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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


Content

Current status

Alternative Research in NIFDC

International collaboration


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


Guideline for the Human Care and Use of

Laboratory Animals（2006）



5

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



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

6


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


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)


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

9

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)


10

我国体外技术发展面临的挑战-体系建设 Guiding opinions on accelerating research of alternative



※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


Guiding opinions on accelerating research of alternative



※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


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/


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)


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

14


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

15


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)


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）

17

Laboratory Technique Training


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

18



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.)

19



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)



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


Alternative Research in NIFDC

In vitro hepatotoxicity assay

In vitro cardiotoxicity assay

Computational toxicity

Cells Co-Culture


23

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


24

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)


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


In Vitro Methods for

Cardiotoxicity Testing

NIFDC

Study 2


In Vitro Methods for Cardiotoxicity Testing Cell Function

Verapamil


In Vitro Methods for Cardiotoxicity Testing Electrophysiology

Verapamil


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


31

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



32

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



33

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


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


35

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


36

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


Co-culture

Absorption of intestine and liver metabolism

NIFDC

Study 4


Effect of TP on Cell


Intestine and liver

ROS induced


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


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


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


Thank You

Email: [email protected]

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