The 23rd Annual Meeting of Japanese Society for … The 23rd Annual Meeting of Japanese Society for...

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The 23rd Annual Meeting of Japanese Society for

Alternatives to Animal Experiments

Basics and Practice of Alternatives to Animal Experiments

December 3 (Friday) – 5 (Sunday) Kitasato University School of Pharmacy (Shirokane Campus)

Date: November 3rd (Friday) － 5th (Sunday), 2010 Kitasato University School of Pharmacy (Shirokane Campus)

Congress Presidents: Prof. Yuji Yoshiyama

Kitasato University School of Pharmacy

The 23rd Annual Meeting of Japanese Society f or Alternatives to Animal Experiments, AATEX 15 (Supplement)

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Program President Lecture : December 4 (Saturday) 8:45-9:00 PL Basics and Practice of Alternatives to Animal Experiments

Yuji Yoshiyama President of The 23rd Annual Meeting of Japanese Society for Alternatives to Animal Experiments Kitasato University School of Pharmacy

Special Lecture 1 : December 4 (Saturday) 9:00-10:00 Chair persons: Hiroyuki Miyazaki (Takata Seiyaku Co., Ltd.) SL-1 Data science on validation studies conducted by JSAAE Takashi Omori Doshisha University Faculty of Culture and Information Science

E-mail: [email protected]

Special Lecture 2 : December 5 (Sunday) 11:00-11:50 Chair persons: Kenji Sugibayashi (Josai university faculty of pharmaceutical sciences) SL-2 Recent topics on chronopharmaceutics focused on molecular clock

Shigehiro Ohdo Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University

Dr. Sugawara memorial lecture : December 4 (Saturday) 13:00-13:30 Symposium 1 : December 4 (Saturday) 15:10-16:50 For a revision of Japanese "Act on Welfare and Management of Animals" Chair persons: Yasuo Ohno (National Institute of Health Sciences), Yuji Yoshiyama (Kitasato University School of Pharmacy) S1-1 Situation at the Ministry of the Environment for the Amendment of the Animal Protection

Law Yoshihiro Hayashi Faculty of Agriculture, Tokyo University of Agriculture

S1-2 Perspectives on amendment of the Animal Law Laboratory animals: To implement

science-based laboratory animal welfare Naoko Kagiyama Hokkaido University Graduate School of Veterinary Medicine

S1-3 Trend of International Advancement of Alternative Program Comparison with Japanese

Legislation and Guidelines Tsutomu Miki Kurosawa OsakaUniversity Graduate School of Medicine Department of Laboratory Animal Medicine

S1-4 Are Animal Experiments the Ultimate Cruelty to Animals?

Koichi Aoki The Japanese Coalition for Animal Welfare


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S1-5 Where Does Humane Science Should Head to? Hiromi Kamekura NPO Japan Anti-Vivisection Association

Symposium 2 : December 5 (Sunday) 9:00-11:00 Bioengineering for Alternatives to Animal Experiments Chair persons: Kikuo Komori (Institute of Industrial Science, University of Tokyo), Koichi Saito (Sumitomo Chemical Co., Ltd.) S2-1 Electrochemical Detachment of Cells and Cell Sheets for Engineering Cellular Tissues

Junji Fukuda, Hiroaki Suzuki Graduate School of Pure and Applied Sciences, University of Tsukuba

S2-2 Microfabrication of co-culture systems

M. Nishizawa1, 2, H. Kaji1, 2, K. Nagamine1, 2, M. Kanzaki2, 3 1Department of Engineering, Tohoku University, 2 JST-CREST, 3Department of Medical Engineering, Tohoku University

S2-3 Perfusion Culture Microchamber Array Chip for High Throughput Drug Dose Response

Assay Toshiyuki Kanamori, Shinji Sugiura, Koji Hattori, Kimio Sumaru National Institute of Advanced Industrial Science and Technology (AIST)

S2-4 Wearable Bio/Chemical Sensor with Soft-MEMS Techniques

Kohji Mitsubayashi Institute of Biomaterials & Bioengineering, Tokyo Medical and Dental University

Symposium 3 : December 5 (Sunday) 9:00-11:30 Biological approaches to development of useful cell based assay system for AATEX - - Toxicological and pharmacological assays in cornea and liver - - Chair persons: Taku Matsushita (Dept. of Applied Life Science, Sojo University), Toshiaki Takezawa (National Institute of Agrobiological Sciences) S3-1 Suggestion of an ideal evaluation system using a corneal epithelial cell from the experience

of CS-088 antiglaucoma ophthalmic solution Takayuki Kikuchi Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd.

S3-2 Development of a human cornea model utilizing a collagen vitrigel membrane as a scaffold

and its advantages Toshiaki Takezawa1, Hiroyuki Yamaguchi1, 2

1National Institute of Agrobiological Sciences, 2Kanto Chemical Co. Inc. S3-3 Induction of drug-metabolizing enzymes by layered co-culture of a human liver cell line

Akiyoshi Taniguchi1, 2, Maki Ohno1

1Biomaterials Center, National Institute for Materials Science, 2Faculty of Science and Engineering, Waseda University.

S3-4 Usability of hepatocyte spheroid culture on Cell-able in DMPK research: results with

human and rat primary hepatocytes Shin Enosawa Division for Advanced Medical Sciences, Clinical Research Center, National Center for Child Health and Development


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S3-5 Development of culture system for induction of hepatic stem cells from human fetal hepatocytes Taku Matsushita Lab. of Biomedical Eng., Dept. of Applied Life Science, Sojo University

S3-6 Application of human stem cells to the safety/toxicity study of drug candidates --- Road to

the guideline plan making Seiichi Ishida National Institute of Health Sciences

Symposium 4 : December 5 (Sunday) Trends of in vitro skin irritation test Chair persons: Shigenobu Hagino (Shiseido Research Center), Yuko Okamoto (Kose Corporation, Fundamental research laboratories) S4-1 “Current status of the alternative to the skin irritation test method”

Yuko Okamoto Kose Corporation, Fundamental research laboratories

S4-2 Cosmetic skin disorders -Focus of irritant contact dermatitis--

Masatoshi Ito, Fukuyoshi Mori Toho University School of Medicine

S4-3 In vivo Researches for Practical Use of Substituting Methods for Skin Irritation Studies

Yasushi Yamada Nihon Bioresearch Inc.

S4-4 Validity of the EpiSkinTM test in Japan; Skin irritancies of cosmetic ingredients tested using

EpiSkinTM Shoichi Yahagi1, Miyuki Fujishiro1, Yukiko Izutsu1, Daisuke Yoshida1, Koji Kurihara2, Yuri Okano1, Hitoshi Masaki1

1 Nikkol group Cosmos Technical Center Co., Ltd., 2 Nikkol group Nikoderm Research Inc. S4-5 Skin irritation test using LabCyte CORNEA-MODEL

Masakazu Katoh Japan Tissue Engineering Co., Ltd.

S4-6 Approach to the current requirements for alternative method of skin irritation testing

Mariko Sugiyama Shiseido Research Center

Symposium 5 : December 5 (Sunday) 14:10-16:50 Development of alternative assay systems for chemical risk evaluation - Carcinogenicity, Developmental toxicity, Immunotoxicity - Chair persons: Noriho Tanaka (Hatano Research Institute, Food and Drug SafetyCenter), Hajime Kojima (National Institute of Health Sciences) S5-L Chemical management policy and chemical risk evaluation in Japan

Shinichi Jitsukuni Chemical Safety Office, Chemical Management Policy Division, METI


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S5-1 Development of alternative assay systems supported by modern technologies S5-1-1 Development of the reporter gene assay system using multicolor luciferase probes

Yoshihiro Nakajima1, Shigeaki Nishii2, Yoshihiro Ohmiya2

1National Institute of Advanced Industrial Science and Technology (AIST), 2Tsuruga Institute of Biotechnology, Toyobo Co., Ltd.

S5-1-2 HTP assay systems using a human artificial chromosome (HAC) vector

TETSUYA Ohbayashi1, MITSUO Oshimura2 1Res. Center. Biosci. Tech., Tottori Univ., 1Grad. Sch. Med., Inst. Regenerative Med. Biofunction., Tottori Univ.

S5-2 Development of alternative assay systems of carcinogenicity, developmental toxicity and

immunotoxicity S5-2-1 A cell transformation assay using Bhas 42 cells: A proposal for guideline and development a

high-throughput screening assay Kiyoshi Sasaki1, Dai Muramatsu1, Shoko Arai1, Nobuko Endou1, Sachiko Kuroda1, Kumiko Hayashi1, Ayako Sakai1, Shojiro Yamazaki1, Yeon-mi Lim1, Makoto Umeda1, Masanori Wada2, Noriho Tanaka1 1Hatano Research Institute, Food and Drug Safety Center, 2ABLE Corporation

S5-2-2 Development of novel alternative tests for developmental toxicity - Reporter gene assays

using mouse ES cells and improvement of rat whole embryo culture - Noriyuki Suzuki1, Koichi Saito1, Masaharu Akita2

1Institution Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 2Kamakura Women's University

S5-2-3 Immunotoxicity: Development of a reporter gene assay system using human cell lines

Setsuya Aiba, Rumiko Saito, Yutaka Kimura, Ikuko Numata, Toshiya Takahashi Department of Dermatology, Tohoku University Graduate School of Medicine

International symposium : December 3 (Friday) 15:00～17:00 P.65 Chair persons Tsutomu Miki Kurosawa (OsakaUniversity Graduate School of Medicine) IS-1 International Symposium for Alternatives to Animal Experimentation. At The 23rs JSAAE

annual meeting. Tsutomu Miki Kurosawa, DVM, M.PHIL, Ph.D, DVCS, DJCLAM Division of Laboratory Animal Medicine, Osaka University Graduate School of Medicine. Japan

IS-2 Comparison of flow cytometry (FCM) and immunohistochemistry (IHC) in

non-radioisotopic murine LLNA using BrdU and the prevalidation of LLNA:BrdU FCM Kyung-Min Lim1, Kyoung-Mi Jung1, Won-Hee Jang1, Bae-Hwan Kim2, Yong-Kyoung Lee3, Young Na Yum3, Soojung Sohn 3

1Pharmaceutical Research Institute, Amorepacific CO R&D Center, Yongin 446-729, Republic of Korea, 2Department of Public Health, Keimyung University, 3National Institute of Food and Drug Safety Evaluation/Korea Food and Drug Administration Korea

IS-3 Current Status of Alternative Study in China

He Zhengming National Institute for the Control of Pharmaceutical and Biological Products, Beijing, P.R. of China.


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IS-4 National Regulations and Standards of Laboratory Animal Welfare in China Guo-jie YANG Peking University Health Science Center，Beijing 100083，China

IS-5 Current activities for enhancing development and establishment of alternatives to animal

experiments in Korea: focusing on an example of bovine corneal opacity and permeability test Yong Heo1

, Yeon Mi Lim1, Joo Hee Han2, Seung Hyeok Seok2, Soon-Young Han3, Soo Jung Sohn3 , Young Na Yum3

1Catholic University of Daegu, College of Natural Sciences, Dept. Occupational Health, Kyongbuk, 2Seoul National University, College of Veterinary Medicine, Seoul, 3National Institute of Food and Drug Safety Evaluation/Korea Food and Drug Administration Korea

Education lecture 1：December 4 (Saturday) 12:10-13:00 Chair persons: Hideto Ariumi (Kitasato University School of Pharmacy) EL-1 Application of Cell-recognizable Matrix-engineering to Tissue Engineering and

Regenerative Medicine. -Frontier of Immobilization of E-Cadhering Chimeric Antibody- Toshihiro Akaik Frontier Research Center, Tokyo Institute of Technology

Education lecture 2 : December 5 (Sunday) 12:00-12:50 Chair persons: Tsuyoshi Yokoi (Faculty of Pharmaceutical Sciences, Kanazawa University) EL-2 State-of-the Art of Hepatic Generation from Stem Cells

Takahiro Ochiya Section for Studies on Metastasis, National Cancer Center Research Institute

The 2nd International Research Promotion Session of Alternative Animal Experiments by Mandom Corp. : December 4 (Saturday) 13:30～14:20 Chair persons: Satoshi Nakahara (Mandom Corporation), Masaharu Akita (Kamakura Women’s University) M-１ Formation and Functional Evaluation of a Minimum Cell Mass for In Vitro Toxicity Tests

Kikuo Komori Institute of Industrial Science, University of Tokyo

M-2 Establishment of chronopharmacokinetic evaluation system in vitro cultured cells:

Molecular basis for rhythmic expression of CYP in serum-shocked HepG2 cell Shigehiro Ohdo, Satoru Koyanagi, Naoya Matsunaga Deapartment of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University

M-3 Establishment of the method for quantitative prediction of detoxification ability and drug

interaction in human liver by using human tissue sample and gene expression systems ~ Utilization of human tissue as alternative experimental systems to animal experiments ~ Kazuya Maeda, Naoki Kotani, Kenta Yoshida, Yuichi Sugiyama Graduate School of Pharmaceutical Sciences, The University of Tokyo


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Challenge contest: December 4 (Saturday) 14:20-15:10 Chair persons: Satoshi Nakahara (Mandom Corporation), Masaharu Akita (Kamakura Women’s University) CC-1 Experiments using water fleas

Sawako Yasuda Yasu Municipal Yasu Elementary School

CC-2 Proposals regarding experiments on leptin in frogs

Yuya Nakamoto, Takahiro Yamashita, Hiroto Kamosaki Hiroshima Prefectural Hiroshima Kokutaiji High School

CC-3 Application of snails’ various sensor functions to medical care

Masaya Ohtsu Toin Gakuen High School

Public Seminar : December 5 (Sunday) 16:00-17:00 Chair persons: Yoshiaki Ikarashi (National Institute of Health Sciences) PS The use of human materials and information is indispensable in biomedical research: in the

era of alternatives of animal experiments. Tohru Masui Office of Policy and Ethics Research Director, Department of Disease Bioresources Research, National Institute of Biomedical Innovation

General Session ( Poster ) Flash oral presentation: December 4 (Saturday) 10:00-12:00 Poster session: December 4 (Saturday) 17:00-18:00 P-1 Accuracy of LLNA: BrdU-ELISA to detect skin sensitization potential of chemicals

Hideki Miyaura1, Masafumi Horiuchi1, Naoaki Yakata1, Masahiro Takeyoshi1, Naomi Kawazu2, Masanori Taruki2

1Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, 2Hita Laboratory, Chemicals Evaluation and Research Institute

P-2 Respiratory sensitization study by quantitative structure-activity relationships (QSAR)

Kazuhiro Sato1, Kohtaro Yuta2 and Yukinori Kusaka 1

1Department of Environmental Health, School of Medicine, University of Fukui, 2In Silico Data Co Ltd.

P-3 Permeability prediction of phthalic esters for cultured human alveolar epithelial A549 cells Genya Tanaka1, Kikuo Komori 1, Takao Fujii 1, Hideto Jinno 2, Yasuyuki Sakai1

1Institute of Industrial Science, University of Tokyo; 2 National institute of Health Sciences P-4 The development of an in vitro assay (ROS assay) to predict skin sensitization based on ROS

production Kazutoshi Saito, Masaaki Miyazawa, Yuko Nukada, Hiroki Sano, Hitoshi Sakaguchi, Naohiro Nishiyama, Kao Corporation


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P-5 Inter-laboratory validation study of in vitro eye irritation test; Short Time Exposure (STE) test (Part 3) Kojima H.1, Kuwahara H.2, Hayashi T.2, Sakaguchi M.3, Toyoda A.3, Goto H.3, Nakamura T.4, Watanabe S.4, Ahiko K.4, Omori T.5, Otoizumi T.5, Sozu T.6, Morimoto T.7, Hayashi K.8, Sakaguchi H.8

1National Institute of health Sciences, 2Kanebo cosmetics Inc., 3POLA Chemical Industries, INC., 4LION Corporation, 5Doshisha University, 6Kyoto University, 7Sumitomo Chemical Co., Ltd., 8Kao Corporation

P-6 Validation of a skin irritation study using a Japanese model; LabCyte EPI-MODEL24, Additional study Kojima, H. 1, Nakamura, M. 2, Yamaguchi, Y. 2, Izumi, R. 3, Suzuki, T. 3, Hagiwara, S. 4, Shinoda, S. 4, Kato, M. 5

1 NIHS, 2KOBAYASHI Pharmaceutical Co., Ltd., 3Fancl Corporation, 4Drug Safety Testing Center Co.,Ltd. 5J-TEC

P-7 Optimal conditions for performance of the comet assay using a three-dimensional human

epidermal model Hajime Kojima, Maki Hojyo National Institute of Health Sciences (NIHS)

P-8 The animal experiment substitute teaching materials in the high school science education Hisashi Iwata Iwami High School tottori prefectural

P-9 Student practice incorporating alternative animal experiments in the faculty of

pharmaceutical sciences Tetsuya Hasegawa1, Hiroshi Kawai1, Kaori Matsumoto1, Atsushi Mitsumoto1, Syunji Horie1, Masayuki Akimoto1

1Faculty of Pharmaceutical Science, Josai International University P-10 Involvement of acetylcholine and response to reduction in phosphorylated connexin43 in drug

development research for ischemic heart disease Hideto Ariumi, Ikuya Imai, Tomoko Miyazaki, Miyoshi Kawakami, Yuji Yoshiyama Division of Community Pharmacy, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmacy, Kitasato University

P-11 Establishment of in vitro test method for peel adhesion of adhesive tapes

Maki Shibuya1, Masako Yabe1, 2, Masaki Uchida1, Hideshi Natsume1, 3, Yasunori Morimoto1, 3

1Faculty of Pharmaceutical Sciences, Josai University, 2Department of Pharmacy Services, Ashikaga Red Cross Hospital, 3 Research Institute of TTS Technology

P-12 Evaluation of the palatability by a taste sensor Toshimi Iizuka, Hiroyuki Miyazaki, Hideto Ariumi, Yuji Yoshiyama

Division of Community Pharmacy Center for Clinical Pharmacy and Clinical Sciences, Kitasato University School of Pharmacy

P-13 Statistical support models for validation studies Taku Otoizumi1, Takashi Omori1

1Doshisha University Faculty of Culture and Information Science


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P-14 Model-based approach to sensitivity, specificity, and accuracy in an interlaboratory validation study Takashi Omori Doshisya University Faculty of Culture and Information Science

P-15 A molecular clock mechanism of cytochromeP450 (CYP) genes expression in serum-shocked

HepG2 cells Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo

Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University P-16 A highly sensitive cell-based screening method for CYP2C9-mediated metabolic activation

with an adenovirus expression system Atsushi Iwamura, Tatsuki Fukami, Hiroko Hosomi, Miki Nakajima, Tsuyoshi Yokoi

Faculty of Pharmaceutical Sciences, Kanazawa University P-17 Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic

activation Hiroko Hosomi, Tatsuki Fukami, Atsushi Iwamura, Miki Nakajima, Tsuyoshi Yokoi

Faculty of Pharmaceutical Sciences, Kanazawa University P-18 A basic study on migration to tissue of site probe drugs by using a cell membrane plate Jin Tokunaga1, Norito Takamura1, Kenji Ogata1, Nao Setoguchi1, Nobunao Ikewaki1, Toyotaka Nishio2, Keiichi Kawai2

1School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 2School of Health Sciences, Kanazawa University

P-19 Characterization of Light emitting-Bhas 42 Cells Using for Short Term Screening of Carcinogens Shin Asada1, Kiyoshi Sasaki1, Ayako Sakai1, Yoshihiro Nakajima2, Yosihiro Ohmiya2, Noriho Tanaka1

1 Hatano Research Institute, Food and Drug Safety Center, 2 National Institute of Advanced Industrial Science and Technology

P-20 An international validation study on a Bhas 42 cell transformation assay using 6-well plates

for the prediction of chemical carcinogenicity Ayako Sakai1, Kiyoshi Sasaki1, Kumiko Hayashi1, Dai Muramatsu1, Shoko Arai1, Nobuko Endou1, Sachiko Kuroda1, Fukutaro Mizuhashi2, Sawako Kasamoto2, Miho Nagai2, Masumi Asakura3, Hideki Hirose4, Nana Ishii4, Kamala Pant5, Shannon W. Bruce5, Jamie E. Sly5, Albrecht Poth6, Susanne Bohnenberger6, Thorsten Kunkelmann6, Shojiro Yamazaki1, Makoto Umeda1, Noriho Tanaka1

1Food and Drug Safety Center, 2Biosafety Research Center, Foods, Drugs and Pesticides, 3Japan Bioassay Research Center, 4Mitsubishi Chemical Medience Corporation, 5BioReliance Corporation, 6Harlan Cytotest Cell Research GmbH 1

P-21 Screening of newly identified anti-cancer compounds using a human MDA-MB-231 cell line

and analysis of their anti-neoplastic mechanism Shuso Takeda1, Kazumasa Matsuo2, Yoshiko Okamoto1, Mitsuru Shindo2, Curtis J Omiecinski3, Hironori Aramaki 1

1Department of Molecular Biology, Daiichi University of Pharmacy, 2Interdisciplinary Graduate School of Engineering Sciences and Institute for Materials Chemistry and Engineering, Kyushu University, 3Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University


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P22 Development of high-performed prediction system for chemical toxicity in the cell using a human artificial chromosome (HAC) vector Shigeyuki Yamaguchi1, Tetsuya Ohbayashi2, Yoshihiro Ohmiya3, Shigeaki Nishii4, Hideo Hoshino3, Tomomi Asai4, Yasuhiro Kazuki1, Mitsuo Oshimura1,2

1Grad. Sch. Med., Inst. Regenerative Med. Biofunction., Tottori Univ., 2 Res. Center. Biosci. Tech., Tottori Univ., 3 Res. Inst. Cell Engineering., AIST., 4 Tsuruga. Bio. Lab., Toyobo.

P-23 Generation of ES cells which monitor the cardiac muscle differentiation using a human

artificial chromosome (HAC) Yuki Yoshimura1, Shigeyuki Yamaguchi1,2, Shiho Endo2, Yoshihiro Nakajima3, Yasuhiro Kazuki4, Yoshihiro Ohmiya5, Mitsuo Oshimura1,4, Tetsuya Ohbayashi2

1Dept. of Biomed. Sci., Inst. of Regenerative Med., Tottori Univ., 2 Div. of Laboratory Animal Sci., Res. Ctr. for Biosci. and Technology, Tottori Univ., 3 Health Research Institute., Advanced Industrial Science And technology., 4Chromo. Eng. Res. Ctr., Tottori Univ., 5Bioproduction Research Institute., Advanced Industrial Science And Technology.

P-24 Establishment and characterization of engineered lymphatic endothelial cells with longer

lifespan M Sugano, R Nakamura, M Kiyono, Y Sone, K Inoue Department of Public Health, School of Pharmacy, Kitasato University

P-25 Investigation of the 3D cell culture system using a shape controlled Titanium coil

Takaki Shma, Kazutaka Yoshino, Akihiro Ametani, Yasuo Seki

HI-LEX Corporation Medical Device Department

P-26 Rapid and spatially controlled formation of bile canaliculi in polarized hepatocyte in micropatterned collagen on oxygen-permeable membrane Hitoshi Matsui1,3, Hiroshi Kimura2, Tomoharu Osada3, Masaru Sekijima3, Teruo Fujii2, Shoji Takeuchi2 and Yasuyuki Sakai2

1BEANS Laboratory, 2Institute of Industrial Science, The University of Tokyo, 3Mitsubishi Chemical Medience Co. Ltd.

P-27 Efficient Reconstruction and Evaluation of a Pseudo-Islet Like Aggregate from an Oxygen Permeable Microwell Sheet Marie Shinohara1, Kikuo Komori1, Teruo Fujii1, Yasuyuki Sakai1

1Institution of Industrial Science, University of Tokyo P-28 Minimization of a Three-dimensional Liver Tissue for an In Vitro Toxicity Test

Kikuo Komori, Hiroaki Suzuki, Teruo Fujii, Yasuyuki Sakai Institute of Industrial Science, University of Tokyo

P-29 Hydrogel electrode for effective electrical stimulation of cells

Y. Ido1, S. Sekine1 , D. Takahashi1, K. Nagamine1,2, T. Miyake1,2 , M. Nishizawa1,2 1Tohoku University, 2JST-CREST

P-30 A basic study of estimation of O2 and CO2 transfer in an oxygenator with a numerical analysis

for minimization of the blood experiment N. Katagiri1, A. Funakubo2, T. Tsukiya1, E. Tatsumi1, T. Mizuno1, Y. Takewa1, K. Shioya1, Y. Taenaka1, Y. Fukui2

1National Cerebral and Cardiovascular Center Research Institute, 2 Division of Electrical and Mechanical Engineering, Tokyo Denki University


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P-31 Evaluation of PDMS/PEG Copolymer Impregnated Membranes as a Human Skin Alternative for In Vitro Skin Permeation Testing Ryotaro Miki1, Soichiro Kimura1, Toshinobu Seki1, Kazuhiko Juni1, Hideo Ueda1, Yasunori Morimoto1, 2

1 Faculty of Pharmaceutical Sciences, Josai University, 2 Research Institute of TTS Technology P-32 Evaluation of skin permeability of volatile substances: establishment of evaluation method

using alternative membrane for animal skin Takeshi Sugama, Takeshi Oshizaka, Hiroaki Todo, Kenji Sugibayashi Faculty of Pharmaceutical Sciences, Josai University

P-33 Effect of skin barrier function and metabolic ability on the in vitro skin irritation using

cultured human skin models Katsunori Furui, Hiroaki Todo, Kenji Sugibayashi Josai university faculty of pharmaceutical sciences

P-34 Alternative animal testing of skin sensitization test using three-dimensional human skin model

involving dendritic cells Tadashi Uchino1, Toshiaki Takazawa2, Yoshiaki Ikarashi1, Tetsuji Nishimura1

1National Institute of Health Sciences, 2National Institute of Agrobiological Sciences

P-35 Investigation of an Eye Irritation Test Using a Human 3D Corneal Model (2nd Report) Satoshi Nakahara

Central Research Laboratories, Mandom Corporation

P-36 Evaluation of acute toxicity using Caenorhaboditis elegan

Maki Nakamura, Yoshihiro Yamaguchi, Hironori Tomi KOBAYASHI Pharmaceutical Co., Ltd. Central R&D Laboratory

P-37 Toxic interactions of clomipramine in chick embryos

Hiroyuki Miyazaki, Toshimi Iizuka, Motohiro Okayasu, Toshio Kouzuki, Takeshi Homma, Takashi Ogura, Yohhei Inada, Takenori Tamaki, Hideto Ariumi, Tomoko Miyazaki, Miyoshi Kido, Yuji Yoshiyama

Division of Community Pharmacy Center for Clinical Pharmacy and Clinical Sciences, Kitasato University School of Pharmacy

P-38 The effect of a biotin deficiency on cultured rat embryos Noriko Ishizuka1, Masaharu Akita2, Sayaka Yanagida2, Atushi Yokoyama 3, Naomi Kashiwazaki 4, Junya Ito 4, Masaru Maebashi 5 Tomo Inomata4

1Kiryu University, 2Kamakura Women's University, 3Kanagawa Life Science Research Laboratory, 4Azabu University, 5Honjo 1st Hospital

P-39 The Effect of chemical compound on Cultured Rat Embryos in S-9mix : Sodium Valproate Masaharu Akita1, Noriko Ishizuka2, Atushi Yokoyama3

1 Department of Nutrition and Dietetics, Kamakura Women’s University, 2 Department of Nutrition, Kiryu University, 3Life Science Laboratory of Kanagawa

P-40 Development of in vitro alternative method for developmental toxicity in using differentiation of embryonic stem cells into neural cell Noriyuki Suzuki, Satoshi Ando, Koichi Saito Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd.


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P-41 Development of in vitro cardiotoxicity assays using human iPS cells (Investigation of assay conditions with doxorubicin) Tomoaki Inoue, Jumpei Kiyokawa, Masaki Honda, Akifumi Shioda, Mitsuyasu Tabo, Masayuki Mishima, and Shuichi Chiba Safety Assessment Department, Fuji Gotemba Research Laboratories, Pharmaceutical Co., Ltd.

P-42 Characterization of human iPS-derived cardiomyocytes using gene expression analysis of

cardiac markers Jumpei Kiyokawa, Masaki Honda, Akifumi Shioda, Mitsuyasu Tabo, Masayuki Mishima, Shuichi Chiba, Tomoaki Inoue Safety Assessment Department, Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd.

P-43 Development of a new system on whole embryo culture with reduced serum

Masaharu Akita1, Noriko Ishizuka2 , Atushi Yokoyama3 1Department of Nutrition and Dietetics, Kamakura Women’s University, 2Department of Nutrition , Kiryu University, 3Life Science Laboratory of Kanagawa

P-44 Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system

Kazuaki Nakamura1, Yoko Ejiri2, Akito Tanoue1 1 Department of Pharmacology, National Research Institute for Child Health and Development, 2 Tsukuba Research Center, Kuraray Co., Ltd 1

P-45 A genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells

Taisei Mizota, Katsutoshi Ohno, Toshihiro Yamada

The Food Safety Research Institute, Nissin Foods Holdings Co., Ltd.

P-46 Evaluation of in vitro micronucleus assay using three-dimensional reconstructed human skin model Toshio Kasamatsu, Katsuyuki Yuki, Hideaki Nakagiri, Shoji Matsumura, Naohiro Ikeda, Naohiro Nishiyama Global R&D - Safety Science, Kao Corporation

P-47 In vitro evaluation system to predict phototoxic potential of water-insoluble chemicals using

three-dimensional dermal skin model Maki Kaneko, Masato Kitagaki, Hirokazu Kouzuki Shiseido Research Center

P-48 Comparative Studies on the Draize Rabbit Eye Test and HET-CAM Test of Eye Irritation Test

So-Young Yang2, Nam-Jin Lee2, Eun Young Jeon2, Mi Sook Jung2, Heung-Mo Bae2, Jae-Myun Ryu2, Seong-Jin Baek2, Jong-Koo Kang1

1Department of Veterinary Medicine, Chungbuk National University, 2Biotoxtech Co. Ltd. P-49 Advantages of the human corneal epithelium model reconstructed in a collagen vitrigel

chamber and its application to eye irritation test . Hiroyuki Yamaguchi1, 2, Toshiaki Takezawa1

1National Institute of Agrobiological Sciences, 2Kanto Chemical Co. Inc. P-50 Assessment of eye irritation potencial using the reconstructed human corneal tissue, LabCyte

CORNEA-MODEL Masakazu Katoh, Noriyuki Uemura, Akemi Usui, Fumiyasu Hamajima, Takahiro Ogasawara, Ken-ichiro Hata Japan Tissue Engineering Co., Ltd


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P-51 Assessment of novel eye irritation test using the reconstructed human corneal tissue LabCyte CORNEA-MODEL Noriyuki Uemura, Masakazu Katoh, Akemi Usui, Ken-ichiro Hata Japan Tissue Engineering Co., Ltd

P-52 Risk assessment for eye irritation using cytotoxicity tests

Shigenobu Hagino, Yuuko Matsumoto, Kitagaki Masato, Tsuyoshi Yoshida, Hiroshi Itagaki Shiseido Research Center

P-53 Discrimination of GHS classification 2A or 2B in modified STE test by neutral red(NR) up-take Yuichiro Goto, Miki Nakanishi, Miho Sakurai, Satoko Kuriwada, Noriyasu Imai, Yuko Okamoto KOSÉ Corporation Fundamental Research Laboratories

P-54 Assessment approach (NI/ I) combining in vitro eye irritation tests

Kenichi Ooshima1, Takayuki Abo2, Takumi Hayashi1, Tomoko Komano1, Kazuhiko Hayashi2, Hitoshi Sakaguchi2, Naohiro Nishiyama2, Hirofumi Kuwahara 1

1 Kanebo cosmetics INC, 2 Kao Corporation P-55 A tiered approach combining the STE test, EpiOcular™ assay, BCOP assay for predicting eye

irritation potential of chemicals Takayuki Abo1, Kenichi Ooshima2, Kazuhiko Hayashi1, Takumi Hayashi2, Tomoko Komano2, Hitoshi Sakaguchi1, Hirofumi Kuwahara2, Naohiro Nishiyama1

1 Kao Corporation, 2 Kanebo cosmetics INC.


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Abstracts


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PL Basics and Practice of Alternatives to Animal Experiments ○Yuji Yoshiyama Division of Community Pharmacy Center for Clinical Pharmacy and Clinical Sciences, Kitasato Uninersity School of Pharmacy E-mail: [email protected] The Japanese Society for Alternatives to Animal Experiments (JSAAE) defines alternatives to animal experiments as the replacement of animal experiments conducted for purposes such as research, education, studies and production with methods that use no animals. The replacement is encouraged to reduce the number of animals used (reduction) and relieve animals from pain (refinement) for humane reasons (3Rs principle: replacement, reduction and refinement). It is significant that this principle was included in the “Act on Welfare and Management of Animals” amended in June 2005. Related regulatory authorities issued guidance on animal experiments in order to ensure the achievement of the law objectives. The Science Council of Japan (SCJ) also issued its own guidelines. With the creation of JaCVAM, the construction of systems necessary to promote the replacement of animal experiments and the assessment of alternatives is being accelerated.

Kitasato University, which will host the 23rd JSAAE annual meeting, is based on the founding spirit that focuses on “development of new frontiers,” “gratitude,” “wisdom and practice,” and “persistence and perseverance,” which were practiced by the founder Shibasaburo Kitasato. Dr Kitasato used to urge his students to “be a pioneer, interact with others with gratitude, and be persistent and perseverant in pursuing practical science with wisdom.” By advocating “wisdom and practice,” Dr Kitasato urged his students to put the acquired knowledge and skills in practice and return the fruit of work to society. Kitasato University has decided to focus on “Basics and Practice of Alternatives to Animal Experiments” as the main theme of the 23rd JSAAE annual meeting in order to reflect his teaching in research on alternatives to animal experiments.

In the special lectures entitled “Statistical considerations on the roles of surrogate markers in clinical research” and “Recent topics on chronopharmaceutics focused on molecular clock” we would like to call for a paradigm shift in order to set new viewpoints for research. The symposium entitled “For a revision of the Japanese Act on Welfare and Management of Animals” and educational lectures are expected to teach us the basics of alternatives to animal experiments. It is necessary to develop alternatives to animal experiments not only in accordance with law and regulations but also paying close attention to the social trend. We are also expected to acquire versatile skills through the symposiums on engineering approaches entitled “Bioengineering for alternatives to animal experiments” and “Biological approaches to development of useful cell based assay system for AATEX.” We will be able to be updated about the latest research trend in “Trends of in vitro skin irritation test” and “Development of alternative assay systems for chemical risk evaluation - carcinogenicity, Developmental toxicity, Immunotoxicity -”. JSAAE members should be central players in all JSAAE events. Because it is very important for them to present the results of their daily research activities at JSAAE meetings, we have introduced a “flush oral presentation session” in addition to poster sessions in order to provide members with more opportunities to share information among them. Researchers of alternatives to animal experiments are urged to develop acceptable methods of animal experiments and alternatives based on the achievements presented at the 23rd JSAAE annual meeting.


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SL-1 Data science on validation studies conducted by JSAAE ○Takashi Omori Doshisha University Faculty of Culture and Information Science E-mail: [email protected] [Objective(s)] Scientific evidence is derived from collected data. The responsibilities of a statistical expert include the design of data collection, validating the collected data, construction of databases to analyze data, data analysis, reporting the results, and so on. Some statisticians prefer to use the term “data science,” rather than “statistics,” as the former seems to better reflect their work. My personal experiences with statistical work in validation studies for the development of alternatives just fits to use this word “data science.” Objective of this lecture is to present what we have learned through the data of the validation studies conducted by the Japanese Society for Alternative Animal Experiments (JSAAE). [Materials and Methods] Statistical work on the following 3 validation studies conducted by JSAAE were presented from the statistical expert point-of-view: (Case 1) a large-scale validation study of cell cytotoxicities to investigate candidates for the Draize eye irritation test; (Case 2) validation of a battery of tests, including red blood cell hemolysis assay and yeast growth inhibition assay, to assess an alternative method for phototoxicity testing; (Case 3) validation of the modification to a local lymph node assay, known as the LLNA-DA. [Results and Discussion] In case 1, over 1500 electronic data files were collected. Unfortunately, these data files required corrections because many of them were not prepared according to our expectations. Thereafter, data management became an important issue. In case 2, 2 graphs of positive control chemicals for pre-experiment and formal-experiment suggested that, the standard operating procedure might be altered at a laboratory level. This graphical display indicated the need for checking the transferability. In case 3, the plot of stimulation index, which is an important measure of the assay, was prepared with its confidence interval as the error bar. This graph showed the results of the approximate statistical tests on the SI scale and successfully provided evidence for inter-laboratory reliability of the LLNA-DA assay.

These cases indicated that statistical work begins at the planning stages of the validation study. Discussion with the members of the management team and the investigators is important for data management and data analysis. To obtain successful results from the validation study, not only a well-designed test method but also a good relationship and understanding between researchers is required.


189

SL-2 Recent topics on chronopharmaceutics focused on molecular clock ○Shigehiro Ohdo Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University E-mail: [email protected]

Mammalians circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN). Clock genes are the genes that control the circadian rhythms in physiology and behavior. Molecular clockworks in peripheral tissue are regulated by a complex interaction of pathways that include extracellular and intracellular signals. However, under certain conditions, such as in the case of tumour, the peripheral clocks could operate independently of the central pacemaker. On the other hand, disruption of circadian rhythm not only increases the risk of tumour development, but also accelerates cancer progression. The cellular processes that are regulated by circadian rhythm are the theoretical foundations for cancer chronotherapy. The antitumor effect and/or toxicity of antitumour drugs vary depending on dosing time associated with 24-hr rhythm of their target enzyme, receptor and pharmacokinetics. For example, the antitumor effect and/or toxicity of irinotecan hydrochloride, interferon and antiangiogenic agents vary depending on dosing time associated with 24-hr rhythm of their target enzyme, receptor, protein and pharmacokinetics. However, many drugs are still given without regard to the time of day. Identification of a rhythmic marker for selecting dosing time will lead to improved progress and diffusion of chronopharmacotherapy. To monitor the rhythmic marker may be useful to choose the most appropriate time of day for administration of drugs that may increase their therapeutic effects and/or reduce their side effects. On the other hand, several drugs can cause alterations to the 24-hr rhythms, which leads to illness and altered homeostatic regulation. The alteration of the clock function, a new concept of adverse effects, can be overcome by devising a dosing schedule that minimizes adverse drug effects on clock function. Furthermore, to produce new rhythmicity by manipulating the conditions of living organs by using rhythmic administration of altered feeding schedules or several drugs appears to lead to the new concept of chronopharmacotherapy. One approach to increasing the efficiency of pharmacotherapy is administering drugs at times during which they are best tolerated. During the process, we have succeeded in the reproduction of circadian rhythm in clock genes, clock-controlled-genes and their target enzyme, receptor and pharmacokinetic factors in vitro cultured cells. Thus we have established the chronopharmacokinetic evaluation system in vitro. The evaluation system leads to the development of chronotherapeutic strategy. The establishment of in vitro drug evaluation system from viewpoints of chronopharmacology is very important for development of alternative to animal experiment methods. In this presentation, I introduce the recent topics on chronopharmaceutics focused on molecular clock.

References 1. Ohdo S (Theme Editor), Chrono-drug-delivery focused on biological clock: Intra- and inter-individual variavility of molecular clock. Advanced Drug Delivery Reviews 62, 885-897, 2010. 2. Ohdo S, Koyanagi S, Suyama H, Higuchi S, Aramaki H. Changing the dosing schedule minimizes the disruptive effects of interferon on clock function. Nature Medicine 7, 3, 356-360, 2001. 3. Matsunaga N, Ikeda M, Takiguchi T, Koyanagi S, Ohdo S. The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver. Hepatology 48, 240-51, 2008. 4. Murakami Y, Higashi Y, Matsunaga N, Koyanagi S, Ohdo S. Circadian clock-controlled intestinal expression of the multidrug-resistance gene mdr1a in mice. Gastroenterology 135, 1636-1644, 2008. 5. Okazaki F, Matsunaga N, Okazaki H, Utoguchi N, Suzuki R, Maruyama K, Koyanagi S, Ohdo S. Circadian rhythm of transferrin receptor 1 gene expression controlled by c-Myc in colon cancer–bearing mice. Cancer Reseach 70(15), 6238–6246, 2010.


190

S1-1 Situation at the Ministry of the Environment for the Amendment of the Animal Protection Law ○Yoshihiro Hayashi Faculty of Agriculture, Tokyo University of Agriculture E-mail: [email protected] The Central Environment Council’s Committee on Animal Protection under the Ministry of the Environment deals with important issues related to the care and management of animals. According to its minutes, its members deliberated on how to enforce the Law on the Protection and Management of Animals amended in 2005 and the Basic Guidance for Animal Protection and Management. At a meeting on October 13, 2006, the committee approved a draft guidance to be submitted to the council. At its meetings held on August 3, 2007 and onward, the committee deliberated on how to amend various standards introduced in accordance with the law and recommended approval of various guides and guidance such as the Guidance on How to Sacrifice Animals. The committee also reviewed the Basic Guidance for Animal Protection and Management and deliberated the amendment of the above-mentioned law on June 16, 2010. As chairman of the committee, Dr. Yoshihiro Hayashi played a central role in leading discussions. In this symposium, he will present the prospects for the amendment of the law. (By the symposium organizer)


191

S1-2 Perspectives on amendment of the Animal Law Laboratory animals (1): To implement science-based laboratory animal welfare

○Naoko Kagiyama Hokkaido University Graduate School of Veterinary Medicine [email protected] The Law for the Humane Treatment and Management of Animals specified the 3R-Principle of humane experimental technique. Based on the 3R-Principle, government ministries promoting science and technology promulgated the Basic Policies on Animal Experiments, and the Science Council of Japan edited the Guidelines for Proper Conduct of Animal Experiments as vital references for the Policies. On the other hand, the Ministry of the Environment established the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain based on the Law.

Scientists have made proactive and cooperative efforts to conduct appropriate care and use of laboratory animals based on these Policies and Standards. As a result, these efforts have brought scientific and ethical animal experimentation without any disadvantages and nuisance to the general public. If regulatory restrictions on laboratory animals had been reinforced, it would have hindered the development of inventive and innovative in vivo research for the health and welfare of the people.

In summary, 1 The Standards and the Basic Policies have been voluntarily implemented by research institutions in collaboration with related organizations. 2 Breeders and research institutions have achieved science-based laboratory animal welfare under the responsibility of the director of the institution. 3 Public and institutional education and qualification systems have been promoting the 3R-Principle, and the effects are verified by assessment and accreditation of outside peer reviews. 4 Western countries respect the policy of science-based laboratory animal welfare in our country where humane treatment of laboratory animals can reach a compromise with scientific rationale of animal experimentation. 5. The National policy on the continuous promotion of science and technology in Japan will be ruined if regulations on the care and use of laboratory animals are reinforced.


192

S1-3 Trend of International Advancement of Alternative Program Comparison with Japanese Legislation and Guidelines ○Tsutomu Miki Kurosawa, DVM, M.Phil, Ph.D, DVCS, DJCLAM OsakaUniversity Graduate School of Medicine Department of Laboratory Animal Medicine E-mail: [email protected] [Objective(s)] The more stringent legislation in animal experimentation is internationally significant. In any of these legislations, the promotion of alternative research is emphasized. However all of legislations state that the importance of animal experiments for the advancement of science. Therefore the more precise descriptions in Refinement (Reduction of Pain and Distress) are prominent. The purpose of this lecture is to propose new opinion in Refinement after the comparison of International trend of legislation with Japanese legislation in refinement. [Materials and Methods] The realistic facts including OIE laboratory animal welfare code, US ILAR Guide (revised) and Protection of animals used for scientific purposes (European Parliament legislative resolution) were selected. Additionally CIOMS Guiding principles for Biomedical Research involving Animals which is revised by ICLAS is discussed. The comparison is made among these international legislations and Japanese Law for the Humane Treatment and Management of Animals, Standards Relating to the Care and Management of Experimental Animals and Guidelines for Proper Conduct of Animal Experiments [Results and Discussion] OIE laboratory animal welfare standard recommends that Members address all the essential elements identified in this chapter in formulating a regulatory framework that is appropriate to their local conditions. Because we do not have regulations to control animal experiments due to scientific freedom which should not require the regulatory control of animal experiment in Japan, we should create a new regulation to control animal experiments. The ILAR Guide is a basic documents for AAALAC International which is a highly recognized an accreditation organization for laboratory animal care and use. The Guide was translated to 13 different languages including Japanese version translated by Dr. Kagiyama et al. The Guide was widely used in particular for housing environment which was not defined in Japan well. However the essence of the Guide is for Veterinary Care in Refinement and the Guide revised describes the detail of Veterinary Care very much. Contrary to this, there is a paucity of veterinary care in Japanese legislation and guides. Eu legislative resolution orders member states to implement their legislation in animal experiments. The sentences of this document emphasize 3Rs principles. ICLAS is also revising CIOMS Guiding Principles which emphasizes the veterinarians role for laboratory animal welfare and refinement in 3Rs. The revised Guiding Principle is more stringent than that of previous version.


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S1-4 Are Animal Experiments the Ultimate Cruelty to Animals? ○Koichi Aoki The Japanese Coalition for Animal Welfare E-mail: [email protected] The Act on Welfare and Management of Animals amended in June 2005 calls for the 3R principle that should be followed in the use of animals for scientific purposes: Replacement of animal experiments with alternatives that use no animals whenever possible (Clause 1, Article 41), Reduction in the number of animals used (Clause 1, Article 41), and Refinement of the way animals are treated to minimize their pain and discomfort (Clause 2, Article 41). In addition, Clause 3, Article 41 calls for the disposal of animals in a way that minimizes their pain.

The Standards for Breeding and Maintenance of Experimental Animals and Relief of Their Pain were issued by the Ministry of Environment in 2006 based on the amended law, followed by the Basic Guidelines for Conducting Animal Experiments at Research Facilities issued by the Ministry of Education, Culture, Sports, Science and Technology, the MHLW Basic Guidelines for Animal Experiments issued by the Ministry of Health, Labor and Welfare, Basic Guidelines for Conducting Animal Experiments at Research Facilities Controlled by the Ministry of Agriculture, Forestry and Fisheries (MAFF) issued by the MAFF, and the Guidelines for Proper Implementation of Animal Experiments issued by the Science Council of Japan. Scientists are required to conduct research activities in accordance with all these guidelines. In addition, the preparatory committee of the liaison council for people involved in animal experiments issued a guidebook designed to ensure the optimization of animal experiments, “Good Health Ensures Tomorrow’s Happiness – with Thanks to Experimental Animals,” in order to help not only professionals involved in animal experiments but also the general public better understand animal experiments.

Although Japan is ruled by law, the Act on Welfare and Management of Animals is the only law related to animal experiments, and there is no law that directly rules animal experiments. The government does not plan to introduce any new law that will specifically rule animal experiments.

When the Act on Welfare and Management of Animals was amended, the liaison council demanded that requirements other than the 3Rs also be included in the new law, but scientists unanimously and systematically presented a strong objection to this demand. The Council’s proposals were neglected and rejected without any discussion or verification. The government accepted the scientists’ demand as legitimate and forced animal right activists to be satisfied with the addition of two Rs to a single R.

As the law will be amended again in 5 years, we would like the government to more seriously consider the requirements we proposed previously in order to ensure greater transparency as to how animal experiments are conducted and how animals are treated. At present, animal experiments are conducted in the dark and there is no way to determine how they are conducted.

It will be necessary to inspect experimental facilities on site beforehand and register them because various problems have been reported such as the escape of recombinant mice from laboratories and problems related to the construction of facilities for animal experiments by Takeda Pharmaceutical.

A number of alternatives to animal experiments have been developed. We hope that the number of experimental animals which suffer from pain can be reduced by taking advantage of these alternatives, but will this dream come true?


194

S1-5 Where Does Humane Science Should Head to? ○Hiromi Kamekura

NPO Japan Anti-Vivisection Association [email protected] JAVA is a citizen’s group established in 1986 that opposes animal testing and seeks abolition of animal experiments. In 1997, JAVA became a supporting member of the Japanese Society for Alternatives to Animal Experiments (hereinafter “the Society”) to support alternatives as a humane and scientific method to replace with prevailing animal experiments.

Together with other major animal protection groups in Europe, Canada and the United States, we have set up ICAPO (International Council on Animal Protection in OECD Programmes) and ICAPPP (International Council on Animal Protection in Pharmaceutical Programmes). Our practical activities, from animal protection activist’s point of view, include proposing to OECD and ICH that alternative methods should be adopted to the test guidelines, in order to abolish animal testing.

In 2005 at the second revision of Act of Welfare and Management of Animals first established in 1973, Principles of 3Rs, all three, were added with regard to animal testing (use of animals for scientific purposes), in which only Refinement was included. With this movement, the Japanese society of scientists as a whole is required to be responsible for promoting and developing alternatives to animals testing in terms of animal welfare.

The area of “alternative methods” studies could not have been born without the existence of citizens who are against animal testing. Naturally, it is mandatory for the Society to come up to their expectations, recognizing that a lot of people think a great deal of the importance of animal welfare.

However, regrettably we sometimes hear that some members of the Society think that “3Rs does not mean ‘abolition of animal testing’.” We must clarify that the ultimate purpose of 3Rs should be to decrease in the incidence or severity of inhumane procedures applied to those animals which have to be used, and replacement as animal experimentation replace methods which use conscious living vertebrates. In other words, it is no other than abolition of animal testing. If we give up the position looking at this issue in perspective, it is impossible to pursue our aim; “the most humane science.”

This announcement represents the voice of citizens who request abolition of animal testing. Now, expecting the 3rd revision of Act on Welfare and Management of Animals, it strongly requests that the Society professing “alternatives to animal experiments”, especially those who will lead the Society into the next generation, firmly decide the destination of alternative method studies, and sincerely wishes the Society to further grow in the right direction based on the standpoint of civil society.


195

S2-1 Electrochemical Detachment of Cells and Cell Sheets for Engineering Cellular Tissues

○Junji Fukuda, Hiroaki Suzuki Graduate School of Pure and Applied Sciences, University of Tsukuba E-mail: [email protected] By adopting electrochemical approaches and using microfabrication techniques, we have been focusing on the development of culture substrates for regenerative medicine as well as microanalysis systems for biochemical examinations. In this presentation, I will introduce three topics that may be related to Alternatives to Animal Experiments. (1) Culture vessel for engineering cell sheets: It is well known that molecules that are bound to a gold surface via a gold-thiolate bond can be reductively desorbed by applying a negative electrical potential. We found that by using this desorption process, even fairly large objects such as cells can be detached from the surface. On the basis of this phenomenon, we developed a culture vessel in which cell sheets can be noninvasively harvested and assembled one above the other to obtain a thicker cell sheet. (2) On-chip cell evaluation system: To evaluate cellular functions using a minimum amount of cells and reagents, we fabricated a microsystem in which cells were cultured in a culture chamber with a volume of 1 µl. The culture medium was extracted at the nanoliter scale with a predetermined interval using electrowetting-based microvalves, and analytes in the medium were detected simultaneously using a sensor integrated into the chip. (3) Plug-type biochemical analysis chip: If solutions are handled in the form of liquid plugs, many solutions can be treated in a single chip for high-throughput biochemical sensing. We demonstrated that a relatively complex procedure of handling solutions used for the enzymatic sensing of an analyte can be carried out efficiently by using our plug-based coulometric microsystem.


196

S2-2 Microfabrication of co-culture systems ○Nishizawa M.1, 2，Kaji H.1, 2，Nagamine K.1 ,2，Kanzaki M.2, 3

1Department of Engineering, Tohoku University，2JST-CREST，3Department of Medical Engineering, Tohoku University E-mail: [email protected] Controlling the environments of cell culture systems is a crucial component of in vitro cell-function studies and of how best to design tissue constructs that mimic the organizational complexity of in vivo tissue architectures. Model systems designed to accurately reflect tissue architectures are urgently needed given the recent campaigns against animal experiments. We have developed the jointed [1] and laminated [2] co-culture systems.

The bioassay system incorporating skeletal muscle cells is required to reveal the complex mechanisms involved in the development and maintenance of type2 diabetes because type2 diabetes is closely associated with defection of glucose uptake in skeletal muscle cells. An assay system, currently available, that is used to monitor skeletal muscle cell contraction and activity consists of myotube monolayer cultured on substrates with a pair of electrodes for stimulation. By applying electric pulse stimulation, sarcomere assembly is accelerated and allows for cell contraction. However, the contracting myotubes were difficult to maintain their structure for a long period of time because they readily detached from the substrate within a few days.

Recently, we prepared contractile C2C12 myotube line patterns embedded in a fibrin gel to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. The frequency and magnitude of myotubes contraction were functions of the pulse frequency and duration, respectively. The patterned C2C12 myotubes, supported by the elastic fibrin gel, showed larger contractile displacement than did a myotube monolayer that was attached on a culture dish. The myotubes/fibrin gel system also maintained the line pattern and contractile activity for a longer period of time (one week) than did the myotubes of the dish system. Now, we are investigating contraction-mediated translocation of the GLUT4 glucose transporter protein in myotubes using this system.

[References] [1] Lab Chip, 9 (2009) 427; Lab Chip, 10 (2010) 2374. [2] Biotechnol. Bioeng., 105 (2010) 1161; J. Am. Chem. Soc., 2010, DOI: 10.1021/ja1062357. Jointed Co-Culture Laminated Co-Culture HeLa HUVECs


197

S2-3 Perfusion Culture Microchamber Array Chip for High Throughput Drug Dose Response Assay ○Toshiyuki Kanamori, Shinji Sugiura, Koji Hattori, Kimio Sumaru National Institute of Advanced Industrial Science and Technology (AIST) E-mail: [email protected] The R&D cost for drug discovery is recently rising more and more, they say, 0.1 billion JPY is needed to launch one new drug, leading to the depression in newly-launched drugs. To overcome this issue, it is very important to streamline and accelerate the earlier stage of drug discovery, especially lead optimization. For this purpose, so-called cell-based assay is utilized in the screening of lead compounds even at this moment, but the reliability is insufficiency because the results obtained with the existing technologies are not well corresponding to the downstream evaluations such as animal and clinical studies. We have pointed out the following two defects of the existing cell-based assay technologies; 1) standard cells for the assay is not obtainable, leading to the variation due to the individual variability of the cells, 2) the natural function of the cells does not develop.

To make the function of the cells closer to that in vivo, we suppose that the micro environment of cell culture should be similar to that in vivo. We are now looking at micro process to cultivate cells, because it is easy to precisely control the cell-culture environment in the micro process. Additionally, high throughput screening, which is necessary for drug discovery, is easily feasible on a microchip, where many micro processes can be integrated.

We have developed the perfusion culture microchamber, which can overcome the existing drug-assay technologies using multi-well plate1), and we have also developed the gradient mixer, by which a set of solutions with different concentrations following a decreasing function can be automatically prepared on a microchip2). Finally, we have developed the perfusion culture microchamber array chip where the perfusion culture microchamber and gradient mixer are integrated. Using the chip, 12 drugs x 8 concentrations x 4 data assays are simultaneously feasible3.. In the near future, the system will be commercialized through a venture business based on AIST technology transfer. References 1) Sugiura S., et al.: Biotech.Bioeng., 100, 1156 (2008) 2) Hattori K., , et al.: Lab Chip, 9, 1763 (2009) 3) Sugiura S., , et al.: Annal.Chem., in press


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S2-4 Wearable Bio/Chemical Sensor with Soft-MEMS Techniques ○Kohji Mitsubayashi Institute of Biomaterials & Bioengineering, Tokyo Medical and Dental University E-mail: [email protected] Wearable bio/chemical sensors have been required for non-invasive physical monitoring in the field of the medical and healthcare sciences. Rapid increasing of diabetes mellitus is now global problem and development of a safe and convenient blood sugar level monitoring technology is strongly required. And, development non-invasive and continuous method of blood sugar monitoring, which is available in daily life within a reasonable cost, is strongly requested. We paid attention to the relationship between the tear glucose level and the blood sugar level. In this study, a contact lens type glucose sensor using biocompatible polymers was fabricated and evaluated. The obtained CL sensor was applied to monitor rabbit tear sugar as the non-invasive approach.

The CL biosensor was designed for continuous glucose monitoring in tear fluids on eye site. In order to achieve flexibility and biocompatibility, the biosensor utilizes hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with 2-ethylhexyl methacrylate (EHMA). Owing to the CL structure of PDMS and poly(MPC-co-PMEH) (PMEH), the sensor fits the rounded shape of human body and has good biocompatibility. The sensor measures the glucose concentration as a current change induced by enzyme reaction at the GOD immobilized PMEH layer. In the MEMS fabrication process, the CL electrodes were formed on PDMS substrate using ion beam sputtering method and patterned using lift-off process. Then, it was coated with PMEH and GOD was immobilized on the sensing region of the CL electrodes by coating a mixture of PMEH solution and enzyme. The CL biosensor was applied directly to Japanese White Rabbit eye without any narcotic treatment for the tear sugar analysis.

The sensor showed linearity in glucose level of 0.05 – 3.00 mmol/l with a correlation coefficient of 0.998. The calibration range includes the reported concentration of tear glucose in normal human subject (0.14 - 0.23 mmol/l). The CL sensor also showed high flexibility and was soft and comfort to the touch. The sensor was attached on the rabbit eye as mentioned before and tear glucose level of the rabbit eye was monitored continuously. The average value of tear glucose level was 0.11 mmol/l. Those glucose concentrations in tear fluids were estimated from the calibration plots. There are no damage on cornea and conjunctiva in medical diagnosis after the examination.


199

S3-1 Suggestion of an ideal evaluation system using a corneal epithelial cell from the experience of CS-088 antiglaucoma ophthalmic solution ○Takayuki Kikuchi Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd. E-mail: [email protected] [Objective(s)] In the development of a drug product, various tests such as pharmacology, toxicology and formulization are required in pre-clinical studies. While in vivo studies using animals are inevitable in some of the tests such as drug mechanism and distribution, researches have been conducted to find alternatives methods considering animal protection and variation of individuals. Our company has experience of developing CS-088 as a novel type antiglaucoma agent1). In the formulization studies of CS-088, an in vitro penetration chamber with excised rabbit cornea was used for formulation screening and elucidation of penetration mechnism2-4). In addition, an attempt was made to evaluate cellular damage from LDH released from cultured cells. [Materials and Methods] In vitro penetration chamber system with excised rabbit cornea was used to evaluate the permeability of CS-088. Rabbit corneal epithelial cells were incubated using a commercially available kit (Kurabo, CornePack®). Cellular damage was estimated by measuring LDH which was released from the cells. [Results and Discussion] Corneal permeability of CS-088 was synergistically enhanced by the addition of boric acid and EDTA. This result correlated well with the effects of IOP (Intraocular Pressure) reduction when CS-088 was administered into rabbits in vivo, indicating that the results obtained in the in vitro penetration study allow us to predict drug efficacy. On the other hand, cellular damage in the presence of CS-088 could be compared with other ophthalmic solutions commercially available by using cultured cells. In my presentation, I would like to suggest replacement with cultured cells and discuss these issues. [References] 1) Inoue, T, et al. (2001) Curr. Eye Res., 23,133-138. 2) Kikuchi, et al. (2005) Int. J. Pharm. 290,83-89. 3) Kikuchi, et al. (2005) Int. J. Pharm. 299,100-106. 4) Kikuchi, et al. (2005) Int. J. Pharm. 299,107-114.


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S3-2 Development of a human cornea model utilizing a collagen vitrigel membrane as a scaffold and its advantages ○Toshiaki Takezawa1、Hiroyuki Yamaguchi1, 2

1National Institute of Agrobiological Sciences, 2Kanto Chemical Co. Inc. E-mail: [email protected] A collagen vitrigel (CV) membrane composed of high density collagen fibrils equivalent to connective tissues in vivo not only possesses excellent transparency and permeability of protein with high molecular weight but also is easily handled with tweezers (1, 2). Therefore, the CV membrane is a useful scaffold to construct various three-dimensional culture models with paracrine interaction between different kinds of cells cultured on both surfaces of it. Also, it has an excellent slow release potential of pre-involved growth factor(s) and consequently it can function as a biomaterial useful for DDS (drug delivery system) (3). Further, the CV can be fabricated into a variety of shapes and can be hybridized with other materials (4, 5). These findings suggest that the culture models utilizing a CV membrane scaffold could be applied to toxicological and pharmacological assays in alternative methods for animal experiments and drug development. Also, it is expected that the utility of the CV would be expanded in regenerative medicine by modifying its function as a DDS and/or architecture (6).

To develop the alternative method for the Draize eye irritation test using rabbits, we established a culture technology to reconstruct a rabbit corneal epithelium model by culturing normal rabbit corneal epithelial cells on the CV membrane scaffold and inducing differentiation to form multilayers of the cells (7). Further, a human corneal epithelial model with barrier function was reconstructed by culturing HCE-T (a human corneal epithelium-derived cell line) cells on the CV membrane scaffold in a Millicell chamber suitable for TEER (transepithelial electrical resistance) measurement. Consequently, eye irritancy of chemicals could be estimated as the time-dependent changes of TEER induced by exposing them to the model (8). However, multi-porous membrane of the chamber is made of polyethylene terephthalate and is inappropriate not only for preparing cryo-sections but also for reconstructing a corneal model reflecting structure and its component in vivo.

Recently, we succeeded in developing a CV chamber that can facilitate the reconstructing process of an organ model composed of epithelium, mesenchyme and endothelium. Also, such a model reconstructed in the CV chamber was easily subjected to TEER assay and cryo-sectioning. The changes of TEER induced by exposing eye-irritant chemicals to the human corneal epithelium model reconstructed in the CV chamber were well correlated with the Draize score. [References] (1) Takezawa T. et al. Cell Transplant. 13: 463-473, 2004. (2) Takezawa T. et al. Cells Tissues Organs 185: 237–241, 2007. (3) Takezawa T. et al. J. Biotechnol. 131: 76-83, 2007. (4) Takezawa T. et al. Tissue Eng. 13: 1357–1366, 2007. (5) Takezawa T. et al. Yakugaku Zasshi 128: 51-60, 2008. (6) Takezawa T. et al. Yakugaku Zasshi 130: 565-574, 2010. (7) Takezawa T. et al. AATEX 13 (Suppl.): 176, 2008. (8) Takezawa T. et al. In submission.


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S3-3 Induction of drug-metabolizing enzymes by layered co-culture of a human liver cell line ○Akiyoshi Taniguchi1, 2、Maki Ohno1

1Biomaterials Center, National Institute for Materials Science, 2Faculty of Science and Engineering, Waseda University. E-mail: [email protected] [Objective(s)] Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells (BPAEC), the expression levels of various CYP genes were significantly increased [1-4]. We examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. [Materials and Methods] We constructed the layered co-culture system using temperature-sensitive polymer grafted surfaces. BPAEC cells were seeded on the PIPAAm-coated dishes and BPAEC sheets grown at 37°C. The temperature was decreased to 20°C to induce the detachment of BPAEC sheets; BPAEC sheets are stratified to HepG2 and assembled into a layered co-culture system. [Results and Discussion] In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital. We also detected CYP3A4 enzyme induction using this co-culture system [5]. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism. [References] 1) Kurosawa, Y., Taniguchi, A. Okano, T. (2005) Tissue Eng. 11, 1650-1657. 2) Takayama, G., Taniguchi, A. Okano, T. (2007) Tissue Eng. 13, 159-166. 3) Ohno M., Motojima, K., Okano, T., Taniguchi, A. (2008) Tissue Eng. 14, 1861-1869. 4) Ohno M., Motojima, K., Okano, T., Taniguchi, A. (2009) J Biochem., 145, 591-597. 5) Ohno M., Motojima, K., Okano, T., Taniguchi, A. (2009) Biol. Pharm. Bull. 32, 813-817.


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S3-4 Usability of hepatocyte spheroid culture on Cell-able in DMPK research: results with human and rat primary hepatocytes ○Shin Enosawa Division for Advanced Medical Sciences, Clinical Research Center, National Center for Child Health and Development E-mail: [email protected] [Objective(s)] Cell-able by Transparent Inc. is a patterned cell culture plate originally developed by Otsuka and Kataoka1, using hydrophilic polymer. Cell-able has approximately 800/well (96-well plate) or 8,000/well (12-well plate) of small circular cell-attaching area (100 micro-m in diameter with 100 micro-m spacing). This novel cell culture platform aims hepatocyte-spheroid culture. From the viewpoint of drug metabolism and pharmacokinetics (DMPK) research, the usability of hepatocyte spheroids formed on Cell-able is discussed. [Materials and Methods] After bovine aortic epithelial cell line, HH (JCRB0099) were seeded on 96-well or 12-well Cell-able, rat or human hepatic parenchymal cells were inoculated. CYP activities, albumin synthesis, transporter activities were evaluated as hepatic functions. Also, observation of transmission electron micrograph observation was performed. [Results and Discussion] 1.Stable CYP activities and albumin synthesis were observed with Cell-able culture compared to conventional monolayer culture. The CYP induction was more distinct with Cell-able than with monolayer. 2.Transmission electron microscopy revealed a Disse-space like structure between endothelial HH cells and hepatocytes, while hepatocytes tightly adhered each other. 3.Cryopreserved hepatocytes with low attaching capability (suspension culture lots) can be used for long-term culture experiment2. 4.Feeder HH cells cultured on Cell-able can be cryopreserved. Also, feeder HH cells were kept static on Cell-able without showing overgrowth. These two methods provide for unscheduled use. 5.Activities of efflux and influx transporter (detected by [3H]-estrone, CDF-DA) were detected. Human primary hepatocytes were indispensable to drug development. Because of poor availability of human hepatocytes, Japanese researcher depends on imported cryopreserved hepatocytes. However, most cryopreserved lots were of low attaching capability and not suitable for culture experiment. By Cell-able culture system, hepatocytes with low attaching capability can be used for culture experiment. In addition, the system keeps hepatic differentiated functions such as CYP activities and albumin synthesis. Taken together, Cell-able culture system is a promising tool for drug discovery. [References] 1. Otsuka et al. ChemBioChem 5; 850-5, 2004 2. Enosawa et al. Drug Metabolism Reviews 39; 342, 2007 3. Miyamoto et al. Cell Transplant 18; 677–681, 2009


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S3-5 Development of culture system for induction of hepatic stem cells from human fetal hepatocytes ○Taku Matsushita Lab. of Biomedical Eng., Dept. of Applied Life Science, Sojo University E-mail: [email protected] [Objective] For the drug development, the establishment of the evaluation test system using human hepatocytes for the prediction of ADMET is strongly desired, because of the species differences of the drug metabolism related enzymes between human and animals. We have succeeded in the induction of human hepatoblasts from fetal hepatocytes, which are known to be hepatic stem cells in fetal liver, and present about the enhancement of CYP3A4 activity of human hepatoblasts by three dimensional (3D) culture. [Materials and Methods] Human fetal hepatocytes (Hc cells) were purchased from DS Pharma Biomedical Co., Ltd. with a certification of informed consent for research and cultured in CS-C complete medium with 10% FBS. Hepatoblast were induced from Hc cells by the treatment with sodium butyrate (SB) and distinguished from other cells by immunostaining for albumin and cytokeratin 19 as protein markers using flow cytometry. 3D culture of hepatoblasts was performed by using poly-L-glutamic acid-coated dishes and porous hydroxyapatite carriers. CYP3A4 activity of the cells was specifically induced by rifampicin and measured as a BROD activity. [Results and Discussion] The delivered Hc cells (primary culture) were serially passaged and the 3rd-7th passaged cells were used in all experiments. Although hepatoblasts were hardly detected in primary Hc cells (about 2%), the population of hepatoblasts clearly increased by the treatment of 5mM SB for 8 days culture (about 90%). The growth of the cells was inhibited by 5mM SB, but maintained under 1mM SB. The hepatoblasts proliferated about 175-fold for 12days culture under 1mM SB and the doubling time of the cells was 38.4h. By the 3D culture of human hepatoblasts using porous hydroxyapatite carrier under 1mM SB, CYP3A4 activity of the cells was enhanced and reached at 4.2 pmol/mg protein/min, which was comparable to that of primary human hepatocytes from adult liver. [References] 1) Ozawa S, Altern. Animal Test. Experiment. 9, 98-105 (2003). 2) Matsushita T, et al., Cytotechnology 42, 57-66 (2003). 3) Matsushita T, et al., J Oral Tissue Engin. 4, 25-31 (2006). 4) Kiyota A, et al., Biol. Pharm. Bull. 30, 2308-2311 (2007). 5) Ijima H, et al., Biochem. Eng. J. 47, 19-26 (2009).


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S3-6 Application of human stem cells to the safety/toxicity study of drug candidates --- Road to the guideline plan making ○Seiichi Ishida National Institute of Health Sciences E-mail : [email protected] In the process of drug development, the safety/toxicity of drug candidates is evaluated, assuming their administration to human, in liver which plays important role in the pharmacokinetics such as absorption, distribution, metabolism, and excretion and on central nervous system, respiratory system and cardiovascular system, so called core battery, which are essential for the maintenance of life support. Many processes of the pharmacokinetics tests in the liver or the safety/toxicity tests in the core battery depends on the animal test. However, the establishment of the evaluation test system using human tissues applicable at the pre-clinical stage is desired, because there exist species differences, which often cause the extrapolation limitation of the results obtained by animal tests to human. Utilization of human primary cells or tissues is preferable, although to get nerve cells or cardiac muscle cells is difficult. There are also problems of human hepatocytes (liver cells) such as a cost or stable supply, because the source of these cells depends on foreign countries nearly 100% due to the regulation in Japan. Many studies have been done so far to get a variety of organ cells by differentiating human stem cells and to use them for the in vitro safety/toxicity test. After the establishment of human iPS cells by Yamanaka et al. in 2007, the speed of the study has been accelerated. Once the iPS derived target tissue cells become available for the pharmacokinetics tests in the liver or the safety/toxicity tests in the core battery, the shortening of the period and the reduction of the cost of drug development are expected.

We have promoted the study to develop in vitro toxicity test using the cells differentiated from human iPS cells under Dr. Hiroyuki Mizuguchi as a project leader from 2008 at National Institute of Biomedical Innovation (http://www.nibio.go.jp/Super Tokku/ips.html). We have already succeeded to differentiate human iPS cells into hepatocytes or cardiac muscle cells. Thus iPS cells are the promising source of an alternative to primary human cells. On the other hand, several difficulties have been clarified during these studies, e.g. it is difficult to go through the complicated differentiation process with reproducibility; it is still insufficient to get the matured cell phenotype by the reported protocol, etc.

Standardization and validation, along with to solve the difficulties above, are required to replace the commonly used safety/toxicity tests with those using iPS derived cells. I will discuss what we need to consider developing the alternative safety/toxicity tests based on iPS derived cells.


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S4-1 Current status of the alternative to the skin irritation test method ○Yuko Okamoto KoseCorporation, Fundamental research laboratories The development of alternative testing for skin irritation has been begun formally in EU since 1998. Through the research for ten years or more, Reconstructed Human Epidermis Test Method has been adopted as the OECD test guideline as the in vitro skin irritation test method for chemicals (TG439) in July, 2010. The skin irritation testing is generally used for the hazard identification caused by chemicals. This Test Guideline (TG439) can be used for the hazard identification of chemicals with UN GHS (The Globally Harmonized System of Classification and Labeling of Chemicals) and EU CLP. In this case, the testing is carried out by the short exposure time (4hr.) and the high concentration.

In the viewpoint of animal welfare, the development of the alternative that can identify the hazard of chemicals to the skin is very important, and is expected to promote the 3Rs. On the other hand, the skin irritation testing is used for the confirmation of safety in chemicals applied to the skin (ingredients of domestic products, ointments and cosmetics). In this case, the testing method should select an appropriate concentration and the exposure time according to the evaluation object.

In Japanese quasi-drug that needs such skin irritation testing as the safety confirmation, "Advisory committee for propriety of safety evaluation including alternative methods in the quasi-drug" concluded that it was insufficient to secure safety in ingredients of the quasi-drug by using in vitro skin irritation testing only in 2009.

It is necessary to develop the in vitro skin irritation testing that can secure safety in order to promote the use of alternatives in Japan. In addition, the establishment of the safety evaluation scheme including the in vitro skin irritation testing and the comprised social mutual agreement are essential.

In this symposium, some lectures of this problem and the correspondence from each standpoint are planned. This symposium is expected to become sharing the current status in Japan and a help of the solution in the future.


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S4-2 Cosmetic skin disorders -Focus of irritant contact dermatitis- ○Masatoshi Ito 1, Fukuyoshi Mori 1 Toho University School of Medicine E-mail: [email protected] Cosmetic skin disorders are classified as follows. 1) Acute irritant contact dermatitis 2) Chronic irritant contact dermatitis 3) Phototoxic contact dermatitis 4) Allergic contact dermatitis 5) Photoallergic contact dermatitis 6) Contact urticaria 7) Contact dermatitis syndrome 8) Pigmentation 9) Depigmentation 10) Abnormal dryness 11) Aggravation of pockmark Cosmetic skin disorders caused by factors such as:

1) Misuse of cosmetic 2) Irritant and allergic contact caused by cosmetic 3) Sales system

Cosmetic ingredients are essential to assess their allergic and irritant reaction in advance, and determine the amount of them. On the other hand, the evaluation of human 4 hour patch test for skin irritation has been documented in CTFA Safety Evaluation Guidelines 2007. In this study, we report the result of 4, 24, and 48 hours human closed patch test as an evaluation of human skin irritation in cosmetic ingredients.


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S4-3 In vivo Researches for Practical Use of Substituting Methods for Skin Irritation Studies ○Yasushi Yamada Nihon Bioresearch Inc.

E-mail: [email protected]

As commonly-used animal test methods in skin irritation studies, there are methods described in OECD test guidelines and the domestic production and distribution guidebook of cosmetics and quasi drugs.

Decreasing the number of animals or not using animals in consideration of animal welfare are described in the OECD test guidelines. Animals are usually exposed to the test article for 24 hours by Draize’s method. However, they are exposed for 4 hours by the OECD test guidelines, and the number of animals used is half of those used by Draize’s method. The exposure period determined in the domestic production and distribution guidebook of cosmetics and quasi drugs is 24 hours; the exposure period is different from that determined in the OECD test guidelines. The 24-hour exposure period is needed for transition to 24-hour occluded patch tests in humans and for assurance of safe use in the market.

On the other hand, development, assessment, and public acceptance of substituting methods for skin irritation studies have been proceeded in terms of animal welfare. Test methods using remodeled human epidermis have been adopted as the OECD methods. To put the substituting methods into practical use, data obtained in in vivo tests in each field are needed to be compared with those obtained by substituting methods and to be substitutable. In vivo data are needed for the assessment of substitutability.

Rabbits are determined to be used in the OECD test guidelines, and rabbits and guinea pigs are determined to be used in the domestic production and distribution guidebook of cosmetics and quasi drugs. However, the skin of rabbits and guinea pigs is highly sensitive, and it is known that some substances which cause mild irritation or no irritation in humans cause severe irritation in rabbits and guinea pigs.

To assess skin irritation potential of products used externally for skin diseases in humans, we used miniature pigs, which have similar skin structures to humans, and the obtained data were compared with those of rabbits and guinea pigs. In comparison of irritancy between animal species using antimycotic drugs, antipruritic drugs, therapeutic drugs for keratosis, and antiallergic drugs, sensitivity to irritancy was as follows: rabbits > guinea pigs > miniature pigs ≒ humans. Data on adverse effects in humans have a high correlation with those in miniature pigs.

It is considered that in vivo data are important to assess the usability of substituting methods.


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S4-4 Validity of the EpiSkinTM test in Japan; Skin irritancies of cosmetic ingredients tested using EpiSkinTM ○Shoichi Yahagi1, Miyuki Fujishiro1, Yukiko Izutsu1, Daisuke Yoshida1, Koji Kurihara2, Yuri Okano1, Hitoshi Masaki1

1 Nikkol group Cosmos Technical Center Co., Ltd., 2 Nikkol group Nikoderm Research Inc., E-mail: [email protected] Objective(s) EpiSkinTM (SkinEthic, France) is a reconstructed human epidermis model. In vitro skin irritation tests using the skin model (which is named as EpiSkinTM test) was approved in OECD as a new Test Guideline 439 to predict skin irritation. However, it is difficult to carry out the EpiSkinTM test in Japan under the same conditions as in the EU, because the skin model is manufactured in France. The topic of this presentation is to validate whether the EpiSkinTM test in Japan is able to give the same results as obtained in the EU. To assess the compatibility of the EpiSkinTM test, we evaluated some reference chemicals which have been evaluated in the EU using different ages of EpiSkinTM after manufacturing. In addition, we introduce the predictability of the EpiSkinTM test on skin irritation using the results of cosmetic ingredients which have been widely formulated. Materials and Methods The skin irritation test was carried out following the standard operation procedure (SOP) of the ECVAM (European Centre for the Validation of Alternative Methods). Skin irritation was judged on the basis of the skin irritation criteria of the ECVAM SOP which are expressed by the results of the cell survival rate and by the amount of secreted IL -1 α. Briefly, the testing protocol is as follows; after 24 hours of cultivation of EpiSkinTM in the maintenance medium, the chemical to be tested was applied on the skin model for 15 minutes. After washing, the skin model was cultured with fresh maintenance medium for 42 hours. The cell survival rate was then evaluated using the MTT assay, and IL -1 α in the culture medium was quantified using a commercially available kit (R & D systems). Results and Discussion In general, the EpiSkinTM skin model gradually builds up a tighter structure of the stratum corneum (SC) depending on the culture period. It is predictable that the structure of the SC influences the potential judgment of a sample on skin irritation by altering the of sample permeability into the living cell layer. Thus, it is possible that the skin irritation of a chemical might be underestimated. Therefore, in order to clarify the influence of differences in the SC conditions on the judgment, we carried out the skin irritation test with two different skin models matured for 13 days (D13 model) and for 16 days (D16 model). The D13 model and the D16 model showed identical results for the chemicals tested. In addition, the classifications by further examinations with the D13 model were consistent with results in the EU. From these results, we conclude that the culture periods of the EpiSkinTM does not influence the classification of chemicals, and that the EpiSkinTM test with the D13 model is suitable in Japan to predict skin irritation. In addition, we discuss the predictability of the EpiSkinTM test on skin irritancy for raw ingredients which are widely used for cosmetics compared with another alternative tests and reactivity in human skin.


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S4-5 Skin irritation test using LabCyte CORNEA-MODEL ○Masakazu Katoh Japan Tissue Engineering Co., Ltd E-mail: [email protected] A material with skin irritation induces the inflammatory effect against keratinocytes in the epidermis after it penetrates through stratum corneum and then it might continuously cause the cytotoxic effect. The skin irritation test using a reconstructed human epidermis model has been developed as an indicator of the cytetoxic effect. After the validation research in the European Centre for the Validation of Alternative Methods (ECVAM) had been accepted, this skin irritation test using the reconstructed human skin model was adopted as OECD test guideline 439 (OECD TG439).

LabCyte EPI-MODEL is one of reconstructed epidermis models, which has been developed by Japan Tissue Engineering Co., Ltd. (J-TEC), and it was started to release from 2005. J-TEC has been also developed the skin irritation test method using LabCyte EPI-MODEL according to the EpiSkinTM

(SkinEthic, France) test method. And then, it has been examined the possibility that the skin irritation test using LabCyte EPI-MODEL is predictable of the in vivo skin irritation, through the validation research in Japanese Society for Alternative Animal Experiments and in the Japanese Center for the Validation of Alternative Methods (JaCVAM).

In this symposium, I would like to present on a current status about the skin irritation test using LabCyte EPI-MODEL including the development research and the validation research.


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S4-6 Approach to the current requirements for alternative method of skin irritation testing ○Mariko Sugiyama Shiseido Research Center E-mail: [email protected] Skin irritation testing is established for two regulatory purposes, i.e., hazard identification of chemicals that may accidentally contact the skin and safety assessment of chemicals, such as household and topical skin products, cosmetics and other quasi drugs, and different methods are used for the two purposes. For hazard identification, Reconstructed Human Epidermis (RhE) has been developed as an alternative in vitro method to OECD TG404 (4 hrs exposure on rabbit) in order to categorize chemicals as irritant or non-irritant according to GHS, and this year, it was adopted as OECD TG439. In Japan, in order to comply with safety assessment requirements, data obtained from primary irritation testing in vitro must be submitted with the application for quasi drugs. Therefore, during 2007~2009, RhE was evaluated by the Advisory Committee for Validation of Alternative Methods on Safety Assessment of Quasi-drugs. The committee concluded that RhE was useful for estimating the potential of chemicals to cause irritation, but it was not sufficiently reliable to be adopted as an alternative method. Furthermore, an objective of primary irritation testing for quasi drugs is not only to evaluate potential to cause irritation, but also to estimate the safety of ingredients in accordance with the planned product composition and directions for use, and therefore a longer exposure time (24 hrs, rabbit) is necessary because some chemicals induce stronger skin reactions with time. Hence, an alternative to OECD TG 404 (4 hrs, rabbit) is not necessarily suitable for predicting actual skin reaction, and will not serve as a guarantee for safe human patch testing.

At present, therefore, we should make an effort to accumulate more information on irritation from skin reactions of the control groups in sensitization tests or cumulative irritation tests (evaluation after 24 hrs). Accumulation of an extensive in vivo database seems to be an essential first step for establishing a reliable in vitro alternative method for predicting primary irritation at 24 hrs.


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S5-L Chemical management policy and hazard assessment of chemical in Japan ○Shinichi Jitsukuni Chemical Safety Office, Chemical Management Policy Division, METI E-mail: [email protected] Nearly 40 years have passed since Chemical Substances Control Law (CSCL) was enacted in 1973. Under this law, the manufacture or import of the PCB similar substances has been regulated. In addition, during this period, there were several important amendments of this law to respond to the social demands of that age.

In 1986, in vitro tests such as Ames test and chromosome aberration test, and repeated dose 28-day toxicity study in rats were introduced as screening tests of assessing effects on human health into CSCL. The reason was that it was needed to efficiently make hazard assessments of chemicals in terms of chronic toxicity and carcinogenicity, and to smoothly classify hazardous chemicals with no high bioaccumulation.

In 2004, it was demanded to assess harmful effects on animals and plants in order to minimize the bad effects on living things in environment. Therefore, hazard tests using daphnia or fish were introduced as screening tests of assessing the effects on living things into CSCL.

Using the above tests, about 10,000 new chemicals have been judged before being manufactured or imported.

There are, however, still some problems as to the limitations on using in vitro tests. On the other hand, the needs for efficient hazard evaluations of chemicals will increase because the risk assessment process for every existing chemical will start next April.

Therefore, more efficient, more effective and more reliable methods of assessing chemical hazards than ever are requested to be established early by interested persons and parties.


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S5-1-1 Development of the reporter gene assay system using multicolor luciferase probes ○Yoshihiro Nakajima1, Shigeaki Nishii2, Yoshihiro Ohmiya2

1National Institute of Advanced Industrial Science and Technology (AIST), 2Tsuruga Institute of Biotechnology, Toyobo Co., Ltd. E-mail:[email protected] [Objective] Reporter assay system using luciferase is widely used as a conventional and powerful tool for quantitative monitoring of gene expression. Recent advances in luciferase technology, involving improvements in both the luciferase and the detection system and a newly cloned luciferase gene, allow us to monitor the expression of multiple genes simultaneously when luciferase are used that induce differently colored emission spectra in the catalysis of a common substrate. Recently, we have developed a multicolor luciferase assay system in which three gene expressions can be simultaneously monitored using green-, orange- and red-emitting luciferases(1､2). To develop rapid- and high reliance-chemical risk analysis assay system, we applied the multicolor luciferase assay system to immunotoxicity test. In this study, we generated a multicolor luciferase cells that carries transgenes encoding green-, orange- and red-emitting luciferases expressed under markers and internal control promoters, and validated accuracy of the assay system. [Materials and Methods] We used the following luciferases as luminescent probes: (i) green-emitting luciferase from click beetle (SLG, max=550 nm): (ii) orange-emitting luciferase that is a point mutant of SLG (SLO, max=580 nm); and (iii) red-emitting luciferase from railroad worm (SLR, max=630 nm). Stable cell line was generated by introducing three reporter vectors (IL2-SLG, IFN-SLO and G3PDH-SLR) into Jurkat cells. We used purified recombinant luciferases as a standard luminescent protein(3). [Results and Discussion] To verify the accuracy of the measurement system, the stable cell lines were seeded on a 96-well plate and respective luciferase activities were measured. We successfully measured expression of two marker genes and one internal control gene (total 288 gene expressions) within 20 min, with a coefficient of variation of less than 15%. In addition, we confirmed that induction of IL2 and IFN expression stimulated by PMA/ionomycin were stably maintained during eight weeks after start of culture. These results suggest that combine use of the measurement system and the stable cell line established in this study are useful tools for the chemical risk analysis. Moreover, absolute responsivity of luminometer could be calibrated using the standard luminescent proteins, allowing us to more high-precision measurement of luminescence. [References] (1) Nakajima Y. et al., A multicolor luciferase assay system, one-step monitoring of multiple gene expressions with a single substrate. BioTechniques 38, 891-894 (2005). (2) Nakajima, Y. and Ohmiya, Y. Bioluminescence Assays -Multicolor luciferase assay, Secreted luciferase assay, and Imaging luciferase assay-. Expert Opin. Drug Discover. 5, 835-849 (2010). (3) Niwa, K et al., Y. Quantum yields and kinetics of the firefly bioluminescence reaction of beetle luciferases. Photochem. Photobiol. 86: 1046–1049 (2010). This work was supported by NEDO grant.


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S5-2-1 A Cell Transformation Assay Using Bhas 42 Cells: A Proposal for Guideline and Development a High-Throughput Screening Assay ○Kiyoshi Sasaki1, Dai Muramatsu1, Shoko Arai1, Nobuko Endou1, Sachiko Kuroda1, Kumiko Hayashi1, Ayako Sakai1, Shojiro Yamazaki1, Yeon-mi Lim1, Makoto Umeda1, Masanori Wada2, Noriho Tanaka1 1Hatano Research Institute, Food and Drug Safety Center, 2ABLE Corporation E-mail: [email protected] [Objective] Bhas 42 cells were established as a clone by transfection with v-Ha-ras oncogene into BALB/c 3T3 cells. The cells are sensitive to contact inhibition of cell growth but transformed foci are induced by treatment with tumor promoters. We aimed to propose Bhas 42 cell transformation assay (CTA) for an OECD guideline and develop a high-throughput Bhas 42 CTA. [Materials and Methods] Characterization of Bhas 42 cells The presence and expression of v-Ha-ras were analyzed by FISH and RT-PCR. Transformation assay 6 or 96 well-plates were used. The cells were treated with chemicals from day 1 to 4 to detect tumor initiators and from day 4 to 14 to detect tumor promoters after plating. On day 21, the cells were fixed and stained with Giemsa’s solution and transformed foci were judged1). Correlation with in vivo Data of 98 chemicals of Bhas 42 CTA were compared with those of human and animals2). Validation One domestic study (6 labs, 9 chemicals) 3). and two international studies (Study 1: 6 labs, 12 chemicals, and Study 2: 4 labs, 23 chemicals) were conducted. Automatic system A robotic system, provides a series of the operation of transformation assay (plating cells, etc.), was developed. Hydrogen peroxide method We found hydrogen peroxide selectively kills normal cells and developed a method to estimate transformation frequencies by OD measurements. [Results and Discussion] Characterization of Bhas 42 cells 100% Bhas 42 cells contained v-Ha-ras. Almost all transformed clones showed higher expression of v-Ha-ras than normal cells. Correlation with in vivo Concordance: 78%, specificity: 84%, positive predictivity: 86%. Validation Interlaboratory agreement of judgment, domestic study: 92%, two international studies: 92 and 98%, and concordance between Bhas 42 CTA and in vivo, domestic study: 89%, two international studies: 100 and 83%. Automatic system Transformation frequencies by this system were similar to those by the manual method. Hydrogen peroxide method Without observation of transformed foci, transformation frequencies were evaluated objectively with a microplate reader. We proposed Bhas 42 CTA to OECD and this assay was accepted as a draft of a test guideline at the WNT meeting in March 2010. And the automatic system and the hydrogen peroxide method may be needed to develop a high-throughput Bhas 42 CTA. This study was supported by the New Energy and Industrial Technology Development Organization (NEDO). [References] 1) Arai, S. et al. (2010) AATEX, 15, 6-13. 2) Sakai, A. et al. (2010) Mutat Res, 702, 100-122. 3) Tanaka, N. et al. (2009) AATEX, 14, 831-848.


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S5-2-2 Development of novel alternative tests for developmental toxicity - Reporter gene assays using mouse ES cells and improvement of rat whole embryo culture - ○Noriyuki Suzuki1, Koichi Saito1, Masaharu Akita2

1Institution Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 2Kamakura Women's University E-mail: [email protected] [Objective(s)] A number of in vitro systems have been proposed as tests for developmental toxicity, however, it could be argued that these tests are unlikely to gain widespread acceptance and use. To establish convenient and accurate in vitro short-term tests for developmental toxicity, we have developed novel reporter assays using mouse ES cells or rat whole embryo culture (WEC) methods with metabolic activation. [Materials and Methods] Reporter gene assays using mouse ES cells Our research plan is as follows: 1) Determination of suitable differentiation methods for mouse ES cells into cardiomyocytes and neurons 2) Analysis of gene expression of ES cells during differentiation into cardiomyocytes and neurons under the differentiation methods 3) Selection of candidates for marker genes by bioinformatics analyses 4) Identification of marker genes by treatment with embryotoxicic and non-embryotoxic compounds 5) Prioritization of the marker genes by gene function and expression profiles 6) Construction of luciferase reporter vectors containing the promoter site of each marker gene upstream of the luciferase gene 7) Establishment of stable transgenic ES cells for detection of the marker gene expression 8) Development of test protocols for reporter gene assays using the transgenic ES cells and investigation of efficacy using some standard compounds Rat whole embryo culture methods with metabolic activation Our research plan is as follows: 1) Determination of rat S-9mix concentration in culture medium 2) Evaluation of effects of test chemicals on growth of cultured embryos with S-9mix 3) Development of new devices to reduce serum volume for WEC 4) Establishment of embryo culture conditions using the new devices. [Results and Discussion] Reporter gene assays using mouse ES cells Thirteen and 22 marker genes were identified during ES cell differentiation into cardiomyocytes and neurons, respectively. Stable transgenic ES cells were established to detect the chemical dependent changes in the marker genes easily and conveniently, and then test protocols for reporter gene assays with the cells were developed. According to the protocols, well-known test compounds were studied to clarify efficacy of the tests, the test showing high predictability and accuracy compared to the original EST. We are planning to evaluate the efficacy of the tests in detail using additional test chemicals. Rat whole embryo culture method with metabolic activation of chemicals Effects of some test compounds on cultured embryos were examined with or without S-9mix. New devices were developed to reduce serum volume for WEC, optimal condition being determined for the embryo growth.

This study was supported by a research grant from the New Energy and Industrial Technology Development Organization (NEDO) in Japan.


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S5-2-3 Immunotoxicity: Development of a reporter gene assay system using human cell lines ○Setsuya Aiba, Rumiko Saito, Yutaka Kimura, Ikuko Numata, Toshiya Takahashi Department of Dermatology, Tohoku University Graduate School of Medicine E-mail: [email protected] Objective(s) We are always exposed to enormous numbers of chemicals, which potentially impact our immune system. The purpose of this study is to discover the common response patterns in dendeitic cells, epithelial cells, or T cells, which are stimulated with different chemicals, by DNA microarray and to develop cell-based screening methods utilizing the obtained information. Materials and Methods Based on the DNA microarray analysis we had conducted for the last 3 years, we decided to focus on the following 7 immune-related genes to evaluate the immunotoxicity of chemicals, HMOX-1, IL-1b, IL-8, IFN-g, IL-10, IL-4, and IL-22. We constracted the plasmid vectors in which the expression of either SLG or SLO luciferase gene was regulated by the promoter of each gene. By electroporation or lipofection of these plasmids, we have established the following reporter cells derived from human keratinocyte cell line, HaCaT, monocyte/macrophage cell line, U937 and THP-1, and T cell line, Jurkat. 1) T cell line (Jurkat): IL-4 reporter cell (2A4), IFN-g reporter cell (2B12), IL-4/IFN-g reporter cell (10C6), IL-2/IFN-g reporter cell (2H4) 2) Monocyte/macrophage cell line (U937): HMOX1 reporter cell (UR2H4117), IL-1b reporter cell (6C12), IL-8 reporter cell (1A10), IL-1b/IL-8 reporter cell (8A4) 3) Monocyte/macrophage cell line (THP-1): IL-1b reporter cell (TCC17bA16.19), IL-8 reporter cell (TGC17EA01), IL-1b/IL-8 reporter cell (TGC17bA16EA26) 4) Keratinocyte cell line (HaCaT): HMOX1 reporter cell (UR38H6)

We first examined the correlation between luciferase activity by the reporter cells and mRNA expression by original cells after various chemical stimulations. Next, we examined whether the luciferase assay using IL-8 reporter cells can be used as an alternative method for skin sensitization testing. We also examined the response of IL-2/IFN-g dual reporter cells to various immunosuppressive drugs. Finally, we tried to evaluate 14 different immunodulatory chemicals by the combination of established reporter cell lines (HMOX1, IL-8, IL-2, IFN-g reporter cells).

Results and Discussion Luciferase activity of established cell lines after chemical stimulation was correlated well with mRNA expression by original cells. The accuracy of the luciferase assay using IL-8 reporter cells as an alternative method for skin sensitization testing was more than 80% in the evaluation of chemicals presented by Casati et al （ATLA 37:305-312, 2009）and Nukada et al. （J Toxicol Sci 33:175-185, 2008）. Consistent with our previous report, the luciferase activity by PMA/Ca ionophore-simulated IL-2/IFN-g reporter cells was more efficiently suppressed by cyclosporine than by dexamethosone. Finally, we combined the reporter cells and established the evaluation system for chemical immunotoxicity. When we evaluated 14 chemicals with well-known imunomodulatory effects, the responses of reporter cells are well correlated with the published immunomodulatory effects of these chemicals. These data strongly suggest the usefulness of our system in the evaluation of chemical immunotoxicity.

This study was supported by a research grant from NEDO in Japan.


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IS-1 International Symposium for Alternatives to Animal Experimentation. At The 23rs JSAAE annual meeting. ○Tsutomu Miki Kurosawa, DVM, M.PHIL, Ph.D, DVCS, DJCLAM Division of Laboratory Animal Medicine, Osaka University Graduate School of Medicine. In not only western countries but also Asian countries, 3Rs for animal experimentation is advancing. JSAAE supports such Asian activities since 2006, one year prior to WC6 held in Tokyo. Because WC6 was a first congress held in Asia, we recognized the importance of the collaboration in Asian countries to advance alternative research together. After the WC6, JSAAE International Communication Committee has organized International symposium regularly. Particularly with Chinese colleagues, we have a special mutual collaboration treaty between two countries and we have invited scientists in alternative research each other to exchange information. As a consequence of this effort, we Chinese and Japanese scientists have had a very intimate connection which may enable for us to exchange information frankly and promptly. The treaty period will be expired this year but we should see a future plan to strengthen this connection. With Korean colleagues, we have invited specialists in this field for their annual meetings. The scientific advancement of alternative research in these countries is significant. Our exchange program may accelerate this achievement. We should look forward to see more intimate relation between Korea and Japan in 3Rs.

However the advancement and enthusiasm of 3Rs in western countries are so rapid and strong. We Asian scientists in this field should exchange information among not only Asian countries but also European and American countries where various regulations and guidelines in laboratory animal welfare are recently revised and published. There are International documents in laboratory animal welfare published such as OIE animal welfare code. The CIOMS Guiding Principle in Biomedical Research will be revised shortly. All of these documents emphasize the importance of 3Rs and require practical aspect of laboratory animal welfare including realistic methods of alternatives. We Asian scientists get together to see these international documents and trends in alternative research all over the world and to strengthen our collaborative effort to advance biomedical research through the advancement of alternative research.


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IS-2 Comparison of flow cytometry (FCM) and immunohistochemistry (IHC) in non-radioisotopic murine LLNA using BrdU and the prevalidation of LLNA:BrdU FCM ○Kyung-Min Lim1,

, Kyoung-Mi Jung1, Won-Hee Jang1, Bae-Hwan Kim2, Yong-Kyoung Lee3, Young Na Yum3, Soojung Sohn 3

1Pharmaceutical Research Institute, Amorepacific CO R&D Center, Yongin 446-729, Republic of Korea, 2Department of Public Health, Keimyung University, 3National Institute of Food and Drug Safety Evaluation/Korea Food and Drug Administration E-mail: [email protected] Non-radioisotopic local lymph node assay (LLNA) employing bromodeoxyuridine (BrdU) with flow cytometric method (FCM) is gaining attention due to a regulatory issue of using radioisotope, 3H-thymidine, in vivo in traditional LLNA. In this study, to compare the performance of two non-radioisotopic endpoints, FCM and immunohistochemistry (IHC), 7 chemicals with known sensitizing potencies were examined in LLNA. Mice were topically treated with chemicals or vehicle on both ears for 3 days. After intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone respectively FCM or IHC to determine BrdU incorporated lymph node cells(LNCs). Weight and histology of treated ear were also examined to evaluate chemical-induced edema and irritation. Both FCM and IHC could successively identify the skin sensitizers from non-sensitizers. Comparison of FCM and IHC with traditional LLNA revealed that FCM has a higher sensitivity although both assays produced comparable sensitivity and performance to traditional LLNA. Moreover, FCM method allows the concomitant measurement of extra markers like B cell/T cell ratio and ex vivo cytokine production. With the defined protocol for LLNA:BrdU FCM, 3 sites (AmorePacific, NiFDS and Keimyung Univ.) participated in the evaluation of transferability, reproducibility and performance of the method for 19 (18+SLS) reference compounds enlisted in the ICCVAM performance standards. The result of the prevalidation suggests that LLNA:BrdU FCM is transferable with a good performance (concordance with tLLNA was 84%). In conclusion, we suggest that non-radioisotopic LLNA using FCM can successfully detect sensitizers with a good correlation to traditional LLNA.


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IS-3 Current Status of Alternative Study in China ○He Zhengming National Institute for the Control of Pharmaceutical and Biological Products

Beijing, P.R. of China. E-mail: [email protected] Laboratory animals have been widely used as raw materials in the production of biological products and as test systems to evaluate the quality (including safety, efficacy and stability etc.) of them. To decrease animal use, reduce animal suffering, the more attention has been paid to the study on alternatives to animal experimentation in recent years, and the progress in development of possible alternatives to in vivo testing has been gradually made in China. In 2006, the Guideline on Humane Treatment to Laboratory Animals was issued by Ministry of Science and Technology (MOST). The 3R research as one part of animal welfare is added in the new edition of the Regulation for Administration of Laboratory Animals, which issued by MOST in 1988, revised in 2000, 2006, 2010 separately. The Regulation (draft) has been referred to the state Council, which will be hopefully issued in force in the near future. In Chinese Pharmacopoeia (2010 edition), it is emphasized that to reduce the animal use, in vitro testing system e.g. chemical, physical or cytological methods, should be used as alternatives to animal experiment for quality control of biological products. More than 200 species of drugs and biologicals are tested for pyrogen materials by bacterial endotoxin test. The research on methodology and validation studies between different labs has been performed. The number of rabbits used for pyrogen test has been reduced by the introduction of Alternative test. “Laboratory Practice for in vitro alternative test of cosmetics” was drafted by Guangdong Entry-Exit Inspection and Quarantine Bureau entrusted by Certification and Accreditation Administration of the PRC, and the document as standard (SN/T2285-2009) was issued in force in 2009. In January, 2010, the first Cosmetics Standards Committee of the State Food and Drug Administration was established. The all above show that the 3R research has been gradually promoted in China in the past a few years.


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IS-4 National Regulations and Standards of Laboratory Animal Welfare in China ○Guo-jie YANG Peking University Health Science Center, Beijing 100083, China For the purpose of strengthening the administration of the laboratory animals and experimental animals and promoting animals’ welfare and ethic review, the Regulations of People’s Republic of China for administration of Laboratory Animals and Guiding Opinions about the Ethical Laboratory Animals was promulgated by the State Technology Supervision Administration in the year of 1988 and 2006 respectively. The National Standards for Laboratory Animal was formulated to guarantee the quality of laboratory animals by the State Administration of Technical Supervision. Up to the present, the administrative regional regulations about laboratory animals were formulated by the 17 provinces, municipality and autonomous regions. Today the regulations about the animal welfare and ethics as well as the replacements of laboratory animals above mentioned will be introduced in the following speech.


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IS-5 Current activities for enhancing development and establishment of alternatives to animal experiments in Korea: focusing on an example of bovine corneal opacity and permeability test ○Yong Heo1

, Yeon Mi Lim1, Joo Hee Han2, Seung Hyeok Seok2, Soon-Young Han3, Soo Jung Sohn3, Young Na Yum3

1Catholic University of Daegu, College of Natural Sciences, Dept. Occupational Health, Kyongbuk, 2Seoul National University, College of Veterinary Medicine, Seoul, 3National Institute of Food and Drug Safety Evaluation/Korea Food and Drug Administration E-mail: [email protected] Since Korean Society of Alternatives to Animal Experiments has formally launched at 2007, various efforts have been made to establish and promote 3R in Korea. In addition to revision of Animal Protection Law by Ministry of Food, Agriculture, Forestry, and Fisheries at 2008, a new law concerning on welfare of experimental animals came into effect by Korea Food and Drug Administration (KFDA) from 2009. Furthermore, establishment of KoCVAM (Korean Center for the Validation of Alternative Methods) in KFDA at 2009 came to be apparently a monumental event. KoCVAM is participating at three international validation studies; CCi MCF-7 cell proliferation assay by ICCVAM (Interagency Coordinating Committee on the Validation of the Alternative Methods), STTA and In vitro alkaline Comet assay by JaCVAM (Japanese Center for the Validation of Alternative Methods). Other two governmental institutes, Korean Academy of Agricultural Science and National Institute of Environmental Research, are also preparing the alternatives for evaluation of pesticide safety and eco-toxicity, respectively. Even though a little premature stage, GLP (good laboratory practice) companies has showed their interest on alternative test methods.

One of Korean efforts for international harmonization on proliferation of alternative test methods is the establishment of BCOP (bovine corneal opacity and permeability assay). BCOP was adopted as a test method for identifying ocular corrosive and severe irritants by OECD at 2009. A project for BCOP establishment in Korea has been funded by KFDA from 2008. Standardization of BCOP method has been completed and introduced to institutes or companies related through various technical seminars, and now inter-laboratory validation is undertaken. Two laboratories are participating at this validation. Fourteen chemicals were used for the study; ammonium nitrate, benzalkonium chloride, chlorhexidine, dibenzoyl-L-tartaric acid, 2,6-dichlorobenzoyl chloride, ethanol, 2-ethyl-1-hexanol, ethyl-2-methyl acetoacetate, glycerol, imidazole, methyl etyhyl ketone, n-hexane, 2,4-pentannediol, trichloroacetic acid. To enhance predictability of BCOP, histopathologic scoring system was newly added to the existing BCOP method. Results on the BCOP validation will be introduced.


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EL-1 Application of Cell-recognizable Matrix-engineering to Tissue Engineering and Regenerative Medicine. -Frontier of Immobilization of E-Cadhering Chimeric Antibody- ○Toshihiro Akaike Frontier Research Center, Tokyo Institute of Technology E-mail: [email protected] Over the last 35 years, synthetic biomaterial design has demonstrated an unquestionable potential for use as extracellular microenvironment to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and growth factors, both for regenerative medicine and tissue engineering. The complexities associated with natural materials, including complex structural composition, purification, immunogenicity and pathogen transmission have driven the development of synthetic biomaterials for use as 2D or 3D extracellular microenvironments [Sci. Tech. Adv. Mat. 11, 014106.2010]. Our recent advances include artificial extracellular matrix formed by immobilizing cell-recognizable molecule (e.g., E-cadherin and N-cadherin) [J. Biol. Chem. 26, 26468–26476,2008, Biomaterials 31, 5287-5296,2010.] or growth factors such as leukocyte inhibitory factor (LIF) [J. Biol. Chem. 26, 26468–26476,2008] for ES cell proliferation and differentiation. E-cadherin is essential for tissue morphogenesis and maintenance of organized solid tissues. We found that mouse embryonic stem (ES) cells cultured on chimera protein (E-cad-Fc)-coated surface had unique single cell morphology, higher proliferative ability and transfection efficiency than those grown under conventional conditions. Moreover, current in vitro cell cultivation strategies in presence of soluble growth factors are not optimal for stem cell proliferation and differentiation studies where frequent media changes are required. We showed that immobilized LIF and E-cadherin can maintain ES cells efficiently with lower dependency of ES cells on LIF. In addition to ES cell proliferation, N-cad-Fc and E-cad-Fc immobilized ECM can be used to induce efficient neural and hepatic differentiation at a single cell level. In summary, precise control over extracellular microenvironment using engineered ECM would be useful to fulfill the huge demand of cells for transplantation in regenerative medicine for development of bioartificial organs as single-celluler cooking plate.


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EL-2 State-of-the Art of Hepatic Generation from Stem Cells ○Takahiro Ochiya Section for Studies on Metastasis, National Cancer Center Research Institute E-mail: [email protected] [Objective(s)] In adults, there is a spectrum of stem cells with a different scale of quantity and potentiality. Among adult stem cells, MSCs, which are present in a range of tissues within the living body, are pluripotent cells similar to ES cells that are currently the subject of much attention. Furthermore, we will have a choice for iPS cells as well on regenerative therapy and utility for substitutable cells for industrial application. [Materials and Methods] To evaluate a possible usefulness of human iPS cells for generation of hepatocyte-like cells, tissue and cells from endoderm origin were used for generation of iPS cells having specific differentiation ability towards hepatocytes. [Results and Discussion] We generated human iPS cells having stemness genes as well as liver-specific genes. We have not yet arrived at the stage when we can claim a full understanding of the unknown possibilities possessed by stem cells, however, and while keeping pace with the progress of research on pluripotent human iPS cells, realizing the potential of stem cells and understanding their molecular mechanisms from a range of angles will require both effort and patience. [References] 1. Banas A, Yamamoto Y, Teratani T, Ochiya T. Stem cell plasticity: learning from hepatogenic differentiation strategies. Dev Dyn, 236:3228-3241, 2007 2. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, Okochi H, Ochiya T. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hapatology, 46:219-228, 2007 3. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, Kato T, Okochi H, Ochiya T. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol, 24: 70-77, 2009 4. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, Kawamata M, Kato T, Okochi H, Ochiya T. In Vivo Therapeutic Potential of Human Adipose Tissue Mesenchymal Stem Cells After Transplantation into Mice with Liver Injury. Stem Cells, 26: 2705-2712, 2008 5. Yamamoto Y, Banas A, Murata S, Ishikawa M, Lim CR, Teratani T, Hatada I, Matsubara K, Kato T, Ochiya T. A comparative analysis of the transcriptome and signal pathways in hepatic differentiation of human adipose mesenchymal stem cells. FEBS J, 275:1260-1273, 2008 6. Ochiya T, Yamamoto Y, Banas A. Commitment of stem cells into functional hepatocytes. Differentiation, 79:65-73, 2010


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M-1 Formation and Functional Evaluation of a Minimum Cell Mass for In Vitro Toxicity Tests ○Kikuo Komori Institute of Industrial Science, University of Tokyo E-mail: [email protected] Cell-based arrays and devices comprising an appropriate number of cells arranged in a physiological manner have become considerably important for in vitro toxicity testing. In addition, to obtain more information from cells, which are nonproliferative and in limited supply, micro-tissues exhibiting physiologically-relevant responses identical to those of their corresponding tissues/organs should be used. In this study, we generated different sizes of two- and three-dimensional micro-tissues of human hepatocarcinoma Hep G2 cells and determined the minimum required size and morphology (2D or 3D) for cytotoxicity analyses by a cell viability test using the indirect mutagen aflatoxin B1 (AFB1).

To control the size of two- and three-dimensional liver tissues, cells were respectively cultured on a cell adhesive/nonadhesive flat surface or a polymer sheet with molded micro-wells. In the case of the flat surface, cells stably adhered and proliferated only on the adhesive regions (63, 200, 630, and 2000 μm in diameters) forming a monolayer cell sheet. In the case of the polymer sheet with micro-wells, cells proliferated upward after adhering in the micro-well (63, 200, 630, and 2000 μm in diameter and ca. 20 μm deep). This way, we successfully obtained different sizes of two- and three-dimensional micro-tissues.

Using these micro-tissues, cytotoxicity of AFB1 was evaluated. AFB1 is known to be metabolized to an ultimate strong toxicant by the intracellular enzyme cytochrome P450 (CYP) 1A1/2, resulting in apoptosis. The cell viability test was performed after 48 h of exposure to AFB1, before which micro-tissues were pretreated with 3-methylcholanthrene to enhance the CYP 1A1/2 activity. As a result, EC50 values for two-dimensional micro-tissues 630 μm in diameter or larger and three-dimensional tissues 200 μm in diameter or larger were nearly equal to that for a conventional 96-well plate-based tissue (6.4 mm in diameter), whereas those for the two-dimensional tissues 200 μm in diameter or smaller and the three-dimensional tissues 63 μm in diameter were one order of magnitude higher than that for the 96-well plate-based tissue. The number of cells was estimated to be about 1000 and 500 cells for the two- and three-dimensional micro-tissues, respectively. This difference is likely due to the fact that cell-cell signaling is accelerated in the three-dimensional micro-tissues because of an increase in cell-cell junctions compared with two-dimensional tissues. Thus, we conclude that for Hep G2 cells, the three-dimensional micro-tissues 200 μm in diameter consisting of about 500 cells are smaller suitable tissues for in vitro toxicity bioassay.

I am grateful to Prof. Y. Sakai, Prof. T. Tatsuma, Prof. T. Fujii, Mr. I. Kameda, Mr. H. Suzuki, and K. Montagne for the supports of this work.


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M-2 Establishment of chronopharmacokinetic evaluation system in vitro cultured cells: Molecular basis for rhythmic expression of CYP in serum-shocked HepG2 cells ○Shigehiro Ohdo, Satoru Koyanagi, Naoya Matsunaga Deapartment of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University E-mail: [email protected] [Objective] Although the pharmacokinetics of several drugs, which are mainly eliminated by CYP metabolism, vary according to their dosing time, mechanism of the variation remains poorly understood. In the present study, we investigate how the 24 hr oscillation in the expression of CYP mRNA is generated in hepatic cells and establish the chronopharmacokinetic evaluation system in vitro cultured cells. [Materials and Methods] HepG2 cells were grown to semi-confluence in DMEM supplemented with 10% fetal bovine serum. Cells were then incubated in serum-starved medium for 12 hr. On the day of serum shock, 50% FCS was added for 2 hr and then changed back to starvation medium. Cells were harvested for RNA and protein extraction at several time points after the serum treatment. The mRNA levels of clock genes and CYP gene were measured by RT-PCR. CYP activity in protein extracts was determined using the P450-Glo assay system. To investigate the effect of molecular components of circadian clock on the mRNA levels of CYP, HepG2 cells were transfected with expression plasmids of clock protein. The transactivation effect of each clock protein on CYP gene was measured using luciferase reporter constructs containing various lengths of 5'-flanking region of CYP gene. An electrophoretic mobility shift assay was performed. To investigate how the rhythmic variation of CYP mRNA expression occured in the serum-shocked HepG2 cells, influence of clock gene protein on the transcriptional activity of CYP gene was assessed using luciferase reporter constructs containing wild-type or mutated fragment of the 5’-flanking region of CYP gene. To explore the temporal binding of endogenous clock gene protein on the CYP promoter in the serum-shocked HepG2 cells, chromatin immunoprecipitation analysis was performed. [Results and Discussion] As brief exposure of HepG2 cells to 50% serum induced the 24 hr oscillation in the expression of clock genes, serum-shocked HepG2 cells were employed as an in vitro model to study the molecular mechanism underlying the circadian clock in human liver cells. Both mRNA levels and metabolic activity of CYP in serum-shocked HepG2 cells fluctuated rhythmically with a period length of about 24 hr. The oscillation in the expression of the CYP gene was the underlying cause of rhythmic change in its metabolic activity. Luciferase reporter gene analysis and electrophoretic mobility shift assay revealed that circadian transcriptional factors, clock gene proteins (a positive component of the circadian clock), activated the transcription of the CYP gene by binding to the DNA sequence near the upstream of transcriptional start site. The transactivation of the CYP gene by a positive component of the circadian clock was repressed by a negative component of the circadian clock. Our findings provide a molecular link between the circadian clock and xenobiotic metabolism. Finally, we can establish the chronopharmacokinetic evaluation system in vitro cultured cells. The establishment of in vitro drug evaluation system from viewpoints of chronopharmacology is very important for development of alternative to animal experiment methods. This research was supported by Mandom International Research Grants on Alternative to Animal Experiments. [References] 1) Takiguchi T, Tomita M, Matsunaga N, Nakagawa H, Koyanagi S, Ohdo S. Molecular basis for rhythmic

expression of CYP3A4 in serum-shocked HepG2 cells. Pharmacogenetics and Genomics 17(12), 1047-1056, 2007.

2) Matsunaga N, Ikeda M, Takiguchi T, Koyanagi S, Ohdo S. The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver. Hepatology 48, 240-251, 2008.


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M-3 Establishment of the method for quantitative prediction of detoxification ability and drug interaction in human liver by using human tissue sample and gene expression systems ~ Utilization of human tissue as alternative experimental systems to animal experiments ~ ○Kazuya Maeda, Naoki Kotani, Kenta Yoshida, Yuichi Sugiyama Graduate School of Pharmaceutical Sciences, The University of Tokyo [email protected] [Objective(s)] For drug discovery and development, to understand the pharmacokinetic properties of drug candidates in humans is one of the most important issues. However, we cannot directly obtain the human pharmacokinetics before clinical trials and we need to predict in vivo pharmacokinetics from in vitro and animal experiments. It is well known that species difference in pharmacokinetics of drugs between animals and humans are sometimes recognized. Thus, the information from animal experiments cannot be directly used for the prediction of human pharmacokinetics. Recently, prediction of human pharmacokinetics from in vitro experiments with human-derived tissue samples has just started. We tried to predict the human hepatic clearance of drugs from in vitro experiments by using cells expressing pharmacokinetics-related genes and human hepatocytes. This kind of approach leads to the decrease in unnecessary animal experiments. [Materials and Methods] Cells expressing several kinds of transporters have already been constructed previously in our group. Human cryopreserved hepatocytes were obtained from commercial sources. For the prediction of hepatic uptake of drugs, the uptake amount of drugs was evaluated simply by incubation human cryopreserved hepatocytes with buffer containing drugs for designated period. For the evaluation of biliary excretion process of drugs, we cultured hepatocytes in sandwich-culture format and observed the transport of drugs into bile pocket after pre-incubation with buffer with or without Ca2+ ion. [Results and Discussion] Human non-renal clearances of several kinds of transporter substrate drugs were well correlated with the predicted values from in vitro uptake clearance into hepatocytes. This result suggested that the rate-limiting step of the hepatic clearance of transporter substrate drugs was mainly hepatic uptake process and hepatic clearance could be predicted from the result of in vitro uptake assay. Since several transporters are involved in the hepatic uptake of drugs, we also performed the estimation of the contribution of each transporter to the hepatic uptake of drugs by using hepatocytes and appropriate probe inhibitors and substrates. Previous reports suggested that the predicted biliary excretion clearance was much smaller than the observed one in rats. We showed this was mainly due to the down-regulation of the expression of hepatic uptake transporters. But in the case of human hepatocytes, the expression levels of uptake transporters were not largely decreased, suggesting that better prediction of biliary excretion clearance of drugs may be possible.


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CC-1 Experiments using water fleas ○Sawako Yasuda Yasu Municipal Yasu Elementary School [Objective] The present study was conducted to determine whether substances with adverse effects on health can be screened using water fleas taking advantage of the fact that the sex of their offsprings changes depending on their living environment. [Methods] 1) Water fleas will be reared in a good condition in aquariums. 2) Water fleas will be allocated to aquariums in the same number. 3) Water fleas will be reared in aquariums for one week in a good condition with and without adding test substances. 4) The number and health condition of water fleas will be observed one week later. [Conclusion and discussions] The effects of the test substances on water fleas can be evaluated by counting male and female water fleas. This method cannot evaluate Effects of substances insoluble in water Effects of gases [References] Potential of water fleas, Iwanami Junior Shinsho, Iwanami Shoten.


227

CC-2 Proposals regarding experiments on leptin in frogs ○Yuya Nakamoto, Takahiro Yamashita, Hiroto Kamosaki Hiroshima Prefectural Hiroshima Kokutaiji High School [Objective] The objective of the present study was to determine whether leptin, which affects weight of fishes and mammals, also affects weight of amphibian species. [Methods] Leptin was injected into the blood stream in frogs to determine whether the frogs loose weight. The relationship between blood leptin concentrations and weight was evaluated using a leptin determination kit in frogs with the same length but different weight. [Conclusion and discussions] Amphibian species can also be considered to be affected by leptin if frogs loose weight upon injection of leptin. In addition, leptin and weight can be considered to be correlated with each other if blood leptin concentrations are higher in lighter frogs and lower in heavier frogs.

Previous studies on leptin usually used mice. However, studies on the effects of leptin on lifestyle-related diseases can be conducted more efficiently using frogs because frogs reproduce more offsprings than mice. [References] ・http://ja.wikipedia.org/wiki/%E3%83%AC%E3%83%97%E3%83%81%E3%83%B3 ・http://www.miobs.com/product/dobutsu/06/index.html ・Reven P , Johnson G, et al.: Biology.


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CC-3 Application of snails’ various sensor functions to medical care ○Masaya Ohtsu Toin Gakuen High School [Objective] It is extremely difficult to determine the effects of drugs, the electromagnetic environment, and the life in space on various functions of living organisms. Various studies have been undertaken to gain better insight into these effects. For this purpose, it is expected to be useful to study the effects on living organisms with various sensor functions. By observing snails’ behaviors, I have found that snails show various sensor functions. The present study was undertaken to evaluate snails’ sensor functions in order to use them in the field of healthcare. [Methods] Euhadra peliomphala was mainly used in experiments. The dependence of various functions on the following parameters was determined in order to evaluate various sensor functions: 1) temperature, 2) humidity, 3) atmospheric pressure, 4) brightness, 5) gravity (external force), and 6) magnetic field. The dependence on parameters 1), 2) and 3) was determined by monitoring the position of snails in the rearing cage. The dependence on parameter 3) was also evaluated by changing atmospheric pressure. The dependence on parameter 4) (1), 2), 3)) was evaluated by changing brightness in the rearing cage. The inclination angle-dependence was determined to evaluate the dependence on parameter 5). A long-term monitoring system was used to evaluate the dependence on parameters 4) and 6). [Conclusion and discussions] Findings obtained in the present study showed that snails have various sensor functions listed below: 1) Humidity sensor: withdrawal of the body from the shell 2) Brightness sensor: move in the dark and immobilization in the light 3) Magnetic field sensor: avoidance of a strong magnetic field 4) Gravity (external force) sensor: move in a direction opposite to the gravity 5) Atmospheric pressure sensor/temperature sensor: determines the migration amount

Findings obtained indicated that snails have various sensor functions and can be used to study the effects of drugs, the electromagnetic field, and the life in space on living organisms. These sensor functions may be used in studies of the effects of drugs and electromagnetic field in the field of healthcare.

References [1] Takuhiro Ohtsu, Masaya Ohtsu: Relationship between the position of snails and atmospheric pressure

and temperature. NTS Inc., Seibutsuno Kagaku: Iden, P79-85, September (2008). [2] Takuhiro Ohtsu, Masaya Ohtsu: Relationship between the position of Euhadra peliomphala and

weather. The 77th Annual Meeting of the Zoological Society of Japan, high school students’ poster, p 12 (2006).

[3] Masaya Ohtsu, Takuhiro Ohtsu: Relationship between the position of Euhadra peliomphala and weather. The 78th Annual Meeting of the Zoological Society of Japan, high school students’ poster 19 (2007).

[4] Takuhiro Ohtsu, Masaya Ohtsu: Effects of external force on behaviors of snails: The 79th Annual Meeting of the Zoological Society of Japan, high school students’ poster HP03 (2008).

[5] Masaya Ohtsu, Takuhiro Ohtsu: Effects of the shell of snails on their behaviors. The 79th Annual Meeting of the Zoological Society of Japan, high school students’ poster HP04 (2008).

[6] Takuhiro Ohtsu, Masaya Ohtsu: Long-term observation of behaviors of snails and slugs in the dark. The 80th Annual Meeting of the Zoological Society of Japan, high school students’ poster 8 (2009).

[7] Masaya Ohtsu, Takuhiro Ohtsu: Effects of the magnetic field on behaviors of snails. The 80th Annual Meeting of the Zoological Society of Japan, high school students’ poster 9 (2009).


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PS The use of human materials and information is indispensable in biomedical research: in the era of alternatives of animal experiments ○Tohru Masui Leader, Office of Policy and Ethics Research Leader, Director, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, Osaka, Japan [email protected] Alternative methods aim to reduce the use of experimental animals in biomedical research especially in R & D of medicines. There are several directions of supporting this movement, i.e. prevention of cruelty to animal to promotion of the use of human materials and information in biomedical research.

In my talk I focused on the latter movement. I have been interested in this issue for more than decade and found that reducing the use of experimental animals directly means increase or encouragement of the use of human materials and information in biomedical studies. In my talk, I will describe our position in the history of human experimentation and alternative methods now established in use of human genome information.

I started my talk in the famous human experimentation of Jenner on smallpox vaccine. I explained his clever observations, human experimentation, and importance of the use of human beings in the biomedical experiments. Though in the era of Salk vaccine, the situation was completely different, he used experimental animals for proving the safety of his vaccine and few hundred thousands of boys and girls were recruited and randomly separated to the experiment and placebo groups in the double blind manner. This was a representative human experiment done in the modern manner. In the development of Helsinki Declaration it has shown very clearly, the establishment of modern clinical studies, i.e. randomization, double blind, and use of placebo.

I then proceed to describe Human genome projects and several projects after this created measures to compare human individual genome information and use them for controlled biomedical experimentations. Since genome information is the basic information of determining biology of human beings, we are very excited this development. These developments create platform of alternative methods of animal experiments.


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P-1 Accuracy of LLNA: BrdU-ELISA to detect skin sensitization potential of chemicals ○Hideki Miyaura1, Masafumi Horiuchi1, Naoaki Yakata1, Masahiro Takeyoshi1, Naomi Kawazu2, Masanori Taruki2

1 Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute ,Japan 2 Hita Laboratory, Chemicals Evaluation and Research Institute ,Japan E-mail: [email protected] [Objective] Local lymph node assay (LLNA-RI) is used for the identification of skin sensitization. It is a method for determining the sensitizing potential of chemicals in which activity is measured as a function of induced proliferative responses in draining lymph nodes. LLNA-RI offers refinement and reduction in the severity of test procedure compared to Guinea Pig Maximization Test/Buhler Test (GPMT/BT), but LLNA-RI has not been widely used because it requires specific procedures of radioisotope (RI). Instead of LLNA-RI, we have developed and validated a modified LLNA based on the BrdU incorporation (LLNA: BrdU-ELISA). LLNA: BrdU-ELISA is useful in situations need to avoid RI. Moreover, it’s developed as OECD Guideline for Testing (OECD TG442B, adopted July 2010). LLNA: BrdU-ELISA expected to become globally used test method for skin sensitization. We report investigations to validate LLNA: BrdU-ELISA by comparative evaluation against LLNA-RI. [Material and Methods] We tested 45 chemicals of LLNA: BrdU-ELISA using CBA mice and compared against the evaluations by Traditional LLNA. The SI (Stimulation index) value represented as ratio of mean absorbance of the incorporated BrdU in the test substance group to the mean absorbance of in the vehicle group ,and the detection criterion of SI > 2 was used to evaluate sensitization. [Results and Discussion] As the result, 97.8% (44/45) of concordance rate was allowed between LLNA: BrdU-ELISA and LLNA-RI. Positive Predictive value and Negative Predictive value was 97.1% (33/34) and 100.0% (11/11), respectively. Additionally, result of ICCVAM Performance Standard 22 chemicals showed high correlation with the EC3 range of LLNA-RI. In conclusion, LLNA: BrdU-ELISA is of considerable benefit for evaluating sensitization in place of LLNA-RI. [References] Takeyoshi M, Yamasaki K, Yakabe Y, Takatsuki M, Kimber I, 2001. Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2’-deoxyuridine (BrdU) incorporation. Toxicol. Lett. 119, 203-208.


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P-2 Respiratory sensitization study by quantitative structure-activity relationships (QSAR) ○Kazuhiro Sato1, Kohtaro Yuta2 and Yukinori Kusaka1

1Department of Environmental Health, School of Medicine, University of Fukui, Fukui 910-1193, Japan 2In Silico Data Co Ltd, Narashino, Chiba275-0025, Japan E-mail: [email protected] 【Objectives】In silico assessment of sensitization is increasingly needed owing to the problems concerning animal welfare, as well as excessive time consumed and cost involved in the development and testing of new chemicals. Respiratory sensitization positive/negative prediction models were generated and parameter analyses were implemented on the basis of QSAR respects. 【Materials and methods】Samples used in respiratory sensitization were selected from the list of “European Chemical Bureau (ECB): R42, R42/43 for positive samples (respiratory sensitizers) and from the classification results of the Japanese Globally Harmonized System of Classification and Labeling of Chemicals (GHS) Inter-ministerial Committee of the National Institute for Technology and Evaluation (NITE) for negative skin sensitizers (controls). The total 214 compounds (61 positive sensitizers and 153 negative sensitizers) of respiratory sensitization were used.

Total 800 parameters were generated from 2-D and 3-D structures of these compounds and these initial parameters were reduced to the 12 parameter of respiratory sensitization by feature selection methods. Various linear and non-linear discriminant analysis methods were applied using this parameter sets. All data analyses were performed using ADMEWORKS/ModelBuilder V3 software (Fujitsu Kyushu Systems Limited, Japan). 【Results】Perfect classification ratio (100%) was achieved in respiratory sensitization. The highest prediction ratio of 97.2% by leave-one-out cross validation was achieved with Support Vector Machine (SVM). 【Dicscussion】The QSAR model for respiratory sensitization are rare. This QSAR based approach is applicable to initial prediction of respiratory sensitization. 【Reference】Sato K et al. AATEX 14(3):940-946, 2009 【Acknowledgement】This work was supported by a Grant –in-Aid from the Ministry of Health, Labour and Welfare, Japan (H20-Labour-009).


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P-3 Permeability prediction of phthalic esters for cultured human alveolar epithelial A549 cells ○Genya Tanaka1, Kikuo Komori 1, Takao Fujii 1, Hideto Jinno 2, Yasuyuki Sakai1

1 Institute of Industrial Science, University of Tokyo, 2 National institute of Health Sciences E-mail: [email protected] [Objectives] Phthalic esters have been widely used as plasticizer for household products. They are released from plastics and are present in the air as gas and/or adsorbed to dirt particles. The released plastic esters are immediately metabolized after being drawn into the body via the lung, resulting in harmful effects to the reproductive system by their metabolites [1]. In this study, we establish a method to predict the permeability of phthalic esters by combing in vitro experiments and mathematical modeling. Herein, we examined the permeability of the human alveolar epithelial cell line A549 for phthalic esters to determine their lung permeation mechanism. [Materials and Methods] A549 cells densely adhere to the apical (Ap) side of a semi-permeable membrane culture insert and are incubated in culture medium containing 0.1-10.0 mM benzylbutyl phthalate (BBP) for 48hours. Phthalic esters permeated from the Ap side to the basolateral (BL) side were detected by GC-MS and HPLC. [Results and Discussion] Although the BBP concentration on the BL side increased as that the Ap side increased, the former was 1-2 orders of magnitude lower than the latter. However, BBP was found to permeate from the Ap side to the BL side through the A549 cell layer. In contrast, since monobutyl phthalate (MBuP) and monobenzyl phthalate (MBeP) were also detected in the culture medium in the BL side, BBP was found to be metabolized by the intracellular enzyme carboxyl esterase (CES). The concentration of MBuP and MBeP was about 10 M in the BL side regardless of the BBP concentration range in the Ap side examined here, indicating that the CES-based metabolic reaction was saturated due to its low activity. Thus, not only BBP but also its metabolites are involved in the lung permeation mechanism. In future work, we will establish the mathematical model based on the above mechanism. [References] [1] Eveillard et al., Toxicol. Appl. Pharmacol., 236, 282–292 (2009).


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P-4 The development of an in vitro assay (ROS assay) to predict skin sensitization based on ROS production ○Saito, Kazutoshi Saito, Masaaki Miyazawa, Yuko Nukada, Hiroki Sano, Hitoshi Sakaguchi, Naohiro Nishiyama Kao Corporation, Tochigi, Japan E-mail: [email protected] [Objective(s)] We have been developing the “human Cell Line Activation Test (h-CLAT)”, an in vitro skin sensitization test, based on augmentation of CD86/CD54 expression in human monocytic leukemia cell line THP-1 cells. From the examination of 106 chemicals, 89 chemicals were identified correctly in h-CLAT 1) and now the pre-validation is under way for the new OECD test guidelines. Recently, Reactive Oxygen Species (ROS) have been shown to induce dendritic cell migration and contact hypersensitivity2). We investigated the use of ROS as a useful marker for assessing skin sensitization in a murine dendritic cell line3). In this study, we developed an in vitro assay to predict skin sensitization based on ROS production in human cell line THP-1 cells. [Materials and Methods] We used fluorescent indicator, CM-H2DCFDA, for ROS detection. CM-H2DCFDA is highly cell-permeant, hydrolyzed within the cell and fluoresces by oxidation. THP-1 cells were pre-loaded with CM-H2DCFDA for 15 min at 37�. After staining, cells were incubated with the test compound under a serum-free condition for 30 min and then fluorescence was measured by flow cytometry. [Results and Discussion] First phase, we treated the THP-1 cells with either a sensitizer, 2,4-dinitrochlorobenzene (DNCB), or a non-sensitizer, benzalkonium chloride (BAC). DNCB but not BAC induced >6-fold ROS production. Next, we evaluated 30 chemicals for their potential to increase ROS production. 15 out of 18 sensitizers induced >2-fold ROS production with a dosage causing >90 % viability, but only 2 out of 12 non-sensitizers did. Consequently, setting the 2-fold ROS production as positive criteria, sensitivity, specificity and accuracy were determined to be 83.3％. Also, 5 compounds (citral, abietic acid, cyclamen aldehyde, ethylene glycol dimethacrylate, butyl glycidyl ether) out of 6 sensitizers categorized as “weak” in local lymph node assay were judged as “positive” in this assay. These results indicated that THP-1 cells-based ROS assay was a sensitive and mechanism-based tool to predict skin sensitization. [References] 1) Ashikaga, T. et. al., Alternatives to Lab Animals , 38 (4), 275-84, 2010 2) Na. et al., Journal of Investigative Dermatology, 127, 1930-1937, 2007 3) Miyazawa. et al., Journal of Investigative Dermatology, 130, S46, 2010


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P-5 Inter-laboratory validation study of in vitro eye irritation test; Short Time Exposure (STE) test (Part 3). ○Kojima H.1, Kuwahara H.2, Hayashi T.2, Sakaguchi M.3, Toyoda A.3, Goto H.3, Nakamura T.4, Watanabe S.4, Ahiko K.4, Omori T.5, Otoizumi T.5, Sozu T.6, Morimoto T.7, Hayashi K.8, Sakaguchi H.8

1National Institute of health Sciences, 2Kanebo cosmetics Inc., 3POLA Chemical Industries, INC., 4LION Corporation, 5Doshisha University, 6Kyoto University, 7Sumitomo Chemical Co., Ltd., 8Kao Corporation E-mail: [email protected] [Objective(s)] Short Time Exposure (STE) test is an easy-to-use in vitro eye irritation test using the cell viability of SIRC (rabbit corneal cell line) cells as an end point following one 5 min treatment. The Japanese Society for Alternative to Animal Experiments organized an Executive Committee and conducted a validation study with five laboratories to assess transferability, inter-laboratory reproducibility, and predictive capacity of the STE test from 2008-2009. These data showed good transferability of the STE test. Assignment of 25 blinded samples to the STE irritation categories “Non-Irritant”(NI) or “Irritant”(I ） showed good inter-laboratory agreement. In this study, we re-evaluated the predictive capacity of the STE test using an additional 40 blinded chemicals. [Materials and Methods] This study was conducted based on the same test protocol of the STE test. Using 40 blinded samples, three experiments for each chemical were evaluated using samples at 5% and 0.05% concentrations in either saline, 5% DMSO in saline or mineral oil as a vehicle. The STE irritation category based on cell viability in 5% sample solutions was compared with GHS category (NI or I). In addition, the STE rank classification was compared with GHS rank (NI; I, Cat. 1 or I: Cat. 2). [Results and Discussion] The validation study is on-going as of September, 2010.

In future, we will compare the STE irritation category with GHS category and the STE rank classification with GHS rank in all laboratories using data from 65 samples including the results from the previous validation study.


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P-6 Validation of a skin irritation study using a Japanese model; LabCyte EPI-MODEL24, Additional study ○Kojima, H. 1, Nakamura, M. 2, Yamaguchi, Y. 2, Izumi, R. 3, Suzuki, T. 3, Hagiwara, S. 4, Shinoda, S. 4, Kato, M. 5

1 NIHS, 2KOBAYASHI Pharmaceutical Co., Ltd., 3Fancl Corporation, 4Drug Safety Testing Center Co.,Ltd. 5J-TEC E-mail: [email protected] [Objective(s)] Based on the EpiSkin statement and its protocol, we created a program to validate the usefulness, relevance and reproducibility (including intra- and inter-laboratory variability and transferability) of a skin irritation study using a Japanese model (LabCyte EPI-MODEL24) from 2008-2009. From these data, a good transferability of the LabCyte EPI-MODEL24 and the original protocol was obtained. The skin irritation categories (Non-Irritant or Irritant) for 25 blinded chemicals using this model and protocol showed good inter-laboratory and good predictivity in each laboratory. However, OECD peer review panel indicated 1-bromohexane, category 2, was misclassified into the “No” category by five of six laboratories, and recommended that the issue be solved. In Accordance with this recommendation, J-TEC revised the protocol. In this study, therefore, our goal was to re-evaluate the predictive capacity of the revised protocol using 20 blinded chemicals from the reference chemical list in the OECD performance standard. [Materials and Methods] Using three laboratories, we are performing a validation study using the revised protocol and 20 blinded chemicals from the reference chemical list in the OECD performance standard. [Results and Discussion] The validation study is on-going as of September, 2010.


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P-7 Optimal conditions for performance of the comet assay using a three-dimensional human epidermal model ○Hajime Kojima, Maki Hojyo National Institute of Health Sciences (NIHS) E-mail: [email protected] [Objective(s)] To evaluate the risk of genotoxicity when human skin is exposed to chemicals, we attempted to determine optimal conditions for the comet assay using a three-dimensional human epidermal model (LabCyte EPI-MODEL: epidermal model, Japan). [Materials and Methods] In this study, the well-characterized genotoxicants, mitomycin C (MMC), methylmethanesulfonate (MMS), and 4-NQO (4-Nitroquinoline 1-Oxide) were utilized as test chemicals to correlate cytotoxicity and the comet assay using this model.

Furthermore, the well-characterized metabolically activated genotoxicants, cyclophosphamide (CP), 2-aminoanthracene (2-AA), and nitrosodimethilamine (NDA), were utilized for the comet assay using an epidermal model to evaluate metabolic activation of chemicals.

[Results and Discussion] Generally, there are the deep correlation with cytotoxicity and genetic endpoint. The results of CP, MMS and 4-NQO show a correlation ship between relative cell growth and tale length. However, the number of tale lengths induced by MMC and NDA did not correlate with cytotoxicity.

We recommend the cytotoxicity test as useful for preliminary testing to determine an adequate dose. However, tales detected with non-cytotoxicity under maximal applicable condition. These results show no strong correlation between cytotoxicity and the comet assay using a 3-dimensional epidermal model.

The comets of 2-AA and NDA increased dose-dependently on the epidermal models treated for 24 hours, while the results were ambiguous when treatment lasted for four hours. CP, on the other hand, did not induce comets.

We consider the comet assay using an epidermal model to be potentially useful in being able to detect indirect genotoxicants regardless of metabolic activation.


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P-8 The animal experiment substitute teaching materials in the high school science education ○Hisashi Iwata

Iwami High School tottori prefectural E-mail: [email protected] [Objective(s)] When students chose biology I in high school, we must perform the instructional activity that can help them to achieve the aim advocated in the course of study for high school effectively. In addition, the education to let them understand life phenomena ethically is possible by learning and thinking about life and death with all the members taking biology in high school. We consider biology education that helps them understand the importance of life and cherish all creatures, not just learning life phenomena. [Materials and Methods] I use a DVD "Human body dissection manual" as a substitute teaching material of animal experiment in high school biology. Then, we can show the principle of 3R especially “Replacement” effectively by showing "Own life and relations with the death", "The significance of the body donation and progress of the anatomy", "The significance of the subject to learn in exchange for the life of the animal", "Medical supplies, cosmetics development and relations with the animal experiment of alternative methods". I am convinced that we can achieve the aim of the course of study for high school enough if we can hit consciousness that biology is formed on the sacrifice of many creatures, and the development of alternative methods (replacement) by such instructional activies.

[Results and Discussion] About 70% of the students enrolled in Iwami Senior High School replied “good” and ”beneficial” for the DVD teaching materials and about 60% are affirmative about the use of the DVD teaching materials. However, there are a lot of students expecting a class only with a textbook and an illustrated book and 30% of them oppose the use of the DVD. It seems that they are influenced by the gossip that biology is subject that needs just memorization and is easy, so the conscious reform to them is urgent. [References]

Shoichi Nakano, Toshitada Yoshioka, Etsuro Tanaka : Illustration physiology(The House of medical book)，Association of Japanese laboratory animal engineer : Collection of illustration laboratory animal technology Ⅰ,Ⅱ(Adthree)


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P-9 Student practice incorporating alternative animal experiments in the faculty of pharmaceutical sciences ○Tetsuya Hasegawa, Hiroshi Kawai, Kaori Matsumoto, Takuya Ishibashi, Kenjiro Matsumoto, Kimihito Tashima, Atsushi Mitsumoto, Yasuo Kodama, Syunji Horie, Masayuki Akimoto

Faculty of Pharmaceutical Science, Josai International University E-mail:[email protected] [Objective(s)] The curriculum of the faculty of pharmaceutical sciences includes student practice courses using experimental animals, such as those of anatomy, pharmacology, and biological pharmaceutics. In pharmacy education, instructors involved in animal experiments should be understood that their activities are approved by agreement of the society, and conduct efficient and effective practice classes. Also, it is important for instructors to inform students majoring in pharmacy of the principles of alternatives to animal experimentation, the “3Rs”, and to advance them through practice courses. In this presentation, cases in which alternatives to animal experiments were incorporated in some on-campus practice classes within the faculty of pharmaceutical sciences and the results of a questionnaire survey about these classes in students are presented. [Materials and Methods] Of the student practice in third-year student provided by the faculty of pharmaceutical sciences, Josai International University, “Effects of Drugs on the Blood Pressure and Heart Rate” and “Actions of Skeletal Muscle Relaxants” among the practice classes of pharmacology and “Pharmacokinetic Analysis using Compartment Models” among the practice classes of pharmaceutics were carried out using alternatives to animal experiments. The 3Rs regarding alternatives to animal experiments were explained before the practice classes were performed using alternatives. As alternative techniques, a PC simulator was used in pharmacology practice and the beaker method was employed in pharmaceutics practice. After the end of each practice course, a questionnaire survey was carried out in the students to evaluate the educational effects and students’ impressions. [Results and Discussion] In the questionnaire survey, the prevention of cruelty to animals and life ethics were commented on most frequently, followed by facilitation of understanding and the procedure involving the use of alternatives. However, some students commented that they could not feel that they were dealing with life science without handling experimental animals, indicating their lack of understanding about the 3Rs. These results suggest that the better arrangement of the contents of practice courses and explanation before them are important to convey not only the usefulness of alternatives to animal experiments, but also the importance of the prevention of cruelty to animals and life ethics.


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P-10 Involvement of acetylcholine and response to reduction in phosphorylated connexin43 in drug development research for ischemic heart disease ○Hideto Ariumi, Ikuya Imai, Tomoko Miyazaki, Miyoshi Kawakami, Yuji Yoshiyama Division of Community Pharmacy, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmacy, Kitasato University E-mail: [email protected] [Objective(s)] Pathological sympathetic overactivation and vagal withdrawal are thought to reduce the survival rate after acute myocardial infarction (AMI). Previously, we tested if aortic depressor nerve stimulation improved the survival rate after AMI in rats. AMI was induced by ligating the left coronary artery. Two minutes after the ligation, 10-Hz stimulation of the intact left ADN was started and continued for 30-min in treatment group, whereas no stimulation was performed in control group. The survival rate at 60 min after AMI was only 6.6 % in the control group, whereas it increased to 76.5 % in the treatment group. With regard to life-threatening arrhythmias in acute ischemia, the effect of vagal nerve stimulation (VS) has been reported to prevent ventricular fibrillation in animals 1). Therefore, we investigated the effect of acetylcholine (ACh), a parasympathetic nerve system neurotransmitter, on the gap junction component Cx43 using H9c2 cells. [Materials and Methods] H9c2 cells, which are spontaneously immortalized ventricular myoblasts from rat embryos, were used due to their conserved electrical and signal transduction characteristics. The cells were cultured in DMEM supplemented with 10% FBS and antibiotics. H9c2 cells were pretreated with 1 mM ACh for 8 h, followed by 1 h of hypoxia (1% of oxygen concentration). [Results and Discussion] When cells were subjected to hypoxia, the total Cx43 protein level was decreased. Hypoxia group showed a marked reduction in the amount of phosphorylated connexin43 (Cx43), whereas the hypoxia-ACh group showed only a slight reduction compared with the hypoxia group. These results suggest that ACh is responsible for restoring the decrease in the Cx43 protein level, resulting in functional activation of gap junctions. [References] 1) Efferent Vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving Connexin43 protein, Circulation, 112. 164-170. 2005


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P-11 Establishment of in vitro test method for peel adhesion of adhesive tapes ○Maki Shibuya1, Masako Yabe1, 2, Masaki Uchida1, Hideshi Natsume1, 3, Yasunori Morimoto1, 3

1 Faculty of Pharmaceutical Sciences, Josai University 2 Department of Pharmacy Services, Ashikaga Red Cross Hospital 3 Research Institute of TTS Technology E-mail: [email protected] [Objective(s)] The adhesive property is one of indispensable examinations in the evaluation of adhesive tapes, because the penetration of bioactive materials in the tapes through the skin is dependent on the area of contact between the application region of the tapes and the applied skin surface.1) In vitro and in vivo tests using the test plate and human skin, respectively have been developed and researched as a peel adhesion test.2) However, the factors influenced the adhesion in in vitro differed from that in in vivo. Hence, in vivo peel adhesion can not be predicted from the data obtained in in vitro. In some cases, in vivo and in vitro tests using animal skin may be required. In the present study, we attempt to establish the in vitro peel adhesion test method, which can predict the adhesion to in vivo human skin. [Materials and Methods] In vitro peel adhesion test : Five adhesive tapes were cut out at 12 mm in width and 100 mm in length under the environment at 25°C and 60% of humidity. Forty millimeters of the length were the adhesion area. In order to remove the influence of stretch of tape, non-stretch tape was put on the backing side. The prepared tapes were adhered to the bakelite (BP) and stainless steel (SSP) plates at 850 g weight of application pressure. Three hours after application, in vitro peel force was measured at 300 mm/min of peel rate and 90° of peel angle using Rheometer (NRM-2002J, Rheotech, Co., Japan) or Tensile and compression testing apparatus (SV-52NA, Imada Seisakusho Co., Ltd, Japan). In vivo peel adhesion test : in vivo peel force was measured under the same conditions as in vitro peel adhesion test. The prepared tape were applied to inside skin of the forearm in volunteers (four males, 22-23 years old). [Results and Discussion] In Rheometer, there was good correlation between in vitro peel force in BP and SSP and in vivo peel force in human skin (BP : r = 0.94, SSP : r = 0.87), but each data varied widely. In Tensile and compression testing apparatus, the better correlation between them was observed than that obtained by Rheometer (BP : r = 0.95, SSP : r = 0.97), and the lower standard deviation value of each date was also obtained. This was due to the improved maintenance of peel angle at 90° during peeling of adhesive tape by using Tensile and compression testing apparatus. Therefore, when the peel force was measured at 300 mm/min of peel rate and 90° of peel angle using Tensile and compression testing apparatus as a test device, the better correlation between them was obtained. These results suggested that the peel force in human skin could be predicted by in vitro peel adhesion test using Tensile and compression testing apparatus without animal experiments, which was a promising method alternative to in vivo peel adhesion test in human skin. [References] 1) P. Minghetti, et al., Boll. Chim. Farm., 140: 63-67 (2001). 2) M. Horgnies, et al., Int. J. Adhes. Adhes., 27: 661-668 (2007).


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P-12 Evaluation of the Palatability by a Taste Sensor ○Toshimi Iizuka, Hiroyuki Miyazaki, Hideto Ariumi, Yuji Yoshiyama Division of Community Pharmacy Center for Clinical Pharmacy and Clinical Sciences, Kitasato University School of Pharmacy E-mail:[email protected] [Objective] The intake of the food is essential to maintain life activity of a human being and an animal. However, there are an allergic material and the harmful food for the creature, and acidity and the bitterness tend to evade a feeding1). The taste is an important factor in the oral formulations. Because adherence is controlled by taste, and the reason is because it influences treatment effect. Brand and value added generic formulations of isosorbide (isosorbide dinitrate or mononitrate) were used for the evaluation of the flavor by a taste sensor, and the measurement of taste said to that reproducibility and objective evaluation are difficult. In addition, investigation of the influence on palatability is performed in animal feed2), but the palatability information of the animal is poor. Therefore we examined whether a taste sensor system helped animal feed choice. [Materials and Methods] The objective taste of brand and value added generic drugs of the isosorbide measured it with the taste sensor system αASTREE® (Alpha M.O.S., France). Seven taste sensors which we used were ZZ, AB, BA, BB, CA, DA and JE. It compared a taste restraint effect by the mineral water and the apple juice dilution. The judgment of the taste was evaluated with Euclidean Distances. [Results and Discussion] The taste of brand was recognized that it was worse than the value added generic drugs by the taste sensor system. Taste of both brand and value added generic drugs were restrained slightly by dilution with a mineral water, but it was restrained largely by dilution with an apple juice. The use of the taste sensor system αASTREE® is the method that is effective to evaluate objective taste. The taste sensor system is possible to measure of acidity and the bitterness, and it may predict the taste that an animal evades. And it may be found the good animal feed of the palatability. [References] 1) Steiner J.E., Glaser D., Hawilo E. et al: Comparative expression of hedonic impact: affective reactions to taste by human infants and other primates. Neurosci. Biobehav. Rev., 2001; 25, 53-74. 2) Akinori O., Hidenori K. et al: The Effects of Differences in Processing Procedure and Harvesting stage of Whole Crop Rice Silage on Palatability in Cattle. Tohoku Agric. Res., 2005; 58, 93-94.


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P-13 Statistical support models for validation studies ○Taku Otoizumi1, Takashi Omori1

1Doshisha University Faculty of Culture and Information Science E-mail: [email protected] [Objective(s)] Statistical expertise is required for analysis and interpretation of data, and a statistician can only serve as an advisor when a biologist needs to apply statistical methods in a study. The Organization for Economic Co-operation and Development (OECD) guidance document (GD)34 (OECD 2005) states that “a statistical advisor (biostatistician) should be a member of, or consultant to, the validation manager/management team and be involved in all phases of the validation of new and revised tests.” The validation study of the Short-Time Exposure test, which is proposed as an alternative to the eye irritation test, is currently in progress, and we provide support in the form of independent statisticians at the planning stage of this study. We collect data from laboratories, construct datasets for analysis, and report the result obtained. Although the need for statistical support in validation studies is determined on a case-by-case basis, the pros and cons of each statistical support model should be considered and examined. Here, we discuss the 3 statistical support models that can be applied in a validation study. [Materials and Methods] We assessed the following 3 statistical support models that can be provided in a validation study: (1) the statistical advisor consuls with and advises the members of the validation management team during all phases of the study, but does not analyze data him/herself, (2) the statistician participants in the study after all the experiments have been completed. He/she collects the data obtained from experiments, analyzes it, and reports the result, and (3) the statistician participates in the study from the planning stage. His/her role is the same as that defined in (2). [Results and Discussion] In model (1), the effort expended by the statistician in analyzing data is relatively small and the statistician would be able to focus on providing his/her statistical expertise. On the other hand, occasionally he/she may be unable to understand the details of the study. In model (2), independent data analysis would ensure validity of the results. However, after collecting data, the statistician must understand the purpose of the study. Therefore, the statistical work, including data analysis, may be time consuming. Usually, the quick response of results rather than construction of a reliable database is a priority for biologists. In model (3), the statistician must understand the details of the study before performing data analysis; however, this procedure would be time consuming for the statistician. Collaboration between the biologist and the statistician is the best means of achieving a successful validation study. The pros and cons of each statistical support model should be taken into account when a validation study is conducted. [Reference] OECD. Guidance document on the validation and international accetance of new or updated test methods for hazard assessment. 2005.


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P-14 Model-based approach to sensitivity, specificity, and accuracy in an interlaboratory validation study ○Takashi Omori Doshisya University Faculty of Culture and Information Science E-mail: [email protected] [Objective(s)] Sensitivity, specificity, and accuracy are proportions of agreement that should be considered when comparing a targeted animal test and an alternative test. These measures are important for the relevance of a validation process. In validation studies, investigators are required to use several different chemicals. On the other hand, the number of experiments performed in a laboratory is often limited. To resolve this problem, a strategy where several laboratories conduct experiments using partially or completely different chemicals is often adopted. However, in such cases, methods to summarize the data and to estimate the sensitivity, specificity, and accuracy have not been examined in detail. In this presentation, we propose a model-based approach to estimate the 3 above-mentioned indices by applying a logistic regression model, and have presented an example of the calculation. [Materials and Methods] Consider a hypothetical validation study of an alternative test for mainly evaluating the relevance by 3 laboratories (A, B, and C). The total numbers of tested chemical was 40; however, only 30 chemicals were tested in each laboratory because of the limitation of time and resources. After we collected the data, the frequencies of agreement of both tests were summarized by the positive or negative findings from the targeted animal test.

The logistic regression model is used to estimate proportions under different conditions. Using this model, we can directly estimate the related 3 measures with a specified coding known as effect coding. Sensitivity is related to the grand mean model and positive animal test results, and specificity to the grand mean model and negative animal test results. Accuracy is related only to the grand mean model.

[Results and Discussion] For the data set such that the sensitivity and specificity were 5/15 and 12/15 for laboratory A, 9/15 and 15/15 for laboratory B, and 8/15 and 13/15 for laboratory C, respectively, the average sensitivity, specificity, and accuracy measures for the laboratories were 0.6, 0.89, and 0.74, respectively. The corresponding values obtained using the logistic regression model approach were 0.60, 0.89, and 0.79, respectively.


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P-15 A molecular clock mechanism of cytochrome P450 (CYP) genes expression in serum-shocked HepG2 cells ○Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University E-mail:[email protected] [Objective(s)] Circadian rhythms have been observed in many biological systems and physiological functions. The effectiveness and toxicity of many drugs vary depending on the dosage in association with 24-hr rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Although the pharmacokinetics of several drugs, which are mainly eliminated by cytochrome P450 (CYP) metabolism, vary according to their dosing time, mechanism of the variation remains poorly understood. In this study, we investigated how the 24 hr oscillation in the expression of CYP was generated in hepatic cells.

[Materials and Methods] As brief exposure of HepG2 cells to 50% serum induced the 24 hr oscillation in the expression of clock genes, serum-shocked HepG2 cells were used as an in vitro model to study the molecular mechanism underlying the circadian clock in human liver. The amount of protein and mRNA were measured by Western blot analysis and RT-PCR. CYP- promoter activity was measured by Dual-Luciferase assay

[Results and Discussion] The present study suggests that temporal variations of CYPs expression were controlled by the transcriptional level in cultured human hepatocyte. Transcription of the CYP promoter was rhythmically controlled by clock genes. Our present findings provide a molecular link between the circadian clock and xenobiotic metabolism. In addition, these cultured cell models may be useful in the analysis of the molecular clock mechanism of the human biological rhythm research.


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P-16 A highly sensitive cell-based screening method for CYP2C9-mediated metabolic activation with an adenovirus expression system ○Atsushi Iwamura, Tatsuki Fukami, Hiroko Hosomi, Miki Nakajima, Tsuyoshi Yokoi Faculty of Pharmaceutical Sciences, Kanazawa University E-mail: [email protected] [Objective(s)] Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s (CYPs) are suggested to be one of the causes. CYP2C9 is the major isoform in hepatic drug metabolism. In the present study, we established a highly sensitive screening method for reactive metabolites generated by CYP2C9 using a cell-based assay. With this method, metabolic activation mediated by 8 hepatotoxic drugs was evaluated. [Materials and Methods] We constructed an adenovirus vector expressing CYP2C9 (AdCYP2C9), and human hepatocarcinoma HepG2 cells with suppressed expression of NF-E2 related factor 2 (Nrf2), a transcription factor of various detoxicification enzymes, by siRNA infected with AdCYP2C9. After infection, these cells were treated with hepatotoxic drugs, and cell viability was evaluated by measuring mitochondrial dehydrogenase activity and the generation of ATP. [Results and Discussion] HepG2 cells infected with AdCYP2C9 were treated with hepatotoxic drugs, resulting in a significant decrease of cell viability by treatment with benzbromarone, tienilic acid and losartan. In addition, this CYP2C9-induced cytotoxicity was enhanced by Nrf2 knockdown. In this study, we first found the metabolic activation of losartan by CYP2C9, and this suggested that the genes regulated by Nrf2 are associated with detoxicification of their cytotoxicity. This highly sensitive screening method would be useful in evaluating drug induced cytotoxicity caused by CYP2C9.


246

P-17 Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation Hiroko Hosomi, Tatsuki Fukami, Atsushi Iwamura, Miki Nakajima, ○Tsuyoshi Yokoi Faculty of Pharmaceutical Sciences, Kanazawa University E-mail: [email protected] [Objective] Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. CYP3A4 is the predominant CYP isoform in human liver and is responsible for the metabolism of more than 50% of clinical drugs. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). [Materials and Methods] We constructed an adenovirus vector expressing CYP3A4 (AdCYP3A4), and human hepatocarcinoma HepG2 cells with suppressed expression of NF-E2 related factor 2 (Nrf2), a transcription factor of various detoxicification enzymes, by siRNA infected with AdCYP3A4. After infection, these cells were treated with hepatotoxic drugs, and cell viability was evaluated by measuring mitochondrial dehydrogenase activity and the generation of ATP. [Results and Discussion] When 7 hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6�-hydroxylase activity (670 pmol/min/mg). Using AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by MTT and ATP assays, which showed that the viabilities of cells treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodon, nitrofurantoin, tacrine, terbinafine, and tolcapone) were significantly decreased. Moreover, the transfection of siNrf2 to decrease the cellular expression level exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of their cytotoxicities activated by CYP3A4. Thus, we constructed a cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical testing and increase our understanding of drug-induced adverse drug reactions associated with CYP3A4. [References] Hosomi H. et al., Toxicol In Vitro, 24: 1032-1038 (2010)


247

P-18 A basic study on migration to tissue of site probe drugs by using a cell membrane plate ○Jin Tokunaga1, Norito Takamura1, Kenji Ogata1, Nao Setoguchi1, Nobunao Ikewaki1, Toyotaka Nishio2, Keiichi Kawai2

1School of Pharmaceutical Sciences, Kyushu University of Health and Welfare、2School of Health Sciences, Kanazawa University E-mail: [email protected] [Objective] We have previously developed a simple, pharmaceutical distribution diagnostic method, to estimate the drug-binding capacity and its factor of each binding site on the human serum albumin (HSA) or α1-acid glycoprotein (AGP) in serum. In this study, to estimate the properties of drugs to migrate to tissue from the drug-binding capacity of HSA, we evaluated the cell membrane permeability of probe drugs used in the pharmaceutical distribution diagnostic method. [Materials and Methods]Porcine kidney-derived LLC-PK1 cells (=cell membrane) were seeded in a Transwell® cell membrane plate and cultured. The upper and lower chambers of the cell membrane plate were assumed to be the tissue and vascular sides, respectively. Phenytoin (PHT) at a final concentration of 100 μM and valproic acid (VPA) at final concentrations of 100 and 200 μM were used as site I and II probe drugs, respectively. [Results and Discussion] HSA at a final concentration of 0, 100, or 600 μM, bucolome (Buc) as a site I inhibitor at a final concentration of 100 or 600 μM, and oleic acid (Ole) as a site II inhibitor at a final concentration of 200 or 1,200 μM were added. In the presence of 100 or 600 μM HSA, 100 or 600 μM Buc caused a significant increase in PHT migration to tissue. Similarly, 200 or 1,200 μM Ole significantly increased VPA migration to tissue. We speculate that Ole inhibited the site for FFA on HSA and then site II. Ole is considered to inhibit the FFA site and site II and then to induce an allosteric change in site I, and, thereby, to increase protein binding; however, in the presence of 600 μM HSA, 1,200 μM Ole did not alter PHT migration to tissue. Further detailed studies on the migration of probe drugs to tissue are needed to estimate such drug migrations based on various clinical laboratory values, such as HSA, AGP, and FFA. [References] 1) Nishio, T., Takamura, N., Nishii, R., Tokunaga, J., Yoshimoto M., Kawai, K.: Influences of haemodialysis on the binding sites of human serum albumin: possibility of an efficacious administration plan using binding inhibition, Nephrol. Dial. Transplant., 23: 2304-2310, 2008.


248

P-19 Characterization of Light emitting-Bhas 42 Cells Using for Short Term Screening of Carcinogens ○Shin Asada1, Kiyoshi Sasaki1, Ayako Sakai1, Yoshihiro Nakajima2, Yosihiro Ohmiya2, Noriho Tanaka1

1 Hatano Research Institute, Food and Drug Safety Center, 2 National Institute of Advanced Industrial Science and Technology E-mail:[email protected] [Objective] The transformation assay using Bhas 42 cells, v-Ha-ras transfected BALB/c 3T3 cells, is known as a method for screening of carcinogenic chemicals. To simplify the transformation assay, we were planning the visualization of cell transformation using light emitting vector-integrated Bhas 42 cells. Previously, we selected matrix metalloproteinase 10 (MMP-10) as a target gene by DNA micro array analysis, and integrated the gene to Bhas 42 cells. In this study, we measure the cell growth rate of clones of the MMP-10 integrated Bhas 42 cells and examined their responsiveness to TPA to characterize them. This study was supported by NEDO project. [Materials and Methods] Promising six clones of the light emitting Bhas 42 cell lines (Clones 3, 4, 8, 15, 20 and 40) were examined. The cells were seeded at a density of 3.5×104 cells/mL onto dishes (diameter 5 cm), and the number of cells were counted using Coulter Counter® for 14 days. Based on this results, time schedule of TPA treatment was determined. The cells were seeded at a density of 1.4×104 cells/mL onto 6-well plate (Day 0), and treated with 50 ng/mL of TPA from Day 4 to Day 21, and the cultures were fixed and stained on Day 28. TPA induced spherical cell morphology and then increase of density in the Bhas 42 cells. Using these features, relative cell growth was measured and morphology of the cells were observed to evaluate responsiveness of the clones to TPA on Day 10.

[Results and Discussion] Doubling time of the parental Bhas 42 cells was approximately 17 hours. In contrast, doubling time of the clone 3, that of clones 4, 8, 15 and 20, and that of clone 40 were approximately 20, 27 and 38 hours, respectively. In the absence of TPA, the cell density of clone 15 was high and that of clone 40 was low In the clones 20 and 40, increased cell density was observed with TPA treatment. TPA caused morphological change in clones 3, 20 and 40.

From the results, we considered that TPA-responsive clones of the light emitting Bhas 42 cells were obtained. A screening method of carcinogens will be established using these clones.


249

P-20 An international validation study on a Bhas 42 cell transformation assay using 6-well plates for the prediction of chemical carcinogenicity ○Ayako Sakai1, Kiyoshi Sasaki1, Kumiko Hayashi1, Dai Muramatsu1, Shoko Arai1, Nobuko Endou1, Sachiko Kuroda1, Fukutaro Mizuhashi2, Sawako Kasamoto2, Miho Nagai2, Masumi Asakura3, Hideki Hirose4, Nana Ishii4, Kamala Pant5, Shannon W. Bruce5, Jamie E. Sly5, Albrecht Poth6, Susanne Bohnenberger6, Thorsten Kunkelmann6, Shojiro Yamazaki1, Makoto Umeda1, Noriho Tanaka1

1Food and Drug Safety Center, 2Biosafety Research Center, Foods, Drugs and Pesticides, 3Japan Bioassay Research Center, 4Mitsubishi Chemical Medience Corporation, 5BioReliance Corporation, 6Harlan Cytotest Cell Research GmbH [email protected] [Objective] The Bhas 42 cell transformation assay (Bhas 42 CTA) is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The assay protocol consists of two assays, the initiation assay and the promotion assay, to detect tumor-initiating activity and tumor-promoting activity, respectively, of chemical carcinogens. This study was carried out to validate Bhas 42 CTA in interlaboratory collaboration. [Methods] Six laboratories joined in the study from three countries. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. [Results and Discussion] On the allover judgment of the assay results obtained from three laboratories, 2-acetylaminofluorene and o-toluidine hydrochloride were positive in both initiation and promotion assays. Dibenz(a,h)anthracene was positive in the initiation assay but negative in the promotion assay. Lithocholic acid, cadmium chloride and methapyrilene hydrochloride were positive in the promotion assay and negative in the initiation assay. Mezerein was positive in the promotion assay and equivocal in the initiation assay. Sodium arsenite was positive in the promotion assay and its judgments in the initiation assay were split between two laboratories except a laboratory which reported the result under an inadequate dosing. Anthracene, D-mannitol, caffeine and L-ascorbic acid were negative in either assay. Consequently, Bhas 42 CTA discriminated all eight carcinogens including tumor-promoters from four non-carcinogens. In the initiation assay, perfect agreement of the judgment by three laboratories was reached for eight out of ten chemicals for which the three laboratories completed their assay under the adequate dosing. In the promotion assay, the perfect agreement was achieved in the judgment for ten of twelve chemicals. Thus, the present study demonstrated that the Bhas 42 CTA is reproducible and applicable to the prediction of chemical carcinogenicity. [This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).]


250

P-21 Screening of newly identified anti-cancer compounds using a human MDA-MB-231 cell line and analysis of their anti-neoplastic mechanism ○Shuso Takeda1, Kazumasa Matsuo2, Yoshiko Okamoto1, Mitsuru Shindo2, Curtis J Omiecinski3, Hironori Aramaki 1

1Department of Molecular Biology, Daiichi University of Pharmacy, 2Interdisciplinary Graduate School of Engineering Sciences and Institute for Materials Chemistry and Engineering, Kyushu University, 3Center for Molecular Toxicology and Carcinogenesis, 306D Life Sciences Building, Pennsylvania State University, University Park E-mail: [email protected] [Objective(s)] Among experimental human breast cancer cell lines, in particular, MDA-MB-231 cells was established as a suitable model for pre-clinical studies since they are highly aggressive, both in vitro and in vivo. We have been studying the isolation of anti-tumor compounds form natural plants, and (-)-xanthatin, one of the xanthanolides sesquiterpene lactones, was found to be an agent having strong anti-cancer potential. When focused on the structure of (-)-xanthatin, a functional group (i.e., a highly reactive group) “exo-methylene lactone” was contained. In the present study, we investigated anti-tumor effects of chemically synthesized xanthanolides sesquiterpene lactones including (-)-xanthatin on MDA-MB-231 cells in vitro, and we studied the anti-tumor mechanism(s). [Materials and Methods] Cell culture conditions and methods were based on procedures described previously (Ref. 1). Of the six xanthanolides used in this study, (-)-xanthatin, (+)-8-epi-xanthatin, and (-)-dihydroxanthatin were chemically synthesized by an original protocol (Ref. 2). Cell proliferation and LDH release were analyzed using a reagent of CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS reagent; Promega, Madison, WI, USA) and a CytoTox96® (Promega), respectively, according to the manufacturer’s instructions. DNA microarray analysis was performed by Hokkaido System Science Co., Ltd (Hokkaido, Japan). [Results and Discussion] We synthesized six xanthanolides and studied their anti-tumor effects on MDA-MB-231 cells with index such as cell growth and change in morphology. Among them, (-)-xanthatin was revealed to be an effective in inhibition of the proliferation with an IC50 value of 5.28 µM. Based on the structure-activity relationship analysis, exo-methylene lactone ring of the (-)-xanthatin was important. (-)-Xanthatin induced a characteristic cell morphological change, followed by caspase-independent cell death. Interestingly, GADD45s, genes induced by stress and cytokines, were highly up-regulated by (-)-xanthatin treatment. Study of the mechanism by (-)-xanthatin-mediated cell death is ongoing. [References] Ref.1: Takeda et al., Toxicology, 259:25 (2009). Ref.2: Matsuo et al., Tetrahedron, 66:8407 (2010).


251

P-22 Development of high-performed prediction system for chemical toxicity in the cell using a human artificial chromosome (HAC) vector ○Shigeyuki Yamaguchi1, Tetsuya Ohbayashi2, Yoshihiro Ohmiya3, Shigeaki Nishii4, Hideto Hoshino3, Tomomi Asai4, Yasuhiro Kazuki1, Mitsuo Oshimura1,2

1Grad. Sch. Med., Inst. Regenerative Med. Biofunction., Tottori Univ., 2 Res. Center. Biosci. Tech., Tottori Univ., 3 Res. Inst. Cell Engineering., AIST., 4 Tsuruga. Bio. Lab., Toyobo. E-mail:[email protected] [Objective(s)] We must be screening a harmful compound from among the chemicals invented every day for the sake of quality of life. Recently, the screening system of the cell base is paid to attention instead of using the screening system of animal base. For the final purpose of this project, we will be creating a high-performed prediction system for the toxicity of harmful chemicals on the cell base assay using multiple bioluminescence probes and a human artificial chromosome (HAC) vector. [Materials and Methods] We developed a HAC vector from human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. The HAC vector enables transgene on the HAC to express under physiological regulation in the host cells. In this project, we constructed the monitor genes for the chemical-toxicological double colored luciferase assay (green luciferase: SLG, red luciferase: SLR). The monitor genes inserted in the HAC vector expressed persistently. [Results and Discussion] We test the reproducibility and precision of this monitor gene by exposure of toxic substance. Base on the results of these experiments, we will report the effectivity of this alternative method compared to conventional system using cultured cell.. [References] 1. Nakajima Y, Kimura T, Sugata K, Enomoto T, Asakawa A, et al. (2005) Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. Biotechniques 38: 891-894. 2. Katoh M, Ayabe F, Norikane S, Okada T, Masumoto H, et al. (2004) Construction of a novel human artificial chromosome vector for gene delivery. Biochem Biophys Res Commun 321: 280-290.


252

P-23 Generation of ES cells which monitor the cardiac muscle differentiation using a human artificial chromosome (HAC) ○Yuki Yoshimura1, Shigeyuki Yamaguchi1, 2, Shiho Endo2, Yoshihiro Nakajima3, Yasuhiro Kazuki4, Yoshihiro Ohmiya5, Mitsuo Oshimura1, 4, Tetsuya Ohbayashi2

1Dept. of Biomed. Sci., Inst. of Regenerative Med., Tottori Univ., 2 Div. of Laboratory Animal Sci., Res. Ctr. for Biosci. and Technology, Tottori Univ., 3 Health Research Institute., Advanced Industrial Science And technology., 4Chromo. Eng. Res. Ctr., Tottori Univ., 5Bioproduction Research Institute., Advanced Industrial Science And Technology. E-mail:[email protected] [Objective(s)] Differentiation of cardiac muscle is regulated by variety of transcriptional factors. The process or stage of the cardiac muscle differentiation is well documented by transcriptional factors specifically expressed by cardiac muscle. To study the cardiac muscle differentiation, we monitored expressions of a key transcriptional factor of cardiac muscle with luminescence. It is important to demonstrate the ‘physiological’ expression of the transcriptional factor. [Materials and Methods] We used Emerald luciferase (ELuc) which allows live imaging to monitor the expression of the transcriptional factor. We designed ELuc to be regulated by promoter region and also by enhancer, repressor and suppressor regions of the transcriptional factor. To introduce this large genomic DNA (175 kbp) into D3 ES cells, we employed human artificial chromosome (HAC) vector. D3 ES cells successfully carrying HAC vector were selected by PCR and FISH. We indused the D3 ES cells to cardiac muscle by hunging drop, conducted luciferase assay at serial time point (every other day). [Results and Discussion] We established D3 ES cells carring a HAC vector monitoring a marker gene specifically expressed in cardiac muscle. We induced D3 to differentiate into cardiac muscle by hunging drop, and conducted luciferase assey with D3 at sorted time points of culturing. We confirmed that the marker gene expression was physiologically demonstrated by luminescence of ELuc encoded HAC in D3. Now, we attempt to produce a mouse carring this HAC monitoring.


253

P-24 Establishment and characterization of engineered lymphatic endothelial cells with longer lifespan ○Sugano M, Nakamura R, Kiyono M, Sone Y, Inoue K Department of Public Health, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. E-mail: [email protected] [Objective(s)] To date, isolated lymphatic endothelial cells have not been considered to be suitable for molecular research, since the lifespan of previously reported lymphatic endothelial cells (LEC) such as normal human dermal lymphatic microvascular endothelial cells are relatively short. Furthermore, the characterization and function of LEC remain unidentified. The aim of this study was to genetically establish an in vitro LEC culture system with a longer lifespan. [Materials and Methods] Normal human dermal lymphatic microvascular endothelial cells were transfected with an expression vector SV 40 large T antigen as conducted previously [1]. The characterization of LEC-SV 40 was examined in the context of shape, surface expression of LEC-related markers, and lifespan. Further, the effect of estrogen stimulation on the induction of molecules related to lymphatic cell maturation/activation in these cells was investigated. [Results and Discussion] The characteristics and functions of LEV-SV were similar to those of LEC. LEV-SV survived almost 2 times longer than LEC. Estrogen stimulation induced several molecules related to LEC activation such as Xlkd1, prox1, and calcrl in LEC-SV. Taken together, the development of this assay system using LEC-SV may shed light on research regarding lymphatic biology. [References] [1] Kim DW, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S. Use of the human elongation factor 1α promoter as a versatile and efficient expression system. Gene 91; 217-223: 1990.


254

P-25 Investigation of the 3D cell culture system using a shape controlled Titanium coil ○Takaki Shima, Kazutaka Yoshino, Akihiro Ametani, Yasuo Seki HI-LEX Corporation Medical Device Department E-mail:[email protected] [Objective(s)] It has been reported that the cells cultured on plastic dish were different from the ones in living organisms. In order to obtain the same result from a test with cultured cell and animal experiment, it is necessary to develop a culture system in which the cells have a same activity compared with organisms. Also, reproduction of the organisms In Vitro is requested for a field of regenerative medicine. Though a lot of materials are used for 3D-cell culture, a problem that the cells are not grown inside of porous scaffold because factors for cell growth are insufficient in the interior of the scaffold is pointed out. In our company, non-woven titanium mesh is developed as scaffold for cell culture and implant. In this presentation, we report titanium coil as a new scaffold for 3D cell culture. [Materials and Methods] Titanium coils that have 0.64mm outer diameter, and gaps between titanium wires are 0, 0.025, 0.05 and 0.1mm, were produced by 0.08mm titanium wire. Autoclaved coils were placed on the 24-well plate, and HEK 293T cells were plated at a density of 1.0x104 cells/cm2. After 3 days, coils were moved to new plate. The cells were observed for 10 days with changing culture medium every 2 or 3 days. After 10 days, cells were fixed with 4%-paraformaldehyde and stained with Alexa488-Phalloidin. The number of cells was calculated from the amount of DNA in the cells. [Results and Discussion] When the cells were confluent on the plastic plate, the cells were not observed on the titanium wire. However, after 3 or 4 days, the coils were moved to new plate, the growing cells were observed. After 7 to 10 days, growth of the cells was completed and they covered allover the coil. The gaps of titanium wires were filled with the cells and the growth of the cells did not depend on the width of titanium wire gaps. Fluorescent staining indicated that the cells formed multilayer around titanium wires. 17.2×104 cells were counted from 5mm coil. It is three times of the cells counted from 96-well plate (5.2×104 cells). These results indicated that the titanium coil was useful scaffold for 3D cell culture. Shape of the titanium coil could be controlled easily. The coil is expected for application as follows: (I) culture numerous cells in a small area, (II) forming a model of living organisms In Vitro, (III) test with the model not carrying out animal experiment. [References] It is unpublished data. No references.


255

P-26 Rapid and spatially controlled formation of bile canaliculi in polarized hepatocyte in micropatterned collagen on oxygen-permeable membrane ○Hitoshi Matsui1, 3, Hiroshi Kimura2, Tomoharu Osada3, Masaru Sekijima3, Teruo Fujii2, Shoji Takeuchi2 and Yasuyuki Sakai2

1BEANS Laboratory, Tokyo, JAPAN, 2Institute of Industrial Science, The University of Tokyo, JAPAN, 3Mitsubishi Chemical Medience Co. Ltd., Ibaraki, JAPAN E-mail:[email protected] [Objective(s)] Polarized hepatocytes that have a specialized compartment called bile canaliculi are an indispensable model for investigating the directional transport of compounds and metabolites. Sandwich-cultured hepatocytes overlaying collagen gel [1] has now become a common approach to construct and examine polarized hepatocytes. However, in a conventional sandwich culture, repolarization normally takes about 4 days, and the form of constituted bile canaliculi demonstrate a wide variation, thus leading to inconsistent results. To overcome these problems, we designed a hepatocyte culture system using a micropatterned collagen gel positioned on an oxygen-permeable PDMS membrane. [Materials and Methods] Figure 1 shows a schematic image of the proposed culture system. Hepatocytes were aligned and induced the polarity in micropatterned collagen gel [2] on oxygen-permeable membrane. We analyzed the formation of polarity by observing the fluorescence of bile canalicular membrane transporter Mrp2 substrate 5-(6)-carboxy-2`,7`-dichlorofluorescein (CDF) accumulated in the bile canalicular lumen between the polarized hepatocytes [Results and Discussion] We showed the bile canaliculi in hepatocytes on oxygen-permeable membranes were detected from culture Day 2. In contrast, almost all of the fluorescence remained inside the hepatocyte cytoplasm on culture Day 2 in conventional sandwich-cultured hepatocytes. When primary hepatocytes were aligned in collagen gel cavities underconditions with abundant oxygen supply through oxygen-permeable fluorocarbon membrane, we succeeded in formation of bile canalicular tube along the channels in which the hepatocytes had been embedded (Figure 2). The culture system demonstrated herein will lead to the accelerated, enhanced and geometrically controlled formation of the bile canalicular network in polarized hepatocytes. This fine hepatic tissue can therefore be a useful tool for performing chemical/drug assays on a chip. [References] 1. "Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration.," J. C. Y. Dunn, R. G. Tompkins, and M. L. Yarmush, Biotechnol Prog, 7, 237 (1991). 2. “Molding of three-dimensional microstructures of gels,” M. D. Tang, A. P. Golden, and J. Tien, J. Am. Chem. Soc, 125, 12988 (2003).

Fig. 1

Fig. 2


256

P-27 Efficient Reconstruction and Evaluation of a Pseudo-Islet Like Aggregate from an Oxygen Permeable Microwell Sheet ○Marie Shinohara1, Kikuo Komori1, Teruo Fujii1, Yasuyuki Sakai1

1Institution of Industrial Science, University of Tokyo E-mail: [email protected] [Objectives] The mouse insulinoma Min6-m91) cell is a useful in vitro pancreatic -cell model to further enhance our understanding of diabetes and discover new drugs. Three-dimensional (3D) micro-tissues of Min6-m9 cells are expected to exhibit physiologically-relevant responses identical to those of Langerhans islets in vivo. In this study, we fabricated different sizes of 3D islet-like cell aggregates using an oxygen permeable sheet with micro-wells and elucidated the relation between their sizes and pancreatic function. In addition, physiological and pancreatic functions of aggregates cultivated on the oxygen permeable (OP) dishes with direct oxygenation from the bottom were compared with those on polystyrene (PS) dishes without direct oxygenation from the bottom. [Materials and Methods] We fabricated polydimethylsiloxane (PDMS) sheets with different sizes of micro-wells (46, 76, 126, 200, and 326 m in diameter and 65, 91, 129, 156, and 225 m deep, respectively) by photolithography and soft lithography techniques. The top surface of the sheet was coated with non-adhesive 2-methacryloxyethyl phosphorylcholine polymer. Min6-m9 cells were seeded on this sheet at a high density of 1-5x105 cells/cm2 and incubated for 30 min before rinsing with medium. These sheets were further cultured for 2 days to obtain Min6-m9 aggregates. Cellular morphology and viability of the aggregates were evaluated by microscopy after cell staining. Respiration of aggregates was estimated by glucose consumption and lactate production using a glucose lactate analyzer. Insulin secretion ability per cell measured by ELISA was determined as an index of pancreatic functions. [Results and Discussion] Different sizes of spherical aggregates were successfully obtained by using PDMS sheets placed on both oxygen permeable (OP) and impermeable (PS) dishes. However, the viability of the aggregate on the former was higher than that on the latter. In contrast, the lactate production in the OP dish was about twice as low as that in the PS dish, due to the fact that aerobic respiration takes place on the OP dish, whereas non-aerobic respiration takes place on the PS. We also found that the intracellular insulin concentration per cell in the aggregates formed in the OP dish was about twice as high as that in the PS dish. Additionally, in the case of PS, the concentration drastically increased from 200 m upwards in diameter and then leveled off. Those results show that oxygen and the aggregate size may play important roles for the formation and functional maintenance of the islet-like aggregate. [References] 1) Minami, K. et al. Am J Physiol Endocrinol Metab, 279: 773-781(2000)


257

P-28 Minimization of a Three-dimensional Liver Tissue for an In Vitro Toxicity Test ○Kikuo Komori, Hiroaki Suzuki, Teruo Fujii, and Yasuyuki Sakai Institute of Industrial Science, University of Tokyo E-mail: [email protected] [Objective(s)] In vitro toxicity tests are widely used to evaluate the effects and toxicity of drugs, cosmetics, and food additives. To obtain more information from cells, which are nonproliferative and in limited supply, micro-tissues exhibiting physiologically-relevant responses identical to those of their corresponding tissues/organs should be used. We have previously examined the minimum required size for in vitro toxicity testing using a two-dimensional micro-tissue made of human hepatocarcinoma Hep G2 cells. In this study, we focused on a three-dimensional micro-tissue of Hep G2 and determined its minimum required size for in vitro toxicity analyses using the cell viability test of an indirect mutagen aflatoxin B1 (AFB1). [Materials and Methods] We fabricated a polydimethylsiloxane (PDMS) sheet with different sizes of micro-wells (63, 200, 630, and 2000 μm in diameter and 20 μm deep) as a template using photolithography techniques. Hep G2 cells were seeded at a high density of 2.0 105 cells cm-2 on the PDMS sheet, after coating its top surface with a non-adhesive polymer, followed by local adsorption of collagen only to the micro-well surface. Based on the EROD assay, the intracellular enzyme cytochrome P450 (CYP) 1A1/2 activity was evaluated as an index of liver functions using tissues cultured for 5 days, which were pretreated with 3-methylcholanthrene to enhance the CYP 1A1/2 activity. A cell viability test was performed after 48 h exposure to AFB1. [Results and Discussion] Hep G2 cells were seeded on the PDMS sheet with micro-wells, resulting in upward proliferation of cells in a smaller diameter of the micro-well for 3days. In contrast, a sheet-like micro-tissue was formed in a larger diameter one. In the case of the smaller micro-well, the autocrine/paracrine action of various growth factors is likely enhanced due to their higher concentration in a small volume, resulting in acceleration of cell proliferation. In the case of the larger micro- well, O2 supply to cells might be limited, resulting in inhibition of cell proliferation.

Using micro-tissues on day 5, we examined the size dependence of drug metabolic activity. As a result, the drug metabolic activity for the micro-tissue 200 μm or larger in diameter was 3-5 times higher than that 63 μm in diameter, indicating a threshold level of the activity appeared between 63 and 200 μm in diameter. Next, we examined cytotoxicity of AFB1, which is well-known to be metabolized by CYP 1A1/2, resulting in the formation of an ultimate strong toxicant, followed by apoptosis. As expected, the cytotoxicity depended on the CYP 1A1/2 activity. EC50 values for micro-tissues 200 μm in diameter or larger is one order of magnitude lower than that 63 μm in diameter and were nearly equal to that for a conventional 96-well plate (6.4 mm in diameter). Thus, in the three-dimensional micro-tissue, the minimum required size for in vitro toxicity tests was determined to be 200 μm in diameter with an estimated number of ca. 500 cells.

[Acknowledgement] This work was supported by 2nd Mandom International Research Grants on Alternative to Animal Experiment.


258

Fig.1 (a) Schematic illustration ofelectropolymerization andelectrochemical actuation to printPEDOT on hydrogel sheet. (b) Picture ofPEDOT/ l d

(a)

(b)

5 mm5 μ

Fig.2 Time course of contractiledisplacements of C2C12 myotubes andthe PEDOT hydrogel electrode.

P-29 Hydrogel electrode for effective electrical stimulation of cells ○Y. Ido1 , S. Sekine1 , D. Takahashi1, K. Nagamine1,2 , T. Miyake1,2 , M. Nishizawa1,2

1 Tohoku University, 2 JST-CREST [email protected] [Objective(s)] Conducting polymers such as poly(3,4-ethylenedioxythiophene)(PEDOT) and polypyrrole (PPy) are attractive electrode materials that have the advantages of biocompatibility, high capacitance, and flexibility. We report herein the micropatterning of PEDOT on a hydrogel, agarose, to provide a fully organic, moist, and flexible electrode[1]. We also present a typical application of the PEDOT/agrose electrode to electrical stimulation for the C2C12 embedded fibrin sheet[2].

[Materials and Methods] The melted 2.8 wt % agarose solution was poured over a Pt microelectrode fabricated on a glass plate. After gelation of the agarose, electropolymerization (1.0 V vs. Ag/AgCl) and electrochemical actuation (±0.5 V vs. Ag/AgCl, 0.02 Hz) for effective peeling the soft gel from master electrode were conducted on the gel-covered electrode in the aqueous monomer solution (Fig.1). [Results and Discussion] Fig.2 shows the electrical stimulation supplied through the PEDOT/agarose electrode induced contraction of the C2C12 myotubes. Importantly, the PEDOT electrode also contracted synchronously with the motion of the cells. We are now conducting long term electrical stimulation for C2C12 myotubes through the PEDOT/collagen electrode which has mechanical strength, transparency and biocompatibility.

[References] [1] Sekine, S.; Ido, Y.; Miyake, T.; Nagamine, K.; Nishizawa,

M., J. AM. GHEM. SOC. 2010, 132, 13174–13175 [2] Nagamine, K.; Kawashima, T.; Ishibashi, T.; Kaji, H.;

Kanzaki, M.; Nishizawa, M., Biotechnol. Bioeng. 2010, 105 (6), 1161-1167.


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P-30 A basic study of estimation of O2 and CO2 transfer in an oxygenator with a numerical analysis for minimization of the blood experiment Katagiri N.1, Funakubo A.2, Tsukiya T.1, Tatsumi E.1, Mizuno T.1, Takewa Y.1, ○Shioya K.1, Taenaka Y.1, Fukui Y.2

1National Cerebral and Cardiovascular Center Research Institute, 2 Division of Electrical and Mechanical Engineering, Tokyo Denki University E-mail: [email protected] [Objective(s)] Applycation of oxygenators has been expanded from cardiopulmonary bypass for several hours at the open heart surgery to a long-term respiratory support for several weeks as an improvement of its gas exchange performance and durability. A mainstream is a hollow fiber membrane oxygenator with extra-capillary blood flow that exchanges gases by the hollow fiber membrane between the blood layer and the gas layers. The outer diameter of the hollow fiber is hundreds of micro meters. The gas exchange in an oxygenator is a transfer phenomenon with minute blood flow among fibers, complexity of chemical reaction of dissolved gases and blood. Therefore, this characteristic transfer makes the estimation of the gas exchange performance at the design stage to be difficult, and frequent blood experiment is assumed to be indispensability at the development. To prevent this problem, some numerical analyses on a computer were examined. However, most of numerical analysis method that can deal the gas transfer of the entire oxygenator required measured mass transfer characteristics of a specific hollow fiber bundle by the blood experiment. In this study, it aimed to estimate oxygen and carbon dioxide transfer rates in a segment of the hollow fiber bundle on a computer without performing the blood experiment by our invented numerical analysis method, and to compare the estimated values with the measured values of the limited part of an object oxygenator that corresponded to the segment model. [Materials and Methods] The invented numerical analysis method combines original programs that calculate membrane transfer source and blood-gas reaction source of both oxygen and carbon dioxide with commercialized computational fluid analysis software that can calculate mass transfer and fluid dynamics. The object oxygenator has a rectangular bundle consists of parallel and staggered arranged hollow fibers. Lengths of a segment model are 30 mm in the blood flow direction (full length of the bundle) and 4 mm in the gas flow direction (sufficient length for comparing with actual data). Velocity conditions were set up with assuming 1, 3 and 5 L/min of the blood flow rate (Q) and 1 of the ratio of gas to blood flow rate (V/Q). Concentration conditions assumed the inflow blood to be a standard venous blood property, and set up the inflow gas 100% oxygen. In addition, the influence of the pH was examined. In the blood experiment for the comparison, the blood sampling port was installed on the blood outflow side of the limited part of an object oxygenator, and the gas transfer rates of the limited part were measured under the same conditions of flow rate and concentration. [Results and Discussion] 1, 3, and 5 L/min to each condition of Q, oxygen transfer rates showed 71.4, 163.6, and 221.9 mmHg at constant pH analysis, and 72.5, 170.4, and 232.0 mmHg at variable pH analysis, and 55.4, 153.1, and 229.4 mmHg at the actual measurement. Carbon dioxide transfer rates showed 101.4, 247.9, and 342.9 mmHg at constant pH analysis, and 66.1, 156.0, and 205.3 mmHg at variable pH analysis, and 99.5, 117.5, and 151.9 mmHg at the actual measurement. It was demonstrated that the influence of the pH was selectively effective on the carbon dioxide transfer analysis, and obtained practicable estimation of gas transfer rates as for both oxygen and carbon dioxide from the variable pH analysis. Thus, the possibility of the estimation of the gas transfer rates in an oxygenator that did not require the blood experiment was shown by using our invented numerical analysis method.


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P-31 Evaluation of PDMS/PEG Copolymer Impregnated Membranes as a Human Skin Alternative for In Vitro Skin Permeation Testing ○Ryotaro Miki1, Soichiro Kimura1, Toshinobu Seki1, Kazuhiko Juni1, Hideo Ueda1, Yasunori Morimoto1, 2

1 Faculty of Pharmaceutical Sciences, Josai University, 2 Research Institute of TTS Technology E-mail: [email protected] [Objective(s)] In vitro skin permeation test is widely conducted using excised human or animal skin to understand skin permeability of various compounds. However, these biological skins have problems such as variation of data, ethical issue, time-consuming preparation. Thus, an alternative instead of human or animal skin has been required for the peameation test. The goal of our study was to prepare a novel artificial membrane that can mimic the human skin permeability behavior for both lipophilic and hydrophilic compounds. [Materials and Methods] The copolymer was synthesized using poly(dimethyl siloxane) (PDMS) macromer (SILAPLANE; FM-0711) as the lipophilic material, and macro-azoinitiator VPE-0601 containing poly(ethylene glycol) 6000 (PEG 6000) units as the hydrophilic material. A homopolymer consisted of PDMS units was also synthesized to deal as the simple lipophilic barrier using FM-0711 and azoinitiator (AMBN). Then, we evaluated molecular weight distribution and chemical composition of the synthetic polymers by gel permeation chromatography (GPC) and 1H-NMR. Flat shape membranes were prepared by impregnating the synthetic polymers into membrane filter. The permeation properties through the impregnated membranes were evaluated using several compounds which have almost the same molecular weight and different lipophilicity. The results were compared with those of human skin. [Results and Discussion] The permeability coefficient (P) of the impregnated membranes were obtained and compared with those of human skin which was previously obtained 1). Linear regression between P of PDMS/PEG 6000 impregnated membrane and human skin showed good correlation and the slope was close to 1.0 that is assumed the ideal value. It seems that PDMS/PEG 6000 impregnated membrane has the potential as the alternative to predict the human skin permeability of both lipophilic and hydrophilic compounds. [References] 1) Morimoto Y., Hatanaka T., Sugibayashi K. and Omiya H. (1992) Prediction of skin permeability of drugs: Comparison of human and hairless rat skin, J. Pharm. Pharmacol., 44, 634-639.


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P-32 Evaluation of skin permeability of volatile substances: establishment of evaluation method using alternative membrane for animal skin ○Takeshi Sugama, Takeshi Oshizaka, Hiroaki Todo, Kenji Sugibayashi Faculty of Pharmaceutical Sciences, Josai University E-mail: [email protected] [Objectives] Skin permeation experiment is important not only for evaluation of efficacy but also safety of chemical compounds. We have previously reported the correlation between skin irritation and skin concentration of chemical compounds1): the skin concentration could be calculated from permeation parameters2). Although many volatile chemical substances are used in our lives, only a few reports were found on the skin permeation of those compounds. In the present study, we selected aromatic volatile chemical compounds and investigated the effect of volatilization rate on the skin permeability. Silicone membrane and poly (2-hydroxyethyl methacrylate) (pHEMA) membrane were also selected as alternatives for skin membrane in addition to hairless rat skin. [Materials and Methods] Sample preparation: Fragrance ingredients (cinnamaldehyde, eugenol and thymol) were selected as volatile chemicals. These fragrance ingredients were dissolved to form saturated solution. Membrane permeation experiment: Permeation experiments were performed with excised hairless rat skin, silicone membrane and pHEMA membrane using a Franz-type diffusion cell. Periodically sampling was conducted over 12 hours and 5 min, for permeation experiments through the rat skin or pHEMA membrane and silicone membrane, respectively. The fragrance concentration in the solution was measured by HPLC. Measurement of membrane concentration: Each membrane was recovered after the permeation experiment. The application area of fragrance was punched out from the membrane to extract the fragrance by ethanol. The fragrance concentration in the solution was measured by HPLC. Measurement of volatilization rate: Upside-open and upside-closed cells were placed on hairless rat skin and pHEMA membrane. Whole set was maintained at 32°C. The volatilization rate of fragrances was calculated from the reduction amount from these cells. [Results and Discussion] The permeabilities of fragrance ingredients through the membranes were represented in the following decreasing rank order; cinnamaldehyde > eugenol > thymol, regardless of the membrane type. On the other hand, the concentration of fragrance ingredient in each membrane showed increasing rank order; cinnamaldehyde ≤ eugenol < thymol. Although the volatilization rate of the fragrance ingredients was eugenol > thymol > cinnamaldehyde, the amount of volatilized fragrance was only less than 0.1%. Therefore, in the present condition, the low volatility of fragrance ingredient might not significantly affect the skin permeability. From these results, fragrance ingredients with low volatility would show almost the same rank order both in skin permeability and skin concentration, regardless of the membrane type. At the present moment, the rank order of permeability and concentration in animal or human skin could be predicted from the permeability through silicone membrane or pHEMA membrane. Further analysis and experiment will be need to allow to predict human or animal skin permeability from the result of permeation experiment using silicone and pHEMA membranes. [References] 1) Kano S., Sugibayashi K., Pharm. Res., 23, 329-335 (2006). 2) Sugibayashi K., Todo H., Oshizaka T., Owada Y., Pharm Res., 27, 134-142 (2010).


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P-33 Effect of skin barrier function and metabolic ability on the in vitro skin irritation using cultured human skin models ○Katsunori Furui, Hiroaki Todo, Kenji Sugibayashi Josai university faculty of pharmaceutical sciences

E-mail: [email protected] [Objective] Three-dimensional cultured human skin models have been used for in vitro skin corrosive/irritation test from the viewpoint of animal welfare. However, false-negative and false-positive reaction were obtained with several chemical compounds. Skin irritation depends on the skin concentration and exposure time of chemical compounds1). Therefore, evaluation of skin concentration would be very important to predict skin irritation of chemical compounds. We have already reported that the skin concentration of chemical compounds could be predicted from skin permeation parameters obtained from skin permeation experiments2). Differences in the skin permeability of chemical compounds must be related to the false-negative and false-positive reaction in the corrosive/irritation test. Several chemical compounds with false-negative and false-positive reactions have high lipophilicity and ester moiety. Therefore, in the present study, we searched physicochemical properties of reference chemicals with false-positive and false-negative reactions and investigated the effect of esterase activity and its distribution in each culture skin models on the concentration-distance profiles of the chemical compounds. [Materials and Methods] Three-dimensional cultured human skin modes; LSE-high and Vitrolife-skin as a full-thickness skin models and LabCyte EPI-MODEL, Episkin and EpiDermTM Epi 606X as an epidermal model were used. Ethyl nicotinate was used as a model compound with an ester moiety. Esterase activity parameters (Km, Vmax) were evaluated using the cultured human skin models, excised human skin and excised hairless rat skin. Skin distribution of fluorescein-5-isothiocyanate was observed using a fluorescent microscope. [Results and Discussion] Carboxylesterase activity resulted in a decreasing order: hairless rat skin > human skin > three-dimensional culture human skin modes. Km and Vmax were different in each skin, although Km values in three-dimensional cultured human skin modes were almost equivalent to each other. Fluorescein-5-isothiocyanate was observed in the viable epidermis in human and animal skins. Esterase distributions in LSE-high, Episkin and EpiDerm were almost the same; the other models LabCyte EPI-MODEL and Vitrolife-skin indicated different distributions. In addition, chemical compounds with high lipophilicity and ester moiety have the possibility to induce a false-negative reaction from list of reference chemicals. These results suggest that the differences of esterase activity and its distribution in skin might induce a false-negative and false-positive reactions for metabolizable chemical compounds with high lipophilicity. [References] 1. Kano S., Sugibayashi K., Pharm. Res., 23, 329-335, (2006). 2. Sugibayashi K., Todo H., Oshizaka T., Owada Y., Pharm. Res., 27, 134-142 (2010).


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P-34 Alternative animal testing of skin sensitization test using three-dimensional human skin model involving dendritic cells ○Tadashi Uchino1, Toshiaki Takazawa2, Yoshiaki Ikarashi1, Tetsuji Nishimura1

1National Institute of Health Sciences, 2 National Institute of Agrobiological Sciences E-mail: [email protected] [Objective] The EU, animal testing will prohibit for safety evaluations of cosmetic ingredients, so establishing an in vitro skin sensitization test method is a matter of great urgency. Recently, in vitro skin sensitization tests using a dendritic cell line [e.g. human cell line activation test (h-CLAT)] have been developed, but it is difficult to test water-insoluble chemicals using these models. So, we established a test method which is a three-dimensional human skin model composed of normal fibroblasts, normal keratinocytes and normal dendritic cells using a collagen vitrigel membrane (VG-KDF-Skin) was made and then cytokine release from VG-KDF-Skin with skin sensitizers was measured by ELISA. In this study, we compared to result of this test method and result of LLNA. In addition, in order to study the possibility of application of this test method to cosmetics, two model cosmetic samples containing skin sensitizer [2,4-dinitrochlorobenzene (DNCB)], were evaluated. [Materials and Methods] Nine skin sensitizers and 5 non-sensitizers were investigated. VG-KDF- Skin was treated with test chemicals for 1 hr. When cytokine (IL-1α, IL-4 and IL-8) release in supernatant further 23 hr incubated was over 150% compared to those in control, the test chemical was determind as positive. In the case of model cosmetic samples, 5 milligrams of cosmetic samples were placed on the applicator pads (0.5 cm2), applied to VG-KDF-Skin. [Results and Discussion] The accuracy, sensitivity and specificity of IL-4 release vs. LLNA were 93%, 89% and 100%, respectively. The accuracy, sensitivity and specificity of IL-1α release vs. LLNA were 50%, 56% and 40%, respectively. Skin sensitizers did not induce significant IL-8 release from VG-KDF-Skin. Milky lotions and creams containing DNCB induced significant IL-4 release. These results suggest that this test method using IL-4 release as an indicator would be applicable for the various description chemicals and cosmetic products of milky lotions and creams.


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P-35 Investigation of an Eye Irritation Test Using a Human 3D Corneal Model (2nd Report) ○Satoshi Nakahara Central Research Laboratories, Mandom Corporation E-mail: [email protected] [Objective(s)] Cosmetic products are generally not used directly in the eye, some are used around the eye or enter the eye by improper use. Therefore, it is necessary to evaluate the eye irritation when such cosmetic products or cosmetic integrants enter the eye. However, we must test cosmetic products by non-animal testing, we should develop estimation of alternative method in a hurry. I reported that there was high correlation between Draize eye irritation test and the eye irritation test using a human 3D corneal model at 30 chemicals.1) This time, I don’t evaluated chemicals but cosmetic products, and I confirmed correlation with Draize eye irritation test. [Materials and Methods] I evaluated the cell viability of 30 cosmetic products with known the eye irritation scores by Draize eye irritation test, using a human 3D corneal model (Japan Tissue Engineering Co., Ltd.) prepared from human corneal epithelial cells, according to the method of Arai et al.2) and Ogasawara et al. 3) (sample exposure, 1 min; post-incubate, 24 h; assessed as irritation for cell viability rate of 50% or less). Furthermore, the inflammatory cytokine IL-1α in the culture medium was analyzed by ELISA. [Results and Discussion] Cosmetic products were evaluated using 100%, 10%, 1% by a human 3D corneal model. This result showed high correlation with Draize eye irritation test, and there was no false-negative. Furthermore, correlation was high with STE test using SIRC cells. So, the eye irritation test using a human 3D corneal model is useful alternative method. In addition, I report that quantity of IL-1αin the culture medium together. [References] 1) Nakahara, S. (2009), 22nd Annual Meeting of the Japanese Society for Alternative to Animal Experiments, Abstracts, poster No., 33. 2) Arai, S., et al. (2007), 6th World Congress on Alternatives & Animal Use in the Life Sciences, Abstracts, p.224. 3) Ogasawara, T., Kato, M., Hamajima, F., Kubo, K., Shigeta, T., Hata, K. (2008), 21st Annual Meeting of the Japanese Society for Alternative to Animal Experiments, Abstracts, poster No., 9.


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P-36 Evaluation of acute toxicity using Caenorhaboditis elegans ○Maki Nakamura, Yoshihiro Yamaguchi, Hironori Tomi KOBAYASHI Pharmaceutical Co., Ltd. Central R&D Laboratory E-mail:[email protected] [Study objective]The number of animals used in toxicity studies has been considerably reduced in recent years in efforts toward animal protection and as a result of abolition of OECD Guideline 401, revision to 420, and other factors. However, there is still a need for use of rodents, though in small numbers. Therefore, in this study, Caenorhaboditis elegans (C. elegans), a nematode, was exposed to test substances, and the maximum exposure doses at which the survival rate was 100% were determined to examine whether this technique could be an alternative to those used in conventional acute oral toxicity studies. [Methods]A temperature-sensitive strain of C. elegans (fer-15) that does not lay egg at 25°C was used. Two thousand L1 larvae of C. elegans on day 1 after hatching by synchronous culture were transferred into S-medium, a liquid medium, containing E. coli OP50, and the test substances were added and incubated at 25°C with shaking. At 18 hours after the start of exposure, live and dead nematodes in 100 μL liquid medium were counted and the maximum exposure dose (MED) at which 100% survival was observed was determined. Seventeen compounds with known LD50 values in rats (133 to 38000 mg/kg body weight) were used as test substances. [Results and Discussion]The MED of polyoxyethylene (20) sorbitan monooleate (Tween 80), with a LD50

of 38000 mg/kg, was about 5%, those of methanol (LD50 = 5600 mg/kg) and ethanol (LD50 = 7000 mg/kg) were about 1%, that of copper sulfate (LD50 = 960 mg/kg) was about 0.03%, and that of caffeine (LD50 = 260 mg/kg) was about 0.02%. It was found that the MEDs of 17 compounds were highly correlated with LD50 values in rats. These findings suggested that evaluation of exposure of C. elegans to a test substance could be used as an alternative to an acute oral toxicity study in rats. In the future, items such as duration of observation period will need to be evaluated.


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P-37 Toxic interactions of clomipramine in chick embryos ○ Hiroyuki Miyazaki, Toshimi Iizuka, Motohiro Okayasu, Toshio Kouzuki, Takeshi Homma, Takashi Ogura, Yohhei Inada, Takenori Tamaki, Hideto Ariumi, Tomoko Miyazaki, Miyoshi Kido, Yuji Yoshiyama Center for Clinical Pharmacy and Clinical Sciences, Kitasato University School of Pharmacy E-mail: [email protected] [Objective(s)] In regard to the use of experimental animals for research and education, alternative methods for animal testing came to be discussed. Alternative test, which comes from mind of animal welfare, is replacement of an animal test with one that uses non-animal systems for purpose of research, education, toxicity test or production (Replacement), and includes reduction of animal use (Reduction) and to lessen or eliminate pain or distress to animals (Refinement). Moreover, alternative test is also useful to the abolition of useless animal experiment, economic evaluation of new compounds, risk evasion with searching a toxic unknown compound in animal experiment, study of the mechanism of action for extrapolation to human. In recent years, from the viewpoints of animal welfare and promotion of efficiency of research and development, the reconsideration of animal experiment, reduction of the used animal number, and international development of the alternative experimental animal which can replace it from mammals, are studied positively. We reported that chick embryo was useful as an alternative of mammals. Clomipramine is known to alter myocardial function manifested by electrocardiogram changes. In this study, we applied electrocardiographic findings of the chick embryo which was alternative experimental animal to pharmacometrics and examined toxic interaction with clomipramine and disopyramide. [Materials and Methods] Fertile eggs of White Leghorns were purchased from Saitama Experimental Animal Supply. These were incubated under 37.6±0.2°C, 65.5RH% in an incubator with automatic turn device, and were used on day 16 of incubation. The clomipramine and the disopyramide which were a test drug were administered into the air sac. The electrocardiograms (ECGs) were measured using three-needle electrode. Two electrodes were inserted into diagonal holes on the equator for using as a bipolar lead, and other 1 was inserted hole on south pole for using as a ground. This bipolar potential was led to a memory oscilloscope via an input box and passed PowerLab, and was recorded as ECGs consecutively. ECGs were recorded for 60 minutes after drug administration, and the heart rates (HRs) were calculated from R-R intervals. [Results and Discussion] After the administration of clomipramine 0.625 mg/egg, the heart rate was not different compared with physiological saline. However, the heart rate was significantly decreased by the administration of 1.25 mg/egg clomipramine. And the heart rate was significantly decreased by combination with clomipramin and disopyramide. In addition, arrhythmia was produced by clomipramin with disopyramide. Clomipramine is a tricyclic antidepressant and may be used in the treatment of depression. Cardiotoxicity of clomipramine was demonstrated in chick embryos. Chick embryo is located in the middle position of in vitro and in vivo, and has the circulatory system, endocrine system, nervous system and interaction of tissue/organ which is impossible of reproduction by in vitro test, thus, elucidation of multifaceted pharmacological action is possible. Furthermore, chick embryo has advantage that management as experimental material is simple, and an experiment under a constant condition is practicable. The evaluation method of toxicological/ pharmacological action utilizing chick embryo is the pharmacological study technique all of above-mentioned 3Rs (Replacement, Reduction and Refinement) is possible. We emphasize that this method can become an alternative test method of very high utility is expected.


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P-38 The effect of a biotin deficiency on cultured rat embryos ○Noriko Ishizuka1, Masaharu Akita2, Sayaka Yanagida2, Atushi Yokoyama 3, Naomi Kashiwazaki 4, Junya Ito 4, Masaru Maebashi 5 Tomo Inomata4

1Kiryu University, Gunma, Japan, 2Kamakura Women's University Kanagawa, Japan, 3Kanagawa Life Science Research Laboratory, Kanagawa, Japan, 4Azabu University, Kanagawa, Japan, and 5 Honjo 1st Hospital, Akita, Japan. E-mail: [email protected] [Objectives] We are attempting an improvement to optimize the embryo culture method as an alternative developmental toxicity test. In the present study, we investigated the effect of a biotin deficiency on cultured embryos. [Materials and Methods] Rat fetuses were carefully removed from mothers on day 11 of gestation. Fetuses were cultured for 48h. The culture was maintained at 37˚C with a gas atmosphere containing 5% CO2:95% O2. Fetuses of the biotin deficient (BD) group were cultured in biotin deficient serum that normal rat serum had had its bitoin removed by using an avidin column. Fetuses of the biotin supplemented (BS) group were incubated in the serum to which biotin had been added. Fetuses of the control (CO) group were incubated in normal rat serum. After 48 h of culturing, fetuses were carefully assessed about the number of heart beets, crown-rump length (CRL), number of somites, amount of protein, and external features in each fetus. [Results and Discussion] The number of heart beets per minute (BD: 181.6±2.85, BS: 186.8±5.99, CO: 172.3±7.21), CRL (mm) (BD: 6.71±0.08, BS: 6.77±0.11, CO :6.87±0.09), number of somites (BD: 14.87±0.35, BS: 15.07±0.32, CO: 15.29±0.42), and amount of protein (mg) per fetus (BD: 2.42±0.02,BS: 2.64±0.02, CO: 2.68±0.03) were not different among the groups. In the biotin deficient group, 7 out of the 15 fetuses showed macroscopic malformations and abnormalities such as cleft lip, procephalic growth retardation, tail hematoma and orbit hematoma. These findings were not observable in the BS and CO groups. These results highly suggest that biotin deficient conditions were teratogenic in fetuses.


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P-39 The Effect of chemical compound on Cultured Rat Embryos in S-9mix : Sodium Valproate ○Masaharu Akita1, Noriko Ishizuka2, Atushi Yokoyama3

1 Department of Nutrition and Dietetics, Kamakura Women’s University， 2 Department of Nutrition, Kiryu University, 3Life Science Laboratory of Kanagawa E-mail : [email protected] [Objective] The aim of our study is to optimize the embryo culture method as an alternative developmental toxicity test. In this report, the effect of sodium valproate on cultured embryos was investigated in presence of S-9 mix in rat embryo culture methods. [Materials and Methods] Rat embryos at 11.5 days of embryonic day (plug day = 0) were cultured for 48 hours, and 25 µg/ml of sodium valproate was administered from 2 hours after culture until the completion of culture for comparison in embryonic anomaly between S-9mix group and no-S-9mix group. The rat liver S-9 and the cofactor mix were combined with a rate of 3:7. [Results and Discussion] For the effect of sodium valproate on cultured embryos, anomaly rates were 13% in the S-9mix group and 50% in the no-S-9mix group after 48 hours of culture. The S-9mix group had a lower anomaly rate by 37% than that of the no-S-9mix group.

These results suggested that, sodium valproate induces anomalies stronger than the metabolite of sodium valproate on cultured rat embryos. These findings suggested that embryo culture method combining metabolic system may be a test method more likely to be able to confirm abnormality due to a chemical, as well as its metabolites, than the existing embryo culture methods.

This work was supported by the grant P06040 from NEDO.


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P-40 Development of in vitro alternative method for developmental toxicity in using differentiation of embryonic stem cells into neural cell ○Noriyuki Suzuki, Satoshi Ando, Koichi Saito Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd. E-mail: [email protected] [Objective(s)] Embryonic stem cell test (EST) is one of the most useful tests for developmental toxicity using mouse embryonic stem cells. However, it is said that the EST has to be improved because other major target tissues such as nervous system and skeletal tissues have to be included in order to get precious information about the embryotoxic potential of chemicals. We reported investigation of novel gene makers for evaluation of embryotoxicity during neural differentiation using mouse ES cells in the last annual meeting. Here, we report the establishment of stable transgenic ES cells to detect the chemical dependent changes in one of the gene markers, Reln, using the reporter-gene easily and conveniently.

[Materials and Methods] A reporter plasmid was generated by insertion of the promoter region of Reln gene up-stream of the luciferase reporter-gene, and the plasmid was transfected into ES cells to develop stable transformant cells. To evaluate availability of the stable transformant cells, expression of Reln gene during ES cell differentiation into neural cells was analyzed periodically by real-time PCR method and measurement of luciferase activities. To establish in vitro short-term assay for prediction of embryotoxicity using the transformant cells, various conditions of the reporter-gene assays were optimized. Then, we evaluated several embryotoxic and non-embryotoxic chemicals by the assays using our transgenic ES cells.

[Results and Discussion] Some stable transgenic ES cell-lines detecting Reln gene expression by luciferase activities were obtained. During differentiation of a selected cell-line (Reln-ES) into neural cells, expression of Reln gene elevated from day 5, changes in Reln gene expression being coincident with those in the luciferase activities. To establish in vitro short-term assays for prediction of embryotoxicity using the Reln-ES cells, we optimized a protocol of the reporter-gene assay using the Reln-ES cells. Inhibition of neuron differentiation were analyzed using some well-known test compounds, being compared with that of cardiomyocyte differentiation obtained by other transformant ES cells (Hand1-ES and Cmya1-ES) reported previously. We are planning to evaluate the efficacy and usefulness of the tests in detail using additional test chemicals.

This study was supported by a research grant from the New Energy and Industrial Technology Development Organization (NEDO) in Japan.


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P-41 Development of in vitro cardiotoxicity assays using human iPS cells (Investigation of assay conditions with doxorubicin) ○Tomoaki Inoue, Jumpei Kiyokawa, Masaki Honda, Akifumi Shioda, Mitsuyasu Tabo, Masayuki Mishima, and Shuichi Chiba Safety Assessment Department, Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd. E-mail: [email protected] [Objective(s)] Human iPS cells (hiPSCs) are pluripotent stem cells that can be used for in vitro assays after differentiation into various organ cells. On the other hand, available primary human cells are limited to a very few cell types. In addition, the organ-specific functions of cell lines are very restricted, so cell lines are often not suitable for in vitro organ toxicity assays. For these reasons, hiPSCs may prove useful for in vitro assays in drug discovery, and contribute to reducing animal use. In this study, we derived cardiomyocytes from hiPSCs and used them for in vitro cytotoxicity assays. Differences of cytotoxicity in different cell types by a known cardiotoxic compound, doxorubicin were investigated. [Materials and Methods] Human iPS cells lines (201B7; Cell, 131, 861, 207 and 253G1; Nat. Biotech., 26, 101, 2008) from Kyoto University were used. Immature hiPSCs were cultured in 96-well plates without feeder cells and incubated with doxorubicin for 24 or 48 hr. After incubation, lactate dehydrogenase (LDH) in the cells and water soluble tetrazolium (WST-1) metabolizing activities were measured. The differentiated cardiomyocytes were obtained by suspension culture of hiPSCs for 4 days to form embryoid bodies followed by adherent culture for several weeks. Cell aggregates containing beating cells were isolated, dispersed by enzyme digestion, seeded in 96-well plated, and cytotoxicity with doxorubicin was measured by the same methods with the immature hiPSCs. The morphology of the cells was also observed. [Results and Discussion] Cytotoxicity with doxorubicin was lower in differentiated cardiomyocytes than in immature hiPSCs, and showed higher IC50 values. The alterations of LDH activities were in parallel with morphological alterations, but WST-1 activities were observed even at concentrations in which cell degeneration is observed. The LDH IC50 values in differentiated cardiomyocytes were almost comparable with the IC50 values in rat primary cardiomyocytes (AATEX, 14, special issue, 457, 2008). From these results, immature hiPSCs are more susceptible to doxorubicin than differentiated cardiomyocytes. Our results agree with the suggestion that a mechanism of doxorubicin cardiotoxicity is the depletion of stem cells in the heart (Circulation, 121, 276, 2010). Thus, the immature and differentiated cells from hiPSCs would, with further refinements, become useful tools for in vitro cytotoxicity assays at the pre-clinical stages of drug discovery to predict clinical toxicities of drug candidates.


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P-42 Characterization of human iPS-derived cardiomyocytes using gene expression analysis of cardiac markers ○Jumpei Kiyokawa, Masaki Honda, Akifumi Shioda, Mitsuyasu Tabo, Masayuki Mishima, Shuichi Chiba, Tomoaki Inoue Safety Assessment Department, Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd. E-mail: [email protected] [Objective(s)] Human induced pluripotent stem (hiPS) cells were established by transfecting human somatic cells with transcription factors. Because these cells have pluripotency like human ES cells, hiPS cells are expected to be applied to in vitro safety assessment in drug discovery and development by differentiating them into various types of cells. This also contributes to a reduction of animal consumption in terms of the 3Rs. The purpose of this study is to differentiate hiPS cells into contracting cardiomyocytes and characterize their properties as cardiomyocytes using gene expression analysis of cardiac markers. [Materials and Methods] We used the hiPS cell line 201B7 from Kyoto University1). For differentiation, the hiPS colonies were dispersed into small cell clumps and the cells were cultured in suspension in plastic petri dishes for 4 days. The embryoid bodies (EBs) were then transferred into adherent culture dishes. Total RNA was extracted from undifferentiated hiPS cells and from contracting areas of differentiated hiPS cells (8, 18 and 38 days after differentiation) and then was reversely transcribed. By real-time PCR analysis, mRNA expression of cardiac marker genes was investigated. [Results and Discussion] The incidence of beating EBs was about 20% in our culture condition. In the contracting areas of differentiated hiPS cells, mRNA expression levels of main cardiac markers and cardiac-related ion channels were upregulated, whereas undifferentiated cell marker genes were downregulated. The mRNA expression peaks of several ion channels appeared on different days after differentiation of hiPS cells; some peaked at the early stage of differentiation, and others were upregulated gradually after differentiation and reached each peak at a later stage. These results indicate that beating EBs contained differentiated cardiomyocytes with appropriate expression of cardiac markers, and mRNA expression profiles of differentiating hiPS-derived cardiomyocytes changed depending on each differentiation stage. Considering appropriate differentiation stages of these hiPS-derived cells would be important when applying them to drug discovery and development in the future. [References] 1) K. Takahashi et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131, 861–872.


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P-44 Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system ○Kazuaki Nakamura1, Yoko Ejiri2, Akito Tanoue1

1Department of Pharmacology, National Research Institute for Child Health and Development, 2Tsukuba Research Center, Kuraray Co., Ltd E-mail:[email protected] [Objective(s)] A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells and primary human hepatocytes cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. [Materials and Methods] We cultured human hepatocarcinoma cell line HepG2 cells and primary human hepatocytes freshly isolated from human livers obtained from operations of liver transplantation in the micro-space culture plate and assessed the formation of 3D architecture, albumin secretion, mRNA expression levels of cytochrome P450 (CYP) enzymes and cell viability after exposure with acetaminophen. [Results and Discussion] HepG2 cells and primary human hepatocytes cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the cells grown in micro-space culture was greater than the damage to the cells grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro. [References] Nakamura. K., Mizutani. R., Sanbe. A., Enosawa. S., Kasahara. M., Nakagawa. A., Ejiri. Y., Murayama. N., Miyamoto. Y., Torii. T., Kusakawa. S., Yamauchi. J., Fukuda. M., Yamazaki. H., Tanoue. A., (in press) Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system. J. Biosci. Bioeng.


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P-45 A genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells ○Taisei Mizota, Katsutoshi Ohno, Toshihiro Yamada The Food Safety Research Institute, Nissin Foods Holdings Co., Ltd. E-mail: [email protected] [Objective(s)] In vitro genotoxicity assays play an important role in predicting potential carcinogenic potency of chemicals. Ames test is the most commonly used assay and some in vitro mammalian cell assays have been used. However, there are some problems of throughput, species difference, and high false positive rates in these assays. For the purpose of constructing an easy-operating genotoxicity test system, we developed a p53R2-dependent luciferase reporter gene assay using human breast cancer cell lines (MCF-7) expressing wild-type p53 gene. Recently, ECVAM provided the recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. In this study, we investigated the reactivity of the assay for the test chemicals recommended by ECVAM using human lymphoblastoid cells (TK6). [Materials and Methods] The p53BS-Luc reporter plasmid was constructed by inserting the sequence for the p53 binding site derived from human p53R2 gene. TK6 cells were transiently co-transfected with p53BS-Luc plasmid and pRL-SV40 internal control plasmid in each assay. After 16 h incubation with test samples, the cells were lysed. Luciferase activities were measured and normalized. Relative p53R2-dependent luciferase activity after treatment with chemicals was expressed as the % of the control cell activity. When the relative luciferase activity of cells treated with the test sample was 130% and over of that treatment with vehicle only in a dose-dependent manner, the test sample was judged to be positive for genotoxicity. [Results and Discussion] The p53R2-dependent luciferase activities induced by 20 chemicals that should be detected as positive, and 41 chemicals (including 19 chemicals have been reported to induce chromosomal aberrations or tk mutation in mouse lymphoma cells) that should give negative results were evaluated. The results of the assay were 85% (17/20) for the sensitivity, 93% (38/41) for the specificity, and 90% (55/61) for the overall agreement. It is suggested that this genotoxicity test system, based on p53R2 gene expression, is applicable to TK6 cells, and can become a valuable tool for predicting the human cancer risk. [References] 1) K. Ohno et al., Mutat. Res. 588, 47-57, (2005). 2) K. Ohno et al., Mutat. Res. 656, 27-35, (2008).


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P-46 Evaluation of in vitro micronucleus assay using three-dimensional reconstructed human skin model ○ Toshio Kasamatsu, Katsuyuki Yuki, Hideaki Nakagiri, Shoji Matsumura, Naohiro Ikeda, Naohiro Nishiyama Global R&D - Safety Science, Kao Corporation E-mail: [email protected] [Objective(s)] It has been concerned with in vitro genotoxicity assay that it produces remarkably high rate of false positive results where it misjudges non-genotoxic chemicals as positive. For example, when a positive result is obtained in in vitro chromosomal aberration (CA) assay, follow-up animal testing (in vivo micronucleus (MN) assay) is necessary to confirm the result. Meanwhile, an animal testing for evaluation of genotoxicity potential of cosmetics has been banned in EU since March 2009. In order to avoid misjudgment on genotoxicity potential of useful chemicals, we evaluated a new in vitro system which can replace in vivo MN assay. [Materials and Methods] As cosmetics are generally applied to the skin, we employed three-dimensional reconstructed skin models, EpidermTM (3D)2), and evaluated 14 chemicals (6 genotoxic chemicals, 3 non-genotoxic chemicals, and 5 chemicals which have shown false-positive results in in vitro CA). Furthermore, we investigated the mechanism by which 3D could judge false positive chemicals as negative correctly. [Results and Discussion] Results of all chemicals evaluated in 3D MN assay conformed to those evaluated in in vivo MN assay so that 3D MN assay was considered promising to replace in vivo MN assay. We further investigated the mechanism by which 3D judge false positive chemical as negative correctly. We focused on the chemical known to generate reactive oxygen species (ROS) under in vitro condition specifically3). Evaluation of gene and enzyme expressions related to antioxidative property revealed that their expression levels of 3D were higher than those of culture cells. This result indicates antioxidative property is one of mechanisms to judge false positive chemicals correctly. In addition, 3D could judge chemicals that require metabolic activation to show genotoxic potential as positive. 3D may also be applicable for evaluation of these chemicals. In order to further confirm usefulness of 3D, we plan to evaluate additional chemicals and mechanisms to judge them correctly. [References] 1) D. Kirkland. et al. Mutation Research, 584, 1-256, 2005 2) R.D. Curren. et al. Mutation Research, 607, 192-204, 2006 3) Takumi-Kobayashi A. et al. Mutation Research 657, 13-18, 2008


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P-47 In vitro evaluation system to predict phototoxic potential of water-insoluble chemicals using three-dimensional dermal skin model ○Maki Kaneko, Masato Kitagaki, Hirokazu Kouzuki Shiseido Research Center E-mail:[email protected] [Objective] The 3T3 neutral red uptake in vitro phototoxicity test (3T3 NRU PT) was recommended by OECD Testing guideline 432 in 2004. This test is regarded as a promising alternative method to animal testing, but is unsuitable for water-insoluble chemicals. Therefore, it is inapplicable to water-insoluble cosmetic ingredients such as UV absorbers and fragrances. Although some work has been done on phototoxicity evaluation of water-insoluble chemicals using human three-dimensional (3-D) cell culture models, an inexpensive, readily available and effective model has not been established. To meet this need, we have developed a prediction method for phototoxic potential of water-insoluble chemicals, using a 3-D dermal skin model consisting of type I collagen and human dermal fibroblasts. [Materials and Methods] Eighteen chemicals were selected from the ECVAM Report, together with non-phototoxic reference compounds (e.g., NPT class). Normal human skin fibroblasts (HF) were seeded in collagen gel and cultured in trans-well plate with medium containing 2% FBS for 14 days. Then, the culture medium was replaced with 0.5 ml of PBS (+). Duplicate plates were prepared for UVA exposure and dark control. Test chemical (50 μl or 50 mg) was topically applied for 1 hour, and the plate was irradiated with a solar simulator (SOL500) at a dose of 10 J/cm2 (confirmed non cytotoxic radiation dose), or not irradiated. Then, the models were incubated overnight at 37°C on fresh maintenance medium. Cell viability was assessed by MTT assay. The percent viability was calculated as (ODsample/ODcontrol)x100. [Results and Discussion]The 3-D in vitro model was able to discriminate efficiently between phototoxic and non phototoxic chemicals. Thus, the 3-D in vitro model is expected to be useful as a screening tool for evaluating the phototoxic potential of water-insoluble chemicals. The next step will be to examine a larger number of chemicals and cosmetic formulations to confirm the robustness of our 3-D in vitro model.


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P-48 Comparative Studies on the Draize Rabbit Eye Test and HET-CAM Test of Eye Irritation Test ○So-Young Yang2, Nam-Jin Lee2, Eun Young Jeon2, Mi Sook Jung2, Heung-Mo Bae2, Jae-Myun Ryu2, Seong-Jin Baek2, Jong-Koo Kang1

1Department of Veterinary Medicine, Chungbuk National University, Gaesin-dong, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Korea, 361-763. 2Biotoxtech Co. Ltd., 58-1 Block, Ochang Scientific Industrial complex, Ochang-eup, Cheongwon-gun, Chungcheongbuk-do, South Korea, 363-883. E-mail: [email protected] [Objective(s)] Using animals in toxicological screening is a controversial issue. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. Hen’s egg chorioallantoic membranes (HET-CAM) tests have become widely used to assess the irritation potential of test substances. The purpose of this study is to report the results of a comparison of Draize rabbit eye test and HET-CAM test. [Materials and Methods] The positive control, 0.1N NaOH and 1% sodium dodecyl sulfate, and negative control, 0.9% NaCl solution, were freshly prepared before the start of each assay. Each egg (on Day 9) was tested with candling light to ensure that all were viable. On Day 10, the CAM of each egg was applied directly with 0.3 ml of the positive/negative control. The reactions for hemorrhage, coagulation and lysis on the CAM were observed over a period of 5 minutes. The time for each reaction was recorded OO seconds and calculated an irritation score (IS). [Results and Discussion] In our study a comparative screening was done with a set of cosmeceuticals to establish parallel data on in vitro and in vivo results. Our study showed good correlation between results obtained by the HET-CAM test and those of the Draize rabbit eye test most cases. The present form of the HET-CAM test can by proposed as a prescreen method of eye irritation tests. [References] Gilleron L, Coecke S, Sysmans M, Hansen E, Van Oproy S, Marzin D, Van Cauteren H, Vanparys P. Evaluation of a modified HET-CAM assay as a screening test for eye irritancy. Toxicol In Vitro. 10(4):431-46, 1996. Kishore AS, Surekha PA, Sekhar PV, Srinivas A, Murthy PB. Hen egg chorioallantoic membrane bioassay: an in vitro alternative to draize eye irritation test for pesticide screening. Int J Toxicol. 27(6):449-53, 2008. Barile FA. Validating and troubleshooting ocular in vitro toxicology tests. J Pharmacol Toxicol Methods. 61(2):136-145, 2010


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P-49 Advantages of the human corneal epithelium model reconstructed in a collagen vitrigel chamber and its application to eye irritation test. ○Hiroyuki Yamaguchi1, 2, Toshiaki Takezawa1

1National Institute of Agrobiological Sciences, 2Kanto Chemical Co. Inc. E-mail: [email protected] [Objective(s)] A collagen vitrigel (CV) membrane is composed of high density collagen fibrils equivalent to connective tissues in vivo and is easily handled with tweezers. Also, it possesses excellent transparency and permeability of protein with high molecular weight and consequently the various researches utilizing it as a cell culture substratum advances so well (1). To develop the alternative method for the Draize eye irritation test using rabbits, we established a reconstruction method of a rabbit corneal epithelium model by culturing normal rabbit corneal epithelial cells on the CV membrane scaffold to the confluent monolayer and subsequently differentiating them in the air-liquid interface culture to form multilayers (2). Further, a human corneal epithelium model with barrier function was reconstructed by culturing HCE-T (a human corneal epithelium-derived cell line) cells on the CV membrane scaffold prepared in a cell culture chamber suitable for TEER (transepithelial electrical resistance) measurement. Consequently, it was suggested that eye irritancy of chemicals could be estimated as the time-dependent changes of TEER induced by exposing them to the model (3). However, a multi-porous membrane of the chamber is made of PET (polyethylene terephthalate) and is inappropriate not only for preparing cryo-sections but also for reconstructing a corneal model reflecting structure and its component in vivo. In this study, we aimed for fabricating a novel CV chamber without the PET membrane and reconstructing a human corneal epithelium model in the chamber. Subsequently, we evaluated its barrier function and the utility of it in eye irritation test. [Materials and Methods] HCE-T cells were seeded in a CV chamber and cultured for 2 days to the confluent stage. Subsequently, a corneal epithelium model was reconstructed by culturing it in the air-liquid interface for 7 days. The model was histologically analyzed after staining its cryo-sections with fluorescence for nuclei and cell junctions. Also, time-dependent changes of TEER after exposing eye-irritant chemicals to the model were measured. [Results and Discussion] Histological observations revealed that the corneal epithelium model was composed of around five cell layers and also tight and gap junctions-related proteins were strongly expressed in the outer 2-3 layers. The decreasing levels of TEER at 10 seconds after exposing chemicals to the model were well correlated with the Draize score of each chemical. These results suggest that the human corneal epithelium model reconstructed in a CV chamber provides a new rapid assay useful for eye irritation test. [References](1) Takezawa T. et al. Yakugaku Zasshi 130: 565-574, 2010. (2) Takezawa T. et al. AATEX 13 (Suppl.): 176, 2008. (3) Nishikawa K. et al. AATEX 14 (Suppl.): 1053, 2009.


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P-50 Assessment of eye irritation potencial using the reconstructed human corneal tissue, LabCyte CORNEA-MODEL ○Masakazu Katoh, Noriyuki Uemura, Akemi Usui, Fumiyasu Hamajima, Takahiro Ogasawara, Ken-ichiro Hata Japan Tissue Engineering Co., Ltd E-mail: [email protected] [Objective(s)] In vitro eye irritation testing alternative to animal testing such as Draize test using rabbits is required from an animal welfare. We have developed the reconstructed human corneal epithelium model, LabCyte CORNEA-MODEL, and then we investigated a test method to evaluate eye irritation effect using this model. In this presentation, we report about the structural characterization of LabCyte CORNEA-MODEL and about the prediction potency of eye irritation test using LabCyte CORNEA-MODEL. [Materials and Methods] For the structural characterization of LabCyte CORNEA-MODEL, it was examined some specific molecules and/or components to express in the tissue by immuno-histchmical analysis and transmission electron microscopy. Reproducibility among inter- or intra-lots of LabCyte CORNEA-MODEL was assessed as the indicators with the cell viability and the barrier function. Moreover, the 61 chemical substances were applied to in vitro eye irritation test using LabCyte CORNEA-MODEL (application period of chemicals: 1 min, post-incubation periods: 24 hours, prediction of eye irritation: < 50% cell viability) and it was evaluated the correlation of in vivo class and the in vitro prediction of eye irritation. [Results and Discussion] Claudin-1, a component of tight junction, E-cadherin, a component of adherence junction, and Desmogrein-3, a component of desmosome, were strongly expressed in the all layer. Additionally, it was confirmed that Mucin-1 and Mucin-16 were expressed in the super-facial layer. These results suggested that this model was correlated with the tissue structure of normal human corneal epithelium. Moreover, when the cell viability and the barrier function of LabCyte CORNEA-MODEL intra- or inter-lots was evaluated, it was showed that the reproducibility between lots was good. Since in vitro results using LabCyte CORNEA-MODEL was correlated with the degree of in vivo eye irritation (sensitivity 83.3%, specificity 80.0%, and agreement rate 82.0%), it suggested that the eye irritation test using this model could be useful for variety of chemicals with irritant potency as an alternative method to Draize test.


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P-51 Assessment of novel eye irritation test using the reconstructed human corneal tissue LabCyte CORNEA-MODEL ○Noriyuki Uemura, Masakazu Katoh, Akemi Usui, and Ken-ichiro Hata Japan Tissue Engineering Co., Ltd E-mail: [email protected] [Objective(s)] In vitro eye irritation tests alternative to animal testing such as Draize test using rabbits are required from the perspective of an animal welfare. Thus, we have developed the reconstructed human corneal epithelium model, LabCyte CORNEA-MODEL, and a test method to evaluate eye irritation effect using WST-8 method. However, WST-8 method only possesses relatively low sensitivity and may underestimate the irritation properties of test reagents although simple and easy to perform. Therefore, the establishment of a novel method with high sensitivity is desirable. In this presentation, we explore the novel sensitive method to predict eye irritation properties of chemicals and cosmetics.

[Materials and Methods]

The apical surface LabCyte CORNEA-MODEL were exposed to the various concentrations (0.0005%-5%) of Benzalkonium Chloride (BAK), cationic surface-acting agent known to cause the puncture of corneal epithelium, for 1 min and washed with PBS ten times. BAK-exposed tissues were then incubated for 24 hours and analyzed histologically with Haematoxylin & Eosin staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunostaining against Claudin-1. TUNEL staining was used to detect apoptosis in the tissue layer. Claudin-1 immunostainig was used to observe the destruction of tight junctions.

[Results and Discussion] According to our histological results of TUNEL staining, we found that BAK induced apoptosis of corneal epithelium in a dose-dependent manner. While lower dose (0.005%-0.3%) of BAK induced apoptosis of only superficial layer of corneal epithelium, relatively higher dose (0.5%-1.0%) of BAK induced apoptosis of both superficial and underlying layer. We also found that tissues exposed to very high concentration (3%-5%) of BAK were negative for TUNEL staining although tissues were extremely damaged, which implied cells underwent necrosis due to extensive tissue damage. We next examined whether BAK affected to tight junction of corneal epithelium using immunostaining with anti-Claudin-1 antibody. We found that exposure to BAK destructed tight junction of LabCyte CORENA-MODEL in a dose-dependent manner, but the sensitivities were less compared to TUNEL staining method. Taken together, we propose the TUNEL staining of LabCyte CORNEA-MODEL as a novel sensitive method to predict the stimulus properties of chemical reagents and cosmetics.


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P-52 Risk assessment for eye irritation using cytotoxicity tests ○Shigenobu Hagino, Yuuko Matsumoto, Kitagaki Masato, Tsuyoshi Yoshida and Hiroshi Itagaki Shiseido Research Center E-mail:[email protected]

[Objective] The SIRC cytotoxicity test using crystal violet staining has been validated as a method to identify non irritants among potential cosmetic ingredients, at the concentration of 10%. We examined the predictive ability of this test for eye irritancy of undiluted chemicals. Furthermore, the combination of this test with a cytotoxicity test using a three-dimensional dermal model was examined to predict eye irritancy at various concentrations. [Materials and Methods] The twenty-seven chemicals classified as non irritant or category 2B in the ICCVAM recommended reference substance list (2006) were used for evaluation of the in vitro tests. Cytotoxicity testing with SIRC cell monolayer culture was performed by application for 72 hours and crystal violet staining was used to obtain the 50%-inhibitory concentration (IC50). Three-dimensional dermal model testing (MATREXTM kit, Toyobo Co.) was performed for 24 hours, together with MTT assay. The results were used, together with previous data (total data: over 100), to analyze predictive ability for eye irritancy. [Results and Discussion] When the predictive ability of the SIRC cytotoxicity test was examined using undiluted chemicals as test samples and triethanolamine as a negative standard, alcohols, ketones, esters, ethers and also other substances with molecular weight less than 180 were false-negative. The false-positive substances did not belong to any particular category. The SIRC cytotoxicity test may be useful for prediction of eye irritancy of undiluted chemicals, as well as 10% solution or suspension, if employed with a clear understanding of its characteristics. On the other hand, the prediction ability of the three-dimensional dermal model was examined using 50% cell viability as a cut-off. This test could predict non irritancy at various concentrations, though false positives were unavoidable. The findings suggest that the cytotoxicity test may be useful for risk assessment of eye irritancy of ingredients intended to be used in cosmetics and medicated cosmetics. [References] 1)Hagino et al., ATLA 36, 641–652, 2008. 2)Hagino et al., ATLA 38, 139–152, 2010.


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P-53 Discrimination of GHS classification 2A or 2B in modified STE test by neutral red(NR) up-take ○Yuichiro Goto, Miki Nakanishi ,Miho Sakurai, Satoko Kuriwada, Noriyasu Imai, Yuko Okamoto KOSÉ Corporation Fundamental Research Laboratories [email protected] [Objective(s)] STE (Short Time Exposure) test1) is an in vitro alternative eye irritation test that assesses the cytotoxicity using SIRC (rabbit corneal cells line) cells followed by a 5 minutes cell exposure. In the original STE test, MTT assay is adopted as the endpoint. In the present study, we modified the cytotoxicity assay to the neutral red (NR) up-take, which is frequently used in our laboratory. Moreover, further investigation was conducted to discriminate GHS classification 2A or 2B substances by several test concentrations using this testing method. [Materials and Methods] Twenty-five chemicals evaluated by the STE test validation study in 20082) and 17 chemicals categorized as GHS classification 2 (2A or 2B) were examined.

For the evaluation of GHS classification 2A or 2B chemicals, test concentrations 2.5%, 2% or 1% was added to the original concentrations, 5% and 0.05%. [Results and Discussion] Twenty-five chemicals were evaluated with modified STE test by NR up-take. A good correspondence with GHS classification (Non-Irritant,Cat.2 or Cat.1) was obtained by the application of 50% cell viability cut-off value.

Next, 17 chemicals were examined by this method. Saline, 5% DMSO in saline, or mineral oil was used as the vehicle. Then we selected the vehicle that shows a lower cytotoxicity if test chemicals were soluble in plural vehicles. Consequently, 13 out of 17 chemicals were classified as GHS classification 2. Furthermore, an additional evaluation was performed for the discrimination of GHS 2A or 2B classification. As a result, 2.5% chemical concentration showed the best accuracy among 3 test concentrations.

Accordingly, this investigation demonstrated that the modified STE test by NR up-take showed almost the same accuracy as the original test. Moreover, it is indicated that the discrimination of GHS classification 2A or 2B would be possible by the application of 2.5% test concentration. [References] 1) Takahashi Y.et al., 2008. Toxicology in Vitro. 22, 760-770. 2) Sakaguchi H. et al., The 21th Annual Meeting of Japanese Society for Alternatives to Animal Experiments (2008).


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P-54 Assessment approach (NI/ I) combining in vitro eye irritation tests ○Kenichi Ooshima 1, Takayuki Abo2, Takumi Hayashi1, Tomoko Komano1, Kazuhiko Hayashi2, Hitoshi Sakaguchi2, Naohiro Nishiyama2, Hirofumi Kuwahara 1

1 Kanebo cosmetics INC, 2 Kao Corporation E-mail: [email protected] [Objective(s)] Although the BCOP test and the ICE test were accepted for the OECD test guideline, both tests are in vitro test methods that classify substances as GHS Cat.1. There is no accepted in vitro test method of eye irritation that can classify Non irritant (GHS NC) to Irritant (GHS Cat.2+Cat.1) by OECD. Since there are many cosmetic ingredients that are classified as GHS (NC), a test method to evaluate GHS (NC) or GHS (Cat.2+Cat.1) is necessary. We previously reported that a good correlation has been confirmed between the eye irritation category (Non-irritant (NI) / Irritant (I)) by the STE test and GHS (NC / Gat.2 + Cat.1) 1). There is a similar report using the EpiOcular™ assay 2). The HET-CAM test can discriminate between positive and negative results with respect to eye irritation evaluating 15 as the discriminative value for MAS 3). In the present study, we evaluated an assessment approach combining these tests (STE, EpiOcular™ and HET-CAM). [Materials and Methods] Materials: Around 50 chemicals were selected with a balanced GHS category and a wide range of chemical classes. STE：SIRC cells were treated with diluted test materials for 5 min and cell viability was measured by the MTT assay1). EpiOcular™: After the test materials were treated on the surface of the tissues for 30 min (liquid) or 90 min (solid), the cell viability was measured by MTT assay2). HET-CAM: Each CAM was observed macroscopically after treatment (20 sec) with the test material. The CAM was then treated with trypan blue3). After the tests, the eye irritation category for the chemical was evaluated in these tests. Moreover, the eye irritation category (NI / I) in these tests was compared with GHS (NC / Gat.2 + Cat.1) and the assessment approach was evaluated. [Results and Discussion] The chemicals that are soluble in the vehicle of the STE test were evaluated by the STE test. The chemicals that were insoluble in the vehicle were evaluated by the EpiOcular™ assay or the HET-CAM test. This approach indicated good ability to predict eye irritation potential of a wide range of chemical substances with the added advantage (rapid, easy, inexpensive) of the STE test. Also, if the chemicals that show false negative (e.g. alcohol, ester etc.) by cell function and cytotoxicity assay were evaluated by the EpiOcular™ assay or the HET-CAM test, the approach showed eye irritation with the most favorable predictivity (accuracy � 85%). Even if the EpiOcular™ assay or the HET-CAM test combined the STE test to predict eye irritation, the accuracy is almost the same. These results suggest that an assessment approach combining these tests may be a useful tool to predict eye irritation potential. [References] 1) Takahashi et al. 2008 Toxicology in Vitro (22) 760-770 2) Harbell et al. 2009 Poster #378, Society of Toxicology, March 3) Hagino et al. 1999 Toxicology in Vitro (13) 99-113


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P-55 A tiered approach combining the STE test, EpiOcular™ assay, BCOP assay for predicting eye irritation potential of chemicals ○Takayuki Abo1, Kenichi Ooshima2, Kazuhiko Hayashi1, Takumi Hayashi2, Tomoko Komano2, Hitoshi Sakaguchi1, Hirofumi Kuwahara2, Naohiro Nishiyama1

1 Kao Corporation, 2 Kanebo cosmetics INC E-mail: [email protected] [Objective(s)] For the assessment of ocular irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the irritation potential for a wide range of chemical classes (Scott et al., Toxicol. In Vitro, 2010). In the present study, the predictive potential of a tiered approach analyzing the results from three in vitro assays (i.e., the STE test with SIRC cells (Takahashi et al., Toxicol. In Vitro, 2008), the EpiOcular™ assay with 3D models (Harbell et al., SOT, 2009) and the BCOP assay with isolated bovine corneas (OECD TG437)) for assessing GHS eye irritation rankings were examined. [Materials and Methods] Around 50 chemicals including the insoluble chemical in the STE test vehicles were selected. The chemicals represented a balance of GHS eye irritation rankings and a wide range of chemical classes. Then, the chemicals were evaluated in either the STE test or the EpiOcular™ assay. Descriptions of the assays were published by Takahashi et al. and Harbell et al. For the last phase of our approach, we adopted the BCOP data from the background review document (BRD, ICCVAM-NICEATM, 2006) to further clarify the irritation classification category of the 50 chemicals being evaluated. The findings in each of the three in vitro assays were reviewed and the accuracy of a tiered approach predicting the GHS eye irritation rankings were estimated. [Results and Discussion] The irritation category of non-irritant (NI) or irritant (I) for the tested chemicals was obtained with either the STE test or the EpiOcular™ assay. If the chemicals were soluble in the STE test vehicle, the results of the STE test were reviewed. However, if not soluble, then the results from the EpiOcular™ were reviewed. If the chemical was classified as a “NI” in any of these assays, it was considered to have a GHS eye irritation ranking of “not classified”. If the chemical was classified as an “I” from either assay, the classification was subsequently confirmed by reviewing the results from the BCOP assay as described in the BRD. If the classification was “severe” in the BRD, the chemical was considered a GHS “Cat. 1”. For those chemicals whose classifications were “non-severe” in the BRD, these were considered to be GHS “Cat. 2”. The tiered (bottom-up) approach combining the STE test or the EpiOcular™ assay and the BCOP assay allowed the determination of the GHS eye irritation rankings for chemicals from a wide range of chemical classes (accuracy > 70%, under prediction rate < 15%). If, however, the EpiOcular™ assay was used instead of the STE test for alcohols and esters etc. the predictivity was markedly improved (accuracy > 75%, under prediction rate < 5%). From these results, this tiered approach might be a promising alternative method for predicting eye irritation.

Date post:	25-Apr-2018
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