Thomas Hartung Doerenkamp-Zbinden Professor and Chair for Evidence-based Toxicology,

Director, Center for Alternatives to Animal Testing (CAAT) Joint appointment: Molecular Microbiology and Immunology

Johns Hopkins University, Baltimore, US

Professor of Pharmacology and Toxicology, University of Konstanz, Germany

Medical device testing in vitro - pyrogenicity testing and beyond -

Promoting for 32 years alternatives to animal testing where they are not fit for purpose

The Bernice Barbour Foundation

About 1/3 funding each from industry, philanthropy and research funding

…and individuals

Examples for medical devices!

Biocompatibility Testing -  Material (solid, contact?) vs.

wash solution (diluted) vs. eluate (long-term, ill defined)

-  Material vs. contaminant vs. degradation product vs. mixture

-  In vivo: Confounder surgery -  In vitro: Problem cell contact,

differentiation -  In silico: Problem too few

positive materials, data avail.

-  3rd dimension: hazard – dose / duration – shape / surface

http://en.wikipedia.org/wiki/Biocompatibility

Involved from about 2000 to 2004 (animal welfare and pyrogen group), then replaced by Albrecht Wendel for pyrogen testing

Biocompatibility!•  Incompatible: Release of substances in toxic

concentrations or of antigens

•  Biotolerant: Release of substances in non-toxic concentrations

•  Bioinert: No release of toxic substances

•  Bioactive: positive interaction leading to reaction of tissue

Effect of Contaminants?!

Medical device testing!•  Material vs. contaminant!•  Surgery vs. material!•  Surface effects!•  Material effects!•  Long-term effects!•  Immune reactions!•  Shape-dependent!•  Batch-dependent!

Pyrogen?

cell associated immune receptors

TLRs

NODs

S.aureus

PAMPs

Pyrogen contamination

killing of bacteria release of inflammatory

mediators

web

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1013 own cells versus 1014 bacteria per person the gut of one person contains 1-2kg of bacteria = 50g of Gram-negative endotoxin = enough to kill one million people by injection = enough to induce fever in one billion people

Pyrogen (bacterial and fungal contamination) testing still consumes more than 300.000 rabbits per year

Limulus assay is restricted to Gram-negative endotoxin, disturbed by many substances and does not reflect human pyrogen potency

1995 Whole blood pyrogen test 2003 and 2004 Validated 2006 Validity statement 2009 Accepted Eur. Pharmacopoeia and with limitations by ICCVAM / FDA PyroDetect (Biotest, now Merck/Millipore)

ALTEX 2013, 30:169-208

Submitted to FDA / ICCVAM

Endotoxin determination on PTFE filters

0.0 50.0 500.0 0

1

2

with filter

without filter

LPS (E. coli O-113) pg/ml

OD

(450

nm

)

TEFLON FILTER (5 µm)

Lessons learned •  alternative methods can outperform animal tests •  importance of commercialization •  15 years from development to acceptance •  Importance International harmonization •  lack of enforcement of implementation •  enabling technology •  importance of public-private partnership

Patient safety: non-endotoxin pyrogens New expression systems in gene technology (Gram-positive bacteria, insect and fungal cells) Medical devices Cellular therapies Environmental pyrogens

New challenges

Risk of Pyrogenic Contamination

short-term

pyrogenic reactions •  inflammatory reactions

long-term

example hemodialysis •  immunodeficiency •  amyloidosis •  erythropoietin response ↓ •  atherosclerosis

contact with tissue / blood consequences

Impairment of the patient reduced biocompatibility

Pyrogen detection on biocompatible materials!

(contaminated)

Biocompatible Material

Rinsing solution

Limulus-Assay

Rabbit Assay

Rinsing solution/ Implant

Blood Cytokine, e.g. IL-1β

ELISA (MAT)

Performance of the MAT for Devices

PERFUSION

STATIC CONTACT IL-1

IL-1

Contamination of steel by manual handling

control A B C D ABCD pooled

1

10

100

1000

10000

donor

IL-1

ß (p

g/m

l)

IL-1β

Static Contact

15-well steel block

Endotoxin response on different materials tested in the 15well steel block

0 12,5 25 50 250 500 1

10

100

1000

10000

100000 polystyrol stainless steel elastomer PTFE

n.d. n.d. n.d.

LPS E.coli O 113 [pg/ml]

IL-1

β [p

g/m

l]

n.d.

Elimination of pyrogenic residuals on titanium plates contaminated by manual handling

1

10

100

1000

10000

100000

IL-1

ß (p

g/m

l)

Test of alginate microcapsules in the whole blood pyrogen test

+ LPS spike [50pg/ml] - LPS

EXTRACT

A l g i n a t e control

O D

[ 4 5 0 n

m ]

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

D E

MICROCAPSULE

A l g i n a t e control

O D

[ 4 5 0 n

m ]

0

1

2

3

D E

NaCl

NaCl

Spider Silk

Spidersilk IL-1β

0

500

1000

1500

2000

2500

pg/ml O113

pg/m

l IL-

1 β

forum Nr.2, August 1999, Greiner Labortechnik

Abb. 2 Abb. 3

32,4 nm 20,6 nm

Abb. 1

LPS is only partially removed

Atomic Force Microscopy of Polypropylen surface LPS Spike (500 ng/ml) and washing

Average peak hight

43,4 nm

Spike noSpike washed

Neurosurgical aneurysm clips

con 0,5 1 2 5 16 240

1000

2000

3000LPS [2 µg/well]LTA [2 µg/well]

coat-incubation time [h]solublestimuli

TNF-α

[pg/

ml]

Presentation of LTA on a surface increases 1000fold its inflammatory potency

Deininger, Immunobiol. 2008

Only 1% of LTA binds to surface

Surface bound LTA is more potent!

Conclusions pyrogen testing!

•  Current tests do not adequately control pyrogen contamination of medical devices

•  Testing on surface is a paradigm shift

•  Human relevant testing is possible

Patents Method for assaying flowing media for microbial toxins EP 02 729 818.9 submitted 25.03.2002, under evaluation US 10/474 694 submitted 10.10.2003, under evaluation JP 2002-581 998 submitted 04.12.2003, granted 10.04.2009 Test procedure with biological system EP 0 741 294 B1 submitted 24.04.1996, granted 05.11.2003 (countries: AT, BE, CH/LI, DE, ES, FR, GB, IT, NL, SE) USP 5 891 728 submitted 02.05.1996, granted 06.04.1999 JPP 3 667 439 submitted 07.05.1996, granted 15.04.2005 Test for determining pyrogenic effect of a material EP 0 851 231 B1 granted 15.12.1997 (countries: AT, BE, CH/LI, DE, ES, FR, GB, IT, NL, SE) US CIP 10/761,237 submitted 23.12.1997 (under evaluation) JP 9-354572 submitted 24.12.1997, granted 04.04.2008

Perspectives for other alternative methods

•  Skin assays (mucosal models?)

•  Genotoxicity •  Sensitization •  Cytotoxicity •  Cell Transformation •  Immunotoxicity

Hartung T and Sabbioni E. Alternative in vitro assays in nanomaterial toxicology.

WIREs Nanomed. Nanobiotechnol. 2011, DOI: 10.1002/wnan.153

Nanoparticles testing – medical device testing on speed?

New hazards from nano?!•  No altered positive effects without

altered negative effects •  Particulate matter is e.g. associated

with arteriosclerosis

•  Inhalation toxicology •  Reactive chemistry goes with surface •  Changed kinetics

Altered kinetics is enough to cause new effects!!!

Cytotoxicity induced by Co-sol and Co-nano in Balb/3T3 cell line at 4h exposure

IC50

Cosol 41µM

Conano 9µM

Cytotoxicity 4h

0

20

40

60

80

100

1 10 100 1000

Concentration (µM)

CFE

(%)

Co-nanoparticlesCoCl2

Ponti et al., Mutagenesis 2009

Co-nano in H2O (SEM

microphotograph)

We have to expect compared to parent compound: -  Altered solubility -  Altered hazard profile -  Altered kinetics

Each and every NP is an individual !!!

These can affect in vitro and in vivo in different ways -  Extrapolation to humans? -  Classical risk factors? -  In vivo extrapolation? -  Low dose extrapolation?

ZrO2-nano in H2O (SEM microphotograph)

Expected suitability of (validated) alternatives (none really evaluated) -  Very useful:

refinement/reduction alternatives pyrogenicity

-  Useful: skin irritation, corrosion, eye irritation, cytotoxicity, genotoxicity, cell transformation

-  Possibly useful: skin absorption, embryotoxicity, myelotoxicity

Workshop in Baltimore, October 2010 ALTEX 2011, 28, 236-241

!

!

Conclusions for nanotox -  The advent of nanotech amplifies

the problems of toxicology (amount of testing, new endpoints, complex kinetics…)

-  We must take advantage of experiences from validation of in vitro tests

-  Tox-21c and ITS approaches should be considered

-  We need quality assurance beyond formal validation (e.g. EBT)

Frontloading of toxicology

“fail early, fail cheap”

Anticipate human or regulatory problems?

“test early, develop clean”

Green Toxicology

InfoDay Green Toxicology Baltimore, 22 Nov 2013 Designing more biocompatible materials from start?

Role Model REACH?!

Endpoint  Informa.on:  Annexes  VI-­‐XI  

(Q)SARs  Read  Across  

In-­‐vitro  

Waiving:  •   technical  •   exposure  

Exis.ng  informa.on  

TESTING  

Last resort

model  

modeling  

model  

model  

HTS  data  genera.on  

All models are wrong, some are useful. George Box

Validation

Sure, animal tests are one-eye blind…

l  Humans are not 70 kg-rats… l  “One suit fits all”-models:

tests can only be either sensitive or specific

l  Statistically underpowered l  Too many endpoints without

statistical correction for multiple testing

l  …

Limitations of in vitro models l Cell identity l Mycoplasma l Dedifferentiation favored by

growth conditions and cell selection

l Cells are bored to death l Lack of oxygen l Tumor origin of many cells l Lack of metabolism and defense l Unknown fate of test

compounds in culture

Tox-20c

Tox-21c EBT

Omics, high-content, HTS Bio-informatics & -engineering

Pathways of Tox (PoT) Human Toxome

Integrated Testing Strategies ITS

Organo-typic cultures Human-on- Chip

http://en.wikipedia.org/wiki/Organ-on-a-chip

Human on Chip Approach •  Prompted by DoD need for

medical countermeasure evaluation

•  Could overcome many test shortcomings, especially using stem cells

•  NAS panel •  $200 million program •  CAAT InfoDay 2012 •  CAAT workshop 3D in 2012 •  10 May 2013 conference FDA /

NIH / DARPA / CAAT •  GCCP for iPSC workshop to come

•  Interim decision points •  Probabilistic / Bayesian approaches •  Modeling and Machine Learning Commisioned white paper: Jaworska, J., and S. Hoffmann. 2010. Integrated Testing Strategy (ITS) - Opportunities to better use existing data and guide future testing in toxicology. ALTEX 27: 231–242.

The future of ITS

Bayesian network ITS for skin sensitization

CAAT workshop Jul 2013

An atmosphere of departure in toxicology

New technologies from biotech and (bio-)informatics revolution

Mapping of pathways of toxicity (PoT)

NAS vision report Tox-21c

“We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments” F. Collins, NIH, 2008

“With an advanced field of regulatory science, new

tools, including functional genomics, proteomics, metabolomics, high- throughput screening, and

systems biology, we can replace current toxicology assays with tests that incorporate the mechanistic underpinnings of disease and of underlying toxic side effects.” M.A. Hamburg, FDA 2011

Initiatives implementing Tox-21c

Organiza.on Approach Purpose Outcome

US  EPA  &  Tox21    (ToxCast  Program)

High-­‐throughput  tes.ng

Chemical  priori.za.on  (ini.ally)

“Biological  signatures”    

Hamner  Ins.tute Case  studies  “Just  do  it”   Proof-­‐of-­‐principle

NIH  project  (CAAT-­‐US) Pathway  mapping Pathway  ID  &  

annota.on Human  Toxome

Mapping the Human Toxome by Systems Toxicology

Hewitt et al., 2005. Science, 307:1572-1573

Endocrine disruption •  Use “omics” to map PoT for endocrine disruption

•  Develop software tools

•  Identify PoT

•  Develop a process for PoT annotation, validation

•  Establish public database on PoT.

PoToMaC - The Pathways of Toxicty Mapping Center Transformative Research Grant: Mapping the Human Toxome by Systems Toxicology

7 companies, 3 stakeholders

European branch?

Evidence-based Toxicology “Evidence-based medicine goes toxicology!”

Hoffmann and Hartung “Toward an evidence-based toxicology”,

Human Exp. Tox., 2006

Mar 2011: US EBTC Oct 2011: Secretariat at CAAT Jan 2012: First conference hosted by

EPA Jun 2012: EU EBTC Diverse working groups Jul 2013: Symposium at IUTOX, Seoul,

Korea Sep 2013: Symposium at EuroTox,

Interlaken, Switzerland Systematic reviews increasingly embraced by IRIS and NTP

EBT  Collabora.on  Steering  CommiXees  United States!

•  Mel Andersen, Hamner*"•  Rick Becker, ACC"•  Kim Boekelheide, Brown"•  Robert Chapin, Pfizer"•  Rodger Curren, IIVS"•  Suzanne Fitzpatrick, FDA"•  Jack Fowle, EPA"•  James Freeman, ExxonMobil"•  Alan Goldberg, CAAT"•  Thomas Hartung, CAAT"•  Michael Holsapple, Battelle"•  Richard Judson, EPA"•  Francis Kruszewski, ACI"•  Martin Stephens, CAAT"•  William Stokes, NIEHS, NIH"•  Raymond Tice, NTP"•  Joanne Zurlo, CAAT"•  + Sebastian Hoffmann (EU EBTC liaison)"

"

Europe!•  Alan Boobis (Imperial College)"•  Neil Carmichael (ECETOC)"•  Thomas Hartung (CAAT)"•  Jan Hengstler (Leibniz Research Centre)"•  Sebastian Hoffmann (seh consulting + services)"•  Philippe Hubert (INERIS)"•  Joanna Jaworska (P&G)"•  Ian Kimber (University of Manchester)"•  Annette Kopp-Schneider (Cancer Research Centre)"•  Marcel Leist (University of Konstanz)"•  Jean-Roch Meunier (L’Oréal)"•  Bennard van Ravenzwaay (BASF)"•  Kai Savolainen (Institute of Occupational Health)"•  Thomas Singer (Hoffmann-La Roche)"•  Nigel Skinner (Agilent)"•  Carl Westmoreland (Unilever)"•  + Martin Stephens (US EBTC liaison)"

"* Affiliations for identification purposes only 56  

The difficulty lies, not in the new ideas,

but in escaping from the old ones.

John Maynard Keynes

(1883 - 1946)