Document ID: EPA-HQ-OPPT-2003-0027-0028
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-13T04:00Z

Research
Triangle
Park,
North
Carolina
Optimization
of
the
Sliced
Testis
Steroidogenesis
Assay
Carol
S.
Sloan,
Amanda
B.
Goodman,
Susan
W.
Pearce
RTI
International
August
2003
This
study
was
performed
under
a
subcontract
to
Battelle
Laboratories,
Columbus,
Ohio
for
prime
contract
No.
68­
W­
01­

023
for
the
U.
S.
Environmental
Protection
Agency
Optimization
of
the
Sliced
Testis
Assay
This
was
Implemented
in
Two
Phases
Basic
Sliced
Testis
Assay

At
first
timepoint
 
designated
baseline
 
media
is
removed
and
discarded

Fresh
media
is
added
and
an
aliquot
is
collected

Half
of
the
samples
are
challenged
with
a
stimulant,
such
as
hCG

Aliquots
of
media
are
collected
at
1,
2,
3
and
4
hours
postchallenge

Medium
samples
are
analyzed
for
testosterone
concentration
in
a
RIA
assay

Medium
samples
are
analyzed
for
Lactate
Dehydrogenase
(
LDH)

as
a
measure
of
cell
viability
in
a
spectrophotometric
assay
Technical
Flow
Illustration
of
Sliced
Testis
Assay
Phase
I
Design
Results
of
Phase
I
Optimizations

Medium­
199
without
phenol
red

Gaseous
atmosphere
of
5%
CO2
/
95%
O2

Rats
that
were
11
weeks
of
age
showed
results
similar
to
those
that
were
15
weeks
of
age,
therefore
rats
11­
15
weeks
can
be
used
in
the
assay;
22
week
old
rats
exhibit
reduced
testosterone
production
Phase
II:
Primary
Experimental
Phase

Factors
that
may
affect
assay
performance
were
tested

These
factors
were
divided
into
four
sections
where
each
section
was
composed
of
factors
that
might
produce
interactions
Phase
II
Design
Phase
II

To
be
tested:

Incubation
Conditions
 
Incubation
Temperature
 
Incubation
Vessel
Type
 
Incubation
Shaker
Speed
 
Incubation
Media
Volume
 
hCG
Concentrations

Testes
Preparation
Conditions
 
Testicular
Fragment
Size
 
Time
Delay
before
starting
the
assay
preparation
 
Organ
Preparation
Technique
 
Sample
Aliquot
Volume
Conclusions
from
Incubation
Factors
Experiments

Optimal
temperature
for
incubation
was
approximately
36
º
C

Optimal
vessel
for
incubation
was
the
scintillation
vial

Optimal
Shaker
speed
was
between
135
and
200
rpm,

approximately
175
rpm

Optimal
media
volume
was
approximately
4­
5
mL

Optimal
hCG
concentration
for
stimulation
was
0.08
to
0.1
IU/
mL
Conclusions
from
Testes
Preparation
Experiments

Optimal
fragment
size
varied
from
100­
200
mg
with
stimulated
versus
non­
stimulated
assays

Time
delay
before
the
start
of
the
incubation
should
be
no
more
than
1
hour
from
the
time
of
testicular
tissue
removal
from
the
male

The
solution
that
the
testes
are
collected
in
may
be
either
cold
DPBS
or
cold
M­
199

The
sample
aliquot
size
removed
for
testing
should
be
around
0.5
mL
Coefficient
of
Variation
Values
(%)
for
Testosterone
Concentrations
by
Sampling
Timepoints
in
the
Optimized
Assay
hCG
Baseline
0.5
hr
1
hr
2
hr
3
hr
4
hr
8
hr
12
hr
24
hr
No
18.9
11.0
6.2
22.1
12.2
14.89
10.52
15.5
19.4
Yes
18.6
15.6
16.4
22.0
38.1
16.1
36.6
28.8
24.5
Means
of
LDH
Concentration
(
mU/
mg)

'

hCG
s
tim
ulat
ion
N
o
Ye
s
0
1000
2000
3000
4000
Timepoint
Base
line
T­
1
(
0.5
h
r)
T­
2
(
1
hr
)
T­
3
(
2
hr
)
T­
4
(
3
hr
)
T­
5
(
4
hr
)
T­
6
(
8
hr)
T­
7
(
12
h
r)
T­
8
(
24
h
r)
Means
of
Testosterone
Concentration
(
ng/
mg)

hCG
s
tim
ulation
No
Ye
s
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0
.1
0.11
0.12
0.13
0.14
Timepoint
Base
line
T­
1
(
0.5
hr
)
T­
2
(
1
hr
)
T­
3
(
2
hr
)
T­
4
(
3
hr)
T­
5
(
4
hr
)
T­
6
(
8
hr
)
T­
7
(
12
h
r)
T­
8
(
24
h
r)
Means
of
LDH
Concentration
(
mU/
mg)

No
hCG
Stimulation
Equilibration
Time
(
minutes)
15
30
45
60
90
120
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
LDH
Concentration
(
mU/
mg)

with
hCG
Stimulation
Eq
uilibra
tion
Time
(
m
inutes)
15
30
45
60
90
120
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
Time
point
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

No
hCG
Stimulation
Equi
libration
Time
(
minu
tes)
15
3
0
45
60
9
0
120
0
.005
0.01
0
0
.015
0.02
0
0
.025
0.03
0
0
.035
0.04
0
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

with
hCG
Stimulation
Equi
libration
Time
(
minu
tes)
15
30
45
60
90
120
0
0.05
0
.1
0.15
0
.2
0.25
0
.3
0.35
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
LDH
Concentration
(
mU/
mg)

No
hCG
Stimulation
Type
of
Injury
Induced
Chemical
Heat
Trauma
500
1000
1500
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
LDH
Concentration
(
mU/
mg)

with
hCG
Stimulation
Type
of
Injury
Induced
Chemical
Heat
Trauma
0
1000
2000
3000
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

No
hCG
Stimulation
Type
of
Injury
Induced
Heat
Trauma
0
0.05
0
.1
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

with
hCG
Stimulation
Type
of
Injury
Induced
Heat
Trauma
0
0.05
0.1
0.15
0.2
0.25
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

No
hCG
Stimulation
Vehicle
Type
DMSO
ETOH
Tween
0
.005
0.010
0
.015
0.020
0
.025
0.030
0
.035
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Means
of
Testosterone
Concentration
(
ng/
mg)

with
hCG
Stimulation
Vehicle
Type
DMSO
ETOH
Tween
0
0.02
0.04
0.06
0.08
Timepoint
Baseline
T­
1
(
1
hr)
T­
2
(
2
hr)
T­
3
(
3
hr)
T­
4
(
4
hr)
Summary

11­
15
week
old
rats
should
be
used

Media
199
without
phenol
red
is
the
preferred
media

Gaseous
atmosphere
of
5%
CO2
/
95%
O2

Optimal
temperature
for
incubation
was
approximately
36
º
C

Optimal
vessel
for
incubation
was
the
scintillation
vial

Optimal
Shaker
speed
was
approximately
175
rpm

Optimal
media
volume
was
approximately
4­
5
mL

Optimal
hCG
concentration
for
stimulation
was
0.08
to
0.1
IU/
mL

Optimal
fragment
size
varied
from
100­
200
mg

Time
delay
before
the
start
of
the
incubation
should
be
no
more
than
1
hour
from
the
time
of
testicular
tissue
removal

The
solution
that
the
testes
are
collected
in
may
be
either
cold
DPBS
or
cold
M­

199

The
sample
aliquot
size
removed
for
testing
should
be
around
0.5
mL
Points
to
Ponder

All
of
these
assays
were
performed
on
control
rat
testes

No
assays
included
any
test
chemicals
except
for
possible
vehicles
that
may
be
used
in
the
pre­
validation
and
validation
assays

It
may
be
necessary
to
measure
specific
cell
viability
(
LDH
is
not
cell
specific)


Use
of
beta­
HSD
(
an
enzyme
specific
to
Leydig
cells)
staining
(
RTI
International)

Ms.
V.
I..
Wilson

Ms.
L.
B.
Pelletier

Ms.
N.
M.
Kuney

Ms.
S.
W.
Pearce

Ms.
K.
D.
Vick

Ms.
L.
McDonald

Ms.
A.
J.
Parham

Mr.
M.
D.
Crews

Ms.
A.
B.
Goodman

Ms.
D.
B.
Bynum

Mr.
C.
Andrew
Clayton

Ms.
Margaret
Zeller
Byron

Ms.
Bonnie
Hamby

Mr.
F.
N.
Ali

Dr.
D.
B.
Feldman,
DVM,
ACLAM,

Veterinarian
Quality
Assurance

Ms.
M.
D.
Phillips
(
RTI)

Mr.
R.
Patterson,
QA
Consultant

Ms.
K.
Cummings
(
ILS)

Battelle

Dr.
Jerry
D.
Johnson

Dr.
David
P.
Houchens

Dr.
Paul
Feder
EPA

Mr.
Gary
Timm

Dr.
Jerome
Goldman
ACKNOWLEDGEMENTS