Final February 2018

Peer Review Panel Evaluation of the Hand1-Luc EST(Embryonic Stem Cell Test) as a Non-Animal Test
for Evaluating the Developmental Toxicity of Chemicals

Japanese Center for the Validation of Alternative Methods
National Institute of Health Sciences
Tokyo, Japan

Report completed by the Peer Review Panel on October, 2nd, 20171

Final February 2018

Table of contents
Executive Summary…………………………………………………………………....3
Peer Review Panel Evaluation
1. Introduction…………………………………………………………………………....5
2. Regulatory Rationale for the Test Method..........................................................5
3. Scientific Rationale and Limitations………………………………………………...7
4. Test Method Protocol……………………………………………………………...…7
5. Between- and within-Laboratory Reproducibility…………………………………. 7
6. Demonstration of the Performance.................................................................. ..8
7. Predictivity of the Test Method ………………………………………………….. …8
8. Availability of Data....………………………………………………………………....8
9. Data Quality & GLP………………………………………………………………. ….9
10. Applicability Domain…………………………………………………………….…..9
11. Proficiency Chemicals…………………………………………………………. …10
12. Performance Standards………………………………………………………. ….10
13. Advantages: Cost, Time, And Animal Welfare………………………………. ...11
14. Completeness of data…………………………………………………………. ....12
15. Validation Study Management and Conduct............................................... ...12
16. Conclusions……………………………………………………………………. ….12
Appendices
1. Appendix 1: Peer Review Panel Members……………………………………. ...13
2. Appendix 2: Acknowledgements……………………………………………..……14
3. Appendix 3: Glossary……………………………………………………………….15
4. Appendix 4: References…………………………………………………………....172

Final February 2018

Executive Summary
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific Peer Review Panel (PRP) to evaluate the validation status of theHand1-Luc EST (Embryonic Stem Cell Test) as a non-animal test for evaluating thedevelopmental toxicity of Chemicals in accordance with established international criteria(OECD, 2005) (1). The Hand1-Luc EST is an in vitro test for developmental toxicity, which
could be used within an “Integrated Approach to Testing and Assessment” (IATA) for
developmental toxicity in vitro.
JaCVAM provided the members of the PRP with the validation report, all supporting
documents of the study and a questionnaire with 14 evaluation criteria were sent to themembers of the PRP before the first meeting of the PRP. In conducting its evaluation, the
PRP addressed each of the evaluation criteria that correspond to internationally harmonizedvalidation and acceptance criteria (OECD GD 34).
On November 22 -23, 2016, in Osaka, Japan, the PRP met with the Validation Management
Team (VMT) of the study and discussed all aspects of the validation study and in particular
the evaluations and questions in the questionnaires submitted by the members of the PRP.
The recommendations of PRP have been taken into account and the validation report ofHand1-Luc EST validation study was revised by the VMT (2, 3, 4, 5).
Overall conclusion: The documents supporting the assessment of the new method arecomplete and allow a sufficient evaluation of the validity of the method. The management
and conduct of the multi-center validation study by the VMT were excellent and they areexceeding the usual standard of multi-center studies as far as communication among
participants. VMT is concerned and also data exchange and analysis and implementing
quality assurance.
Compared to in vivo testing for developmental toxicity the test does not require to sacrificeanimals, considerably reduces cost, time and required expertise. With the high positive
predictive value of the validated prediction model the new in vitro test is ideally fit to identify
chemicals with a high potential of developmental toxicity within an IATA for assessing
developmental toxicity.
Regulatory rationale: At present, there is no in vitro test guideline available to assess
developmental toxicity. Therefore, the Hand1-Luc EST has been developed to meet therequirements, which the original mEST could not satisfy (6). The validation study showedthat Hand1-Luc EST test allows to identify positive developmental toxicants with highconfidence and can be used as a high-throughput test for screening purposes. However, topredict developmental toxicity more accurately, this test will need to be combined with otherassays within an IATA for in vitro developmental toxicity testing.
Scientific rationale: In the Hand1-Luc EST mouse Hand1-ES (KOB1) cells are used, which
are transfected with a vector containing the luciferase gene monitored by the Hand1
promoter. Test chemicals are applied to the Hand1-ES cells from the start of differentiation,
when the three primordial tissues are not yet formed (day 0= day 3.5 in vivo) until day 5 (=8.5
in vivo), when ESC are differentiating into cardiomyocytes (mesoderm induction) (2, 3, 4, 5).
Thus, depending on the mode of action of chemicals, it is possible to cover a large number oforgans formed or specific organs only. Indeed, if the genes playing important roles in thethree germ layer formation are affected by the chemical, then all the downstream genes
governed may affect the ect-, endo- and mesoderm layer formation.The Hand1 gene isinvolved in the development and differentiation of heart, limbs and facial bones (7, 8, 9, 10).
Therefore, chemicals triggering malformations in these organs may be detected with theHand1-Luc assay.
Limitations: Differentiation is measured by luciferase activity and thus chemicals interferingwith luciferase protein should not be tested. Protease inhibitors cannot be detected in the3

Final February 2018
Hand1-Luc EST, since protease activity is used to assess cell viability. The Hand1-Luc EST
can also not evaluate the effect of metabolites due to the inability of ES cells to metabolize
compounds.
Validation study reference chemicals: A sufficiently number of representative, codedchemicals (28) was used in the validation study to evaluate the performance of method.
Details of the chemical selection procedure are described in detail in the “chemical selection
report”. The experts of the PRP concluded that the selection of test chemicals todemonstrate performance of the assay was appropriate.
Assay Reproducibility: The experts of the PRP concluded that the validation reportprovides excellent information on variability (e.g. coefficient of variation, CV) of the data thatwere measured in the individual laboratories and on the CV of the within- and betweenlaboratory reproducibility.
Test method predictivity: The prediction model has been established with a high number ofembryotoxicants (71) with different toxicological mechanisms (11). The experts of the PRP
were satisfied with the determination of accuracy, predictive capacity and the way in which
existing data on developmental toxicity of relevant species have been taken into account.
Data quality: During the validation study quality checks were carefully conducted by anindependent expert. All of the participating laboratories are GLP certified and the study was
conducted in the spirit of Good Laboratory Practice (GLP). All data were available to theexperts of the PRP for review.
Test method protocols: As far as the identification of positive and negative results wasconcerned, detailed information is provided in the revised version of the protocol, including
the acceptance criteria and the calculation used to fit curves to obtain IC50 and ID50 values.
Therefore, the experts of the PRP concluded that the protocol is complete and sufficiently
detailed for new laboratories to conduct the Hand1-Luc EST assay.
Applicability domain: There is some evidence that the Hand1 gene and its functions arevery similar between human and mouse. The applicability of the Hand1-Luc EST is restricted
to the examination of all the pathways related to the Hand1 gene (formation of the heart,limbs and craniofacial bones) and the early period of development (from 3.5 to 8.5 days after
fertilization in the mouse). The Hand1-Luc EST can also detect embryotoxicity by disruptionof genes involved in the development of the three germ layers (Ectoderm, Mesoderm andEndoderm) due to the exposure to chemicals in and after the undifferentiated stage.

4

Final February 2018

Introduction
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific Peer Review Panel (PRP) to evaluate the validation status of theHand1-Luc EST (Embryonic Stem Cell Test) as a non-animal test for evaluating thedevelopmental toxicity of chemicals in accordance with established international criteria(OECD, 2005) (1). The Hand1-Luc EST is an in vitro test for developmental toxicity, which
may be used within an “Integrated Approach to Testing and Assessment” (IATA) for
developmental toxicity.
JaCVAM provided the members of the PRP with the validation report and all supporting
documents of the validation study and provided the members of the PRP with aquestionnaire of 14 evaluation criteria before the first meeting of the PRP. During theevaluation the PRP discussed each of the evaluation criteria, which are addressing
acceptance criteria of international validation studies.
On November 22 -23, 2016, in Osaka, Japan, the PRP met with the Validation Management
Team (VMT) of the Hand1-Luc EST validation study and discussed all aspects of thevalidation study and in particular the topics addressed in the questionnaires submitted to the
PRP. The comments and recommendations of the PRP were taken into account in therevised version of the validation report and discussed during a teleconference of the PRP
and the VMT on May 27, 2017.
This report summarizes the final evaluation and conclusions of the experts of the PRP
following the 14 criteria order submitted by JaCVAM.
Evaluation Criterion 1: A rationale for the test method should be available, including
description of toxicological mechanisms, a clear statement of scientific need, andregulatory application.
Chemicals that exhibit the potential for developmental toxicity should be identified andeliminated in the early stages of the development of new chemicals to which consumers,
patients and workers are exposed. All of the current guidelines for reproductive toxicity(OECD 414, 415, 416, 421, 422, 426, 443) are requiring to sacrifice a high number ofpregnant animals. In addition, the cost triggered by all those experiments is high and specificexpertise to conduct them is required. With the implementation of Directive 2010/63/EU, theneed for validated alternative methods, in particular in vitro replacement methods, for thesafety evaluation of cosmetic substances and products became crucial. This is maintained in
the EU Cosmetics Directive (EC) No 1223/2009. However, there is presently no in vitro test
guideline available to assess one of the endpoints, which are covered by in vivo reproductive
toxicity tests, e.g. developmental toxicity. Therefore, the Hand1-Luc EST in vitro test fordevelopmental toxicity has been developed to meet the requirements, which the original
mEST could not satisfy (6).
The validated protocol is using Hand1-ES (KOB1) cells transfected with a vector containing
the luciferase gene monitored by the Hand1 promoter. Hand1-ES (KOB1) cells aredifferentiated into cardiomyocytes during 120 hrs of culture (5 days). The proposed protocol
includes assays for estimating cytotoxicity followed by measurement of Hand1 promoter
activity (differentiation toxicity). This test does not require to sacrifice any pregnant animals,
and considerably reduces cost, time and required expertise (2). With the high positive
predictive value of the validated prediction model the new in vitro test is ideally fit to identify
chemicals with a high potential of developmental toxicity within a future IATA for in vitro
developmental toxicity in vitro.
Compared to the original EST, the Hand1-Luc EST is easier to perform, requires lower
quantity of chemicals, has a wider applicability (because the prediction model is based on ahigher number of chemicals and Hand1 gene alteration data can be used to create AOP) and5

Final February 2018considers concentrations in the culture medium close to the saturated free concentration infetuses measured in developmental toxicity studies in vivo. Therefore, the test allows to
identify positive embryotoxicants with high confidence and could become a high-throughput
test for eliminating true developmental toxicants and serve as a tool for screening purposes.
However, to predict developmental toxicity more accurately, this test will need to be
combined with other in vitro assays in an IATA.
A short summary is provided to describe the mechanism of developmental toxicity, which iscovered by the Hand1-Luc assay.
Figure 1 shows the possible pathways and genes related to Hand1.

If a chemical affects one of these genes, it may be detectable by measuring Hand1 gene
expression. In downstream pathways, Hand1 is related to the development of different
organs and the depletion or reduction of its expression is reported to have a negative effecton development. Chemicals have different individual modes of action, since some may affectspecific pathways (enzyme inhibition), others may affect a particular physiological function(for example, ion channel inhibition), and others that may affect DNA synthesis (enzyme
inhibition or intercalating agent).
In the Hand1-Luc EST, chemicals are applied to the cells from the beginning of differentiation
when the three primordial tissues are not yet formed (day 0= day 3.5 in vivo) until day 5 (=8.5
in vivo) when mESC are differentiated into cardiomyocytes (mesoderm induction). Thus,
depending on the mode of action of chemicals, it would be possible to cover a large numberof organs formed or specific organs only. Indeed, if the genes playing important roles in thethree germ layer formation are affected by the chemical, then all the downstream genes
governed may affect the ect-, endo- and mesoderm layer This may be measured by analteration of the Hand1 gene expression since the mesoderm would be also affected.
In this assay, the measurements are performed 120 hrs after inducing the ES cells into
cardiomyocytes. During this time window, the heart, the placenta, the neural tube and the ear
are formed. The somite segmentation has begun and the first branchial arch has maxillary6

Final February 2018
and mandibular components at day 8.5. The limb buds are however not yet visible until day 9.
Hand1 gene is involved in the development and differentiation of heart, limbs and facialbones (7, 8, 9, 10). Thus, chemicals triggering malformations in these organs may be
detected with the Hand1 assay. However, some chemicals may alter genes expressed 120
hrs. (5 days) after the beginning of differentiation and may not be detected by this assay.
Concerning species differences, the Hand1 gene is well preserved among different mammal
species and the amino acid sequence homology between mouse and human is 92% (NCBI
homology alignment browser; Basic Local Alignment Search Tool (BLAST)).
As suggested before, a combination with other tests analyzing the development of otherorgans (bones, neurons, digestive gut) will be crucial to be able to cover prenatal
development. For example, three different tests covering the three different germ layers(endo-, meso-, ectoderm) would allow for the understanding of the possible pathways
involved in abnormal development and provide information for identifying AOPs.
Evaluation Criterion 2: The relationship between the test method endpoint(s) and thebiological effect and to the toxicity of interest should be addressed, describing
limitations of the test methods.
Differentiation is measured by luciferase activity and thus chemicals interfering withluciferase protein should not be tested. Protease inhibitors cannot be detected in the Hand1Luc EST since protease activity is used to assess cell viability.
Another limitation is that the Hand1-Luc EST can not evaluate the effect of metabolites dueto the inability of ES cells to metabolize compounds. Metabolism is an important issue of allin vitro toxicity tests.
The experts of the PRP concluded that users of the test must be aware of these general
limitations of all of the currently available in vitro developmental toxicity tests.
Evaluation Criterion 3: A detailed test method protocol should be available
Questions of the experts addressed the appropriateness of the final volume of the vehicles.
This point has been detailed in the revised version of the protocol. The highest concentrationof PBS (-) that does not alter differentiation is 5% and for DMSO it is 0.1% (lead laboratorydata). Moreover, in many in vitro tests the highest test concentration is 1000μg/ml. Thus, if
the molecular weight of a test chemical is 200, then the final concentration would beequivalent to 5mM, a very high concentration.
As far as the identification of positive and negative results is concerned, detailed information
is provided in the revised version of the protocol, including the acceptance criteria and thecalculation used to fit curves to obtain the IC50 and ID50 values.
The experts of the PRP concluded that the protocol appears complete and adequate in detailfor a laboratory to conduct the study.
Evaluation Criterion 4: Within- and between-laboratory reproducibility of the test
method should be demonstrated
Since in the first draft provided no measure of variability (e.g. coefficient of variation), all CV
values have been added in the appropriate appendix to the revised validation report.
Moreover, the report mentioned several times discrepancies between the laboratories
regarding the establishment of the maximum dose. Therefore, a paragraph has been added
to warn the experimenter to pay particular attention during the precipitation evaluation
especially for DMSO dissolved liquids where small bubbles could be hard to distinguishinside the assay medium.
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Final February 2018
The experts of the PRP concluded that the additional improvements provide excellent
information on the within- and between-laboratory reproducibility.
Evaluation Criterion 5: Demonstration of the test method’s performance should bebased on testing of representative, preferably coded reference chemicals
The PRP concluded that a sufficiently high number of representative, coded chemicals (28)was used in the validation study to evaluate the performance of the method. Details of thechemical selection procedure are described in detail in the “chemical selection report”.
Some misclassifications have been noted when comparing in vitro and in vivo data. The mostobvious reason is when the chemical interferes with a pathway not related to Hand-1.
Another reason is when the chemical disturbs the development during a period which is notcovered by the Hand-1 Luc test. However, this cannot yet be taken into account due to lackof information on AOPs in the field of developmental toxicity both in experimental animals
and humans.
The experts of the PRP concluded that the selection of test chemicals to demonstrateperformance of the assay was appropriate.
Evaluation Criterion 6: Accuracy or predictive capacity should be demonstrated using
representative chemicals. The performance of test methods should have been
evaluated in relation to existing relevant toxicity data as well as information from therelevant target species.
The comments of the experts were quite positive, e.g., the prediction model uses only one
equation and the solubility of the chemicals is taken into account for the prediction whichbecomes a way to express the solubility of chemicals in the body fluids. The prediction model
has been established with a high number embryotoxicants (71) covering a wide spectrum of
mechanisms of toxicity (11).
An evaluation in relation to the target species is quite difficult since the developmental toxicity
of chemicals is very variable among different animal species and humans. However, in thecurrent validation study all data banks and publications that are publicly available andcovering the endpoint developmental toxicity have been properly taken into account by the
managers of the validation study in order to determine the performance of the test method.
Thus, the members of the PRP were satisfied with the determination of accuracy, predictive
capacity and the way in which existing data on developmental toxicity data from relevant
species were taken into account.
Evaluation Criterion 7: All data supporting the assessment of the validity of the test
method should be available for expert review
All data were available to the experts for review. The experts noted that, in several
experiments, ID50 values were higher than IC50 and that this this particular issue was notdiscussed in the validation study. Therefore, the relevance of such data should be explained
and their relevance for determination of the performance of the test.
It was explained that this phenomenon was also observed in the original EST especially for
negative chemicals that do not show a gap between the IC50 and the ID50. The VMT tried to
improve this point by operating measurements in the same plate which was proved to be agood way. During the validation study, they also faced this problem and tried to correct it asmuch as possible by implementing the curve fitting (2 and 3 parameter curve fittings)
according to recommendations by VMT members. As a result, a better ratio IC50/ID50 (see thevalidation report) was obtained. However, in some cases the IC50 value was still lower thanthe ID50. This may be due to the fact that since two different devices were used (luminometerand fluorometer) measuring two different endpoints (cytotoxicity and luciferase activity), the8Final February 2018
resulting measurements were not identical for IC50 and ID50. Although this may happen, thegap observed when the ID50 was higher than the IC50 is not dramatically high and does notcompromise the validity of the data.
Thus the members of the PRP were satisfied with the availability of the data and the
explanations given by the VMT.
Evaluation Criterion 8: Ideally, all data supporting the validity of a test method shouldhave been obtained in accordance with the principles of Good Laboratory Practice(GLP)
Since there were some doubts that the validation study had been conducted according toprinciples of GLP, the VMT provided the following explanation:
The validation report has been corrected in part 2.5 (Quality Check monitoring). According to
the OECD guidance 34, p21: “Clear and comprehensive standardized test method protocols,
preferably in compliance with GLP Principles, together with standard operating procedures(SOP), as appropriate. This should include a description of the test system, exposure
conditions, dose selection procedures, endpoint(s) assessed, measurements taken,
specialized equipment or supplies that may be needed, measures of variability, the way inwhich the results are calculated and expressed, and the use of positive and negative controls
and other performance checks.” (1)During the validation, the quality check was carefully operated by an independent expert. An
example of an empty quality check sheet is available for each phase. The QC sheet, theprotocol and the calculation sheet contain, what is required by the OECD GD 34:- Description of the test system: Described in the protocol and in the validation report- exposure conditions: described in the protocol- dose selection procedures: described in the protocol- endpoint(s) assessed: described at the end of the protocol and revealed by the calculation
sheet provided- measurements taken: described in the protocol- specialized equipment or supplies that may be needed: described in the protocol- measures of variability: defined and calculated automatically in the calculation sheet- the way in which the results are calculated and expressed: described at the end of the
protocol and the calculations used in the excel sheet were validated- the use of positive and negative controls and other performance checks: 5-FU was used asa positive control. No negative control was used.
Finally, since all the participating laboratories possess the GLP certification, the following
sentence was added at the end of part 2.5:”Finally, all the participating laboratories possess
the GLP certification. The present study was conducted in the spirit of Good Laboratory
Practice (GLP).”Taking into account these detailed explanations, the experts of the PRP concluded it appears
that the answer is “yes.”
Evaluation Criterion 9: Applicability domain of the validity of the test method should
be defined for expert review
There is some evidence that the Hand1 gene and its function is very similar between humanand mouse. The applicability of the Hand1-Luc EST is restricted to the examination of all thepathways related to the Hand1 gene (formation of the heart, limbs and craniofacial bones)
and the early period of development (from 3.5 to 8.5 days after fertilization in the mouse).
The Hand1-Luc EST can also detect embryotoxicity by disruption of genes involved in thedevelopment of the three germ layers (Ectoderm, Mesoderm and Endoderm) due to the9Final February 2018
exposure to chemicals in and after the undifferentiated stage.
The PRP experts concluded that the documents provided are demonstrating that the VMT of
the validation study have extensively evaluated the applicability domain of the test method
and that the applicability domain is sufficiently described in the revised validation report andthat, therefore, the answer is “yes”.
Evaluation Criterion 10: Proficiency chemicals should be set up in the proposed
protocolApparently due to language problems the VMT misunderstood this point. As a consequence,a list of proficiency chemicals has been added to the protocol. Therefore, the PRP experts
concluded that the apparent error has been corrected properly in the revised protocol.
Evaluation Criterion 11: Performance standard should be set up with proposed
protocolAs described for the proficiency chemicals definition, apparently due to language problems
the VMT misunderstood this point. As a consequence, the performance standards have been
added in Appendix 44 of the revised validation report.
Therefore, the PRP experts concluded that the apparent error has been corrected properly inthe revised protocol.

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Final February 2018
Evaluation Criterion 12: Advantages in terms of time, cost and animal welfare
Since this information is missing in the validation report, the VMT provided the following
information on the Hand1-Luc EST, which the PRP experts accepted as adequate:
The following table shows the approximate cost for one run with one chemical
Material

Original EST

Hand1-Luc EST

D3 (600$/vial)

600$

/

3T3 (600$/vial)

600$

/

KOB1-ES (Hand1)(2200$/vial)

/

2200$

1$/Petri dish8$/

1$/ 60cm plate

8$

11$/ 24 well plate88$/

6$/ 96 well plate

100$

/

15$/96U well white
plate

/

15$

Chemical

50$/100mg~300 mg(=150$)~30 mg (=15$)

Assay Medium

2$/ml

500 ml (=1000$)

30ml 0$ (contained in
the 2200$ kit)

Maintenancemedium3$ +1$(LIF)/ml/

50 ml 0$ (contained in
the 2200$ kit)

2$ + 1$(LIF)/ml

30$

MTT assay

70$/100mg

15mg=21$/

Cell titer fluor

8$/µl

/

80$

Steady glo

13$/ml/104$

Total with cells
purchase

2605$

2414$

Total without cells
purchase

1405$

214$ + ~300$ (medium)

price
Cells

Plates/= 514$

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Final February 2018
Furthermore, with the time that can be saved the human resources becomes much cheaper
for the Hand1-Luc EST as shown below.
Time
Human
resources ($)

1hr ~ 90$

Original EST

Hand1-Luc EST

4 days with 4 hours
per day: 16 hours

2 days, 1 hours perday: 2 hours

1440$

180$

In a near future, the kit should be cheaper than it is and an option available is to sell cells
only once by conclusion of Material Transfer Agreement that allows the user to create its own
stock.
Finally, raising the number of chemicals does not affect very much the time to conduct theHand1-Luc EST especially during cell seeding.
Evaluation Criterion 13: Completeness of all data and documents supporting theassessment of the test method
The data and documents supporting the assessment of the new method are complete andallow a sufficient evaluation of the validity of the method. The experts of the PRP havetherefore concluded that in general the documents are complete.
Evaluation Criterion 14: Validation management and conduct
The management and conduct of the multi-center validation study of the Hand1-Luc EST by
the VMT were excellent and they are exceeding the usual standard of multi-center studies as
far as communication among participants and VMT is concerned and also data exchange
and analysis and implementing quality assurance.
Therefore, the experts of the PRP are concluding that the answer is “yes” based on thesummaries of the face-to-face meetings and the conference calls.

Conclusions
The panel concluded that the reproducibility and predictivity of the Hand1-Luc EST assay issufficient to support its use as an in vitro test to identify with high confidence chemicals which
are toxic to prenatal development. It could become a high-throughput test for eliminating
developmental toxicants early within the screening of chemicals to be used in patents andconsumers and to which workers and the environment are exposed. The panel also
concluded that compared to in vivo testing for developmental toxicity the new in vitro test
does not require to sacrifice pregnant animals, considerably reduces cost, time and required
expertise. The panel proposed that with the high positive predictive value of the validated
prediction model the new in vitro test ideally fits to identify chemicals with a high potential ofdevelopmental toxicity within an IATA for assessing developmental toxicity in vitro. In thisintegrated strategy, negative results in the Hand1-Luc EST assay would require further
testing in vitro or in vivo animals.
Thus, it should not be used as a stand-alone assay.

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Final February 2018

Appendix 1
Independent Peer Review Panel Members
Horst Spielmann MD, Peer Review Panel Chairman,
Professor, Inst. Pharmacy, FU Berlin, Germany, horst.spielmann@fu-berlin.de
Horst Spielmann is Professor for Regulatory Toxicology in the Institute for Pharmacy, Facultyof Biology, Chemistry, and Pharmacy at the Freie Universität Berlin in Berlin, Germany. He is
the former and first head of the National German Center for the Validation of Alternative
Methods (ZEBET) at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany,
where he directed the standardization and international validation of several in vitro methods
and also served as the head of the Department of Scientific Services. He served asGermany’s first national representative on the Scientific Advisory Committee for theEuropean Centre for the Validation of Alternative Methods at the European Commission’s
Joint Research Centre in Ispra, Italy, and has served as an expert for many years for theEuropean Commission’s Framework Programs on alternatives to animal testing and in vitro
toxicology. In December 2012, he was appointed as the Animal Welfare Officer for the Stateof Berlin.
Roland Buesen DVM, BASF SE, Ludwigshafen, Germany, roland.buesen@basf.com,
Roland Buesen, Ph.D., is a veterinary specialist for pharmacology and toxicology withextensive experience in basic and applied health science research areas such as toxicology
and cell biology. Since 2007, he works as a study director for several in vivo toxicity assays
according to OECD and other guidelines including teratogenicity and reproductive toxicology
assays. Before 2007, he gained qualified practical experience in all aspects of cell culture
techniques, immunology assays, high performance liquid chromatography, as well as
development and experimental improvement of in vitro assays. He holds several publications
in refereed journals dealing with in vitro as well as in vivo toxicology issues. Dr. Buesen has
been speaker at national and international meetings.
Elise Grignard PhD, EURL ECAM, EU JRC, Ispra, Italy, Elise.GRIGNARD@ec.europa.eu
Elise Grignard is a Scientific Officer at the, at the European Union Reference Laboratory foralternatives to animal testing (European Commission, Directorate General Joint ResearchCentre), contributing to the identification and evaluation of in vitro methods for their potential
validation, in the field of endocrine disruption and reproductive toxicity. She is a member of
the ICCVAM Developmental and Reproductive Toxicology Working Group, and contributes to
the OECD work through the Validation Management Group on Non-Animal Testing and theAdvisory Group on Endocrine Disrupters Testing and Assessment.
Pertti Hakkinen PhD, National Library of Medicine, National Institutes of Health,Bethesda, USA, hakkinenp@mail.nlm.nih.gov,
Pertti Hakkinen is the Senior Toxicologist and the Toxicology and Environmental Health
Science Advisor (to the Director) in the Division of Specialized Information Services of the
National Institutes of Health’s National Library of Medicine (NIH NLM). He provides
leadership in the development of new resources in exposure science, toxicology, risk
assessment, and risk communication, and enhancements to existing resources in thesefields. Also, he is NIH NLM’s Principal Agency Representative on ICCVAM. In addition, he is
the project leader for the Chemical Hazards Emergency Medical Management (CHEMM) tool,
an online information resource for alternatives to animal testing (ALTBIB), and the recently
updated and enhanced ToxTutor® educational resource. Further, he is an Adjunct Associate
Professor in Preventive Medicine & Biostatistics in the F. Edward Hébert School of Medicine
13Final February 2018at the Uniformed Services University of the Health Sciences (USU) in Bethesda and a coleader of the Environmental Health Sciences graduate level course offered by theFoundation for Advanced Education in the Sciences (FAES) at the NIH.
Koichi Imai, Professor, Osaka Dental University, Osaka, Japan,
imai@cc.osaka-dent.ac.jp
Professional career2014 Professor of Osaka Dental Univ. (Dept. of Biomaterials), Osaka, Japan
2010 Asso. Prof. of Osaka Dental Univ. (Dept. of Biomaterials)
2005 Head of Analytical Instrument Facility, Inst. Dental Research, Osaka Dental Univ.
Head of Low Temperature Facility, Institute of Dental Research, Osaka Dental Univ.
Head of Laser Facility, Institute of Dental Research, Osaka Dental Univ.
1989 Head of Tissue Culture Facility, Institute of Dental Research, Osaka Dental Univ.
Scientific positions
2017 Board member of ISO/TC106 (Dentistry)
2016 Vice-president of the "Japanese Association of Regenerative Dentistry"
2016 President of the "Nano Biomedical Society"2014 Director of the "Japanese Society for Dental Materials and Devices"
2014 Director of the "Osaka Odontrogical Society"2012 Director of the "Bio-Integration Society"2007 Director of the "Japanese Society for Alternative to Animal Experiments"
2003 Board member of ISO/TC194 (Biological evaluation of medical devices)

Norihito Matsumoto PhD, Ono Pharmaceutical Co., Ltd., Osaka, Japan,

no.matsumoto@ono.co.jp,
Norihito Matsumoto is the head of Mechanistic Toxicology Group in Safety ResearchLaboratories of Ono Pharmaceutical Co., Ltd. His areas of expertise are mechanism
research in toxicology and exploratory toxicology including hepatotoxicity, gen-toxicity,
phototoxicity and embryotoxicity during the early stage of drug development. He is thecouncilor of the Japanese Society of Toxicology and the Japanese Society for Alternatives toAnimal Experiments.

Hyung Sik Kim, Professor, Sungkyunkwan University, Soul, Korea, hkims@skku.edu
Professional career

Since 2013 Professor, School of Pharmacy, SungKyunKwan University
2012 - 2013 Professor, College of Pharmacy, Pusan National University.
2007- 2012 Associate Professor, College of Pharmacy, Pusan National University.
2006 - 2009 Vice Dean, College of Pharmacy, Pusan National University.
2003 - 2006 Assistant Professor, College of Pharmacy, Pusan National University
2002 - 2003 Visiting Fellow in National Institutes of Health (NIH), USA
1997 - 2003 Senior Scientist, National Institute Tox. Research (NITR), Koran FDA, Korea
1994 -1996 Teaching Assistance, College of Pharmacy, SungKyunKwan U., Suwon, Korea
Research areas: Chemical carcinogenesis/mechanism of action, Epigenetic transgenerational study for cancer development, Screening & testing for novel anticancer agents(HDAC inhibitor, Topo-II inhibitors), Development of biomarkers associated with chemical
carcinogenesis, Metabolomics profiling associated with aging and inflammation, Action
mechanism for toxicities of environmental xenobiotics in molecular and cellular levels
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Appendix 2
Acknowledgements
The Peer Review Panel members gratefully acknowledge Florian Le Coz from Sumitomo
Chemical Co Ltd. Osaka, for his invaluable assistance with translation and editing during thepeer review process. The Panel also acknowledges the members of the Validation
Management Team for the completeness of the validation study reports, their cooperation
and responsiveness to requests for additional information and analyses, and their responsive
consideration of suggestions and updating of the validation study reports and test method
protocol. Finally, the Panel expresses its appreciation to Dr. Hajime Kojima and his staff at
the National Institute of Health Sciences for their excellent support and arrangements for thepeer review panel meetings

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Final February 2018

Appendix 3
Glossary
5-FU

5-Fluorouracil

3T3

BALB/c 3T3 cells derived from mouse embryonic fibroblast cells(American Type Culture Collection)

BfR

Bundesinstitut für Risokobewertung, Federal Institute for Risk
Assessment (G)BASF

Badische Anilin und Soda Fabrik AG, Ludwigshafen (G)CellTiter fluor

Cell Viability Assay is a non-lytic, single-reagent-addition fluorescenceassay that measures the relative number of viable cells in a population.

D3

Mouse embryonic stem cells clone D3

DMSO

Dimethyl Sulfoxide

EST

Embryonic Stem Cell Test

EURL ECVAM

European Union Reference Laboratory at the JRC, Ispra (I)

Hand1

Heart and neural crest derivatives expressed 1

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative
Methods

IC50

Cytotoxicity was expressed as the concentration of chemical that
reduces the viability of cells to 50%

ID50

The inhibition of differentiation was expressed as the concentration of
the test chemical that reduces the luminescence by 50%

JaCVAM

Japanese Center for the Validation of Alternative Methods

KoCVAM

Korean Center for the Validation of Alternative Methods

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

Steady-Glo

Product designed for a high-throughput quantitation of luciferase
expression in mammalian cells is commonly performed by batchprocessing of 96- and 384-well plates.

VMT

Validation Management Team

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Final February 2018

Appendix 4
References1.

OECD. Guidance Document No. 34: Guidance Document on the Validation andInternational Acceptance of New or Updated Test Methods for Hazard Assessment.
Organization for Economic Cooperation and Development, Paris, France, 2005
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2005)14&doclanguage=en

2.

Le Coz F., Suzuki N., Nagahori H., Omori T., Saito K. (2015): Hand1-Luc embryonicstem cell test (Hand1-Luc EST): a novel rapid and highly reproducible in vitro test forembryotoxicity by measuring cytotoxicity and differentiation toxicity using engineeredmouse ES cells. J. Toxicol. Sci. 40(2): 251-261

3.

Hand1-Luc EST Validation Management Team. Hand1-Luc Embryonic Stem Cell Test
(Hand1-Luc EST), Draft Protocol Ver.10 E, 2017, February 1st

4.

Hand1-Luc EST Validation Management Team. Validation Study Report on Hand1Luc EST (Embryonic Stem Cell Test) As a Non-animal Test Evaluating
Developmental Toxicity of Chemicals, March 2017.

5.

Hand1-Luc EST Validation Management Team. Validation Study Report on Hand1Luc EST (Embryonic Stem Cell Test) As a Non-animal Test Evaluating
Developmental Toxicity of Chemicals, October 2016.

6.

Bremer S., Hartung T. (2004): The use of embryonic stem cells for regulatory
developmental toxicity testing in vitro- the current status of test development. Curr.
Pharm. Des. 10(2): 2733-2747

7.

Firulli BA., Fuchs RK., Vincentz JW., Clouthier DE., Firulli AB. (2014): Hand1
phosphoregulation within the distal arch neural crest is essential for craniofacial
morphogenesis. Development 141(15): 3050-3061

8.

Laurie LE., Kokubo H., Nakamura M., Saga Y., Funato N. (2016): The transcription
actor Hand1 is involved in Runx2-Ihh-regulated endochondral ossification. PLOS
ONE doi 10.1371: 1-10

9.

McFadden DG., Barbosa AC., Richardson JA., Schneider MD., Srivastava D., Olson
EN. (2005): The Hand1 and Hand2 transcription factors regulate expansion of theembryonic cardiac ventricles in a gene dosage-dependent manner. Development132(1): 189-201

10.

Reamon-Buettner, SM., Ciribilli, Y., Traverso, I., Kuhls, B., Inga, A., and Borlak, J.
(2009): A functional genetic study identifies HAND1 mutations in septation defects of
the human heart. Hum. Mol. Genet. 18(19): 3567-3578

11.

Nagahori H., Suzuki N., Le Coz F., Omori T., Saito K. (2016). Prediction of in vivo
developmental toxicity by combination of Hand1-Luc embryonic stem cell test andmetabolic stability test with clarification of metabolically inapplicable candidates.
Toxicol. Lett. 259: 44-51

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