수의학박사학위논문 Sprague Dawley 랫드에서 Zinc oxide 나노물질의표면처리에 따른독성에관한연구 2015 년 8 월 서울대학교대학원 수의학과실험동물의학전공 박학수

Transcription

1 저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할수없습니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 수의학박사학위논문 Sprague Dawley 랫드에서 Zinc oxide 나노물질의표면처리에 따른독성에관한연구 2015 년 8 월 서울대학교대학원 수의학과실험동물의학전공 박학수

3 Sprague Dawley 랫드에서 Zinc oxide 나노물질의표면처리에따른 독성에관한연구 지도교수박재학 이논문을수의학박사학위논문으로제출함 년 월 서울대학교대학원 수의학과실험동물의학전공 박학수 박학수의박사학위논문을인준함 년 월

4 나노물질은 100 nm 이하의크기를갖는물질을의미하며, 이를포함한제품에는자동차용품, 가전제품, 생활주방용품, 문구용품, 화장품등매우다양할뿐만아니라생활주변에서쉽게접할수있다. 다양한나노물질중산화아연 (zinc oxide, ZnO) 은식품첨가제, 식품포장재, 식품보존제, 플라스틱, 페인트, 배터리첨가제, UV 차단제선크림화장품등상업적으로다양하게사용되고있다. 나노물질은표면처리, 코팅을통해물리화학적특성이변화하며, 그에따라독성이나효력이다양하게관찰된다. 그러나표면처리에따른나노입자의독성연구는아직미흡한수준이며, 세포수준의연구가주를이루고있고동물을이용한중장기적인노출에따른독성연구는아직이루어지지않고있다. 이에본연구에서는 ZnO 나노물질의표면을음전하와양전하로처리하여 90일간의중장기적인노출을통해독성의차이를확인하고향후식품분야에폭넓게사용될경우표면개질에따른안전성에대한자료를제시하고자한다. 음전하를띠는 20 nm 산화아연나노입자 (20 nm, negative charged ZnO nanoparticle, 이하 ZnO(-) 로표시함 ) 와양전하를띠는 20 nm 산화아연나노입자 (20 nm, Positive Charged ZnO nanoparticle, 이하 ZnO(+) 로표시함 ) 의독성변화와무독성량을조사하기위하여, SD 랫드를대상으로 14일용량결정시험및 90일반복투여경구독성시험을실시하였다. 125, 250 및 500 mg/kg의투여용량을설정하여 SD 랫드에 90일간매일반복하여경구로투여하였다. 또한독성반응의지속성및회복성을평가하기위해 14일간회복기간을두어관찰하였다.

5 ZnO(-) 과 ZnO(+) 모두 90일반복투여경구독성시험결과, 시험기간동안동물이치사에이르지는않았으나췌장, 위, 안구에서시험물질에의한병변이관찰되었고빈혈과관련된인자의변화를보였다. 125 mg/kg 투여용량까지유의할만한독성학적인변화가관찰되어무독성량 (NOAEL) 은암 수모두찾지못했으나, 최소독성량은암 수모두 125 mg/kg 이었다. 기존의표면전하가개질되지않은 ZnO 나노입자의 90일반복투여독성시험결과와비교한결과 MCV, MCH, MCHC의감소량이더적고, 혈소판및전해질의변화가관찰되지않았다. 이는표면개질로인하여독성이감소된결과로판단되었다. 음전하와양전하로표면전하를개질한결과, ZnO(+) 투여군이더욱강한부작용을보여표면개질에따른독성의차이가현저함을보여주었고표면전하가개질되지않은 ZnO을투여하였을경우보다혈액학적, 혈청학적영향이감소하여표면개질로인하여조혈기계및전해질독성이감소된것으로판단되었다.

6 목 차 초록 ⅰ 목차 iii 표목차 ⅴ 그림목차 xii 사진목차 xiv 약어표 xvii General Introduction 1 제 1 장 ZnO(-) 의 90일반복경구투여연구 7 제 1 절 14일용량결정시험 7 1. 시험물질의물리화학적특성파악 7 2. 시험방법 9 3. 결과 고찰 18 제 2 절 90일반복경구투여연구 시험방법 결과 고찰 94 제 2 장 ZnO(+) 의 90 일반복경구투여연구 9 7 제 1 절 14일용량결정시험 시험물질의물리화학적특성파악 시험방법 100

7 3. 결과 고찰 109 제 2 절 90일반복경구투여연구 시험방법 결과 고찰 180 최종결론 182 참고문헌 Abstract 1 9 1

8 1 ZnO(-) 90 TABLE 1. Experimental Design in the 14 Days Study 9 TABLE 2. Urinalysis in the 14 Days Study 11 TABLE 3. Microscopic Examination of Urinary Sediments in the 14 Days Study 11 TABLE 4. Hematological Analysis in the 14 Days Study 13 TABLE 5. Blood Biochemical Analysis in the 14 Days Study 14 TABLE 6. Survival and Body Weights for Rats 19 TABLE 7. Organ Weights and Organ-Weight-to-Body-Weight Rations for Male and Female Rats in the 14 Days Gavage Study of 20 nm, Negative Charged ZnO 21 TABLE 8. Experimental Design in the 90-Day Study 24 TABLE 9. Urinalysis in the 90-Day Study 28 TABLE 10. Microscopic Examination of Urinary Sediments 28 TABLE 11 Hematological Analysis in the 90-Day Study 29 TABLE 12 Blood Biochemical Analysis in the 90-Day Study 30 TABLE 13 Clinical Signs for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 36 TABLE 14 Survival and Body Weights of Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 37 TABLE 15 Individual Sensory Reactivity Assessments Data for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 40 TABLE 16 Individual and Group Mean Functional Performance Test Values and Standard Deviations for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 41

9 TABLE 17 Feed Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 42 TABLE 18 Water Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 43 TABLE 19 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 44 TABLE 20 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 46 TABLE 21 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 48 TABLE 22 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 50 TABLE 23 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study(Main group) of 20 nm, Negative Charged ZnO 53 TABLE 24 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 54 TABLE 25 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 55 TABLE 26 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 57 TABLE 27 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 59

10 TABLE 28 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 61 TABLE 29 Hematology Data of Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 64 TABLE 30 Hematology Data of Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 65 TABLE 31 Hematology Data of Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 66 TABLE 32 Hematology Data of Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 67 TABLE 33 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 68 TABLE 34 Clinical Chemistry Data for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 69 TABLE 35 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 70 TABLE 36 Clinical Chemistry Data for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 71 TABLE 37 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO 73 TABLE 38 Summary of the Incidence of Nonneoplastic Lesions in

11 Female Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO 76 TABLE 39 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 79 TABLE 40 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 81 TABLE 41 Summary of toxicokinetic parameters for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 91 TABLE 42 Tissue distribution Data or Rats in the 90-Day Gavage Study of 20 nm, Negative Charged Zn 93 TABLE 1 Experimental Design in the 14 Days Study 101 TABLE 2 Urinalysis in the 14 Days Study 102 TABLE 3 Microscopic Examination of Urinary Sediments in the 14 Days Study 102 TABLE 4 Hematological Analysis in the 14 Days Study 104 TABLE 5 Blood Biochemical Analysis in the 14 Days Study 105 TABLE 6 Survival and Body Weights for Rats in the 14 Days Gavage Study of 20 nm, Negative Charged ZnO 110 TABLE 7 Experimental Design in the 90-Day Study 114 TABLE 8 Urinalysis in the 90-Day Study 118 TABLE 9 Microscopic Examination of Urinary Sediments 118 TABLE 10 Hematological Analysisin the 90-Day Study 119 TABLE 11 Blood Biochemical Analysis in the 90-Day Study 120

12 TABLE 12 Clinical Signs for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 125 TABLE 13 Survival and Body Weights of Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 126 TABLE 14 Individual Sensory Reactivity Assessments Data for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 129 TABLE 15 Individual and Group Mean Functional Performance Test Values and Standard Deviations for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 130 TABLE 16 Feed Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 131 TABLE 17 Water Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 132 TABLE 18 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 133 TABLE 19 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 135 TABLE 20 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 137 TABLE 21 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 139 TABLE 22 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study(Main group) of 20 nm, Negative Charged ZnO 142

13 TABLE 23 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 143 TABLE 24 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 144 TABLE 25 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 146 TABLE 26 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 148 TABLE 27 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 150 TABLE 28 Hematology Data of Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 153 TABLE 29 Hematology Data of Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Positive Charged ZnO 154 TABLE 30 Hematology Data of Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 155 TABLE 31 Hematology Data of Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Positive Charged ZnO 156 TABLE 32 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO 157 TABLE 33 Clinical Chemistry Data for Female Rats in the 90-Day

14 Gavage Study (Main Study) of 20 nm, Positive Charged ZnO 158 TABLE 34 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 159 TABLE 35 Clinical Chemistry Data for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Positive Charged ZnO 160 TABLE 36 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO 162 TABLE 37 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO 166 TABLE 38 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 168 TABLE 39 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO 170 TABLE 40 Tissue distribution Data or Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO 179

15 FIGURE 1 (A) Transmission Electron Microscopy (TEM) and (B) Scanning Electron Microscopy (SEM) Images of 20 nm, Negative Charged ZnO Nanoparticles 8 FIGURE 2 Hydrodynamic Size of 20 nm, Negative Charged ZnO Nanoparticles 8 FIGURE 3 Zeta Potential of 20 nm, Negative Charged ZnO Nanoparticles 8 FIGURE 4 Growth Curves for Male Rats Administered 20 nm, Negative Charged ZnO by Gavage for 14 Days 20 FIGURE 5 Growth Curves for Female Rats Administered 20 nm, Negative Charged ZnO by Gavage for 14 Days 20 FIGURE 6 Growth Curves for Male Rats Administered 20 nm, Negative Charged ZnO by Gavage for 90 Days 38 FIGURE 7 Growth Curves for Female Rats Administered 20 nm, Negative Charged ZnO by Gavage for 90 Days 38 FIGURE 8 Zinc concentration in plasma after 90 day-repeated oral administration to rats. 90 FIGURE 1 (A) Transmission electron microscopy (TEM) and (B) Scanning Electron Microscopy (SEM) Images of 20 nm, Positive Charged ZnO Nanoparticles 98 FIGURE 2 Hydrodynamic size of 20 nm, Positive Charged ZnO nanoparticles 98 FIGURE 3 Zeta potential of 20 nm, Positive Charged ZnO

16 nanoparticles 99 FIGURE 4 Growth Curves for Male Rats Administered 20 nm, Positive Charged ZnO by Gavage for 14 Days. 111 FIGURE 5 Growth Curves for Female Rats Administered 20 nm, Positive Charged ZnO by Gavage for 14 Days. 111 FIGURE 6 Growth Curves for Male Rats Administered 20 nm, Positive Charged ZnO by Gavage for 90 Days 128 FIGURE 7 Growth Curves for Female Rats Administered 20 nm, Positive Charged ZnO by Gavage for 90 Days 128

17 PLATE 1. Normal limiting ridge of stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 82 PLATE 2. Sqaumous cell hyperplasia (H) and squamous cell vacuolation (arrow) in limiting ridge of forestomach of SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day. 82 PLATE 3. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 83 PLATE 4. Eosinophilic chief cell (EC) in glandular stomach of male SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day 83 PLATE 5. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 84 PLATE 6. Intracytoplasmic hyaline droplet (arrows) in glandular stomach of male SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day 84 PLATE 7. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 85 PLATE 8. Submucosal inflammatory cell infiltration (SI) in glandular stomach of male SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day 85 PLATE 9. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 86 PLATE 10. Mucous cell hyperplasia (arrows) in glandular stomach of male SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day 86

18 PLATE 11. Normal microscopic structure of the pancreas of a male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 87 PLATE 12. Chronic inflammation (A) and acinar cell apoptosis (arrows) and prominent acinar cells (P) in the pancreas of SD rat administered 500 kg/mg ZnO(-) by gavage for 90-Day 87 PLATE 13. Normal microscopic structure of the eye of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day 88 PLATE 14. Retinal atrophy is remarkable in the eye of male SD rat administered 500 mg/kg ZnO ZnO(-) by gavage for 90-Day. 88 PLATE 1. Normal limiting ridge of stomach of male SD rat administered 0 mg/kg ZnO(+) by Gavage for 90-Day 172 PLATE 2. Squamous cell hyperplasia (H) and squamous cell vacuolation (arrows) in limiting ridge of forestomach of SD rat administered 500 mg/kg ZnO(+) by Gavage for 90-Day 172 PLATE 3. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(+) by Gavage for 90-Day 173 PLATE 4. Eosinophilic chief cell (arrows) in glandular stomach of male SD rat administered 500 mg/kg ZnO(+) by Gavage for 90-Day 173 PLATE 5. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(+) by Gavage for 90-Day 174 PLATE 6. Submucosal inflammatory cell infiltration (SI) in glandular stomach of male SD rat administered 500 mg/kg ZnO(+)

19 by Gavage for 90-Day 174 PLATE 7. Normal glandular stomach of male SD rat administered 0 mg/kg ZnO(+) by gavage for 90-Day 175 PLATE 8. Mucous cell hyperplasia (arrows) in glandular stomach of male SD rat administered 500 mg/kg ZnO(+) by gavage for 90-Day 175 PLATE 9. Normal pancreas of male SD rat administered 0 mg/kg ZnO(+) by Gavage for 90-Day 176 PLATE 10. Acinar cell atrophy (A) and acinar cell apoptosis (arrows) and an interstitial inflammatory cell infiltration (I) in pancreas of SD rat administered 500 kg/mg ZnO(+) by Gavage for 90-Day 176 PLATE 11. Normal eye of male SD rat administered 0 mg/kg ZnO(+) by Gavage for 90-Day 177 PLATE 12. Retinal atrophy in eye of male SD rat administered 500 mg/kg ZnO(+) by Gavage for 90-Day 177

20 ZnO ZnO(-) ZnO(+) Hct MCV MCH MCHC NOAEL PAA HEPES TEM SEM DLS ICP-AES ICP-AAS AUC ANOVA SPSS DETA PDAC zinc oxide 20 nm, Negative charged ZnO nanoparticle 20 nm, Positive charged ZnO nanoparticle Hematocrit Mean Cell Volume Mean Cell Hemoglobin Mean Cell Hemoglobin Concentration No Observed Adverse Effect Level Poly-acrylic acid 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Transmission Electron Microscopy Scanning Electron Microscopy Dynamic Light Scattering Inductively Coupled Plasma Atomic Emission Spectroscopy Inductively Coupled Plasma Atomic Absorption Spectrometry Area Under the Curve Analysis of variance Statistical Package for the Social Sciences Diethylenetriamime Poly-dimethyldiallylammonium

21 나노기술은지난수년간다양한연구분야에서급격한속도로발전되어왔다 ( 예를들면재생의학, 세포공학, 약학, 화장품 ). 그러나나노물질이경구, 경피, 흡입, 주사등의다양한경로를통해체내로흡수되며인간의건강에위해요소가되고있다. 따라서나노물질에대해농도, 투여량, 반응성에대한주의깊은연구가필요하다. 이런이유로산업계뿐만아니라의약학응용분야에서도나노입자및다른나노물질과관련하여건강과안전에대한관심이증가하고있다. 1,2 산화아연 (ZnO) 나노입자 (NP:Nano particle) 는최근들어기체와액체상합성및졸-겔처리과정으로합성되고있다. 3 또한비액화졸-겔방법을기반으로벤질아마이드의전구체와아연아세틸화를이용해서산화아연나노입자를합성하고있다. 4 나노입자는물과혼합될때독특한특성을가지고있다. 기체및액체와반응성이매우좋을뿐아니라표면적이넓고생물분자와유사한크기를가지고있다. 5,6 나노입자는 1 100nm 크기사이에서적어도 1차원적으로원자혹은분자적으로응집되는데크기가더큰물질과비교하여물리화학적성질이다르게변화한다. 7-9 나노입자들은다양한거대원료물질에서만들어지는데, 반응이화학적조성과크기혹은모양에기반한다. 10 최근들어나노기술을이용하여의학과건강, 화장품, 의류, 개인용품, 스포츠용품및자외선차단제등이뜨거운이슈가되어왔다. 11 ZnO와이산화티타늄 (TiO 2 ) 같은나노물질은화장품과의약품에오랫동안사용되어왔다. 11 ZnO 나노입자는자외선을흡수하는능력이있고, 가시광선을투과하기때문에자외선차단제, 파운데이션, 로션, 반사혹은번들거림방지코팅제같은수많은개인관리용품및페인트등에폭발적으로사용되었다. 12 현대화장품산업에서가장인기있는나노입자는자외선차

22 단에사용되는 ZnO일것이다. 13 나노입자의흥미로운물리적특성이피부보호제품에사용하기에이상적인것이다. 14 ZnO 나노입자는자외선자외선차단기능을이용하여자외선차단제로섬유에도사용된다. 15 나노기술은식품산업에도폭넓게사용되는데, 예를들면식품의포장, 전달, 조제, 평가, 식품처리등에도사용되고있고항세균효과로인해앞으로도더폭넓게사용될가능성이높다. 16 새로운나노입자가사용되는또다른잠재력이높은응용분야는의학분야인데, 탐지, 진단, 모니터링, 처치등에많이사용된다. 17 그러나이런활용성과우수성이있음에도불구하고, 인체에대한나노입자의위해성노란은계속되어왔다. 12 더욱이나노물질은기존의일반적인화학물질과는다른고유의표면의특성, 크기, 그리고물성의다양성으로인해기존의독성평가방법으로는그위해성을정확히평가할수없다는문제점을가짐으로써, 나노물질및나노제품이인체와환경에미칠잠재적위해성에대한우려가전세계적으로증가하는추세에있다. 경구적으로노출되는경우는직업적, 환경적인경로로써물이나음식을섭취하거나흡입된입자를삼키게되는경우다. ZnO 나노물질은상업적인용도로자외선차단제생산공정상에서공기중으로노출될가능성이잠재적으로높아서섭취의가능성도높은것으로알려져있다 13,18. 생산공정상에서갈아내는공정, 마찰, 혼합, 분사공정에서많이생산되고작업자들에게노출된다 19. 이들경로에대한연구를살펴보면, 동물시험의경우랫드에서이리듐 (iridium) 과플러렌 (fullerene) 입자는최소의전신성흡수를보인다. 18,19 다른예로써랫드에서음전하라텍스나노입자는점막층을통과해서확산되고, 상피세포와반응하여다른장기로운반된다. 20 최근에유채 [Brassica rapa(turnip)] 에서유래한나노입자는간에서항간섬유화효과를보여주었다. 21 이들결과를보면나노입자의전신작용은예상하기어렵고위험할수도있음을보여주고있다. 나노물질과나노입자는호흡기, 신경계, 소화기계, 면역계및순환기에독성을가지고있다. 최근에 14일간시험한 ZnO 나노입자의독성시험에서간, 신장, 폐, 비장및췌장에서조직병리학적인

23 병변이관찰되었고간기능이상인자의상승이관찰되었다. 22 이들나노입자의독성은투여량, 크기, 표면적, 입자의화학적성질, 결정의구조그리고표면코팅과연관성을가진다. 23 나노기술과관련된독성과환경적인영향이존재하는데, 세포주및동물모델을이용한연구뿐만아니라박테리아, 선충류 (nematodes), 조류 (algae), 고등식물그리고어류등다양한실험모델을이용하여확인되고있다. 24 ZnO가포함된금속성나노물질을대상으로하여이들나노물질의세포독성을평가한연구에서 20 nm 크기의 ZnO, TiO 2, SiO 2, Al 2 O 3 중 ZnO가사람폐섬유아세포에서세포독성이가장강한것으로확인되었다. 25 또다른연구에서사람신장세포주를대상으로 ZnO, TiO 2, CdS의세포독성을평가한결과, 사람신장사구체와세뇨관배양세포를대상으로물리생화학적특성과금속조성과관련성을가지는 ZnO와 CdS의세포독성이확인되었다. 26 이러한 ZnO 나노물질의세포독성과관련된다수의연구에서 ZnO NP의세포독성은세포내활성산소 (reactive oxygen, ROS) 및유리기 (free radical) 생성증가에따른산화스트레스 (oxidative stress) 에의한것으로밝혀졌다. 27,28 일례로, 사람간세포주에 ZnO NP를노출시키면활성산소를생성하여미토콘드리아경로를통한세포자멸사 (apoptosis) 를유도할뿐만아니라, 산화스트레스로인해 DNA 손상을야기함으로써세포독성과유전독성을동시에갖는것으로확인되었다. 29 특히, 사람간세포주에서 ZnO NP에의해증가된활성산소가유도하는세포자멸사는암억제유전자 (tumor suppressor gene) 인 p53 신호경로전달경로를따르는것으로밝혀졌다. 30 다른연구자는사람피부섬유아세포에서는 ZnO NP에의해증가된활성산소가직간접적으로 DNA 손상을유도하며, 이로인해 p53 신호전달경로가활성화되고최종적으로세포자멸사에이른다고보고하였다. 31 사람피부섬유아세포를대상으로한또다른연구에서 ZnO NP에의해유도되는세포자멸사과정에 p53 신호전달경로외에 p38 mitogen-activated protein kinase (MAPK) 신호전달경로가함께관여하는것으로확인되었다. 32

24 나노물질은고유의물리화학적특성에따라세포독성이다르게나타나며, 특히나노물질의표면화학에있어서표면개질 (modification) 에사용된리간드가세포반응에영향을미치는중요한요소로알려져있다. Yin et al. 은 ZnO NP를 oleic acid(oa) 와 poly-methacrylic acid(pmaa), 배지에서흡착된물질로표면을개질하여서로다른표면전하를띄게한후, 사람림프아세포주에서독성을평가한바있다. 독성평가결과, 표면을개질하지않은 ZnO NP를노출한경우에활성산소생성및세포생존도가가장많이영향을많았다. 그러나유전독성의경우에는 PMAA로표면을개질한 ZnO NP가가장높은것으로분석되는등, 동일한 ZnO NP에서도표면개질물질에따라독성이달리나타나는것을확인할수있었다. 33 나노물질이인체에노출되는경로는크게경구 (oral), 경피 (dermal), 그리고흡입 (inhalation) 의 3 가지경로로나뉘고있으며, 노출경로에따른인체위해성을예측하기위하여동물모델을이용한독성연구결과가보고되고있다. 랫드에 50 nm ZnO 나노입자를 14일동안경구투여한결과, 신장세포에서에너지대사불균형및미토콘드리아와세포막손상이일어나는것으로확인되었다. 34 경피노출을통한독성연구를살펴보면, 랫드에 20 nm ZnO 나노입자를 28일동안경피투여를수행한후에꼬리에서의콜라겐단백질양을분석한결과, ZnO 나노입자를투여한군에서콜라겐농도가낮은것을확인하였고, 이러한원인을작은크기의 ZnO 나노입자가접촉부위피부층을투과하여세포내산화스트레스를야기하였기때문으로예측하였다. 35 이러한결과를종합하여볼때, 동물의조직세포에서독성은세포내산화스트레스증가에의한것으로예상되며, 이는 in vitro 연구를통해밝혀진 ZnO 나노입자의독성기전과유사한것으로판단된다. 앞선 in vitro toxicity에서언급한바와마찬가지로, 나노물질은고유의물리화학적특성에따라다른독성을나타낼수있다. 이와관련하여랫드에 20 nm ZnO 입자와마이크로크기의 ZnO를 14일동안농도별로 (5-2000

25 mg/kg) 경구투여한결과, 고농도투여군에서마이크로크기의 ZnO를투여한군에비해 ZnO 나노입자를투여한군의간, 췌장, 심장, 위의조직병변발생정도가높은것으로관찰되었다. 또한동일조직에대해 ZnO 나노입자투여군내에서도고농도투여군에비해저농도투여군의조직병변발생정도가높은것으로관찰되었다. 36 또다른연구에서랫드를대상으로 20 nm와 250 nm 크기 ZnO 나노입자의흡입독성을평가한결과, 물질의질량농도와표면적농도가폐독성과높은상관성이있는것으로분석되었다. 37 앞서살펴본활용도와연구에비해일반소비자들은나노물질의위해성에대하여잘알지못하고있으나, 알고자하는욕구는강하다는연구조사가있다. ZnO 나노물질은다양한나노물질중에서가장일반적으로많이사용되는나노물질의하나로페인트에서부터화장품, 선크림등사용처도매우다양하다. 뿐만아니라다수의연구를통해금속성나노물질중에서나노독성이높은물질에속하는것으로여겨지고있다. 또한나노물질은물질이가진고유한물리화학적특성으로인하여동일한나노물질이라도서로다른물리화학적조건을가진다면독성도다르게나타날수있다는가능성이제기되고있다. 따라서나노물질에대한독성평가에있어서물질의크기와농도및표면개질에따른독성규명은중요한의미가있을것으로예상된다. 나노물질은표전의전하처리에따라다양한독성양상을보인다. 표면전하를가진금나노입자는중성인상태의나노입자보다더강한세포독성을보이는경향이있다 40. 양전하 ZnO, silica, silica-titania는비탐식성세포에서음전하나노물질보다더강한세포독성을보인다 금속산화나노물질인 ZnO의경우 ZnO 20nm(+) 가 ZnO 20nm(-) 보다강한세포독성을보인다는연구가있다 44. 그러나반대로나노물질의표면처리로독성으로부터보호해준다는효과가있다는연구사례도발표되고있다. 표면처리된 ZnO 나노물질은사람의후각망울세포에대해보호효과를높여준다는연

26 구가있고, 다른연구에서는표면전하처리된 ZnO가간세포독성을막아준다는연구가있어표면처리에따른나노입자의독성연구를통해보다안전하게나노물질을사용하는데중요한지침이될수있을것이라판단된다 45,46. 더욱이이들연구는세포수준의연구가주를이루고있고동물을이용한중장기적인노출에따른독성연구는아직이루어지지않고있다. 이에본연구에서는 ZnO 나노물질의표면을음전하와양전하로처리하여 90일간의중장기적인노출을통해독성의차이를확인하고향후식품분야에폭넓게사용될경우표면개질에따른안전성에대한결과를확인하고자한다.

27 시험물질은 20 nm 크기의 ZnO 나노물질 (Ultra fine Zinc Oxide ZnO-310, SUMITOMO OSAKA CEMENT, Japan) 을분말상태로구매하여사용하였다. 음성대조물질로써멸균증류수 ( 대한약품공업 ( 주 ), 한국 ) 와시험물질의표면개질을위해 sodium citrate를사용하였다. 음전하를띠는 ZnO를제조하기위해서리간드를사용하여나노물질표면을개질하였다. 후보리간드물질로는 poly-acrylic acid (PAA), citrate (sodium citrate) 등이있으나, sodium citrate는분산성이매우좋으며입자크기가작고일정하므로우선적으로분산성및입자크기를확인하였다. 실험결과를종합하여음전하를띠는 ZnO 제조를위하여 HEPES ph 7 조건에서 citrate가가장적합한리간드물질로결정하였다. ZnO(-) 의물리화학적특성을분석을위하여 TEM 및 SEM을사용하여입자크기및입형을관찰한결과, 평균적으로 29 ± 3 nm와 33 ± 8nm의크기를갖는구형으로확인되었다 (Figure 1). 또한 DLS 분석결과, hydrodynamic 크기는 ± 11.3 nm이었으며, 제타전하는 ± 1.0 mv로음의표면전하를갖는것으로확인하였다 (Figures 2 and 3).

28 FIGURE 1 (A) Transmission Electron Microscopy (TEM) and (B) Scanning Electron Microscopy (SEM) Images of 20 nm, Negative Charged ZnO Nanoparticles FIGURE 2 Hydrodynamic Size of 20 nm, Negative Charged ZnO Nanoparticles FIGURE 3 Zeta Potential of 20 nm, Negative Charged ZnO Nanoparticles

29 2. 시험방법 2.1 실험동물, 시험물질조제및투여군설정 실험동물은 SD-rat(CrjBgi:CD(SD)IGS, 오리엔트바이오, Korea) 를사용하 였다. 90 일반복경구투여용량을설정하기위하여 ZnO(-) 을 SD 랫드에 14 일간매일반복경구로투여하였다. 시험물질최종농도가 2000 mg/ml 인 고용량이되도록 HEPES-citrate Buffer 를가해 vortex mixer 를이용하여 1 분간균질화조제하였다. 그리고멸균증류수를이용하여중용량군과저용 량군이되도록각각 1000 mg/ml 과 500 mg/ml 으로조제하였다. 조제한시 험물질은투여직전 30 초간 vortex 하여재균질화를시켜주었다. 각군별 시험물질의조제는투여기간동안매일조제하였다. 예비시험 2000 mg/kg 용량에서유의할만한영향이관찰되지않아, 2000 mg/kg 을고용량투여 군으로선정한후공비 2 로하여 1000 mg/kg 을중용량투여군, 500 mg/kg 의저용량투여군을각각설정하였다. 대조군으로는멸균증류수를투여하 는음성대조군과부형제를투여하는부형제대조군을설정하였다 (Table 1). 본연구는한국화학융합시험연구원헬스케어연구소의실험동물의사용 과관리에관한규정 ( 실험동물윤리위원회개정, ) 을준수하여 실시하였다. TABLE 1. Experimental Design in the 14 Days Study ZnO(-) ZnO(-) ZnO(-)

30 2.2 투여및증상관찰, 사료및음수측정, 안검사시험물질인 ZnO(-) 는임상예정경로인경구로투여하고, 7일 / 주, 1회 / 일로하여 14일간투여하였다. 투여액량은체중측정을기준으로 10 ml/kg로산출하여모든군을동일하게투여하였다. 시험물질투여후매일 1회일반증상, 매주체중측정, 매주 1회사료및음수량을측정하였다. 모든동물에대하여군분리전및투여최종주에동물눈의외관을육안으로관찰하였다. 대조군과고용량투여군의암 수각 5마리씩에대해서는투여최종주에산동제 (Ocutropine, Samil pharm CO., LTD, Korea) 를이용하여동공을확장시킨후, 안저카메라 (Genesis, Kowa, Japan) 를이용하여안저를관찰하였다. 2.3 요검사각군마다암 수각 5마리씩에대해서는투여최종주에 Multistix 10 SG (Siemens, USA) 스트립과요측정기 (Clinitek 500, USA) 를이용하여항목에대해요검사를실시하였다 (Table 2). 또한대조군과고용량투여군의암 수각 5 마리씩에대해서는대사케이지를이용하여 3 시간동안채뇨한신선뇨에대해서는아래 (Table 3) 의검사를실시하였고, 요량은 24시간동안채뇨한축뇨를이용하여측정하였다.

31 TABLE 2. Urinalysis in the 14 Days Study 비중 (specific gravity) - ph - 백혈구 (white blood cell) - 단백질 (protein) mg/dl 당 (glucose) mg/dl 케톤체 (ketone body) mg/dl 유로빌리노젠 (urobilinogen) E.U./dL 빌리루빈 (bilirubin) mg/dl 잠혈 (occult blood) - 아질산 (nitrite) - 색상및투명도 (color) - TABLE 3. Microscopic Examination of Urinary Sediments in the 14 Days Study 요량 (urine amount) ml 메스실린더측정 적혈구 (red blood cell, RBC) - 백혈구 (white blood cell, WBC) - 상피세포 (epithelial cell) - 광학현미경 원주체 (cast) - 결정체 (crystal) -

32 2.4 안검사, 부검, 혈액학적, 혈청생화학적검사모든동물에대하여군분리전및투여최종주에동물눈의외관을육안으로관찰하였다. 대조군과고용량투여군의암 수각 5마리씩에대해서는투여최종주에산동제 (Ocutropine, Samil pharm CO., LTD, Korea) 를이용하여동공을확장시킨후, 안저카메라 (Genesis, Kowa, Japan) 를이용하여안저를관찰하였다. 빈사및폐사동물의경우에는발견즉시부검을실시하고시험종료시생존동물은이소플루란을이용하여심마취시켜채혈하고안락사시킨다음체표및모든체공 (external surface & all orifices), 두개강 (cranial cavity), 흉강및복강의모든장기 (thoracic and abdominal & their contents) 에대하여육안적검사를실시하였다. 투여가종료된후모든동물에대해부검시육안적검사를실시한후에뇌, 심장, 간장, 비장, 신장에대한습장기중량을측정하였고, 절식체중에대한상대장기중량을측정하였다. 부검전날절식시킨동물을이소플루란으로마취한다음복대동맥으로부터채혈한혈액중약 3 ml의혈액은 EDTA가함유된 CBC bottle (EDTA 3K, BD vacutainer) 에취하여자동혈구측정기 (ADVIA 120E, Siemens, USA) 를이용하여측정하였고 (Table 4.) 혈액응고시간의측정은채혈한후 sodium citrate가함유된 vacutainer (9NC sodium citrate, BD vacutainer) 에취하여원심분리 (3000 rpm, 10분 ) 하여얻은혈장을자동응고시간측정기 (ACL 7000, Instrumentation Laboratory, USA) 를이용하여측정하였다. 채혈한혈액중혈액검사와혈액응고검사에사용된혈액을제외한나머지의혈액을실온에서응고시킨다음원심분리 (3000 rpm, 10분 ) 하여혈청을취한후자동혈청분석기 (HITACHI 7060, HITACHI, Japan) 와전해질분석기 (EasyLyte PLUS Na/K/Cl Analyzer, MEDICA USA) 를이용하여측정하였다 (Table 5).

33 TABLE 4. Hematological Analysis in the 14 Days Study 총백혈구수 (total leucocyte count) 감별백혈구수 (differential leucocyte count) 호중구 (neutrophil) 림프구 (lymphocyte) 단핵구 (monocyte) 호산구 (eosinophil) 호염기구 (basophil) 총적혈구수 (total erythrocyte count) 10 3 cells/μl 레이저광학법 % 레이저광학법 10 6 cells/μl 레이저광학법 혈색소량 (hemoglobin concentration) g/dl cyanmethaemoglobin 헤마토크리트치 (hematocrit) % RBC MCV/10 평균적혈구용적 (mean cell volume) fl 레이저광학법 평균적혈구헤모글로빈량 (mean cell hemoglobin) 평균적혈구헤모글로빈농도 (mean cell hemoglobin concentration) pg g/dl Hb/RBC 10 Hb/(RBC MCV) 1000 망상적혈구비율 (reticulocyte) % 레이져광학법 혈소판수 (platelet) 10 3 cells/μl 레이져광학법 프로트롬빈시간 (prothrombin time) secs 광산란검출방식 부분활성트롬보플라스틴시간 (activated partial thromboplastin time) secs 광산란검출방식

34 TABLE 5. Blood Biochemical Analysis in the 14 Days Study 총단백질 (total protein-) g/dl Biuret 법 알부민 (albumin-) g/dl BCG 법 A/G ratio (albumin/globulin) 총빌리루빈 (total bilirubin-) mg/dl Azobilirubin 법 알칼라인포스파타제 ( a l k a l i n e phosphatase-) 아스파테이트아미노기전이효소 (aspartate aminotransferase-) U/L U/L Albumin/Globulin 산출측정 p-nitrophemyl phosphate 법 UV-rate 법 알라닌아미노기전이효소 (alanine aminotransferase, -) U/L UV-rate 법 크레아티닌 (creatinine--) mg/dl Jaffe 법 혈액요소질소 (blood urea nitrogen-) mg/dl Urease GLDH 법 총콜레스테롤 (total cholesterol-) mg/dl COD-HDAOS 법 트리글리세라이드 (triglycerides-) mg/dl GPO-HDAOS 법, Glycerol blanking 법 혈당 (glucose-) mg/dl Hexokinase-G-6-PDH 법 칼슘 (calcium-) mg/dl OCPC 법 인 (inorganic phosphorus-) mg/dl Fiske Subbarow 법 크레아틴키나아제 (creatine kinase-) IU/L JSCC 법 나트륨 (Sodium-) 칼륨 (potassium-) 염소 (chloride-) mmol/l 이온전극선택법 mmol/l 이온전극선택법 mmol/l 이온전극선택법 2.5 조직병리학적검사모든투여군을개체별로다음의장기를적출한다음 10 % 중성완충포르말린용액 (neutral buffered formalin: NBF) 에고정하고, 그중, 고환, 부고환은 Bouin 고정액에, 안구는 Davidson 고정액에고정하였다. 조직병리학적검사는대조군과고용량군에대해서만실시하였다.

35 간 (liver) 신장 (kidney) 부신 (adrenal gland) 심장 (heart) 폐 (lung) 뇌 (brain) 뇌하수체 (pituitary gland) 비장 (spleen) 정낭 (seminal vesicle) 고환 (testis) 난소 (ovary) 부고환 (epididymis) 전립선 (prostate gland) 자궁 (uterus) 질 (vagina) 혀 (tongue) 기관 (trachea) 식도 (esophagus) 흉선 (thymus) 갑상선 (thyroid gland) 위 (stomach) 방광 (urinary bladder) 소장 / 대장 (small/large intestine) 눈 (eye) 악하선 (submandibular gland) 췌장 (pancreas) 피부 (skin) 유선 (mammary gland) 흉골 (sternum) 대퇴골 (femur) 척수 (spinal cord) 좌골신경 (sciatic nerve) 장간막림프절 (mesenteric lymph node) 2.6 통계처리체중측정, 사료섭취량측정, 음수섭취량측정, 혈액학적검사, 혈액생화학적검사및장기중량측정의자료에대해서는분산의동질성을평가하기위해 Levene's 검사를실시한후, one way ANOVA 검사를하여유의성검정을실시하였다. 분산의동질성및시험군간유의성이인정된경우, 사후검정으로 Scheffe 다중검정을실시하였고, 분산이이질적이며시험군간유의성이인정된경우사후검정으로 Dunnett's T3 다중검사를실시하였다. 모든통계분석은 SPSS (Ver 19.0) 통계프로그램을이용하여수행하였다.

36 3. 결과 3.1 증상관찰, 체중, 사료및음수측정시험기간동안에일반소견관찰결과에서는시험물질투여군에서폐사례및시험물질투여에의한일반소견은관찰되지않았다. 수컷의시험물질투여군에서대조군과비교하여체중이감소하였으며, 2000 mg/kg 투여군에서통계학적으로유의하게감소하였다. 체중은암컷의경우, 시험물질투여군에서유의성있는변화는나타나지않았으나, 2000 mg/kg 투여군에서대조군과비교시체중이감소하였다 (Table 6, Figures 4 and 5). 사료섭취량은대조군과비교하여유의성있는변화는나타나지않았다. 음수섭취량은수컷의경우, 7일차에 1000 mg/kg 에서, 14일차에 2000 mg/kg 에서통계학적으로유의성있게 (P<0.05) 증가하였다. 3.2 임상병리학적검사요검사의경우유의성있는변화는나타나지않았다. 혈액학적검사결과대조군과비교시수컷 2000 mg/kg 에서호중구의증가 (P<0.01), 림프구 (P<0.01) 및헤마토크리트치 (P<0.05) 의감소, 수컷 1000 및 2000 mg/kg 에서혈색소량이감소 (P<0.05), 1000 및 2000 mg/kg 에서혈소판수가통계학적으로유의성있게 (P<0.05) 증가하였다. 암컷의경우, 대조군과비교시 2000 mg/kg 에서총백혈구수 (P<0.01), 호중구가통계학적으로유의하게 (P<0.01) 증가하였고, 림프구가통계학적으로유의하게 (P<0.05) 감소하였다 및 2000 mg/kg 투여군에서혈색소량, 평균적혈구용적, 평균적혈구헤모글로빈량, 프로트롬빈시간이통계학적으로유의성있게 (P<0.05또는 P<0.01) 감소하였고, 혈소판수의증가가통계학적으로유의성 (P<0.05또는 P<0.01) 있게나타났다. 혈액생화학적검사결과대조군과비교하여수컷의 1000 mg/kg 투여군에서총단백질, 알부민, A/G ratio가통계학적으로유의성있게 (P<0.05) 감소하였으며, 2000 mg/kg 투여군에서총빌리루빈및알칼라인포스파타제가통계학적으로유의성있게 (P<0.05또는 P<0.01) 증가하였다. 대조군과비

37 교하여암컷의 2000 mg/kg 투여군에서알부민및 A/G ratio 가통계학적으 로유의성 (P<0.05 또는 P<0.01) 있게감소하였으며, 알칼라인포스파타제 및혈액요소질소가통계학적으로유의성 ( P<0.01) 있게증가하였다. 3.3 안검사결과, 부검, 장기중량및조직병리학적검사안검사결과암수의시험물질투여군에서대조군과비교하여유의성있는변화는나타나지않았다. 부검시수컷 1000 및 2000 mg/kg 에서장점막의암적색변화, 위벽및장벽의얇아짐, 정낭위축, 비장종대및신장암적색변화등이각각 3례및 2례관찰되었고, 암컷 1000 및 2000 mg/kg 에서는장점막및신장의암적색변화, 자궁팽대, 비장종대등의소견이각각 4례및 5례에서관찰되었다. 절대장기중량은수컷의경우, 대조군과비교시 2000 mg/kg 투여군에서심장이통계학적으로유의하게감소하였다. 상대장기중량은수컷의경우, 대조군과비교시 1000 mg/kg 투여군에서심장이통계학적으로유의하게감소하였다. 암컷의경우, 1000 및 2000 mg/kg 투여군에서간장이, 2000 mg/kg 투여군에서뇌가통계학적으로유의하게증가하였다 (Table 7). 위에서염증성병변이주로관찰되었다. 이들병변은수컷과암컷모든투여군에서관찰되었으며, 용량의존적인경향을보였다. 점막하조직에서광범위한호산구의중등도침윤, 선위점막의미란, 전위부과각화, 점막하부종, 선위와전위의경계부위인 limiting ridge에서편평상피의과다형성소견도관찰되었다. 비장에서골수외조혈이관찰되었는데, 수컷과암컷모두 2000 mg/kg 투여군에서중등도와심한강도의골수외조혈이관찰되었다. 이들병변은대조군과비교하여현저한차이를보였다.

38 4. 고찰 ZnO(-) 를 14일간반복경구투여한결과표적장기는수컷과암컷의모든시험물질투여군의위에서용량의존적인염증성병변을보인위로사료된다. 또한 1000 mg/kg 용량까지부작용이관찰되어 90일반복투여독성시험을위한용량은위염을유발한조직병리소견을고려하여 1000 mg/kg 이하를고용량으로설정하는것이바람직하다고판단하였다.

39 TABLE 6. Survival and Body Weights for Rats in the 14 Days Gavage Study of 20 nm, Negative Charged ZnO Dose (mg/kg) al a Surviv Mean Body Weight b (g) Initial Final Change Male Negative control Ve h ic le control 2/ ± ± ± 8.1 5/ ± ± ± / ± ± ± / ± ± ± / ± ± 20.2* 49.4 ± 6.1 Female Negative control Ve h ic le control 2/ ± ± ± 6.7 5/ ± ± ± / ± ± ± / ± ± ± / ± ± ± 6.9 * Significantly different (P<0.05) from the vehicle control group by Scheffe's test a Number of animals surviving at day 90/number initially in group b Weights and weight changes are given as mean ± standard error

40 MEAN BODY WEIGHT IN GRAMS G1-0 mg/kg G2-0 mg/kg G3-500 mg/kg G mg/kg G mg/kg DAYS ON STUDY FIGURE 4 Growth Curves for Male Rats Administered 20 nm, Negative Charged ZnO by Gavage for 14 Days MEAN BODY WEIGHT IN GRAMS G1-0 mg/kg G2-0 mg/kg G3-500 mg/kg G mg/kg G mg/kg DATS ON STUDY FIGURE 5 Growth Curves for Female Rats Administered 20 nm, Negative Charged ZnO by Gavage for 14 Days

41 TABLE 7. Absolute and retative organ weight for Male Rats in the 14 Days Gavage Study of 20 nm, Negative Charged ZnO * Negative Vehicle 500 mg/kg 1000 mg/kg n** Male Liver Absolute Relative Kidney (Left) Absolute Relative Kidney (Right) Absolute Relative Spleen Absolute Relative Brain Absolute Relative 8.26 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± mg/kg 7.08 ± ± ± ± ± ± ± ± ± ± 0.10 Heart 1.19 ± 1.24 ± 1.11 ± 0.97 ± 0.85 ± Absolute d Relative 0.44 ± 0.43 ± 0.39 ± 0.38 ± 0.37 ± c 0.03 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weights/g body weight (mean ± standard error) ** Number of animals Significantly different from negative control (a:p<0.05, b:p<0.01) and vehicle control (c:p<0.05, d:p<0.01) Scheffe's test

42 TABLE 7. Absolute and retative organ weight for Female Rats in the 14 Days Gavage Study of 20 nm, Negative Charged ZnO * Negative Vehicle 500 mg/kg 1000 mg/kg n** Female Liver Kidney (Left) Kidney (Right) Spleen Brain Heart Absolute Relative Absolute Relative Absolute Relative Absolute Relative Absolute Relative Absolute Relative 5.17 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.08a 0.86 ± ± ± ± ± ± ± ± ± ± mg/kg 6.35 ± ± 0.09 b 0.91 ± ± ± ± ± ± ± ± 0.10 c 0.78 ± ± 0.05 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weights/g body weight (mean ± standard error) ** Number of animals Significantly different from negative control (ap<0.05, bp<0.01) and vehicle control (cp<0.05, dp<0.01) Scheffe's test

43 1. 시험방법 1.1 시험물질시험물질을칭량하여최종농도가 500 mg/ml인고용량이되도록 HEPES-citrate buffer 를가해조제하였다. 그리고멸균증류수를이용하여중용량군과저용량군이되도록각각 250 mg/ml과 125 mg/ml이되도록 vortex mixer를이용하여 1분간균질화조제하였다. 조제한시험물질은투여전에 30초간 vortex로흔들어균질화시켰다. 각군별시험물질의조제는투여기간동안매일조제하였다. 조제물의분석은 ICP-AES (HORIBA JOBIN YVON ULTIMA2, France) 로수행하였다. 안정성및균질성분석은 ICP를이용한 ZnO formulation의분석법검증 (Study No.: TBH-1367) 에서실시하였으며, 1 mg/ml 및 100 mg/ml의조제물은균질하였으며, 실온 24시간동안안정하였다. 조제시험물질의함량측정은 1일과 45일및 90일의조제물에대해투여전각각 1회씩실시한결과, 1일차는 %, % 및 %, 45일차는 %, % 및 %, 90일차는 %, % 및 % 로모두 100 ± 15 % 이내로적합하였다. 1.2 실험동물및시험계실험동물은 SPF 랫드를 (Crl:CD(SD), 오리엔트바이오, 경기도가평군, Korea) 사용하였다. 동물을구매한후약 7 ~ 8일동안헬스케어연구소의동물사육실의환경하에서검역및순화시키면서일반건강상태를관찰한후건강한개체를선별하여시험에사용하였다. 본연구는한국화학융합시험연구원헬스케어연구소의실험동물의사용과관리에관한규정 ( 실험동물윤리위원회개정, ) 을준수하여실시하였다. 순화기간을거친후각군간평균체중및표준편차가균일하도록군분리를실시하였다. 개체식별은유성매직을이용하여각동물의꼬리에표시하였다. 사육상자에시험번호, 시험물질명, 시험항목, 입수일, 순화기간, 군분리일, 시험기간,

44 성 / 동물번호및시험책임자를기재한개체식별카드를부착하여식별하였 다. 사료는방사선 (2.0 Mrad) 멸균된쥐사료 ( 카길애그리퓨리나, 경기도 평택시, Korea) 를자유섭취시켰다. 음수는 UV 조사된여과수를폴리카보 네이트제음수병 (500 ml) 으로자유섭취시켰다. 투여시 6 주령동물을사용하였으며온도 21.1 ~ 23.0, 습도 48.9 ~ 60.8 %, 환기횟수 10 ~ 15 회 / 시간, 명암주기 ( 조명시간 ) 12 시간 / 일 ( 오전 8 시 ~ 오후 8 시 ), 조도는 150 ~ 300 Lux, 스텐인레스제사육상자 (270 W 500 D 200 H (mm)) 에케이지당 2 마리씩사육하였다. 순화기간을거친 후각군간평균체중및표준편차가균일하도록군분리를실시하였다. 앞서수행한 14 일반복경구투여용량결정시험의결과를바탕으로 2000, 1000 mg/kg 용량에서유의할만한영향이관찰되어, 본시험에서는 500 mg/kg 을고용량투여군으로선정한후공비 2 로하여 250 mg/kg 을중 용량투여군, 125 mg/kg 의저용량투여군을각각설정하였다. 대조군으로는멸균증류수를투여하는음성대조군과부형제를투여하는 부형제대조군을설정하였다. 또한주시험군외에독성반응의지속성및회 복성을평가하기위하여음성대조군, 부형제대조군과고용량군에는회복 군 ( 암수각각 5 마리씩 ) 및조직분포시험군 ( 암수각각 2 마리 ) 을두며, 회복 기간은투여종료후 2 주간더관찰한후부검을수행하였다 (Table 8). TABLE 8. Experimental Design in the 90-Day Study 군 투여용량 투여액량 수컷 동물수 개체번호 암컷 G1 G2 HEPES-citrate buffer G3 ZnO(-) G4 ZnO(-) G5 ZnO(-) Note * : Groups include ten animals for the primary 90-day oral toxicity study, five animals for the 14-day recovery study, and two animals for the organ distribution study.

45 1.3 투여, 관찰및사료, 음수측정, 감각반응및기능평가투여경로는경구로투여하였다. 조제된시험물질은투여하는동안 Vortex 하면서경구투여용주사기 ( 존데 ) 를이용하여위내에강제투여하였다. 투여개시직전의체중을측정하고, 주 1회체중을측정하여이를기준으로투여액량을계산하였다. 투여액량은 10 ml/kg 로산출하였다. 모든시험동물에대하여 7일 / 주, 1회 / 일로하여 90일간투여하였다. 전동물에대하여시험기간동안당일투여후매일 1회이상씩일반소견의변화, 독성소견및폐사의유무를관찰하였다. 주시험모든동물에대하여간대성및강직성운동, 반복적형태 (grooming의항진, 반복적인 circling) 또는이상한행동, 공격성, 운동협응, 걸음걸이, 자세와취급시의반응변화를 1주일에 1회씩아래항목별로관찰하였다. gait hyperthemia / hypothermia tremors skin colour twitches respiration convulsions palpebral closure bizarre behaviour urination salivation defecation pilo-erection transfer arousal exophthalmia tail elevation lachrymation 주시험모든동물에대하여감각반응평가를시험물질투여종료시점에 아래항목별로측정하였다.

46 grasp response touch escape vocalisation pupil reflex toe pinch blink reflex tail pinch finger approach 주시험대조군및고용량군에서암수각 5마리의동물을선정하여시험물질투여종료시점에랫드용악력측정장치 (Grip strength, 1027 CSX, Columbus Instrument, USA) 를이용하고, 앞다리및뒷다리에대해 3회측정해, 각각평균을측정하였다. 그리고자발운동량관찰시스템 (Motor activity, Columbus Instrument, USA) 을이용하여자발운동량을측정하였다. 체중은입수시와군분리할때를포함하여투여개시후매주 1회, 측정하였다. 사료및음수섭취량은투여개시후매주 1회측정하였다. 측정방법은사료및음수를급여한후익일에잔량을측정하여마리당평균섭취량 (g/rat/day) 을산출하였다. 1.4 요검사, 안검사, 혈액학적, 혈액생화학적검사주시험각군마다암 수각 5 마리씩에대해서는투여최종주에 Multistix 10 SG (Siemens, USA) 스트립과요측정기 (Clinitek 500, USA) 를이용하여아래 Table 9와같이요검사를실시하였다. 대조군과고용량투여군의암 수각 5마리씩에대해서는대사케이지를이용하여 3시간동안채뇨한신선뇨에대해서는아래 Table 10과같은검사를실시하였고, 요량은 24시간동안채뇨한축뇨를이용하여측정하였다. 1.5 ml Micro tube에담겨진요를 Micro 고속원심분리기를이용하여 1500 rpm / 5 min 동안원심분리하였다. 상층액은버리고요침사염색시약 (Jorvet Urine Sediment Stain / Jorgensen Laboratories, Inc. / USA) 을이용하여염색하였다. 염색된요침사를 micro pipette으로슬라이드에떨어뜨린후 cover slip으로덮고현미경으로검경하였다. 대조군과고용량투

47 여군의암 수각 5마리씩에대해서는투여최종주에산동제 (Ocutropine, Samil pharm CO., LTD, Korea) 를이용하여동공을확장시킨후, 안저카메라 (Genesis, Kowa, Japan) 를이용하여안저를관찰하였다. 혈액학적검사를위해채혈전에전날절식시킨동물을 Isoflurane으로마취한다음복대동맥으로부터채혈한혈액중약 3 ml의혈액은 EDTA가함유된 CBC bottle (EDTA 3K, BD vacutainer) 에취하여자동혈구측정기 (ADVIA 120E, Siemens, USA) 를이용하여측정하였고혈액응고시간의측정은채혈한후 sodium citrate가함유된 vacutainer (9NC sodium citrate, BD vacutainer) 에취하여원심분리 (3000 rpm, 10분 ) 하여얻은혈장을자동응고시간측정기 (ACL 7000, Instrumentation Laboratory, USA) 를이용하여측정하였다 (Table 11). 혈액생화학적검사는채혈한혈액중혈액검사와혈액응고검사에사용된혈액을제외한나머지의혈액을실온에서응고시킨다음원심분리 (3000 rpm, 10분 ) 하여혈청을취한후자동혈청분석기 (HITACHI 7060, HITACHI, Japan) 와전해질분석기 (EasyLyte PLUS Na/K/Cl Analyzer, MEDICA USA) 를이용하여측정하였다 (Table 12).

48 비중 (specific gravity) - ph - 백혈구 (white blood cell) - 단백질 (protein) 당 (glucose) 케톤체 (ketone body) 유로빌리노젠 (urobilinogen 빌리루빈 (bilirubin) mg/dl mg/dl mg/dl E.U./dL mg/dl Multistix 10G (Siemens, USA) 요측정기 (Clinitek 500, USA) 잠혈 (occult blood) - 아질산 (nitrite) - 색상및투명도 (color) - 육안관찰 요량 (urine amount) ml 메스실린더측정 적혈구 (red blood cell, RBC) - 백혈구 (white blood cell, WBC) - 상피세포 (epithelial cell) - 원주체 (cast) - 결정체 (crystal) -

49 TABLE 11. Hematological Analysis in the 90-Day Study 총백혈구수 (total leucocyte count) 감별백혈구수 (differential leucocyte count) 호중구 (neutrophil) 10 3 cells/μl 레이저광학법 림프구 (lymphocyte) 단핵구 (monocyte) % 레이저광학법 호산구 (eosinophil) 호염기구 (basophil) 총적혈구수 (total erythrocyte count) 혈색소량 (hemoglobin concentration) 10 6 cells/μl g/dl 레이저광학법 cyanmethaemoglobin 측정 헤마토크리트치 (hematocrit) % RBC MCV/10 평균적혈구용적 (mean cell volume) fl 레이저광학법 평균적혈구헤모글로빈량 (mean cell hemoglobin) 평균적혈구헤모글로빈농도 (mean cell hemoglobin concentration) pg g/dl Hb/RBC 10 Hb/(RBC MCV) 1000 망상적혈구비율 (reticulocyte) % 레이져광학법 혈소판수 (platelet ) 10 3 cells/μl 레이져광학법 프로트롬빈시간 (prothrombin time) secs 광산란검출방식 부분활성트롬보플라스틴시간 (activated partial thromboplastin time) secs 광산란검출방식

50 TABLE 12. Blood Biochemical Analysis in the 90-Day Study

51 1.5 부검, 장기중량, 조직병리학적검사 시험종료시 ( 시험이종료될때 ) 생존동물은이소플루란으로마취하에채 혈하고안락사시킨다음체표및모든체공, 두개강, 흉강및복강의모든 장기에대하여육안적검사를실시하였다. 주시험모든동물에대하여모 든동물에대해부검할때육안적검사를실시한후에다음장기에대한 습장기의중량을측정하고, 절식한동물의체중에대한상대장기중량을측 정하였다. 좌우가있는장기는좌우를합하여무게를측정하였다. 조직병리학적검사는모든투여군을개체별로다음의장기를적출하여 10% 중성완충포르말린용액 (neutral buffered formalin) 에고정하였으며, 안구는 Davidson 액에, 고환및부고환은 Bouin 액에고정하였다. 조직병 리학적검사는대조군과고용량군에대해서만실시하였다. 단, 고용량에서 이상이발견된경우전군에대해서병리조직학적검사를실시하였다.

52 1.6 독성동태시험투여개시일, 투여 28일및 90일차에투여전 (0, blank), 투여후 0.5, 1, 2, 4, 6, 10, 24시간까지혈액을채취하였다. 독성동태시험의분석은각군당시간대별로암수각 3마리에대해실시하였다. 투여군은군당처음 3마리는투여전, 2, 10시간에채혈하였다. 다음 3마리는 0.5, 4, 24시간에, 다음 3 마리는 1, 6 시간에채혈하였다. 채혈은미정맥으로부터채혈하였고, 1 ml 주사기 (25G) 로약 1mL의혈액을채취하여헤파린처리된 1.5 ml microtube 에넣어빙냉상태로유지하고, rpm으로원심분리하여혈장을분리하였다. 혈장은 1.5 ml microtube 2개로나누어분석시까지 -80 에냉동보관하였다. 독성동태시험군용동물은 90일째채혈이종료된후안락사

53 하였다. Plasma의경우측량후일정부피의 vial에넣고, 질산 3 ml를넣어분해하고, 과산화수소 1 ml을첨가하여 160 에서가열분해후, 5 ml의 3차증류수에희석하여 0.45 μm filtering하여 ICP-AES (Inductively coupled plasma atomic emission spectroscopy) 로서울여대에서분석하였다. 독성동태해석은시간별혈중농도데이터에근거한비모델해석법을사용하였다. 최고혈중농도 (Cmax), 최고혈중농도도달시간 (Tmax), 24시간까지의시간별혈중농도곡선하면적 (AUC) 및소실기의속도상수로부터구한반감기 (t1/2) 등의체내독성동태파라미터를 Kinetica (Thermo Electron Co., USA) 프로그램을이용하여산출하였다. 1.7 조직분포시험부검전에주사기로미정맥에서약 1 ml의혈액을채혈하였고, 일정량의변을채취하였다. 부검후, 적출한뇌, 간, 신장, 정소, 난소, 비장, 폐, 위장, 소장, 대장에대해지방을제거하고생리식염수로세정한후중량을측정하여 50 ml의튜브에넣어드라이아이스가담긴아이스박스에임시보관하였고, 측정이전에전처리과정을실시하였다. 측정장비는 ICP-AAS (HORIBA JOBIN YVON ULTIMA2, JAPAN) 및 Balance (Satorius, ME254S, German) 를사용하여분석하였다. 1.8 자료의통계처리체중측정, 사료섭취량측정, 음수섭취량측정, 혈액학적검사, 혈액생화학적검사및장기중량측정의자료에대해서는음성대조군및부형제대조군과의분산의동질성을평가하기위해 Levene's 검사를실시한후, one way ANOVA 검사를하여유의성검정을실시하였다. 분산의동질성및시험군간유의성이인정된경우, 사후검정으로 Scheffe 다중검정을실시하였고, 분산이이질적이며시험군간유의성이인정된경우사후검정으로 Dunnett's T3 다중검사를실시하였다. 모든통계분석은 SPSS (Ver 19.0) 통계프로그램을이용하여수행하였다. 조직병리학적검사결과에대한자료

54 는음성대조군및부형제대조군과의유의성을검사하는비모수검정법인 Fisher s exact test 를실시하였다.

55 2. 결과 2.1 사망률및일반관찰, 체중, 감각반응, 기능평가시험기간동안암 수모든시험물질투여군에서빈사및폐사동물은관찰되지않았다. 일반소견관찰결과, 시험물질투여와관련된일반소견의변화는투여직후에관찰된유연소견이었다. 암수시험물질투여군의유연소견은 125 mg/kg 투여군에서투여 33일차부터관찰되다가 63일차부터투여종료시까지관찰되었고, 250 mg/kg 투여군에서투여 25일차부터투여종료시까지관찰되었다. 500 mg/kg 투여군에서투여 14일차부터관찰되어투여종료시까지빈도가높게관찰되었다. 그밖에관찰된소견으로는입모소견이암수투여군에서투여 8일차부터관찰되었고, 탈모소견은투여 13일차부터시험종료일까지관찰되었다. 또한, 일부시험물질투여군동물에서는연변, 설사, 상처, 구강주위오염, 피모오염및안구혼탁소견이산발적으로관찰되었다 (Table 13). 시험기간동안주 1회행동평가를실시한결과, 특이할만한이상행동은관찰되지않았다. 체중변화시험기간중생존동물에대한체중변화에있어서는암수의시험물질투여군에서대조군과비교하여통계학적으로유의성있는변화는나타나지않았다 (Table 14, Figures 6 and 7).

56 Table 13. Clinical Signs for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO Negative Vehicle 125 mg/kg 250 mg/kg n a mg/kg Male Mortality b Clinical signs Piloerection Salivation Fur loss Wound Opacity of eyeball Abdominal distension Diarrhea Staining around mouth Female Mortality b Clinical signs Piloerection Salivation Soft stool Fur loss Wound Soiled fur a b Number of animals Number of dead animals/number of tested animals

57 TABLE 14 Survival and Body Weights of Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO Final weight c Mean Body Weight b (g) Relative to Dose Survival a Negative (mg/kg) Initial Final Change s (%) Male Negative control 15/15 d ± ± ± 13.7 Vehicle control 15/15 d ± ± ± / ± ± ± / ± ± ± /15 d ± ± ± a b c d Female Negative control 15/15 d ± ± ± 5.5 Vehicle control 15/15 d ± ± ± / ± ± ± / ± ± ± /15 d ± ± ± Number of animals surviving at day 90/number initially in group Weights and weight changes are given as mean ± standard error Day 90 Main study group + Recovery Groups

58 700 MEAN BODY WEIGHTS IN GRAMS G1 (0 mg/kg) G2 (0 mg/kg) G3 (125 mg/kg) G4 (250 mg/kg) G5 (500 mg/kg) WEEKS ON STUDY FIGURE 6 Growth Curves for Male Rats Administered 20 nm, Negative Charged ZnO by Gavage for 90 Days 450 MEAN BODY WEIGHTS IN GRAMS G1 (0 mg/kg) G2 (0 mg/kg) G3 (125 mg/kg) G4 (250 mg/kg) G5 (500 mg/kg) WEEKS ON STUDY FIGURE 7 Growth Curves for Female Rats Administered 20 nm, Negative Charged ZnO by Gavage for 90 Days

59 감각반응및기능평가시험물질투여마지막주에감각반응을평가한결과, 특이할만한이상반응은관찰되지않았다 (TABLE 15). 대조군과고용량군에대해서악력및운동량을평가한결과특이할만한변화는관찰되지않았다 (TABLE 16). 사료섭취량측정수컷의경우 125 mg/kg 투여군에서투여 6, 9, 12 및 13주차에통계학적으로유의한증가가관찰되었고, 250 mg/kg 투여군에서는투여 5, 8, 9, 10, 11, 12 및 13주차에통계학적으로유의한증가가관찰되었다. 500 mg/kg 투여군에서는투여 3, 4, 5, 6, 8, 9 및 13주차에또한회복군그룹에서 1 및 2주차에통계학적으로유의한증가가관찰되었다. 암컷의경우 250 mg/kg 투여군에서투여 1주차에통계학적으로유의한증가가관찰되었고, 500 mg/kg 투여군에서는투여 1주차부터 13주차까지또한회복군에서 2주차에통계학적으로유의한증가가관찰되었다 (Table 17). 음수섭취량측정시험기간동안음수섭취량에서는수컷의경우 125 mg/kg 투여군에서투여 4주차에통계학적으로유의한증가가관찰되었고, 250 mg/kg 투여군에서는투여 3 및 4주차에통계학적으로유의한증가가관찰되었다. 500 mg/kg 투여군에서는투여 1, 2, 3, 4 및 12주차에또한회복군그룹에서 2 주차에통계학적으로유의한증가가관찰되었다. 암컷의경우 500 mg/kg 투여군에서투여 1, 2, 3, 4 및 5주차에또한회복군그룹에서 2주차에통계학적으로유의한증가가관찰되었다 (Table 18). 요검사 모든투여군에서특이한변화는관찰되지않았다 (Table 19~22).

60 TABLE 15 Individual Sensory Reactivity Assessments Data for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO* Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Male Grasp response 1.2 ± ± ± ± ± 0.8 Vocalisation 0.7 ± ± ± ± ± 1.0 Toe pinch 5.2 ± ± ± ± ± 1.0 Tail pinch 0.6 ± ± ± ± ± 0.0 Finger approach 1.8 ± ± ± ± ± 1.0 Touch escape 0.8 ± ± ± ± ± 1.3 Pupil reflex 4.0 ± ± ± ± ± 0.0 Blink reflex 4.0 ± ± ± ± ± 0.0 Female Grasp response 3.0 ± ± ± ± ± 1.0 Vocalisation 1.4 ± ± ± ± ± 0.3 Toe pinch 4.4 ± ± ± ± ± 2.2 Tail pinch 0.8 ± ± ± ± ± 0.8 Finger approach 2.0 ± ± ± ± ± 0.6 Touch escape 2.6 ± ± ± ± ± 1.7 Pupil reflex 4.0 ± ± ± ± ± 0.0 Blink reflex 4.0 ± ± ± ± ± 0.0 * Data are given as mean ± standard error a Number of animals

61 TABLE 16 Individual and Group Mean Functional Performance Test Values and Standard Deviations for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO* Main Study Negative Vehicle 500 mg/kg na Male Grip strength (kg) Forelimb ± ± ± Hindlimb ± ± ± Motor activity Total Movements 77 ± ± ± 10 Total Movement Time (second) 431 ± ± ± 129 Ambulatory Move Time (second) 330 ± ± ± 141 Ambulatory Distance (cm) 1142 ± ± ± 705 Female Grip strength (kg) Forelimb ± ± ± Hindlimb ± ± ± Motor activity Total Movements 75 ± 9 76 ± ± 4 Total Movement Time (second) 470 ± ± ± 22 Ambulatory Move Time (second) 384 ± ± ± 29 Ambulatory Distance (cm) 1938 ± ± ± 202 * Data are given as mean ± standard error a Number of animals

62 TABLE 17 Feed Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO * Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n ** Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.90 a,c ± ± ± ± ± 2.32 a,c ± ± ± ± 1.99 b,d ± 1.53 b,d ± ± ± 3.11 c ± ± 3.31 c ± ± ± ± ± ± ± ± ± 2.58 c ± 1.14 c ± ± ± 3.21 a,c ± 2.44 a,c ± 4.58 a,c ± ± ± ± 2.10 a ± ± ± ± ± 4.08 a,c ± ± ± ± 2.81 a ± 3.35 a ± ± ± ± 4.30 a ± 2.93 a ± 1.82 a ± ± ± 2.33 a,c ± ± ± 1.77 a,c WEEK Female ± ± ± ± 1.05 a,b ± 2.46 a,c ± ± ± ± ± 1.90 b,d ± ± ± ± ± 1.10 a,c ± ± ± ± ± 2.99 a ± ± ± ± ± 1.96 a,c ± ± ± ± ± 2.39 b,d ± ± ± ± ± 3.59 a ± ± ± ± ± 2.13 a,c ± ± ± ± ± 2.48 b,d ± ± ± ± ± 2.28 c ± ± ± ± ± 2.18 a ± ± ± ± ± 3.56 b,d ± ± ± ± ± 1.33 b,d ± ± ± ± ± c ± 3.04 Significantly different from negative control ( a P<0.05, b P<0.01) and vehicle control ( c P<0.05, d P<0.01) Scheffe's test WEEK * Data are given as mean ± standard error ** Number of animals

63 TABLE 18 Water Consumptions for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO * Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n ** Male ± ± ± ± ± b,d ± ± ± ± ± b,d ± ± ± ± 4.70 a,c ± 8.06 a,c ± ± ± ± ± ± ± ± a ± a ± a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 8.42 a,c ± ± ± ± ± ± ± ± ± ± ± 2.90 a Female ± ± ± ± ± 4.92 b,d ± ± ± ± ± 7.83 b,d ± ± ± ± ± 6.85 b,d ± ± ± ± ± a ± ± ± ± ± a,c ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.60 b Significantly different from negative control ( a P<0.05, b P<0.01) and vehicle control ( c P<0.05, d P<0.01) Scheffe's test WEEK WEEK * Data are given as mean ± standard error ** Number of animals

64 TABLE 19 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Male Volume (ml) 18.2 ± ± ± 5.8 Color Yellow Red blood cell, RBC White blood cell, WBC Epithelial cell Cast Crystal ~ ~ Specific gravity ± ± ± ± ± Glucose (mg/dl) Negative Bilirubin (mg/dl) Negative Ketone body (mg/dl) Negative Blood (Ery/uL) Negative Trace

65 TABLE 19 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a ph Protein (mg/dl) Negative Trace Urobilinogen (E.U./dL) Nitrite Negative White blood cell(leu/ul) Negative a Number of animals

66 TABLE 20 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Female Volume (ml) 12.2 ± ± ± 2.8 Color Yellow Red blood cell, RBC White blood cell, WBC Epithelial cell ~ Cast Crystal ~ ~ Specific gravity ± ± ± ± ± Glucose (mg/dl) Negative Bilirubin (mg/dl) Negative Ketone body (mg/dl) Negative Blood (Ery/uL) Negative Trace a Number of animals

67 TABLE 20 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a ph Protein (mg/dl) Negative Trace Urobilinogen (E.U./dL) Nitrite Negative White blood cell(leu/ul) Negative a Number of animals

68 TABLE 21 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a Male Volume (ml) 18.4 ± ± ± 2.6 Color Yellow Red blood cell, RBC White blood cell, WBC Epithelial cell Cast Crystal ~ ~ Specific gravity ± ± ± Glucose (mg/dl) Negative Bilirubin (mg/dl) Negative Ketone (mg/dl) Negative Blood (Ery/uL) Negative Trace a Number of animals

69 TABLE 21 Urinalysis Results for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a ph Protein (mg/dl) Negative Trace Urobilinogen (E.U./dL) Nitrite Negative White blood cell(leu/ul) Negative a Number of animals

70 TABLE 22 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a Female(continued) Volume (ml) 11.6 ± ± ± 2.7 Color Yellow Red blood cell, RBC White blood cell, WBC Epithelial cell Cast Crystal ~ ~ Specific gravity ± ± ± Glucose (mg/dl) Negative Bilirubin (mg/dl) Negative Ketone (mg/dl) Negative Blood (Ery/uL) Negative a Number of animals

71 TABLE 22 Urinalysis Results for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a ph Protein (mg/dl) Negative Trace Urobilinogen (E.U./dL) Nitrite Negative White blood cell(leu/ul) Negative a Number of animals

72 수컷 500 mg/kg 투여군 1례에서안구혼탁소견이관찰되었고, 그외특이한이상소견은관찰되지않았다. 수컷 250 mg/kg 투여군에서흉선결절이 1례, 500 mg/kg 투여군에서고환위축및정낭응고선위축이 1례가관찰되었고, 암컷음성대조군에서는우측부신에검은반점이 1례가, 500 mg/kg 투여군에서는간의외측좌엽의갈색변화 1례가관찰되었다 (Table 23 and 24). 절대장기중량에서는암수의시험물질투여군에서대조군과비교하여통계학적으로유의성있는변화는나타나지않았으나, 수컷 500 mg/kg 회복군에서악하선중량이대조군에비해통계학적으로유의하게 (P<0.05) 증가하였다 (Table 7). 상대장기중량에서는수컷의경우 500 mg/kg 투여군에서부신중량이대조군에비해통계학적으로유의하게증가하였고, 암컷의경우 500 mg/kg 회복군에서난소및자궁중량이통계학적으로유의하게증가하였다 (Table 25~28).

73 TABLE 23 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study(Main group) of 20 nm, Negative Charged ZnO Negative Vehicle 125 mg/kg 250 mg/kg n a mg/kg Male External No gross findings Internal No gross findings Mass in thymus Testicular atrophy Seminal vesicular atrophy Female External No gross findings Internal No gross findings Black spot in right adrenal gland Brown discoloration in left lateral lobe of liver a Number of animals

74 TABLE 24 Summary Incidence of Necropsy Findings Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg na Male External No gross findings Internal No gross findings Female External No gross findings Internal No gross findings a Number of animals

75 TABLE 25 Absolute and retative organ weight for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Male Necropsy body wt ± ± ± ± ± 58.7 Liver Absolute ± ± ± ± ± Relative 2.51 ± ± ± ± ± 0.12 Kidney Absolute 3.33 ± ± ± ± ± 0.43 Relative 0.65 ± ± ± ± ± 0.05 Spleen Absolute 0.78 ± ± ± ± ± 0.08 Relative 0.16 ± ± ± ± ± 0.02 Adrenal gland Absolute ± ± ± ± ± Relative ± ± ± ± ± b Testis Absolute 3.28 ± ± ± ± ± 0.70 Relative 0.64 ± ± ± ± ± 0.18 Brain Absolute 2.27 ± ± ± ± ± 0.12 Relative 0.45 ± ± ± ± ± 0.08 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a Number of animals b Significantly different (P<0.05) from the vehicle control group by Scheffe's test

76 TABLE 25 Absolute and retative organ weight for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Male Pituitary gland Absolute ± ± ± ± ± Relative ± ± ± ± ± Lung Absolute 1.75 ± ± ± ± ± 0.13 Relative 0.34 ± ± ± ± ± 0.03 Heart Absolute 1.63 ± ± ± ± ± 0.16 Relative 0.32 ± ± ± ± ± 0.02 Thymus Absolute 0.41 ± ± ± ± ± 0.11 Relative 0.08 ± ± ± ± ± 0.02 prostate gland Absolute 0.75 ± ± ± ± ± 0.26 Relative 0.15 ± ± ± ± ± 0.04 Submaxillary gland Absolute 0.86 ± ± ± ± ± 0.11 Relative 0.17 ± ± ± ± ± 0.03 Epididymis Absolute 1.45 ± ± ± ± ± 0.24 Relative 0.29 ± ± ± ± ± 0.04 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a Number of animals

77 TABLE 26 Absolute and retative organ weight for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Female Necropsy body wt ± ± ± ± ± 20.7 Liver Absolute 6.98 ± ± ± ± ± 0.48 Relative 2.39 ± ± ± ± ± 0.16 Kidney Absolute 1.82 ± ± ± ± ± 0.15 Relative 0.63 ± ± ± ± ± 0.04 Spleen Absolute 0.50 ± ± ± ± ± 0.09 Relative 0.17 ± ± ± ± ± 0.03 Adrenal gland Absolute ± ± ± ± ± Relative ± ± ± ± ± Ovary Absolute ± ± ± ± ± Relative ± ± ± ± ± Brain Absolute 1.99 ± ± ± ± ± 0.06 Relative 0.68 ± ± ± ± ± 0.05 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a Number of animals b Significantly different (P<0.05) from the vehicle control group by Scheffe's test

78 TABLE 26 Absolute and retative organ weight for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Female Pituitary gland Absolute ± ± ± ± ± Relative ± ± ± ± ± Lung Absolute 1.17 ± ± ± ± ± 0.08 Relative 0.40 ± ± ± ± ± 0.03 Heart Absolute 1.02 ± ± ± ± ± 0.07 Relative 0.35 ± ± ± ± ± 0.02 Thymus Absolute 0.29 ± ± ± ± ± 0.06 Relative 0.10 ± ± ± ± ± 0.02 Uterus Absolute 0.73 ± ± ± ± ± 0.34 Relative 0.25 ± ± ± ± ± 0.15 Submaxillary gland Absolute 0.51 ± ± ± ± ± 0.06 Relative 0.18 ± ± ± ± ± 0.02 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a Number of animals

79 Recovery Groups Negative Vehicle 500 mg/kg n a Male Necropsy body wt ± ± ± 58.7 Liver Absolute ± ± ± 1.33 Relative 2.29 ± ± ± 0.26 Kidney Absolute 3.28 ± ± ± 0.32 Relative 0.61 ± ± ± 0.09 Spleen Absolute 0.77 ± ± ± 0.09 Relative 0.14 ± ± ± 0.02 Adrenal gland Absolute ± ± ± Relative ± ± ± Testis Absolute 3.3 ± ± ± 0.29 Relative 0.64 ± ± ± 0.03 Brain Absolute 2.21 ± ± ± 0.04 Relative 0.41 ± ± ± 0.05 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a Number of animals b TABLE 27 Absolute and retative organ weight for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO* Significantly different (P<0.05) from the vehicle control group by Scheffe's test

80 TABLE 27 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO* Recovery Groups Negative Vehicle 500 mg/kg n a Male Pituitary gland Absolute ± ± ± Relative ± ± ± Lung Absolute 1.79 ± ± ± 0.08 Relative 0.34 ± ± ± 0.04 Heart Absolute 1.59 ± ± ± 0.24 Relative 0.30 ± ± ± 0.04 Thymus Absolute 0.33 ± ± ± 0.09 Relative 0.06 ± ± ± 0.02 Prostate gland Absolute 0.88 ± ± ± 0.14 Relative 0.17 ± ± ± 0.03 Submaxillary gland Absolute 0.82 ± ± ± 0.06b Relative 0.15 ± ± ± 0.02 Epididymis Absolute 1.54 ± ± ± 0.16 Relative 0.29 ± ± ± 0.02 * a b Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) Number of animals Significantly different from negative control (P<0.05) by Scheffe's test

81 TABLE 28 Absolute and relative organ weight for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO * Recovery Groups Negative Vehicle 500 mg/kg n a Female Necropsy body wt ± ± ± 24.0 Liver Absolute 7.48 ± ± ± 0.55 Relative 2.54 ± ± ± 0.12 Kidney Absolute 1.96 ± ± ± 0.25 Relative 0.67 ± ± ± 0.08 Spleen Absolute 0.57 ± ± ± 0.09 Relative 0.19 ± ± ± 0.02 Adrenal gland Absolute ± ± ± Relative ± ± ± Ovary Absolute 0.09 ± ± ± Relative ± ± ± Brain Absolute 2.00 ± ± ± 0.10 Relative 0.68 ± ± ± 0.07 * a b Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) Number of animals Significantly different from negative control (P<0.05) by Scheffe's test

82 TABLE 28 Absolute and relative organ weight for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO * Recovery Groups Negative Vehicle 500 mg/kg n a Female Pituitary gland Absolute ± ± ± Relative ± ± ± Lung Absolute 1.34 ± ± ± 0.31 Relative 0.46 ± ± ± 0.10 Heart Absolute 1.10 ± ± ± 0.15 Relative 0.37 ± ± ± 0.04 Thymus Absolute 0.31 ± ± ± 0.09 Relative 0.10 ± ± ± 0.03 Uterus Absolute 0.73 ± ± ± 0.22 Relative 0.25 ± ± ± 0.05 Submaxillary gland Absolute 0.55 ± ± ± 0.04 Relative 0.19 ± ± ± 0.01 * Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weights (relative weights) are given as mg organ weighs/g body weight (mean ± standard error) a b Number of animals Significantly different from vehicle control (P<0.05) by Scheffe's test

83 수컷의경우대조군과비교시 250 및 500 mg/kg 투여군에서혈색소량, 헤마토크리트치, 평균적혈구용적, 평균적혈구헤모글로빈량및평균적혈구헤모글로빈농도가통계학적으로유의하게감소하였고, 250 mg/kg 투여군에서호산구수치가통계학적으로유의하게증가하였다. 암컷의경우에는대조군과비교시 500 mg/kg 투여군에서혈색소량, 평균적혈구용적, 평균적혈구헤모글로빈량및평균적혈구헤모글로빈농도등이통계학적으로유의하게감소하였고, 총적혈구수수치는통계학적으로유의하게증가하였다 (Table 29~32). 회복군에서혈색소량과평균적혈구용적은정상수치를보였고, 총적혈구수, 헤마토크리트치, 평균적혈구헤모글로빈량및평균적혈구헤모글로빈농도는회복되지않았다. 수컷의경우대조군과비교시 250 및 500 mg/kg 투여군에서총단백질및알부민수치가통계학적으로유의하게 (P<0.05) 감소하였고, 500 mg/kg 투여군에서는크레아틴키나아제수치가통계학적으로유의하게 (P<0.05) 증가하였다. 암컷의경우에는대조군과비교시 250 및 500 mg/kg 투여군에서총단백질및알부민수치가통계학적으로유의하게 (P<0.05) 감소하였다 (Table 33~36). 이들수치는회복군에서모두회복되었다.

84 TABLE 29 Hematology Data of Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n** Male Total white cells/ul) blood cell count ( ± ± ± ± ± 1.80 Neutrophils (%) 17.5 ± ± ± ± ± 5.8 Lymphocytes (%) 78.7 ± ± ± ± ± 6.2 Monocytes (%) 1.8 ± ± ± ± ± 0.6 Eosinophils (%) 1.4 ± ± ± ± 1.3a 2.6 ± 1.2 Basophils (%) 0.3 ± ± ± ± ± 0.1 Total erythrocyte (106 cells/μl) 8.52 ± ± ± ± ± 0.73 Hemoglobin concentration (g/dl) 14.7 ± ± ± ± 0.6c 13.9 ± 1.2c Hematocrit (%) 43.7 ± ± ± ± 1.9c 43.1 ± 2.7 Mean cell volume (fl) 51.3 ± ± ± ± ± 2.2c Mean cell hemoglobin (pg) 17.3 ± ± ± ± 0.6a,c 15.3 ± 0.8a,c Mean cell hemoglobin concentration (g/dl) 33.8 ± ± ± ± ± 1.1a,c Reticulocytes (%) 1.71 ± ± ± ± ± 0.49 Platelet (103 cells/ul) 1033 ± ± ± ± ± 220 Prothrombin time (sec) 14.7 ± ± ± ± ± 1.1 Activated partial thromboplastin time (sec) 16.8 ± ± ± ± ± 3.3 Significantly different from negative control (ap<0.05, bp<0.01) and vehicle control (cp<0.05, dp<0.01) Scheffe's test *Data are given as mean ± standard error ** Number of animals

85 TABLE 30 Hematology Data of Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n** Female Total white cells/ul) blood cell count ( ± ± ± ± ± 2.12 Neutrophils (%) 22.0 ± ± ± ± ± 6.0 Lymphocytes (%) 72.7 ± ± ± ± ± 6.6 Monocytes (%) 2.2 ± ± ± ± ± 0.9 Eosinophils (%) 2.0 ± ± ± ± ± 1.1 Basophils (%) 0.2 ± ± ± ± ± 0.1 Total erythrocyte (106 cells/μl) 7.86 ± ± ± ± ± 0.34a,c Hemoglobin concentration (g/dl) 14.1 ± ± ± ± ± 0.3c Hematocrit (%) 40.9 ± ± ± ± ± 1.3 Mean cell volume (fl) 52.0 ± ± ± ± ± 2.1b,d Mean cell hemoglobin (pg) 18.0 ± ± ± ± ± 0.8b,d Mean cell hemoglobin concentration (g/dl) 34.6 ± ± ± ± ± 0.6a,c Reticulocytes (%) 1.97 ± ± ± ± ± 0.33 Platelet (103 cells/ul) 1030 ± ± ± ± ± 149 Prothrombin time (sec) 15.0 ± ± ± ± ± 0.8 Activated partial thromboplastin time (sec) 15.7 ± ± ± ± ± 2.1 Significantly different from negative control (ap<0.05, bp<0.01) and vehicle control (cp<0.05, dp<0.01) Scheffe's test *Data are given as mean ± standard error ** Number of animals

86 TABLE 31 Hematology Data of Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle n** mg/kg Male Total white blood cell count (103 cells/ul) 8.18 ± ± ± 1.87 Neutrophils (%) 17.3 ± ± ± 1.8 Lymphocytes (%) 77.6 ± ± ± 3.9 Monocytes (%) 2.7 ± ± ± 1.7 Eosinophils (%) 1.5 ± ± ± 0.3 Basophils (%) 0.3 ± ± ± 0.1 Total erythrocyte (106 cells/ul) 8.69 ± ± ± 0.46c Hemoglobin concentration (g/dl) 14.4 ± ± ± 0.5 Hematocrit (%) 43.1 ± ± ± 1.5 Mean cell volume (fl) 49.6 ± ± ± 1.7 Mean cell hemoglobin (pg) 16.6 ± ± ± 0.8 Mean cell hemoglobin concentration (g/dl) 33.4 ± ± ± 0.7 Reticulocytes (%) 1.64 ± ± ± 0.46 Platelet (103 cells/ul) 992 ± ± ± 137 Prothrombin time (sec) 14.9 ± ± ± 0.6 Activated partial thromboplastin time (sec) 18.4 ± ± ± 2.0 Significantly different from negative control (a:p<0.05, b:p<0.01) and vehicle control (c:p<0.05, d:p<0.01) Scheffe's test * Data are given as mean ± standard error ** Number of animals

87 TABLE 32 Hematology Data of Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle n** Female Total white blood cell count (103 cells/ul) 500 mg/kg 2.90 ± ± ± 1.18 Neutrophils (%) 18.3 ± ± ± 4.7 Lymphocytes (%) 76.2 ± ± ± 5.2 Monocytes (%) 2.7 ± ± ± 0.5 Eosinophils (%) 2.1 ± ± ± 1.1 Basophils (%) 0.3 ± ± ± 0.1 Total erythrocyte (106 cells/ul) 7.64 ± ± ± 0.46a,c Hemoglobin concentration (g/dl) 13.8 ± ± ± 0.4 Hematocrit (%) 40.4 ± ± ± 1.2a Mean cell volume (fl) 52.9 ± ± ± 2.0 Mean cell hemoglobin (pg) 18.1 ± ± ± 0.8a,c Mean cell hemoglobin concentration (g/dl) 34.2 ± ± ± 0.4c Reticulocytes (%) 1.74 ± ± ± 0.36 Platelet (103 cells/ul) 990 ± ± ± 129 Prothrombin time (sec) 14.9 ± ± ± 0.6 Activated partial thromboplastin time (sec) 15.5 ± ± ± 1.6 Significantly different from negative control (a:p<0.05, b:p<0.01) and vehicle control (c:p<0.05, d:p<0.01) Scheffe's test * Data are given as mean ± standard error ** Number of animals

88 TABLE 33 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Male Total protein (g/dl) 6.2 ± ± ± ± 0.4b,c 5.6 ± 0.3b,c Albumin (g/dl) 2.5 ± ± ± ± 0.1b,c 2.4 ± 0.1 A/G ratio 0.7 ± ± ± ± ± 0.0 Total bilirubin (mg/dl) 0.05 ± ± ± ± ± 0.02 Alkaline phosphatase (U/L) 297 ± ± ± ± ± 272 Aspartate aminotransferase (U/L) 105 ± ± ± ± ± 27 Alanine aminotransferase (U/L) 35 ± 4 38 ± ± 6 34 ± ± 6 Creatinine (mg/dl) 0.5 ± ± ± ± ± 0.1 Blood urea nitrogen (mg/dl) 14.6 ± ± ± ± ± 7.9 Total cholesterol (mg/dl) 70 ± ± ± ± 9 71 ± 9 Triglycerides (mg/dl) 75 ± ± ± ± ± 45 Glucose (mg/dl) 142 ± ± ± ± ± 18 Calcium (mg/dl) 10.3 ± ± ± ± ± 0.5 Inorganic phosphorus(mg/dl) 7.5 ± ± ± ± ± 0.7 Creatine kinase (IU/L) 483 ± ± ± ± ± 291a,c Sodium (mmol/l) ± ± ± ± ± 1.5 Potassium (mmol/l) 4.46 ± ± ± ± ± 0.26 Chloride (mmol/l) ± ± ± ± ± 1.7 Significantly different from negative control (a:p<0.05, b:p<0.01) and vehicle control (c:p<0.05, d:p<0.01) by Scheffe's test * Data are given as mean ± standard error a Number of animals

89 TABLE 34 Clinical Chemistry Data for Female Rats in the 90-Day Gavage Study (Main Study) of 20 nm, Negative Charged ZnO * Main Study Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Female Total protein (g/dl) 7.0 ± ± ± ± 0.6b 5.9 ± 0.3b,d Albumin (g/dl) 3.2 ± ± ± ± ± 0.2b,c A/G ratio 0.8 ± ± ± ± ± 0.1 Total bilirubin (mg/dl) 0.10 ± ± ± ± ± 0.03 Alkaline phosphatase (U/L) 205 ± ± ± ± ± 51 Aspartate aminotransferase (U/L) 113 ± ± ± ± ± 40 Alanine aminotransferase (U/L) 34 ± ± ± ± ± 8 Creatinine (mg/dl) 0.6 ± ± ± ± ± 0.1 Blood urea nitrogen (mg/dl) 17.7 ± ± ± ± ± 5.2 Total cholesterol (mg/dl) 82 ± ± ± ± ± 11 Triglycerides (mg/dl) 22 ± ± ± ± ± 24 Glucose (mg/dl) 139 ± ± ± ± ± 9 Calcium (mg/dl) 10.9 ± ± ± ± ± 0.2 Inorganic phosphorus(mg/dl) 6.1 ± ± ± ± ± 0.8 Creatine kinase (IU/L) 493 ± ± ± ± ± 273 Sodium (mmol/l) ± ± ± ± ± 1.1 Potassium (mmol/l) 4.01 ± ± ± ± ± 0.30 Chloride (mmol/l) ± ± ± ± ± 1.3 Significantly different from negative control (a P<0.05, b P<0.01) and vehicle control (c P<0.05, d P<0.01) by Scheffe's test * Data are given as mean ± standard error a Number of animals

90 TABLE 35 Clinical Chemistry Data for Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO * Recovery Groups Negative Vehicle 500 mg/kg n a Male Total protein (g/dl) 5.9 ± ± ± 0.2 Albumin (g/dl) 2.4 ± ± ± 0.1 A/G ratio 0.7 ± ± ± 0.0 Total bilirubin (mg/dl) 0.09 ± ± ± 0.02 Alkaline phosphatase (U/L) 279 ± ± ± 32 Aspartate aminotransferase (U/L) 119 ± ± ± 24 Alanine aminotransferase (U/L) 41 ± 9 38 ± ± 8 Creatinine (mg/dl) 0.6 ± ± ± 0.1 Blood urea nitrogen (mg/dl) 15.1 ± ± ± 4.7 Total cholesterol (mg/dl) 76 ± ± 5 68 ± 6 Triglycerides (mg/dl) 92 ± ± ± 35 Glucose (mg/dl) 154 ± ± ± 13 Calcium (mg/dl) 10.0 ± ± ± 0.3 Inorganic phosphorus(mg/dl) 6.8 ± ± ± 1.0 Creatine kinase (IU/L) 618 ± ± ± 148 Sodium (mmol/l) ± ± ± 1.1 Potassium (mmol/l) 4.60 ± ± ± 0.38 Chloride (mmol/l) ± ± ± 0.7 * Data are given as mean ± standard error a Number of animals

91 TABLE 36 Clinical Chemistry Data for Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO * Recovery Groups Negative Vehicle 500 mg/kg n a Female Total protein (g/dl) 6.4 ± ± ± 0.3 Albumin (g/dl) 2.9 ± ± ± 0.1 A/G ratio 0.8 ± ± ± 0.1 Total bilirubin (mg/dl) 0.09 ± ± ± 0.02 Alkaline phosphatase (U/L) 163 ± ± ± 29 Aspartate aminotransferase (U/L) 108 ± ± ± 13 Alanine aminotransferase (U/L) 46 ± ± 8 26 ± 5 Creatinine (mg/dl) 0.7 ± ± ± 0.1 Blood urea nitrogen (mg/dl) 17.4 ± ± ± 2.0 Total cholesterol (mg/dl) 82 ± 4 83 ± ± 18 Triglycerides (mg/dl) 26 ± ± ± 19 Glucose (mg/dl) 141 ± ± ± 15 Calcium (mg/dl) 10.2 ± ± ± 0.2 Inorganic phosphorus(mg/dl) 4.3 ± ± ± 0.4 Creatine kinase (IU/L) 339 ± ± ± 96 Sodium (mmol/l) ± ± ± 1.7 Potassium (mmol/l) 3.92 ± ± ± 0.21 Chloride (mmol/l) ± ± ± 2.5 * Data are given as mean ± standard error a Number of animals

92 본시험은위, 췌장및안구에서유의성있는변화가관찰되었다. 먼저위는전위, 전위와선위부로나누어병변을관찰하였다. 주시험군 500 mg/kg 투여군에서의위의병변은다음과같다. 전위의병변은, 500 mg/kg 투여군에서대체로편평상피의공포화가미약하게관찰되었다. 또한, 편평상피의과형성및상피하의염증세포침윤이대체로미약하게관찰되었다. 선위부의병변은, 점막상피의미란, 세포질내유리질작은방울, 점막하조직의부종및염증세포침윤, 호산성의주세포출현, 점액세포의과형성등이관찰되었는데대체로용량의존적으로유의성있게증가하였다. 회복군에서는주시험군에서관찰된위의병변이대부분관찰되었으나, 그정도에있어서미약하게회복되는경향을보였다. 또한주시험군 500 mg/kg 투여군에서의췌장의병변은다음과같다. 만성췌장염, 췌장샘포의세포자멸사등이관찰되었고, 회복군에서는주시험군에서관찰된췌장의병변대부분이관찰되었으나, 미약하거나약한정도로회복되는경향을보였다. 췌장에서의병변역시용량의존적으로유의성있게증가하였다. 마지막으로주시험군 500 mg/kg 투여군에서의안구의병변은다음과같다. 안구에서의망막의위축이중등도및현저한정도로관찰되었고회복군에서는동일한망막위축병변이유사한비율의빈도수로관찰되었다. 500 mg/kg 투여군의위, 췌장및안구에서대조군과비교하여유의한병변이관찰되어 125 mg/kg 및 250 mg/kg 투여군의위, 췌장및안구를관찰하였다. 위의경우, 500 mg/kg 투여군에서관찰되었던병변이 125 mg/kg 및 250 mg/kg 투여군에서대부분관찰되면서대체로용량의존적인경향을나타냈다. 췌장역시, 500 mg/kg 투여군에서관찰되었던병변이 125 mg/kg 및 250 mg/kg 투여군에서대부분관찰되면서대체로용량의존적인경향을나타냈다. 안구의망막위축병변은, 암수모두 125 mg/kg 및 250 mg/kg 투여군에서관찰되지않았다.(Table 37~40, PLATE 1~14)

93 TABLE 37 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Main group Negative Vehicle 500 mg/kg n a Liver Necrosis 1 b (1.0) c 0 0 Hepatocyte vacuolation 0 2(1.0) 0 Inflammatory cell infiltration 1(1.0) 3(1.0) 2(1.0) Kidney Cortical tubular basophillia 4(1.0) 2(1.0) 1(1.0) Tubular cast 0 0 1(1.0) Inflammatory cell infiltration 2(1.0) 0 2(1.0) Adrenal gland Cortical vacuolation 7(1.3) 3(1.0) 4(1.3) Cystic degeneration Heart Myocardial inflammatory cell infiltration 3(1.0) 5(1.2) 4(1.5) Lung Perivascular inflammatory cell infiltration 2(1.0) 2(1.5) 1(1.0) Pituitary Cyst Seminal vesicle Atrophy Testis Tubular atrophy Epididymis Atrophy Prostate gland Interstitial inflammatory cell infiltration 5(1.6) 5(1.2) 7(1.3) Intra-luminal inflammatory cells 0 0 2(2.5) Atrophy a Number of animals with tissue examined microscopically b c Number of animals with lesion Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe

94 TABLE 37 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Main group Negative Vehicle 500 mg/kg n a Spleen Extramedullary Hematopoiesis (EMH) 0 3(1.0) 2(1.0) Thymus Atrophy Thyroid gland Cyst Lymphoid hyperplasia 0 1(1.0) 0 Inflammatory cell infiltration 1(1.0) 0 0 Duodenum Submucosal gland hyperplasia 0 0 3(1.7) a Number of animals with tissue examined microscopically b c Number of animals with lesion Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe

95 TABLE 37 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Forestomach Squamous cell vacuolation 0 b 0 3(1.0) c 6 e,g (1.0) 5 d,f (1.0) Squamous cell hyperplasia 0 1(1.0) 10 e,g (2.6) 10 e,g (3.1) 10 e,g (3.3) Subepithelial inflammatory cell infiltration e,g (1.0) 10 e,g (1.0) 9 e,g (1.0) Glandular stomach Submucosal inflammatory cell infiltration 1(1.0) 3(1.0) 10 e,g (2.1) 10 e,g (2.8) 9 e,g (2.8) Submucosal edema 0 1(1.0) 5 d (1.8) 8 e,g (1.3) 7 e,g (1.3) Superficial epithelial degeneration / regeneration 0 1(1.0) 3(1.0) 3(1.0) 1(1.0) Mucous cell hyperplasia e,g (1.4) 10 e,g (1.5) 10 e,g (1.3) Epithelial erosion 0 0 2(1.0) 2(1.0) 2(1.5) Intracytoplasmic hyaline droplet e,g (2.7) 10 e,g (2.6) 9 e,g (2.0) Eosinophilic chief cell 0 0 2(2.0) 4 d,f (1.5) 8 e,g (1.8) Eye Retinal atrophy (1.0) 4 d,f (3.8) Pancreas Acinar cell apoptosis 3(1.0) 3(1.0) 9 e,g (1.4) 10 e,g (1.9) 10 e,g (3.2) Peri-islet cell pigment 1(1.0) Fat replacement 1(1.0) 1(1.0) 0 0 1(1.0) Chronic inflammation e,g (1.5) 10 e,g (1.2) 10 e,g (2.6) a Number of animals with tissue examined microscopically, b Number of animals with lesion c Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe Significantly different from at ( d, f : P<0.05, e,g : P<0.01) respectively by Fisher s exact test

96 TABLE 38 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Main group Negative Vehicle 500 mg/kg n a Liver Inflammatory cell infiltration 1 b (1.0) 1(1.0) 0 Granuloma 0 0 1(2.0) Periportal vacuolation 1(1.0) 0 6 e (1.2) Kidney Cortical tubular basophillia Tubular cast Inflammatory cell infiltration 0 0 2(1.0) Adrenal gland Cystic degeneration 1(2.0) 0 2(1.0) Heart Myocardial inflammatory cell infiltration Lung Perivascular inflammatory cell infiltration Pituitary Cyst Spleen Extramedullary Hematopoiesis (EMH) 1(1.0) 0 0 Thymus Atrophy Thyroid gland Cyst 0 0 2(1.0) Duodenum a b c Submucosal gland hyperplasia Number of animals with tissue examined microscopically Number of animals with lesion Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe Significantly different from at ( d, f P<0.05, e, g : P<0.01) respectively by Fisher s exact test

97 TABLE 38 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Main group Negative Vehicle 500 mg/kg n a Forestomach Squamous cell vacuolation 0 b 0 2(1.0) c Squamous cell hyperplasia e,g (2.5) Subepithelial inflammatory cell infiltration 0 0 3(1.0) Glandular stomach Submucosal inflammatory cell infiltration 1(1.0) 1(2.0) 8 e,g (1.9) Submucosal edema 0 0 1(1.0) Superficial epithelial degeneration / regeneration a b c Mucous cell hyperplasia e,g (1.5) Epithelial erosion 0 0 3(1.0) Intracytoplasmic hyaline droplet e,g (2.0) Eosinophilic chief cell Number of animals with tissue examined microscopically Number of animals with lesion Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe Significantly different from at ( d, f P<0.05, e, g : P<0.01) respectively by Fisher s exact test

98 TABLE 38 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Main group) of 20 nm, Negative Charged ZnO Negative Vehicle 125 mg/kg 250 mg/kg 500 mg/kg n a Female Eye Retinal atrophy d,f (3.6) Pancreas Acinar cell apoptosis 1(1.0) 0 7 e,g (1.1) 10 e,g (1.7) 10 e,g (3.5) Chronic inflammation d,f (1.0) 3(1.0) 4 d,f (1.0) Fat replacement 2(1.0) (1.5) a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe Significantly different from at ( d, f : P<0.05, e,g : P<0.01) respectively by Fisher s exact test

99 TABLE 39 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a Male Liver Hepatocyte vacuolation 0 b 1(1.0) c 0 Inflammatory cell infiltration 2(1.0) 2(1.0) 3(1.0) Focal necrosis 0 1(1.0) 0 Kidney Cortical tubular basophillia 0 1(1.0) 0 Pelvic mineralization 1(1.0) 0 0 Tubular cast 0 1(1.0) 0 Inflammatory cell infiltration 2(1.0) 1(1.0) 1(1.0) Adrenal gland Cortical vacuolation 1(1.0) 4(1.0) 2(1.5) Cyst Lung Pigmented histiocytes Epididymis Inflammatory cell infiltration 1(1.0) 0 0 Prostate gland Interstitial infiltration inflammatory cell 4(1.3) 3(1.3) 1(1.0) Concretion Thymus Hemorrhage / congestion Atrophy a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe

100 TABLE 39 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a Male Forestomach Squamous cell hyperplasia 1(1.0) 0 4(2.3) Squamous cell vacuolation 0 0 2(1.0) Submucosa infiltration inflammatory cell 0 0 2(1.0) Glandular stomach Submucosa infiltration inflammatory cell 0 0 2(1.5) Submucosal edema Superficial epithelial degeneration / regeneration 0 0 1(1.0) Erosion Intracytoplasmic hyaline droplet 0 0 3(1.0) Eosinophilic chief cell 0 0 1(3.0) Eye Retinal atrophy 0 0 3(3.3) Pancreas Acinar cell apoptosis 1(2.0) 3(1.0) 4(1.0) Chronic inflammation 1(2.0) 1(1.0) 0 Basophilic focus 1(1.0) 0 2(1.0) Fat replacement 2(1.0) 2(1.0) 1(1.0) a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe

101 TABLE 40 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 90-Day Gavage Study (Recovery Groups) of 20 nm, Negative Charged ZnO Recovery Groups Negative Vehicle 500 mg/kg n a Female Liver Inflammatory cell infiltration 0 b 2(1.0) c 0 Kidney Tubular cast Inflammatory cell infiltration Adrenal gland Cortical vacuolation 0 1(1.0) 0 Cyst 1(1.0) 0 0 Lung Pigmented histiocytes 0 0 1(1.0) Eosinophilic histiocyte 0 0 1(2.0) Thymus Atrophy Forestomach Squamous cell hyperplasia 0 0 2(2.0) Squamous cell vacuolation 0 0 3(1.3) Submucosa infiltration inflammatory cell 0 0 2(1.0) Glandular stomach Submucosal edema 0 0 2(1.0) Erosion 0 0 1(1.0) Intracytoplasmic hylaine droplet 0 0 2(1.0) Eosinophilic chief cell 0 0 1(1.0) Eye Retinal atrophy 0 0 3(3.3) Pancreas Acinar cell apoptosis 2(1.0) 2(1.0) 3(1.0) Basophilic focus 0 0 1(1.0) Fat replacement 0 0 1(1.0) a Number of animals with tissue examined microscopically b Number of animals with lesion c Average severity grade of lesion in affected animals: 1=minimal, 2=slight, 3=moderate, 4=marked, 5=severe

102 PLATE 1. Normal limiting ridge of stomach of male SD rat administered 0 mg/kg ZnO(-) by gavage for 90-Day. Note the normal epithelium (NE) in forestomach. H&E PLATE 2. Sqaumous cell hyperplasia (H) and squamous cell vacuolation (arrow) in limiting ridge of forestomach of SD rat administered 500 mg/kg ZnO(-) by gavage for 90-Day. Note the squamous cell hyperplasia (H) in forestomach. H&E

103 PLATE 3. PLATE 4. Normal glandular stomach of male SD Eosinophilic chief cell (EC) in glandular rat administered 0 mg/kg ZnO(-) by stomach of male SD rat administered gavage for 90-Day. Note the normal 500 mg/kg ZnO(-) by gavage for mucosa (N) in glandular stomach. H&E 90-Day. Note the eosinophilic chief cell (EC) in glandular stomach. 83 H&E

104 PLATE 5. PLATE 6. Normal glandular stomach of male SD Intracytoplasmic hyaline droplet (arrows) rat administered 0 mg/kg ZnO(-) by in glandular stomach of male SD rat gavage for 90-Day. Note the normal administered 500 mg/kg ZnO(-) by mucosa (N) in glandular stomach. H&E gavage for 90-Day. Note the intracytoplasmic hyaline droplet (arrows) in glandular stomach. 84 H&E

105 PLATE 7. PLATE 8. Normal glandular stomach of male SD Submucosal inflammatory cell infiltration rat administered 0 mg/kg ZnO(-) by (SI) in glandular stomach of male SD gavage for 90-Day. Note the normal rat administered 500 mg/kg ZnO(-) by submucosa (NS) in glandular stomach. gavage for 90-Day. Note the H&E submucosal inflammatory cell infiltration (SI) in glandular stomach. H&E 85

106 PLATE 9. PLATE 10. Normal glandular stomach of male SD Mucous cell hyperplasia (arrows) in rat administered 0 mg/kg ZnO(-) by glandular stomach of male SD rat gavage for 90-Day. Note the normal administered 500 mg/kg ZnO(-) by parietal cell distribution (arrow) and in gavage for 90-Day. Note the mucous glandular stomach. Alcian blue cell hyperplasia (arrows) stained with blue in glandular stomach. Alcian blue 86

107 PLATE 11. PLATE 12. Normal microscopic structure of the Chronic inflammation (A) and acinar pancreas of a male SD rat administered cell apoptosis (arrows) and prominent 0 mg/kg ZnO(-) by gavage for 90-Day. acinar cells (P) in the pancreas of SD Note the normal acinar cell (NA) in rat administered 500 kg/mg ZnO(-) by the pancreas. H&E gavage for 90-Day. Note chronic inflammation in the pancreas. H&E 87

108 PLATE 13. PLATE 14. Normal microscopic structure of the Retinal atrophy is remarkable in the eye eye of male SD rat administered 0 of male SD rat administered 500 mg/kg mg/kg ZnO(-) by gavage for 90-Day. ZnO ZnO(-) by gavage for 90-Day. Note the normal outer layer of retina Note retinal atrophy (R) in the outer (N) in the eye. H&E layer of the retina in the eye. Compare with the normal eye in male SD control rat shown in PLATE 13. H&E 88

109 시험물질을투여한후시간별혈중농도로부터산출한독성동태파라미터들은다음과같았다. 투여첫날 Cmax는 125, 250 및 500 mg/kg 용량증가에따라수컷에서는각각 51.84, 54.80, μg /ml, 암컷에서각각 54.14, 47.38, μg /ml였으며, 투여 28일차에는수컷에서각각 32.51, 94.17, μg /ml, 암컷에서는각각 36.69, 76.94, μg /ml로나타났다. 투여마지막 90일차에는수컷에서각각 37.65, 70.43, μg /ml, 암컷에서는각각 40.20, 86.65, μg /ml로나타났다. 각용량에서투여첫날 AUC는수컷에서각각 , , μg hr/ml, 암컷에서는각각 , , μg hr/ml였으며, 투여 28일차의 AUC는수컷에서각각 , , μg hr/ml, 암컷에서는각각 , , μg hr/ml를나타냈다. 투여마지막 90일차에는수컷에서각각 , , μg hr/ml, 암컷에서는각각 , , μg hr/ml를나타냈다. 최고혈중농도도달시간 (Tmax) 은 4 ~ 6 시간이었으며, 1 ~ 13시간의평균소실반감기를나타내며혈중에서소실되었다 (Table 41, Figure 8). 본연구와함께연구한결과에따르면 PET 이미징기법으로형광물질 Cy5.5를표지하여 ex-vivo NIRF optical imaging을이용하여나노입자의이동경로와분포를확인한결과 24시간내에위장관을통해변으로배출되며, 간과신장에서도일부검출되었다. 이는본시험에서수행한분포시험과일치한다.

110 Figure 8. Zinc concentration in plasma after 90 day-repeated oral administration to rats. A, 125mg/kg; B, 250mg/kg; C,500mg/kg. The data are presented as increased zinc levels after subtraction of the basal zinc level in the control group

111 Table 41 Summary of toxicokinetic parameters for Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO Day Dose mg/kg Sex M F M F M F M F M F M F M F M F M F Cmax Tmax AUC MRT T 1/ Note: Toxicokinetic parameters include maximum observed peak serum concentration (Cmax, mg/ml); time at which Cmax was observed (Tmax, h); area under the serum concentration time curve (AUC, h ug/ml); mean residence time (MRT, h);and half-life (T 1/2,h).

112 검증된분석법에따라 Rat의혈장, 변, 뇌, 간, 신장, 정소, 난소, 비장, 폐, 위장, 소장, 대장을분석하였다. 분석결과암수간의차이는암컷이약간높은농도로관찰되는경향을보였으나차이는미미하였다. 음성대조군, 부형제대조군과비교하여간, 신장, 대장, 소장, 혈장의경우투여농도에따라검출농도가증가하여해당장기에흡수및분포되어있고투여된물질의평균 95% 가변으로배출되는것이관찰되었다. 그러나뇌, 정소, 난소, 비장, 위, 폐의경우음성대조군, 부형제대조군과비교투여농도에따른증가량이미미하거나증가량이보이지않았다 (Table 42.).

113 Table 42 Tissue distribution Data For Rats in the 90-Day Gavage Study of 20 nm, Negative Charged ZnO Male Female V-control 125 mg/kg 250 mg/kg 500 mg/kg V-control 125 mg/kg 250 mg/kg 500 mg/kg Plasma Liver Kidney Large intestine Small intestine Feces Stomach Lung Spleen Brain Testes Ovary Note: V-control, vehicle control, Unit:ug/g

114 3. 고찰 ZnO(-)(20 nm, Negative Charge) 을 125, 250 및 500 mg/kg의용량으로 90일간반복적으로경구로투여하여일반소견, 기능관찰, 체중, 음수 / 사료섭취량, 뇨검사, 혈액학적검사, 혈액생화학적검사, 장기중량, 부검및조직병리학적검사를실시하였고, 14일간회복성을관찰하였다. 시험기간동안모든투여군에서시험물질투여로인한폐사동물은관찰 되지않았으며, 기능관찰, 체중변화, 사료 음수섭취량, 요검사, 부검소견, 장기중량에서는시험물질투여와관련된독성변화는관찰되지않았다. 일반관찰결과, 시험물질투여와관련된일반소견은투여직후에관찰된유연이었다. 암수투여군의유연소견은투여 14일차부터 500 mg/kg 투여군에서처음관찰되기시작하여모든투여군에서시험종료일까지관찰되었다. 이소견은 ZnO의가스를흡입시유연이나타나는특징이있다는주장과관련있는것으로사료된다. 38 안과학적검사결과, 수컷 500 mg/kg 투여군 1례 ( 동물번호 :1505) 에서안구혼탁이관찰되었다. 이소견은조직병리학적검사에서망막의위축소견이두드러지게관찰되어안과학적검사결과와부합하는소견으로확인되었다. 혈액학적검사결과, 암수투여군에서통계학적으로유의성을나타냈던혈색소량, 헤마토크리트치, 평균적혈구용적, 평균적혈구헤모글로빈량그리고평균적혈구헤모글로빈농도의감소는대체로군간용량의존성이인정되므로시험물질로인한변화로판단한다. 이러한적혈구지표의변화는 Zn 이온이철결핍성및철적아구성빈혈과골수기능억제를유도한다는주장과관련있는것으로사료된다. 39,40 혈색소량과, 평균적혈구용적은정상적으로회복되었고다른수치는유의성을보이고있어본연구의회복기간중에는정상수치로회복되지않았다.

115 조직병리학적검사결과, 암수동물에서음성대조군및부형제대조군과 비교하여현저한변화를나타낸장기는췌장, 안구그리고위의병변이었 다. 췌장에서선방세포의세포자멸사와도관주변의임파구침윤이암수투여군모두에서용량의존적으로관찰되었고, 미약한병변부터심한정도까지도관상피세포의과형성과선방세포재생이다수관찰되었다. 이러한췌장의병변은제조 ZnO 나노입자를 90일간투여한랫드에서췌장염을유발한다는보고에서도사례를찾을수있다. 41 또다른연구에서 20nm의 ZnO 를 14일간경구투여시심각한위점막의손상과함께그보다는미약한정도의염증세포침윤과같은병변이간과췌장에서관찰되었다는보고가있다. 42 또한, ZnO를나노사이즈와마이크로사이즈로 14일간경구투여하였을때, 간, 췌장, 심장과위에서조직학적병변이관찰되었는데, 나노사이즈에서는저용량으로갈수록병변의빈도수가높아졌고, 마이크로사이즈에서는고용량으로갈수록병변의빈도수가높아졌다는보고가있다. 36 표적장기로판단한안구의망막위축소견이암수몇례에서관찰되었는데, 대조군에서는해당소견이관찰되지않았고, 250, 500 mg/kg 투여군에서만관찰되어시험물질에의한영향을받은장기로판단되었다. 그러나반대로저농도로아연을투여할경우망막질환을예방해준다는보고가있어상반되는결과를보여주었다. 43 결국아연에의한망막위축소견은농도에따른변화라고판단된다. 암수동물의시험물질투여군에서주로관찰된선위와전위부경계부위 (limiting ridge) 의상피세포과형성, 선위부의상피하의염증세포침윤, 세포질내호산성과립을가진주세포유사세포병변은대조군에서관찰되지않아시험물질에의한영향으로판단되었다. ZnO의경구투여에의한위의

116 병변은 20 nm와 120 nm의 ZnO를투여한마우스위의점막고유판, 점막하및장막층에서염증을일으킨다는보고가있어 ZnO에의한특이적병변으로판단된다. 42,44 회복군에서이들조직병변의회복을관찰한결과, 췌장, 위의병변은회복되었지만망막병변은회복되지않았다. 최근연구에서 ZnO 나노입자는사람의암세포와미토콘드리아에독성이있다고한다. 45 다른연구에서빠르게분화하는세포에대하여선택적인독성을유발한다는보고도있다. 46 동일선상에서본시험에서관찰된조직병리학적소견은 ZnO 나노입자가위의상피세포나췌장의재생성선방세포같은활발하게분화증식하는세포와관련성이있음을뒷받침한다. 독성동태시험및조직분포시험자료에서도암수모두의간, 신장, 대장, 소장에서유사한용량, 시간의존적으로증가된아연의흡수및누적결과를보여준다. 반면에뇌, 고환, 난소, 비장, 위, 폐에서는대조군과비교하여암수모두통계학적으로유의한결과가관찰되지않았다. 따라서음전하 ZnO 나노입자는성적특이성은갖지않는것으로판단된다. 나아가췌장, 위, 눈에서관찰된주된조직병리학적소견은분포시험자료와연관성이없는것으로나타났다. 종합적으로보면 125 mg/kg 투여용량까지유의할만한독성학적인변화가관찰되어무독성량 (NOAEL) 은암 수모두찾지못했으나, 최소독성량은암 수모두 125 mg/kg 이었다. 전신성부작용으로유연, 췌장의선방세포자멸사와만성염증성병변, 염증성위병변, 망막의위축그리고빈혈과관련된지표의변화가관찰되었다.

117 제 2 장 ZnO(+) 의 90 일반복경구투여연구 제 1 절 14 일용량결정시험 1. 시험물질및물리화학적특성파악시험물질은 20 nm 크기의 ZnO 나노물질 (Ultra fine Zinc Oxide ZnO-310, SUMITOMO OSAKA CEMENT, Japan) 을분말상태로구매하여사용하였다. 음성대조물질로써멸균증류수 ( 대한약품공업 ( 주 ), Korea) 와시험물질의표면개질을위해 L-Serine을사용하였다. 양전하를띠는 ZnO를제조하기위해서리간드를사용하여나노물질표면을개질하였다. 후보리간드물질로는 4 종류의아미노산 thiamine, arginine, lysine, serine과 diethylenetriamime (DETA), poly-dimethyldiallylammonium (PDAC) 를사용하여분산성및입자크기를확인하였다. 실험결과를종합하여양전하를띠는 ZnO 제조를위하여 HEPES ph 6 조건에서 serine이가장적합한리간드물질로결정하였다. ZnO(+) 의물리화학적특성을분석을위하여 TEM 및 SEM 방법을사용하여입자크기및입형을관찰한결과, 평균적으로 35 ± 5 nm와 32 ± 7 nm 의크기를갖는구형으로확인되었다 (Figure 1). 또한 DLS 분석결과, hydrodynamic 크기는 ± 17.8 nm이었으며, 제타전하는 ± 0.5 mv로양의표면전하를갖는것으로확인하였다 (Figure 2, 3).

118 FIGURE 1 (A) Transmission electron microscopy (TEM) and (B) Scanning Electron Microscopy (SEM) Images of 20 nm, Positive Charged ZnO Nanoparticles FIGURE 2 Hydrodynamic size of 20 nm, Positive Charged ZnO nanoparticles