Validation Study Report on Hand1-Luc EST (Embryonic Stem Cell Test)
As a Non-animal Test Evaluating Developmental Toxicity of Chemicals

Prepared by
The validation management team of the Hand1-Luc EST
October, 2016

1

CONTENTS
Contents .............................................................................................................................................. 2
List of abbreviations ........................................................................................................................... 7
Modular approach ............................................................................................................................... 9
Summary ........................................................................................................................................... 10
1.

Rational for the proposed test .................................................................................................... 121.1. Introduction ............................................................................................................................ 121.2. Objectives and intended use................................................................................................... 14
1.3. Patent ..................................................................................................................................... 15

2. Organization of the study .............................................................................................................. 16
2.1. Validation management team ................................................................................................. 16
2.2. Participating laboratories ....................................................................................................... 16
2.3. Sponsors ................................................................................................................................. 16
2.4. Supporters .............................................................................................................................. 17
2.5. Quality check monitoring ...................................................................................................... 17
2.6. Meeting held .......................................................................................................................... 17
2.7. Chemical Selection ................................................................................................................ 20
2.8. Chemical distribution management ....................................................................................... 20
2.9. Statistical analysis .................................................................................................................. 21
3. Pre-validation study ...................................................................................................................... 22
3.1. Preliminary studies................................................................................................................. 22
3.1.1. Cell characterization ....................................................................................................... 22
3.1.1.1. Objectives ................................................................................................................ 22
3.1.1.2. Study design ............................................................................................................. 22
3.1.1.3. Results ...................................................................................................................... 23
3.1.2. Protocol amendment ....................................................................................................... 24
3.1.2.1. Objectives ................................................................................................................ 24
3.1.2.2. Study design ............................................................................................................. 25
3.1.2.3. Results ...................................................................................................................... 26
3.1.3. Conclusion ...................................................................................................................... 28
3.2. Phase 0 study ......................................................................................................................... 29
3.2.1. Goal................................................................................................................................. 29
3.2.2. Study design .................................................................................................................... 29
3.2.2.1. Protocol .................................................................................................................... 29
3.2.2.2. Data analysis ............................................................................................................ 292

3.2.3. Chemicals........................................................................................................................ 29
3.2.4. Results ............................................................................................................................. 30
3.2.4.1. Lead laboratory ........................................................................................................ 30
3.2.4.2. Lab A........................................................................................................................ 30
3.2.4.3. Lab B........................................................................................................................ 31
3.2.4.4. Lab C........................................................................................................................ 31
3.2.5. Conclusion ...................................................................................................................... 32
3.3. Phase 1 study ......................................................................................................................... 32
3.3.1. Goal................................................................................................................................. 32
3.3.2. Study design .................................................................................................................... 32
3.3.2.1. Protocol .................................................................................................................... 32
3.3.2.2. Data analysis ............................................................................................................ 33
3.3.3. Chemicals........................................................................................................................ 34
3.3.4. Results ............................................................................................................................. 34
3.3.4.1. IC50 and ID50 values variation and final judgment ................................................... 34
3.3.4.2. Summary of the within-laboratory reproducibility .................................................. 39
3.3.5. Conclusion ...................................................................................................................... 40
4. Validation study ............................................................................................................................ 41
4.1. Phase 2a study ........................................................................................................................ 41
4.1.1. Goal................................................................................................................................. 41
4.1.2. Study design .................................................................................................................... 41
4.1.2.1. Protocol .................................................................................................................... 42
4.1.2.1.1. Preparation ........................................................................................................ 43
4.1.2.1.2. Hand1-Luc EST assay ....................................................................................... 44
4.1.2.2. Prediction model ...................................................................................................... 47
4.1.2.3. Positive and negative judgment ............................................................................. 48
4.1.2.4. Acceptance criteria ................................................................................................... 49
4.1.3. Chemicals........................................................................................................................ 50
4.1.4. Results ............................................................................................................................. 50
4.1.4.1. IC50 and ID50 values variation and final judgment ................................................... 50
4.1.4.2. Summary of the between-laboratory reproducibility ............................................... 54
4.1.5. Conclusion ...................................................................................................................... 54
4.2. Phase 2b/2c studies ................................................................................................................ 54
4.2.1. Goal................................................................................................................................. 54
4.2.2. Study design .................................................................................................................... 55
4.2.3. Chemicals........................................................................................................................ 55
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4.2.4. Results ............................................................................................................................. 56
4.2.4.1. IC50 and ID50 values variation and final judgment ................................................... 56
4.2.4.2. Summary of the between- and within-laboratory reproducibilities.......................... 65
4.2.5. Conclusion ...................................................................................................................... 66
4.3. Phase 3 study ......................................................................................................................... 66
4.3.1. Goal................................................................................................................................. 66
4.3.2. Study design .................................................................................................................... 66
4.3.2.1. Protocol .................................................................................................................... 66
4.3.2.2. Data analysis ............................................................................................................ 67
4.3.3. Chemicals........................................................................................................................ 68
4.3.4. Results ............................................................................................................................. 69
4.3.4.1. IC50 and ID50 values variation and final judgment ................................................. 69
4.3.4.2. Summary of the between-laboratory reproducibility ............................................. 79
4.3.5. Conclusion ...................................................................................................................... 80
5. Discussion ..................................................................................................................................... 82
5.1. Test definition ........................................................................................................................ 82
5.2. Definition of the performance standards ................................................................................ 82
5.3. Predictive capacity ................................................................................................................. 82
5.3.1. Prediction model ............................................................................................................. 82
5.3.2. Predictive capacity .......................................................................................................... 85
5.4. Transferability ........................................................................................................................ 85
5.5. Within-laboratory reproducibility .......................................................................................... 86
5.6. Between-laboratory reproducibility ....................................................................................... 86
5.7. Applicability domain.............................................................................................................. 87
5.7.1. Limitations due to the material ....................................................................................... 87
5.7.2. Limitations due to the measured biological parameter ................................................... 87
5.7.3. Justifications on misclassified chemicals ........................................................................ 88
5.7.3.1. False negative chemicals .............................................................................................. 88
5.7.3.1.1. Boric acid .............................................................................................................. 88
5.7.3.1.2. Dexamethasone ..................................................................................................... 89
5.7.3.1.3. Dimethadione ........................................................................................................ 89
5.7.3.1.4. Methoxyacetic acid ............................................................................................... 90
5.7.3.1.5. Phenytoin .............................................................................................................. 90
5.7.3.2. False positive chemicals............................................................................................... 91
5.7.3.2.1. Diphenhydramine .................................................................................................. 91
6. CONCLUSIONS .......................................................................................................................... 92
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7. ACKNOWLEDGMENTS............................................................................................................. 93
8. REFERENCES ............................................................................................................................. 94
APPENDIX
Appendix 1 Suzuki et al, 2011aAppendix 2 Suzuki et al, 2011bAppendix 3 Suzuki et al, 2012
Appendix 4 Le coz et al, 2015
Appendix 5 Nagahori et al, 2016
Appendix 6 Quality check sheet（Hand1-Luc Ph0 study)
Appendix 7 Quality check sheet（Hand1-Luc Ph1 study)
Appendix 8 Quality check sheet（Hand1-Luc Ph2a study)
Appendix 9 Quality check sheet（Hand1-Luc Ph2b study)
Appendix 10 Quality check sheet（Hand1-Luc Ph2c study)
Appendix 11 Quality check sheet（Hand1-Luc Ph3 study)
Appendix 12 Quality check statement for each validation phases
Appendix 13 Minutes of Hand1-Luc EST 1st VMT meeting
Appendix 14 Minutes of Hand1-Luc EST 2nd VMT meeting
Appendix 15 Minutes of Hand1-Luc EST 3rd VMT meeting
Appendix 16 Minutes of Hand1-Luc EST 4th VMT meeting
Appendix 17 Minutes of november 6th (2013) Hand-1
Appendix 18 Minutes of Hand1-Luc EST 5th VMT meeting
Appendix 19 Minutes of Hand1-Luc EST 6th VMT meeting
Appendix 20 Minutes of Hand1-Luc EST 6th VMT meeting conference callAppendix 21 Minutes of Hand1-Luc EST 7th VMT meeting
Appendix 22 Minutes of Hand1-Luc EST 8th VMT meeting
Appendix 23 Minutes conference call 20150528
Appendix 24 Minutes of Hand1-Luc EST 9th VMT meeting
Appendix 25 Minutes conference call 20160406
Appendix 26 Chemical selection report
Appendix 27 Statistical analysis report
Appendix 28 Hand1protocol_ver04E
Appendix 29 Hand1protocol_ver05-1E
Appendix 30 from 6 days to 5 days measurement investigation and result
Appendix 31 Hand1-Luc EST Study plan
Appendix 32 Hand1protocol_ver06E5

Appendix 33 Hand1_Data Sheet_verE08.3
Appendix 34 Hand1 protocol_ver07E
Appendix 35 Hand1 protocol_ver08E
Appendix 36 Hand1 71 chemicals data
Appendix 37 Hand1protocol_ver09E
Appendix 38 Hand1 related pathway references

6List of abbreviations
AFC

Aminofluorocoumarin

AIST

The National Institute of Advanced Industrial Science and Technology

BG

Background

BfR

Bundesinstitut Riskobewertung(German Federal Institute for Risk Assessment)

DF

Dose finding study

DMSO

Dimethyl Sulfoxide

DS

Definitive study

ECVAM

European Centre for the Validation of Alternative Methods

EST

Embryonic Stem cell Test

EURL

European Union Reference Laboratory

FDSC

Food and Drug Safety Center

Hand1

Heart and neural derivatives 1

HRI

Hatano Research Institute

ICATM

International Cooperation on Alternative Test Methods

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative
Methods

JaCVAM

Japanese Center for the Validation of Alternative Methods

JCIAJapan Chemical Industry Association

KAO

Kao Corporation

KoCVAM

Korean Center for the Validation of Alternative Methods

LRILong Range Research InitiativeMC

Medium control

MD

Maximum dose

METI

Ministry of Economy Trade and Industry

NEDO

New Energy Development Organization

NICEATMNTP Interagency Center for the Evaluation of Alternative
Toxicological Methods

OECD

Organization for Economic Co-Operation and development

PBS

Phosphate Buffer Saline

RKI

Robert Koch Institute

ROC

Receiver Operating Characteristic

SC

Sumitomo Chemical Co., Ltd.

SCAS

Sumika Chemical Analysis Service, Ltd.

VC

Vehicle control
7

VMT

Validation Management Team

WEC

Whole Embryo Culture8Modular approach
In 2004, ECVAM proposed a modular approach to the validation of alternative methods (Hartung etal., 2004), according to which the various information requirements for peer-review and as generated
during the validation process are broken down into seven independent modules as mentioned below:
Module 1: Test definition
Module 2: Within-laboratory reproducibility
Module 3: Transferability
Module 4: Between-laboratory reproducibility
Module 5: Predictive capacity
Module 6: Applicability domain
Module 7: Definition of performance standards
All the modules are summarized and discussed in part 5.

9

Summary
Risk assessment of developmental toxicity is essential for chemicals to be distributed on the market.
Long established developmental toxicity assays with experimental animals are still used inlaboratories because they are, with the current human knowledge, the most accurate test to predict
developmental toxicity of chemicals. Since the end of the 20th century, regulatory authorities
promote the development of alternative test methods to reduce the use of animals and the cost interms of time and money induced by the current available protocols. The development of thesemethods is supervised by the ICATM, composed by worldwide associations (ICCVAM for the USA,ECVAM for Europe, JaCVAM for Japan, KoCVAM for Korea and Health up Canada).
In this context, the embryonic stem cell test (EST) had been developed to respond to the needs of the
3Rs (replace, refine, reduce) in 1997 by the group of Dr. Horst Spielmann. In this test,
embryotoxicity of chemicals was evaluated by measuring cytotoxicity and differentiation toxicity
using mouse ES D3 cells. Cytotoxicity was measured in both 3T3 cells and D3 cells with the MTT
assay. Differentiation toxicity was analyzed by microscopically counting the beating of embryoid
bodies after 10 days of culture. It was validated by ECVAM in 2002 (Genschow et al, 2004) but thisprotocol did not reach the status of an official OECD test guideline due to some limitations in the
manipulations, in the applicability domain or in the reliability and relevance of the system (Buesen etal, 2004). Thus, Sumitomo Chemical Co., Ltd. has developed the Hand1-Luc EST to improve theoriginal EST. Hand1 (Heart and neural crest derivatives 1) is a key gene for the development of theheart and some other organs (facial bones, limbs). The test measures both cytotoxicity anddifferentiation toxicity (Hand1 expression alteration by luciferase activity monitoring) withengineered mouse KOB1-ES cells being stable transformants of a vector containing the promoter ofHand1 gene upstream of luciferase reporter gene. The cells are differentiated in the presence of thechemical during 120 hr (5 days) into cardiomyocytes. This protocol is much easier than the original
EST, by simply culturing cells in 96 well round bottom plate and measuring cytotoxicity anddifferentiation toxicity consequently in the same plate.
The validation study began in February 2013, lasted 3 years. It was applied in a step wise manner(pre-validation phase (phases 0 and 1) and validation phase (phases 2 and 3). The phase 0 consisted
in the technical transfer to three different participant laboratories (Kao Corporation, SCAS, Ltd and
HRI/FDSC). Each laboratory tested three chemicals with one dose finding study and three definitive
studies. At this step, chemicals were incubated 6 days with the cells. The analysis of phase 0 led to
improve the protocol, establish acceptance criteria and confirm the technical transfer. At this step,
the further phase studies to verify within- and between-laboratory reproducibility were planned andthe acceptance criterion for those two parameters was decided to be more than or equal to 75%.
Between phase 0 and phase 1, the incubation time of chemicals with the cells has been changed from10

6 days to 120 hr (5 days) so as to improve the IC50/ID50 ratio that sometimes was lower than one.
Phase 1 consisted in three coded chemicals tested once, each with one dose finding study and one
definitive study to test the within-laboratory reproducibility. With a newly created prediction model
based on the modification implemented at the end of phase 0, the result obtained was 88.9% thatlargely fulfilled the criterion (75%). A more precise analysis of phase 1 led to fit the curve (twoparameter curve fitting) for the determination of the IC50 and ID50 values, to establish the majority
judgment for the evaluation of embryotoxicity and the implementation of the solubility of thechemicals in the medium. The validation proceeded to phase 2 that was separated into threesubphases 2a, 2b and 2c. Phase 2a was used to check the newly established improvements and toverify the between-laboratory reproducibility. The prediction model was improved according to thechanges applied for phase 2 (acceptance criteria, two parameter curve fitting and improved
prediction model). Phase 2a (four coded chemicals tested with one dose finding study and two or
three definitive studies in only one set) gave very good results with a between-laboratory
reproducibility of 100%. Thus, phases 2b and 2c were launched. Those phases consisted in eightcoded chemicals tested three times to verify both between- and within-laboratory reproducibility.
The results obtained were both within- (phase 2b and 2c results) and between- (phase 2a, 2b and 2c
results) laboratory reproducibility of 83.3%. This phase ended with a new recommendation about
fitting curves that should be done not with two parameters but with three parameter curve fitting.
This change resulted in the improvement of the prediction model and phase 3 was started. This lastphase consisted in sixteen chemicals tested only once with a dose finding study and two or threedefinitive studies. The results gave a between-laboratory reproducibility of 75% fulfilling theacceptance criterion. These results proved that Hand1-Luc EST is robust, transferable between
laboratories, reproducible both within- and between-laboratories and applicable to the prediction ofchemical embryotoxicity.

11

1

Rational for the proposed test

1.1

Introduction

In our society, consumers are more and more intensively exposed to chemicals produced by theindustry. The different possible toxicities triggered by those compounds need to be accurately tested
before putting chemicals on the market. In the context of 3R (Replacement, Reduction, Refinement),
there is a high demand for a rapid, reliable and cost-effective alternative test methods.
Developmental toxicity, due to complicated biological mechanisms that are at stake, is still taking its
first faltering steps in in vitro experiments. Presently, only three in vitro developmental toxicity tests
are validated by ECVAM: the Whole Embryonic Culture assay (WEC), the Micromass
embryotoxicity assay and the Embryonic Stem cell Test (EST) but none of them reached theguideline status (Spielmann et al, 2006). Among those three tests, the WEC and the Micromass test
although being recognized as alternative test methods are still demanding killing animals. Some say
1R (replacement) should be aimed instead of 3R and thus, those methods may not be sufficient.
However, the EST, using mouse embryonic stem cells, does not require any animal killing which led
it to be a test with high expectancies. The original test developed in 1997 (Spielmann et al, 1997)and validated by ECVAM in 2002 (Genschow et al, 2004) investigated the inhibition of cardiac
differentiation by chemicals from ES cells within 10 days by microscopic observation of beating
cardiomyocytes derived from mouse ES cells. The endpoints used for the prediction were IC50 ofboth mouse D3 ES cells and 3T3 cells, and ID50 of mouse ES cells. The IC50, representing theconcentration of chemicals for the one cell viability is reduced by 50%, was measured with the MTT
assay. The ID50 was expressed as the concentration of chemicals inhibiting the development ofcontracting cardiomyocytes by 50% (number of well containing beating cardiomyocytes). The
prediction model consisted in three different equations and the chemical was classified as three
categories (strong, week and non embryotoxic). The accuracy of the test reached 78% (Genschow etal, 2004). The heart being the one of the main organs to be developing during embryogenesis, theEST was still a reliable tool to test embryotoxicity. However, it was not accepted as an OECD test
guideline for the following reasons (Marx-Stoelting et al, 2009): a lack of clarity in discrimination
between non- and weak/moderate embryotoxicants with a necessity of improving the prediction
model, the applicability domain that should be widen with a integration of a metabolic system andthe test being time consuming and requiring high experimental expertise.
To face those problems, the Hand1-EST was first developed by Sumitomo Chemical, Co. Ltd (SC)since 2011 supported by the NEDO (New Energy and industrial technology Development
Organization) project. The first step of the development consisted in determining the genes involvedin ES cell differentiation into cardiomyocytes. 120 marker genes were picked up (Suzuki et al, 2011a,
12see appendix 1) by DNA chip and quantitative PCR. Among those, thirteen early embryogenesis
expressed marker genes expression was analyzed after treatment with embryotoxicants andnon-embryotoxicants. According to their role in the development process, their expression pattern
and their altered expression after chemical exposure, the Hand1 gene was chosen. This gene has a
key role for cardiac differentiation process (McFadden. et al., 2005; Reamon-Buettner et al., 2009).
It is also involved in cranial morphogenesis (Firulli et al, 2014) and limbs formation (Laurie et al.,2016). The next step consisted in developing a novel high-throughput EST (Suzuki et al., 2011b, seeappendix 2). In that study, the same type of cells was used as in the original EST (D3 and 3T3 cells).
D3 cells were stably transfected with Hand1 promoter upstream of luciferase reporter gene, whichallowed to see the influence of chemicals on the gene expression translating differentiation toxicity.
The conclusion of the study was high predictability and accuracy with reduced test duration andmanpower compared to the original EST. Furthermore, in 2012, another study was published (Suzuki
et al., 2012, see appendix 3) in which Hand1-Luc EST was investigated to explore reproducibility bycomparing a set of 6 well-known test chemicals in four different laboratories. Good correspondence
was obtained among all four laboratories, proving the high potency of transferability,
within-laboratory variability, and between-laboratory variability of the protocols. In 2016, thepresent Hand1-Luc EST was established (Le Coz et al, 2015; Nagahori et al, 2016, see appendix 4
and 5 respectively).
The test has been developed, after having analyzed the weak points of the original EST, to be a moreaccurate test with a wider applicability and with much easier manipulations. The test is using
Hand1-ES (KOB1) cells. These cells are transfected with a vector containing the luciferase gene
monitored by the Hand1 promoter. Thanks to the validation process, the test has been improved anda summary of the latest version of the protocol is presented below:
Hand1-ES (KOB1) cells are seeded in a 96 well round bottom plate. Chemicals are dissolved in
PBS(-) or DMSO and their solubility in the assay medium is checked to determine the maximum
dose (MD) of the experiment. Between 2 and 4 hrs after cell seeding, different concentrations ofchemical are added to the wells. The plate is then incubated for 120 hr (5 days). After 120 hr (5 days)
after the beginning of differentiation, cytotoxicity is measured with CellTiter-Fluor™ Cell Viability
Assay and followed by measurement of Hand1 promoter activity through luciferase activity with
Steady-Glo® Luciferase Assay System. Both endpoints are measured consecutively in the same plate(Figure1).

13

Day
0

120
Hr
(Day 5)

Hand1-ES (KOB1)cells
Preparation of test compound andEstablishment of the maximum dose
white 96 well roundbottom plate

・Cell viability assay(CellTiter Fluor)

ES-IC50 (cytotoxicity)

・Luc-activity(Steady-Glo)

ES-ID50 (Differentiation toxicity)

Data analysis

Figure 1 Experiment outline of Hand1-Luc EST
The raw data are then copied in an excel data sheet automatically calculating IC50 and ID50 results
through a three parameter curve fitting. Then, the IC50, the ID50 and the MD values are inserted in
the prediction model which gives then the predicted positivity or negativity of the chemical.
Compared to the original EST, the Hand1-Luc EST is thus easier, requires low quantity of chemicals,has a wider applicability and considers solubility in the medium that could be considered to be close
to the saturated free concentration in fetus that was observed in the reproductive toxicity studies.
Finally, through the prediction model, the test allows to predict positive embryotoxicants with highconfidence. This test is therefore a high-throughput test for eliminating true embryotoxicants andthus a very powerful tool for screening purposes.
The validation study of the Hand1-Luc EST was started from February 2013. It was applied in a step
wise manner (pre-validation phase (phases 0 and 1) and validation phase (phases 2 and 3) in threedifferent laboratories. Twenty eight different coded chemicals were used to verify the reproducibilityof the test along the three phases.
The current report, which was prepared by SC, with the support of the VMT members, presents thevalidation process and the validation outcome of the Hand1-Luc EST.
1.2

Objectives and intended use

The aim of the Hand1-Luc EST validation study was to scientifically validate the Hand1-Luc EST informal between-laboratory studies in order to assess the within- and the between-laboratory
reproducibility of the assay, its transferability and its predictive capacity. Twenty-eight different
chemicals with different mode of action were chosen to justify the relatively wide applicability
14domain of the assay. The ultimate goal is to demonstrate its reliability for adoption as an OECD test
guideline that could be used as a component of a test battery for the prediction of chemicaldevelopmental toxicity.
This report will also show the limitation of the test in terms of applicability by providing different
biological concrete explanations leading to think that the test should be in the future combined toother tests to predict accurately developmental toxicity of chemicals.
1.3 Patent
The test method and the cells employed in this study are covered by Japanese Patent Number
5428527, and International Unexamined Patent Application Publication Number WO/2009/148177.

15

2

Organization of the study

2.1

Validation management team

The Validation management was composed of the following members:
Trial coordinator, VMT Chair

Noriho Tanaka (HRI/FDSC/ OTIP, Kanagawa, Japan)

Lead Laboratory

Koichi Saito, Noriyuki Suzuki, Hirohisa Nagahori,
Florian Le Coz (SC, Osaka, Japan)

JaCVAM representative

Hajime Kojima (JaCVAM, NIHS, Tokyo, Japan)

Biostatisticians

Takashi Omori, Azusa Mori, Mayumi Kobayashi, Aoi
Maruya (Faculty of Medicine of Kobe, Kobe University,
Hyogo, Japan)

Consultant

Kazuhiko Matsumoto (Nagoya-City University, Nagoya,Japan)

In vivo teratogenicity expert

Makiko Kuwagata (HRI/FDSC, Kanagawa, Japan)

In vitro teratogenicity expert

Andrea Seiler (RKI, Berlin, Germany)

Quality Check monitoring

Yoshihiro Ohmiya (AIST, Ibaraki, Japan)

ICCVAM representative

David Allen (NICEATM/ICCVAM, North Carolina, USA)ECVAM Representative

Michael Schaeffer (EURL ECVAM, Ispra, Italy)

KoCVAM Representative

Eui-Bae Jeung (KoCVAM, Chungcheongbuk-do, Korea)

2.2

Participating laboratories

The validation study was conducted in three participating laboratories as follows;
Laboratory 1: HRI/FDSC (Mika Watanabe, Mayu Ikezumi; Kanagawa, Japan)
Laboratory 2: SCAS (Kazunori Yanagi, Takeshi Izukawa; Osaka, Japan)
Laboratory 3: KAO (Toshio Kasamatsu, Naohiro Ikeda, Joshou Ryuu, Yuichi Ito; Tochigi, Japan)
2.3 Sponsors
Ministry of Economy, Trade and Industry (METI), Japan
Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI)
Partially supported by National Institute of Advanced Industrial Science and Technology(AIST)
16

2.4 Supporters
National Institute of Health Sciences (NIHS)
Japanese Center for the Validation of Alternative Methods (JaCVAM)
2.5 Quality Check monitoring
The quality check sheet has been filled in by every laboratory before and during each experiment(see Appendix 6 to 11) to verify that the protocol was correctly respected.
Dr. Ohmiya was responsible for the QC monitoring during the validation study. All the statements
submitted for each phases can be found in appendix 12.
2.6 Meetings held
The minutes of the VMT meetings described below can be found in appendix 13 to 25.
Title: 1st VMT meeting (21-22/02/2013, Osaka)
Members: Lori Rinckel (ICCVAM), Andrea Seiler (BfR), Eui-Bae Jeung (KoCVAM), Noriho
Tanaka (HRI), Hajime Kojima (NIHS), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Kumiko
Kobayashi, Sachiko Kitamoto (SC), Takashi Omori, Maruya Nishio, Aoi Maruya, Mayumi
Kobayashi (Doshisha University), Makiko Kuwagata (HRI), Yoshihiro Ohmiya (AIST), Shojiro
Yamazaki (HRI) and participating laboratories.
Subject: Discussion and planning of test protocol, judgment criteria and outline of validation studyfor Hand1-Luc ESTMinutes: Appendix 13
Title: 2nd VMT meeting with conference call (30/04/2013-01/05/2013, Kyoto)
Members: Lori Rinckel (ICCVAM), Andrea Seiler (BfR), Eui-Bae Jeung (KoCVAM), Micheal
Schaeffer (ECVAM), Noriho Tanaka (HRI), Hajime Kojima (NIHS), Koichi Saito, Hirohisa
Nagahori, Noriyuki Suzuki (SC), Takashi Omori, Manabu Nishio, Aoi Maruya, Mayumi Kobayashi,
Azusa Mori, Fuminari Yoshifuji (Doshisha University), Kazunori Yanagi (SCAS), Shojiro Yamazaki(HRI).
Subject: Justification on points raised in the 1st VMT meeting and planning of phase 0 study. Changeof 2D to 3D cell culture, protocol improvement, two plates to one plate change
Minutes: Appendix 1417

Title: 3rd VMT meeting with conference call (18-19/07/2013, Kyoto)
Members: Lori Rinckel (ICCVAM), Andrea Seiler (BfR), Hajime Kojima (NIHS), Noriho Tanaka(HRI), Yoshihiro Omiya (AIST), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Florian Le Coz(SC), Takeshi Omori, Aoi Maruya, Manabu Nishio, Mayumi Kobayashi, Azusa Mori, Kamoda,
Takayana (Doshisha University), Kazunori Yanagi, Takeshi Izukawa (SCAS), Liu Josho (KAO),
Mayu Ikezumi, Shojiro Yamazaki (HRI).
Subject: Improvement of the protocol. Discussion to proceed to phase 0.
Minutes: Appendix 15
Title: Lecture and training for technical transfer (19/07/2013, Osaka)
Members: Noriyuki Suzuki, Florian Le Coz (SC), Ikezumi Mayu (HRI), Liu Josho, Naohiro Ikeda(KAO), Takeshi Izukawa (SCAS)
Subject: Training of the participating laboratories for the protocol
Title: 4th VMT meeting with conference call (22-23/09/2013, Kyoto) followed by anotherteleconference call (06/11/2013)
Members: Lori Rinckel (ICCVAM), Andrea Seiler (BfR), Eui-Bae Jeung (KoCVAM), Micheal
Schaeffer (ECVAM), Hajime Kojima (NIHS), Noriho Tanaka (HRI), Yoshihiro Omiya (AIST),
Makiko Kuwagata (HRI), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Florian Le Coz (SC),
Takeshi Omori, Aoi Maruya, Manabu Nishio, Mayumi Kobayashi, Azusa Mori, Kamoda, Takayana(Doshisha University), Kazunori Yanagi, Takeshi Izukawa (SCAS), Liu Josho, Ikeda (KAO), Mayu
Ikezumi, Mika Watanabe, Shojiro Yamazaki (HRI).
Subject: Analysis of phase 0 data, improvement of the protocol and preparation for phase1.
Minutes: Appendix 16 and 17
Title: 5th VMT meeting with conference call (19-21/02/2014, Kyoto)
Members: Andrea Seiler (BfR), Eui-Bae Jeung (KoCVAM), Michael Schaeffer (ECVAM), Warren
Casey (ICCVAM), Noriho Tanaka (HRI), Hajime Kojima (NIHS), Koichi Saito, Hirohisa Nagahori,
Noriyuki Suzuki, Florian Le Coz (SC), Takashi Omori, Mayumi Kobayashi, Azusa Mori, Manabu
Nishio, Aoi Maruya (Doshisha University), Yoshihiro Ohmiya (AIST), Kazuhiko Matsumoto(Nagoya-City University), Makiko Kuwagata (HRI), Liu Shujie, Naohiro Ikeda (KAO), Kazunori
Yanagi, Takeshi Izukawa (SCAS), Mayu Ikezumi, Mika Watanabe, Shojiro Yamazaki (HRI)
Subject: Analysis of phase 1 data, protocol improvements and preparation of phase 2
Minutes: Appendix 18

18

Title: 6th VMT meeting (10/05/2014, Kyoto)
Members: Noriho Tanaka (HRI), Hajime Kojima (NIHS), Koichi Saito, Hirohisa Nagahori, Noriyuki
Suzuki, Florian Le Coz (SC), Takashi Omori, Mayumi Kobayashi, Azusa Mori, (Doshisha
University), Yoshihiro Ohmiya (AIST), Kazuhiko Matsumoto (Nagoya City University), Makiko
Kuwagata (HRI), Liu Shujie, Naohiro Ikeda (KAO), Kazunori Yanagi, Takeshi Izukawa (SCAS),
Mayu Ikezumi, Mika Watanabe, Shojiro Yamazaki (HRI), Takao Ashikaga
Subject: Analysis of phase 2a results and establishment of phase 2b, 2c plan.
Minutes: Appendix 19
Title: Conference call following the 6th VMT meeting (21/05/2014)
Members: Andrea Seiler (BfR), Eui-Bae Jeung (KoCVAM), Warren Casey, David Allen (ICCVAM),
Noriho Tanaka (HRI), Hajime Kojima (NIHS), Makiko Kuwagata (HRI), Koichi Saito, Hirohisa
Nagahori, Noriyuki Suzuki, Florian Le Coz (SC), Shojiro Yamazaki (HRI)
Subject: Analysis of phase 2a and discussion for the promotion of phase 2b and 2c
Minutes: Appendix 20
Title: 7th VMT meeting (25/09/2014, Tokyo)
Members: Noriho Tanaka (HRI), Hajime Kojima (NIHS), Takashi Omori, Mayumi Kobayashi,
Azusa Mori, Aoi Maruya (Doshisha University), Koichi Saito, Noriyuki Suzuki, Le Coz Florian(SC), Joshou Ryuu, Shota Nakagawa (KAO), Kazunori Yanagi, Takeshi Izukawa (SCAS), Mika
Watanabe, Mayu Ikezumi, Shojiro Yamazaki (HRI)
Observers: Tomoka Hisaki(Shiseido Co. Ltd.), Tatsuya Mizukoshi(JCIA), Kohji Yamakage(HRI)
Subject: Analysis of phase 2b and discussion for phase 2c
Minutes: Appendix 21
Title: 8th VMT meeting with conference call (20-21/02/2015, Kyoto)
Members: Noriho Tanaka (HRI), Hajime Kojima (NIHS), Warren Casey (ICCVAM), Andrea Seiler(BfR), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Le Coz Florian (SC), Takashi Omori,Mayumi Kobayashi, Azusa Mori, Aoi Maruya (Doshisha University), Yoshihiro Ohmiya (AIST),
Makiko Kuwagata (HRI), Kazuhiko Matsumoto (Nagoya-City University), Joshou Ryuu (KAO),
Kazunori Yanagi, Takeshi Izukawa (SCAS), Mika Watanabe, Mayu Ikezumi, Shojiro Yamazaki(HRI)
Observers: Tomoka Hisaki (Shiseido Co. Ltd.), Tatsuya Mizukoshi (JCIA), Kohji Yamakage (HRI)
Subject: Analysis of phase 2 and preparation of phase 3 with protocol improvements
Minutes: Appendix 22

19

Title: Conference call (28/05/2015, Kyoto)
Members: Noriho Tanaka (HRI & OTIP, Chair), Hajime Kojima (JaCVAM,NIHS,Co-chair), David
Allen (ICCVAM), Andrea Seiler (ZEBET), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Le
Coz Florian (Sumitomo Chemical), Takashi Omori, Azusa Mori (Doshihsa university), Makiko
Kuwagata (HRI), Shojiro Yamazaki (OTIP)
Subject: Discussion on 3 parameters curve fitting models for the phase 3 validation study
Minutes: Appendix 23
Title: 9th VMT meeting with conference call (19-20/02/2016, Kobe)
Members: Andrea Seiler (BfR), David Allen (ICCVAM), Noriho Tanaka (HRI), Hajime Kojima(NIHS), Koichi Saito, Hirohisa Nagahori, Noriyuki Suzuki, Florian Le Coz (SC), Takashi Omori(Kobe University), Mayumi Kobayashi, Azusa Mori (Doshisha University), Eui-Bae Jeung(KoCVAM), Yoshihiro Ohmiya (AIST), Makiko Kuwagata (HRI), Kazuhiko Matsumoto(Nagoya-City University), Yuichi ito, Joshou Ryuu (KAO), Kazunori Yanagi, Takeshi Izukawa(SCAS), Mika Watanabe, Mayu Ikezumi, Shojiro Yamazaki (HRI)
Advisors: Horst Spielmann (Freie Universität Berlin), Shihori Tanabe (NIHS)
Observers: Akiko Tamura, Tomoka Hisaki (Shiseido Co. Ltd.)
Subject: Analysis of phase 3 and final decision on the validation
Minutes: Appendix 24
Title: Conference call (6/04/2016)
Members: Noriho Tanaka (HRI), Hajime Kojima (NIHS), David Allen (ICCVAM), Horst Spielmann(Freie Universität Berlin), Takashi Omori (Kobe University), Koichi Saito, Hirohisa Nagahori,
Noriyuki Suzuki, Le Coz Florian (SC), Shojiro Yamazaki (HRI)
Subject: Comparison of prediction models and curve fitting: choice for the best combination andfuture schedule for the validation.
Minutes: Appendix 25
2.7 Chemical selection
The chemical selection was done under the recommendations of Dr Kuwagata. The details on thecriteria can be found in Appendix 26.
2.8 Chemical distribution management
Coding and distribution of chemicals were performed by JaCVAM. The coded chemicals were sent20

to the safety officer, who was not involved in the experiments, together with a sealed envelope
containing the material safety data sheets (MSDS). Since the chemicals were coded, the laboratories
did not know their identity and therefore all chemicals were treated as hazardous chemicals. The
disclosure of codes was performed at a VMT meeting, after the data had been finalized per QC
confirmation.
2.9

Statistical analysis

The details about the statistical analysis performed for all the phases can be found in Appendix 27.

21

3

Pre-validation study

This pre-validation study consisted in preliminary, phase 0 (transferability) and phase 1 studies.
3.1

Preliminary studies

Prior to the phase 0 study, the preliminary studies lasted 6 months (From February 2013 to July2013) according to comments of the VMT members in the 1st and 2nd VMT meetings.
3.1.1 Cell characterization
3.1.1.1 Objectives
Basic concept of the Hand-Luc EST is originated from the NEDO (New energy and industrial
technology development organization) EST project to quantify marker genes by luciferase reporter
gene assays (Suzuki et al, 2011a,b). In the Hand-Luc EST, new engineered cells named Hand1-ES(KOB1) cells are used due to the limited use for mouse ES (D3) cells and international general use in
the future. The objective of the study was to obtain background data (cell growth, induction of theluciferase gene and number of vector copy per cells) of the Hand1-ES (KOB1) cells to certify thequality control (QC) of the cells.
3.1.1.2 Study design
Cell growth and induction of the luciferase gene by Hand1 promoter
Three experiments without chemical treatment (medium control) have been done by two different
researchers. The study design is illustrated in Figure 2. Materials and methods used in this study
were basically described in the previous report (Suzuki et al, 2011b).
1. Cell viability

Cell counting with
Trypan-Blue staining at day 0

2. Cell growth
Cytotoxicity assay

3. Cell differentiation
Differentiation assay

Medium control at day 0 vs at day 6
using Cell-titer Glo (n=6)

Medium control at day 0 vs at day 6
using Steady-Glo (n=6)

Foldproliferation

Fold
induction

Figure 2 Study design to obtain background data of Hand1-ES (KOB1) cells
22

Copy number of vector containing luciferase gene governed by the Hand1 promoter
Copy numbers of exogenous DNA in Hand1-ES (KOB1) cells has been determined by Real-time
PCR using SYBR® Green. Two samples were used:- Plasmid (pGL4.17/Hand1-promoter Luc) for calibration curve- Extracted genome DNA from Hand1-ES (KOB1) cells
3.1.1.3 Results
Cell growth and induction of the luciferase gene by Hand1 promoter
Cell viability at day 0 was more than 93% in all experiments (data not shown). The results of cell
growth and luciferase gene inducement of Hand1-ES (KOB1) cells are summarized in Table 1.
In cytotoxicity assays, cell proliferation showed variations from 55 to 479 fold, proving the cells had
been correctly growing between day 0 and day 6. The luciferase gene was also correctly induced(fold induction of luciferase activity varying from 1075 to 5726) in the differentiation assays. CV
values were below 48% suggesting that the cytotoxicity and differentiation assays using Hand1-ES(KOB1) is stable and robust.
Table 1 Measurement of cell growth and luciferase gene inducement of Hand1-ES (KOB1) cells
Experiment

Cytotoxicity assay
Count Meana ± SD

Run 1b
Run 2
Run 3
Run 1c
Run 2
Run 3
a

Differentiation assay
CV (%)

Day 0

426259 ± 28881

7

Day 6

23537940 ± 3910573

17

Day 0

480807 ± 36579

8

Day 6

47033807 ± 522981

1

Day 0

516313 ± 68767

13

Day 6

53997767 ± 916170

2

Day 0

85987 ± 17792

21

Day 6

41197103 ± 406601

1

Day 0

128140 ± 13354

10

Day 6

41292740 ± 1444272

3

Day 0

186093 ± 16252

9

Day 6

42436440 ± 1651403

4

Fold
55
98
105
479
322
228

Count Meana ± SD

CV (%)

235 ± 6526272338 ± 130170

48

163 ± 1610269655 ± 61080

23

213 ± 3718257849 ± 100480

39

101 ± 88580269 ± 173219

30

122 ± 2420

511333 ± 100871

20

110 ± 98171247 ± 25452

15

Fold
1075
1653
1210
5726
4180
1559

Mean values of six wells (counts of luminescence).

b,cData obtained by two independent researchers.

Copy number of vector containing luciferase gene governed by the Hand1 promoter
The ratio copy of plasmid and genome DNA sample gave 20517 copies. Furthermore, the genomic
23

DNA sample weighs 0.00387 μg and the mouse genome/cell weighs 5.479 x 10-12 g. By dividing the
genomic DNA sample and the mouse genome per cell, a cell number of 706.3 was obtained. The
number of copy found previously divided by the number of cells gave a number of 29 copies percells.
3.1.2 Protocol amendment
The original protocol (Figure 3) was developed as part of the NEDO (New energy and industrial
technology development organization) EST project to quantify marker genes by luciferase reporter
gene assays (Suzuki et al, 2011a,b). In this protocol, cytotoxicity was measured with CellTiter-Glo®
Luminescent Cell Viability Assay and Luciferase activity (differentiation toxicity) with Steady-Glo®
Luciferase Assay System in separate plates.

Hand1-ES (KOB1) cells
Test compoundsDay
0

Differentiation assay

white U96 well plateDay
6

Cytotoxicity assay

96well plateLuc-activity(luminescence)

Cell viability assay(luminescence)

ES-ID50(Differentiation toxicity)

ES-IC50(ES/cytotoxicity)

Figure 3 Schematic overview of the original Hand1-Luc EST protocol
The main point raised during the 2nd VMT meeting was to improve the way of culturing cells where
it would be better to grow cells the same way (round bottom well plate to form embryoid bodies) for
both cytotoxicity and differentiation toxicity. Measurements in the same plate would be ideal for not
only scientific standpoints but also shortening of test duration and cost performance. Also, for somechemicals, the ID50 value was higher than the IC50 value which should not be because luciferase
cannot be expressed if the cells are dead. Preliminary studies of SC showed that the improvementwith measuring the cytotoxicity and differentiation toxicity in the same plate should solve theproblem (data not shown).
Then amendment of the original protocol was proposed. Schematic overview of the new protocol isshown in Figure 4. To measure cytotoxicity, CellTiter-Fluor™ Cell Viability Assay was used since
this reagent does not exhibit toxicity to the cells and thus, the next step with the measurement with24

Steady-Glo® Luciferase Assay System could be operated.

Hand1-ES(KOB1) cells
Test compounds

Day
0
Day
6

white U96 well plate
Cell viability assay(CellTiter Fluor)
Luc-activity(Steady-Glo)

IC50(Cytotoxicity）
ID50(Differentiation toxicity)

Figure 4 Schematic overview of the new Hand1-Luc EST protocol
3.1.2.1 Objectives
The objectives of the study was not only to prove reproducibility of the protocols but also to fix theprotocol that would be used in the validation study by comparing the data from the original (Figure3) and the new (Figure 4) protocols.
3.1.2.2 Study design
Twenty chemicals were tested three times independently by two different researchers in SC.
Variation of IC50 and ID50 values were evaluated.
ProtocolTwo protocols (Figures 3 and 4) were used. Designs of the test plates in the original and new
protocols are illustrated in Figure 5.

25

Design of the plates (Original protocol):Cytotoxicity assay

Differentiation assay

Design of the plate (New protocol):
MC: Medium control
VC: Vehicle control
BG: BackgroundFigure 5 Design of test plates in the original and new protocols
Chemicals
Twenty chemicals were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). All chemicals
were reagent grades (purity> 97%). 5-Fluorouracil was used as a standard chemical in this study.
Data analysis
The IC50 and ID50 values were calculated using two experimental values at two tested concentrations
across 50% activity rate in a concentration-response curve. Using the value of higher and lower sidesof 50% of concentration and cell viability, a linear equation was created as follows (Kishi et al.,2015).
IC50=10^(log(A/B)×(50-C)/(D-C)+log(B))
A: the concentration of higher side of 50% of cell viability, B: the concentration of lower side of
50% of cell viability, C: cell viability at the concentration of B, D: cell viability at the concentrationof A, ^: symbol of power in Excel software.
Similarly, suppression of mESCs differentiation by 50% was also calculated using the same
formula described above.
ID50=10^(log(A’/B’)×(50-C’)/(D’-C’)+log(B’))
A’: the concentration of higher side of 50% of luciferase activity, B’: the concentration of lower sideof 50% of luciferase activity, C’: luciferase activity at the concentration of B’, D’: luciferase activity
at the concentration of A’, ^: symbol of power in Excel software.
3.1.2.3 Results
Data from twenty chemicals tested by the original protocol are summarized in Table 2.
26

The results show not only that the Hand1-Luc EST is reproducible (CV<54%, IC50 value for ascorbicacid) for both the parameters IC50 and ID50 but also that two different researchers obtain results witha low variability when performing three tests with the same chemical.
The results from the new protocol are shown in Table 3. When comparing the results to Table 2, very
similar IC50 and ID50 values were found. Retinoic acid gave the highest CV (86%). The reason is that
the IC50 and ID50 values are exceptionally low. CV values of the other chemicals are all under 44%
showing the protocol is highly reproducible.
The relationship between the IC50 and ID50 was also improved especially for isoniazid, ascorbic acid,and acrylamide. Although for some cases ID50 values higher than the IC50 ones especially forcytosine arabinoside where the ratio the ID50/IC50 is 1.4, the gap between the IC50 and the ID50 is
considered as reasonable.
Table 2 Results of twenty chemicals tested by the original protocol (two plates)
In vivo

Test chemicals

categorya

IC50
Meanb ± SD

ID50
CV(%)

(μg/ml)Meanb ± SD

CV(%)

(μg/ml)Strong5-Fluorouracilc

0.06 ± 0.01

8

0.06 ± 0.01

13Strong5-Fluorouracild

0.06 ± 0.00

5

0.04 ± 0.00

3StrongHydroxyuread

5.69 ± 0.71

12

4.31 ± 0.164Strong6-Aminonicotinamided

0.93 ± 0.18

19

1.12 ± 0.26

23StrongCytosine arabinosided

0.30 ± 0.00

7

0.06 ± 0.01

20Strong

Methotrexated

0.02 ± 0.00

20

0.02 ± 0.00

2StrongRetinoic acidd

0.0006 ± 0.0002

37

0.0003 ± 0.0001

46Strong5-Bromo-2’-deoxyuridined

0.29 ± 0.06

21

0.43 ± 0.08

19

Weak

Valproic acidc

151.56 ± 34.41

23

134.69 ± 54.47

40

Weak

Dexamethasonec

32.22 ± 3.89

12

20.42 ± 1.66

8

Weak

Boric acidc

135.39 ± 1.91

152.59 ± 6.37

12

Weak

Methoxyacetic acidc

618.89 ± 24.89

4

480.34 ± 6.44

1

Weak

Lithium chloridec

777.46 ± 32.43

4

384.65 ± 145.29

38

Weak

Dimethadionec

>1000

-

>1000

-

Weak

Diphenylhydantoin(Free)c

47.75 ± 8.61

18

6.43 ± 0.58

9

Weak

Caffeinec

197.05 ± 30.37

15

83.45 ± 13.84

17

Non

Isoniazided

99.86 ± 2.92

3384.22 ± 44.39

12

Non

Ascorbic acidd

40.98 ± 22.07

54

164.04 ± 15.81

10

Non

Diphenhydramine HCld

22.05 ± 3.00

14

15.60 ± 4.66

30

Non

Dimethyl phthalatec

>500

-

35.50 ± 2.71

8

NonAcrylamidec

43.02 ± 6.40

15

71.60 ± 10.72

15

27

a

Brown et al, 2002

Mean values of three studies (μg/ml).

bc,dData from two independent researchers.

- Values for the ones the CV could not be determined

Table 3 Results of twenty chemicals tested by the new protocol (one plate)
IC50

In vivocategorya

Test chemicals

ID50

Ratio
ID50/IC50

Meanb ± SD

CV

Meanb ± SD

CV

(μg/ml)(%)(μg/ml)(%)

Strong5-Fluorouracilc

0.03 ± 0.002

8

0.04 ± 0.002

5

1.33

Strong5-Fluorouracild

0.03 ± 0.01

27

0.04 ± 0.01

16

1.33

StrongHydroxyuread

5.61 ± 0.65

124.50 ± 0.45

10

0.80

Strong6-Aminonicotinamided

1.13 ± 0.03

3

0.96 ± 0.12

13

0.85

StrongCytosine arabinosided

0.05 ± 0.002

3

0.07 ± 0.01

12

1.40

Strong

Methotrexated

0.04 ± 0.01

21

0.03 ± 0.01

31

0.75

StrongRetinoic acidd

0.005

-

0.00006±0.00005

86

0.12

Strong5-Bromo-2’-deoxyuridined

0.37 ± 0.06

15

0.41 ± 0.03

6

1.11

Weak

Valproic acidc

117.05 ± 26.71

2395.40 ± 35.80

38

0.82

Weak

Dexamethasonec

26.15 ± 1.35

531.40 ± 5.18

17

1.20

Weak

Boric acidc

65.08 ± 6.42

10

63.28 ± 22.10

35

0.97

Weak

Methoxyacetic acidc

400.30 ± 12.33

3475.85 ± 4.42

1

1.19

Weak

Lithium chloridec

>1000

-

950.18 ± 41.62

4

<0.95

Weak

Dimethadionec

>1000

-

>1000

-

(1.0)

Weak

Diphenylhydantoinc

>250

-

11.59 ± 2.43

21

<0.046

Weak

Caffeinec

199.56 ± 6.23

3

111.07 ± 25.06

23

0.56

Non

Isoniazided

339.95 ± 59.59

18

410.91 ± 49.53

12

1.21

Non

Ascorbic acidd

634.27 ± 148.07

23804.31 ± 182.36

23

1.23

Non

Diphenhydramine HCld

21.05 ± 5.38

26

14.38 ± 5.67

39

0.68

Non

Dimethyl phthalatec

432.62 ± 58.80

1440.89 ± 17.83

44

0.095

Non

Acrylamidec

116.76 ± 3.72

386.14 ± 2.57

3

0.74

a Brown et al, 2002

Mean values of three studies (μg/ml).

bc,dData for two independent researchers.

- Cases where the CV could not be determined

3.1.3 Conclusion
Thanks to the analysis of the cells’ characteristics and the usefulness of the protocol obtained thepreliminary phase, the relevancy of the protocol was verified. Phase 0 was thus launched to verify
28the transferability of the test by using the new protocol in three participating laboratories.
3.2 Phase 0 study
This phase began in July 2013 and ended in September 2013.
3.2.1

Goal

The goal of phase 0 was to prove that Hand1-Luc EST is transferable to other laboratories byanalyzing the IC50 and ID50 variability of three non-coded chemicals.
3.2.2

Study design

The technical transfer was done in SC in Osaka with the three participant laboratories.
3.2.2.1 Protocol transfer
All the details of the protocol based on Figure 4 (one plate method) were taught by a SC’s researcher.
The protocol version used for this phase was 04E (see Appendix 28). For each chemical, threedefinitive studies (DS) were applied.
3.2.2.2 Data analysis
IC50 and ID50 values were calculated by the method described in 3.1.2.2

Study design (Dataanalysis).
3.2.3 Chemicals
For this phase, the positive control 5-Fluorouracil (5-FU) and three non-coded test chemicals were
tested (Table 4).
Table 4 Information about the test chemicals used for phase 0
Test

CAS No.

Supplier

Physical property

Solventchemicals

In vivoclassification

Ascorbic acid50-81-7

Sigma

Solid

PBS

Negative

Boric acid

10043-35-3

Sigma

Solid

PBS

Positive

Hydroxyurea

127-07-1

Sigma

Solid

PBS

Positive29

3.2.4 Results
3.2.4.1 Lead laboratory
Table 5 shows the background data obtained in the lab with the chemicals used for phase 0. The
chemicals have been tested with three definitive studies and the variance remained very low(CV<35%) showing the test to be highly reproducible. The IC50 and ID50 values were the highest forascorbic acid followed by boric acid and then by hydroxyurea .
Table 5 Background data on test chemicals used in phase 0 study(technical transfer confirmation study) in lead laboratory (SC)Test chemicals

IC50

ID50

Meanb ± SD (μg/ml)

CV (%)

Meanb ± SD (μg/ml)

CV (%)

Ascorbic acid634.27 ± 148.07

23804.31 ± 182.36

23

Boric acid65.08 ± 6.42

10

63.28 ± 22.10

35

Hydroxyurea

5.61 ± 0.65

124.50 ±

10

0.45

Lead lab.: SCaMean values of three definitive studies.

b

3.2.4.2 Lab A
Table 6 shows the data obtained in phase 0 for lab A for three different test compounds. The IC50 and
ID50 values obtained for the three chemicals tested in three definitive studies revealed a very low
variance (CV<38%). IC50 and ID50 values differ from the lead lab from two to three times forascorbic acid but for the two other chemicals the results are very close to the ones obtained in thelead lab. The IC50 and ID50 values were the highest for ascorbic acid followed by boric acid and thenby hydroxyurea as observed in the lead lab.

Table 6 Results on Lab Aa in phase 0 study (technical transfer confirmation study)
Test chemicals

IC50
Meanb ± SD (μg/ml)

Ascorbic acid

ID50
CV (%)

Meanb ± SD (μg/ml)

CV (%)

289.50 ± 76.54

26251.29 ± 96.58

38

Boric acid

83.73 ± 10.36

12

92.07 ± 6.57

7

Hydroxyurea

4.30 ± 0.27

6

4.91 ± 0.43

9

Lab A: FDSCa

Mean values of three definitive studies.

b30

3.2.4.3 Lab B
Table 7 shows the data obtained in phase 0 for lab B for three different test compounds. The IC50 and
ID50 values obtained for the three chemicals tested in three definitive studies revealed a very low
variance (CV<28%). All of the data are very close to the ones obtained in the lead lab. The IC50 and
ID50 values were the highest for ascorbic acid followed by boric acid and then by hydroxyurea as
observed in lab A and the lead laboratory.
Table 7 Results on Lab Ba in phase 0 study ( technical transfer confirmation study)
Test chemicals

IC50
Meanb ± SD (μg/ml)

Ascorbic acid

ID50
CV (%)

Meanb ± SD (μg/ml)

CV (%)

552.29 ± 153.85

28932.86 ± 116.26

12

Boric acid58.80 ±

4.10

7

87.61 ± 6.51

7

Hydroxyurea

4.44 ±

0.33

7

3.47 ± 0.86

25

Lab B: KAOaMean values of three definitive studies.

b

3.2.4.4 Lab C
Table 8 shows the data obtained in phase 0 for lab B for three different test compounds. The IC50 and
ID50 values obtained for the boric acid and hydroxyurea tested in three definitive studies revealed avery low variance (CV<27%). However, the CV values obtained for ascorbic acid were much higher(73% for IC50 and 87% for ID50) and the IC50 and ID50 values obtained were three to four times
lower than the one obtained in the lead lab. All of the other data are very consistent and the IC50 and
ID50 values are close to the ones obtained in the lead lab. The IC50 and ID50 values were the highest
for ascorbic acid followed by boric acid and then by hydroxyurea as observed in lab A, B and thelead lab.
Table 8 Results on LabCa in phase 0 study (technical transfer confirmation study)
Test chemicals

IC50
Meanb ± SD (μg/ml)

Ascorbic acid

ID50
CV (%)

Meanb ± SD (μg/ml)

CV (%)

212.79 ± 155.62

73

178.78 ± 154.73

87

Boric acid

88.22 ±

3.33

4

109.35 ± 6.79

6Hydroxyurea

5.32 ±

1.43

27

5.54 ± 0.45

8

Lab C: SCASaMean values of three definitive studies.

b31

3.2.5 Conclusion
Except for ascorbic acid, the data of the two chemicals were close with a very low variance. Lab C,
with ascorbic acid, obtained data with a bit higher variance and IC50 and ID50 values were a bit lower
than the ones obtained in lab A and B. Given the fact that the historical data of the lead lab (see Table2) for this chemical show a high variance, then it may be possible this variance is due to the intrinsic
properties of the chemical.
Phase 0 showed a successful transferability of the protocol in the three participant labs with highly
reproducible results obtained with the test and phase 1 was started.
3.3

Phase 1 study

Phase 1 study began in November 2013 and ended in March 2014.
3.3.1

Goal

The goal of phase 1 was to verify the within-laboratory reproducibility.
3.3.2

Study design

All chemicals were tested in three sets, a set consisting in one dose finding study (DF) and one
definitive study (DS) to test the within-laboratory reproducibility of Hand1-Luc EST. The DF
consists in testing the chemical with a 10 dilution ratio to find the range where both IC50 and ID50 aresituated. The DS is then implemented with a lower dilution ratio within the range defined by the DF
to improve accuracy of the results.
3.3.2.1 Protocol
The protocol version used for this phase was version 05-1E (see Appendix 29). The incubation timeof the cells for differentiation was changed from 6 days to 120 hr (5 days). The reason for thischange is explained in detailed in Appendix 30. The schematic overview is presented in Figure 6.
After having obtained the IC50 and ID50 values, the positivity or the negativity of chemicals wascalculated thanks to the prediction model detailed in part 3.3.2.2. The condition for the success ofphase 1 is a between-laboratory reproducibility higher than 75% as fixed in the study plan (seeAppendix 31).

32

Hand1-ES(KOB1) cells
Test compoundsDay0120 hr(Day5)

white U96 well plate
Cell viability assay(CellTiter Fluor)
Luc-activity(Steady-Glo)

IC50(Cytotoxicity）
ID50(Differentiation toxicity)

Figure 6 Schematic overview of the modified new protocol
3.3.2.2 Data analysis
IC50 and ID50 values were calculated by the method described in 3.1.2.2 Study design (Dataanalysis).
The prediction model for phase 1 was constructed based on the data of twenty-one chemicals
obtained in the lead lab (each chemical tested three times independently) (boric acid, carbamazepine,
cefotaxime, chlorthalidone, cytosine arabinoside, dimethadione, diphenylhydantoin, glyburide,hydrochlorothiazide, indomethacin, isoniazid, lithium chloride, methotrexate, penicillin G, saccharin
sodium salt, Sant-1, sulfasalazine, tomatidine hydrochloride, trazadone hydrochloride, valproic acid,warfarin) (Figure 7).
The general concept of the prediction consisted in using both IC50 and ID50 along with watersolubility. The first two parameters (IC50 and ID50) were determined by the Hand1-Luc EST and thewater solubility by a free software called EPIsuite. Since the participating labs were not able to get
the water solubility values (software not provided), those values were given by the lead lab to thebiostatisticians so the calculation for prediction can be made. The model was based on two notions:- The larger is the gap between the IC50 and the ID50, the higher is the potential for thechemical to be embryotoxic (while the chemical does not kill the cells, there is an effect on Hand1
gene that would lead to embryotoxicity).
- The larger is the gap between the ID50 and the water solubility, the higher is the potential
to be embryotoxic (if the chemical is highly soluble then it will have more chances to reach the fetus
cells and have an effect).
Discriminant equation is as follows:
Score = 1.279 × log (IC50/ID50) + 0.33 × log (WSol/ID50) - 0.67
33

Probability was calculated as an inverse logit transform of the score. When the probability shows
more than 0.5, the chemical is evaluated as “Positive”. When the probability shows less than 0.5, thechemical is evaluated as “Negative”. The fifty percent line was decided because this was the best
cut-off found to separate the in vivo positive and in vivo negative chemicals (Figure 7).

50% line
Probability
High

Low

Figure 7 Development of equation for prediction model for phase 1
3.3.3 Chemicals
The chemicals used for phase 1 are presented in Table 9.
Table 9 Coded test chemicals used for phase 1 study
Set

1

2

3

Code No.

Test Chemicals

SCAS

KAO

FDSC

HA01

HB02

HC03

HA02

HB03

HA03

HB01

HA06
HA04

CAS

SupplierNo.

PhysicalSolvent

property

6-Aminonicotinamide

329-89-5

TCI

Solid

HC01

Isoniazid

54-85-3

Sigma(Fluka)

HC02

5,5-Diphenylhydantoin

57-41-0

Wako

HB04

HC05

6-Aminonicotinamide

329-89-5

HB05

HC06

Isoniazid

54-85-3

HA05

HB06

HC04

5,5-Diphenylhydantoin

57-41-0

HA08

HB09

HC07

6-Aminonicotinamide

329-89-5

HA09

HB07

HC08

Isoniazid

HA07

HB08

HC09

5,5-Diphenylhydantoin

In vivoclassification

DMSO

Positive

Solid

PBS

Negative

Solid

DMSO

Positive

TCI

Solid

DMSO

Positive

Sigma(Fluka)

Solid

PBS

Negative

Wako

Solid

DMSO

Positive

TCI

Solid

DMSO

Positive54-85-3

Sigma(Fluka)

Solid

PBS

Negative57-41-0

Wako

Solid

DMSO

Positive

3.3.4 Results
3.3.4.1 IC50 and ID50 values variation and final judgment
For each chemical, one dose finding study (DF) and one definitive study (DS) was applied.
34

6-Aminonicotinamide:
Table 10 shows the results of IC50 and ID50 values for 6-aminonicotinamide in phase 1. For eachstudies (dose finding and definitive), the values were very close to each other between the three sets.
This is supported by figure 8 where no dispersion was observed. Furthermore, the judgment was thesame for all the sets in every lab verifying again the within-laboratory reproducibility.
Table 10 Results of IC50 and ID50 values and final judgment obtained in each sets for
6-aminonicotinamide in phase 1
IC50 values (μg/ml)
Lab

A

B

C
a

Set No.

ID50 values (μg/ml)

Final

Dose finding

Definitive study

Dose finding

Definitive study

(DF)

(DS)

(DF)

(DS)

1

1.13

1.71

1.11

0.70

P

2

1.91

1.28

1.87

1.01

P

3

1.93

1.47

1.88

1.10

P

1

1.60

1.01

1.45

0.83

P

2

1.27

1.24

0.80

0.91

P

3

1.90

1.14

0.47

0.95

P

1

0.83

1.45

0.52

1.02

P

2

1.07

0.89

0.42

0.70

P

3

1.37

1.40

0.62

0.67

P

Judgment

Lab A: SCAS, Lab B: KAO, Lab C: FDSC

Figure 8 IC50 (circle) and ID50 (diamond) values obtained in the definitive study for
6-aminonicotinamide (Lab A: SCAS, Lab B: KAO, Lab C: FDSC)
35

Isoniazid:
Table 11 shows the results of IC50 and ID50 values for isoniazid in phase 1. The ID50 values of lab A
were much lower compared to the values of labs B and C (Figure 9) which led to get a positive final
judgment. However, the within-laboratory reproducibility was verified with each lab having thesame results for each set.
Table 11 Results of IC50 and ID50 values and final judgment obtained in each sets for isoniazid inphase 1
IC50 values (μg/ml)
Lab
A

B

C

a

Set No.

ID50 values (μg/ml)

Final

Dose finding

Definitive study

Dose finding

Definitive study

(DF)

(DS)

(DF)

(DS)

1

>500

291.36

28.90

37.99

P

2

314.83311.72

53.13

30.83

P

3

365.15564.78135.56<15.63P

1

108.54353.40149.59418.08N2207.74354.36190.42374.32

N

3

186.28325.12161.81390.01N

1282.90344.03324.28402.20N

2

254.14315.63414.69394.64N

3

266.80324.21484.44402.14N

judgment

Lab A: SCAS, Lab B: KAO, Lab C: FDSC

Figure 9 IC50 (circle) and ID50 (diamond) values obtained in the definitive study for isoniazid(Lab A: SCAS, Lab B: KAO, Lab C: FDSC).
36

As shown in Figure 9, the IC50 and the ID50 values did not vary between the sets in each laboratory
verifying the within-laboratory reproducibility. In the case of lab A, the differentiation toxicity isdifferent from the other labs, but the same type of curve was observed in each laboratory (Figure 10)
supporting the success of the within-laboratory reproducibility. The reason why the values of lab A
are much lower than the other labs could not be explained.
DS1

DS2

DS3

DS1

DS2

DS3

DS1

DS2

DS3

Figure 10 Differentiation toxicity curves obtained in each lab for three sets for isoniazid
5, 5-Diphenylhydantoin:
Table 12 shows the results of IC50 and ID50 values for 5, 5-diphenylhydantoin in phase 1. All the data
within each lab are close to each other and do not vary. However, there are two cases where the IC50
value was very low compared to the other runs (see also Figure 11). This was observed for the 3rd setof Lab B (10.96 μg/ml) and the 1st set of lab C (34.95 μg/ml). These values are explained in figure
12. Indeed, the cytotoxicity curves, with a V shape, crossed two times the 50% line and thus, a very
low IC50 value was triggered. When entering the data in the prediction model, a negative final
judgment for the 3rd set of lab B was obtained because the gap between the IC50 and the ID50 was
reduced. A positive result was still triggered for the 1st set of lab C because of a slight larger gap
between the IC50 and ID50 values compared to the 3rd set of lab B. Although the curves generated
very low IC50, when looking at the shape of the curve, they are the same in each laboratory (Figure12). This supports again the success of the within-laboratory reproducibility although the judgment
between set 3 and sets 1/2 for Lab B was different.

37

Table 12 Results of IC50 and ID50 values and final judgment obtained in each sets for 5,
5-Diphenylhydantoin in phase 1
IC50 values (μg/ml)
Lab

A

B

C

a

Set No.

ID50 values (μg/ml)

Final
judgment

Dose finding

Definitive study

Dose finding

Definitive study

(DF)

(DS)

(DF)

(DS)

1

>250

>250

6.79

2.46

P

2

>250

>250

1.38

3.63

P

3

>250

>250

8.25

2.37

P

1

>500

>500

27.03

32.72

P

2

>500

>500

9.33

8.67

P

3

>500

10.96

12.87

9.69

N

1

>500

34.85

5.48

8.59

P

2

>500

>500

4.73

8.74

P

3

>500

>500

8.42

4.98

P

Lab A: SCAS, Lab B: KAO, Lab C: FDSC

Figure 11 IC50 (circle) and ID50 (diamond) values obtained in the definitive study for
5, 5-diphenylhydantoin (Lab A: SCAS, Lab B: KAO, Lab C: FDSC).

38

Figure 12 Cytotoxicity curves obtained in each lab for 3 sets with diphenylhydantoin(Lab A: SCAS, Lab B: KAO, Lab C: FDSC)
3.3.4.2 Summary of within-laboratory reproducibility
The summary of the final judgment is shown in Table 13.
Table 13

Summary of the judgment obtained for each chemical in each laboratory results ofphase 1 study

Laboratorya

A

B

C
a

Chemicals

Set No.

Final

In vivo1

2

3

judgment

classification

6-Aminononicotinamide

P

P

P

P

P

Isoniazid

P

P

P

P

N

5, 5-Diphenylhydantoin

P

P

P

P

P

6-Aminononicotinamide

P

P

P

P

P

Isoniazid

N

N

N

N

N

5, 5-Diphenylhydantoin

P

P

N

P

P

6-Aminononicotinamide

P

P

P

P

P

Isoniazid

N

N

N

N

N

5, 5-Diphenylhydantoin

P

P

P

P

P

Lab A: SCAS, Lab B: KAO, Lab C: FDSC

The obtained within-laboratory reproducibility was 89% (8/9 chemicals).
The within-laboratory reproducibility of phase 1 satisfied then the criterion of 75% fixed in the study
39

plan (see Appendix 31). For two chemicals, Isoniazid and 6-aminonicotinamide all the labs obtained
the same results for all the three sets. However, for 5,5 diphenylhydantoin, lab B did not satisfy the
reproducibility (reason explained before).
3.3.5 Conclusion
All the results obtained in phase 1 show highly reproducible results. The cases where the results
differ are due to curves with a V shape (Figure 12). Furthermore, there were cases where
precipitation of the chemical at the highest concentration led to curves with shapes as shown in
Figure 13 (next part). Those problems were discussed and solutions were provided for the next phase.
The within-laboratory reproducibility (89%) of phase 1 satisfied the criterion of 75% fixed in the
study plan (see Appendix 31). Given those good results, the validation study with phase 2 was set inmotion.

40

4

Validation study

The validation study consisted in phases 2 and 3.
Phase 2 was separated into three subphases, phase 2a, 2b and 2c. Phase 2a began in March 2014 andended in May 2014.
4.1
4.1.1

Phase 2a study
Goal

Phase 2a was established to verify the between-laboratory reproducibility with four coded chemicals.
4.1.2

Study design

For this phase, each chemical was tested in only one set composed of one dose finding study and two
definitive studies. The dose finding study was taken into account for the judgment.
In phase 1, some curves had a V shape leading to truncated IC50 or ID50 value (see figure 12). Thus,
the curves were fitted with a two parameter curve fitting to avoid this case. This fitting would also
erase the problem of curve rising from the concentration 0 and then decreasing along with theincreasing concentration of chemicals (see Figure 10, lab C). The prediction model was revised
according to this improvement (see part 4.1.2.2).
Finally, during phase 1, there were some cases of chemical precipitation that truncated the curves(Figure 13).

Figure 13 Differentiation toxicity curve obtained for 5, 5-diphenylhydantoin.
In this case, there are chances that the chemical precipitated on the tips or the chemical was not41

correctly pipetted, leading to the concentration to be lower than expected triggering a non-toxic
result at this concentration. To avoid precipitation, the protocol was improved by implementing the
precipitation evaluation and the setting of the maximum dose (MD).
4.1.2.1 Protocol
Thanks to the pre-validation process, the protocol set until phase 1 has been improved. The protocol
version used for this phase was the 06E version. The detailed version can be found in Appendix 32.
Hand1-Luc EST consists in using mouse ES cells stably transformed with a vector containing theluciferase gene governed by the Hand1 gene promoter and make them differentiate into
cardiomyocytes for 120 hr (5 days). The chemical is added to the cells from the beginning of thedifferentiation (day 0). After 120 hr (5 days) of incubation, cytotoxicity (measured with
CellTiter-Fluor™ Cell Viability Assay, IC50) and differentiation toxicity (Hand1 gene expression
inhibition measured with Steady-Glo® Luciferase Assay System through luciferase activity, ID50) aremeasured. The highest concentration of chemical tested is the one for the chemical dissolves in theassay medium and is defined as the maximum dose (MD). IC50, ID50 and MD are used for theprediction model to determine the positivity or the negativity of the chemical (see Figure 14).

Figure 14 Outline of the Hand1-Luc EST

42

4.1.2.1.1

Preparation

Cells
Transgenic mouse embryonic stem (ES) cell, named as Hand1-ES (KOB1), containing firefly
luciferase gene under the control of Hand1 (heart and neural crest derivatives expressed transcript 1)
promoter were established by SC as a previous method (Suzuki et al., 2011b) with minor
modifications. An embryonic stem cell line (named as KOB1-ES) from C57/B6 mouse establishedby SC was used for development of the Hand1-ES (KOB1) cells. For the validation, the cells usedwere provided by the lead lab from frozen tubes.
Materials
Two different media are used for the Hand1-Luc EST. The first one is the maintenance medium used
for cell passage (Table 14) where the cells are maintained in an undifferentiated state. The second
one is the assay medium (Table 15) used for the assay when the cells are differentiated into
cardiomyocytes.
Table 14 Composition of the maintenance medium
Content

VolumeStemMedium

10 mL

100mM 2-ME solution

10 mL

ESGRO (107unit/mL)

1 mL

50 mg/mL G418 solution

20 mL

Table 15 Composition of the assay medium
Content

Volume

DMEM

82 mL

FBS (Heat inactivated)

15 mL

GlutaMaxI solution (100× conc.)

1 mL

100mM NEAA solution

1 mL

Penicillin/Streptomycin solution

1 mL

100mM 2-ME solution

100 mLAll of the necessary ingredients for the assay were distributed to the participant laboratories by thelead lab before each phase.
Materials are detailed in the protocol that can be found in Appendix 32.

43Maintenance of cells
Hand1-ES (KOB1) cells are thawed and seeded in 6 cm diameter gelatin coated plates and arepassaged every 2 or 3 days. The day before each assay, cells are passaged one more time and mustreach confluence the day of the experiment (80 to 90%). As a rule, cells must be used within two
passages.
4.1.2.1.2

Hand1-Luc EST assay

Cell preparation
The assay is done in a 96 round bottom well white plate to allow cells to aggregate and form
embryoid bodies autonomously. The design of the plate is shown in Figure 15.

MC: medium Control
VC: Vehicle Control
Column 11: BG: BackgroundFigure 15 Hand1-Luc EST’s plate design
The cells (750 per well) are seeded. The cell viability must be higher or equal to 90%. MC consists
in medium and cells only, VC consists in cells, assay medium and solvent (0.1% for DMSO and 1%
for PBS) and BG consists in medium only. All those three columns are used for the final calculation
to obtain IC50 and ID50 values but also to set acceptance criteria to evaluate the quality of theexperiment. From column 10 to 4, a gradient of chemical is prepared according to the dilution ratio
decided. Column 10 is the column with the highest chemical concentration and column 4 with thelowest.
Preparation of test chemicals and maximum dose (MD) setting
Chemicals are first dissolved in PBS or DMSO according to the step described in Figure 16.
44

Figure 16 Vehicle selection and preparation of test chemicals
If the chemical does not dissolve in PBS(-) until a concentration of 25 mg/ml then DMSO is used todissolve the chemical until complete dissolution. Once the chemical is dissolved, the precipitation
test is done. It consists in adding 1 μl of chemical in 999 μl of assay medium and evaluate if
precipitation occurs. If not, the concentration is determined as the maximum one tested for the assay.
If it precipitates, then the concentration is divided by 2 in the solvent and the precipitation evaluationin assay medium is done again. This is repeated until the chemical dissolves. All the steps aredescribed in Figure 16. The maximum dose (MD) is therefore defined as the highest dissolvable
concentration.
Chemical dilution for dose finding and definitive studies
Chemicals, after being dissolved in the appropriate solvent are diluted in a 96 well storage roundbottom plate from the maximum dose determined as described previously. The chemicals are addedin the assay medium (20 μl in 980 μl of assay medium if the chemical is dissolved in PBS (-) (1%v/v) and 2 μl in 998 μl of assay medium if the chemical is dissolved in DMSO (0.1%)). For the dose
finding study, used to determine the concentration range for the one both IC50 and ID50 are
45

observable, a 10 dilution ratio is systematically applied.
After obtaining the results, the dilution ratio is lowered to get more accurate values of the IC50 and
ID50.
Measurement of cytotoxicity and differentiation toxicity
Chemicals can be added to the cells between 2 hrs and 4 hrs after seeding (50 μl of medium
containing chemicals added to the already seeded 50 μl of cells. The final volume in each well is 100
μl/well). The cells are incubated for 120 hr (5 days) at 37ºC, 5% CO2.
After 120 hr (5 days) of incubation, cytotoxicity is measured followed by differentiation toxicity.
Cytotoxicity is expressed as the concentration of chemicals that reduced the viability of cells to 50%of the control level against the vehicle control. The reagent used to determine the value is
CellTiter-Fluor® Cell Viability Assay. The principle of this product is the following: Thecell-permeant substrate enters the cell, where it is cleaved by the live-cell protease activity to
produce the fluorescent AFC. The live-cell protease is labile in membrane-compromised cells andcannot cleave the substrate. After having determined cytotoxicity, differentiation toxicity is measured
in the same plate by adding 100 μl of Steady-Glo® Luciferase Assay System. This product contains
beetle luciferin which is cleaved by the firefly luciferase express by the cells which produces
oxyluciferin being luminescent. The inhibition of differentiation was expressed as the concentrationof the test chemical that reduces the luminescence by 50%.
Estimation of IC50 and ID50 values
The algorithm for the estimation of IC50 and ID50 values was implemented in a developed Microsoft
Excel data sheet through all phases. An example of the data sheet used in this validation study isshown in Appendix 33. The data sheet files in all phases remain separated into three parts: the facecover sheet, the calculation sheets and the summary sheet. Once a user enters data into the face coversheet, the IC50 and ID50 values are estimated with the concentration-response plots and theacceptance criteria are also calculated in the calculation sheets. Finally, the summarized resultsappear in the summary sheet. IC50 and ID50 values were estimated by fitting the curve to these data
(two parameter fitting)
Two parameter curve fitting:Phase 1 showed some cases where the IC50 was lower than the ID50. Thus, to correct all those
problems, a curve fitting method was proposed by VMT members.
Two parameter curve fitting was implemented in the calculation sheet by Dr. Omori, biostatistician.
Figure 17 shows that the new IC50 and ID50 obtained after curve fitting is not significantly different
from the non-fitted data. Furthermore, thanks to this fitting, the IC50 and ID50 relationship was46

improved and the cases where the curve crossed the 50 % line (V curve) were no longer possible.

Figure 17 Plotting of the IC50 and ID50 value obtained in both way of calculating(with or without curve fitting)
4.1.2.2 Prediction model
In addition to the implementation of the curve fitting, the evaluation of precipitation was included in
the protocol. Those two significant changes led to the revision of the prediction model because theIC50 and ID50 values are slightly different and the MD (maximum dose) is used for the model. The
Figure 18 shows the new prediction model which was established by the analysis of the previously
used twenty-one chemicals data revised with their respective MD, IC50 and ID50 values with the new
two parameters curve fitting. The equation is as follows:
Score = 1.252 x log(IC50/ID50)+ 0.548 x log(MD/ID50) – 0.80547And the probability is obtained by applying the inverse logit to the score.
36%
line

Sensitivity

ROC curve

62% line

Positive(P) area

Probability: 36%

Probability: 62%
1-Specificity

Negative(N)area

Equivocal (E)area

Figure 18 Development of equation for prediction model for phase 2a
The ROC curve showed cut-off criteria at 36% and 62% and the chemical with a probability lower
than 0.36 are considered negative. Chemicals with a probability situated between 0.36 and 0.62 areconsidered as equivocal and the chemicals with a probability higher than 0.62 are considered
positive. After discussion about the prediction model, the chemicals situated in the equivocal area
were determined as positive because this led to a higher predictive power for positive chemicals anda better accuracy when comparing to the in vivo results.
4.1.2.3 Positive and negative judgment
The positivity or the negativity of the chemical was decided with one dose finding study (DF) and 2
definitive studies (DS). The dose finding study result was used for the final judgment as shown in
the table below (Table 16).

48

Table 16

Majority judgment and number of studies needed according to the different possibilities
for phase 1
Dose finding study

Definitive study 1

Definitive study 2

Final judgment

Case 1N

N

N

N

Case 2N

N

P

N

Case 3

N

P

N

N

Case 4P

N

N

N

Case 5PP

P

P

Case 6P

P

N

P

Case 7P

N

P

P

Case 8N

P

P

P

4.1.2.4 Acceptance criteria
Acceptance criteria were established for i) quality control of cell condition, ii) quality control ofdifferentiation and cytotoxicity assays, iii) performance standard of the assay and iv) quality control
for the effect of the vehicle (PBS(-) or DMSO). The criteria described in the protocol are thefollowings:
Quality controli) Quality control of cell condition
Check viability of the cells by staining an aliquot of the cell suspension with Trypan
blue. A viability of 90% or greater is acceptable.
ii) Quality control of differentiation and cytotoxicity assays
To verify the cell growth and cell differentiation, the comparison of the medium
control (MC) and background (BG) is used.
- The lower limit of the 95% confidence intervals of the ratio of MC / BG should beabove 1 for the cytotoxicity assay- The lower limit of the 95% confidence intervals of the ratio of MC / BG should beabove 10 for the differentiation assayiii) Performance standard of the assay
The quality of the assay must be controlled using 5-FU as a positive-reference
chemical.
- The range of ID50 for 5-FU should be within 0.003 and 0.067 μg/mL(according to phase 0 study).
- The range of IC50 for 5-FU should be within 0.003 and 0.065 μg/mL(according to phase 0 study).
iv) Quality control for effect of vehicle49

To verify the effect of vehicles, the comparison of the medium control (MC) andvehicle control (VC) is used.
- The lower limit of the 95% confidence intervals of the ratio of VC / MC shouldbe above 0.2 for the cytotoxicity and differentiation assays.
- In addition to the above criterion
CV (SD/mean x 100%) of VC should be below 100%.
4.1.3. Chemicals
Information about the coded chemicals used for phase 2a is shown in Table 17.
Table 17 Coded chemicals used for phase 2aNo

Code No.
FDSC

SCAS

KAO

1

HA11

HB19

HC27

2

HA12

HB20

HC28

3

HA13

HB21

HC29

4

HA14

HB22

HC30

Chemical name
Cytosinearabinoside
Lithium Chloride
Cefotaxime sodium
salt
Penicillin G

CAS
No.

Supplier

Physicalproperty

Solvent

In vivoclassification

69-74-9

Sigma

Solid

PBS

Positive

7447-41-8

Wako

Solid

PBS

Positive

64485-93-4

TCI

Solid

PBS

Negative69-57-8

TCI

Solid

PBS

Negative

4.1.4 Results
4.1.4.1 IC50 and ID50 values variation and final judgment
The results are presented for each chemical.
Cytosine arabinoside
Table 18 shows the results obtained with cytosine arabinoside during phase 2a.

50

Table 18 Results obtained with cytosine arabinoside during phase 2a showing the IC50 and ID50
values with the final judgment obtained for the DF and the DS studies
Chemical nameLab

A

Cytosinearabinoside

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DF

0.02

0.03

P

DS1

0.03

0.04

P

DS2

0.05

0.05

P

DF

0.06

0.03

P

DS1

0.44

0.71

P

DS2

0.41

0.43

P

DF

0.13

0.03

P

DS1

0.03

0.03

P

DS2

0.04

0.04

P

P

P

P

The IC50 and ID50 values are close to each other in each laboratory. However, higher values for lab B
were found, around 10 times higher than the other labs. Each lab obtained a positive result for thechemical showing the between-laboratory reproducibility to be verified.
Lithium Chloride:
Table 19 shows the results obtained with lithium chloride during phase 2a.
Table 19 Results obtained with lithium chloride during phase 2a showing the IC50 and ID50 values
with the final judgment obtained for the DF and the DS studies
Chemical nameLab

A

Lithium chloride

B

C

Final

judgment

judgment

IC50 value (μg/ml)

ID50 value (μg/ml)

DF106.12

246.71NDS1569.71454.90P(E)aDS2

522.23455.71P(E)a

DF

214.87252.91

P(E)aDS1

640.11295.99P(E)aDS2

450.04276.00P(E)aDF

459.26228.60P(E)aDS1

493.04318.69P(E)a310.24

P(E)a

DS2
a

Runs

Run

471.03

PPP

P(E) is used to show the chemical that is in the equivocal area (see Figure 19) but is considered as positive.

As shown in Table 19, all the labs for each run obtained the same positive judgment except for DF of
Lab A where the IC50 value (106.12 μg/ml) is lower than the values obtained in the other labs for the
DF studies. The difference is due to the fact that those dose finding studies are done with a 1051

dilution ratio (to find the range of the IC50 and ID50). This triggers less accurate results than the DS
studies and thus tends to show variation. The curves obtained have the same shape for all the labs
and thus the between-laboratory reproducibility has been verified (see Figure 19).

IC50=106.12 μg/ml

IC50=214.87 μg/ml

IC50=459.26 μg/ml

Figure 19 Dose finding studies in the three participant laboratories for lithium chloride
Cefotaxime sodium salt
Table 20 shows the results obtained with cefotaxime sodium salt during phase 2a.
Table 20 Results obtained with cefotaxime sodium salt during phase 2a showing the IC50 and ID50
values with the final judgment obtained for the DF and the DS studies
Chemical nameLab

A

Cefotaxime
sodium salt

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DF

55.64

228.56

NDS1

256.18332.07NDS2

280.23320.37

N

DF

51.77

358.95

NDS1

353.57570.78NDS2

342.07621.18NDF155.03294.18NDS1

221.23521.54

N

DS2

49.95

370.29N

N

N

N

The definitive studies for cefotaxime sodium salt reveal high reproducibility between the labs.
However, the DS2 of lab C the IC50 value was much lower. This is explained in Figure 21.
At the concentration 2 of chemical, the counts in the very lower well (743.419) were much lower
than the other well (~1500) of the same concentration of chemical (Figure 20A). This made thecytotoxicity curve decreasing at the lowest concentrations and thus triggering a very low IC50 valueof 49.95 μg/ml (Figure 20B).
All the laboratories for each run obtained the same judgment (N) confirming the between-laboratory
reproducibility.
52

Figure 20 Data obtained with Cefotaxime sodium salt in lab C during DS2
Penicillin G
Table 21 shows the results obtained with penicillin G during phase 2a.
Table 21

Results obtained with penicillin G during phase 2a showing the IC50 and ID50 values
with the final judgment obtained for the DF and the DS studies

Chemical nameLab

A

Penicillin G

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DF

>1000

>1000

N

DS1

942.75

>1000

N

DS2

>1000

>1000

N

DF

>1000

>1000

N

DS1

>1000

>1000

N

DS2

>1000

>1000

N

DF

>1000

>1000

N

DS1

902.64

>1000

N

DS2

>1000

>1000

N

N

N

N

Penicillin G showed highly reproducible results (IC50 and ID50 values and the judgment) for both DF
and DS studies in the three participating lab confirming the between-laboratory reproducibility.
Furthermore, the judgment was the same for all the laboratories supporting the between-laboratory
reproducibility.

53

4.1.4.2 Summary of the between-laboratory reproducibility
The result of the between-laboratory reproducibility is shown in Table 22.

Chemical name
Cytosine arabinoside

Laboratory
A

B

C

P

P

P

Lithium chloride

P

P

P

Cefotaxime sodium salt

N

N

N

Penicillin G

N

N

N

Table 22

Probability values obtained for each chemical of phase 2a and their classification

As shown in table 22, the between-laboratory reproducibility was verified for all the chemicals
where all the labs managed to have the same classification (4/4 = 100%).
4.1.5 Conclusion
The new prediction model and the improvement of the protocol along with the acceptance criteria
showed high between-laboratory reproducibility (100%) with a low variability between the labs
concerning the IC50 and ID50 values along with a correct classification of the chemicals.
The next phase was then launched given the good results.
4.2 Phase 2b/2c studies
The second part of phase 2 was again divided into two: phases 2b and 2c. Phase 2b was implemented
between May and September 2014 followed by phase 2c from September 2014 to February 2015.
The data presented below show the mix of both phases 2b and 2c (2b/2c).
The protocol version used for this phase was the 07E version (see Appendix 34).
4.2.1

Goal

The goal of phase 2b/2c was to verify the within- and the between-laboratory reproducibility of the
protocol with more chemicals than the previous phase (eight chemicals).
4.2.2

Study design

The prediction model used for phases 2b/2c is the same as the one used for phase 2a. The curve
54

fitting used to determine IC50 and ID50 value is the two parameter curve fitting.
For each chemical, one dose finding study and two or three definitive studies were applied. However,from this phase the dose finding study is not taken into account for the final judgment. The majority
judgment and its possibilities are shown in Table 23.
Table 23

Majority judgment applied from phase 2b, 2c detailing the possible cases obtainable
Dose finding study

Definitive study

Definitive study

Definitive study

1

2

3

Final judgment

Case 1

-

P

P

-

P

Case 2

-

N

P

P

P

Case 3

-

N

P

N

N

Case 4

-

N

N

-

N

Case 5

-

P

N

P

P

Case 6

-

P

N

N

N

To verify the within-laboratory reproducibility, each set (DF+ two or three DS) was repeated threetimes.
4.2.3 Chemicals
The chemicals used in phases 2b/2c and their information is shown in Table 24.

55

Table 24
Set

5

6

7

8

9

10

11

12

Code No.

Information of chemicals used in phases 2b/2c
Test chemicals

FDSC

SCAS

KAO

HA101

HB106

HC208

HA110

HB206

HC105

HA201

HB109

HC108

HA102

HB101

HC211

HA111

HB209

HC106

HA204

HB110

HC109

HA103

HB212

HC101

HA207

HB102

HC110

HA112

HB111

HC203

HA104

HB201

HC102

HA210

HB103

HC111

HA107

HB112

HC206

HA202

HB104

HC103

HA105

HB107

HC209

HA108

HB204

HC112

HA205

HB105

HC104

HA106

HB108

HC212

HA109

HB207

HC107

HA208

HB210

HC201

HA211

HB202

HC204

HA203

HB205

HC207

HA206

HB208

HC210

HA209

HB211

HC202

HA212

HB203

HC205

CAS

SupplierNo.

PhysicalSolvent

property

In vivoclassification

Caffeine

59-08-2

Sigma

Solid

PBS

P

Glibenclamide

10238-21-8

Sigma

Solid

DMSO

N

Acetazolamide

59-66-5

Sigma

Solid

DMSO

P

Valproic acid1069-66-5

Sigma

Solid

PBS

P

82385-42-0

Sigma

Solid

PBS

N

(+)-Camphor

464-49-3

TCI

Solid

DMSO

N

Ascorbic acid50-81-7

Sigma

Solid

PBS

N

BrdU

59-14-3

TCI

Solid

DMSO

P

Saccharin sodium
salt

4.2.4 Results
4.2.4.1 IC50 and ID50 values variation and final judgment
The results are presented for each chemical.
Caffeine
Table 25 shows the results obtained with caffeine during phase 2b/2c in the three participating labs.

56

Table 25. IC50 and ID50 values and the judgment obtained during phase 2b/2c with caffeine(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Test chemical

Lab

Set
1

A

2
3
1

Caffeine

B

2

3

1
C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

> 125

124.48

DS2

> 125

> 125

DS1

175.0989.74

DS2

158.83

80.00

DS1

194.3

100.63DS2

210.92105.44

DS1

> 125

> 125

DS2

> 125

> 125

DS1

> 250

163.32

DS2

> 250

> 250

DS3

> 250

> 250

DS1

> 125

> 125

DS2

> 125

92.17

DS3

> 125

112.27DS1> 250

127.88DS2> 250

144.08

DS1

> 7.8

> 7.8

DS2

> 7.8

> 7.8

DS1

> 62.5> 62.5

DS2> 62.5> 62.5Set Judgment

aFinal judgment

N
P

P

P
N

N

N

N

P
N

N

N

a. Judgment decided with the majority judgment of the 3 sets

Within-laboratory reproducibility (see Table 25, set judgment)
The within-laboratory reproducibility has been verified only for lab B where all the IC50 and ID50
values are close to each other in each set. However, the first set of lab A showed a negative result
while the two other sets give a positive result. This is due to a different MD chosen. Indeed set 2 and3 have an MD set at 250 μg/ml but the MD of the first set was determined at 125 μg/ml. In this caseonly, set 2 and set 3 managed to reach the cytotoxic dose and to trigger an IC50. When entering thevalues in the prediction model, set 2 and set 3 give a positive prediction because the gap between the
IC50 and the ID50 is higher than in set 1 which triggered a negative prediction.
For the same reason (MD determination), the first set of lab C triggered a positive prediction while
the two other set triggered a negative prediction for caffeine.
Between-laboratory reproducibility (see Table 25, final judgment)
For the same reason as mentioned above (MD determination), the between-laboratory reproducibility
was not satisfied for caffeine due to the positive prediction obtained for Lab A while the two other57

laboratories got a negative result.
Glibenclamide
Table 26 shows the results obtained with glibenclamide during phase 2b/2c in the three participatinglabs.
Table 26.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with glibenclamide(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2
3
1

Glibenclamide

B

2
3
1

C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

86.78

31.69

DS2

91.77

18.35

DS1

57.31

17.98

DS2

36.16

25.13

DS1

> 62.5

23.69

DS2> 62.5

24.47

DS1

91.32

18.81

DS2

100.04

31.93

DS1

> 125

10.62

DS2

> 125

31.98

DS1

> 250

17.09

DS2

23.45

2.58

DS1

> 125

12.55

DS2

64.34

32.26

DS1

> 62.5

33.01

DS2> 62.5

22.46

DS1

87.81

46.79

DS2

89.80

38.32

Set Judgment

aFinal judgment

P
P

P

P
P
P

P

P
P
P

P

P

a. Judgment decided with the majority judgment of the 3 sets

Within-laboratory reproducibility (see Table 26, set judgment)
Each lab obtained the same prediction (positive) in each set confirming the reproducibility of theresults within the labs. The ID50 values did not vary between the studies. However, the IC50 values
showed a higher variance between the studies which is due to different MD chosen. Furthermore, inthe case of the third set of lab B DS2, the IC50 value is much lower than the other ones (23.45
μg/ml). This is due to a dilution ratio of 10 that was used during the experiment greatly lowering theaccuracy of the results. The reason why no cytotoxicity was observed for the third set of lab B DS1
could not be explained.

58

Between-laboratory reproducibility (see Table 26, final judgment)
Each lab obtained the same prediction (positive) confirming the reproducibility of the results
between the labs.
Acetazolamide
Table 27 shows the results obtained with acetazolamide during phase 2b/2c in the three participatinglabs.
Table 27.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with acetazolamide(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2
3

1
Acetazolamide

B

2
3
1

C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

322.52

27.77

DS2

> 500

17.99

DS1

124.39

24.40

DS2

88.97

11.95

DS1

> 250

24.72

DS2

> 250

20.22

DS1

> 25

>25

DS2

> 25

0.23

DS3

>25

4.49

DS1

> 250

1.37

DS2

> 250

1.09

DS1

> 250

2.01

DS2

> 250

1.46

DS1

> 250

8.02

DS2

139.58

17.72

DS1

> 125

31.73

DS2

> 125

14.3

DS1

> 250

10.4

DS2

> 250

2.45

Set Judgment

aFinal judgment

P
P

P

P

P

P

P

P
P
P

P

P

a. Judgment decided with the majority judgment of the 3 sets

Within-laboratory reproducibility (see Table 27, set judgment)
Each lab obtained the same prediction (positive) in each set confirming the reproducibility of theresults within the labs. The ID50 values did not highly vary between the studies. However, for the
first step of Lab B, the IC50 is set at > 25 μg/ml. Although it did not affect the final judgment, thiscase is worth noticing. The reason is that the laboratory set the highest concentration tested at 25
μg/ml, maybe to try to reduce the concentration range to get a better accuracy of the results (due tothe very low ID50 values). However, since no cytotoxicity was triggered at 250 μg/ml (DF, data not59

shown), the highest concentration should not have been lowered.
Between-laboratory reproducibility (see Table 27, final judgment)
Each lab obtained the same prediction (positive) confirming the reproducibility of the results
between the labs.
Valproic acid
Table 28 shows the results obtained with valproic acid during phase 2b/2c in the three participatinglabs.
Table 28.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with valproic acid(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2

3

1
Valproic acid

B

2
3
1

C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

146.15

127.1

DS2

130.34122.84DS1

140.94110.5

DS2

149.78134.23

DS1

102.39236.78DS2

100.3

104.77DS3

116.86123.44DS1

156.5537.91

DS2

120.9665.89

DS1

161.85

27.54

DS2

126.1867.7

DS1

122.64

92.18

DS2

131.4886.1

DS1

119.8965.36

DS2

149.0139.58

DS1

156.87

66.43

DS2

103.9160.27

DS1128.93115.31

DS2

126.42

46.3

Set Judgment

aFinal judgment

P
P

P

P

P
P

P

P
P
P

P

P

a. Judgment decided with the majority judgment of the 3 sets

Within-laboratory reproducibility (see Table 28, set judgment)
Each lab obtained the same prediction (positive) in each set confirming the reproducibility of theresults within the labs. The IC50 and ID50 values did not vary between the studies.

60

Between-laboratory reproducibility (see Table 28, final judgment)
Each lab obtained the same prediction (positive) confirming the reproducibility of the results
between the labs.
Saccharin sodium salt
Table 29 shows the results obtained with saccharin sodium salt during phase 2b/2c in the threeparticipating labs.
Table 29.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with saccharin
sodium salt (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2
3
1

Saccharin
sodium salt

B

2
3
1

C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000> 1000DS1> 1000> 1000DS2> 1000a. Judgment decided with the majority judgment of the 3 sets

> 1000Set Judgment

aFinal judgment

N
N

N

N
N
N

N

N
N
N

N

N

Within-laboratory reproducibility (see Table 29, set judgment)
Each lab obtained the same prediction (negative) in each set confirming the reproducibility of theresults within the labs. The IC50 and ID50 values did not vary between the studies.
Between-laboratory reproducibility (see Table 29, final judgment)
Each lab obtained the same prediction (negative) confirming the reproducibility of the results
between the labs.
61(+)-Camphor
Table 30 shows the results obtained with (+)-camphor during phase 2b/2c in the three participatinglabs.
Table 30.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with (+)-camphor(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2
3
1

(+)-Camphor

B

2

3
1
C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

> 250

> 250

DS2

> 250

> 250

DS1

> 250

> 250

DS2

> 250

> 250

DS1

> 250

> 250

DS2

> 250

> 250

DS1

> 250

> 250

DS2

> 250

> 250

DS1
DS2

> 250
> 250

127.02
> 250

DS3

> 250

> 250

DS1

> 250

> 250

DS2

> 250

> 250

DS1

> 250

> 250

DS2

> 250

> 250

DS1

> 125

> 125

DS2

> 125

> 125

DS1

> 125

> 125

DS2

> 125
a. Judgment decided with the majority judgment of the 3 sets

> 125

Set Judgment

aFinal judgment

N
N

N

N
N

N

N

N
N
N

N

N

Within-laboratory reproducibility (see Table 30, set judgment)
Each lab obtained the same prediction (negative) in each set confirming the reproducibility of theresults within the labs. The IC50 and ID50 values did not vary between the studies. The maximum
dose chosen for lab C set 2 and 3 varies from the other studies and labs but this did not affect the
final judgment.
Between-laboratory reproducibility (see Table 30, final judgment)
Each lab obtained the same prediction (negative) confirming the reproducibility of the results
between the labs.
62

Ascorbic acid
Table 31 shows the results obtained with ascorbic acid during phase 2b, 2c in the three participatinglabs.
Table 31.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with ascorbic acid(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2
3
1

Ascorbic acid

B

2

3

1
C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

552.44216.15DS2

538.77470.80DS1

462.28548.47DS2

225.34630.78DS1

545.64681.89DS2

324.62597.13DS1

62.71

195.45

DS2

119.77293.70

DS1

74.55

60.26

DS2

13.52

18.39

DS1

630.97749.75DS2

848.43389.97DS3

736.1

621.97DS1

668.39445.86DS2

964.70255.66

DS1> 10009.13

DS2

359.14222.02

DS1> 100041.95

DS2770.06
a. Judgment decided with the majority judgment of the 3 sets

5.14

Set Judgment

aFinal judgment

P
N

N

N
N
P

N

N

P
P

P

P

Within-laboratory reproducibility (See table 31, set judgment)
The reproducibility has been verified in lab C where all the sets triggered the same judgment(positive).
However, for lab A and B, the results were different. For lab A, the first set gave a positive result
while the two other ones gave a negative result and in the case of lab B, the second set gave apositive result while the two other ones gave a negative result. Even when looking at the cytotoxicity
and differentiation toxicity curves, no peculiar reason could be found. However, when referring tophase 0 and the lead lab results, it may be possible that this chemical has a tendency to give resultswith a high variation. This may be due to the intrinsic properties of the chemical.

63

Between-laboratory reproducibility (See Table 31, final judgment)
The between-laboratory reproducibility could not be verified for ascorbic acid where lab C had adifferent result compared to the two other labs. The reason is the same as the one proposed in thewithin-laboratory reproducibility part.
BrdU
Table 32 shows the results obtained with BrdU during phase 2b/2c in the three participating labs.
Table 32.

IC50 and ID50 values and the judgment obtained during phase 2b/2c with BrdU
(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)

Test chemical

Lab

Set
1

A

2

3
1
BrdU

B

2
3
1

C

2
3

Run

IC50 (μg/ml)

ID50 (μg/ml)

DS1

0.41

0.28

DS2

0.38

0.3

DS1

0.35

0.34

DS2

> 0.5

0.21

DS3

0.35

0.24

DS1

0.44

0.44

DS2

0.25

0.27

DS1

0.22

0.53

DS2

0.19

0.11

DS1

0.33

0.3

DS2

0.49

0.53

DS1

0.26

0.34

DS2

0.29

0.87

DS1

0.04

0.37

DS2

0.32

0.14

DS1

0.51

0.13

DS2

0.69

0.37

DS1

1.09

0.25

DS2

0.54
a. Judgment decided with the majority judgment of the 3 sets

0.41

Set Judgment

aFinal judgment

P

P

P

P
P
P

P

P
P
P

P

P

Within-laboratory reproducibility (see Table 32, set judgment)
Each lab obtained the same prediction (positive) in each set confirming the reproducibility of theresults within the labs. The positivity result is due to the very low values of both IC50 and ID50 values.
The IC50 and ID50 values did not vary between the studies.

64

Between-laboratory reproducibility (see Table 32, final judgment)
Each lab obtained the same prediction (positive) confirming the reproducibility of the results
between the labs.
4.2.4.2 Summary of the between- and within-laboratory reproducibilities
Phase 2b/ 2c allowed to verify the within- and the between-laboratory reproducibility of the protocol.
The summary of the results is presented in Table 33.
Table 33

Judgment results in each lab along the sets for the eight chemicals tested in phases 2b/2c

Test chemical

Caffeine

Glibenclamide

Acetazolamide

Valproic acid

Saccharin
sodium salt(+)-camphor

Ascorbic acid

BrdU

Laboratory

Set

Concordance

Mean

1

2

3

between sets

A

N

P

P

2/3

P

B

N

N

N

3/3

N

C

P

N

N

2/3

N

A

P

P

P

3/3

P

B

P

P

P

3/3

P

C

P

P

P

3/3

P

A

P

P

P

3/3

P

B

P

P

P

3/3

P

C

P

P

P

3/3

P

A

P

P

P

3/3

P

B

P

P

P

3/3

P

C

P

P

P

3/3

P

A

N

N

N

3/3

N

B

N

N

N

3/3

N

C

N

N

N

3/3

N

A

N

N

N

3/3

N

B

N

N

N

3/3

N

C

N

N

N

3/3

N

A

P

N

N

2/3

N

B

N

P

N

2/3

N

C

P

P

P

3/3

P

A

P

P

P

3/3

P

B

P

P

P

3/3

P

C

P

P

P

3/3

P

Concerning the within-laboratory reproducibility, twenty coded chemicals out of twenty-foursatisfied the reproducibility (same judgment obtained three times for the same chemical) which65raises the within-laboratory reproducibility to 83.3%, satisfying the criterion fixed in the study plan(75%).
As for the between-laboratory reproducibility, it has been verified by compiling the results obtainedin phases 2a, 2b and 2c. During phase 2a, four chemicals out of four satisfied the between-laboratory
reproducibility (see part 4.1.4.2) and phase 2b/2c six out of eight which leads to a general
between-laboratory reproducibility of 10/12= 83.3% satisfying again the study plan’s criterion of
75%.
4.2.5 Conclusion
The revised prediction model and the improved the protocol showed high between- andwithin-laboratory reproducibilities (83.3%). There were still some concerns about the twoparameters curve fitting applied (where sometimes the IC50 value was lower than the ID50 one) and athree parameter curve fitting was advised at the end of phase 2c. The prediction model was revised(see 4.3.2.2) and phase 3 was then launched with sixteen other coded chemicals thanks to the goodresults obtained during phase 2.
4.3 Phase 3 study
Phase 3 study started in February 2015 and lasted one year.
4.3.1

Goal

The goal of phase 3 was to verify the between-laboratory reproducibility with the new prediction
model and the three parameter curve fitting analysis with sixteen coded chemicals (Table 34).
4.3.2

Study design

4.3.2.1 Protocol
The Hand1-Luc EST protocol used for this phase was the 08E version (see Appendix 35).
Each chemical was tested in one set. One set consisted in one dose finding study followed by two or
three definitive studies. The decision on positivity or negativity of the chemical was taken with the
majority judgment.

66

4.3.2.2 Data analysis
Three parameter curve fitting
The results of phase 2 showed that there were some cases where the IC50 remained lower than the
ID50 which led to think that the curve fitting method could be improved by implementing a threeparameter curve fitting method instead of a two parameter curve fitting method and use the raw data
instead of the normalized one. The data from phase 2a were then analyzed in two different ways. The
first one was to use the Graphpad PRISM software and the second one was to use a curve fitting
developed by Dr. Omori. PRISM software gave good results but for some cases, the software could
not fit the curves (data not shown). The three parameter curve fitting developed by Dr. Omori gave
good results and they were described in a teleconference call where the IC50 and ID50 relationshipwas improved. This is the method used for phase 3.
Prediction model
The prediction model was changed from phase 2 to phase 3 for two main reasons.
The first reason is that the IC50 and ID50 values used for the establishment of the model are changed
because of the new curve fitting. Furthermore, the lead lab had new chemicals data which led themodel to be also adapted. Indeed, the previous one was based on the data of twenty-one chemicals
but the new one is now based on seventy-one chemicals.
This prediction model is the last one and is the one to be proposed for validation. A more detailed
explanation of the prediction can be found in the published report (Nagahori et al, 2016). For a better
understanding the prediction model is shortly described below:
For the prediction, three parameters are used: the IC50, the ID50 and the MD (maximum dose). The
equation is as follows:
Score = 1.474 x log(IC50/ID50)+ 0.714 x log(MD/IC50) − 1.109
And the probability is obtained by applying the inverse logit to the score.

67

Figure 21 A: ROC curve; B: Ideal separation (0.52) between the in vivo positive and negative
chemicals
The data shown in Figure 21B are based on the data of seventy-one chemicals obtained in the leadlab (see Appendix 36).
The plot of sensitivity versus 1-Specifity is called receiver operating characteristic (ROC) curve.
This curve plays a central role in evaluating diagnostic ability of tests to discriminate the true state ofsubjects, finding the optimal cut off values, and comparing two alternative diagnostic tasks wheneach task is performed on the same subject (Karimollah Hajian-Tilaki, 2013).
The ROC curve (Figure 21A), allowed to fix a cut-off probability of 0.52 which was confirmed to be
the best one by placing the seventy-one chemicals in a graph with in abscissa Log (MD/IC50) and in
ordinate Log (IC50/ID50) (Figure 21B).
4.3.3 Chemicals
The sixteen chemicals used for phase 3 and their information are presented in Table 34.

68

Table 34 Information of chemicals used in phase 3
No

Code No.

CAS

Test chemical

SupplierNo.

PhysicalSolvent

property

In vivoclassification

FDSC

SCAS

KAO

1

HA301

HB310

HC314

Methotrexate

59-05-2

TCI

Solid

DMSO

P

2

HA306

HB315

HC319

6-Aminonicotinamide

329-89-5

TCI

Solid

DMSO

P

4

HA316

HB301

HC310

Dexamethasone

50-02-2

TCI

Solid

DMSO

P

6

HA307

HB311

HC320

Diphenylhydantoin

57-41-0

Wako

Solid

DMSO

P

7

HA312

HB316

HC301

Hydroxyurea

127-07-1

Sigma

Solid

PBS

P

9

HA303

HB307

HC311

Boric acid

10043-35-3

sigma

Solid

PBS

P

10

HA308

HB312

HC316

Methoxyacetic acid625-45-6

Wako

Liquid

PBS

P

11

HA313

HB317

HC302

Dimethadione695-53-4

aldrich

Solid

PBS

P

12

HA318

HB303

HC307

Dimethyl phthalate131-11-3

TCI

Liquid

DMSO

N

13

HA304

HB308

HC312

Cimetidine

51481-61-9

TCI

Solid

PBS

N

14

HA309

HB313

HC317

119-61-9

Sigma

Solid

DMSO

N

15

HA314

HB318

HC303

109-86-4

TCI

Liquid

PBS

N

16

HA319

HB304

HC308

54-85-3

Sigma

Solid

PBS

N

18

HA310

HB314

HC318

34381-68-5

Sigma

Solid

PBS

N

19

HA315

HB319

HC304

Benzophenone
Ethylene glycol
methyl ether
Isoniazid
Acebutololhydrochloride
Diphenhydramine HCl

147-24-0

Sigma

Solid

PBS

N

20

HA320

HB305

HC309

Acrylamide

79-06-1

Wako

Solid

PBS

N

4.3.4 Results
4.3.4.1 IC50 and ID50 values variation and final judgment
The results are presented for each chemical from tables 35 to 50.
Methotrexate
Table 35 shows the results obtained with methotrexate during phase 3.
Table 35 Results obtained with methotrexate during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Test chemicalLab
A

Methotrexate

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

0.04

0.02

P

DS2

0.06

0.02

P

DS1

0.11

0.03

P

DS2

0.05

0.003

P

DS1

0.04

0.05

P

DS2

0.04

0.03

P

P
P
P

69

The IC50 and ID50 values remain close to each other. Given the fact that all the values are very low(lower than 0.11), the insertion in the prediction model gave a positive result. All the labs obtained
the same judgment, verifying the between-laboratory reproducibility for methotrexate.
6-aminonicotinamide
Table 36 shows the results obtained with 6-aminonicotinamide during phase 3.
Table 36 Results obtained with 6-aminonicotinamide during phase 3 showing the IC50 and ID50
values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLabA

6-aminonicotinamide

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

1.40

0.58

P

DS2

1.46

0.88

P

DS1

2.03

0.67

P

DS2

3.02

0.65

P

DS1

0.68

0.71

P

DS2

>1.25

>1.25

P

P
P
P

The IC50 and ID50 values remain close to each other. Given the fact that all the values are very low(lower than 3μg/ml), the insertion in the prediction model gave a positive results. All the labs
obtained

the

same

judgment,

verifyingthebetween-laboratory

reproducibility

for

6-aminonicotinamide.
Dexamethasone
Table 37 shows the results obtained with dexamethasone during phase 3.
Table 37 Results obtained with dexamethasone during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLabA

Dexamethasone

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

31.56

20.27

N

DS2

34.69

27.92

N

DS1

37.35

13.98

N

DS2

52.23

24.13

N

DS1

13.72

21.82

N

DS2

> 125

> 125

N

N
N
N

70

The IC50 and ID50 values remained close to each other confirming the reproducibility of the assay.
All the labs obtained the same judgment, verifying the between-laboratory reproducibility for
dexamethasone. The DS of Lab C was however worth noticing where no toxicity was triggered.
After having checking with the lab responsible for the experiment, they acknowledged that thechemical was not added in the medium.
Diphenylhydantoin
Table 38 shows the results obtained with diphenylhydantoin during phase 3.
Table 38 Results obtained with diphenylhydanthoin during phase 3 showing the IC50 and ID50
values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Diphenylhydantoin

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

> 125

9.96

P

DS2

> 125

14.33

P

DS1

> 15.625

3.81

N

DS2

> 15.625

4.08

N

DS1

41.65

33.4

N

DS2

19.46

38.14

N

P
N
N

The close data observed between the run confirms the reproducibility of the test. However, there
were different judgment observed and thus, the between-laboratory reproducibility could not be
verified. The ID50 values obtained in each lab do not vary dramatically. However, due to the different
MD chosen between lab A and lab B, the gap between the IC50 and the ID50 varies and triggers then adifferent judgment (Positive for lab A and negative for lab B). Lab C, exhibiting a cytotoxic
concentration of chemical at 41.65μg/ml for DS1 and 19.46μg/ml for DS2, triggers a negative
judgment because of a smaller gap between the IC50 and ID50 values.
Hydroxyurea
Table 39 shows the results obtained with hydroxyurea during phase 3.

71

Table 39 Results obtained with hydroxyurea during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Hydroxyurea

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

3.06

2.14

P

DS2

6.31

3.39

P

DS1

3.44

2.36

P

DS2

2.97

1.83

P

DS1

1.51

1.00

P

DS2

1.29

1.78

P

P
P
P

The IC50 and ID50 values remained close to each other confirming the reproducibility of the test.
Given the fact that all the values are very low (lower than 6), the insertion in the prediction modelgave positive results. All the labs obtained the same judgment, verifying the between-laboratory
reproducibility for hydroxyurea.
Boric acid
Table 40 shows the results obtained with boric acid during phase 3.
Table 40 Results obtained with boric acid during phase 3 showing the IC50 and ID50 values withthe final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Boric acid

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

62.59

47.40

N

DS2

83.99

55.32

N

DS1

115.41

78.26

N

DS2

66.07

48.76

N

DS1

94.06

40.98

N

DS2

94.5

31.32

P

DS3

97.37

44.60

N

N
N

N

The IC50 and ID50 values remain close to each other confirming the reproducibility of the test. All thelabs obtained the same judgment, verifying the between-laboratory reproducibility for boric acid.
Methoxyacetic acid
Table 41 shows the results obtained with methoxyacetic acid during phase 3.

72

Table 41 Results obtained with methoxyacetic acid during phase 3 showing the IC50 and ID50
values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Methoxyacetic acid

B
C

Runs

Final

judgment

judgment

586.88NN

382.57342.91NDS1

656.17475.35

NDS2

544.62738.53NDS1

585.04747.86NDS2> 1000

>1000

N

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

392.88DS2NNThe IC50 and ID50 values remain close to each other confirming the reproducibility of the test. All thelabs obtained the same judgment, verifying the between-laboratory reproducibility for methoxyaceticacid
Dimethadione
Table 42 shows the results obtained with dimethadione during phase 3.
Table 42 Results obtained with dimethadione during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Dimethadione

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

925.95> 1000N

DS2> 1000535.55

NDS1> 1000

459.3

N

DS2

> 1000228.34

NDS1

902.68727.71NDS2> 1000> 1000N

N
NN

The IC50 and ID50 values remained close to each other between the runs confirming the
reproducibility of the test. All the labs obtained the same judgment, verifying the between-laboratory
reproducibility for dimethadione
Dimethyl phtalate
Table 43 shows the results obtained with dimethyl phtalate during phase 3.

73

Table 43 Results obtained with dimethyl phthalate during phase 3 showing the IC50 and ID50
values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Dimethyl phtalate

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

> 250

130.57

N

DS2

> 250

69.56

N

DS1

> 250

28.41

P

DS2

> 250

24.17

P

DS1

10.57

> 1000

N

DS2

94.98

> 1000N

NPN

The IC50 and ID50 values remained close to each other between the runs confirming the
reproducibility of the test.
It seems the ID50 values situated between 20 and 70 μg/ml when an IC50 and MD are at 250 μg/ml,
are in the critical window, a slight variation of ID50 could trigger a positive or a negative result
according to the prediction model. This is what happens between lab A and lab B which triggers a
different judgment.
In the case of lab C, the IC50 values are greatly lower than ID50 (Figure 22 and Table 43). This can be
explained as follows: Dimethyl phthalate is a liquid chemical that only dissolves in DMSO andprecipitate (or forms small bubbles) in the assay medium at high concentration. Lab C set the highest
concentration to 1000 μg/ml which consists in almost the pure substance. It is possible that during
the precipitation evaluation the bubbles of DMSO that did not dissolve were not clearly observable.
When pipetting assay medium from the 2ml assay block, the chemical may have stayed (bubbles on
the top of the liquid and pipetting was done in the middle) in the assay block due to its incapacity todissolve at 1000 and 500 μg/ml leading to non toxic effect at highest concentration.

DS2

DS1
Non fitted curve

Non fitted curve

Figure 22 Results of cytotoxicity for dimethyl phtalate obtained in lab C(Comparison of cytotoxicity curves between DS1 and DS2)
74

Cimetidine
Table 44 shows the results obtained with cimetidine during phase 3.
Table 44 Results obtained with cimetidine during phase 3 showing the IC50 and ID50 values withthe final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Cimetidine

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

> 250

> 250

N

DS2

> 250

> 250

N

DS1

> 250

> 250

N

DS2

> 250

> 250

N

DS1

> 250

> 250

N

DS2

> 250

> 250

N

N
N
N

The IC50 and ID50 values remained close to each other between the runs confirming the
reproducibility of the test. All the labs obtained the same judgment, verifying the between-laboratory
reproducibility for cimetidine.
Benzophenone
Table 45 shows the results obtained with benzophenone during phase 3.
Table 45 Results obtained with benzophenone during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Benzophenone

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1> 62.5> 62.5

NDS2> 62.5> 62.5

N

DS1

> 250

3.32

P

DS2

103.47

7.51

P

DS1

16.92

143.13

N

DS2

50.79

208.65NNPNThe IC50 and ID50 values remain close to each other between the runs confirming the reproducibilityof the test. Lab B obtained a positive result while the two other labs obtained a negative result. The
positive results obtained in lab B is due to a larger gap between the IC50 and the ID50 values
compared to lab A (where no gap was observed) and an IC50 value lower than the ID50 value for lab
C. For the latter, the fact that IC50 values that were considerably lower than the ID50 values is due to75

precipitation. Indeed, as shown in Figure 23, the concentration of 166.66 and 250 μg/ml do notcompletely kill the cells leading to a flat curve at the end triggering a curve fitting shifted to the right.
DS1 displayed the same pattern (data not shown).

Non fitted curve

Non fitted curve

Figure 23 Results obtained by lab C (DS2) with benzophenone
Ethylene glycol methyl ether
Table 46 shows the results obtained with ethylene glycol methyl ether during phase 3.
Table 46 Results obtained with ethylene glycol methyl ether during phase 3 showing the IC50 and
ID50 values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Ethylene glycol
methyl ether

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

>1000

>1000

N

DS2

>1000

>1000

N

DS1

>1000

>1000

N

DS2

>1000

>1000

N

DS1

>1000

>1000

N

DS2

>1000

>1000

N

N
N
N

No toxicity was observed between the runs for all the laboratories confirming the reproducibility ofthe test. All the labs obtained the same judgment, verifying the between-laboratory reproducibility
for ethylene glycol methyl ether.
Isoniazid
Table 47 shows the results obtained with isoniazid during phase 3.

76

Table 47 Results obtained with isoniazid during phase 3 showing the IC50 and ID50 values withthe final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Isoniazid

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

287.04376.41

NDS2

269.78356.65

NDS1

406.45421.92NDS2263.94312.65

NDS1

177.82280.12NDS2

303.54385.88NNNN

The IC50 and ID50 values remain close to each other between the runs confirming the reproducibilityof the test. All the labs obtained the same judgment verifying the between-laboratory reproducibility
for isoniazid.
Acebutolol HCl
Table 48 shows the results obtained with acebutolol HCl during phase 3.
Table 48 Results obtained with acebutolol HCl during phase 3 showing the IC50 and ID50 values
with the final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Acebutolol HCl

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

182.16220.38NDS2

206.92202.01

N

DS1

95.46

38.42

P

DS2

95.84

74.24

N

DS3

95.48

69.06

N

DS1

9.45

49.08

N

DS2

35.89

94.33

N

N

N

N

The IC50 and ID50 values remain close to each other between the runs confirming the reproducibilityof the test. However, for lab C the ID50 values of DS1 and DS1 are much higher (49.08 and94.33μg/ml respectively) than the IC50 values (9.45 and 35.89μg/ml respectively) which is not
relevant. This is due to the fact that the 10 dilution fold has been chosen where a lower fold dilution
would have shown more accurate results (see Figure 24)
All the labs obtained the same judgment verifying the between-laboratory reproducibility forisoniazid.

77

DS1

DS2
Non fitted curve

Non fitted curve

Figure 24 Results of acebutolol HCl obtained in lab C. Comparison of cytotoxicity curves between
DS1 and DS2
Diphenhydramine
Table 49 shows the results obtained with diphenhydramine during phase 3.
Table 49 Results obtained with diphenhydramine during phase 3 showing the IC50 and ID50
values with the final judgment obtained in the DS studies(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Diphenhydramine

B

C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

5.01

7.67

P

DS2

17.36

6.31

P

DS1

7.81

2.78

P

DS2

10.82

1.43

P

DS1

10.20

6.14

P

DS2

2.36

12.59

N

DS3

4.8

4.15

P

P
P

P

The IC50 and ID50 values remained close to each other between the runs confirming the
reproducibility of the test. All the labs obtained the same judgment verifying the between-laboratory
reproducibility for diphenhydramine.
Acrylamide
Table 50 shows the results obtained with acrylamide during phase 3.

78

Table 50 Results obtained with acrylamide during phase 3 showing the IC50 and ID50 values withthe final judgment obtained in the DS studies (Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical nameLab
A

Acrylamide

B
C

Runs

Final

judgment

judgment

Run

IC50 value (μg/ml)

ID50 value (μg/ml)

DS1

40.81

64.23

N

DS2

41.53

48.74

N

DS1

43.61

14.86

P

DS2

34.04

13.52

P

DS1

14.02

33.61

N

DS2

32.88

48.00

N

N
P
N

The IC50 and ID50 values remain close to each other between the runs confirming the reproducibilityof the test. Acrylamide did not satisfy the between-laboratory reproducibility because lab B obtained
a positive result. This seems to be due to a lower accuracy because they used a 5 dilution ratio while
the other lab chose a lower one (2 and 3). This triggered an ID50 value lower than the other labs andthus, a positive result.
4.3.4.2 Summary of the between-laboratory reproducibility
Since each chemical was tested in only one set, only the between-laboratory reproducibility wastested with the new parameter curve fitting (three parameters) and the new prediction model.
As shown in Table 51, between-laboratory reproducibility reached 75% (12/16) which again satisfies
the criterion fixed in the study plan (75%).

79

Table 51. Results on the between-laboratory reproducibility obtained in phase 3(Lab A: FDSC, Lab B: SCAS, Lab C: KAO)
Chemical name

Laboratory

In vivoA

B

C

classification

Methotrexate

P

P

P

P

6-aminonicotinamide

P

P

P

P

Dexamethasone

N

N

N

P

5,5 diphenylhydantoin

P

N

N

P

Hydroxyurea

P

P

P

P

Boric acid

N

N

N

P

Methoxyacetic acid

N

N

N

P

Dimethadione

N

N

N

P

Dimethyl phthalate

N

P

N

N

Cimetidine

N

N

N

N

Benzophenone

N

P

N

N

EGME

N

N

N

N

Isoniazid

N

N

N

N

Acetobutolol HCl

N

N

N

N

Diphenhydramine

P

P

P

N

Acrylamide

N

P

N

N

Among the sixteen chemicals tested in phase 3, four of them (5,5 diphenylhydantoin, dimethyl
phthalate, benzophenone and acrylamide) did not satisfied the between-laboratory reproducibility.
The global between-laboratory reproducibility for phase 3 is therefore raised at 75% (12/16) which
satisfies the criteria fixed in the study plan confirming the relevancy of the Hand1-Luc EST.
4.3.5 Conclusion
Phase 3, with the three parameter curve fitting and the new prediction model safely satisfied the
between-laboratory reproducibility criterion (75%). However, some problems were pointed out andthe protocol was improved with minor changes to avoid this kind of problems to happen again. The
improvement are listed below- Since precipitation was observed, especially for liquid chemicals, the precipitation
evaluation in the medium should be even more taken care of (watch for DMSO bubbles appearance
for example).
- Once the dose finding study is done, the dilution ratio chosen shall be lowest whenpossible (1.5 or 2).
- When the IC50 and/or the ID50 of D1 and DS2 are different from a factor 7 (value decided
80

by analyzing the historical data), then another experiment shall be done and the data used will be theones the closest to the ones previously obtained. This point was considered to avoid the cases where
manipulation errors (for example, forgetting to add chemicals in the medium or dilution mistake) are
analyzed for the final judgment.
- The quality of the cells was also pointed out by the participant laboratories. Thus, before
sending each lot, one tube shall be thawed and the quality of cells shall be verified.
All those improvements should raise the reproducibility of the protocol. The very final version of the
protocol can be found in Appendix 37.

81

5

DISCUSSION

The discussion is proposed following the modular approach as recommended by ECVAM (Hartung,2004).
5.1 Test definition
As previously described in this report, the Hand1-Luc EST is defined as a developmental toxicity
test in vitro assessing the cytotoxicity and the differentiation toxicity by using engineered mouseembryonic stem cells and make them differentiate into cardiomyocyte during 120 hrs (5 days). The
cells, called KOB1 ES cells, are stably transfected with a vector containing the luciferase gene
modulated by the Hand1 gene promoter. The Hand1 gene being a significant gene at the first steps ofthe development of the organism is used to reveal the gene disruption of embryotoxic chemicals. The
endpoints of the test are the IC50, the ID50 which are measured at 120 hr (day5) and the maximum
dose (MD) consisting of the concentration of the chemical that dissolves in the assay medium(assessed before the test). The IC50 is measured thanks to a commercial kit and represents theconcentration of chemical for the one 50% of the cells die. The ID50 is measured via a luciferase
assay system and shows the concentration of chemical 50% of the signal is inhibited.
The test has been developed to respond to needs of the 3Rs. It finds its origins in trying to solve theproblems faced by the original EST validated by ECVAM in 2002 which failed to be raised as anOECD guideline. Among those, Hand1-Luc EST improved the time, the cost and the difficulty of theoriginal test.
5.2 Definition of the performance standards
The performance standards are evaluated at each experiment with the in vivo positive chemical 5-FU.
The acceptance criteria for this chemical are the following:- IC50: 0.0345 μg/mL ± 5SD (0.003 - 0.065 μg/mL) and- ID50: 0.0355 μg/mL ± 5SD (0.003 - 0.067 μg/mL)
5.3 Predictive capacity
5.3.1 Prediction model
The prediction model consists in a formula using IC50, ID50 and MD values. The score given by theformula is then converted into a probability. If the probability is higher than 0.52 then the chemical82

is considered as positive. If the probability is lower than 0.52 then the chemical is considered as
negative.
Score= 1.474 x log (IC50/ID50) + 0.714 x log (MD/IC50) − 1.109
Probability = Inverse logit (Score)
Positive: Probability ≥ 52%
Negative: Probability < 52%
The improvements the prediction model undertook are described in the manuscript. The first version
is available in section 3.3.2.2, the second one in section 4.1.2.2 and the final version in 4.3.5. Shortly,
the prediction model was improved and adapted because of the following main reasons:- IC50 and ID50 values obtained in the lead lab were changed because of theimplementation of the curve fitting and the prediction model needed to be adapted to those changes- New data from other chemicals (21 chemicals for the first and the second prediction
model and 71 chemicals for the last version) were measured in the lead lab and those data were used
to improve the prediction model so as to widen the applicability range of the Hand1-Luc EST (seeAppendix 36).
- Maximum dose (MD) was implemented and used in the prediction model.
Furthermore, for some chemicals, no IC50 or ID50 were triggered given values higher than the
maximum dose. Actually, those values do not indicate real IC50 or ID50 ones. However, as describedin the formula, the developmental toxicity is still evaluated thanks to the two ratios IC50/ID50 and
MD/IC50. If the ID50 is higher than the MD, then both ratios will be equal to 1 (IC50=ID50=MD) andthe score will be -1.109 leading to a negative results (not toxic). However, there are cases where theIC50 is equal to the MD but the ID50 is triggered and then lower than the MD. In this case, the part0.714 x log (MD/IC50) of the formula will be erased and the evaluation of the toxicity will be done
only by the ratio IC50/ID50 (gap between cytotoxicity and Hand1 disruption). For those reasons, theevaluation of developmental toxicity is still possible even though some values are higher than theMD.
The other point was to prove that Hand1-Luc EST is not just simply a cytotoxicity test. The
explanation can be given by analyzing in details the discriminant equation of the prediction model:
Two main aspects are analyzed in the equation that are the ratio IC50/ID50 (Ratio A) and MD/IC50(ratio B).
Ratio A is used to define the potency of a chemical to disrupt the Hand1 gene compared tocytotoxicity. If there is a gap between the IC50 and the ID50 values, then at non cytotoxic
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concentration, the chemical is affecting the expression of the Hand1 gene and the greater is the ratio
A, the greater would be the ability for the chemical to induce genetic disruption that would lead tofetus malformations. However, it is important to keep in mind, that even if the concentration ofchemicals is not cytotoxic, a disruption in the gene expression balance can still lead to the fetus
death due to organ function suppression or disruption (protein expression or cell migration
disruption).
When ratio A is close to 1 (IC50≃ID50), cells are dying before the effect on the Hand1 gene
expression can be observed. In this case, Hand1-Luc EST may be thought not to be sufficient topredict embryotoxicity. However, cytotoxic compounds can also be embryotoxic by preventing the

fetus to grow up normally or even lead to fetal death. Toxicology is based on the dose applied to theorganism. A dose that kills for example 20% of the cells may induce malformations but notnecessary fetus death. However, a dose that kills 80% of the cells has greater chances to induce fetal
death.
The other parameter is ratio B which is expressed as the ratio maximum dose (MD), IC50. The
maximum dose, as previously defined as the concentration of chemical that dissolves in the assay
medium, can be thought to be soluble in blood and therefore have greater chances to reach the fetus
and exert its effect. With the same way of thinking as in the case of ratio A, the greater is the gap
between the MD and the IC50 the greater is the possibility for the chemical to be cytotoxic. If theratio B is close to one, then the chemical, at the IC50 dose, may not necessarily be soluble in bloodand thus may have fewer chances to reach the fetus by crossing the blood vessel wall and/or theplacental barrier to exert its toxic effect on the fetus.
In other available in vitro developmental toxicity tests, the evaluation of chemical precipitation is notdescribed in the protocol. Precipitation may affect the results and thus the results obtain at theconcentration precipitation occur may not be the correct ones. Hand1-Luc EST is therefore moreaccurate concerning this aspect.
In addition, the cut-off criterion to separate positive and negative chemicals has been set to 52%
because first, this value was proposed on the ROC curve (see Figure 21) and also because this value
best separated the in vivo positive and the in vivo negative chemicals used for the prediction model.
5.3.2 Predictive capacity
The predictive capacity of the Hand1-Luc EST estimated with seventy-one chemicals is presented inTable 52.

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Table 52.

Predictive capacity of the Hand1-Luc EST with the most recent prediction model

As shown in Table 52, sensitivity and negative predictive value is low which means that Hand1-Luc
EST negative predicted chemicals are predicted, as such, with a low confidence. However, the highspecificity and positive predictive value shows that a Hand1-Luc EST positive predicted chemical isvery likely to be actually positive in vivo. This result supports that the Hand1-Luc EST is powerfultool to predict embryotoxic positive chemicals in vitro.
Accuracy sets at 60.6% which is considered as insufficient according to Spielmann et al., 2006. The
other protocols that evaluate embryotoxicity in vitro and that undertook a validation process had
better accuracy. Indeed, the accuracy reached 70% for the rat limb bud micromass test, 80% for thewhole embryo culture test (by combining 2 PMs) and 78% for the original EST (Spielmann et al.,2006). However, they only tested twenty chemicals while our predictive capacity is determined byseventy-one chemicals. All those seventy-one chemicals have different mode of action and therefore
different effect on development. For chemicals that do not affect Hand1 gene expression will be hardto detect. Furthermore, developmental toxicity involves complex mechanisms and establishingcriteria about accuracy to know whether an embryotoxicity test is reliable or not (Spielmann et al,2006) should be revised according to the test definition. The discussion about developmental
mechanisms and applicability domain is described in details in part 5.7.
5.4 Transferability
The three participant laboratories, although having expertise in tests involving cell culture andmanipulation, were given training. All the participant laboratories got the training the same time at85

the same place (SC, Osaka). Hand1-Luc EST aiming to be an easy test with no complicatedmanipulation requiring special expertise, the training consisted only in showing the test steps and thepoints to be taken care of. Phase 0, as described in part 3.2, showed reproducible results with low
variation of the IC50 and ID50 values.
5.5

Within-laboratory reproducibility

Within-laboratory reproducibility of the protocol was confirmed in phases 1, 2b, 2c where a test
substance was tested in three sets (a set consisting in one dose finding study and one definitive study
for phase 1 and for phase 2b, c, one dose finding study and two or three definitive studies). The
acceptance criterion for within-laboratory reproducibility was set in the study plan as 75% (seeAppendix 31)
As shown in part 3.3.4.2, phase 1 showed a within-laboratory reproducibility of 89% (8/9 tests) andphase 2b, 2c showed a within- laboratory reproducibility of 83.3% (20/24 tests) (part 4.2.4.2) whichlargely fulfills the acceptance criterion of 75%. The content of the test for phase 1 is however
different from phases 2b and 2c. Indeed, at that time, no curve fitting was used and the evaluation of
precipitation was not done either. However, it is reasonable to think that the reproducibility would
still remain as a high level if the same chemicals were tested since the protocol was improved to
avoid the cases that triggered one result that did not fulfill the reproducibility.
The protocol for phase 2 and phase 3 is basically the same except minor changes leading to thinkthat the reproducibility would be better thanks to the improvements applied (see part 4.3.5) in thelast version of the protocol.
5.6 Between-laboratory reproducibility
The between-laboratory reproducibility has been tested in phases 2 and 3 (parts 4.1, 4.2 and 4.3).
Each compound was tested in one dose finding study and two or three definitive studies and thepositive or the negative judgment was decided thanks to the majority judgment. Twelve codedchemicals were tested in phase 2 and sixteen coded chemicals in phase 3.
The between-laboratory reproducibility of phase 2 was 83.3% (10/12 chemicals) and the one forphase 3 was 75% (12/16 chemicals). The reason for discrepancy of some chemical is described indetails in parts 4.2.4 and 4.3.4. The protocol has been improved according to the problem raised
when analyzing the cases and thus, with the present protocol, although the reproducibility alreadyattained the 75%, it is expected to be much better in the future.

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5.7 Applicability domain
As many current in vitro available tests, Hand1-Luc EST has limitations. Those limitations can be
due to the material used or the biological parameter evaluated.
5.7.1 Limitations due to materials
The evaluation of the cytotoxicity is evaluated thanks to the CellTiter-Fluor™ Cell Viability Assay.
Viability of cells is measured via the protease activity happening in living cells. Thus, all thechemicals with a protease inhibitory activity cannot be tested in the Hand1-Luc EST.
5.7.2 Limitations due to the measured biological parameters
Hand1-Luc EST is evaluating developmental toxicity by the disruption of Hand1 gene expression.
Therefore, chemicals that do not affect directly or indirectly Hand1 gene expression may not be
detected. However, high cytotoxic chemicals would still be predicted as positive (see part 5.3.1).
Figure 25 shows the possible pathways and gene related to Hand1. If a chemical affects one of thesegenes then it could be detectable via Hand1 gene expression measurement. This scheme has been
established thanks to all the papers enumerated in Appendix 38.

Figure 25 Hand1 gene related pathways (references can be found in Appendix 38)
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Chemicals have their own mode of action. Some chemicals could affect some specific pathways(enzyme inhibition), some others could affect a particular physiological function (for example, ion
channel inhibition) or some others could affect directly DNA synthesis (enzyme inhibition orintercalating agent). When tested in vivo, the appearance of malformations maybe very selective tosome special organs while some other may possibly affect a relatively larger number of organs or
eventually lead to fetal death. Hand1 gene is reported to be involved in the heart, the limb and thefacial bones development (see introduction). Thus, chemicals reported to trigger these organs’malformations may be detected with Hand1. However, it is important to keep in mind that somechemicals may alter genes expressed after 120 hr (5 days) after the beginning of differentiation. In
this case, Hand1 may not be able to detect the effect of those chemicals.
As previously emphasized when the other in vitro developmental toxicity test were assessed, effectsof metabolites should be assessed. Unfortunately, Hand1-Luc EST cannot evaluate metabolites
developmental effects of chemicals due to the incapacity of the ES cells to metabolize compounds.
Another point which is important to consider, is the placental barrier. Chemicals that do not cross thebarrier will be considered as negative. However, the Hand1-Luc EST does not provide this systemand therefore misclassification may occur because of this aspect.
Development is a very complex phenomenon that involves different mechanisms. The seventy-one
chemicals we used have their own mode of action and they can disrupt many different genes, somerelated to Hand1 and some unrelated.
Therefore, given all the points developed above, the accuracy of 60.6% for the Hand1-Luc EST isreliable for the assessment of the developmental toxicity of chemicals in screening stages.
5.7.3

Justifications on misclassified chemicals

Phase 3 revealed six chemicals misclassified and the possible biological reason is detailed below foreach of them.
5.7.3.1 False negative chemicals
5.7.3.1.1 Boric Acid
Boric acid can be used as an antiseptic, insecticide, flame retardant, neutron absorber, or precursor toother chemical compounds.
Boric acid is positive in vivo but negative in the Hand1-Luc EST. In rats and mice, the most88

frequently observed malformations were enlarged lateral ventricles of the brain and agenesis or
shortening of the thirteenth rib (Heindel, 1992). Boric acid is also reported to affect the developmentof the skeleton axis by altering the expression of Hox genes (Hox 4,5,6) leading to cervicalmalformations (Narotsky, 2004).
Moreover, boric acid can inhibit the activity of HDAC (Histone deacetylases) as can do valproic acid
(VPA). This inhibition is usually related to teratogenicity and the malformations have been classified
as fusions and homeotic transformation of the axial skeleton segments, quite similar to those induced
by VPA (Di Renzo, 2007).
There is no paper showing cardiac teratogenicity for the chemical. Therefore, it can be suggested thatboric acid does not affect the development of the heart and thus consequently the expression of theHand1 gene.
In addition, in a novel neural EST that assesses the expression of two different marker genes (Reln
and Tubb) (results soon to be published), boric acid is classified as positive chemical supporting thefact this chemical is actually toxic for neuronal development.
5.7.3.1.2 Dexamethasone
Dexamethasone, synthetic glucocorticoid, is widely used to treat inflammatory conditions such asallergies, skin conditions, ulcerative colitis, arthritis and breathing disorders. This chemical is
positive in vivo but predicted as negative in the Hand1-Luc EST.
In rodents, dexamethasone elicited cleft palate in vivo and prevented neural tube closure in vitro(Hansel, 1999). According to in-house data (DNA chip analysis), the glucocorticoid receptor is not
yet expressed in the ES cells after 120 hrs of differentiating. Therefore, the effect of dexamethasone
on Hand1 may not be observable.
5.7.3.1.3 Dimethadione
Dimethadione is used as an antiepileptic drug. It inhibits T-type calcium channel in the thalamus. It
is predicted as negative in the Hand1-Luc EST but positive in vivo. The main defects observed in thefetus are facial dysmorphism, cardiac defect and brain retardation (Van Boxtel, 2008).
Dimethadione is reported to have a toxic effect on the placenta and not on embryonic tissues that
may link to the malformations observed in the fetus (Ozolins et al, 2015). This correlates with ourobservation where dimethadione did not exhibit any cytotoxicity (IC50>1000μg/ml). Another fact
seems to be the ability by dimethadione to produce free radicals that could lead to embryotoxicity(Hood, 2006). Hand1 gene being not related to this phenomenon, Hand1-Luc EST may thus not beable to detect the embryotoxicity of dimethadione.
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5.7.3.1.4

Methoxyacetic acid

Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene
glycol monomethyl ether.
This chemical is positive in vivo but predicted as negative in the Hand1-Luc EST.
In utero, 2-MAA exposure increases the incidence of hydrocephalus, hydronephrosis, heart, tail andlimb malformations in species ranging from rodents to nonhuman primates (Brown, 1984; Scott etal., 1989). In 1986, another study reports open or irregular fusion of the neural tube and irregularsomite segmentation (Yonemoto, 1986). More recent studies show that limb formation is greatly
disrupted and the cell cycle is affected by effect on histones proteins (Dayan at al, 2014). Finally,methoxyacetic acid is also reported to disrupt protein kinase pathway, oxidative stress and DNA
metabolic process. This chemical has more effect on neural tube, limbs and branchial arches than in
the heart (Robinson et al, 2010).
In the article of Brown (1984), two types of experiments have been done. The first was to inject
MAA in pregnant rat from D8 to D14 and see the effect at D20. The second experiment was to inject
MAA to pregnant rats at D10 of pregnancy and see the effect on the fetuses at D12.
In the first experiment, the earlier was the injection, the greater was the fetus’ death. The
malformations observed were skeletal malformations, hydrocephalus, dilated kidney pelvis andbladder atrophy. At observation days 10 and 12, heart and renal malformations were observed but
those are likely to be the results of fetal resorption or growth retardation. For the second experiment,
the main malformations observed were incomplete neural suture and forelimb bud malformations.
Given those observations, the effect of MAA seems to be more likely happening in the latter stage ofdevelopment (D12 to D20). Hand1-Luc EST assessing the gene perturbation at the earlier stages
may therefore be not adequate to assess the developmental toxicity of MAA. As described for boric
acid, in the neural EST results we are going to submit soon, MAA is also classified as positive
stressing again its toxicity for neuronal development. According to the above, Hand1-Luc EST
cannot detect the embryotoxicity of MAA because of the early expression of Hand1 gene and MAA
specific effect on neuronal and skeletal development.
5.7.3.1.5 Phenytoin
Phenytoin is an antiepileptic drug which can be useful in the treatment of epilepsy. It is used to block
sodium channels.
This chemical is positive in vivo but predicted as negative in the Hand1-Luc EST.
According to many papers, the main reason for phenytoin to be embryotoxic is the reactive oxygen
species generation due to metabolism (Winn et al., 2003; Azarbayjani et al., 2006). It is also reported
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that phenytoin altered genes and pathway related to Hand1 such as TGF-β and the proto-oncogene
Wnt-1 (Musselman et al., 1994). An inhibition of the Hand1 gene with no cytotoxicity was actuallyobserved. The non cytotoxicity may be explained by the absence of ROS due to the missing of
metabolism in the system. The alteration of Hand1 would therefore be linked to TGFβ and Wnt-1
pathways disruption. The fact that the negativity is triggered may be due to the low solubility of thechemical lowering the IC50 (=MD)/ID50 ratio of the prediction model.
5.7.3.2 False positive chemicals
5.7.3.2.1 Diphenhydramine
Diphenhydramine is an antagonist of the histamine receptor H1. It is predicted as positive in the
Hand1-Luc EST but is negative in vivo.
The EST also misclassified this chemical suggesting that this type of in vitro system may be moresensitive to this compound class (Genschow et al, 2004; Panzica-Kelly et al, 2012). Indeed, in the
Hand1-Luc EST, a relatively high cytotoxicity was observed (IC50~15μg/ml).
In addition, as published in 2012, the positivity of diphenhydramine in the original EST was
discussed (Riebeling et al, 2012). It seems the origin of the positivity could be the inhibition ofpotassium voltage gated channel (type H) affecting the beating potential of the differentiatedcardiomyocytes. This effect is reversible when diphenhydramine is withdrawn. The Hand1-Luc EST,
although it does not assess cardiomyocytes beating, shows a certain inhibition of the Hand1 genesuggesting that the positivity of the chemical in vitro may also have its origin via other mechanisms.
The in vivo negative results may also be due to metabolism.

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6

CONCLUSIONS

The aim of the study was to validate Hand1-Luc EST, in a formal between-laboratory reproducibility
study, following the modular approach of Hartung et al. (2004).
During the three years of validation, the protocol has been improved and reproducibility (between
and within) has been verified. Furthermore, thanks to the many data obtained by the lead lab and therecommendations of the VMT members, a prediction model using the IC50, ID50 and MD values wasestablished and improved.
Hand1-Luc EST in its current form has been confirmed to be a transferable and reproducible (>75%)
protocol. Also, with a positive predictive value of 80.8% along with an accuracy of 60.6%, theHand1-Luc EST is defined as a new a powerful tool to detect embryotoxicants and can be used forscreening chemicals.
Also, compared to the original EST, Hand1-Luc EST has been recognized to improve the following
points:- Time of the experiment (10 days reduced to 120 hr (5 days))- Manipulations: it is a very easy protocol that does not require medium change, hanging
drops method or subjective microscopic observations- Chemical quantity: just a few milligrams (50mg maximum) of chemicals are needed- Prediction model: The prediction model uses only one equation and the solubility of thechemicals is taken into account for the prediction which becomes a way to express the solubility ofchemicals in the body fluids. The prediction model has been established with a high number
embryotoxicants (71) with different toxicological mechanisms.
With the conclusions asserted by the VMT and considering that developmental toxicity is quite acomplicated phenomenon, we consider that the current protocol, mainly focusing on heartdevelopmental toxicity, could be in the future combined with similar protocol evaluating thedevelopmental toxicity of other organs, or some other protocols evaluating metabolism in vitro.
This protocol also respecting the 3R and could considerably reduce the use of animals, the VMTrecommend that an OECD test guideline for Hand1-Luc EST to be drafted to encourage regulatory
acceptance and universal implementation of this validated, reliable, cost- and time-efficient test
method.

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7

ACKNOWLEDGMENTS

The VMT members would like to thank the participant laboratories for their precious collaborationand efforts to obtain valuable data to allow the present protocol reach the current validation status.

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8.

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