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1 별지제 14 호서식 자체연구개발과제최종보고서 단위과제명나노독성평가를위한기술개발과제번호 나노독 601 연구과제명 제조나노물질의안전성지원센터설립을위한기반구축연구 (II) 책임자소속독성연구과성명강태석 연구자및참여율연구결과 책임자강태석 10% 연구자이규식 25% 연구자이종권 10% 연구자송치원 25% 연구자곽승준 5% 연구자김지명 5% 연구자조영희 5% 연구자강경묵 5% 연구자조샛별 5% 연구자신유지 5% 필요성 및 목표 방법 및 결과 기대성과 나노기술은차세대성장동력사업으로선정되어국가에서지속적으로제품화를위한연구비를지원하고있음 현재대부분의연구결과가제품화로이루어지기전에안전성문제가해결되어야함 국외나노기술, 안전성자료및제품동향등에대한정보수집을통해향후안전성지원에필요한정보체계를구축하는것이필요함 국내외나노안전성관련기관간기술협력체계구축 국내외나노제품출시현황과연구개발동향분석 제조나노물질의안전성, 유효성시험또는나노물질의특성분석지침검토및제정 표면처리금나노물질의표적장기에미치는영향연구 제조나노물질안전성평가관련전문연구원육성 국내나노물질안전성전문인력육성에기여 나노물질의안전성평가에새로운독성평가연구방법제시 활용계획 나노소재식품ㆍ의약품등의허용 / 규제기준제정시과학적근거자료로활용 연구개발비 ( 천원 ) 책정액 150,000 사용액 150,000 발표 ( 게재 ) 계획연보학술지명학술대회명비고보안성유 ( ) 무 ( O ) 위와같이자체연구개발과제최종보고서를제출합니다 년 12 월 31 일 독성연구과장성명 : 강태석 ( 서명혹은인 ) 식품의약품안전평가원장귀하

2 편집순서 1 : 겉표지 ( 뒷면 ) ( 측면 ) ( 앞면 ) 자체최종연구보고서 주의 ( 주의내용기재 ) ( 글 14 point 고딕체 ) 제조나노물질의안전성지원센터설립을위한기반구축연구 (Ⅱ) 식품의약품안전평가원 과제번호 :10181 나노독 601 제조나노물질의안전성 지원센터설립을위한 기반구축연구 (Ⅱ) Fundamental study for establishment of safety evaluation center of manufatured nanomaterials(Ⅱ) 독성평가연구부 독성연구과

3 주의내용 주 의 1. 이보고서는식품의약품안전평가원에서시행한자체연구개발과제의 최종보고서입니다. 2. 이보고서내용을발표할때에는반드시식품의약품안전평가원에서 시행한자체연구개발과제의연구결과임을밝혀야합니다. 3. 국가과학기술기밀유지에필요한내용은대외적으로발표또는공개 하여서는아니됩니다. 4. 이보고서내용을신문, 방송, 참고문헌, 세미나등에인용시에는 해당주관부서또는연구책임자와사전에상의하여주시기바랍 니다.

4 편집순서 2 : 보고서요약문 요약문 연구과제명 중심단어 제조나노물질의안전성지원센터설립을위한기반구축연구 나노안전성, 산학연관협의체, 나노기술및독성, 금나노 연구기간 이프로젝트에서는나노물질제조의안전성지원센터설립에기여하기위해여러목적을가진과제를수행하였다. 이과제는 (1) 시판되고있는나노물질및제품동향조사 (2) 나노물질안전성에대한연구동향및결과조사 (3) 나노물질안전성평가연구보고들을통하여나노물질및제품중나노물질의안전성평가에적당한시험평가방법의수집및분석 (4) 각국의나노안전성관련정책동향조사 (5) 표면전하 30nm 금나노입자의 in vivo 독성시험및 (6) 인력양성으로구성되어있다. (1) 시장에출시되어있는나노제품에대한연구는특허검색을포함한인터넷검색및문헌조사를통해수행하였다. (2) 현재까지 OECD WPMN 선정 14개물질에대한안전성연구에대한연구동향및그결과조사를수행하였다. (3) 14개나노물질의안전성연구결과를도출하기위하여수행한대표적실험방법을수집하고분석하였다. (4) 각국의나노안전성관련정책동향조사를위하여일본및영국의나노기술관련보고서번역및발간을수행하였으며, 미국등각국의나노안전성관련정책동향을조사하였다. (5) 표면전하를띠는 30nm 금나노입자의 in vivo 안전성시험을수행하였다. 사용한실험기법은 2DE를통한나노물질과결합하는혈액단백질분석, ICP-MS, LM 및 TEM 영상분석이다. 각기법을통한결과는아래와같다. 1 금나노물질과결합하는혈액단백질은총종이었으며, 금나노 (+) 군에서보다많은양의단백질을확인하였다. 2 금나노물질의체내분산은주로간및비장에서축적됨이확인되었으며, 특히간에집중적으로축적됨을확인하였다. 또한간및비장에서는금나노 (-) 군이금나노 (+) 군보다더많거나비슷한양이축적됨이관찰되었으나, 타장기에서는금나노 (+) 군이더많은축적을나타내었다. 3 광학현미경상에서모든장기에서세포사와관련된양상은나타나지않았으며, 간장에서는 kupffer cell 및 sinusoid에서축적양상이관찰되었다. 비장에서금나노 (-) 군은 PALS 또는적수부위대식세포에서색소침착소견이관찰되었으나, 금나노 (+) 군은책소침착소견이관찰되지않았다. 기타장기에서는투여물질과관련된소견이관찰되지않았

5 다. 4 투과전자현미경 (TEM) 영상분석에서는간및비장에금나노물질이집중적으로축적되어있음을확인하였으며, 간에서는주로 Kupffer cell 또는 Sinusoid의내피세포에축적되어있음을관찰하였고, 비장에서는대식세포에축적되어있었으며, 세포소기관중주로용해소체에분포하고있었다. (6) 나노안전성영역의전문인력양성을위해서전문연구원을채용하여본과제를수행하도록하였다. 따라서본과제의연구를통하여국내나노물질안전성전문인력육성에기여하였고, 나노물질의국제협력및안전성평가연구방법에기여하였으며, 안전성지원센터설립을위한기반을마련하였다.

6 편집순서 3 : 영문요약서 Summary Title of Project Keywords Fundamental study for establishment of safety evaluation center of manufatured nanomaterials Nano safety, Nanobiotechnology and Toxicology, gold nanoparticle Project Period Several purpose of studies were performed in this project to contribute to esablish an evaluation center of manufactured nanomaterials. Those studies are (1) the study of present condition about release of nanotechnology related products (2) the trend and results of nanomaterials safety studies (3) the analysis of method for tests of nanomaterials safety (4) the pulse-taking of politics for nanomaterial safety in several nations (5) in vivo safety tests of surface charged 30nm gold nanoparticles, and (6) human resource development for nanosafety area. (1) The study about nonoproducts in the market had been performed through internet searching (including patent survey) and literature survey. (2) The study about nonoproducts safety studies had been performed through internet searching(oecd homepage) and literature survey(pubmed). (3) The analysis of method for tests of nanomaterials safety had been performed literature survey(pubmed). (4) The pulse-taking of politics for nanomaterial safety in several nation had been performed by following that 1) the japan and UK nanotechnoloy reports had transcript and publish 2) the pulse-taking of politics for nanomaterial safety in USA and several nation. (5) In vivo safety tests of surface charged 30nm gold nanoparticles were performed. Used assays were TEM, LM, ICP-MS, and 2DE. no significant toxic effects were observed in clinical sign, mortality, body weight. naunp and paunp were found in the tissues of liver, spleen, mesentric lymph node, kidney, lung, heart, brain, testis, tyroid gland and blood by inductively coupled plasma-mass spectrometry (ICP-MS). And, the toxicity evidence of n- and paunp by LM and TEM was not found. In the results suggest that naunp and paunp did not show severe toxicity, and it was distributed and accumulated in many kind of organs. (6) For human resource development for nanosafety area, research scientists were hired than, these projects had been carried out by them. Therefore, this research contributes human resource development for nanosafety area and new opportunities for assessment of nanomaterial safety about nanotechnology and research about nanotechnology.

7 편집순서 4 : 목차 목 차 Ⅰ. 연구개발과제연구결과 제 1 장연구개발과제의개요 1 제 2 장연구개발과제의국내 외연구개발현황 4 제 3 장연구개발과제의연구수행내용및결과 7 제 4 장연구개발과제의연구결과고찰및결론 143 제 5 장연구개발과제의목표달성도및관련분야에의기여도 145 제 6 장연구개발과제연구개발결과활용계획 146 제 7 장참고문헌 147

8 편집순서 5 : 연구개발과제연구결과 연구개발과제연구결과 제1장연구개발과제의개요제1절연구개발과제의목표 가 ) 연구개발의최종목표 나노제품및제품화나노물질에대한동향정보수집 나노물질의안전성에대한연구결과및관련정책동향수집 표면처리금나노물질이표적장기에미치는영향연구 나노안전성시험연구수행을통한나노안전성전문인력양성 나 ) 연차별연구개발목표 연도구분 연구개발목표 국내외나노물질안전성평가기술동향분석을통한새로운독성평가적용기술개발 제조나노물질의 in vitro 안전성평가수행및전문인력양성 나노물질안전성평가체계구축을위한추진전략및나노물질의안전성평가가이드라인 ( 안 ) 작성 나노제품및제품화나노물질에대한동향정보수집 나노물질의안전성에대한연구결과및관련정책동향수집 나노안전성시험연구수행을통한나노안전성전문인력양성 연구개발비 ( 천원 ) 인원 (M/Y) 150, , 합계 300,

9 제 2 절연구개발과제의필요성 현재차세대성장산업으로부각되고있는 21세기미래기술로써나노기술, 정보기술, 바이오기술, 환경기술, 문화기술등을손꼽을수있다. 특히나노크기의재료를기반으로하는나노기술이전자, 재료, 의약, 에너지등의기술분야로응용성이확대됨에따라 21세기기술개발의중추핵심분야로등장하여국내외연구자들로부터많은주목을받고있다. 미국, 일본, 독일등의선진국들은정보기술, 바이오기술, 나노기술을새로운전략분야로이미설정하고중장기기술개발계획에많은예산을들여추진하고있으며, 구체적으로미국은 National Nanotechnology Initiative (NNI) 라는전략사업으로나노기술을국가개발사업 3대주요프로그램의하나로중점육성하고있다. 이러한각국의전폭적인지원에힘입어나노입자들의개발이활발해짐에따라이에대한위해성또한문제되고있다. 나노물질에대한유해성연구는국제적으로는 2003 년미국항공우주국 (NASA) 존슨우주센터연구팀이 mg 의탄소나노튜브를용액형태로쥐의폐에주입하고관찰한결과폐조직을손상시켰다고발표한것을시발점으로연구되기시작되었다. 최근의문헌조사에의하면 10,000 건의 peer review 보고서가검토중에있는데주로초미세입자들이감수성있는사람들의호흡기와순환기에장애를일으킨다고하고, 초미세입자인용접물질, 제련물질및베릴륨같은산업물질에대한안전성과위해성평가도수행되고있다. 또한 metal oxide나 carbon 초미세입자에대한동물실험도수행되고있는데이들이 blood-brain barrier 를통과한다는점이문제시되고있다. 또한 SWCNT(nanotube 의일종 ) 는 carbon 초미세입자와는다른조직반응을보이는데이러한물질은대식세포에의해서제거되지않는다. 그리고 quantum dot의하나인 cadmium selenide 나 fullerenes 는광독성이있다고알려져있다. 따라서, 이러한나노입자의유해성에대한연구작업이매우절실해지고있는시점이다. 최근선진연구기관에서는산업에많이이용되는실리카와알루미늄등의금속나노입자들의세포독성에대한연구를비롯하여나노물질의유해성연구가진행되고있으며우리에게도잘알려진 C60-Fulleren 과같은카본나노튜브의세포내축적에대한연구도활발하게진행되고있다. 나노물질의유해성은매우작은크기와나노물질특유의벌크물질 (bulk materials) 과크게다른입자의물리화학적특성으로인해발생하며석면이나유리섬유와같은침상구조를갖는물질들의암유발가능성과같은유해성에대해서는이미오래전부터잘알려져왔다. 나노물질의경우석면과유리섬유와는다르게형상적특성뿐만아니라매우작은크기로인한유해성이더주목을받고있다. 높은표면반응력과세포막투과능은나노물질들이쉽게생체에유입될수있도록하고, 세포수준의스트레스유발가능성을높이고더나아가석면처럼생체에축적되고지속적인영향을미칠수있는것으로보고되고있다. 생체를대상으로한나노물질의유해성연구는대부분은주사로인한주입이나세포배양을통해이루어지며주로금속나노물질을대상으로한다. 일반적으로나노입자는주사보다는호흡기, 구강, 피부에의해서도인체에유입되는것으로알려져있으나실질적으로유입되는경로와축적되는기관과이에대한거동연구는아직도매우기초적인단계로시작단계에불가한실정이다 년에는국내에서나노기술에관한영향평가가실시되었는데, 크게산업 경제적측면과사회 문화적측면으로나눠전문가들의평가가이루어졌으며, 나노기술가치를 35조원으로산정하고해마다 26% 씩성장하여 15년뒤면 593 조원에이른다고보고하고있고나노입자독성의연구가의무화되어야함을권고하고있다. 이에따라나노기술영향평가위원회는나노물질의안전성 유해성 독성등에대한연구를의무화하고, 평가방법을개발할것을주문하였고정부부처별역할분담에서식약청 / 복지부를나노물질독성평가의주무기관으로지정하였다. 이에따라식품의약품안전평가원 / 식약청은 2007 년부터나노물질독성기반연구사업을추진하고있다. 국제적으로 JECFA, US EPA, FDA 등에서도나노물질에대하여기존물질과다른평가방법의필요성을강조하고있으나아직까지국제적으로공인된평가방법은없는실정이다. 나노기술에대한국제적인위상을확보하기위해서는나노물 - 2 -

10 질의독성평가에대한기술및노하우를확보하는것이무엇보다중요하다하겠다 년나노기술발전시행계획을참고하여볼때 ' 09년나노기술분야에대한정부투자는 2,598 억원이다. 그리고 2010 년의나노안전성분야각부처의예산추정치는약 84억원으로 2009 년도예산을대비하여도아직은비율이 5% 이내이나나노기술제품의산업화와함께나노기술의환경 보건 안전 (EHS : Environment, Health and Safety) 및윤리적 법적 사회적영향 (ELSI : Ethical, Legal and Social Issues) 문제에대한정부의대응을가속화하여선진국수준인 5% 이상으로확대할계획이다. 이에따라식품의약품안전평가원에서는나노기술이함유된제품으로식약청의허가를필요로하거나식약청의향후관리대상물질의인체안전성확보를위한평가방법제시등전문적인지원을위하여나노제품의안전성지원센터설립을목표로본연구를 2009 년부터진행하기시작하였다. 하지만국내나노분야의산업화진행과정에서식약청관리대상물질및정식허가를필요로하는나노기술함유제품의수가현재많지않고, 아직국제적으로나노물질의안전성에대한정책적인규제는없는상황이다. 따라서국내 외나노기술, 나노물질의안전성연구자료, 나노제품동향등에대한정보수집, 국외나노물질의안전성정책동향의조사와이를효과적으로전달할수있는정보전달체계구축을통하여향후안전성지원센터의설립을위한기반을지속적으로구축하고자한다. 이러한조사연구는 2010 년부터시작되는나노안전성평가기반연구사업단의연구방향및대상물질선정에많은도움이될것으로사료된다. 또한최근나노기술은바이오분야에서매우다양하게활용되고있는데나노물질이생체내에서진단의민감도와특이도가뛰어나약물전달, 암진단및치료같은의학분야에서특히관심사가되고있다. 나노기술을이용한진단또는치료용의약품의투약은비경구투여를통하여이루어지는경우가많아정맥으로투여된나노물질은혈액이나혈청에서유래된단백질등과결합하여 corona" 를유도시켜생물적합성에영향을미치게된다. 이단백질 corona" 는매우복잡하고다양한구조를형성한다. 따라서혈액과의생물적합성은나노물질의생체내환경에서중요한요소가된다. 나노물질과혈청단백질의결합은나노물질의크기와표면성질을변화시키고이에인지, 탐식, 염증과같은원하지않는반응을유발한다. 따라서나노물질의표면처리를통하여나노물질의인지와탐식과정을막는것이대상장기로원하는약물이나유전자를전달하는데반드시필요하다. 그리하여나노물질의생체내투여에따라생체외와는다른생체내에서의물리화학적특성분석및혈액단백질과의상호작용의연구사업수행을통하여나노물질의생체내환경에서의변화를조사함으로써일반독성실험결과의예측및실험결과의해석에도움이되고자한다. 그리고이러한기전연구는향후다른나노물질의연구에있어서연구방법및결과에참고가될수있다. 마지막으로연구사업수행을통하여나노물질안전성평가를향후담당하게될전문인을양성하려고한다

11 제 2 장연구개발과제의국내 외연구개발현황 나노기술이가지는잠재적성장력은매우큰것으로여겨지고있어서전세계적으로기술선점을통한국가의산업발전및성장을이루기위해이분야에매우많은국가적인연구투자가이루어지고있고또매년증가하고있다. 우리나라도나노기술촉진법제정및나노기술종합발전계획수립등을통해전폭적인지원이이루어지고있다 년현재나노기술자체에대한최근국내분석에따르면우리나라의기술수준은미국, 일본, 독일에이어네번째수준의위치에도달해있는것으로나타났다 ( 나노위클리 319 호 pp.6~7). 표 1. 기술분야별나노기술상대수준 이렇게나노기술이발전함에따라나노기술이산업및사회에미치는영향에대한관심도따라서높아지고있다. 이분야에대해 OECD에서도 2009 년부터 2010 년에걸쳐나노기술작업반 (WPN) 2단계 (phase II) 활동을위해국제적협력을통한연구를진행하고있는데, 특히미국도관망적이었던 1단계와는달리 2단계에서는적극적인참여를통해영향력을확대해가고있다 ( 나노위클리 319 호 pp.1~ 4). 현재나노기술을응용한제품이시장에본격적으로상업화가되면서산업및사회에대한영향뿐만아니라안전성에대한관심또한날로증가하고있다. 미국의나노기술개발은국가나노기술전략 (NNI) 을통하여부처간조정을거친후추진되고있다 년에는기존의 7개프로그램구성영역 (PCA) 에서사회적차원 (Social Dimensions) 을환경, 보건및안전 (EHS) 부분과교육및사회적차원부문으로분리하여안전성연구를강화하였다 년 2월 1 1일나노기술개발수정법안 (National nano technology Initiative Amendment Act of 2009, H.R.554) 이하원을통과하여나노기술의안전성, 산업화및교육문제에초점을맞추고있고더나아가 2010 년 1월 21일에는나노물질을함유하는제품의건강및안전에대한잠재적위험에대처하기위한 2010 년나노기술안전법 (Nanotechnology Safety Act of 2010, S.2942) 을상원에제출하였다. 법안주요내용으로는미국 FDA를나노물질, 나노제품의안전성을평가하는주관기관으로정하고 FDA내에안전성관련프로그램을설치하여제품중나노기술과관련한건강및안전성을평가하고기업들에게최상의사용방법제공을목표로하고있다. 미국은향후환경 / 보건 / 안전분야연구비를현재의 5% 수준을전체나노기술분야연구비총액의 10% 까지확대할예정이라고한다. 유럽연합의나노관련연구개발은 1984 년부터추진되고있는프레임워크프로그램 (FP) 을통해추진 - 4 -

12 되고있다. Project on Emerging Nanotechnologies 의보고서에따르면, 나노안전성에관한연구에대해서 EU에서는 7차프레임워크프로그램 (FP7, 2007~13) 기간동안나노연구분야에총예산 35억유로규모의연구비를지원하는데, 이는연간약5.14 억유로 ( 약 8,889 억원 ) 규모이며, 위험평가및관리연구분야를위해최근 2년동안 (2007~2008) 5,000 만유로이상으로확대되었다. 주요영역은나노물질특성화, 인간건강과노출, 환경적영향을포함시키고있고특히특성화와독성학분야에서발전이진행되고있다. 이는전체기금중 5% 정도로나노기술에대한 EH&S 분야연구비로는세계최고규모의공공지원연구비규모라고한다 ( 향후 FP는개별나노물질의독성연구에서전반적인위험의안전평가와관리를위한보다광범위한접근방식으로이전될것이며독성연구, 직업상의노출평가와위험감소를위한합의된방법, 기술이중요한활동부분이될것이다. 일본의나노기술개발은과학기술기본계획에의해추진된이래현재제 3기과학기술기본계획 (2006 ~2010) 을수립하여연구가진행되고있다일본의나노기술개발전략중주목할만한사항은일본산업계의적극적인대응과집중적으로육성해야하는분야의선정이다. 초기의나노기술추진전략에는사회환경영향연구에대한구체적인계획이포함되어있지않았지만 2005 년부터문부과학성, 경제산업성이주축이되어나노물질이인체와환경에미치는영향, 나노기술의안전성등나노기술의사회적영향에대한관심이활발해졌다. 현재나노기술연구개발을추진하고있는기관으로는산업기술종합연구소 (AIST), 물질재료연구기관, 국립환경연구소, 국립의약품식품위생연구소등이있고이중나노기술의사회적영향문제에주목하는기관으로 AIST가대표적이다. 이외에신에너지산업기술종합개발기구 ( NEDO ) 의 나노입자의특성평가기법연구개발 에 2006 년부터 2010 년까지 5년간 20억엔, 연간 4 억엔을지원하고있으며, 후생노동성에서는나노재료의안전성평가방법과안전대책방법등에대한검토를실시하고있다 중국은기존의전통적인물리화학적연구기반체계를나노기술로의체계로비교적빨리전환시킨성공적인나라이다. 중국국무원이발표한국가중장기과학및기술발전계획강령 ( ) 에따르면나노연구를단백연구, 양자제어연구, 발육및생식연구와더불어 4대중대과학연구로꼽고있다. 이같은정부의지원아래 2009 년부터는미국보다많은수의논문발표와함께발명특허건수에서도약 4년사이에 5.7 배성장을하고있다. 나노연구분야에있어서핵심적인역할을수행하고있는중국과학원나노기술센터외에 2006 년에 나노생물반응과안전성연합실험실 을설립하여나노생물반응과안전성에관련된기초연구와나노제품안전성평가방법및안전성인증체계확립을위한연구개발을수행하고있다 년 11월영국에서는나노안전성연구센터 (NNRC: National Nanotoxicology Research Centre) 를설립하여나노물질의특성을규명하고인체, 환경, 생식과미생물안전성에대한연구를통합적으로연구하게되며, 협업연구를통해광범위한나노물질의제품의호흡, 섭취및피부를통해인체에흡수될때의그영향에대한독성시험을실시하고있다. 향후 NNRC는나노물질로인한인체건강의위해함에대한이해를증진시킴으로써영국의나노안전성관련정책마련에있어공헌하게될것으로기대하고있다 ( 영국 ( 에딘버러 ) The Journal지 Chris Grainger; 온라인판 (Issue로게재 ) ( ). 우리나라의경우는나노연구사업전체예산은 2008 년 2,429 억 4 천 9 백만원, 2009 년에 2,457 억 7 천 7 백만원이었다. 이중 EH&S 분야연구비는미미한수준으로, 환경부와식약청예산이이분야에해 - 5 -

13 당한다고보고추정하면, 2008 년에 53억 5천4백만원 (0.022%), 2009 년에 113 억1천만원 ( 약 0.055%) 이다. 이는 2년에걸쳐연간약 83억 3천2백만원정도가나노기술 EH&S분야에지원된것으로생각할수있는데, 선진국에비해서는절대금액뿐만아니라그비율에서도많이미치지못하는것이다. 그럼에도불구하고, 비록영국보다는그설립이늦어졌으나, 우리나라에서도향후나노안전성센터설립을위해기반구축연구를통해기술적 인적인프라를구축하여나노물질 / 제품의안전성연구를지원함으로써식약청관련나노제품의산업화를지원하는데이연구의목적이있다. 현재까지는나노기술및나노기술특성분석 / 검증, 안전성평가등나노기술관련전분야에대한국제적인표준이부족한상황에서식품의약품안전평가원은 OECD와의공동협력연구참여및각부처와의협력을강화하여표준화작업에참여하고있다특히 ISO/TC 229에용어에대한표준가이드라인으로 2009 년현재까지 2개의확정된표준안을제시하고있다. 이두개의표준은 (1) ISO/TR :2008 Nanotechnologies -- Health and safety practices in occupational settings relevant to nanote chnologies 와 (2) ISO/TR 27687:2008 Nanotechnologies -- Terminology and definitions for nano-ob jects -- Nanoparticle, nanofibre and nanoplate 으로출간되어유료 ( 각각 및 스위스프랑 ) 로제공되고있다

14 제 3 장연구개발과제의연구수행내용및결과 제 1 절국외나노안전성관련정책동향 미국의나노안전성관련정책동향 1. 배경 미국정부는 2011 회계연도 ( ) 정부예산액으로 National Nanotechnology Initiative(NNI) 의연구개발비로 17억 6,000 만달러를요청하였고이중작년대비예산이크게증가한분야는 EHS 부분인 PCA7( 환경, 보건, 안전 ) 로 1.2 억달러가투자된다. 작년대비 28% 가증가하였고예산증가는 EPA, 국립산업안전보건원 (NIOSH), 및 FDA의예산증가에기인한다 년 1월 21일나노물질을함유하는제품의건강및안전에대한잠재적위험에대처하기위한 2010 년나노기술안전법 (Nanotechnology Safety Act of 2010) 이상원에제출됨에따라미국FDA의나노물질및나노제품의안전성에대한연구및감독이더욱강화될예정이다 미국 EPA 연구개발국 (ORD) 에서나노물질을연구하고그결과를근거로제조나노물질의개발및사용으로인한잠재적악영향을최소화하고이익을극대화할규제기준마련하고자전략보고서발간하고있다. 2. 나노기술안전법및 FDA 나노제품관리방안 이법안의공식명칭은 나노기술프로그램의신설을위한연방식품ㆍ의약품ㆍ화장품 (Federal Food, Drug, and Cosmetic Act) 을수정하는법 이다. 주요골자는 2011 년부터 2015 년까지연 2,500 만달러를책정하여 FDA를나노물질ㆍ나노제품의안전성평가를수행하는주관기관으로정하고 FDA내에안전성관련프로그램을설치하여일상제품중나노기술과관련한건강및안전성을평가함과동시에기업들에게최적의사용방법을개발하며제공하고자한다. 수정내용은식품ㆍ의약품ㆍ화장품법제 10장의마지막에나노기술프로그램섹션 (SEC.1011 Nanotechnology Program) 을추가하였다 신설프로그램내용소개 일반나노물질의생물시스템과의상호작용및 FDA 관리제품의나노물질에대한과학적문헌및데이터의평가 각나노물질의생물시스템과의거동에대한일반화된원리의공식화를위해 DB와모델을이용한정보의개발과조직 부처내프로그램의촉진, 공동연구참여, 나노스케일에서독성에기여할수있는신규물성에대한과학적이해의심화 나노크기물질의측정과검출의이해를위해부처간협력촉진 나노물질과생물시스템의상호작용과관련한과학적정보와데이터의수집, 해석및전파 FDA 내에나노물질에대한전문지식축적 진행중인훈련과새로운정보가부처내및부처간에시의적절한전파 국제적ㆍ국가적표준활동참여

15 2006 년미국 FDA에서는나노기술에대한과학적현실과관련하여 FDA의규제권한문제를파악하기위해 Nano Task Force 를출범시켜과학적문제와규제정책에대한권고사항을제시하였다. 권고내용으로시판전에당국의규제를받지않는제품 ( 식품보조제, 화장품식품등 GRAS군제품 ) 및규제대상제품 ( 의약품, 생물학적제품, 식품, 의료기기생물학제품, 의료기기 ) 과관련하여 FDA가규제정책을효과적으로시행할수있도록나노기술에대한지식을함양하는데초점을두고있다. 과학적기반을둔안전성과위해평가에필요한연구를수행하는유일한미국정부기관으로서 FDA는 2011 년부터진보된규제과학적주도권을유지하기위해산ㆍ학ㆍ연과협력할수있도록 Nanotechnology Collaborative Opportunities for Research Excellence(CORE) 프로그램등을통해 FDA 주요관리제품에대한안전성확보를계획하고있다. 주요내용으로첫번째로나노기술제품과관련안전성을평가하기위한실험실구축을주된내용으로나노물질을포함한제품의안전성, 효능성을시험할수있는방법개발및표준화작업, 두번째로나노제품의안전성을적절하게평가할수있는직원양성과교육프로그램개발, 세번째로나노물질측정과감지방법및물질특성에따른영향평가등을포함하고있다 3 EPA 의나노물질연구전략 나노물질과관련된환경보호과제는나노물질을개발하고사용하는과정에서인간및생태계에예기치않게발생하는노출을예방하거나최소화하는일이다. 이와관련하여 EPA가나노물질과환경에관한정보를개발하기위한일환으로관련정부부처, 기업, 학계, NGO 등과함께 1) 경제협력개발기구 (OECD) 제조나노물질작업반 (WPMN) 참여 2) 나노기술관련환경ㆍ보건ㆍ안전 (EHS) 연구를위한국가나노기술이니셔티브 (NNI) 전략수행 3)EPA 나노물질연구전략 (Nanomaterial Research Strategy, NRS) 개발 4) 오염방지및산업계등여러분야에서나노물질의환경ㆍ보건ㆍ안전정보를자발적으로제출하도록하는 EPA 나노물질관리프램 (Nanomaterial Stewardship Program, NMSP) 시작등 4가지역할을수행하고있다. 현재매우다양한나노물질이존재하며그종류가증가하는추세이나예산등을감안하여단일벽탄소나노튜브 (SWCNT), 다중벽탄소나노튜브 (MWCNT), 플러렌 (Fulleren), 은 (Silver), 산화세륨 (cerium oxide), 이산화티티늄 (Titanium oxide), 영가철 (zero-valent iron) 7개제조나노물질을선정하여각물질에대해다른국가와도공동협력연구를통해집중적으로연구하는데초점을두고있으며예측모델및도구의개발을최종목표로삼고있다. EPA는아래와같은 4개연구주제로분류하고이에따른 8개주요세부연구과제를수행함으로써제조나노물질의개발및사용으로발생하는잠재적악영향을최소화하고이익을극대화할규제를마련하고자한다 3.1 연구주제 오염거동및노출연구 환경및생물샘플에서의제조나노물질검출 정량화를위한기술개발연구 - NIST, NCI, DOE와협력하여나노물질분석법개발 - 환경매트릭스에서탄소나노물질및비탄소나노물질을검출하기위한분석법개발 - 타기관과협력하여다양한환경매트릭스에대한기준시험물질개발 제조나노물질의거동및변형에영향을주는주요프로세스및물리 화학적특성과의관계연구 - 제조나노물질의거동 ( 흡착, 분산, 확산, 응집, 분해등 ) 을좌우하는프로세스규명 나노물질의화학적 표면적특성은자연적으로발생한성분들과상호작용하면서변화하여나노물질의거동에변화를가져옴 - 제조나노물질의물리 화학적특성및그것이거동프로세스에영향을미치는과정파악 - 8 -

16 - 제조나노물질의거동에대한예측모델개발 제조나노물질의노출평가연구 - 연구대상 ( 사람, 생태계 ) 의수용체에대한주요노출경로규명 - 나노물잘의노출정보를얻기위해필요한물리하학적특성규명 - 유전체학, 단백질체학, 대사체학을적용하여노출지표규명 위해성평가및실험방법고시를위한인체및생태계영향연구 제조나노물질의인체영향및정량화방법연구 - 물리화학적분석 ( 나노물질의순도확인및특성검증, ADME을규명하기위해세포및조직에서검출및정량화수행 ) - 나노물질독성평가를위한대체실험방법 ( 세포및비세포 ) 개발노력지원 - 다양한노출경로에따른생체내독성실험 제조나노물질의생태계영향및정량화방법연구 - 제조나노물질의유해성평가시기존실험법의적합성여부검사 - 생태계에대한나노물질영향의근본매커니즘을파악하고유해성평가절차의잠재적격차확인 - 생태계유해성또는위해성을예측할방법및모델개발 위해성평가방법개발연구 제조나노물질의특성에따른위해성평가방법의개선연구 - 나노기술은새로운분야이므로종합환경평가 (comprehensive environmental assessment, CEA) 같은신중하고체계적인평가방법이필요함. CEA의경우나노물질에관한위해성평가를대기나수질, 독성, 폐기물중어느특정분야에만국한하지않고전반적인영역을다루고있음 ( 나노물질의 life cycle, 사례연구등 ) 위해성예방및관리연구 배출가능성이높은제조나노물질의위해성을최소화하기위한규제및시행방법연구 - 나노물질의잠재적영향을이해하고노출감소방법을강구하려면나노물질의환경진입점 (entry point) 이될수있는것들을파악해야함 - 나노물질의환경배출가능성을보다명확히파악하기위해제조, 사용, 최종처리형태에대한체계적평가실시 - 나노물질및제품의제조과정에서나노물질배출을최소화하거나예방하고에너지소비를줄일수있는제조공정설계 제조나노물질의친환경적이용방법연구 - 나노기술을적용하여환경문제를해결해야할필요성대두로나노기술을산업공정에이용한폐기물및부산물의최소화 - 기존오염물질의저감및오염환경의복원에나노물질을사용할경우어떠한실용성, 성과, 장점이있는지보고할계획 - 9 -

17 영국나노안전성관련정책동향 1. 배경 영국정부는나노기술의잠재적편익을인식하고 2004 년왕립학회와왕립공학아카데미 (Royal Society and Royal Academy of Engineering) 의권고에따라범부처간나노기술관계장관회의 (Ministerial Group on Nanotechnologies), 연구프로그램의우선순위를결정하는나노기술연구조정그룹 (Nanotechnology Research Coordination Group) 결성, 나노기술이해관계자회의및환경ㆍ보건ㆍ안전 (EHS) 지식기반마련등다양한방식으로안전한사용을촉진하기위해노력하여왔다. 영국이나노기술개발의편익을최대한누릴수있도록마련한구체적실행계획으로는첫번째로산업계및학계나노연구를지원, 나노기술인력양성및교육강화를위한비즈니스, 혁신과산업분야육성, 두번째로나노소재의사용과노출리스크에대한이해증진을위한환경ㆍ보건ㆍ안전 (EHS) 연구강화, 세번째로나노소재관련정책및규제개선을목표로한규제마련, 네번째로세계무대에서영국의주도권유지를목표로한국제활동강화등이다 나노기술의개발과사용단계에서보건과환경을지키는것이정부의역할이며 EHS 연구는적절한규제마련에필수적이며소비자들이출시된제품을안심하고구입할수있는지식기반을제공한다. 2. 영국의나노기술현황 영국나노기술현황나노기술기업수에있어미국과독일에이어 3위, 특허출원수는 4위, 나노광학과나노재료에있어상당한강점을유지하고있다. 영국은기업혁신기술부 (BIS) 를통하여나노기술표준개발을이끌어내는데중요한역할을하고있으며국제및유럽표준화기구의대다수나노화이니셔티브는영국이제안한것들이다. 영국은매우견실한나노기술기반을확보하고있으나나노기술애플리케이션을개발하는기업대다수가중소기업이라전략적방향을제시하는데한계가있다. 이러한문제를해결하기위해서는부분간의문제를조율할수있는응집력있는리더쉽이필요하다. 마지막으로연구의중복을피하고산업계와학계간의커뮤니케이션개선및나노기술의개발을지속하고혁신을추구하기위해서는충분한기술력을갖춘인재육성이필수적이나현재적절한교육프로그램부재와고비용교육경비가걸림돌이다. 3. 영국의환경, 보건및안전 (EHS) 연구 나노물질이인간의건강과보다광범위한환경에미치는영향이얼마나다른지를이해하려면이분야에대한연구가필수적이다. 정부및관련이해관계자들도 EHS 분야에대한정부활동조율이불충분하며이분야에대한투자를늘려야한다고인식하고있다. 3.1 국내활동 2009 년 11월에 HPA(Health Protection Agency) 는국가나노독성연구센터 (National Nanotoxicology Research Center) 를설립하여나노물질의특성을규명하고나노섬유 / 와이어 / 튜브를포함하는광범위한나노에어로졸로흡입된나노물질의생역동학 (biokinetics) 을조사하여 2010 년 12월에완료할예정이다. 식품기준청 (FSA) 에서는음식물소비에따른소화기관에서의나노입자의작용과동태를조사하기위해 in vitro 흡수모델과독성동태를조사할예정이다. 한편 Food and Environmental Research Agency 에서는식품내의제조나노물질검출을위한여과법

18 개발에중점을둘것이다. 보건성 (DH) 에서는나노물질이건강에미치는영향연구와관련하여탄소나노튜브, 흡입된나노입자의독성, 외과수술에쓰이는나노입자및죽상혈전증 (atherothrombosis) 과나노독성등 5개연구를지원하고있다. 보건안전성 (HSE) 은나노기술이야기하는위험을이해하여기존의직업관련규제프레임워크와새로운위험의규제에대한타당성을알릴수있는연구프로그램을지원하고있다. 특히제조나노물질에노출될수있는근로자, 공장, 연구소및소매환경과같은작업현장에서발견되는노출수준에주안점을둘계획이다. 3.2 국제활동영국은환경식품농업부 (Defra) 가주도하고보건안전청 (HSE) 의지원하에 OECD WPMN 프로그램에서중요한역할을맡고있다. 영국은산화세륨과산화아연등두가지우선순위나노물질의조사를주도하고있다정부기관과영국산업계가공동으로자금을지원하는 PROSPECT 프로젝트에서는두가지물질에관한기초과학연구수준을해소하고측정문제에대한혁신적인개발을위해 370만파운드를제공하고있다. 미국 EPA와공동협력연구로나노물질의전달, 동태및생체유용성과생체및생태계와의상호작용을정확하게예측하는모델개발사업으로제출하여 2010 부터 4년간 500 만파운드를투입할예정이다. 지금까지영국은유럽연합공동연구지원사업 (FP7) 프로젝트의 Nanosciences, Nanotechnologies, Materials and New production Technologies 분야로부터 1,000 만파운드이상의자금을유치하였다. 정부는 FP7을통하여이루어질향후결정의범위와내용에대한자문을제공하면서영향력을행사하기위한영국의협상지위를높이고있다 3.3 독성학기술 2008 년 Royal Commission on Environmental Pollution(RCEP) 보고서에서는신소재및제품의성공적인개발에있어독성연구가극히중요하므로새로운독성학자의부족을나노기술분야에대한위험으로파악하였다. 파악된주요관심사로신입사원의기초실무부족과계산능력결여및직업선택에있어독성학의가시성을언급하고있다. 기업혁신기술부 (BIS) 는아동교육가족부, 과학기술공학및수학네트워크 (Science, Technology, Engineering and Mathematics Network : STEM-NET) 와협력하여독성학학위의지식을보다광범위한과제의일부로포함시켜 STEM 자격요건에포함시키도록장려하고있다 4. 규제 바람직한규제는투명하고책임을지며, 적절하며, 일관성이있으며대상이분명해야한다. 나노물질을포함한물질의규제는부작용이없이그물질을제조사용하고폐기함이매우중요하다. 나노기술에대하여정비된규제가가져다줄이익으로는산업계가프레임워크내에서위험을효과적으로관리할수있다는사실을인지시켜혁신적인제품을개발할수있도록하고한편일반대중들은제품을신뢰할수있음과동시에자신들에게충분한선택권이있다는것을느끼해할수있다는점이다. 나노물질은국가및유럽수준에서단일법제가아닌다수부분또는제품특유의규제하에서관리되고있다. 이는각나노물질을특정용도에따라적절하게규제하는것이그목적이다. 정부부처와기관들은모든분야의규제와정책이나노물질에적절하게적용되도록협력하고있으며 BRASS(Center for Business Relationships, Accountability, Sustainability and Society) 가문제해결을위하여노력하고있다. 아래각항에는나노물질이인체에접촉할가능성이높은분야에적절한규제가이루어지기위한활동들이기술되어있다

19 4.1 식품나노물질이함유된식품은 EU 일반식품법규정의안전요구사항의대상이며출시되는식품이안전해야하고신개발성분, 첨가제및식품포장재에대한특정법률도있다. 식품기준청 (FSA) 은식품에영향을미칠가능성이매우높은나노기술의사용에는사용허가전에어떤형태의승인과정이필요하다고밝히고있다 신개발식품영국은 1997 년이전에식품으로판매되지않았던제품들을신개발식품 ( 식품첨가제제외 ) 의범위에포함시켜출시전안전평가를거치도록제안서에제조나노물질을포함하는것을지지하고있다. 유럽의회는 2010 년에제안된개정검토를하여채택될경우 EU 규제가 2012 년에전면적으로실시될것이며영국식품기준청에서는유럽위원회와회원국간의협상에적극적으로개입하여지속적으로필요한수준의소비자보호가이루어지도록추가개정에대한압력을가할예정임 식품첨가제 2010 년 1월부터시행된유럽의회및유럽이사회규제에따라관리하고있다. 이개정에는나노기술이관련된기존식품첨가제의제조방법에대한변화가있을경우 EFSA(European Food Safety Authority) 의재평가를받아야한다는점을명시하고있다 식품포장재유럽위원회나회원국에는 EFSA가식품포장재의제조에사용되는물질이나화합물에대한독립적인전문인체건강위험평가를요구하는규정이있다. 따라서 EFSA의위험평가를거친후에나노물질을포장재로사용할수있다. 영국도나노형태의개별물질에대한식품포장재사용을허가하기전에안전에대한충분한평가를거치도록하고안전한사용의정착에필요한규제를실시하기위한사례별접근법을장려할계획이다. 4.2 의약품및의료기기 2006 년의약품위원회 (Commission on Human Medicines) 과유럽의약품기준청 (EMA) 가나노기술에대한특정규제의필요성을제기하지않았으나 EMA는전담나노기술그룹을신설하여체계적인지침을마련할충분한자료가수집중이고필요시구체적인지침을마련할것으로보인다. 유럽의료기기전문가실무그룹 (European Medical Devices Expert Working Group) 은특정나노기술태스크포스가제시한정보에기초한의료기기규제프레임워크의타당성을평가하고있다. 의약품건강관리제품규제청 (MHRA) 는의약품및의료기기에대한 EU 규제가충분히엄격하며나노기술과관련된위험을포함할만큼범위가넓다고판단하고있다 4.3 화장품화장품안전에관한 EU지침 (Directive on the safety of Cosmetic Products) 하에서모든화장품은소비자가사용하기에안전한제품인지를확인하는평가를거쳐야하지만나노물질은구체적으로언급되어있지않다. 평가에는아래와같은세부사항이포함되어야하며제조업체가보고한부작용을고려해야한다 - 사용된각성분의일반독성프로파일 - 각성분의화학적구조 - 각성분의노출기준 - 화장품이사용되는부위의특정노출특성 - 화장품을사용하는개인별부류의특정노출특성 2013 년 7월 11일부터개정된화장품에관한 EU 규제 (Regulation on Cosmetic Products) 가시행될예정이며이규제에는필수안전성평가가상술되어있지만제조업체가나노물질이함유된신규화장품출시 6개월전에이사실을유럽위원회에통지할것을요구하고있다. 제조업체가유럽위원회에보고해야할나노물질에관한보고에는나노물질의식별, 나노입자의크기, 물리화학적성질, 시장에출시할예상수량, 노출조건을예측할수있는독성프로파일과안전데이터등이있다

20 독일나노안전성관련정책동향 1. 배경 나노기술의잠재적인위험성을보다효과적으로평가하기위해서는나노입자의방출이환경과건강에주는영향에대한지식과정보를확대하는것이중요하다. 독일연방교육연구부 (BMBF) 는건강과환경에대한영향조사와관련하여 Nanocare 프로젝트를발족시켰으며이프로젝트는개발초기단계에새로운나노물질또는나노구조물질의취급으로인한위험성을조사하고그결과를이해관계가있는기업과일반대중에게전달하기위해기업이나산업분야의참여자가함께수행토록하고있다. 2. 독일의나노기술추진전략 독일나노기술현황다른국가와의경쟁력에있어독일은나노기술잠재력의효율적활용이절대적으로필요한시기에직면하고있음. 현재독일에서활동중인나노기업은약 750 개이며 2007 년 R&D분야의투자는약 47억유로로미국, 일본다음으로세계세번째로큰규모이다. 나노기술에있어독일의강점은잘구조화된 R&D 인프라와다양한세부나노기술분야의높은연구개발수준의확보에있으며이들연구개발결과를활용할수있는산업적기반도함께보유하고있다. 독일은나노기술을활용할수있는훌륭한토대를가지고있음에도불구하고, 미래에기술적그리고경제적도전에대한요구가증가하고있음을인정하고대처하고있다. 미국과아시아권에비해독일은연구개발결과를제품화시키는데취약하나이것은실질적으로나노기술이점과위험요인에대한평가, 홍보와소비자요구에대한대응, 규제와표준화과정에대한필요뿐만아니라연구결과를이용한노력의증대에대한강한도전에독일이직면하고있음을의미한다. 3. Nanocare 활동 리스크연구 Nanocare consortium 은 2005 년설립하여산업적으로생산된나노물질의건강위해지식과정보를위한연구를수행하고있다.( 2009 년까지 8백만유로지원 ) EU REACH 제도하에서나노물질의등록이이루어지더라도새로운나노물질은현재의시험방법에감지되지않아영향을평가하지못하는경우가발생할수있다. 이런관점에서 Nanocare 는영향을정확히평가할수있는표준화절차를개발하고이과정에서새롭게적용되는나노기술에대한안전성문제를제기하고더안전한나노물질을확보하기위한해결책을찾아내려고노력하고있다. 물질특성에대한이해가높을수록잠재적위해요소에대한평가가가능해지고예방도할수있기때문에연구자들은 Nanocare 프로젝트를통해이목적을달성할수있을것으로기대한다. 관심대상물질 Nanocare 의중점연구대상물질은크게 3가지로구분된다. 첫번째로산업적카본블랙, 두번째로산화아연, cerium oxide, 이산화티타늄으로대표되는 Metal oxides, 세번째로 Barium sulfate 및 Stronium carbonate 등이다.

21 화학물질명영문명 ALOOH BaSO 4 CeO 2 SrCO 3 ZnO ZrO 2, mixed oxides(ti-al-zr, Ti-Zr) TiO 2 Boehmite Barium sulfate Cerium dioxide Stromtium carbonate Zinc oxide Zirconium dioxide Titanium oxide Carbon Black(reference material) Nanocare Organization chart 주요연구분야로나노입자생산 (WP1), 입자표준화및물리화학적특성분석 (WP2), 적절한 in vitro model 개발 (WP3a), 동물모델에서의 in vivo validation(wp4), 작업장에서의노출평가, 새로운측정방법의개발 (WP5), Nanocare 에서수집된자료의분석 (WP6), 국민과전문인을상대로한소통등지식정보전달연구 (WP7) 로구성되어있다. Generation of Knowledge Management of Knowledge Transfer of Knowledge Project Management WP6 Knowledge Basis Portal WP7 Transfer Dialogue Meetings WP1 NP Production WP2 NP Characteri- Zation WP3a in vitro Models WP3b in vitro End points WP4 in vivo Validation WP5 Reality Occupat Exposure 4. Nanocare 성과 프로젝트참여자에게는표준작업수순서 (SOP) 에준하여비교할수있는결과생산을요구함 나노입자와세포와의상호작용연구 - in vitro 시험으로기존의시험방법표준화및새로운나노독성측정방법개발에집중 - 랫드를이용한흡입독성연구에서 pulmonary tissue나 fluid에서효소의활성과단백질량분석연구 이산화티타늄연구성과

22 - 나노구조이산화티타늄은백색이지는않지만투명성이우수해자외선차단제로많이사용된다. 현재식품에는사용되고있지않다. - 독일은 OECD 안전성시험에서이물질에대한 lead sponsor 국가로서흡입독성, 피부통과여부및관련독성기전등광범위한연구결과를생산하고있다 - 광범위한 European NANODERM 프로젝트결과를통해나노입자는건강한피부를투과할수없는것으로알려져있다. - in vivo 연구를통해나노이산화티타늄은낮은농도에서폐에병리학적반응을유발시키지않았으나, 고농도에서는 in vitro 시험결과에일치하는염증반응을유발하였다. 그러나염증반응은장시간지속되기보다는며칠후에진정되는양상을보여주었다. - 현재의 Nanocare 시험은고농도로비교적단기간에대한영향을관찰한것으로장기간저농도노출에대한시험이필요한상황이다. - 폐이외의다른장기 ( 간, 신장, 비장, 뇌림프절 ) 에서의잔류량에대한연구도함께수행되고있음 작업장에서의노출평가성과 Nanocare 프로젝트의중요한연구는나노물질을생산또는공정하는작업장에서의노출연구분야로 Dusting testing, 사고방출시입자분산에대한컴퓨터모델링, 측정기술과방법의개발, 작업장에서의나노입자의농도측정등 4가지로세분하여프로젝트를수행하였다. 그결과 4개산업소재지를선정하여 11개작업장에서 Nanocare 가개발한측정전략을적용한결과어떤작업장에서도나노입자가심각한나노집합체나응집체를증가시키지않는것으로관찰되었다. Nanocare consorcium 15개의대학및비대학연구기관으로구성 - BASF, -Bayer MaterialScience - DECHEMA - EVONIK - SOLVAY - SusTech - Nanomat - VDI Technologiezentrum - Westfalische Wilhelms-Universitat - ItN - Universitat Des Saarlandes - Universitat Bielefeld - IGF - Forschungszentrum Karls - IBE Nanocare 프로젝트내에서 11개물질을대상으로독성시험을수행한결과, 거의모든물질에대해독성영향을보이지않는역치 (threshold) 를결정할수있었다. 그러나고동도로존재시염증을일으키고있으나이증상은전형적으로나노물질이아닌경우에도관찰되고있는것으로대부분시간이경과됨에따라회복되었다. 시험한물질에따라서로다른생물학적영향을나타내고있어나노의활동을일반적으로방법으로평가하기보다는물질의구성, 크기, 구조등이고려되어져야할것같다

23 5. 향후추진사업 독일이국제적으로나노기술의위해성평가에선도국가가되기위해서독일연방교육연구부 (BMBF) 는 Nanocare 프로젝트가종료되기전이라도환경 (NanoNature) 과건강 (NanoCare) 에집중할수있는 2개의예산프로그램을추진하기로결정했다. 향후프로젝트는제조나노물질의흡수, 분포, 배설및세포구성물과의상호작용을이해함으로서위해평가를지원할수있을것으로기대하고있다

24 프랑스나노안전성관련정책및규제동향 1. 배경 오늘날치열한국제경쟁가운데나노기술은수많은가능성과동시에논란을제시하고있고최첨단연구개발대상으로부상하고있다. 5년전부터프랑스에서는관계부처와과학기술분야의우수기업들이참여한가운데연구와활용에중점을둔다양한프로젝트를실시하고있다 2009 년프랑스정부가공공토론위원회에요청하여토론을통해국민을대상으로정보를제공하는한편의문점이나기대및건의사항을파악하여정책적으로활용할평가요소를마련하고자환경및지속가능발전부, 경제산업부, 노동부, 연구부, 국방부, 보건부, 농업부가공동으로참여하여객관적인시각에서투명하게나노기술의문제점을제시하였다 2. 프랑스나노기술적용분야현황 프랑스는영국과같이기초연구는매우발달하였으나특허나기업창출을통해경제적부가가치를창출하지못하는상아탑범주국가에속한다 년까지세계 5위권이상으로진출하지는못할것으로예상하고있으며, 빠른시일내에재도약하지못한다면한국, 중국에도뒤쳐진마이너그룹으로후퇴할것으로우려하고있다. 아래에서는프랑스나노기술응용분야중한국식약청관리제품과관련된유사분야에한하여소개하고자한다. 2.1 보건분야향후수십년간변화중평균수명연장과유목민적인삶의방식확대가예상되는가운데이두가지변화가보건의료체계에도뚜렷한영향을미칠것으로예측하고있음. 질병의조기발생가능성에대해의료비의지속적인증가가따라갈수없는상황에서전체의료사이클 ( 예방검진, 진단, 치료 ) 을고려한혁신이이루어져야되는상황에서나노기술이러한사이클을축소최적화하면서혁신적인제품개발에기여할수있다. 나노구조물의극민감 (ultrasensible) 탐지를통해분석력이향상될전망인데일례로암세포성장을조기에알려주는분자를탐지할수있어합리적비용으로환자의조기치료를통한생존율향상을도모할수있다. 기존의약물투약은거동부위로운송되어야하는제한성때문에어려움이있으나나노미터규모의빈캡슐에약물을넣어용해성을향상시키고질병세포에만투약이가능하다. 의료기기업계에는항균작용에효과적인은나노결정체를이용하기도하고보철임플란트등의료장비발전에기여한다. 나노기술은머지않아대상질병세포의효율적파괴, 치료효과극대화, 부작용감소, 재생의학및맞춤식치료법을구상하기위한과정에서결정적인요소를제공할것이다. 2.2 식품및농업분야나노소재는음식물자체에구성분의형태로첨가되거나위생안전및관리향상목적에사용되는저장및포장자재로서이용된다. 농업분야에서는식물보호와수의약품및살충제등가축건강을위해사용또는사료등에서응용된다. 자연에존재하는나노물질은잠재적으로인간이섭취하는농산품의오염을유발할수있기때문에농업활동에있어매우중요한해결과제이다. 식품응용분야로서현재유럽및국제식품시장에서는나노캡슐형비타민이나오메가3 등건강보조제와

25 기능식품등에서응용되고있다. 나노기술은불용성물질의분산을유도하고가공공정에취약한분자를보호하며식품의맛과색을형성하는화합물을방출하는등의목적으로활용된다. 나노기술은목적별로식품의다양한구조화를가능케하고이를통해얻어진활성소재는향상된감각적특성과안전성을보이고효과적인확산성과전반적으로향상된생물가용성을나타낸다. 이러한방식으로활성소재가더많이첨가되면전체적으로필요한소재의양이최소화시킬수있다 2009 년프랑스식품위생안전청 (Afssa) 의조사결과와 2008 년경제재정산업부산하경쟁소비자문제부방지총국 (DGCCRF) 조사에따르면나노물질을포함한식품은연구단계로몇명제품을제외한상품화를제외하고는프랑스국내식품시장에서상용화되지못하고있는실정이다. 유럽지역내나노식품에대한구정으로인해보급은매우제한적이며엄격한규정을통해소비자를대상으로무해성보증이이미실시되고있다. 나노물질이포함되는경우판매에앞서프랑스또는유럽식품위생관련기관의전문평가를거쳐당국의허가를취득해야한다. 포장의경우에는식품과의접촉에있어적합할수있도록불활성포장이이루어져야한다 포장및식품이력추적나노기술은현재대부분포장, 절단면, 냉장고저장칸등식품과접촉하게되는소재분야에서활용된다. 활성소재라불리는신소재에는식품내박테리아의확산을막아소비자의중독위험을감소시키기위해항박테리아작용을하는은나노입자 ( 박테리아대사에필수과정인철고정및제거작용 ) 가이용된다. 포장제품라이프사이클분석은나노물질의환경적영향을평가하는데있어매우중요하다. 활성포장의경우지속적으로활동하는항박테리아인자를포함하고있기때문에세균생태에영향을미칠수있다. 지능형소재에나노센서를포함시켜살모넬라균등세균감염이나과일등식품의숙성정도를나타내는향을탐지할수있는센서를이용하면식품의질변화를지속적으로관찰할수있다 농업및축산업에서의응용식물재배분야에서도생산조건을최적화하고환경에미치는영향을최소화하기위해비닐하우스재배나수처리분야에도중금속등을여과해주는필름이나멤브레인을이용하게될예정이다. 나노기술을활용하여생산한제품과그과정에서발생한모든종류의부산물이물, 토양, 생물다양성등자연환경과관련작업환경등에미치는영향에대한심도있는연구가필요하다. 가축용영양보충사료에서도나노구조의수산화규산염과녹색조류등이해독제나독소흡착제등으로활용된다. 유럽당국은허가대상물질인신종첨가제에대하여특별평가조사를요청하고있다. 건강제품과마찬가지로수의약분야에서의나노기술응용잠재성도매우크다. 유럽에서는아직나노기술을활용한수의약에대한허가신청이이루어진것이없다. 2.3 일상용품나노기술은이미스포츠용품, 화장퓸, 물감, 메니큐어, 섬유, 건설자재등우리의일상에서다양한방식으로활용되고있다 자외선차단용화장품나노물질의특성과리포좀과같은구조물은자외선차단제와같은제품에서다양하게응용되고있다. 이산화티타늄, 이산화세륨, 이산화아연등과같은입자의경우자외선을차단하고가시광선을거의통과하지않는다는특징이있다. 노화방지제등과같은화장품의경우에도규산이나아연산입자를포함시켜피부를건조하게만드는효소를포집할수있다. 치위생분야에서도나노입자의규소를일부치약에첨가시키는방식으로활용한다. 화장품에관한유럽신규규정은나노물질에대한내용을따로포함하고있다

26 2.3.2 스포츠용품및섬유단순하고안정적인구조를가진탄소나노튜브는그강도가철강보다백배높으면서무게는여섯배낮다는특성을지닌다. 무게를감소시킬수있는나노튜브를테니스, 배드민턴라켓, 골프클럽과공, 하키스틱, 자전거구조물등의제작에활용한다. 열에대한특성과구김방지, 물과불, 마모등에대한저향력을개선하기위해나노기술을이용한다. 스포츠의류분야에서도은등금속나노입자를섬유에포함시켜살균성을높이고악취를방지한다. 섬유산업에서는나노기술이용을통해의복과패션을초월한기타다양한응용이가능하다. 미래의섬유는분자단계에서의처리과정을거쳐의약품개발에활용될수있으며특수조명하에서만나타나는발광나노물질을첨가함으로써불법복제예방에활용될수있다. 3. 인체및환경위해성 산업, 보건및환경적위해성을평가하기위해서는인간환경이위험에처하는상황등에대해명확히파악하는것이필요하다. 이후인체노출을감소시킴으로써위해성을줄일수있는예방대책을마련할수있다. 3.1 위험과노출의특성규명 2005 년국제표준화기구나노기술위원회 (ISO/TC229) 가구성되어나노물질을규명및분류할수있는변수의목록을작성하고이를바탕으로독성및위해성연구를위한표준화작업이진행중이다. 의료분야에서나노소재의빠른발전과식품분야에서의무한한발전가능성으로인해인체에서의확산에대한문제가제기된다. 나노입자는막을순환하는단백질에연결되어그작은크기와표면에너지로인해세포속으로침투하여세포핵이나 DNA를파괴하는요인으로작용할수있다. 이에따라프랑스는국제사회에나노소재별로영향을미칠수있는신체장기를판별하기위한독성동태연구및 ADME연구와모순정보결여와발암성및생식기에미칠수있는영향등을고려하여유전독성연구를우선적으로실시할것등을제안하였다. 3.2 독성평가일반적으로독성은크기와형태, 표면의특성등에기인한다. 이산화티타늄, 카본블랙의경우나노입자가마이크로크기입자비해독성이더강하게나타난다 년프랑스의약품안전청 (Afssaps) 에서실시한조사에따르면화장품은비용해성이나생체지속성의특징을띤이산화티타늄, 이산화철, 아연산, 금, 은산화물등의나노물질을포함하고있을가능성이높은것으로나타났다. 아직나노물질독성평가를위한공인된 in vitro 시험법이없는가운데지난 2009 년 3 월부터유럽연합규정 76/768/EEC 7차수정안에따라최종재와구성분의동물시험이금지된상황이다. 전체적으로인체에미치는나노입자의독성에관해서는충분히파악되지못한상태이다. 한편암발생위험성파악을위한제조나노물질에대한정보도불충분한상태이다. 플러렌, 이산화티타늄의경우 in vitro 결과에따르면자외선작용에의해발암성을나타내는반면에 in vivo 결과에서는암발생률의증가가전혀나타나지않아혼란스러운상태이다. 결과적으로나노입자의독성연구에있어서는일반연구처럼나노입자의화학구성이나최대수용가능수준평가만을고려하는것으로충분치않으며신체기관에침투한경우어떻게되는지, 어떠한반응을나타내는지, 어떠한방식으로제거되는지등을파악하는작업도수반되어야한다. 기존의화학물질과비교했을때적절한측정방법이나수단의결여로인해인체노출의특성과같은인증된역학및독성자료를확보하지못한상태로인체위해성에대한적절한평가를실시한다는것은불가능하다. 3.3 작업장노출나노물질과나노기술의급속한발전은생산및산업현장에서일하는많은근로자들의노출위험문제를

27 발생시키고있다 년부터프랑스환경부산하예방및대비위원회는복합물의무해성이나독성파악에앞서나노물질을취급하는작업장에대해일련의근로자보호책실시를권고하였다. 폐쇄공간에서의복장착용, 임신및수유여성들의관련작업장근무면제, 개별및집단보호장비마련, 폐기물의안전한수집및처리, 감시기관및관계자들에대한정보제공, 특수의료감시지원등이있다 년 8월프랑스노동위생환경국 (Afsset) 나노입자의인체위해성에대한불확실성이존재하는가운데나노입자를미지의위험 (unknown danger) 수준으로공표하고나노물질을취급하는경우위험물질노출을감소하기위해안전보건수칙도입이필요하다고강조했다. 4. 나노연구및과제 역사적으로환경과사회, 그리고인간에게큰영향을미치는기술의발전은초국가적인범위로다루어지기때문에최종적으로공공당국만이소비자와국민보호의책임을지는경우오늘날과같이국제교류가활발한시기에자국의산업주체들과고용이직면하게될어려움을막기위해서는개별국가단위로문제를다룰수없다. 나노과학은나노크기물질의거동을이해하기위해다양한분야의정보를활용한다. 나노과학과나노기술과관련된주요 3개분야로나노전자공학, 나노바이오기술, 나노물질분야로구분할수있다. 이중나노바이오기술은생명체의거시분자와나노물체와의결합을의미한다. 따라서구성물에서다양하고복잡한시스템에이르기까지상당한개발및시험영역이라할수있다. 유럽공중보건계획 에따르면프랑스의제안에따라나노물질안전에관해 2009 년 2월발표된공동액션에서유전독성을발생시키는분자탐지방법수립에관한 Nanogenetox 프로그램수립을위한자료를발표하였다. 여기에서이산화규소, 이산화티타늄, 탄소나노튜브등다양한제품에서이용되고있는 15개나노입자에대해서는그특성이물리 화학적으로규명되고시험이예정되어있고 3년간 17개기관과 13개회원국이참여한다. 나노기술은생물학과보건분야에서도활용할수있을것이다. 이를위해후기유전체학 (post-genomics) 시대의생명과학과제라할수있는나노미터크기세포의기능파악및시스템생물학 (systems biology) 과의융합등에있어서의의료계와의협력이계획중에있다. 5. 규제 나노기술은의학, 정보, 기계, 자원이용최적화, 신생에너지생산등많은산업분야에장밋빛미래를보장해주기도하지만동시에건강과환경에미치는영향, 정보의전송및저장이나생물분야에서의새로운이용가능성여부등많은의문점을제기하기도한다. 잠재적위험물질특성규명을위해사용되는수단과방법은충분하지않으며현재까지는나노크기분자의특성을규명하거나그위해성에대한정보를파악할수없는상태이다. 이런상황에서입법자들은경제발전과더불어대비책마련의의무가있으며지식과전문성, 정보확보등새로운법안과기준을마련하는데노력이필요하다. 독성관점에서보면다양한독성유발요인들로인해기존의실험기준으로는나노입자의위험성을완전히규명하는것이불가능하다. 이러한요인을반영한새로운기준을정립하기위해서국제표준기구 (ISO) 와 OECD의협력작업이진행중이다. 프랑스노동부는나노물질의위험성이불확실한경우에도화학위해성예방에관한노동법규칙에동일하게적용하고있고이러한규칙은노동법 R 및 R 에규정되어있는발암성 돌연변이성 생식독성물질 (CMR) 관리대책에관한사항을포함하고있다. 제조및사용되는나노물질의 CMR 물질에대한강력한리스크관리대책을적용하며예방책을채택할것이권고된다

28 5.1 식품에대한규제 EU 규정과사전허가시스템등에따라식품이나식품포장물질에서의나노기술이용이규제되고있다. 나노형태의새로운제조물에대해서는관련사항을담은문건을당국에제출해야되고이를바탕으로위생청이무해성평가를실시후허가결정을내린다. 이러한규제는식품첨가물에서도마찬가지로명시적인대책도입을통해강화되고있으며현재진행중인새로운식품이나식품구성분규정수정작업또한나노물질형태로구성된것에대한새로운평가규정을포함시키게될예정이다. 식품위생안전청 (Afssa) 은 2006 년 6월보건국의요청에따라인간및동물이섭취하는음식물과의접촉가능성이있는물질중나노입자를포함하는제품을판별하고분류하는역할을하는전문기관을구성하였다. 현재까지나노물질포함식품에대한표기가의무사항은아니지만새로운식품에관한수정안에따르면상황별로추가적인표기가의무화될수있다. 특히유럽의회는 2009 년 3월 25일결의안을통해나노형태의식품성분에대하여의무화할것을청구한바있다. 5.2 화장품에대한규제 화장품에대해현재도입중인새로운유럽규정은공표이후늦어도 42 개월내에적용될것인데이규정은 생산에앞서사전통지를통해안전평가와사용정당화등을밝힐것을담고있다. 5.3 EU 신화학물질관리제도 (REACH) 신 REACH 제도의경우특별히위험하다고판단되지않는한년 1톤이하로제조되는물질이나나노입자상태물질의특수성을고려하지않는것을보완하기위해국가적차원의대책마련이계획중에있다. 5. 향후안전성관련연구계획 나노입자에관한산업연구프로그램에있어서는보건 환경안전성평가작업이함께수반되어야한다. 리스크평가에관한나노기술공공연구재정지원을강화해야할필요가있는데프로젝트별재정지원이많이투입되는연구소별로프로그램을보완하는대안이될수있다. 이외에도유럽집행위원회산하에나노연구를조정감독할수있는특수기관을만드는것도바람직할것같다. 활성화시킬분야로다음과같은것들이있다. - 인체및환경에미칠수있는영향을파악하기위해나노물질독성, 환경독성등에관한물리 화학적특성규명등계측법과리스크에관한연구활성화 - 나노물질의안전성에관한연구개발 - 나노물질의특성규명및안전성연구에관한감시보장 - OECD WPMN 및 ISO 나노기술기준마련작업등을중심으로유럽및국제공동연구에대한활발한참여보장 - 나노물질안전연구전략방향수립및민관연구파트너쉽수립등을통한관계자협력활성화 - 정부부처요청에따라위생안전연구소및관계기관의나노기술을활용한제품영향연구브리핑실시 나노과학은전문가들이나국민들에게있어투명하고이해가능해야한다. 나노기술을신뢰하기위해서 는소비제품에의존재여부파악이나무해성분석이선결되어야한다. 이러한목적에서나노물질의위험 성과리스크를규명ㆍ관리하기위해안전성연구는지속ㆍ강화되어야한다

29 일본나노안전성관련대책동향 1. 배경 일본노농후생성의약식품국에서는급속도로발전하는나노기술에대비하여 2009 년 3월나노물질안전대책에관한검토회보고서를발표하였다. 한편 2009 년 8월에과학기술진흥기구 (JST) 의연구개발전략센터에서나노기술분야당면문제분석보고서에서는제3기과학기술기본계획을위해나노연구를국가적으로어떻게진척시켜나가야할것인지를제시하였다 2. 나노기술의현상의식과일본의과제 재료과학의중요성증대 - 사용재료원소 :80 년대 11종류 60종류, 기능성재료에의한다기능화 일본의위치 : 화학, 물리, 재료과학, 소재, 전자부품이강함. 중국, 한국이일본을급속히추격하고미국유럽은비즈니스모델과연결한 IT, 솔루션에서압도적우세 나노기술조직화에대한선행이필요 일본의약점 - 공학계지망자반감, 글로벌화, 연구거점, 이노베이션을유발할펀딩 (funding), 장기전략이부족 3. 주요연구현황 제3기과학기술기본계획 ( ) 에서나노기술ㆍ재료가기존의생명공학, 정보통신, 환경과함께 4대중점분야로선정되었다. 일본의나노기술연구개발의정책적분류는 나노전자영역, 재료영역, 나노바이오ㆍ생체재료영역, 나노기술추진기반영역, 나노사이언스ㆍ물질과학영역등 5개분야로나뉘어추진되고있다. 정부부처내에서는문부과학성, 경제산업성, 후생노동성, 환경성, 농림수산성이관련연구사업을추진하고있다

30 4. 규제현황 화학물질의안전성규제에관한법을관장하고있는후생노동성에서도나노재료에특화된법률이나화학물질의크기에주안점을둔규제는없다. 나노재료에대한정보가부족한상황에서더많은정보수집에기초하여법규제나운용을재검토할필요성은제기되고있다. 5. 안전대책관련과제와향후방향 나노기술의 EHS 문제에대응하는일본의활동은부처간의공동협력에기반하고있다. 4.1 안전대책관련과제앞으로나노재료를사용한제품이개발되어나노재료가다양한용도로이용될것으로예상된다. 따라서나노재료의위험성관리라는관점에서사용실태에관한정보나생체에미치는영향등에관한정보를수집하면서생체에미치는영향이나물성에관한시험방법등을개발하고개발된시험방법에의거하여 in vivo, in vitro 시험등을실시해야한다. 아울러나노재료와관련하여정부, 사업자, 소비자간의정보교환도중요한과제이다. 4.2 안전대책방향나노재료의개발상황과사용실태등을주시하여나노재료가인체의건강에미치는영향에관한정보를수집하고수집한정보를소비자에게적극적으로제공하면서국민의건강을확보한다는관점에서어떤대책이필요한지검토해야한다. 제조자책임이라는관점에서사업자가개발단계에서부터주체적으로안전대책을마련해야하지만정부측도사업자와협력하여안전대책마련에적극적으로대처해야한다. 4.3 향후의구체적대응전략 나노재료의안전성및사용실태등에관한정보수집아직까지나노재료에특화하여안전성정보등을수집하는법률상의규정은없다. 그러나의약품이나화장품에대해서는부작용보고등을통해각제품의부작용정보를수집하고있다. 식품이원인이라고의심되는건강피해의사례는도도부현등을통해, 가정용품을비롯한소비생활용제품으로인한사망, 위해등중대한제품사고는도도부현등이나경제산업성을통해후생노동성에보고하고있다 나노재료의안전성에관한시험및연구의추진후생노동과학연구비보조금화학물질리스크연구사업을통해나노재료의안전성확인을비롯해건강영향평가방법의확립, 풀러렌, CNT, 산화티타늄의경피독성평가방법의개발, 표면코팅나노실리카등나노재료의체내동태연구, 각종재료의설치류ㆍ영장류대상체내동태ㆍ조직이행성노출연구등을추진하고있다 관계부처등과연계강화관계부처와의연계는종합과학기술위원회산하에설치된 나노기술의연계개발추진과사회수용에관한기반연구 를비롯한과학기술제휴시책등을통해내각부를중심으로각관청간에정보를교환하거나공유하는데힘써야한다 국제기관등과협력후생노동성은관계부처와연계하면서 OECD의스폰서쉽프로그램정책에적극적으로협력하고미국 FDA 등해외기관과도정보를교환등국제협력을꾸준히강화해야한다 소비자와의리스크커뮤니케이션에충실사업자는소비자가필요로하는정보에대한의견을받아들이면서제품에사용된나노재료가인체의

31 건강에미치는영향등에대해정보를수집하고그정보를알기쉬운형태로공개하거나제공하여소비자의 신뢰성을확보하고정부도정보를일반에제공하는홈페이지를개설하고알기쉬운형태로공개해투명성 있게대응해야한다. 제 2 절국내외연구개발동향분석과나노제품출시현황 OECD WPMN에서선정한 14개나노물질의연구현황을확인하기위하여 OECD 홈페이지의 DB를이용하여각국별수행한프로젝트와각프로젝트별분야를검색하여정리하였다. 또한나노제품출시현황조사를위해서인터넷검색을사용하였다. 인터넷기사검색을통해출시제품에대한기사를검색한후해당제품의제조사홈페이지를찾아가제품에사용된나노물질이나나노기술을확인하는방법으로조사하였다. FDA승인의약품은마찬가지로인터넷기사검색후제조사홈페이지를방문하여확인하였고여기에더하여 FDA승인의약품조회를통하여최종확인하였다. 이런방법으로접근하였을때어떤경우는해당제품제조사에서홍보하는내용 ( 제품에적용된나노물질또는나노기술에대한정보 ) 을얻을수있으나, 반면어떤제품에대해서는전혀알수없는경우도있었다 OECD WPMN 선정 14 개나노물질연구현황 OECD DB 검색결과 (

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46 나노물질및나노기술을이용한나노제품의개발동향 현재나노기술을응용한제품이연구개발단계이거나시판되고있다. 본보고서에서는나노기술이광범위하게응용되는분야 ( 전자, 재료, 에너지, 환경, 기계및바이오산업 ) 중식품의약품안전청과관련있는소관제품군 ( 식품, 의약품, 화장품및의료기기등 ) 등에초점을맞춰조사하였다. 대상물질로는 OECD 안전성프로그램지원사업에서리스트된물질 ( 표 2) 과관련된제품개발동향 ( 표 3) 과현재국내에서연구개발이부진하고및전문가양성이시급한의약품제품화분야에서미국 FDA의의약품관련제품의허가현황 ( 표 4) 을제시하였다. 1) 주요나노물질의특성및용도 표 2 번호물질명용도 나노물질 이용의장점 특성및장래용도 단층탄소나노튜브는탄소에의한 6 원환네트워크 1 SWCNTs MWCNT 와 유사한용도로 사용됨 경량화, 전도성부여 가단층의동축관형상으로된물질이다. 평면의그라파이트를둥글게해서통모양으로만든구조이다. 지름은1nm전후임. 향후트랜지스터, 연료전지, 수소가스흡수등에활용. 2 MWCNTs 반도체트레이 :90% 기타 : 10% ( 전도페이스트등 ) 고강도, 성형품의표면평활성, 열전달성, 전자파차단성 다층탄소나노튜브는탄소에의한 6원환네트워크가다층의동축관형상으로된물질이다. 지름은 nm 정도임. 현재높은전도성을살려반도체공장에서사용되는반도체나실리콘웨이퍼의이송용기에정전방지를위해사용. 향후연료전지, 의류, 의료분야에서카테터로이용. 3 TiO2 화장품, 토너, 자동차도료기타 : 아이스크림, 설탕, 사탕, 껌의색소 자외선차단, 전하조정, 자동차도료, 토너광촉매 ( 아나타제형 ) 등. 다이아몬드보다굴절률이높고가시광을흡수하지않으며화학적안정성이우수하다는특징이있어백색안료나자외선흡수제로서도료, 화장품등의원료로널리사용됨. 결정구조에따라루틸형과아나타제형이공업적으로이용된다. 화장품분야에서는루틸형이사용되고아나타제형은광촉매기능 ( 태양광으로셀프클리닝, 공기청

47 정, 수질정화, 항균, 곰팡이방지기능이있다 ) 이 있어각종도료에널리사용됨. 향후토너용도에 도확대될것으로기대됨 4 Ag 은나노입자 : 전자장치의접합 _ 배선재료로 100% 사용 < 구체적인예 > 섬유, 일용품, 피혁, 가전, 냉장고, 스포츠, 기타 은나노입자 : 소량으로도전도성을얻을수있다. 은 + 무기미립자 : 비표면적확대에의한향균효과의향상 식품밀봉용기섬유 : 의류, 침구마스크일용품 : 주방용품, 세면용품, 우산슬리퍼수처리 : 자동판매기, 배관등가전 : 에어컨필터, 환기팬, 세탁기, 냉장고, 청소기, PC, 전화스포츠 : 스포츠웨어, 신발기타 : 장난감, 의료, 위생, 화장품용기, 건축재료 반도체회로 5 CeO2 연마 제 :70~80% 기타 :30~20% 폭의미세화를통한연마성능향상에나노크기화의장점이있다. 산소저장능력과자외선차폐효과가높은우수한기능을가지고있다. 특히반도체관련연마제로나노크기가이용된다. 향후연료전지의전해질용도나자외선차단성능을살린화장품용도등이검토되고있다 6 Fe 식품첨가물환경수질정화및오염에영가철이사용 60 개의탄소로구성되며 20 면체 ( 축구공모양 ) 의 구조를가지고있다. C60 의분자지름은 1nm 이 다. 7 Fulleren 스포츠용도 :100% 라켓, 골프클럽등 반발성능의향상, 경량화, 강도향상. 전자를쉽게받아들이고높은전도성을가진다. 수지와조합함으로써강도를높여경량화를실현할수있으므로배드민턴이나테니스라켓에사용. 또한 DNA를절단하는세포독성을이용해암세포를공격, 활성산소제거기능을살려화장품등에응용하고있다. 향후연료전지, 태양전지, 바

48 이오의약, 화장품으로도유망함. 8 ZnO 화장품 ( 땀띠치료제 ), 섬유, 도료, 식품포장재의자외선차단등 자외선차단, 투명성향상, 비표면적확대에의한항균탈취효과 금속아연을기화하여공기로연소시켜제조한다. 물에용해되지않고전도성과압전성이있다. 산화티탄에비해투명성이우수하기때문에화장품용도로산화티탄과조합하여이용함. 건식실리카는수지의보강, 도료등의점성증 9 Silica 실리콘고무충전, 도료, 토너, 잉크, 의약품 ( 약물전달체 ), 화장품 ( 계면활성제 ), 농약 고무강도향상, 점성증가제, 침강방지제, 절연성, 유동성, 내수성향상및안정제 가, 반도체웨이퍼의연마와같은다양한제품용도로사용됨. 실리콘고무의첨가제로온도변화에대한내성이높아지므로자동차엔진의패킹등에이용됨. 전기절연성이나내구성을살려서전자제품에이용되고있음. 향후식품용도 ( 식품첨가물 ) 로도확대될가능성이높음. 10 Alumina 화장품 ( 데오드란트, uv차단제, 피부세정제 ), 밀봉제, 세라믹부품, 전자부품 화학적안정성, 전기절연성 높은화학적안정성과기계적강도, 전기절연저항이큰점등다양한특성을가지고있어세라믹스재료, 연삭재, 연마제및내화물등의분야에이용되고있음. 향후안료용도, 연료전지로도확대될가능성이있음. 11 Gold 의약품, 화장품, 식품 촉매활성향상과촉매수명연장, 향산화작용 촉매, 연료전지촉매, 반도체세척용초순수등에 이용될것으로기대된다

49 2) 나노물질이사용되고있는제품 표 3 물질명용도국가회사제품명 SWCNTs MWCNTs 기타 Korea hanwhananotech Applied Therapeutics 백라이트, 평면램프, 화학적센서, 나노바이오재료, 전도성히팅필름등 전자파차폐, 전도성도료, 각종센서, 강도 보강재등 Applied Therapeutics Sunscreens Mustela Bebe/Enfant High Protection SPF 50 Burt s Bees Inc. Chemical-Free Sunscreen SPF 15 Fallene Cotz SPF 58 USA Dermatone Laboratories Dermatone SPF 20 Natural Formula Colorescience Face Brushes--After Glow Brush Brush Colores and TiO2 화장품 Innovative Skincare IS Clinical SPF 20 Moisturizing Treatment Sunscreen Banana Boat Kids Tear Free SPF 30 SkinCeuticals Physical UV Defense SPF 30 Boots and Oxonica Ltd. Soltan Facial Sun Defence Cream - Optisol UK Coppertone Spectra3 SPF 50 Ag 치약 China SunVex Quan Zhou Hu Zheng Nano SunVex Dailywear Lotions Nano-silver Toothpaste

50 Technology Co., Ltd. Natural Korea Company, Ltd. CosilNanoBeautySoap Cosil Whitening Mask Korea Claypia Co,.Ltd Daejanggum Yellow Earth Soap Phlogopite Summitek Inc. Nano-silver Aroma Soap GNS Nanogist NANOVER Mask Pack 화장품 Gaia Infonet Co., Ltd. Natural Ingredients ( Model : Face Foam ) Platinum Silver Nanocolloid Cream Japan DHC Skincare Platinum Silver Nanocolloid Milky Essence New Zealand Skybright Natural Health Colloidal Silver Liquid Colloidal Silver Cream Shanghai 의약품 China Huzheng Nano Technology CO.,LTD Antibacterial for Necessities, Ceramics, Medicine 의료기기 Korea Duhkha Human Tech Yes Ear ( 보청기 ) CeO 2 촉매제 UK Oxonica Ltd. Envirox Fuel Borne Catalyst filter UK NB International Limited Aqulic AQ-001SF natural shower filter Fe 2O 3 기타 China Galaxia Nano Technology Limited Galaxia Nano Technology Limited Nano Oxygen Supply Living East Cup

51 의료 tumor 진단을위한표지로많이사용되기도함. White Out Sircuit Cosmeceuticals Inc. O.M.G. SERUM Sircuit Addict version 1.0 Fulleren 화장품 USA Fullerene C-60 Day Cream Zelens Fullerene C-60 Eye Cream Fullerene C-60 Night Cream Applied Therapeutics Applied Therapeutics Sunscreens Mustela Bebe/Enfant High Protection SPF 50 Fallene Cotz SPF 58 ZnO MOXIEformen D-Fense Antioxidant Moisturizer with SPF 17 화장품 USA Daily Sun Defense SPF 20 SkinCeuticals Physical UV Defense SPF 30 Sport UV Defense SPF 45 ZnO 화장품 USA Dermatone SPF 20 Natural Formula Dermatone Laboratories Lips n Face Protection Creme and Sunblock Crème Colorescience Face Brushes-After Glow Brush and Brush Colores Innovative IS Clinical SPF 20 Moisturizing

52 Treatment Sunscreen Skincare SPF 20 Sunscreen Powder Banana Boat Kids Tear Free SPF 30 DERMAdoctor International Cosmeceuticals, Inc. POUTlandish Hyper Moisturizing Paint Q-SunShade SPF 30+ Tinted Oxide Sunscreen with Z-COTE Lip Zinc Rosacea Care Rosacea Care Sunscreen 30 Keys Soap Solar Rx SPF 30+ Nano-Zinc Oxide Sunblock ImageSkinCare Solar Defense Creme and Moisturizer Organic Coppertone Spectra SPF 50 China Australia NuCelle Inc. ShenzhenBecomeI ndustry&tradeco.,ltd. Crown Laboratories, Inc. SunSense SPF 30+Sunscreen Self Cleaning Coating Blue Lizard Regular Rénergie Flash Lifting Silica 화장품 France Lancome Rénergie Microlift Rénergie Neck Rénergie Microlift Eye UK LEOREX Leorex Up-LiftingWash

53 Leorex Up-Lifting Serum LEOREX Neck & Décolleté LEOREX Booster Plus LEOREX Booster HWNB Microhydrin Products 보조식품 USA RBC Life Sciences Microbright Tooth Powder 치약 Nanorama - Gold Toothpaste Alumina 화장품 파우더 Australia A d v a n c e d Nanotechnology Limited Alusion Alumina Powders Chantecaille Chantecaille Nano Gold Energizing Cream Lancome Flash Bronzer Glow 'N Wear USA Neiman Marcus Nano Gold Energizing Cream Gold Gold 화장품 USA Lexon Na. notech, Inc Nanorama - Nano Gold Essence & Additive Nanorama - Nano Gold Mask Pack 화장품 Taiwan SongSing Nano Technology Co., Ltd. Nano Cosmetics Nano Engine Oil NanoPlasma Nanoseal G.N.C. Korea Center Company, Ltd Nanoseal G.S.E

54 Quan Zhou Hu Zheng Nano Technology Quan Zhou Hu Co., Ltd. Gold Nanoparticle soaps China Zheng Nano Technology Co., Ltd. Quan Zhou Hu Zheng Nano Technology Co., Ltd. Nano-gold Mask 치약 USA Lexon Nanotech, Inc. Nanorama - Gold Toothpaste 의약품 USA Carter-Wallace First Response Home Pregnancy Test 3) 미국 FDA 승인나노의약품등현황 표 4 Category 제품명 성분 기능 회사명 Anti-nausea drug Nanocrystal Emend for chemotherapy nanoparticle patient 승인년도 Merck 2003 nanoparticle made American Cancer Abraxane of the human Breast cancer Pharmaceu therapy protein albumin ticle Partner Lipid nanoparticle -Ovarian cancer Doxil that incorporate -AIDS related ALZA 2005 PEG Kaposi"s Sarcoma Immunosupressant Rapamune Cholesterol TriCor control Nanocrystal nanoparticle Nanocrystal nanoparticle convinient administration and storage Cholesterol lowering drug Wyeth 2000 Abott laboratories 2004 Hormone therapy Estrasorb estrogen Micellar nanoparticle Novavox 2003 drug delivery platform Surgical mesh Medical tools TiMESH Titanium with polypropylene properties including biocompatibility and resistence to Gfe Medizintechnik 2004 infection

55 EnSeal Laparoscopic Vessel Fusion System elctrode consist of nanometer sized conductive particles embeded in temparaturesensitive material Allow surgeons to seal and transect vessels SurgRx 2004 Imaging Appetite control Bone Replaceme -nt Acticoat siver nanoparticle Wound dressing Antimicrobial surface SilvaGard siver nanoparticle treatment for medical devices TriLite Imaging and alloyed nanocrystal diagnostic long-term photostability for Qdot nanometer-scale live cell imaging Nanocrystals atom cluster and dynamic studies improve the rate of Megace ES Megestrol acetate dissolution and bioavailability Repairing bone Vitoss 3-D β-tcp defect and enhence resortion and new bone growth Zirconium nanosized zirconium Oxide oxide Dental application Smith & 2005 Nephew Acry Med 2005 Crystalplex Corporation Invirogen Corporation Par pharmaceutical 2004 Orthovita 2004 Altair nanotechnologies 나노물질독성연구동향조사 금 다양한나노입자중에서금나노 (AuNPs) 는생체에거부반응이없어조영제, 약물전달체, 초기상태진단및암방사선치료분야에서의료목적으로가능한안전한물질이라고알려져있다입자크기에따른생체내조직분포경향에차이가있을수있음, 비교적큰입자는간비장등에분포되나작은사이즈는뇌등을포함한모든장기에분포되는경향을보이고있다. in vivo 노출에의한금나노독성의경우염증자극을매개로한 2차적면역독성유발가능성을제외하고심각한장기독성영향이없는것으로알려져있다. 그러나최근 in vitro 결과보고에의하면작은크기의금나노의세포독성을조사한결과금나노가섬유모세포, 상피세포, 대식세포및생식세포에서의독성유발잠재성은여전히논란의대상이되고있다

56 OECD 조사 Title : In vitro reactivity of fine and ultrafine particles Country : Switzerland Start : 2007 End : 2010 Summary : Toxicity of particulates may be due to the reactivity of their surface when entering in contact with cells. Among other, their potential to generate reactive oxygen species seems to be very important. This work intends to use a simple method in order to determine the ability of some particles (ultrafine TiO2, crocidolite, diesel SRM 1650 and particles from real occupation situations) to catalyse the oxygen reduction by ascorbic acid. The results obtained indicated that: It is possible to detect such reaction with simple equipment (specific oxygen electrode) In the experimental conditions, diesel SRM 1650 reacts about 10 times faster than crocidolite and that ultrafine TiO2 didn t showed any reactivity. Particles sampled in a bus depot indicated a very high reactivity. Heavy metals (Fe, Cu) could be potential important elements to explain this reactivity. Experimental improvement has to be done mainly for getting a particulate suspension without artefacts. 입자의독성은세포와접촉하여들어갈때그표면의반응에의한것일수있다. 또다른것은 ROS를강력하게발생시키는것이매우중요한것같다. 아스코빅산에의하여산소환원을촉매하는몇몇입자 ( 극미세 TiO2, 청석면, 디젤 SRM 1650 및작업장상황의입자 ) 의기능을확인하기위해단순한방법을사용하겠다는것이다. 이결과 crocidolite 는 diesel SRM 1650 보다 10분더빠른반응을보였으며 TiO2 에서는어떠한반응도나타나지않았다. 버스차고에서채집된입자에서가장높은반응을나타내었다. 무거운중금속들 (Fe, Cu) 은이반응을설명할수있는잠재적인중요한요소가될수있다. 실험의개선은주로입자가인공산물없이입자의부유물을얻기위한방법을얻기위한것이다 Title : Cytotoxicity of Nanoparticles Country : United States Start : 2004 End : 2005 Summary : Catherine J. Murphy and Michael Wyatt, University of South Carolina, are supported by the Inorganic, Bioinorganic and Organometallic Chemistry Program for exploratory research into the cytotoxicity of nanoparticles. Gold nanoparticles with sizes ranging from four to 200 nm will be functionalized with amino acids, small peptides and other bioactive groups. The functionalized nanoparticles will be characterized by electronic absorption spectroscopy, surface-enhanced Raman spectroscopy and transmission electron microscopy and then tested for cytotoxicity. Further pharmacological characterization will include determining the mechanism of cell death and subcellular localization studies. This Small Grant for Exploratory Research (SGER) supports high risk/high impact research in nanobiotechnology. Chemists and pharmacologists will conduct preliminary studies

57 on the interactions of nanoparticles with cells. Because nanoparticles are being considered as cellular imaging and drug-delivery agents, it is important to understand the inherent bio-activity of these nanoparticles. The nanoparticle may mimic the size, shape and surface functionality of protein aggregates that have also shown cytotoxic responses. Project URL : 4에서 200nm 크기의금나노입자가아미노산, 작은펩티드및기타생활성그룹들을기능화하는것이다. 기능화된나노입자들은 EAS( 전자흡수분광학 ), Surface-enhaced Raman Spectroscopy 와전자현미경에의하여특징짓고, 그리고, 세포독성을실험한다. 더나아가약리학특성화는세포사기전및세포구역화연구결정이포함된다. 이작Small Grant for Exploratory Research (SGER) 는 nanobiotechnology 에높은위험 / 높은영향연구를지원한다. 화학자및 pharmacologists 는세포들과나노입자간의상호작용에대한예비연구를수행할것이다. 나노입자는세포형상과약물수송물질로고려되고있으며, 이들나노입자들의내제된바이오-활성을이해하는데매우중요하다. 그나노입자는크기, 형태, 단백질응집의표면기능을모방할수있으며, 세포독성반응을나타낸다 금 (Au) 나노물질에대한독성연구논문요약 연 Reference Test 결과 번 1 Thomas M., Klibanov A., -2~4nm % viability after transfection 2003, Conjugation to gold -PEI2 coating nanoparticles enhances -COS-7 cell line polyethylenimine's transfer of -MTT test plasmid DNA into mammalian cells. PNAS. 100, Tkachenko A. et al nm Multifunctional gold -BSA, 4 targeting peptide nanoparticle-peptide coating complexes for nuclear -HepG2 targeting., J. Am. Chem. -LDH test Soc. 2003, 125, Viability slightly compromised 3 Goodman C. et al. Toxicity of -2nm core particle LD50(Cos-1) : anionic~1um and gold nanoparticles -NH3, COOH cating cationic>7.37 um; similar for functionalized with cationic -Cos-1, Red blood cells, other cell types and anionic side chains. E.coli Bioconjugate. Chem. 2004, 15, -MTT, Trypan blue, Top agar assay, hemolysis assay, vesicle leakage assay 4 Tkachenko A. et al, Cellular -22nm Cell viability는 Hela cell 은 20% 감 trajectories of -BSA, 4 targeting peptides 소, 3T3/NIH는 5% 감소 peptide-modified gold particle -Hela, 3T3/NIH, HepG2 complexes: comparison of -LDH

58 nuclear localization signals and peptide transduction domains Bioconjugate Chem. 2004,15, Shukla R. et al. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview.langmuir 2005, 21, Pernodet N. et al. Adverse effects of citrate/gold nanoparticles on human dermal fibroblasts. Small 2006, 2, Pan Y. et al., Size-Dependent Cytotoxicity of Gold nanoparticles, Small,2007,3,No.11, Renault S. et al. Impacts of gold Nanoparticle exposure on two freshwater species: a phytoplanktonic alga (Scenedesmus subspicatus) and a benthic (Corbicula fluminea), bivalve GoldBulletin2008,41/2, Gannon CJ.et al, Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells, JournalofNanobiotechology,200 8, 6:2-3~8nm -lysine, PLL, FITC coating -RAW macrophage cell -MTT 100uM-72h후에 cell viability 10% 감소 -13nm -citrate -Human dermal fibroblast -microscopy -Au0.8TPPMS, Au1.2TPPMS, Au1.4TPPMS, Au1.4TPPTS, Au1.8TPPMS -TPPMS (tris-sulfonated triphenylphosphine P(C 6 H 4 SO 3 Na) 3 ),TTPTS -HeLa, SSK-Mel-28 melanoma cell, L929 mouse fibroblast, J774A1 -TEM,MTT assay, FACS -10nm -amine coating -Algae -TEM, MS-ICP, RT-PCR -5nm -AuNPs ver. RFA treated AuNPs -Panc-1, Hep3B cell -MTT, PI-FACS, TEM Dose-dependently cell area, density 감소. Many vaculoe -입자의크기별로그세포독성이나타남. (1.4MS> 1.8MS>15MS) -특히 1.4nm 크기에서 12h 이내에빠른 necrosis 에의한세포죽음이관찰. -1.2nm는 apoptosis로인한세포죽음관찰. -최소농도인 1.6x10 2 에서 20% 의치사율을보였으며, TEM 결과 cell wall에강하게금나노입자가흡흡착되어있는것을관찰. -이것은 intracellular와 wall의형성에방해를유도. 또한 oxidative stress유도. -일반금나노입자의독성관찰은없었으나, RF (radiofrequency) 로 heating 시킨금나노입자에서는 cytotoxicity 관찰,

59 10 Li JJ. Et al, Gold Nanoparticles Induce Oxidative Damage in Lung Fibroblasts In Vitro. 2008,AdvancedMaterial,20, nm -MRC-5 fetal lung fibroblasts -trypan blue, real-time PCR, HPLC, TEM, EDX 11 Sereemaspun A. et al, Effect of gold nanopaartilce on Renal cell : An implication for exposure risk, Renalfailure,2008,20: Wang S. et al. Challenge in understanding size and shape dependent toxicity of gold nanomaterials in human skin keratinocytes. ChemicalPhysicsLetters,2008,46 3, nm -Renal cell(urine saple with renal cell) -microosopy -AuNPs(GNP): 5,12,20,30,50,70nm Gold nanorod(gnr):2.5, 3.0, 3.5 (aspect ratio) -CTAB, PSS coating -HaCaT keratinocyte cell line -MTT, absorption spectroscopy, TEM -대부분의금나노입자는세포내로흡수되었으며, vesicles 주변에많이관찰되었다. 그러나몇몇은 cytosol에도관찰. -cytotoxicity 관찰, cell proliferation의저해, oxidative stress 관찰없음, DNA damage는관찰. -renal cell 안에금나노입자발견. -아직그독성여부는모르나, 다른나노입자들의독성이나타나는것으로보아금나노입자역시 risk가있을것이다. -GNP는 human skin cell에독성없음 -GNR경우 CTAB coating으로높은독성관찰. CTAB로인하여,media에 aggregation으로존재. 따라서 gold nanomaterial의 cytotoxcity를이해하기어려움. 13 Wiwanitkit V. et al, Effect of gold nanoparticles on spermatozoa: the first world report, FertilSteril.2009 Jan;91(1):e Chen Y et al. Assessment of the In Vivo Toxicity of Gold nanoparticles. Nanoscale Res Lett. (2009)4: nm -spermatotoxicity -Single-donorfreshsemensa mple -3,5,8,12,17,37,50,100nm -naked AuNPs -BALB/c mice -ELISA,CARS Microscopy -25% 운동성감소 -정자머리와꼬리부분내에서금나노입자발견. -8~37nm 크기의금나노입자에서쥐의병리증상이관찰됨. -다수의쥐들은 21일내로사망. 이쥐들을병리판독한결과간장에서 Kupffer cell의증가가관찰되었으며, 완전한폐구조형성의감소, 비장에서는 white pulp의확산이관찰되었다. -Immunogenicity관찰

60 15 Uboldi C. et al. Gold nanoparticles induce cytotoxicity in the alveolar type-ii cell lines A549 and NCIH441. ParticleandFibreToxicology2009, 6: nm -sodium citrate residues on AuNPs -Humal alveolar type-ii (ATII) like cell lines A549, NCIH441. -MTT,LDH,Ki-67,TEM -cell viability 감소, cellular proliferation 영향, LDH 증가. -처리 3hr이내에금나노입자세포내관찰. -cell의 membrane-bound vesicles 에축적되어있으나, cytoplasm내퍼져있는것은관찰못했음. 16 Wiwanitkit V. et al, Effect of gold nanoparticle on the microscopic morphology of white blood cell. Cytopathology. 20(2): , April nm -white blood cell -white blood cell에금나노입자가축적되어있음관찰. -따라서 cytotoxic drug treatment 연구에도움이될것이다. 은 은나노물질은현재도의약품, 의약부외품, 화장품, 식품용기, 건강보조제, 섬유, 전자제품, 의료기기등다양한소비자제품이생산되어지기때문에나노물질독성연구중가장연구많은결과가보고되었다. 은나노독성은일반적으로사이즈에비례해서독성이강하게나타난다고알려져있지만생체내에서은나노이온의방출인한독성유발가능성, 사이즈가동일하더라도 surface chemistry 변화에의한영향등이고려되어지고있다. 장기별독성으로는은자체의항박테리아특성때문에폐독성, 피부독성, 신경독성, 유전독성, 생식독성, ADME 등전장기를대상으로독성연구가진행되고있다. 연구대상사이즈의범위는 4-100nm 가대부분이고평균 20nm 이하크기의은나노독성결과가많이보고되었다. 한편보고된은나노제품의평균은나노사이즈는 24nm 로알려져있다. 은나노입자는위장관, 피부, 폐배리어 (barrier) 를통과해서혈액내로순환될수있기때문에전체생체내장기에도달할수있는것으로알려져있고축적되는주요장기로는간 (liver), 노, 비장등으로알려져있다. OECD 조사 Title : Buccal permeability studies of nanostructured materials Country : Austria Start : January

61 End : August 2011 Summary : Deposition of drugs on the buccal mucosa is suited for local and systemic treatment. This mucosa is a nonceratinized squamous epithelium with a thickness of approximately µm, lining the cheek area of the oral cavity. It is known that the main penetration barrier for drugs lies in the outermost quarter of to one third of the epithelium. Possible effects of nanomaterials (NMs) on the oral mucosa have not been investigated yet. Thus, for a responsible production and use of NMs, the knowledge about biological actions within the oral exposure route is highly important. The aim of this project is to build up a standardized permeability model (Franz-cells) to investigate the penetration/permeation of (i) carboxy-polystyrol-particles, (ii) silver-particles and (iii) titanium-dioxide-particles across the mucosa of the pig. Up to now it appears that the mucus together with the upper third of the oral epithelium forms a strong barrier for CPP. The role of the mucus through increases in the size of the CPP by coating with proteins and repulsion of the slightly hydrophobic particles by the hydrophilic mucus disserves further attention. 구강상피에약물의침착은국소및체계적인치료를위해적합하다. 이점막은비각화편평상피이며 µm 의두께를가졌고, 구강의뺨영역을피복한다. 그것은상피의가장바깥쪽 1/3 지점에있는약물에대한주요침투장벽으로알려져있다. nanomaterials (NMs) 의가능한영향들은아직조사되지않았다. 따라서 NMs의사용과생산의책임을위하여, 노출경로간의생물학적활동에대한지식은매우중요하다. 이프로젝트의목적은돼지의점막을투과하는 (i) carboxy-polystyrol- 입자, (ii) 실버-입자그리고, (iii) 티타늄-이산화탄소-입자의투과 / 침투를연구하여표준침투모델 (Franz-cells) 을구축하는것이다. 지금까지그것은 CPP를위한강한장병을형성하는구강상피상부 1/3을포함한점막을나타난다. 단백질코팅에의한 CPP의크기증가와수용성점막에의한약한비수용성입자의반발력을통한점막의역할에해로움을줄지관심이기울어진다 Title : Dispersion and retention of ultrafine and nanoparticles in the lung Country : Germany Start : February 2009 End : December 2010 Summary : Ultrafine particles emerge from thermal processes like welding and combustion. They often form larger agglomerates and aggregates that are composed of ultrafine primary particles. Engineered nanoparticles smaller than 100 nm also tend to aggregate and agglomerate. It is not clear to what extent these agglomerates and aggregates resolve into primary particles after inhalation. The size of particles is discussed as a trigger of toxicity, so there is a need to clarify a possible process of de-agglomeration and de-aggregation within the lung. Furthermore other physico-chemical parameters are discussed to influence toxicity. In appropriate testing systems (in vivo, in vitro) it is intended to elucidate agglomeration and aggregation of nanomaterials in a biological environment. In addition toxicity and transfer of nanoparticles is investigated. 초미세입자는용접및연소와같은열프로세스에서생성된다. 종종큰응집의형태는미세일차입자들로형성된다. 기계적으로 100nm 보다작은나노입자역시어느정도의덩어리응집되는경향이있다. 이것은흡입후에일차입자들로응집이나타나는지명확하지않다. 이입자의사이즈는독성을촉발시키는것으로폐내에서 de-agglomeration 과 de-aggregation 의가능한프로세스를명확하게할필요가있다. 또한다른물리화학매개변수독성영향을설명한다. 적절한테스트시스템에서 (in

62 vivo, in vitro) 생물학적환경내에서나노물질의응집을명료하게하는것이만들어졌다. 나노의독성 및전송에대한조사를한다 Title : Study of transport of inhalated nano-sized particles (Ag, Pb, Cd) and their allocation in organs Country : Czech Republic Start : 2000 End : 2010 Summary : The study will be focused on the only transport of fine particles of elements or oxides. The nonbiogenous elements (Ag, Cd and Pb) have been selected as products of nanotechnological processes. The research will give us more information for a proper understanding of risks of technologies producing Ag, Cd and Pb nano-sized particles as well as their oxides, which can have health impact for animals and humans or the impact on the environment. 이연구는성분또는산화물의미세입자이동에초점을맞춘것이다. 생물에서생기지않는성분은나노기술과정의생산물에의해선택되어졌다. 이조사는 Ag, Cd and Pb 나노크기입자즉환경의영향또는동물과인간건강에대한영향또는환경에의한영향을준건강을줄수있는산화물의기술적생산위해성에대한이해를증진시키기위한더많은정보를우리에게줄것이다. (2) 은 (Ag) 나노물질에대한독성연구논문요약 연 Reference Test 결과 번 1 Sung JH et al., Subchronic Inhalation Toxicity of Silver Nanoparticles -18~19nm -Rat -90일간노출 -Liver 안에서의 bile-duct hperplasia는 dose dependent하게증가되었으며, 조직병리학적결과 dose TOXICOLOGICALSCIENCES, -Inhalation test, TEM, dependent하게조직손상결과가 나 2009, 108(2), Biochemistry, Hematology, Histopathology 타났다. 또한 infalammatory cell infiltrate, chronic alveolar inflammation, small granulomatous In vivo lesion이 포함되어있다. 은나노입 자의주요표적장기는암컷, 수컷모두 간과폐였다. 2 Ji JH et al., Twenty-Eight-DayInhalationToxi -12.6nm -Rat(specific - 수컷, 암컷의무게변화는없었음. -blood biochemical value 는고농도

63 citystudyofsilvernanoparticlesin Sprague-DawleyRats.,2007,Inhal ahtiontoxicology,19(10): In vivo 3 Sung JH et al., Lung function changes in Sprague-Dawley rats after prolonged inhalation exposure to silver nanoparticles. InhalahtionToxicology,2008,20(6 ): In vivo pathogen-free(spf) Sprague-Dawley rat) -28일간노출 -Inhalation test, Blood Biochemistry, Hematology, -18nm -90일간노출 -lung function test measurement (cellular differsntial count, inflammatory measurement(albumin, LDH, total protein), in BAL fluid 그룹에서 calcuim 증가. 저농도그룹에서는 gamma-gt 증가. Hematology변화없음. -은나노입자의주요분포장기는폐, 간장, 뇌, Olfactory bulb에분포. -부검결과간에서암, 수컷모두 cytoplasmic vacuolization이관찰, 고농도에서는 hepatic focal necrosis 관찰. 다른장기인콩팥, 비장, 폐, 심장, 생식기관, 뇌등등뚜렷한변화를찾을수없음. -ACGIH(American Conference of Governmantal Industrial Hygiensts) 에서제시한 silver dust limint (100ug/m 3 ) 만큼의뚜렷한영향관찰이없었음. -inflamation response : Albumin, LDH, total protein 증가 -폐의 fuction test결과고농도흡입 그룹에선감소, tidal volume이농도의존적으로감소되는경향확인. -부검결과: infalammatory cell infiltrate, chronic alveolar inflammation, small granulomatous lesion이나타남, alveolar 벽이두꺼워지고, alveolar macrophage 축적. 4 Kim YS et al., Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of silver nanoparticles in spraque-dawley rats. Inhalationtoxicology,2008,20: In vivo 5 Laura Braydich-Stolle et al., In Vitro Cytotoxicity of Nanoparticles in Mammalian -60nm (NAMATECH) -rat -28일간반복경구투여. -biochemistry, hematology, organ weights, histopathology, rat bone marrow micronucleus test -저농도(30mg/kg), 중농도 (300mg/kg), 고농도 (1000mg/kg) -AgNP (15nm), MoO 3 NP(30nm),AlNP(30nm) -C18-4 line -암, 수컷 bone marrow에서 genetic toxicity 없음. -농도의존적으로각조직에축적관찰. -특히, kidney에서는암컷이수컷보다 2배나축적되어있는것을관찰. C: AgNP(15nm, 10ug/ml) D: AlNP(30nm, 10ug/ml) E: AoO 3NP(30nm,10ug/ml)

64 Germline Stem Cells TOXICOLOGICAL SCIENCES, 2005, 88(2), In vitro -Inhalation chamber monitoring, TEM, Lung function test, BAL cell 측정 Cytotoxicity 순서 : AgNP>AlNP>MoO 3 NP AgNP는 mitochondria 기능이 5ug/ml농도이하에서저하되었음이관찰되었고, 반변 LDH leakage는증가하였다. 6 Kim S et al., Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells,toxicologyinvitro,23(2009) In vitro -<10nm -HepG2 cell line -dark-field microscopy, TEM -MTT, LDH assay, DCFH-DA, DNA damage (DAPI) -AgNP ver. AgNO3 (ion effect때문에 ) -Ag ion 만큼 AgNP도독성이강하다. -Ag ion과는독립적으로 oxidative stress를야기시킨다. 7 Ofek Bar-Ilan et al.,toxicity Assessments of Multisized Gold and Silver Nanoparticles in Zebrafish Embryos, small 2009, 5, No. 16, zebrafish embryo -3,10,50,100nm -INAA, ICP-MS -120시간이지난후 100% 치사 -embryo morphologycal malformation -Ag ion 발생으로 in vivo상태에서불안정. In vitro 8 Lee KJ et al. Invivoimagingoftransportandbio compatibilityofsinglesilvernano particlesinearlydevelopmentofz ebrafishembryos.acsnano,2007,1(2), In vivo (eco 관련 ) -5-46nm -Zebrafish embryo -in vivo imaging, biocompatibility, toxicity test -cleavage-stage embryo 에서은입자이동이관찰되었음. ( 수정 120h이후 ) -in vivo imaging을위한 biocompatibility test에서는저농인 <0.08nM농도면괜찮음. -농도가증가할수록정상적인발달을가진 zebrafish수가감소하며, 죽는수가증가함. 9 Kim YS et al., Subchronic oral - Fisher 344 male and

65 toxicity of silver nanoparticles. Part Fibre Toxicol female rats - 56nm - 13 weeks(oral) 10 Lankveld DP et al., The kinetics of the tissue distribution of silver nanoparticles of different sizes. Biomaterials ;31(32): Braydich-Stolle et al. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. toxicol Sci 88(2005) Li PW et al., Induction of cytotoxicity and apoptosis in mouse blastocysts by silver nanoparticles. Toxicol Lett. 2010, 16;197(2): Wistar male rats - 20, 80, 100nm - ICP-MS - Mouse spermatogonia stem cell line c nm ug/ml - mouse blastocysts - 15nm - 25, 50μM - TUNEL assay, Annexin V staining 간의조직병리학적소견 (A) 대조군, (B) 염증세포및산호성백혈구가다발성병소에침착. 특히중심정맥과문맥주위에서관찰 (C) 담관의과형성 (D) 염증세포의침착이몇몇병소에서관찰되는데, 특히중심정맥및간소엽의동모양혈관에서관찰됨. - NOAEL : 30mg/kg - LOAEL : 125mg/kg - 20nm particle은주로간에분포되어있었으며, 다음으로신장과비장에도관찰됨 - 크기가큰입자는주로비장에서관찰되었으며, 다음으로간및폐에도관찰됨 - 반복투여시간, 폐, 비장에서축적됨. - 10μg /ml과그이상의용량에서세포괴사및세포자멸사가증가함. - 10μg /ml에서사립체의기능및세포생존능이감소함. - 5μg /ml에서 LDH leakage가증가함 - 5μg /ml에서세포자멸사가증가함. - 은나노물질은 in vitro 상에서 blastocyst에서세포자멸사를유도 - 초기 post-implantation을지연유도 - 은나노물질은은이온에서유도되는배아세포독성이낮게관찰되었음

66 13 kim Y et al., Twenty Eight day oral toxicity, Genotoxicity, and Gender-related tissue distribution of Silver nanoparticles in sprague-dawley rats. Inhal toxicol (6): Ji JH et al., Twenty Eight day oral toxicity, Genotoxicity, and Gender-related tissue distribution of Silver nanoparticles in sprague-dawley rats. Inhal toxicol : Sung JH et al., Subchronic inhalation toxicity of silver nanoparticles. Toxicol Sci : Samberg ME et al., Evaluation of Silver nanoparticle toxicity in skin in vivo and keratinocytes in vitro. Environ Health Perspect 2010;118(3) Wang J et al., Expression changes of dopaminergic system-related genes in PC12cells induced by manganese, silver, or copper - SD rats - 60nm - Oral - SD rats - <100nm - Inhalation - SD rats nm - Inhalation - Human epidermal keratinocytes, porcine skin - unwashed 20,50,80nm - washed 20,50,80nm - carbon coated 25,35nm - DLS, TEM, zeta potential - MTT, alamarblue, - PC12cells - 15nm - TEM, DLS, LDV - 10μg / ml,24h incubation - RNA isolation and 위, 간, 신장, 폐, 고환, 뇌및혈액에서축적이관찰됨. - 은나노입자의조직내분포는용량의존적으로조직에축적되어관찰됨. - 숫컷신장에서보다암컷신장에서은나노입자가두배증가가관찰됨. - 축적부위는폐, 간및뇌임 - ACGIH의은나노입자노출한계인 100microg/m3에서는독성이관찰되지않음 - 축적부위는폐, 간, 비장및뇌임 - 용량의존적으로혼합된염증세포의침착, 만성폐렴및육아종성병변관찰됨. - NOAEL : 100 microg/m(3) - HEKs의세포질내공포에모든은나노입자가위치함 - unwashed : 용량의존적으로세포생존능이감소 -washed : IL-1β, IL-6, IL-8 and TNFα의농도가증가 - porcine skin에서가려움증관찰않됨 - 현미경적관찰에서국소적염증영역과피부의상부각질층과표면상에은나노입자의위치함을관찰 - Gpx1은감소됨 - 이결과는금속나노입자가지속적으로산화성스트레스를유도함

67 nanoparticles. Neurotoxicol. 2009; 30, Kim SH et al., Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicology in vitro 2009, 23; Rahman MF et al. Expression of genes related to oxidative stress in the mouse brain after exposure to silver-25 nanoparticles. Toxicol Lett 2009, 187; Asharani PV et al. cytotoxicity and genotoxicity of silver nanoparticles in human cells. Acsnano 2009, 3(2); Hsin YH et al., The apoptotic effect of nanosilver is mediated by a ROS-and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett 2008, 179; Arora S et al., Cellular responses induced by silver nanoparticles: In vitro studies RT-PCR - human hepatoma cells - 10nm - TEM, ICP-spectrometry - Deionization - MTT, alamarblue, LDH - C57BL/6N mice - 25nm - I.P, 100,500,1000mg/kg - TEM, DLS, LDV - RT-PCR - human lung fibroblast cells, human glioblastoma cells - 6~20nm - TEM, SCGE, CBMN - NIH3T3 cells nm - 50, 100μg /ml - TEM - MTT, Western blot, Annexin-V, Confocal microscope, RT-PCR - A431(human skin carcinoma), T-180(human fibrosarcoma) nm - TEM, XTT, - 은나노입자의처치세포의세포질과핵에서응집및세포산화성스트레스를유도. - 은나노입자의세포독성은일차적으로산화성스트레스의결과이며, 은이온의독성에비의존적임. - AgNPs는산화성스트레스, 특정유전자발현에의해유도된자유기, 변경된유전자발현, 세포자멸사등에의해신경독성을유발함. - AgNPs는 ATP를감소시킴 - SCGE와 CBMN는용량의존적으로나타남. - G2/M기에서세포주기가정지 - 사립체에서 Bax의위치변경과 cytochrome C의세포질내로방출 - nanosilver와연계된세포사는사립체의존적. - nanosilver에의해유도된세포사는 ROS 발생과 JNK의활성화에관계가있음. - nanosilver는 ROS와 JNK를통하여사립체경로를경유한세포사를유발함 - caspase 3 assay에서은나노의세포자멸사농도는 0.78μg /ml이며, 세포괴사의농도는12.5μg /ml 임 TiO 년미국 FDA 가나노산화티타늄을화장품성분으로사용한허가한이래로안전성연구의초점 은각질층통과로인해혈액을통한산화티타늄의염증및 DNA 손상을통한발암성유발가능성이제 시되고있음. 현재대부분의독성연구는 피부와흡입시폐독성과피부독성의영향평가임. 기관의구조특징에따라

68 조직내투과정도는달라질수있으나피부의경우통과하지못하는것으로알려져있으나손상된피부의장기노출시혈액내로침투할수있는가능성이제시되고있음. 특히이산화티타늄은매우낮은독성과비발암성물질로알려져있으나 2006 년 IARC에서 pigment-grade 이산화티타늄의경우사람에게발암성을유발시킬수있는물질로 Group 2로등급분류되어졌다 2002 년식품에대한색소첨가제로이산화티타늄을규제한이래로경구투여에의한독성연구는감소하고있음. OECD 조사 Title : Toxicokinetics of titanium dioxide nanoparticles Project Ref. : DEU6193 Last Update : 02/07/2010 Status : Completed Checked by National Designated Contact Point? Yes Country : Germany Start : December 2007 End : June 2009 Summary : The aim of this project was the determination of the biokinetics of TiO2 nanoparticles (NP) in the whole body of healthy adult rats after NP administration to the respiratory tract either via inhalation or instillation. We developed an own methodology to freshly synthesize and aerosolize TiO2-NP in our lab for the use of inhalation studies. These NP underwent a detailed physical and chemical characterization providing pure polycrystalline anatase TiO2-NP of about 20 nm (geometric standard deviation 1.6) and a specific surface area of 270 m²/g. In addition, we developed techniques for sufficiently stable radioactive 48V labelling of the TiO2 NP. The kinetics of solubility of 48V was thoroughly determined. The methodology of quantitative biokinetics allows for a quantitative balance of the retained and excreted NP in control of the administered NP dose and provides a much more precise determination of NP fractions and concentrations of NP in organs and tissues of interest as compared to spotting biokinetics studies. 이 project 의목표는건강한 adult rats의기도에 NP를투입하거나흡입또는주입시켜 TiO2 nanoparticles 의 biokinetics 을측정하는것이다. 우리는 lab에서 inhalation 연구에사용하기위하여 aerosolize TiO2-NP과 freshly synthesize한방법론을개발하였다

69 크기가 20nm와표면적이 270m²/g 인순수다결정 anatase TiO2 나노입자의세부적인물리화학적특성제공을진행하였다. 또한 TiO2 나노입자에안정적인 radioactive 48V 표지를위한기술도개발하였다. 48V 의용해도역학은확인되었다. 양적 biokinetics 의방법론은 quantitative balance 를유지하고, NP 배설과 NP dose 의통제를허용하고, NP fractions 을훨씬더정확하게결정하고, 장기내 NP 농도및 spotting biokinetics 연구를비교하여흥미로운조직을제공한다 Title : Use of Toxicogenomics for toxicity evaluation of nanoparticles with S. cerevisiae and Japanese Medaka Project Ref. : KOR5157 Last Update : 14/01/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : Korea Start : 2007 End : 2009 Summary : Elucidation of environmental hazardous effects of several nanoparticles by toxicogenomics study with S. cerevisiae and Japanese Medaka / suggestion of guideline for toxic effects of nanoparticle being exposed to environmental and ecosystems. S. cerevisiae and Japanese Medaka 의 toxicogenomics 연구로몇몇 nanoparticles 의환경유해효과에 대해설명하고 environmental and ecosystems 에노출되기시작한 nanoparticle 의독성효과에대한 guideline 을제안했다 Title : NANOTOX (Toxicology of nanoparticles : influence of size, chemical composition and surface reactivity on lung and kidney) Project Ref. : FRA2949 Last Update : 30/06/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : France Start : 2005 End : 2009 Project information is attached

70 English summary : The development of nanotechnologies will lead to an important production of nanoparticles (NP) and consequently to a potential human exposure. The effects of these NP on health are still misunderstood. The main route of exposure to NP is the respiratory tract but secondary targets are possible because NP seem able to cross epithelium and to get access to the blood stream. The aim of this project was to study according to the NP-physico-chemical characteristics (size, chemical composition, surface reactivity), (1) the abilities and ways of NP-accumulation in the main respiratory target cells (epithelium, endothelium, macrophages, pulmonary fibroblasts) and the response of renal cells that have in charge NP elimination (2) and their oxidative and inflammatory effects susceptible to drive lung inflammatory diseases (asthma and chronic obstructive pulmonary disease (COPD)). Due to their potential translocation in blood, their effects on both the pulmonary circulation and the kidney involved in their elimination were investigated. We have used nanosoots specially generated and characterized for this study by one of the partners ( EPLF, Lausanne) that will be compared to commercially available black carbon particles. We have also used TiO2 NP for an identification by electron microscopy as well as fluorescent NP for a detection by confocal microscopy and flow cytometry. Biological effects were studied both in vivo in normal animals and in animals exhibiting respiratory diseases (models of allergic rats or suffering of COPD) and in vitro on the different target cells. We have determined the molecular mechanisms involved in the adaptative or pathological responses: (1) the signalling pathways and transcription factors activation, the activation of proinflammatory and proapoptotic genes using a genomic approach(2) the characterisation and the role of the oxidative stress in the observed responses. It has been shown (1) that the size and the surface of NP are important in uptake, cytotoxicity, oxidative stress and proinflammatory response (2) that the different tested cell types have not the same sensitivity to NP. We have developed a method to quantify the uptake using flow cytometry and we have compared with the data obtained by TEM. About 80 to 90% of the cells accumulate NP, mostly in vesicles, sometimes free in the cytoplasm and nucleus, never in mitochondria. All the tested NP induce the cytoplasmic production of ROS and a proinflammatory response ( IL6, IL8, GM-CSF, TNF) dependent on the NP and the cell type. It was observed that the cytokines are able to be adsorbed on NP and that it depends on the NP and the cytokine. The studies on normal or compromised rats (asthma, COBP) have shown that CB NP can play an adjuvant role in asthma and modify the lung vascular tonus on contrary than TiO2 NP. 나노기술의성장은나노입자의생산으로이어질것이며, 그결과잠재적으로인간에게노출될것이다. 이 nanoparticles 이건강에미치는영향은여전히오해를받고있다. nanoparticles 의노출 main route는 respiratory tract 이지만상피를통과하여혈류를타고이동하기때문에이차적인표적도가능하다. 이 project 목표는 nanoparticles 의물리적화학적특징에따라 (size, chemical composition, surface reactivity) (1) 능력및 main respiratory target cells의 NP 축적의방법과 charge NP elimination 을가지는 renal cells 의반응과 (2) 산화및염증성효과와 drive lung inflammatory diseases ( 천식과만성폐쇄성폐질환 (COPD)) 를연구했다. 혈액에의한잠재적인 translocation 때문에폐순환및나노입자의제거에관련된신장효과조사했다. 우리는상업적으로이용가능한 black carbon particles 와비교

71 하기위해 partners 중하나를특별생산및특성화한 nanosoots 를연구에사용했다. 또한우리는 TiO2 나노입자검증을위하여전자현미경과형광나노입자의검증을위하여공초점현미경및 flow cytometry 를이용하였다. 생물학적효과는정상동물과호흡기질환 ( 알레르기또는 COPD 쥐모델 ) 동물로 Iv vivo 연구와다른표적세포를통한 In vitro 연구를수행했다. 우리는적응및병적반응을포함한분자메커니즘 : (1) 신호경로및전사요소활성화, 게놈접근법을이용한proinflammatory 및 proapoptotic 유전자활성화 (2) 관찰된반응에서산화적 stress 의역할을특성화등을결정하였다. (1) 크기및나노입자의표면은흡수, 세포독성, 산화적 stress 및 proinflammatory 반응이중요하며, (2) 다른실험세포의형태는나노입자의같은민감도를가지지않는것으로보인다. 우리는 flow cytometry 를사용하여흡수를수량화하는방법을개발하였고 TEM에서가져온데이터와비교하였다. 나노입자는세포의 vesicles 에약80~90% 가축적되었고때때로 cytoplasm 와 nucleus 축적되며 mitochondria 에는축적되지않는다. 실험된모든나노입자는세포질에서 ROS 생산하였고, proinflammatory 반응은나노입자및세포의타입에따라의존적으로유도되었다. cytokines 은 NP과흡수가가능하였고 cytokin 와 NP에의존하는것으로관찰되었다. 정상또는손상된쥐의연구는 CB NP 이 TiO2 NP보다천식보조제역할과폐혈관의긴장을완화시켜준다는것을보여준다 Title : Health Effects of Inhaled Nanomaterials Project Ref. : WWC0138 Last Update : 06/12/2006 Status : Underway Checked by National Designated Contact Point? No Country : United States Start : 2004 End : 2007 Summary : Objective: The potential health impact of engineered nanomaterials is unknown. Nanomaterials are particles less than 100 nm in diameter. It is anticipated there will be an exponential increase in the commercial use of these materials in society as carbon nanotubes, nanowires, and silicon/metal alkoxides. This use will lead to a concomitant increase in exposure of the general population to nanomaterials in products and the environment through incidental introduction to the soil, water and air. Little is known what the environmental fate of these particles will be. Epidemiological and toxicological studies on the effects of particulate air pollution support the premise that ultrafine or nanosize particles cause pulmonary inflammation as well as systemic effects. Therefore, we propose to test the hypothesis that inhaled nanomaterials cause respiratory effects in the form of oxidative stress and inflammation. We further propose such events will lead to release of pro-inflammatory cytokines as well as other mediators to induce cell proliferation and alterations

72 in the normal cellular milieu of the airways and alveoli of the lungs. We will test whether these health impacts of nanomaterials on the respiratory system are driven in large measure by (1) particle size, (2) particle composition and/or (3) trace contaminants associated with the manufacturing process of nanomaterials. Approach: These studies will be done at the Center for Health and the Environment, and the Department of Chemistry, University of California, Davis. Nanomaterials will be obtained from commercial sources as well as generated in our laboratories at the University of California, Davis. To study the health effects of nanomaterials, a novel exposure system will be used to generate aerosols of these materials requiring only small quantities (100 mg) for short-term inhalation in rodents. Exposures will take place for up to 3 consecutive days, with exposures being 6 hours/day. Exposures will use specific types of carbon nanotubes (single-walled, bundled and multiwalled) of known size and composition. Ultrafine titanium dioxide (TiO2) and ultrafine carbon black (CB) will also be aerosolized to compare their potential health effects to these various forms of nanotubes in the respiratory tract. Cellular, biochemical and histological assays will be measured in the respiratory tract to determine potential changes due to nanoparticle exposure. These measures include indicators of oxidative stress, inflammation, cell injury and repair and metabolic change. The role of metal contaminants (present in catalysts used in the manufacturing process) will be studied using nanotube preparations with these trace metals/contaminants removed. Expected Results: (1) Characterization of aerosolized nanotubes, ultrafine TiO2. and CB under environmentally relevant conditions found in the workplace. (2) The influence of uniquely distinct forms of nanotubes to produce health effects in the respiratory system. (3) The impact of trace metals associated with nanotubes to enhance/cause health effects due to inhalation. 공학적으로제조된나노물질의강력한건강에대한영향은알려져있지않다. 나노물질의직경이 100nm 이하의입자이다. silicon/metal alkoxide 와 nanowire, 탄소 nanotube 같은사회에서일반적으로사용되는물질들로급격히증가될것으로예상된다. 이나노물질들의사용은제품에나노물질의일반적인노출증가와토양, 공기, 물에부수적인노출된환경을초래한다. 이들입자들의환경적잔류에대하여는아주적게알려져있다. 특별히공기오염의영향에대한위생학적및독성학적연구는미세또는나노크기의입자가전신적영향과같은호흡기염증이원인이란것을지지해준다. 그러므로우리는산화적 stress 와염증의형태로호흡기영향의원인이흡입된나노물질이라는가설을실험하는것을제안한다. 우리는폐의폐포와기도의정상적세포환경에서의변화와세포증식을유도하는다른중재자인 pro-inflammatory cytokies 의방출을초래할것이라는것도제안한다. 호흡기계에서나노물질의건강에대한영향이 (1) 입자크기, (2) 입자구성인지 (3) 나노물질조제과정과관련되는오염에의하여유도하는지를시험하고자한다. 이연구는 University of California, Davis의 Department of Chemistry 의환경및 Health 센터에서수행된다. nanomaterials 의 health effect 를연구하기위하여이물질은 rodents 의단기흡입을위하여소량필요하다. 노출은하루 6시간씩연속 3일, size and composition 잘알려진 carbon nanotubes 사용

73 titanium dioxide (TiO2) 와 carbon black (CB) 도이러한다양한형태의나노튜브호흡기관에서그들의잠재적인건강효과비교할 aerosolized 될것이다. 기대결과 : (1) 환경적으로관련있는상태하에비말나노튜브, 미세 TiO2 그리고 CB의특성은작업장에서만들어졌다. (2) 나노튜브의독특한형태의영향은호흡기계의건강에미치는영향을야기한다. (3) 나노튜브와관련된미량의금속영향은흡입에의해건강에미치는영향의원인이다. (3) 티타니아 (TiO 2 ) 나노물질에대한독성연구논문요약 Reference 연구방법 연구결과 1 Wu J et al. -Anatase:4,10nm -주요표적장기: 피부, 간장 2009,Toxicityandpenetratio noftio2nanoparticlesinhairl essmiceand porcine skin after subchronic dermal exposure,toxicology,inpres s 피부독성 2 Hussain et al. 2009,Oxidativestressandpro inflammatoryeffectsofcarbo nblackandtitaniumdioxide nanoparticles: Role of particle surface area and internalized amount, Toxicology 260, Rutle: 25,60,90nm Degussa P25: 21nm <in vivo> 1) 돼지귀에 10, 60nm 크기 를 30 일동안도포. 2)pathogen-free-mutant hairless mice : 60 일반복. 10,25,P25,60nm 3)5% dorsal skin 투여 SOD,MDA,HYP analysis(skin,liver), 조직병 리학검사,EM 확인,ASS( 조직 내분포확인 ) <in vitro> -Porcine skin 세포주 -4,10,25,60,90nm, 5% -ASS, 투과관찰,tape stripping,tem -Anatase:15nm Anatase+rutile : 평균 50nm -16HBE 세포주 -DTT, real time PCR(GM-CSF), uptake 관찰 <in vivo> 1) 돼지귀 : 10, 60nm 각질층투과 관찰. 심층부위까지투과. 2)60 일반복 : 피부통과하여다른장 기 ( 간장 ) 에서발견. 조직손상관찰. 21nm 는 넓게분포되어뇌에서관 찰됨. 그러나병리학적이상소견없 음. 피부, 간장조직에서 SOD,MDA 증가 à 산화적손상관찰. <in vitro> - 산화적손상관찰, 10nm,P25(21nm) 에 서 HYP level 증가. -free radical 발생 à 산화적손상, -collagen 감소 à 피부노화야기가능성. -15nm 는세포내에서관찰. 다양한 크기의 endosome, cytoplasm 에서 응집된형태로관찰. -TiO2 노출시킨후 4 시간경과 후 RAS 로측정한결과 15nmà2280±140nm, 50nmà2640±260nm 크기로응집되어 관찰. - 산화적손상관찰.(15nm>50nm) - 염증반응유발 (15nm>50nm) -15nm 에서 GM-CSF 증가, pro-inflammation유발. 3 Brian A. Koeneman et al. -<40nm -<40nm크기의나노입자는 media

74 2009,Toxicityandcellularres ponsesofintestinalcellsexpo sedto titanium dioxide,cellboiltoxicol.olin epublish 4 Kobayashi et al. 2009,Comparative pulmonary toxicity study of nano-tio 2 particlesofdiffere ntsizesandagglomerationsi nrats:differentshort-andlon g-termpost-instillationresul ts, toxicology,inpress 폐장독성 5 Eric Fabian et al., 2008, Tissuedistributionandtoxicit yofintravenouslyadminister edtitaniumdioxidenanopart iclesinrats,archtoxicology,8 2, Caco-2세포주 -1,20,100,1000ug/ml -TEER,EM,confocal image,mass balance 실험1 -anatase:5(uf), 24(SF), 154nm (F) -CD(SD)rat -intratracheal (5mg/kg) -1,3,14,21,28일: 몸무게측정 -3일,1달: 폐무게측정 -1일,1주: BALF검사 -1일,3일,1주(7일),1달(28일): 병리검사 ( 폐, 간장, 비장, 뇌 ) 실험2-5nm를 DSP의 2~13mg/ml농도에서분산시킨결과각 18, 65, 300nm 크기로응집. -negative control: DSP -positive control: Min-U-Sil5 (pulmonary inflammation유발물질 ) -CD(SD)rat -intratracheal (5mg/kg) -1, 3, 7, 14, 21, 28, 35, 42, 56, 63, 70, 77, 84, 91일 : 몸, 폐무게측정 -1일, 3일, 1주 (7), 1달 (28), 3달 (91):BALF, 조직병리검사 -20~30nm -Wistar rat -IV 투여 (5mg/kg, 1,14,28일후관찰 ) -장기별무게측정, 분포확인, 혈액검사 (serum) 상태로들어가면 80~350nm크기로응집. -TEER결과 10,100ug/ml농도에서 epithelial integrity를저해하지않는다. -세포생존율검사결과세포죽음관찰없음. -10ug/ml에서는 apical surface관찰, 세포질내 free calcium야기. ** 최종결론 : 그러나 TiO 2 는세포죽음, 다른영향의암시같은결과는보이지않음. -입자의 characterization결과 solution상태에서응집된다. -실험1) 1주일까지는염증관련인자증가 : UF>SF>F 그러나 1달후관찰결과전체적으로회복상태따라서 short term에서입자가작을수록면역반응잘유도. -실험2) 1일 (24h) 때만염증반응관찰. 1달후회복 -Min-U-Sil partilce노출그룹은 3달동안폐에서지속적인 pulmonary inflamation관찰. ** 최종결론 Short-term에서 pulmonary inflammatory반응. 1주일까지는작은크기일수록더잘유도된다. Long-term( 1달 ) 에서는회복단계. -주요표적장기 : 간장, 비장, 폐, 신장그중간장에서가장높은분포, 1일 째가장많이발견. 간에서는 28일까지축적. - serum결과이상관찰없음.

75 6 Kiss et al., 2008,Investigationofmicron izedtitaniumdioxidepenetra tioninhumanskinxenografts anditseffectoncellularfuncti onsofhumanskin-derivedce lls,experimental Dermatology, 17, 피부독성 -in vivo : TiO 2 가들어있는친수성에멀젼선크림 (Anthelios XL SPF 60, La Roche Posay, La Roche Posay, France). In vitro: 9nm anatase -SCID mice, HaCaT keratinocyte 세포주, HDFs, SZ95세포주, primary human melanocyte -MTT, AnnexinV,WB,Nuclear microanalysis, calcium image -invivo: 다양한 nuclear microscopy method결과상피층투과되지않음. -in vitro: 세포생존율, 증식, 아포토시스, 분화같은기능에영향줌. 7 Wang J et al., Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration Toxicology Letters Volume 168, Issue 2, 30 January 2007,Pages 경구독성 8 Vicki H. Grassian et al., 2007,InhalationExposureSt udyoftitaniumdioxidenano particleswithaprimaryparticl esizeof2to5nm.,environme ntalhealthperspectives,115, 흡입독성 -25nm, 80nm, 150nm -CD-1(ICR) mice -oral gavage, 5g/kg -주요장기무게측정 ( 간, 비장, 신장 ), blood biomarker assay, 조직병리학적검사, 조직별분포측정 (ICP-MS) -2-5nm ( 평균 3nm) -C57B1/6 mice -Inhalation, ( 단회 :0.77mg/m 3,7.22mg/m 3 10일간 4시간씩반복 : 8.88±1.98 mg/m 3,,154ug/mice 각각 1주, 2주, 3주경과후관찰 ) 주간급성독성은이상없음. -간장의무게변화에서암컷의 25,80nm 처리그룹에서다른그룹보다유의하게증가된결과를보임. -blood biomarker assay결과암컷의 25,80nm 처리그룹에서 ALT/AST, LDH의증가와 LDH,α-HBDH 증가 를보임. 따라서간장과심근의손상손상이관찰. 그러나조직병리검사결과심장, 폐, 생식기, 비장에서이상결과없음. - 조직병리검사결과 80nm 처리에서뇌, 신장, 간장부분에서이상소견관찰. -조직별분포측정결과간장, 비장, 신장, 폐에서관찰. -2-5nm크기의나노입자가 aerosol 상태에서는 128nm 로 aggregation 됨. -단회흡입시 : total cell 뿐만아니라대식세포수현저히증가, 그러나호중구백혈구와 total protein, LDH, 폐의병리검사결과이상없음. -반복흡입시 : 대식세포수증가, cytokine은매우낮게측정, 조직병리검사결과이상없음.

76 9 Chen HW. et al., 2006,Titaniumdioxidenano particlesinduceemphysema -likelunginjuryinmice, FASEB,20 (13): Fedulov AV et al. Pulmonary exposure to particles during pregnancy causes increased neonatal asthma susceptibility. Am J Respir Mol Biol ; BAL fluid 측정, 폐조직병리검사, Dark field micrographs nm ( 평균 21nm) -ICR mice, THP-1과 A549 세포주 -intratracheal 단회투여 (0.1, 0.5mg) (3d, 1w, 2w) -폐조직형태검사, TUNEL, 면역침강염색법, cdna microarray, real-time PCR, WB, ELISA, pathway 분석. 폐장독성 - BALB/c mice GD 14 - Respirable-size - 50ug/mouse - intranasal insufflation - gene chip microarray ** 최종결론급성독성결과가나타나지만시간이지날수록회복되는것으로보임. -주요표적장기 : 폐 -투여 1주경과후폐의모양과조직구조가유의한변화관찰. 시간과농도의존적으로폐기종변화관찰. -대식세포 accumulation, alveolar septa 광범위한저해,2형 pneumocyte hyperlasia, epithelial cell apoptosis 야기. -PIGF,CXCL1,5, CCL3발현으로폐기종, 세포자살유도. ** 최종결론심각한폐기종유발과 PIGF, 면역반응야기될것이다. - 임신 mice에서급성세포성염증증가 - 차산자에서알러지감수성증가 11 Shimizu M et al. Maternal exposure to nanoparticle titanium dioxide during the prenatal period alters gene expression related to brain development in the mouse. Part Fibre Toxicol ; Slc:ICR mice GD nm - 100μg /mouse/day - SC injection - cdna microarray - 수컷차산자의중추신경기능및발달과 관련있는유전자발현변화있음 12 Takeda K et al. Nanoparticles transferred frompregnant mice to their offspring can damage the - Slc:ICR mice GD nm - 100μg /mouse/day - SC injection - 차산자체중감소 - 차산자일일정자생산률감소 - 차산자부고환정자운동성감소 - 차산자지지세포수감소

77 genital and cranial nerve system. J Health Sci ; Komatsu et al. The effects of nanoparticles on mouse testis Leydig cells in vitro. Toxicol in Vitro ; FE-SEM, TEM - 차산자고환의조직병리학적변화관찰 - Mouse testis Leydig cell line TM nm μg /ml - incubation - TEM, FE-SEM, EDS - RT-PCR 14 Zucker RM et al. Detection of TiO2 nanoparticles in cells by Flow cytometry. Cytometry Part A (A); Hussain S et al. Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells. particle and fibre toxicology ; Li N et al. Spleen injury and apoptotic pathway n mice caused by titanium dioxide nanoparticles. Toxicology letters ; Shi Y et al. Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase-8/t-bid-independe nt mitochondrial pathway. Toxicology letters human-derived retinal pigment epithelial cells (ARPE-19) nm μg /ml - TEM, DLS, SEM, XRD, Light scatting technique - bronchial epithelial cells - 15nm μg /cm -2 - DLS, TEM, - Flow cytometry, caspase assay - ICR female mice - 6-7nm mg/kg, 45day - TEM, XRD, BET(Brunauer- Emmett-Teller), ICP-MS - RT-PCR, ROS assay, ELISA - BEAS-2B cells ±0.031nm μg /ml - EDS, SEM, DLS - MTT assay, Apoptosis assay, ROS assay, Western blot assay, PCR analysis, sirna knockdown μg /ml처치시 TM3 cell의생존률및증식율감소 - 100μg /ml처치시 HO-1 및 StAR mrna 발현변화없음 μg /ml 농도에서 flow cytometer 이용시 5-10개의입자를관찰됨. - 핵주위의 ER에높은농도로관찰됨. - lipid peroxidation, lysosomal membrane destabilization 및 cathepsin B 방출 - TiO2는마우스비장에축적되며, 울혈, 비장조직의림프절증식과비장세포의세포사유발. - caspase-3, caspase-9 및 Bcl-2 가활성화 - 사립체관련세포자멸사기전을통함 - ROS 및형태학적세포자멸사용량의존적증가 - 세포생존률용량의존적감소 - caspase-3 및 PARP는형태학적세포자멸사증가에비례해서증가 - caspase-9증가한반면caspase-8 및 t-bid는변화가없음

78 196; 사립체관련세포자멸사기전을통하 지만 caspase-8/t-bid 기전을통하지 않음 Silica 나노실리카는현재의약품첨가제, 식품성분, 질병진단, 약물전달체, 화장품등에광범위하게응용되고있다. 실리카의경우도일반적으로크기에비례한독성이보고되어지나크기의존적으로중대한독성영향은보고되지않았다. 실리카의경우크기외에형태 (shape) 변화에의한다양한독성이제시될수있음을제시하고있다. 주요독성기전으로는 ROS 유발에따른광범위한장기별독성가장가능성이높은것으로제시되고있다. OECD 조사 Title : Health effects of fine and ultrafine particle toxicity in the lung - genotoxicity Country : Germany Start : January 2009 End : December 2010 Summary : Inhalative application of ultra-fine particles in animal experiments has shown a carcinogenic effect in the lung. With particles categorised as inhalable a carcinogenic effect was also observed. Currently, no clear decision can be made whether these effects are based on mutagenicity or not. Within the project the mutagenic potency of some particulate substances as well as the underlying mechanism has to be investigated in test systems (in vitro and/or in vivo) preferably using lung epithelial cells. 동물실험에서미세한입자들의흡입시험은폐에발암성을나타내었다. 흡입발암효과와같은분류의입자로써또한관찰이되었다. 현재이러한효과가돌연변이를유발하는지또는아닌지에대한여부는불명확하다. 근본적인기전과마찬가지로일부특별한물질의돌연변이원성프로젝트내에서폐상피세포들을이용한시험을조사하였다 Title : Description and evaluation of a carcinogenicity study with 19 dusts in rats Country : Germany Start : October 2000 End : December 2002 Summary : There is a lack of scientific knowledge on the properties of insoluble particles which are responsible for the toxic effects in the lung of human and experimental animals (e.g. size, surface, density). To

79 contribute to the identification of relevant parameters determining particle carcinogenicity a study with 19 different dust samples was evaluated. The results aid in the evaluation of published data on insoluble dusts and finally could help in establishing adequate worker protection. Project URL : 인간과실험동물의폐에서원인이될수있는독성효과와극도의미세한입자의과학적지식에대해서부족한실정이다. 관련매개변수를식별하는데기여할수있는 carcinogenicity 연구는다른 19가지먼지샘플을평가했다. 이결과불용해성의먼지들의데이터평가와작업자의보호를설정하는데도울수있었다 Title : Carcinogenicity studies of granular dusts in rats - results and interpretations Country : Germany Start : July 2006 End : July 2007 Summary : A large intratracheal carcinogenicity study in rats with 19 granular dusts has been performed. The histological diagnoses of the experimental groups have been finished. These data were used for a statistical analysis of tumour incidences and dose-responses and to derive an optimal metric for the carcinogenic potency of granular bio-durable particles. In addition the results will be discussed with respect to recent publications on the issue. Project URL : 19 granular dusts를가지고 rats를이용하여대규모의기도내발암성연구를하였다. 이런실험적인그룹의조직학적진단은종료하였다. 이들의데이터는암발생의통계학적분석과용량반응성그리고 granular bio-durable particles 의발암성을위한최적의계량을이끌어내는데사용되었다. () Silicon dioxide 나노물질에대한독성연구논문요약 연번 Reference materials and Methods 결과 1 Sohaebuddin S.K et al. - 30nm Nanomaterial cytotoxicity is - Cytotoxicity composition, size, and cell type dependent. - DLS, MTS, ROS test Particle and Fibre Toxicology 2010, 7:22 - MitoProbe DilC1 - Caspase-3 and Caspase-7 activation, 2 Yang XF et al. SiO2 nanoparticles induce Annexin V/Propidium iodide assay - 15, 30nm - HaCaT Cell - 세포자멸사, 세포괴사는세포타입에따라다양함. - 나노물질을구성과흡입에따라다양함

80 cytotoxicity and protein expression alteration in HaCaT cells. Particle and Fibre Toxicology 2010, 7:1 3 Ye Y.Y et al. Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line. Toxicology in Vitro 24 (2010) D-DIGE anakysis - MALDI-TOF-MS - L-20 cell - Colloids (21,48,86nm) - LDH, TEM, ROS, GSH, V-FITC/PI assay - HaCaT cell의생존능력이감소함. - SiO2 particle보다작은사이즈의 cell은 apoptotic rate 속도가높아짐. - 위사진은노출시간에따른변화 - Colloid의세포독성은크기, 양, 시간에의존함. Alumina 나노입자가일반적으로화장품용도로 흡습제, 연마제로사용되는외에전지, 소방재로사용되고있 다. 제한적인응용범위로인해독성평가연구결과가부족한상태이다. OECD 조사 Title : EVALUATION AND CONTROL OF OCCUPATIONAL HEALTH RISKS FROM NANOPARTICLES Project Ref. :WWC5669 Last Update :28/07/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Denmark Start :2006 End :2007 Summary : Gather and structure the existing Nordic knowledge and experience on nanoparticle risk assessment, and enhance the use of existing data, to guide future data collection, Provide a knowledge base on evaluation and control of the occupational health risk of engineered nanoparticles as input to the

81 Nordic Working Environment Authorities for their development of polices and strategies for controlling nanoparticle exposures. Strengthen the Nordic research potential in the field and facilitate Nordic participation in EU sponsored research. 모인기존 nordic 지식과 nanparticle 위험평가의경험. 미래의데이터수집품을이끌기위해기존데이터의사용을강화는평가에대한지식기초와그들의개발에대한환경권한을움직이게하는것은단속하는 nordic의입력으로서의설계된 nanoparticle 의직업건강위험의통제그리고 nanoparticle 노출을통제하기위한전략을제공한다. 필드에서잠재적인 nordic 연구를강화하고 EU에후원받는연구에 nordic 참가를용이하게한다 Title : NanoKem: Nanoparticles in the paint- and lacquer industry. Exposure and toxic properties. Project Ref. : WWC5671 Last Update : 28/07/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : Denmark Start : 2007 End : 2010 Summary : Evaluation of exposure risks while handling nanoparticles and while sanding paint and lacquer that contain nanoparticles. Physical and chemical characterization of nanoparticles used in paint and lacquer and characterization of dust created by sanding paint and lacquer that contain nanoparticles. Identification and evaluation of the toxic effects of nanoparticles used by the paint- and lacquer industry and comparison to the effects created by sanding dust from paint products. Translocation of particles across barriers, carcinogenicity, effects on cardiac system, embriogenic effects and allergenic effects will be part of the study. Development of model for risk assessment of nanoparticles, for use or expected use in the paint- and lacquer industry. 노출의평가는 nanoparticle 은사포로닦은것을칠하고 nanoparticle 을포함한래커와 nanoparticle 을다룰때위험하다. Nanoparticle 의물리화학적특징은 nanoparticle 로구성된래커와사포로닦고페인트한것에의해만들어진먼지의특징과페인트와래커가사용되어진다. 페인트에의해사용되어지는 nanoparticle 의유해한효과의식별및평가-그리고래커산업과페인트제품으로부터나온사포먼지를비교하였다. 내벽으로입자들의이동, 발암성, 강심제시스템의효과, embriogenic 효과와알레르기효과가연구의부분일것이다. paint 와래커산업의사용또는예상되는사용에대해 nanoparticle 의위험평가의모델을개발한다 Title : Risk analysis and governance of nanomaterials Project Ref. : WWC5676 Last Update : 28/07/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : Denmark

82 Start : 2005 End : 2008 Summary : To identify and assess the potential risks of nanotechnologies; Explore and further develop current decision-making tools for dealing with complex and uncertain emerging risks, and; provide recommendations on how to govern nanotechnologies within a precautionary framework focused at protecting human health and the environment without hindering innovation. 나노기술의잠재적인위험을확인하고평가하는것 ; 복잡하고불확실한신흥위험을다루기위해탐구하고현재의의사결정도구를한층더개발한다. 그리고 ; 지배하는방식으로인간의건강과환경을혁신을저해하지않고보호하는것에집중하는예방체재내의나노기술에서권고를제공한다 Title : Fate of nanoparticles in mammalian cells: effect of composition, shape, size and surface charge Project Ref. : WWC0178 Last Update : 17/12/2008 Status : Underway Checked by National Designated Contact Point? Yes Country : Canada Start : 2005 End : 2007 Summary : The purpose of this two-year study is to examine the effect of inorganic nanoparticle morphology, size, composition and surface charge, as well as the particle presentation method, for use with mammalian cells. The proposed experiments are designed 이번 2년동안의연구목적은포유류의세포를사용하여 nanoparticle 형태학, 규모, 구성요소와표면전하의효과뿐만아니라입자프리젠테이션방법을조사하는것이다. 제안된실험은디자인되었다 Title : CHARACTERIZATION AND TOXICOLOGICAL EVALUATION OF NANOPARTICLES FROM LIQUID-BASED NANOFILM PRODUCTS Project Ref. :WWC5670 Last Update :28/07/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Denmark Start :2006 End :2010 Summary : To understand the atmosphere-chemical reactions risk of exposure to chemicals and nanoparticles during use of nanoparticle-base pump and spray products as well as screening their potential cellular and respiratory effects in vitro and in vivo at different user scenarios

83 nanoparticle 을기반으로한펌프의사용동안에화학제품과 nanoparticle 의대기화학반응위험과 vitro 에서그리고다른사용자시나리오에서의체내에서그들의잠재적인세포와호흡기효과를가리는 것뿐만아니라스프레이제품을이해한다 Title : Framework Development for Adaptive Environmental Risk Assessment and Uncertainty Analysis for Nanomaterials Project Ref. : WWC5678 Last Update : 28/07/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : Denmark Start : 2007 End : 2010 Summary : Develop and apply uncertainty analysis tools for environmental risk assessment of nanomaterials in order to identify and document critical areas of uncertainty; Develop a framework for adaptive environmental risk assessment for nanomaterials in various forms; Evaluate nano-based environmental technologies with respect to environmental risks and the applicability of the developed frameworks on these technologies. 불확실성의비판적인지역을확인하고문서화하기위해개발하고 nanometerial 의환경위험평가를위해불확실성분석도구를사용한다. 여러가지형식에서 nanomaterial 의적응성환경위험평가를위해체재를개발한다. 이기술에서환경위험에관한 nano를기반으로한환경기술과선진체재의적용성을평가한다 Title : In vitro and in vivo evaluation of the ecotoxicity of nanoparticles Project Ref. : BEL9939 Last Update : 01/03/2010 Status : Not-Completed Checked by National Designated Contact Point? No Country : Belgium Start : October 2006 End : October 2010 Summary : The ecotoxicity of engineered nanoparticles of various types and sizes is evaluated. Furthermore, central hypotheses in nanotoxicology are tested, i.c. the importance of surface area, the ecotoxicity relative to bulk material, the occurrence of indirect effects due to light absorption or nutrient adsorption. Ecotoxicological effects assessment for the aquatic environments is performed using the standard test organisms Pseudokirchneriella subcapitata and Daphnia magna. Cytotoxicity tests are peformed with the fish gill cell line RTGill-W1. 타입과규모의설계된 nanoparticle 의생체독성은평가된다. 게다가, 나도독성의중앙가설즉, 표면지

84 역, 크기물질과관계가있는생체독성, 가벼운흡수에의한간접효과의발생또는영양의흡착의중요성은테스트되어진다. 수중환경을위한생체독성효과평가는표준테스트유기체인 Pseudokirchneriella subcapitata 와 Daphnis magna를사용하면서수행된다. 세포독소테스트는물고기아가미세포라인인 RTGill-W1으로실행하였다 Title :Characterization of substances in naoscale as a basis for their assessment under Regulation (EC) No 1907/2006 (REACH) Project Ref. :DEU5155 Last Update :08/06/2010 Status :Underway Checked by National Designated Contact Point? Yes Country :Germany Start :May 2010 End :December 2011 Summary : Literature research considering the available information for physical chemical characterization of nanomaterials. Evaluating which physical chemical methods listed in Regulation (EC) No 440/2008 can be applied to substances in nanoscale. Evaluating which of the physical chemical data of substances in microscale can be transferred to substances in nanoscale. Identification of systematic differences concerning substances in nanoscale and microscale. Identification of additional physical chemical methods which are necessary to characterize nanomaterials. These methods should satisfy international standards to be accplicable under the REACH Regulation. Nanomaterials 의이용가능한정보특성화를고려하고있는문학연구 NO440/2008 이할수있는규칙 (EC) 에리스트되어진물리화학적방법을평가하는것은 nanoscale 의물질에적용되어질것. microscale 의물질의물리화학적인데이터중 nanoscale 에서물질로이동해질수있는가평가하는것 nanoscale와 microscale 의물질에관한체계적인차이의식별 nanomaterial 을특징짓기위해필요한추가적인신체의화학방법의식별 이방법은정규의범위의아래의적용할수있는국제표준을만족시켜야한다 Title : Histological diagnosis of rat lungs after exposure to granular, biopersistent dusts Project Ref. : DEU3648 Last Update : 08/06/2010 Status : Completed Checked by National Designated Contact Point? Yes Country : Germany Start : November 2003 End : October 2005 Summary : There are scientific discussions whether the existing Occupational Exposure Limit for granular dusts

85 is sufficiently health protective for workers. In this respect, a carcinogenicity study in rats with intratracheal administration of 19 different granular dusts plays a major role. The lungs of these rats have been evaluated only partly histologically. In this project the remaining rat lungs will be evaluated for the presence of tumours. In addition the degree of fibrosis and inflammation will be examined in selected rat lungs. The results are expected to contribute to the evaluation of the carcinogenic activity of granular, biopersistent dusts and to improve the health protection of workers. 입상의먼지를위한기존직업한계가충분히근로자를위한건강보호재에대해과학적인논의가있다. 이런점에서, 19가지다른입상먼지의기관내투여로쥐에관한발암성연구는주요한역할을한다. 쥐의폐는단지부분적으로조직학적으로평가되었다. 이프로젝트에서나머지쥐의폐는종양의존재를위해평가될것이다. 또한섬유증과염증의정도는선택된쥐의폐에서조사될것이다. 결과는입상과 biopersistent 한먼지의발암성의활동의평가에공헌하고작업의건강보호를개선할것으로예상된다 Title : Carcinogenicity studies of granular dusts in rats - results and interpretations Project Ref. : DEU6694 Last Update : 08/06/2010 Status : Completed Checked by National Designated Contact Point? Yes Country : Germany Start : July 2006 End : July 2007 Summary : A large intratracheal carcinogenicity study in rats with 19 granular dusts has been performed. The histological diagnoses of the experimental groups have been finished. These data were used for a statistical analysis of tumour incidences and dose-responses and to derive an optimal metric for the carcinogenic potency of granular bio-durable particles. In addition the results will be discussed with respect to recent publications on the issue. 19 가지입상의먼지로쥐에큰기관내발암성연구를수행되었다. 실험적그룹의조직학의진단은완료되었다. 이데이터는결과에대한통계적분석과농도-반응생물학적내구성입자를위해최적조건거리를도출하기위해사용되었다. 게다가결과는주제에대한최근의출판물에관해서논의될것이다 Title : Description and evaluation of a carcinogenicity study with 19 dusts in rats Project Ref. : DEU1371 Last Update : 08/06/2010 Status : Completed Checked by National Designated Contact Point? Yes Country : Germany Start : October

86 End : December 2002 Summary : There is a lack of scientific knowledge on the properties of insoluble particles which are responsible for the toxic effects in the lung of human and experimental animals (e.g. size, surface, density). To contribute to the identification of relevant parameters determining particle carcinogenicity a study with 19 different dust samples was evaluated. The results aid in the evaluation of published data on insoluble dusts and finally could help in establishing adequate worker protection Rat 에서 19 가지먼지의발암성연구의기술과평가 인간과실험적동물의폐에독성효과의원인이되는물에녹지않는입자들의특성에대한과학적지식은부족하다 ( 크기, 표면, 농도등 ). 입자발암성을결정하고있는관련매개변수의식별에공헌하기위해 19가지의다른먼지샘플이있는연구는평가되었다. 결과보조물은물에녹지않는먼지에관한발표된데이터의평가를. 그리고적절한근로자보호를확립할때에마침내거들수있었다 Title : Research and Development of Nanoparticle Characterization Methods - Evaluating Risks associated with Manufactured Nanomaterials - Project Ref. : JPN2691 Last Update : 13/07/2010 Status : Underway Checked by National Designated Contact Point? Yes Country : Japan Start : June 2006 End : March 2011 Summary : This project consists of development of methods for preparation and characterization of test samples, toxicity tests using well-characterized samples, exposure assessment, and research on the societal aspects of nanomaterials. 이프로젝트는준비의방법과테스트샘플의특성화의개발, 잘특징화된샘플을사용하고있는독성테스트, 노출평가와 nanomaterials 의사회의방향에관한연구로구성되었다 Title : NanoPIST - Intentionally producted nanoparticles: toxicological signatures Project Ref. : FRA7696 Last Update : 08/03/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : France Start : November 2008 End : November

87 Summary : Nanosciences and nanotechnologies lead to a deeply increasing production of nanomaterials, underlining the necessity to consider their potential health effect. The surface reactivity of nanomaterials could render them toxic since they enter easily in 나노과학과나노기술은 nanomaterial 의매우증가하고있는생산, 그들의잠재적인건강효과를고려하기위한필요성을강조하고있다. 그들이쉽게안에서들어옴으로 nanomaterial 의표면반응성은그들을유해하도록할수있었다 Title : Chemical Fate, Biopersistance, and Toxicology of Inhaled Metal Oxide Nanoscale Materials Project Ref. : WWC0752 Last Update : 06/04/2007 Status : Underway Checked by National Designated Contact Point? No Country : United States Start : 2005 End : 2008 Summary : Objective: Observations of unpredicted biological disposition, increased biopersistence, and increased toxicity of the same inhaled materials as they decrease in size (and increase in surface area) has sparked interest in the developing field of nanotechnology. There is an immediate need to confirm and extend those observations as they apply to nanoscale materials (NSMs), including NSMs of different composition. This proposed research will directly compare the biological disposition, persistence, and toxicity of two commercial nanoscale and non-nanoscale metal oxide classes. We will determine the impact of particle class, particle size, and surface area on NSMs that are of significant commercial relevance for nanotechnology (SiO2 and Al2O3). The proposed studies will test the hypothesis that the biological disposition, persistence, and toxicity of metal oxides change with size (comparisons between nanoscale and micron size) and composition (comparisons between SiO2 and Al2O3) of metal oxide powders. Specifically, we will test the hypothesis that metal oxide particles that are manufactured in the nanoscale have (a) longer biological persistence and (b) increased pulmonary and systemic toxicity relative to the same metal oxides of larger sizes. Approach: Two primary objectives will be executed after repeated inhalation studies (F344 rats, 6 hours/day, 5 days/week for 6 weeks to nanoscale- and micron-size materials of the same composition): (1) determine biological disposition (translocation/elimination/persistence) by measurement of residual metal oxide in plasma and six target organs (lung, brain, liver, kidney, spleen, intestine), and (2) determine local (lung/respiratory tract) and systemic toxicity by measurements of sensitive biochemical markers of inflammation and oxidative stress in addition to histopathological analysis. For both (1) and (2), measurements will be made immediately post exposure, 4 weeks post exposure, and 17 weeks post exposure to complement previous studies in our laboratory on micron-size SiO2 (Langley et al., 2004). Expected Results: We expect to see extrapulmonary disposition of both types and sizes of metal oxide powders. Because both metal oxides will have partial solubility in vivo, we expect some of the retained and eliminated material

88 will be in a soluble form (this not tested directly). We expect that the NSM will have a longer biopersistence and its increased surface area will lead to increased pulmonary and systemic toxicity. However, we expect that the effect will diminish with decreasing dose and that if doses in the range of plausible human exposure (not yet known) are studied, the toxic effects will diminish 목표 : 예측하지못하는생물학적배열, 증가된 biopersistence 와그것들의크기 ( 그리고표면지역의증가 ) 가감소함으로써같이흡입된물질의독성이증가되어진것을관찰하는것은나노기술이발전하고있는지역의관심에활기를불어넣고있다. 다른구성요소중에 NSMs를포함한, nanoscale materials (NSMs) 를적용이됨으로써그것들의관찰을연장하고확장이직접필요하다. 이제안된연구는생물학적배열을직접비교할것이다. 이제안된연구는생물학적배열, 지속과두가지상업상의 nanoscale 와비 nanoscale 금속 oxide 종류의독성을직접비교할것이다. 나노기술 (SiO2와 Al2O3) 를위해중요한상업상의의미에서인 NSM에서우리는입자종류, 입자규모와표면지역의영향을결정할것이다. 제안된연구는생물학적배열, 지속과크기 (nanoscale와 micron 크기비교 ) 와함께금속 oxides 의독성이전하를가진바뀐다는것그리고금속 oxide 가루의구성요소 (SiO와 Al2O3 사이비교 ) 의가설을테스트할것이다. 특히, 우리는 nanoscale 에서제작된 oxide 입자에금속을씌우는가설을테스트할것이다 (a) 지속적으로긴생물학 (b) 큰규모의같은금속 oxides 와관계가있는증가되어진폐와침투적인체계의독성. 접근 : 2가지주요한목적은반복적인흠입연구를진행후실행될것이다 ( 같은구성요소중 nanoscale- 과 micron- 크기의물질들을 F344 rat에 6시간 / 일, 5일 / 주 6주동안 ). : (1) 혈장과 6개의목표기관 ( 폐, 뇌, 간, 콩팥, 비장, 창자 ) 에서잔여금속 oxide의측정에의해생물학적배열을결정한다. (2) 병리조직학적분석을더하여염증과 oxidative stess의민감한생화학표시의측정에의해지역과체계의독성을결정한다. (1) 과 (2) 을위해, 측정은즉시후노출, 4 주후노출과 micron- size SiO2에서우리실험실에관한이전의연구를보충하는 17 주의후노출이될것이다 (Langly et al., 2004). 예상되는결과 : 우리는금속 oxide 가루의규모와타입의폐외배열를볼것으로예상한다. 양쪽금속 oxides 가생체내여부분적인용해도를가질것이기때문에, 우리는보관되고제거된물질의일부가증대시키게되는폐와침투적인독성에선도일것이라고예상한다 ( 직접적으로실험을하지는않았다 ). 우리는 NSM이긴 biopersistence 와증가하고있는농도와인간의노출의범위의복용량 ( 아직알려지지않았다 ) 을연구하게되면, 유해한효과가감소될것이라고예상한다 Title :Characterisation of ultrafine particles (nano particles) for workplace protection (part 2) Project Ref. :DEU2847 Last Update :02/07/2010 Status :Completed Checked by National Designated Contact Point? Yes Country :Germany Start :July 2004 End :December 2007 Summary : To assess the increased biological effects of ultrafine particles 100 nm in diameter not only the free primary particles, but also the aggregates and agglomerates (A+A) of these primary particles, the stabilities of these A+A and the solubility of the primary particles have to be considered. According to environmental measuring programs workplace measurements of ultrafine aerosols are performed with instruments like the scanning mobility particle sizer (SMPS), that classify particles according

89 to their diameter, but do not distinguish between massive particles and A+A which consist of ultrafine primary particles. Therefore, in comparison with measurements from SMPS, a method for the characterisation of ultrafine aerosols by transmission electron microscopy (TEM) has been developed. In addition to free primary particles and aggregates the number and the size of primary particles within the A+A can be registered. It is intended - to optimize this measuring method until it generates reproducible results for a duration of sampling of 1 hour and to apply it to relevant work places, - to characterise the stability of the A+A within aqueous suspension (i.e. to detect differences as they already have been observed between the A+A from diesel soot and from fume of metal inert gas welding) and - to analyse 6 of the ultrafine samples of the 19 granular dust samples that were tested on rats in the carcinogenicity study of Pott and Roller Free primary particles and aggregates are registered with the condensation particle counter. Simultaneously the ultrafine particles are sampled on nuclepore filters. TEM-analysis is performed at magnification x 40,000 in 10 to 25 TEM-fields using direct and indirect preparation. Information is obtained on - the morphology of the ultrafine particles - their composition of A+A - the number and the surface estimate of all primary particles - the size distribution of the A+A - distinction between liquid and solid particles - on the solubility of the ultrafine particles - on the stability of the A+A in aqueous suspension, when adequate dispersion agents are used. It is expected that the results on the concentrations of primary particles per mg or per A+A, which are generated for ultrafine aerosols in certain work places are useful to characterise the ultrafine aerosols and may be generalized for these work places. 자유로운주된입자들뿐만아니라이들의주된입자들의함계와덩어리들 (A+A) 안에직경이 100nm 의초미립자의증가되어진생물학적효과를평가한다, 이 A+A 의안정성과주요한입자의용해도는고려되어야한다. 초미세에어로졸의작업장측정의환경측정프로그램은이동성입자치수 (SMPs) 와같은기구로수행되어지고, 그들의직경에따르는입자를분류하다. 그러나초미세주된입자로구성되는방대한입자와 A+A 를구별하지않는다. 그러므로, SMPs로부터측정과비교해볼때, 전달전자현미경검사 (TEM) 에의한초미세에어로졸의묘사의방법은개발되었다. 자유로운주된입자와합계에더하여 A+A 내의주된입자의수와규모는등록될수있다. 그것은의미하게된다. - 1시간의견본추출기간과그것을관련직업에적용하기하여재생가능한결과를생성할때까지이것을측정하는방법을최적화하는것, - 수성중지내에 A+A 의안정성을묘사하는것 ( 그들이디젤매연으로부터그리고금속불활성가스의연기로부터 A+A 사이에이미관찰되었을때차이를발견하기위해용접한다 ) 그리고 - Pott와 Poller 2003 에관한발암성연구에서쥐에서테스트되었는 19가지의입상의먼지샘플의초미세샘플중의 6가지를분석한다. 자유로운주된입자와합계는응축입자수로등록된다. 동시에초미립자 nuclepore 필터에서샘플로추출한다. TEM-analysis 는 10~25 TEM-field 에직접적으로사용되고모든주된입자들의표면을확대 *40000 으로행한다. 정보는적절한분산물질이사용되어질때얻어진다. - 형태학의초미립자 - A+A 의구성 - 모든주요한입자의수와표면추정치 - A+A 의규모배포 - 유동적이고고체의입자의차이 - 용해도에서의초미립자 - 수성중지의 A+A 의안정성이다, mg당또는 A+A 당주된입자의결과는기대되어진다. 어떤작업장소에서초미립에어로졸을위해생성된것은초미립에어로졸을묘사하는것에도움이되고이작업장소를위해일반화될지도모른다 Title :Study of the transfer of aluminium to the brain via the olfactory route (INRS) Project Ref. :FRA7491 Last Update :30/06/2009 Status :Underway

90 Checked by National Designated Contact Point? Yes Country :France Start :2009 End :2011 Summary : Neurotoxic effects of aluminium have turned up among patients suffering from kidney disorders and treated by dialysis. In addition, certain epidemiological studies mention neurological disorders among employees highly exposed to aluminium (welders, aluminium powder production workers). Occupational exposure to aluminium exists in the metal production and metalworking sectors (foundry, powder production, recycling, welding). It occurs primarily via the inhalatory route. Depending on the sector of activity, employees can be exposed to different forms of aluminium. The presence of fine and ultra fine particles at the workplace (welding fumes) is a subject of great concern in occupational health. We propose to study, in the rat, the potential transportation of aluminium to the central nervous system via the olfactory route by short-circuiting the hematoencephalic barrier. We shall study the influence of the solubility and granulometry of the compounds studied using soluble (aluminium chloride, AlCl3) and not very soluble (aluminium fluoride, AlF3) salts as well as insoluble aluminium particles (Al2O3) of micrometric and nanometric size. The aluminium will be quantitatively analysed in different cerebral structures by atomic absorption spectrometry in a graphite furnace after intranasal instillation of different doses of aluminium (5, 10 and 20 µg of metal Al). A kinetic study will be conducted by analysing the aluminium distribution in the different structures at different time intervals after the intranasal instillation treatment 알루미늄의신경독성의결과는콩팥장애와투석에의해치료되어지고있는환자들중에서찾았다. 또한, 어떤역학연구는알루미늄 ( 용접공, 알루미늄가루생산근로자 ) 에매우노출되는근로자중에신경학적인장애에대해언급한다. 알루미늄의직업노출은금속생산과금장부문 ( 주조, 가루생산, 재활용, 용접하는것 ) 의안에존재한다. 그것은흡입을통하여주로발생한다. 활동의부분에따라, 근로자들은알루미늄의다른형식에노출될수있다. 작업장 ( 연기를용접하는것 ) 에서의미세하고초미세입자의존재는직업건강의엄청난원인이다. 우리는 rat에서 hematoencephalic 장벽을단락시키는것에의한 olfactory 의길을거쳐중추신경계에알루미늄의잠재적인운송에서연구를제안한다. 우리는용해도의영향과연구하게될혼합물의 granulometry 를연구는 micrometric 과 nanometric 크기의물에녹지않는알루미늄입자 (Al2O3) 뿐만아니라잘녹지않는소금 ( 염화알루미늄, AlCl3) 과물에녹일수있는 ( 알루미늄불소, AlF3) 것을사용하였다. 알루미늄을다른농도 (5,10, 20ug) 로비강점적투여한후흑연용접의원자흡수분광분석법에의해다른소뇌구조를정량적으로분석하게될것이다. 운동의연구는비강의점적투여처리후에다른시간간격으로다른구조에서알루미늄배포를분석하여실시될것이다. (5.2.) Aliminum Oxide 나노물질에대한독성연구논문요약 연 번 Reference materials and Methods 결과

91 1 Hussain S.M et al. In vitro toxicity of nanoparticles in BRL 3A rat liver cell. Toxicology in vitro 19(2005) , 103nm - BRL 3A rat liver cells - MTT, LDH, ROS, GSH test 2 Braydich-Stolle et al. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. toxicol Sci 88(2005) Kim IS et al. Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells. J Nanosci Nanotechnol (5): Di Virgilio AL, et al. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells. J Hazard Mater ;177(1-3): nm - Mouse spermatogonia stem cell line c Human lung epithelial cell, A549 carcinoma cell, L-132 normal cell - clonogenic assay - CHO-K1 cell - Neutral red uptake assay - MTT assay - Uptake of NPs by TEM 30 및 103nm 크기의 Al nanoparticles 에서는독성을나타내지않음 - 세포의형태적인관찰에서는 10μg /ml아래의용량에서세포자멸사, 세포괴사및위축이없으나, Al particle이세포질에축적되어있음. - LDH leakage : 5μg /ml에서부터 LDH leakage가증가함 - Apoptosis Assay : 5 and 10μg /ml 에서세포자멸사증가 - Al2O3는세포증식및세포생존에약간의부작용발생 5 Balasubramanyam A et al., Evaluation of genotoxic effects of oral exposure to aluminum oxide nanomaterials in rat bone marrow. Mutat Res ;676(1-2): Al 2O 3(30-40 nm) - Wistar rats bone marrow cells - size conform TEM - dose : 5~2000mg/kg - micronuleus and chromosomal analysis. - ICP-MS aberration - 50μg /ml를 CHO-K1 cells에노출된 TEM 사진 - TiO2가관찰되는것보다적은양의응집된 Al2O3 관찰 - 모든처치군에서사망없음 - Al 2 O 3 (30-40nm) : 소핵의빈도증가는 mg/kg bw에서관찰 - Al 2O 3(30nm) : 염색체이상의증가는 mg/kg bw에서관찰 - Al 2 O 3 (40nm) : 염색체이상의증가는 2000mg/kg bw에서관찰

92 Zinc Oxide 나노산화아연은화장품, 페인트, 약물전달체, 충진제등에광범위하게응용되고있다. 산화아연은화장품용도로이산화티타늄에비해투명성이우수해 자외선차단제및도료로사용되고 있으나산화과정에서촉매활성반응성이높아사용에제한적인단점이있다. 이산화티타늄과같이활성산소형성을경유한 DNA 에잠재적독성과피부독성에서의나노입자의표피 투과성여부이다. OECD 조사 Title : In vitro and in vivo evaluation of the ecotoxicity of nanoparticles Country : Belgium Start : October 2006 End : October 2010 Summary : The ecotoxicity of engineered nanoparticles of various types and sizes is evaluated. Furthermore, central hypotheses in nanotoxicology are tested, i.c. the importance of surface area, the ecotoxicity relative to bulk material, the occurrence of indirect effects due to light absorption or nutrient adsorption. Ecotoxicological effects assessment for the aquatic environments is performed using the standard test organisms Pseudokirchneriella subcapitata and Daphnia magna. Cytotoxicity tests are peformed with the fish gill cell line RTGill-W1. 다양한유형들과크기의제조된나노의환경독성을평가할수있다. 또한, 나노독성학의중심가설은시험되었으며, 노출영역의중요성, 대량소재와관련된환경독성, 광흡수또는영양흡수의따른간접적영향발생등이다. 수생환경을위한환경독성학적효과평가는표준테스트생물인 Pseudokirchneriella subcapitata 및 Daphnia magna를사용하여수행됩니다. 세포독성테스트는물고기의아가미세포인 RTGill-W1으로수행한다 ZnO 나노물질에대한독성연구논문요약 연 Reference materials and Methods results and conclusion 번 1 Beyerle A et al., Toxicity - Murine alveolar epithelial-like - 세포생존능은 5μg / ml에서급격히감 pathway focused gene type II cells(la4) 소하였으며, 세포독성역시 5μg / ml급격 expression profiling of - 70nm 히증가하였음. PEI-Based polymers for , 2.5, 5, 10μg / ml - 관련유전자는 Heat shock 관련유전 pulmonary applicatoins. - LDH, cell viability, RNA 자및산화스트레스유전자인 Hspb1, mol pharmaceutics isolation, Gene Expression Hmox1, Mt2, Ugt1a2가처치 6시간후 7(3) profiling 에관찰되었음. 2 Heng BC et al., Toxicity of - Human bronchial - ZnO 10μg / ml농도에 H2O2를무처치 zinc oxide(zno) nanoparticles epithelial cells(beas-2b) 시 99% 이상생존 on human bronchial - <10nm - ZnO 10μg / ml이상에서는급격한생존 epithelial cells (BEAS-2B) μg / ml 률이감소됨. is accentuated by - WST-8 assay - H2O2 5와 10μM을처치하였을때

93 oxidative stress. Food and chem Toxicol ; Yin H et al., Effects of surface chemistry on cytotoxicity, genotoxicity and the generation of reactive oxygen species induced by ZnO nanoparticles. Langmuir (19); Moos PJ et al., Zinc oxide particulate matter requires cell contect for toxicity in human colon cancer cells. Chem Res Toxicol ; Yuan JH et al., Determination, characterization and cytotoxicity on HELF cells of ZnO nanoparticles. Colloids and Surfaces B: Biointerfaces ; Palomaki J et al., Engineered nanomaterials cause cytotoxicity and activation on mouse antigen presenting cells. Toxicology ; Huang CC et al. Oxidative stress, calcium homeostasis and altered gene expression in human lung - WIL-2S human lymphoblastoid cells mg/ml - Oleic Acid coating, Poly(methacrylic acid) coating, Cell culture medium coating - MTT assay, Measurement of ROS, TEM, XPS - RKO colon cance cells - nzno : mean 45nm - mzno : mean 330nm μg / cm2 - ICP-S, TEM, SEM, DLS - Cell viability(cck-8) assay, Apoptotic assay(cell Lab Quanta SC flow cytometer), Mitochondrial potential(mitoprobe JC-1 Assay kit) - HELF cells - 20±5, 30±10, 40±10nm mg/L -1 - TEM, XRD, MTT, SEM - RAW cell line and bmdc cells. - 20nm ug/ml(RAW 264.7) ug/ml(bmDC cells) - Tryphan blue - BEAS-2B cell - 20nm ug/ml. - SEM, TEM, BET, XRD, ZnO 10μg / ml이하에서도급격한생존률감소가나타남. - ZnO 10μg / ml에 10μM H2O2 처치시생존률은 40.6±2.0% 이었으며, 5μM 처치시 72.8±2.0% 와무처치시 99.9%±1.1% 보다낮게관찰됨 - Coating을하지않은 ZnO는 ROS 발생증가와세포생존률을감소를유발하였지만, Coating 된모든 ZnO는 ROS 생성과세포독성을감소시켰음. - 감소유발은 cell culture medium과 PMAA가강하게나타났으며, OA가그다음으로관찰되었음. - LC50는 nzno는 15±1이며, mzno는 29±4μg / cm2로관찰되었음. - RKO 세포가있는배지내에서고형의형태로부터 Zn로변형은시간및농도의존적임 - 하지만입자와세포의직접적인접촉이 RKO 세포의독성유발에필요함. - 세포사의기전은사립체기능차단을포함함 - 세포독성은농도의존적으로발생 - 20mg/L -1 이상의농도에서세포생존률이 10% 이라로관찰됨 mg/L -1 이하에서안전함. - ZnO가가장드라마틱하게면역효과를나타내었음. 주로 proinflammatory cytokine, IL-1β 그리고중성호성백혈구의화학주성인자인 CXCL-9을증가시킴 - ZnO는산화적스트레스상승과세포막손상을유발하는동시에농도및시간의존적세포독성을유도함. - 5에서 10ug/ml의농도일때농도와

94 epithelial cells exposed to ZnO nanoparticles. Toxicology in vitro ; Nair S et al., Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells.. j Mater Sci: Mater Med ; Jingxia Z et al. Influences of nanoparticle zinc oxide on acutely isolated rat hippocampal CA3 pyramidal neurons. Neurotoxicol ; Yoshida R et al. Mutagenicity of water-soluble ZnO nanoparticles in Ames test. J. Toxicol Sci (1); ICP-MS - MTS assay, ROS measurement, LDH, measurement, measure intrecellular Ca++ - human osteoblast cancer cell line(mg-63) - 40nm-1.2 μm - XRD, SEM, DLS - MTT assay - single rat hippocampal CA3 pyramidal neuron - 20nm-80nm g/ml - TEM, DLS - Whole-cell patch-clamp - Salmonella typhimurium strains TA98, TA100, TA1535, TA Escherichia coli strain 독성간의현저한관계가나타남 - 나노입자의독성은 membrane 관련임 g/ml의 ZnO 나노입자는 sodium channel의 opening number를증가시키고, potassium channel의조정을늦춤으로써 Ina와 Ik 흐름의증대를상승시키는역할을수행함. - 모든종에서유전독성관찰되지않음. Fullerene 일반적으로카본나노튜브, 카본블랙에비해독성이약한것으로알려져있다. free radical scavenger 와항산화작용으로화장품에활용된다. 그러나활성산소생성과동시에제거작용으로인한유전독성유발가능성에대한연구결과가보고되고있다. 독성동태학적으로노출후흡수를억제하기위해다수의생체내배리어가 2차장기로로분포를억제하나노출양에따라독성효과를나타난다. 경구노출에의해서는흡수가매루제한적으로나타났고표피층은추과하지못하는것으로알려져있다. 노출경로에따라주로간에축적되나신장폐, 비장, 심장, 뇌에도축적이보고된다. 깬형성에의해간접적유전독성효과가보고되며한편으로특정타잎의플러렌자체는항염증및항암효과가보고된다. OECD 조사

95 Title :Toxicology of nanomaterials Project Ref. :FRA3332 Last Update :08/03/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :2008 End :2012 Summary : the projects aims at gathering the effort to evaluate the potential toxicity of nanomaterials. The following nanomaterials are considered: carbon nanotubes, SiO2, SiC, Pt and semi-conducting QD. The workplan include, biodistribution and pharmacoki Nanomaterial 의잠재적인독성평가를모으는것이이번과제의목표이다. 다음 nanomaterial 에따라 고려된다 : 탄소 nanotube, SiO2, SiC, Pt 와중간조건의 QD. 작업계획은생물분배와약동학을포함한 다 Title :Development of fullerene derivatives as novel lead compounds for medicine Project Ref. :WWC0748 Last Update :15/02/2010 Status :Completed Checked by National Designated Contact Point? Yes Country :Japan Start :1996 End :2009 Summary : Fullerene and its derivatives show interesting biological activities based on their unique physical properties and chemical reactivity. We intend to develop fullerene derivatives as a new type of lead compound to be used as medicine and have reported that the anionic fullerene derivatives show antioxidant activities, the cationic derivatives have antibacterial and antiproliferative activities, and amino-acid types of fullerene derivatives have Human immunodeficiency virus-reverse transcriptase inhibition activities, and sulfonium type derivative have HCV RNA polymerase inhibition activity. Our data suggest that the fullerene derivatives are promising novel lead compounds for anti-neurodegenerative disease, anti-cancer, anti-bacterial, and anti-hiv, and anti-hcv drug. fullerene 과그파생물은그들의유일한신체의소유물과화학반응성을근거로하는흥미있는생물학적활동을보여준다. 약으로사용되기위해우리는선도하는합성물중새로운형태로써 fullerene 파생물을개발하는것을의도하고있고, 그리고 anionic fullerene 파생물이산화방지제활동을보여준다라고보고했다. cationic 파생물은항균성과항증식성활동을가지고, fullerene 파생물의아미노산타입은인체면역결핍바이러스-역전사효소억제활동을가지고, 끌어내는 sulfonium 타입이 HCV RNA 중합효소억제활동을가지고있다. 우리데이터는 fullerene 파생물이항신경퇴행성질병, 항암, 항세균성과항 HIV와항 HCV 약물에대하여새롭게선도하는합성물일것이라고제안한다

96 Title :NER: Fullerene-Microbe Interactions: Implications for Disinfection and Risk Assessment Project Ref. :WWC0771 Last Update :10/04/2007 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2005 End :2007 Summary : Understanding the bio-reactivity of fullerenes, which are C60 nano materials, is important because of the many applications for which these nanomaterials are being considered. Fullerenes are known to have a high electron affinity and high reactivity with nucleophiles. They may serve as photosensitizers to produce reactive oxygen species such as hydroxyl radicals, superoxide radical anions, and peroxyl species, all of which are highly bio-reactive species. The potential biological effects of fullerenes that could result from these characteristics are not well understood. They may cause a wide variety of toxic effects to various organisms, but they also have the potential to be developed into engineering strategies to control biofouling or disinfect water. This project is studying the chemistry of reactive oxygen production by fullerene-based nanomaterials in the context of assessing the associated effects of fullerenes on bacteria and viruses. Specifically, the investigators are evaluating three hypotheses: (1) that fullerenes can act photocatalytically or through dark reactions to produce reactive oxygen species that inhibit or inactivate microbial growth; (2) that the reactive oxygen species formed from fullerenes hinder heterotrophic and photosynthetic activities and cause population shifts reflecting differential responses and protective mechanisms used by different species; and (3) that oxidative stress of cell membranes through peroxidation and epoxidation of phospholipids is an important mechanism responsible for cell death. 이러한 nanomaterials 의많은응용이고려되어지고있기때문에 C60 nano 물질인 fullerenes 의생물학적활동을이해하는것은중요하다. Fullerenes 는 nucleophiles 를가진높은전자친화력과높은반응성을가지는것을알고있다. 수산화기, 과산화물기음이온그리고 peroxyl 종, 높은생물학적활동종인것과같은활동적인산소종들을생성하기위해그들은감광성의역할을할수있다. 이특성으로부터기인할수있는 fullerenes 의잠재적인생물학적효과는잘이해될수없다. 그들은여러가지유기체에다양한유해한효과를초래할수있다. 그러나그들도생물부착을통제할엔지니어링전략으로개발된잠재성또는물을소독한다. 이프로젝트는세균과바이러스에서 fullerenes 의연합효과를평가하는것의환경에서 fullerene 를기본으로하는 nanomaterials 에의해반응적인산소생산의화학을연구하고있다. 특히, 조사자는 3가지의가설을평가하고있다. (1) microbial 성장을방해하거나, 억제하는종이라는반응적인산소를생성하기위해그 fullerenes 는광분해또는완전히어두운반응을작동할수있다. (2) fullerenes 에서만들어지는반응적인산소종이종속영양성과광합성활동을저해하고차동응답을반영하고있는원인인구이동과다른종에의해사용되는보호용메커니즘 (3) 과산화를통한세포막과인지질의에폭시화는세포의죽음에대한책임있는중요한메커니즘를통하여세포막의 oxidative 스트레스 Title :In vitro evaluation of fine/ultrafine particulate reactivity

97 Project Ref. :FRA8245 Last Update :22/01/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :December 2006 End :March 2010 Summary : This research intends to set up and compare three different methods devoted to the measurement of PM reactivity toward oxygen. This comparison will allow to give some recommandations toward the use and applicability of such tests for the NP reactivit 이연구는산소쪽으로 PM 반응성의측정에바쳐지는 3 가지다른방법을설립하고비교하는것을 의도합니다. 이비교는사용을향한약간의권고와 NP 반응성에대한그런테스트의적용성을줄것이 다 Title :NanoPIST - Intentionally producted nanoparticles: toxicological signatures Project Ref. :FRA7696 Last Update :08/03/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :November 2008 End :November 2011 Summary : Nanosciences and nanotechnologies lead to a deeply increasing production of nanomaterials, underlining the necessity to consider their potential health effect. The surface reactivity of nanomaterials could render them toxic since they enter easily in nanopist - 의도적으로생산되어진 nanoparticle : 독성학적특성 나노과학과나노기술은 nanomaterial 의매우증가하고있는생산, 그들의잠재적인건강효과를고려 하기위한필요성을강조하고있다. 그들이쉽게안에서들어옴으로 nanomaterial 의표면반응성은그들 을유해하도록할수있었다 Title :Recognition and Physicochemical Characterization of Nanomaterial-Peptide Interactions Project Ref. :WWC0182 Last Update :17/12/2008 Status :Underway Checked by National Designated Contact Point? Yes Country :Canada Start :2005 End :

98 Summary : The interface between biological systems and nanomaterials is of critical importance to the understanding of how the properties of nanomaterials can be affected by their molecular interactions with biomolecules. This research is targeted to better un 생물학적시스템과 nanomaterials 사이의인터페이스는 nanomaterials 의속성방법을이해하는것에 대한비판적인중요성은생물분자를가진그들의분자의상호작용에의해영향을받을수있다. 이연 구는더좋은목표가될것이다 Title :Translocation of Nanoparticles and Ultrafine Particles across Tissue Barriers in Mice. Project Ref. :WWC5673 Last Update :28/07/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Denmark Start :2007 End :2009 Summary : Understanding fate of nanoparticles in mice and ultimately in humans 마우스들과근본적인인간에서의 nanoparticle 운명을이해하는것 Title :RESPINTTOX Project Ref. :FRA3163 Last Update :22/01/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :January 2006 End :December 2008 Summary : Examinantion of toxic effects of carbon nanotubes in the respiratory system in relation with their phsyico-chemical characteristics 그들의물리화학적특성과의관계의호흡기시스템의탄소 nanotubes의유해한효과의검사 Title :NANOTOX (Toxicology of nanoparticles : influence of size, chemical composition and surface reactivity on lung and kidney) Project Ref. :FRA2949 Last Update :30/06/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France

99 Start :2005 End :2009 Summary : NANOTOX(ANR Edition 2005 du programme Santé-Environnement Santé-Travail SEST) Toxicologie des Nanoparticules : Influence de la taille, de la composition chimique et de la réactivité de surface sur leurs effets pulmonaires et rénaux France NANOTOX(ANR 년건강프로그램 - 환경건강 - SEST 의일 ) nanoparticles 의독성학 : 증가한전체규모, 화학구성과폐와신장의프랑스인에대한그들의효과의표면반응성 Title :Pulmonary Toxicity Screening Studies with Nano vs. Fine-Sized Particles in Rats. Project Ref. :WWC0737 Last Update :19/02/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Business and Industry Advisory Committee (BIAC) Start :2005 End :2009 Summary : The full text of summary is available in outcome and outputs section (see "Objectives and Specific Aims" section of the document attached in outcome and outputs section). Please note that project is underway with no planned end-date. 요약문의전체텍스트는결과와출력부분안에서사용가능합니다. ( " 목적과특별한목표 " 의결과및 출력부분에첨부된문서의부분참조 ). 계획종료일함께진행되는계획을참고하길바랍니다 Title :Research on the establishment of health risk assessment methodology for nanomaterials Project Ref. :WWC0749 Last Update :09/03/2010 Status :Completed Checked by National Designated Contact Point? Yes Country :Japan Start :2005 End :2006 Summary : In order to collect the basic data for further evaluation, we investigated measurement methods from the biological samples, dispersion methods in vitro system, inhalation exposure system, ADME, and international trend survey for fullerene and titanium nanoparticle. 그이상의평가를위한기초적인데이터를수집하기위해, 우리는생물학적샘플로부터측정방법을 조사했다. fullerene 과티타늄 nanoparticles 를위해체외시스템의분산방법, 흡입노출시스템, ADME

100 와국제경향조사 Title :Structure-function Relationships in Engineered Nanomaterial Toxicity Project Ref. :WWC0755 Last Update :06/12/2006 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2005 End :2008 Summary : Objective: As nanotechnology develops into a mature industry the environmental and health effects of its core materials are of increasing importance. A significant challenge for this area of research is that for every class of engineered nanoparticle (nanotubes, metal nanocrystals) there are literally thousands of possible samples with various sizes, surfaces and shapes. This huge parameter space cannot be narrowed by focusing only on commercial materials, as few systems are in commerce at this point. Indeed, most nanotechnology companies are optimizing and evaluating hundreds of material prototypes for possible commercial use. In such a climate, all stakeholders benefit from an understanding of how fundamental nanoparticle characteristics (e.g. surface chemistry, size, shape) control their biological effects. This aim is the overarching objective of this proposal, which stated another way, will provide the first structure-function relationships for nanoparticle toxicology. This information benefits industry in that it will suggest material modifications that may produce systems with minimal environmental and health impact. It also benefits regulators by not only indicating whether information on one nanoparticle type can be used to predict the properties of a related material, but also by setting a framework for evaluating newly developed nanoparticle variants. Finally, a correlation between biological effects and nanoparticle structure will enable the development of chemical methods to alter more toxic nanomaterial species into less toxic materials upon disposal. To realize these structure-function relationships requires that we develop new analytical tools as well as evaluate material datasets with systematic changes in fundamental properties. Our specific objectives are 1) to expand the characterization of nanoparticle structure in biological media and 2) to characterize the effects of nanoparticles on cell function. This data will be used to test the hypothesis that nanoparticle structure (e.g. size and shape) controls directly cytotoxicity. A secondary hypothesis is that of the four major materials parameters in engineered nanoparticles (size, shape, composition and surface) surface will be the most important in governing cellular effects. These hypotheses will be tested in several major classes of nanoparticles. Approach: This work exploits recent advances in nanochemistry which allow for the production of highly size and surface controlled nanoparticles from a variety of materials. These model systems provide the systematic variations in nanoparticle structure required for structure-function relationships. Our model systems will include engineered carbon nanoparticles, both C60 and single-walled carbon nanotubes; up to eight distinct sizes of nanoscale iron oxides; and a wide variety of nanoscale titania with varying surface coatings. All of these materials have been reported to generate oxygen radicals under some circumstances; thus we expect to correlate our structures with the acute cellular toxicity in three human cell lines. This overarching objective is strongly supported by

101 ongoing efforts to expand the characterization of nanoparticle structure directly in biological media (objective #1). Additionally, structure-function trends are made much more general if they can be rationalized by some basic mechanism. Thus, objective #2 aims to both characterize nanoparticle-cell interactions as well as put forward a mechanism to explain any observed acute toxicity. Expected Results: The introduction of a new class of materials into consumer products will require information about the potential behavior and risks these systems pose to the environment and people. Risk management will be improved with the information provided in this grant particularly in that we will establish structure-function relationships for several major classes of nanomaterials. 나노기술로써중심이되는물질의중요성이증가되어지고있는것중에환경과건강의효과가성숙한산업으로발달하고있다. 이지역의연구를위한중요한도전은설계된 nanoparticle(nanotubes, 금속 nanocrystals) 의모든학급을위해가능한수천의샘플이여러가지규모, 표면과형체를가진문자그대로있다라는것이다. 이거대한매개변수공간은거의시스템이상업안에이점에서그렇지않을때단지상업상의물질에집중하여한정될수없다. 실제로, 대부분의나노기술회사는가능한상업상의사용에대해최적화하고있고수많은물질원형을평가하고있다. 그런기후에서, 모든이해관계자는근본적인 nanoparticles 의특징 ( 표면화학, 크기, 형태 ) 조건울그들의생물학적효과로통제하는방법에대한이해로부터이익을얻는다. nanoparticle 독물학을위한구조기능관계를제공할것인다른방법을대신할수있는이제안이대단히중요한이번목표이다. 그것이최소의환경과건강영향을가진시스템을생산할수있는물질수정을제안할것이다고하는점에서이정보는산업에도움이된다. 그것은또한관련되어진물질의속성을예측할수있는하나의 nanoparticle 타입에대한정보를나타내줄뿐만아니라새로운 nanoparticle 변화를평가하기위한뼈대를만들어주는유익한규제기관이다. 마지막으로생물학적효과와 nanoparticle 구조사이의상관관계는적은독성안에더유해한 nanomaterial 종을바꾸는화학적방법의개발뿐만아니라금속처리를평가하는것을개발할수있을것이다. 근본적인소유지에있어서의이구조기능관계변화를실현한다. 우리의특별한목표는 1) 생물학적메디아안에 nanoparticle 구조의특징을확장시키는것, 2) 세포의기능에서 nanoparticles 의효과를특징화시키는것이다. 이자료는 nanoparticle 구조 ( 크기와형태 ) 조건을직접적으로세포독성의가설을테스트하는데사용될것이다. 두번째가설은설계되어진 nanoparticles ( 크기, 형태, 구성요소와표면 ) 4개의중요한금속한도는관리하고있는세포의효과에가장중요할것이다. 이들가설은 nanoparticles 의몇몇의중요한것들에서테스트되어질것이다. 접근 : 이번작업은규모의생산을매우고려하고여러가지물질로부터통제된 nanoparticles 를포장하는 nano화학의최근의진보를개척한다. 이모델시스템은구조기능관계를위해필요한 nanoparticle ' 구조 ' 에서의체계적인변화를제공한다. 우리의모델시스템은계획되어진탄소 nanoparticles 가포함될것이다. C60과하나의내벽화된탄소 nanotube; 산화철의 8개까지구분되어지는 nanoscale 의크기 ; 그리고변화하는표면코팅과함께티타니아의광범위하게다양한 nanoscale 이다. 이물질들모두약간의환경아래근본적인산소가보편적으로보고되어지고있다 : 따라서우리는 3개의인간의세포라인에서급성세포독성과함께우리의 ' 구조 ' 를연관시키는것을예상한다. 이대단히중요한목적은생물학적미디어에서 nanoparticle 구조의특성화를직접확대시키기위한계속적인노력으로강하게지지되어진다 ( 목표 #1). 부가적으로, 그들이기초적인약간의메커니즘에서의합리화되어질수있으면구조기능경향은훨씬일반적이게된다. 목적 #2 는 nanoparticle- 세포상호작용을특징지은것만큼이나어떤관찰된급성독성을설명하기위해메커니즘을앞에내려고한다. 예측결과 : 소비자생산에서물질의새로운급의소개는잠재적인행동에관한정보를필요로할것이고환경과사람에게이시스템자세를위태롭게한다. 위험관리는우리가 nanomaterials 의주요한몇명의클래스를위해구조기능관계를확립할것이라고하는점에서특별하게이번승인에서제공된정보와함께개선되어질것이다

102 Title :Comprehensive study concerning assessment method for health effects of nanomaterials Project Ref. :JPN3388 Last Update :09/03/2010 Status :Underway Checked by National Designated Contact Point? Yes Country :Japan Start :April 2008 End :March 2011 Summary : In order to investigate human health effects of nanomaterials, we intend to develop a new hazardous assessment method for the cerebral nervous system, the respiratory system, and the genital system including influence to future generations of mice and rats. Then, we will identify ADME of nanomaterials in rodent and rhesus monkey body, and develop an evaluation method which enable us to extrapolate results of human from results of experimental animals such as rodent and primate. nanomaterials 의인간의건강효과를조사하기위해정리한다. 우리는마우스와렛드의미래세대에영향을포함하여생식계, 뇌의신경계, 호흡계의새로운위험한평가방법을개발하는것을의도한다. 그다음, 우리는설치류와붉은털원숭이신체에서 nanomaterials 의 ADME을확인할것이다그리고, 우리가설치류와영장류와같은실험적동물의결과로부터인간의결과를추론하는것을가능하게하는평가방법을개발할것이다 Title :Study on development of methods for thel assessment of dermal toxicity of nanomaterials Project Ref. :JPN6794 Last Update :09/03/2010 Status :Underway Checked by National Designated Contact Point? Yes Country :Japan Start :April 2007 End :March 2010 Summary : We are investigating chronic influences of metal nanoparticles on dermal absorption, immune system, and carcinogenic properties in order to identfy essential indicators and develop test methods to evaluate dermal toxicity of nanomaterials 우리는피부의흡수, 면역시스템, 그리고발암성의속성의본질적인지표를확인하고 nanomaterials 의 피부의독성을평가하는시험방법을개발한다 Title :Development of Alternative In Vitro Methods to Assess Pulmonary Toxicity of Inhaled Fine and Nano-sized Particles Project Ref. :WWC

103 Last Update :19/02/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Business and Industry Advisory Committee (BIAC) Start :2005 End :2009 Summary : The full text of summary is available in outcome and outputs section (see "Objectives and Specific Aims" section of the document attached in outcome and outputs section). Please note that project is ongoing with no planned end date. 요약에대한전체문서는결과와출력부분에서이용할수있다 ( 결론과출력에첨부된문서의 목표와 특별한목적 부분을보시오 ). 프로젝트는끝나는시기가계획되어있지않고계속진행중이다.) Title :Evaluated Nanoparticle Interactions with Skin Project Ref. :WWC0137 Last Update :06/12/2006 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2004 End :2007 Summary : Objective: The focus of this research is to assess the nature of interaction between manufactured nanoparticles and the skin, including dermal absorption, cutaneous toxicity as well as the ability to distribute to the skin after systemic exposure. These studies will utilize iron oxide nanocrystals, cadmium selenide nanocrystals and carbon fullerene nanoparticles which are representative of the broad spectrum of nanoparticles presently being used by industry. Eight particle types selected from these commercially relevant manufactured nanoparticles will be studied to allow assessment of size, shape and composition on absorption, distribution or toxicity to the skin. Approach: All studies will be conducted in three well-characterized in vitro skin models: human skin keratinocyte cell cultures, porcine skin flow-through diffusion cells, and the isolated perfused porcine skin flap (IPPSF). Nanoparticles will be applied topically in three exposure scenarios (neat, water, mineral oil) at two doses to assess potential dermal absorption in the diffusion cell studies and to assess cellular toxicity (light and electron microscopy, viability) and irritation (IL-8 release) in cell culture. Those particles which penetrate skin or cause direct irritation will then be completely characterized in IPPSF studies which have previously been shown to be predictive of in vivo absorption in humans. Similarly, to model nanoparticle uptake into skin after systemic exposure, nanoparticles will be infused into the arterial blood supply of the IPPSF to assess ability to distribute out of the vasculature into the skin. Deposition of particles in epidermal tissue after both infusion and topical exposure will be evaluated using high-resolution electron microscopy. Expected Results: Presently, there are minimal data available on the interaction between manufactured nanoparticles and

104 biological tissues. The basic requirement for any risk assessment includes information on hazard (e.g. toxicity) and exposure (e.g. absorption). This proposal focuses on the health effects of nanoparticle interactions with the skin. This integrated research program will generate data on the ability of nanoparticles to be toxic to keratinocytes as well as assess the ability of nanoparticles to either be absorbed into skin after topical exposure, or distribute into skin as would occur after systemic exposure by an alternate route of administration. At the conclusion of the research, the boundaries of a dermal risk assessment for manufactured nanoparticle exposure will be available. 목표 : 이번연구의요점은재료화된 nanoparticle 과체계적인노출이후피부에분배하는능력뿐만아니라피부흡수, 피부의독성을포함한피부사이에상호작용의성질을평가이다. 이번연구는현재산업에의해사용되고있는 nanoparticles 의넓은스펙트럼의대표인산화철 nanoparticle, 카드늄셀레니드 nanocrystals 과탄소 fullerene nanoparticles 를활용할것이다. 이상업상관련제작된 nanoparticles 에서선택된 8의입자타입은피부에흡수, 배포또는독성에서규모, 형태와구성요소의평가를인정하기위해연구하게될것이다. 접근 : 모든연구는 3가지특징화가잘되어있는체외피부모델 : 사람의피부켈틴생성세포배양, 확산세포를통한돼지의피부흐름, 분리되어지고관류되어진돼지피부플랩 (IPPSF) 으로구성되어질것이다. Nanoparticle 은확산세포연구와세포독성 ( 빛과전자현미기술, 생존능력 ) 그리고세포배양를자극 (IL-8 방출 ) 하는것을평가하는것으로잠재적인피부의흡수를평가하는 2가지농도에 3가지노출시나리오 ( 니트, 물, 미네럴오일 ) 로제목별로응용하게될것이다. 피부에스며들거나또는직접적인자극이원인이되는그들입자들은인간의체내흡수를예측되도록보였다는것이이전에 IPPSF연구에서완전하게특징화되었을것이다. 마찬가지로피부로혈관계에서분배하는능력을평가하기위해체계적인노출이후피부의 nanoparticle 이해를모델화하기위해, nanoparticles 는 IPPSF의동맥피공급에주입될것이다. 주입과주된노출후표피조직안의입자들의퇴적물은고해상도의전자현미경을사용하여평가되어질것이다. 예측결과 : 현재, 재료화된 nonoparticle 과생물학적조직사이의상호작용은이용가능한최소의데이터이다. 어떤위험평가를위해기초적인필요한조건은위험 ( 독성 ) 과노출 ( 흡수 ) 의정보를포함한다. 이러한제안은피부에서 nanoparticle 의상호작용중건강효과가주요하다. 이번통합적인연구프로그램은주된노출이후피부에흡수또는투여의교체경로에의해체계의노출이후발생함으로써피부에분해를통해 nanoparticles 의능력을평가하는것만큼 keratinocytes 에유해한 nanoparticles 의능력에관한데이터를만들어낼것이다. 이번연구의결론은재료화된 nanoparticle 의노출을위한피부의위험평가의경계선이다 Fullerene 나노물질에대한논문요약 연 번 1 Reference materials and Methods 결과 Park EJ et al. Carbon fullerenes (C60s) can induce - ICR male mice (25±1 g) - 단회기관지내투여, 2mg/kg, inflammatory responses in the - C60 2mg/kg control, 1day, 14day, 28day lung of mice. Toxicology and - TEM, ELISA, RT-PCR Applied Pharmacology IL-1, TNF-α, IL-6 같은

105 (2010) proinflammatory cytokines 증가 - IL-12, IFN-γ 같은 Th1 cytokine 2 Folkmann JK et al. Oxidatively Damaged DNA in Rats Exposed by Oral Gavage to C 60 Fullerenes and Single-Walled Carbon Nanotubes. Environ Health Perspect 117: (2009). - Fisher 344 rats - 84 female - 0.7nm ㆍ응집에의해 salines 내에서는 407nm(low dose), 621, 5,117nm(high dose) 로변화ㆍCorn oil에서는 234nm(low dose), 40, 713과 3,124(high dose) 로변화 mg, 0.64mg/kg - DLS, ROS, RNA-PCR, MTT 증가 - 8-oxodG가간에서증가 - 폐에 8-oxodG만높은복용량을생성 3 Hideyuki Yamawaki, Naoharu Iwai. Cytotoxicity of water-soluble fullerene in vascular endothelial cells. Am J Physiol Cell Physiol 290: , Human umbilical vein epithelial cell - 7.1±2.4nm - 1,10,100μg/mL - TEM, LDH - 고용량에서세포손상, 세포사및세포성장정지관찰 - 탐식세포기능활성화 Iron oxide 산화철나노입자는초상자성 (superparamagnetic) 특성과약물전달, MRI 갗은생의료공학, 암조직선 택적파괴에활용가능성때문에관심이높다. 과다한산화철입자는세포내철의공급과다를유발 시킴으로써세포내독성영향을미친다. 코팅되지않은입자는낮은용해도를갖기때문에 dextran, PEG 등으로코팅하여생체내적합성과분포도를증가시킨다. 이들코팅물질변화에따른독성평가 가보고되고있다 OECD Title :Evaluated Nanoparticle Interactions with Skin Project Ref. :WWC0137 Last Update :06/12/

106 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2004 End :2007 Summary : Objective: The focus of this research is to assess the nature of interaction between manufactured nanoparticles and the skin, including dermal absorption, cutaneous toxicity as well as the ability to distribute to the skin after systemic exposure. These studies will utilize iron oxide nanocrystals, cadmium selenide nanocrystals and carbon fullerene nanoparticles which are representative of the broad spectrum of nanoparticles presently being used by industry. Eight particle types selected from these commercially relevant manufactured nanoparticles will be studied to allow assessment of size, shape and composition on absorption, distribution or toxicity to the skin. Approach: All studies will be conducted in three well-characterized in vitro skin models: human skin keratinocyte cell cultures, porcine skin flow-through diffusion cells, and the isolated perfused porcine skin flap (IPPSF). Nanoparticles will be applied topically in three exposure scenarios (neat, water, mineral oil) at two doses to assess potential dermal absorption in the diffusion cell studies and to assess cellular toxicity (light and electron microscopy, viability) and irritation (IL-8 release) in cell culture. Those particles which penetrate skin or cause direct irritation will then be completely characterized in IPPSF studies which have previously been shown to be predictive of in vivo absorption in humans. Similarly, to model nanoparticle uptake into skin after systemic exposure, nanoparticles will be infused into the arterial blood supply of the IPPSF to assess ability to distribute out of the vasculature into the skin. Deposition of particles in epidermal tissue after both infusion and topical exposure will be evaluated using high-resolution electron microscopy. Expected Results: Presently, there are minimal data available on the interaction between manufactured nanoparticles and biological tissues. The basic requirement for any risk assessment includes information on hazard (e.g. toxicity) and exposure (e.g. absorption). This proposal focuses on the health effects of nanoparticle interactions with the skin. This integrated research program will generate data on the ability of nanoparticles to be toxic to keratinocytes as well as assess the ability of nanoparticles to either be absorbed into skin after topical exposure, or distribute into skin as would occur after systemic exposure by an alternate route of administration. At the conclusion of the research, the boundaries of a dermal risk assessment for manufactured nanoparticle exposure will be available. 목표 : 이번연구의요점은재료화된 nanoparticle 과체계적인노출이후피부에분배하는능력뿐만아니라피부흡수, 피부의독성을포함한피부사이에상호작용의성질을평가이다. 이번연구는현재산업에의해사용되고있는 nanoparticles 의넓은스펙트럼의대표인산화철 nanoparticle, 카드늄셀레니드 nanocrystals 과탄소 fullerene nanoparticles 를활용할것이다. 이상업상관련제작된 nanoparticles 에서선택된 8의입자타입은피부에흡수, 배포또는독성에서규모, 형태와구성요소의평가를인정하기위해연구하게될것이다. 접근 : 모든연구는 3가지특징화가잘되어있는체외피부모델 : 사람의피부켈틴생성세포배양, 확산세포를통한돼지의피부흐름, 분리되어지고관류되어진돼지피부플랩 (IPPSF) 으로구성되어질것이다. Nanoparticle 은확산세포연구와세포독성 ( 빛과전자현미기술, 생존능력 ) 그리고세포배양

107 를자극 (IL-8 방출 ) 하는것을평가하는것으로잠재적인피부의흡수를평가하는 2가지농도에 3가지노출시나리오 ( 니트, 물, 미네럴오일 ) 로제목별로응용하게될것이다. 피부에스며들거나또는직접적인자극이원인이되는그들입자들은인간의체내흡수를예측되도록보였다는것이이전에 IPPSF연구에서완전하게특징화되었을것이다. 마찬가지로피부로혈관계에서분배하는능력을평가하기위해체계적인노출이후피부의 nanoparticle 이해를모델화하기위해, nanoparticles 는 IPPSF의동맥피공급에주입될것이다. 주입과주된노출후표피조직안의입자들의퇴적물은고해상도의전자현미경을사용하여평가되어질것이다. 예측결과 : 현재, 재료화된 nonoparticle 과생물학적조직사이의상호작용은이용가능한최소의데이터이다. 어떤위험평가를위해기초적인필요한조건은위험 ( 독성 ) 과노출 ( 흡수 ) 의정보를포함한다. 이러한제안은피부에서 nanoparticle 의상호작용중건강효과가주요하다. 이번통합적인연구프로그램은주된노출이후피부에흡수또는투여의교체경로에의해체계의노출이후발생함으로써피부에분해를통해 nanoparticles 의능력을평가하는것만큼 keratinocytes 에유해한 nanoparticles 의능력에관한데이터를만들어낼것이다. 이번연구의결론은재료화된 nanoparticle 의노출을위한피부의위험평가의경계선이다 Title :Development of Alternative In Vitro Methods to Assess Pulmonary Toxicity of Inhaled Fine and Nano-sized Particles Project Ref. :WWC0739 Last Update :19/02/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Business and Industry Advisory Committee (BIAC) Start :2005 End :2009 Summary : The full text of summary is available in outcome and outputs section (see "Objectives and Specific Aims" section of the document attached in outcome and outputs section). Please note that project is ongoing with no planned end date. 요약에대한전체문서는결과와출력부분에서이용할수있다 ( 결론과출력에첨부된문서의 목표와 특별한목적 부분을보시오 ). 프로젝트는끝나는시기가계획되어있지않고계속진행중이다.) Title :Development of a visualization methods for ADME of nanomaterial Project Ref. :JPN4272 Last Update :09/03/2010 Status :Completed Checked by National Designated Contact Point? Yes Country :Japan Start :April 2006 End :March 2009 Summary :

108 To reveal the internal behavior of nanoparticles, used already for cosmetic products and others, and essential for the assessment of their risk/safety problems, the convergent X-ray probe elemental mapping method and laser ablation/mass spectrum mapping method are newly developed and investigated for the visualization of internal motion of nanoparticles in the body. 화장품과다른것들을위해항상사용되어지고있는 nanoparticles 의내부행동과그것들의위험 / 안전문제의평가를위해필수적인것, 수렴성의 X선탐측기요소의매핑방법과레이저제거 / 질량스펙트럼매핑방법을밝히는것은새롭게개발되고신체에서 nanoparticles 의내부움직임의시각화를위해조사하고있다 Title :Pulmonary Toxicity Screening Studies with Nano vs. Fine-Sized Particles in Rats. Project Ref. :WWC0737 Last Update :19/02/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :Business and Industry Advisory Committee (BIAC) Start :2005 End :2009 Summary : The full text of summary is available in outcome and outputs section (see "Objectives and Specific Aims" section of the document attached in outcome and outputs section). Please note that project is underway with no planned end-date. 요약문의전체텍스트는결과와출력부분안에서사용가능합니다. ( " 목적과특별한목표 " 의결과및출력부분에첨부된문서의부분참조 ). 계획종료일함께진행되는계획을참고하길바랍니다 Title :Structure-function Relationships in Engineered Nanomaterial Toxicity Project Ref. :WWC0755 Last Update :06/12/2006 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2005 End :2008 Summary : Objective: As nanotechnology develops into a mature industry the environmental and health effects of its core materials are of increasing importance. A significant challenge for this area of research is that for every class of engineered nanoparticle (nanotubes, metal nanocrystals) there are literally thousands of possible samples with various sizes, surfaces and shapes. This huge parameter space cannot be narrowed by focusing only on commercial materials, as few systems are in commerce at this point. Indeed, most nanotechnology companies are optimizing and evaluating hundreds of material prototypes for possible commercial use. In such a climate, all stakeholders benefit from an understanding of how fundamental nanoparticle characteristics (e.g. surface chemistry, size, shape)

109 control their biological effects. This aim is the overarching objective of this proposal, which stated another way, will provide the first structure-function relationships for nanoparticle toxicology. This information benefits industry in that it will suggest material modifications that may produce systems with minimal environmental and health impact. It also benefits regulators by not only indicating whether information on one nanoparticle type can be used to predict the properties of a related material, but also by setting a framework for evaluating newly developed nanoparticle variants. Finally, a correlation between biological effects and nanoparticle structure will enable the development of chemical methods to alter more toxic nanomaterial species into less toxic materials upon disposal. To realize these structure-function relationships requires that we develop new analytical tools as well as evaluate material datasets with systematic changes in fundamental properties. Our specific objectives are 1) to expand the characterization of nanoparticle structure in biological media and 2) to characterize the effects of nanoparticles on cell function. This data will be used to test the hypothesis that nanoparticle structure (e.g. size and shape) controls directly cytotoxicity. A secondary hypothesis is that of the four major materials parameters in engineered nanoparticles (size, shape, composition and surface) surface will be the most important in governing cellular effects. These hypotheses will be tested in several major classes of nanoparticles. Approach: This work exploits recent advances in nanochemistry which allow for the production of highly size and surface controlled nanoparticles from a variety of materials. These model systems provide the systematic variations in nanoparticle structure required for structure-function relationships. Our model systems will include engineered carbon nanoparticles, both C60 and single-walled carbon nanotubes; up to eight distinct sizes of nanoscale iron oxides; and a wide variety of nanoscale titania with varying surface coatings. All of these materials have been reported to generate oxygen radicals under some circumstances; thus we expect to correlate our structures with the acute cellular toxicity in three human cell lines. This overarching objective is strongly supported by ongoing efforts to expand the characterization of nanoparticle structure directly in biological media (objective #1). Additionally, structure-function trends are made much more general if they can be rationalized by some basic mechanism. Thus, objective #2 aims to both characterize nanoparticle-cell interactions as well as put forward a mechanism to explain any observed acute toxicity. Expected Results: The introduction of a new class of materials into consumer products will require information about the potential behavior and risks these systems pose to the environment and people. Risk management will be improved with the information provided in this grant particularly in that we will establish structure-function relationships for several major classes of nanomaterials. 나노기술로써중심이되는물질의중요성이증가되어지고있는것중에환경과건강의효과가성숙한산업으로발달하고있다. 이지역의연구를위한중요한도전은설계된 nanoparticle(nanotubes, 금속 nanocrystals) 의모든학급을위해가능한수천의샘플이여러가지규모, 표면과형체를가진문자그대로있다라는것이다. 이거대한매개변수공간은거의시스템이상업안에이점에서그렇지않을때단지상업상의물질에집중하여한정될수없다. 실제로, 대부분의나노기술회사는가능한상업상의사용에대해최적화하고있고수많은물질원형을평가하고있다. 그런기후에서, 모든이해관계자는근본적인 nanoparticles 의특징 ( 표면화학, 크기, 형태 ) 조건울그들의생물학적효과로통제하는방법에대한이해로부터이익을얻는다. nanoparticle 독물학을위한구조기능관계를제공할것인다른방법을대신할수있는이제안이대단히중요한이번목표이다. 그것이최소의환경과건강영향을가진시스템을생산할수있는물질수정을제안할것이다고하는점에서이정보는산업에도움이된다. 그것은또한관련되어진물질의속성을예측할수있는하나의 nanoparticle 타입에대한정보를나타내줄뿐

110 만아니라새로운 nanoparticle 변화를평가하기위한뼈대를만들어주는유익한규제기관이다. 마지막으로생물학적효과와 nanoparticle 구조사이의상관관계는적은독성안에더유해한 nanomaterial 종을바꾸는화학적방법의개발뿐만아니라금속처리를평가하는것을개발할수있을것이다. 근본적인소유지에있어서의이구조기능관계변화를실현한다. 우리의특별한목표는 1) 생물학적메디아안에 nanoparticle 구조의특징을확장시키는것, 2) 세포의기능에서 nanoparticles 의효과를특징화시키는것이다. 이자료는 nanoparticle 구조 ( 크기와형태 ) 조건을직접적으로세포독성의가설을테스트하는데사용될것이다. 두번째가설은설계되어진 nanoparticles ( 크기, 형태, 구성요소와표면 ) 4개의중요한금속한도는관리하고있는세포의효과에가장중요할것이다. 이들가설은 nanoparticles 의몇몇의중요한것들에서테스트되어질것이다. 접근 : 이번작업은규모의생산을매우고려하고여러가지물질로부터통제된 nanoparticles 를포장하는 nano화학의최근의진보를개척한다. 이모델시스템은구조기능관계를위해필요한 nanoparticle ' 구조 ' 에서의체계적인변화를제공한다. 우리의모델시스템은계획되어진탄소 nanoparticles 가포함될것이다. C60과하나의내벽화된탄소 nanotube; 산화철의 8개까지구분되어지는 nanoscale 의크기 ; 그리고변화하는표면코팅과함께티타니아의광범위하게다양한 nanoscale 이다. 이물질들모두약간의환경아래근본적인산소가보편적으로보고되어지고있다 : 따라서우리는 3개의인간의세포라인에서급성세포독성과함께우리의 ' 구조 ' 를연관시키는것을예상한다. 이대단히중요한목적은생물학적미디어에서 nanoparticle 구조의특성화를직접확대시키기위한계속적인노력으로강하게지지되어진다 ( 목표 #1). 부가적으로, 그들이기초적인약간의메커니즘에서의합리화되어질수있으면구조기능경향은훨씬일반적이게된다. 목적 #2 는 nanoparticle- 세포상호작용을특징지은것만큼이나어떤관찰된급성독성을설명하기위해메커니즘을앞에내려고한다. 예측결과 : 소비자생산에서물질의새로운급의소개는잠재적인행동에관한정보를필요로할것이고환경과사람에게이시스템자세를위태롭게한다. 위험관리는우리가 nanomaterials 의주요한몇명의클래스를위해구조기능관계를확립할것이라고하는점에서특별하게이번승인에서제공된정보와함께개선되어질것이다 Title :Comparison of the cytotoxic, genotoxic and transforming effects of metal oxide particles in micro- and nano-particulate form (INRS) Project Ref. :FRA4384 Last Update :30/06/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :2007 End :2009 Summary : Recent development of nanotechnology is a subject of concern as regards the potential occupational health risk related to nanoparticle exposure (particles for which one dimension is less than 100 nm). Several animal studies have pointed out the specificity of nanoparticles in lung toxicity when compared to fine particles (of micrometric size). As an hypothesis, the very high surface area of nanoparticles could greatly increase their capacity to produce radical species. Furthermore, recent in vitro studies have shown that toxic and genotoxic activities of certain metallic oxide nanoparticles are correlated with their capacity to generate radical species. In order to characterize the carcinogenic potential of nanoparticles, the objectives of this study are as follows:

111 i) to compare the carcinogenic potential of nanoparticles and microparticles of same chemical and structural composition; ii) to examine whether this potential is correlated with their capacity to generate radical species 나노기술의최근의개발은 nanoparticle 노출과관계가있는잠재적인직업적인건강위험에관심에있다 ( 하나의크기가 100 nm 미만인입자 ). 미세한입자 ( 마이트로미터크기 ) 와비교하여몇몇의동물연구는폐독성에서 nanoparticles 의특성를지적했다. 가설로써, nanoparticles 의매우높은표면부분은근본적인종을생산하는그들의능력을상당히증대시킬수있었다. 게다가최근에체외연구에서어떤금속성 oxide nanoparticles 의독성과유전독성활동을보이는것은근본적인종을생성하는그들의능력과연관성을보여주고있다. nanoparticles 의발암성의잠재성을특징짓기위해, 이연구의목적은다음과같다. 1) nanoparticles 의발암성의잠재성과같은화학의microparticles 과구조적구성비교하는것 2) 이잠재성이근본적인종을생성하는그들의능력과관련시키게되는지조사하는것 Title :Evaluation of the pulmonary toxicity of nanometric and sub-micrometric iron oxide particles by intratracheal instillation in the rat (INRS) Project Ref. :FRA2993 Last Update :30/06/2009 Status :Underway Checked by National Designated Contact Point? Yes Country :France Start :2009 End :2011 Summary : The growing use of nanomaterials is leading to an increasing exposure of workers to nanoaerosols, the toxic properties of which are not always known. The experimental results currently available suggest that the pulmonary toxicity of nanoparticles can be influenced by the structure of the particle, its size, its specific surface area, and its surface reactivity, but probably also by its oxidation state. Numerous metal oxides exist in different oxidation states, this is the case in particular for iron oxides which can be present as ferric oxide (Fe2O3) and ferrous ferric oxide or magnetite (Fe3O4 or FeO Fe2O3). Despite the role played by iron on the onset of fibrosis and pulmonary cancers, and the multiple industrial applications of these oxides, to date few studies have looked at the pulmonary toxicity of nanometric iron oxides and the influence of the redox potential on the toxicity of these nanoparticles. In order to gain insight into the pulmonary toxicological properties of iron oxides, in particular the influence of their size and their state of oxidation, it is proposed to administrate these particles to rats by intratracheal instillation. Following exposure of the animal, parameters related to inflammatory response, cell proliferation, oxidative stress and genotoxicity induced by these particles will be studied. Since these pulmonary markers are associated with the carcinogenic potential of fibres and particles, the results will provide additional knowledge regarding the toxicity of these particles and allow an evaluation of the hazard associated with the exposure to such chemicals. nanomaterials 의사용의성장은거의알려지지않은독성의특성인 nanoaerosols 가근로자에게노출의 증가를유발하고있다. Nanoparticle 의폐독성의현재이용가능한실험적결과를제안하는것은입자

112 의표면, 크기, 특별한표면부분에의해영향을받을수있으나아마도산화상태일것이다. 다수의금속 oxides 는다른산화상태의안에존재한다, 이것은 ferric oxide(fe2o3) 또는철을함유한 erric oxide 또는자철광 (Fe3O4 또는 FeO Fe2O3) 으로되기위해특별한경우이다. 섬유증과폐암의시작그리고이러한 oxides 의다양한산업응용에서철에의해진행되는역할에도불구하고예전의연구에서는 nanometric iron oxides의폐독성과이것들의 nanoparticles 의독성이산화환원잠재성에의해증가되어진다고보고있다. 철의 oxides 의폐의독물학의특성, 특히그들의크기와그것들의산화상태의증가대한통찰을얻기위한순서로기관내점적주사에의해이입자들을 rat에게주는것이제안되었다. 동물의노출후에, 염증반응과세포확산, oxidative 스트레스그리고이들에의해유발되어진독성의한도와관계가있다는것을연구하게되어질것이다. 이들의폐의마커는섬유와입자의발암성의잠재성과관련되므로, 결과는이입자의독성에관한추가적인지식을제공할것이고그런화학제품에의노출과관련하여위험의평가가가능하게될것이다 Title :Iron Oxide Nanoparticle-Induced Oxidative Stress and Inflammation Project Ref. :WWC0142 Last Update :06/12/2006 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2004 End :2007 Summary : Objective: The manufacture of nanoparticles (<100 nm diameter) of varying shapes and compositions has exploded over the last several years with applications ranging from diagnostic imaging to nanoscale molecular construction. Given the vast potential use of these materials, both intentional and unintentional human exposures are likely. Although much has recently been learned about their synthesis, very little is known about cellular or organ responses upon contact with nanoparticles. A defining feature of nanoparticles is their large specific surface area; thus, it is possible that current concepts of dose expressed as mass concentration, which is very low for nanoparticles, may fail in predicting exposure outcomes if this feature is not taken into account. We hypothesize that the small size of nanoparticles contributes to their evasion of normal particle clearance mechanisms, increases the likelihood of contact with cells of many types, particularly epithelial cells, and allows their translocation to sites distant from the original exposure. We hypothesize further that this contact results in inflammation and oxidant stress and that the large surface area of the nanoparticles potentiates their effects. We will address these hypotheses with the following objectives to determine if nanoparticles: 1) induce oxidative stress and toxicity in cultured epithelial and endothelial cells; 2) cause lung inflammation or extrapulmonary effects after in vivo exposure; and 3) are translocated to extrapulmonary sites. Approach: This project, through the Principle and Co-Investigators, will be conducted in a multidisciplinary manner, combining expertise in the fields of toxicology and materials science. We will primarily use nanoparticles composed of iron oxide (Fe2O3, 3-25 nm), but for specific experiments, bulk composition and surface coatings will be varied. Nanoparticles may gain access to tissues in humans through inhalation or via the blood (i.e. oral, intravenous, or inhalation exposure). Thus, for in vitro studies, we will use human alveolar epithelial and umbilical vein endothelial cells and monitor their uptake of nanoparticles as well as

113 inflammation- and oxidative stress-related responses using a range of doses. For in vivo studies, we will use F-344 rats for intratracheal instillation and intravenous injection exposures. Endpoints related to lung inflammation, inflammatory cell activation, and oxidative stress as well as those indicating vascular endothelial injury and acute phase responses will be assessed. Comparisons will be made with ultrafine (20 nm) TiO2 particles. Tissues from exposed rats (lung, liver, and olfactory bulb) will also be examined for the presence of the particles following intratracheal and intranasal instillations and intravenous injection exposures. Expected Results: This project will provide dose-, size- and composition-related toxicological information about nanoparticles and will also explore their translocation to extrapulmonary tissues. The insight gained regarding mechanisms of response to nanoparticles and their disposition after exposure can be used to predict outcomes of human exposures in environmental, occupational, and therapeutic settings. 목표 : 형체와구성요소를바꾸는 nanoparticles(<100 nm 직경 ) 의제조업은진단의이미징부터 nanoscale 분자의구조까지미치고있는응용으로지난몇년동안폭발적이였다. 인간이의도했던의도하지않했던이물질의광대한잠재적인사용을줄것이라예상한다. 비록많은것이그들의통합에대하여최근배우게되었지만, 아무것도 nanoparticles 와의접촉에세포의기관응답에대하여알려져있지않다. nanoparticles 의정의하고있는특징은그들의큰특정의표면지역이다. 따라서, nanoparticles 를위해매우낮은이러한특징이고려되지않으면노출결과를예측이실패할있는질량의농도로써표현되어지는투여량은현재의개념으로가능하다. nanoparticles 의작은규모는보통의입자간격메커니즘의그들의회피에공헌하고, 많은타입의세포특히 epithelial 세포와의접촉의가능성을증대시켜주고, 원래의노출로부터멀리떨어져있는위치에그들의전좌가일어나는것을가능하게한다는것이우리의가설이다. 우리는이러한접촉이염증과 oxidative stess 를초래하는것과 nanoparticles 의큰표면지역이그들의효과를강화할것이라고한층더가설을세운다. 우리는이러한가설들만약 nanoparticles 이 : 1) oxidative stess 의유도와배양되어진 epithelial 과 endothelial 세포의독성 ; 2) 체내노출후폐염증또는폐외의영향에원인이된다 ; 3) 폐외부분이전좌되어진다. 에따라과제를결정하였다. 접근 : 주되고공통연구자를통한이번프로젝트는독성과물질과학의범위에서전문지식을결합한종합적인방식으로구성되어질것이다. 우리는 iron oxide(fe2o3, 3-25nm) 의구성되어진 nanoparticle 을첫번째로사용할것이다. 그러나특별한실험을위해대부분구성요소와표면코팅은서로다른것이될것이다. Nanoparticles 는흡입과피 ( 경구, 동맥투여, 흡입확산 ) 를통하여사람의조직에서평가한것을얻을것이다. 그러므로체외연구를위해우리는사람의치경음 epithelial 과탯줄정맥 endothelial 세포, 그리고염증과 oxidative stress 과관련되어진투여량의범위를사용하여반응을보는것만큼 nanoparticles 의활용을모니터할것이다. 체내연구를위해우리는기관내점적주입과혈관내투여확산을위해 F-344 rat을사용할것이다. 종료시점은폐염증, 염증세포활동그리고그들이표시하고있는혈관의 endothelial 상처와급성단계반응을평가되어질뿐만아니라 oxidative 스트레스와관련되어진다. 비교는초미세 (20nm) Tio2 입자로만들것이다. 노출되어진 rat( 폐, 간, 그리고 olfactory bulb) 로부터얻은조직은기관내, 비도점적주사그리고혈관내주사흡입을통해입자들의존재를증명되어질것이다. 예측결과 : 이번프로젝트는투여량, 크기그리고구성요소가 nanoparticle 에관한독성정보와관련되어질것이고, 폐외조직의전좌를조사하게될것이다. 통찰은 nanoparticles 에대한반응의메커니즘에관해서얻게될것이고, 노출후의그들의성질은환경과직업과치료설정에서인간의노출의결과를예측하기위해사용될수있다 Title :Size Dependent Neuronal Translocation of Nanoparticles

114 Project Ref. :WWC0162 Last Update :07/06/2007 Status :Underway Checked by National Designated Contact Point? No Country :United States Start :2004 End :2007 Summary : There is increasing concern about potential adverse effects of engineered nanoparticles (NP) on the environment and on human health. Because of their small size of <100 nm in diameter, NP are likely to elicit much greater biological activities than larger-sized particles. Distribution of NP from the portal of entry to other organs may induce serious adverse effects. In particular, the findings of nano-sized particles translocating via sensory neurons from the respiratory tract (nasal region) to structures of the Central Nervous System (CNS) raise questions about potential short-term and longterm health consequences. Are uptake and translocation in sensory nerves controlled by the size of NP? Does NP chemical composition and surface coating influence neuronal uptake and translocation? Does the translocation of NP induce signs of toxicity in target areas of the brain? This proposed research will address these fundamental questions within the SGER program of NSF. The research is considered exploratory and high risk because it is a first attempt to systematically examine NP size-dependent translocation along neuronal pathways, representing a project of an emerging area of nanotoxicology. Results will likely catalyze nanotechnological engineering with respect to developing nanostructured materials based on health effects. The studies will be performed in rats using intranasal instillation of NP. The Objective 1a studies (limit of NP size for translocation) will be performed with gold nanoparticles of 5 different sizes, 2 nm. 100 nm. This will be followed by Objective 1b studies on the impact of NP chemistry using different types of NP (nanogold with different coatings; TiO2; SiO2; Cd-based quantum dots and rods and Pt-Fe core-shell nanoparticles). This research necessitates close collaborations between materials scientists and toxicologists; the proposed studies will, therefore, be performed in a multidisciplinary team approach involving scientists in the disciplines of classical toxicology, cellular/molecular toxicology, and materials science at the University of Rochester. Results of this exploratory research will form the basis for a subsequent comprehensive research program to assess biological effects of NP, whether they are adverse or benign, and to elucidate underlying cellular and molecular mechanisms. 환경과사람의건강에서계획화된 nanoparticle(np) 의잠재적인부정적효과가관한영향이증가하고있다. 지름 100nm 이하의작은크기이기때문에 NP는큰크기의입자들보다생물학적활동이더크게끌어낼것으로예상된다. 다른기관들에들어가정맥으로부터 NP의분배는심각하게부정저인효과를유발하고있다. 특히, 잠재적으로짧은기간과오랜기간의건강결과에관한질문이올라가고있는 CNS 에서발생하는호흡계 ( 코부분 ) 의구조로부터감각의신경세포를통하여 nano크기의입자들이전좌를한다는것을발견하였다. 감각신경의활용과전좌를 NP의크기에의해통제되어질수있는가? NP화학구성요소와표면코팅이신경세포활용과전좌에영향을줄것인가? NP의전좌는뇌의목표지역의독성징후를유도할것인가? 이번에제안된연구는 NSF의 SGER프로그램에서근본적인질문을고심할것이다. 연구는 nano독성의최근에만들어진부분의프로젝트를나타내면서신경경로를따라 NP 크기 -의존전좌를조직적으로조사할첫번째시도인때문에탐사와위험율이높은것을고려하였다. 결과는

115 건강효과를기본으로한 nano구조화된물질들을발달한점에서 nano기술을계획하게하는촉매역할을하게되었다. 연구에서는 NP의비강점적투여를사용한 rat을이용하였다. 1a 연구의목표 ( 전좌를위한 NP 크기의제한 ) 는 5개의다른크기, 2nm, 100nm 의금 nanoparticles 로수행하였다. 이것은 NP( 다르게코팅한 nanogold : TiO2; SiO2; 양자점그리고막대가기본이된 Cd 그리고 Pt-Fe 코어셀 ) 의다른형태를사용하고있는화학적 NP의영향인 1b의연구목표에따른것이다. 이연구는물질과학자와독물학자사이에정밀한협력을필요로하다. ; 그러므로제안된연구는고전적인독물학, 세포의 / 분자의독물학그리고 Rochester 대학의물질과학의단련에과학자들을포함시키고있는다방면으로훈련되는팀접근에서수행될것이다. 이탐구를위한연구의결과는그것들이부정적이거나또는양석적이고세포와분자의메커니즘기초가되는것을해명하기위해 NP의생물학적효과를평가하는이후의포괄적인연구계획의기초를형성할것이다. Carbon nanotube(cnt) 이들나노물질의구조적특성때문에 장기간염증반응을유방할수있는 high-aspect ratio 및 biopersistenc 에기인한독성관찰이요구되고탄소나노튜브의경우여러가지세포타잎에서세포독성이많이보고되었다. 특히석면과같이유전독성도함께고려되어져야한다. MWCNT의경우농도의존적으로발암가능성이제시되는보고도있으나정확한기전규명은명확하지않다. 이에반해 SWCNT에서는 MWCNT와유사한유전독성결과가나타나지않고있다. 탄소나노튜브의경우도그자체가 ROS 생성과물리학적특성에따른 surface chemistry 변화가잠재적독성효과를결정하는주요인자가될수있다. OECD 조사 Title : Toxicology of nanomaterials Project Ref. : FRA3332 Last Update : 08/03/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : France Start : 2008 End : 2012 Summary : the projects aims at gathering the effort to evaluate the potential toxicity of nanomaterials. The following nanomaterials are considered: carbon nanotubes, SiO2, SiC, Pt and semi-conducting QD. The workplan include, biodistribution and pharmacoki 이 project 는 nanomaterials 의잠재적인 toxicity 를평가하기위한수집활동을목표로한다

116 nanomaterials : carbon nanotubes, SiO2, SiC, Pt and semi-conducting QD. biodistribution and pharmacoki 를포함한다 Title : C' NanoTubBio Project Ref. : FRA4236 Last Update : 12/02/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : France Start : January 2009 End : January 2009 Summary : Investigation of the toxic effects of carbon nanotubes in the respiratory system by using an interdisciplinary approach (physics and biology). Influence of the health status of the exposed organism. interdisciplinary approach 에서이용중인 respiratory system 내의 carbon nanotubes 의독성효과를조사한다. exposed organism은건강상태에영향을미친다 Title : TOXICOLOGICAL ASSESSMENT OF NANOPARTICLES ON HUMAN HEALTH USING IN VITRO, EX VIVO AND IN VIVO MODELS Project Ref. : WWC5667 Last Update : 05/03/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : Belgium Start : 2006 End : 2010 Principal Investigator : Name : Dr. Olivier Toussaint Institution : University of Namur - Research Unit on Cellular Biology Address : Rue de Bruxelles, 61 B-5000 Namur Country : Belgium Contact Summary :

117 The Nanotoxico project is a multidisciplinary platform which main goal is the toxicological assessment of manufactured nanoparticles (carbon nanotubes, silicon and titanium carbides) using in vitro, ex vivo and in vivo assays. Nanotoxico project 는 in vitro, ex vivo and in vivo assays 을이용하여 manufactured nanoparticles (carbon nanotubes, silicon and titanium carbides) 의독성학적으로평가하는종합적인 platform 이다 Title : Study on development of methods for thel assessment of dermal toxicity of nanomaterials Project Ref. : JPN6794 Last Update : 09/03/2010 Status : Underway Checked by National Designated Contact Point? Yes Country : Japan Start : April 2007 End : March 2010 Summary : We are investigating chronic influences of metal nanoparticles on dermal absorption, immune system, and carcinogenic properties in order to identfy essential indicators and develop test methods to evaluate dermal toxicity of nanomaterials. 우리는피부흡수, 면역체계, 면역체계, 필수지표를동일화하기위한발암성특성에서금소나노물질의만성적영향을조사하고, 나노물질의피부독성을평가하기위한방법을개발한다 Title : Cytotoxicity and transforming ability of commercial carbon nanotubes on Syrian hamster embryonic cells (INRS) Project Ref. : FRA7928 Last Update : 30/06/2009 Status : Underway Checked by National Designated Contact Point? Yes Country : France Start : 2007 End : 2009 Carbon nanotubes (CNTs) are materials with at least one dimension in the nanometer scale. Due to their physicochemical properties, they are becoming materials used more and more in the commercial, environmental and medical sectors. Taking into account their physicochemical properties and their insolubility, they can be regarded as fibers; they may be potentially dangerous

118 The first in vivo and in vitro studies suggest that carbon nanotubes are potentially toxic for humans. However, data are still insufficient to assess their carcinogenic potential. In this study, cytotoxicity and transforming abilities of commercial carbon nanotubes will be evaluated in the Syrian hamster embryonic cells (SHE) model. Results should give insights regarding the carcinogenic and toxic potential of CNTs, and may open perspectives for future studies, with the purpose to improve prevention in the industrial sectors concerned. CNTs는나노미터척도에서최소의크기를가진물질이다. 물리화학적특성덕택에일상적, 환경적그리고의학적분야에점점더사용되고있다. 그들의물리화학적특성과불용성을참고하여섬유처럼간주할수있으며, 매우위험하다. 먼저 carbon nanotubes가사람에게잠재적유독성이있어 in vivo and in vitro 연구를제안한다. 그러나, data는발암잠재력을평가하기에는부족하다. commercial carbon nanotubes 의 cytotoxicity and transforming abilitie을 Syrian hamster embryonic cells model로평가했다. CNTs의발암및잠재적독성에관한통찰력을제공해야하며, 미래연구를위한견해와산업분야와관련하여예방향상을목적으로해야한다 SWCNTs 나노물질에대한독성연구논문요약 연 Reference materials and Methods 결과 번 1 Yang Z et al. Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) Kunming mice - 5, 50, 100, 300, 400, and 500 mg/kg - RFS-100/S Raman spectrometer, TEM, ROS 2 Albini A et al. Interactions of single-wall carbon nanotubes with endothelial cells. Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) Human Umbilical Vein Epithelial cells - 1-5nm - 5, 10, 25, or 50 μg/ml - MTT assay, Neutral red uptake assay (NRU) - 회장에주로분포하고있었으며, 점막상피에주로분포 - pristine 과 oxidased SWCNT 의차이 는없었음

119 3 Belyanskaya L et al. Effects of carbon nanotubes on primary neurons and glial cells. NeuroToxicology 30 (2009) Dong L et al. Cytotoxicity Effects of Different Surfactant Molecules Conjugated to Carbon Nanotubes on Human Astrocytoma Cells. Nanoscale Res Lett (2009) 4: Yang ST et al. Long-term accumulation and low toxicity of single-walled carbon nanotubes in intravenously exposed mice. Toxicology Letters 181 (2008) Chicken embryo(neuron, glial cell) - 0, 7.5, 15, 30 μg / ml - SEM, TEM, ICP-OES, ELISA - Human 1321N1 astrocytoma cells - 0.5mg/ml - uv-vis spectrophotometer, epifluorescence microscopy - Male CD-ICR mice ( 25 g) - 40, 200, 1000 μg /mouse - TEM, TNF-α assay, ELISA - 나노튜브과응집되는시간의존적으로독성의수준구분됨. - 중추신경계와말초신경계모두영향 - BBB를통과할수있기때문에뇌및중추신경계진단및치료등에사용가능 - 주요축적장기는간, 폐및비장임 M W CNTs 나노물질에대한논문요약 연 번 Reference materials and Methods 결과 - 3T3 fibroblasts, RAW 1 2 Sohaebuddin SK et al. Nanomaterial cytotoxicity is composition, size, and cell type dependent. Sohaebuddin et al. Particle and Fibre Toxicology 2010, 7:22 Kim JS et al. Aspect ratio has no evect on genotoxicity of macrophages, telomerase-immortalized bronchiolar epithelial cells. - MWCNTs - 8, 20-30,50nm length - DLS, MTT, ROS, Annexin V/Propidium iodide assay - CM-95, rats, CHO-k1, ICR mice - 세포독성은 ROS의발생량, lysosomal membrane 불안성및사립체의투과성으로특징지을수있음 - 노출된세포의타입에따라다양한독성이나타남 - 모든유전독성시험에서독성을

120 - diameter : nm, - length : high-aspect multi-wall carbon nanotubes. Arch Toxicol ratio MWCNT(>10μm ), - low-aspect ratio MWCNT(>150nm) - Ames test, micronuclei assay, 관찰하지못했음. - 반면 high-aspect ratio MWCNT 는직접적유전독성을유발하지않고, 간접적으로산화적스트레스와염증을통하여유전독성을유발함 Coccini T et al. Effects of 3 water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cellstoxicology 269 (2010) Human A549 pneumocytes와 D384 astrocytoma cells μg / ml - MTT, calcein/pi assays (calcein/propidium iodide (PI) - water solubility, 높은분산도, 낮은응집경향등이세포독성을조절하는요소임 4 Patlolla A et al. Evaluation of cell viability, DNA damage, and cell death in normal human dermal fibroblast cells induced by functionalized multiwalled carbon nanotube. Mol Cell Biochem (2010) 338: Carbon nanotubes, Annexin-V - (outer diameter of nm, lengths of 15 0 μm, purity >95%) - 40, 200, 400μg / ml - LDH, MTT, TEM, - 용량의존적으로독성나타남. - DNA 손상과세포자멸사기전을통한세포생존능감소 5 6 Haniu H et al. Proteomics-based safety evaluation of multi-walled carbon nanotubes. Toxicology and Applied Pharmacology 242 (2010) Vittorio O et al. Influence of purity and surface oxidation on cytotoxicity of multiwalled carbon nanotubes with human neuroblastoma cells. Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) - Human monoblastic leukemia cells - As-grown( nm), HTT1800( nm), HTT2800( nm) - FE-SEM, Raman spectroscopy, MTT, - SH-SY5Y cells - 97% purity : 20-40nm - 99% purity : 35-40nm - 97% purity treated with nitric acid : 20-40nm - 5,10,100,500μg / ml - SEM, TEM, ROS, FIB, - 세포증식은 As-grown에서만확인됨 - HSP β-1, Neutral α-glucosidase AB, DNA mismatch repair protein Msh2 등의 peptide 발현 - parkinson's disease와 Cancer와관련된 Dj-1 protein도발현 - ED25는 48, 34.4, 18.4μg / ml μg / ml에서 gene 및약물치료에

121 MTT, WST-1, Hoechst, Oxidative stress assays. 사용될수있을것으로판단됨 7 Mitchell LA et al. Pulmonary and Systemic Immune Response to Inhaled Multiwalled Carbon Nanotubes. TOXICOLOGICAL SCIENCES 100(1), (2007) - Male C57BL/6 mice - Diameter : 10-20nm, - Length : 5-15μm - 0.3, 1, 5mg / m3 - SEM, TEM, ICP MS, Real-time RT-PCR, ELISA - macrophage에검은색입자가관찰 - 폐포액에서입자를가진 macrophage 관찰 - 혈구세포에는증가되지않음 - 14일째 0.3이상에서 nonmonotonic 전신성면역억제관찰 - 1mg/ m3에서 NK cell의기능감소 제 3 절정보제공사이트제작 그림 1. 나노물질안전성정보시스템

122 그림 2. 나노물질안전성정보시스템 ( 세부항목 ) 그림 3. 나노물질안전성정보시스템 ( 세부항목 )

123 제 4 절표면처리금나노물질의표적장기에미치는영향연구 1 연구수행방법 1. 시험의목적 2009 년에 PEG 표면처리한금나노물질나노물질의독성시험연구를자체연구과제로수행한바있으며본과제를통해 30 nm크기의금나노물질에의한 in vivo 단회투여독성을확인한바있다. 또한본과제를수행함에있어서표면처리한금나노물질에따라세포독성의정도나독성이진행되는순서혹은독성이발생하는기작이다를수있음을가정해볼수있다. 따라서, 나노물질에대한 in vivo 독성연구를시행할때다각도로접근해야할필요가있다. 이에, 본연구에서는표면처리금나노물질에대한 14 일반복투여독성을통한표적장기및혈 액결합단백질을분리동정하고자하였다. 2. 실험방법 가. 금나노입자의합성및특성분석금나노입자는한국생명공학연구원에의뢰하여, (+)30, (-)30 nm 로합성하였으며, PEG를코팅하여 PBS 조건에서안정화시켰다. 합성된금나노입자는 TEM으로크기와형태를확인하였으며, UV/Vis-NIR spectrophotometer 를이용하여흡수곡선의변화를검사하였다. DLS를이용하여표면전하의변화와입자의평균크기를측정하였다. 상세한합성및분석방법은다음과같다. (1) 양전하및음전하를표면처리한금나노입자의합성및특성분석 ( 가 ) 음전하로표면처리된골드나노입자합성 재료 : HAuCl 4 3H 2 O (hydrogentetrachloroaurate, Aldrich) 11-mercaptoundecanoic acid mpeg-sh (5kDa) 실험방법 1 제조에사용할삼각플라스크를세척용액으로완전히세척한후, 3차증류수로한번더세척한후오븐에건조시킨다 ml 0.01 wt % HAuCl 4 용액을교반상태에서가열시킨다. 3 위의용액이끓기시작하면 2 ml 1wt % sodium citrate를한번에빨리첨가한다. 4 색깔이레드와인색이되며, 15분더가열시키고, 반응완료후상온에서냉각시킨다. 5 mercaptoundecanoic acid를에탄올에녹인후골드나노입자에넣고 4시간동안교반시킨다. 6 음이온으로코팅된골드나노입자용액 10 ml에 5kDa의 SH-mPEG 0.1g/2ml 을한방울씩넣어상온에서 10시간동안교반하면서반응시킨다. 7 반응이끝난후, 위의용액을원심분리기 (8,000rpm, 4, 30분 ) 를이용하여상층액을버리고증류수에재분산시킨다

124 8 원심분리과정을 3 회반복하고마지막에는 PBS 에분산시킨후 0.2 μm의 filter 에통과시켜일 반세균오염을차단한다.. ( 나 ) 양전하로표면처리된골드나노입자합성 재료 : HAuCl 4 3H 2O (hydrogentetrachloroaurate, Aldrich) Amiundecanethiol (C 2H 7NS) mpeg-sh (5kDa) Sodiumborohydride (NaBH 4) 실험방법 mM HAuCl 4 / 40 ml DW 용액에 Aminoundecanethiol 213mM 을물과에탄올에녹인것을넣고상온에서 400 rpm으로 20분동안교반시켜준다 ul 10mM Sodiumborohydride 를물과에탄올에녹인후위의용액에재빨리넣고 1000rpm 에서 10분동안더교반시켜준다 rpm 에서 overnight으로교반시켜준다. 4 반응시킨위의용액 20 ml에 mpeg-sh (5kDa) g/4ml (DW) 를한방울씩떨어뜨리면서 10시간동안교반시켜준다. 5 TFF cassette 을이용하여 100,000 kda의멤브레인을통해미반응된물질을제거하고, centrifuge 를이용하여 8000rpm 에서 20분씩두번의과정을통해상층액을버린후마지막에는 PBS로재분산시킨후 0.2 μm의 filter에통과시켜일반세균오염을차단한다.. ( 다 ) PEG(polyethyleneglycol) 을이용한골드나노입자의표면처리 SH-mPEG의구조 H 3C-(OCH 2CH 2)n-SH SH : 티올기 (Thiol group) 는골드표면에부착됨, 황 (sulfur) 원자들이골드표면에에피택셜결합 (epitaxial bonding) 으로층 (layer) 을형성. PEG chain : 물에대한용해도및생체적합성 (biocompatible) 을부여. SH-mPEG와골드표면의반응골드-티올 (gold-thiol) 반응은일반적으로골드에 SH결합에의한산화적첨가반응 (oxidative addition) 으로써볼수있다. 티올화된골드입자는콜로이드안정성 (colloid stability) 을얻으며, PBS에서골드의응집 (aggregation) 을방지하기위함이다. ( 라 ) 합성시주의사항 골드나노입자의크기는일반적으로 HAuCl 4 의농도와환원제및표면코팅물질로사용되는 Sodium citrate, Sodium borohydride 의농도에따라크게달라지므로, 원하는크기의골드나노입자를제조하기위해서는, 이러한점을고려해야한다. 균일한크기의골드나노입자를얻기위해서는 Sodium citrate, Sodium borohydride 용액을한번에빨리넣어줘야한다

125 표면처리된골드나노입자를세포실험에서응집되지않게하기위해티올화된친수성고분자인 PEG로처리해주어콜로이드안정성 (colloid stability) 을얻고, PBS에서골드의응집 (aggregation) 을방지할수있다. 골드나노입자표면에부착되지않은미반응 PEG를효과적으로제거하기위해서는적어도 3-5 회의원심분리실험을수행하는것이바람직하다. 초고속으로회전하는원심분리기를이용하여분리할경우, 골드나노입자끼리비가역적으로뭉칠수있으니주의해야한다. SH-mPEG를코팅하기위해서골드나노입자의표면에원자가몇개가있는지계산을한후 PEG가붙을수있는적당량을넣어주면된다. ( 마 ) 합성결과에대한분석 1 골드입자용액의색깔 : 육안상색깔의변화를관찰하여골드나노입자가형성되었음을확인할수있다. 합성시반응시간에따른색깔의변화는 pare yellow-black-purple-blue-red 와같다. 2 전자현미경 (TEM) 을이용하여골드입자의크기및형태를관찰할수있다. 3 동력학적광산란 (DLS) 을이용하여나노입자의크기및표면전하 (zeta potential) 를확인할수있다. 4 흡광도계 (UV-Vis_NIR spectrophotometer) 를이용하여 maximum absorption peak 로골드입자의크기를추정할수있다. 높은파장은큰입자와관계되며, 골드입자의뭉침현상 (aggregation) 은흡수파장이 600~800nm 사이의장파장영역에서관찰되어지며 peak 의모양을보면확인할수있다. (2) 합성한금나노입자의농도측정유도결합플라즈마질량분석법 (ICP-MS: Inductively coupled plasma - massspectroscopy, 이하 ICP-MS 로칭함 ) 은원자의고유한질량의차이를이용한극미량원소분석장비다. 따라서골드나노입자의농도를기초과학연구소에있는 ICP-MS를이용하여분석하였다. 또한, 골드나노입자의개수를 fcc 골드밀도와제조된골드나노입자의부피를이용하여계산하였다

126 표 5. 수령받은금나노입자의종류별농도 농도 ml당 particle 갯수 (+) charge 30 nm 5 mg/ml 13.8X10 12 (-) charge 30 nm 5 mg/ml 17.6X10 12 나. 동물실험 제조된금나노물질을투여하기위하여아래와같이군을분리하여실험을실시하였다. 실험동물은 BALB/C 마우스 (6주령) 총 75마리를사용하였으며, 실험전 1주일간순화시킨후실험을수행하였다. (1) 투여용량 군 농도 마리수 / 군 비고 control PBS 5개군 (5마리/ 군 ) (+) charge 30 nm 5 mg/ml 5개군 (5마리/ 군 ) (-) charge 30 nm 5 mg/ml 5개군 (5마리/ 군 ) 표 6. BA LB/C 마우스군구성 (2) 체중분석 실험실시전 1주일간각개체의체중을분석하여군분리를실시하였으며, 매일투여전개체의체중을측정한후 14일간체중변화를분석하였다. (3) 육안검사 투여전 후실험동물의외양및행동형태에대한검사를실시하였으며, 그결과를매일기록지에기록하였다. (4) 부검 투여 14일후익일절식을실시하였으며, 부검실로이동하여케타민으로마취를유도하였으며, 혈액채취후부검을실시하였으며, 실험에필요한각장기별무게를측정하였다. 다. 광학현미경분석 각장기별무게를측정한후광학현미경용슬라이드를만들기위하여 10% 중성포르말린으로고정하였으며, 48시간고정후수세하였으며, 탈수과정및파라핀포매과정을거쳐조직슬라이드를제작하였다. 제작된조직슬라이드를광학현미경으로각장기별변화를관찰하였다. 라. 전자현미경분석 부검후전자현미경용장기는 1mm크기로잘게자른후 2.5% glutaraldehyde 에고정시킨후연세대학교의과학연구원에전자현미경촬영을의뢰하여장기내침착위치를확인하였다. 마. ICP-MS 분석 부검후 ICP-MS용샘플은혈액을포함하여각장기에대하여 15ml conical tube에담아한국기초과학연구원에분석을의뢰하였다

127 바. 혈액단백질분석 부검을통하여채취한혈액들을군별로 pooling 하여연세대학교 ( 주 ) 프로테옴텍에 2DE 분석을의뢰한 후그결과를분석하였다. 다. 2DE 분석실험 (1) 나노물질투여마우스혈청준비금나노물질을 2주간투여한마우스 3군 ( 각20마리 ) 의각군별로 pooling 된혈청약 6ml을수령하였다. 대조군은 Control 마우스 (20마리 ) 에서획득한혈청이고, Nano(-) 나노입자와 Nano(+) 나노입자를각각주입한마우스군 ( 각20마리 ) 에서획득한혈청이었다. (2) 혈중나노입자에비특이적으로결합하는단백질제거수령한 6ml 정도의혈청시료군을 3000rpm 으로 5분간원심분리하여잔여혈구를제거하고, 상등액을취하여 34,000rpm 으로 15분간원심분리한다. 상등액은조심스럽게따라버리고, 침전물을 1 mm EDTA가포함된 10 mm Tris-Cl (ph 7.5) 버퍼 (TE 버퍼 ) 를넣고잘흔들어준후 34,000rpm 으로 15분간원심분리한다. TE버퍼를넣고잘혼합한후에다시원심분리한다. 상등액을조심스럽게따라버리고침전물을회수한다. 대조군도시료군과동일한방법으로실험을진행하여, 침전물을회수한다. (3) 나노입자에결합한단백질회수회수한침전물에 100ul 의 50mM Tris-HCl ph 8.8, 6 M Urea, 30% glycerol, 2% SDS가들어있는 urea lysis buffer 에흔들어잘섞는다. 잘섞인시료를 34,000rpm 에서 15분간원심분리한다. 나노입자에서떨어져나간단백질이들어있는상등액을조심스럽게회수하여 Bradford 법으로단백질의양을측정한다. (4) 회수된나노입자결합단백질의이차원전기영동법으로의분리 4.1. 시료의 solubilization 과 in-gel rehydration 각각의시료를총부피가 125ul 가되도록 6M urea, 2M Thiourea, 4% CHAPS, 0.4% DTT로희석한후, 7 ml의 IPG buffer, 3 ml의 1% bromophenol blue (BPB) 를가한다. 이들을 Immobiline ph gradient Drystrip 으로 rehydration tray위에서시료를얹은후, 시료의증기화와 urea의결정화를방지하기위하여 2 ml의 Covering fluid를가하고, 대략 24시간정도실온에서 rehydration 한다 First dimension Isoelectric focusing (IEF) 과 IEF gel strips의평형 Rehydrated IPG strips 을제조회사가권장하는방법에따라 Multiphor II unit를이용하여 20 C 에서 isoelectric focusing (IEF) 을실시한다. Focusing 조건은각각의 ph3-10(nl) 7cm Immobiline strip에따라 V에서 1시간, 300V 에서 1시간, 500V 에서 6시간실시한다. Isoelectric focusing 후각각의 strip을 20mM Tris-HCl(pH 8.8), 6M urea, 2% SDS, 20% glycerol, 2.5% acrylamide 그리고 5 mm TBP에서 15분간평형에이르도록가볍게흔들어준다. 4.3 The second dimensional polyacrylamide gel electrophoresis 본실험에서사용된 gel들은 12% 7cm gel을사용한다. strip의평형이완료된후, 각각의 strip을 second dimensional gel위에얹고 0.5% low melting agarose 와 0.001% BPB가들어있는 SDS로

128 봉합한다. 전기영동은 Bio-rad 사의미니젤전기영동장치를사용하며, dye 가 gel 의바닥까지오도 록약 90 시간동안 120 volt 로실시한다. 4.4 Gel staining 필요시실험에서의 silver staining방법을간략히기술하면, 전기영동후 gel을 45% methanol, 5% acetic acid로고정시키고, 증류수로 1시간동안 acetic acid를씻어낸다. 0.02% sodium thiosulfate 로 2분간 sensitization 시키고증류수를이용하여 1분씩 2번세척하고, gel을 0.1% silver nitrate와 20분간반응한다음다시증류수로세척한다. 이후 2%(w/v) sodium carbonate, 0.04%(v/v) formaldehyde 로현상하여원하는세기의 spot이나타나면 1% acetic acid로반응을정지시킨다. 마지막으로 gel을증류수로세척한후, sealed plastic bag속에저장후 4 C에서보관한다. 코마시로염색할경우에는 gel을꺼낸후 30% methanol과 10% acetic acid로 1시간동안고정하고증류수로간단히씻은후 colloidal Coomassie brilliant G250 으로 12시간동안염색한다. 10% methanol 과 7% acetic acid로 4시간동안탈염색한다. 코마시나실버염색된 2D gel을증류수로세척한후 Bio-Rad GS-710 Calibrated Densitometer (Bio-Rad, Hercules, USA) 스캐너를이용하여이미지를디지털화한후에 sealed plastic bag속에저장후 4 C에서보관한다 전개된 2D gel의이미지분석법개발디지털화한이미지는 ImageMaster V (GE healthcare, US) 프로그램을이용하여단백질점의탐색, 일치화, 정량화 (%v) 를시행하여유효한단백질점들을표준화하고그위치가일치되는단백질점간의 %v값을비교하여그강도의차이가증가하거나감소하는단백질점및서로짝을이루지못하여특이하게나타나는단백질점을분석한다. 시료군 Nano(+), 과대조군 Control, 시료군Nano(-) 과대조군 Control 를비교하여특이적이거나정량적으로 2배이상차이가나는단백질 spot 을검출한다. 라. 통계학적분석 모든실험결과는통계프로그램 Sigma-stat (SAS Institute, Inc.) 를이용하여등분산검정후 one way ANOVA에서유의차가인정되는경우 Dunnett' s 또는 Duncan' s Multiple Range Test 를실행하여대조군과투여군간의통계학적유의성 (p< 0.05, p< 0.01, p< 0.001) 을검증하였다

129 2 연구수행의결과 1. 금나노물질의합성및 QC 결과 가. 양전하및음전하를가지는금나노입자의합성에따른특성분석결과 (1) 자외 - 가시선분광광도계 그림 4. 양이온및음이온으로코팅된골드나노입자의흡광도 두입자모두 1X PBS (ph 6.8) 에분산되어있으며, 두나노입자모두 absorbance maximum 값이 530 nm 로유사하게나타났다. In DW In PBS (Ph6.8) (+)AuNP (-)AuNP 그림 5. 양이온및음이온으로코팅된골드나노입자의분산안전성확인 두입자의분산의안전성을 14 일간 DW 와 PBS 에서확인하였으며, 530nm 로유사하게확인되었다

130 (2) 투과전자현미경 (transmission electron microscope) 그림 6. 양이온및음이온으로코팅된골드나노입자의 TEM 이미지 TEM 이미지를통해서골드나노입자의원형모양과코팅이되었는지를확인할수있다. (3) 입자의크기 골드나노입자의크기는 DLS 장비를이용하여분석하였으며, 30.9nm 의평균값을얻었다. 그림 7. DLS 분석을통한음이온및양이온으로표면처리된금나노입자의평균크기측정결과 (4) Zeta potential meter 를이용한나노입자의표면전위분석 표 7. 양이온표면처리금나노입자의제타포텐셜값 Sample Zeta potential (mv) Aminoundecanethiol + gold nanoparticles 33.85±1.23 mercaptoundecanoic acid + gold nanoparticles -36.8±

131 그림 8. 음이온및양이온표면처리금나노입자의제타포텐셜값 (5) 합성한금나노입자의농도측정유도결합플라즈마질량분석법 (ICP-MS: Inductively coupled plasma - massspectroscopy, 이하 ICP-MS 로칭함 ) 은원자의고유한질량의차이를이용한극미량원소분석장비다. 따라서골드나노입자의농도를기초과학연구소에있는 ICP-MS를이용하여분석하였다. 또한, 골드나노입자의개수를 fcc 골드밀도와제조된골드나노입자의부피를이용하여계산하였다. 표 8. 수령받은금나노입자의종류별농도 Sample n mol mol/l n-aunp 1.16 * * *10-9 p-aunp 2.28 * * *10-9 사. 나노입자의오염여부확인 그림 9. Blood agar plate 에나타나는오염세균콜로니모양의예시 수령받은금나노입자세종류를각각 20ul 씩채취하여 Blood Agar Plate (Komed 社 ) 에도말한후, 48h 동안 37, CO 2 5% 조건하에배양한다. Blood Agar Plate 는미생물을배양과분리목적으로사용하

132 는배지로서혈액을첨가하여균종의용혈성을알아본다. 그러므로혈액한천평판배지는영양강화배지이면서용혈성을구분하는배지이다. Streptococcus pyogenes 의용혈을찾기위해이배지를사용할때는배지백금이등으로배지바닥까지여러번천자한후배양을한다. 천자된부위는산소압이낮아져산소에불안정한 hemolysin O의용혈을증가시킨다. S. pyogenes 의선별을위해서혐기성배양을시행할수있다. 그조성은 Heart Muscle, Infusion from(solids) 2.0g, Pancreatic Digest of Casein 5.0g, Yeast Extract 5.0g, Sodium Chloride 5.0g, Agar 15.0g, Sheep Blood, Defibrinated 50ml 이며, Final ph : 7.3 ± 0.2 at 25 조건하에만든다. 2. 체중변화측정 그림 일간금나노물질 ( 양전하, 음전하 ) 투여한쥐의체중변화 군분리후투여 14 일간매일개체별체중을특정하여군별체중의변화를관찰한결과군간의차이 는관찰할수없었다. 3. 장기별무게변화측정 표 일간금나노물질 ( 양전하, 음전하 ) 투여한쥐의각장기무게변화 각장기별무게를측정한결과군간의유의한결과를관찰할수없었다

133 그림 일간금나노물질 ( 양전하, 음전하 ) 투여한쥐의각장기무게변화 4. 부검결과 부검결과육안적으로투여군 ( 양이온및음이온 ) 간및비장에서색깔변화를관찰할수있었다. 그림 일간금나노물질 ( 양전하, 음전하 ) 투여한쥐부검후간및비장의색깔변화

134 5. 광학현미경분석결과 그림 13. 간조직의광학현미경사진. negative charge 금나노입자가주로동모양모세혈관 (sinusoid) 에 침착됨을확인할수있었으며, positive charge 에서보다많은양이침착됨을확인할수있었다

135 그림 14. 비장조직의광학현미경사진. negative charge 금나노입자가주로비장세포에침착됨을확 인할수있었으며, positive charge 에서보다많은양이침착됨을확인할수있었다

136 그림 15. 폐조직의광학현미경사진. positive charge 금나노입자가폐포의 alveolar type I 세포에침 착됨을확인할수있었다

137 그림 16. 신장조직의광학현미경사진. positive charge 금나노입자가신장의사구체에침착됨을확인 할수있었다

138 6. 투과전자현미경분석결과 그림 17. 간조직의투과전자현미경사진. negative charge 금나노입자가주로동모양모세혈관 (sinusoid) 주위에침착됨을확인할수있었으며, positive charge 에서보다많은양이침착됨을확인할 수있었고, 침착되는세포소기관은 lysosome 으로관찰되었다

139 그림 18. 비장조직의투과전자현미경사진. negative 및 positive charge 금나노입자가주로 lysosome 에침착됨을관찰하였다

140 그림 19. 폐조직의투과전자현미경사진. negative charge 금나노입자가폐포에서관찰할수없었고, positive charge 금나노입자가주로 lysosome 에침착됨을관찰하였다

141 그림 20. 신장조직의투과전자현미경사진. negative charge 금나노입자는사구체에서관찰할수없 었고, positive charge 금나노입자가주로 lysosome 에침착됨을관찰하였다

142 7. 혈액단백질분석결과 3.1. 제공된마우스혈액에서나노입자분리혈청에함유된나노입자 Nano(+), Nano(-) 를완전히회수하기위해서 34,000rpm 으로 15분간원심분리를하였다. 실험결과 34,000rpm 에서 15분간원심분리하면나노입자가완전히침강되어침전물형태로튜브의바닥에있고상등액에는남아있지않은것으로관찰되었다. Control 혈청시료도본방법으로원심분리하여침전물을얻었다 분리된나노입자에서비특이적으로결합하는단백질제거법개발미국국립암센터에서는회수된나노입자를 PBS로 3번세척을하였다. PBS에는 salt들이많기때문에이방법으로회수된나노입자에는 salt가남아있을우려가있어 2DE를수행하는데어려움이따르게된다. 그러므로본연구에서는 salt가적게들어가는 500ul TE버퍼를사용하여나노입자를 vortexing 하여혼합하고, 34,00rpm 에서 15분동안원심분리하고, 이과정을두번더반복하여, 비특이적으로약하게나노입자에결합되어있는단백질을제거하였다. 만약에 detergent 를쓰면나노입자에비교적강하게달라붙어있는단백질도제거될우려가있기때문에미국국립암센터방법과같이세척과정에서 detergent 를사용하지않았다. 대조군인 Control 혈청침전물도동일한방법으로처리하였다 나노입자에붙은혈청단백질회수완전히세척된나노입자로부터붙어있는단백질을분리하기위해강한 detergent 를사용하였다. 방법에서기술한 urea lysis buffer 는통상적인이차원전기영동을위해시료로부터모든단백질을녹이는데사용하는버퍼이다. 그러므로나노입자에비교적강하게붙어있는단백질이라도이버퍼에녹아서나노입자로부터분리될수있다. 더불어이버퍼는이차원전기영동에서사용하는버퍼에상응하여, 이차원전기영동을위한별도의처리과정이필요없다. 이방법으로실험에서대조군을포함하여, 각나노입자로부터약 210ug 의나노입자로부터분리된단백질을얻어 2차원전기영동을실행하였다. 다른전처리없이이시료를이차원전기영동을했을때, 양호한이미지의 2D gel사진을얻을수있었다 나노입자로부터회수한단백질의이차원전기영동법개발실험에서얻은 210ug 의단백질시료를, 나노입자를투여하지않은혈청시료를대조군 (Control) 으로, Nano(+) 나노입자와, Nano(-) 나노입자를투여한마우스를시료군으로하여, 이러한나노입자에결합하는혈청단백질을가지고 2DE를진행하였다. 혈청으로부터나노입자의회수, 나노입자의세척, 나노입자로부터이에결합하는단백질회수, 회수된단백질의이차원전기영동법을확립하였다. 이차원전기영동을진행하고코마시염색을했을때, 그림1와그림2와같은양호한 2D gel 이미지를얻을수있었다

143 그림 21. 마우스 Control 혈청침전물에서얻은단백질과, Nano(-) 나노입자에서회수한단백질의 2D 젤 코마시블루염색이미지. Control Nano(-) 그림 22. 마우스혈청침전물로부터얻은 Control ( 대조군 ) 단백질과, Nano(+)( 시료군 ) 로부터분리한단백 질의코마시블루염색 2D 젤이미지. Control Nano(+) 2D gel 을프로그램으로분석한결과표 1 와같이단백질스팟개수를셀수있었다. 2D gel 이미지는 ImageMaster 로충분히비교분석할수있음을보여주었다. 이로써시료들의 표 10. 본시료 2D gel 에서관찰된단백질의총스팟개수. Gel Name 시료 Control 162 시료 Nano(-) 122 시료 Nano(+) 136 Spots

144 3.5. 대조군 (Control) 과대비로 Nano(-) 시료에서감소한단백질점들각 2D gel에서 ImageMaster로단백질점으로지정된이미지를가지고, Control를대조군으로하여 Nano(-) 2D gel과정량적으로비교분석하고, Control를대조군으로하여 Nano(+) 시료의 2D gel 이미지를비교분석하였다. 단백질스팟 pairing과각스팟을 %Volume 값으로환산하여정량적으로분석하였고, 두 pairing된스팟의정량적인차이가 2배이상감소하거나, 2배이상증가하거나, 한쪽 Gel에특이하게나타나는단백질점을찾아내었다. 그림 23. Control 를대조군으로 Nano(-) 에서감소한단백질점의 2D gel 상의프로파일 Control Nano(-) Control 를대조로 Nano(-) 2D gel 에서분자량이작은단백질점의양이감소한새로운스팟이관찰 되었다 대조군 (Control) 과대비로 Nano(-) 시료에서증가한단백질점들 Control 를대조로 Nano(-) 시료의 2D gel 을분석하여시료군에서 2 배이상증가하는단백질점을찾 아내었다 ( 그림 24). 그림 24. Control 대비로 Nano(-) 시료에서증가한단백질점들의 2D gel 프로파일 Control Nano(-) Nano(-) 에서단백질점이증가한양으로, 분자량이 30kDa 이하, 산성쪽에서관찰되었다 ( 그림 24)