The EpiOcular™ Model Protocol, Performance and Experience

The EpiOcular™ Model
Protocol, Performance and
Experience
Rodger Curren, Ph.D., John Harbell, Ph.D.
and Kevin Trouba, Ph.D.
Institute for In Vitro Sciences, Inc.
EpiOcular™
Outline
•
Background
•
Protocol
•
Performance
•
Experience
EpiOcular™
1.
The EpiOcular™ tissue model is produced by The MatTek Corporation,
located in Ashland, Massachusetts.
2.
The model estimates the potential ocular irritation of a test article by
measuring cytotoxicity (MTT dye conversion) by the EpiOcular™ tissue
construct after topical exposure to the test article.
3.
The endpoint of the assay, the ET50 value, is the time of exposure to
test article required to reduce cell viability to 50% of negative control
levels.
4.
The EpiOcular™ assay assumes that in vitro cytotoxicity is directly
related to in vivo damage that a toxicant would inflict upon an exposed
cornea.
5.
This is based in part on the hypothesis by Maurer and Jester1 that the
level of ocular irritation is related to the extent of initial injury, and that
regardless of the processes leading to tissue damage, the extent of
initial injury is the principal factor determining the outcome of ocular
irritation.
1Extent
of initial corneal injury as the mechanistic basis for ocular irritation: key findings and
recommendations for the development of alternative assays. Regul Toxicol Pharmacol. 2002 Aug;36(1):106-17.
EpiOcular™
Its Position in a Continuum of Sensitivity
Rabbit
EpiOcular
BCOP
Extreme
Severe
Moderate Mild
Household
Very Mild
Color Cosmetics
Personal Care
Industrial Chemicals
EpiOcular™
Cornea
http://medicalimages.allrefer.com/large/cornea.jpg
http://www.drmingwang.com/Publications/book/22.jpg
EpiOcular™
Histology
Upper squamous cells
Central squamous cells
Lower rounded cells
NOTE: The EpiOcular™ tissue construct models only the top epithelial
layer and not the stromal and endothelial layers of the cornea.
3
EpiOcular™
Tissue Culture Technique: Air/Liquid Interface
Tissue Culture Well
Culture Insert
Tissue
Membrane
Medium
EpiOcularTM
Reagents & Equipment
EpiOcularTM
Basic Procedure
1 min – 24 hr
Dose & Incubate
3 hr
MTT Addition
Reduction
2 hr
Quantification
Extraction
Reagents and Initial Tissue Preparation
EpiOcularTM tissues and reagents
are shipped overnight cold, and all
materials are stored at 4°C. Tissues
can be used within 48 hours of receipt.
The tissues are examined for obvious
defects and may be rejected based
on blistering, excess fluid, air bubbles
below the tissue insert, etc..
Prior to test article dosing, tissues
are transferred (using sterile
technique) to 6-well plates that contain
fresh assay medium.
The tissues are incubated at standard
conditions (5% CO2, 37°C, 95%
humidity) for at least 1 hour.
Reagents and Initial Tissue Preparation
Unused EpiOcularTM tissues should
be equilibrated with 5% CO2 and
stored at 4°C. We perform this step
by gassing with 5% CO2 in a storage
bag.
Our experience indicates that
normal equilibration at 5% CO2,
37°C, 95% humidity (i.e., in a tissue
culture incubator) may produce
variability in tissue performance.
Prior to dosing with TA or controls,
the tissues are refed with fresh, prewarmed assay medium and
generally dosed within 30 minutes of
refeeding.
Dosing
Dosing of aqueous or semi-viscous
test articles (TA) is performed with a
positive displacement pipette.
Solid materials are “sprinkled” onto
the surface of the tissue (millicell
removed temporarily from well).
A dosing device (e.g. the flat end of
a sterile push pin) can be used to
ensure that the TA covers the
complete tissue surface.
The tissues are incubated at
standard conditions for a specified
amount of time.
Dosing times generally range from
1 minute to 24 hours.
Dosing Device
Rinse and Soak
The tissues are removed from the
incubator and the TA is removed
using phosphate buffered saline
(PBS). The PBS is sprayed against
the millicell wall (not directly on the
tissue) to create a gentle vortex which
aids in TA removal.
The tissues are “soaked” in
medium at room temperature
to ensure a more complete removal
of any remaining TA.
Following the soak process, the
tissues are rinsed again with PBS
prior to the MTT reduction step.
Complete TA removal is necessary
to prevent prolonged exposure and
overestimated toxicity.
MTT Reduction and Extraction
Individual tissues are placed into
wells containing unreduced
3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)
solution.
The tissues are incubated at standard
conditions for 3 hours.
A dark blue or purple color signifies
the presence of reduced MTT/viable
tissue.
Following incubation, the tissues are
removed and placed into isopropanol
for 2 hours with shaking at room
temperature to extract the reduced
MTT.
Transfer of MTT/Isopropanol and Quantification
Extracted MTT is thoroughly mixed
and transferred to a 96-well plate.
The 96-well plate/MTT-isopropanol
samples are quantified using a
microplate reader.
Raw OD550 values are used to
to calculate the final ET50 values.
0.3% TRITON®-X-100
Percent of Control
100& Interpretation
Graph
80
60
40
Data
20
0
0
10 20 30 40 50 60
Exposure Time (minutes)
EpiOcular™ Reproducibility
Model Inception = 1996
Positive control: 0.3% Triton-X-100
(Historical Control Range = 27 minutes +/- 2 std. dev.)
Negative control: Sterile DI H20
Test Material
Mean ET50
Value
(min)
Standard
Deviation
(min)
CV
26.1
5.4
20.7%
27.0
6.0
22.2%
0.3% Triton X-100
(Combined data from MatTek and IIVS)
0.3% Triton X-100
(IIVS only - through Oct., 2004)
SD RAN G E
1997
1998
1999
2000
2001
2002
2003
2004
2005
0.0
0.5
1.0
0.0
1.5
41%
35%
20%
95%
5%
52%
26%
17%
95%
5%
36%
31%
24%
92%
8%
29%
25%
27%
81%
18%
35%
36%
20%
91%
9%
32%
22%
31%
85%
15%
36%
26%
25%
87%
13%
33%
27%
19%
79%
21%
47%
35%
15%
97%
3%
1997-2005
YTD
38%
29%
22%
89%
11%
>50
>50
>50
>50
>50
>50
>50
>50
>50
>500
22.9
25.0
22.1
20.7
22.9
22.5
24.1
22.2
24.77
23.00
T O 0.5
T O 1.0
T O 1.5
T O 1.5
T O 2.0
#
Production
Lots
AVE ET 50
(m in)
Correlation of In Vitro and In Vivo Results
Draize - EpiOcular™
Draize Score1
Classification
Example
EpiOcular ET50
(min)2
0-15
Non-irritating, Minimal
PEG-75 Lanolin
> 256-26.5
15.1-25
Mild
3% SDS
26.5-11.7
25.1-50
Moderate
5% Triton X-100
11.7-3.45
50.1-110
Severe, Extreme
5% BAK
< 3.45
Based on 20% dilutions for materials with density > 0.95
1Kay,
J.H. and Calandra, J.C., Interpretation of eye irritation tests, J. Soc. Cosmetic
Chem., 13, 281-289 (1962)
2MatTek
Assessment of Ocular Irritation Ranges of Market-Leading Cosmetic
and Personal-Care Products Using an In Vitro Tissue Equivalent
N E McCain, R R Binetti, S D Gettings, and B C Jones
Avon Products, Inc., Suffern, NY, USA
Formulation Category
Surfactant Formulas
Adult Shampoo
Children’s Shampoo
Baby Shampoo
Baby Wash
Eye Area Cosmetics
Liquid Eyeliner
Eye Cream
Mascara
Pencil Eyeliner
Creams/Lotions
Adult Lotion/Cream
Sunscreen
Baby Lotion
Non-Surfactant Haircare
Children’s Styling Gel
Adult Conditioner
Children’s Conditioner
Children’s Hair Detangler
Number
of
Samples
Mean
ET50
(min)
Range of
ET50 Scores
(min)
9
7
4
4
27.0
58.2
74.9
114.5
17.1-35.1
37.9-135.4
31.9-107.1
76.9-155.9
15
11
20
3
532.2
581.8
655.1
1440
64-1440
102-1200
199-1080
1440
17
15
2
511.4
673.6
1200
234-1440
252-1440
1200
2
5
2
3
172.7
279.6
728.0
868.4
126-219.3
100-530
256-1200
205.1-1200
10%
100%
Test Article and Other Effects
Hydroscopic Test Article
Direct MTT Reduction
Air Bubble
0% Tissue Kill
~ 25% Tissue Kill
~ 60% Tissue Kill
Test Article and Other Effects
Robust tissue peripherally
Residual Test Article
Inconsistent MTT reduction- air bubble or
test article artifact?
EpiOcularTM
MTT Reduction Throughout the Tissue
(Hines et al, 2003)
Verification that MTT reduction occurs in all layers of the EpiOcular™
tissue construct and reasonably reflects cytotoxicity. EpiOcularTM tissue was
treated for the indicted time with 0.3% Triton X-100. MTT reduction is
dependent on the presence of glucose in the MTT medium.
EpiOcular™
Summary & Acknowledgments
•
•
•
•
Background
Protocol
Performance
Experience
The IIVS lab team