EDVMS
MEETING
DEC
10­
12,
2003
PUBERTAL
MALE
AND
FEMALE
RAT
ASSAYS
EDSTAC
TIER
1
Endocrine
Disrupter
Screening
and
Battery
1998
In
Vitro
ER
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
Steroid
Hormone
Synthesis
In
Vivo
Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

Hershberger
Assay
with
Castrate­
immature­
male
rat
(
10d)

Frog
Metamorphosis
Assay
Short­
term
Assessment
of
the
Fish
Reproductive
System
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
Questions?

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

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

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

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

Should
we
be
running
concurrent
dose­
range
finding
studies?

Can
we
reduce
the
sample
size
in
these
assays?
Comments
Data
analyses
presented
used
SAS
Proc
GLM
with
BW21
as
a
covariate.

Post
hoc
t­
tests
were
used
to
compare
means
to
control,
by
block,
when
F­
value
significant.

We
had
several
a
priori
hypotheses
for
many
of
the
chemicals
because
they
had
been
tested
prior
to
the
RTI
and
TI
2003
studies
Analyses
done
by
LEG
at
EPA
Use
of
relative
organ
weights
is
not
appropriate
for
EDC
screening
as
the
results
can
be
very
misleading.
ANCOVA
not
much
better
when
treatment
affects
body
weight.

Need
MTD
of
10%
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.

Pubertal
Assessment
of
the
LE
versus
the
SD
rats
TI­
2000.
For
each
sex
there
were
12
rats/
sex/
strain,
run
in
two
complete
blocks
to
determine
intralab
variability
using
6
treatment
groups
(
a
control
and
5
chemicals
at
a
single
dose).

Factorial
design
­
2
Strains
x
6
Treatments
x
2
Blocks
for
each
sex
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
Published
studies
that
have
used
the
pubertal
female
assay
Atrazine
­
2
labs
Nonyphenol
DES
­
2
labs
Methoxychlor
­
VO
and
cycles
Estradiol
Octylphenol
­
VO
and
cycles
Nonylphenol
­
VO
and
cycles
Tamoxifen
Fadrazole
Ketoconazole
Finasteride
Testolactone
Flutamide
Perchlorate
­
in
prep
PBDE
­
submitted
PTU
MBC
­
VO
and
cycles
DBP
­
VO
and
cycles
Chemicals
studied
this
year
in
the
pubertal
female
assay.
New
data
RTI
­
completed
Atrazine
75
or
150
mg/
kg/
d
Fenarimol
50
or
250
mg/
kg/
d
Methoxychlor
25
and
50
mg/
kg/
d
Bisphenol
A
400
or
600
mg/
kg/
d
Ketoconazole
50
and
100
mg/
kg/
d
PTU
2
and
25
mg/
kg/
d
TherImmune
2000
(
2x2x6
factorial.

Blocks,
LE
versus
SD
and
chemicals.

n=
6/
strain/
block)
­
completed
Ethynyl
estradiol
.005
mg/
kg/
d
Tamoxifen
10
mg/
kg/
d
PTU
240
mg/
kg/
d
Ketoconazole
100
mg/
kg/
d
Pimozide
30
mg/
kg/
d
Methoxychlor
100
mg/
kg/
d
TherImmune
2003
­
completed
Ethynyl
estradiol
0.0025
and
0.005
mg/
kg/
d
Methoxychlor
12.5,
25
and
50
mg/
kg/
d
Phenobarbital
50
and
100
mg/
kg/
d
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
and
delayed
age
at
VO
70
80
90
100
Percent
of
control
body
weight
­
6
­
5
­
4
­
3
­
2
­
1
0
1
2
3
4
5
6
7
Delay
in
VO
in
Days
FR
PTU2
PTU
25
BPA400
BPA600
MET25
MET50
KETO50
KETO100
FEN50
FEN250
ATR75
ATR150
Estrogens
in
the
pubertal
female
assay
Easily
detected
by
accelerated
VO
if
they
are
effective
with
oral
administration
in
the
rat
Effect
of
Ethynyl
Estradiol
on
puberty
in
the
SD
female
rat
TI­
SD­
2003
31.9
31.1
28.5
Oil
EE0025
EE005
25
30
35
AGE
AT
VO
**
**

33.4
32.5
28.7
Oil
EE0025
EE005
AGE
AT
Estrus
80
67
20
Oil
EE0025
EE005
%
Cycling
Normally
**
**
Effect
of
Ethynyl
estradiol
on
AGE
AT
VO
in
the
SD
female
rat
TI­
SD­
2003.
Note:
Ovarian
and
Uterine
histological
changes
noted
in
almost
all
females
in
TI­
SD/
LE­
2000
(
SD
more
affected)
but
no
affects
noted
in
TI­
SD­
2003
study.

31.9
31.1
28.5
Oil
EE0025
EE005
26
28
30
32
34
36
38
TI­
SD­
2003
**
34.9
25.9
36.3
26
Oil
EE005
24
26
28
30
32
34
36
38
SD
LE
TI­
SD/
LE­
2001
**
Effects
of
Methoxychlor
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
32.9
29.8
27.4
Con
A
MET
25
MET
50
22
24
26
28
30
32
34
36
Age
VO
128
105
89.5
CON
A
MET
25
MET
50
80
90
100
110
120
130
140
Wt
at
VO
*
*
*

35.1
31.1
28.8
Con
A
MET
25
MET
50
25
30
35
40
Age
1st
P/
E
*
*
*
Effects
of
Methoxychlor
ON
AGE
AT
VO
in
the
Female
RTI­
1994­
LE,
EPA­
1989
and
RTI­
SD­
2003
rat
studies
32.9
29.8
27.4
Con
A
MET
25
MET
50
22
24
26
28
30
32
34
36
RTI­
SD­
2003
*

*
32
26.3
25.4
Con
A
MET
25
MET
50
EPA­
LE­
1989
29
25.6
Con
A
MET
50
RTI­
LE­
1994
*

*
*
Effects
of
Methoxychlor
in
the
Pubertal
Female
SD
rat
assay
94
85
126
106
87
91
107
94
89
91
100
100
95
90
111
102
77
91
102
98
92
92
100
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
Histo
Normal
thyroid
Histo
80
90
100
110
120
Con
A
MET
25
MET
50
Lack
of
effect
of
BPA
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
on
age
and
weight
at
VO.
Octylphenol
and
Nonylphenol
accelerate
VO
in
this
assay.
BPA
is
positive
in
the
in
vitro
and
uterotropic
assays
but
negative
here
32.3
33
32.3
CON
B
BPA
400
BPA
600
30
31
32
33
34
35
36
Age
VO
123
116
108
CON
B
BPA
400
BPA
600
100
110
120
130
140
Wt
at
VO
*
Effects
of
BPA
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
86
66
74
68
94
81
86
88
86
80
100
100
89
73
82
83
88
83
87
90
86
85
100
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
Histo
Normal
thyroid
Histo
60
70
80
90
100
110
Con
A
BPA
400
BPA
600
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
ovary
and
uterine
weights
80
85
90
95
100
Percent
of
control
body
weight
60
70
80
90
100
110
Percent
OF
CONTROL
weight
FR
BPA400
BPA600
Ovarian
weight
80
85
90
95
100
Percent
of
control
body
weight
60
70
80
90
100
110
Percent
OF
CON
weight
FR
BPA400
BPA600
Uterine
Weight
Daily
oral
(
mg/
kg/
d)
Octylphenol
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).
Also
see
Stoker
et
al.

and
Goldman
et
al.,
2001.

0
50
100
150
200
0
1
2
3
4
5
6
78
Days
VO
is
accelerated
Octylphenol
"
Antiestrogens"

and
Inhibitors
of
Steroidogenesis
Tamoxifen­
mixed
agonist/
antagonist
ICI
and
ZM
­
ER
antagonists
Antralex
­
GnRH
antagonist
Fadrazole
­
Aromatase
inhibitor
Fenarimol
­
fungicide
that
inhibits
aromatase
in
vivo
and
in
vitro
Ketoconazole
­
like
fenarimol,
but
less
specific
for
P450
aromatase
Results
as
expected,
or
better,
except
Fenarimol
Effects
of
TAMOXIFEN
10
MG/
KG
in
the
SD
AND
LE
Pubertal
Female
rat
from
TI
2000
26.7
26.5
34.9
36.3
SD
LE
20
25
30
35
40
CON
TAM
AGE
VO
64.2
66.2
111
130
SD
LE
50
60
70
80
90
100
110
120
130
140
CON
TAM
Weight
at
VO
Effects
of
TAMOXIFEN
10
MG/
KG
in
the
SD
and
LE
Pubertal
Female
rat
from
TI
2000
36.8
44.2
64.7
81.7
SD
LE
20
30
40
50
60
70
80
90
CON
TAM
OVARIAN
WT
111
102
292
275
SD
LE
50
100
150
200
250
300
CON
TAM
Uterine
Weight
Effect
of
"(
anti)
estrogens"
on
age
at
VO
in
the
Female
rat.
Data
from
Ashby
et
al.,
2002;
Marty
et
al.,
1999;
and
Kim
et
al.,
2002.
ZM
189,154
and
ICI
182,780
are
ER
antagonists.
Tamoxifen
is
a
mixed
ER
agonist
and
antagonist.
Antralex
is
a
GnRH
antagonist.

­
14
­
12
­
10
­
8
­
6
­
4
­
2
0
2
4
6
8
FAD
.6
FAD
1.2
FAD
6
ANTARELIX
DES
5
TAM
50
TAM
200
Deviation
in
days
from
Control
80
0
10
0
0
6
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
Control
ZM
oral
ICI
oral
ANTERELIX
sc
%
VO
day
33
Effects
of
FENARIMOL
in
the
Pubertal
Female
SD
rat
assay
RTI­
SD­
2003
34.2
33.7
32.9
Age
VO
30
31
32
33
34
35
36
Con
A
Fenarimol
50
Fenarimol
250
115
133
128
Wt
at
VO
100
110
120
130
140
Con
A
Fenarimol
50
Fenarimol
250
*
Lack
of
effect
of
FENARIMOL
on
VO
in
the
Female
rat
from
RTI­
SD­
2003
and
EPA­
LE­
1990
studies
34.2
33.7
32.9
26
28
30
32
34
36
Con
A
Fenarimol
50
Fenarimol
250
RTI­
SD­
2003
29.3
31.6
30.4
26
28
30
32
34
36
Con
Fenarimol
35
Fenarimol
70
EPA­
LE­
1990
Effects
of
FENARIMOL
in
the
Pubertal
Female
SD
rat
assay
RTI­
SDS­
2003
90
88
84
81
78
146
102
95
82
86
100
100
101
109
101
103
80
118
121
106
100
101
100
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
H
Normal
thyroid
70
80
90
100
110
Con
A
Fenarimol
50
Fenarimol
250
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
ovary
and
uterine
weights
80
85
90
95
100
Percent
of
control
body
weight
60
70
80
90
100
110
120
Percent
OF
CONTROL
weight
FR
FEN50
FEN250
Ovarian
weight
80
85
90
95
100
Percent
of
control
body
weight
60
70
80
90
100
110
Percent
OF
CON
weight
FR
FEN50
FEN250
Uterine
Weight
Effects
of
KETOCONAZOLE
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay.
OVARIAN
HISTOPATH
IS
KEY
FOR
THIS
CHEMICAL
32.3
33
33.3
Con
A
KETO
50
KETO
100
30
31
32
33
34
35
36
Age
VO
123
127
123
CON
B
KETO
50
KETO
100
100
110
120
130
140
Wt
at
VO
Effects
of
KETOCONAZOLE
on
age
at
VO
in
the
Female
rat
RTI­
SD­
2003,
TI­
SD/
LE­
2000
and
MSM­
1999
32.3
33
33.3
Con
A
KETO
50
KETO
100
30
31
32
33
34
35
36
RTI­
2003
32.3
33
33.3
Con
A
KETO
50
KETO
100
TI­
2000
33.5
36
39.7
Con
A
KETO
50
KETO
100
MSM­
1999
*
Effects
of
KETOCONAZOLE
in
the
RTI­
SD­
2003
and
TI­
SD/
LE­
2000
Pubertal
Female
SD
rat
assay.

Percent
of
females
with
ovarian
histologic
lesions
100
80
0
0
20
40
60
80
100
CON
KETO
50
KETO
100
0
100
0
100
0
20
40
60
80
100
SD
LE
RTI­
SD­
2003
TI­
SD/
LE­
2000
Effects
of
KETOCONAZOLE
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
92
84
73
73
100
108
109
91
89
0
100
99
96
91
89.3
103
110
167
108
100
98
10
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
Histo
Normal
thyroid
Hist
70
80
90
100
110
CON
KETO
50
KETO
100
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
ovary
and
uterine
weights
80
85
90
95
100
Percent
of
control
body
weight
65
70
75
80
85
90
95
100
105
Percent
OF
CONTROL
weight
FR
KETO50
KETO100
80
85
90
95
100
Percent
of
control
body
weight
65
70
75
80
85
90
95
100
105
Percent
OF
CON
weight
FR
KETO50
KETO100
Effects
of
CNS
active
chemicals
on
puberty
in
the
female
Atrazine
­
alters
CNS
dopamine,

prolactin
and
LH
surges,
also
may
affect
aromatase
and
etc.
Results
reproducible
Phenobarbital
­
alters
CNS,
pituitary
and
liver
function,
enhancing
hormone
metabolism
and
secretion.

Results
robust
and
reproducible
Effects
of
ATRAZINE
in
the
Pubertal
RTI­
SD­
2003
Female
rat
age
and
weight
at
VO
and
age
at
first
estrus.
Note:
EPA
needs
to
provide
more
guidance
on
analysis
of
estrous
cycle
data
32.9
34.2
36.4
Con
A
ATR
75
ATR
150
30
31
32
33
34
35
36
37
38
39
Age
VO
128
128
130
CON
A
ATR
75
ATR
150
100
110
120
130
140
Wt
at
VO
*
35.1
35.8
38.1
Con
A
ATR
75
ATR
150
32
33
34
35
36
37
38
39
40
41
Age
1st
P/
E
**
Effects
of
ATRAZINE
at
100­
150
mg
in
the
Pubertal
Female
rat
assay.
RTI
(
SD)
versus
Laws
(
Wistar)

and
Ashby
et
al
(
SD
and
AP
strains).

32.9
34.2
36.4
Con
A
ATR
75
ATR
150
30
31
32
33
34
35
36
37
38
39
Age
VO­
RTI
*
32.5
35.9
37.1
32.8
34.3
36.3
Con
­
Laws
ATR
50
ATR
100
30
31
32
33
34
35
36
37
38
39
Age
VO­
EPA­
1
2
*

*
38
41
39
42.2
Con
ATR
100
35
36
37
38
39
40
41
42
43
44
45
SD
AP
*

*
Effects
of
ATRAZINE
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
86
81
87
86
84
90
96
93
78
80
100
100
92
95
97
96
84
93
111
97
94
89
100
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
Histo
Normal
thyroid
Hist
80
90
100
110
Con
A
ATR
75
ATR
150
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
ovary
and
uterine
weight
80
85
90
95
100
Percent
of
control
body
weight
65
70
75
80
85
90
95
100
105
Percent
OF
CONTROL
weight
FR
ATR75
ATR150
Ovarian
Weight
80
85
90
95
100
Percent
of
control
body
weight
65
70
75
80
85
90
95
100
105
Percent
OF
CON
weight
FR
ATR75
ATR150
Uterine
Weight
Effects
of
Phenobarbital
on
female
rat
puberty
TI­
SD­
2003
31.9
33.1
32.8
34.5
Oil
PB25
PB50
PB100
29
30
31
32
33
34
35
36
37
AGE
AT
VO
*
**
118
123
121
128
Oil
PB25
PB50
PB100
115
120
125
130
135
Wt
at
VO
*
Effects
of
Phenobarbital
on
female
rat
puberty.

TI­
SD­
2003
8.67
9.79
10.76
11.32
Oil
PB25
PB50
PB100
8
9
10
11
12
13
LIVER
WT
41.4
52
52.6
51.8
Oil
PB25
PB50
PB100
30
35
40
45
50
55
60
ADRENAL
WT
**

**
**

**
**
**
Antithyroid
effects
on
puberty
in
the
female
PTU
and
the
corresponding
growth
retardation
have
surprisingly
little
affect
on
pubertal
landmarks
Effects
of
PTU
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
32.3
33.3
33.1
CON
B
PTU
2
PTU
25
30
31
32
33
34
35
36
Age
VO
123
127
118
CON
B
PTU
2
PTU
25
100
110
120
130
140
Wt
at
VO
Effects
of
PTU
in
the
RTI­
SD­
2003
Pubertal
Female
SD
rat
assay
72
75
104
99
433
60
58
69
86
60
100
97
88
105
104
268
93
87
95
97
96
100
BWT
OVWT
UT
WET
UT
DRY
THYROID
LIVER
ADRENAL
KID
PIT
BW
GAIN
Normal
Ovary
Histo
Normal
thyroid
Hist
10
100
1000
Con
A
PTU
2
PTU
25
OBSERVATIONS
ON
THE
PUBERTAL
FEMALE
ASSAY
Responds
very
well
to
estrogens
Responds
very
well
to
GnRH
antagonist
and
potent
aromatase
inhibitors
Responds
well
to
ketoconazole,
but
effects
not
the
pubertal
landmarks
Reproducible
responses
to
Atrazine
Reproducible
responses
to
Phenobarbital
Negative
for
Fenarimol
at
doses
tested.

Need
a
study
at
the
MTD
with
doses
from
dose­
range
finding,
not
guessing
EDVMS
MEETING
DEC
10­
12,
2003
PUBERTAL
MALE
AND
FEMALE
RAT
ASSAYS
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
Alternative
Assays
for
Possible
Inclusion
in
T1S
the
Pubertal
Male
assay
was
an
Alternative
assay
that
EDSTAC
wanted
developed
and
evaluated
for
screening
If
standardized
and
validated
and
found
to
be
functionally
equivalent,
or
better,

then
an
alternative
assay
could
be
included
in
T1S.
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
Our
Immature
(
21
­
52
Days
of
Age)
Intact
Male
Rat
Protocol
to
Evaluate
Pubertal
Development
and
Thyroid
Function.
Detects
Evaluate
Pubertal
Development
and
Thyroid
Function.
Detects
inhibition
of
steroidogenesis,
antithyroid
and
(
anti)
androgenic
inhibition
of
steroidogenesis,
antithyroid
and
(
anti)
androgenic
activities
and
altered
HPG
maturation.

activities
and
altered
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
The
serum
androgen
profile
changes
dramatically
during
changes
dramatically
during
puberty
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
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
Adult
Male
rats
measured
repeatedly
at
ten
minute
intervals.

Large
CVs
require
large
effects
of
large
sample
sizes
(
Fail
et
al.,
1996)
Modified
USEPA
Endocrine
Disrupter
Screening
and
Testing
Battery
with
the
pubertal
male
assay
replacing
the
Hershberger
assay
In
Vitro
ER
Binding
and/
or
Transcriptional
Activation
AR
Binding
and/
or
Transcriptional
Activation
Steroid
Hormone
Synthesis
In
Vivo
Uterotropic
Assay
in
Ovariectomized
Adult
Rat
(
3d)

Pubertal
Female
Rat
Assay
including
Thyroid
(
20d)

Pubertal
Male
Rat
Assay
including
Thyroid
(
30d)

Frog
Metamorphosis
Assay
Short­
term
Assessment
of
the
Fish
Reproductive
System
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
Chemicals
investigated
in
the
pubertal
male
Chemicals
investigated
in
the
pubertal
male
assay
at
RTI
and
TI
this
year
assay
at
RTI
and
TI
this
year
RTI
­
completed
RTI
­
completed
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
Vinclozolin
30
and
100
mg/
kg/
d
mg/
kg/
d
Methoxychlor
25
and
50
Methoxychlor
25
and
50
mg/
kg/
d
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)
­

n=
6/
strain/
block/
chemical)
­

completed
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
­
completed
TherImmune
2003
­
completed
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
pubertal
male
assay
p,
p'
DDE
­
several
labs
Vinclozolin
­
2
labs
Linuron
­
PPS
Cyproterone
acetate
Flutamide
­
3
labs
Finasteride
­
2
labs
Ketoconazole
­
2
labs
Testolactone
TP
BBP
DBP
­
2
labs
Methoxychlor
­
2
labs
BPA
DES
PTU
Perchlorate
­
in
prep
PBDE
­
submitted
Phenobarbital
Fenitrothion
MBC
­
PPS
Atrazine
­
2
labs
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
and
delay
in
age
at
PPS
80
85
90
95
100
Percent
of
control
body
weight
­
10
­
5
0
5
10
Delay
in
PPS
in
Days
Feeding
Vinclozolin
Linuron
DDE1
DDE2
DBP
DBP2
KETO­
TH
MT­
TH
FLUT­
TH
FR­
SMEC
AR
antagonists
Flutamide
Vinclozolin
p,
p'
DDE
Linuron
Results
­

all
chemicals
produced
expected
robust
responses,

easily
detected
all
of
the
above.
Reproducible
41.1
53.3
54
Oil
F25
F50
35
40
45
50
55
60
AGE
AT
PPS
DAYS
Effects
of
flutamide
at
25
and
50
mg/
kg
on
puberty
in
the
male
rat
TI­
SD­
2003
**
**
214
319
311
Oil
F25
F50
150
200
250
300
350
400
WT
AT
PPS
DAYS
**
**
698
175
135
Oil
F25
F50
0
200
400
600
800
1000
SD
TI
2003
SV
WT
Effects
of
flutamide
at
25
and
50
mg/
kg
In
the
male
rat
TI­
SD­
2003
(
RED)
and
TI­
SD/
LE­
2000
**
**
2.71
3
3.5
Oil
F25
F50
2.6
2.8
3
3.2
3.4
3.6
TESTES
WT
**
**

371
80
477
76
Oil
F50
0
100
200
300
400
500
SD
LE
SV
WT
**
2.67
3.12
3.09
3.52
Oil
F50
2.6
2.8
3
3.2
3.4
3.6
SD
LE
TESTES
WT
**
Effects
of
Vinclozolin
in
male
pubertal
SD
rat
assay­
RTI
2003
41.4
43.8
46.8
0
30
100
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
45.5
46
46.5
47
47.5
48
Age
at
PPS
219
243
260
0
30
100
180
200
220
240
260
280
Weight
at
PPS
**

**

**

**
Effects
of
Vinclozolin
on
AGE
AT
PPS
in
male
SD
and
LE
rats
41.4
43.8
46.8
0
30
100
40
41
42
43
44
45
46
47
48
49
50
RTI­
SD­
2003
**

**

**

**
40.7
44.4
47.8
0
25
100
RTI­
LE­
1994
**

**

41.1
42.7
45.5
0
30
100
EPA­
LE­
1996
**
**
204.9
232
270
0
25
100
RTI­
LE­
1997
Effects
of
Vinclozolin
in
Weight
at
PPS
in
male
LE
and
SD
rats
219
243
260
0
30
100
180
200
220
240
260
280
300
RTI­
SD­
2003
**

**
**
204
208
232
0
30
100
EPA­
LE­
1996
**
Effects
of
Vinclozolin
in
male
RTI
2003
pubertal
rat
assay
96
55
77
77
82
107
102
84
97
103
94
108
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
50
60
70
80
90
100
110
120
30
100
%
OF
CONTROL
WEIGHTS
**
**

**

**

**

**

*
Effects
of
Vinclozolin
on
male
reproductive
organ
weights
in
the
RTI­
SD­
2003
study
compared
to
EPA­
LE­
1996
pubertal
rat
assay
at
100
mg/
kg/
d
96
71
100
80
86
96
55
107
77
82
BWT
SV
TESTIS
PROSTATE
EPIDIDYMIS
50
60
70
80
90
100
110
120
RTI
EPA
%
OF
CONTROL
WEIGHTS
**
**

**

**

**

**
The
intact
male
assay
is
much
less
sensitive
to
the
effects
of
vinclozolin
on
ASO
than
are
seminal
vesicle
weights
in
our
Hershberger
or
pubertal
male
assays.

0
50
100
150
200
250
300
30
40
50
60
70
80
90
100
Percent
of
Control
Intact
Male
Hershberger
Pubertal
2
labs
The
Adult
Intact
Male
assay
is
much
less
sensitive
to
the
Effects
of
vinclozolin
on
ventral
prostate
weight.
Males
treated
with
TP
in
the
Hershberger
Assay.

0
50
100
150
200
250
300
0
20
40
60
80
100
%

Control
Intact
Male
Hershberger
Pubertal
2
Labs
It
is
critical
that
the
T1
Screening
assay
be
able
to
detect
we
ak
antiandrogens
like
DDE
and
linuron
because
they
cause
reproductive
tract
malformation
in
utero.
Exposure
to
100
mg
p,
p'
DDE/
kg­
dam's
body
weight
on
gestational
days
14­
18
alters
development
of
the
External
Genitalia.
Gray
et
al,
1999.

Control
SD
Male
Rat
Treated
SD
Male
with
Cleft
Phallus,
Exposed
Os,
Hypospadias
and
Slight
Vaginal
Pouch
Effects
of
p,
p'
DDT
and
p,
p'
DDE
treatment
during
sexual
Effects
of
p,
p'
DDT
and
p,
p'
DDE
treatment
during
sexual
differentiation
in
Dutch
Belted
rabbits.
(
Veeramachaneni
et
differentiation
in
Dutch
Belted
rabbits.
(
Veeramachaneni
et
al.,
1996;
2000;
Palmer
et
al.,
2000)

al.,
1996;
2000;
Palmer
et
al.,
2000)

Does
exposed
weekly
to
p,
p'
DDT
at
25
mg/
kg
Does
exposed
weekly
to
p,
p'
DDT
at
25
mg/
kg
from
GD
1
through
6
weeks
postpartum,

from
GD
1
through
6
weeks
postpartum,

followed
by
10
mg/
kg
directly
to
male
up
to
followed
by
10
mg/
kg
directly
to
male
up
to
12
weeks
of
age.

12
weeks
of
age.

Effects
Effects
Cryptorchidism
Cryptorchidism
Atypical
germ
cells
in
undescended
testes
inclduing
Atypical
germ
cells
in
undescended
testes
inclduing
Carcinoma­
in­
situ
like
cells
Carcinoma­
in­
situ
like
cells
At
8
and
12
weeks
At
8
and
12
weeks
serum
p,
p'
DDT
at
231
and
38
ppb
serum
p,
p'
DDT
at
231
and
38
ppb
serum
p,
p'
DDE
at
187
and
37
ppb
serum
p,
p'
DDE
at
187
and
37
ppb
Effects
of
p,
p'
DDE
in
male
pubertal
rat
assay
RTI­
SD­
2003
41.4
44.9
45.7
0
50
100
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
45.5
46
46.5
47
47.5
48
Age
at
PPS
219
251
259
0
50
100
180
200
220
240
260
280
Weight
at
PPS
**
**
**

**
Effects
of
p,
p'
DDE
in
male
pubertal
rat
assay
41.4
44.9
45.7
0
50
100
40
41
42
43
44
45
46
47
48
49
50
RTI­
SD­
2003
**
**
**

**
43.3
43.5
48
0
30
100
40
41
42
43
44
45
46
47
48
49
50
EPA­
LE­
1994
**
Effects
of
p,
p'
DDE
in
male
pubertal
rat
assay
RTI­
SD­
2003
98
88
88
97
90
101
100
102
96
109
96
102
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
80
90
100
110
50
100
%
OF
CONTROL
WEIGHTS
*
**
100
97
101
105
100
100
70
37
34
100
102
88
0
50
100
150
200
250
300
350
30
40
50
60
70
80
90
100
110
Intact
Male
Assay
Hershberger
Pubertal
M
%
control
The
Adult
intact
male
assay
is
insensitive
to
the
Effects
of
p,
p'
DDE
at
100
mg/
kg/
d
on
SV
versus
ASO
weight.
Males
treated
with
TP
in
the
Hershberger
Assay.
100
88
115
97
99
0
50
100
150
200
250
300
350
30
40
50
60
70
80
90
100
110
120
IM
H
Pubertal
%
Control
The
intact
male
assay
is
insensitive
to
the
Effects
of
p,
p'
DDE
at
100
mg/
kg/
d
on
ventral
prostate
weight.

Males
treated
with
TP
in
the
Hershberger
Assay.
Effects
of
gestational
day
14­
18
treatment
with
linuron
(
75
mg/
kg/
d)
and/
or
benzyl
butyl
phthalate
(
BBP)
on
fetal
male
rat
testicular
testosterone
production
in
3
h,
fetal
testis
and
whole
body
testosterone
levels,
and
nipple/
areola
induction.
All
values
differ
significantly
from
control
(
Hotchkiss
in
prep)

CON
LIN
BBP
LIN+
BBP
0
2
4
6
8
10
Numbers
of
areolas/
nipples
CON
LIN
BBP
LIN+
BBP
0
1
2
3
4
5
TESTICULAR
T
PRODUCTION
(
ng/
testis/
3
CON
LIN
BBP
LIN+
BBP
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
TESTICULAR
T
(
ng/
testis)

CON
LIN
BBP
LIN+
BBP
0
0.05
0.1
0.15
0.2
0.25
WHOLE
BODY
T
(
ng/
fetus)

Low­
dose
Effects
of
Linuron
on
Sexual
Differentiation
of
the
male
rat
(
McIntyre
et
al,
2000,
Toxicol
Appl
Pharm
167,
87­
99).
Dams
were
dosed
with
linuron
at
12.5,
25
or
50
mg/
kg/
d
from
GD
12
to
21.
No
direct
exposure
to
pups.
This
pesticide
is
negative
in
published
teratology
and
multigenerational
tests.
Could
the
current
tests
detect
these
malformations
in
only
20
F1
males/
group?

0
0
3.6
1.8
11.6
11.6
11.4
4.6
0
12.5
25
50
Dose
of
Linuron
mg/
kg/
d
0
2
4
6
8
10
12
14
Hypoplastic
Testis
Hypoplastic
Epididymal
Percent
of
Male
Offspring
Malformed
Effects
of
Linuron
in
male
pubertal
rat
assay
RTI­
SD­
2003
39.6
43.6
45.5
0
50
100
38
38.5
39
39.5
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
45.5
46
46.5
47
Age
at
PPS
207
227
224
0
50
100
190
200
210
220
230
Weight
at
PPS
**

**

**
*
Effects
of
Linuron
on
PPS
in
the
EPA­
LE­
1987
and
RTI­
SD­
2003
male
rats
39.6
43.6
0
50
38
39
40
41
42
43
44
45
46
47
RTI­
SD­
2003
43
45.5
0
40
38
39
40
41
42
43
44
45
46
47
EPA­
LE­
1987
**
*
Effects
of
Linuron
in
male
pubertal
rat
assay
RTI­
SD­
2003
84
56
71
70
84
93
93
73
85
74
92
97
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
50
60
70
80
90
100
50
100
%
OF
CONTROL
WEIGHTS
*
*
**

**
**
**
**
**
**
100
94
104
93
81
0
50
100
150
60
70
80
90
100
110
Intact
Male
Hershberger
Pubertal
M
%
Control
The
Adult
intact
male
assay
is
much
less
sensitive
to
the
Effects
of
Linuron
at
100
mg/
kg/
d
on
ventral
prostate
weight.
Males
treated
with
TP
in
the
Hershberger
Assay.
100
105
104
96
89
0
50
100
150
200
50
60
70
80
90
100
110
Intact
Male
Hershberger
Pubertal
%
Control
The
Adult
intact
male
assay
is
much
less
sensitive
to
the
Effects
of
Linuron
at
100
mg/
kg/
d
on
SV
versus
ASO
weight.
Males
treated
with
TP
in
the
Hershberger
Assay.

MTD
The
Phthalate
Esters
Inhibitors
of
Fetal
Male
Rat
Testicular
Hormone
Synthesis.
Not
an
AR
antagonist
Negative
in
Developmental
Toxicology/
Teratology
Studies
Negative
in
some
Multigenerational
Studies
Results
in
Transgenerational
Studies
DEHP
++

BBP
++

DBP
++

DINP
+

DEP
negative
DMP
negative
DOTP
negative
Effects
of
DBP
in
male
rat
TI­
2000­
SD/
LE
43
44.2
0
1000
40
41
42
43
44
45
46
47
48
49
50
SD
AGE
at
PPS
**
**

47.5
51.8
0
1000
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
LE­
AGE
at
PPS
**
**
Effects
of
DBP
in
male
rat
EPA­
LE­
1985,
1986
42.4
48.4
0
1000
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
LE­
EPA­
1985
AGE
at
PPS
**
**
**

39.5
42.6
43.4
44.4
0
250
500
1000
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
LE­
EPA­
1986
AGE
at
PPS
*
**
**
3.09
2.44
OIL
DBP
2.00
2.50
3.00
Testes
weights
477
382
OIL
DBP
350
400
450
500
Seminal
Vesicle
weights
623
514
OIL
DBP
500
550
600
650
LABC
weights
5.9
21.1
20
17.5
BWT
TESTES
SV
LABC
0
5
10
15
20
25
%
REDUCTION
INDUCED
BY
DBP
DBP
reduces
reproductive
tissue
weights
in
the
pubertal
male
assay
using
SD
rats,
the
least
affected
strain.
TI­
2000
0.62
1.01
1.76
1.33
Testis
SV
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
0
1000
LE­
EPA­
1985
Nec
at
82
days
Effects
of
1000
mg/
kg
DBP
on
testis
weight
in
the
LE
and
SD
male
rat
from
studies
TI­
SD/
LE­
2002,
EPA­
LE­
1985
and
EPA­
SD­
1981
0.45
0.35
1.44
0.75
Testis
SV
SD­
EPA­
1981
Nec
at
52
days
**

**
*
0.37
0.174
1.34
0.37
Testis
SV
LE­
TI­
2000
Nec
at
53­
54
days
**
1.22
0.381
1.55
0.477
Testis
SV
SD­
TI­
2000
Nec
at
53­
54
days
*

**

**

**

Strain*
Treatment
Interaction
p<
0.01
SD
less
affected
SD
less
affected
SD
not
less
affected
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.
EFFECTS
OF
AN
ESTROGENIC
AND
POTENTIALLY
ANTIANDROGENIC
PESTICIDE
NO
EFFECT
ON
PPS,
AS
EXPECTED
FROM
EARLIER
WORK
REDUCED
ANDROGEN­
DEPENDENT
TISSUE
WEIGHTS
POSITIVE
IN
THE
PUBERTAL
MALE
Effects
of
Methoxychlor
in
male
pubertal
rat
assay
RTI­
SD­
2003
41.4
41.8
41.8
0
25
50
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
Age
at
PPS
219
219
220
0
25
50
170
180
190
200
210
220
230
240
Weight
at
PPS
Effects
of
Methoxychlor
on
PPS
in
the
male
rat
from
EPA­
LE­
1989
study
verus
RTI­
SD­
2003
41.4
41.8
41.8
0
25
50
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
RTI­
SD­
2003
42.4
40.6
43.4
0
25
50
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
EPA­
LE­
1989
NO
EFFECT
ON
PPS
AT
THESE
DOSES
IN
EITHER
STUDY
Effects
of
Methoxychlor
in
male
pubertal
rat
assay
96
73
86
95
96
100
96
91
97
94
101
101
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
70
80
90
100
25
50
%
OF
CONTROL
WEIGHTS
**
*
ANDROGENIC
EFFECTS
IN
THE
PUBERTAL
MALE
ASSAY
VERY
POSITIVE
RESPONSES
EXACTLY
AS
EXPECTED
ON
PPS,
TESTIS
AND
SEX
ACCESSORY
TISSUES
47.5
32.9
43
36.8
CON
MT
25
30
35
40
45
50
SD
LE
AGE
AT
PPS
DAYS
Effects
of
METHYLTESTOSTERONE
at
80
mg/
kg
on
puberty
in
the
male
rat
TI­
SD/
LE­
2000
**
185
144
238
119
CON
MT
100
150
200
250
WT
AT
PPS
**
**

**
477
710
371
729
CON
MT
200
300
400
500
600
700
800
900
1000
SD
LE
SV
WT
Effects
of
METHYLTESTOSTERONE
at
80
mg/
kg
In
the
male
rat
TI­
SD/
LE­
2000
**
2.67
0.6
3.09
0.89
CON
MT
0
1
2
3
TESTES
WT
**

221
335
182
374
CON
MT
0
100
200
300
400
500
VP
WT
**
AN
INHIBITOR
OF
P450
ENZYMES,
INCLUDING
STEROIDOGENESIS
VERY
POSITIVE
REPRODUCIBLE
REPRODUCTIVE
AND
OTHER
ENDOCRINE
(
ADRENAL)
AND
ORGAN
(
LIVER)

EFFECTS
Effects
of
Ketoconazole
in
male
pubertal
rat
assay
RTI­
SD­
2003
39.6
42.3
44.1
0
50
100
38
38.5
39
39.5
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
Age
at
PPS
207
227
235
0
50
100
190
200
210
220
230
240
250
260
Weight
at
PPS
**

**

**

**
Effects
of
Ketoconazole
ON
THE
AGE
AT
PPS
39.6
42.3
44.1
0
50
100
38
38.5
39
39.5
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
RTI­
SD­
2003
**

**
47.5
48.9
43
46.3
0
100
38
39
40
41
42
43
44
45
46
47
48
49
50
SD
LE
TI­
SD/
LE­
2000
**
Effects
of
Ketoconazole
on
reproductive
organs
in
TI­
SD/
LE­
2000
and
RTI­
SD­
2003.
Also,
consistent
increases
in
adrenal
and
liver
weights
at
both
doses.

**

**
638
546
567
448
623
471
0
100
400
450
500
550
600
650
700
**

639
423
367
223
477
261
0
100
150
200
250
300
350
400
450
500
550
600
650
700
SD­
TI
LE­
TI
SD­
RTI
266
208
181
123
221
137
0
100
100
150
200
250
300
**

**
VP
SV
LABC
**
*
Relationship
between
reduction
in
body
weight
due
to
restricted
feeding
versus
ventral
prostate
weight
80
85
90
95
100
Percent
of
control
body
weight
60
70
80
90
100
110
120
Percent
OF
CONTROLventral
prostate
weigh
Feeding
Vinclozolin
KETO­
TH
FR­
SMEC
R­
square
=
0.819
#
pts
=
5
y
=
­
36.6
+
1.42x
Effects
of
Atrazine
in
male
pubertal
rat
assay
41.4
42
42.9
0
75
150
40
40.5
41
41.5
42
42.5
43
43.5
44
Age
at
PPS
219
209
200
0
75
150
170
180
190
200
210
220
230
240
Weight
at
PPS
*
**
ATRAZINE
A
CNS
ACTIVE
CHEMICAL
DELAYS
PPS
IN
ALL
STUDIES
VARIABLE
EFFECTS
ON
BODY
WEIGHT
Effects
of
ATRAZINE
in
the
Pubertal
Male
rat
assay
RTI
versus
Stoker
al.

41.4
42
42.9
Con
A
ATR
75
ATR
150
40
41
42
43
44
45
Age
PPS­
RTI
42.1
43.8
43.6
43.4
Con
­
Stoker
ATR
50
ATR­
100
ATR­
150
40
41
42
43
44
45
46
Age
PPS­
EPA
*

*

*
*

1.5
d
0.6
d
1.7
d
1.3
d
1.5
d
Effects
of
Atrazine
in
male
pubertal
rat
assay.
RTI
versus
Stoker
data
at
150
mg/
kg/
d
81
76
79
81
87
103
90
84
75
94
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
70
80
90
100
Stoker
RTI
%
OF
CONTROL
WEIGHTS
PHENOBARBITAL
AFFECTS
CNS,
LIVER
METABOLISM
AND
ENDOCRINE
ORGANS
DIRECTLY
ROBUST,
REPRODUCIBLE
RESPONSES
ON
MALE
PPS
AND
REPRODUCTIVE
ORGAN
WEIGHTS
Effects
of
Phenobarbital
in
male
pubertal
rat
assay
39.6
41.3
43
0
50
100
38
38.5
39
39.5
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
Age
at
PPS
207
220
224
0
50
100
190
200
210
220
230
Weight
at
PPS
**
**
*

*
Effects
of
Phenobarbital
in
male
pubertal
rat
assay
39.6
41.3
43
0
50
100
38
38.5
39
39.5
40
40.5
41
41.5
42
42.5
43
43.5
44
44.5
45
RTI
SD
2003
**
**
41.1
41.1
43.6
Oil
PB50
PB100
38
39
40
41
42
43
44
45
TI­
SD­
2003
**
Effects
of
Phenobarbital
in
male
pubertal
rat
assay
92
76
82
88
88
92
119
97
100
95
97
99
97
117
BWT
SV
LABC
PROSTATE
EPIDIDYMIS
TESTIS
Thyroid
70
80
90
100
110
120
130
50
100
%
OF
CONTROL
WEIGHTS
*
**
**
**
*
*
OBSERVATIONS
ON
THE
PUBERTAL
MALE
ASSAY
Produced
expected
results
for
all
chemicals
Both
SD
and
LE
strains
responded
significantly
Appears
to
detect
all
the
activities
as
expected
More
sensitive
than
the
Adult
Intact
Male
Assay
More
sensitive
than
the
Hershberger
to
DBP
but
not
to
the
AR
antagonists
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
