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Nome societagrave

International Symposium on

Alternative in vitro methods to characterize the role of

Endocrine Active Substances (EASs)

in hormone-targeted tissues

December 17th

2012

Istituto Superiore di Sanitagrave mdash ISS

Aula Pocchiari

Rome Italy

Nome societagrave

Welcome address

Dear Colleagues

it is a great pleasure to welcome you at the CAAT-IPAM-ISS Symposium on ldquoAlternative in vitro meth-

ods to characterize the role of Endocrine Active Substances (EASs) in hormone-targeted tissuesrdquo jointly

organised by the Department of Veterinary Public Health and Food Safety and the Department of Envi-

ronment and Primary Prevention whose activities include the use of alternative in vitro methods to in-

vestigate key issues of the role of EASs in endocrine-regulated targets

The interest in alternative in vitro methods for toxicity testing has raised in the last years within the sci-

entific community and has been moved forward also by the increasing societal demand on the reduction

of animal use coupled with the requirements in high quality evaluations of chemical safety In particular

the processes of screening and prioritization of chemicals may be strongly supported by an integrated

use of alternative (either in silico or in vitro) methods

To this end the European Commission recently adopted new legislative tools aimed to improve the use

of alternative methods as the Registration Evaluation Authorization and Restriction of Chemicals

(REACH EC No 19072006) the Revised Directive on the Protection of Animals used for Scientific Pur-

poses (EU 632010) and the new Cosmetics Regulation (EC No 12232009) In all of them the 3Rs

(Refinement Reduction Replacement) principle is emphasized in many articles as part of the new toxi-

cological requirements

Therefore we are grateful to the Center for Alternatives to Animal Testing (CAAT) Europe and the Ital-

ian Platform on Alternative Methods (IPAM) for their efforts to promote and disseminate alternative

methods and to contribute to the successful organization of this Symposium

Along with a pleasant stay in Rome we wish all participants a fruitful exchange of ideas

Umberto Agrimi Head of the Department of Veterinary Public Health and Food Safety

Loredana Musmeci Head of the Department of Environment and Primary Prevention

Nome societagrave Aims of the Symposium

This Symposium is aimed to emphasize the role of alternative methods in search for potential Endocrine

Disrupting Chemicals (EDCs) or Endocrine Active Substances (EASs)

Under the REACH Regulation EAS identification is one of the main concern since within the definition of

Substances of Very High Concern (SVHC Article 57 of Regulation (EC) No 19072006 ) are included sub-

stances which are i) Carcinogenic Mutagenic or toxic to Reproduction (CMR) ii) Persistent Bioaccumu-

lative and Toxic (PBT) or very Persistent and very Bioaccumulative (vPvB) and iii) identified on a case-

by-case basis from scientific evidence as causing probable serious effects to human health or environment

of an equivalent level of concern as those above (eg endocrine disrupters)

Nowadays EASs are challenging classical concepts in toxicology due to their suggested ldquolow dose ef-

fectsrdquo andor ldquonon monotonic dose responsesrdquo leading for these reasons innovative approaches in risk

assessment

The Symposium programme is divided in four sessions aimed to give an overview of the state-of-art in EAS

investigation by alternative methods highlighting the academic regulatory and industrial points of view as

well presenting critical issues in human targets of endocrine disrupters and their metabolic fate

In particular recent advances in the field of biokinetics as well as in the characterization of new molecular

and cellular biomarkers of different reproductive- and hormone-targeted tissues will be presented by in-

vited international experts

A general discussion among all participants at the end of the symposium is essential part of the program

Isabella De Angelis Department of Environment and Primary Prevention

Stefano Lorenzetti Department of Veterinary Public Health and Food Safety

Acknowledgements

We acknowledge the Scientific Committee and the Scientific Secretariat for supporting the Symposium or-

ganization

Nome societagrave Scientific Committee

Isabella De Angelis Istituto Superiore di Sanitagrave - ISS Rome Italy

Franca Fassio MerckSerono Ivrea Italia

Simonetta Gemma Istituto Superiore di Sanitagrave - ISS Rome Italy

Stefano Lorenzetti Istituto Superiore di Sanitagrave - ISS Rome Italy

Laura Narciso Istituto Superiore di Sanitagrave - ISS Rome Italy

Costanza Rovida Center for Alternatives on Animal Testing - CAAT Europe Konstanz Germany

This event has been supported by unrestricted grants of

Scientific Secretariat


SCIENTIFIC PROGRAMME

INTRODUCTIVE SESSION Chairpersons Isabella de Angelis (ISS-Rome) and Stefano Lorenzetti (ISS-Rome)

850-910 Thomas Hartung (CAAT-Baltimore)

Endocrine disruption as the pilot of mapping the human toxome

910-930 Costanza Rovida (CAAT-Konstanz)

Implementation of regulatory issues

930-950 Alberto Mantovani (ISS-Rome)

Endocrine Active Substances EASs understanding modes of action for risk assessment

950-1010 Johann Steinkellner (EFSA-Parma)

Exploration of alternative methods for toxicity assessment of pesticide metabolites

1010-1030 Serena Cinelli (RTC-Pomezia Rome)

Improving test methods in the spirit of the 3Rs the point of view of a contract research

organization

1030-1100 Coffee break

SESSION 1 EASs in reproductive-targeted tissues Chairpersons Simonetta Gemma (ISS-Rome) and Marcello Spanograve (ENEA-Rome)

1100-1120 Stefano Lorenzetti (ISS-Rome)

A prostate perspective on male fertility and EASs from toxicogenomics to phenotypic

anchoring

1120-1140 Marcello Spanograve (ENEA-Rome)

Human sperm (epi)genetic biomarkers to assess the impact of EASs on male reproducti-

ve function

1140-1200 Luana Paulesu (Univ Siena-Siena)

In vitro effects of EASs in human placenta

SESSION 2 EASs in different hormone-targeted tissues Chairpersons Alberto Mantovani (ISS-Rome) and Paolo Marzullo (Univ Novara-Novara)

1200-1220 Igor Bendik-Falconnier (DSM-Basel)

Endocrine active nutrients explored in human bone cell cultures

1220-1240 Robert A Smith (Univ Glasgow-Glasgow)

The use of cell models in determining neuronal responses to EASs

1240-1300 Arti Ahluwalia (Univ Pisa-Pisa)

Dynamic in-vitro organ models of metabolism

1300-1330 General discussion

1330-1430 Lunch

SESSION 3 EASs and kinetics Chairpersons Thomas Hartung (CAAT-Baltimore) and Emanuela Testai (ISS-Rome) 1430-1450 Emanuela Testai (ISS-Rome)

The role of biokinetics in in vitro tests and in the interpretation of results

1450-1510 Freacutedeacuteric Yves Bois (Univ Compiegne-Compiegne)

Physiologically-based modeling of ovarian steroid hormones synthesis for EASs health risk

assessment

1510-1530 Daniel R Dietrich (CAAT-Konstanz)

EASs contra human amp environmental health relevant or playground for merchants of do-

om


INTRODUCTIVE SESSION

Thomas HARTUNG

CAAT Johns Hopkins University Baltimore US amp CAAT-Europe University of Konstanz Germany

thartungjhsphedu

Thomas Hartung MD PhD is professor and chair for toxicology pharmacol-

ogy molecular microbiology and immunology at Johns Hopkins Bloomberg

School of Public Health Baltimore MD and University of Konstanz Ger-

many He directs the Centers of Alternative to Animal Testing (CAAT) of

both universities From 2002-2008 he led the European Center for the Valida-

tion of Alternative Methods (ECVAM) of the European Commissions Joint

Research Centre in Ispra Italy He has authored more than 350 articles

ENDOCRINE DISRUPTION AS THE PILOTE OF MAPPING THE HUMAN TOXOME

The US National Academies National Research Council report from 2007 Toxicity Testing in the 21st Century A vi-

sion and a strategy has created an atmosphere of departure in the US It suggests moving away from traditional (animal)

testing to modern technologies based on pathways of toxicity These pathways of toxicity could be modeled in relatively

simple cell tests which can be run by robots The goal is to develop a public database for such pathways the Human

Toxome to enable scientific collaboration and exchange

There is a continuously growing awareness about Tox-21c in all stakeholder groups It was first embraced by scientists

and in the US Most importantly the US agencies followed fast on the 2007 NASNRC report the Tox-21 alliance in

2008 (paper in Science first-authored by now NIH head Francis Collins EPA made it their chemical testing paradigm in

2009 FDA followed most evidently with the Science article by FDA head Margret Hamburg in 2011) Chemical and con-

sumer product industry got engaged eg with the Human Toxicology Project Consortium In Europe all this is rather

delayed with some adaptation of the vocabulary but not necessarily grasping the new approach This is not alternative

methods under a new name However interest is lately increasing strongly in Europe

Tox-21c suggests moving to a new resolution ie pathways of toxicity The problem is that the respective science is only

emerging What will be needed is the Human Toxome as the comprehensive pathway list an annotation of cell types spe-

cies toxicant classes and hazards to these pathways an integration of information in systems toxicology approaches the

in-vitro-in-vivo-extrapolation by reversed dosimetry and finally making sense of the data most probably in a probabilistic

way The NIH is funding since September 2011 by a transformative research grant The Human Toxome project led by

CAAT The project involves US EPA ToxCast the Hamner Institute Agilent and several members of the Tox-21c panel

The new approach is shaped around pro-estrogenic endocrine disruption as a test case

Early on the need for quality assurance for the new approaches as a sparring partner for their development and implemen-

tation has been noted Formal validation as developed for the first generation of alternative methods can only partially

serve this purpose For this reason the Evidence-based Toxicology Collaboration (EBTC) was created in the US and

Europe in 2011 and 2012 respectively This collaboration of representatives from agencies industry academia and stake-

holder groups aims to develop tools of Evidence-based Medicine for toxicology The secretariat is run by CAAT the first

conference was held in early 2012 hosted by US EPA and working groups have started to address pertinent issues and

methodologies All together Tox-21c and its implementation activities including the Human Toxome and the EBTC

promise a credible approach to revamp regulatory toxicology

References Hartung T van Vliet E Jaworska J Bonilla L Skinner N and Thomas R Systems Toxicology ALTEX 2012 29 119-

128

Hartung T From alternative methods to a new toxicology Eur J Pharmaceutics Biopharmaceutics 2011 77338ndash349

Hartung T and McBride M Food for thoughthellip on mapping the human toxome ALTEX 2011 28 83-93

Leist M Hasiwa M Daneshian M and Hartung T Validation and quality control of replacement alternatives ndash current

status and future challenges Toxicological Research DOI 101039C2TX20011B

Hartung T Evidence based-toxicology ndash the toolbox of validation for the 21st century ALTEX 2010 27241-251

Hartung T Toxicology for the twenty-first century Nature 2009 460208-212

Hartung T A toxicology for the 21st century Mapping the road ahead Toxicol Sci 2009 109(1)18-23

Notes

Costanza ROVIDA

CAAT-Europe University of Konstanz Germany

costanzarovidachimiciit

Costanza Rovida is scientific Officer at the Center for Alternatives to Animal Testing in Europe

(CAAT-Europe) and project manager for REACH Mastery She took a degree

in Chemistry with specialisation in Analytical Chemistry in 1989 After a pe-

riod focussed on the optimisation of analytical techniques in the area of food

and environmental analyses she moved to a pharmaceutical Industry where

she learned about method validation plus toxicology and drug efficacy Con-

vinced that in vivo methods are not the right scientific answer to our toxico-

logical questions in the period 2005-2008 she worked at ECVAM (European

Centre for Validation of Alternative Methods) where she gained experience of

alternative methods applied in the area of skin and respiratory sensitisation

and she had the opportunity to participate in the working groups organised by

the European Commission for the implementation of REACH the latest

Regulation on the evaluation and authorisation of chemical substances She

was one of the pioneer members of CAAT Europe and since 2009 she has strived for the application of

in vitro methods for regulatory purposes Publications on this topic are many including a publication

on Nature (Hartung T and Rovida C Opinion Chemical regulators have overreached 2009 Nature

460) She is engaged in many scientific Committees that are active in the field of alternative methods

by organizing symposia and workshops to disseminate the 3R strategies She is also still very much

involved in the REACH process by following clients to be compliant with all the provisions of that

Regulation

IMPLEMENTATION OF REGULATORY ISSUES

The term ldquoendocrine disrupterrdquo (ED) was introduced in the early 1990s and later defined as (WHO 2002) ldquohellip an exogenous

substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact

organism or its progeny or (sub)populationsrdquo Possibility of perturbation of the endocrine system has been considered for a

long time since both the uterothophic bioassay (OECD TG 440) and the anti-androgenic screening test (OECD TG 441) were

originated in the 30rsquos

In the latest 20 years concern about endocrine disruptors is definitely increasing and in the European Union there are at the

moment three important Regulation that specifically ask to address this effect The first one was probably REACH

(Regulation 19072006) which includes the endocrine disruptor activity as a condition to include a substance in the authoriza-

tion list In addition both Regulation 11072009 for Plant Protection Products and proposed new Regulation for Biocidal

products ask that approved substances should not have endocrine disruptor activity and for the definition both refers to Article

57(f) of REACH which does not provide a clear definition for getting to a unique and generally approved decision Regula-

tion 12722008 (CLP) adds the endocrine disruptor activity in Annex II regarding the description of the procedure to assign

the classification as reproductive toxicant in the area where ldquoweight of evidencerdquo is described

Even if endocrine disruptor activity is generally recognized as a serious effect there is no agreement about the methods that

should be applied for the assessment and when the activity for a substance is universally recognized as the case for Bisphenol

A the discussion is still open for the definition of the no effect dose

One of the problem is that endocrine disruption is not a toxicological endpoint in itself but rather a class of modes and

mechanisms of action There are a battery of in vitro tests available that must be used for initial screening but in the end con-

firmation in an intact organism is always required ie in vivo in rats even though it is well recognized that endocrine activity

is very different among species

State of the art in this topic has been recently presented in a document issued by an expert panel charged by the European

Commission Regardless the ambiguous conclusion of this report there is an urgent need to define a set of methods for this

class of substances Within the scope of REACH 18 substances have been now selected due to their concern of being endo-

crine disruptors Decision is expected in two years

Notes

Alberto MANTOVANI

Food and Veterinary Toxicology Unit Dept of Fodd Safety and Veterinary Public Health Istituto

Superiore di Sanitagrave Rome Italy

albertomantovaniissit

Alberto Mantovani (AM 1956 DVM MSc) is currently director of the Food

and Veterinary Toxicology Unit at the Italian National Health Institute (ISS)

The main research topics of the Unit are endocrine disrupters (ED) natural bio-

active substances trace elements and nanotoxicology the research activities

pivot on translating mechanistic research into risk (and risk-to-benefit) assess-

ment AM co-authored about 50 scientific papers on toxicology and risk as-

sessment in the 2006-12 period he co-ordinated the pilot national project on

ED (2000-2003)the research area IV (cross-cutting technologies) of the EU

project ReProTect (2005-10) on non-animal testing strategies for reproductive

toxicants and the national PREVIENI project (2008-11) on the development

and use of biomarkers to assess the impact of ED exposure Since 2003 AM is

an expert of the European Food Safety Authority (EFSA) member of

FEEDAP Panel (substances used in feeds) on 2003-12 member of PPR panel (pesticides) since July

2012 and expert collaborating to EFSA opinions on non-animal testing bisphenol A and TTC-

thresholds of toxicological concerns

ENDOCRINE ACTIVE SUBSTANCES UNDERSTANDING PATHWAYS FOR RISK ASSESSMENT

Alberto Mantovani Stefano Lorenzetti Cinzia La Rocca Laura Narciso Sabrina Tait

Endocrine disrupters (ED see the dedicated ISS web area httpwwwissitinte) are natural or man-made chemicals in

diet andor environment able to cause adverse effects in exposed organisms or their progeny by altering endocrine homeo-

stasis In a broader sense endocrine active substances (EAS) include all compounds modulating endocrine functions thus

EAS may also exert beneficial or protective effects The so-called ldquophytoestrogensrdquo are a recognized example of EAS that

may exert beneficial or adverse effects depending on the exposure level lifestage sex as well as on the interactions with

other EAS (see the EDID database at httpwwwissitinte) The endocrine system is a network regulating most body

functions the pre- and post-natal lifestages are particularly sensitive and long-term persistent effects may result from

early exposures to EAS From the toxicologist standpoint rather than an effect per se endocrine disruption is a group of

modes of action highly relevant to risk assessment due to their complexity and serious long-term (even trans-generation)

impact As a consequence the assessment of ED (and EAS) would likely progress together with the development of new

testing approaches that exploit system biology and integrate pathways into the classical hazard characterization More-

over up to now the toxicology of ED has largely pivoted around the effects on fertility reproductive development and

thyroid whereas comparatively limited attention has been paid till now to other relevant aspects such as the neuroendo-

crine and endocrine-immune interfaces and especially the impact on metabolic programming which might represent a

field of considerable public health significance The development of conceptual frameworks supported by robust scientific

is also needed to deal with long-debated issues in EDEAS assessment such as i) the relevance of low-dose effects and

non-monotonic responses (an EAS eliciting different hits with partly overlapping dose-response relationships ) or ii)

combined exposures to different EAS (is there something more than dose addition ) We present two examples of pub-

lished ISS studies providing mechanistic insights into risk assessment issues namely the use of transcriptomics to charac-

terize components of mixtures and the use of clinically relevant biomarkers to characterize EAS potentially interfering

with the same pathways

Acknowledgement the abstract has been elaborated within the frame of the Ministry of Health grant ldquoReach endocrine

disruptersrdquo

Notes

Johann STEINKELLNER

European Food Safety Authority EFSA - Pesticides Unit Parma Italy

JohannSTEINKELLNERefsaeuropaeu

Between 1994 and 2002 I have worked in the Department of En-vironmental Toxicology of the Institute for Cancer Research of the University of Vienna in the field of genetic toxicology (eg gene mutation assays with Salmonella typhimurium chromoso-mal aberration and micronucleus tests with plant cells cultivated hepatic cells in rats and primary human lymphocytes Comet As-says with a variety of cell types spectrometric and biochemical enzyme measurement methods and determination of GST geno-typing of humans (eg with PCR) I have been teaching in the field of genetic toxicology at the University of Vienna I have obtained a PhD in the field of anticarcinogensisantimutagenesis in humans I hold a master degree in toxicology and am a Eurotox registered member of the Austrian Society of Toxicology

From 2002 to 2007 I have worked as a toxicologist in the field of health effects and environmental ef-fects of ldquoExisting Chemicalsrdquo ldquoNew Chemicalsrdquo and Pesticides (Regulation 67548EEC and adapta-tions thereof Directive 91414EC) Risk Assessment of ldquoExisting Chemicalsrdquo (Regulation 79393EEC) and have contributed to the implementation of REACH Between 2007 and 2009 I have worked at the European Food Safety Authority (EFSA) as a toxicolo-gist responsible for human health risk assessment in the field of peer review of plant protection prod-ucts under Directive 91414EC

Since 2009 I am working as a toxicologist in EFSArsquos Pesticide Unit in support of EFSAs Panel on Plant Protection Products and their Residues (PPR) I have been involved beyond many other activi-ties in support of pesticide risk assessment for instance in the drafting of scientific opinionsguidance documents on neurotoxicity of deltamethrin dermal absorption exposure assessment for workers op-erators bystanders and residents cumulative risk assessment of pesticides and the toxicological rele-vance of pesticide metabolites

EXPLORATION OF ALTERNATIVE METHODS FOR TOXICITY ASSESSMENT OF PESTICIDE METABO-

LITES

The framework of evaluation and authorisation of chemical plant protection products and the active substances they contain

in the EU is laid down in Regulation (EC) No 11072009 and appertaining regulations Assessment of the risk for the

consumer requiring also the identification of metabolites and of degradates of the active substances is a major part of this

process

One of the outcomes of the evaluation of an application for use of an active substance on a crop is the establishment of two

residue definitions one for monitoring and one for dietary risk assessment While the residue definition for monitoring has

regulatory purposes for the enforcement of the MRLs (Maximum Residue Levels) and must reflect analytical practicalities

the residue definition for dietary risk assessment may be wider as its purpose is to assess consumer safety and it should

therefore include all metabolites and degradates of toxicological relevance

However in practice only the toxicological properties of the active substance are investigated through the range of

toxicological studies while only very limited information about the toxicological properties of metabolites and degradates is

available in the majority of cases In this context it is notable that subjecting all metabolites to the full testing scheme ap-

plied for active substances appears not feasible due to the sheer number of metabolites identified in many cases In addition

this would lead to a significant increase in the use of test animals in the field of pesticide risk assessment

Therefore EFSArsquos PPR Panel has developed a scientific opinion in which alternative testing methods were explored in re-

gard to their applicability for testing of pesticide metabolites

The opinion identifies the Threshold of Toxicological Concern (TTC) concept as and appropriate screening tool for assess-

ment of toxicity of metabolites and identified three critical steps in its application which are 1) estimation of the metabolite

level 2) evaluation of genotoxity alerts and 3) detection of potentially neurotoxic metabolites A TTC concept for acute

exposures was also established based on pesticide active substances to which an Acute Referenced Dose (ARfD) has been

allocated Assessment schemes both for chronic and acute dietary risk assessment are presented in the opinion using a com-

bination of the TTC approach with QSAR modelsread across and targeted testing

In addition to proposals for assessment of metabolites the opinion also describes how the proposed assessment tools could

be utilised for assessment of differential toxicity of pesticide isomers

The results obtained from the work on this opinion will be the basis for the development of a guidance document for toxic-

ity assessment of metabolites and isomers

Notes

Serena CINELLI

RTC - Research Toxicology Centre Rome ndash Italy

SCinellirtcit

Dr Serena Cinelli is Associate Scientific Director at Re-

search Toxicology Centre (RTC) a Contract Research

Organisation specialised in non-clinical safety studies

located in Pomezia (Rome) Italy She has more than 25

years experience in genetic and in vitro toxicology testing

for industry and she is author of numerous toxicology

reports submitted to international regulatory authorities

Dr Cinelli also acts as Contract Professor in Environ-

mental Mutagenesis and is author of numerous peer re-

viewed publications

IMPROVING TEST METHODS IN THE SPIRIT OF THE 3RS THE POINT OF VIEW OF A CONTRACT RE-

SEARCH ORGANIZATION

Serena Cinelli Germano Oberto Isabella Andreini

A multiple-level tiered approach to identify and characterize the hazards of Endocrine Active Substances (EAS) is suggested

by the most important international organisations which provide guidance for industry for the safety assessment of chemi-

cals Both OECD and EPA indicate that non-clinical development should include a screening phase in which in silico in

vitro and in vivo assays are performed to provide mechanistic data for hazard identification followed by an in vivo testing

phase to better characterize the identified risk

Based on current evidence scientists and regulators acknowledge that stand alone in vitro methods are not sufficient to relia-

bly predict in vivo effects due to the complicated nature of hormonal systems Nevertheless several of the available in vitro

methods can provide extremely important data to clarify in vivo mechanisms of action of EAS and can be used as alterna-

tives to in vivo assays traditionally employed for single endocrine mechanism and effect (eg Uterotrophic assay)

In order to obtain an effective reduction of animal use with the application of alternative in vitro methods it is necessary to

satisfy different steps such as a successful validation process inclusion into regulatory requirements acceptance by indus-

try and wide application in non-clinical Contract Research Organizations (CROs)

The role of CRO is increasingly important in application of alternative methods since the general trend of industry is to con-

tract out most of the non-clinical development In this respect CRO is the ideal candidate not only to run but also to validate

new alternative methods since understanding the needs of industry and regulatory framework becomes a key factor

However one strong hurdle for the CRO is the balance between the immediate investments and the delayed validation pro-

ject payback The CRO must keep in mind that industry may have a conservative approach driven by the risk aversion fear

that results from alternative in vitro approaches might be not readily accepted by regulatory authorities as the ones from in

vivo conventional studies

Different scenarios will be presented by the author with possible situations and proposed solutions to manage safety assess-

ment programs aware that a complete fulfilment of the 3Rs philosophy is not possible but a wise strategy of in vitro testing

selection and expert data interpretation can help to reduce and refine animal testing

Notes

Notes

SESSION 1

EASs IN REPRODUCTIVE TARGET TISSUES

Stefano LORENZETTI



stefanolorenzettiissit

Stefano Lorenzetti graduated in Biological Sciences (1991) and in Human

Nutrition Sciences (2003) is employed since 2006 at the Dpt of Food

Safety and Veterinary Public Health of the Italian National Health Institute

- ISS (Istituto Superiore di Sanitagrave) in Rome He has been lecturer (2006-

09) in Molecular Biology and Toxicology at the Faculty of Medicine of

University Tor Vergata of Rome at the post-graduated course of Sciences

of Nutrition Since January 2012 he has been appointed as a member of

the Italian national expert group of ldquoAlternative methods to animal experi-

mentationrdquo

He participated to different international project on the hazard and risk

characterization of environmental and dietary contaminants such as the EU

Integrated Project ReProTect (ldquoDevelopment of a novel approach in hazard and risk assessment or repro-

ductive toxicity by a combination and application of in vitro tissue and sensor technologiesrdquo) to develop

an integrated in vitro approach linking toxicogenomics to clinical biomarkers (phenotypic anchoring)

His research is focused on the set up of in vitro alternative methods to animal experimentation to study

the role of endocrine disruptorsEDCs or Endocrine Active SubstancesEASs such as bioactive com-

pounds of plant origin (eg polyphenols) and environmental and dietary contaminants (eg plasticizers

pesticides and biocides)

A PROSTATE PERSPECTIVE ON MALE FERTILITY AND Endocrine Active Substances FROM TOXI-

COGENOMICS TO PHENOTYPIC ANCHORING

Stefano Lorenzetti

Although prostate function is critical for male fertility in reproductive toxicology it is still an overloo-

ked target (1) Indeed LNCaP cell line may represent an alternative in vitro method of the human pro-

state epithelium to screen bioactive chemicals affecting male fertility (1-3) within the EU Integrated

Project ReProTect LNCaP cell line has been used as a cellular model to investigate androgen receptor

(AR)-dependent signaling to perform toxicogenomic studies of (anti)androgen-like chemicals (1-5) and

a cell-based bioassay was employed to provide a phenotypic anchoring to gene expression profiling da-

ta (4) The selected cell-based cell-specific clinically used biomarker of effect has been the Prostate-

Specific Antigen (PSA) monitored as secreted protein Besides to be a supportive tool for the toxicoge-

nomic approach the PSA secretion assay has been thus implemented as an independent tool to investi-

gate prostate-mediated effects on male reproduction (4)

References 1 Lorenzetti S Narciso L Marcoccia D Altieri I 2012 J Biol Res 1(LXXXIV)36-41

2 Lorenzetti S Altieri I Arabi S Balduzzi D Bechi N Cordelli E Galli C Ietta F Modina SC Narciso L Pacchierotti F

Villani P Galli A Lazzari G Luciano AM Paulesu L Spanograve M Mantovani A 2011 Annals Ist Super Sanitagrave 47(4)429-

444

3 Lorenzetti S Marcoccia D Narciso L Mantovani A 2010 Reprod Toxicol 30(1)25-35

4 Lorenzetti S Lagatta V Marcoccia D Aureli F Cubadda F Aricograve E Canini I Castiello L Parlato S Gabriele L Maran-

ghi F Mantovani A 2008 Toxicol Lett 180S(S1)S123-S124

5 Lorenzetti S Narciso L 2012 In Computational approaches to nuclear receptors edited by Pietro Cozzini and Glen E

Kellogg Drug Discovery series of Royal Society of Chemistry Cambridge (UK) DOI 1010399781849735353

Notes

Marcello SPANOrsquo

Laboratory of Toxicology ENEA Casaccia Rome Italy

Marcellospanoeneait

Marcello Spanograve (MS) is senior scientist at the Toxicology

lab Unit of Radiobiology and Human Health ENEA

(Italian National agency for New technologies Energy and

sustainable economic development) His main interest is

represented by the study of sperm DNAchromatin (epi)

genetic integrity after environmental exposures (endocrine

interferers ionizing and not ionizing electromagnetic radia-

tion) MS has more than 25 year experience in the field of

automated cytology development use and application of

methods to detect damage in mammalian germ cells induced

by radiation and chemicals also exploring the impact of

sperm DNA damage on human reproductive capabilities

MS has been involved in EU projects on reproductive toxi-

cology since 1991 and co-authored more than 130 peer re-

view papers and book chapters

HUMAN SPERM (EPI)GENETIC BIOMARKERS TO ASSESS THE IMPACT OF ENDOCRINE ACTIVE SUB-

STANCES ON MALE REPRODUCTIVE FUNCTION

Marcello Spanograve Eugenia Cordelli and Francesca Pacchierotti

Infertility is a common disorder affecting about 15 of all couples trying to conceive and subfertility has become a relevant

growing problem in affluent countries Temporal and spatial trends toward decreasing semen quality increasing rates of repro-

ductive tract abnormalities and a well documented worldwide testicular cancer incidence increase pointed to possible environ-

mental causes of male reproductive system impairment Even if it is difficult to disentangle the responsibility of endocrine

stressors from other lifestyle factors potentially impairing human fertility it is believed that a high level of environmental en-

docrine active substances (EASs) especially during fetal life may disturb the hormone control of male urogenital tract organo-

genesis with lifelong consequences From a toxicological point of view spermatogenesis is expected to be vulnerable to repro-

ductive toxicants because of the large number of cell divisions and cell diffentiation processes continuously occurring through-

out the whole reproductive life In addition the final stages of gamete differentiation in male mammals are sensitive targets of

DNA-reactive chemicals because they are repair deficient Human biomonitoring studies of South African and Mexican popu-

lations living in endemic malarias areas have consistently indicated impaired semen quality associated with high levels of envi-

ronmental DDT-exposure There are limited but sound epidemiological data indicating adverse effects of EASs on human

sperm DNA Low-level exposure to PCB-congeners apparently interferes with sperm chromatin integrity (and sperm motility

as well) in humans ndash findings that are consistent with experimental studies The need to establish alternative methods model-

ling mammalian spermatogenesis is an issue of high priority In this context direct in vitro assays on spermatozoa addressing

motility and genetic damage are relevant since they can detect effects on terminally differentiated male gametes It has recently

been proposed that EASs exposure can affect sperm chromatin integrity by a mechanism involving epigenetic changes to the

paternal genome Rodents exposed in-utero to the fungicide vinclozolin or the insecticide methoxychlor showed abnormal

DNA methylation patterns in spermatozoa and impaired male fertility Once established these epigenetic changes may be per-

manent and thus paternally passed to subsequent generations Following human population studies showing an association be-

tween EASs exposures and altered global methylation levels in blood lymphocytes biomonitoring studies are in progress to

determine sperm DNA methylation patterns in relation to EASs exposures It is probable that in the near future reproductive

risk assessment from EASs exposure could benefit from more affordable high-throughput technologies providing a more com-

plete evaluation of the possible genetic and epigenetic effects

Notes

Luana PAULESU Department of Physiology University of Siena Siena Italy

paulesuunisiit

Luana Ricci married Paulesu is Professor of Physiology and the Director of

the Center ldquoBiology of Reproductionrdquo at the University of Siena (Italy) She

carried out research on human placenta mainly focusing on the secretion and

action of cytokines She also carried out studies on animal species with differ-

ent types of placenta In the last few years she has been a partner of RePro-

Tect an Integrated project Supported by the European Sixth framework

studying the effect of estrogen-like compounds in in vitro models of human

placenta She is author or co-author of 90 full-papers published in Interna-

tional Journals and of numerous chapters in scientific volumes She is the Edi-

tor of the book ldquoSignal molecules in animal and human gestationrdquo Research

Signpost Trivandum Kerala India She has attended numerous congresses

also as an invited speaker

Bechi N Ietta F Romagnoli R Focardi S Corsi I Buffi C Paulesu L Toxicol Sci 200693(1)75-81

Ietta F Wu Y Romagnoli R Soleymanlou N Orsini B Zamudio S Paulesu L Caniggia I Am J

Physiol Endocrinol Metab 2007 Jan292(1)E272-80 Bremer S Brittebo E Dencker L Knudsen LE Mathisien L Olovsson M Pazos P Pellizzer C Pau-

lesu LR Schaefer W Schwarz M Staud F Stavreus-Evers A Vaumlhaumlnkangas K Altern Lab Anim 200735(4)421-39

Ferro EA Mineo JR Ietta F Bechi N Romagnoli R Silva DA Sorda G Bevilacqua E Paulesu LR 2008172(1)50-8

Ietta F Bechi N Romagnoli R Bhattacharjee J Realacci M Di Vito M Ferretti C Paulesu L Am J Physiol Endocrinol Metab 2010 298 (3)E411-8

Bechi N Ietta F Romagnoli R Jantra S Cencini M Galassi G Serchi T Corsi I Focardi S Paulesu L Environ Health Perspect 2010118(3)427-31

Moslashrck TJ Sorda G Bechi N Rasmussen BS Nielsen JB Ietta F Rytting E Mathiesen L Paulesu L Knudsen LE Reprod Toxicol 2010 30(1)131-7

Bhattacharjee J Ietta F Giacomello E Bechi N Romagnoli R Fava A Paulesu L Placenta 2010 31(5)423-30

Paulesu L Bhattacharjee J Bechi N Romagnoli R Jantra S Ietta F Curr Pharm Des 201016 (32)3601-15 Review

de Oliveira Gomes A de Oliveira Silva DA Silva NM de Freitas Barbosa B Franco PS Angeloni MB Fermino ML Roque-Barreira MC Bechi N Paulesu LR Dos Santos MC Mineo JR Ferro EA

IN VITRO EFFECT OF EASs IN HUMAN PLACENTA

Luana Paulesu Nicoletta Bechi Roberta Romagnoli Francesca Ietta

Endocrine disrupter chemicals (EDCs) are environmental pollutants of agricultural or industrial origin which may influence

human reproductive health These compounds are able to interfere with the delicate balance of the endocrine system by mim-

icking the action of the steroid hormones They can also be transferred from the mother to the embryo and cause reproduc-

tive and developmental toxicity We investigated the effect of selected EDCs on in vitro models of human placenta In par-

ticular we examined para-nonylphenol (p-NP) and Bisphenol A (BPA) The choriocarcinoma BeWo cell line and the HTR-8

Sv-neo cells were used to identify chemical lethal concentration able to reduce 50 of cell viability (cell toxicity) and the

chorionic villous explants from fresh human placenta were used to perform functional studies of chemicals at non-toxic but

environmentally relevant concentrations Vehicle-treated cultures were used as negative controls

We found toxicity of the chemicals tested at concentrations in the order of microM Lower non-toxic concentrations in the order

of nM were able to interfere with the hormone (β-human chorionic gonadotropin β-hCG) secretion as well as inducing tro-

phoblast differentiation and apoptosis

These results raise concern about maternal exposure to environmental factors and the potential involvement of these chemi-

cals in pregnancy disorders

Notes

Notes

SESSION 2

EASs IN DIFFERENT HORMONE TARGET TISSUES

Igor BENDIK-FALCONNIER

Igor Bendik-Falconnier DSM Nutritional Products Ltd NIC-RD-HN CH-4002 Basel Switzerland

igorbendikdsmcom

Igor Bendik-Falconnier obtained his Bio II diploma in molecular genetics

and his PhD in cell biology at the University of Basel Switzerland Subse-

quently he spent his post-doc and associate professor years at the Burnham

Institute La Jolla and the Sidney Kimmel Cancer Center La Jolla California

Back in Switzerland he was a deputy group leader at the department of re-

search (ZLF) at the medical faculty of Basel where he was awarded several

grants to explore cancer formation In 1998 he started his career in industry

at Hoffmann-La Roche Ltd later in Roche Vitamins Basel and explored the

efficacy of natural healthndashbeneficial food ingredients In the VHF depart-

ment he led the bone biology group During his career he has successfully

trained and supervised a number of diploma and PhD students Since the merger with DSM in October

2003 he has continued in his role as a senior scientist to support and lead different teams in nutritional

research with the focus on vitamins and nutraceuticals During his industrial commitment he has led

different research teams and was responsible for different cellular and molecular research technologies

Currently he is heading the biostatistics and bioinformatics group at DSM Nutritional Products Ltd

ENDOCRINE ACTIVE NUTRIENTS EXPLORED IN HUMAN BONE CELL CULTURES

Many natural food-borne compound classes interact with the human body in an endocrine hormone-like manner These nutri-

ents fulfil important physiological roles mediated through healthy nutrition An example is genistein the major soy isofla-

vone which is well known to be active in bone tissue a common target for endocrine hormones DSM has explored the

function of genistein in bone cell biology using available omics and primary cell culture technologies DSM has develop

geniVidatrade the nature-identical aglycone as a product for postmenopausal bone health in the field of nutrition This presen-

tation will cover the use of cell culture technology for the characterization of an endocrine active nutrient in a given bio-

logical concept and the strategy to develop it as a nutraceutical for postmenopausal bone health Besides efficacy also the

industrial toolbox for testing of endocrine disruption properties and the regulatory framework will be discussed

Notes

Robert A SMITH

School of Life Sciences University of Glasgow Glasgow G12 8QQ UK

RobertSmithglasgowacuk

DO CELL MODELS OFFER REALISTIC ALTERNATIVES FOR DETERMINING POTENTIAL NEURONAL RE-

SPONSES TO ENDOCRINE ACTIVE SUBSTANCES

The complexity of the human nervous system comprised of a multitude of interacting neural and non-neuronal cell popula-

tions not to mention the plasticity changes occurring during its development and throughout adult life presents a difficult task

when attempting to determine perturbation following potential toxic insults This is particularly so when employing in vitro

approaches Emphasis has focussed on monitoring a number of neuronal-specific endpoints rather than merely reporting gen-

eral cytotoxic responses leading to cell death for example quantifying changes in neural and glial specific marker proteins

including receptor expression analysing effects on neurite outgrowth functional competency and synaptogenesis Primary neu-

rons mainly from rodent species have been successfully cultured from numerous regions of the brain spinal cord and periph-

eral systems and have been widely applied to toxicological studies for many years (Smith 2009) A number of transformed

neural lines including those established from human tumours such as SH-SY5Y cells have also been routinely used both in

immature and differentiated states with promising outcomes in screening programmes During the last decade exciting develop-

ments in molecular biology have resulted in the generation of human embryonic neural stem cell lines derived from umbilical

blood (Buzanska et al 2010) and recently induced pluripotent stem cell (transduced from adult dermal fibroblasts) have be-

come available (Kumar et al 2012) Although issues of variablility and high costs may limit their current usefulness such

technologies have potential as future in vitro model systems for toxicological assessments The pros and cons of utilising these

in characterising the role of endocrine active substances relevant to neural tissues will be discussed References Buzanska L Zychowicz M Ceriotti L Coecke S Rauscher H Sobanski T Whelan M Domanska Janik K Colpo P

Rossi F (2010)Toxicology 270 35-42 Kumar KK Aboud AA Bowman AB (2012) Neurotoxicology 33 518-529 Smith RA (2009)

ATLA 37 367-375

Professor of Cellular Neuroanatomy (Personal Chair awarded January 2007)

Honorary Senior Research Fellow (September 2011)

Degrees Held Master of Arts (1974) University of Oxford (Zoology Honours)

Master of Letters (2010) University of Glasgow

Doctor of Philosophy (1978) University of Southampton (Cell

Biology)

Previous Posts Postgraduate MRC studentship awarded by the MRC Toxicology

Unit Carshalton Postdoctoral Research Assistant Southampton University Lecturer amp Senior Lecturer

in Anatomy

Major International Commitments

WHO Temporary Adviser Geneva 1989 Member of Consul ta t ion on In vi tro techniques fo r assess-

ment o f neurotoxicchemica ls

EuroTox 1990 Leipzig Invited Speaker and Chairman of Symposium of In Vitro Neurotoxicology

FRAME London 1990 Discussant of 2nd Working Party Report on Neurotoxicology

INVITOX 92 1992 De Haan Invited Speaker

ECVAM Ispra 1994 Member of In Vitro Neurotoxicology Working Party

IPCS Consultation on In vitro neurotoxicology Research Triangle Park USA 1995

German Clinical Pharmacology amp Experimental Toxicology Meeting 1996 Dresden Plenary lecture

NIH Bethesda USA September 1997 Invited Seminar

Advisory Editorial Board of the International Review of Cell amp Molecular Biology 1998-present

University of South Bohemia Czech Republic 2000 Invited Seminar

NRC Laboratories Ottawa Canada 2002 2005 2006 Visiting Scientist (funded by British Council

EuroTox 2009 Dresden Invited Speaker

Approximately 70 peer reviewed publications in cell biology neurobiology toxicology gonadal differentiation and human

anatomy

Major Reviews SMITH RA Ord MJ (1983) Mitochondrial form and function relationships in vivo Their potential in toxi-

cology and pathology Int RevCytol 83 63-134 SMITH RA Jiang Z-G (1994) Neuronal modulation and plasticity in vi-

tro Int RevCytol 153 233-296 Fatokun AA Stone TW SMITH RA (2008) Oxidative stress in neurodegeneration and

available means of protection Frontiers in Bioscience 13 3288-3311 Hou ST Jiang SX SMITH RA (2008) Permissive

and repulsive cues and signalling pathways of axonal outgrowth and regeneration Int Rev Cell Mol Biol 267 125-181

SMITH RA (2009) Twenty-first century challenges for In Vitro Neurotoxicity ATLA 37 367-375 Roberts RA SMITH

RA Safe S Szabo C Tjalkens RB Robertson FM (2010) Toxicological and patho-physiological roles of reactive oxygen

and nitrogen species Toxicology 276 85-94

Notes

Associate Professor of Bioengineering at the Department of Chemical Engineer-

ing Faculty of Engineering University of Pisa and affiliated with the Interde-

partmental Research Center ldquoE Piaggiordquo where she is Vice Director and head

of the MCB Group (wwwdionisiocentropiaggiounipiit and

wwwcentropiaggiounipiit) Prof Ahluwalia is also an associate of the National

Council of Research Institute of Clinical Physiology (CNR-IFC) and head the

NanoBioscopy Lab at the Institute

Although highly multidisciplinary in nature her research has centered on the

interaction between biological systems and man-made devices or structures fo-

cused towards the creation of organ and system models in-vitro She has coordinated and participated

in projects on tissue engineering biosensing bioreactors neural cell culture cardiovascular engineer-

ing nanotoxicology and robots for autism She has several papers published in international scientific

journals (over 90) and is author of 13 patents on microfabrication and on microfabricated multicom-

partmental bioreactors A total of 4 patents have been industrialized to date

Arti AHLUWALIA

Interdepartmental Research Center EPiaggio University of Pisa Italy

artiahluwaliacentropiaggiounipiit

DYNAMIC IN-VITRO ORGAN MODELS OF METABOLISM

Unraveling the complexity of inter-organ and inter-tissue cross talk in vivo is a complex and challenging task Intelligent in

vitro models able to recapitulate the physiological interactions between tissues in the body connected by the bloodstream have

enormous potential as they enable detailed studies on specific two-way or higher order organ-organ and tissue interactions

These models are the first step towards building an integrated picture of systemic signaling in physiological or pathological

conditions Using a scaled down fluidic system we have developed in vitro models of endogenous metabolism and toxicity

through ingestion composed of 3-tissue connected cultures The metabolic model represents the central abdominal region and

is composed of hepatocytes endothelial cells and visceral adipose tissue Closely paralleling in vivo nutrient balance in normo-

glycemic media the model is able to maintain homeostatic glucose and lipid equilibrium Moreover it expresses systemic in-

flammation as well as endothelial specific stress in the presence of hyperglycemic and hypoinsulinemic conditions A similar

model of nanomaterial ingestion developed within the InLiveTox project is composed of an epithelial barrier and down-

stream endothelial cells and hepatocytes Exposure of the epithelial barrier causes downstream inflammation and stress even in

the absence of nanoparticles indicating that the model can be used to study multiple pathways and systemic responses to me-

tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS

The expertise of dr Testai is focused on mammalian toxicology toxicokinet-

ics and risk assessment She has been involved not only in research but also

in regulatory activities in the area of risk assessment for human health associ-

ated to exposure to natural and synthetic chemicals with different field of ap-

plication as outlined below Education Emanuela Testai received her degree in Biological Sciences from the University

of Pisa (Italy) in 1981 Her experimental training was carried out at the National Research

Council - Institute of Mutagenesis and Differentiation in Pisa (1979-1981) Her post-doctoral

training was completed at the Istituto Superiore di Sanitagrave (National Institute for Health)

Rome (1982-1984)

Professional experience Current Position Senior Scientist at Istituto Superiore di Sanitagrave -

Rome Italy- Department of Environment and Primary Prevention (since 2007) Head of the Mechanisms of Toxicity

Unit (since 2009) Young Researcher (1985-1994) and Researcher (1994-2007) at Istituto Superiore di Sanitagrave

(Comparative Toxicology and Ecotoxicology Laboratory-Biochemical Toxicology Unit then Dept Environment and

Primary Prevention) Between 2001-2004 responsible for the Biochemical Toxicology Unit

Member of Scientific Committees within the European Community (Scientific Committee for Health and Environmental

Risks ndashSCHER since 2004) of WG of the European Food Safety Authority (EFSA) of National Committees for Plant Pro-

tection Products and for Biocides National Inspector for Good Laboratory Practise expert within the OECD Test Guide-

lines Program consultant for the Italian Ministry of Health

Scientific Leader of NationalInternational Research Projects on xenobiotics metabolism and toxicity granted by the Italian

Ministry of Health the National Research Council NATO EUResearch Interests Molecular mechanisms of toxicity bio-

transformation toxicokinetics and toxicity of xenobiotics (environmental pollutants pesticides natural toxins) enzymology

of drug-metabolizing systems in hepatic and extrahepatic tissues metabolic biomarkers of individual susceptibility to toxic

compounds application of mechanistic and toxicokinetic data to risk assessment molecular epidemiology (genotyping of

populations to identify metabolic biomarkers of individual susceptibility to toxic compounds) risk assessment associated to

exposure to cyanotoxins and study of cyanobacterial communities Reviewer activity for Drug Metabolism and Disposition

Life Science Toxicology Environmental Toxicology and Pharmacology Environmental Toxicology Environmental Sci-

ence and Technology Toxicology in Vitro Journal of Biochemical and Molecular ToxicologyPharmacological Research

Chemico-Biological Interactions Reproductive Toxicology Science of Total Environment Food and Chemical Toxicology

Analytica Chimica Acta Annali ISS Water Research Critical Review in Toxicology Archives of Toxicology

Scientific Publications Peer-reviewed international journals and book chapters 87 Other publications 38 Congress

Communications140

Emanuela Testai

Department of Environment and Primary Prevention - Istituto Superiore di Sanitagrave ndash Rome Italy

emanuelatestaiissit

THE ROLE OF BIOKINETICS IN IN VITRO TESTS AND IN THE INTERPRETATION OF RESULTS

When developing testing strategies kinetics is considered the crucial body of information for the design and performance of

toxicological tests and for toxicity data interpretation Indeed following exposure to a chemical its uptake bioavailability and

biotransformation determine the actual in vivo target dose which is one of the most relevant parameter in quantitative risk as-

sessment A typical example is the oral pharmacokinetics for bisphenol A (BPA) showing lt1 of total BPA in the unconju-

gated and biologically active form in serum from experimental animals and humans at peak levels due to a relevant pre-

systemic detoxication when compared with the higher values attained after parenteral administration

Despite the above consideration is even more relevant for alternativenon animal testing strategy in vitro biokinetics is gener-

ally neglected it is a matter of fact that the nominal applied concentration is generally associated to observed effects rather

than the actual level of cell exposure This causes a high level of uncertainty in the in vitro concentration-effect relationship it

has been considered one of the major causes for in vitroin vivo differences and make it difficult to translate an in vitro concen-

tration into an in vivo dose (in vitrondashin vivo extrapolation) The actual intracellular concentration may be affected both by

abiotic processes (ie interactions with mediumplate chemical instability) or by interaction with cells (ie transport across the

membranes biotransformation to reactiveinactive metabolites bioaccumulation) altering in vitro bioavailability after acute

and even more after repeated treatments Physiologically based toxicokinetic (PBTK) models can be developed for the integra-

tion of dynamic and kinetic data produced from in vitro methods into a biologically meaningful framework for the extrapola-

tion to in vivo conditions This approach could make possible to derive a NOEC in in vitro experimental models from which

extrapolate the corresponding in vivo dose Examples will be discussed evidencing the impact of kinetic behaviour on the ef-

fects of chemicals including endocrine active substances

The work has been partially supported by the FP7-EU funded Project PredictIV Grant ndeg 202222

Notes

Freacutedeacuteric Y Bois is an internationally known expert in quantitative toxicology He was

trained and worked at Harvard University UCSF UC Berkeley the Lawrence Berke-

ley Laboratory INSERM and INERIS He is currently Professor at the Compiegne

Technology University and Research Director at the Institut National de lrsquoEnvironne-

ment Industriel et des Risques (INERIS) He has coordinated and participated to inter-

national research projects in the areas of statistics and mathematical modelling for

pharmacology toxicology epidemiology and health risk assessment (funders US-

FDA US-NIH US-EPA US-OSHA European Commission INSERM French Mi-

nistry of Research) Recipient of the American Statistical Association Outstanding Statistical Application

Award and of the French Epidaure Prize for Environmental Health Research EDUCATION 1993 Habilitation Universiteacute de Nancy France1988 Doctorat egraves Sciences Universiteacute de Metz France1981 Doctorat de

Pharmacie Universiteacute de Nancy France

POSITIONS HELD 2009-Professor Chair of Mathematical Modelling for Systems Toxicology UTC and INERIS France1999-09 Re-

search Scientist Head of the Toxicology unit then Scientific Officer INERIS France 1991-99 Staff Scientist Lawrence Berkeley Labora-

tory Berkeley USA and INSERM U444 Paris 1987-90 Post-Doctoral Researcher Biologist UC San Francisco and UC Berkeley USA

1986-87 Research Associate Energy and Environmental Policy Center Harvard University USA

SCIENTIFIC COMMUNICATIONS Cheng S Bois F Environmental Health Perspectives in press

Adler S Bois F et al 2011 2010 Archives of Toxicology 85367-485

Bois F Jamei M Clewell HJ 2010 Toxicology 278256-267

Bois F 2010 Basic and Clinical Pharmacology and Toxicology 106 154-161 doi 101111j1742-7843200900488x

Bois F 2009 GNU MCSim Bioinformatics 251453-1454 doi 101093bioinformaticsbtp162

Micallef S Bois F et al 2007Clinical Pharmacokinetics 4659-74

Jonsson F Jonsson EN Bois F Marshall S 2007 Journal of Biopharmaceutical Statistics 1765-92

Amzal B Bois F et al 2006 Journal of the American Statistical Association 101773-785

Mezzetti M Bois F et al 2003 Journal of the Royal Statistical Society Series C 52291-305

Bernillon P Bois F 2000 Environmental Health Perspectives 108(suppl5)883-893

Bois F Maszle D 1997 Journal of Statistical Software 2(9)httpwwwstatuclaedujournalsjssv02i09

Gelman A Bois F Jiang J 1996 Journal of the American Statistical Association 911400-1412 Bois F Compton-Quintana P 1992 Journal of Theoretical Biology 159361-375

Freacutedeacuteric Y BOIS

Chair of Mathematical Modeling for Systems Toxicology Bioegineering Department Universiteacute de Tech-

nologie de Compiegravegne Royallieu Research Center Compiegne France

fredericboisutcfr

PHYSIOLOGICALLY-BASED MODELING OF OVARIAN STEROID HORMONES SYNTHESIS FOR ENDO-

CRINE ACTIVE SUBSTANCES HEALTH RISK ASSESSMENT

Freacutedeacuteric Y Bois and Nadia Quignot

A finely tuned balance between estrogens and androgens controls reproductive functions and the last step of steroidogenesis

plays a key role in maintaining that balance Environmental toxicants are a serious health concern and numerous studies have

been devoted to studying the effects of endocrine active substances (EASs) The effects of EASs on steroidogenic enzymes may

influence steroid output and thus lead to reproductive toxicity We first review the most significant quantitative modeling efforts

aimed at predicting the EASs effects Those examples clearly show that a tight coupling of systems toxicology with quantitative

in vitro to in vivo extrapolation through physiological pharmacokinetic modeling is required for improving predictive capacity

Our most recent work is an illustration of that approach to predict hormonal balance disruption on the basis of data on aroma-

tase activity and mRNA levels modulation obtained in vitro on granulosa cells we developed a mathematical model for the last

gonadal steps of the sex steroids synthesis pathway The model can simulate the ovarian synthesis and secretion of estrone es-

tradiol androstenedione and testosterone and their response to endocrine disruption The model is able to predict ovarian sex

steroid concentrations under normal estrous cycle in female rat and the ovarian estradiol concentrations in adult female rats

exposed for 6 hours to the parent compound and the metabolites of atrazine bisphenol A methoxychlor and vinclozolin Re-

sults are presented and discussed in the framework of the next generation of risk assessment for EASs

Notes

Date of birth July 27 1959

Citizen of Zurich Switzerland

Profession Registered Toxicologist (Dr nat sci ETH) DGPT SSTP Eurotox ATS Aug

2005 ndash Nov 2006 adjunct judge Court of Mediation and Appellation at the Swiss Supreme

Court Bern Switzerland (wwwrekoadminch)

Jan 2002 - Professor of Toxicology Head of Human and Environmental Toxicology Univer-

sity of Konstanz Germany Consultant toxicologist for SwissMedic (httpwwwswissmedicch)

Jan 1996 -2002 Professor of Toxicology Head of Environmental Toxicology University of Kon-

stanz Germany Adjunct Professor Department of Environmental and Occupational Health Gradu-

ate School of Public Health University of Pittsburgh USA Adjunct Associate Professor Swiss

Federal Institute of Technology (ETH) Zurich Switzerland

May 1995 ndash Dec 1995 Visiting Associate Professor University of Pittsburgh Department of

Environmental and Occupational Health and Toxicology University of Pittsburgh USA

April 1989 ndash July 1991 Post-doctoral fellow University of North Carolina and Glaxo Research Laboratories Research Tri

angle Park North Carolina USA

Nov 1985 ndash Oct 1988 PhD ndash Thesis Institute of Toxicology Zurich Switzerland

Registered Board Member Swiss Board of Toxicologists (SSPT) German Board of Toxicologists (GSPT) EUROTOX

Regitered Toxicologist Fellow of the Academy of Toxicological Sciences (ATS)

Memberships Academy of Toxicological Sciences (ATS) Society of Environmental Toxicology and Chemistry (SETAC)

Society of Toxicology (SOT) Swiss Society of Pharmacology and Toxicology (SGPT) German Society of Pharmacology and

Toxicology (DGPT) Society of Toxinology Society for General and Applied Microbiology (VAAM) Federation of European

Microbiological Societies (FEMS) European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC)

Publications Peer-Reviewed 120 Books and Bookchapters 13 Book reviews Reports and Laymen Communications 14

Abstracts of presentations at intl meetings gt214 Invited lectures gt122

Science Advisory Panels (5 of 19) European Union Institute of Consumer Health and Safety ECVAM JRC Ispra I (http

wwwjrcceceuintdefaultaspsidsz=who_we_arehtm) Chair of the Endocrine Disruption Task Force (ED-TF) ldquoIn-Vitro

Technologies for the Detection of Endocrine Effectsrdquo Endocrine Disruption and Ecotoxicology Expert of the EDTA-

VMG non-animal OECD (httpwwwoecdorgenvehs) Paris France Co-Chair EDTA-VMG non-animal OECD April

2007 ndash December 2010 Expert Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) for the

European Commission DG-SANCO Bruxelles Belgium Chair of the expert peer-review panel of US-EPAs draft Toxicolo-

gical Reviews of Cyanobacterial Toxins Anatoxin-a Cylindrospermopsin and Microcystins LR RR YR and LA January 10

2007 Peer review meeting in Cincinnati OH USA Expert IARC (WHO International Agency for Research on Cancer

wwwiarcfr) Member of the Scientist team to develop IARC Monograph Volume 94 Ingested Nitrates and Nitrites and

the Blue-green Algae Toxins Microcystin-LR and Nodularin Lyon 14-21 June 2006

Editorships Chemical Biological Interactions Editor-in-Chief (92012-) Toxicology in Vitro Editor Asia and Asia-Pacific

(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH

Human and Environmental Toxicology University of Konstanz Konstanz Germany

danieldietrichuni-konstanzde

EASrsquoS CONTRA HUMAN amp ENVIRONMENTAL HEALTH RELEVANT OR PLAYGROUND FOR MERCHANTS

OF DOOM Endocrine Active Substances (EAS) have been made responsible for increased incidences of nearly all human diseases imagin-

able and especially for the demise of populations of certain species Moreover unfounded suggestions such as the ldquolow-dose

hypothesisrdquo or ldquono-threshold hypothesisrdquo for adverse effects have provided for a media and research hype that created an atmos-

phere of biased and emotional science that has all the ingredients that current ldquomerchants of doomrdquo in science need to thrive

without having to prove any of the hypotheses or suggestions put forth The latter is exactly contrary to what is needed namely

a through assessment of facts in a bigger context a factual and critical assessment of what mechanistic toxicology hazard char-

acterization and risk assessment can achieve to properly estimate the dangers of EAS Moreover this assessment needs to be

compared to available epidemiological data for each of the diseases within the context of verified EAS exposure to ensure that

predictions from in vitro and in vivo mechanistic assessments are realistic and target the population(s) at highest risk While the

environmental impact of EAS appears to restricted to locations of highest contamination eg sewage treatment plant effluents

and thus can be remediated by technical improvement of sewage treatment more generalized adverse effects of EAS in wildlife

has not been convincingly documented to allow causal relationships between presumed exposure and population effects Simi-

larly although in vitro assays would predict a potential for EAS to interact with human endocrine receptors and associated sig-

nal transduction pathways and some in vivo experiments purport the existence of transgenerational effects at low doses of EAS

or effects on development of endocrine organs fertility or fecundity at high doses of EAS in surrogate animals so far very little

of the latter evidence has been found to withstand scrutiny with regard to scientific quality On the contrary real-life exposures

with EAS eg BPA in humans specifically dosed demonstrate that absence of biologically available and thus active form of

BPA and therefore the absence of potential endocrine activity Corroborating the latter findings an extremely thorough epidemi-

ological study from Denmark annihilated the association of inhibited sperm counts in Danish men with exposure to EAS de-

spite that the latter was purported to be the case even in top journals for nearly two decades These examples demonstrate 2 main

issues 1) only a rigorous scientific approach will provide analytical results that allow any predictions of risk in humans 2)

most epidemiological studies provide for wrong associations of exposure to EAS and adverse effects primarily due to faulty

design too small cohorts and especially due to biased politically motivated or financially driven approaches at the outset

Notes

Notes

Notes

Notes

Notes

Nome societagrave

Welcome address

Dear Colleagues

it is a great pleasure to welcome you at the CAAT-IPAM-ISS Symposium on ldquoAlternative in vitro meth-

ods to characterize the role of Endocrine Active Substances (EASs) in hormone-targeted tissuesrdquo jointly

organised by the Department of Veterinary Public Health and Food Safety and the Department of Envi-

ronment and Primary Prevention whose activities include the use of alternative in vitro methods to in-

vestigate key issues of the role of EASs in endocrine-regulated targets

The interest in alternative in vitro methods for toxicity testing has raised in the last years within the sci-

entific community and has been moved forward also by the increasing societal demand on the reduction

of animal use coupled with the requirements in high quality evaluations of chemical safety In particular

the processes of screening and prioritization of chemicals may be strongly supported by an integrated

use of alternative (either in silico or in vitro) methods

To this end the European Commission recently adopted new legislative tools aimed to improve the use

of alternative methods as the Registration Evaluation Authorization and Restriction of Chemicals

(REACH EC No 19072006) the Revised Directive on the Protection of Animals used for Scientific Pur-

poses (EU 632010) and the new Cosmetics Regulation (EC No 12232009) In all of them the 3Rs

(Refinement Reduction Replacement) principle is emphasized in many articles as part of the new toxi-

cological requirements

Therefore we are grateful to the Center for Alternatives to Animal Testing (CAAT) Europe and the Ital-

ian Platform on Alternative Methods (IPAM) for their efforts to promote and disseminate alternative

methods and to contribute to the successful organization of this Symposium

Along with a pleasant stay in Rome we wish all participants a fruitful exchange of ideas

Umberto Agrimi Head of the Department of Veterinary Public Health and Food Safety

Loredana Musmeci Head of the Department of Environment and Primary Prevention












assessment










Acknowledgements


ganization























organization





anchoring



ve function











1330-1430 Lunch





assessment



om



Thomas HARTUNG


thartungjhsphedu





































128








Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes












assessment










Acknowledgements


ganization























organization





anchoring



ve function











1330-1430 Lunch





assessment



om



Thomas HARTUNG


thartungjhsphedu





































128








Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes























organization





anchoring



ve function











1330-1430 Lunch





assessment



om



Thomas HARTUNG


thartungjhsphedu





































128








Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes









1330-1430 Lunch





assessment



om



Thomas HARTUNG


thartungjhsphedu





































128








Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes


Thomas HARTUNG


thartungjhsphedu





































128








Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Costanza ROVIDA






















Regulation


























Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Alberto MANTOVANI











































disruptersrdquo

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Johann STEINKELLNER







LITES




process























Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Serena CINELLI


SCinellirtcit










viewed publications


SEARCH ORGANIZATION
























Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

SESSION 1


Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Stefano LORENZETTI












mentationrdquo












Stefano Lorenzetti














444






Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Marcello SPANOrsquo


Marcellospanoeneait











































Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes


paulesuunisiit







































Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

SESSION 2




igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes



igorbendikdsmcom

























Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Robert A SMITH





















ATLA 37 367-375






Biology)



in Anatomy
















anatomy









Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes















Arti AHLUWALIA
















tabolites or drugs

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















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fredericboisutcfr

















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(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

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SESSION 3

EASs AND KINETICS









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes









Rome (1982-1984)






















Communications140

Emanuela Testai


emanuelatestaiissit





















Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes































fredericboisutcfr

















Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes




































(32004-82012) Reviews Editor ( 82003-82012)

Daniel R DIETRICH



























Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

Notes

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