The LUSH Black Box Prize and the skin sensitisation adverse outcome
pathway
Dr Gill Langley, a science judge on the Lush Prize judging panel, asks whether thePrize judges could be making the first Black Box Award in 2015 for work on skinsensitisation.
March 2015
Introduction
The Lush Black Box Prize offers, in any one year, the full £250,000 Lush prize fund for a
key breakthrough in human toxicity pathways research using 21 st-century techniques. It’s
called the Black Box Prize because 20th-century product safety testing, relying on animal
tests, explains little about whether and how chemicals cause adverse health effects inhumans: it treats how chemicals cause toxic effects as a ‘black box’. The Lush Prize wants
to ‘open the box’ by aiding the development of advanced human-relevant tests that explain
toxic effects, replace the use of animals and improve the science of safety testing.
The aim of the Black Box Prize is to stimulate a worldwide research and training focus onhuman toxicity pathways, with a view to replacing animal tests in toxicology. This would
drive forward the culture change in the world of toxicology, which has already started as aresult of the U.S. National Research Council’s 2007 report [Committee on Toxicity Testingand Assessment of Environmental Agents, National Research Council (2007). Toxicity
Testing in the 21st Century: A Vision and a Strategy]. See Appendix 1 for an explanation of
21st-century toxicology.
What’s a toxicity pathway?
A toxicity pathway is the molecular sequence of events inside the body’s cells that follow
when a toxic chemical first interacts with the body. Toxicity pathways develop when normal
healthy cell pathways are damaged, resulting in changes in gene function, cell signalling orprotein production, and injuring the healthy function of the cells. The eventual result isseen as a toxic effect in the whole human, such as when a chemical carcinogen causes atumour.
Although most toxic effects ultimately involve a complex interplay of different tissues,organs and body systems, toxicity pathway research focuses on the early events at themolecular and cellular levels (see Figure 1, below). The Adverse Outcome Pathway (AOP)
concept is a related approach that looks at the entire chain of events from chemical
exposure through to effects at the level of the whole organism or groups of individuals(populations).

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Figure 1

Flow diagram showing different pathway concepts in skin sensitisation (skinallergy)
Key toxicity pathways are being researched using computational models and humanmolecular, cellular and tissue studies in the test tube. When pathways are understood,
non-animal tests can be developed which identify whether or not chemicals trigger thesepathways and eventually lead to toxic effects.
21st-century toxicology offers dramatic advantages over traditional animal testing: speed,
human relevance, cost-effectiveness, understanding the causes of toxicity, predicting
human variability, testing chemical mixtures, and the replacement of animal testing, whichcauses suffering to many thousands of animals every year.
Why are toxicity pathways so important?
There are expected to be hundreds of toxicity pathways, variously associated with different
tissues and toxic endpoints (e.g. skin, liver, kidney, nervous system) in the body and withdifferent classes of chemicals. A single chemical may activate several toxic pathways, butthere is probably a smaller core of cellular events that will provide the key targets for new
safety tests.
Modern thinking is that new non-animal tests will be best developed on the basis ofpathways.

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The process of pathway elucidation, test development, validation and acceptance typicallytakes many years’ work by many researchers and regulatory experts, and roughly
comprises:
1. Elucidate the pathways causing a particular toxicity
2. Identify the key cellular steps
3. Devise complementary non-animal tests (chemical, computational and/or in vitro) to
detect and measure the key steps
4. Conduct multi-laboratory assessments of all the tests and devise a way ofinterpreting the data
5. Achieve successful validation studies
6. Develop a weight-of-evidence (or integrated testing) strategy that combines thetests in a logical framework allowing decisions to be made about toxicity; or achemical categories-based assessment (grouping chemicals based on both upstream chemical and down-stream biological processes)
7. Achieve regulatory acceptance for the tests (e.g. European Commission, OECD).
Research that could win a Black Box Prize
The Lush Prize website says:“Research is eligible for the Black Box Prize if it fully elucidates and describes a humanadverse outcome pathway, with experimental evidence to demonstrate all the necessary
key steps from the first interaction of one or more chemical molecules to the full effects atthe cellular, tissue or individual level. Research must have led to the development andregulatory acceptance, ideally at OECD level, of one or more non-animal tests that replacesome or all existing animal tests related to this pathway. The research should have been
completed and published within five years prior to the annual award.”
The research should have been completed and published within five years prior to theannual award.”
The skin sensitisation pathway
The toxic endpoint of skin sensitisation (SS) is allergic contact dermatitis. Whensomeone’s skin is repeatedly exposed to an allergenic chemical, the following symptoms
usually follow: a skin rash that can ooze, drain or crust and become raw, scaled or
thickened. Itching, skin redness or inflammation and localised swelling follow, and the skin
may blister. If left untreated, the skin may darken and become leathery and cracked. Oncesomeone has become allergic to a substance they will always react to it.
The first fully explained and described toxicity pathway now exists (see Figure 1 andAppendix 2), for skin sensitisation caused by chemicals via a reaction with skin proteins.
There are other pathways of skin sensitisation (SS) caused by other kinds of allergens(e.g. metals) and knowledge of the underlying biology of some of the key steps for theseother allergens still remains incomplete.
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Work to elucidate the pathways leading to SS has been underway for very many years,
and like most research, progress has come gradually. Chemically induced SS involves
complex mechanisms with many levels of fine control. Several different kinds of cells
interact including the skin and the local lymph nodes of the immune system (see Appendix
3 for diagram and explanation).
The full toxicity pathway and AOP for SS caused by chemicals via a reaction with skinproteins are now sufficiently understood, and non-animal tests are under development orare being assessed for their validity (see Appendix 4).
The SS AOP is summarised as eleven steps with four key steps (see Figure 1 andAppendix 2):
 Key step 1 is the molecular interaction of the chemical with skin proteins Key step 2 is an inflammatory response and change in cell signalling pathways
inside skin cells
 Key step 3 is activation of dendritic cells
 Key step 4 is T-lymphocyte proliferation
It will not be possible to assess all SS with only one non-animal test. Several
complementary tests will be needed, which might target any or all of these steps (andother steps identified as important for different kinds of allergens, in the future). The tests
will also need to provide a measure of how allergenic substances are, i.e. their potency.
But without knowing the toxic pathways, the relevant non-animal tests could not be
developed with any confidence in their reliability.

Significance of the skin sensitisation pathway
Since the Lush Prizes were launched in 2012, the SS pathway caused by chemicals via areaction with skin proteins is the first to be elucidated. Understanding the first SS pathway
is a highly significant achievement, for these reasons:
1. SS isn’t a simple ‘topical’ toxicity endpoint (like skin irritation) – it involves asystemic response because the skin and the immune system play a part.
2. ‘Opening the black box’ for SS sets a vitally important precedent by showing that
discovering human toxicity pathways for other, more complex toxic endpoints reallyis achievable – as envisaged by the US National Research Council in 2007.
3. The achievability of understanding toxicity pathways is shown by the fact that theOECD has set up an international AOP Development Programme.
4. This signals that replacing animal tests for all the other endpoints using toxicity
pathways is practical, and in this sense does indicate the beginning of the end foranimal tests.
5. At present there are no validated non-animal tests for SS although several arebeing developed (see Appendix 4). Tests on mice (the Local Lymph Node Assays)
and guinea pigs (the Guinea Pig Maximisation Test and the Buehler Test) are theLush Black Box Prize Background Paper – April 20154

only accepted methods. Thousands of these animal tests are conducted every year
(nearly 1,000 in Britain alone in 2013).
6. SS is a significant human health problem caused not only by cosmetics andfragrances, but also by a wider range of chemicals, metals and drugs. The
understanding gained in research into the first SS pathway will help understandingof other SS pathways – and therefore the faster replacement of animal tests.
Should a Black Box Prize be awarded for the SS pathway?
According to the Lush Prize policy (see Research that could win a Black Box Prize,
above), the SS adverse outcome pathway (AOP) appears to be eligible for a Black Box
Prize,and includes kep steps at the system and organ levels, not just the cellular level (seeFigure 1 and Appendix 2).
It is difficult to pinpoint the date of publication of the complete pathway, because so many
researchers were involved in a long developmental process. To win the Black Box Prize for2015 the research should have been published not earlier than 2010.
Certainly, the OECD published a report in 2012 [The Adverse Outcome Pathway for Skin
Sensitisation Initiated by Covalent Binding to Proteins, Part 1: Scientific Evidence[www.oecd.org/officialdocuments/displaydocumentpdf/ ?
cote=env/jm/mono(2012)10/part1&doclanguage=en] which concluded that the steps in thepathway are sufficiently well understood to devise appropriate non-animal tests for SS.
The OECD is the major player in producing test guidelines for chemical testing, agreed
internationally.
This could be considered as an official “approval” of the value of the SS pathway. The
OECD has also accepted the AOP concept as a framework for human health toxicology
within its AOP Development Programme.
Lush Prize welcomes comment and debate around this issue. Please use the contact us
link at the bottom of all our web pages.

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APPENDIX 1
What is 21st century toxicology?
21st century toxicology is so called because it applies new and emerging techniques andmodels developed in this century, such as genomics, proteomics and the other ’omics,
three-dimensional human tissue models, modern analytical microscopy, mathematicalmodelling and organs-on-chips. Computational systems are used to integrate and interpret
data from human test-tube studies to create understanding of complex biologicalprocesses at the level of the whole human.

‘Organs-on-chips’ can be used to study human cells
working in concert with each other as in real life

A transformation in toxicology has been unfolding since the publication in 2007 of the U.S.
National Research Council’s report which recommended a “21 st century paradigm” forsafety testing, involving a radical move away from animal tests towards a completely new
framework based on understanding toxicity pathways within human cells.

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APPENDIX 2
The 2012 OECD Summary of the AOP for skin sensitisation caused by chemicals
binding to skin proteins(Steps 1 – 6 comprise the toxicity pathway contained within cells, and 7 – 11 are part of
the AOP with organ-and system-level steps)
Knowledge of the AOP for skin sensitisation elicited by covalent binding of
substances to proteins has evolved rapidly over the past decade and may besummarized as:
Step 1) The target substance must be bioavailable (i.e. it must penetrate the stratum
corneum of the skin).
Step 2) The target substance must be a direct-acting electrophile, be converted from a
non-reactive substance (pro-electrophile) to a reactive metabolite via metabolism, or beconverted from a nonreactive substance (pre-electrophile) to a reactive derivative via an
abiotic process, typically oxidation.
Step 3) The molecular sites of action are targeted nucleophilic sites in proteins (e.g.
cysteine and lysine residues) in the epidermis.
Step 4) The molecular initiating event is the covalent perturbation of dermal proteins,which is irreversible (i.e. formation of the hapten-protein complex or complete antigen). In
vivo, this event is associated with the production of a specific memory T-cell response.
Step 5) Biochemical pathways affected by the definitive electrophile’s action on themolecular targets are incompletely known but often include inflammation-related pathways,
including the mitogen-activated protein kinase signalling pathway and the oxidative stressresponse pathway, especially in keratinocytes and dendritic cells.
Step 6) The cellular/tissue-level outcomes are incompletely known but include epidermal
responses such as: 1) immune recognition of chemical allergens by keratinocytes,
specialized epidermal dendritic cells (i.e. Langerhans cells) and dermal dendritic cell; 2)
responses in the form of expression of specific cell surface markers, such as adhesion
molecules, chemokines, and cytokines such as IL1β or IL-12p70 are typically taken as
evidence of dendritic cell maturation.
Step 7) The organ-level responses include:a) Dendritic cell migration to the lymph node, where they present major histocompatibility
complex (MHC) molecules to naive T-lymphocytes (T-cells), andb) T-cell differentiation and proliferation as allergen-specific memory T-cells.
Step 8) The target organ(s) are the skin and local lymph nodes; the target cell populations
are the immune cells, especially effector T-cells.
Step 9) The key physiological response is acquisition of sensitivity.
Step 10) The key organism response is dermal inflammation upon receiving the substancechallenge in the elicitation phase. This response is associated with stimulation of specificmemory T-cell produced in the induction phase.
Step 11) The overall effect on mammals is allergic contact dermatitis in humans, or its
rodent equivalent contact hypersensitivity.

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APPENDIX 3
Brief summary of the skin sensitisation reaction
The process is sequential. First is sensitisation (or induction) when first exposed to achemical, then elicitation of the skin condition which reacts on repeat exposure to thechemical. The skin is exposed to an allergy-causing chemical (or metabolises a chemical
to a reactive form) and dendritic cells in the skin are activated. These take the chemicaland present it to the T-lymphocyte cells in the nearest lymph nodes.
These T-cells then multiply so that if the chemical allergen is experienced again by theindividual, these T-cells will respond more quickly and more aggressively. The reaction
they create by releasing cytokines (which cause inflammation) in the skin leads to allergic
contact dermatitis.

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APPENDIX 4
Progress with non-animal tests for skin sensitisation
A number of non-animal tests are under consideration for assessing the SS hazards ofchemicals, but none has yet been fully approved by regulatory agencies. Some of the tests
have been worked on for 10 years, but until the understanding of the SS toxicity
pathway/AOP, it was difficult to prove that any or all of them were reliable and relevant inpredicting chemical toxicity for humans.
The formal concept of 21st-century pathway-based toxicology has only existed since 2007.
Enormous progress has been made since then. For example, most of the evidence
indicating that the Nrf2-Keap1-ARE regulatory pathway is a toxicity pathway for SS was
accumulated between 2007 and 2009. The Direct Peptide Reactivity Assay (DPRA), a
chemical test, was first developed in 2007 and has gained much credibility in recent years.
With understanding of the SS pathways, and with subsequent pathway elucidation forother toxic endpoints, non-animal test methods are progressing faster and will gain theconfidence of scientists and regulators more quickly.
Non-animal tests currently under discussion at, and recently approved by, the OECD(considered the ultimate stamp of approval) include:The Direct Peptide Reactivity Assay (DPRA) is now an official OECD test guideine
the OECD (since 2012) as a new OECD test guideline. It is based on key steps 3
and 4 of the skin sensitisation AOP and quantifies the reactivity of chemicals tosynthetic peptides.
The KeratinoSens (or in vitro ARE-Nrf2 Luciferase) test is based on the second key
step in the AOP. The OECD has drafted a new test guideline for this test.
There is also a draft proposal for a new test guideline on in vitro skin sensitisation
testing using the human Cell Line Activation Test (h-CLAT). The test detects thethird key step (dendritic cell activation) of the skin sensitisation AOP.

Yet other tests are under development as this is an active research field, thanks to thestimulus provided by acceptance of the 21st-century toxicology concept.

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