Fish replacement: Current status minimising the use of fish in tests

Dr Stewart Owen

Current trends in fish in vitro toxicology: Applications of 3Rs principles SETAC Brussels.

10th May, 2017

Peddling an alternative path

Standing on giant shoulders…

Prof Awadhesh Jha

Dr Matt Baron

Dr Richard Maunder

Dr Laura Langan

Prof John Sumpter

Dr Luigi Margiotta-Casaluci

Prof Mariann Rand-Weaver

Dr Nic Bury, Prof Christer Hogstrand

Dr Leon Barron, Dr Tom Miller

Dr Lucy Stott, Elisabeth Chang

Prof Charles Tyler

Dr Lina Gunnarsson

Dr Jon Green

Prof Kevin Chipman

Dr Chibuzor Uchea

And many more……

Dr Tim Martin

Dr Russell Davenport

Dr Andrew Goodhead

Prof Alistair Boxall

Dr Philipp Antczak

How could you use fewer fish?

A few approaches that we have been trying:

Build a virtual fish

In vitro alternatives – Gills, Guts and Livers

Build a fish that isn’t really a fish

In vivo early life stages

Build a prediction

Get it right first time with qAOP predictions

What about: Could we save fish in standard designs?

What is the point of a freshwater and solvent control?

Other than EDC’s

Get the biodegradation assays right

Prof Jason Snape (AZ)

Newcastle University

Dr Tim Martin

Dr Russell Davenport

Dr Andrew Goodhead

80% of chemicals and 95% of APIs

currently fail the key biodegradation

screening tests (OECD 301 series tests)

used to assign chemical persistence

Climb the mountain…

Highest Priority

Lowest Priority

Focus Effort

RQ = 0.000001

RQ = 1

Could we have a reduced fish strategy?

Do we need fish at all?

Leon Barron, Tom Miller

Many are looking…

Find a route to the top…

Pharmacology is equivalent:

fish plasma concentration within

pharmacological range

Validation of PECs and in vivo

plasma concentrations and effects in fish

No Risk

Risk prioritisation

In vitro fish

metabolism models

Bio-

informatic

read-across

Fish plasma

concentration

Target

conservation in fish

Human and

mammalian data

Pharmaceutical

s enter fish

In silico

uptake models

In vitro

gill cell uptake

models

Below human Cmax No Risk

Dilution &

%

connectivit

y

Average

amount

of API per

capita per day

PECExcreta

PECInfluent

PECEffluent

PECSurface-water

% metabolism &

metabolite

identification

Dilution & %

connectivity

Dilution, mixing and

degradation (½ life)

Partitioning (Kd)

and degradation (½

life)

PECSediment

PECSludge PECSoil

PECGroundwater

Partitioning (Kd)

and degradation (½

life)

Partitioning (Kd),

leaching &

degradation (½ life)

Applicatio

n rate and

mixing

depth

MECSediment MECGroundwater

MECSoil

MECSurfacewater

Pharmacology is equivalent:fish plasma concentration within

pharmacological range

Validation of PECs and in vivoplasma concentrations and effects in fish

Low Risk

Risk prioritisation

In vitro fish metabolism models

Bio-informaticread-across

Fish plasmaconcentration

Targetconservation in fish

Human andmammalian data

Pharmaceuticals enter fish

In silicouptake models

In vitrogill cell uptake

models

Below human CmaxLow Risk

Dilution & % connectivity

Average amount

of API per

capita per day

PECExcreta

PECInfluent

PECEffluent

PECSurface-water

% metabolism & metabolite identification

Dilution & % connectivity

Dilution, mixing anddegradation (½ life)

Partitioning (Kd)and degradation (½ life)

PECSediment

PECSludgePECSoil

PECGroundwater

Partitioning (Kd)and degradation (½ life)

Partitioning (Kd), leaching &

degradation (½ life)

Application rate and

mixing

depth

MECSedimentMECGroundwater

MECSoil

MECSurfacewater

Do the tools exist?

The Virtual Fish - In Vitro

Nature Protocols 11 (3), 490-498.

Functional gills

Uptake

The Virtual Fish - In Vitro

Gut uptake model

Laura Langan (Plymouth)

The Virtual Fish - In Vitro

3D tissue culture

Build a fish that isn’t a fish

Greenfish Environ. Sci. Technol., 2016, 50 (12), pp 6536–6545

18

Any more clear for projector?

Build a prediction

TR128: Guidance on assessment and application of Adverse

Outcome Pathways (AOPs) relevant to the Endocrine System

ISSN-2079-1526-128 (online)

ECETOC 2016

Can we use AOPs?

Can an AOP be predictive?

Can an AOP be quantitative?

Can a Path be a Network?

Quantitative AOP might be predictive

Margiotta-Casaluci L., et al. (2016). Internal exposure dynamics drive the

Adverse Outcome Pathways of synthetic glucocorticoids in fish. Nature

Scientific Reports 6: 21978.

How do you use fewer fish?

Could you try:

Build a virtual fish

In vitro alternatives – Gills, Guts and Livers

Build a fish that isn’t really a fish

In vivo early life stages

Build a prediction

Get it right first time with qAOP predictions

What about:

Could we save fish in standard designs?

What is the point of a freshwater and solvent control?

Fin

Confidentiality Notice

This file is private and may contain confidential and proprietary information. If you have received this file in error, please notify us and remove

it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the

contents of this file is not permitted and may be unlawful. AstraZeneca PLC, 1 Francis Crick Avenue, Cambridge Biomedical Campus,

Cambridge, CB2 0AA, UK, T: +44(0)203 749 5000, www.astrazeneca.com

24

Thank you:

To all our collaborators, colleagues and funding partners.

BBSRC/AstraZeneca IPA (BB/I00646X/1) ‘Mode-Of-Action Ecotoxicology of

Pharmaceuticals’. PIs Prof John Sumpter and Dr Mariann Rand-Weaver. Dr Luigi

Margiotta-Casaluci. July 2011-June 2014. Brunel University.

BBSRC/AstraZeneca IPA (BB/L01016X/1) ‘Moving up a dimension: 3D in vitro models

as effective alternatives to live fish studies’. PIs Prof Awadhesh Jha and Prof Simon

Jackson. Dr Matthew Baron and Dr Richard Maunder March 2014-Feb 2017.

Plymouth University.

Innovative Medicines Initiative (IMI) under grant agreement no.115735—iPiE: Intelligent

led assessment of Pharmaceuticals in the Environment; resources of which are

composed of financial contribution from the European Union's Seventh Framework

Programme (FP7/2015-2018) and European Federation of Pharmaceutical Industries

and Associations (EFPIA) companies' in kind contribution

Refs

• Baron M.G., Mintram K.S., Owen S.F., Hetheridge M.J., Moody J.A., Purcell

W.M., Jackson S.K., Jha J.N. (2017). Pharmaceutical metabolism in fish: using

a 3-D hepatic in vitro model to assess clearance. PLoS one 12(1), e0168837.

doi:10.1371/journal.pone.0168837

• Patel A., Panter G.H., Trollope H.T., Glennon Y.C., Owen S.F., Sumpter J.P.,

Rand-Weaver M. (2016). Testing the “read-across hypothesis” by investigating

the effects of ibuprofen on fish. Chemosphere 163, 592-600

• Miller T.H., Baz-Lomba J.A., Harman C., Reid M.J., Owen S.F., Bury N.R.,

Thomas K.V., Barron L.P. (2016). The first attempt at non-linear in silico

prediction of sampling rates for polar organic chemical integrative samplers

(POCIS). Environmental Science & Technology 50 (15), 7973-7981. [Editors

choice award and cover of issue]

• Margiotta-Casaluci L., Owen S.F., Huerta B., Rodríguez-Mozaz S., Kugathas

S., Barceló D., Rand-Weaver M., Sumpter J.P. (2016). Internal exposure

dynamics drive the Adverse Outcome Pathways of synthetic glucocorticoids in

fish. Nature Scientific Reports 6: 21978.

• McCarthy I.D., Owen S.F., Watt P.W., Houlihan D.F. (2016). Individuals

maintain similar rates of protein synthesis over time on the same plane of

nutrition under controlled environmental conditions. PloS one 11 (3),

e0152239.

• Langan L.M., Dodd N.J.F., Owen S.F., Purcell W.M., Jackson S.K., Jha A.N.

(2016). Direct measurements of oxygen in the middle of 3D tissue cultures

using electron paramagnetic resonance spectroscopy. PloS one 11 (2),

e0149492.

• Green J.M., Metz J., Lee O., Trznadel M., Takesono A., Brown A.R., Owen

S.F., Kudoh T., Tyler C.R. (2016). High-Content and Semi-Automated

Quantification of Responses to Estrogenic Chemicals Using a Novel

Translucent Transgenic Zebrafish. Environmental Science & Technology 50

(12), 6536–6545.

• Schnell, S., Stott, S.C., Hogstrand, C., Wood, C.M., Kelly, S.P., Pärt, P., Owen,

S.F., & Bury N.R. (2016). Procedures for the reconstruction, primary culture

and experimental use of rainbow trout gill epithelia. Nature Protocols 11 (3),

490-498.

• Miller T.H., McEneff G.L., Stott L.C., Owen S.F., Bury N.R., & Barron L.P.

(2016). Assessing the reliability of uptake and elimination kinetics modelling

approaches for estimating bioconcentration factors in the freshwater

invertebrate, Gammarus pulex. Science of The Total Environment 547, 396-

404.

• Brown A.R., Owen S.F., Peters J., Zhang Y., Soffker M., Paull G.C., Hosken

D.J., Wahab M.A., Tyler C.R. (2015). Climate change and pollution speed

declines in zebrafish populations. Proceedings of the National Academy of

Sciences 112 (11), E1237-E1246.

• Uchea C., Owen S.F., Chipman K. (2015). Functional xenobiotic metabolism

and efflux transporters in trout hepatocyte spheroid cultures. Toxicology

Research 4 (2), 494-507.

• Miller T.H., McEneff G.L., Brown R.J., Owen S.F., Bury N.R., Barron L.P.

(2015). Pharmaceuticals in the freshwater invertebrate, Gammarus pulex,

determined using pulverised liquid extraction, solid phase extraction and liquid

chromatography–tandem mass spectrometry. Science of The Total

Environment 511, 153-160.

• Stott L.C., Schnell S., Hogstrand C., Owen S.F., Bury N.R. (2015). A primary

fish gill cell culture model to assess pharmaceutical uptake and efflux:

Evidence for passive and facilitated transport. Aquatic Toxicology 159, 127–

137.

• Corcoran J., Winter M.J., Lange A., Cumming R., Owen S.F, Tyler C.R. (2015).

Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene

transcript levels in common carp (Cyprinus carpio) at pharmacological and

environmental exposure levels. Aquatic Toxicology 161, 127-137.

• Margiotta-Casaluci L., Owen S.F., Cumming R.I., de Polo A., Winter M.J.,

Panter G.H., Rand-Weaver M., Sumpter J.P. (2014). Quantitative cross-species

extrapolation between humans and fish: The case of the anti-depressant

fluoxetine. PloS one 9 (10), e110467.

• Parker T., Libourel A-P., Hetheridge M.J., Cumming R.I., Sutcliffe T.P.,

Goonesinghe A.C., Ball J.S., Owen S.F., Chomis Y., Winter M.J., (2014). A

multi-endpoint in vivo larval zebrafish (Danio rerio) model for integrated

cardiovascular functional assessment. Journal of Pharmacological and

Toxicological Methods 69(1):30-38.

• Corcoran J., Lange A., Cumming R., Owen S.F., Ball J.S., Tyler C.R., Winter

M.J. (2014). Bioavailability of the imidazole antifungal agent clotrimazole and

its effects on key biotransformation genes in the common carp (Cyprinus

carpio). Aquatic Toxicology 152, 57-65.

• Rand-Weaver M., Margiotta-Casaluci L., Patel A., Panter G.H., Owen S.F.,

Sumpter J. (2013). The Read-Across Hypothesis and Environmental Risk

Assessment of Pharmaceuticals. Environmental Science and Technology 47

(20), 11384–11395.

• Readman G.D., Owen S.F., Murrell J.C., Knowles T.G. (2013). Do fish perceive

anaesthetics as aversive? PLoS ONE 8(9): e73773.

• Uchea C., Sarda S., Schulz-Utermoehl T., Owen S., Chipman, K. (2013). In

vitro models of xenobiotic metabolism in trout for use in environmental

bioaccumulation studies. Xenobiotica. 43(5), 421-431.

• Bickley L.K., Brown A.R., Hosken D.J., Hamilton P.B., Le Page G., Paull G.C.,

Owen S.F., and Tyler C.R. (2012). Interactive effects of inbreeding and

endocrine disruption on reproduction in a model laboratory fish. Evolutionary

Applications. 6(2): 279-289.

• Baron M.G., Purcell W.M., Jackson S.K., Owen S.F., Jha A.N. (2012). Towards

a more representative in vitro method for fish ecotoxicology: morphological and

biochemical characterisation of three-dimensional spheroidal hepatocytes.

Ecotoxicology 21(8):2419-2429.

• Wilkes, L., Owen, S.F., Readman, G.D., Sloman, K.A. and Wilson, R.W. (2012).

Does structural enrichment for toxicology studies improve zebrafish welfare?

Applied Animal Behaviour Science 139(1): 143-150.

• Brown A.R., Bickley L.K., Ryan T.A., Paull G.C., Hamilton P.B., Owen S.F.,

Sharpe A.D. and Tyler C.R. (2012). Differences in sexual development in

inbred and outbred zebrafish (Danio rerio) and implications for chemical

testing. Aquatic Toxicology. 112–113: 27–38.

• Bartram, A.E., Huggett, D.B., Hutchinson, T.H., Hetheridge, M.J., Kinter, L.B.,

Ericson, J.F., Constantine, L.B., Sumpter, J.P. Owen, S.F. (2012). Liver and gill

EROD activity in rainbow trout (Oncorhynchus mykiss) exposed to the beta-

blocker Propranolol in vivo and in vitro. Environmental Toxicology 27(10): 573–

582.

• Brown, A.R., Bickley, L.K., Le Page, G., Hosken, D.J., Paull, G.C., Hamilton,

P.B., Owen, S.F., Robinson, J., Sharpe, A.D., Tyler, C.R. (2011). Are

toxicological responses in laboratory (inbred) Zebrafish representative of those

in outbred (wild) populations? A case study with an endocrine disrupting

chemical. Environmental Science and Technology 45: 4166–4172.

• Owen, S.F., Huggett, D.B., Hutchinson, T.H., Hetheridge, M.J., Kinter, L.B.,

Ericson, J.F., Constantine, L.B. and Sumpter, J.P. (2010). The value of

repeating studies and multiple controls: replicated 28‐day growth studies of

rainbow trout exposed to clofibric acid. Environmental Toxicology and

Chemistry 29(12): 2831–2839.

• Winter M.J., Owen S.F., Murray-Smith R., Panter G.H., Hetheridge M.J, Kinter,

L.B. (2010). Using preclinical data to aid the aquatic Environmental Risk

Assessment of human pharmaceuticals: concepts, considerations and

challenges. Integrated Environmental Assessment and Management 6(1): 38–

51.

• Brown, A.R., Hosken, D.J., Balloux, F., Bickley, L.K., LePage, G., Owen, S.F.,

Hetheridge, M.J., Tyler, C.R. (2009) Genetic variation, inbreeding and chemical

exposure - combined effects in wildlife and critical considerations for

ecotoxicology. Philosophical Transactions of the Royal Society B 364: 3377-

3390.

• Owen S.F., Huggett D.B., Hutchinson T.H., Hetheridge M.J., Kinter L.B.,

Ericson J.F., Sumpter J.P. (2009) Uptake of propranolol, a cardiovascular

pharmaceutical, from water into fish plasma and its effects on growth and

organ biometry. Aquatic Toxicology 93(4): 217-224.

• Williams T.D., Readman G.D., Owen S.F. (2009). Key issues concerning

environmental enrichment for laboratory-held fish species. Laboratory Animals

43:1-14.

• Winter M.J., Redfern W.S., Owen S.F., Valentin J-P., Hutchinson T.H. (2008).

Validation of a larval zebrafish locomotor assay for assessing the seizure

liability of early-stage development drugs. Journal of Pharmacological and

Toxicological Methods 57(3): 176 –187 [Most cited article in journal]

• Owen, S.F., Giltrow, E., Huggett, D.B., Hutchinson, T.H., Saye, J.-A, Winter,

M.J., and Sumpter, J.P. (2007). Comparative physiology, pharmacology and

toxicology of β-blockers: mammals versus fish. Aquatic Toxicology 82: 145–

162.