.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

The aims of this project are:

(I) to provide tools for risk assessment and decision making instruments;

(II) to develop, for the long term, new testing strategies adapted to innovation 

requirements;

(III) to establish a close collaboration among authorities and industry to come to 

efficient Risk Management approach and to bring in ”safe by design” in the 

application development phase.

verktyg strategi tillsammans med industrin

”safe by design” – ”value chains”‐ tänkandet inbildat

NANoREG‐Work package list‐ vad det innehåller

Work packageNo

Work package title Type of activity

Lead participantNo

Lead participant short name

Person‐months

Startmonth

Endmonth

WP1 Scientific answers to regulatory issues RTD 2 JRC 174,6 1 42

WP2 Synthesis, supplying and characterization RTD 4 NRCWE 621,20 1 42

WP3 Exposure through life cycle analysis RTD 7 CEREGE 535,90 1 42

WP4 Biokinetics and toxicity testing in vivo RTD 3 BAuA 680,90 1 42

WP5 Advancement of Regulatory Risk Assessment and Testing

RTD 12 NIA 1231,60 1 42

WP6 Keeping pace with innovation

RTD 5 RIVM 124 1 42

WP7 Interaction, Dissemination and Exploitation

OTH 10 TEMAS 170 1 42

WP8 Project Management MGT 1 Min I&M 30 1 42

total 3568,8027.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

Nanocellulosa och REACH

27.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

Bleached pulp cellulose (CAS‐nro:65996‐61‐4):

Exempted from REACH registration according to REACH Annex IV If grinded mechanically to NFC (without chemical modifications), 

should probably (?) stay exempted. The rationale should be given by the manufacturer.

If chemically modified e.g. with covalent bounds, would be consideredas a new substance,  and this would lead to obligation to register

NFC nanocellulose is nanomaterial by definition

Om så här nanocellulosa behövs inte registreras Men om den formuleras med kemikaliska metoden? Och vad ska man då registreras när det är en polymer? Inget nytta att registrera monomer

NANoREG och nanocellulosa (Nano Fibril Cellulose)

27.11.2014 | Kemi myndighetsmöte, Jukka Ahtiainen 

Finska koordinering på Säkerhets‐ och kemikalieverket (Tukes)

Samarbetet med industrin, forskning och myndigheten

UPM Corporation och Stora Enso (pengar finns här)

Toksikologiska (andningsorgan) studier på Arbetshälsoinstitutet i

Helsingfors

Målet: “… to assess the in vivo and in vitro genotoxic and

immunotoxic effects of nanofibrillar cellulose (NFC)”

Tack!

Box 2, 172 13 Sundbyberg 08-519 41 100

Besöks- och leveransadress Esplanaden 3A, Sundbyberg

kemi@kemi.se www.kemikalieinspektionen.se