Validation
Plan
for
the
Validation
Plan
for
the
EDSP
Mammalian
EDSP
Mammalian
Tier
2
Assay
Tier
2
Assay
Presentation
to
the
Presentation
to
the
Endocrine
Disruptor
Methods
Validation
Subcommittee
Endocrine
Disruptor
Methods
Validation
Subcommittee
June
6,
2003
June
6,
2003
J.

J.
Kariya
Kariya
2
Overview
of
presentation
Overview
of
presentation

Validation
plan
Validation
plan

Concerns
Concerns

Questions
for
EDMVS
Questions
for
EDMVS
3
Validation
plan
Validation
plan

Accept
the
OPPTS
Guideline
Accept
the
OPPTS
Guideline
for
Reproductive
for
Reproductive
Toxicity,
with
additional
thyroid
endpoints,
as
Toxicity,
with
additional
thyroid
endpoints,
as
valid
for
EDSP
Tier
2
purposes.

valid
for
EDSP
Tier
2
purposes.


Include
clarifications
Include
clarifications
of
certain
procedures
and
of
certain
procedures
and
endpoints
already
in
the
Guideline
as
part
of
the
endpoints
already
in
the
Guideline
as
part
of
the
EDSP
assay.

EDSP
assay.


Ask
for
independent
Ask
for
independent
peer
review
peer
review
of
the
validity
of
of
the
validity
of
the
Guideline
and
clarifications
for
EDSP
the
Guideline
and
clarifications
for
EDSP
purposes.

purposes.
4
Additional
plans
Additional
plans

Encourage
one
Encourage
one­
generation
extension
generation
extension
of
F1
of
F1
when
indicated
by
Tier
1,
and
in
all
Tier
1
when
indicated
by
Tier
1,
and
in
all
Tier
1
bypasses.

bypasses.


Develop
additional
information
Develop
additional
information
on
the
on
the
relevance
and
reliability
of
the
one
relevance
and
reliability
of
the
one­

generation
extension,
for
possible
future
generation
extension,
for
possible
future
inclusion
in
the
EDSP
assay.

inclusion
in
the
EDSP
assay.
5
The
1998
The
1998
Reprotox
Reprotox
Guideline
is
Guideline
is
generally
accepted
as
valid 

generally
accepted
as
valid 


 
for
regulatory
risk
assessment
of
for
regulatory
risk
assessment
of
reproductive
toxicity
reproductive
toxicity

Reproductive
toxicity
can
be
"

Reproductive
toxicity
can
be
"
an
effect
produced
by
a
naturally
occurring
estrogen"
and
can
be
an
indicator
of
can
be
an
indicator
of
endocrine
effects
endocrine
effects


the
Guideline
is
valid
for
EDSP
the
Guideline
is
valid
for
EDSP
purposes.

purposes.
6
Additional
endpoints
Additional
endpoints

EDSTAC:
consider
specific
additional
EDSTAC:
consider
specific
additional
endpoints,
for
non
endpoints,
for
non­
reprotox
reprotox
endpoints
endpoints

SVTF:
adding
all
of
these
would
be
SVTF:
adding
all
of
these
would
be
impractical
and
in
some
cases
not
useful;

impractical
and
in
some
cases
not
useful;

focus
on
thyroid
focus
on
thyroid­
specific
endpoints
specific
endpoints

Relevance
of
most
of
the
thyroid
Relevance
of
most
of
the
thyroid­
specific
specific
endpoints
was
shown
in
the
PTU
study
endpoints
was
shown
in
the
PTU
study
which
EDMVS
reviewed
(
but
drop
T
which
EDMVS
reviewed
(
but
drop
T
3)
7
Table
1:
Additional
endpoints
Table
1:
Additional
endpoints
proposed
for
EDSP
purposes
proposed
for
EDSP
purposes
"
For
F1
and
F2
weanlings,
histopathological
examination
of
treatment­
related
abnormalities
noted
at
macroscopic
examination
should
be
considered,
if
such
evaluation
were
deemed
appropriate
and
would
contribute
to
the
interpretation
of
the
study
data."

(
Whole­
mount
not
specified)

Whole­
mount
histology
of
mammary
tissue
in
males,
triggered
if
abnormalities
are
seen
in
gross
examination.
AGD
only
for
F2
and
only
when
triggered
by
a
treatmentrelated
effect
in
F1
sex
ratio
or
sexual
maturation.
At
birth
(
PND
0)

Anogenital
distance,
all
animals
in
both
F1
and
F2,
at
birth
(
PND
2)
Differences
are:
addition
of
a
new
timepoint
for
examination
(
pnd
13);
and
number
of
animals
(
all,
vs.
3/
sex/
litter).

"
At
the
time
of
termination
or
death
during
the
study,
all
parental
animals
(
P
and
F1)
and
when
litter
size
permits
at
least
three
pups
per
sex
per
litter
from
the
unselected
F1
weanlings
and
the
F2
weanlings
should
be
examined
macroscopically
for
any
structural
abnormalities
or
pathological
changes.
Special
attention
should
be
paid
to
the
organs
of
the
reproductive
system."

Areola/
nipples:

what,
where,
how
many,

in
both
males
and
females,
F1
and
F2,
PND
13.
Also
at
necropsy
for
F1
only.
Declined
to
add
T3
on
the
basis
of
PTU
study
results.

Add
 
or
change
 
T4/
TSH
measurement
to
PND
21?

Not
in
current
Guideline
TSH,
T4,
thyroid
weight,
thyroid
histology,
all
at
necropsy
8
Clarifications
to
Clarifications
to
current
Guideline
current
Guideline

Table
2:
Specification
of
endpoints
for
Table
2:
Specification
of
endpoints
for
mammalian
two
mammalian
two­
generation
study"
(
in
handout)

generation
study"
(
in
handout)
9
Concerns
Concerns

Strain/
species
White
Paper
(
August)

Strain/
species
White
Paper
(
August)


Interlab
Interlab
variability
variability

Thyroid:
other
mechanisms;
sensitivity
Thyroid:
other
mechanisms;
sensitivity

Extension
of
F1:
additional
studies
Extension
of
F1:
additional
studies

Timing,
if
other
studies
are
needed
Timing,
if
other
studies
are
needed
10
Questions
for
EDMVS
Questions
for
EDMVS
#
1
of
5
#
1
of
5

Does
the
EDMVS
agree
that
the
additional
endpoints/
clarifications
proposed
for
the
2­
generation
assay
(
Table
1)
are
well­
characterized
and
that
further
validation
of
this
set
of
endpoints
for
use
in
EDSP
Tier
2
is
unnecessary?
11
Questions
for
EDMVS
Questions
for
EDMVS
#
2
of
5
#
2
of
5

Does
the
EDMVS
agree
that
the
endpoints
in
the
Tier
2
assay
will
allow
a
compound
to
be
identified
as
possibly
having
"
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen"
(
or
androgen/
anti­
androgen
or
thyroid
mimic/
inhibitor)
in
the
absence
of
Tier
1
data?
If
not,
what
other
endpoints
should
be
included,

or
what
supplemental
testing
would
be
appropriate?
12
Questions
for
EDMVS
Questions
for
EDMVS
#
3
of
5
#
3
of
5

Does
the
EDMVS
agree
that
the
procedures
and
endpoints
in
Table
2
of
the
handout
should
be
listed
explicitly,
to
ensure
adequate
examination?
13
Questions
for
EDMVS
Questions
for
EDMVS
#
4
of
5
#
4
of
5

If
EDMVS
advises
EPA
to
validate
additional
endpoints,


can
"
new"
endpoints
be
validated
separately
from
endpoints
already
in
the
reproductive
toxicity
assay?


is
it
necessary
to
validate
all
new
endpoints
in
a
twogeneration
study,
or
can
relevance
and
reliability
be
established
in
a
shorter
assay

how
many
laboratories
should
be
required
for
interlaboratory
comparability?


how
many
chemicals
per
mode
of
endocrine
activity
should
be
tested
in
validation?
(
e.
g.,
ER/
AR
binding,

each
step
of
steroidogenesis,
thyroid
hormone
transport
protein
binding,
thyroid
hormone
metabolism,
etc.)
14
Questions
for
EDMVS
Questions
for
EDMVS
#
5
of
5
#
5
of
5

Does
the
EDMVS
agree
that
the
onegeneration
extension
study
shows
increased
sensitivity
and
provides
greater
precision
in
dose/
response
assessment,

which
will
be
of
use
in
risk
assessment,

when
the
F1
animals
are
allowed
to
mature
to
PND
95
than
when
they
are
sacrificed
at
PND
21?
