USEPA
USEPA
EDMVS
EDMVS
August
2003
August
2003
PUBERTAL
RAT
ASSAYS
PUBERTAL
RAT
ASSAYS
L
E
GRAY
JR
L
E
GRAY
JR
Endocrinology
Branch
Endocrinology
Branch
RTD,
NHEERL,
ORD,
USEPA
RTD,
NHEERL,
ORD,
USEPA
gray.
earl@
epa.
gov
gray.
earl@
epa.
gov
Pubertal
Female
and
Male
Rat
Assays
Pubertal
Female
and
Male
Rat
Assays
Role
of
Each
Assay
in
Tier
1
Screening
History
­
Current
Protocols
Current
Status
of
Data
Base
chemicals
labs
(
EPA
studies,
contractor
studies,
industry
studies
in
literature)

Results
Sensitivity
­
rat
strains
­
other
assays
Next
Steps
Chemicals­
mechanisms
examined
Concurrent
dose­
range
finding
studies
Additional
endpoints
to
strengthen
detection
of
EAT
USEPA
Endocrine
Disrupter
Screening
and
Testing
Advisory
USEPA
Endocrine
Disrupter
Screening
and
Testing
Advisory
Committee
Recommended
T1S
Battery
(
1998)

Committee
Recommended
T1S
Battery
(
1998)

In
Vitro
In
Vitro
ER
Binding
and/
or
Transcriptional
Activation
ER
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
Steroid
Hormone
Synthesis
Steroid
Hormone
Synthesis
In
Vivo
In
Vivo
Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

(
Anti)
androgen
assay
in
castrate­
T­
treated
male
rat
(
10d)

(
Anti)
androgen
assay
in
castrate­
T­
treated
male
rat
(
10d)

Frog
Metamorphosis
Assay
Frog
Metamorphosis
Assay
Short­
term
Assessment
of
the
Fish
Reproductive
System
Short­
term
Assessment
of
the
Fish
Reproductive
System
Objectives
of
Screening
and
Objectives
of
Screening
and
Testing
for
EAT
Testing
for
EAT
Objectives
of
Screening
(
T1S)

Objectives
of
Screening
(
T1S)
Fast,
cost
effective,
and
sensitive
means
of
Fast,
cost
effective,
and
sensitive
means
of
determining
which
chemicals
should
be
subject
to
the
more
determining
which
chemicals
should
be
subject
to
the
more
comprehensive
Tier
2
Testing
(
T2T).
Screening
consists
of
in
vitro
and
in
comprehensive
Tier
2
Testing
(
T2T).
Screening
consists
of
in
vitro
and
in
vivo
assays
designed
to
screen
for
activity
in
EAT
hormone
systems.

vivo
assays
designed
to
screen
for
activity
in
EAT
hormone
systems.

However,

However,
if
is
not
designed
to
quantify
such
activity
(
i.
e.
dose­
response).

if
is
not
designed
to
quantify
such
activity
(
i.
e.
dose­
response).

EDSTAC
Final
Report,
1998.
6
­
7.

EDSTAC
Final
Report,
1998.
6
­
7.

Detect
(
anti)
Estrogens
(
E)

Detect
(
anti)
Estrogens
(
E)

Detect
Antiandrogens
(
A)

Detect
Antiandrogens
(
A)

Detect
Antithyroid
(
T)

Detect
Antithyroid
(
T)

Provide
information
use
in
Tailoring
Testing
(
T2T)

Provide
information
use
in
Tailoring
Testing
(
T2T)

Identify
Potential
EDCs
that
Act
via
HPG
on
EAT
Identify
Potential
EDCs
that
Act
via
HPG
on
EAT
Objectives
of
Testing
(
T2T)

Objectives
of
Testing
(
T2T)

Dose­
response
Dose­
response
Adverse
Adverse
Include
Most
Sensitive
Endpoints
and
Life
Stages
Include
Most
Sensitive
Endpoints
and
Life
Stages
EDSTAC
Screening
Battery:

EDSTAC
Screening
Battery:

Detects
Estrogens
by
sc
and
oral
routes
in
vivo
and
in
vitro
Detects
Androgens
and
Anti­
in
a
senstive
assay
using
simple
endpoints
(
organ
weights)
and
in
vitro.

Detects
HPG/
CNS
alterations
related
to
FSH,
LH,
prolactin,

dopamine,
GH
with
simple,
precise
developmental
landmark
without
RIAs.

Detects
Inhibition
of
Steroidogenesis
in
vivo
(
in
female)
with
simple,
precise
endpoints
and
in
vitro
Detects
Thyroid
effects
in
intact
Female
Rat
(
RIAs)

and
Frog
Metamorphosis
Uses
extensively
utlized
in
vivo
endpoints/
assays
0
10
20
30
40
50
60
70
80
90
Time
(
min)

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Serum
LH
829
837
965
855
0
10
20
30
40
50
60
70
80
90
Time
(
min)

0.0
0.5
1.0
1.5
2.0
Serum
Testosterone
829
837
965
855
Basal
Serum
LH
and
Testosterone
levels
(
ng/
ml)
in
Control
LE
Basal
Serum
LH
and
Testosterone
levels
(
ng/
ml)
in
Control
LE
Adult
Male
rats
measured
repeatedly
at
ten
minute
intervals.

Adult
Male
rats
measured
repeatedly
at
ten
minute
intervals.

(
Fail
et
al.,
1996)

(
Fail
et
al.,
1996)
Alternative
Assays
for
Possible
Alternative
Assays
for
Possible
Inclusion
in
T1S
Inclusion
in
T1S
the
the
Pubertal
Male
Pubertal
Male
assay
was
and
assay
was
and
Alternative
assay
that
EDSTAC
Alternative
assay
that
EDSTAC
wanted
developed
and
evaluated
for
wanted
developed
and
evaluated
for
screening
screening
If
If
standardized
and
validated
standardized
and
validated
and
found
to
be
and
found
to
be
functionally
equivalent,
or
better,
then
an
functionally
equivalent,
or
better,
then
an
alternative
assay
could
be
included
in
T1S.

alternative
assay
could
be
included
in
T1S.
Modified
USEPA
Endocrine
Disrupter
Screening
and
Testing
Modified
USEPA
Endocrine
Disrupter
Screening
and
Testing
Battery
with
the
Battery
with
the
pubertal
male
pubertal
male
assay
replacing
the
assay
replacing
the
Hershberger
assay
Hershberger
assay
In
Vitro
In
Vitro
ER
Binding
and/
or
Transcriptional
Activation
ER
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
Steroid
Hormone
Synthesis
Steroid
Hormone
Synthesis
In
Vivo
In
Vivo
Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

Pubertal
Male
Rat
Assay
including
Thyroid
(
30d)

Pubertal
Male
Rat
Assay
including
Thyroid
(
30d)

Frog
Metamorphosis
Assay
Frog
Metamorphosis
Assay
Short­
term
Assessment
of
the
Fish
Reproductive
System
Short­
term
Assessment
of
the
Fish
Reproductive
System
EDSTAC
Screening
Battery:

EDSTAC
Screening
Battery:

Detects
Estrogens
by
sc
and
oral
routes
and
in
vitro
Detects
Androgens
and
Anti­
in
the
pubertal
male
assay
using
simple
endpoints
(
PPS
and
organ
weights)

and
in
vitro.

Detects
HPG/
CNS
alterations
related
to
FSH,
LH,
prolactin,

dopamine,
GH
with
simple,
precise
developmental
landmark
without
RIAs
in
pubertal
female
and
male.

Detects
Inhibition
of
Steroidogenesis
in
vivo
(
in
male
and
female)
with
simple,
precise
endpoints
and
in
vitro
Detects
Thyroid
effects
in
intact
pubertal
Male
and
Female
Rat
using
RIAs
and
Frog
Metamorphosis
Uses
extensively
utlized
in
vivo
endpoints/
assays
Many
endpoints
from
the
pubertal
assays
already
are
included
in
the
1998
EPA
Multigenerational
Test
Guidelines
and
have
been
used
to
set
NOAELs
including
delayed
VO
(
PFOA,
Atrazine),

delayed
PPS
(
vinclozolin,

on
the
right),
reduced
ventral
prostate
and
epididymal
weights
(
vinclozolin),
altered
LH
(
Atrazine),
and
inhibition
of
aromatase
resulting
in
delayed
delivery
(
fenarimol),
for
example.
Examples
of
Developmental
DERs
(
Diagnostic
Examples
of
Developmental
DERs
(
Diagnostic
Endocrine­
mediated
Responses)
in
pubertal
assays
that
cannot
Endocrine­
mediated
Responses)
in
pubertal
assays
that
cannot
be
confused
with
delayed
growth
or
nonspecific
toxicity.

be
confused
with
delayed
growth
or
nonspecific
toxicity.

Methyltestosterone,
TP
and
trenbolone
in
male
Methyltestosterone,
TP
and
trenbolone
in
male
Accelerated
PPS
Accelerated
PPS
Larger
sex
accessory
glands,
but
smaller
testes
and
low
LH
Larger
sex
accessory
glands,
but
smaller
testes
and
low
LH
Flutamide
and
vinclozolin
in
male
Flutamide
and
vinclozolin
in
male
Larger
testes,
increased
serum
T
and
LH
Larger
testes,
increased
serum
T
and
LH
Delay
in
PPS
with
no
affect
on
somatic
growth
Delay
in
PPS
with
no
affect
on
somatic
growth
PTU
and
other
antithyroid
agents
in
male
or
female
PTU
and
other
antithyroid
agents
in
male
or
female
absent
T3
and
T4
with
elevated
TSH
absent
T3
and
T4
with
elevated
TSH
increased
thyroid
weight
and
histopathology
increased
thyroid
weight
and
histopathology
Weak
estrogens
in
female
Weak
estrogens
in
female
Pseudoprecocious
puberty
Pseudoprecocious
puberty
Constant
estrus
Constant
estrus
Questions?

Questions?

Given
the
number
of
chemicals
run
in
more
than
one
lab,
is
a
Given
the
number
of
chemicals
run
in
more
than
one
lab,
is
a
formal
interlab
study
still
needed?

formal
interlab
study
still
needed?

Is
the
pubertal
male
assay
sensitive
enough
to
replace
the
Is
the
pubertal
male
assay
sensitive
enough
to
replace
the
Hershberger
assay
in
T1S?

Hershberger
assay
in
T1S?

Should
we
evaluate
the
adrenal
or
other
endpoints
in
more
Should
we
evaluate
the
adrenal
or
other
endpoints
in
more
detail?

detail?

What
mechanisms
of
action
need
further
evaluation
to
establish
What
mechanisms
of
action
need
further
evaluation
to
establish
the
sensitivity
of
the
assay?

the
sensitivity
of
the
assay?

Should
we
be
running
concurrent
dose­
range
finding
studies?

Should
we
be
running
concurrent
dose­
range
finding
studies?

Can
we
reduce
the
sample
size
in
these
assays?

Can
we
reduce
the
sample
size
in
these
assays?
Endocrine­
Disrupting
Chemicals:
Prepubertal
Exposures
and
Effects
on
Sexual
Maturation
and
Thyroid
Activity
in
the
Female
Rat.
A
Focus
on
the
EDSTAC
Recommendations
Jerome
M.
Goldman,
Susan
C.

Laws,
Sharon
K.
Balchak,
Ralph
L.
Cooper,
and
Robert
J.
Kavlock.

Critical
Reviews
in
Toxicology,
30(
2):
135­
196
(
2000)

Modified
Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Juvenile
Female
Rats.

Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
procedures
to
quantify
the
effects
of
environmental
compounds
on
pubertal
development
and
thyroid
function
in
the
intact
juvenile
female
rat.
This
assay
detects
agents
that
display
antithyroid,
estrogenic,
antiestrogenic
(
estrogen
receptor
[
ER]
or
steroid­
enzyme­
mediated)
activity,
or
alter
puberty
via
changes
in
luteinizing
hormone
(
LH),
follicle
stimulating
hormone
(
FSH),
prolactin
(
PRL)
and
growth
hormone
(
GH)
secretion,
or
via
alterations
in
hypothalamic
function.

Required
Endpoints:

Growth
(
body
weight).
Liver,
kidney,
pituitary,
and
adrenal
weights
Age
and
weight
at
vaginal
opening
Serum
thyroxine
(
T4),
thyroid
histology
and
thyroid
stimulating
hormone
(
TSH)

Uterine
and
ovarian
weights
and
histology
Vaginal
cytology
Optional
Endpoints:

Serum
tri­
iodothyronine
(
T3),
estradiol
(
E2),
and
prolactin
Thyroid
weight
Liver,
kidney,
pituitary,
adrenal
and
vaginal
histology
Ex
vivo
ovarian
and
pituitary
hormone
production
Hypothalamic
neurotransmitter
concentrations
Estrous
cycle
length
(
requires
extension
of
dosing)
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Assign
to
Treatments
based
upon
body
weight.

15/
group
Dose
Female
Necropsy
Our
Immature
(
21
­
43
Days
of
Age)
Intact
Female
Rat
Protocol
to
Evaluate
Pubertal
Development
and
Thyroid
Function.
Detects
inhibition
of
steroidogenesis,
antithyroid
and
(
anti)
estrogenic
activities
and
altered
HPG
maturation.

Wean
Days
of
age
Endpoints:
Growth
Age
and
weight
at
vaginal
opening
(
VO)

Vaginal
cytology
Serum
thyroxine
and
thyroid­
stimulating
hormones
Ovarian
and
uterine
weights
and
histology
Liver,
kidney,
pituitary
and
adrenal
weights
Dose
daily
Daily
examination
for
VO
and
lavage
LEGJR
Chemicals
under
investigated
or
being
Chemicals
under
investigated
or
being
studied
in
the
pubertal
female
assay
studied
in
the
pubertal
female
assay
RTI
­
ongoing
RTI
­
ongoing
Atrazine
75
or
150
mg/
kg/
d
Atrazine
75
or
150
mg/
kg/
d
Fenarimol
50
or
250
mg/
kg/
d
Fenarimol
50
or
250
mg/
kg/
d
Methoxychlor
25
and
50
mg/
kg/
d
Methoxychlor
25
and
50
mg/
kg/
d
Bisphenol
A
400
or
600
mg/
kg/
d
Bisphenol
A
400
or
600
mg/
kg/
d
Ketoconazole
75
and
150
Ketoconazole
75
and
150
mg/
kg/
d
mg/
kg/
d
PTU
75
and
150
mg/
kg/
d
PTU
75
and
150
mg/
kg/
d
TherImmune
2000
(
2x2x6
factorial.

TherImmune
2000
(
2x2x6
factorial.

Blocks,
LE
versus
SD
and
chemicals.

Blocks,
LE
versus
SD
and
chemicals.

n=
6/
strain/
block)
­
completed
n=
6/
strain/
block)
­
completed
Ethynyl
estradiol
0.005
mg/
kg/
d
Ethynyl
estradiol
0.005
mg/
kg/
d
Tamoxifen
10
mg/
kg/
d
Tamoxifen
10
mg/
kg/
d
PTU
240
mg/
kg/
d
PTU
240
mg/
kg/
d
Ketoconazole
100
mg/
kg/
d
Ketoconazole
100
mg/
kg/
d
Pimozide
30
mg/
kg/
d
Pimozide
30
mg/
kg/
d
Methoxychlor
100
mg/
kg/
d
Methoxychlor
100
mg/
kg/
d
TherImmune
2003
­
ongoing
TherImmune
2003
­
ongoing
Ethynyl
estradiol
0.0025
and
0.005
Ethynyl
estradiol
0.0025
and
0.005
mg/
kg/
d
mg/
kg/
d
Methoxychlor
12.5,
25
and
50
Methoxychlor
12.5,
25
and
50
mg/
kg/
d
mg/
kg/
d
Phenobarbital
50
and
100
mg/
kg/
d
Phenobarbital
50
and
100
mg/
kg/
d
Published
studies
that
have
used
Published
studies
that
have
used
the
pubertal
female
assay
the
pubertal
female
assay
Atrazine
­
2
labs
Atrazine
­
2
labs
Nonyphenol
Nonyphenol
DES
­
2
labs
DES
­
2
labs
Methoxychlor
­

Methoxychlor
­
VO
and
cycles
VO
and
cycles
Estradiol
Estradiol
Octylphenol
­

Octylphenol
­
VO
and
cycles
VO
and
cycles
Nonylphenol
­

Nonylphenol
­
VO
and
cycles
VO
and
cycles
Tamoxifen
Tamoxifen
Fadrazole
Fadrazole
Ketoconazole
Ketoconazole
Finasteride
Finasteride
Testolactone
Testolactone
Flutamide
Flutamide
Perchlorate
­

Perchlorate
­
in
prep
in
prep
PBDE
­

PBDE
­
submitted
submitted
PTU
PTU
MBC
­

MBC
­
VO
and
cycles
VO
and
cycles
DBP
­

DBP
­
VO
and
cycles
VO
and
cycles
Importance
of
Strain
Effects
in
Screening
Assays???

The
OECD
program
to
validate
the
rat
uterotrophic
bioassay:
Phase
Two
­
Dose
Response
Studies.
Jun
Kanno,
Lesley
Onyon,
Shyamal
Peddada
,

John
Ashby,
Elard
Jacob
and
William
Owens.
doi:
10.1289/
ehp.
5780
(
available
at
http://
dx.
doi.
org/).
Online
23
January
2003
Abstract:

The
Organisation
for
Economic
Co­
operation
and
Development
has
completed
Phase
2
of
an
international
validation
program
for
the
rodent
uterotrophic
bioassay.

The
purpose
of
the
validation
program
was
to
demonstrate
the
performance
of
two
versions
of
the
uterotrophic
bioassay,
the
immature
female
rat
and
the
adult
ovariectomized
rat,
in
four
standardized
protocols.

.
........
It
is
noteworthy
that
these
results
were
reproducible
under
a
variety
of
different
experimental
conditions
(
e.
g.,

animal
strain,
diet,
housing,
bedding,
vehicle,
animal
age,

and
so
on),
indicating
that
the
bioassay's
performance
as
a
screen
is
robust.
Daily
oral
(
mg/
kg/
d)
Methoxychlor
treatment
induces
pseudoprecocious
puberty
in
the
21
day
old
female
rat.
Data
are
expressed
as
the
number
of
days
that
VO
was
accelerated
(
Gray
and
Ostby,
1998).

0
50
100
150
200
mg/
kg/
d
by
gavage
0
1
2
3
4
5
6
7
8
Days
that
VO
was
accelerated
Pubertal
Female
Assay
Data
Pubertal
Female
Assay
Data
All
but
two
values
differ
from
control
All
but
two
values
differ
from
control
20
25
30
35
40
Age
at
VO
Oil
KETO
MET
EE
TAM
PTU
PIM
35.6
36.4
26.6
25.9
26.6
36.1
37.1
ns
ns
ns
Pubertal
Female
Assay
Data
Pubertal
Female
Assay
Data
100
150
200
250
300
Uterine
Wet
Weight
(
mg)

Oil
KETO
MET
EE
TAM
PTU
PIM
238
180
222
261
97
127
160
ns
ns
p<.
0001
p<
0.0009
ns
Pubertal
Female
Assay
Data
Pubertal
Female
Assay
Data
20
30
40
50
60
70
80
Adrenal
Weight
(
mg)

Oil
KETO
MET
EE
TAM
PTU
PIM
40.3
77.9
39.5
40.4
32.9
24.9
31.5
ns
ns
Pubertal
Female
Assay
Data
Pubertal
Female
Assay
Data
20
30
40
50
60
70
80
Ovaries
Weight
(
mg)

Oil
KETO
MET
EE
TAM
PTU
PIM
73.2
68.4
64.5
54.4
40.5
40
50.9
p<
0.01
ns
ns
ns
Endocrine­
Disrupting
Chemicals:
Prepubertal
Exposures
and
Effects
on
Sexual
Maturation
and
Thyroid
Function
in
the
Male
Rat.
A
Focus
on
the
EDSTAC
Recommendations.
Tammy
E.
Stoker,
Louise
G.
Parks,
L.
Earl
Gray,
and
Ralph
L.
Cooper.
Critical
Reviews
in
Toxicology,
30(
2):
197­
252
(
2000)

Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Immature
(
23­
to
53­
Day­
Old)
Male
Rats
Purpose
and
applicability
The
purpose
of
this
protocol
is
to
outline
procedures
to
quantify
the
effects
of
environmental
compounds
on
pubertal
development
and
thyroid
function
in
the
intact
juvenile/
peripubertal
male
rat.
This
assay
detects
compounds
that
display
antithryoid,
estrogenic,
androgenic,

antiandrogenic
[
androgen
receptor
(
AR)
or
steroid
enzyme
mediated]
activity,
or
alters
puberty
via
changes
in
follicle
stimulating
hormone
(
FSH),
luteinizing
hormone
(
LH),
prolactin,
growth
hormone
(
GH)
or
hypothalamic
function.

Required
endpoints
Growth
(
body
weight)

Age
and
weight
at
preputial
separation
Serum
thyroxin
(
T4),
thyroid
histology
and
TSH
Seminal
vesicle
plus
coagulating
gland
weight
(
with
and
without
fluid),
Ventral
prostate
weight,

Levator
ani
plus
bulbocavernosus
weight,
Epididymal
and
testis
weights
and
histology
Optional
measures
Serum
testosterone,
estradiol,
LH,
prolactin
and
tri­
iodothyronine
(
T3)

Liver,
kidney,
adrenal
and
pituitary
weights
and
histology
ex
vivo
testis
and
pituitary
hormone
production,
Hypothalamic
neurotransmitter
levels
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Assign
to
Treatments
based
upon
body
weight.

15/
group
Dose
Male
Necropsy
Our
Immature
(
21
­
52
Days
of
Age)
Intact
Male
Rat
Protocol
to
Evaluate
Our
Immature
(
21
­
52
Days
of
Age)
Intact
Male
Rat
Protocol
to
Evaluate
Pubertal
Development
and
Thyroid
Function.
Detects
inhibition
of
Pubertal
Development
and
Thyroid
Function.
Detects
inhibition
of
steroidogenesis,
antithyroid
and
(
anti)
androgenic
activities
and
altered
steroidogenesis,
antithyroid
and
(
anti)
androgenic
activities
and
altered
HPG
maturation.

HPG
maturation.

Wean
Days
of
age
Required
endpoints:

Growth
Age
and
weight
at
preputial
separation
(
PPS)

Serum
thyroxine
and
thyroid­
stimulating
hormones
Thyroid
Histology
Seminal
vesicle
plus
coagulating
gland
weight
(
with
fluid)

Ventral
prostate
weight,
Levator
ani/
bulbocavernosus
muscle
weight
Testis
and
epididymal
weights
and
histology
Liver,
kidney,
adrenal
and
pituitary
weights
Optional
endpoints
Serum
hormones
and
ex
vivo
testis
and
pituitary
hormone
production
Dose
daily
Daily
examination
for
PPS
LEGJR
20
30
40
50
Age
in
days
0
1
2
3
4
ng/

ml
Serum
T
Serum
DHT
Serum
Androstanediol
The
serum
androgen
profile
changes
The
serum
androgen
profile
changes
dramatically
during
puberty
dramatically
during
puberty
20
30
40
50
Age
in
days
10
100
1000
10000
mg
Epididymis
Testes
Ventral
Prostate
Seminal
Vesicle
Reproductive
organ
weights
grow
Reproductive
organ
weights
grow
rapidly
during
puberty
rapidly
during
puberty
Chemicals
under
investigated
or
being
Chemicals
under
investigated
or
being
studied
in
pubertal
male
assay
studied
in
pubertal
male
assay
RTI
­
ongoing
RTI
­
ongoing
Atrazine
75
and
150
mg/
kg/
d
Atrazine
75
and
150
mg/
kg/
d
p,
p'
DDE
50
and
100
mg/
kg/
d
p,
p'
DDE
50
and
100
mg/
kg/
d
Vinclozolin
30
and
100
mg/
kg/
d
Vinclozolin
30
and
100
mg/
kg/
d
Methoxychlor
25
and
50
mg/
kg/
d
Methoxychlor
25
and
50
mg/
kg/
d
PTU
2
and
25
mg/
kg/
d
PTU
2
and
25
mg/
kg/
d
Ketoconazole
50
and
100
Ketoconazole
50
and
100
mg/
kg/
d
mg/
kg/
d
Linuron
50
and
100
mg/
kg/
d
Linuron
50
and
100
mg/
kg/
d
Phenobarbital
50
and
100
Phenobarbital
50
and
100
mg/
kg/
d
mg/
kg/
d
TherImmune
2000
(
2x2x6
factorial.

TherImmune
2000
(
2x2x6
factorial.

Blocks,
LE
versus
SD
and
chemicals,

Blocks,
LE
versus
SD
and
chemicals,

n=
6/
strain/
block/
chemical)
­
completed
n=
6/
strain/
block/
chemical)
­
completed
Flutamide
50
mg/
kg/
d
Flutamide
50
mg/
kg/
d
Methyl
Testosterone
80
mg/
kg/
d
Methyl
Testosterone
80
mg/
kg/
d
PTU
240
mg/
kg/
d
PTU
240
mg/
kg/
d
Ketoconazole
100
mg/
kg/
d
Ketoconazole
100
mg/
kg/
d
Pimozide
30
mg/
kg/
d
Pimozide
30
mg/
kg/
d
DBP
1000
mg/
kg/
d
DBP
1000
mg/
kg/
d
TherImmune
2003
­
ongoing
TherImmune
2003
­
ongoing
Phenobarbital
25,
50
and
100
Phenobarbital
25,
50
and
100
mg/
kg/
d
mg/
kg/
d
Vinclozolin
10,
30
and
100
mg/
kg/
d
Vinclozolin
10,
30
and
100
mg/
kg/
d
Flutamide
25
and
50
mg/
kg/
d
Flutamide
25
and
50
mg/
kg/
d
Published
studies
using
the
Published
studies
using
the
pubertal
male
assay
pubertal
male
assay
p,
p'
DDE
­
several
labs
p,
p'
DDE
­
several
labs
Vinclozolin
­
2
labs
Vinclozolin
­
2
labs
Linuron
­
PPS
Linuron
­
PPS
Cyproterone
acetate
Cyproterone
acetate
Flutamide
­
3
labs
Flutamide
­
3
labs
Finasteride
­
2
labs
Finasteride
­
2
labs
Ketoconazole
­
2
labs
Ketoconazole
­
2
labs
Testolactone
Testolactone
TP
TP
BBP
BBP
DBP
­
2
labs
DBP
­
2
labs
Methoxychlor
­
2
labs
Methoxychlor
­
2
labs
BPA
BPA
DES
DES
PTU
PTU
Perchlorate
­
in
prep
Perchlorate
­
in
prep
PBDE
­
submitted
PBDE
­
submitted
Phenobarbital
Phenobarbital
Fenitrothion
Fenitrothion
MBC
­
PPS
MBC
­
PPS
Atrazine
­
2
labs
Atrazine
­
2
labs
Vinclozolin
(
Two
Studies)

Vinclozolin
(
Two
Studies)

These
data
were
used
to
set
a
These
data
were
used
to
set
a
NOAEL
by
EPA
in
2001
NOAEL
by
EPA
in
2001
40.9
40.9
42.7
45.5
40.6741.08
41.83
44.42
47.8
0
5
15
25
100
0
10
30
100
Dose
of
Vinclozolin
(
mg/
kg/
d)

35
40
45
50
Age
in
Days
RTI
EPA
Age
at
Preputial
Separation
0
50
100
Dose
of
Vinclozolin
(
mg/
kg/
d)

2
2.5
3
3.5
Testes
Weights
(
g)
0
50
100
Dose
of
Vinclozolin
(
mg/
kg/
d)

440
460
480
500
520
540
Epididymal
Weight
(
mg)

p<
0.04
p<
0.0003
Pubertal
vinclozolin
administration
delayed
PPS
and
Pubertal
vinclozolin
administration
delayed
PPS
and
reduced
weights
of
androgen­
dependent
tissues
without
reduced
weights
of
androgen­
dependent
tissues
without
affecting
testis
weight.
Serum
T
and
LH
were
increased.

affecting
testis
weight.
Serum
T
and
LH
were
increased.
p,
p'­
DDE
(
Two
Studies)

p,
p'­
DDE
(
Two
Studies)

Kelce
et
al.,
1995
Kelce
et
al.,
1995
45.8
46.7
47.7
50.6
43.3
42.3
43.5
48
0
10
30
100
0
10
30
100
Dose
of
p,
p'­
DDE
(
mg/
kg/
d)

40
42
44
46
48
50
52
Age
in
Days
Normal
Runts
Age
at
Preputial
Separation
Linuron
(
Gray
et
al.,
1999)

Linuron
(
Gray
et
al.,
1999)

43
43.4
43.6
45.5
0
10
20
40
Dose
of
Linuron
(
mg/
kg/
d)

40
41
42
43
44
45
46
47
Age
in
Days
Age
at
Preputial
Separation
Dibutyl
Phthalate
(
Two
Studies)

Dibutyl
Phthalate
(
Two
Studies)

Gray
et
al.,
1999
Gray
et
al.,
1999
42
48
39.5
42.6
43.4
44.4
0
250
500
1000
0
1000
Dose
of
DBP
(
mg/
kg/
d)

38
40
42
44
46
48
50
Age
in
Days
Age
at
Preputial
Separation
Age
at
PPS
is
delayed
and
Wt
at
PPS
is
Age
at
PPS
is
delayed
and
Wt
at
PPS
is
increased
with
pubertal
DEHP
treatment.

increased
with
pubertal
DEHP
treatment.

Gray
et
al.
The
Toxicologist,
2003
Gray
et
al.
The
Toxicologist,
2003
234
244
245
254
252
0
11
33
100
300
220
230
240
250
260
Wt
At
Full
PPS
45.6
46.7
47.2
47.2
49.1
0
11
33
100
300
44
45
46
47
48
49
50
Age
At
Full
PPS
Methoxychlor
(
Data
from
Five
Methoxychlor
(
Data
from
Five
Studies)
Gray
et
al.,
1989;
1999.

Studies)
Gray
et
al.,
1989;
1999.

41.1
41
44
43.9
49.5
68
74
0
25
50
100
200
300
400
Dose
of
Methoxychlor
(
mg/
kg/
d)

40
50
60
70
80
Age
in
Days
Age
at
Preputial
Separation
The
detection
of
estrogens
can
be
confounded
by
their
direct
effect
on
the
brain,
inhibiting
food
consumption
and
growth.
Methoxychlor
which
is
estrogenic
in
vivo
accelerates
VO
at
dosage
levels
that
affect
weight
by
<
10%
but
PPS
in
the
male
is
only
affected
at
levels
with
>
10%
growth
retardation.
Methoxychlor
was
administered
from
weaning
throughout
puberty
at
0,
25,
50,
100,
or
200
mg/
kg/
d.
As
anticipated,
the
pubertal
female
assay
is
a
more
diagnostic
assay
for
xenoestrogens
than
is
the
male
assay
85
90
95
100
Percent
of
Control
Weight
at
day
42
0
1
2
3
4
5
6
7
8
9
10
VO
Acceleration
in
Days
Female
VO
60
70
80
90
100
110
Percent
of
Control
Weight
at
day
63
­
5
0
5
10
15
PPS
Delay
in
Days
Male
PPS
Diagnostic
estrogenic
response
area
Diagnostic
endocrine
response
area
Gray
et
al.,
1989.

Above
MTD
Relationship
between
reduction
in
body
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
and
weight
due
to
restricted
feeding
versus
and
delay
in
age
at
PPS
delay
in
age
at
PPS
80
85
90
95
100
Percent
of
control
body
weight
­
10
­
5
0
5
10
15
Delay
in
PPS
in
Days
Feeding
Vinclozolin
Linuron
DDE1
DDE2
DBP
DBP2
KETO­
TH
MT­
TH
FLUT­
TH
Relationship
between
reduction
in
body
weight
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
testis
weight
due
to
restricted
feeding
versus
testis
weight
80
85
90
95
100
Percent
of
control
body
weight
20
40
60
80
100
120
Percent
of
control
testis
weight
Feeding
MT­
TH
FLUT­
TH
DBP­
TH/
sd
DBP­
TH/
le
Relationship
between
reduction
in
body
weight
due
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
seminal
vesicle
weight
to
restricted
feeding
versus
seminal
vesicle
weight
80
85
90
95
100
Percent
of
control
body
weight
0
50
100
150
200
Percent
OF
CONTROL
SV
weightt
Feeding
Vinclozolin
KETO­
TH
MT­
TH
FLUT­
TH
DBP­
TH­
le
TherImmune
Pubertal
Male
Assay
Data
TherImmune
Pubertal
Male
Assay
Data
from
2000
from
2000
All
values
differ
sign
from
control
All
values
differ
sign
from
control
30
35
40
45
50
55
AGE
AT
PPS:

Oil
KETO
MT
DBP
FL
PTU
PIM
43.6
46.9
35.3
46.3
54.5
52.8
51.7
TherImmune
Pubertal
Male
Assay
TherImmune
Pubertal
Male
Assay
Data
from
2000
Data
from
2000
All
values
differ
signicantly
from
control
All
values
differ
signicantly
from
control
200
250
300
350
400
450
500
550
Epididymides
WT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
488
415
440
421
288
249
362
0
500
1000
1500
2000
2500
3000
3500
TESTIS
WEIGHT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
2893
2757
747
1647
3318
1854
2271
ns
TherImmune
Pubertal
Male
Assay
TherImmune
Pubertal
Male
Assay
Data
from
2000.

Data
from
2000.

All
values
but
one
differ
from
control
All
values
but
one
differ
from
control
TherImmune
Pubertal
Male
Assay
Data
TherImmune
Pubertal
Male
Assay
Data
from
2000
from
2000
All
values
differ
sign
from
control
All
values
differ
sign
from
control
100
200
300
400
500
600
700
800
LABC
WT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
600
461
687
428
300
140
380
TherImmune
Pubertal
Male
Assay
Data
TherImmune
Pubertal
Male
Assay
Data
from
2000
from
2000
All
values
differ
sign
from
control
All
values
differ
sign
from
control
0
100
200
300
400
VP
WT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
202
134
355
167
61
57
134
Pubertal
Male
Assay
Data
Pubertal
Male
Assay
Data
All
values
differ
sign
from
control
All
values
differ
sign
from
control
0
100
200
300
400
500
600
700
800
SV
WT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
427
244
720
282
78
92
228
Pubertal
Male
Assay
Data.
Flutamide,
PTU
and
PIM
Pubertal
Male
Assay
Data.
Flutamide,
PTU
and
PIM
animals
had
sig
smaller
adrenals
if
the
Keto
group
animals
had
sig
smaller
adrenals
if
the
Keto
group
is
deleted
from
the
analysis
for
variance
problems.

is
deleted
from
the
analysis
for
variance
problems.

0
20
40
60
80
100
120
140
ADRENALS
WT
(
mg)

Oil
KETO
MT
DBP
FL
PTU
PIM
45.4
117
42.4
45.4
50.1
27.2
44.4
p
<.
0001
EDSTAC
Final
Report
comments
EDSTAC
Final
Report
comments
on
assays
for
androgens
on
assays
for
androgens
The
Hershberger
Assay
compared
to
other
Assays
for
Antiandrogens
The
Hershberger
Assay
compared
to
other
Assays
for
Antiandrogens
"
In
the
castrated
male
rat,
the
gonads
have
been
removed
and
effects
on
"
In
the
castrated
male
rat,
the
gonads
have
been
removed
and
effects
on
androgen­
dependent
accessory
sex
organs
and
tissues
are
likely
to
be
direct
and
androgen­
dependent
accessory
sex
organs
and
tissues
are
likely
to
be
direct
and
not
a
result
of
pituitary
or
gonadal
secretion.
....

not
a
result
of
pituitary
or
gonadal
secretion.
....

For
a
non­
in
utero
assay,
this
assay
robustly
detects
androgens
and
For
a
non­
in
utero
assay,
this
assay
robustly
detects
androgens
and
antiandrogens
with
a
dynamic
response
that
typically
exceeds
that
of
the
intact
antiandrogens
with
a
dynamic
response
that
typically
exceeds
that
of
the
intact
adult
male
(
Raynaud,
1984).
Most
of
the
studies
are
able
to
detect
significant
adult
male
(
Raynaud,
1984).
Most
of
the
studies
are
able
to
detect
significant
effects
with
only
five
animals
per
group.
.....

effects
with
only
five
animals
per
group.
.....

in
one
study
which
used
10­
15
drugs,
the
Hershberger
assay
was
more
in
one
study
which
used
10­
15
drugs,
the
Hershberger
assay
was
more
responsive
than
was
the
intact
male
for
every
chemical
examined
(
Wakeling
et
al.,

responsive
than
was
the
intact
male
for
every
chemical
examined
(
Wakeling
et
al.,

1981).

1981).

The
power
of
the
castrate­
male
assay
arises
from
the
fact
that
castration
creates
The
power
of
the
castrate­
male
assay
arises
from
the
fact
that
castration
creates
a
"
fetal­
like"
endocrine
system
with
regard
to
the
regulation
of
androgen
a
"
fetal­
like"
endocrine
system
with
regard
to
the
regulation
of
androgen
secretion,
as
the
HPG
axis
can
no
longer
compensate
for
the
effect
of
the
secretion,
as
the
HPG
axis
can
no
longer
compensate
for
the
effect
of
the
chemical
on
the
AR.
For
example,
p,
p'
DDE
reduces
sex
accessory
gland
weights
chemical
on
the
AR.
For
example,
p,
p'
DDE
reduces
sex
accessory
gland
weights
in
this
assay,
but
not
when
administered
to
intact
male
rats
(
Kelce
et
al.,
1997).
"

in
this
assay,
but
not
when
administered
to
intact
male
rats
(
Kelce
et
al.,
1997).
"
It
is
critical
that
the
T1
Screening
assay
be
able
to
detect
weak
It
is
critical
that
the
T1
Screening
assay
be
able
to
detect
weak
antiandrogens
like
DDE
and
linuron
because
they
cause
reproductive
antiandrogens
like
DDE
and
linuron
because
they
cause
reproductive
tract
malformation
in
utero.

tract
malformation
in
utero.
Exposure
to
100
mg
p,
p'
DDE/
kg­
dam's
body
Exposure
to
100
mg
p,
p'
DDE/
kg­
dam's
body
weight
on
gestational
days
14­
18
alters
development
of
the
External
weight
on
gestational
days
14­
18
alters
development
of
the
External
Genitalia.
Gray
et
al,
1999.

Genitalia.
Gray
et
al,
1999.

Control
Male
Treated
Male
with
Cleft
Phallus,

Exposed
Os,
Hypospadias
and
Slight
Vaginal
Pouch
100
37
100
97
101
105
100
0
50
100
200
300
dose
mg/
kg/
d
0
20
40
60
80
100
120
Intact
Male
Assay
Hershberger
%
control
SV
In
the
pubertal
male
assay
DDE
delays
PPS
by
about
4
days.

The
intact
male
assay
(
n=
75)
is
insensitive
as
compated
to
the
Hershberger
assay
(
n=
12)
to
the
effects
of
p,
p'
DDE
at
100
mg/
kg/
d
on
ASO
versus
VP
and
SV
weight.
This
chemical
is
a
false
negative
in
the
adult
male
assay
100
26
100
88
115
97
99
0
50
100
200
300
dose
mg/
kg/
d
0
20
40
60
80
100
120
%
Control
VP
The
intact
male
assay
is
much
less
sensitive
to
the
effects
of
The
intact
male
assay
is
much
less
sensitive
to
the
effects
of
vinclozolin
on
ASO
than
are
seminal
vesicle
weights
in
our
vinclozolin
on
ASO
than
are
seminal
vesicle
weights
in
our
Hershberger
or
pubertal
male
assays.

Hershberger
or
pubertal
male
assays.

0
50
100
150
200
250
300
40
50
60
70
80
90
100
Percent
of
Control
Intact
Male
Hershberger
Pubertal
50%
Hypospadias
100
101
102
101
105
100
86
65
48
0
200
400
600
800
1000
40
50
60
70
80
90
100
Percent
of
Control
Intact
Male
Assay
Hershberger
Pubertal
Male
The
intact
male
assay
is
less
sensitive
to
the
effects
of
the
phthalate
ester
DBP
at
the
limit
dose
of
1
g/
kg/
d
on
ASO
weight
than
is
seminal
vesicle
weights
in
our
Hershberger
or
pubertal
male
assays.
The
pubertal
assay
was
the
most
sensitive
to
this
chemical
50%
epi
or
testis
abnormalities
100
105
104
96
89
0
25
50
100
150
0
20
40
60
80
100
120
Intact
male
assay
as
%
Control
100
31
0
100
0
20
40
60
80
100
120
LEG
data.
SV
(
mg)

Linuron
delays
PPS
in
the
pubertal
male
assay
by
more
than
two
days.
The
Linuron
delays
PPS
in
the
pubertal
male
assay
by
more
than
two
days.
The
intact
male
assay
is
much
less
sensitive
to
the
Effects
of
Linuron
at
100
intact
male
assay
is
much
less
sensitive
to
the
Effects
of
Linuron
at
100
mg/
kg/
d
on
SV
versus
ASO
weight
than
males
in
the
Hershberger
Assay.

mg/
kg/
d
on
SV
versus
ASO
weight
than
males
in
the
Hershberger
Assay.

n=
75
rats
n=
12
rats
MTD
56%
epididymal
agenesis
at
this
dose
A
comparison
of
the
Male
Assays
with
Antiandrogens.
Developmental
A
comparison
of
the
Male
Assays
with
Antiandrogens.
Developmental
Toxicants
are
detected
in
the
Hershberger
and
PPS
assays.
Sex
accessory
Toxicants
are
detected
in
the
Hershberger
and
PPS
assays.
Sex
accessory
gland
size,
serum
T
and
LH
are
not
always
altered.
When
affected,
seminal
gland
size,
serum
T
and
LH
are
not
always
altered.
When
affected,
seminal
vesicle
weights
are
more
reduced
in
immature
than
adult
animals
with
vesicle
weights
are
more
reduced
in
immature
than
adult
animals
with
intact
animals
being
least
affected
intact
animals
being
least
affected
AGD
areola
++
NO
NO
??

+
+++
4
DAY
NO
NO
+++
+++
7
DAY
+++
+

+++
+++
2
DAY
++
VARIABLE
RESULTS
++
+
5
DAY
+++
??

++++
+++
LONG
++++
++

p,
p'
DDE
Vinclozolin
DBP
Linuron
Flutamide
Develop
Malform.
Hersh.
Necropsy
Pubertal
male
PPS
Pubertal
male.
OTHER
Intact
Adult
Fenitrothion
Should
EPA
run
dose­
range
finding
studies
as
part
of
the
ongoing
validation
effort
of
the
Pubertal
assays?
Propose
to
follow
the
model
of
a
dose
range
finding
study
for
in
utero
study
presented
to
EDMVS
in
2002.

Dosing
would
extend
for
the
full
duration
of
each
protocol
(
20
or
30
d
for
female
and
male
rat,
respectively)

Chemical
administered
during
gestation
for
the
duration
planned
for
the
full
study.

Doses
selected
based
upon
what
information
about
the
chemical
and
other
members
of
the
class
was
available.
Solubility
and
cost
also
were
considered.

Five
dosage
levels.
0,
62.5,
125,
250
and
500
mg/
kg/
d
by
gavage
in
corn
oil
Two
pregnant
females
per
dose
level.
Weighed
and
dosed
daily
Additional
endpoints,
as
per
a
full
study
were
collected
Effects
of
oral
"
unknown"
treatment
on
maternal
weight
and
weight
gain
during
treatment
in
the
SD
rat.

32.3
28.7
19.9
24.9
­
1.25
0
62.5
125
250
500
Dose
0
10
20
30
40
Weight
gain
(

g)
342
344
322
328
302
0
62.5
125
250
500
Dose
260
280
300
320
340
360
Weight
(

g)

In
utero
example
Additional
endpoints
for
potential
inclusion
during
Additional
endpoints
for
potential
inclusion
during
assay
validatation
or
follow­
up
T1.5
studies
assay
validatation
or
follow­
up
T1.5
studies
ex
vivo
gonad
hormone
production
ex
vivo
gonad
hormone
production
serum
E2,
T,
corticosterone,
T3,
LH
and
etc
serum
E2,
T,
corticosterone,
T3,
LH
and
etc
estrous
cyclicity
in
extended
female
assay
estrous
cyclicity
in
extended
female
assay
adrenal
weight
histology
and
function
adrenal
weight
histology
and
function
Pubertal
Male
Assay
Data
Pubertal
Male
Assay
Data
0
20
40
60
80
100
120
ADRENALS
(
mg)

Oil
KETO
45.4
117
Pubertal
Female
Assay
Data
Pubertal
Female
Assay
Data
0
20
40
60
80
100
120
ADRENALS
(
mg)

Oil
KETO
40.3
77.9
p
<.
0001
p
<.
0001
PRIORITIZATION
FOR
SCREENING
BASED
UPON
QSAR,
HPTS,
VOLUME,
ETC
TIER
1
SCREENING
About
100
rats
per
chemical
(
about
10,000
Rats/
100
chemicals)

HOLD
95%
Negative
No
Testing
90%
(­)
10%(+)
True
+
(
5%)

False
+
(
5%)

(
Type
II
Error)

(
10
chemicals
per
100
screened)

Replicate
Tier
1
Repeat
False
positives
5%

(
about
50
rats
per
repeat
or
500
rats
per
100
chemicals
screened)
Positives
5%
Tier
2
Testing
(
about
1,500
rats
per
chemical,

or
7,500
rats
per
100
screened)

Estimate
of
Animal
Use
in
EDSP
(
Assuming
5%
of
Chemicals
are
endocrine
active)

18,000
Rats
per
Hundred
Chemicals
Evaluated
Gray
et
al.,
in
press.
Toxicology
Letters.
ICT
IX
LEGJR
P
dose
V
dose
BWT
g
Mean
(
adj)
p­
value
(
adj)
Mean
p­
value
0
0
161
7.8
(.
18)
­
7.8
(.
48)
­

0
25
160
8.1
.2
8.2
.6
0
50
156
8.3
.04
8.1
.7
25
0
159
8.5
.005
8.5
.3
25
25
155
8.5
.02
8.2
.6
50
0
163
8.7
.002
8.8
.14
50
50
163
8.9
.0001
9.2
.055
ANCOVA.
Proper
and
effective
use
to
analyze
effects
of
vinclozolin
and
procymidone
on
liver
weight
in
the
Hershberger
assay.
ANOVA
(
adj)
df
SS
ANOVA
(
unadj)
df
SS
P
dose
2
8.04
2
8.04
V
dose
2
.36
2
.36
P
*
V
2
.81
2
.81
BWT
1
57.8
­
­

Model
7
67.0
6
0.5
Error
41
9.45
42
67.2
Total
48
76.4
48
76.4
ANCOVA.
Proper
and
effective
use
to
analyze
effects
of
vincolzolin
and
procymidone
on
liver
weight
in
the
Hershberger
assay.

ANOVA
tables
Organ
weight
Standard
error
with
ANCOVA
Standard
error
without
ANCOVA
Liver
g
.18
.48
Kidney
mg
32
57
Adrenals
mg
2.0
2.3
LABC
mg
7.1
8.6
SV
mg
6.9
6.9
VP
mg
3.6
3.7
Effect
of
ANCOVA
using
body
weight
on
standard
errors
of
tissue
weights
in
the
Hershberger
assay.
Using
relative
weights
confuses
interpretation
of
effects
on
hormone
Using
relative
weights
confuses
interpretation
of
effects
on
hormone
responsive
tissues.
When
body
weight
is
reduced
by
a
treatment
relative
responsive
tissues.
When
body
weight
is
reduced
by
a
treatment
relative
organ
weights
can
appear
elevated.
Question:
are
estrogens
stimulating
organ
weights
can
appear
elevated.
Question:
are
estrogens
stimulating
growth
of
androgen
dependent
tissues
in
a
Hershberger
assay?

growth
of
androgen
dependent
tissues
in
a
Hershberger
assay?

(
data
from
Yamasaki
et
al.,
2003,
Toxicology
183,
93)

(
data
from
Yamasaki
et
al.,
2003,
Toxicology
183,
93)

286
283
270
243
294
261
251
238
V+

TP
0.5
2
10
V+

TP
0.5
2
10
200
220
240
260
280
300
320
Body
Weight
Estrone
Equilin
62
76
96
108
72.4
96.5
98.8
109.2
V+

TP
0.5
2
10
V+

TP
0.5
2
10
50
60
70
80
90
100
110
120
SV
Weight
(
mg/
100
g
BWT)

Estrone
Equilin
Interpretation
of
effects
these
estrogens
on
SV
weight
would
be
clear
from
Interpretation
of
effects
these
estrogens
on
SV
weight
would
be
clear
from
an
analysis
of
the
actual
weights
and
the
rest
of
the
results
of
the
an
analysis
of
the
actual
weights
and
the
rest
of
the
results
of
the
Screening
battery,
which
would
indicate
that
the
chemicals
are
estrogens
Screening
battery,
which
would
indicate
that
the
chemicals
are
estrogens
and
estrogens
interact
with
TP
affecting
the
SV.

and
estrogens
interact
with
TP
affecting
the
SV.

(
data
from
Yamasaki
et
al.,
2003,
Toxicology
183,
93)

(
data
from
Yamasaki
et
al.,
2003,
Toxicology
183,
93)

V+

TP
0.5
2
10
V+

TP
0.5
2
10
200
220
240
260
280
300
320
Body
Weight
Estrone
Equilin
177
215
259
262
213
252
248
260
V+

TP
0.5
2
10
V+

TP
0.5
2
10
160
180
200
220
240
260
280
SV
Weight
Estrone
Equilin
