Mammalian
2­
generation
assay
validation:

History,
Plan,
and
Questions
for
the
Endocrine
Disruptor
Methods
Validation
Subcommittee
June
6,
2003
A.
Validation
History
B.
Validation
Plan
C.
Questions
for
the
EDMVS
D.
Tables
1
and
2
(
Endpoints
for
consideration)
E.
Attachment
1:
Mammalian
Working
Group
notes
F:
Attachment
2:
SVTF
notes
G:
Attachment
3:
Mammalian
Working
Group
reconsideration
notes
1Historical
information
about
the
SVTF
can
be
found
at
http://
www.
epa.
gov/
scipoly/
oscpendo/
history/
standards.
htm
2Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
Final
Report:
Volume
1.
August
1998.
Table
5.3:
Mammalian
Tier
2
Test
Endpoints.
pp.
5­
56
and
5­
57
(
http://
www.
epa.
gov/
scipoly/
oscpendo/
history/
chap5v14.
pdf
)
.
See
also
Federal
Register
63(
248):
71542­
71568,
Dec.
28,
1998,
USEPA,
Endocrine
Disruptor
Screening
Program;
Proposed
Statement
of
Policy,
Table
2.
(
http://
www.
epa.
gov/
scipoly/
oscpendo/
endofr.
htm
)

3SVTF
members
present
were:
T.
Colborn
and
R.
Liroff
(
WWF),
P.
DeFur
(
VCU),
W.
Kelce
(
Monsanto),
R.
Miller
(
Dow),
T.
Schettler
(
PSR),
W.
Owens
(
P&
G),
T.
Kubiak
(
USFWS),
J.
Stevens
(
Novartis),
R.
Combes
(
for
PETA,
HS,
and
DDF),
L.
Touart
(
OPP),
W.
Stokes
(
NIEHS),
K.
Hamernik
(
OPP),
J.
Seed
(
OPPT),
R.
Kavlock
(
NHEERL),
P.
Fenner­
Crisp
(
OPP),
and
G.
Timm
(
OSCP).
Also
present
were
D.
Roush
(
AAAS
Fellow),
T.
Green
(
VCU),
and
J.
Kariya
(
OSCP).

Page
3
of
31
Validation
History
EPA's
approach
to
validating
a
mammalian
2­
generation
assay
for
use
in
the
Endocrine
Disruptor
Screening
Program
(
EDSP)
follows
a
path
prepared
by
the
Standardization
and
Validation
Task
Force
(
SVTF)
1
in
January/
February
2000.

Mammalian
Workgroup
proposal
The
SVTF's
Mammalian
Workgroup
(
MWG)
prepared
a
proposal
for
validation
of
a
mammalian
Tier
2
assay
for
the
EDSP,
which
is
summarized
in
Attachment
1.

The
MWG
concluded
the
following:
8.
Adding
all
the
additional
endpoints
recommended
by
EDSTAC2
is
not
feasible
in
the
laboratory.
Decisions
must
be
made
about
which
endpoints
are
most
appropriate
to
add.
9.
Given
the
history
of
use
of
the
2­
generation
assay,
especially
after
the
new
Guidelines
went
into
effect
in
1998,
validation
in
multiple
laboratories
is
not
necessary.

The
MWG
discussed,
but
did
not
come
to
a
conclusion
about,
whether
any
further
validation
studies,
if
needed,
could
be
limited
to
new
endpoints
or
should
include
all
endpoints,
both
"
old"
and
"
new".

Standardization
and
Validation
Task
Force
comments
The
SVTF3
reviewed
the
MWG's
proposal.
Specific
comments
are
listed
in
Attachment
2.
These
concerns
were
to
be
considered
by
the
MWG,
whose
final
recommendations
would
then
be
reviewed
by
the
SVTF
so
that
work
could
proceed.

Mammalian
Workgroup
reconsideration
The
MWG
discussed
the
SVTF's
comments.
The
MWG
this
time
recommended
an
in­
utero­
through­
lactation
assay
and
a
two­
generation
extension
study.
See
Page
4
of
31
Attachment
3.

Dissolution
of
the
SVTF
and
its
Workgroups
A
few
days
after
the
Mammalian
Workgroup's
conference
call,
the
SVTF
and
its
Workgroups
were
dissolved
due
to
a
concern
that
the
SVTF
did
not
comport
with
the
requirements
for
a
Federal
Advisory
Committee.
Thus
it
was
not
possible
to
take
the
MWG's
recommendations
back
to
the
SVTF
as
planned.

EPA
decisions
After
much
internal
EPA
discussion
(
ORD
and
OSCP)
it
was
decided
that
a
study
to
address
the
question
of
whether
there
are
effects
that
would
be
seen
at
PND
45/
60
that
cannot
be
evaluated
at
PND
21
did
indeed
need
to
be
done.
A
full
2­
generation
study,
however,
was
not
considered
necessary;
a
one­
generation
study
would
suffice.
Details
of
the
study
were
decided
upon
at
this
and
a
subsequent
meeting
which
included
the
NIEHS
Project
Officer
for
a
contract
under
which
the
study
might
be
accomplished.
(
OSCP's
contract
was
not
available
yet.)
It
was
agreed
that
in
the
future,
additional
chemicals
would
need
to
be
tested.

In
addition,
it
was
agreed
to
move
forward
with
the
full
2­
generation
study
of
propylthiouracil
that
the
SVTF
had
endorsed.
4OPPTS
Health
Effects
Test
Guideline
870.3800:
Reproduction
and
Fertility
Effects.
EPA
712­
C­
98­
208.
August,
1998.
(
http://
www.
epa.
gov/
opptsfrs/
OPPTS_
Harmonized/
870_
Health_
Effects_
Test_
Guidelines/
Series/
870­
380
0.
pdf
)

Page
5
of
31
Validation
Plan
1.
Accept
the
existing
mammalian
2­
generation
reproductive
toxicity
assay4
as
validated
based
on
OECD
acceptance,
review
by
SAP
for
OPP
use,
acceptance
by
the
SVTF,
and
use
of
the
Guideline
in
40+
studies
for
OPP.

2.
Accept
the
additional
endpoints/
clarifications
in
Table
1
as
validated
based
on
the
PTU
study
presented
to
the
EDMVS
in
July,
2002
for
the
thyroid
endpoints,
and
on
experience
with
and
SVTF
review
of
the
other
endpoints.

3.
Accept
the
clarifications
in
Table
2
as
validated
because
they
are
simply
more
detailed
descriptions
of
the
items
covered
in
the
general
2­
generation
reproductive
toxicity
assay
that
is
generally
recognized
as
validated.

4.
Continue
to
develop
information
that
will
allow
quantification,
using
an
actual
example
rather
than
simple
power
calculations,
of
the
usefulness
of
extending
additional
members
of
the
F1
generation
to
PND
45/
60
or
beyond.
a.
Weak
antiandrogen
(
malformations
plus
histology)
b.
Weak
estrogen
(
malformations
plus
histology)

Until
such
information
is
available,
encourage
the
optional
extension
of
one
or
more
additional
F1
animals
per
sex
per
dose
to
adulthood
in
all
cases
where
Tier
1
is
bypassed,
and
of
one
or
more
additional
F1
animals
per
dose
of
the
appropriate
sex(
es)
where
Tier
1
information
indicates
interaction
of
the
test
chemical
with
the
estrogen,
androgen,
and/
or
thyroid
systems.
Page
6
of
31
Page
7
of
31
Questions
for
the
EDMVS
1.
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?

2.
Does
the
EDMVS
agree
that
the
endpoints
in
the
Tier
2
assay
(
including
the
endpoints
proposed
in
Table
1)
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?
(
That
is,
for
chemicals
which
voluntarily
bypass
Tier
1,
will
a
Tier
2
assay
that
includes
these
endpoints
allow
identification
of
a
chemical
as
meeting
the
requirements
of
FQPA?)
If
not,
what
other
endpoints
should
be
included,
or
what
supplemental
testing
would
be
appropriate?

3.
Does
the
EDMVS
agree
that
the
procedures
and
endpoints
in
Table
2
should
be
listed
explicitly
(
even
though
already
covered
in
the
Guideline),
to
ensure
adequate
examination?

4.
If
EDMVS
advises
EPA
to
validate
additional
endpoints,
a.
can
"
new"
endpoints
be
validated
separately
from
endpoints
already
in
the
reproductive
toxicity
assay?
(
I.
e.,
is
it
scientifically
acceptable
to
examine
the
relevance
and
reliability
of
endpoints
or
must
we
validate
the
entire
assay?)
b.
is
it
necessary
to
validate
all
new
endpoints
in
a
2­
generation
study,
or
can
relevance
and
reliability
be
established
in
a
shorter
assay,
such
as
a
onegeneration
protocol
or
an
in­
utero­
through­
lactation
protocol?
c.
how
many
laboratories
should
be
required
for
interlaboratory
comparability?
d.
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.)

5.
Does
the
EDMVS
agree
that
the
one­
generation
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
90
than
when
they
are
sacrificed
at
PND
21?
Page
8
of
31
Page
9
of
31
Table
1
Proposed
additions
to
and
clarifications
of
endocrine
endpoints
for
the
Tier
2
mammalian
assay
Based
on
discussions
from
Standardization
and
Validation
Task
Force
and
EDMVS.

Start
with
OPPTS
Guideline
870.3800
(
2­
generation
reproductive
toxicity
assay)

Add
Proposed
addition
Current
Guideline
Comments/
Questions
Anogenital
distance,
all
animals
in
both
F1
and
F2,
at
birth
(
PND
2)
AGD
only
for
F2
and
only
when
triggered
by
a
treatment­
related
effect
in
F1
sex
ratio
or
sexual
maturation.
At
birth
(
PND
0)

Areola/
nipples:
what,
where,
how
many,
in
both
males
and
females,
F1
and
F2,
PND
13.
Also
at
necropsy
for
F1
only.
"
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."
Differences
are:
addition
of
a
new
timepoint
for
examination
(
pnd
13);
and
number
of
animals
(
all,
vs.
3/
sex/
litter).

TSH,
T4,
thyroid
weight,
thyroid
histology,
all
at
necropsy
Not
in
current
Guideline
Declined
to
add
T3
on
the
basis
of
PTU
study
results.

Add
 
or
change
 
T4/
TSH
measurement
to
PND
21?
Page
10
of
31
Whole­
mount
histology
of
mammary
tissue
in
males,
triggered
if
abnormalities
are
seen
in
gross
examination.
"
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)
Clarify
Proposed
clarification
Current
Guideline
Comments/
Questions
Testis
location
at
necropsy
(
descended/
undescended
attached/
floating)
"
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."

Malformation,
agenesis,
or
inappropriate
presence
of
any
of
the
sex
organs
(
e.
g.,
prostate
agenesis,
presence
of
uterus
in
male)
See
above
Number
of
days
until
the
plug
is
observed
should
be
analyzed
as
an
indirect
indicator
of
sexual
behavior.
"
Each
day
[
during
the
mating
period],
the
females
should
be
examined
for
presence
of
sperm
or
vaginal
plugs."
Current
Guideline
focus
is
on
identifying
Day
0
of
pregnancy,
not
analyzing
days
until
plug.
Page
11
of
31
Not
resolved
Proposed
addition
Current
Guideline
Comments/
Questions
Prostate
weight
by
lobe
(
ventral
and
dorsolateral)
Whole
prostate
weight
Lobe
weights
would
be
in
addition
to
total
weight
Histology
on
testis/
ovary
for
2
females
and
2
males
per
litter
of
the
F1
generation
(
that
is,
the
pair
used
for
breeding
the
F2,
and
one
additional
pair)
(=
40
animals
per
sex,
assuming
20
litters)
Histopathology
for
"
ten
randomly
chosen
high
dose
and
control
P
and
F1
animals
per
sex,
for
those
animals
that
were
selected
for
mating.
Organs
demonstrating
treatment­
related
changes
should
also
be
examined
for
the
remainder
of
the
high­
dose
and
control
animals
and
for
all
parental
animals
in
the
low­
and
mid­
dose
groups.
Additionally,
reproductive
organs
of
the
low­
and
mid­
dose
animals
suspected
of
reduced
fertility...
should
be
subjected
to
histopathological
evaluation."
Proposed
addition
would
apply
to
all
dose
groups?

F1
dosing
to
be
continued
to
necropsy
Not
clear
if
this
was
intended
to
be
a
version
of
the
one­
gen
extension.
It
was
separate
from
the
onegen
extension
proposal.

Items
considered
and
specifically
declined
to
add
6.
Sex
hormones
(
androgens,
estrogens,
LH,
FSH)
7.
Accessory
sex
organ
function.
8.
Neurobehavioral
toxicity
(
declined
for
practicality
reasons,
not
a
reflection
of
appropriateness).
May
wish
to
require
a
separate
request
for
neurobehavioral
study
if
indicated
by
other
available
information.
9.
Cageside
observation
of
sexual
behavior.
(
Days
until
vaginal
plug
is
sufficient
indirect
indicator
of
mating
behavior.)
10.
Age
at
testis
descent.
11.
Pinna
detachment,
eye
opening.
12.
Brain
histology.
Page
12
of
31
5Op.
cit.,
p.
5­
47
Page
13
of
31
Table
2:
Specification
of
endpoints
for
mammalian
two­
generation
study
The
following
specific
endpoints
are
already
covered
by
the
current
Reproductive
Toxicity
Guideline
in
the
sections
quoted
in
Table
1
above,
but
are
listed
for
greater
clarity
and
to
ensure
adequate
attention
to
important
details.

EDSTAC
suggested
that
it
may
be
appropriate
to
"
tailor"
Tier
2
tests
if
previously
collected
information
of
appropriate
quality
indicates
no
interaction
with
one
or
more
of
the
endocrine
systems
examined
in
the
Endocrine
Disruptor
Screening
Program5.
Using
EDSTAC's
example:
in
cases
where
Tier
1
results
are
available
and
weight­
of­
theevidence
evaluation
indicates
that
interaction
with
the
thyroid
system
is
unlikely,
it
might
be
acceptable,
for
EDSP
purposes,
to
forego
the
collection
of
information
related
solely
to
thyroid
effects
in
the
Tier
2
mammalian
assay.
The
following
list
of
clarifications
may
be
an
appropriate
starting
point
when
such
tailoring
is
being
considered.

Suggestions
for
portions
of
in­
life
and
necropsy
procedures
are
included
to
show
how
important
endpoints
can
be
measured.

MALES:

Necropsy
after
puberty
1.
body
weight,
any
unusual
malformations
or
anomalies,
euthanize
2.
shave
ventral
surface
from
inguinal
region
to
neck
and
count
nipples
and
areolas
(
observer
blind
to
treatment),
record
position
of
areolas
and
nipples
3.
check
animals
for
hypospadias,
epispadias,
cleft
phallus
and
measure
AGD
4.
note
if
testes
obviously
undescended
5.
note
if
inguinal
region
soiled
with
urine
6.
note
if
prepuce
partially
or
entirely
detached
from
glans
penis,
especially
if
a
persistent
thread
of
tissue
is
present
along
frenulum
7.
for
animals
on
study
past
puberty,
record
age
at
onset
of
preputial
separation
and
age
at
complete
PPS
(
if
different).
Also
record
weights
at
PPS.
8.
Weights
of
animals
after
weaning,
at
least
twice
a
week.

Internal
endpoints
9.
location
of
each
testis
(
scrotal,
abdominal,
gubernaculum
attached
to
abdominal
wall)
10.
gubernacular
cords,
present
or
absent,
and
length
in
mm
11.
note
if
present,
cranial
suspensory
ligaments
12.
note
if
testes
are
small,
absent,
fluid
filled,
enlarged,
appear
infected
or
other
13.
note
if
epididymides
are
small,
absent,
or
infected
(
record
region
of
effects)
Page
14
of
31
14.
note
if
ventral
prostate
is
small,
absent
or
infected
15.
note
if
dorsolateral
prostate
is
small,
absent,
or
infected
16.
note
if
seminal
vesicles
are
small,
absent,
infected,
or
one
side
larger
than
the
other
17.
note
if
coagulating
glands
are
small
absent,
infected,
one
side
larger
than
the
other,
or
detached
from
seminal
vesicles
18.
note
if
kidneys
display
hydronephrosis,
calcium
deposits
19.
note
presence
of
hydroureter
20.
note
presence
of
bladder
stones
or
blood
in
bladder
Weights
and
histology
21.
each
testis
individually
(
histo
for
both
or
one
for
sperm
numbers
and
one
histo
)
22.
each
corpus
plus
caput
epididymis
(
histo
for
both
or
one
for
sperm
numbers
and
one
for
histo)
23.
each
cauda
epididymis
(
histo
for
both
or
one
for
sperm
numbers
and
one
for
histo)
24.
entire
seminal
vesicle,
plus
coagulating
glands
with
fluid
as
a
unit,
if
possible
25.
entire
ventral
prostate,
if
possible
(
histo)
26.
each
kidney
27.
paired
adrenals
28.
liver
29.
levator
ani
plus
bulbocavernosus
30.
Cowper's
glands
as
a
pair,
if
possible
31.
glans
penis
32.
dorsolateral
prostate
(
histo)
33.
brain
34.
pituitary
35.
thyroid
weight
after
fixation
and
histology
36.
heart
weight
and
histo
if
the
chemical
is
suspect
antithyroid
FEMALES
Necropsy
37.
body
weight,
any
unusual
malformations
or
anomalies,
euthanize
38.
shave
ventral
surface
from
inguinal
region
to
neck
and
count
nipples
and
areolas
(
observer
blind
to
treatment),
record
position
of
areolas
and
nipples
39.
check
animals
for
female
rat
hypospadias,
cleft
phallus
and
measure
AGD,
AVD
40.
note
if
vaginal
opening
not
present.
41.
note
if
vaginal
thread
is
present
42.
note
if
mammary
tumors
are
present
(
histo
if
present)
Note:
females
can
have
the
estrous
cycle
staged
and
all
killed
on
the
same
day
of
estrous,
but
if
not
a
terminal
vaginal
smear
should
be
taken
to
distinguish
proestrus
(
when
the
uterus
is
large)
from
other
stages
(
when
it
is
smaller)

Internal
observations
Page
15
of
31
43.
position,
size
and
color
of
ovaries
44.
presence
of
cranial
suspensory
ligaments
45.
presence
of
follicular
cysts
on
ovary
or
atrophy
of
ovary
46.
absence
of
lower
vagina
47.
uterine
abnormalities,
including
bi­
or
unilateral
agenesis
of
oviducts
of
uterine
horns,
infections,
hydrometrocolpos,
etc.
48.
presence
of
any
male
tract
tissues,
including
ventral
prostate,
seminal
vesicles,
Cowper's
glands,
levator
ani
or
bulbocavernosus
muscles.
(
Save
any
for
histological
confirmation)

Necropsy
weights
and
histology
49.
body,
liver,
kidneys,
adrenals,
brain,
pituitary,
heart
(
if
antithyroidal)
weights
and
histology
on
abnormalities.
50.
ovaries
(
histo)
51.
oviducts
(
histo
not
weight,
if
abnormal)
52.
uterine
weight
and
histology
Page
16
of
31
Page
17
of
31
Attachment
1
SVTF/
Mammalian
Workgroup
proposal:
endpoints
to
include
in
a
Tier
2
mammalian
assay,
and
studies
to
be
performed
in
validation
January
6,
2000
Page
18
of
31
1Asterisked
items
are
included
in
the
feasibility
study
but
their
usefulness
will
be
re­
evaluated
after
the
feasibility
study.

Page
19
of
31
Mammalian
Workgroup
proposal:
endpoints
to
include
in
a
Tier
2
mammalian
assay,
and
studies
to
be
performed
in
validation
January
6,
2000
6.
Use
the
EPA/
OPPTS
revised
Reproductive
Toxicity
Test
Guideline
(
1998)
as
base.

7.
TSH,
T3*
1,
T4,
thyroid
weight*,
and
thyroid
histology
for
P
and
F1
breeders,
both
control
and
high
dose.

8.
(
Neurobehavioral
study
done
separately
if
needed.)

9.
AGD
for
both
F1
and
F2
(
not
triggered).

10.
Testis
location
at
necropsy
(
descended/
undescended,
attached/
floating)*.
Age
at
descent
is
not
needed.

11.
Note
malformation,
agenesis,
or
inappropriate
presence
of
any
of
the
sexual
organs
(
e.
g.,
prostate
agenesis,
presence
of
uterus
in
male).

12.
Prostate
weights
both
whole
and
by
lobe*
(
ventral
and
dorsolateral).

13.
Nipple
retention/
areola:
"
where,
what,
and
how
many"
a.
F1
and
F2,
day
13,
males
and
females.
b.
whole­
mount
histopathology
of
mammary
tissue
if
abnormalities
seen
c.
nipple
observation
at
necropsy
in
F1
males
and
females.

14.
No
measurement
of
sex
hormone
levels
(
androgen,
estrogen,
LH,
FSH).

15.
Number
of
days
until
vaginal
plug
is
sufficient
as
an
indicator
of
"
mating
and
sexual
behavior".
Behavioral
observation
per
se
is
not
required.

16.
Carry
all
F1
females
to
PND
45,
and
all
F1
males
to
PND
60.
Examine
all
internal
and
reproductive
tract
tissues
for
structural
malformations/
dysgenesis.
(
Checklist
to
be
included.)

17.
Histology
on
testis/
ovary
for
2
females
and
2
males
per
litter
of
the
F1
generation
(
viz.,
the
pair
used
for
breeding
F2,
and
one
additional
pair).
Dosing
to
be
included
between
PND
21
and
PND
45/
60
for
both
pairs.
2Feasibility
was
placed
at
higher
priority
than
sensitivity
because
practicality
was
seen
to
be
the
greatest
potential
obstacle
to
implementation
of
new
endpoints.
Sensitivity
was
also
regarded
as
critical,
however.

Page
20
of
31
18.
Sprague­
Dawley
rats
should
be
used
for
all
studies
as
this
is
the
strain
for
which
most
historical
2­
generation
study
data
are
available.

19.
First
perform
two
feasibility
studies:
a.
Methoxychlor
(
tech
grade,
50
mg/
kg),
vinclozolin
(
100
mg/
kg),
ethinyl
estradiol
(
dose
to
be
determined),
each
in
corn
oil
and
by
gavage.
(
May
need
to
be
by
diet
for
practicality
reasons.)
b.
Propylthiouracil
via
drinking
water,
3
doses
+
water
control.

20.
If
the
additional
endpoints
are
practical,
examine
the
sensitivity
of
the
additional
nonthyroid
endpoints
using
three
doses
for
vinclozolin,
methoxychlor,
and
ethinyl
estradiol.
2
This
sensitivity
test
might
be
accomplished
by
a
protocol
shorter
than
a
full
2­
generation
protocol.
Page
21
of
31
Attachment
2
Standardization
and
Validation
Task
Force
comments
on
Mammalian
Workgroup
proposal
for
mammalian
Tier
2
assay
endpoints
and
validation
studies
January
12,
2000
Page
22
of
31
Page
23
of
31
Attachment
2
Standardization
and
Validation
Task
Force
comments
on
Mammalian
Workgroup
proposal
for
mammalian
Tier
2
assay
endpoints
and
validation
studies
January
12,
2000
1.
The
Working
Group
should
explicitly
state
that
Developmental
Neurobehavioural
Tests
were
not
included
in
this
version
as
they
were
considered
to
make
the
entire
test
unfeasible.
It
should
be
noted
that
this
was
a
practicality
issue,
and
not
an
appropriateness
issue.
If
these
tests
are
needed,
they
should
be
run
as
separate
tests.

2.
The
nipple
retention
observation
(#
8
in
the
MWG
proposal)
should
include
where
the
nipples
are
located,
as
well
as
presence
and
number.
This
should
be
observed
in
both
males
and
females.

3.
There
was
concern
that
excluding
sex
hormone
levels
(#
9)
in
lab
animals
(
because
of
high
variability
and
expense)
would
implicitly
negate
the
utilization
of
these
endpoints
for
animals
in
the
field.
There
was
also
concern
about
the
use
of
hormone
levels
(
including
thyroid)
as
endpoints
in
themselves,
since
normal
levels
of
hormones
change
throughout
development.
(
Some
evidence
indicates
that
thyroid
levels
are
affected
only
at
birth,
and
adjust
later
in
development.)
However,
as
blood
is
being
collected
for
the
thyroid
levels,
it
was
suggested
that
blood
be
saved
for
later
analysis
of
sex
hormone
levels
as
well.
This
was
indicated
as
one
point
where
clarification
concerning
rationalization
was
needed
from
the
working
group.

4.
Indicate
that
the
measurement
of
sexual
behavior
(#
10)
is
indirect,
and
that
no
observation
is
being
required.

5.
#
11:
It
was
recommended
that
a
flow
chart
be
created
indicating
where
all
the
animals
will
be
utilized
for
the
2­
gen.
The
general
consensus
was
that
the
recommendation
to
carry
the
animals
out
to
PND
45
and
60
(
female
and
male
respectively)
was
unnecessary.
It
was
suggested
that
this
extension
represented
a
revision
of
the
entire
2­
gen
protocol,
and
was
not
appropriate
for
the
SVTF
to
get
into.
Not
only
could
this
create
feasibility
problems,
but
it
would
not
appropriately
address
the
concerns
of
potential
`
minor
and
occasional'
long
term
effects
(
i.
e.,
would
these
small
percentage
effects
be
seen
significantly
in
this
number
of
animals?).
This
extension
would
also
create
the
need
for
inter­
laboratory
comparison.
There
are
no
historical
2­
gen
studies
carried
out
to
45/
60
days,
and
comparison
to
21
day
2­
gen
studies
was
considered
insufficient.
In
addition,
this
would
place
a
large
burden
on
animal
usage.
The
working
group
was
asked
to
find
a
use
for
the
potentially
500­
600
animals
(
estimated
from
breeding
120
pairs)
culled
from
the
initial
litters
if
this
extension
is
carried
out.
Page
24
of
31
One
suggestion
was
to
carry
some
of
these
animals
to
the
normal
PND
21
(
at
which
time
they
would
be
sacrificed).
This
could
serve
for
historical
2­
gen
inter­
laboratory
comparisons
as
mentioned
in
the
previous
paragraph.
It
was
suggested
that
perhaps
the
45/
60
day
extension
would
be
better
explored
in
the
in
utero/
lactation
protocol,
as
an
extension
is
already
incorporated
into
that
protocol.

6.
#
14:
The
PTU
study
was
authorized
to
continue
forward.
However,
the
study
utilizing
one
dose
each
of
methoxychlor,
vinclozolin
and
ethinyl
estradiol
(+
control)
was
questioned.
Two
(
and
possibly
three)
complete
chemical
studies
with
three
doses
each
was
considered
a
better
alternative.
These
complete
studies
should
include
E,
A
and
T
effects.
Antiandrogens
should
also
be
addressed.
A
third
study
could
potentially
be
done
in
another
lab
supported
by
industry
if
EPA
lab
capacity
is
insufficient.
DDE
(
antiandrogen)
was
suggested
as
one
chemical.
Perchlorate
is
being
done
by
the
Department
of
Defense.

The
doses
of
vinclozolin
and
methoxychlor
were
considered
to
hit
different
areas
of
their
respective
dose­
response
curves.
It
might
be
appropriate
to
do
rangefinding
studies
before
launching
into
the
full
studies.
In
addition,
dosing
the
animals
by
diet,
rather
than
by
gavage,
would
allow
easier
comparison
with
previous
2­
gen
studies.

If
the
45/
60
day
extension
is
excluded,
the
only
significant
proposed
addition
to
the
2­
gen
is
the
thyroid
endpoints.
Validation
of
these
endpoints
using
multiple
chemicals
and
multiple
labs
might
then
be
feasible
if
only
the
new
endpoints
need
to
be
included
in
a
validation
study.
Page
25
of
31
Attachment
3
Mammalian
Working
Group's
response
to
SVTF
comments
on
mammalian
two­
generation
assay
February
2,
2000
Page
26
of
31
Page
27
of
31
Attachment
3
Mammalian
Working
Group's
response
to
SVTF
comments
on
mammalian
two­
generation
assay
February
2,
2000
Difference
in
design
stems
from
difference
in
purpose
of
study
The
Workgroup
felt
that
the
SVTF's
comments
stem
from
a
different
understanding
of
the
purpose
of
the
demonstration
study
than
what
the
Workgroup
intended.
The
Workgroup's
intention
was
to
do
an
initial
study
to
determine
how
many
F1
animals
need
to
be
carried
past
PND
21
in
order
to
pick
up
low­
incidence
effects
which
have
been
identified
in
nonguideline
research
investigations
and
which
suggest
the
potential
for
adverse
consequences.
Effects
such
as
prostate
agenesis
and
changes
in
sperm
production
cannot
be
observed
at
PND
21
and
require
extension
of
the
observation
period
to
manifest
themselves.
This
initial
study
is
not
intended
to
be
used
for
standardization/
prevalidation.
The
Workgroup
agreed
at
this
meeting
that
it
would
fall
to
a
later
study
to
demonstrate
the
practicality
of
carrying
whatever
number
of
F1
animals
is
eventually
determined
to
be
necessary.

The
SVTF,
on
the
other
hand,
had
focused
on
the
study
as
simply
a
demonstration
of
the
practicality
of
modifying
the
2­
generation
reproduction
study
to
include
additional
endocrine
endpoints
 
that
is,
as
a
standardization/
prevalidation
exercise.
The
Task
Force
was
concerned
about
the
inclusion
of
such
a
major
change
to
the
protocol
in
a
standardization/
prevalidation
exercise.
The
SVTF
noted
that
carrying
over
a
large
number
of
F1
animals
past
PND
21
would
not
be
a
reasonable
test
of
practicality
of
the
final
study
design
if
the
high
number
of
carryover
animals
were
not
in
the
final
design.

Reiteration
of
need
for
extension
from
PND
21
to
PND
45/
60
for
all
F1
animals
in
one
study
The
Workgroup
reaffirmed
its
belief
that
carrying
only
one
animal
per
sex
of
the
F1
generation
past
PND
21
provides
insufficient
power
to
detect
effects
with
low
incidence.
For
example,
it
would
not
be
able
to
pick
up
effects
that
have
a
10%
incidence.
Although
this
is
termed
"
low"
incidence,
the
Workgroup
noted
that
a
10%
incidence
of
reproductive
effects
would
probably
be
considered
extremely
high
in
a
regulatory
setting,
and
thus
one
needs
to
be
able
to
detect
effects
that
are
seen
even
less
frequently
than
10%.
Carrying
all
F1
animals
out
to
PND
45/
60
will
allow
better
determination
of
the
incidence
of
certain
effects
in
compounds
of
known
toxicity,
and
will
thus
allow
calculation
of
the
number
of
animals
needed
to
detect
such
effects
in
compounds
of
unknown
toxicity.

Alternative:
use
an
in
utero
assay
instead
of
a
2­
gen
assay
to
decide
number
of
animals
needed
It
would
be
more
efficient
to
use
the
shorter
in­
utero­
through­
lactation
assay
(
currently
under
development)
to
determine
the
number
of
F1
animals
needed
in
the
extension.
Whatever
number
of
animals
is
decided
upon
via
the
in
utero
study
could
then
be
run
Page
28
of
31
through
a
2­
generation
protocol
to
determine
the
practicality
of
carrying
that
number
of
animals
routinely.

Implementation
issues:
lab
is
available
to
start
2­
gen
now,
but
might
not
be
available
if
delayed
There
was
reluctance
to
lose
the
opportunity
to
begin
a
2­
generation
study
now
since
a
lab
is
currently
available.
It
was
agreed
that
the
study
would
have
to
be
done
with
the
extension
to
make
it
worthwhile;
there
would
be
little
point
in
doing
the
2­
generation
study
with
only
the
thyroid
additions
and
other
minor
adjustments
for
endocrine
endpoints.

Options
for
proceeding
The
Workgroup
decided
on
four
options,
outlined
in
the
attached
table.
Although
Option
2
is
the
preferred
option
scientifically,
Option
4
is
the
recommended
one
given
that
a
laboratory
is
currently
available
to
begin
a
2­
gen
study.
Also,
the
length
of
the
2­
gen
argues
for
as
early
a
start
on
standardization/
validation
as
possible.

Chemicals
and
number
of
doses
Given
that
the
Mammalian
Workgroup
is
focusing
on
whether
low
incidence
effects
can
be
picked
up
in
the
2­
gen­
with­
extension,
the
"
three­
chemical/
one­
dose­
each"
design
is
still
preferred
by
the
Workgroup.
It
is
more
important
to
cover
E,
A,
and
T
than
to
get
dose­
response
information
when
the
issue
is
whether
the
assay
will
detect
the
effect
at
all.
The
Workgroup
notes
that
the
in
utero
assay
development
will
provide
relevant
doseresponse
information
for
the
new
endpoints
being
considered
as
additions
to
the
2­
gen
study.

If
the
purpose
of
the
study
is
to
establish
how
low
a
dose
will
elicit
effects,
then
of
course
the
3­
dose­
per­
chemical
design
is
necessary.
However,
at
this
stage
the
question
seems
to
be
whether
the
effect
can
be
detected,
not
at
what
level
it
first
appears.

If
the
decision
is
made
that
a
3­
dose­
per­
chemical
design
must
be
used,
the
Workgroup
recommends
using
[
p,
p'­]
DDE
because
it
is
a
weak
anti­
androgen.

Since
the
effects
to
be
investigated
in
the
extension
of
the
F1
generation
to
PND
45/
60
are
not
related
to
the
thyroid
(
and
since
the
extension
is
the
main
focus
of
the
Workgroup's
concern),
it
does
not
appear
to
be
appropriate
to
test
propythiouracil
at
this
time.
PTU
is
more
appropriately
tested
in
the
validation
step
for
the
2­
gen,
after
the
extension
is
examined.

The
availability
of
vinclozolin
is
questionable.
Based
on
EPA­
RTP's
recent
experience,
there
may
be
problems
(
delay
and
cost)
obtaining
this
chemical
for
study.
The
Workgroup
recommends
using
flutamide
in
this
study.
Page
29
of
31
It
appears
that
in
the
end
the
Workgroup
recommended
Option
4
(
in
utero
in
parallel
with
a
2­
gen­
with­
extension)
using
flutamide
at
2
doses
plus
control
for
the
in
utero
study
and
3
doses
plus
control
for
the
2­
gen­
with­
extension.

What
the
results
at
45/
60
days
will
be
compared
to
The
SVTF
noted
that
if
the
entire
F1
generation
were
extended
out
to
45
days
for
the
female
and
60
days
for
the
male,
there
would
be
no
results
at
21
days
to
compare
to.
This
is
acceptable
because
there
is
expected
to
be
only
an
increase
in
the
types
of
effects
seen;
the
effects
already
known
to
occur
by
PND
21
do
not
go
away
by
day
45/
60.
Thus
there
is
no
need
to
have
a
comparison
group.

PND4
culls
The
Workgroup
considered
possible
uses
for
the
animals
culled
at
PND
4.
Gene
arrays
could
be
done,
but
interpretation
of
the
results
would
be
a
problem.
Similarly,
one
could
look
at
the
development
of
the
reproductive
tract,
but
interpretation
of
this
would
also
be
difficult.
The
Workgroup
believes
that
at
this
time
there
is
no
useful
purpose
for
the
animals,
although
there
are
procedures
in
development
which
eventually
will
be
able
to
make
use
of
these
animals.

Interlaboratory
comparisons
Assuming
any
option
other
than
Option
1,
the
interlaboratory
comparison
is
more
appropriately
dealt
with
at
the
validation
step
rather
than
at
the
examination
of
the
extension.
Page
30
of
31
Options
for
validation
of
mammalian
2­
generation
reproduction
assay
with
additional
endocrine
endpoints
Option
Pros
Cons
1.
2­
gen,
no
extension,
but
with
the
minor
endocrine
modifications
(
i.
e.,
2­
gen
validation)
1.
Starts
2­
gen
immediately
2.
Shortest
time
to
completion
of
validation:
22
months?
3.
Cannot
detect
low
incidence
effects
4.
Does
not
provide
any
new
information
(
may
not
even
be
needed
for
validation?)

2.
In
utero
 
>
2­
gen
practicality
demo
 
>

2­
gen
validation
3.
Most
scientifically
thorough.
Results
of
in
utero
will
determine
number
of
F1
that
need
to
be
carried
to
PND
45/
60
in
2­
gen
practicality
demo.

4.
Potentially
fewer
animals
and
more
practical
than
if
2­
gen­
with­
extension
is
done
w/
o
benefit
of
in
utero
results
(
that
is,
Option
3
below).
1.
Misses
opportunity
to
start
2­
gen
now
2.
Longest
time
to
completion
of
validation
(
12
+

22
+
22)
=
56
months
=
4
b
years?

3.
2­
gen­
with­
extension
 
>
2­
gen
validation
Options
for
2­
gen­
with­
extension:
either
2
chemicals
(
3
doses
each)
or
(
1
chemical
@
3
doses)
+
(
3
chemicals
@
1
dose
each)

(
In
utero
begins
up
to
a
year
later,
when
the
master
support
contract
is
in
place)
1.
Starts
2­
gen
immediately
a.
Large
number
of
animals
in
F1
extension;
might
be
difficult
to
perform.

b.
Takes
44
months
to
complete
validation
(
3
b
years)

4.
In
utero
and
2­
gen­
with­
extension
in
parallel
 
>
2­
gen
validation
(
In
utero
begins
immediately)
a.
Starts
2­
gen
immediately.

b.
2­
gen
validation
has
the
benefit
of
in
utero
results
as
well
as
2­
gen­
with­
extension
a.
Large
number
of
animals
in
F1
extension;
might
be
difficult
to
perform.

b.
Takes
44
months
to
complete
validation
(
3
b
years)

"
 
>
"
means
"
followed
by"
Page
31
of
31
Option
Chemical,
Number
of
Doses
1.
2­
gen,
no
extension
(
Not
discussed)

2.
In
utero
 
>
2­
gen
practicality
demo
 
>
2­
gen
validation
In
utero:
vinclozolin
or
flutamide,
2
doses
+
control
2­
gen
practicality:
(
Not
discussed)

2­
gen
validation:
(
Not
fully
explored,
but
at
least
PTU
­
3
doses
+
control)

3.
2­
gen­
with­
extension
 
>
2­
gen
validation,
in
utero
in
master
support
contract
In
utero:
DDE,
2
doses
+
control?
or
vinclozolin
or
flutamide,
2
doses
+
control?

2­
gen
with
extension:
vinclozolin
or
flutamide,
3
doses
+
control
(?)
,
methoxychlor,
1
dose
*
or
DDE,
3
doses
ethinyl
estradiol,
1
dose
­
+
control
2­
gen
validation:
(
Not
fully
explored,
but
at
least
PTU
­
3
doses
+
control)

4.
In
utero
and
2­
gen­
withextension
in
parallel
 
>
2­
gen
validation
In
utero:
DDE,
2
doses
+
control?
or
vinclozolin
or
flutamide,
2
doses
+
control?

2­
gen
with
extension:
vinclozolin
or
flutamide,
3
doses
+
control
(?)
,
methoxychlor,
1
dose
*
or
DDE,
3
doses
ethinyl
estradiol,
1
dose
­
+
control
2­
gen
validation:
[
Not
fully
explored,
but
at
least
PTU
­
3
doses
+
control]
