In Vitro Alternatives to In Vivo
Toxicity Testing
Presented to The Northland Chapter
of The Society of Toxicology
October 7, 2010
Proprietary to CeeTox, Inc.
Presentation Objectives
• International drivers behind the development of
alternative methods
• Significant changes in the EU and USA
• Validation Committees
• Provide an overview of in vitro models that address
• Skin Sensitization
• Dermal irritation, corrosion
• Ocular toxicity
• Limitations and Issues
• List of Validated Methods
Proprietary to CeeTox, Inc.
Formation of the European Union (EU) and
Legislative Actions That Stimulated the Development
of Alternative Methods
• Treaties Rome (1957/58)
• European Economic Communities (EEC)
• William Russell and Rex Burch’s: 3Rs (1959)
• “The Principles of Humane Experimental Technique”
• Cosmetics Directive (1978)
• Directive 76/768/EEC
• Main European law on the safety of cosmetics
• Maastricht Treaty (1992/93)
• Formation of the European Union (EU)
• European Commission (EC)
Proprietary to CeeTox, Inc.
Significant Changes in the European Union Have
“Jump Started” the Development of Alternative
Methods
• Amendment/Annex VII
• Bans the use of animals for evaluating the safety of cosmetics
• Bans the sale of cosmetics if ingredients were tested on animals
Proprietary to CeeTox, Inc.
Implementation and Timing of Amendement VII
Recognized methods
For 2009
For 2013
SkinEthic
laboratories
Proprietary to CeeTox, Inc.
EU Implements Ban on Animal Testing: Phase I
Today's the Day: Animal Testing Ban Initiated in Europe
Posted: March 11, 2009
Ban 1: testing on animals to assess the safety of ingredients
Ban 2: prohibits the sale of cosmetic products containing
ingredients tested on animals
Proprietary to CeeTox, Inc.
The REACH Initiative Could Cost Billions of
Dollars and Use Millions of Animals
• Registration, Evaluation, and Authorization of
Chemicals (REACH)
• Industry is responsible for chemical risk (production, transport,
labeling)
• Relevant toxicology:
Proprietary to CeeTox, Inc.
Safety Testing Required by REACH
1 to 10 Tons/year
•
•
•
•
•
•
•
Physical and chemical
Skin corrosion
Skin irritation
Eye irritation
Skin sensitization
Mutagenicity
Acute oral toxicity
10 Tons/yr
•
•
•
•
•
•
Thousands of chemicals
Require millions of animals
billions of dollars
Skin irritation
Eye irritation
Mutagenicity
Acute toxicity
Repeat dose toxicity
Reproductive toxicity
In Vitro methods welcomed
Does REACH Allow for In Vitro Data?
•
•
•
•
Article 13
General requirements for generation of information
on intrinsic properties of substances
In particular for human toxicity, information shall be
generated whenever possible by means other than
vertebrate animal tests, through the use of
alternative methods, for example, in vitro methods or
qualitative or quantitative structure-activity relationship
models or from information from structurally related
substances (grouping or read-across).
The Value of In Vitro Data For Assessing Drug and
Chemical Safety Issues is Now Recognized as a Viable
Approach
NAS Report
Toxicity Testing in the 21st Century:
A Vision and Strategy
Animal studies—time consuming and
expensive
Lack of predictability of animal tests
Use of human cells in culture
Systems biology and pathways =
mechanisms
Proprietary to CeeTox, Inc.
US EPA Launched ToxCast in 2007
The ToxCast Program for Prioritizing Toxicity Testing of
Environmental Chemicals
David J. Dix1, Keith A. Houck, Matthew T. Martin, Ann M. Richard,
R. Woodrow Setzer and Robert J. Kavlock
Computational Chemistry
High Throughput Screening
Toxicogenomic Technologies
Predict the potential for toxicity
Prioritize chemicals for animal testing
Toxicol. Sci. (2007) 95 (1): 5-12.
Proprietary to CeeTox, Inc.
Both The United States, Japan, and Europe Have
Agreed to Support Development of In Vitro Methods
European Commission: ECVAM – European Center for the
Validation of Alternative Methods
•Japanese Center for Validation of Alternative Methods: JaCVAM
•US National Institutes of Environmental Health Sciences (NIEHS):
National Toxicology Program Interagency Center for the Evaluation
of Alternative Toxicological Methods (NICEATM)
•ICCVAM – Interagency Coordinating Committee on the Validation
of Alternative Methods
• Organization for Economic Co-operation and Development
(OECD) – Global Harmonization and Oversight
Proprietary to CeeTox, Inc.
An Alternative for Determining Chemical
Sensitization
Proprietary to CeeTox, Inc.
Building of Good Science
•
•
•
•
•
•
Frank Gerberick
Ian Kimber
David Basketter
Cindy Ryan
Andreas Natsch
Roger Emter
Proprietary to CeeTox, Inc.
Sensitization is a Multiple Step Process Involving
Non-specific and Specific Mechanisms
Induction Phase
Elicitation Phase
Chemical
Edema and Erythema
Langerhans Cell (LC)
Exaggerated
Required for Immune
response
Response on
reexposure
Cellular
Influx
Cytokines,
Costimulatory,
Adhesion
Molecules
Increase
Migration to Local
Lymph Node
Transports
antigen
LC and
Lymphocyte
Interaction
Specific
Response
Lymphocyte
Proliferation
Difficult to model both phases in vitro
Proprietary to CeeTox, Inc.
Guinea Pig Skin Sensitization Test Was First Animal
Test for Sensitization: Qualitative
Guinea Pig
Maximization
Test
Topical antigen
application: ID
injection w/ or
w/o FCA
Days 5-8
Buehler
Assay
Topical antigen
application:
Closed Patch
Days 0, 6-8, and
13-15
20 animals/group
Induction
Day 20-22 topical
application
Challenge
Day 27-28 topical
Application (untreated
flank for 6 h)
48, 72 h after
challenge
Endpoint Analysis
21, 24, 48 h after
removing patch
erythema
Modified from Sailstad, 2002
Proprietary to CeeTox, Inc.
Murine Local Lymph Node Assay (LLNA) Provides a
Quantitative Approach and Reduces Animal Use
Days 1, 2 and 3
Agent applied to ears
Day 6
Day 6: 5 h post IV
injection
Lymph nodes removed
IV tail injection of isotope
Proliferation measured:
Isotope incorporation expressed as
disintegration per minute (dpm)
Modified from Sailstad, 2002
LLNA = EC3 = % concentration that produces a 3X increase in proliferation
Proprietary to CeeTox, Inc.
What Are The In Vitro Assays Attempting to
Replace?
• Guinea pig maximization test (GMPT)
• Mouse local lymph node assay (LLNA)
• Human Patch test
Proprietary to CeeTox, Inc.
In Vitro Models Should be as Good as The
Currently Accepted In Vivo Tests
HRIPT Should
Verify NOELS
Estimate NESILS
Not to Identify
Hazard
+
Alternative
Methods Should
GMPT
LLNA
Identify Hazard and Provide
Indication of Potency
Estimate NOEL
Proprietary to CeeTox, Inc.
Desired Outcomes of In Vitro Sensitization
Testing
•
•
•
•
•
•
•
•
Identify chemicals that are true skin sensitizers
Good Sensitivity and Specificity
Identify the correct potency category
Discriminate irritants from sensitizers
Detect metals that are sensitizers
Detect sensitizers in mixtures
Quantitative/NOEL
Identify chemicals that cause skin sensitization in
humans (clinical allergic contact dermatitis)
Proprietary to CeeTox, Inc.
How Will The In Vitro Data Be Used?
• Identify a potential skin sensitizer
• To understand potency
• Risk Assessment Models
• Hazard identification
• Concentration response
• NOELs and NESILs
• Exposure scenarios
• Risk
• Go-No-Go Decision
Proprietary to CeeTox, Inc.
When You Take Away the Lymph Node: The
Chemical and Two Cell Types Remain
Induction Phase
Peptide binding
Chemical
Keratinocytes
Langerhans Cells
Required for Immune
response
Keratinocytes
Identification of unifying events
Characteristic of chemical sensitizers
Biochemical, molecular, chemical
HaCaT
3D Skin Models
Dendritic cells
Identification of unifying events
Characteristic of chemical sensitizers
Biochemical, molecular, chemical
Dendritic cells from blood
Cell lines derived from blood
Proprietary to CeeTox, Inc.
Assays That Focus on Human Dendritic Cells and Cell
Lines

Dendritic cells (DCs) in vitro
 Prepared from human monocytes
 CD-34+ cord blood progenitors
 Immature DCs acquire a mature phenotype when exposed to sensitizers:
Mediated via MAPK


Trompezinski et al. (2008) Toxicol Appl Pharmacol, 230, 397-406
Gene expression changes dendritic cells

Ryan, C. A., L. A. Gildea, et al. (2004), Toxicol Letts 150, 301-316.

Difficult to isolate and culture, donor availability, interindividual variations,

Human Cell Line Activation Test (hCLAT)




THP-1 monocytic leukemia cell
U-937 histiocytic lymphoma cell line
Monitor CD86 and CD54 expression levels
Sakaguchi et al. (2006) Toxicol In vitro 20, 774-784
Proprietary to CeeTox, Inc.
NICEATM/ICCVAM
Chemical Reactivity and Peptide Binding
Assay
• Chemical allergens are reactive (electrophilic)
• Tested multiple substrates:
•
•
•
•
Glutathione
Lysine
Cysteine
Histidine
Synthetic peptides
• Good correlation between potency and depletion?
• GSH >> Cysteine >> Lysine (Not) Histidine
• Analyzed depletion of GSH or synthetic peptides by HPLC/UV
Gerberick, G. F., J. D. Vassallo, et al. (2004) Tox Sci 81, 332-343.
Proprietary to CeeTox, Inc.
In Order to Detect Chemical Sensitizers There Must a Unifying or
Common Property Between the Chemicals and the Test System Must
Possess a Means of Detecting This Property
What do we know?








Most sensitization processes involve a reactive form (electrophile)
Bind to cellular proteins
Electrophiles can induce specific gene expression profiles
ARE/EpRE control transcription of genes associated with cell protection,
AKR, and NQO1
Discovered through studies focused on PAH metabolism
Identified a cellular response mechanism to reactive intermediates
Example PAHs include: Benzo(a)Pyrene, Menadione
Most chemicals with electrophilic sites are capable of being sensitizers



J. Ashby et al. (1995) Toxicology, 103, 177-194.
K.H. Schulz et al. (1977) Arch Derm Res, 258, 41-52.
J. Ashby et al. (1993) Environ Hlth Persp, 101, 62-67.
Proprietary to CeeTox, Inc.
Monitoring Key Genes Controlled by Three
Signaling Pathways
Reactive
Metabolites
Proteosomal
Degradation
of Nf2
Chemical
Sensitizers
GSH
ROS
Metals
NrF2
Menadione
NrF2
MTF1
Maf
Nrf1
Maf
CYP1A1/2
AhR
Arnt
XRE
NrF2
Maf
ARE/EpRE
NQ01
AKR
MTF1
TXN
IL8
Proprietary to CeeTox, Inc.
MRE
MT1
MT2
Nrf1
Maf
ARE/EpRE
MT1
MT2
Eleven Genes Monitor Three Separate But
Related Signaling Pathways
• NADPH Quinone
oxidoreductase
• Aldoketoreductase
• Thioredoxin
• Interleukin 8
• Aldehyde dehydrogenase
• Hemeoxygenase 1
• Glutamate cysteine ligase
catalytic subunit C
•
•
•
•
CYP1A1
MafF
Metallothionein 1
Metallothionein 2
Proprietary to CeeTox, Inc.
When You Take Away the Lymph Node There
Are Essentially Two Cell Types
Induction Phase
Chemical or hapten
Chemical Reactivity
Protein binding
ARE-gene expression
Hapten-protein
Keratinocytes
Identification of unifying events
Characteristic of chemical sensitizers
Biochemical, molecular, chemical
Gerberick et al. (2004) Toxicol Sci 81, 332-343
Divkovic et al. (2005) Contact Dermat 53, 189-200
Natsch and Emter (2008) Toxicol Sci 102, 110-119
Langerhans Cell (LC)
Required for Immune
response
Dendritic cells
Identification of unifying events
Characteristic of chemical sensitizers
Biochemical, molecular, chemical
Proprietary to CeeTox, Inc.
Highest Predictive Power From a Tiered In Vitro System
Solubility
Chemical Reactivity
Direct and Indirect
Cytotoxicity
Gene expression
Human Cell Models
HaCaT cells
Human 3D Skin Models
Proprietary to CeeTox, Inc.
Chemical Reactivity Inherent to the Molecule is a Key
Characteristic of Chemical Sensitizers
% GSH Compared to Control
GSH Levels of Glycerol and p-Benzoquinone
120
Non-Sensitizer
Extreme Sensitizer
100
80
60
40
20
0
Control
0.2 mM Glycerol
2 mM Glycerol
20 mM Glycerol
0.2 mM pBenzoquinone
Compounds and Concentrations
Proprietary to CeeTox, Inc.
2 mM pBenzoquinone
20 mM pBenzoquinone
Learning to Interpret Gene Expression Data by
Building a Training Set of 39 Chemicals
Glycerol
Benzoic acid
Non
1-Butanol
Perillaldehyde
SDS
2- hydroxy-ethyl-acrylate
Lactic acid
Isoeugenol
Limonene
Vanillin
Weak
Phenylacetaldehyde
Phthalic anhydride
Salicylic acid
2-aminophenol
Hydroxcitronellal
1,4-phenylenediamine
Phenyl benzoate
Propyl Gallate
Benzyl cinnamate
Eugenol
Citral
trans-2-hexanal
Strong
1-chloro-2,4-dinitrobenzene
5-chloro-2-methyl-4-isothiozolin-3one
Diphenylcyclopropenone
Diethyl maleate
Diethyl sulfate
Moderate
p-Benzoquinone
Proprietary to CeeTox, Inc.
Extreme
Evaluations Based on Potency, Magnitude, Number,
and Reactivity
HaCaT cells used as model
Glycerol
2-Hydroxy-Ethyl-Acrylate
30.0
30.0
25.0
20.0
AKR1C2
NQO1
15.0
TXN
IL8
10.0
30.0
Moderate
25.0
20.0
AKR1C2
NQO1
15.0
TXN
IL8
10.0
5.0
5.0
10
50
100
500
1000
Exposure Concentration (uM)
2500
Strong
20.0
AKR1C2
NQO1
15.0
TXN
IL8
10.0
5.0
0.0
0.0
0.0
Fold Induction
Non-very weak
Fold Induction
Fold Induction
25.0
2-Aminophenol
5
10
25
50
75
Exposure Concentration (uM)
1
2.5
25
50
250
Exposure Concentration (uM)
Detailed concentration response data expressed as mass per unit area may allow
the NOEL to be estimated for human exposure.
Proprietary to CeeTox, Inc.
Rules Based Gated Logic Algorithm to Analyze
Data and Provide a Toxicity Score
(1) PTI score =  {(V + i (R) + i (GARE) + i (GXRE) + i (GMRE)) }
Where V = viability, R = reactivity, G = gene expression,
i = weighting factor
Weighting Factors
I takes into account
Cell viability
Gene expressed
Signaling pathway
Magnitude of induction
Concentration response
Potency of induction
PTI Score
N = 0-1
W = 2-3
M = 4-5
ES = 6-8
Proprietary to CeeTox, Inc.
Assigning Potency Categories Based on In
Vitro Data
120
Accuracy (%)
100
80
60
40
20
0
ES
M
W
Potency Category
Proprietary to CeeTox, Inc.
N
Number of Compounds High Positive
Frequency Histogram: Gene Response Pattern
or Sensitivity
18
16
14
12
10
8
6
4
2
0
NQ AKR TXN
IL8 CYP ALD HMO MAF GCL MT2 MT1
Marker Genes
Proprietary to CeeTox, Inc.
Blinded Study With 58 Test Compounds
SenCeeTox™ Showed Good Predictivity
+ In vivo
- In Vivo
P+
25
3
P-
6
33
Totals
31
Totals
28
89%
PPV
39
85%
NPV
36
Sensitivity = 81 % How good the assay is at predicting a sensitizer
Specificity = 92 % How good the assay is at predicting a non-sensitizer
Proprietary to CeeTox, Inc.
Relationship Between LLNA EC3 and PTI: Extreme
Variability in EC3 Values Limits Direct Extrapolation
Relationship Between PTI and LLNA
10000
LLNA (mM)
1000
PTI vs LLNA mM
Plot 1 Regr
100
10
1
0.1
0.01
0
2
4
6
8
10
Predicted Toxicity Index (PTI)
Proprietary to CeeTox, Inc.
GPMT ?
HRIPT ?
Proprietary to CeeTox, Inc.
Alternative Methods For Determining
Skin Corrosion and Irritation
Applying Skin Models to Identify Chemicals
That are Corrosives and Irritants
 Skin Corrosion: “the production of irreversible damage
to the skin; namely, visible necrosis through the
epidermis and into the dermis, following the application
of a test substance for up to 4 hours [in at least one of
three test animals]" (UNECE, 2004).
 Skin Irritation: “production of reversible inflammatory
changes in the skin” (ECETOC, 1990; OECD, 1992)
In Vitro Models for Prediction of Dermal Toxicity
SkinEthic
laboratories
Photo courtesy of SkinEthic
Structure and Function of Skin


Largest Organ
Layers:
 Stratum Corneum- nonviable functions as barrier
(bacteria, UV, chemical)
 Epidermis: tough, outer layer of skin. Primarily
keratinocytes originating from basal layer, slowly
migrate toward surface. Langerhans’ cells located
here
 Dermis: thick layer of fibrous and elastic tissue
(primarily collagen, elastin, and fibrillin) gives the skin
flexibility and strength
 Fat Layer (subcutaneous): extra energy storage

Function

Barrier (protection)
3-Dimensional In Vitro Dermal Models Used by CeeTox: EpiSkin and
Epiderm Reconstructed Human Epidermis
•Normal human keratinocytes
•Similar to human epidermis
by histology and
immunohistochemistry
In vivo
In vitro
•Validated by ECVAM for
dermal irritation (4/2007)
•Validated by ICCVAM and
OECD for corrosion
•Internal validation at CeeTox
SkinEthic
laboratories
Keratin 10
From www.skinethic.com
Filaggrin
In Vitro 3-Dimensional Human Dermal Models Have
Similar Lipid Profile to Human Skin
•MatTek EpiDerm has similar
lipid profile to human skin
(phospholipids, ceramides,
cholesterol)
•In vitro skin models can be
used for studies related to
dermal barrier function
•Useful substrate for
transdermal drug delivery
investigations
From www.mattek.com
Cells Grown at an Air-Liquid Interface Allow Insoluble
Products to be Tested
•In vitro HE has metabolic activity similar to that of human skin
•P450 activity
•Phase II biotransformation enzymes
Apply
compound to
skin and
measure
metabolites
in media
From Bolmarcich, 2006
Xenobiotic Biotransformation Capacity of In Vitro
Dermal Models
3-dimensional human
skin models express a
variety of cytochrome
P450 enzymes at similar
levels as human
epidermal tissue
•Cyp1A1, 1B1
•Cyp 2A, 2B, 2C, 2D, 2E
•Cyp 3A4, 3A5
From Bolmarcich, 2006
Induction of CYP1A in 3-Dimensional Dermal Models
From Bolmarcich, 2006
Treatment of EpiDerm with 3-MC or b-NF leads to induction of Cyp1A1 and 1A2
In Vitro Reconstructed Human Epidermis Is a Well
Validated Model for Predicting In Vivo Outcome
•Multiple validation tests by ICCVAM and ECVAM
•>20 peer-reviewed publications
•Internal validation studies at CeeTox
From ICCVAM Proposed Minimum Performance Standards (06/2003)
From ECVAM Statement on Validity of In-Vitro Tests for Skin Irritation (04/2007)
Protocol for The MTT Assay
yellow
purple
 MTT or 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide is a simple
inexpensive method for measuring the activity of living cells via mitochondrial
dehydrogenases
 Simple, accurate and reproducible assay of cell viability
 Assay Procedure

Dissolve MTT(5mg/mL) in medium (yellow color solution); working solution 1mg/mL

Mitochodrial dehydrogenases of viable cells cleave the tetrazolium ring yielding
purple formazan crystals which are insoluble in aqueous solutions (3 hr incubation)

Dissolve crystals in acidified isopropanol (purple color)

Transfer to 96 well plate in duplicate

Measure using spectrophotometer (OD570)
Multiple Endpoint Analysis With Histology Improves
Data Interpretation
De Wever et al. (2004) In Dermatoxicology 6th Edition, Chap 43, Taylor and Francis
2 % Dithranol and MTT Conversion
1
2
3
1. MTT solution
2. PBS
4
5
6
3. H20
4. 2% Dithranol
5. Compound a
6. Compound b (7.5%)
Dithranol: Hydroxyanthrone anthracene derivative,
medicine applied to the skin for treatment of psoriasis
Compound Interference in MTT Assay
Sample compounds demonstrating intrinsic ability to convert MTT in the absence of cells
Protocol for In Vitro Skin Corrosion: Human
Skin Model Test OECD 431
• EpiDerm® or EpiSkin®
• Barrier must resist rapid penetration (ET50 >2 hr: 1%
Triton X-100)
• Dose usually neat: liquids 25 µL/cm2; solids ground to
fine powder
• MTT reduction is the recommended assay
• Corrosive = viability after 3 min < 50% or if viability after
3 min is > 50% and viability after 1 hr is < 15%
• Non Corrosive = viability after 3 min > 50% and viability
after 1 hr is > 15%
U
KO ntr
H eat
0.
8 3 m ed
8% N
K in
Na OH (C
)
0. C l 3 1h
8
(
H % N min C)
2S
O aCl (NC
4
1
)
H 10% h (N
2
n- S O 3m C )
he
4
1 in
pt
yl 0% (C
n- am
1h )
i
he ne
(C
pt
3
)
y
m
SD l a
i
n
m
S
20 ine (C)
%
1
SD
3m h (C
Su
S
)
lfa
2 in
m 0% (N
C
ic
Su Ac 1h ( )
lfa id 3 NC
t-b mic m )
in
ut
(
yl Aci
ph
d C)
t-b eno 1h
(
C uty l 3m C)
ap
l
ry phe in
(C
lic
n
A c ol 1 )
C
Is
os apr id 3 h (C
te ylic m
in )
ar
A
Is ic A cid (C)
os
c
te id 1h
ar 3m (C
ic
)
Ac in (
id NC
1h )
(N
C
)
8N
Viability (percent of control)
Example of Dermal Corrosion Assay
CeeTox Dermal Corrosion Model
140
120
100
80
60
40
20
0
Treatment: C = corrosive, NC = non-corrosive
Skin Models: Corrosion and Irritation
 Skin Corrosion: “the production of irreversible damage
to the skin; namely, visible necrosis through the
epidermis and into the dermis, following the application
of a test substance for up to 4 hours [in at least one of
three test animals]" (UNECE, 2004).
 Skin Irritation: “production of reversible inflammatory
changes in the skin” (ECETOC, 1990; OECD, 1992)
Skin Irritation Protocols Vary Depending on
Model Used
Application
Time 0
Exposure
Wash
Exposure time
EpiSkin 15 min
SkinEthic (RHE) 42 min
EpiDerm (1 hr)
Recovery
42 hr
Viability = MTT
IL-1 release
Histology
What if Cells Die During Exposure?
Mean tissue viability is < 50%
Irritant (I) R38
Mean tissue viability is > 50% and IL-1 is > 50 pg/mL
Mean tissue viability is > 50%
Non Irritant (NI)
Mean tissue viability is > 50% and IL-1 is < 50 pg/mL
CeeTox Validation: Irritation
Epi-200 (MatTek)
Relative viability %
100
50
0
PBS
PC
Water
Caprylic Acid
propyl disulfide
diethyl phthalate
isopropanol
Eugenol
Classification
Criteria
NC
NI
qualified
PC
I
qualified
Water
NI
qualified
Caprylic Acid
I
qualified
no compound
NI
qualified
propyl disulfide
I
qualified
diethyl
phthalate
NI
qualified
isopropanol
NI
qualified
Eugenol
I
qualified
NC: PBS
PC: 5% SDS
Qualified: Standard deviation is < 20%
NI: Non-irritant
I: Irritant
Current Validated Protocols are Not Based on a
Mechanism of Toxicity
• All models whether skin or ocular use single endpoint
(MTT)
• Viability assay: separation of endpoints is based on
exposure time and recovery
• Seems to work well for corrosivity or strong irritants
• Need to evaluate chemical in both models if viability low
• Not mechanism based
• For dermal irritation additional endpoints based on
mechanism should improve the methods ability to
discern a weak or mild irritant from a strong
Multiple Endpoints Can be Measured in a Single CeeTox
In Vitro Dermal Experiment
Adapted from www.skinethic.com
Viability (MTT) and IL-1 endpoints have been validated by
ICCVAM and ECVAM
Alternative Models For Ocular
Toxicity
Rabbit Draize Test for Ocular Toxicity
•
•
•
•
•
Test was developed 1944 by Draize et al.
Qualitative endpoints or scoring system
Test reliable for identifying moderate to severe irritants
Weak to mild irritants often not identified correctly
Three areas of the eye are evaluated
• Cornea
• Iris
• Conjuctivae
Interpreting Draize Ocular Data
Cornea
A. Opacity
Scattered
Opaque
Score
Conjunctivae
A. Redness
Score
1
2
3
4
1
2
3
B. Chemosis
B. Area Involved
25%
>75%
Any swelling
1
2
3
4
Swelling lids closed
1
2
3
4
C. Discharge
Score = A x B x 5: total maximum = 80
Iris
A. Values
Still responds to light
No reaction to light
1
2
Any amount of discharge
Discharge with moistening lids
and hairs
Discharge with moistening lids
and considerable area around
the eye
(A + B + C) x 2: total maximum = 20
Score = A X 5; total maximum = 10
Blazka and Hayes (2008) in Princples and Methods of Toxicology
1
2
3
Modified Maximum Average Score (MMAS)
• Multiple time points for observations
• 4, 24, 48 hr each animal
• Maximum Average Score (MAS)
• Average of individual animal scores at each time of observation
then selecting the maximum of these averages.
• Modified Maximum Average Score (MMAS)
• Not all of the tests used to compile databases included times
less than 24 hr
• Maxima calculated at 24 hr or more after instillation
Bagley DM et al. (1992) Toxic in vitro, 6, 487-491.
Validation of Alternative Methods Requires a
Reliable Reference Chemical Set
• Progress towards acceptance can be hindered
when:
• Chemicals not freely available or lacking purity data
• Non-standardized in vivo test protocols are used
• Idea was to set up an internationally accepted chemical bank
Bagley DM et al. (1992) Toxicol In Vitro 6, 487-491
In Vitro Alternatives to Animal Eye Irritation/Toxicity
Testing
• Organotypic Models Already Evaluated by ICCVAM
•
•
•
•
Bovine corneal opacity and permeability Test (BCOP)
Isolate rabbit eye (ICE) test
Isolated chicken eye (ICE) test
Hens egg test on chorio-allantoic membrane (HET- CAM)
ICCVAM endorsement as valid methods for severe eye toxicity
• Reconstituted Human Tissue Models
• EpiOcular- Immortalized keratinocytes (MatTek, Inc)
• SkinEthic-Reconstituted Human Corneal Epithelium (HCE)
model (SkinEthic)
Cells Grown at an Air-Liquid Interface Allow Insoluble
Products to be Tested
•In vitro HE has metabolic activity similar to that of human skin
•P450 activity
•Phase II biotransformation enzymes
Apply
compound to
skin and
measure
metabolites
in media
From Bolmarcich, 2006
Human Corneal Epithelial (3D) Cell Model for
Evaluating Ocular Toxicity: SkinEthic
• In vitro model for assessing:
• Xenobiotic challenges to corneal epithelium
• Corneal epithelial biology/physiology
• Alternative to animal testing
• Relevant, reliable, consistent
• Biological relevance
• Human corneal epithelial origin
• Avoid loss of phenotype associated with permanent cell lines
Species and Tissue
Specific
SkinEthic® Human Corneal Epithelial Model For
Ocular Irritation
24 hr Exp
24 hr Exp + 24 hr Recovery
Non keratinized flattened
PBS
Lateral cytoplasmic
Extension
Regular column cuboidal
0.01% BAK
0.1% BAK
Pauly et al. (2009) Invest Ophthal Visual Sci, 50, 1644-1652
Data obtained in the HCE Model Shows Good
Correlation with Draize Test
Tes Substance
Type of
Surfactant
Benzalkonium chloride Cationic
Cetylpyridium bromide Cationic
Exp Conc Draize Score
(%)
(MMAS)
0
Measured ET50
(Min)
0.01
0.1
0.3
1
5
10
9
45
84
108
>240
32.6
9.6
3.5
2.2
1.5
0.01
1
2.7
36
>240
13.1
Sodium lauryl sulfage
Cationic
1
3
10
1
16
38
18.2
10.5
1.8
Triton X-100
Cationic
1
5
10
1.7
32.3
68.7
20.6
5.3
Dose and Time Relationships Combined with
Reference Compounds
Corneal Epithelial Cells Exposed to Benzalkonium Chloride (BAC):
MTT Response Over Time
120
100
% Control
80
10 MIN BAC
20 MIN BAC
60
30 MIN BAC
60 MIN BAC
40
20
0
0.01%
0.1%
EXPOSURE CONCENTRATION (% )
1%
Evaluations Based on Determining ET50
Corneal Epithelial Cells Exposed to
1% Benzalkonium Chloride:
MTT Response Ov er Time
Corneal Epithelial Cells Exposed to
0.01% Benzalkonium Chloride:
MTT Response Ov er Time
120.0
y = 0.0152x2 - 1.7129x +
118.56
100.0
% Control (MTT)
% Control (MTT)
120.0
80.0
60.0
40.0
20.0
80.0
60.0
40.0
20.0
0.0
0.0
0
20
40
60
80
Corneal Epithelial Cells Exposed to
0.1% Benzalkonium Chloride:
MTT Response Ov er Time
120.0
y = -0.0116x2 - 0.5247x +
93.793
100.0
80.0
60.0
40.0
20.0
0.0
0
20
40
Time (min)
0
20
40
Time (min)
Time (min)
% Control (MTT)
y = 0.0159x2 - 2.3x +
91.39
100.0
60
80
60
80
MatTek Corporation’s EpiOcular® Model
10% Cetylpyridinium bromide
3 min
9 min
20 min
EpiOcular System Showed Strong Concordance with In
vivo Draize Test
Stern et al. (1998) Toxicol In vitro 12, 455
Comparison of MatTek Corporation EpiOcular
Model to In Vivo Data
Test Material
Type of
Draize
Surfactant Score
3.2% Benzethonium chloride
C10-12 Alchohol ethoxylate
Cetyl alcohol (50% w/w paste)
10% Cetylpyridinium bromide
50% Didecyldimonium chloride
Quatemium-18 (100% neat)
3% Sodium Lauryl Sulfate
Cationic
Nonionic
Nonionic
Cationic
Cationic
Cationic
Anionic
39% Sodium methyl 2-sulfonate
& disodium 2-sulfolaurate
Anionic
Predicted
Draize
95%
Prediction
Interval
Results
27.5
42.3
0
89.7
110
66.3
16
31.2
63.8
<1.8
20.5
>97.0
<13.8
23.1
5.0 to 57.5
37.1 to 90.4
0 to 27.7
0 to 46.6
>69.9
0 to 39.9
0 to 49.3
Agreed
Agreed
Agreed
Differed
Agreed
Differed
Agreed
39
41.2
14.8 to 67.6
Agreed
75% Accuracy
Blazka, ME et al. (2005) World Congress on Alternatives and Animal Use in the Life Sciences
Methods Validated by ICCVAM, ECVAM, and
Accepted for Regulatory Use by OECD
Endpoint
Method
Validated and Recommended for Regulatory Use:
Skin Penetration
In Vitro Skin absorption
OECD 428
EPISKIN with MTT
Reduction and IL-1α release
ECVAM: as a replacement
ICCVAM: as a screen in a tiered-testing strategy
EpiDerm with MTT
Reduction and IL-1α release
ECVAM: as a replacement (a negative result may require further testing)
ICCVAM: as a screen in a tiered-testing strategy
Corrositex
ECVAM: as a replacement
ICCVAM: as a screen in a tiered-testing strategy
OECD 435
In Vitro Skin Corrosion: Human Skin Model Test
OECD 431
Ames-Bacterial Reverse Mutation Test
OECD 471
In Vitro Mammalian Chromosome Aberration Test
OECD 473
In Vitro Mammalian Cell Gene Mutation Test
OECD 476
LLNA in mice
OECD 429
Skin Irritation
Skin Corrosivity
Genotoxicity
Skin Sensitization
Proprietary to CeeTox, Inc.
Methods Validated by ICCVAM or ECVAM and
Accepted for Regulatory Use
Endpoint
Method
Eye Corrosion
Bovine Corneal
Opacity and
Permeability (BCOP) ICCVAM; ECVAM; JaCVAM
OECD 437
Eye Corrosion
Isolated Chicken Eye
ICCVAM;ECVAM;JaCVAM
Assay (ICE)
OECD 438
Eye Corrosion
Eye Irritation
Fluorescein
Leakage, INVITTOX
Protocol 71
Cytosensor
Microphysiometer
Test Method,
INVITOX Protocol
102 modified
Validated and Recommended for Regulatory Use:
ECVAM
ECVAM
In Summary
• New global initiatives make alternatives mandatory
• We have made significant progress
• Still have a long way to go
• Future Directions
•
•
•
•
•
Integrative approach
Functional biochemistry to confirm gene expression changes
Mechanistic endpoints
Improved cell models
More efficient validation and acceptance
Proprietary to CeeTox, Inc.
Thank You and Thanks to
A Great Team of Scientists at
CeeTox, Inc