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TRANSCRIPT
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中国食品药品检定研究院 食品药品安全评价研究所
Alternative Study in Life
Science in China
Jufeng Wang
Zhengmin He, Bo Li
National Center for Safety Evaluation of Drugs
National Institutes for Food and Drug Control
China
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中国食品药品检定研究院 食品药品安全评价研究所
Content
Current status
Alternative Research in NIFDC
International collaboration
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中国食品药品检定研究院 食品药品安全评价研究所
Overview of Progress in China
Present Status of AAT Research (Cosmatics,
drug as a case) • Policies & Regulatory
• Standards
Future Perspectives for Research and Application of AAT • To set up research system for alternative methods
• To create ChCVAM
• To set up Information and data sharing platform
• International Collaboration
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中国食品药品检定研究院 食品药品安全评价研究所
Guideline for the Human Care and Use of
Laboratory Animals(2006)
Overview of Progress in China
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中国食品药品检定研究院 食品药品安全评价研究所
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Chapter 1: General Provision
Chapter 2: Requirement for humane
care of laboratory animals in breeding
Chapter 3: Requirement for humane care of
laboratory animals in experimentation
Chapter 4: Requirement for humane care of
laboratory animals in transportation
Chapter 5: Relative measures
Chapter 6: Supplementary Provision
Overview of Progress in China
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中国食品药品检定研究院 食品药品安全评价研究所
China Food and Drug Administration
launched “System Construction of
Alternative Methods to Cosmetic
Animal Study " in August, 2011
National Institutes for Food and Drug Control
(NIFDC)
Guangdong Entry-Exit Inspection and
Quarantine Bureau
Guangdong Provincial Center for Disease
Control and Prevention
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Overview of Progress in China
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中国食品药品检定研究院 食品药品安全评价研究所 7
我国体外技术发展面临的挑战-体系建设 Research Report of the System Construction of Alternative Methods to Animal Test for Cosmetic
I. Research on alternative methods to cosmetic toxicological animal study abroad 1. Research status 2. Development trend II. Research on alternative methods to cosmetic toxicological animal study in China 1. Research status 2. Main problems 3. Development need III. Research of alternative methods to cosmetic animal study and validation system construction 1. Guiding ideology 2. Overall goal 3. Main study IV. Implementation steps 1. To make regulations and technical standards 2. To Establish organizational framework of alternative methods to cosmetic animal study in China 3. To carry out research and validation of alternative methods to cosmetic animal study
V. Safeguard Measures VI. Expected results and Prospect
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中国食品药品检定研究院 食品药品安全评价研究所 8
I. Guiding ideology
II. Overall goal
III. Main missions
IV. Implementation steps
V. Safeguard Measures
Guiding Opinions on Accelerating Research of Alternative
Methods to Animal Test for Cosmetic and Validation System
Construction (discussion draft)
(Experts seminar June, 2012)
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中国食品药品检定研究院 食品药品安全评价研究所
III. Main missions
To enhance the establishment of “Validation Center” :China Center for
Validation of Alternative Methods of animal experiment (ChCVAM)
To enhance the establishment of “Technical subject”: Validation
technical system of Alternative Methods
To build up“two platform”
• Information and data sharing platform
• Technical training and academic communication platform
To make technical regulations and technical standards
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我国体外技术发展面临的挑战-体系建设 Guiding opinions on accelerating research of alternative
methods to animal test for cosmetic and validation system
construction (discussion draft)
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中国食品药品检定研究院 食品药品安全评价研究所
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我国体外技术发展面临的挑战-体系建设 Guiding opinions on accelerating research of alternative
methods to animal test for cosmetic and validation system
construction (discussion draft)
※Name:China Center for Validation of Alternative Methods of Animal Experiment (ChCVAM) ※ Main responsibilities : Responsible for developing work plan and implementation plan to validate
alternative methods to cosmetics toxicological animal test Undertaking organization and implementation, coordination and
management, as well as technical guidance of validation of the alternative
methods
Following up the progress and trend of international alternative
methods to animal experiments; organizing international cooperation
and technical exchanges; enhancing the domestic and international
acceptance of the alternative methods
Validation Center
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中国食品药品检定研究院 食品药品安全评价研究所 11
Guiding opinions on accelerating research of alternative
methods to animal test for cosmetic and validation system
construction (discussion draft)
※Composition: Food and drug test facilities, high-quality inspection and testing resources in the society
※Main responsibilities: To carry out and undertake research and validation of alternative
methods to animal experiment
Technical Support
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中国食品药品检定研究院 食品药品安全评价研究所 12
Website of CCARE platform
http://www.vitrotox.com
Guidance on the Construction of the Research and
Validation System for the Promotion of Cosmetic Alternative
Toxicological Method(discussion)
※Academic Exchange Platform: To establish technical and academic communication system on alternative
methods for experimental animals, to strengthen the
building of talent team, to promote the technical
development, and to provide support for conquer the
possible problem of alternative methods research and
validation.
※Information and Data Sharing Platform: To establish the research information and basic database for alternative
methods for experimental animals, to provide related
information, data and technique standards of home and
abroad novel techniques and methods , and to realize the
information and data resource sharing.
Information and Data Sharing
Platform
http://www.vitrotox.com/
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中国食品药品检定研究院 食品药品安全评价研究所
To Develop Technical Specifications and Standards
※ Combing with the actual work, to develop the guidelines for alternative methods for animal experiments, and related
validated evaluation technical specifications, management
methods, information and data sharing management methods,
etc.
※According to the actual needs, to promote the equivalent transformation of international standards (guidelines) as well
as the upgradation of industry standards to the national
standards. To improve the standardized system of alternative
methods for animal experiments gradually, by working closely
with related department on the basic researches for standard
formulation.
Guidance on the Construction of the Research and
Validation System for the Promotion of Cosmetic Alternative
Toxicological Method(discussion)
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中国食品药品检定研究院 食品药品安全评价研究所
No. of Standard Standard Title Lab of
Completion
GB/T 21827-2008 Chemicals-Skin Allery Reaction Test- Local Lymph-node Method Shanghai CIQ
GB/T 21787-2008 Chemicals-Skin Corrosion and Nerve Development Toxicity Shanghai CIQ
SN/T 2285-2009 Cosmetics-Specifications on In Vitro Alternative Method Laboratory Guangdong CIQ
SN/T 2328—2009 Cosmetics-Acute Keratinocyte Toxicity Test Guangdong CIQ
SN/T 2329-2009 Cosmetics-Eye Irritation/Corrosive Chick Embryo Chorioallantoic Test Guangdong CIQ
SN/T 2330-2009 Cosmetics-Mouse Embryonic Stem Cell Test Method for Embryo and
Development Toxicity
Guangdong CIQ
GB/T 21769-2008 Chemicals-In Vitro 3T3 Neutral Red Extraction Light Toxicity Test Guangdong CIQ
GB/T 21757-2008 Chemicals-Acute Oral Toxicity Test-Acute Toxicity Classification
Method
Guangdong CIQ
GB/T 21804-2008 Chemicals-Acute Toxicity Test-Fixed Dose Method Tianjin CIQ
GB/T 21826-2008 Chemicals-Acute Toxicity Test-Increasing or Decreasing Dosage
Method
Shenzhen CIQ
Published National & Industry Standards
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中国食品药品检定研究院 食品药品安全评价研究所
Standards Undertaking Approval or
Research
Grade Standard Title Lab of Completion
National Chemicals- Skin Corrosion: In Vitro Membrane Barrier Method (draft) Shanghai CIQ
Industry Cosmetics- In Vitro Developmental Toxicity Test: Rat Whole Embryo Assay
(draft)
Shanghai CIQ
Industry Cosmetics: Surface Reactive Agents for Eye Irritation of the Corneal Cell Test
(in the study)
Shanghai CIQ
Industry Chemicals-Skin Allergy Reaction Local Lymph-node Test:Brdu-ELISA(in the study)
Shanghai CIQ
Industry Cosmetics- Local Lymph-node Skin Sensitization Test (completed) Guangdong CIQ
Industry Cosmetics- Eye Irritation 3T3 Cell Toxicity Prediction Assay (completed) Guangdong CIQ
Industry Cosmetics- Justification Regulations on Risk Analysis and Decision Tree (draft) Guangdong CIQ
Industry Cosmetics- Combined Red Cell Assay for Light Toxicity (draft) Beijing CIQ
Industry Cosmetics- Specifications on In Vitro Alternative Methods Good Laboratory
Practice for Cell Culture and Sample Preparation (darft)
Guangdong CIQ
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中国食品药品检定研究院 食品药品安全评价研究所
Title Nature Type
Skin Photosensitive Reaction Test Recommend Method
Skin Allergy Test:Local Lymph-node Test,Brdu-ELISA Recommend Method
In vitro Mammalian Cell Micronucleus Test Recommend Method
Strategies for Alternative Eye Irritation Test Recommend Method
In vitro Skin Irritation Test Using Recombinant Human Skin Model Recommend Method
Isolated Eye Model Recommend Method
Cosmetics Severe Eye Irritation and Corrosive Bovine Corneal Opacity
and Permeability Test
Recommend Method
Cosmetics Eye irritation and Chick Embryo Chorioallantoic Membrane
Assay
Recommend Method
Cosmetics In Vitro Alternative Safety Testing Recommend Method
Specifications for In Vitro Alternative Testing Laboratory Recommend Method
The "12th Five-Year" Plan for the Construction of
Cosmetic Standard System (draft)
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中国食品药品检定研究院 食品药品安全评价研究所
China National Accreditation Service for Conformity Assessment, National Institutes for Food
and Drug Control, Guangdong Inspection and Quarantine Technology Center, Chinese Academy
of Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau, Xinjiang
Inspection and Quarantine Technology Center, MB Research Laboratories, Estee Lauder
Shanghai R&D center, Guangdong Laboratory Animal Monitoring Institute; Sun Yat-Sen
University Laboratory Animal Center
Animal Alternative Testing Workshop ,2012.6.3-6,Guangdong CIQ
Demonstration & practice on CAMVA/BCOP(OECD TG437)
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Laboratory Technique Training
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中国食品药品检定研究院 食品药品安全评价研究所
Animal Alternative Testing Workshop , 2014.5.4-8(Guangdong, CIQ)
Demonstration & practice on CAMVA/BCOP/3T3-NRU-PT
Host: Guangdong Inspection and Quarantine Technology Center
Support:Institute for In Vitro Sciences (IIVS), L’Oreal (China) Co., Ltd, International
Humane Society (HIS)
More than 90 participators from 40 enterprises
and institutions
Scope:Academic discussion and laboratory training
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Laboratory Technique Training
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中国食品药品检定研究院 食品药品安全评价研究所
Training for techniques of cosmetic
alternative toxicological skin and eye
irritation test, include EpiDerm skin
irritation test, EpiDerm MTT ET-50 assay,
EpiOcular eye irritation test, EpiOcular
MTT- ET-50 assay.
2013.9.7-9.15
Beijing Institute for Drug Control
MatTek Corporation (Boston, U.S.A),
specialized in developing, production and
testing of in vitro alternative model skin,
eye, respiratory system etc.)
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Laboratory Technique Training
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中国食品药品检定研究院 食品药品安全评价研究所 20
Establishment of Alternative Methods for
Cosmetic Toxicity Testing
CFDA,2011,8
Joint validation of 3T3 Neutral Red Uptake
Phototoxicity Test in December, 2011. (NIFDC,
Beijing Institute, Shenzhen Institute and Guangdong
Institute)
Laboratory Technique Training
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中国食品药品检定研究院 食品药品安全评价研究所
Activity in China
Society of Alternative method in toxicology
2014, President: Peng Shuangqing, Ph.D
International Conference in Alternative study
2015, Xian, over 200 scientists including 10
international speakers
Formed a AOP work group
2016, Conference in Alternative study
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中国食品药品检定研究院 食品药品安全评价研究所
Alternative Research in NIFDC
In vitro hepatotoxicity assay
In vitro cardiotoxicity assay
Computational toxicity
Cells Co-Culture
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中国食品药品检定研究院 食品药品安全评价研究所
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Preliminary Screening on Drugs with
DILI Potential in Four Liver Cell Lines
Methods: Sixteen drugs were employed as positive controls corresponding with the
livertox and four drugs without any reports or clinical observations associated with DILI
were utilized as negative control drugs. the CCK-8 assay was conduct in evaluating every
liver cell line in discriminating DILI in vitro.
Conclusion: HepaRG cell line was superior to other cell lines in
discriminating drugs associated with DILI potential. The abilities to predict
drugs associated with DILI were HepaRG > L-02 > hiHeps > HepG2
NIFDC
Study 1
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中国食品药品检定研究院 食品药品安全评价研究所
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Applications of High-Content Screening
Assay in Four Liver Cell Lines
Method: A HCS assay was conducted based on five parameters (oxidative
stress, mitochondrial damage, endoplasmic reticulum stress, disorders of
neutral lipid metabolism and calcium homeostasis).
Note: Representative high content images from the HepaRG cell
line treated with diclofenac sodium, buspirone hydrochloride and
danazol, respectively.
Conclusion: The HepaRG cell line was more sensitive to DILI screening in vitro
compared to other cell lines and was considered as a superior cell line model in
multi-parametric screening assay.
L-02 HepG2
HepaRG hiHeps
(a) (b)
(c) (d)
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中国食品药品检定研究院 食品药品安全评价研究所
Characterization of Liver-related
Biomarkers Assay in Four Liver Cell Lines
Drugs Cell lines
L-02 HepG2 HepaRG hiHeps
Diclofenac
sodium - -
AST*, LDH*,
GLDH*,
MAO**, MDH* -
Buspirone
hydroclorid
e
AST*,
GLDH* GLDH*
AST**, LDH**,
MDH** MDH*
Danazol 5’-NT* - AST**, ALP**,
LDH**, MDH**
ALT*, AST*,
GGT*, LDH*,
GLDH*,
MDH** *P
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中国食品药品检定研究院 食品药品安全评价研究所
In Vitro Methods for
Cardiotoxicity Testing
NIFDC
Study 2
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中国食品药品检定研究院 食品药品安全评价研究所
In Vitro Methods for Cardiotoxicity Testing Cell Function
Verapamil
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中国食品药品检定研究院 食品药品安全评价研究所
In Vitro Methods for Cardiotoxicity Testing Electrophysiology
Verapamil
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中国食品药品检定研究院 食品药品安全评价研究所
In Vitro Methods for Cardiotoxicity Testing Electrophysiology
Verapamil
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中国食品药品检定研究院 食品药品安全评价研究所
WGCNA
Results based on Affymetrix data from 208 cardiac tissues
Weighted correlation network
analysis (WGCNA)
NIFDC
Study 3 Properties of Drug Induced Cardiotoxicity
Characterization using WGCNA
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中国食品药品检定研究院 食品药品安全评价研究所
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DrugMatrixGene
Expression
Profiles Benchmark
Drugs and
Compounds
Pathology
Profiles
Gene
Expression
Profiles
Pharmacology
Profiles
Literature
Profiles
Benchmark
Drugs and
Compounds
Gene Expression• CodeLink RU1 10K
rat array
• Affymetrix Whole Genome Arrays
Pharmacology Assays• Binding, Enzyme, ADME
Drug Literature • Pharmacology
• Toxicology
• Structure
• Pathways
Pathology Assays• Histopathology
• Clinical chemistry
• Hematology
• Body and organ weights
Results:34 cardiotoxicity specific histopathological indicators including 20 indicators with severity scores in one or more experiments >0;
Results :88 different compounds Corresponding to active structure categories and 32 active categories.
Search: Affymetrix data from 208 cardiac tissue(31042 probe);
Properties of Drug Induced Cardiotoxicity
Characterization using WGCNA
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中国食品药品检定研究院 食品药品安全评价研究所
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UNC93B1、CYBB、CYBA、Aif1 can be used as candidate genes for drug induced cardiotoxicity by comparing major genes in Blank/Salmon
modules and recent research progress
Properties of Drug Induced Cardiotoxicity
Characterization using WGCNA
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中国食品药品检定研究院 食品药品安全评价研究所
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A B
C D
n=10
Fig. 2.2 Changes in the serum cTnI¥H-FABP¥CK-MB¥NOX2 concentrations during the process of ISO induced cadiotoxicity at
different time points using different dose. P < 0.05 n=6/group
The generation and identification of NOX2 in the model
Changes in serum cTnI、H-FABP、CK-MB、NOX2 concentration
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中国食品药品检定研究院 食品药品安全评价研究所
Changes of the heart function after ISO treated hESC-CM
Real-time Cell analysis the effect on amplitude and beat rate regularity of hESC-
CM treated by ISO
B C
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中国食品药品检定研究院 食品药品安全评价研究所
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ISO promoted oxidative stress in hESC-CM
Con
1h
1nM 10nM
4h
Con 1nM 10nM
4h
1h
A:ISO enhanced ROS production . B: ISO inhibited MMP P < 0.05 vs. vehicle group
ISO promoted ROS production and inhibited MMP.
A B
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中国食品药品检定研究院 食品药品安全评价研究所
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NOX2 expression changes in the hESC-CM and mRNA level
(a) qPCR analysis NOX2 mRNA expression. (b) The levels of NOX2 concentration in hESC-CM
. Data are mean ± SEM (n=6 per group). *P < 0.05 versus saline-treated group.
A B
NOX2 expression was elevated after ISO treated
hESC-CM
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中国食品药品检定研究院 食品药品安全评价研究所
Co-culture
Absorption of intestine and liver metabolism
NIFDC
Study 4
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中国食品药品检定研究院 食品药品安全评价研究所
Effect of TP on Cell
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中国食品药品检定研究院 食品药品安全评价研究所
Intestine and liver
ROS induced
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中国食品药品检定研究院 食品药品安全评价研究所
High Content Screening
TP induced ROS
using DCF-DA kit
% C
on
tro
l
0 .0
0 .5
1 .0
1 .5
2 .0
1 g /m L T P
5 g /m L T P
5 g /m L T P + 1 0 g /m L D G
5 g /m L T P + 1 0 0 g /m L D G
*
*
24 h DG pretreatment
before 1 h TP treatment
DCF Hoechst
DG significantly
decreased TP induced
ROS
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中国食品药品检定研究院 食品药品安全评价研究所
2014 May
Cooperation Memorandum signed
between IIVS and NIFDC
2014-2015: 3 times training
courses
2014 September
Cooperation Memorandum signed
between Berlin University of
Science and Technology, Germany
and NIFDC
2015: 2 scientists visited, 40 days
International Collaboration
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中国食品药品检定研究院 食品药品安全评价研究所
On August 28-30th, 2014, visiting the EU joint research center at Ispra, Italy
Cooperative Intent: to establish a mutual contact mechanism and multiple cooperation,
which will start from the alternative methods for animal experiments.
We have completed the Chinese translation : part of the principle EU
guideline/regulation.
International Collaboration
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中国食品药品检定研究院 食品药品安全评价研究所
Thank You
Email: [email protected]