Lush Training Prize 2014
Background Paper
Prepared by TJM Consultancy July 2014

Lush Science Prize 2014 – Background Paper

11. Executive Summary
1.1 What is the Lush Science Prize?
Now in its third year, the Lush Prize supports animal-free testing by awarding money
prizes totalling £250,000 to the most effective projects and individuals who have beenworking towards the goal of replacing animals in product or ingredient safety testing.
Prizes are awarded for developments in five strategic areas: science; lobbying; training;
public awareness; and Young Researcher Awards. Should there be a major
breakthrough in 21st Century Toxicology - the area which holds out most hope for a‘Eureka’ moment leading to the replacement of animal tests – a Black Box Prizeequivalent to the entire annual fund of £250,000 will be awarded to the individual orteam responsible.
1.2 Methodology
In order to obtain an overview of developments in the field of animal replacement in
toxicity pathway research, we firstly reviewed the recent work of the relevant scientificinstitutions and projects in this area, including AXLR8; OECD; CAAT; Hamner Institutes;
Human Toxome Project; ECVAM; ICCVAM; US Tox21 Programme, the ToxCast
programme; and the Human Toxicology Project Consortium (see section 4). We also
assessed recent developments in toxicity pathway research by reviewing the relevant
literature (see section 5).
In our search for candidate prize winners, we identified conferences focusing on animal
replacement in toxicity testing that have been held in the preceding 12 months. These
included EUSAAT 2013 and SEURAT -1. From these we obtained 170 abstracts fororal and poster presentations. These we scored using the system derived in previous
years (see section 3) in which 3 points are awarded for projects identifying new toxicity
pathways, 2 points for reporting new knowledge or tools, and 1 point for abstracts which
stand out in some other way. We then performed literature searches using Pub Med,
Google Scholar and Terkko Feed Navigator to identify projects describing recent
advances in toxicity pathway research. A further two relevant projects were identified
directly from the Tox21 and ToxCast programme websites. In all, searches yielded inexcess of 1000 projects which we assessed as described (see Section 3).
Overall, from all sources combined, from 68 abstracts which scored 1 or more, 7 scored1; 39 scored 2; 14 scored 2 +1 (i.e. total 3); and 7 scored 3 because they appeared tobe reporting a new toxicity pathway. A single project scored the maximum possible
marks of 3 for reporting a new toxicity pathway plus an additional mark for combining
methodologies and for using human non-embryonic iP stem cells. The titles andauthors of those abstracts scoring 1 or more are shown in section 6, whilst full abstractsof those projects scoring a total of 3 (either as 3 or as 2+1) or more are given in
Appendix 1.
1.3 Projects recommended for the shortlist
There were 8 projects which received the highest scores of either 3 or 3+1 for reporting
new pathways of toxicity. The full abstracts are given in Section 6.4. We consider all
worthy of being considered by the judges as potential prize winners. We have notincluded those scoring 2+1 because the emphasis of the award was on new pathways,
Lush Science Prize 2014 – Background Paper2rather than on bringing new knowledge or tools to previously understood toxicity
pathways.

Lush Science Prize 2014 – Background Paper

32. Background
The objectives of 21st Century Toxicology research can be summarised as: improving
safety testing of chemicals and contaminants by using more relevant and predictive
human models; and simplifying and automating tests so that many more chemicals canbe tested for safety. The Science background papers for the 2012 and 2013 LushPrizes provide an overview, and links to further resources, describing the concept of
21st Century Toxicology.
The 2014 Lush Science Prize seeks to reward those researchers making 'outstanding
contributions' to 21st Century Toxicology research. In previous years, the Science prize
has been £50,000 which, in 2013, was divided equally between two winning groups.
The focus of the award has been on research aimed at elucidating key pathways inwhich perturbation results in toxicity. For 2014, we have continued this approach, andfurther extended it to endeavours to build up a map of all known substances and theireffects on the human organism. The vision is that, when new substances become
available, their molecular similarity to other substances on the map, and the pathways
they take, will enable computer simulations to give greater predictive accuracy of
toxicity than any animal model, with extremely rapid results. Once this map is built andworking, animal testing should become a thing of the past.
The Brief for prize applicants, taken from the Lush prize website http://www.lushprize.org/
was as follows:
Science Prize
For individuals, research teams or institutions for work conducted on relevant toxicity
pathways. Outstanding research producing an effective non-animal safety test based on
an approach other than toxicity pathways, where none existed before, may also be
considered.
There is a £50,000 prize fund shared between all the winners of the Science Prize.
21st Century Toxicology is a new approach to safety testing which is exciting regulators,
toxicologists, campaigners and companies around the world. It has become possible
because of advances in biology, genetics, computer science and robotics.
It offers better relevance to humans (rather than using mice, rats and rabbits), and will
explain the underlying causes of toxicity. Unlike animal methods, the new tests will help
predict human variability and differential effects on embryos, children and adults. And
as the superior scientific basis of the new approach is recognised, outdated animal
tests will be replaced.

Lush Science Prize 2014 – Background Paper

43. Methodology
A primary purpose of this report is to identify key projects making major contributions to
the field of animal-free toxicology research from which the Lush Prize judging panel will
select the winners.
Of the “3 Rs”, Lush is interested primarily in Replacement, so our search for potential
prize winners focused on projects working towards the replacement of animals in
product testing. We did not include research aimed at either Refining or Reducing theuse of animals in experimentation, and we excluded research that was linked to animal
testing in other ways. Whilst we gave consideration to work taking place anywhere in
the world, as far as possible we only included work reported in the year preceding theaward (i.e. July 2013 – July 2014).
At each stage of our search, research projects were excluded based on carefulselection criteria, in order to achieve a manageable shortlist of excellent work which
fully met the prize brief. Our selection procedure was a three stage process, starting byreviewing the project title and rejecting any articles which were clearly reviews or whichwere obviously unsuitable through using animal models or being overly focused on
disease. In the second stage, assessment of the abstracts which passed the initial filter
permitted further elimination of those reporting findings from clinical trials, those
focusing on cancer research, all research including animal subjects, or work conducted
on non-human cell lines. In the third stage, projects were scored using a systemdevised and successfully applied in previous years. In this system, points are awarded
according to the following criteria:
Does the work appear to be reporting a new pathway discovery?

Score 3

If it is working with apparently previously understood pathway research,does it bring new knowledge or tools?

Score 2

Does it stand out in any other way?

Score 1

As source material from which to identify key developments in the area of toxicology
research, and begin the search for candidate prize winners, we started by reviewing therecent research of some key institutions and collaborative projects working in the areaof animal replacement in toxicity pathway research. These included AXLR8; OECD;
CAAT; Hamner Institutes; Human Toxome Project; ECVAM; ICCVAM; US Tox21
Programme, ToxCast Programme; and the Human Toxicology Project Consortium.
Secondly, we identified relevant conferences held in the preceding 12 months andassessed abstracts, where available, for oral and poster conference presentations.
Scientific conferences provide the forum in which the most up-to-date science isshared, reporting on recent developments and work-in-progress, without the lag timerequired for formal presentation as a journal publication. The relevant conferences for
2013-2014 included the European Society for Alternatives to Animal Testing (EUSAAT)
2013 (Linz, September 2013) and the EU SEURAT-1 Project meeting 2014 (Barcelona,February 2014). None of these abstracts was currently available online (mid-July2014). Abstracts for the EUSAAT 2013 conference were made available to us inadvance of their public dissemination through the kind assistance of Dr. Ursula G.
Sauer, Secretary General of EUSAAT. Proceedings from the SEURAT-1 2014 meetingwere kindly provided by Sara Vinklatova, Administrator, COACH SEURAT-1. Due to aconfidentiality issue, one of the 31 SEURAT abstracts was not available for our review.
The 9th World Congress on Alternatives & Animal Use in the Life Sciences (9WC)

Lush Science Prize 2014 – Background Paper5meeting will be held in Prague in August of this year: clearly proceedings were notavailable so far ahead of time and so they should be included in the 2015 Lush Prize
Science Review.
Thirdly, we conducted a review of the recent literature. For this we used three sources.
Firstly, we searched PubMed for research published from 01/07/2013 to date, withsearch term “toxicity pathways” and restricting the subject matter to “humans”. As asecond literature source, we searched Google Scholar for relevant papers published in
the period 01/07/2014 to date, using the term “toxicology pathway” and excluding
review articles. As a final literature source, we searched Terkko Feed Navigator using
the terms “toxicology” and “pathway.”

Lush Science Prize 2014 – Background Paper

64. Significant Institutional and Project Developments
This section summarises significant events or news focussing on 21st century toxicology
from selected Institutions and major collaborative projects, reported within the last year.
4.1 Tox21
Tox 21 is a collaborative effort among NIH, the Environmental Protection Agency (EPA),
and the Food and Drug Administration. NIH partners include NCATS (National Center
for Advancing Translational Sciences) and the National Toxicology Program,administered by the National Institute of Environmental Health Sciences. The Tox21
initiative is designed to improve current toxicity assessment methods, which are slow
and costly.
Tox21 scientists are currently testing a library of more than 10,000 chemical compounds(Tox21 10K) in NCATS' high-throughput (large-scale) robotic screening system. To date,the team has produced nearly 50 million data points from screening the Tox21 10K
library against cell-based assays. Data generated from 12 of these assays form thebasis of the 2014 challenge described below.
Tox21 partner NCATS has recently launched a Chemical Toxicity Data Model
Competition. NCATS describes the Toxicology in the 21st Century (Tox21) Data
Challenge 2014 as “a crowdsourcing competition to develop computational models thatcan better predict chemical toxicity”. Participants who submit the winning models, as
judged by the Tox21 Data Challenge Committee, will have the opportunity to submit apaper for publication in a special thematic issue of Frontiers in Environmental Science.
NCATS will also recognize winning submissions in national communications, including
on the NCATS website and in social media channels. Selected models will become partof the Tox21 programme’s arsenal of tools which help researchers assess how various
chemicals might disrupt biological processes in the human body and lead to negative
health effects.
Tox21 has come under some criticism because critics have claimed that its assays do
not necessarily predict human or animal toxicity very accurately. Marchan et al 1
summarise some of these criticisms, but observe that the Tox21 approach is an elegant
one and that future projects may benefit from a combination of Tox21's high-throughput
technology and Europe's 'in vivo relevant' in vitro systems and systems biology
approaches.
4.2 ToxCast
The US Environmental Protection Agency (EPA)'s ToxCast (Toxicity Forecaster)
programme aims to use high-throughput screening tools to test many thousands ofchemicals in in vitro assays.
Completed in 2013, the second phase of ToxCast evaluated over 2,000 chemicals froma broad range of sources, including industrial and consumer products, food additives,and potentially “green” chemicals that could be safer alternatives to existing chemicals.
These chemicals were evaluated in more than 700 high-throughput assays covering a
range of high-level cell responses and approximately 300 signaling pathways. ToxCast
1

R Marchan, C van Thriel, & HM Bolt. Recent developments in in vitro toxicology: perspectives of European
research and Tox21. (2013). Arch. Toxicol 87, 2043-2046

Lush Science Prize 2014 – Background Paper

7research is ongoing to determine which assays, and under what conditions, may lead totoxicological responses. The results of this research then can be used to propose thecontext in which decision makers can use the data. The EPA’s Endocrine Disruptor
Screening Program has already begun the scientific review process necessary in orderthat ToxCast data may be used to prioritize the thousands of chemicals that needtesting for potential endocrine-related activity.
4.3 AXLR8The EU AXLR8 Project’s mission was to “accelerate the transition to a toxicity pathwaybased paradigm for chemical safety assessment through internationally co-ordinatedresearch and technology development”. It published its final report in December 2013.
The report highlighted a number of overarching recommendations that have arisen from
AXLR8 workshops as suggestions for optimising future EU research under the Horizon
2020 funding programme, including:▪ A need for effective, early, co-ordination in order to▪ maximise potential synergies within and between
projects▪ define relationships, milestones, deliverables, andresponsibilities clearly▪ undertake “horizon-scanning” and long-range
planning▪ ensure accountability of all participants.
▪ Use a hybrid of European and international funding
models. Europe tends to fund research on the basisof individual scientific merit, independently of a wider
strategy, whereas programmes in other regions haveadopted a science-driven 'top-down' approach.
AXLR8 recommends a novel framework thatreconciles these approaches in the future.
▪ Maintain a policy focus on key health-related societal
challenges, while incorporating '21st century' tools ascore building blocks of the research strategy.
▪ Recognise that classical 'integrated projects' may notprovide the best return on investment, since funds are
divided in too many directions. Alternative funding
models should be considered under Horizon 2020.
▪ Dedicate funding to support '21st century' researchand testing infrastructures throughout EU member
states.
▪ Encourage 'value-added' collaborations among
established research teams in other parts of theworld, to share the workload and develop synergies
without duplication.

Lush Science Prize 2014 – Background Paper

8AXLR8 recommends that the EU invest €250 million over the seven years of the
Horizon 2020 funding round, focussing on three pillars of work:▪ Adverse Outcome Pathway (AOP) discovery andinformatics▪ Enabling technologies and infrastructures▪ Implementation
This work should be co-ordinated by a strong, central, multiagency Steering Group.
AXLR8 goes on to suggest a number of priority targets for Horizon 2020 funding,grouped under the three “pillar” headings. They hope these targets will provide afoundation upon which the Steering Group can build in developing calls for proposals
under Horizon 2020. They will allow capitalisation on already ongoing innovations,while also offering perspectives for longer term research and development, with
opportunities to exploit synergies within and across different pillars.
Source: The AXLR8 Project: Final Report & Recommendations. December 2013 http://
axlr8.eu/

4.4 The Organisation for Economic Co-operation and Development (OECD)
As noted in the 2013 Lush Prize Science paper, the OECD launched a programme todevelop AOPs for the testing of chemicals. The AOP concept has been broadly
adopted within the field of toxicity sciences. The OECD reports that its AOP
development programme has 18 projects completed or in progress – 3 of these arebased on animal models (2 rat, 1 fish). In addition, 3 AOP case studies are being
developed, one of which uses animals (aquatic organisms).
Source: www.oecd.org/env/ehs/testing/listsofprojectsontheaopdevelopmentprogrammeworkplan.htm

4.5 Hamner Institutes
Based in North Carolina, USA, The Hamner Institutes for Health Sciences brings
together safety assessments for chemicals and drugs, world-class education andtraining, and accelerated technology development.
In December 2013, The Hamner Institutes and the European Union Reference
Laboratory for alternatives to animal testing (EURL EVCAM) published a report on theoutcome of a joint workshop (held in September 2011) on how Toxicity Pathways
concepts are being applied. The Hamner press release said “The US National
Research Council's report (2007), "Toxicity Testing in the 21st Century: A Vision andStrategy”, represented a step-change in thinking surrounding the safety assessment ofenvironmental chemicals. The take-home message was that, by understanding toxicity
in terms of Toxicity Pathways, we'll be able to use intelligent combinations ofcomputational and in vitro methods to assess the potential hazard and risk that achemical may pose to humans and the environment, without resorting to animal testing.
Shifting to this new paradigm promises more efficient, comprehensive and cost effective
testing strategies for every chemical in commerce and will provide a convincing
scientific basis to move away from animal testing. But 7 years on from the publication of
the NRC landmark report, much still needs to be done to translate Toxicity Pathway
concepts into safety assessment practice.”

Lush Science Prize 2014 – Background Paper

9Dr. Melvin Andersen, Chief Science Officer at The Hamner Institutes, commented, “Ourreport remains timely as international risk assessment and toxicology communities
debate new ideas regarding definitions and use of toxicity pathway and network biology
concepts for making regulatory and public health decisions about chemical safety.
Adoption of cell-based, high throughput tools would signal a new era for responsible
regulation and more efficient use of resources in supporting regulation. Thesemeetings of experts, allowing opportunity for wide-ranging discussions, will help gain
clarity and consensus on the value of these tools”.
Source: www.thehamner.org/news-events/entry/the-hamner-and-eurl-ecvam-publish-report-onhow-toxicity-pathway-concepts-c

4.6 European Union Reference Laboratory for alternatives to animal testing(EURL EVCAM)
The role of EURL EVCAM is the validation of methods which reduce, refine or replace
the use of animals for safety testing and efficacy/potency testing of chemicals,
biologicals and vaccines.
In June 2014, EURL EVCAM announced formation of the EU Network of Laboratories
for the Validation of Alternative Methods (EU-NETVAL) to improve the validation
process of alternative methods.
In March 2014 EURL EVCAM hosted a Workshop on "Advancing Adverse Outcome
Pathways for Integrated Toxicology and Regulatory Applications", with the Joint
Research Centre (JRC), Environment Canada, US EPA, US Education Research &Data Center (ERDC), and US, Swiss, & Norwegian academic institutes.
“The workshop brought more than 50 experts from regulatory agencies, academia andindustry together to discuss the Adverse Outcome Pathway concept as a framework tocharacterise, organise, and define predictive relationships between measurable key
events that reflect the progression from a chemical-induced perturbation to an adverse
outcome considered relevant to regulatory decision-making. The workshop progressed
ongoing efforts (e.g. at the level of the OECD) and provided consideration and expert
opinion on the critical next steps required to advance the use and acceptance of the
AOP framework to support integrated toxicology and regulatory decision-making. In
addition, discussions fed into further refinements of the AOP wiki – an IT system jointly
led by the EC Joint Research Centre and the US Environmental Protection Agency forthe purpose of the OECD programme on the development of AOPs, which was firstreleased in August 2013. The AOP wiki is intended to facilitate the development andevaluation of Adverse Outcome Pathways, providing developers with a structured wiki
type environment to capture the scientific information in a user-friendly manner inaccordance with the elements of an AOP.”
Source: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/workshop-on-advancing-adverseoutcome-pathways-for-integrated-toxicology-and-regulatory-applications

4.7 HSI-Human Toxicology Project Consortium (HTPC)The HSI-Human Toxicology Project Consortium is a group of stakeholders currently
drawn from the corporate and public interest communities that share the objective of
accelerating implementation of a biological pathway-based approach to toxicology as

Lush Science Prize 2014 – Background Paper

10described in the National Research Council’s 2007 report on “Toxicity Testing in the
21st Century.”
The HTPC is co-sponsoring training in non-animal cosmetics testing in China. China ispreparing to end mandatory animal testing for domestically-produced cosmetics.

Lush Science Prize 2014 – Background Paper

115. Literature Highlights
We have undertaken an extensive literature search to identify potential Lush Prizenominees. Details of the search and the results are given elsewhere in this document(see Sections 3 and 6). In the course of the research for this Science Prize paper twoparticular reports that did not specifically meet the criteria of the prize stood out. The
first was a workshop report from the transatlantic think tank for toxicology (t4) entitled“Consensus Report on the Future of Animal-Free Systemic Toxicity Testing” by Leist etal2. The second was an article in 'The Biochemist' (the magazine of the UK Biochemical
Society) in which Unilever scientists describe several case studies showing how they
are actively implementing alternative methods in safety testing of consumer products
and their constituents3.
The landmark t4 report is the consensus of three years of international collaboration anddiscussion by 200+ experts in the field. The report makes several important
recommendations on how alternative testing methodologies can be improved, andprovides a scientific roadmap for future risk assessment approaches. It builds on the
Basketter paper mentioned in the 2013 Lush Science paper, and makes several key
recommendations:▪ focus on improving existing methods rather thanfavouring de novo design▪ combine hazard testing with toxicokinetics predictions▪ develop integrated test strategies▪ incorporate new high-content endpoints to classicalassays▪ evolve test validation procedures▪ promote collaboration and data-sharing of different
industrial sectors▪ integrate new disciplines, such as systems biology
and high-throughput screening▪ involve regulators early on in the test development
process.
The Leist et al. report emphasised the importance of focussing on data quality and thescientific background of test methods.
The Unilever article is an example of how a major multinational consumer goods
manufacturer is implementing animal replacement strategies in its safety testing work,
utilising the concepts and tools of 21st century toxicology, toxicity pathways, and AOPs.
One other significant report in the ALTEX journal was a t4 Workshop on Pathways of
Toxicity, held in October 20124. More than 30 front-line researchers and experts spent
three days outlining the benefits of mapping Pathways of Toxicity (PoT, aka AOP), andclarifying the meaning and definition of PoT. The workshop came up with a preliminary
2

Leist et al 2014, ALTEX 31, 3/14, p341-356

3

Reynolds F, Westmoreland C and Fentem J (2014). Non-animal approaches and safety science.
Biochemist, 36, 19-25
4

t4 workshop report: Pathways of Toxicity. A Kleensang, A Maertens et al. Altex 31, 1/14, 53-612014 DOI:
10.14573/altex.1309261

Lush Science Prize 2014 – Background Paper

12definition of PoT as “A molecular definition of cellular processes shown to mediate
adverse outcomes of toxicants”. It further recognized that normal physiologicalpathways exist that maintain homeostasis and these, if sufficiently perturbed, canbecome PoT. Secondly, the workshop sought to define the adequate public andcommercial resources for PoT information, including data, visualization, analyses, tools,
and use-cases, as well as the types of efforts that will be necessary to enable thecreation of such a resource. Thirdly, the workshop explored ways in which systems
biology approaches could inform pathway annotation, and which resources are needed
and available that can provide relevant PoT information to the diverse user
communities.

Lush Science Prize 2014 – Background Paper

13

6. Toxicity Pathway Abstracts
6.1 Conference Abstract Selection
As described in the Methodology, we reviewed abstracts from the EUSAAT 2013conference and the SEURAT-1 2014 conference. From the 140 abstracts which
comprised the EUSAAT 2013 conference presentation and poster proceedings, we
identified 59 abstracts which we passed through our selection process to the scoring(3rd) stage. Of these, 37 scored 1 or more. From the 30 SEURAT-1 abstracts, 10scored 1 or more.
6.2 Published Abstract Selection
The PubMed search identified a total of 470 articles, and these were further narrowed
to 112 by reviewing the titles and rejecting reviews and articles which were obviously
unsuitable through using animal models or being overly focused on disease. These 112
were then further whittled down through review of the abstracts in the second stage ofthe selection process as described above. The final 53 abstracts were passed to thethird assessment stage, to be scored as potential prize winners. Of these, 12 scored 1
or more. The Google Scholar search yielded a further 24 relevant abstracts, of which 7
were passed to the scoring stage, a final 3 scoring 1 or more. The Terkko
FeedNavigator search yielded 504 papers of which we identified 10 novel papers topass through to the scoring stage, of which 4 scored 1 or more. We also scored two
papers identified via the Tox21 and ToxCast websites.
6.3 Scores
Of the total 68 abstracts scoring 1 or more, 7 scored 1 for standing out in some way (forexample for providing opportunities for data sharing, or for combining methodologies togive “added value”); 39 scored 2 for bringing new knowledge or tools to a previously
identified pathway of toxicity; 14 scored 2 for bringing new knowledge or tools to apreviously identified pathway of toxicity but with an additional 1 (i.e. total 3) because
they stood out in some way; and 7 scored 3 because they appeared to be reporting a
new toxicity pathway. One scored the maximum possible marks of 3 for reporting a new
toxicity pathway with an additional mark for standing out by combining methodologies
and for using human non-embryonic iP stem cells.
The Table lists details (Title, Authors, source, and score) of all the abstracts scoring 1 ormore. All of the abstracts for those projects scoring a total of 3 (either as 3 or as 2+1)
or more are shown in full in Appendix 1.

Title

Authors

Profiling of the Tox21 10K compound library R Huang, S
for agonists and antagonists of the estrogen Sakamuru et al
receptor alpha signaling pathway

Lush Science Prize 2014 – Background Paper

14

Source

Score

Tox21
website2

TitleAuthors

SourceScore

Phenotypic screening of the ToxCast
chemical library to classify toxic andtherapeutic mechanisms

N C Kleinstreuer, J
Yang et al.

ToxCastwebsite2

Genomic Allergen Rapid Detection InHouse Validation—A Proof of Concept

H Johansson, F
Rydnert et al.

GoogleScholar

2+1

Dose response modeling of etoposideinduced DNA damage response

Z Li, B Sun et al.

GoogleScholar

2+1

Assessing dose-dependent differences in
DNA-damage, p53 response andgenotoxicity for quercetin and curcumin

B Sun, SM Ross etal.

GoogleScholar2

Implementing Toxicity Testing in the 21stCentury (TT21C): Making safety decisions
using toxicity pathways, and progress in a
prototype risk assessment

Y Adeleye, M
Andersen et al.

TerkkoFeedNavigator

2+1

Proposal of an in silico profiler for
categorisation of repeat dose toxicity dataof hair dyes.

MD Nelms, G Ates
et al.

TerkkoFeedNavigator

2+1

Molecular modelling study of the PPARγ
receptor in relation to the mode of action/
adverse outcome pathway framework forliver steatosis.

I Tsakovska, M Al
Sharif et al.

TerkkoFeedNavigator

2+1

Thimerosal induces apoptotic and fibroticchanges to kidney epithelial cells in vitro

MF Hornos
Carneiro, C Morais
et al.

TerkkoFeedNavigator3

Monomethylarsonous acid inhibited
endogenous cholesterol biosynthesis in
human skin fibroblasts

Guo L et al

PubMed1

Effect of myeloperoxidase inhibition on
gene expression profiles in HL-60 cells[human] exposed to 1,2,4,-benzenetriol.

E Miyahara, T
Nishikawa et al.

PubMed1

DON shares a similar mode of action as the PC Schmeits, MR
ribotoxic stress inducer anisomycin while
Katika et al.
TBTO shares ER stress patterns with the
ER stress inducer thapsigargin based oncomparative gene expression profiling in
Jurkat T cells.

PubMed2

Effect of zinc oxide nanomaterials-induced
oxidative stress on the p53 pathway.

MI Setyawati et al.

PubMed1

Building predictive models for mechanismof-action classification from phenotypic
assay data sets.

EL Berg et al.

PubMed2

Lush Science Prize 2014 – Background Paper

15TitleAuthors

SourceScore

An integrated metabonomics andtranscriptomics approach to understanding
metabolic pathway disturbance induced byperfluorooctanoic acid.

S Peng, L Yan etal.

PubMed2

miRNA expression profiling in a humanstem cell-based model as a tool fordevelopmental neurotoxicity testing.

G Pallocca, M
Fabbri et al.

PubMed2

Activation of Egr-1 in human lung epithelial
cells exposed to silica through MAPKs
signaling pathways.

L Chu, T Wang etal.

PubMed3

Toxicogenomics-based identification of
mechanisms for direct immunotoxicity.

J Shao, MR Katika
et al.

PubMed3

Evaluation of a human neurite growth assay AK Krug, NV
as specific screen for developmental
Balmer et al.
neurotoxicants.

PubMed2

Glutathione metabolism modeling: amechanism for liver drug-robustness and a
new biomarker strategy.

S Geenen, FB duPreez et al.

PubMed2

Titanium dioxide nanoparticles activate the
ATM-Chk2 DNA damage response in
human dermal fibroblasts.

RY Prasad, PD
Chastain et al.

PubMed3

Human 3D corneal models for a detailed
quantification of the initial depth of injury as
an indicator for cellular damage in thehuman eye

M. Bartok, D.
Gabel, et al

EUSAAT
2013

2+1

Further refinement of the reconstructed skin R. Curren, S.
micronucleus genotoxicity assay (RSMN)
Pfuhler et al.

EUSAAT2013

1

hSKP-derived hepatocyte-like cells for
toxicity testing

J. De Kock, R.
Rodrigues et al.

EUSAAT
2013

2

The way forward in case of a false positivein vitro genotoxicity result for a cosmetic
substance?

T. Doktorova, G.
Ates et al.

EUSAAT
2013

2+1

Testing neurodevelopmental toxicity on
differentiating human embryonic stem cells

A. C. Feutz and C.
de Geyter

EUSAAT
2013

1

Development and application of an
S. Foerster and M.
algorithm to determine statistically-valid
Leist
non-cytotoxic concentrations from imperfectin vitro cytotoxicity data sets

EUSAAT2013

2+1

Lush Science Prize 2014 – Background Paper

16TitleAuthors

SourceScore

Human neural progenitor cell (hNPC) aging
is mimicked by 3D cultures in vitro: an“Adverse Outcome Pathway” gains
importance at susceptible life stages

E. Fritsche

EUSAAT
2013

3

Immortalized primary-like human cells asnovel model systems in nephrotoxicity

J. Grillari

EUSAAT2013

2

Dynamic culture of human liver equivalents
inside a micro-bioreactor for long-term
substance testing

T. Hasenberg, E.
M. Materne et al.

EUSAAT
2013

2

Assessing the effects of repeated cigarettesmoke exposure using human organotypic
systems reproducing the respiratory tract invitro

J. Hoeng, C.
Mathis et al.

EUSAAT2013

2

In vitro phototoxicity screening assay orsystemically administered pharmaceuticals
using a reconstructed skin model EpiDerm

Y. Kaluzhny, M. W.
Kinuthia et al.

EUSAAT
2013

2

Optimization of the EpiOcular eye irritation
test for hazard identification and labelling ofchemicals in response to the requirementsof the EU Cosmetic Directive and REACH
Legislation

Y. Kaluzhny, H.
Kandarova et al.

EUSAAT
2013

2

Analysis of the validated Epiderm Skin
Corrosion Test (EpiDerm SCT) and a
prediction model for sub categorizationaccording to the UN GHS and EU CLP

H. Kandarova, S.
Letasiova et al.

EUSAAT2013

1

The diXa Project

J Kleinjans

EUSAAT
2013

2+1

Alternative method in practice:
postvalidation experience of the skinsensitization in vitro test strategy

S. N. Kolle, A.
Mehling et al.

EUSAAT2013

2

Defining compound subcytotoxic effects on
epigenetic determinants of humanembryonic stem cell renewal and lineage
commitment

E. Koutsouraki and
P. De Sousa

EUSAAT
2013

2

Integrating transcriptomics andmetabolomics to identify pathways of
toxicity of the parkinsonian toxin MPP+

A. Krug et al.

EUSAAT2013

2+1

Use of transcriptome profiling in stem cellbased test systems for reproductive toxicity

M. Leist

EUSAAT2013

2+1

Lush Science Prize 2014 – Background Paper

17TitleSourceScore

Modelling local and systemic toxicity:
J. Madden, M.
incorporation of in silico predictions in the
Hewitt et al.
development of adverse outcome pathways

EUSAAT
2013

2

The COSMOS project – developing
integrated computational approaches topredict repeated dose toxicity

J. Madden, A.
Richarz et al.

EUSAAT2013

1

Characterization of lymphoblastoid cell lines T. Markovic, M.
as a novel in vitro test system to predict
Gobec et al.
immunotoxicity of xenobiotics

EUSAAT
2013

2

A multi-organ-chip platform for long-term
maintenance and substance testing of
human tissue co-culture

E. M. Materne, A.
Lorenz et al

EUSAAT2013

2

Human embryonic stem cells differentiation
reveals toxicity signatures for HDAC
inhibitors and mercuric toxicants

K. Meganathan, S.
Jagtap et al

EUSAAT2013

3

Enhancing the readout of the embryonicstem cell test with molecular approaches

A. Piersma

EUSAAT
2013

2

Neurotoxicity in vitro: assessment of thepredictivity of neuronal networks coped tomicroelectrode arrays for identification ofneurotoxicants

T. Ramirez
Hernandez, T.
Weisschu et al

EUSAAT
2013

2

Inter-laboratory validation of the yeast
estrogen and yeast androgen screens for
identification of endocrine active
substances

T. Ramirez
Hernandez, C.
Woitkowiak et al.

EUSAAT2013

2+1

Identification of thalidomide-specifictranscriptomics and proteomics signaturesduring differentiation of human embryonicstem cells

A. Sachinidis

EUSAAT2013

2

Construction of an impedance monitoring
system for cell seeding and drug screening
in a 3D cell culture model

C. Schäfer, W.
Hilber et al.

EUSAAT
2013

2

New perspectives for realistic and efficientin vitro screening for inhalable drugs

O. Schmid, A. G.
Lenz et al.

EUSAAT
2013

2

Development of a microfluidic biochip forchronic monitoring of 3D neural tissues
derived from human embryonic stem cells

L. Stoppini et al.

EUSAAT
2013

2

Cerium oxide nanoparticles air exposure: a F. van Acker, I.
comparison study using a human 3D airway Kooter et almodel, A549 and BEAS-2B cell lines

EUSAAT
2013

2

Lush Science Prize 2014 – Background Paper

Authors18

TitleAuthors

SourceScore“Skin on a Chip” – Perfused long term
culture of skin tissue

I. Wagner, B. Atacet al.

EUSAAT
2013

2

Phase I biotransformation of testosterone
by human skin and reconstructed skintissues

G. Weindl, W.
Klipper et al.

EUSAAT2013

2

Evaluation of the sub-classification ofdermal corrosives in vitro using the epiCS®
(CellSystems) reconstructed human skinmodel

D. Weisensee, O.
Engelking et al

EUSAAT
2013

2

Monitoring of multilayer development of
human 3D cornea constructs by transepithelial impedance measurement

J. Wiest et al

EUSAAT
2013

2

Elucidation of perturbed pathways by using
stem cell derived neural progenitors

D. Zagoura, F.
Pistollato et al

EUSAAT
2013

2

Development and characterization of abioengineered conjunctiva model on thebasis of immortalized cells

M. Zorn-Kruppa, M. EUSAAT
Bartok et al.
2013

2

Towards a High Throughput Microscopy
Pathway in Toxicity Reporter Platform for
Chemical Safety Assessment.

S. Wink et al.

SEURAT1 2014

2

Infection of stem cell-derived hepatocyteswith hepatotropic viruses.

N Helsen, J
Paeshuyse et al.

SEURAT1 2014

2

Electrochemical Microsensors for
Monitoring the Functions of Liver Cells in a
Modular Based Microfluidic System.

J Bonne la Cour, S SEURATGenerell et al.
1 2014

2

Use of in silico tools to perform
extrapolation for risk assessment ofchemicals

M Gajewska, A
Pain et al

SEURAT1 2014

2+1

Hepatic Toxicity Evaluation Using Oxygenbased Phosphorescent Particles andMicrofluidics-based Glucose and Lactate
sensors

D Bavli, S Prill etal.

SEURAT1 2014

2

TK /TD modelling to analyze real time
hepatotoxicity data for cosmetics

S. Teng, S.
Barcellini, R. et al

SEURAT1 2014

2+1

COSMOS Database: Public Availability of
Repeated Dose Toxicity Data andCollaborative Interoperability with theToxBank Data Warehouse Supporting
Integrated Data Analysis

D Hristozov, N
Jeliazkova et al.

SEURAT1 2014

2

Lush Science Prize 2014 – Background Paper

19TitleAuthors

SourceScore

Doxorubicin irreversibly inhibits the
hypoxia-inducible factor (HIF) pathway inhuman cardiomyocytes

J Louisse, M
Barilari, et al.

SEURAT1 2014

3+1

Alerting Chemotypes for Liver Steatosis,
Steatohepatitis and Fibrosis Identified by
Mining COSMOS DB

A MostragSzlichtyng, V
Vitcheva et al.

SEURAT1 2014

2

ToxBank Integrated Analysis of SEURAT-1
Reference Compounds

P Kohonen, R
Grafström et al.

SEURAT1 20143

6.4 Nominated Abstracts
There were 8 projects which received the highest scores of either 3 or 3+1 for reporting
new pathways of toxicity. The abstracts are given below. We consider all worthy of
being considered by the judges as potential prize winners. We have not included thosescoring 2+1 because the emphasis of the award was on new pathways, rather than onbringing new knowledge or tools to previously understood toxicity pathways. Due tospace constraints, we have given the affiliations of the first author only, although manyof these projects were collaborative efforts between international partners. For
abstracts from published work, we provide the digital object identifier (DOD) whichallows the original document to be located online.
Doxorubicin irreversibly inhibits the hypoxia-inducible factor (HIF) pathway inhuman cardiomyocytes
J Louisse, M Barilari, U Chaudhari, J Antonydas Gaspar, J Hengstler, A Sachinidis, S
Hoffmann- Bremer. Systems Toxicology Unit / EURL- ECVAM, Institute for Health andConsumer Protection, Joint Research Centre, European Commission, Ispra, Italy
SEURAT-1 2014
Score 3+1
Abstract
We aimed to identify mechanisms underlying doxorubicin-induced delayed
cardiotoxicity. To this end we used transcriptomic and high content imaging (HCI) dataof iPSC-derived human cardiomyocytes that had been exposed to doxorubicin at a low,
clinically relevant, concentration of 150 nM using both single and repetitive dosing
regimen, including washout (recovery) studies. Analysis of the transcriptomic data
indicates that besides well-known pathways related to doxorubicin's DNA-damaging
effect; also genes controlled by the hypoxia- inducible factor (HIF) are affected.
Interestingly, modulations of the HIF pathway were not seen in doxorubicin exposed
primary liver cells. Currently, HCI analyses are being performed in order to assess thederegulation of the HIF-pathway in cardiomyocytes at the protein level. The findings
indicate that the observed delayed cardiotoxicity of doxorubicin may result from thedown-regulation of the HIF pathway that leads to an impairment of blood vessel
formation. Therefore, the selection of biomarkers for predictive cardiotoxicity testingshould include those related to an impaired hypoxia response.

Lush Science Prize 2014 – Background Paper

20Thimerosal induces apoptotic and fibrotic changes to kidney epithelial cells invitro
M F Hornos Carneiro, C Morais, D M. Small, D A. Vesey, F Barbosa Jr. Aand G C.
Gobe. Environmental Toxicology. Article first published online: 18 JUN 2014 DOI:
10.1002/tox.22012. Centre for Kidney Disease Research, School of Medicine,
University of Queensland, Translational Research Institute, Kent Street,
Woolloongabba, Brisbane, Queensland, Australia
Score 3
Abstract
Thimerosal is an ethyl mercury-containing compound used mainly in vaccines as abactericide. Although the kidney is a key target for mercury toxicity, thimerosal
nephrotoxicity has not received the same attention as other mercury species. The aimof this study was to determine the potential cytotoxic mechanisms of thimerosal on
human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposedfor 24 h to thimerosal (0–2 µM), and assessed for cell viability, apoptosis, and cellproliferation; expression of proteins Bax, nuclear factor-κB subunits, and transforming
growth factor-β1 (TGFβ1); mitochondrial health (JC-1, MitoTracker Red CMXRos); andfibronectin levels (enzyme-linked immunosorbent assay). Thimerosal diminished HK2cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability
transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion.
This is the first report about kidney cell death and pro-fibrotic mechanisms promoted bythimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces
kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic
pathway, and (2) thimerosal is a potential pro-fibrotic agent in human kidney cells. We
suggest that new evidence on toxicity as well as continuous surveillance in terms offibrogenesis is required concerning thimerosal use. © 2014 Wiley Periodicals, Inc.
Environ Toxicol, 2014.
Activation of Egr-1 in human lung epithelial cells exposed to silica through
MAPKs signaling pathways.
Chu L, Wang T, Hu Y, Gu Y, Su Z, Jiang H. PLoS One. 2013 Jul 18;8(7):e68943. doi:
10.1371/journal.pone.0068943. Department of Pathology, Third Xiangya hospital,
Central South University, Changsha, Hunan, PR China
Score 3
Abstract
The alveolar type II epithelial cell, regarded historically as a key target cell in initial
injury by silica, now appears to be important in both defense from lung damage as wellas elaboration of chemokines and cytokines. The molecular basis for silica-induced
epithelial cell injury is poorly understood. In this study we explored the activation of
nuclear factor Egr-1 and related signal pathway. Human II alveolar epithelial line A549
cells were exposed to silica for indicated time to assay the expression and activation of
Egr-1 and upstream MAPKs. Immunofluorescence, western-blot techniques, RT-PCR,
Electrophoretic mobility shift assay (EMSA), transient transfection assay, kinase
inhibitor experiments were performed. It was found that the expression of Egr-1 at
mRNA and protein level was significantly increased in A549 cells after administration

Lush Science Prize 2014 – Background Paper

21with silica and the activity of Egr-1 peaked by silica treatment for 60 minutes.
Furthermore, phosphorylated-ERK1/2, P38 MAPKs (the upstream kinase of Egr-1)
ballooned during 15-30minutes, 30-60minutes respectively after silica exposure in A549
cells. By administration of ERK1/2, P38 inhibitor, the expression and transcription of
Egr-1 were both markedly decreased. But PKC inhibitor did not prevent the increase of
Egr-1. These results indicated Egr-1 played a critical role in silica-induced pulmonary
fibrosis in an ERK1/2, P38 MAPKs-dependent manner, which suggests Egr-1 is an
essential regulator in silicosis, and underlines a new molecular mechanism for fibrosis
induced by silica.
Toxicogenomics-based identification of mechanisms for direct immunotoxicity.
Shao J, Katika MR, Schmeits PC, Hendriksen PJ, van Loveren H, Peijnenburg AA,
Volger OL. Toxicol Sci. 2013 Oct;135(2):328-46. doi:10.1093/toxsci/kft151. RIKILTInstitute of Food Safety, Wageningen University and Research Centre, 6700 AE
Wageningen, The Netherlands
Score 3
Abstract.
Compounds with direct immunotoxic properties, including metals, mycotoxins,
agricultural pesticides, and industrial chemicals, form potential human health risks due
to exposure through food, drinking water, and the environment. Insights into themechanisms of action are currently lacking for the majority of these direct
immunotoxicants. Therefore, the present work aimed to gain insights into the molecularmechanisms underlying direct immunotoxicity. To this end, we assessed in vitro theeffects of 31 test compounds on the transcriptome of the human Jurkat T-cell line.
These compounds included direct immunotoxicants, immunosuppressive drugs withdifferent mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of
the microarray data allowed us to identify canonical pathways and Gene Ontology
processes that were transcriptionally regulated in common by immunotoxicants (1) withstructural similarities, such as tributyltin chloride and tributyltin oxide that activated theretinoic acid/X receptor signaling pathway and (2) without structural similarities, such as
As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9 treated OTA, S9treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded
protein response, and FTY720, lindane, and propanil, which activated the cholesterol
biosynthesis pathway. In addition, processes uniquely affected by individual
immunotoxicants were identified, such as the induction of Notch receptor signalling andthe downregulation of acute-phase response genes by OTA. These findings werevalidated by quantitative real-time PCR analysis of genes involved in these processes.
Our study indicated that diverse modes of action are involved in direct immunotoxicity
and that a set of pathways or genes, rather than one single gene, can be used toscreen compounds for direct immunotoxicity.
Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in
human dermal fibroblasts.
Prasad RY, Chastain PD, Nikolaishvili-Feinberg N, Smeester L, Kaufmann WK, Fry RC.
Nanotoxicology. 2013 Sep;7(6):1111-9. doi: 10.3109/17435390.2012.710659.
Department of Environmental Sciences and Engineering, Gillings School of Global
Public Health, University of North Carolina, Chapel Hill, NC, USA

Lush Science Prize 2014 – Background Paper

22

Score 3
Abstract
The use of nanoparticles in consumer products increases their prevalence in theenvironment and the potential risk to human health. Although recent studies haveshown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a moredetailed view of the underlying mechanisms of this response needs to be established.
Here, the effects of nano-TiO2 on the DNA damage response and DNA replication
dynamics were investigated in human dermal fibroblasts. Specifically, the relationshipbetween nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2
and ATR/Chk1 was examined. The results show increased phosphorylation of H2AX,
ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA
synthesis and frequency of replicon initiation events in DNA-combed fibres. Taken
together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2
DNA damage response pathway.
Human neural progenitor cell (hNPC) aging is mimicked by 3D cultures in vitro:an “Adverse Outcome Pathway” gains importance at susceptible life stages.
E. Fritsche. Leibniz Research Institute of Environmental Medicine, Duesseldorf,
GermanyEUSAAT 2013.
Score 3
Abstract
The “Adverse Outcome Pathway” (AOP) concept is currently promoted by the OECDbecause it puts existing information, e.g. the linkage between a direct molecular
initiating event and an adverse outcome at a biological level of organization, into aframework that is applicable for risk assessment. Here, I present an AOP forneurotoxicity, which links an excess of reactive oxygen species (ROS) to impaired
neurogenesis. This exercise gathers existing information on the molecular and key
events involved in ROS-induced disturbance of this process, which is fundamental
during development as well as for neuronal regeneration in the adult. In the elderly,
cognition declines during the process of aging, also referred to as normal brain aging.
which is the adverse outcome. One reason for this loss of organ function is decline inregenerative capacities of NPCs. We have mimicked this decline of NPC function
during the aging process with human neurospheres in vitro and show that the key
player in adaptation against ROS, the transcription factor Nrf2, is playing a causal rolein this age-related susceptibility towards oxidative stress. In summary, we describe an
AOP for neurotoxicity and show that we can mimic the cellular responses of this AOP
with a 3D in vitro model. Moreover, we identify a susceptibility factor for a vulnerable life
stage, which is Nrf2 in NPCs of the aging brain.
Human embryonic stem cells differentiation reveals toxicity signatures for HDAC
inhibitors and mercuric toxicants.
K. Meganathan, S. Jagtap, S. Perumal Srinivasan, V. Shindev, V. Wagh, J. Hescheler,
and A. Sachinidis. Center of Physiology and Pathophysiology, Institute of
Neurophysiology, Cologne, Germany

Lush Science Prize 2014 – Background Paper

23

EUSAAT 2013.
Score 3
Abstract.
Differentiation of human embryonic stem cells (hESCs) has been aimed to use as a tool
for many clinical applications, previously we have used hESC differentiation to show themechanism of developmental toxicants. In the present study we have attempted toshow the toxicity mechanisms of HDAC inhibitors (Valproic acid (VPA), SAHA,
Trichostatin A (TSA)) and mercuric toxicants (Methyl mercury (MeHg), Mercury Chloride(HgCl2), thimerosal) using hESC differentiation in combination with genome wide
expression profiling. Gene expression analysis showed that VPA, reduced neural tube
(OTX2, DNMT3A) and forebrain (ISL1, and EMX2) related transcripts which have been
proved previously with in-vivo studies. In addition VPA over expressed axonogenesis
related markers such as SLIT1, SLITRK3, and SEMA3A. GO enrichment score analysis
reveals dysregulation of neuronal related biological processes such as neurogenesis
and neuronal differentiation with all three HDAC inhibitors. The mRNA and miRNAcorrelation analysis explains many miRNA signatures for VPA toxicity including
miR-20a, miR-302, miR-301, miR-155, and its corresponding mRNA targets. Also we
could demonstrate a set of neuronal related markers (including transcription factors)
that has been dysregulated by all HDAC inhibitors and mercuric toxicants preferably
MeHg and thimerosal. Together this multilineage differentiation system attempts torepresent the toxicity of the positive developmental neuronal toxicants with substantiate
molecular evidence.
References
Meganathan, K., Jagtap, S., Wagh, V., et al. (2012). Identification of thalidomidespecific transcriptomics and proteomics signatures during differentiation of humanembryonic stem cells. PLoS One 7, e44228.
Colleoni, S., Galli, C., Gaspar, J. A., et al. (2011). Development of a neuralteratogenicity test based on human embryonic stem cells: response to retinoic acid
exposure. Toxicol Sci 124, 370-377.
Copp, A. J. and Greene, N. D. (2010). Genetics and development of neural tube
defects. J. Pathol 220, 217-230.
Cotariu, D. and Zaidman, J. L. (1991). Developmental toxicity of valproic acid. Life Sci
48, 1341-1350.
Krug, A. K., Kolde, R., Gaspar, J. A., et al. (2013). Human embryonic stem cell-derived
test systems for developmental neurotoxicity: a transcriptomics approach. Arch Toxicol
87, 123-143.
Jagtap, S., Meganathan, K., Gaspar, J., et al. (2011). Cytosine arabinoside induces
ectoderm and inhibits mesoderm expression in human embryonic stem cells duringmultilineage differentiation. Br J Pharmacol 162, 1743-1756.
ToxBank Integrated Analysis of SEURAT-1 Reference Compounds
P Kohonen, R Grafström, G Myatt, and B Hardy. Karolinska Instituet, Sweden
SEURAT-1 2014
Score 3

Lush Science Prize 2014 – Background Paper

24

Abstract
The SEURAT-1 strategy is to adopt a mode-of-action framework to describe repeateddose toxicity to derive predictions of in vivo toxicity responses. ToxBank is the crosscluster infrastructure project which provides a web-accessible shared repository of
research data and protocols. Experiments generate dose response data over multiple
timepoints using different omics platforms including transcriptomics, proteomics,
metabolomics, and epigenetics over different cell lines and a common set of referencecompounds (details available at wiki.toxbank.net). Data is also generated fromfunctional assays and bioreactors and supplemented with in silico approaches. This
complex and heterogeneous data is consolidated and harmonized through theToxBank data warehouse in order to perform an integrated data analysis. We describe
for 14 reference compounds the meta-analysis of multiple types of time- dependent
dose response omics and functional data combined with in vitro and in vivo
background knowledge including consideration of modeling variations in biokinetic
responses. Open TG-GATEs human in vitro liver data of the reference compounds
includes reactive compounds (e.g., acetaminophen, CCl4), mitochondrial disruptors(e.g., Rotenone), promiscuous binders (e.g., valproic acid, amiadarone), nuclear
hormone receptor ligands (e.g., tamoxifen, WY14643), selective binders (e.g.
fluoxetine) and cardiotoxins (e.g., Doxorubicin, Nifedipine). Adverse events of interest
that are represented include cytotoxicity, fibrosis, steatosis, cholestasis andphospholipidosis. Overall we obtained 31,717 differential expression results with 14
compounds from the 45 comparisons, with Doxorubicin providing over 5000 results.
Pathway enrichment analysis of Doxorubicin identified a number of key pathways
including mismatch repair after 24 hours treatment and TNF-signaling at high doses
after 24 hours.

Lush Science Prize 2014 – Background Paper

25

7. Conclusions
The review of the most recent EUSAAT and SEURAT-1 conference proceedings, andan extensive literature search, yielded eight abstracts describing projects which arepotential candidates for the Lush Science Prize shortlist - see Section 6.
In the course of the research for this year's prize, three trends were noted. Firstly, there
is a significant amount of research centred around the potential toxicity of nanoparticles,
including pathways of toxicity. Given the ubiquity of nanoparticles in cosmetics andpersonal care products, it may be worth considering giving particular attention to thisarea of research in future years.
Secondly, we noted that research efforts are emerging which address the development
and validation of toxicity assays, and the identification of biomarkers of toxin exposure.
These are the key elements of Phase II of the seminal 2007 “Toxicity Testing in the 21stCentury: A Vision and Strategy” paper – briefly outlined in the 2012 Lush Science Prizebackground paper. The 2012 Science prize identified Phase I of the 21st century toxicity
roadmap – identifying toxicity pathways – as the focus of its efforts. This emphasis was
also applied in 2013 and 2014. We recommend that the 2015 Science Paper expands
its remit to include research that falls within Phase II of the adoption of 21st century
toxicity – assay development and validation, and identification of biomarkers of toxin
exposure.
Thirdly, while not directly relevant to the Lush Science prize, we came across several
EUSAAT conference abstracts that highlighted the efforts of developing countries toreplace animals in toxicity testing, and to educate scientists and educators on theimportance of replacing animal testing in their work. Examples include introducing the
RFFIT test to check for antibody development following rabies vaccinations, instead ofa mouse innoculation test, in Sri Lanka; also the work of Interniche, 2012 Lush Science
prize winners, who have supported humane science teaching and the replacement ofanimal experiments in a number of countries including Uzbekistan and Kyrgyzstan. In
passing, we also noted political efforts in major world economies to ban the use ofanimals for cosmetics testing, including in India, China, and the USA. Thesedevelopments may be suitable for consideration for other categories of the Lush Prize.

Lush Science Prize 2014 – Background Paper

26

Appendix 1. Abstracts of projects scoring 3 or more.
Genomic Allergen Rapid Detection In-House Validation—A Proof of Concept
H Johansson, F Rydnert, J Kühnl, A Schepky… - Toxicol. Sci. (June 2014) 139 (2):362-370. doi: 10.1093/toxsci/kfu046
Score 2+1
Abstract
Chemical sensitization is an adverse immunologic response to chemical substances,inducing hypersensitivity in exposed individuals. Identifying chemical sensitizers is ofgreat importance for chemical, pharmaceutical, and cosmetic industries, in order toprevent the use of sensitizers in consumer products. Historically, chemical sensitizers
have been assessed mainly by in vivo methods, however, recently enforced European
legislations urge and promote the development of animal-free test methods able topredict chemical sensitizers. Recently, we presented a predictive biomarker signature in
the myeloid cell line MUTZ-3, for assessment of skin sensitizers. The identified genomic
biomarkers were found to be involved in immunologically relevant pathways, induced byrecognition of foreign substances and regulating dendritic cell maturation andcytoprotective mechanisms. We have developed the usage of this biomarker signature
into a novel in vitro assay for assessment of chemical sensitizers, called Genomic
Allergen Rapid Detection (GARD). The assay is based on chemical stimulation of
MUTZ-3 cultures, using the compounds to be assayed as stimulatory agents. The
readout of the assay is a transcriptional quantification of the genomic predictors,
collectively termed the GARD Prediction Signature (GPS), using a complete genome
expression array. Compounds are predicted as either sensitizers or nonsensitizers by aSupport Vector Machine model. In this report, we provide a proof of concept for thefunctionality of the GARD assay by describing the classification of 26 blinded and 11
nonblinded chemicals as sensitizers or nonsensitizers. Based on these classifications,the accuracy, sensitivity, and specificity of the assay were estimated to 89, 89, and88%, respectively.
Dose response modeling of etoposide-induced DNA damage response
Z Li, B Sun, RA Clewell, ME Andersen, Q Zhang- Toxicol. Sci. (2013) doi: 10.1093/
toxsci/kft259.
Score 2+1.
Abstract
The 2007 NRC Report “Toxicity Testing in the 21st Century: A Vision and A Strategy”,
recommended an integrated, toxicity pathway-oriented approach for chemical testing.
As an integral component of the recommendation, computational dose responsemodeling of toxicity pathways promises to provide mechanistic interpretation andprediction of adverse cellular outcomes. Among the many toxicity pathways, the DNA
damage response is better characterized and thus more suited for computationalmodeling. In the present study, we formulated a minimal mathematical model of thispathway to examine the dose response for etoposide (ETP), an anti-cancer drug thatcauses DNA double strand breaks (DSBs). In the model, DSB results from inhibition of
topoisomerase by ETP and p53 is activated by a bistable switch composed of a positive

Lush Science Prize 2014 – Background Paper

27

feedback loop between ATM and γH2AX. Our stochastic model recapitulated the dose
response for several molecular biomarkers measured with flow cytometry in HT1080
cells, including phosphorylated p53, ATM, γH2AX, and micronuclei. Model simulations
were consistent with a bimodal pattern of p53 activation and a graded populationaveraged response at high ETP concentrations. The graded response was a result ofheterogeneous activation of individual cells due to molecular stochasticity. This workshows the value of combining data collection on single cell responses and mechanistic,
stochastic modeling to develop and test hypothesis for the circuitry of important toxicity
pathways. Future studies will determine how well this initial modeling effort agrees witha broader set of experimental studies on pathway responses by examining a morediverse group of DNA-damaging compounds.
Implementing Toxicity Testing in the 21st Century (TT21C): Making safety
decisions using toxicity pathways, and progress in a prototype risk assessment.
Yeyejide Adeleye, Melvin Andersen, Rebecca Clewell, Michael Davies, Matthew Dent,
Sue Edwards, Paul Fowler, Sophie Malcomber, Beate Nicola, Andrew Scott, Sharon
Scott, Bin Sun, Carl Westmoreland, Andrew White, Qiang Zhang, Paul L. Carmichael.
Toxicology. In press, DOI: 10.1016/j.tox.2014.02.00
Score 2+1
Abstract
Risk assessment methodologies in toxicology have remained largely unchanged for
decades. The default approach uses high dose animal studies, together with human
exposure estimates, and conservative assessment (uncertainty) factors or linear
extrapolations to determine whether a specific chemical exposure is ‘safe’ or ‘unsafe’.
Although some incremental changes have appeared over the years, results from all
new approaches are still judged against this process of extrapolating high-dose effects
in animals to low-dose exposures in humans. The US National Research Council
blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy
called for a transformation of toxicity testing from a system based on high-dose studies
in laboratory animals to one founded primarily on in vitro methods that evaluatechanges in normal cellular signalling pathways using human-relevant cells or tissues.
More recently, this concept of pathways-based approaches to risk assessment has
been expanded by the description of ‘Adverse Outcome Pathways’ (AOPs). The
question, however, has been how to translate this AOP/TT21C vision into the practical
tools that will be useful to those expected to make safety decisions. We have sought toprovide a practical example of how the TT21C vision can be implemented to facilitate asafety assessment for a commercial chemical without the use of animal testing. To thisend, the key elements of the TT21C vision have been broken down to a set of actions
that can be brought together to achieve such a safety assessment. Such components ofa pathways-based risk assessment have been widely discussed, however to-date, noworked examples of the entire risk assessment process exist. In order to begin to test
the process, we have taken the approach of examining a prototype toxicity pathway(DNA damage responses mediated by the p53 network) and constructing a strategy forthe development of a pathway based risk assessment for a specific chemical in a casestudy mode. This contribution represents a ‘work-in-progress’ and is meant to both
highlight concepts that are well-developed and identify aspects of the overall process
which require additional development. To guide our understanding of what a pathwaysbased risk assessment could look like in practice, we chose to work on a case study

Lush Science Prize 2014 – Background Paper

28chemical (quercetin) with a defined human exposure and to bring a multidisciplinary
team of chemists, biologists, modellers and risk assessors to work together towards asafety assessment. Our goal was to see if the in vitro dose response for quercetin could
be sufficiently understood to construct a TT21C risk assessment without recourse torodent carcinogenicity study data. The data presented include high throughput pathway
biomarkers (p-H2AX, p-ATM, p-ATR, p-Chk2, p53, p-p53, MDM2 and Wip1) andmarkers of cell-cycle, apoptosis and micronuclei formation, plus gene transcription in
HT1080 cells. Eighteen point dose response curves were generated using flow
cytometry and imaging to determine the concentrations that resulted in significant
perturbation. NOELs and BMDs were compared to the output from biokinetic modelling
and the potential for in vitro to in vivo extrapolation explored. A first tier risk assessment
was performed comparing the total quercetin concentration in the in vitro systems withthe predicted total quercetin concentration in plasma and tissues. The shortcomings ofthis approach and recommendations for improvement are described. This paper
therefore describes the current progress in an ongoing research effort aimed atproviding a pathways-based, proof-of-concept in vitro-only safety assessment for a
consumer use product.
Proposal of an in silico profiler for categorisation of repeat dose toxicity data of
hair dyes
Nelms MD1, Ates G, Madden JC, Vinken M, Cronin MT, Rogiers V, Enoch SJ. Arch
Toxicol. 2014 Jun 3..
Score 2+1
Abstract.
This study outlines the analysis of 94 chemicals with repeat dose toxicity data takenfrom Scientific Committee on Consumer Safety opinions for commonly used hair dyesin the European Union. Structural similarity was applied to group these chemicals into
categories. Subsequent mechanistic analysis suggested that toxicity to mitochondria ispotentially a key driver of repeat dose toxicity for chemicals within each of thecategories. The mechanistic hypothesis allowed for an in silico profiler consisting of fourmechanism-based structural alerts to be proposed. These structural alerts related to anumber of important chemical classes such as quinones, anthraquinones, substituted
nitrobenzenes and aromatic azos. This in silico profiler is intended for groupingchemicals into mechanism-based categories within the adverse outcome pathway
paradigm.
Molecular modelling study of the PPARγ receptor in relation to the mode ofaction/adverse outcome pathway framework for liver steatosis
Tsakovska I, Al Sharif M, Alov P, Diukendjieva A, Fioravanzo E, Cronin MT, Pajeva I.
Int J Mol Sci. 2014 May 5;15(5):7651-66. doi: 10.3390/ijms15057651.
Score 2+1
Abstract.
The comprehensive understanding of the precise mode of action and/or adverse
outcome pathway (MoA/AOP) of chemicals has become a key step toward thedevelopment of a new generation of predictive toxicology tools. One of the challengesof this process is to test the feasibility of the molecular modelling approaches to explore
Lush Science Prize 2014 – Background Paper

29

key molecular initiating events (MIE) within the integrated strategy of MoA/AOP
characterisation. The description of MoAs leading to toxicity and liver damage has been
the focus of much interest. Growing evidence underlines liver PPARγ ligand-dependent
activation as a key MIE in the elicitation of liver steatosis. Synthetic PPARγ full agonists
are of special concern, since they may trigger a number of adverse effects not observedwith partial agonists. In this study, molecular modelling was performed based on the
PPARγ complexes with full agonists extracted from the Protein Data Bank. The receptor
binding pocket was analysed, and the specific ligand-receptor interactions wereidentified for the most active ligands. A pharmacophore model was derived, and themost important pharmacophore features were outlined and characterised in relation totheir specific role for PPARγ activation. The results are useful for the characterisation of
the chemical space of PPARγ full agonists and could facilitate the development of
preliminary filtering rules for the effective virtual ligand screening of compounds withPPARγ full agonistic activity.
Thimerosal induces apoptotic and fibrotic changes to kidney epithelial cells invitro
M F Hornos Carneiro, C Morais, D M. Small, D A. Vesey, F Barbosa Jr. Aand G C.
Gobe. Environmental Toxicology. Article first published online: 18 JUN 2014 DOI:
10.1002/tox.22012.
Score 3
Abstract
Thimerosal is an ethyl mercury-containing compound used mainly in vaccines as abactericide. Although the kidney is a key target for mercury toxicity, thimerosal
nephrotoxicity has not received the same attention as other mercury species. The aimof this study was to determine the potential cytotoxic mechanisms of thimerosal on
human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposedfor 24 h to thimerosal (0–2 µM), and assessed for cell viability, apoptosis, and cellproliferation; expression of proteins Bax, nuclear factor-κB subunits, and transforming
growth factor-β1 (TGFβ1); mitochondrial health (JC-1, MitoTracker Red CMXRos); andfibronectin levels (enzyme-linked immunosorbent assay). Thimerosal diminished HK2cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability
transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion.
This is the first report about kidney cell death and pro-fibrotic mechanisms promoted bythimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces
kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic
pathway, and (2) thimerosal is a potential pro-fibrotic agent in human kidney cells. We
suggest that new evidence on toxicity as well as continuous surveillance in terms offibrogenesis is required concerning thimerosal use. © 2014 Wiley Periodicals, Inc.
Environ Toxicol, 2014.
Activation of Egr-1 in human lung epithelial cells exposed to silica through
MAPKs signaling pathways.
Chu L, Wang T, Hu Y, Gu Y, Su Z, Jiang H. PLoS One. 2013 Jul 18;8(7):e68943. doi:
10.1371/journal.pone.0068943. Print 2013.
Score 3

Lush Science Prize 2014 – Background Paper

30

Abstract
The alveolar type II epithelial cell, regarded historically as a key target cell in initial
injury by silica, now appears to be important in both defense from lung damage as wellas elaboration of chemokines and cytokines. The molecular basis for silica-induced
epithelial cell injury is poorly understood. In this study we explored the activation of
nuclear factor Egr-1 and related signal pathway. Human II alveolar epithelial line A549
cells were exposed to silica for indicated time to assay the expression and activation of
Egr-1 and upstream MAPKs. Immunofluorescence, western-blot techniques, RT-PCR,
Electrophoretic mobility shift assay (EMSA), transient transfection assay, kinase
inhibitor experiments were performed. It was found that the expression of Egr-1 at
mRNA and protein level was significantly increased in A549 cells after administration
with silica and the activity of Egr-1 peaked by silica treatment for 60 minutes.
Furthermore, phosphorylated-ERK1/2, P38 MAPKs (the upstream kinase of Egr-1)
ballooned during 15-30minutes, 30-60minutes respectively after silica exposure in A549
cells. By administration of ERK1/2, P38 inhibitor, the expression and transcription of
Egr-1 were both markedly decreased. But PKC inhibitor did not prevent the increase of
Egr-1. These results indicated Egr-1 played a critica role in silica-induced pulmonary
fibrosis in an ERK1/2, P38 MAPKs-dependent manner, which suggests Egr-1 is an
essential regulator in silicosis, and underlines a new molecular mechanism for fibrosis
induced by silica.
Toxicogenomics-based identification of mechanisms for direct immunotoxicity.
Shao J, Katika MR, Schmeits PC, Hendriksen PJ, van Loveren H, Peijnenburg AA,
Volger OL. Toxicol Sci. 2013 Oct;135(2):328-46. doi: 10.1093/toxsci/kft151.
Score 3
Abstract.
Compounds with direct immunotoxic properties, including metals, mycotoxins,
agricultural pesticides, and industrial chemicals, form potential human health risks due
to exposure through food, drinking water, and the environment. Insights into themechanisms of action are currently lacking for the majority of these direct
immunotoxicants. Therefore, the present work aimed to gain insights into the molecularmechanisms underlying direct immunotoxicity. To this end, we assessed in vitro theeffects of 31 test compounds on the transcriptome of the human Jurkat T-cell line.
These compounds included direct immunotoxicants, immunosuppressive drugs withdifferent mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of
the microarray data allowed us to identify canonical pathways and Gene Ontology
processes that were transcriptionally regulated in common by immunotoxicants (1) withstructural similarities, such as tributyltin chloride and tributyltin oxide that activated theretinoic acid/X receptor signaling pathway and (2) without structural similarities, such as
As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9 treated OTA, S9treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded
protein response, and FTY720, lindane, and propanil, which activated the cholesterol
biosynthesis pathway. In addition, processes uniquely affected by individual
immunotoxicants were identified, such as the induction of Notch receptor signaling andthe downregulation of acute-phase response genes by OTA. These findings wereLush Science Prize 2014 – Background Paper

31validated by quantitative real-time PCR analysis of genes involved in these processes.
Our study indicated that diverse modes of action are involved in direct immunotoxicity
and that a set of pathways or genes, rather than one single gene, can be used toscreen compounds for direct immunotoxicity.
Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in
human dermal fibroblasts.
Prasad RY, Chastain PD, Nikolaishvili-Feinberg N, Smeester L, Kaufmann WK, Fry
RC. Nanotoxicology. 2013 Sep;7(6):1111-9. doi: 10.3109/17435390.2012.710659.
Score 3
Abstract
The use of nanoparticles in consumer products increases their prevalence in theenvironment and the potential risk to human health. Although recent studies haveshown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a moredetailed view of the underlying mechanisms of this response needs to be established.
Here, the effects of nano-TiO2 on the DNA damage response and DNA replication
dynamics were investigated in human dermal fibroblasts. Specifically, the relationshipbetween nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2
and ATR/Chk1 was examined. The results show increased phosphorylation of H2AX,
ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA
synthesis and frequency of replicon initiation events in DNA-combed fibres. Taken
together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2
DNA damage response pathway.
Human 3D corneal models for a detailed quantification of the initial depth of
injury as an indicator for cellular damage in the human eye.
M. Bartok, D. Gabel, M. Engelke, M. Zorn-Kruppa3, J. M. Brandner, K. Reisinger, K.
Daton, and K. Mewes EUSAAT 2013
score 2+1
Abstract
Currently, the definite prediction of all GHS categories for the eye-irritating potential ofchemicals in one single test system relevant to the human eye is not possible. Instead,chemicals can only be classified according to the GHS in the framework of an extensive
battery.
Our study aims at the complete replacement of the Draize Eye Irritation Test by a new
test system which is based on biotechnologically produced hemi-cornea equivalents(Engelke et al., 2013). The sophisticated structure of the hemi-cornea model comprises
both an epithelium and stroma compartment. Hence, this two-compartment model
offers the potential to analyze the initial depth of injury (DoI) after substance applicationand to discriminate between damages induced in the epithelium and/or the stroma. We
developed different approaches for the analysis of the corneal DoI in the hemi-cornea
model:
1. A collagen membrane is inserted between stroma and epithelium during production
as an artificial Bowman’s

Lush Science Prize 2014 – Background Paper

32membrane. This membrane allows the detachment of the epithelium from the stroma
and the individual quantification of the cell viability in both compartments after chemicaltreatment.
2. A TUNEL assay is performed on cryosections of the hemicornea in order to label all
cells which have undergone
apoptosis after topical treatment with the respective test substances. The TUNEL assay
can be combined with thedetection of cleaved-caspase 3, another biomarker for apoptosis. The border between
the area of fluorescentlabelled cells and non-labelled cells indicates the DoI.
3. In order to specifically label the viable cells within the treated tissues, the MTT
viability assay is combined with image analysis on cryosections. Quantitativeinterpretation of the cryosection images is based on ImageJ software analysis tools. By
using this method we can distinguish between substances of all three GHS categories
by means of their depth of injury.
By using one single method or a combination of the abovementioned methods we aimat the quantification of the initial depth of injury within epithelium and stroma of thehemi-cornea as a stand-alone test system for the reliable prediction of the eye irritating
potential of substances according to the GHS system.
The way forward in case of a false positive in vitro genotoxicity result for acosmetic substance?
T. Doktorova, G. Ates, C. Chesne, T. Vanhaecke, and V. Rogiers.
EUSAAT 2013.
Score 2+1.
Abstract.
The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a highsensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant
positive results (i.e., low specificity) thereby imposing the need for additional follow-up
by in vitro and in vivo genotoxicity tests. This has a major impact on the cosmetic
industry in Europe, seen the imposed animal testing and marketing bans on cosmetics
and their ingredients. Afflicted but safe substances will be lost. Using the example oftriclosan, a cosmetic preservative, proofof- principle for the potential applicability of a
toxicogenomics based in vitro assay as a potential follow-up test for positive in vitro
genotoxicity results, is described. Triclosan shows a positive in vitro chromosomal
aberration test but is negative during in vivo follow-up tests. Toxicogenomics analysis
unequivocally shows that triclosan is identified as a compound acting through non-AND
reactive mechanisms. This study illustrates the potential of genome-wide
transcriptomics data in combination with in vitro experimentation as a possible weightofevidence follow-up approach for de-risking a positive outcome in a standard
mutagenicity/genotoxicity battery. As such, in the future a substantial number ofcompounds wrongly identified as genotoxicants could be saved.
Development and application of an algorithm to determine statistically-valid noncytotoxic concentrations from imperfect in vitro cytotoxicity data sets.

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S. Foerster and M. Leist.
EUSAAT 2013.
Score 2+1
Abstract.
Many in vitro test systems for chemicals or bioactive agents measure sophisticatedfunctional or biochemical endpoints. However, such data can be hard to interpret, or beeven meaningless, when cells are dying under the chosen assay conditions. Therefore,
the determination of the non-cytotoxic concentration range for each test compound is
crucial. A simple and widely used approximation is to define concentrations below thosethat trigger a 10% reduction in cell viability as non-cytotoxic (EC10 of the full cytotoxicity
curve). However, a statistically valid approach to define non cytotoxicity is based on
three conditions: first, a benchmark response (BMR) level (e.g. 10% cell death) needs
to be defined; second, the benchmark concentration (BMC), i.e. the concentration of thetest compound at which the BMR is reached, needs to be defined. This requires
selection of a mathematical model, and its adaptation to the data points of thecytotoxicity curve. Third, the lower limit of the confidence interval of the BMC (BMCL)
needs to be calculated, because only concentrations smaller than the BMCL can beconsidered to be non-cytotoxic with a defined minimum confidence level. An automatedprocedure to calculate the BMCL corresponding to a BMR of 15% cell death (BMCL15)
has been developed here. For many data sets of average quality this BMCL15
corresponded to the EC10, but it provided the additional advantage of taking the overall
data quality into account and thereby increases the reliability of the cytotoxicity border.
The concept of BMC and BMCL is an adaptation of the benchmark dose (BMD) concept
developed for in vivo toxicology studies. Several software programs are available forthe purpose of BMD calculations, but the adaptation to in vitro tests is difficult for benchscientists, and the concept has still not found its way into most of the toxicology
laboratories. A further reason that the BMC concept is not widely implemented yet isthat this statistical approach has to be adapted to real-life problems that are typical forcomplex cellular assays. For instance, the data may not cover the full range of
toxicities, data variation may be large, assays with different test concentrations mayneed to be pooled and normalizations may result in imperfect baseline data. We show
here that the software solution developed by us automatically copes with such types ofconcentration-response data from various stem cell-based assays from the ESNATS
consortium. To define cytotoxicity in these test systems it was crucial to develop a
reliable and reproducible method that allows interlaboratory comparisons. Aninterdisciplinary programming approach was chosen by combining biologicalassumptions with a statistical BMC estimation concept. Using this approach it waspossible to determine the highest non-cytotoxic concentration for all compounds tested
in the different stem cell based test systems.
Human neural progenitor cell (hNPC) aging is mimicked by 3D cultures in vitro:an “Adverse Outcome Pathway” gains importance at susceptible life stages.
E. Fritsche
EUSAAT 2013.
Score 3
Abstract

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34

The “Adverse Outcome Pathway” (AOP) concept is currently promoted by the OECDbecause it puts existing information, e.g. the linkage between a direct molecular
initiating event and an adverse outcome at a biological level of organization, into aframework that is applicable for risk assessment. Here, I present an AOP forneurotoxicity, which links an excess of reactive oxygen species (ROS) to impaired
neurogenesis. This exercise gathers existing information on the molecular and key
events involved in ROS-induced disturbance of this process, which is fundamental
during development as well as for neuronal regeneration in the adult. In the elderly,
cognition declines during the process of aging, also referred to as normal brain aging.
which is the adverse outcome. One reason for this loss of organ function is decline inregenerative capacities of NPCs. We have mimicked this decline of NPC function
during the aging process with human neurospheres in vitro and show that the key
player in adaptation against ROS, the transcription factor Nrf2, is playing a causal rolein this age-related susceptibility towards oxidative stress. In summary, we describe an
AOP for neurotoxicity and show that we can mimic the cellular responses of this AOP
with a 3D in vitro model. Moreover, we identify a susceptibility factor for a vulnerable life
stage, which is Nrf2 in NPCs of the aging brain.
The diXa Project.
J Kleinjans.
EUSAAT 2013.
Score 2+1.
Abstract
The EU nowadays witnesses increasing demands with regard to chemical safety. In
particular, animal-based test models need to be replaced preferably by robust, nonanimal assays in vitro/ in silico which better predict human toxicity in vivo, are lesscostly, and are socially better acceptable. Consumer’s and patient’s health will benefit
and competitiveness of EU’s chemical manufacturing industry will be increased. For
developing such assays, FP6/FP7 Research Programmes are exploiting the revenuesof data-dense genomics technologies. However, till date, there is no infrastructure
foreseen which aims at capturing all data produced by toxicogenomics (TGX) projects,
in a standardized, harmonized and sustainable manner. Data may thus evaporate. The
lack of such an infrastructure also prevents innovative breakthroughs from metaanalyses of joint databases and systems modeling. Driven by these needs of the TGX
research community, diXa focuses on networking activities, for building a web-based,
openly accessible and sustainable e-infrastructure for capturing TGX data, and forlinking this to available data bases holding chemico/physico/toxicological information,and to data bases on molecular medicine, thus crossing traditional borders between
scientific disciplines and reaching out to other research communities. To advance data
sharing, diXa ensures clear communication channels and delivers commonly agreed
core service support to the TGX research community, by providing SOPs for seamlessdata sharing, and by offering quality assessments and newly developed tools andtechniques for data management, all supported by hands-on training. Through its joint
research initiative, by using data available from its data infrastructure, diXa will
demonstrate the feasibility of its approach by performing cross-platform integrative
statistical analyses, and cross-study meta-analyses, to create a systems model for
predicting chemical- induced liver injury.

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35

Integrating transcriptomics and metabolomics to identify pathways of toxicity ofthe parkinsonian toxin MPP+.
A. Krug, T. Hartung, and M. Leist.
EUSAAT 2013
Score 2+1
Abstract.
Chemicals targeting the nervous system may favour the risk of the development ofbrain disorders such as autism or Parkinson’s disease (PD). Therefore test systems areneeded to first of all identify neurotoxicants and in a subsequent step to classify themaccording to their mode of action. Individual “omics” technologies are of rising interest in
the field of toxicology for classifications, but they cannot unravel all the complexities of
toxicant effects on human beings. As new alternative strategy, we integrated two“omics”-technologies, transcriptomics and metabolomics, to obtain a more precise
picture of all cellular processes in an established model system of basal ganglia
neurotoxicity. We exposed human dopaminergic neurons to the parkinsonian toxin MPP+, to test the usefulness of this combined “omics” technology. With the combination oftranscriptomics and metabolomics we wanted to answer the question, which of theevents triggered by MPP+, including mitochondrial respiration deficits, oxidative stressand energy failure, was most upstream. After 24 h of treatment with 5 µM MPP+, ATPdepletion was rather minor, but pronounced changes were already seen on thetranscriptome level. Gene ontology comparison did not support our expectation ofaltered glycolysis, but revealed changes which were mainly involved in cellular aminoacid and amine metabolic processes (upregulated), as well as reactive oxygen species
metabolic processes (upregulated) and DNA conformation changes (downregulated).
On the metabolic level changes of glutathione, homocysteine or methionine sulfoxide
confirmed the role of oxidative stress as initial pathway of toxicity. Combining data ofboth approaches, the transsulfuration pathway seemed to be strongly implicated in
MPP toxicity. This pathway contributes to the conversion of methionine to cysteine,
which is the rate limiting amino acid for glutathione synthesis. Intermediates of thispathway, such as S-adenosyl-methionine and S adenosyl-homocysteine are important
contributors in DNA methylation processes, possibly explaining the changes on AND
conformation. Our data suggests that combined omics analysis is more sensitive forpathway identification than individual approaches and we found that effects on DNA
level as well as oxidative stress are observable before strong mitochondrial respiration
deficits or energy failure were detected.
References
Ramirez, T., Daneshian, M., Kamp, H., et al. (2013). Metabolomics in toxicology andpreclinical research. ALTEX 30, 209 225.
Pöltl, D., Schildknecht, S., Karreman, C., and Leist, M. (2012). Uncoupling of ATPdepletion and cell death in human dopaminergic neurons. Neurotoxicology 33, 769-779.
Scholz, D., Pöltl, D., Genewsky, A. et al. (2011). Rapid, complete and large-scale
generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem
119, 957- 971.

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36

Use of transcriptome profiling in stem cell based test systems for reproductive
toxicity.
M. Leist.
EUSAAT 2013.
Score 2+1
Abstract
Neurodevelopmental toxicity is usually characterized by relatively subtle changes ofcellular phenotype that result in altered connectivity and network function of the nervous
system. One approach to characterize in a relatively comprehensive way the changesof cells exposed to toxicants, is whole genome transcript profiling. Many developmental
toxicants exert effects even after their exposure has ceased. A potential explanation are
epigenetic changes, left as “memory traces” in the genome. We have used here several
human embryonic stem cell (hESC)-based models of early neural development to study
transcriptional and epigenetic changes exerted by several different mercurial
compounds and histone deacetylase inhibitors (HDACi). For instance, one system(UKN1) modelled the directed differentiation of pluripotent hESC to PAX6-positive
neural precursors, and another system (UKK) modelled the same period of early tissue
specification, but allowed generation of cells of all germ layers. In all systems,
transcriptome profiling clearly distinguished mercurials from HDACi and negative
control compounds. The transcriptome patterns were used to predict the modes ofaction of unknown compounds in a “blind testing” part of this study. The transcriptome
patterns were also analysed for underlying biological themes. The overrepresentation oftranscription factor binding sites in different transcriptome sets indicated an astonishingoverlap of the damage response to different toxicants, but also indicated “pathways”
triggered only by one class of toxicant, but not by another. The idea of exploring
pathways of toxicity on the basis of the altered transcriptome patterns was followed
further by time-series analyses and epigenetic profiling. Preliminary results suggest that“pathways-of-toxicity” may not be constant for a given toxicant, but that they are highly
dependent on the model system and the time point of exposure.
Background reading of previous work Krug, A. K., Kolde, R., Gaspar, J. A., et al. (2013).
Human embryonic stem cell-derived test systems for developmental neurotoxicity: atranscriptomics approach. Arch Toxicol 87, 123-143.
Human embryonic stem cells differentiation reveals toxicity signatures for HDAC
inhibitors and mercuric toxicants.
K. Meganathan, S. Jagtap, S. Perumal Srinivasan, V. Shindev, V. Wagh, J. Hescheler,
and A. Sachinidis.
EUSAAT 2013
Score 3
Abstract
Differentiation of human embryonic stem cells (hESCs) has been aimed to use as a tool
for many clinical applications, previously we have used hESC differentiation to show themechanism of developmental toxicants. In the present study we have attempted toshow the toxicity mechanisms of HDAC inhibitors (Valproic acid (VPA), SAHA,
Trichostatin A (TSA)) and mercuric toxicants (Methyl mercury (MeHg), Mercury Chloride

Lush Science Prize 2014 – Background Paper

37

(HgCl2), thimerosal) using hESC differentiation in combination with genome wide
expression profiling. Gene expression analysis showed that VPA, reduced neural tube
(OTX2, DNMT3A) and forebrain (ISL1, and EMX2) related transcripts which have been
proved previously with in-vivo studies. In addition VPA over expressed axonogenesis
related markers such as SLIT1, SLITRK3, and SEMA3A. GO enrichment score analysis
reveals dysregulation of neuronal related biological processes such as neurogenesis
and neuronal differentiation with all three HDAC inhibitors. The mRNA and miRNAcorrelation analysis explains many miRNA signatures for VPA toxicity including
miR-20a, miR-302, miR-301, miR-155, and its corresponding mRNA targets. Also we
could demonstrate a set of neuronal related markers (including transcription factors)
that has been dysregulated by all HDAC inhibitors and mercuric toxicants preferably
MeHg and thimerosal. Together this multilineage differentiation system attempts torepresent the toxicity of the positive developmental neuronal toxicants with substantiate
molecular evidence.
References
Meganathan, K., Jagtap, S., Wagh, V., et al. (2012). Identification of thalidomidespecific transcriptomics and proteomics signatures during differentiation of humanembryonic stem cells. PLoS One 7, e44228.
Colleoni, S., Galli, C., Gaspar, J. A., et al. (2011). Development of a neuralteratogenicity test based on human embryonic stem cells: response to retinoic acid
exposure. Toxicol Sci 124, 370-377.
Copp, A. J. and Greene, N. D. (2010). Genetics and development of neural tube
defects. J. Pathol 220, 217-230.
Cotariu, D. and Zaidman, J. L. (1991). Developmental toxicity of valproic acid. Life Sci
48, 1341-1350.
Krug, A. K., Kolde, R., Gaspar, J. A., et al. (2013). Human embryonic stem cell-derived
test systems for developmental neurotoxicity: a transcriptomics approach. Arch Toxicol
87, 123-143.
Jagtap, S., Meganathan, K., Gaspar, J., et al. (2011). Cytosine arabinoside induces
ectoderm and inhibits mesoderm expression in human embryonic stem cells duringmultilineage differentiation. Br J Pharmacol 162, 1743-1756.
Inter-laboratory validation of the yeast estrogen and yeast androgen screens for
identification of endocrine active substances.
T. Ramirez Hernandez, C. Woitkowiak, H. A. Huener, C. Schoenlau, H. Hollert, S.
Broschk, O. Zierau, G. Vollmer, M. Jaeger, A. Poth, E. Higley, M. Hecker, M. Liebsch, S.
Hoffmann, B. van Ravenzwaay, and R. Landsiedel.
EUSAAT 2013
Score 2+1
Abstract
Endocrine disruptor compounds (EDCs) are a group of natural or synthetic compounds
that have the capacity to interact with the endocrine system of living organisms andconsequently causes adverse health effects in an intact organism, or its progeny, or(sub)populations. Due to the impact that this interaction could have on human health,there is an increasing interest in assessing the risk of the exposure to EDCs. Currently,

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38

several in vitro and in vivo assays have been developed and few of them validated andregulatory accepted. For instance, the US EPA developed the Endocrine Disruptor
Screening Program, which has been recently implemented. For the programme a large
number of experimental animals will be still use used even for testing some of the invitro assays. Herein, we performed the inter-laboratory validation of two robust models
that addresses agonistic and antagonist effect at the human hormone receptor, the YES(Yeast Estrogen Screen) and the YAS (Yeast Androgen Screen). Both assays are nonanimal alternatives to the estrogen and androgen receptor binding assays proposed in
the EDSP and OECD Conceptual Framework. The ring trial is the final experimental
part of the validation process at the European Center for Validation of Alternative
Methods (ECVAM). A set of 24 compounds with estrogenic, anti-estrogenic, androgenicor anti-androgenic activity were tested in five different laboratories in a blinded fashion.
The analysis of the data demonstrates a high reproducibility for both methods among
the different participating laboratories. Most importantly, both assays exhibited a goodpredictivity. In conclusion, the methods have been successfully transferred to otherlaboratories and they exhibit a high accuracy to identify EDCs that interact with sex
hormone receptors.
Use of in silico tools to perform extrapolation for risk assessment of chemicals
M Gajewska, A Paini, J Vicente, S Benito, A Worth.
SEURAT-1 2014
Score 2+1
Abstract
The aim of this work is to develop a computer-assisted toxicokinetic/ toxicodynamic
strategy for risk assessment of cosmetic ingredients promoting alternatives to animal
testing. A physiologically based toxicokinetic-toxicodynamic (PBTK-TD) model and avirtual cell-based assay (VCBA) for humans have been developed to simulate acuteexposure to cosmetics via inhalation, oral and dermal absorption. The models havealso been coupled to perform in vitro- to-in vivo (IVIVE) and route-to-route (RR)
extrapolations. The IVIVE is a multiscale approach that joins in vitro information at celllevel with in vivo organ level kinetics. RR is useful in cases when toxicity data for one
exposure route are available, but others are lacking. As an example, a case study on
caffeine is presented. Blood concentrations following oral and dermal exposure are
simulated at the oral no-observed-effect-level (NOEL) dose derived from animal studies
to predict internal systemic concentrations and extrapolated to a dermal safe limit. The
difference in resulting toxicokinetic parameters (Area Under Curve, bioavailability, peakconcentration, percentage of dose absorbed) is shown for two exposure routes. The
coupled dynamic model at organ level links the calculated blood levels with so-called"effect" compartment to quantify the development of caffeine tolerance and VCBA
calculates the estimated concentration inside the liver cell (HepaRG). The entire
analysis including data access, model simulation in R, visualisation and reporting havebeen implemented in a KNIME workflow which is freely available for use.
TK /TD modelling to analyze real time hepatotoxicity data for cosmetics
S. Teng, S. Barcellini, R. Beaudouin, R. Rahmani, A. Pery
SEURAT-1 2014

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39

Score 2+1
Abstract
Recent in vitro improvements permit to monitor cell responses in real time. It thenbecomes straightforward to assess not only to the level of effects but also to its
dynamics. However, these new tools require a change of paradigm in the way the data
are analyzed, i.e. moving from descriptive statistics towards the calibration of toxicodynamic models able to account for the time-course of the responses.
Here, we modelled Cell Index (CI) values derived from the electric impedance
measured on electronic sensor plates containing adherent hepatocytes (HepaRG)
exposed to three hepatotoxic cosmetics (coumarin, isoeugenol and benzophenone-2).
Impedance values reflected real time cyto-morphological modifications and cell viability.
We calibrated a mechanistic toxicokinetic/toxicodynamic (TK/TD) model, which
incorporates different phenomena: i) cosmetics uptake and elimination by the cell ii)
decrease of exposure concentration (due, for example, to evaporation, binding to well
walls, or metabolism by the cells) iii) cell morphological modifications due to exposure
to cosmetics iv) heterogeneity of sensitivity between cells.
For the three cosmetics tested, we could adequately fit acute data (for 48h exposure)with only a small number of parameters. We showed that equilibrium between medium
and cell concentrations was quasi-instantaneous and that no difference of sensitivity
was expected between the cells.
We then challenged the model with data obtained for the same substances during 4
weeks of exposure and showed that long-term effects were generally under-estimated.
The calibration of our model to analyze these data is ongoing and will permit toinvestigate the increase of sensitivity occurring with time.
Doxorubicin irreversibly inhibits the hypoxia-inducible factor (HIF) pathway inhuman cardiomyocytes
J Louisse, M Barilari, U Chaudhari, J Antonydas Gaspar, J Hengstler, A Sachinidis, S
Hoffmann- Bremer
SEURAT-1 2014
Score 3+1
Abstract
We aimed to identify mechanisms underlying doxorubicin-induced delayed
cardiotoxicity. To this end we used transcriptomic and high content imaging (HCI) dataof iPSC-derived human cardiomyocytes that had been exposed to doxorubicin at a low,
clinically relevant, concentration of 150 nM using both single and repetitive dosing
regimen, including washout (recovery) studies. Analysis of the transcriptomic data
indicates that besides well-known pathways related to doxorubicin's DNA-damaging
effect; also genes controlled by the hypoxia- inducible factor (HIF) are affected.
Interestingly, modulations of the HIF pathway were not seen in doxorubicin exposed
primary liver cells. Currently, HCI analyses are being performed in order to assess thederegulation of the HIF-pathway in cardiomyocytes at the protein level. The findings
indicate that the observed delayed cardiotoxicity of doxorubicin may result from thedown-regulation of the HIF pathway that leads to an impairment of blood vessel

Lush Science Prize 2014 – Background Paper

40formation. Therefore, the selection of biomarkers for predictive cardiotoxicity testingshould include those related to an impaired hypoxia response.
ToxBank Integrated Analysis of SEURAT-1 Reference Compounds
P Kohonen, R Grafström, G Myatt, and B Hardy
SEURAT-1 2014
Score 3
Abstract
The SEURAT-1 strategy is to adopt a mode-of-action framework to describe repeateddose toxicity to derive predictions of in vivo toxicity responses. ToxBank is the crosscluster infrastructure project which provides a web-accessible shared repository of
research data and protocols. Experiments generate dose response data over multiple
timepoints using different omics platforms including transcriptomics, proteomics,
metabolomics, and epigenetics over different cell lines and a common set of referencecompounds (details available at wiki.toxbank.net). Data is also generated fromfunctional assays and bioreactors and supplemented with in silico approaches. This
complex and heterogeneous data is consolidated and harmonized through theToxBank data warehouse in order to perform an integrated data analysis. We describe
for 14 reference compounds the meta-analysis of multiple types of time- dependent
dose response omics and functional data combined with in vitro and in vivo
background knowledge including consideration of modeling variations in biokinetic
responses. Open TG-GATEs human in vitro liver data of the reference compounds
includes reactive compounds (e.g., acetaminophen, CCl4), mitochondrial disruptors(e.g., Rotenone), promiscuous binders (e.g., valproic acid, amiadarone), nuclear
hormone receptor ligands (e.g., tamoxifen, WY14643), selective binders (e.g.
fluoxetine) and cardiotoxins (e.g., Doxorubicin, Nifedipine). Adverse events of interest
that are represented include cytotoxicity, fibrosis, steatosis, cholestasis andphospholipidosis. Overall we obtained 31,717 differential expression results with 14
compounds from the 45 comparisons, with Doxorubicin providing over 5000 results.
Pathway enrichment analysis of Doxorubicin identified a number of key pathways
including mismatch repair after 24 hours treatment and TNF-signaling at high doses
after 24 hours.

Lush Science Prize 2014 – Background Paper

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