Lush Prize 2016
Science Prize
Research Paper

Prepared by Drs T & J McCann, TJM Consultancy

Lush Prize 2016 – Science Prize Research Paper

1Note from Lush Prize: This Research Paper was commissioned to help identify potential
science projects that may be suitable for nomination for the Lush Prize 2016, but which
may not have been aware of the Lush Prize through our outreach activities.
Additional publications research discovered that some of the projects highlighted in thispaper did not meet the strict Eligibility Criteria for the Lush Prize(http://lushprize.org/awards/eligibility/); those that did were contacted and it wassuggested that they submit a nomination.
All nominations firstly go through a shortlisting process before being passed to the
independent panel of judges who select winners of the Lush Prize 2016.

Lush Prize 2016 – Science Prize Research Paper

21. Executive Summary
1.1 What is the Lush Science Prize?
Now in its fifth 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 Researchers. 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 Prize equivalent to the entire
annual fund of £250,000 will be awarded to the individual(s) or team(s) responsible. In
2015, the judges awarded a Black Box prize for the development of the skinsensitisation AOP and associated, approved, in vitro assays.
The Science Prize is awarded to the group (or groups) whose work the judging panel
believe has made the most significant contribution(s) to the replacement of animal
testing in the preceding year. This 2016 Science Background paper identifies 29 pieces
of work that we believe constitute potential candidates for the Judge's shortlist.
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 the OECD; CAAT; Hamner Institutes;
Human Toxome Project; ECVAM; UK NC3Rs; US Tox21 Programme; the ToxCast
programme; the Human Toxicology Project Consortium; and EU-ToxRisk (see section
4). We also assessed recent developments in toxicity testing research by reviewing therelevant literature (see section 5 for some highlights).
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 the EUSAAT-Linz conference, the Society of Toxicology annual conference,and the SEURAT-1 annual conference. There were a total of 2930 abstracts from oral
and poster presentations from these conferences, but many were not relevant to theLush Science prize. We then performed literature searches using PubMed, Google
Scholar and Terkko Feed Navigator to identify projects describing recent advances intoxicity testing research. Three additional relevant projects were identified directly fromthe Tox21, ToxCast, and Human Toxicology Project Consortium websites. In all,searches yielded in excess of 3,100 projects which we assessed as described in
Section 3. Relevant abstracts were scored using the system derived in previous years(see section 3 for details) in which 3 points are awarded for projects identifying new
toxicity pathways, assays, or biomarkers; 2 points for reporting new knowledge or tools;
and 1 point for abstracts which stand out in some other way.
Overall, from 85 abstracts which scored 1 or more: 5 scored 1; 51 scored 2; 7 scored 2+ 1 (i.e. total 3); and 17 scored 3 because they appeared to be reporting a new toxicity
pathway, assay, or biomarker. Finally, 5 projects scored the maximum possible marksof 3 for reporting a new toxicity assay, pathway, or biomarker, plus an additional markfor standing out as well. The titles and authors of those abstracts scoring 1 or more areshown in section 6.3, whilst full abstracts of those projects scoring at least 3 (either as
2+1, 3, or 3+1) are given in Section 6.4.

Lush Prize 2016 – Science Prize Research Paper3

1.3 Projects recommended for the shortlist
There were 29 projects which received scores of at least 3 for reporting new pathways,
assays, or biomarkers of toxicity. The full abstracts are given in Section 6.4. We
consider all to be worthy for consideration by the judges as potential prize winners.

Lush Prize 2016 – Science Prize Research Paper

42. Background
There are two compelling reasons for ending animal testing: it is cruel and it isineffective1. The objectives of 21st Century Toxicity Testing (tt21c) research can besummarised as: improving safety testing of chemicals and contaminants by using more
relevant and predictive human models; and simplifying and automating tests so thatmany more chemicals can be tested for safety. The Science background papers for the
2012, 2013, and 2014 Lush Prizes provide an overview, and links to further resources,
describing the concept of 21st Century Toxicology.
The Lush Prizes aim to focus attention on toxicity testing for consumer products andingredients, in a way which complements those projects which address the use ofanimals in medical testing. The Lush Science Prize seeks to reward those researchers
making 'outstanding contributions' to tt21c research. In previous years, the focus of theaward has been on research aimed at elucidating key pathways in which perturbation
results in toxicity. This approach was continued in 2015, when it was further extended
to cover additional aspects of tt21c, beyond toxicity pathways. In particular, in 2015, we
also sought to identify significant contributions to the development of new in vitro
assays that advance tt21c, and to the discovery of biomarkers that signal early
activation of toxicity pathways. Here, for the 2016 Science Prize paper, we haverepeated this three-pronged approach to identify projects that are candidates for theLush Science Prize.
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.

1

http://www.lushprize.org/background/animal-testing/Lush Prize 2016 – Science Prize Research Paper

53. Methodology
The main aim of this report is to assist the Lush Prize judging panel by identifying key
projects that are making major contributions to the field of animal-free toxicology
research. From these, the panel may select the winners for the 2016 Lush Science
Prize. In this section we describe how we identified projects that might be worthy of
consideration as potential prize winners, and then how we scored each project to createa shortlist for the panel’s consideration.
Of the “3 Rs”, Lush’s interest focuses primarily on Replacement, so our search forpotential prize winners targeted projects working towards the replacement of animals in
product testing, and we excluded research aimed at either Refining or Reducing the useof animals in experimentation. Since the focus of the Lush prize is on generalpathways of compound safety testing, we excluded research that focuses on specificdiseases, including cancer. We considered projects based anywhere in the world, but
only considered work written in the English language. As far as possible, we restricted
the search to work reported in the year preceding the award (i.e. June 2015 – June2016).
In the identification of key developments in the area of toxicology research, and in the
search for candidate prize winners, we followed three separate strands of investigation.
We started firstly by reviewing the recent research of some key institutions andcollaborative projects working in the area of animal replacement in toxicity pathway
research. These included the OECD; CAAT; Hamner Institutes; Human Toxome
Project; ECVAM; UK NC3Rs; US Tox21 Programme; the ToxCast programme; the
Human Toxicology Project Consortium; and EU-ToxRisk (see section 4).
Secondly, we identified relevant conferences held in the preceding 12 months andassessed abstracts, where available, for oral and poster presentations. Scientificconferences provide the forum in which the most up-to-date science is shared, reportingon recent developments and work-in-progress, without the lag time required for formal
presentation as a journal publication. The relevant conferences for 2015 – 2016, forwhich abstracts were available, included: the 16th annual congress of EUSAAT (theEuropean Society for Alternatives to Animal Testing), held in September 2015 in Linz;
the EU SEURAT-1 Project’s Symposium held in December 2015 in Brussels; and finally
The Society of Toxicology 55th Annual Meeting held in March 2016, in New Orleans,Louisiana.
Thirdly, we conducted a review of the recent literature. For this we used three separate
sources. Firstly, we searched PubMed for research published from 01/06/2015 to
17/06/2016, combining search terms “toxicity pathway,” “toxicity assay” and “toxicity
biomarker”, excluding any review articles and clinical trials, and restricting the subject
matter to “humans”. As a second literature source, we searched Google Scholar for
relevant papers published in the period 2015 to 2016, combining search terms “toxicity
pathway,” “toxicity assay” and “toxicity biomarker”, and restricting the subject matter to“humans”. As a final literature source, we searched Terkko Feed Navigator, combining
search terms “toxicity pathway,” “toxicity assay” and “toxicity biomarker”.
As in previous years, our selection procedure was a three stage process. At eachstage of our search, research projects were carefully excluded based on our selection
criteria, in order to achieve a manageable shortlist of excellent work which fully met the
prize brief. In the first stage, we reviewed the title of the work and rejected any whichwere clearly reviews or which were obviously unsuitable through either using animalmodels or through being overly focused on disease. We also rejected any which wereLush Prize 2016 – Science Prize Research Paper

6not written in the English language, and those for which abstracts were unavailable. In
the second stage, we assessed the abstracts of projects which passed the initial filter
and further eliminated those which reported findings from clinical trials, those focusing
on disease research, and all research that included animal subjects. In the third stage,
projects identified as potentially relevant based on the abstract were scored using asystem devised and successfully applied in previous years. In this system, points areawarded according to the following criteria:
Does the work appear to be reporting: discovery of a new pathway; asignificant advance in assay technology or approach; or a new biomarker
for early activation of toxicity?

Score 3

If it is working with an apparently previously understood pathway, assay
technology, or biomarker, does it bring new knowledge or tools?

Score 2

Does it stand out in any other way?

Score 1

Projects awarded a score of 2 or 3 could also receive an extra 1 point if they also stoodout in some other way, so the maximum possible score is 4.

Lush Prize 2016 – Science Prize Research Paper

7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
Tox21 is a collaborative effort among the National Institutes of Health (NIH), theEnvironmental Protection Agency (EPA), and the Food and Drug Administration (FDA).
NIH partners include NCATS (National Center for Advancing Translational Sciences)and the National Toxicology Program, administered by the National Institute ofEnvironmental Health Sciences. The Tox21 initiative is designed to improve current
toxicity assessment methods, which are slow and costly, by utilising robotic systems
and high-throughput technologies to screen thousands of compounds.
In January 2016, Tox21 launched the Tox Testing Challenge2, with an award fund of up
to $1 million to improve the relevance and predictivity of data generated fromautomated chemical screening technology used for toxicity testing. The Challenge is
co-sponsored by the Tox21 consortium members. In May 2016 Tox21 announced thatten selected semi-finalists will each receive $10,000 to help develop prototypes of theirideas.
4.2 ToxCast
The US EPA's ToxCast (Toxicity Forecaster) programme aims to use high-throughput
screening tools to test many thousands of chemicals in in vitro assays. It is organised
under the umbrella of Tox21, and has made some significant progress in the last year3.
•

A 17-centre, transatlantic, collaborative, large-scale modelling project has
demonstrated the efficacy of using predictive computational models trained onhigh-throughput screening data to evaluate over 32,000 chemical structures foroestrogen receptor activity.

•

Updates of the ToxCast and Tox21 high-throughput in vitro database, available
to download, with associated summary files, for thousands of chemicals andhundreds of assays.

•

Publication of a network model of oestrogen receptor activation, based on
integration of data from 18 in vitro assays for various aspects of oestrogen
receptor function4.

4.3 EU-ToxRisk
EU-ToxRisk is a consortium of 39 partner organisations from academia, SMEs, large
corporations, and regulatory bodies, co-funded by Horizon 2020 and Cosmetics
Europe. The project has a budget of €30 million, to be spent over six years, and is the2 http://transformtoxtesting.com/
3 https://www.epa.gov/chemical-research/toxicity-forecaster-toxcasttm-data
4 Judson et al Integrated model of chemical perturbations of a biological pathway using 18 in vitro highthroughput screening assays for the estrogen receptor. Toxicol Sci 148, 137-54 doi: 10.1093/toxsci/kfv168

Lush Prize 2016 – Science Prize Research Paper8successor to the SEURAT-1 programme. SEURAT-1's budget was €50 million, spentover five years, so EU-ToxRisk is funded at half the level of SEURAT-1. EU-ToxRisk
aims to integrate new concepts for efficient, animal-free regulatory chemical safetyassessment. The project was started in January 2016 and is co-ordinated by Bob vande Water, Professor of Toxicology at Leiden University, The Netherlands.
4.4 The Organisation for Economic Co-operation and Development (OECD)
Animal-free Test Guidelines published by the OECD since the 2015 Lush Science Prizeresearch paper was prepared include:•

TG 491 in vitro test for identifying chemicals inducing serious eye damage using
an established rabbit cornea cell line;•

TG 492 in vitro test for identifying chemicals NOT requiring classification for eye
irritation or serious eye damage, using reconstructed human cornea-likeepithelium (RhCE). We have included the Kaluzhny et al paper describing thevalidation of this test in our list of potential Lush Prize winners, with a score ofscore 3+1;•

TG 493 describes in vitro binding assays for identifying human oestrogen
receptor binding chemicals;•

The hCLAT test, recommended for approval by EURL ECVAM last year, hasbeen approved as a test guideline by the OECD Working Group (April 2016),
although the official publication of the Test Guideline (TG 442E) has not yet
appeared.

4.5 Hamner Institutes
The Hamner Institutes suddenly and unexpectedly closed on 31st December 2015, after
more than 40 years of non-profit research into chemical safety testing. One division,
the Institute for Chemical Safety Sciences, was sold to SciMetrika, when the rest of the
Hamner Institutes were closed down. The Hamner Institutes' website is still running,and makes no mention of the Institutes' closure. It is not clear what has happened to
the DILIsym project, although its website is still running.
4.6 Cosmetics Europe
Cosmetics Europe is the trade association for the European cosmetics industry. In
addition to its support for the EU-ToxRisk programme, mentioned above, Cosmetics
Europe has independently created a Long Range Science Strategy that will fundresearch focussed on repeat dose toxicity, bioavailability (ADME), and systemic toxicity.
The primary goal of the strategy is animal-free prediction of risk of human toxicity (forcosmetic ingredients and products) with broad scientific and regulatory acceptance.
Currently funding (amount unspecified) is allocated for the period 2016 to 2020.
4.7 European Union Reference Laboratory for alternatives to animal testing(EURL ECVAM)

Lush Prize 2016 – Science Prize Research Paper

9The role of EURL ECVAM is the validation of methods which reduce, refine, or replace
the use of animals in safety testing and in efficacy/potency testing of chemicals,
biologicals and vaccines.
In November 2015, an expert meeting arranged by EURL ECVAM determined thatnegative data from approved in vitro skin sensitisation assays should be accepted as
evidence of low risk, unless there was additional information to suggest that a chemical
is likely to be a pro-hapten5.
In February 2016, EURL ECVAM conducted a public survey to identify knowledge
sources relevant to 3Rs. The results of the survey will be available by the end of 2016.
In April 2016, EURL ECVAM announced that EU member states had modified REACH
regulations to make the newly approved in vitro skin sensitisation assays TG 442-C, -D,
and the hCLAT assay, the default sources of information for skin sensitisation risk
under REACH, rather than the animal-based LLNA that was previously the default test.
4.8 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 the implementation of a biological pathway-based approach to toxicology,
as described in the National Research Council’s 2007 report on “Toxicity Testing in the
21st Century.” Its work in the 2015/2016 period has focussed mainly on disseminationof advances in assays and technologies for tt21c, such as the development of brainorganoids, and high content screening tools for compound safety testing. The HTPC
website highlighted two interesting papers, discussed in Section 5.
4.9 National Centre for the Replacement, Refinement, & Reduction of Animals in
Research (NC3Rs)
In November 2015, NC3Rs published a roadmap for the UK on developing and applying
non-animal technologies (NATs) to improve the methods and tools available forchemical safety testing6. The roadmap was prepared in collaboration with the MRC,BBSRC, EPSRC, Innovate UK, and the Defence Science & Technology Laboratory. It
outlines plans for how the UK might invest in NATs over a 15 year period to 2030.
While the roadmap offers direction for UK investment, it does not accurately reflect thecurrent state of regulatory approvals, highlighting, as it does, that there are no nonanimal OECD test guidelines for skin sensitisation. Such OECD guidelines were
published in February 2015 and had been recommended for adoption before that.

5 https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-status-reports/pre-prohapten-workshop-report
6 https://www.nc3rs.org.uk/sites/default/files/documents/NonAnimalTechCO082_RYE_4_nrfinal2.pdf

Lush Prize 2016 – Science Prize Research Paper

105. Literature Highlights
We have undertaken an extensive literature search to identify potential Lush Science
Prize nominees. Details of the search and our results are given elsewhere in thisdocument (see Sections 3 and 6). In the course of the research for this Science Prizepaper, we noted some trends in the nature of research undertaken to develop
alternatives for animal tests, which the Lush Prize organisation might consider relevantto the future scope of the Science Prize.
Firstly, two papers7,8 highlighted the potential for tt21c techniques and assays to beextended to drug and food ingredient safety testing. While the Lush Prize focusses on“toxicity testing for consumer products and ingredients, to complement projects
addressing the use of animals in medical testing”, the reality is that many more animals
are used in preclinical drug development and medical research than in chemical testing.
Steps to extend the principles and accumulated knowledge of tt21c to other areas andindustries where animals are widely used are to be welcomed.
Secondly, we came across very many reports and descriptions of specific in vitro
models for detecting chemical-induced perturbations in cellular systems. These range
from newly developed cell lines, through 3D culture models and perfusion systems, toorgans-on-a-chip and even humans-on-a-chip. Such systems are based on ideas thathave been developed over quite some time, and incremental changes are being made.
These tools are developed by research teams and companies approaching the
problems in different ways, and it is very difficult to predict which systems will be
successful in the long-term. As an example, hiPSCs can be differentiated tohepatocyte-like cells. Unfortunately, these cells express a foetal hepatocyte phenotype
and are therefore not suitable for testing chemical hepatotoxicity in adults. Growing
hiPSC-hepatocytes in a 3D culture system shifts the phenotype towards a more adultpattern, but only incompletely. One of the reports that we have proposed be considered
for a prize9 noted that hepatocytes only acquire an adult phenotype post-partum. Avior
et al treated hiPSC-hepatocytes with nutritional factors and successfully induced an
adult hepatocyte phenotype. Significant as such work appears to be, it will be difficult toassess its impact on tt21c for some time. The process of incremental improvements intechnology, following significant leaps such as the development of iPSCs, make it
difficult to identify individual steps as worthy of awards without the benefit of hindsight.
Finally, and depressingly, we were struck by the number of papers that continue to
conduct comparative studies between animal and human models – examples include
comparing mouse and human neuroprogenitor cells during assay developemnt10,
comparing human and rat lung slices for testing the toxicity of cigarette smoke11, and7

Langley et al, Lessons from Toxicology: Developing a 21st-Century Paradigm for Medical Research. Environ
Health Perspect. 2015 Nov;123(11):A268-72. doi: 10.1289/ehp.1510345

8

Rovida et al, Toxicity testing in the 21st century beyond environmental chemicals. ALTEX. 2015;32(3):171-81.
doi: 10.14573/altex.1506201.

9

Avior et al, Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem
cells-derived and fetal hepatocytes. Hepatology. 2015 Jul;62(1):265-78. doi: 10.1002/hep.27803 Score 2+1

10 Druwe et al, Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitablefor high-throughput screening. Toxicology. 2015 Jul 3;333:14-24. doi: 10.1016/j.tox.2015.03.011
11 Obernolte et al EUSAAT 2015, Abstract 161

Lush Prize 2016 – Science Prize Research Paper

11

comparing transported activities in primary human and Cynomolgus monkey
hepatocytes12. Clearly there remains a significant drive to fund and conduct animal
experiments without prior stringent ethical review.

12 Ullrich et al EUSAAT 2015, Abstract 59

Lush Prize 2016 – Science Prize Research Paper

12

6.

Toxicity Pathway Abstracts

6.1 Conference Abstract Selection
As described in the Methodology, we reviewed abstracts from the 16th annual congressof EUSAAT, the EU SEURAT-1 Project’s 2015 Brussels Symposium, and The Societyof Toxicology’s 55th Annual meeting, 2016.
From the 333 abstracts which comprised the EUSAAT 2015 conference presentationand poster proceedings, we identified 18 abstracts which passed through our selection
process to the final (3rd) scoring stage. Of these, 12 scored 1 or more.
From the SEURAT-1 2015 meeting, 40 abstracts were available, of which 7 passed tothe scoring stage. Of these, 6 scored 1 or more.
From the total of 2557 abstracts presented at the Society of Toxicology’s 2016 meeting,and identified as potentially relevant based on the Abstract book keyword index, 96
were identified by the keyword “biomarker”, of which 3 were scored; 61 were identified
by the keyword “mechanism”, of which 2 were scored; and 117 were identified by the
keywords “alternatives to animals,” of which 12 were scored. Of the total 17 abstracts
which passed through to the scoring, stage, 11 scored 1 or more.
6.2 Published Abstract Selection
From the PubMed search, we identified 1842 relevant titles from the “Toxicity assay”
search, a further 114 relevant projects from the “Toxicity biomarker” search, and finallyan additional 173 titles from the “toxicity pathway” search: a combined total of 2129
articles.
Stages 1 and 2 of the selection process (review of titles and, if necessary abstracts, toreject review articles, articles not written in English, results of clinical trials, articles
reporting use of animal subjects, or those focused on cancer research or otherdisease), reduced these 2129 titles to just 80 abstracts: 58 from the Toxicity assay
search; 7 from the Toxicity biomarker search; and 15 from the Toxicity pathway search.
These final 80 abstracts were passed to the third assessment stage, to be scored as
potential prize winners. Of these, 45 scored 1 or more (36 from the Toxicity assay
search, 6 from the Toxicity biomarker search, and 3 from the Toxicity pathway search).
The Google Scholar search for the period 2015 to 2016 identified 383 possibly relevant
abstracts additional to those identified by PubMed described above. Of these, 329
were identified by the Toxicity pathway search, a further 14 from the Toxicity biomarker
search, and 40 from the Toxicity pathway search. Of the 383, only 9 survived selection
stages 1 and 2 (4 from the Toxicity assay search and 5 from the Toxicity pathway
search) and were passed on to the scoring stage. Of these 9, a final 6 scored 1 or
more.
The Terkko FeedNavigator search yielded no further relevant papers beyond thosealready identified by the other literature searches.
We also identified three further relevant projects, directly from the Tox21, ToxCast, andHTPC websites, which scored 1 or more.
6.3 Scores

Lush Prize 2016 – Science Prize Research Paper

13

From the three separate sources of potential shortlisted projects, we identified a total of
85 abstracts describing work which scored at least one point according to our given
criteria. Of these, 5 scored 1 for standing out in some way (for example by providing
opportunities for data sharing, or for combining methodologies to give “added value”);
51 scored 2 for bringing new knowledge or tools to a previously identified pathway,
assay, or biomarker of toxicity; 7 scored 2 for bringing new knowledge or tools to apreviously identified pathway, assay, or biomarker of toxicity but with an additional 1(i.e. total 3) because they stood out in some other way; and 17 scored 3 because they
appeared to be reporting a new toxicity pathway, assay, or biomarker. Only 5 scoredthe maximum possible 4 marks, with 3 awarded for reporting a new toxicity pathway,
assay, or biomarker plus an additional mark for standing out in some other way.
The Table lists details (Title, Authors, contribution (pathway, assay, or biomarker),source, and score) of all the abstracts scoring 1 or more. The Table is ordered bysource of abstract – websites, PubMed, Google Scholar, EUSAAT, SEURAT-1, and
SoT. All of the abstracts for those projects scoring a total of 3 or more are shown in fullin Section 6.4. For abstracts identified in the PubMed searches, the abstract title is a
hyperlink to the PubMed index for that paper.
Title

Authors

Category

SourceScore

Modelling the Tox21 10 K chemical profiles
for in vivo toxicity prediction and mechanism
characterization

Huang et al

Assay

Tox21

3+1

High-throughput imaging-based
nephrotoxicity prediction for xenobiotics withdiverse chemical structures

Su et al

Assay

HTPC

3

Lush Prize 2016 – Science Prize Research Paper

14TitleAuthors

Category

SourceScore

Integrated Model of Chemical Perturbationsof a Biological Pathway Using 18 In Vitro
High-Throughput Screening Assays for the
Estrogen Receptor

Judson et al

Assay

ToxCast2+1

Characterization of the modes of action of
deoxynivalenol (DON) in the human Jurkat
T-cell line

Katika et al

Pathway

PubMed3

Toxicological responses of environmental
mixtures: Environmental metal mixtures
display synergistic induction of metalresponsive and oxidative stress genes inplacental cells

Adebambo et Biomarker
al

PubMed2

Exposure to coplanar PCBs induces
endothelial cell inflammation throughepigenetic regulation of NF-κB subunit p65

Liu et al

Pathway

PubMed3

Identification of TGF-β receptor-1 as a key
regulator of carbon nanotube-induced
fibrogenesis

Mishra et al

Pathway

PubMed2

Roles of oxidative stress and the ERK1/2,
PTEN and p70S6K signaling pathways inarsenite-induced autophagy

Huang et al

Pathway

PubMed2

Action of methyl-, propyl- and butylparaben
on GPR30 gene and protein expression,cAMP levels and activation of ERK1/2 andPI3K/Akt signaling pathways in MCF-7
breast cancer cells and MCF-10A nontransformed breast epithelial cells

Pathway
Wróbel &Gregoraszcz
uk

PubMed2

Cadmium delays non-homologous end
joining (NHEJ) repair via inhibition of DNAPKcs phosphorylation and downregulation of
XRCC4 and Ligase IV

Li et alPathway

PubMed2

Tributyltin induces mitochondrial fission
through NAD-IDH dependent mitofusindegradation in human embryonic carcinoma
cells

Yamada et al Pathway

PubMed2

Molecular mechanisms of human thyrocyte
dysfunction induced by low concentrations ofpolychlorinated biphenyl 118 through the
Akt/FoxO3a/NIS pathway

Guo et al

Pathway

PubMed2

Concurrent acetylation of FoxO1/3a and p53
due to sirtuins inhibition elicit Bim/PUMA
mediated mitochondrial dysfunction andapoptosis in berberine-treated HepG2 cells

Shukla et al

Pathway

PubMed2

The plasticizer BBP selectively inhibits
epigenetic regulator sirtuins.

Zhang et al

Pathway

PubMed3+1

Lush Prize 2016 – Science Prize Research Paper

15TitleAuthors

Towards in vitro DT/DNT testing: Assaying
chemical susceptibility in early differentiating
NT2 cells.

SourceScore

Menzner et al Assay

PubMed3

A fluorescence high throughput screening
method for the detection of reactive
electrophiles as potential skin sensitizers.

Avonto et al

Assay

PubMed2

Development of an in Silico Profiler for
Mitochondrial Toxicity.

Nelms et al

Assay

PubMed2+1

Transcriptomic Analysis of Human Primary
Bronchial Epithelial Cells after Chloropicrin
Treatment

Pesonen etal

Pathway

PubMed2+1

Eye Irritation Test (EIT) for Hazard
Identification of Eye Irritating Chemicals
using Reconstructed Human Cornea-like
Epithelial (RhCE) Tissue Model

Kaluzhny et
al

Assay

PubMed

3+1 –
new
OECD
test

Integrating High-Dimensional
Transcriptomics and Image Analysis Tools
into Early Safety Screening: Proof ofConcept for a New Early Drug Development
Strategy

Verbist et al

Assay

PubMed1

A transcriptome-based classifier to identify
developmental toxicants by stem cell testing:design, validation and optimization forhistone deacetylase inhibitors

Rempel et al

Assay

PubMed2+1

Human Pluripotent Stem Cell Based
Developmental Toxicity Assays for Chemical
Safety Screening and Systems Biology Data
Generation

Shinde et al

Assay

PubMed3Morphological and Functional
Characterization and Assessment of iPSCDerived Hepatocytes for In Vitro Toxicity
Testing

Lu et alAssay

PubMed1

Selective Sensitization of Zinc Finger Protein
Oxidation by Reactive Oxygen Species
through Arsenic Binding

Zhou et al

Pathway

PubMed2

Nephron Toxicity Profiling via Untargeted
Metabolome Analysis Employing a High
Performance Liquid Chromatography-Mass
Spectrometry-based Experimental andComputational Pipeline

Ranninger et Assayal

PubMed2+1

Non-animal photosafety screening forcomplex cosmetic ingredients withphotochemical and photobiochemicalassessment tools

Nishida et al

PubMed2

Lush Prize 2016 – Science Prize Research Paper

Category

Assay

16

TitleAuthors

Category

SourceScore

Mimicking enzymatic effects of cytochrome
P450 by an efficient biosensor for in vitro
detection of DNA damage

Jalalvand et
al

Assay

PubMed3

Toxicogenomic responses of human liver
HepG2 cells to silver nanoparticles

Sahu et al

Pathway

PubMed2

Development of the adverse outcome
pathway "alkylation of DNA in malepremeiotic germ cells leading to heritable
mutations" using the OECD's users'
handbook supplement

Yauk et al

Pathway

PubMed3–
new
AOP

Gene Expression Regulation and Pathway
Analysis After Valproic Acid andCarbamazepine Exposure in a Human
Embryonic Stem Cell-Based
Neurodevelopmental Toxicity Assay

Schulpen etal

Pathway

PubMed2

Mechanism of neutrophil activation andtoxicity elicited by engineered nanomaterials

Johnston etal

Pathway

PubMed2

Investigation of acetaminophen toxicity in
HepG2/C3a microscale cultures using asystem biology model of glutathione
depletion

Leclerc et al

Pathway/
Assay

PubMed2

Multiparametric assay using HepaRG cells
for predicting drug-induced liver injury

Tomida et al

Assay

PubMed2

Extension of the Dermal Sensitisation
Threshold (DST) approach to incorporatechemicals classified as reactive

Safford et al

Assay

PubMed2

Arsenic Inhibits DNA Mismatch Repair by
Promoting EGFR Expression and PCNA
Phosphorylation

Tong et al

Pathway

PubMed2

Particle-induced cell migration assay(PICMA): A new in vitro assay for
inflammatory particle effects based on
permanent cell lines

Westphal et
al

Assay

PubMed3

Endoplasmic Reticulum Stress and StoreOperated Calcium Entry Contribute to Usnic
Acid-Induced Toxicity in Hepatic Cells

Chen et al

Pathway

PubMed2

Evaluation of an optimized protocol using
human peripheral blood monocyte derived
dendritic cells for the in vitro detection ofsensitizers: Results of a ring study in fivelaboratories

Reuter et al

Assay

PubMed3

Perfluorooctanoic acid (PFOA) affects distinct
molecular signalling pathways in humanprimary hepatocytes

Burhke et al

Pathway

PubMed2

Lush Prize 2016 – Science Prize Research Paper

17TitleAuthors

Category

SourceScore

The Myeloid U937 Skin Sensitization Test
(U-SENS) addresses the activation ofdendritic cell event in the adverse outcome
pathway for skin sensitization

Piroird et al

Assay

PubMed2

Microbial-derived lithocholic acid and vitamin
K2 drive the metabolic maturation ofpluripotent stem cells-derived and fetal
hepatocytes

Avior et al

Assay

PubMed2+1

Contribution of membrane trafficking
perturbation to retinal toxicity

Khoh-Reiter
et al

Pathway

PubMed2

Development of a toxicogenomics signaturefor genotoxicity using a dose-optimizationand informatics strategy in human cells

Li et alPathway

PubMed2Genes specifically modulated in sensitizedskins allow the detection of sensitizers in areconstructed human skin model.
Development of the SENS-IS assay

Cottrez et al

Assay

PubMed2+1

Use of HPLC/UPLC-spectrophotometry for
detection of formazan in in vitro
Reconstructed human Tissue (RhT)-based
test methods employing the MTT-reduction
assay to expand their applicability to strongly
coloured test chemicals

Alépée et al

Assay

PubMed2

Successful validation of genomic biomarkers
for human immunotoxicity in Jurkat T cells in
vitro

Schmeits et
al

Assay

PubMed3

Identification of cornifelin and early growth
response-1 gene as novel biomarkers for invitro eye irritation using a 3D reconstructed
human cornea model MCTT HCE™

Choi et al

Biomarker/assay

PubMed2High-content screening of drug-induced
mitochondrial impairment in hepatic cells:
effects of statins

Tolosa et al

Assay

PubMed3

Development of an in vitro test to identify
respiratory sensitizers in bronchial epithelial
cells using gene expression profiling

Dik et al

Pathway

GoogleScholar

2

Contact dermatitis: in pursuit of sensitizer’s
molecular targets through proteomics

Guedes et al Pathway

GoogleScholar2

A fluorescence high throughput screening
method for the detection of reactive
electrophiles as potential skin sensitizers

Avonto et al

Assay

GoogleScholar2

MicroRNA-122: A Novel HepatocyteEnriched in vitro Marker of Drug-Induced
Cellular Toxicity

Kia et al

Pathway

GoogleScholar2

Lush Prize 2016 – Science Prize Research Paper

18

TitleAuthors

Category

SourceScore

Assessment of the eye irritation potential ofchemicals: A comparison study between two
test methods based on human 3D hemicornea models

Tandon et al

Assay

GoogleScholar3

RNA transcripts for the quantification ofdifferentiation allow marked improvements inthe performance of embryonic stem cell test
(EST)

Romero et al Assay

GoogleScholar2

Conversion of the draft OECD guideline
for the testing of chemicals: theCytosensor Microphysiometer test
methodEggert et al

Assay

EUSAAT2015 #39

2

CERST-NRW – a platform for developing
human in vitro methods for (neuro)developmental toxicity testing

Goniwiecha
et al

Assay

EUSAAT2015 #87

1

Stem cell-derived human dorsal root
ganglia-like cells to identify peripheral
neurotoxicants

Hoelting et al AssayEUSAAT2015 #136

3

Development, optimization andstandardization of an in vitro skinirritation test for medical devices using
the reconstructed human tissue model
EpiDerm

Kandarova et Assayal

EUSAAT2015 #233

2

Identification of transcriptome signaturesand biomarkers specific for migrationinhibiting potential developmental
toxicants in human neural crest cells

Pallocca et al Biomarker

EUSAAT2015 #188

2

BoNT activity determination by stimulusdependent release of neuro-secretory
vesicle-targeted luciferase from neuronalcell lines

PatheNeuschäferRube et al

Assay

EUSAAT2015 #16

3+1

Evaluation of developmental
neurotoxicity triggered by N-methyl-Daspartate receptor (NMDAR) antagonists
based on the Adverse Outcome Pathway(AOP) concept

Price et al

PathwayEUSAAT2015 #63

2

Intra- and inter-laboratory validation of
LuSens: a reporter gene-cell line todetect keratinocyte activation by skinsensitizers

Ramirez et al Biomarker

EUSAAT2015 #194

2

Metabolomics in vitro: a new approachfor systemic toxicity – first applications
for mode of action identification andchemical grouping

Ramirez et al AssayEUSAAT2015 #196

1

Lush Prize 2016 – Science Prize Research Paper

19TitleAuthors

Category

SourceScore

Micellar liquid chromatography as analternative for in vivo and expensive in
vitro tests commonly used in predictionof human intestinal absorption

Shokry et al

Assay

EUSAAT2015 #54

3

In vitro assays for the potency
determination of botulinum neurotoxin
serotypes A and B

Wild et al

Assay

EUSAAT2015 #17

3+1

Evaluation of an in vitro human dermal
Sensitization test for use with medical
device extracts

Willoughby

Assay

EUSAAT2015 #315

2

In silico modeling for the prediction ofdose and pathway related adverse
effects in humans from in vitro repeateddose studies

Klein et al

Assay

SEURAT-1
2015
NOTOX #2

2

A 3D in vitro HepaRG model for theidentification and study of compoundswith cholestatic liability

Puigvert et al Pathway

SEURAT-1
2015
NOTOX #5

2

A High Content Imaging (HCI) BAC-GFP
Toxicity Pathway Reporter Platform for
Mechanism-based Assessment of DrugInduced Liver Injury (DILI)

Wink et al

Assay

SEURAT-1
2015
Detective#1

2

In vitro verification of an adverse
outcome pathway of cholestatic liver
injury using “omics” technologies

Wink et al

Assay

SEURAT-1
2015
Detective#2

2

In vitro based prediction of humanhepatotoxicity

Stöbe et al

Assay

SEURAT-1
2015
Detective#3

2

Identification of genomic biomarkers foranthracycline-induced cardiotoxicity inhuman iPSC-derived cardiomyocytes:
an in vitro repeated exposure toxicity
approach for safety assessment

Chaudhari et Biomarkers
al

SEURAT-1
2015
Detective#4

2

Migration of Human Neural Crest Cells
as Functional Endpoint to Screen for
Developmental Neurotoxicity

Nyffeler et al

Assay

SoT 2016#1171

3

Human Relevant Intra-Laboratory
Validated In Vitro Vasculo/Angiogenesis
Test

Heinonen et
al

Assay

SoT 2016#2176

3

Lush Prize 2016 – Science Prize Research Paper

20TitleAuthors

Category

SourceScore

High Density Human Stem Cell
Micromass to Model Teratogen-Induced
Limb Malformations

Yu et alAssay

SoT 2016#2178

3

Quantifiable 3D In Vitro Vasculogenesis
Assay Emphasizes Importance of CellMatrix Interactions When Screening
Putative Developmental Toxins

Thiede et al

Assay

SoT 2016#2180

2Multivariate Models for Prediction of
Human Skin Sensitization Hazard

Strickland et
al

Assay

SoT 2016#2202

1

Development of Photo-Direct Peptide
Reactivity Assay

Nishida et al

Assay

SoT 2016#2204

2

Identification of Compounds That
Modulate Retinol Signaling Using a CellBased qHTS Assay

Chen et al

Assay

SoT 2016#2462

2

Development of an In Vitro Test forAllergenic Potency of Proteins

Tsukumo et
al

Assay

SoT 2016#2471

2

In Vitro Model Development for Safety
Assessment of Acutely Ingested
Proteins

Wezalis et al Assay

SoT 2016#3064

2

In Vitro Developmental Neurotoxicity
Testing Models Using HiPSC-Derived
Neural Stem Cells

Yi et alAssay

SoT 2016#3074

2

Thymic Stromal Lymphopoietin Induced
by Protein and Chemical Allergens in
Cultured Human Keratinocytes: A
Potential Biomarker for Immediate-Type
Hypersensitivity

Kuroda et al

Biomarkers

SoT 2016#2732

2

Bile Canaliculi Dynamics Alterations as
Early Preclinical Predictive Markers ofDrug Induced Cholestasis

Burbank et al Biomarkers

SoT 2016#1970

2

In Vitro Toxicity and Resilience of A 3D
Human Dopaminergic Model toRotenone

Harris et al

SoT 2016#2340

2

Pathway

6.4 Nominated Abstracts
This year 29 projects received the highest scores of either 3 or 4 for reporting new
pathways, assays, or biomarkers of toxicity. The 29 abstracts are given below. For
abstracts from published work, we provide the digital object identifier (DOI) which allows
the original document to be located online. For conference abstracts, we give theabstract or poster number for identification.
We consider all worthy of being considered by the judges as potential prize winners.
Lush Prize 2016 – Science Prize Research Paper

21Modelling the Tox21 10 K chemical profiles for in vivo toxicity prediction and mechanism
characterization
R Huang, M Xia, S Sakamuru, J Zhao, S A Shahane, M Attene-Ramos, T Zhao, C P Austin, A
Simeonov.
Division of Pre-clinical Innovation, National Center for Advancing Translational Sciences,
National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, USA.
Nat Commun. 2016 Jan 26;7:10425. doi: 10.1038/ncomms10425.
Score 3 + 1 (New assay/pathway + scale)
Abstract
Target-specific, mechanism-oriented in vitro assays post a promising alternative to traditional
animal toxicology studies. Here we report the first comprehensive analysis of the Tox21 effort, alarge-scale in vitro toxicity screening of chemicals. We test 10,000 chemicals in triplicates at
15 concentrations against a panel of nuclear receptor and stress response pathway assays,
producing more than 50 million data points. Compound clustering by structure similarity andactivity profile similarity across the assays reveals structure-activity relationships that are usefulfor the generation of mechanistic hypotheses. We apply structural information and activity data
to build predictive models for 72 in vivo toxicity end points using a cluster-based approach.
Models based on in vitro assay data perform better in predicting human toxicity end points than
animal toxicity, while a combination of structural and activity data results in better models thanusing structure or activity data alone. Our results suggest that in vitro activity profiles can beapplied as signatures of compound mechanism of toxicity and used in prioritization for more indepth toxicological testing.
High-throughput imaging-based nephrotoxicity prediction for xenobiotics with diverse
chemical structures
R Su, S Xiong, D Zink, L H Loo.
Department of Pharmacology, Yong Loo Lin School of Medicine, National University ofSingapore, 10 Medical Drive, Singapore, 117597, Singapore. Email: loolh@bii.a-star.edu.sg.
Arch Toxicol. 2015 Nov 27. [Epub ahead of print]
Score 3 (Assay)
Abstract
The kidney is a major target for xenobiotics, which include drugs, industrial chemicals,
environmental toxicants and other compounds. Accurate methods for screening large numbersof potentially nephrotoxic xenobiotics with diverse chemical structures are currently notavailable. Here, we describe an approach for nephrotoxicity prediction that combines highthroughput imaging of cultured human renal proximal tubular cells (PTCs), quantitative
phenotypic profiling, and machine learning methods. We automatically quantified 129 imagebased phenotypic features, and identified chromatin and cytoskeletal features that can predictthe human in vivo PTC toxicity of 44 reference compounds with ~82 % (primary PTCs) or 89 %(immortalized PTCs) test balanced accuracies. Surprisingly, our results also revealed that a
DNA damage response is commonly induced by different PTC toxicants that have diverse
chemical structures and injury mechanisms. Together, our results show that humannephrotoxicity can be predicted with high efficiency and accuracy by combining cell-based andcomputational methods that are suitable for automation.
Keywords: dna damage response; high-content screening; in vitro model; nephrotoxicity;
phenotypic profiling; toxicity prediction

Lush Prize 2016 – Science Prize Research Paper

22Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro
High-Throughput Screening Assays for the Estrogen Receptor.
R S Judson1, F M Magpantay2, V Chickarmane3, C Haskell4, N Tania5, J Taylor6, M Xia7, R
Huang7, D M Rotroff8, D L Filer9, K A Houck10, M T Martin10, N Sipes11, A M Richard10, K
Mansouri9, R W Setzer10, T B Knudsen10, K M Crofton10, R S Thomas10.
1

U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; Email:judson.richard@epa.gov.
2
Department of Mathematics, University of Manitoba, Winnipeg, MB, Canada;
3
Division of Biology, California Institute of Technology, Pasadena, California 91125;
4Department of Mathematics, University of Southern California, Los Angeles, California 90089;
5Department of Mathematics, Smith College, Northampton, Massachusetts 01063;
6
Courant Institute, New York University, New York New York 10012;
7
NIH Chemical Genomics Center, National Center for Advancing Translational Sciences,
Rockville, Maryland 20892;
8Department of Statistics and Bioinformatics Research Center, North Carolina State University,
Raleigh, North Carolina 27607;
9
ORISE Fellow at the U.S. EPA, Research Triangle Park, North Carolina 27711;
10
U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711;
11
NIH National Toxicology Program, Research Triangle Park, North Carolina 27711.
Toxicol Sci. 2015 Nov;148(1):137-54. doi: 10.1093/toxsci/kfv168. Epub 2015 Aug 13.
Score 2+1
(Assays + scale)
Abstract
We demonstrate a computational network model that integrates 18 in vitro, high-throughput
screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding,transcriptional activation, and ER-dependent cell proliferation. The network model uses activity
patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist,or is otherwise influencing the assays through a manner dependent on the physics andchemistry of the technology platform ("assay interference"). The method is applied to a library of1812 commercial and environmental chemicals, including 45 ER positive and negativereference chemicals. Among the reference chemicals, the network model correctly identified theagonists and antagonists with the exception of very weak compounds whose activity wasoutside the concentration range tested. The model agonist score also correlated with the
expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated,
111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset
and model were also used to begin a systematic investigation of assay interference. The mostprominent cause of false-positive activity (activity in an assay that is likely not due to interactionof the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set ofimportant environmental chemicals with human exposure potential for additional in vivo
endocrine testing. Finally, this model is generalizable to any molecular pathway for which there
are multiple upstream and downstream assays available.
Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is
written by US Government employees and is in the public domain in the US.
Keywords: EDSP; In vitro; biological modeling; estrogen receptor; high-throughput screening;prioritization
Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat Tcell line.
Lush Prize 2016 – Science Prize Research Paper

23

MR Katika, PJ Hendriksen, H van Loveren, A Peijnenburg.
RIKILT-Institute of Food Safety, Wageningen University & Research Centre, Wageningen, The
Netherlands .
J Immunotoxicol. 2015 Jul-Sep;12(3):206-16. doi: 10.3109/1547691X.2014.925995.
Score 3 (Pathway)
Abstract
Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals
and grains. As such, DON poses a risk for many adverse health effects to human and animals. In
particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being abinding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study
aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2)understand why immune cells are more sensitive to DON than most other cell types. Previously publishedmicroarray studies have described the effects of DON on immune cells. To build upon these findings,
here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells
that were exposed for 3 h to 0.5 µM DON. Induction of ER stress by DON was confirmed byimmunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and
DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT,
activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are
known inducers of the T-cell activation response. Induction of an oxidative stress response was also
confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1,
as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that
DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, theseresults allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding toeukaryotic 60S ribosomal subunit  →  ribotoxic stress  →  ER stress  →  calcium release from the ER intocytoplasm  →  T-cell activation and oxidative stress  →  apoptosis. It is proposed that immune cells are moresensitive to DON than other cell types due to the induction of a T-cell activation response by increased
intracellular calcium levels.
Keywords: Apoptosis; ER stress; Jurkat cells; NF-κB; NFAT; deoxynivalenol; immunotoxicity; oxidative
stressExposure to coplanar PCBs induces endothelial cell inflammation through epigenetic regulationof NF-κB subunit p65.

D Liu, JT Perkins, MC Petriello, B Hennig.
Superfund Research Center, University of Kentucky, Lexington, KY 40536, United States;
Department of Animal and Food Sciences, College of Agriculture, Food and Environment,
University of Kentucky, Lexington, KY 40536, United States. Email: bhennig@uky.edu.
Toxicol Appl Pharmacol. 2015 Dec 15;289(3):457-65. doi: 10.1016/j.taap.2015.10.015.
Score 3 (Pathway)
Abstract
Epigenetic modifications of DNA and histones alter cellular phenotypes without changing
genetic codes. Alterations of epigenetic marks can be induced by exposure to environmental
pollutants and may contribute to associated disease risks. Here we test the hypothesis that
endothelial cell dysfunction induced by exposure to polychlorinated biphenyls (PCBs) is
mediated in part though histone modifications. In this study, human vascular endothelial cells
were exposed to physiologically relevant concentrations of several PCBs congeners (e.g.,
PCBs 77, 118, 126 and 153) followed by quantification of inflammatory gene expression andchanges of histone methylation. Only exposure to coplanar PCBs 77 and 126 induced theLush Prize 2016 – Science Prize Research Paper

24expression of histone H3K9 trimethyl demethylase jumonji domain-containing protein 2B(JMJD2B) and nuclear factor-kappa B (NF-κB) subunit p65, activated NF-κB signalling as
evidenced by nuclear translocation of p65, and up-regulated p65 target inflammatory genes,
such as interleukin (IL)-6, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1),
vascular cell adhesion molecule-1 (VCAM-1), and IL-1α/β. The increased accumulation of
JMJD2B in the p65 promoter led to a depletion of H3K9me3 repression mark, which accounts
for the observed up-regulation of p65 and associated inflammatory genes. JMJD2B geneknockdown confirmed a critical role for this histone demethylase in mediating PCB-induced
inflammation of the vascular endothelium. Finally, it was determined, via chemical inhibition,that PCB-induced up-regulation of JMJD2B was estrogen receptor-alpha (ER-α) dependent.
These data suggest that coplanar PCBs may exert endothelial cell toxicity through changes inhistone modifications.
Copyright © 2015 Elsevier Inc. All rights reserved.
Keywords: ER-α; Epigenetics and vascular inflammation; H3K9me3; JMJD2B; PCBs

The plasticizer BBP selectively inhibits epigenetic regulator sirtuins.
J Zhang, H Ali, YS Bedi, M Choudhury.
Dept of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health
Science Center, Kingsville, TX, USA. Email: mchoudhury@pharmacy.tamhsc.edu.
Toxicology. 2015 Dec 2;338:130-41. doi: 10.1016/j.tox.2015.10.004.
Score 3 + 1 (Pathway + combination of human in vitro assay and in silico molecular binding
analysis)
Abstract
The plasticizer benzyl butyl phthalate (BBP) is a well-known endocrine disruptor. Widespread
human exposure to phthalates has raised substantial public concern due to its detrimental
health effects. However, molecular mechanisms of the phthalates effect require elucidation. In
this study, we analyzed: 1) the binding interaction of several phthalates and persistent organic
pollutants with epigenetic regulator sirtuins and 2) the effect of BBP on the sirtuins in HepG2
cells. AutoDock molecular docking analysis showed that BBP binds to Sirt1 and Sirt3 proteins
similarly to the native ligands with shortest binding free energies (ΔGb) of -7.35 and -8.3
kcal/mol, respectively; and inhibition constants (Ki) of 4.07 µM and 0.82 µM, respectively.
Furthermore, BBP was superimposed onto the co-crystallized ligands within the least rootmean-square deviation (RMSD) of 0.96Å and 1.55Å for Sirt1 and Sirt3, respectively, and boundinto the sites with a sufficient number of hydrogen bonds, implying the best fit compared toother sirtuins. In HepG2 cells, BBP significantly down-regulated Sirt1 and Sirt3 (p<0.05) gene
expression at a concentration as low as 10nM; other sirtuins remained unaffected. Consistent
with decreased gene expression, Sirt1 and Sirt3 protein levels were significantly decreased at
48 h (p<0.05). In addition, mitochondrial biogenesis regulators PGC-1α, NRF-1, and NRF-2,
were decreased (p<0.05). SiRNA studies showed that BBP did not regulate PGC-1α via sirtuin
and BBP requires sirtuin's presence to regulate NRF-1 or NRF-2. BBP significantly increased
ROS production (p<0.05) and ROS may be chiefly regulated by NRF-1 and NRF-2 in HepG2
cells under Sirt1 and Sirt3 silenced condition. This is the first report to demonstrate that BBP
selectively disrupts specific sirtuins in HepG2 cells. In conclusion, our study suggests that BBP
can impair two vital epigenetic regulators and mitochondrial biogenesis regulators in liver cells.

Lush Prize 2016 – Science Prize Research Paper

25

Keywords: Aging; Benzyl butyl phthalate (BBP); Endocrine disruptor; Metabolic syndrome;
Mitochondria; Sirtuins

Towards in vitro DT/DNT testing: Assaying chemical susceptibility in early differentiating NT2cells.

AK Menzner, S Abolpour Mofrad, O Friedrich, DF Gilbert.
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg,
Erlangen, Germany; Erlangen Graduate School in advanced Optical Technologies (SAOT),Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany. Email:daniel.gilbert@fau.de.
Toxicology. 2015 Dec 2;338:69-76. doi: 10.1016/j.tox.2015.10.007.
Score 3 (Assay)
Abstract
Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitablemodel for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT)
studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) andpermit toxicity testing at different stages of maturation. NT2 cells have recently been reported toshow specific changes in dielectric resistance profiles during differentiation which can beobserved as early as 24h upon RA-stimulation. These observations suggest alteredsusceptibility to chemicals at an early stage of differentiation. However, chemical susceptibilityof early differentiating NT cells has not yet been studied. To address this question, we haveestablished a cell fitness screening assay based on the analysis of intracellular ATP levels and
we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early
differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19
chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether
any of the identified drugs have previously been associated with DT/DNT, we conducted aliterature search on the identified molecules and quantified the fraction of chemicals assigned to
the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X
in the hit list and the small molecule library. While the fractions of the categories N and B weredecreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27fold) in the hit list compared to the chemical library. From these data as well as from theliterature review, identifying large fractions of chemicals being directly ( 42%) and indirectly
associated with DT/DNT ( 32%), we conclude that our method may be beneficial to systematicin vitro-based primary screening for developmental toxicants and neurotoxicants and we
propose cell fitness screening in early differentiating NT2 cells as a strategy for evaluatingchemical susceptibility at different stages of differentiation to reduce animal testing in thecontext of the 3Rs.
Keywords: Cell viability screening; Chemical susceptibility; Developmental toxicity andneurotoxicity testing; Differentiation; Human pluripotent embryonal carcinoma cells

Development of an in Silico Profiler for Mitochondrial Toxicity.
MD Nelms, CL Mellor, MT Cronin, JC Madden, SJ Enoch.

Lush Prize 2016 – Science Prize Research Paper

26

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University , Byrom
Street, Liverpool L3 3AF, U.K.
Chem Res Toxicol. 2015 Oct 19;28(10):1891-902. doi: 10.1021/acs.chemrestox.5b00275.
Score 2 + 1 (Assay + in silico tool for identifying MIEs)
Abstract
This study outlines the analysis of mitochondrial toxicity for a variety of pharmaceutical drugs
extracted from Zhang et al. ((2009) Toxicol. In Vitro, 23, 134-140). These chemicals weregrouped into categories based upon structural similarity. Subsequently, mechanistic analysis
was undertaken for each category to identify the molecular initiating event driving mitochondrial
toxicity. The mechanistic information elucidated during the analysis enabled mechanism-based
structural alerts to be developed and combined together to form an in silico profiler. This profiler
is envisaged to be used to develop chemical categories based upon similar mechanisms as partof the adverse outcome pathway paradigm. Additionally, the profiler could be utilized inscreening large data sets in order to identify chemicals with the potential to induce
mitochondrial toxicity.

Transcriptomic Analysis of Human Primary Bronchial Epithelial Cells after Chloropicrin
Treatment.

M Pesonen, M Storvik, T Kokkola, J Rysä, K Vähäkangas, M Pasanen.
Research and Development, Centre for Military Medicine, Finnish Defence Forces ,Tukholmankatu 8A, PL 50, 00301 Helsinki, Finland.
Chem Res Toxicol. 2015 Oct 19;28(10):1926-35. doi: 10.1021/acs.chemrestox.5b00123.
Score 2 + 1 (Pathway + gene analysis)
Abstract
Chloropicrin is a vaporizing toxic irritant that poses a risk to human health if inhaled, but themechanism of its toxicity in the respiratory tract is poorly understood. Here, we exposed humanprimary bronchial epithelial cells (HBEpC) to two concentrations of chloropicrin (10-50 µM) for 6
or 48 h and used genomic microarray, flow cytometry, and TEM-analysis to monitor cellularresponses to the exposures. The overall number of differentially expressed transcripts with afold-change > ± 2 compared to controls increased with longer exposure times. The initial
response was activation of genes with a higher number of up- (512 by 10 µM and 408 by 40 µM
chloropicrin) rather than down-regulated transcripts (40 by 10 µM and 215 by 40 µM
chloropicrin) at 6 h seen with both exposure concentrations. The number of down-regulatedtranscripts, however, increased with the exposure time. The differentially regulated transcripts
were further examined for enriched Gene Ontology Terms (GO) and KEGG-pathways.
According to this analysis, the "ribosome" and "oxidative phosphorylation" were the KEGGpathways predominantly affected by the exposure. The predominantly affected (GO) biologicalprocesses were "protein metabolic process" including "translation," "cellular protein complex
assembly," and "response to unfolded protein." Furthermore, the top pathways, "NRF2activated oxidative stress" and "Ah-receptor signaling," were enriched in our data sets by IPAanalysis. Real time qPCR assay of six selected genes agreed with the microarray analysis. In
addition, chloropicrin exposure increased the numbers of late S and/or G2/M-phase cells as
analyzed by flow cytometry and induced autophagy as revealed by electron microscopy. The

Lush Prize 2016 – Science Prize Research Paper

27

targets identified are critical for vital cellular functions reflecting acute toxic responses and arepotential causes for the reduced viability of epithelial cells after chloropicrin exposure.

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed
Human Cornea-like Epithelial (RhCE) Tissue Model.

Y Kaluzhny, H Kandárová, L d'Argembeau-Thornton, P Kearney, M Klausner.
MatTek Corporation; ykaluzhny@mattek.com.
J Vis Exp. 2015 Aug 23;(102):e52979. doi: 10.3791/52979.
Score 3 + 1 (Assay + new OECD test)
Abstract
To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH
legislation, validated non-animal alternative methods for reliable and accurate assessment ofocular toxicity in man are needed. To address this need, we have developed an eye irritation
test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE)
tissue model that is based on normal human cells. The EIT is able to separate ocular irritants
and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling(GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals
and a second, similar protocol for solid test articles. The EIT prediction model uses a single
exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as
determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids)
EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&A) for liquids,
100.0/68.4/ and 84.6% SS&A for solids, and 97.6/68.3/ and 83.1% for overall SS&A. The EIT
will contribute significantly to classifying the ocular irritation potential of a wide range of liquidand solid chemicals without the use of animals to meet regulatory testing requirements. The
EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

A transcriptome-based classifier to identify developmental toxicants by stem cell testing: design,validation and optimization for histone deacetylase inhibitors.

E Rempel1, L Hoelting, T Waldmann, NV Balmer, S Schildknecht, M Grinberg, JA Das Gaspar,
V Shinde, R Stöber, R Marchan, C van Thriel, J Liebing, J Meisig, N Blüthgen, A Sachinidis, J
Rahnenführer, JG Hengstler, M Leist.
1

Department of Statistics, TU Dortmund University, 44139, Dortmund, Germany.

Arch Toxicol. 2015 Sep;89(9):1599-618. doi: 10.1007/s00204-015-1573-y.
Score 2 + 1 (Assay + use of informatics)
Abstract
Test systems to identify developmental toxicants are urgently needed. A combination of humanstem cell technology and transcriptome analysis was to provide a proof of concept that toxicantswith a related mode of action can be identified and grouped for read-across. We chose a test
system of developmental toxicity, related to the generation of neuroectoderm from pluripotent
stem cells (UKN1), and exposed cells for 6 days to the histone deacetylase inhibitors (HDACi)
valproic acid, trichostatin A, vorinostat, belinostat, panobinostat and entinostat. To provide
insight into their toxic action, we identified HDACi consensus genes, assigned them toLush Prize 2016 – Science Prize Research Paper

28superordinate biological processes and mapped them to a human transcription factor network
constructed from hundreds of transcriptome data sets. We also tested a heterogeneous groupof 'mercurials' (methylmercury, thimerosal, mercury(II)chloride, mercury(II)bromide, 4chloromercuribenzoic acid, phenylmercuric acid). Microarray data were compared at the highest
non-cytotoxic concentration for all 12 toxicants. A support vector machine (SVM)-basedclassifier predicted all HDACi correctly. For validation, the classifier was applied to legacy data
sets of HDACi, and for each exposure situation, the SVM predictions correlated with thedevelopmental toxicity. Finally, optimization of the classifier based on 100 probe sets showedthat eight genes (F2RL2, TFAP2B, EDNRA, FOXD3, SIX3, MT1E, ETS1 and LHX2) aresufficient to separate HDACi from mercurials. Our data demonstrate how human stem cells andtranscriptome analysis can be combined for mechanistic grouping and prediction of toxicants.
Extension of this concept to mechanisms beyond HDACi would allow prediction of humandevelopmental toxicity hazard of unknown compounds with the UKN1 test system.
NOTE: The UKN1 human stem cell line used in this study seems to require differentiation using
conditioned medium from mouse embryonic cells – check with authors whether these are
primary cells or a cell line.

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety
Screening and Systems Biology Data Generation.

V Shinde1, S Klima2, PS Sureshkumar1, K Meganathan1, S Jagtap1, E Rempel3, J
Rahnenführer3, JG Hengstler4, T Waldmann2, J Hescheler1, M Leist2, A Sachinidis 5.
1

Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of
Cologne.
2

Department of Biology, University of Konstanz.

3

Department of Statistics, Technical University of Dortmund.

4

Leibniz Research Centre for Working Environment and Human Factors, Technical Universityof Dortmund.
5

Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of
Cologne; a.sachinidis@uni-koeln.de.
J Vis Exp. 2015 Jun 17;(100):e52333. doi: 10.3791/52333.
Score 3 (Assay)
Abstract
Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination
with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs.
To provide a solid scientific basis for such assays, it will be important to gain quantitative
information on the time course of development and on the underlying regulatory mechanisms bysystems biology approaches. Two assays have therefore been tuned here for theserequirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent
cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generationof cells of all three germ layers. This system recapitulates key steps of early human embryonicdevelopment, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells
are exposed to chemicals during differentiation. The UKN1 test system is based on hESC
differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system
recapitulates early neural development and predicts early developmental neurotoxicity andepigenetic changes triggered by chemicals. Both systems, in combination with transcriptome
Lush Prize 2016 – Science Prize Research Paper

29

microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be usedin combination to generate input data for systems biology analysis. These test systems haveadvantages over the traditional toxicological studies requiring large amounts of animals. The
test systems may contribute to a reduction of the costs for drug development and chemicalsafety evaluation. Their combination sheds light especially on compounds that may influence
neurodevelopment specifically.
NOTE: The UKN1 human stem cell line used in this study seems to require differentiation using
conditioned medium from mouse embryonic cells – check with authors whether these are
primary cells or a cell line.

Nephron Toxicity Profiling via Untargeted Metabolome Analysis Employing a High Performance
Liquid Chromatography-Mass Spectrometry-based Experimental and Computational Pipeline.

C Ranninger1, M Rurik2, A Limonciel3, S Ruzek1, R Reischl1, A Wilmes3, P Jennings3, P Hewitt4,
W Dekant5, O Kohlbacher2, CG Huber6.
1

Department of Molecular Biology, Division of Chemistry and Bioanalytics, University ofSalzburg, 5020 Salzburg, Austria.
2

Applied Bioinformatics Group, Center for Bioinformatics, Quantitative Biology Center and
Department of Computer Science, University of Tübingen, Tübingen 72076, Germany.
3

Division of Physiology, Department of Physiology and Medical Physics, Medical University of
Innsbruck, Innsbruck 6020, Austria.
4

Merck KGaA, Merck Serono, Nonclinical Safety, Darmstadt 64293, Germany, and.

5

Department of Toxicology, University of Würzburg, Würzburg 97078, Germany.

6

Department of Molecular Biology, Division of Chemistry and Bioanalytics, University ofSalzburg, 5020 Salzburg, Austria, c.huber@sbg.ac.at.
J Biol Chem. 2015 Jul 31;290(31):19121-32. doi: 10.1074/jbc.M115.644146.
Score 2 + 1 (Metabolomics assay + linked to proteomics & transcriptomics)
Abstract
Untargeted metabolomics has the potential to improve the predictivity of in vitro toxicity models
and therefore may aid the replacement of expensive and laborious animal models. Here we
describe a long term repeat dose nephrotoxicity study conducted on the human renal proximal
tubular epithelial cell line, RPTEC/TERT1, treated with 10 and 35 µmol·liter(-1) of
chloroacetaldehyde, a metabolite of the anti-cancer drug ifosfamide. Our study outlines theestablishment of an automated and easy to use untargeted metabolomics workflow for HPLChigh resolution mass spectrometry data. Automated data analysis workflows based on open
source software (OpenMS, KNIME) enabled a comprehensive and reproducible analysis of thecomplex and voluminous metabolomics data produced by the profiling approach. Time- andconcentration-dependent responses were clearly evident in the metabolomic profiles. To obtaina more comprehensive picture of the mode of action, transcriptomics and proteomics data werealso integrated. For toxicity profiling of chloroacetaldehyde, 428 and 317 metabolite features
were detectable in positive and negative modes, respectively, after stringent removal ofchemical noise and unstable signals. Changes upon treatment were explored using principal
component analysis, and statistically significant differences were identified using linear models
for microarray assays. The analysis revealed toxic effects only for the treatment with 35µmol·liter(-1) for 3 and 14 days. The most regulated metabolites were glutathione andLush Prize 2016 – Science Prize Research Paper

30

metabolites related to the oxidative stress response of the cells. These findings arecorroborated by proteomics and transcriptomics data, which show, among other things, anactivation of the Nrf2 and ATF4 pathways.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords: KNIME; OpenMS; bioinformatics; chloroacetaldehyde; high performance liquid
chromatography (HPLC); mass spectrometry (MS); metabolomics; oxidative stress; toxicity
profiling

Mimicking enzymatic effects of cytochrome P450 by an efficient biosensor for in vitro detection of
DNA damage.

AR Jalalvand1,2,4, MB Gholivand1, HC Goicoechea2, T Skov3, K Mansouri4.
1

Department of Analytical Chemistry, Faculty of Chemistry, Razi University, Kermanshah
671496734, Iran. Email: mbgholivand2013@gmail.com.
2

Laboratorio de Desarrollo Analítico y Quimiometría (LADAQ), Cátedra de Química Analítica I,
Universidad Nacional del Litoral, Ciudad Universitaria, CC 242 (S3000ZAA), Santa Fe,
Argentina.
3

Quality & Technology, Department of Food Science, Faculty of Science, University of
Copenhagen, Copenhagen, Denmark.
4

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah,
Iran.
Int J Biol Macromol. 2015 Aug;79:1004-10. doi: 10.1016/j.ijbiomac.2015.05.047.
Score 3 (Assay)
Abstract
A novel biosensor for detecting DNA damage induced by benzo(a)pyrene (BP) and its
metabolite was presented in this work. The nafion-solubilized single wall carbon nanotubesionic liquid (SWCNTs-NA-IL) composite film was prepared and then horseradish peroxidase(HRP) and double-stranded DNA were alternately assembled on the composite film by thelayer-by-layer method. The biosensor was characterized by cyclic voltammetry (CV),electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), scanningelectron microscopy (SEM) and computational methods. UV-vis spectrophotometry was also
used to investigate DNA damage induced by BP and its metabolites in solution. The DNA
biosensor was treated separately in BP, hydrogen peroxide (H2O2) and in their mixture,
respectively. The EIS analysis showed a decrease in the charge transfer resistance at the
DNA/HRP/SWCNTs-NA-IL/GCE incubated in a mixture of HRP and H2O2, because HRP in thepresence of H2O2 could mimic enzymatic effects of cytochrome P450 (CYP450) to metabolize
BP which could cause significant DNA damage and the exposed DNA bases reduced theelectrostatic repulsion of the negatively charged redox probe and leads to Faradaic impedancechanges. Finally, a novel biosensor for BP determination was developed and this method
provided an indirect, and quantitative estimation of DNA damage in vitro.
Copyright © 2015 Elsevier B.V. All rights reserved.
Keywords: Benzo(a)pyrene; Biosensor; DNA damage

Lush Prize 2016 – Science Prize Research Paper

31Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells
leading to heritable mutations" using the OECD's users' handbook supplement.

CL Yauk1, IB Lambert2, ME Meek3, GR Douglas1, F Marchetti1.
1

Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario,
Canada.
2

Department of Biology, Carleton University, Ottawa, Ontario, Canada.

3

R. Samuel McLaughlin Centre for Population Health Risk Assessment, University of Ottawa,
Ottawa, Ontario, Canada.
Environ Mol Mutagen. 2015 Dec;56(9):724-50. doi: 10.1002/em.21954.
Score 3 (Pathway - new AOP)
Abstract
The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome
Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity
that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently
released to provide supplementary guidance on AOP development. This article describes one
AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. Thisoutcome is an important regulatory endpoint. The AOP describes the biological plausibility andempirical evidence supporting that compounds capable of alkylating DNA cause germ cell
mutations and subsequent mutations in the offspring of exposed males. Alkyl adducts are
subject to DNA repair; however, at high doses the repair machinery becomes saturated. Lack ofrepair leads to replication of alkylated DNA and ensuing mutations in male premeiotic germ
cells. Mutations that do not impair spermatogenesis persist and eventually are present inmature sperm. Thus, the mutations are transmitted to the offspring. Although there are some
gaps in empirical support and evidence for essentiality of the key events for certain aspects ofthis AOP, the overall AOP is generally accepted as dogma and applies broadly to any species
that produces sperm. The AOP was developed and used in an iterative process to test andrefine the Users' Handbook, and is one of the first publicly available AOPs. It is our hope that
this AOP will be leveraged to develop other AOPs in this field to advance method development,
computational models to predict germ cell effects, and integrated testing strategies.
© 2015 Her Majesty the Queen in Right of Canada.
Keywords: adverse outcome pathway; alkylation; germ cell; heritable mutation; weight ofevidence

Particle-induced cell migration assay (PICMA): A new in vitro assay for inflammatory particle
effects based on permanent cell lines.

GA Westphal1, I Schremmer1, A Rostek2, K Loza2, N Rosenkranz1, T Brüning1, M Epple2, J
Bünger1.
1

Institute for Prevention & Occupational Medicine of the German Social Accident Insurance,
Institute of the Ruhr-University Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789 Bochum,
Germany. Emails: westphal@ipa-dguv.de. schremmer@ipa-dguv.de. rosenkranz@ipadguv.de Bruening@ipa-dguv.de & buenger@ipa-dguv.de .

Lush Prize 2016 – Science Prize Research Paper

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2

Inorganic Chemistry & Center for Nanointegration Duisburg-Essen (CeNIDE), University ofDuisburg-Essen, Universitaetsstr. 5-7, 45117 Essen, Germany. Emails: alexander.rostek@unidue.de kateryna.loza@uni-due.de & matthias.epple@uni-due.de
Toxicol In Vitro. 2015 Aug;29(5):997-1005. doi: 10.1016/j.tiv.2015.04.005.
Score 3 (Assay)
Abstract
Inflammation is a decisive pathophysiologic mechanism of particle toxicity and accumulation ofneutrophils in the lung is believed to be a crucial step in this process. This study describes an invitro model for investigations of the chemotactic attraction of neutrophils in response to particles
using permanent cell lines. We challenged NR8383 rat macrophages with particles that werecharacterized concerning chemical nature, crystallinity, and size distribution in the dry state andin the culture medium. The cell supernatants were used to investigate migration of differentiatedhuman leukemia cells (dHL-60 cells). The dose range for the tests was determined using animpedance-based Real-Time Cell Analyzer. The challenge of NR8383 cells with 32-96 µg cm(2) coarse and nanosized particles resulted in cell supernatants which induced strong and dosedependent migration of dHL-60 cells. Quartz caused the strongest effects - exceeding thepositive control "fetal calf serum" (FCS) several-fold, followed by silica, rutile, carbon black, andanatase. BaSO4 served as inert control and induced no cell migration. Particles caused
NR8383 cells to secrete chemotactic compounds. The assay clearly distinguished between theparticles of different inflammatory potential in a highly reproducible way. Specificity of the test issuggested by negative results with BaSO4.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Keywords: Cell migration; NR8383 cells; Neutrophilic inflammation; Particles; dHL-60 cells

Evaluation of an optimized protocol using human peripheral blood monocyte derived dendritic
cells for the in vitro detection of sensitizers: Results of a ring study in five laboratories.

H Reuter1, S Gerlach1, J Spieker2, C Ryan2, C Bauch4, C Mangez5, P Winkler4, R Landsiedel3,
M Templier5, A Mignot5, F Gerberick2, H Wenck1, P Aeby6, A Schepky1.
1

Research and Development, Beiersdorf AG, Unnastrasse 48, D-20245 Hamburg, Germany.
Email: Hendrik.Reuter@beiersdorf.com.
2

The Procter & Gamble Company, Mason Business Center, 8700 Mason Montgomery Road,
Mason, OH 45040, USA.
3

BASF Product Safety - Experimental Toxicology and Ecology, BASF SE, GV/TB - Z570, D67056 Ludwigshafen, Germany.
4

Johnson & Johnson GmbH, Johnson & Johnson Platz 2, D-41470 Neuss, Germany.

5

Institut de Recherche Pierre Fabre, Centre Expérimental et Pharmacocinétique de Campans,
Bel Air - Route de Lautrec, F-81106 Castres cedex, France.
6

Independent Consultant, Ch. de Messidor 59, CH-1723 Marly, Switzerland.

Toxicol In Vitro. 2015 Aug;29(5):976-86. doi: 10.1016/j.tiv.2015.03.021.
Score 3 (Assay)
Abstract

Lush Prize 2016 – Science Prize Research Paper

33

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with
relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay)
are still supplying most of the data used to assess the sensitization potential of new chemicals.
However, the 7th amendment to the EU Cosmetic Directive have introduced a testing ban forcosmetic ingredients after March 2013. We have developed and optimized a stable andreproducible in vitro protocol based on human peripheral blood monocyte derived dendritic cells
to assess the sensitization potential of chemicals. To evaluate the transferability and thepredictivity of this PBMDCs based test protocol, a ring study was organized with fivelaboratories using seven chemicals with a known sensitization potential (one none-sensitizer
and six sensitizers, including one pro-hapten). The results indicated that this optimized test
protocol could be successfully transferred to all participating laboratories and allowed a correct
assessment of the sensitization potential of the tested set of chemicals. This should allow awider acceptance of PBMDCs as a reliable test system for the detection of human skinsensitizers and the inclusion of this protocol in the toolbox of in vitro methods for the evaluationof the skin sensitization potential of chemicals.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Keywords: Human dendritic cells; In vitro test system; Peripheral blood monocyte derived
dendritic cells; Ring study; Skin sensitization

Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent
stem cells-derived and fetal hepatocytes.

Y Avior1,2, G Levy1,2, M Zimerman1,2 D Kitsberg1,2 ,, R Schwartz3, R Sadeh2,4 , A Moussaieff1,2,
M Cohen1,2, J Itskovitz-Eldor5, Y Nahmias1,2,4.
1

Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem,
Israel.
2

Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.

3

Department of Medicine, Weill Cornell Medical College, New York, NY.

4

School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem,
Israel.
5

Stem Cell Center, Technion, Haifa, Israel.

Hepatology. 2015 Jul;62(1):265-78. doi: 10.1002/hep.27803.
Score 2 + 1 (Assay + significant advance in use of hPSCs for hepatocyte model)
Abstract
The liver is the main organ responsible for the modification, clearance, and transformational
toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes.
However, the scarcity and variability of primary hepatocytes currently limits their utility. Humanpluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes;
however, current protocols still produce fetal-like hepatocytes with limited mature function.
Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggestingthat nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid,a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4
and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells
produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes,

Lush Prize 2016 – Science Prize Research Paper

34while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon
receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist
rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear
factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format.
Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained instem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally,stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including
steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins.
Conclusion: Our work provides fresh insights into liver development, suggesting that microbialderived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues
results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology.
© 2015 by the American Association for the Study of Liver Diseases.

Genes specifically modulated in sensitized skins allow the detection of sensitizers in areconstructed human skin model. Development of the SENS-IS assay.

F Cottrez, E Boitel, C Auriault, P Aeby , H Groux.
ImmunoSearch, Les Cyclades, Chemin de Camperousse, Grasse, France. Email:hgroux@immunosearch.fr.
Toxicol In Vitro. 2015 Jun;29(4):787-802. doi: 10.1016/j.tiv.2015.02.012.
Score 2 + 1 (Assay/biomarker + predictivity)
Abstract
Analysis of genes modulated during the sensitization process either on mice (LLNA) or human(blisters) combined with data mining has allowed the definition of a comprehensive panel ofsensitization biomarkers. This set of genes includes already identified markers such as the ARE
family and others not yet associated with the sensitization process (the so-called SENS-IS genesubset). The expression of this set of genes has been measured on reconstituted humanepidermis models (Episkin) exposed to various sensitizers and non-sensitizers. Fine analysis of
their expression pattern indicates that it is the number of modulated genes rather than theintensity of up-regulation that correlates best with the sensitization potential of a chemical.
Moreover, sensitizers that are weak inductors of ARE genes tend to be relevant modulators of
the SENS-IS subset. By combining the expression data obtained with both gene subsets, it is
now possible to identify a wide variety of sensitizers on a test system (in vitro reconstructed
human epidermis) that is very similar to the in vivo situation and compatible with a large variety
of test substance characteristics.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Keywords: Alternatives to animal tests; Reconstituted epidermis; SENS-IS; Skin sensitization;
Toxicogenomics

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

PC Schmeits1,2, J Shao1,2, DA van der Krieken1, OL Volger1, H van Loveren2,3 , AA
Peijnenburg1, PJ Hendriksen1.

Lush Prize 2016 – Science Prize Research Paper

35

1

RIKILT Institute of Food Safety, Wageningen University and Research Centre, Wageningen,
The Netherlands.
2

Department of Toxicogenomics, Maastricht University, the Netherlands.

3

Centre for Health Protection, National Institute for Public Health and the Environment (RIVM),
Bilthoven, The Netherlands.
J Appl Toxicol. 2015 Jul;35(7):831-41. doi: 10.1002/jat.3079.
Score 3 (Assay)
Abstract
Previously, we identified 25 classifier genes that were able to assess immunotoxicity using
human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose,
Jurkat cells were exposed for 6  h to subcytotoxic doses of nine immunotoxicants, five nonimmunotoxicants and four compounds for which human immunotoxicity has not yet been fully
established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR
analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine
immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%,
respectively, which is better than the performance in our previous study. Only one compound
was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants,
chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow andimidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in themolecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloropropanol and cypermethrin, although structurally different, affected protein metabolism andcholesterol biosynthesis and transport. In addition, four compounds, i.e.  chlorpyrifos, aldicarb,benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein
metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar
processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25
immunotoxic classifiers performed very well in a screening with new non-immunotoxic andimmunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be
applied within a tiered approach for animal free testing of human immunotoxicity.
Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: Fluidigm; Jurkat; ToxPi; biomarker; classifier; genomic; high throughput;
immunotoxicity; prediction; qRT-PCR

High-content screening of drug-induced mitochondrial impairment in hepatic cells: effects ofstatins.

L Tolosa1, A Carmona1, JV Castell1,2,3, MJ Gómez-Lechón1,3, MT Donato1,2,3.
1

Unidad de Hepatología Experimental, Torre A, Instituto Investigación Sanitaria La Fe (IIS LaFe), Bulevard Sur s/n, 46026, Valencia, Spain. laiatolosa@hotmail.com. gomez_mjo@gva.es.
2

Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de
Valencia, 46010, Valencia, Spain.
3

CIBERehd, FIS, 08036, Barcelona, Spain. gomez_mjo@gva.es

Arch Toxicol. 2015 Oct;89(10):1847-60. doi: 10.1007/s00204-014-1334-3.
Score 3 (Assay)

Lush Prize 2016 – Science Prize Research Paper

36

Abstract
A frequent mechanism for drug-induced liver injury (DILI) is mitochondrial impairment, and early evaluation of
new drugs for their potential to cause mitochondrial dysfunction is becoming an important task for drug
development. To this end, we designed a high-content screening assay to study mitochondrial-induced
hepatotoxicity in HepG2 cells in detail. Simultaneous assessment of mitochondrial mass and cell viability in cells
exposed for 24 h to compounds provides preliminary information on the mitochondrial- or nonmitochondrial-related
hepatotoxic potential of compounds. To fully address the mechanisms implicated in mitochondrial impairment,
prelethal changes in mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial
permeability transition, intracellular calcium concentration and apoptotic cell death were studied in cells incubatedfor 1 h with compounds. The assay correctly classified a set of well-known mitochondrial toxicants and negative
controls and revealed high sensitivity for the detection of mitochondrial DILI and the establishment of different
mitochondrial toxicity risks (low to high). This procedure was used for analysing the potential mitochondrial
impairment of six statins to determine their clinical risk. All the tested statins produced mitochondrial impairment,
although they showed different levels of toxicity (low-medium toxicity risk). The results suggest that this cell-based
assay is a promising in vitro approach to predict the potential of drug candidates to induce

mitochondrial-associated hepatotoxicity.
Keywords: Hepatotoxicity; High-content screening; Mitochondrial injury; Statins

Assessment of the eye irritation potential of chemicals: A comparison study between two test
methods based on human 3D hemi-cornea models

R. Tandon, M. Bartok, M. Zorn-Kruppa, J.M. Brandner, D. Gabel, M.Engelkea
Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
dr.melinda.bartok@gmail.com
Toxicology in Vitro Vol 30, Issue 1, Part B, 25 December 2015, Pages 561–568
doi:10.1016/j.tiv.2015.09.003
Score 3 (Assay)
Abstract
We have recently developed two hemi-cornea models (Bartok et al., Toxicol in Vitro 29, 72, 2015; Zorn-Kruppa etal. PLoS One 9, e114181, 2014), which allow the correct prediction of eye irritation potential of chemicals
according to the United Nations globally harmonized system of classification and labeling of chemicals (UN GHS).
Both models comprise a multilayered epithelium and a stroma with embedded keratocytes in a collagenous matrix.
These two models were compared, using a set of fourteen test chemicals. Their effects after 10 and 60 minutes(min) exposure were assessed from the quantification of cell viability using the MTT reduction assay. The first
approach separately quantifies the damage inflicted to the epithelium and the stroma. The second approachquantifies the depth of injury by recording cell death as a function of depth. The classification obtained by the twomodels was compared to the Draize rabbit eye test and an ex vivo model using rabbit cornea (Jester et al. Toxicol in
Vitro. 24, 597–604, 2010). With a 60 min exposure, both of our models are able to clearly differentiate UN GHS
Category 1 and UN GHS Category 2 test chemicals.

Keywords: Human 3D hemi-cornea model; Relative MTT-depth of injury; Globally harmonizedsystem of classification and labelling of chemicals; The Draize eye irritation test

Stem cell-derived human dorsal root ganglia-like cells to identify peripheral neurotoxicants

L Hoelting1,2, S Klima1, C Karreman1,M Grinberg3, J Meisig4,5, M Henry6, T Rotshteyn6, J
Rahnenführer3, N Blüthgen4,5, A Sachinidis6, T Waldmann1,M Leist1

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1

Doerenkamp-Zbinden Chair of in-vitro Toxicology & Biomedicine, University of Konstanz,
Germany;
2

Konstanz Graduate School Chemical Biology KORS-CB, Germany;3

Faculty of Statistics, TU Dortmund University, Germany;

4

Institute of Pathology, Charité-Universitätsmedizin, Germany;

5

Integrative Research Institute for the Life Sciences & Institute for Theoretical Biology,
Germany;
6

Institute of Neurophysiology & Center for Molecular Medicine Cologne (CMMC), Germany

EUSAAT 2015 Abstract 136
Score 3 (Assay)
Safety sciences and the identification chemical hazard have been seen as one of the mostimmediate practical applications of human pluripotent stem cell technology. Protocols for thegeneration of many desirable human cell types have been developed, but optimization ofneuronal models for toxicological use has been astonishingly slow, and the wide, clinicallyimportant field of peripheral neurotoxicity is still largely unexplored. Here, a 2-step protocol togenerate large lots of identical peripheral human neuronal precursors was characterized andadapted to the measurement of peripheral neurotoxicity. High content imaging allowed an
unbiased assessment of cell morphology and viability. The computational quantification ofneurite growth as functional parameter highly sensitive to disturbances by toxicants was usedas endpoint reflecting specific neurotoxicity. The differentiation of cells towards dorsal root
ganglia-like neurons was tracked in relation to a large background data set based on gene
expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed
as first toxicological assay that harnesses the potential of human pluripotent stem cells togenerate cell types/tissues that are not otherwise available for prediction of human systemicorgan toxicity. Testing of more than 30 chemicals showed that human neurotoxicants, as well
as neurite growth enhancers, were correctly identified. Various classes of chemotherapeutics
causing human peripheral neuropathies were identified, while they were missed when tested onhuman central neurons. The PeriTox-test established here shows the potential of human stem
cells for clinically-relevant safety testing of drugs in use and of new emerging candidates.

BoNT activity determination by stimulus-dependent release of neuro-secretory vesicle-targetedluciferase from neuronal cell lines

A Pathe-Neuschäfer-Rube, F Neuschäfer-Rube, L Genz, Gerhard Püschel
University of Potsdam, Institute of Nutritional Science, Nuthetal, Germany
EUSAAT 2015 Abstract 16
Score 3 + 1 (Assay + potential high impact on reducing animal tests)
Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons andthereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments
and,more frequently, for cosmetic medical interventions. Botulinum toxin for pharmacologicalapplications is isolated from bacterial cultures. Due to partial denaturation of the protein, thespecific activity of these preparations shows large variations. Because of its extreme potential
toxicity, pharmacological preparations must be carefully tested for their activity. For the current
gold standard, the mouse lethality assay, several hundred thousand mice are killed per year.

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Alternative methods have been developed that suffer from one or more of the following deficits:
In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic andcell based immunological assays are specific for just one of the different serotypes. The current
study takes a completely different approach that overcomes these limitations: Neuronal celllines were stably transfected with plasmids coding for luciferases of different species, whichwere N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory
vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The
depolarization-dependent release was efficiently inhibited by of botulinum toxin in aconcentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay
might thus be an alternative to the mouse lethality assay and the immunological assays alreadyin use.
Note: This work has now been published as a full paper - DOI:
http://dx.doi.org/10.14573/altex.1503061

Micellar liquid chromatography as an alternative for in vivo and expensive in vitro tests
commonly used in prediction of human intestinal absorption

D Shokry, L Waters, G Parkes
School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom
EUSAAT Abstract 54
Score 3 (Assay)
Oral administration is one of the most important and abundant routes of drug administration,
therefore estimation of intestinal absorption is crucial for pharmaceutical compounds, especiallyfor drugs with poor solubility [1]. That is why it is important to determine the extent of drug
absorption for new drug entities (NDE) in preformulation studies. This study proposes a novel,simple, economic, accurate and precise way to predict intestinal absorption using a modified
chromatographic method.
Introduction: Animal testing is the standard method currently used to predict the extent and rateof intestinal absorption [2,3], i.e. through in vivo testing, for pharmaceutical compounds. In thisstudy a form of chromatography, known as MLC, has been applied to predict passive intestinal
absorption with a selection of model compounds through measurement and calculation of thepartition coefficient, Pmw. In this method bile salts were used as a mobile phase (instead of themore standard head and tail surfactants) to provide an environment more closely simulating thehuman intestinal environment.
Methods and Reagents: Two bile salts were considered separately, namely sodium
deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC) no buffers or organic modifiers
were added during the study to simulate intestinal conditions as closely as possible. Low
concentrations of 13 model drugs were prepared in the corresponding mobile phase
concentration to avoid saturation of the bile salt mobile phase. The samples were injected into
the chromatographic system with capacity factors obtained by analysis of the retention data
recorded for the model drugs used. Modelling of Human intestinal absorption (% HIA), intestinal
permeability of Caco-2 and PAMPA was performed by multiple linear regression. The models
obtained confirmed the ability of MLC to predict human intestinal absorption (% HIA), Caco-2
and PAMPA intestinal permeability coefficients. Conclusion: The use of NaTDC in MLC was
found to be a better method in the prediction of Human intestinal absorption while NaDC was

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found to be better for prediction of PAMPA and Caco-2 intestinal permeability coefficients
although both bile salts provided a superior prediction for both the in vivo (% HIA) and in vitro(Caco-2 and PAMPA) methods for intestinal absorption determination compared with current
methods. Therefore, it was concluded that the MLC method was found to be a successful tool in
prediction of human intestinal absorption (% HIA), Caco-2 and PAMPA intestinal permeability.
Since MLC is a more economic [4], time saving [5], eco-friendly [6] method that does notinclude the use of animals, it can be considered to be a superior method over all available
alternative options for prediction of intestinal absorption.
References[1] Williams, H. D., Trevaskis, N. L., Charman, S. A. et al. (2013). Pharmacol Rev 65, 315-499.
[2] Fagerholm, U., Johansson, M. and Lennernäs, H. (1996). Pharm Res 13, 1336-1342.
[3] Fotaki, N. (2009). Expert Rev Clin Pharmacol 2, 195-208.
[4] Armstrong, D. W. (1985). Separation & Purification Methods 14, 213-304.
[5] Khaledi, M. G. (1997). J Chromatography A 780, 3-40.
[6] Kanakaiah, B. (2013). Int J Bio-Pharma Res 2, 117-122.

In vitro assays for the potency determination of botulinum neurotoxin serotypes A and B

E Wild1, H Behrensdorf-Nicol, U Bonifas, J Klimek, B Krämer, B Kegel
1Paul-Ehrlich-Institut, Langen, Germany

EUSAAT Abstract 17
Score 3 + 1 (Assay + potential high impact on reducing animal tests)
Botulinum neurotoxins (BoNTs) have a paralytic effect, which is broadly applied in the fields of
medicine and cosmetics. The toxins produced by the bacterium Clostridium botulinum have to
be tested for their specific activity and potency, what is currently mostly done with LD50 tests inmice. As these tests cause a high distress for the animals, it is desirable to find alternatives tothe LD50 test, for which at least 600,000 mice are used annually [1]. The combined assay
presented here could be a suitable candidate for this task [2]. BoNTs use an elaborate
intoxication process leading to a high toxicity with a human lethal dose of approximately 1 ng
per kilogram body weight. During intoxication, BoNTs specifically recognize receptors on
efferent nerves preceding neuromuscular junctions. In consequence of the binding, the toxin istaken up via endocytosis. Conformational changes caused by alterations of the pH and redox
milieu in the endosome allow the light toxin subunit containing the protease domain to traverse
the endosomal membrane and to simultaneously separate from the receptor bound toxin
subunit. This leads to an activation of the protease, which then starts to cleave specific proteins
involved in the release of the neurotransmitter acetylcholine. The blocked neurotransmitter
release from the efferent nerve causes a flaccid paralysis of the respective muscle. The
combined assay for potency determination of BoNTs which has been developed in ourlaboratory is based on the binding capacity as well as the proteolytic activity of the toxins. As
the medically relevant BoNT serotypes A and B differ in their receptor proteins as well as intheir substrates, two separate combined assays were developed. In these assays, BoNTs arebound to receptor molecules coated on a microtiter plate. As receptor molecules, theganglioside GT1b as well as a receptor peptide (derived from synaptic vesicle protein 2c for
BoNT/A, and synaptotagmin for BoNT/B) are applied. After binding, the protease domain is
separated from the residual molecule by reduction and transferred to a second microtiter plate
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40coated with the substrate for the protease (synaptosomal-associated protein 25 for BoNT/A,
and synaptobrevin for BoNT/B). Finally the cleaved substrate is detected with antibodies
directed against the cleavage site. The development of the assay for the activity determinationof BoNT/B has already been completed. In-house validation studies have shown that the assay
is highly sensitive, with a detection limit below 1 pg/ml. For BoNT/A, assay optimization is
ongoing. In addition to being preferable from an ethical point of view, the combined assay isalso easier to perform, cheaper and faster than the LD50 test.
References[1] Bitz, S. (2010). ALTEX 27, 114-116.
[2] Behrensdorf-Nicol, H. A., Bonifas, U., Kegel. B. et al. (2010). Toxicol In Vitro 24, 988-994.

Migration of Human Neural Crest Cells as Functional Endpoint to Screen for
Developmental Neurotoxicity
J. Nyffeler, X. Dolde, M. Leist.
Doerenkamp-Zbinden Chair for In Vitro Toxicology and Biomedicine, University of Konstanz,
Konstanz, Germany.
Society of Toxicology 2016 Abstract 1171
Score 3 (Assay)
Introduction: Neural crest cells (NCCs) are a transient stem cell population arising at the time ofneurulation. NCCs migrate to various body parts and differentiate into cells of the peripheral
nervous system, melanocytes as well as craniofacial bones and other structures. Therefore,
failure of NCC to migrate can lead to severe developmental defects, so calledneurocristopathies. Aim: We developed an in vitro test system using human NCCs andscreened an 80-compound library of potential environmental toxicants for compounds
interfering with NCC migration.
Methods: NCCs were differentiated from human pluripotent stem cells and cryopreserved until
further use. For the cMINC migration assay, cells were seeded in plates containing silicon
stoppers to create a cell-free circular area. Removal of the stoppers allowed the start ofmigration. After 48 h, the number of viable cells in the circular zone was quantified by highcontent image analysis. The whole library, originally assembled and published by the national
toxicology program of the US (NTP), was tested in three independent biological experiments at
the highest non-cytotoxic concentration (≤ 20 µM). Compounds causing > 20% migration
inhibition were re-ordered for confirmation and concentration-response curves were obtained.
Results: Internal quality controls lead to consistent results and all negative controls werecorrectly classified. The screen identified 26 compounds as potentially migration-inhibiting, of
which 22 were confirmed upon retesting. Among these migration-inhibiting compounds, seven
were organophosphates, five were drug-like compounds, three were organochlorines and threewere polybrominated flame retardants. Moreover, there were three other pesticides, and an
industrial chemical. Conclusions: The cMINC assay yielded stable and reproducible results.
Comparison to data already available on the library compounds from in vivo testing and otherscreens suggested a high sensitivity of our assay. We expect that a battery of related
developmental toxicity tests may be used to generate alerts on potential toxicants, and toprioritize them for further testing.

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Human Relevant Intra-Laboratory Validated In Vitro Vasculo/Angiogenesis Test
T. Heinonen, O. Huttala, T. Toimela.
FICAM, University of Tampere, Tampere, Finland. Sponsor: T. Knudsen.
Society of Toxicology 2016 Abstract 2176
Score 3 (Assay)
Formation of vascular network is a crucial process during embryonic development, and it also
contributes to the pathogenesis of numerous disorders such as cancer and diabetes mellitus.
One of the principal teratogenic mechanisms suspected to be associated with medication isvascular disruption (van Gelder et al.,Therapie 69(1), 13-24. 2014). Vascular network is formed
either by vasculogenesis (in situ differentiation and growth of blood vessels) and angiogenesis(growth of new blood vessels from the preexisting ones). The vasculogenesis/angiogenesis testdeveloped in FICAM utilizes primary human adipose stem cells and primary human umbilicalcord vein endothelial cells. The 3D coculture formed contains 3D endothelial tubule structure(vWF) with lumen, younger and more mature vessels, basement membrane (colIV), adherens
junctions (ve-cadherin, occludin) between endothelia, pericytes (PDGFRb, SMMHc, SMA) andendothelial cells characterized by immunostaining and electron microscopy. Further, thematuration process covering vasculogenesis and angiogenesis was confirmed by measuring
the expression of the key marker genes (e.g. ANGPT1/2, FGF2, VEGF, PDGF, VEGFR1/2,
TIE-2, PDGFRb). The test passed successfully intra-laboratory validation according to theOECD 34 guideline: reproducibility, repeatability and relevance were proven. There was no
statistical difference between the results of different technicians (P=0.44, ANOVA) or different
repeats of the same technician (CV<15%). The relevance to inhibit tubule formation was shownwith drugs with published human data e.g. Erlotinib, Levamisole, 2-methoxyestradiol,acetylsalicylic acid, anti-VEGF, Cladribine, and Doxorubicin resulting in a good concordance(Pearson R=0.6921). This indicates that this human cell based test has potential be used as
predictive in vitro test in assessing vascular disrupting potential of chemical substances in man.
The applicability covers development of vascular-disrupting drugs as well as testing ofchemicals for their potential to disrupt vasculature formation during embryogenesis.

High Density Human Stem Cell Micromass to Model Teratogen-Induced Limb
Malformations
S. Yu, J. Tan, K. Clark, P. G. Alexander, R. Tuan.
Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Sponsor: L. Vernetti.
Society of Toxicology 2016 Abstract 2178
Score 3 (Assay)
Limb malformations are a major feature of exposure to many developmental teratogens.
Embryonic limb mesenchyme high density micromass culture is an excellent system forstudying skeletal tissue development in vitro. We have developed micromass cultures withhuman adult bone marrow-derived mesenchymal stem cells (hBMSC), a more homogenous cell
population, that can undergo chondrogenesis and chondrocyte hypertrophy in a predictable andphysiological manner. In this study, we challenge hBMSC-based micromass with three
toxicants, thalidomide, warfarin and mifepristone, to test the possibility that this hBMSC-based

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42culture may be used to study the effects of teratogenic compounds on limb development.
hBMSCs micromass were induced to undergo chondrogenesis in TGF-β1 containing medium.
On day 10, cultures were either analyzed for chondrogenic differentiation or induced to undergo
hypertrophy by TGF- β1 withdrawal and T3 supplementation. Candidate teratogens were added
throughout the culture period and the effect on chondrogenic and hypertophic markers wereassayed histologically and by qRT-PCR as a function of dose. Control micromasses
demonstrated robust chondrogenesis and hypertrophy, indicated by increased Alcian Blue
staining and alkaline phosphatase (ALP), activity, respectively, and stage-specific gene
expression. Thalidomide did not show any detectable impact on chondrogenesis andhypertrophy at any dose. Warfarin had minimal impact on chondrogenic gene expression but asignificant effect on hypertrophic gene expression, matrix elaboration and ALP activity.
Mifepristone significantly reduced chondrogenic and subsequent hypertrophic gene expression.
The effects of both warfarin and mifepristone occurred at doses near reported maternal serum
levels. The 3 toxicants tested affected hBMSC micromasses in ways related to their mechanismof action. Thalidomide, an inhibitor of angiogenesis, did not show any detectable impact onchondrogenesis or hypertrophy at any dose in this avascular model Warfarin, an inhibitor of
post-translational γ-carboxylation critical for cartilage matrix production, affected mainly
hypertrophy. Mifepristone, a glucocorticoid antagonist, strongly inhibited both chondrogenesis
and hypertrophy. We conclude that high density, 3-dimensional hBMSC-based micromass
cultures represent a reproducible and controlled model to screen for potential limb teratogens.

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7. Conclusions
The review of the most recent EUSAAT, SoT, and SEURAT-1 conference proceedings,
and an extensive literature search, yielded 29 abstracts describing projects which we
believe the Judges should consider as potential candidates for the Lush Science Prizeshortlist. These are given in Section 6.
Once again we have reviewed many more papers and abstracts than in the previous
year (over 3,100 this year compared with around 2,200 in 2015). We also scored more
abstracts this year than last (85 vs 50 in 2015). It was pleasing to find a larger
proportion of high scoring (3 and 4) abstracts this year – 34% - compared with 2015(10%).
The nominated abstracts are very diverse, and cover developing models for new toxicity
assays, improvements in the phenotypic status of human iPSC-hepatocytes, highthroughput- and high content- screening, in silico analyses, and demonstrations of theutility of modern tt21d approaches. We believe that they are all worthy candidates forthe 2016 Science Prize.

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