Incorporating Alternative Methods Into Medical Device Safety Testing

Strategies

Recently Available In Vitro Models

Presented  by:  James  McKim,  Ph.D.,  DABT  Chief  Science  Officer  for  CeeTox,  Inc.    

Medical  Devices  Cover  a  Wide  Range  of  Products  

New Devices: Artificial Trachea Grown From Patient’s Stem Cells

A large number of medical device safety studies are negative There are alternative methods that are being used now New alternative methods are becoming available

A � � �B � � �C � � �A � � �B � � � 0 0 0

C � � � 0 � � 0 0

A � � � 0

B � � � 0 0 0

C � � � 0 � � 0 0

A � � � � �B � � � � 0 �C � � 0 � � � 0 � � �A � � � 0

B � � 0 0 0 �C � � 0 0 0 � 0 �A � � � � �B � � � � 0 � 0 �C � � � � � � 0 � � �A � � � 0 0

B � � 0 0 0 � �C � � 0 0 0 � � � �A � � � � � �B � � � � 0 � � �C � � � � � � � � � �

A=  Limited  exposure  5  24  hours  )  B=Prolonged  exposure  (24  hours  -­‐  30  days  C=Permanent  contact  (>  30  days)�  FDA  and  ISO  evaluation  tests      0  =  Additional  tests  for  FDA

Initial  EvaluationSupplemental  Evaluation

Device  Categories

Implan

t  Devices

Externa  Co

mmun

icating  Devices

Surface  Devices

Blood

Bone/Tissue

System

ic  to

xicity  

(acute)/py

rogenicity

Circulating  Blood

Tissue/bone  dentin  communicating

Blood  path  indirect

     Breached/compromised  surface

Mucosal  membrane

Skin

Body

 Con

tact

Contact  D

uration

Cytotoxicity

Sensitizatio

n

Irrita

tion  or  in

tracutan

eous  

reactiv

ity

Carcinog

enicity

Subchron

ic  to

xicity

Geno

toxicity

Implan

tatio

n

Hemocom

patib

ility

Chronic  toxicity

ISO/FDA Test Chart

Cytotoxicity Acute LD50/systemic toxicity Genotoxicity Hemocompatibility Sensitization Irritation

New Models For Predicting Systemic Toxicity are Available

Acute oral LD50 Subchronic toxicity Important considerations for medical devices

Extracts (polar and non-polar) Mixtures Active chemicals in low concentration Exposure/time Concentration-response Development of device controls

What is Systemic Toxicity and What do We Want From Alternative Methods?

§  Toxicity that occurs after a chemical is absorbed into general circulation •  Acute systemic toxicity

Single  dose,  and  short  exposure  time  Intrinsic  toxicity  of  a  chemical,  LD50,  organ  effects    

•  Subacute systemic toxicity Repeated-­‐dose  study,  typically  14  day  Information  on  toxicity  following  repeated  exposure  Helps  establish  doses  for  subchronic  studies      

•  Subchronic systemic toxicity Repeated-­‐dose,  typically  28  and  90  days  Organ  specific  effects  Establish  NOAEL  and  LOAEL  Regulatory  implications  FDA  and  EPA    

•  Chronic systemic toxicity

The Goal is to Predict Human Systemic Toxicity

What  data  are  required  for  risk  assessment    NOAEL,  LOAEL    RfD,  Benchmark  dose    

Most  value  =  Subchronic  

Ideally  

Realistically  

Biochemical Function Membrane  Integrity  Mitochondrial  Function  Cell  Proliferation  Redox-­‐State  Oxidative  Stress  Apoptosis    

Focus on Cell Biology and Physical Chemical Properties Improves the Model

Physical-Chemical Properties Solubility  Partition  coefficients  Pka  Protein  binding  Metabolic  stability  Metabolic  activation  Transporter  interaction    

Pharmacology CNS  receptors  Cardiovascular  receptors  and  ion  channels  

Pharmacokinetic Parameters Clearance  Bioavailability  Volume  of  distribution    

Multiple Endpoints Are Essential For Correct Interpretation of In Vitro Data

CMPD H4IIE 24HR 20050817

0

20

40

60

80

100

120

140

1 10 100 1000

FIG. A: Exposure Concentration (µM)

% C

ontr

ol

Cell#  MemTox  MTT  ATP  GSH  

Cell Line

Development of an Acute Toxicity Model: A Collaboration With LOREAL Paris

Merging Thresholds Significantly Improved Predictive Power

Incorporation of Physical-Chemical Properties and Receptor Binding Improved Predictive Power

Manuscript is Being Prepared Working on improving efficiency for screening

Alternative Methods for Predicting Skin Sensitization

NAMSA/CeeTox  Collaborative  Project  

Proprietary  to  CeeTox,  Inc.  

Induction  Phase          Chemical  

 

Required  for  Immune  response  

 Keratinocytes  Identification  of  unifying  events  Characteristic  of  chemical  sensitizers  Biochemical,  molecular,  chemical  

Dendritic cells Identification  of  unifying  events  Characteristic  of  chemical  sensitizers  Biochemical,  molecular,  chemical  

Most In Vitro Approaches Focus on Chemical Reactivity, Keratinocytes, or Dendritic Cells as the Test System

Peptide binding

Keratinocytes  

Langerhans  Cells  

HaCaT  3D  Skin  Models  

Dendritic  cells  from  blood  Cell  lines  derived  from  blood  

Alternative Methods Currently in The Validation Process  

 §  Direct peptide reactivity assay (DPRA)

ú  Synthetic peptides with cysteine or lysine ú  Gerberick,  G.  F.,  J.  D.  Vassallo,  et  al.  (2004)  Tox  Sci  81,  332-­‐343.  

§  MUSST ú  Myeloid U-937 Skin Sensitization Test ú  Monitors expression of CD86 by flow cytometry

§  hCLAT ú  Human cell line activation test (THP-1) ú  CD86 and CD54 by flow cytometry ú  Sakaguchi et al., (2009) Cell Biol Toxicol 25, 109-125

§  KeratinoSens ú  HaCaT cells transfected with a luciferase reporter construct under the control of ARE

§  SenCeeTox® (In Vitro Sensitization Assay) ú  Combines cell viability, Nrf2/ARE gene expression, and direct reactivity ú  Uses both HaCaT and 3D skin models ú  Express dose as mass per unit area of skin ú  Provides potency category (non-weak, moderate, strong/extreme)

Solubility  Exposure  

Solubility  Finished  products  

Natsch,  A  and  Emter,  R  (2008)  Tox  Sci    102,  110-­‐119.      

New Method (SenCeeTox®) is Based on Known Steps in the Sensitization Process

§  Dose (mass/unit area) §  Penetration into skin (exposure) §  Chemical binding to protein (haptenization) §  Activation of essential signaling pathways in skin

epidermis (signaling in Epidermis) §  Recruitment of Langerhans cells §  Transport to local lymph node §  Proliferation of T-cells

Proprietary  to  CeeTox,  Inc.  

Proprietary  to  CeeTox,  Inc.  

   

             

                         

Chemcical  Reactivity          Direct  and  Indirect  

Cytotoxicity    

Gene  expression  

Solubility  

Human  Cell  Models                    HaCaT  cells  (high  solubility)  Human  3D  Skin  Models  (low  solubility)  

The In Vitro Sensitization Method (IVSA) Incorporates Key Events Occurring in the Epidermis

SenCeeTox®   IVSA  

Figure 2 from Bolmarcich, 2006

RHE or

Reconstructed Human Skin For Determining Chemical Sensitization Models Penetration

and Early Activation of Key Signaling Pathways

Critical  Factors  Chemical  size  Partition  coefficient  Solubility  Penetration  Finished  product  testing  

Direct  Application  to  dendritic  cell  models  may  increase  false  positive  responses    

Multiple Signaling Pathways are Required In Order to Identify Potency Categories

NQ01AKRTXNIL8

Chemical Sensitizers

Maf

Keap 1

NrF2

NrF2

ARE/EpRE

NrF2

Proteosomal Degradation

of Nf2

Maf

Keap 1

NQ01AKRTXNIL8

Chemical Sensitizers

Maf

Keap 1

NrF2

NrF2

ARE/EpRE

NrF2

Proteosomal Degradation

of Nf2

MafMaf

Keap 1

MafNrf1

MTF1

GSHROS

Metals

MRE

Nrf1

ARE/EpRE

MTF1

MafMT1 MT2

MT1 MT2

MafMafNrf1

MTF1

GSHROSGSHROS

Metals

MRE

Nrf1

ARE/EpRE

MTF1

MafMT1 MT2MT1 MT2

MT1 MT2MT1 MT2XRE

Menadione

CYP1A1/2

Reactive Metabolites

ArntAhR

XRE

Menadione

CYP1A1/2

Reactive Metabolites

ArntArntAhR

McKim  JM  et  al.  (2010)  Cutan  Ocular  Toxicol,  29  171-­‐192.  

Proprietary  to  CeeTox,  Inc.  

Eleven Genes Monitor Three Separate But Related Signaling Pathways

§  Nrf2/ARE  Genes  •  NADPH  Quinone  oxido  reductase  •  Aldoketoreductase  •  Thioredoxin  •  Interleukin  8  •  Aldehyde  dehydrogenase  •  Hemeoxygenase  1  

•  Glutamate  cysteine  ligase  catalytic  subunit  C  

§  AhR/XRE  •  CYP1A1  

§  Nrf1/MRE  •  Metallothionein  1  •  Metallothionein  2  

 

Proprietary  to  CeeTox,  Inc.  

Learning to Interpret Gene Expression Data by Building a Training Set of 39 Chemicals

Glycerol Benzoic acid 1-Butanol SDS Lactic acid Limonene Vanillin Salicylic acid Hydroxcitronellal Phenyl benzoate Benzyl cinnamate Eugenol Citral trans-2-hexanal Diethyl maleate Diethyl sulfate

Phenylacetaldehyde Perillaldehyde 2- hydroxy-ethyl-acrylate Isoeugenol Phthalic anhydride 2-aminophenol 1,4-phenylenediamine Propyl Gallate 1-chloro-2,4-dinitrobenzene 5-chloro-2-methyl-4-isothiozolin-3-

one Diphenylcyclopropenone p-Benzoquinone

Moderate  

Strong  

Extreme  

Weak  

Non  

Proprietary  to  CeeTox,  Inc.  

Chemical Reactivity is a Key Characteristic of Chemical Sensitizers

GSH Levels of Glycerol and p-Benzoquinone

0

20

40

60

80

100

120

Control 0.2 mM Glycerol 2 mM Glycerol 20 mM Glycerol 0.2 mM p-Benzoquinone

2 mM p-Benzoquinone

20 mM p-Benzoquinone

Compounds and Concentrations

% G

SH C

ompa

red

to C

ontr

ol

Non-Sensitizer Extreme Sensitizer

Direct Peptide Reactivity Assay

Chemicals TestedBlank LA BA pBQ HyEtAcrylate

Pept

ide

Rem

aini

ng (m

M)

0

200

400

600

800

LYS-Conc CYS-Conc

LA = Lactic Acid

BA = Benzoic Acid

pBQ = p-Benzoquinone

HyEtAcr = Hexylethylacrylate

Multiple  Methods  Can  Improve  Predictive  Power    

Gerberick,  GF  et  al.,  (2004)  Tox  Sci    81,  332-­‐343  Gerberick,  GF  et  al.,  (2007)  Tox  Sci    97,    417-­‐  427  

0.0

5.0

10.0

15.0

20.0

25.0

30.0

10 50 100 500 1000 2500

Exposure Concentration (uM)

Fold

Indu

ctio

n

AKR1C2NQO1TXNIL8

0.0

5.0

10.0

15.0

20.0

25.0

30.0

5 10 25 50 75

Exposure Concentration (uM)

Fold

Indu

ctio

n

AKR1C2NQO1TXNIL8

0.0

5.0

10.0

15.0

20.0

25.0

30.0

1 2.5 25 50 250

Exposure Concentration (uM)

Fo

ld In

du

ctio

n

AKR1C2NQO1TXNIL8

Glycerol   2-­‐Hydroxy-­‐Ethyl-­‐Acrylate   2-­‐Aminophenol  

Multiple Genes Are Required In Order to Accurately Predict Potency Categories

Proprietary  to  CeeTox,  Inc.  

Blinded Study With 58 Test Compounds Showed Good Predictivity

25

33 6

3 P+

+ In vivo - In Vivo

P-

31

89%

85%

36

39

28

Totals

Totals

PPV

NPV

Sensitivity = 81 % How good the assay is at predicting a sensitizer Specificity = 92 % How good the assay is at predicting a non-sensitizer

Allergic Contact Dermatitis (Sensitization) Requires Activation of Key Signaling Pathways

McKim  JM  et  al.  (2012)  Cutan  Ocular  Tox,  Early  On-­‐line,  Informa  Press    

Evaluation of Down Stream Effector Molecules Implicates TNF and CCL5

Induction of signaling genes by sensitizers at 100 µM

p-benzo

quinone

2-aminophen

ol

4-vinylp

yridine

2,3-butan

edione

eugen

ol

benzo

ic Acid

glycero

l0.02.55.07.5

10.04050607080

p38 MAPKTNFCCL27CCL5CCL17

Fold

indu

ctio

n

Figure 2 from Bolmarcich, 2006

RHE or

Reconstructed Human Skin Provides a Viable Model for Testing Medical Device Extracts

-5 5

15 25 35 45 55 65 75

GSH

Dep

letio

n (%

) Reactivity Assay Comparing Positive Control Response With Different Vehicles

Nrf2/ARE Mediated Gene Expression Following Exposure to Extracts from Dental Cement Without 2-HEMA

Nrf2/ARE Mediated Gene Expression Following Exposure to Extracts from Dental Cement Containing 2-HEMA

Summary of In Vitro Versus In Vivo Testing

Application of a New In Vitro Sensitization Assay for the Evaluation of Medical Device Extracts J.M. McKim, Jr1, D.J. Keller1, J.R. Gorski2, and L.H. Moilanen3

1CeeTox Inc., Kalamazoo, MI, 2NAMSA, Northwood, OH and 33M Medical Department, 3M Center, St. Paul, MN

Summary of Research Evaluating Reconstructed Skin Models For Use With SenCeeTox®

Evaluation  of  Skin  Irritation  

Skin Irritation : EPI-200-SIT OECD TG439 SOP V7

§  Skin  irritation:    §  MTT  viability  assay  (n=3)  §   IL-­‐1α  release  (n=3)  §  Histology  (n=2)  

§  Criteria  for  In  vitro  Interpretation  

Application  

0   60  min  

Wash  

incubation   42  hours  

Cytotoxicity  (MTT)  

IL-­‐1a  Release  

24  hr  

Note:  Endpoints  in  yellow  above  are  not  required,  but  may  add  interpretive  value  

A � � �B � � �C � � �A � � �B � � � 0 0 0

C � � � 0 � � 0 0

A � � � 0

B � � � 0 0 0

C � � � 0 � � 0 0

A � � � � �B � � � � 0 �C � � 0 � � � 0 � � �A � � � 0

B � � 0 0 0 �C � � 0 0 0 � 0 �A � � � � �B � � � � 0 � 0 �C � � � � � � 0 � � �A � � � 0 0

B � � 0 0 0 � �C � � 0 0 0 � � � �A � � � � � �B � � � � 0 � � �C � � � � � � � � � �

A=  Limited  exposure  5  24  hours  )  B=Prolonged  exposure  (24  hours  -­‐  30  days  C=Permanent  contact  (>  30  days)�  FDA  and  ISO  evaluation  tests      0  =  Additional  tests  for  FDA

Initial  EvaluationSupplemental  Evaluation

Device  Categories

Implan

t  Devices

Externa  Co

mmun

icating  Devices

Surface  Devices

Blood

Bone/Tissue

System

ic  to

xicity  

(acute)/py

rogenicity

Circulating  Blood

Tissue/bone  dentin  communicating

Blood  path  indirect

     Breached/compromised  surface

Mucosal  membrane

Skin

Body

 Con

tact

Contact  D

uration

Cytotoxicity

Sensitizatio

n

Irrita

tion  or  in

tracutan

eous  

reactiv

ity

Carcinog

enicity

Subchron

ic  to

xicity

Geno

toxicity

Implan

tatio

n

Hemocom

patib

ility

Chronic  toxicity

Summary and Conclusions

Cytotoxicity Acute LD50/systemic toxicity Genotoxicity Hemocompatibility Sensitization Irritation

Medical Device Challenges  Sensitivity  Cell  or  tissue  model  selected  Controls  Exposure  of  cells  Exposure  time  Solubility      

Thank You