Document ID: EPA-HQ-OPPT-2003-0027-0024
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-14T04:00Z

Prevalidation
Study
Plan
for
Sliced
Testes
Assay
Gary
Timm
Presented
to
EDMVS
August
20,
2003
Sliced
Testes
Assay
Prevalidation/
Validation
Study
Plan
°
June
EDMVS
Meeting
Discussed:

 
Objectives
of
validation
study
 
Data
interpretation
procedure
 
Basic
sliced
testes
protocol
 
Prevalidation
study
design
 
Reference
chemicals
 
Laboratory
selection
 
Validation
study
design
 
Measurements
of
reliability
 
Data
analysis
and
reporting
June
Proposal
for
Prevalidation
Studies
°
Conduct
prevalidation
studies
in
two
laboratories
 
Baseline
study
 
Pilot
study
 
Multichemical
study
June
Proposal
for
Prevalidation
Studies
(
2)

°
Baseline
study
 
Run
optimized
protocol
 
3
runs
without
hCG
 
3
runs
with
hCG
challenge
 
Measure
testosterone
formation
and
LDH
 
No
test
chemical
 
Three
replicate
studies
June
Proposal
for
Prevalidation
Studies
(
3)

°
Pilot
studies
of
positive
controls
 
Aminoglutethimide
(
positive
control)

 
Ethane
dimethanesulfonate
(
Leydig
cell
toxicant)

 
Two
labs
 
Three
replicates
June
Proposal
for
Prevalidation
Studies
(
4)

°
Multichemical
studies
 
9
chemicals
 
2
laboratories
 
2
replicate
studies
°
Validation
in
6
laboratories
would
begin
after
the
successful
conclusion
of
these
studies
EDMVS
Responses
to
Questions
1.
Does
the
EDMVS
agree
with
the
stated
objectives
and
data
interpretation
in
the
proposed
Validation
Study
Plan?
Yes,
but:

°
Concerns
expressed
about
the
assay
 
Accuracy
and
sensitivity
of
sliced
testes
assay
 
Potentially
better
assays
on
the
horizon;
don't
invest
too
heavily
in
sliced
testes
assay
 
Need
a
reliable
means
to
detect
Leydig
cell
toxicity
EDMVS
Responses
to
Questions
2.
Does
the
EDMVS
agree
with
the
structure
of
the
prevalidation
and
validation
Program?

°
Yes,
agreed
that
two
laboratories
was
a
reasonable
choice
for
prevalidation.

°
Concern
that
program
should
be
more
efficient
 
Do
we
need
to
look
at
data
from
multiple
time
points
or
is
end
of
assay
OK?

 
Use
data
from
prevalidation
to
pick
number
of
labs
for
validation,
do
not
rely
on
literature
values
EDMVS
Responses
to
Questions
°
Concerns
about
positive
control
chemicals
 
Aminoglutethimide
may
not
be
a
good
positive
control
chemical
 
EDS
may
not
be
a
good
cytotoxicant
reference
chemical
°
Concerns
about
reference
chemicals
 
Should
not
attempt
to
cover
every
known
mode
of
action,
use
phamacologic
agents
of
known
mode
of
action
 
Ideally
should
choose
only
chemicals
with
a
single
mode
of
action,
however
selectivity
seems
to
be
dose
dependent.

 
Should
include
several
cytotoxicants
during
prevalidation
EDMVS
Responses
to
Questions
3.
Have
we
selected
appropriate
measures
of
reliability?

 
Yes,
but
the
endpoints
being
used
for
the
power
calculations
need
to
be
clearly
stated
 
Need
to
verify
linearity
in
baseline
Testosterone
production
curve
 
Should
estimate
potency
of
test
chemicals
by
calculating
an
EC10
or
EC50.
EDMVS
Responses
to
Questions
4.
Are
the
number
of
replicates
taken
over
both
prevalidation
and
validation
sufficient
to
generate
robust
statistics?

 
Yes,
three
replicates
seems
sufficient
in
prevalidation.
If
the
variability
is
small,

consider
reducing
to
2
in
validation
 
Need
to
add
more
negative
chemicals
 
Negatives
should
include
evaluation
of
influence
of
pH
and
osmolality
EDMVS
Responses
to
Questions
5.
Should
dosing
solutions
be
prepared
centrally
or
on
site?

 
The
stock
solution
should
be
prepared
centrally
 
Dilutions
should
be
made
on
site
with
instructions
provided
by
the
lead
lab
or
chemical
repository
EDMVS
Responses
to
Questions
6.
Do
doses
need
to
be
confirmed
by
analytical
chemistry?

 
The
identity
and
purity
of
the
neat
test
substance
should
be
checked
by
the
chemical
repository
 
The
suitability
and
solubility
of
the
test
substance
should
be
checked
and
the
concentration
of
stock
solution
should
be
confirmed
by
the
repository
 
Labs
should
save
aliquots
of
dosing
solutions
should
for
analysis;
analysis
should
be
performed
only
"
for
cause"
EDMVS
Responses
to
Questions
6.
Naïve
labs/
trained
labs
issue
 
No
naïve
labs!
Training
is
necessary
to
minimize
interlaboratory
difference
in
techniques
 
Competency
of
labs
should
be
demonstrated
by
conducting
a
positive
control
prior
to
full
validation
effort
 
Incompetent
laboratories
should
be
excluded
Issue:
Leydig
cell
toxicity
°
Concerns:

 
Don't
want
false
positive
by
artificially
high
in
vitro
dosing
that
kills
cells
 
Don't
want
general
cytotoxicants
to
trigger
positive,
2
gen
is
wrong
follow­
up
for
general
cytotoxicants
 
May
be
"
red
herring."
If
cytotoxic
to
Leydig
cells
at
biologically
relevant
concentration
levels,
isn't
this
a
legitimate
positive?
Leydig
cell
toxicity
(
cont)

°
EPA
Options:

1.
Use
LDH
assay
for
cell
viability
2.
Use
3 ­
HSD
staining
specific
for
Leydig
cell
viability
instead
of
the
more
general
LDH
assay
Issue:
Choice
of
reference
chemicals
°
EPA
confirmed
by
search
of
literature
that
selectivity
is
not
unique
but
is
dose
dependent.

°
EPA
will
select
reference
chemicals
that
work
at
key
stages
of
steroidogenesis
°
EPA
will
run
4
known
cytotoxicants
to
select
the
best
positive
control
for
cytotoxicity
EPA
Listened
and
Redesigned
Prevalidation
Program
°
Focus
only
on
prevalidation;
validation
will
be
a
separate
work
assignment.

°
Redesigned
the
prevalidation
program:
lead
lab
and
3
participating
labs
°
Lead
lab:

 
Baseline,
testes
variability
study
 
Test
of
positive
control
 
Cytotoxicity
studies
 
Multichemical
studies
 
Training
of
participating
laboratories'
personnel
Redesigned
Prevalidation
Program
°
Participating
Laboratories
(
3)
and
lead
lab
 
Baseline
studies
in
triplicate
°
measure
testosterone
with
and
without
hCG
°
no
test
chemical
 
Positive
control
studies
°
Positive
control
(
aminoglutethimide)

°
Reference
cytotoxicant
Baseline
and
Testes
Variability
Study
(
Lead
Lab)

°
Purpose:

 
To
demonstrate
competence
of
lead
lab
using
optimized
assay
 
To
obtain
testosterone
production
data
as
a
function
of
time
in
the
absence
of
inhibitors
 
To
estimate
Leydig
cell
density
after
4
hr
incubation
 
To
evaluate
the
variability
in
contralateral
testes
fragments
to
decide
on
future
experimental
design:

°
Randomized
fragments
°
Block
design
using
single
testes
Baseline
and
Testes
Variability
Study
(
Lead
Lab)

°
Study
Design:

 
5
animals
 
24
runs
(
a
run
corresponds
to
an
incubation
vial
containing
1
testes
fragment)

 
Incubate
for
4
hours
with
and
without
hCG
 
3
replicate
studies
with
measurements
(
samples)
at
5
time
points
for
runs
1­
6.
(
A
replicate
study
is
an
independent
study.)

 
2
replicate
studies
with
measurements
at
2
and
4
hrs
(
incubation
times)
for
fragments
7­
24
Baseline
and
Testes
Variability
Study
23,24
1,2
B
5
Y
2,4
21,22
1,2
A
5
Y
2,4
19,20
1,2
B
4
Y
2,4
17,18
1,2
A
4
Y
2,4
15,16
1,2
B
3
Y
2,4
13,14
3,4
A
3
Y
2,4
11,12
1,2
B
2
Y
2,4
9,10
3,4
A
2
Y
2,4
7,8
1,2
B
1
Y
2,4
4­
6
2
A
1­
3
Y
0­
5
1­
3
1
A
1­
3
N
0­
5
Run
Fragment
number
Testis
Animal
hCG
Sample
time
Baseline
Study
Design
4­
6
3
Y
Media
1­
3
3
N
Media
Testes
fragment
Number
runs
hCG
Sample
Type
13­
15
3
Y
Media
+
AG
(
high)
10­
12
3
Y
Media
+
AG
(
med)
7­
9
3
Y
Media
+
AG
(
low)
4­
6
3
Y
Media­
vehicle
control
1­
3
3
N
Media­
vehicle
control
Testes
fragment
Number
runs
hCG
Sample
type
Positive
Control
Study
Design
Cytotoxicity
Study
Design
25­
27
3
Y
Toxicant
B­
high
22­
24
3
Y
Toxicant
B­
med
19­
21
3
Y
Toxicant
B­
low
16­
18
3
Y
Toxicant
A­
high
13­
15
3
Y
Toxicant
A­
med
10­
12
3
Y
Toxicant
A­
low
7­
9
3
Y
Positive
control
4­
6
3
Y
Media
control
1­
3
3
N
Media
control
Testes
fragment
Number
runs
hCG
Sample
type
Multichemical
Study
Design
34­
36
3
N
Chem
B­
high
31­
33
3
Y
Chem
B­
high
28­
30
3
Y
Chem
B­
med
25­
27
3
Y
Chemical
B­
low
22­
24
3
N
Chem
A­
high
19­
21
3
Y
Chem
A­
high
16­
18
3
Y
Chem
A­
med
13­
15
3
Y
Chemical
A­
low
10­
12
3
Y
Cytotox
control
7­
9
3
Y
Positive
control
4­
6
3
Y
Media
control
1­
3
3
N
Media
control
Testes
fragment
Number
runs
hCG
Sample
type
Reference
Chemicals
by
Mode
of
Action
Dimethoate
StAR
Lindane
cAMP
inhibitor
ER
binder
Bisphenol
A
cAMP
inhibitor
P450c17,
arom
Ketoconazole
P450SCC
Aromatase
Aminoglutethimide
P450SCC
Mode
2
Chemical
Mode
of
Action
Reference
Chemicals
by
Mode
of
Action
Aromatase
Econazole
P450c17
AR
antagonist
Flutamide
P450c17
Epostane
3 ­
HSD
ER
binder
Genestein
3 ­
HSD
Mode
2
Chemical
Mode
of
Action
Reference
Chemicals
by
Mode
of
Action
Ethane
dimethanesulfonate
Leydig
cell
toxicant
Mode
2
Chemical
Mode
of
Action
AR
binder
Vinclozolin
Negative
chemical
Finasteride
5 ­
reductase
Prochloraz
Aromatase
Summary
°
Lead
lab:

 
Baseline,
testes
variability
study
 
Test
of
positive
control
 
Cytotoxicity
studies
 
Multichemical
studies
 
Training
of
participating
laboratories'
personnel
°
Participating
Laboratories
(
3)
and
lead
lab
 
Baseline
studies
in
triplicate
 
Positive
control
studies
in
triplicate
°
Estimated
completion
4­
30­
2004