Document ID: EPA-HQ-OPPT-2003-0016-0003
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-05-23T04:00Z

REPORT
ON
ONE­
GENERATION
EXTENSION
STUDY
OF
VINCLOZOLIN
AND
DI­
N­
BUTYL
PHTHALATE
ADMINISTERED
BY
GAVAGE
ON
GESTATIONAL
DAY
6
TO
POSTNATAL
DAY
20
IN
CD
©
(
SPRAGUE­
DAWLEY)
RATS
EPA
CONTRACT
NUMBER
68­
W­
01­
023
WORK
ASSIGNMENT
2­
10
May
7,
2002
Prepared
for
JAMES
P.
KARIYA
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
DC
BATTELLE
505
King
Avenue
Columbus,
Ohio
43201
08055.001.012
1
5/
5/
03
1
FINAL
REPORT
TITLE:
One­
Generation
Extension
Study
of
Vinclozolin
and
Di­
n­
Butyl
Phthalate
Administered
by
Gavage
on
Gestational
Day
6
to
Postnatal
Day
20
in
CD
®
(
Sprague­
Dawley
)
Rats
AUTHORS:
Julia
D.
George,
Ph.
D.
Rochelle
W.
Tyl,
Ph.
D.,
DABT
Bonnie
T.
Hamby,
B.
S.
Christina
B.
Myers,
M.
S.
Melissa
C.
Marr,
B.
A.,
RLATG
PERFORMING
LABORATORY:
Center
for
Life
Sciences
and
Toxicology
Chemistry
and
Life
Sciences
Group
RTI
International
P.
O.
Box
12194
Research
Triangle
Park,
NC
27709­
2194
SPONSOR:
Battelle
Memorial
Institute
505
King
Avenue
Columbus,
OH
43201­
2693
SPONSOR'S
REPRESENTATIVE:
David
P.
Houchens,
Ph.
D.
EDSP
Project
Manager
Battelle
STUDY
INITIATION
DATE:
April
25,
2002
IN­
LIFE
PERFORMANCE
DATES:
July
23,
2002
­
November
22,
2002
EXPERIMENTAL
DATES:
July
29,
2002
­
November
22,
2002
FINAL
REPORT
DATE:
May
5,
2003
RTI
IDENTIFICATION
NUMBER:
65U­
08055.001.012
Author:
Approved:

_______________________________
________________________________

Julia
D.
George,
Ph.
D.
Date
Satinder
K.
Sethi,
Ph.
D.
Date
Study
Director
Senior
Research
Vice
President
Center
for
Life
Sciences
and
Toxicology
Chemistry
and
Life
Sciences
Group
RTI
International
RTI
International
08055.001.012
2
5/
5/
03
2
TABLE
OF
CONTENTS
Page
ABSTRACT
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6
OBJECTIVES
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9
MATERIALS
AND
METHODS
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9
Test
Material
and
Dose
Formulations
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9
Animals
and
Husbandry
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9
Text
Table
1.
Study
Schedule
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10
Figure
1.
Study
Design
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14
F0
Females
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15
Progeny
(
F1)
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15
Necropsy
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16
F0
Females
and
PND
21
F1
Females
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16
Necropsy
of
Pnd
4
Culled
Pups
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17
Necropsy
of
F1
Males
on
PND
21
and
95
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17
External
and
Internal
Examination
of
F1
Males
at
Necropsy
.
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17
Statistical
Analyses
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19
Personnel
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21
Analytical
Report
and
Protocol/
Amendments
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21
Storage
of
Records
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21
Compliance
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21
RESULTS
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22
Dose
Formulations
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22
Vinclozolin
Results
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22
F0
Female
Observations
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22
Fate
of
F0
Females
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22
F0
Female
Gestation
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23
F0
Female
Lactation
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23
F0
Female
Reproductive
Indices
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24
Unscheduled
F0
Female
Necropsy
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24
Scheduled
F0
Female
Necropsy
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24
F1
Observations
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24
Fate
of
F1
Animals
During
Lactation
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24
Observations
of
F1
Pups
During
Lactation
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25
Unscheduled
F1
Pup
Necropsy
During
Lacatation
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25
Scheduled
F1
Pup
Necropsy
on
Pnd
21
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25
Fate
of
Pnd
95
F1
Males
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27
F1
Male
Post
Wean
Observations
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27
Unscheduled
Post
Wean
F1
Male
Necropsy
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28
Scheduled
Necropsy
of
F1
Males
on
Pnd
95
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28
08055.001.012
3
5/
5/
03
3
TABLE
OF
CONTENTS
Page
DBP
Results
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29
F0
Female
Observations
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29
Fate
of
F0
Females
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29
F0
Female
Gestation
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29
F0
Female
Lactation
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30
F0
Female
Reproductive
Indices
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31
Unscheduled
F0
Female
Necropsy
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31
Scheduled
F0
Female
Necropsy
.
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31
F1
Observations
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31
Fate
of
F1
Aninals
During
Lactation
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31
Observations
of
F1
Pups
During
Lactation
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32
Unscheduled
Necropsy
of
F1
Pups
During
Lacatation
.
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32
Necropsy
of
F1
Pups
at
Pnd
21
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33
Fate
of
Pnd
95
F1
Males
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34
F1
Males
Postwean
Observations
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34
Unscheduled
Necropsy
of
Postwean
F1
Males
.
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35
Scheduled
Necropsy
of
F1
Males
on
Pnd
95
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35
DISCUSSION
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37
Text
Table
2
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38
Text
Table
3
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39
Text
Table
4
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41
Text
Table
5
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43
Text
Table
6
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48
Text
Table
7
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52
Text
Table
8
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54
Text
Table
9
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57
CONCLUSIONS
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59
REFERENCES
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60
PROTOCOL
DEVIATIONS
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64
Appendices
Individual
Animal
Data
Tables
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Appendix
I
Analytical
Chemistry
Report
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Appendix
II
Protocol
and
Two
Amendments
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Appendix
III
08055.001.012
4
5/
5/
03
4
LIST
OF
TABLES
Table
Title
Page
1
Analyses
of
Dosing
Formulations
66
2
Summary
of
the
Fate
of
the
F0
Females
69
3
Summary
and
Statistical
Analysis
of
F0
Female
Body
Weights
and
Weight
Change
During
Gestation
70
4
Summary
and
Statistical
Analysis
of
F0
Female
Feed
Consumption
During
Gestation
73
5
Summary
of
the
F0
Female
Clinical
Observations
During
Gestation
77
6
Summary
and
Statistical
Analysis
of
F0
Female
Body
Weights
and
Weight
Changes
During
Lactation
and
Sacrifice
Weight
84
7
Summary
and
Statistical
Analysis
of
F0
Female
Feed
Consumption
During
Lactation
87
8
Summary
of
the
F0
Female
Clinical
Observations
During
Lactation
and
Necropsy
Findings
90
9
Summary
and
Statistical
Analysis
of
F0
Reproductive
and
Lactational
Indexes
for
the
F1
Litters
102
10
Summary
and
Statistical
Analysis
of
the
F1
Litter
Size,
Pup
Body
Weights,
Anogenital
Distance
and
Nipples
During
Lactation
106
11
Summary
of
the
F1
Pup
Clinical
Observations
on
Postnatal
Days
0
Through
21
113
12
Summary
of
the
F1
Pup
Necropsy
Findings
on
Postnatal
Days
0
Through
21
and
Sex
Confirmation
for
Pups
Culled
on
Postnatal
Day
4
115
13
Summary
and
Statistical
Analysis
of
F1
Male
Pup
Necropsy
Data
and
Necropsy
Weights
on
Postnatal
Day
21
118
14
Summary
of
the
F1
Pup
Necropsy
Findings
on
Postnatal
Day
21and
Sex
Confirmation
for
F1
Female
pups
on
Postnatal
Day
21
128
08055.001.012
5
5/
5/
03
5
15
Summary
of
the
Fate
of
the
F1
Males
130
16
Summary
and
Statistical
Analysis
of
the
F1
Male
Preputial
Separation
Data
131
17
Summary
and
Statistical
Analysis
of
the
F1
Male
Body
Weights
and
Weight
Changes
During
the
Post
Wean
Period
133
18
Summary
and
Statistical
Analysis
of
the
F1
Male
Feed
Consumption
During
the
Post
Wean
Period
142
19
Summary
of
the
F1
Male
Clinical
Observations
During
the
Post
Wean
Period
151
20
Summary
and
Statistical
Analysis
of
the
F1
Male
Necropsy
Data
and
Necropsy
Weights
on
Postnatal
Day
95
169
21
Summary
of
the
F1
Male
Necropsy
Findings
for
Scheduled
and
Unscheduled
Necropsy
on
Postnatal
Day
95
181
08055.001.012
6
5/
5/
03
6
RTI
Project
No.:
65U­
08055.001.012
RTI
Protocol
No.:
RTI­
807
FINAL
REPORT
One­
Generation
Extension
Study
of
Vinclozolin
and
Di­
n­
Butyl
Phthalate
Administered
by
Gavage
on
Gestational
Day
6
to
Postnatal
Day
20
in
CD
®
(
Sprague­
Dawley)
Rats
ABSTRACT
In
the
standard
two­
generation
test
(
U.
S.
EPA,
1998),
most
F1
animals
are
sacrificed
and
examined
at
postnatal
day
(
pnd)
21;
only
one
animal
per
sex
per
litter
is
usually
allowed
to
continue
to
maturity.
Although
the
basic
two­
generation
study
design
was
developed
to
provide
information
on
insult
to
the
reproductive
tract,
there
is
concern
that
certain
effects
may
be
missed,
simply
because
the
reproductive
tract
has
not
had
sufficient
time
to
develop
before
the
observations
are
made
on
the
vast
majority
of
offspring.
The
study
design
tested
through
this
work
assignment
examined
whether
or
not
allowing
more
of
the
F1
generation
males
to
continue
through
puberty
to
adulthood
will
provide
additional
information
in
detecting
endocrine­
mediated
effects
than
in
a
standard
two­
generation
design.
The
objectives
of
this
one­
generation
extension
study
are
to
determine:
1)
whether
some
of
the
effects
from
perinatal
exposure
to
Di­
n­
butyl
phthalate
(
DBP)
or
to
vinclozolin
(
VIN),
that
can
be
easily
detected
after
puberty,
are
missed
in
weanling
animals
of
the
F1
generation;
and
2)
whether
some
of
these
effects
occur
at
an
incidence
that
would
go
undetected
if
only
one
male
per
litter
is
retained
past
puberty
and
examined
at
adulthood.
In
summary,
the
hypothesis
being
tested
is
that
adverse
reproductive
effects
will
be
detected
if
three
or
more
F1
males
per
litter
are
examined
at
or
after
puberty
but
will
be
missed
if
most
of
the
F1
males
are
examined
only
at
weaning,
and
only
one
male/
litter
is
retained
to
adulthood.
The
hypothesis
will
be
tested
using
two
known
and
well­
characterized
anti­
androgens,
DBP
and
VIN,
each
at
two
doses,
one
a
known
effect
level
and
one
a
LOAEL
(
lowest
observable
adverse
effect
level)
identified
by
basic
research
protocols.

Sperm­
positive
female
CD
®
(
Sprague­
Dawley)
rats
(
the
F0
generation)
were
administered
VIN
(
CAS
No.
5­
0471­
44­
8)
or
DBP
(
CAS
No.
84­
74­
2)
orally
by
gavage
from
gestational
day
(
gd)
6
(
sperm
detection
=
gd
0)
to
pnd
20
(
date
of
birth
designated
pnd
0),
at
50
or
100
mg/
kg/
day,
or
100
and
500
mg/
kg/
day,
respectively
at
a
dose
volume
of
5
ml/
kg/
day
in
Mazola
®
corn
oil.
A
vehicle
control
group
dosed
with
corn
oil
was
run
concurrently.
Twenty­
five
sperm­
positive
F0
females
were
assigned
to
each
treatment
group.
Body
weights
and
feed
consumption
for
the
F0
females
were
recorded
for
F0
females
during
gestation
and
lactation,
and
F1
offspring
from
birth
through
scheduled
sacrifice.
Clinical
signs
were
recorded
at
least
once
daily
for
all
animals,
with
twice
daily
observations
during
the
treatment
period.
On
the
day
of
birth
(
pnd
0),
individual
anogenital
distance
(
AGD)
and
body
weights
were
recorded
for
all
live
F1
pups
in
all
litters.
F1
litters
were
standardized
on
pnd
4
to
yield
ten
pups,
maximizing
the
number
of
male
pups
retained.
Natural
litters
with
ten
or
fewer
pups
were
not
adjusted.
The
culled
F1
pups
were
weighed,
euthanized,
and
examined
08055.001.012
7
5/
5/
03
7
internally
to
confirm
sex.
The
remaining
F1
pups
were
counted,
(
and
survival
indices
were
calculated
weekly),
sexed
and
weighed
throughout
lactation
to
weaning
(
pnd
21).
The
presence
or
absence
of
retained
nipples
and
areolae
on
the
ventrum
was
recorded
for
all
F1
males
at
approximately
pnd
11­
13.
Males
with
one
or
more
nipples
or
areolae
were
uniquely
marked
until
weaning.
At
weaning,
AGD
at
body
weight
was
documented
for
all
pups.
Males
were
weight
ranked
within
litters
and
pairs
matched
by
weight.
Pairs
were
assigned
either
to
the
pnd
21
necropsy
group
or
the
pnd
95
retention
group.
If
the
litter
had
an
odd
number
of
male
pups
or
only
one
male
pup,
they
were
weighed
and
assigned
to
the
retention
group.
On
pnd
21,
all
males
assigned
to
the
pnd
21
necropsy
group
were
euthanized
and
necropsied.
All
remaining
female
pups
were
euthanized
and
examined
internally
to
confirm
sex.
In
addition,
all
F0
maternal
females
were
euthanized,
necropsied,
and
examined
for
gross
lesions.
For
each
retained
F1
male
offspring,
observations
for
the
cleavage
of
the
balanoprepreputial
gland
(
preputial
separation)
began
at
35
days
of
age
and
continued
until
acquisition
of
preputial
separation.
Body
weight
and
feed
consumption
data
were
collected
weekly
from
pnd
21
to
pnd
95.
At
scheduled
sacrifice
on
pnd
95,
the
males
were
euthanized
and
necropsied.
Body
and
organ
weights
were
recorded,
nipples/
areolae
were
counted,
and
AGD
was
measured.

Based
on
the
observations,
the
following
conclusions
can
be
made:

°
Specific
male
offspring
malformations
were
detected
on
pnd
95
but
not
on
pnd
21.
Examples
include
prostate
dorsal
lobe
abnormal/
reduced
in
size
(
VIN,
both
doses;
DBP,
high
dose),
prostate
ventral
lobe
abnormal/
reduced
in
size
(
both
compounds,
both
doses),
and
epispadias
(
VIN,
both
doses).

°
The
incidence
of
specific
male
offspring
malformations
detected
on
pnd
95
was
higher
than
the
incidence
of
the
same
malformation
observed
on
pnd
21.
Examples
include
agenesis
of
all
or
parts
of
the
epididymis(
des)
(
high
dose
of
both
VIN
and
DBP),
hypospadias
(
low
dose
VIN),
and
missing/
reduced
in
size/
abnormal
seminal
vesicles
(
high
dose
of
both
VIN
and
DBP).

°
The
effects
of
VIN
on
the
incidence
of
hypospadias
and
ventral
prostate
agenesis
were
more
obvious
at
pnd
95
than
at
pnd
21.
This
effect
was
more
apparent
at
the
low
dose
than
at
the
high
dose.
Specifically,
hypospadias
was
observed
in
9.7%
vs
15.8%
of
the
animals
on
pnd
21
and
95,
respectively,
whereas
high
dose
animals
exhibited
hypospadias
at
80.0%
vs
98.6%
on
pnd
21
and
95,
respectively.

°
The
effects
of
DBP
(
high
dose)
on
the
incidence
of
epididymal
agenesis
on
pnd
95
was
approximately
twice
that
observed
on
pnd
21,
and
thus
were
more
obvious
on
pnd
95
than
on
pnd
21.

°
Adverse
effects
on
the
weight
of
some
male
reproductive
tissues
were
more
apparent
at
pnd
95
than
on
pnd
21.
Examples
include
adjusted
right
or
left
testis
weight
(
high
dose
VIN),
absolute
right
cauda
epididymis
weight
(
low
dose
VIN),
adjusted
right
cauda
epididymis
weight
(
low
dose
VIN
and
DBP),
absolute
LABC
weight
(
low
dose
VIN),
adjusted
LABC
weight
(
high
dose
VIN
and
DBP),
and
absolute
and
adjusted
Cowper's
gland
weight
(
high
08055.001.012
8
5/
5/
03
8
dose
VIN).

°
Adverse
reproductive
system
effects
in
toto
(
structural
malformations
and
other
abnormalities)
of
the
low
and
high
doses
of
VIN
and
the
high
dose
of
DBP
on
F1
adult
male
offspring
would
most
likely
be
statistically
significant
with
either
one
or
three
adult
males/
litter,
and
would
have
been
detected
with
either
study
design.

°
Adverse
reproductive
system
structural
effects
in
toto
at
the
low
dose
of
DBP
on
F1
adult
male
offspring
were
clearly
biologically
significant
but
not
necessarily
or
likely
statistically
significant,
with
either
one
or
three
adult
males/
litter,
and
provide
an
example
of
effects
that
would
not
be
detected
with
either
study
design.

°
The
more
males
examined
per
litter,
the
better
the
characterization
of
the
litter
as
responding
or
not
responding
adversely
to
exposure,
and
the
smaller
the
variance
term
for
pooled
litters
within
each
treatment
group.
The
enhanced
sensitivity
with
more
males
examined
per
litter
would
increase
the
likelihood
of
detection
of
effects
as
statistically
and
biologically
significant.
Also,
for
effects
with
low
incidence,
such
as
in
the
low
dose
DBP
group
in
this
study,
the
risk
with
fewer
males
examined
per
litter
is
that
the
effect
might
be
missed,
i.
e.,
the
litter
would
be
designated
as
not
responding,
on
the
basis
of
the
one
male
examined,
if
that
male
did
not
exhibit
the
effect.
08055.001.012
9
5/
5/
03
9
OBJECTIVES
The
study
design
tested
through
this
work
assignment
examined
whether
or
not
allowing
more
of
the
F1
generation
males
to
continue
through
puberty
to
adulthood
would
provide
additional
information
in
detecting
endocrine­
mediated
effects
than
in
a
standard
two­
generation
design.
Secondary
hypotheses
to
be
tested
included
whether
some
of
the
effects
easily
detected
in
adults
would
be
missed
in
the
weanling
sacrifice,
especially
those
diagnostic
of
the
two
potent
antiandrogens
(
see
below)
and
whether
examining
more
males
at
the
weanling
and
adult
necropsy
would
identify
effects
and
incidences
of
these
effects
not
identified
using
the
number
of
weanlings
and
adults
specified
in
the
current
EPA
OPPTS
reproductive
toxicity
testing
guidelines.
Assessment
of
male
offspring
survival,
growth
and
development
through
lactation,
and
weaning
through
reproductive
development
until
adulthood
were
evaluated
using
VIN
(
50
or
100
mg/
kg/
day)
and
DBP
(
100
or
500
mg/
kg/
day),
two
well
known
male
endocrine
disruptors,
administered
to
the
dams
from
gd
6
to
pnd
20.

MATERIALS
AND
METHODS
Test
Material
and
Dose
Formulations
VIN
(
CAS
No.
50471­
44­
8)
was
procured
by
the
sponsor
from
Chem
Service,
Inc.
(
Lot
No.
270­
71B).
GC­
FID
purity
analysis
by
Battelle­
Sequim
indicated
a
purity
of
99.75%
(
Final
Chemical
Report
for
WA
2­
10,
Battelle,
February
12,
2003,
Appendix
II).
DBP
(
CAS
No.
84­
74­
2)
was
procured
by
the
sponsor
from
Sigma­
Aldrich,
Inc.
(
Lot
No.
080K1023).
GC­
FID
purity
analysis
by
Battelle­
Sequim
indicated
a
purity
of
99.72%
(
Final
Chemical
Report
for
WA
2­
10,
Battelle,
February
12,
2003,
Appendix
II).
Mazola
®
corn
oil
(
expiration
dates
4­
03
and
9­
03)
was
purchased
by
Battelle­
Sequim
from
retail
outlets.
Peroxide
determination
of
the
corn
oil
was
2.07
meq/
kg
(
expiration
date
4­
03)
and
1.38
meq/
kg
(
expiration
date
9­
03).
The
corn
oil
was
stored
frozen.

Dose
formulations
were
mixed
in
corn
oil
for
administration
at
5
ml/
kg.
One
vehicle
formulation
was
mixed
to
be
administered
to
the
control
group
animals
concurrently
with
both
DBP
and
VIN.
Stability
analysis
of
dose
formulations
of
VIN
in
corn
oil
(
10
and
20
mg/
ml)
indicated
that
the
formulations
were
stable
for
at
least
eight
weeks.
Formulations
assayed
(
triplicate
average)
between
90.1
and
99.6%
of
the
target
concentration
(
Appendix
II,
Table
7).
Stability
analysis
of
DBP
formulations
in
corn
oil
(
20
and
100
mg/
ml)
indicated
that
these
formulations
were
also
stable
for
at
least
eight
weeks,
with
assayed
concentrations
between
91.2
and
98.6%
of
the
target
concentration
(
Appendix
II,
Table
6).

Animals
and
Husbandry
One
hundred
seventy
(
170)
nulliparous
female
and
110
male
outbred
albino
CD
®
(
Sprague­
Dawley)
rats
(
Crl:
CD
®
[
SD]
IGS
BR)
were
received
from
Charles
River
Breeding
Laboratories
(
Raleigh,
NC)
on
July
15,
2002
(
Text
Table
1).
Both
the
females
and
males
were
70
days
old
on
arrival.
08055.001.012
10
5/
5/
03
10
Text
Table
1.
Study
Schedule
Event
Datesa
Animals
Arrive
07/
15/
02
Eartag
Male
(
breeders)
and
Female
Rats
07/
16­
18/
02
Mate
Females
07/
22/
02
F0
Gestation
(~
3
weeks)
07/
23/
002
 
08/
20/
02
F0
First
Day
of
Dosing
(
gd
6)
07/
29/
02
F0
Lactation
(~
3
weeks)
08/
13/
02
­
09/
10/
02
F1
Anogenital
Distance
Measurements
(
pnd
0)
08/
13/
02
­
08/
20/
02
F1
Cull,
Retain
all
Males
and
Enough
Females
to
=
10
pups
(
pnd
4)
08/
17/
02
­
08/
24/
02
F1
Culled
Pups
Visceral
to
Confirm
Sex
(
pnd
4)
08/
17/
02
­
08/
24/
02
F1
Male
Nipple
Retention
(
pnd
11­
13)
08/
24/
02
­
09/
02/
02
F1
Anogenital
Distance
Measurements
(
pnd
21)
09/
03/
02
­
09/
10/
02
F0
Last
Day
of
Dosing
(
pnd
20)
09/
10/
02
F0
Female
Necropsy
09/
03/
02
­
09/
10/
02
F1
Pnd
21
Necropsy
(~
1
weeks)
09/
03/
02
­
09/
10/
02
F1
Pnd
21
Weaning
of
Males
Only
(~
1
weeks)
09/
03/
02
­
09/
10/
02
F1
Post
Wean
Holding
(
10
weeks)
09/
03/
02
 
11/
23/
02
F1
Preputial
Separation
(
pnd
35
­
~
pnd
50)
09/
17/
02
­
10/
02/
02
F1
Male
Sacrifice
(
pnd
95)
11/
16/
02
­
11/
22/
02
F1
Male
Nipple
Retention
(
at
sacrifice)
11/
16/
02
­
11/
22/
02
F1
Anogenital
Distance
Measurements
(
at
sacrifice)
11/
16/
02
­
11/
22/
02
The
animals
were
quarantined
for
one
week,
during
which
time
they
were
weighed
and
examined
by
a
veterinarian.
Representative
animals
were
subjected
to
fecal
examination
and
serum
viral
antibody
analysis.
For
serum
viral
antibody
analysis,
within
one
day
after
receipt,
five
female
rats
were
arbitrarily
chosen
from
the
shipment
of
animals,
sacrificed,
and
blood
collected
for
assessment
of
viral
antibody
status.
Heat­
inactivated
serum
was
sent
to
BioReliance
(
Rockville,
MD)
for
their
Level
1
Rat
Antibody
Screen.
The
viral
screen
consisted
of
evaluation
for
the
presence
of
antibodies
against
the
following:
Toolan
H­
1
virus
(
H­
1),
Sendai
virus,
pneumonia
virus
of
mice
(
PVM),
rat
coronavirus/
sialodacryoadenitis
(
RCV/
SDA),
Kilham
rat
virus
(
KRV),
CAR
Bacillus
(
CARB),
and
Mycoplasma
pulmonis
(
M.
Pul.)
and
parvo
(
PARVO).
Results
of
the
physical
examination,
serology,
and
parasitology
were
negative
for
signs
of
infectious
disease;
one
animal
was
equivocal
for
CARB.
The
animals
were
considered
to
be
in
good
health
and
suitable
for
use
in
this
study.

After
mating
was
completed,
four
additional
rats
were
randomly
selected
and
designated
as
sentinels.
They
were
singly
housed
in
the
study
room(
s)
in
polycarbonate
solid­
bottom
cages
with
bedding
and
provided
feed
and
water
ad
libitum
(
as
described
below
for
study
animals).
They
were
examined
once
daily
by
cage­
side
observation
for
morbidity
or
mortality
at
the
same
time
as
clinical
observations
or
morbidity/
mortality
checks
for
the
study
animals.
No
sentinels
exhibited
any
morbidity
or
mortality.
At
the
time
of
necropsy
of
retained
F1
offspring,
the
sentinels
were
terminated,
blood
samples
collected,
and
serum
samples
prepared.
All
sentinel
serum
samples
08055.001.012
11
5/
5/
03
11
were
submitted
to
BioReliance
(
Rockville,
MD)
for
serological
evaluation
(
see
above).
Analysis
of
serum
(
as
described
above)
from
sentinels
sacrificed
during
the
necropsy
of
the
F0
females
was
negative,
as
was
the
analysis
from
serum
taken
during
the
retained
F1
necropsy.

F0
females
were
individually
identified
by
eartag.
One
hundred
ten
(
110)
male
rats
of
the
same
strain
from
the
RTI
breeding
colony,
originally
from
the
same
supplier,
were
used
to
generate
timed­
mated
females.
For
breeding,
individual
females
were
placed
in
the
home
cage
of
singly­
housed
males
(
i.
e.,
one
male
and
one
female).
On
the
following
morning
and
each
morning
thereafter,
the
females
were
examined
for
the
presence
of
vaginal
sperm
or
a
vaginal
copulation
plug
(
Hafez,
1970).
The
day
on
which
vaginal
sperm
or
plugs
were
found
was
designated
as
gd
0.
These
females
were
presumed
pregnant.
The
sperm­
positive
females
(
dams),
designated
the
F0
generation,
were
housed
individually
or
with
their
litters
until
scheduled
sacrifice.
Sperm­
negative
females
were
retained
in
the
same
male's
cage
and
checked
for
sperm
or
vaginal
plug
on
successive
mornings
until
insemination
occurred
or
the
treatment
groups
were
filled.
When
all
treatment
groups
were
filled,
the
remaining
sperm­
negative
females
were
sacrificed
by
asphyxiation
with
CO2.
The
fate
of
all
animals
was
fully
documented.

A
total
of
25
timed­
mated
females
per
group
were
assigned
to
this
study.
Confirmed­
mated
females
were
assigned
to
treatment
groups
by
stratified
randomization
for
body
weight
on
gd
0,
so
that
mean
body
weight
on
gd
0
did
not
differ
among
treatment
groups.
Selected
F1
weanlings
were
identified
by
eartag,
and
F1
pups
prior
to
weaning
were
not
uniquely
identified.
The
method
and
numbers
for
identification
were
documented
in
the
study
records.

All
adult
animals
were
euthanized
by
CO2
asphyxiation.
F1
pups
culled
on
pnd
4
were
sacrificed
by
decapitation.
Animals
received
with
the
initial
shipment,
but
not
used
in
the
study,
were
removed
from
the
study
room
prior
to
the
start
of
the
treatment
period
and
used
for
methods
development
and
training
of
the
RTI
staff.
Records
were
kept
documenting
the
fate
of
all
animals
received
for
the
study.

The
experiment
was
carried
out
under
standard
laboratory
conditions.
The
animals
were
individually
housed
during
the
quarantine
period
and
upon
the
initiation
of
the
treatment
period
in
solid­
bottom
polycarbonate
cages
with
stainless­
steel
wire
lids
(
Laboratory
Products,
Rochelle
Park,
NJ)
with
Sani­
Chip
®
cage
litter
(
P.
J.
Murphy
Forest
Products
Corp.,
Montville,
NJ).
F0
females
were
housed
in
monogamous
breeding
pairs
during
the
mating
period.
Females
were
caged
separately
and
individually
once
they
were
successfully
mated
(
or
at
the
end
of
the
mating
period).
F0
females
were
housed
with
their
F1
litters
during
lactation.
Postwean,
retained
F1
males
were
housed
singly
until
necropsy.
All
animals
were
housed
in
the
RTI
Animal
Research
Facility
for
the
duration
of
the
study.
All
animal
rooms
were
on
a
14:
10
hour
(
light:
dark)
light
cycle
per
day
and
were
airconditioned
temperature
and
relative
humidity
(
RH)
were
continuously
monitored,
controlled,
and
recorded
using
an
automatic
system
(
Siebe/
Barber­
Colman
Network
8000
System,
Version
4.4.1,
Loves
Park,
IL).
One
light
cycle
deviation
occurred
(
see
deviation
list).
The
protocol­
mandated
temperature
range
was
66­
77
°
F
(
22+
3
°
C),
and
the
RH
range
was
30­
70%
(
NRC,
1996).
The
F0
animals
(
and
F1
pups
during
lactation)
were
housed
in
Room
303
of
the
Animal
Research
Facility,
and
the
F1
animals
postweaning
were
housed
in
Room
403,
404,
407,
and
503.
Temperature
and
08055.001.012
12
5/
5/
03
12
RH
readings
for
the
animals
rooms,
excluding
transient
deviations
(
as
noted
in
the
Protocol
Deviation
list)
are
presented
here.
Temperature
and
RH
readings
for
Room
303
from
July
23,
2002
to
September
25,
2002,
were
70.7
to
76.8
°
F
and
45.4
to
63.7%
RH.
Temperature
and
RH
readings
for
Room
403
from
September
3,
2002
to
November
19,
2002
were
70.0
to
74.8
°
F
and
39.3
to
62.2%
RH.
Temperature
and
RH
readings
for
Room
404
from
September
4,
2002
to
November
21,
2002
were
69.6
to
74.5
°
F
and
41.2
to
69.8%
RH.
Temperature
and
RH
readings
for
Room
503
from
September
6,
2002
to
November
22,
2002
were
70.4
to
74.5
°
F
and
44.6
to
60.5%
RH.
Temperature
and
RH
readings
for
Room
407
from
September
25,
2002
to
November
22,
2002
were
69.6
to
75.3
°
F
and
46.3
to
63.5%
RH.
Two
deviations
occurred
in
Rooms
303,
403,
404,
and
503,
and
one
deviation
occurred
in
Room
407,
in
which
the
RH
was
above
that
specified
in
the
protocol
for
one
hour
on
each
occasion
(
see
Protocol
Deviations).

Purina
Certified
Rodent
Chow
(
No.
5002,
PMI
Feeds,
Inc.,
St.
Louis,
MO;
batch
numbers
documented
in
the
study
records)
was
available
ad
libitum.
All
animals
in
all
groups
received
the
same
batch/
lot
(
lot
#
JUN
24
02
1B)
of
Purina
Certified
Rodent
Chow
at
all
times.
The
analyses
of
each
feed
batch
for
nutrient
levels
and
possible
contaminants
were
performed
by
the
supplier,
examined
by
the
Study
Director,
and
maintained
in
the
study
records.
The
feed
was
also
analyzed
at
the
manufacturer
for
the
phytoestrogens
daidzein,
genistein,
and
glycitein.
Analysis
indicated
that
the
total
phytoestrogens
in
this
lot
of
feed
ranged
from
341
to
358
ppm
(
Appendix
II;
Chemistry
report
to
be
amended
to
include
correct
feed
analysis).

Deionized
water
(
generated
in­
house
from
tap
water;
source:
City
of
Durham,
Department
of
Water
Resources,
Durham,
NC)
was
available
ad
libitum
by
plastic
water
bottles
with
butyl
rubber
stoppers
and
stainless­
steel
sipper
tubes.
Contaminant
levels
of
the
Durham
City
water
were
measured
at
regular
intervals
by
the
supplier
per
EPA
specifications.
The
deionized
water
was
analyzed
by
Balazs
Analytical
Laboratories,
Inc.
(
Sunnyvale,
CA).
There
were
no
known
contaminants
that
may
have
affected
the
outcome
of
this
study.

Study
Design
A
graphic
representation
of
the
study
design
is
presented
in
Figure
1.
The
study
began
with
25
timed­
mated
females/
group.

Exposure
began
for
F0
females
on
gd
6
beginning
on
July
29,
2002,
when
they
were
approximately
12
weeks
old.
The
doses
were
chosen
based
on
results
in
the
literature.
For
VIN,
Gray
et
al.
(
1994)
showed
that
administering
VIN
in
corn
oil
by
gavage
once
daily
on
gd
14
through
pnd
3
in
rats
at
0,
100,
or
200
mg/
kg/
day
caused
dose­
related
incidences
and
severities
of
male
reproductive
tract
malformations
and
renal
system
malformations
in
the
offspring.
In
another
dose
response
publication
(
Ostby
et
al.,
1997),
maternal
rats
were
dosed
by
gavage
on
gd
14
through
pnd
3
with
VIN
at
0,
100,
and
200
mg/
kg/
day
(
first
study),
with
expected
male
offspring
reproductive
malformations
observed
at
both
doses.
In
a
second
study,
the
doses
were
0,
3.125,
6.25,
12.5,
25,
50,
and
100
mg/
kg/
day.
In
offspring
males,
reduced
AGD
was
observed
at
>
3.125
mg/
kg/
day,
retained
areolas
were
observed
at
>
6.25
mg/
kg/
day,
and
reduced
ventral
prostate
weight
and
hypospadias
were
observed
at
50
mg/
kg/
day.
Ectopic
testes
were
only
observed
at
100
and
200
08055.001.012
13
5/
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03
13
mg/
kg/
day.
Effects
on
serum
testosterone
levels
and
spermatogenesis
were
only
observed
at
$
100
mg/
kg/
day.
Finally,
in
a
study
by
Hellwig
et
al.
(
2000),
Wistar
and
Long­
Evans
rats
were
orally
dosed
with
VIN
from
gd
14
to
pnd
3
at
200,
12,
6,
3,
1,
or
0
mg/
kg/
day.
The
high
dose
(
200
mg/
kg/
day)
was
maternally
toxic,
and
male
offspring
from
both
strains
exhibited
reduced
AGD;
retained
nipples/
areolas;
hypospadia;
penile
hypoplasia;
development
of
a
vaginal
pouch;
hypoplasia
and
chronic
inflammation
of
the
epididymides,
prostate,
seminal
vesicles,
and
coagulating
glands;
testicular
tubule
atrophy;
and
chronic
inflammation
of
the
urinary
bladder.
At
12
mg/
kg/
day,
retained
nipples/
areolas
were
present
in
both
strains
in
preweanling
males
but
persisted
only
in
Long­
Evans
rats.
Long­
Evans
rats
(
but
not
Wistar)
also
exhibited
a
low
incidence
of
hypoplasia
of
accessory
sex
organs.
Based
on
the
results
from
Gray
et
al.
(
1994),
Ostby
et
al.
(
1997),
and
Hellwig
et
al.
(
2000)
in
this
study,
VIN
in
corn
oil
was
administered
by
oral
gavage
once
daily
on
gd
6
through
pnd
20
at
0
mg/
kg/
day
(
vehicle
control;
the
same
control
group
as
for
DBP
since
same
vehicle
is
used
for
both
chemicals),
50
mg/
kg/
day
(
considered
the
LOAEL
for
male
reproductive
tract
malformations),
and
100
mg/
kg/
day
(
a
clear
effect
level).
An
oral
dose
of
50
mg/
kg/
day
DBP
has
been
defined
as
the
NOAEL
(
no
observable
adverse
effect
level)
by
Mylchreest
et
al.
(
1998a,
b,
1999).
Male
malformations,
including
shortened
AGD,
small
flaccid
testes,
agenesis
of
portions
(
caput,
corpus,
cauda)
of
or
the
entire
epididymis,
delayed
puberty,
retained
nipples
and
areolae,
etc.,
have
been
observed
after
doses
of
100
to
760
mg/
kg
DBP
(
Gray
et
al.,
1998;
2000).
Interestingly,
in
utero
(
gd
15­
29)
exposure
of
Dutch­
Belted
rabbits
to
DBP
at
400
mg/
kg
in
corn
syrup
by
gavage
also
resulted
in
reduced
testis
and
accessory
gland
weights
and
reduced
ejaculated
sperm
in
adult
F1
offspring
males;
F1
males
also
exhibited
reduced
serum
testosterone,
a
slight
increase
in
histological
alterations
of
the
testis,
a
doubling
of
the
percentage
of
abnormal
sperm,
and
one
F1
male
(
out
of
17)
manifested
hypospadias,
hypoplastic
prostate
and
cryptorchid
testes
with
carcinoma
in
situ­
like
cells
(
Higuchi,
et
al.,
2003).
Therefore,
for
this
study,
DBP
in
corn
oil
was
administered
by
oral
gavage
once
daily
on
gd
6
through
pnd
20
at
0
(
vehicle
control),
100
mg/
kg/
day
(
the
LOAEL;
lowest
observed
adverse
effect
level),
and
at
500
mg/
kg/
day
(
an
obvious
effect
level).

F0
females
were
assigned
to
the
different
groups
by
means
of
randomization
stratified
by
body
weight,
such
that
the
body
weights
of
all
groups
were
homogeneous
at
study
initiation.
The
range
for
F0
all
females
on
gd
0
was
211.2
to
294.6
g.
08055.001.012
14
5/
5/
03
14
F0
females
Q
M
gd
0
G
L
F1
gd
6
N1
G
L
W
retained
F1
males
N4
RN
AGD
P
pnd
0
AGD
AGD
RN
N3
PPS
S
N2
RN
Key:
Direct
dosing
of
F0
parental
females,
gd
6
­
pnd
20
Possible
indirect
exposure
of
F1
offspring
in
utero
and
during
lactation
from
transplacental
and/
or
translactional
transfer
No
dosing
of
retained
F1
males
from
weaning
on
pnd
21
to
scheduled
necropsy
on
pnd
95
±
5
Q
=
quarantine
(
one
week)
L
=
lactation
(
three
weeks)
M
=
mating
(
one
week)
pnd
=
postnatal
day
G
=
gestation
(~
three
weeks)
P
=
parturition
(
date
of
birth,
pnd
0)
gd
=
gestational
day
AGD
=
anogenital
distance
W
=
wean
on
pnd
21
PPS
=
acquisition
of
preputial
separation
S
=
standardize
litters
to
10
(
with
maximum
number
of
males)
on
pnd
4
RN
=
examination
of
males
for
retained
nipples
N1
=
necropsy
of
F0
parental
females
at
weaning
of
F1
litters
N2
=
necropsy
culled
females
to
confirm
sex
N3
=
necropsy
any
remaining
F1
females
(
and
confirm
sex),
and
necropsy
three
F1
males
per
litter
at
weaning
on
pnd
21
N4
=
necropsy
of
retained
F1
males
at
pnd
95
±
5
Figure
1.
Study
Design
08055.001.012
15
5/
5/
03
15
F0
Females
F0
females
were
dosed
with
vehicle
control,
50
or
100
mg/
kg
of
VIN,
or
100
or
500
mg/
kg
DBP
in
Mazola
®
corn
oil
at
5
ml/
kg,
adjusted
with
respect
to
the
most
recent
body
weight.
Dosing
was
done
once
daily
by
oral
gavage
with
an
appropriate­
sized
syringe
fitted
with
a
16
g
two­
inch
stainless­
steel
curved
dosing
needle
(
Perfektum
®
,
Popper
and
Sons,
New
Hyde
Park,
NY).
F0
females
were
dosed
daily,
from
gd
6
through
pnd
20,
and
necropsied
after
weaning
of
their
litters.

Observations
for
mortality
were
made
twice
daily
(
a.
m.
and
p.
m.),
and
the
general
condition
of
all
animals
was
checked
daily.
Clinical
examinations
were
conducted
and
recorded
daily
throughout
the
course
of
the
study.
This
record
included
the
degree
and
duration
of
symptoms.
These
cage­
side
observations
included,
but
were
not
limited
to,
changes
in:
skin
and
fur,
eyes,
mucous
membranes,
respiratory
and
circulatory
system,
autonomic
and
central
nervous
system,
somatomotor
activity,
and
behavioral
pattern.

The
body
weights
of
the
F0
female
rats
were
determined
and
recorded
upon
assignment
to
dose
groups.
During
gestation,
F0
females
were
weighed
daily
from
gd
6
through
gd
20.
Dams
producing
litters
were
weighed
daily
during
lactational
(
pnd
0
through
pnd
21),
and
body
weight
gains
were
computed.

During
pregnancy
of
F0
females,
feed
weight
was
recorded
for
gd
0,
6,
9,
12,
15,
18,
and
20.
During
lactation
of
the
F1
litters,
maternal
feed
weight
was
recorded
for
pnd
0,
4,
7,
14,
and
21,
although
maternal
feed
consumption
after
pnd
14
was
confounded
by
the
contribution
from
the
pups
since
the
pups
were
self­
feeding
by
this
time.
(
The
contribution
of
self­
feeding
pups
on
"
maternal"
feed
consumption
during
the
last
week
of
lactation
has
been
estimated
at
30­
40%,
e.
g.,
Hanley
and
Watanabe,
1985;
Tyl
et
al.,
2002).

Beginning
on
gd
20,
each
female
was
observed
twice
daily
(
a.
m.
and
p.
m.)
for
evidence
of
littering.
On
the
day
of
birth
(
pnd
0),
AGD
was
measured
and
body
weight
recorded
for
all
live
F1
pups
in
all
litters.
Body
weight
was
recorded
for
all
live
pups
on
pnd
4
prior
to
culling
and
euthanasia.
F0
females
that
had
not
delivered
by
gd
26
were
euthanized
and
examined
internally
for
pregnancy
status.
The
F0
dams
with
litters
were
allowed
to
rear
their
young
to
pnd
21.
On
pnd
21,
each
litter
was
weaned.
All
F0
females
in
all
groups
were
subjected
to
a
complete
gross
necropsy
on
pnd
21.
No
tissues
were
weighed
or
retained.

Progeny
(
F1)
All
F1
pups
were
counted,
sexed,
weighed,
and
examined
as
soon
as
possible
after
birth
(
date
of
birth
designated
pnd
0)
to
determine
the
number
of
viable
and
stillborn
pups
from
each
litter.
Thereafter,
litters
were
evaluated
for
survival
on
pnd
4,
7,
14,
and
at
weaning
(
pnd
21).
Individual
AGD
and
body
weight
were
recorded
on
pnd
0
for
all
F1
offspring.

On
pnd
4,
the
size
of
each
litter
was
adjusted
to
ten
by
eliminating
extra
pups
by
random
selection
to
maximize
the
number
of
males.
Natural
litters
with
ten
or
fewer
pups
were
not
culled.
F1
pups
culled
to
standardize
litters
on
pnd
4
were
euthanized
and
subjected
to
a
visceral
examination
to
confirm
the
internal
sex.
No
tissues
were
retained.
08055.001.012
16
5/
5/
03
16
All
live
pups
were
counted,
sexed,
weighed
individually,
and
examined
grossly
at
birth
(
pnd
0),
pnd
4,
7,
14
and
at
weaning
(
pnd
21).
The
body
weights
and
sexes
were
recorded
on
an
individual
basis,
with
male
pups
with
nipples/
areolae
uniquely
identified
on
pnd
11­
13.
AGD
was
recorded
with
the
individual
pup
weight
on
pnd
0
for
all
F1
pups,
and
the
presence
or
absence
of
retained
nipples
and
areolae
on
the
ventrum
was
recorded
for
F1
male
offspring
at
approximately
pnd
11­
13.
All
pups
were
examined
for
physical
abnormalities
at
birth
and
throughout
the
preweaning
and
postwean
period.
All
pups
dying
during
lactation
were
necropsied,
when
possible,
to
investigate
the
cause
of
death.
At
weaning,
the
F1
males
were
weight
ranked
within
litters,
and
pairs
were
matched
and
assigned
to
either
the
pnd
21
necropsy
group
or
the
pnd
95
retention
group
(
e.
g.,
one
each
of
the
two
heaviest
males
from
each
litter
went
into
the
pnd
21
necropsy
group
or
the
retention
group,
etc.).
For
litters
with
an
odd
number
of
males
or
with
more
than
six
males,
the
extra
males
were
assigned
to
the
pnd
95
retention
group.
All
males
(
weight
matched
to
the
retained
males)
designated
for
pnd
21
examination
were
sacrificed
on
pnd
21,
and
any
remaining
pnd
21
females
were
sacrificed.
The
remaining
males
in
each
litter
(
weight
matched
to
the
pnd
21
necropsy
males)
and
any
extra
males
were
retained
until
scheduled
sacrifice
on
pnd
95.
F1
males
assigned
to
the
pnd
95
retention
group
were
held
without
dosing
until
scheduled
sacrifice.

F1
male
postweaning
body
and
feed
weights
were
recorded
on
pnd
21,
25,
28,
32,
35,
39,
42,
46,
49,
53,
56,
60,
63,
67,
70,
74,
77,
81,
84,
88,
91,
and
95.
Body
weight
gain
and
feed
consumption
were
calculated
for
pnd
21
to
25,
25
to
28,
28
to
32,
32
to
35,
35
to
39,
39
to
42,
42
to
46,
46
to
49,
49
to
53,
53
to
56,
56
to
60,
60
to
63,
63
to
67,
67
to
70,
70
to
74,
74
to
77,
77
to
81,
81
to
84,
84
to
88,
88
to
91,
91
to
95,
and
21
to
95.
F1
postweaning
observations
and
procedures
for
each
retained
male
offspring
included
observations
for
the
cleavage
of
the
balanoprepreputial
gland
(
preputial
separation),
which
began
at
35
days
of
age
and
continued
until
acquisition
of
preputial
separation.
Individual
body
weights
were
recorded
at
acquisition.
All
retained
F1
postweanling
males
were
weighed
as
indicated
above
(
with
clinical
observations
daily)
until
scheduled
necropsy
on
pnd
95.
On
pnd
95,
F1
males
were
euthanized
and
the
number
of
nipples
or
areolae
counted.
They
were
then
given
a
complete
external
and
internal
examination.
No
tissues
were
saved.

Necropsy
F0
Females
and
PND
21
F1
Females
F0
females
that
were
moribund
were
sacrificed
by
CO2
asphyxiation,
necropsied,
and
discarded.
F0
females
that
failed
to
deliver
by
pnd
27
were
sacrificed
and
necropsied.
Uteri
from
any
F0
females
who
appeared
nonpregnant
were
stained
with
10%
ammonium
sulfide
(
Salewski,
1964)
for
confirmation
of
pregnancy
status
and
a
count
of
implantation
sites.
F0
females
found
dead
were
necropsied.
Intact
fetuses
(
in
utero,
delivered
prior
to
the
death
of
the
dam)
were
examined
externally
and
viscerally
(
with
focus
on
the
reproductive
system)
and
discarded.
Scheduled
sacrifice
of
the
F0
maternal
animals
occurred
after
F1
litters
were
weaned.
Maternal
females
were
examined
internally
for
gross
lesions.
Female
F1
offspring
were
sacrificed
at
weaning
and
necropsied
to
confirm
sex
by
internal
examination,
then
discarded.
No
tissues
or
organs
were
saved.
08055.001.012
17
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17
Necropsy
of
Pnd
4
Culled
Pups
All
F1
pups
culled
on
pnd
4,
when
the
litters
were
standardized,
were
subjected
to
a
complete
external
examination
and
an
internal
examination
of
the
reproductive
organs
to
determine
sex.

Necropsy
of
F1
Males
on
Pnd
21
and
95
±
5
The
F1
males
selected
for
necropsy
on
pnd
21
or
retained
until
pnd
95
were
euthanized
by
CO2
asphyxiation
and
then
weighed.
AGD
was
measured
by
Vernier
calipers
(
precision
to
0.1
mm)
at
necropsy.

External
and
Internal
Examination
of
F1
Males
at
Necropsy
Each
male
selected
for
pnd
21
necropsy
or
pnd
95
adult
necropsy
was
examined
externally
at
necropsy.
Any
unusual
malformations
or
anomalies
were
noted.
Then,
the
ventral
surface
was
shaved
from
inguinal
region
to
neck,
the
nipples
and
areolae
were
counted,
and
the
number
and
position
of
the
areolae
and
nipples
was
recorded.
The
animals
were
checked
for
hypospadias,
epispadias,
and
cleft
phallus,
and
the
AGD
was
measured.
The
animals
were
also
examined
for
obviously
undescended
testes
and
preputial
separation.
The
reproductive
organs
were
carefully
observed
for
the
following:

C
Location
of
each
testis
(
scrotal,
abdominal,
gubernaculum
attached
to
abdominal
wall)
C
Gubernacular
cords,
present
or
absent,
and
length
in
mm
C
Presence
of
cranial
suspensory
ligaments
C
Testes
which
were
small,
absent,
fluid
filled,
enlarged,
appeared
infected,
or
other
C
Epididymides
which
were
small,
absent,
or
infected
(
including
region
of
effects)
C
Ventral
prostate
which
was
small,
absent,
or
infected
C
Dorsolateral
prostate
which
was
small,
absent,
or
infected
C
Seminal
vesicles
which
were
small,
absent,
infected,
or
one
side
larger
than
the
other
C
Coagulating
glands
which
were
small,
absent,
infected,
one
side
larger
than
the
other,
or
detached
from
seminal
vesicles
In
addition
the
urinary
system
was
evaluated
as
follows:

C
Kidneys
with
hydronephrosis
or
calcium
deposits
C
Hydroureter(
s)
C
Urinary
bladder
stones
or
blood
in
urinary
bladder
The
following
organs
were
weighed:

C
Each
testis
individually
C
Each
corpus
plus
caput
epididymides
C
Each
cauda
epididymides
C
Entire
seminal
vesicle,
plus
coagulating
glands
with
fluid
as
a
unit,
if
possible
08055.001.012
18
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03
18
C
The
prostate
ventral
and
dorsolateral
lobes
separately
C
Paired
adrenals
C
Liver
C
Levator
ani
plus
bulbocavernosus
muscle
complex
C
Cowper's
(
bulbourethral)
glands
as
a
pair
C
Glans
penis
(
only
if
preputial
separation
has
occurred)

Because
the
male
necropsies
on
pnd
21
and
95
were
neither
routine
nor
standard,
details
are
provided
below.
The
procedures
were
the
same
for
both
the
pnd
21
and
95
necropsies.
Each
animal
was
terminated
by
asphyxiation
with
CO2
and
then
weighed.
The
ventrum
was
shaved
and
the
numbers
of
nipples
and/
or
areolae
were
recorded.
The
presence
or
absence
of
a
soiled
perineal
area
was
noted.
Anogenital
distance
was
then
measured
by
a
vernier
caliper
(
precision
to
0.2
mm).
The
animal
was
then
opened
with
a
ventral
midline
incision
and
the
specified
organs
removed,
trimmed
and
weighed
(
to
0.0001g).
At
the
pnd
21
necropsy,
the
males
had
not
yet
achieved
puberty
(
preputial
separation,
PPS),
so
the
status
of
the
phallus
was
determined
by
careful
dissection
and
removal
of
the
foreskin.
At
the
pnd
95
necropsy,
PPS
had
been
achieved
before
the
necropsy,
so
the
phallus
could
be
assessed
by
gentle
retraction
of
the
foreskin.
Findings
of
the
phallus
included
cleft
phallus
(
defined
as
ventral
midline
cleft
from
tip;
care
was
taken
to
distinguish
the
cleft
from
a
prominent
ventral
midline
furrow
along
the
"
seam"),
hypospadias
(
ventral
opening
was
not
on
the
tip
but
below
it,
somewhere
along
the
ventral
midline
seam,
usually
associated
with
cleft
phallus),
and
epispadias
(
ventral
opening
is
not
on
the
tip
but
below
it
on
the
dorsal
side
of
the
phallus).
Any
other
gross
lesions
were
also
noted.

On
pnd
21,
the
testes
had
just
descended
into
the
scrotal
sacs;
on
pnd
95,
the
testes
had
descended
long
before
the
necropsy.
For
both
necropsies,
the
testes
were
each
gently
retracted
from
the
scrotal
sacs,
and
the
presence
of
the
gubernacular
cords
was
noted.
If
the
cords
remained
attached
to
the
caudal
base
of
the
scrotal
sacs,
they
were
not
measured.
If
the
cords
had
detached
from
the
scrotal
sac
(
so
they
could
be
measured),
they
were
measured
(
to
0.1mm).
The
presence
or
absence
of
cranial
suspensory
ligaments
(
normally
observed
only
in
females,
attached
to
the
ovaries
and
the
underside
of
the
diaphragm)
was
also
noted.
Any
organs
to
be
retained
were
stored
accordingly
(
e.
g.,
pituitaries
were
frozen);
nonretained
organs
and
the
carcass
were
then
discarded.

The
whole
pituitary
was
frozen
and
delivered
to
Dr.
Ralph
Cooper
at
the
U.
S.
EPA's
National
Health
and
Environmental
Effects
Research
Laboratory
(
NHEERL)
(
RTP,
NC).
All
other
organs
were
examined
and
weighed,
as
described
above,
and
discarded.

Two
decisions
were
made
for
the
materials
and
methods
on
this
study:

1.
Andrology
(
e.
g.,
cauda
epididymal
sperm
number,
morphology,
and
motility,
and
testicular
homogenization­
resistant
spermatid
head
counts
to
calculate
daily
sperm
production
and
efficiency
of
daily
sperm
production)
was
not
included
for
the
F1
male
offspring
during
the
pnd
95
necropsy.
The
rationale
is
that
andrology
is
not
a
sensitive
endpoint
for
VIN
and
DBP
in
rodents.
AGD,
retention
of
nipples/
areolae,
organ
weights
and
male
reproductive
system
malformations
detected
by
careful
dissection
and
examination
are
much
more
sensitive
to
08055.001.012
19
5/
5/
03
19
these
potent
anti­
androgens.
Interestingly,
DBP
did
cause
reduced
ejaculated
sperm
counts,
as
well
as
reproductive
malformations
in
male
offspring
rabbits
exposed
in
utero
to
400
mg/
kg/
day
(
Higuchi
et
al.,
2003).
These
andrology
assessments
are
also
very
labor
intensive
(
and
therefore
expensive)
and
require
close
timing
between
the
demise
of
each
male
and
his
andrologic
assessment.

2.
Histopathology
of
the
F1
offspring
male
reproductive
organs
from
the
pnd
95
(
or
pnd
21)
necropsy
was
not
performed.
The
rationale
was
that
it
was
anticipated
that
differences
in
incidence
and
severity
of
effects
on
male
reproductive
system
organs,
at
the
two
different
time
points,
pnd
21
and
95,
for
the
two
different
chemicals,
VIN
and
DBP,
and
for
the
two
different
doses
per
chemical,
could
be
adequately
detected
from
the
gross
dissections
and
examinations,
and
by
absolute
and
adjusted
(
for
body
weight)
organ
weights
(
and
because
of
the
expense
of
and
time
for
the
histology
procedures
and
pathological
examinations).

Statistical
Analyses
The
unit
of
comparison
was
the
pregnant
female,
the
F1
pup,
or
the
retained
F1
male
offspring,
as
appropriate.
Treatment
groups
for
each
chemical
were
compared
to
the
concurrent
control
group
using
either
parametric
ANOVA
under
the
standard
assumptions
or
robust
regression
methods
(
Zeger
and
Liang,
1986;
Royall,
1986;
Huber,
1967)
that
do
not
assume
homogeneity
of
variance
or
normality.
The
homogeneity
of
variance
assumption
was
examined
via
Levene's
Test
(
Levene,
1960),
which
is
much
more
robust
to
the
underlying
distribution
of
the
data
than
the
traditional
Bartlett's
Test.
If
Levene's
Test
indicated
lack
of
homogeneity
of
variance
(
p<
0.05),
robust
regression
methods
were
used
to
test
all
treatment
effects.
The
robust
regression
methods
used
variance
estimators
that
make
no
assumptions
regarding
homogeneity
of
variance
or
normality
of
the
data.
They
were
used
to
test
for
linear
trends
across
dose
within
chemicals,
as
well
as
overall
treatment
group
differences
(
via
Wald
chi­
square
tests).
Significant
overall
treatment
effects
were
followed
by
single
degree­
of­
freedom
t­
tests
for
exposed
vs.
control
group
comparisons,
if
the
overall
treatment
effect
was
significant.
If
Levene's
Test
did
not
reject
the
hypothesis
of
homogeneous
variances,
standard
ANOVA
techniques
were
applied
for
comparing
the
treatment
groups
within
chemicals.
The
GLM
procedures
in
SAS
®
8
(
SAS
Institute
Inc.,
1999a,
b,
c,
d,
e,
2000)
were
used
to
test
for
linear
trend,
evaluate
the
overall
effect
of
treatment
and,
when
a
significant
treatment
effect
was
present,
to
compare
each
exposed
group
within
chemicals
to
the
concurrent
control
group
via
Dunnett's
Test
(
Dunnett,
1955,
1964).
Standard
ANOVA
methods,
as
well
as
Levene's
Test,
are
available
in
the
GLM
procedure
of
SAS
®
Version
8,
and
the
robust
regression
methods
are
available
in
the
REGRESS
procedure
of
SUDAAN
®
Release
8.0
(
RTI,
2001).

The
F0
maternal
post­
implantation
loss
percentage
across
dose
groups
within
chemicals
was
compared
using
weighted
ANOVA
techniques.
Since
percentage
data
derived
from
litters
tend
to
have
unequal
variances,
the
arcsine
of
the
square
root
transformation
to
the
litter
percentages
was
applied
prior
to
analysis.
The
ANOVA
was
weighted
by
the
number
of
implants
(
denominator
of
the
post­
implantation
loss
percentage)
in
order
to
further
stabilize
the
variances.
In
the
presence
of
significant
treatment
effects,
Dunnett's
Test
was
used
for
pairwise
comparisons
to
control.
The
08055.001.012
20
5/
5/
03
20
average
post­
implantation
loss
percentage
(
prior
to
transformation)
was
presented
for
each
dose
group
within
chemical,
with
sample
size
and
standard
error.

Independent
binary
endpoints,
such
as
the
F0
maternal
reproductive
indices
(
e.
g.,
mating,
fertility,
and
live
litter
indices),
were
analyzed
by
Fisher's
Exact
Test
for
overall
heterogeneity
among
treatment
groups
and
by
an
exact
version
of
the
Cochran­
Armitage
Test
for
linear
trend
on
proportions
(
Cochran,
1954;
Armitage,
1955;
Agresti,
1990).
When
the
overall
Fisher's
Exact
Test
was
significant
(
p<
0.05),
pairwise
comparisons
of
individual
exposed
groups
within
chemicals
vs.
control
were
performed
using
pairwise
Fisher's
Exact
Tests.
All
of
these
tests
were
obtained
via
the
FREQ
procedure
in
SAS
8.
The
SAS
MULTTEST
procedure
was
used
to
obtain
p­
value
adjustments
for
the
multiple
treatment
comparisons
resulting
from
repeated
applications
of
Fisher's
Exact
Test.
The
p­
value
adjustments
were
based
on
the
bootstrap
and
permutation
resampling
techniques
of
Westfall
and
Young
(
1993).

Cluster­
correlated
data,
such
as
F1
periodic
pup
body
weights
during
lactation,
the
periodic
pup
survival
indices,
the
lactation
index,
the
percentage
of
stillborns
and
live
births,
the
sex
ratio,
the
percentage
of
male
pups
with
areolae
and/
or
nipples
on
pnd
11­
13
and
21,
and
the
AGD
were
analyzed
using
GEE
regression
methods
(
Zeger
and
Liang,
1986;
Liang
and
Zeger,
1986)
in
SAS
®
8
or
SUDAAN
®
8.0
to
evaluate
overall
significance,
test
for
linear
trend
across
dose
groups
within
chemicals,
and
test
pairwise
comparisons
to
the
control
group
values.
For
AGD,
a
body
weight
covariate
was
included
in
the
regression
model.
Some
of
these
outcomes
were
continuous
(
e.
g.,
body
weights,
AGD)
and
some
were
binary
(
e.
g.,
periodic
pup
survival).
Ordinal
outcomes
included
those
measured
on
a
severity
scale,
such
as
none,
mild,
moderate,
and
severe.
The
analyses
compared
the
results
for
adult
offspring
with
those
of
weanlings.
No
matter
what
type
of
endpoint
was
examined,
when
data
from
multiple
offspring
from
the
same
litter
were
used
in
the
analysis,
the
resulting
correlation
was
adjusted
for
responses
within
litters.

Developmental
landmarks,
for
example,
F1
pup
age
at
preputial
separation,
and
the
AGD
were
analyzed
using
either
parametric
ANOVA
under
the
standard
assumptions
or
robust
linear
regression
methods,
with
and
without
including
body
weight
at
acquisition
as
a
covariate
in
the
regression
model.

A
test
for
statistical
outliers
was
performed
in
the
UNIVARIATE
procedure
of
SAS
®
8
on
F0
maternal
body
weights,
feed
consumption
(
in
g/
day),
and
retained
F1
male
body
and
organ
weights.
When
examination
of
pertinent
study
data
did
not
provide
a
plausible,
biologically
sound
reason
for
inclusion
of
the
data
flagged
as
"
outlier,"
the
data
were
excluded
from
summarization
and
analysis
of
the
data,
and
the
report,
and
were
designated
as
outliers.
When
feed
consumption
data
for
a
given
animal
for
a
given
observational
interval
(
e.
g.,
pnd
0­
4,
4­
7
or
7­
14
during
the
lactational
exposure
period)
were
designated
outliers
or
unrealistic,
then
summarized
data
for
this
animal
encompassing
this
period
(
e.
g.,
pnd
0­
20
for
the
lactational
exposure
period)
also
did
not
include
this
value.
For
all
statistical
tests,
p
#
0.05
(
one­
or
two­
tailed)
was
used
as
the
criterion
for
significance.
08055.001.012
21
5/
5/
03
21
Personnel
This
study
was
conducted
at
RTI
International,
Research
Triangle
Park,
NC,
under
contract
to
Battelle,
Columbus,
OH.
Dr.
David
P.
Houchens,
EDSP
Project
Manager,
was
the
Sponsor's
Representative.
Dr.
R.
W.
Tyl
served
as
Project
Toxicologist.
Dr.
Julia
D.
George
served
as
Study
Director.
Reproductive
and
Developmental
Toxicology
personnel
included
Ms.
M.
C.
Marr
(
Laboratory
Supervisor),
Ms.
C.
B.
Myers
(
Reproductive
Toxicity
Study
Supervisor
and
Data
Analyst),
Mr.
W.
P.
Ross,
Ms.
M.
C.
Rieth,
Ms.
V.
I.
Wilson,
Ms.
L.
B
Pelletier,
Ms.
M.
P.
Gower,
Ms.
N.
M.
Kuney,
Ms.
R.
T.
Krebs,
Ms.
S.
W.
Pearce,
Ms.
K.
D.
Vick,
Ms.
L.
McDonald,
Ms.
A.
J.
Parham,
Mr.
M.
D.
Crews,
Mr.
C.
G.
Leach,
Ms.
A.
Goodman,
and
Mr.
T.
W.
Wiley.
Bulk
chemical
analysis
and
handling,
dose
formulation,
and
dose
formulation
analysis
were
provided
by
the
sponsor
through
Dr.
E.
A.
Crecelius,
PNNL,
Battelle
Marine
Sciences
Laboratory,
Sequim,
WA.
Mr.
M.
M.
Veselica
(
Supervisor,
RTI
Materials
Handling
Facility),
Mr.
D.
L.
Hubbard,
and
Mr.
R.
A.
Price
provided
receipt
and
disbursement
of
dose
formulations
at
RTI.
Animal
care
was
provided
by
Dr.
D.
B.
Feldman,
DVM,
ACLAM,
Veterinarian,
and
Mr.
F.
N.
Ali,
Manager
of
RTI
Animal
Research
Facility.
RTI
Quality
Assurance
personnel
were
Ms.
D.
J.
Smith,
Ms.
M.
D.
Phillips,
and
Ms.
T.
M.
Kenney.
Ms.
Christine
Sexsmith,
QA
Consultant,
audited
the
draft
report.

The
final
report
was
prepared
by
Dr.
J.
D.
George
and
Dr.
R.
W.
Tyl,
with
assistance
from
Ms.
C.
B.
Myers
on
data
compilation
and
statistical
analyses,
and
by
Mr.
T.
W.
Wiley
on
data
entry.
Ms.
C.
B.
Myers
was
responsible
for
all
activities
concerning
organization
and
custody
of
the
study
records.
Ms.
M.
C.
Marr
was
responsible
for
archiving
the
study
records.
Ms.
D.
B.
Bynum
provided
secretarial
assistance.

Analytical
Report
and
Protocol/
Amendments
The
bulk
chemical
and
dose
formulation
analytical
reports
were
prepared
and
signed
by
the
author(
s)
and
included
as
appendices
to
the
final
report.
The
protocol
and
two
amendments
detailing
the
design
and
conduct
of
the
study
are
presented
in
Appendix
III
of
this
final
report.

Storage
of
Records
All
original
data
sheets
and
records
collected
during
the
present
study
will
be
stored
in
the
RTI
Archives,
under
the
control
of
the
RTI
Chemistry
and
Life
Sciences
Archivist,
and
remain
the
responsibility
of
RTI.
Worksheets
and
computer
printouts,
which
were
generated
in
the
statistical
analysis
of
data,
are
stored
in
the
RTI
Archives.
Copies
of
this
report
are
filed
with
the
RTI
Archives
and
with
Battelle.
All
remaining
dose
formulations
were
shipped
back
to
the
sponsor.
Records
and
samples
from
this
study
in
RTI
Archives
may
be
released
to
the
Sponsor
upon
written
request.

Compliance
All
records,
data,
biological
specimens,
and
reports
will
be
maintained
in
storage
for
the
time
period
specified
by
the
contract
or
for
as
long
as
the
quality
of
the
preparation
affords
evaluation,
whichever
is
less.
Quality
control
(
QC)
and
quality
assurance
(
QA)
procedures
followed
those
08055.001.012
22
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03
22
outlined
in
the
Quality
Assurance
Project
Plan
(
QAPP)
prepared
for
this
study,
and
in
accordance
with
the
Quality
Management
Plan
(
QMP)
for
this
project.
The
RTI
Animal
Research
Facility
is
fully
accredited
by
the
Association
for
Assessment
and
Accreditation
of
Laboratory
Animal
Care
(
AAALAC),
International.
At
all
times,
animals
were
housed,
handled,
and
used
according
to
the
NRC
Guide
(
NRC,
1996).

RESULTS
Dose
Formulations
Predosing
analysis
of
VIN
in
corn
oil
indicated
that
the
formulations
were
97.5­
97.7%
of
the
target
concentrations,
whereas
DBP
formulations
assayed
at
94.1­
94.3%
of
the
target
concentrations
(
Table
1).
Aliquots
of
the
dosing
solutions
and
the
control
formulation
were
scheduled
to
be
taken
on
the
first
day
of
dosing
(
first
gd
6),
and
on
the
first
pnd
0,
7,
14,
and
20.
Aliquots
for
gd
6,
pnd
7,
and
14
were
taken
on
the
first
day
of
dosing
for
each
designated
pnd
day.
Due
to
an
oversight,
the
first
pnd
0
and
20
samples
were
not
taken
until
pnd
1
and
21,
respectively.
This
was
not
considered
to
have
an
adverse
effect
on
the
study.
In
addition,
the
dosing
bottles,
with
the
remainder
of
the
dosing
solutions,
were
saved
after
dosing
was
completed.
These
first­
day
aliquots
and
postdosing
samples
in
the
dosing
bottles
were
shipped
back
to
the
sponsor
and
were
analyzed
for
test
chemical
concentration.
The
VIN
first­
day
samples
assayed
at
93.6­
94.6%
of
the
target
concentrations,
whereas
the
postdosing
samples
assayed
at
94.8­
95.8%
of
the
target
concentration
(
Table
1).
The
first
dosing
day
samples
of
DBP
assayed
at
94.4­
94.5%
of
the
target
concentrations,
whereas
the
postdosing
samples
assayed
at
94.1­
94.3%
of
the
target
concentrations
(
Table
1).
The
estimated
limits
of
detection
are
0.57
:
g/
ml
for
DBP
and
0.11
:
g/
ml
for
VIN.
Additional
analytical
data
are
presented
in
Appendix
II.

Vinclozolin
Results
F0
Female
Observations
(
VIN)

Fate
of
F0
Females
(
VIN)

One
F0
female
in
the
100
mg/
kg/
day
VIN
group
was
found
dead
on
pnd
0
due
to
dystocia,
with
three
delivered
pups,
and
ten
pups
retained
in
utero
(
Tables
2
and
A­
1).
In
addition,
one
dam
in
the
control
group
was
euthanized
on
pnd
3
because
her
entire
litter
was
missing
and
presumed
dead
on
pnd
1.
Females
that
were
euthanized
on
gd
26/
27,
because
they
had
not
delivered,
included
one
in
the
vehicle
control
group
and
two
in
the
100
mg/
kg/
day
VIN
group.
Therefore,
there
were
23
females
in
the
vehicle
control
group,
and25
and
22
females
in
the
50
and
100
mg/
kg/
day
VIN
group,
respectively,
that
delivered
and
reared
litters
to
scheduled
sacrifice
on
pnd
21
(
Tables
2
and
A­
1).
08055.001.012
23
5/
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23
F0
Female
Gestation
(
VIN)

There
were
no
significant
differences
in
the
F0
maternal
body
weights
for
the
VIN­
treated
animals
compared
to
the
control
animals
on
gd
6,
9,
15,
18,
or
20
(
Tables
3
and
A­
2).
On
gd
12,
a
decreasing
linear
trend
was
noted,
but
there
was
no
treatment­
related
effect
by
pairwise
comparisons
to
the
concurrent
control
group
value.
Maternal
body
weight
change
exhibited
a
decreasing
linear
trend
for
gd
6
to
9,
9
to
12,
15
to
18,
18
to
20,
and
6
to
20,
with
the
high
dose
animals
gaining
significantly
less
weight
for
the
periods
of
gd
6
to
9,
18
to
20,
and
6
to
20
(
gestational
treatment
period).
For
gd
15
to
18,
the
high
dose
VIN
animals
gained
more
weight
than
the
control
animals.
No
significant
treatment­
related
effects
were
observed
across
groups
for
body
weight
change
during
the
gestational
period
(
gd
0
to
20)
(
Table
3).
There
were
no
treatment­
related
effects
across
groups
for
maternal
feed
consumption
(
g/
day
or
g/
kg/
day)
prior
to
initiation
of
treatment
(
Tables
4
and
A­
3).
Feed
consumption
values
expressed
as
g/
day
or
g/
kg/
day
were
significantly
decreased
in
the
100
mg/
kg/
day
VIN
group
on
gd
6
to
9,
9
to
12,
6
to
20
(
gestational
treatment
period),
and
gd
0
to
20
(
gestational
period),
and
in
the
50
mg/
kg/
day
group
on
gd
9
to
12.
For
gd
12
to
15,
15
to
18,
or
18
to
20,
there
were
no
treatment­
related
effects
across
groups
for
maternal
feed
consumption
when
expressed
as
g/
day
or
g/
kg/
day
(
Table
4).
Clinical
observations
(
Tables
5
and
A­
4)
in
the
control
and
VIN­
treated
groups
during
gestation
included
weight
loss
in
five
females,
alopecia
in
four
females,
efflux
of
dosing
compound
in
two
females,
and
piloerection
in
one
female
in
the
control
group;
rooting
postdosing
in
ten
females,
alopecia
in
four
females,
piloerection
in
three
females,
weight
loss
in
two
females,
and
efflux
of
dosing
compound
and
salivation
prior
to
dosing
in
one
female
each
in
the
50
mg/
kg/
day
VIN
group;
and
rooting
postdosing
in
15
females,
weight
loss
in
12
females,
piloerection
in
eight
females,
alopecia
in
seven
females,
efflux
of
dosing
compound,
salivation
prior
to
dosing,
and
scabs
on
one
female
each
at
100
mg/
kg/
day
VIN.

F0
Female
Lactation
(
VIN)

F0
maternal
lactational
body
weights
in
the
VIN­
treated
animals
exhibited
a
dose­
related
decreasing
trend
on
pnd
0,
4,
7,
and
14
(
Tables
6
and
A­
5).
Maternal
body
weight
was
significantly
decreased
in
the
100
mg/
kg/
day
VIN
group
on
pnd
0,
4,
and
7,
and
in
the
50
mg/
kg/
day
group
on
pnd
7
and
14.
Maternal
lactational
body
weight
was
not
significantly
affected
by
VIN
treatment
on
pnd
21.
Maternal
lactational
body
weight
change
did
not
exhibit
any
consistent
treatment
effect
for
pnd
0
to
4,
4
to
7,
7
to
14,
or
14
to
21
(
Table
6).
However,
when
body
weight
change
was
calculated
for
the
entire
lactational
period
(
pnd
0
to
21),
the
100
mg/
kg/
day
VIN
group
exhibited
a
significantly
larger
change
in
body
weight
(
i.
e.,
increase)
compared
to
the
control
group.
Examination
of
the
data
suggests
that
this
increased
weight
gain
in
the
high
dose
group
was
a
rebound
effect.
F0
maternal
lactational
feed
consumption,
expressed
as
g/
day
and
g/
kg/
day,
was
largely
unaffected
by
VIN
treatment,
with
only
feed
consumption
from
pnd
0
to
4
(
g/
day)
exhibiting
a
decreasing
dose­
related
trend
(
Tables
7
and
A­
6).
Maternal
clinical
observations
(
Tables
8
and
A­
7)
during
lactation
included
alopecia
in
six
females,
rust­
colored
fur
in
four
females,
and
piloerection,
reddish
vaginal
discharge,
and
weight
loss
due
to
a
faulty
water
bottle
in
one
female
each
at
0
mg/
kg/
day;
alopecia
in
eight
08055.001.012
24
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03
24
females,
rooting
postdosing
in
seven
females,
and
efflux
of
the
dosing
compound,
piloerection,
and
salivation
prior
to
dosing
in
two
females
each
at
50
mg/
kg/
day;
and
rooting
postdosing
in
11
females,
alopecia
and
salivation
prior
to
dosing
in
nine
females
each,
piloerection
in
seven
females,
rustcolored
fur
in
two
females,
chromodacryorrhea,
efflux
of
the
dosing
solution,
reddish
vaginal
discharge,
and
overgrown
teeth
in
one
female
each
at
100
mg/
kg/
day
(
Table
8).
One
female
was
found
dead
on
pnd
0
(
Table
8).

F0
Female
Reproductive
Indices
(
VIN)

Pregnancy
was
confirmed
in
24,
25,
and
23
F0
females
in
the
0,
50,
and
100
mg/
kg/
day
VIN
groups,
respectively
(
Tables
9
and
A­
8).
F0
females
in
the
control
and
VIN­
treated
groups
were
similar
with
respect
to
fertility
index
(
Table
9).
In
addition,
there
was
no
effect
of
exposure
to
VIN
on
gestational
index
or
gestational
length.
There
were
also
no
differences
across
groups
for
the
number
of
total
implantation
sites
per
litter,
percent
postimplantation
loss
per
litter,
or
number
of
total,
live,
or
dead
pups
per
litter
at
birth
(
pnd
0)
(
Table
9).

Unscheduled
F0
Female
Necropsy
(
VIN)

The
one
F0
female
in
the
high
dose
group
that
was
found
dead
on
pnd
0
apparently
died
of
dystocia.
Tan
patches
on
the
liver,
mottled
lungs,
multiple
red
foci
on
the
thymus,
and
reddened
trachea
were
noted
(
Tables
8
and
A­
7).
Blood­
stained
fur
around
the
mouth
and
vagina,
and
no
food
in
the
stomach
were
also
noted;
she
had
ten
retained
pups
in
her
uterus.

Scheduled
F0
Female
Necropsy
(
VIN)

Necropsy
findings
for
F0
females
at
scheduled
sacrifice
were
minimal
and
consisted
of
alopecia
on
different
parts
of
the
body,
occurring
in
one
to
three
animals
in
each
treatment
group
(
Tables
8
and
A­
7).
Rust­
colored
fur
was
noted
in
two
control
animals
and
two
high
dose
animals
(
Table
8
and
A­
7).

F1
Observations
(
VIN)
Fate
of
F1
Animals
During
Lactation
(
VIN)

There
were
24,
25,
and
22
live
litters
on
pnd
0
at
0,
50,
and
100
mg/
kg/
day,
respectively
(
Tables
9
and
A­
8).
F1
pup
mortality
for
pnd
0­
21
was
14,
13,
and
23
(
including
three
pups
euthanized
because
their
dam
was
found
dead)
pups
at
0,
50,
and
100
mg/
kg/
day,
respectively.
By
weaning
on
pnd
21,
there
were
23,
25,
and
22
F1
litters
at
0,
50,
and
100
mg/
kg/
day,
respectively
(
Tables
10
and
A­
8).
08055.001.012
25
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25
Observations
of
F1
Pups
During
Lactation
(
VIN)

Live
birth
and
stillbirth
indices
were
unaffected
across
all
groups,
as
were
the
survival
indices
for
pnd
4,
7,
14,
and
21,
and
the
lactational
index
for
pnd
4
to
21
(
Table
9
and
A­
8).
The
mean
number
of
live
pups
per
litter
for
pnd
0,
4,
7,
14,
and
21
was
unaffected
across
all
groups
(
Tables
10
and
A­
8).
Mean
F1
female
AGDs
(
absolute
or
adjusted
for
body
weight)
per
litter
on
pnd
0
were
equivalent
across
all
groups.
However,
mean
F1
male
AGDs
(
absolute
or
adjusted
for
body
weight)
exhibited
a
significant
dose­
related
decrease,
with
AGDs
in
both
the
50
and
100
mg/
kg/
day
VIN
groups
significantly
shorter
than
the
control
group
(
Tables
10
and
A­
9).
Average
absolute
F1
male
AGD
was
2.18
mm
in
the
control
group,
compared
to
1.96
and
1.56
mm
in
the
50
and
100
mg/
kg/
day
VIN
groups,
respectively.
Average
adjusted
F1
male
AGD
was
2.15
mm
in
the
control
group,
compared
to
1.94
and
1.58
mm
in
the
50
and
100
mg/
kg/
day
VIN
groups,
respectively.
Mean
F1
pup
body
weights
per
litter
(
sexes
combined
or
separately)
were
significantly
depressed
in
the
100
mg/
kg/
day
dose
group
on
pnd
0,
4,
7,
and
14
(
Tables
10
and
A­
10).
On
pnd
21,
mean
F1
pup
body
weight
per
litter
for
the
sexes
combined
and
for
males
was
significantly
decreased
at
the
high
dose.
The
sex
ratio
(
percent
male
pups/
litter)
was
significantly
decreased
at
100
mg/
kg/
day
VIN
on
pnd
0
but
not
on
pnd
4
(
Table
10).
Average
number
of
nipples/
areolae
per
male
pup
and
the
percent
male
pups
with
one
or
more
nipples/
areolae
were
significantly
increased
in
both
of
the
VIN­
treated
groups
compared
to
the
control
group
(
Tables
10
and
A­
11).
Clinical
signs
observed
for
pnd
0
through
21,
other
than
dead
or
missing
(
presumed
dead)
pups,
were
minimal
and
included
a
white
area
on
the
abdomen
(
1)
in
the
vehicle
control
group,
hematoma
(
1),
not
using
hind
limbs
(
1),
and
left
eye
not
completely
open
on
pnd
14
(
1)
in
the
50
mg/
kg/
day
group,
and
bruised
hindlimbs
(
1),
necrotic
tail
tip
(
1),
and
dehydration
(
7)
in
the
100
mg/
kg/
day
group
(
Tables
11
and
A­
12).

Unscheduled
F1
Pup
Necropsy
During
Lactation
(
VIN)

Necropsy
findings
of
F1
pups
found
dead
or
euthanized
moribund
on
pnd
0­
21
included
the
usual
findings:
pups
that
died
on
pnd
0
exhibited
open
(
fetal
state)
or
closed
ductus
arteriosus
(
postnatal
state),
no
air
(
fetal
state)
or
air
(
postnatal
state)
in
lungs,
no
or
little
milk
in
stomach,
and
autolysis
of
abdominal
organs
(
Tables
12
and
A­
13).
Necropsy
findings
for
F1
pups
culled
on
pnd
4
indicated
that
the
number
of
pups
missexed
in
the
high
dose
group
was
7.8
(
3.95
±
2.19%,
out
of
75
pups),
compared
to
0
in
the
control
and
low
dose
groups
(
Tables
12
and
A­
13).

Scheduled
F1
Pup
Necropsy
on
Pnd
21(
VIN)

After
selection,
there
were
74,
82,
and
65
F1
male
offspring
evaluated
at
scheduled
sacrifice
on
pnd
21
at
0,
50,
and
100
mg/
kg/
day,
respectively
(
Tables
13,
A­
15,
and
A­
16).
F1
male
body
weight
at
sacrifice
on
pnd
21
was
significantly
reduced
at
100
mg/
kg/
day
VIN
(
Table
13).
AGD
(
absolute
or
adjusted
for
body
weight)
was
significantly
reduced
in
both
VIN
dose
groups.
The
average
number
of
areolae
or
nipples
per
pup
and
the
percent
male
pups
with
one
or
more
nipples
or
areolae
was
increased
in
both
VIN­
treated
groups.
The
percentages
of
male
pups
necropsied
on
pnd
21
with
hypospadias
(
83%)
or
cleft
phallus
(
44%)
were
increased
at
the
high
dose,
compared
08055.001.012
26
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26
to
0%
for
both
findings
in
the
control
group
(
Tables
13
and
A­
15).
No
effect
of
VIN
treatment
was
seen
on
the
incidence
of
epispadias
or
soiled
inguinal
region
(
0%
incidence
for
control
and
treated
groups).
In
addition,
no
pnd
21
F1
males
were
observed
to
have
a
partially
or
entirely
detached
prepuce,
and
100%
of
the
males
at
0,
50
and
100
mg/
kg
were
observed
to
have
at
least
one
gubernacular
cord,
which
are
appropriate
findings
at
this
age
(
Tables
13
and
A­
15).
There
was
no
treatment
effect
for
the
percent
males
with
at
least
one
cranial
suspensory
ligament
or
on
the
length
of
the
right
or
left
gubernacular
cord
(
Table
13).
Observation
of
at
least
one
cranial
suspensory
ligament
occurred
in
0,
1.3,
and
0%
of
the
male
pups
at
0,
50
and
100
mg/
kg
VIN,
respectively,
on
pnd
21.

Mean
absolute
weights
for
liver
and
paired
adrenal
glands
were
equivalent
across
treatment
groups
for
the
F1
males
on
pnd
21.
Absolute
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
levator
ani
plus
bulbocavernosus
complex
(
LABC),
and
paired
Cowper's
gland
weights
were
significantly
decreased
at
100
mg/
kg/
day
VIN.
Right
corpus
plus
caput
epididymis,
seminal
vesicle
with
coagulating
glands,
ventral
prostate,
whole
prostate,
and
LABC
weights
were
also
decreased
at
50
mg/
kg/
day
VIN.
Mean
adjusted
weights
(
adjusted
with
respect
to
body
weight)
for
paired
adrenal
glands,
left
testis,
and
Cowper's
gland
were
equivalent
across
treatment
groups
for
the
F1
males
on
pnd
21
(
Tables
13
and
A­
16).
Adjusted
liver
weight
was
increased
compared
to
the
controls,
whereas
adjusted
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
and
LABC
were
significantly
decreased
at
100
mg/
kg/
day
VIN.
Adjusted
right
testis
weight
was
increased,
whereas
adjusted
right
corpus
plus
caput
epididymis,
seminal
vesicle
with
coagulating
glands,
ventral
prostate,
whole
prostate,
and
LABC
weights
were
decreased
at
50
mg/
kg/
day
VIN
(
Table
13).

Necropsy
findings
for
F1
pups
on
pnd
21
at
scheduled
necropsy
included,
most
notably,
missing
Cowper's
glands
(
bilateral)
in
0,
5,
and
43
pups
in
the
0,
50,
and
100
mg/
kg/
day
VIN
groups
and
missing
dorsal
prostate
gland,
which
occurred
in
21
of
the
high
dose
animals,
compared
to
0
in
the
control
and
low
dose
groups
(
Tables
14
and
A­
17).
Missing
right
or
left
Cowper's
glands
was
observed
in
one
or
two
animals
(
left;
50
or
100
mg/
kg/
day,
respectively)
or
in
three
or
two
animals
(
right;
50
or
100
mg/
kg/
day,
respectively).
Findings
observed
only
in
the
high
dose
animals
included
missing
right
caput
(
1),
missing
left
corpus
and
caput
(
1),
missing
or
spongy
LABC
(
1
each),
penis
reduced
in
size
or
reduced
in
size
and
soft
(
3
and
1),
missing
ventral
prostate
(
4),
missing
left
lobe
of
ventral
prostate
(
1),
misshapen
seminal
vesicles
(
6),
misshapen
and
reduced
seminal
vesicles
(
1),
and
testis
in
the
abdominal
cavity
(
1­
2)
(
Table
14).
Necropsy
of
F1
females
on
pnd
21
indicated
that
4%
of
the
female
pups
in
the
high
dose
group
were
missexed
(
i.
e.,
identified
as
females,
but
internally
were
males).
F1
females
in
the
control
and
50
mg/
kg/
day
VIN
group
were
all
correctly
sexed
(
Tables
14
and
A­
18).
08055.001.012
27
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27
Fate
of
Pnd
95
F1
Males(
VIN)

During
the
postwean
period,
one
high
dose
F1
male
was
euthanized
moribund
on
pnd
64,
and
another
high
dose
F1
male
was
found
dead
on
pnd
80
(
Tables
15
and
A­
19).
There
were
82,
95,
and
74
F1
males
at
0,
50,
and
100
mg/
kg/
day,
respectively,
evaluated
at
scheduled
necropsy
on
pnd
95
(
Table
15).

F1
Male
Post
Wean
Observations
(
VIN)

Absolute
F1
male
age
at
acquisition
of
preputial
separation
exhibited
a
dose­
related
increasing
trend,
with
both
the
50
and
100
mg/
kg/
day
VIN­
treated
males
achieving
preputial
separation
later
than
the
control
males
(
Tables
16
and
A­
20).
The
absolute
mean
postnatal
day
of
preputial
separation
was
40.6
days
for
the
50
mg/
kg/
day
group
and
42.2
days
for
the
100
mg/
kg/
day
dose
group,
compared
to
40.0
days
for
the
vehicle
control
group.
When
postnatal
day
of
preputial
separation
was
adjusted
for
body
weight,
only
the
high
dose
group
was
significantly
delayed
compared
to
the
control
group
(
42.1
vs.
40.1
days,
respectively;
Table
16).
F1
male
body
weight
on
the
day
of
acquisition
of
preputial
separation
increased
in
a
dose­
related
manner
and
was
significantly
greater
at
the
high
dose.

F1
male
body
weight
was
significantly
reduced
at
100
mg/
kg/
day
VIN
throughout
the
postwean
period,
up
until
pnd
95
(
Tables
17,
A­
21,
and
A­
22).
Differences
in
body
weight
change
were
minimal,
with
the
high
dose
group
gaining
significantly
less
weight
than
the
control
group
for
the
periods
of
pnd
25
to
28,
32
to
35,
and
42
to
46.
A
dose­
related
increasing
trend
was
observed
for
body
weight
change
on
pnd
84
to
88,
but
there
was
no
overall
treatment
effect.
Body
weight
change
for
pnd
46
to
49
exhibited
an
overall
treatment
effect
but
no
dose­
related
pattern
or
pairwise
differences
between
the
treated
and
the
control
groups
(
Table
17).

Feed
consumption
(
expressed
as
g/
day)
exhibited
a
decreasing
trend
and
overall
treatment
effect
on
pnd
21­
25,
with
the
high
dose
group
slightly
but
significantly
lower
than
the
controls
(
10.5
g/
day
vs.
11.2
g/
day;
Tables
18,
A­
23,
and
A­
24).
A
decreasing
trend
was
also
noted
for
feed
consumption
(
g/
day)
for
pnd
42
to
46,
but
there
was
no
overall
treatment
effect
or
pairwise
differences
between
the
treated
groups
and
the
contols.
Feed
consumption
for
pnd
25
to
28
exhibited
a
significant
overall
treatment
effect
but
no
dose­
related
trend
(
Table
18).
There
were
no
significant
treatment
effects
on
feed
consumption
(
g/
day)
from
pnd
28
through
pnd
42,
pnd
46
through
pnd
74,
pnd
77
to
81,
or
for
the
postwean
period
as
a
whole
(
pnd
21
to
95).
However,
feed
consumption
(
g/
day)
exhibited
a
dose­
related
increasing
trend
and
overall
treatment
effect
for
pnd
74
to
77,
81
to
84,
84
to
88,
88
to
91,
and
91
to
95,
with
the
high
dose
group
significantly
higher
compared
to
the
control
group
(
Table
18).
When
feed
consumption
was
expressed
as
g/
kg/
day,
feed
consumption
was
significantly
increased
over
the
control
group
at
every
time
period
except
pnd
21­
25
(
Table
18).
Feed
consumption
was
increased
at
the
lower
dose
for
pnd
25
to
28,
but
not
at
any
other
time
point.
Clinical
observations
included
sore(
s)
in
four
males,
dehydration
or
rustcolored
fur
in
two
males
each,
alopecia,
chromodacryorrhea,
cleft
phallus,
torn
ear,
and
weight
loss
due
to
a
sipper
tube
malfunction
in
one
male
each
at
0
mg/
kg/
day;
cleft
phallus
in
48
males,
hypospadias
in
14
males,
sore(
s)
in
six
males,
dehydration
in
two
males,
and
torn
ear
in
one
male
08055.001.012
28
5/
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03
28
at
50
mg/
kg/
day;
and
cleft
phallus
in
75
males,
hypospadias
in
74
males,
vaginal
pouch
in
40
males,
undescended
testes
in
17
males,
torn
ear
or
sore(
s)
in
three
males,
chromodacryorrhea
in
two
males,
alopecia,
dehydration,
red
ear,
morbundity,
pinpoint
pupils
and
tearing,
lethargy,
bleeding
penis,
prostration,
rectal
discharge,
labored
breathing,
and
death
in
one
male
each
at
100
mg/
kg/
day
(
Tables
19
and
A­
25).

Unscheduled
PostWean
F1
Male
Necropsy
(
VIN)

Necropsy
of
the
one
high
dose
male
sacrificed
moribund
and
the
one
male
found
dead
indicated
that
they
both
had
cleft
phallus,
hypospadias,
and
reduced
seminal
vesicle
size
(
Table
21).
Missing
Cowper's
glands,
missing
LABC,
missing
or
reduced
prostate,
undescended
testes,
and
vaginal
pouch
were
also
observed
in
one
or
the
other
of
these
pups
(
Tables
21
and
A­
28).

Scheduled
Necropsy
of
F1
Males
on
PND
95
(
VIN)

At
necropsy
on
pnd
95,
the
average
body
weight
of
the
F1
males
was
significantly
lower
at
100
mg/
kg/
day
(
Tables
20
and
A­
27).
Average
AGD
(
absolute
and
adjusted
for
body
weight)
exhibited
a
dose­
related
decreasing
trend
and
overall
treatment
effect,
and
was
significantly
decreased
at
both
50
and
100
mg/
kg/
day.
The
average
adjusted
AGD
was
35.8
mm
and
30.0
mm
for
the
50
and
100
mg/
kg/
day
groups,
compared
to
37.4
mm
for
the
control
group
(
Tables
20
and
A­
26).
The
incidence
of
control
males
with
one
or
more
nipples
or
areolae,
hypospadias,
epispadias,
or
soiled
inguinal
region
was
0%.
However,
the
incidence
of
these
parameters
and
cleft
phallus
(
control
=
1.2%)
was
clearly
increased
after
treatment
with
VIN.
The
number
of
males
with
one
or
more
nipples
or
one
or
more
areolae
exhibited
a
dose­
related
increasing
trend
and
overall
treatment
effect,
with
significant
increases
at
both
50
and
100
mg/
kg/
day.
Approximately
50
­
59%
of
the
low
dose
animals
and
95
­
97%
of
the
high
dose
animals
had
one
or
more
nipples
or
areolae
on
pnd
95,
compared
to
0%
for
the
control
group
(
Tables
20
and
A­
26).
The
percent
males
with
hypospadias,
epispadias,
cleft
phallus,
or
soiled
inguinal
region
also
increased
in
a
dose­
related
manner.
A
significant
increase
in
the
percent
affected
males
in
both
VIN­
treated
groups
compared
to
the
control
group
was
observed
for
hypospadias
and
cleft
phallus,
reaching
99
­
100%
affected
at
the
high
dose
(
Table
20).
Increased
incidence
of
epispadias
and
soiled
inguinal
region
of
15
and
51%,
respectively,
was
observed
at
100
mg/
kg/
day,
but
not
at
50
mg/
kg/
day.
The
percent
males
with
a
partially
or
entirely
attached
prepuce
was
not
affected
by
VIN
treatment,
nor
was
there
a
treatment
effect
for
the
percent
males
with
at
least
one
cranial
suspensory
ligament
(
Table
20).
The
incidence
of
at
least
one
cranial
ligament
was
0,
0,
and
1.35%
in
the
0,
50,
and
100
mg/
kg/
day
groups,
respectively.
The
percent
males
with
at
least
one
gubernacular
cord
exhibited
an
increasing
dose­
related
trend
but
no
pairwise
differences
between
the
treated
groups
and
the
control
group
(
Table
20).
The
percent
males
with
at
least
one
gubernacular
cord
was
6.2,
11.6,
and
17.6
in
the
0,
50,
and
100
mg/
kg/
day
groups,
respectively.
The
average
length
of
the
gubernacular
cord
was
not
affected
by
treatment.
Mean
absolute
weights
for
liver
and
paired
adrenal
glands
were
equivalent
across
treatment
groups
for
the
F1
males
on
pnd
95
(
Tables
20
and
A­
27).
08055.001.012
29
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03
29
Absolute
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
levator
ani
plus
bulbocavernosus
complex
(
LABC),
and
paired
Cowper's
gland
weights
exhibited
a
decreasing
linear
trend
and
overall
treatment
effect,
and
were
significantly
decreased
at
100
mg/
kg/
day
VIN.
Decreased
right
cauda
epididymis
and
LABC
weight
were
also
observed
at
50
mg/
kg/
day
VIN.
Mean
adjusted
weights
(
adjusted
with
respect
to
terminal
body
weight)
for
liver,
paired
adrenal
glands,
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
LABC
and
Cowper's
glands
also
exhibited
a
decreasing
trend
and
overall
treatment
effect
and
were
significantly
decreased
at
the
higher
dose.
Adjusted
LABC
weight
was
also
decreased
at
50
mg/
kg/
day
VIN.
No
other
significant
differences
from
controls
were
observed
for
adjusted
organ
weights
at
50
mg/
kg/
day,
although
all
the
values
at
this
dose
were
decreased
slightly
(
Table
20).

Necropsy
findings
for
F1
pups
at
scheduled
necropsy
on
pnd
95
were
more
varied
than
those
observed
at
pnd
21,
and
included
missing
Cowper's
glands
(
bilateral)
in
0,
3,
and
49
pups,
reduced
seminal
vesicles
in
0,
3,
and
50
pups,
vaginal
pouch
in
0,
2,
and
43
pups,
reduced
ventral
prostate
in
0,
4,
and
40
pups,
LABC
with
reduced
size
in
0,
2,
and
36
pups,
glans
penis
not
entirely
detached
in
0,
3,
and
20
pups,
reduced
dorsal
prostate
in
0,
2,
and
19
pups,
missing
dorsal
prostate
in
0,
0,
and
17
pups,
missing
ventral
prostate
in
0,
0,
and
12
pups,
and
testis
reduced
and/
or
undescended
in
1,
2,
and
19
pups
at
0,
50,
and
100
mg/
kg/
day,
respectively
(
Tables
21
and
A­
28).
Additional
observations
are
presented
in
Table
21
and
A­
28).

DBP
Results
F0
Female
Observations
(
DBP)

Fate
of
F0
Females
(
DBP)

Females
that
were
euthanized
on
gd
26/
27,
because
they
had
not
delivered,
included
one
in
the
vehicle
control
group
(
as
noted
above),
two
in
the
100
mg/
kg/
day
DBP
group,
and
one
in
the
500
mg/
kg/
day
DBP
group.
One
female
at
0
mg/
kg
was
euthanized
on
pnd
3
since
her
entire
litter
was
missing
on
pnd
1
and
presumed
dead
and
cannibalized
(
Tables
2
and
A­
1).
Therefore,
there
were
23,
23,
and
24
F0
females
in
the
vehicle
control
group,
100,
and
500
mg/
kg/
day
DBP
group,
respectively,
that
delivered
and
reared
litters
to
scheduled
sacrifice
on
pnd
21.

F0
Female
Gestation
(
DBP)

There
were
no
significant
differences
in
the
F0
maternal
body
weights
for
the
DBP­
treated
animals
compared
to
the
control
animals
on
gd
0,
6
,
9,
or
12
(
Tables
3
and
A­
2).
On
gd
15,
a
decreasing
linear
trend
was
noted,
but
there
were
no
significant
treatment­
related
effects
by
pairwise
comparisons
compared
to
the
control
group
rats.
Maternal
body
weight
significantly
decreased
in
a
dose­
related
manner
on
gd
18
and
20
also,
with
the
500
mg/
kg/
day
DBP
group
significantly
below
the
control
group
(
Table
3).
Maternal
body
weight
change
exhibited
a
08055.001.012
30
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30
decreasing
linear
trend
for
gd
6
to
9,
15
to
18,
18
to
20,
6
to
20
(
treatment
period),
and
0
to
20
(
gestation
period),
with
the
high
dose
animals
gaining
significantly
less
weight
for
the
periods
of
gd
18
to
20,
6
to
20,
and
0
to
20.
There
were
no
treatment­
related
effects
across
groups
for
maternal
feed
consumption
(
g/
day
or
g/
kg/
day)
prior
to
initiation
of
treatment
(
Tables
4
and
A­
3).
Feed
consumption
values
expressed
as
g/
day
exhibited
a
decreasing
trend
for
gd
6
to
9,
9
to
12,
12
to
15,
15
to
18,
6
to
20,
and
0
to
20
(
Table
4).
Feed
consumption
(
g/
day)
was
significantly
decreased
in
the
500
mg/
kg/
day
DBP
group
on
gd
6
to
9,
15
to
18,
and
6
to
20
(
treatment
period).
Feed
consumption
on
gd
18
to
20
exhibited
no
treatment­
related
effects.
When
maternal
feed
consumption
was
expressed
as
g/
kg/
day,
a
decreasing
trend
was
noted
for
gd
6
to
9,
12
to
15,
15
to
18,
and
6
to
20
(
Table
4).
Feed
consumption
(
g/
kg/
day)
was
significantly
decreased
at
500
mg/
kg/
day
DBP
for
gd
6
to
9
(
Table
4).
Maternal
feed
consumption
(
g/
kg/
day)
was
increased
compared
to
the
control
group
at
100
mg/
kg/
day
DBP
for
gd
15
to
18.
No
effect
of
DBP
treatment
was
observed
for
maternal
feed
consumption
(
g/
kg/
day)
on
gd
9
to
12,
18
to
20,
or
0
to
20
(
Table
4).
Clinical
observations
in
the
control
and
DBP­
treated
groups
during
gestation
included
weight
loss
in
five
females,
alopecia
in
four
females,
efflux
of
dosing
compound
in
two
females,
and
piloerection
in
one
female
in
the
control
group;
alopecia
in
nine
females,
weight
loss
in
five
females,
efflux
of
dosing
compound
or
rooting
postdosing
in
four
females
each,
piloerection
in
three
females,
and
sore(
s)
or
struggling
during
dosing
in
one
female
each
in
the
100
mg/
kg/
day
DBP
group;
and
rooting
postdosing
in
nine
females,
alopecia
in
seven
females,
weight
loss
in
six
females,
piloerection
in
five
females,
efflux
of
dosing
compound,
salivation
prior
to
dosing,
scabs,
sore(
s),
rough
coat
or
umbilical
hernia
in
one
female
each
at
500
mg/
kg/
day
DBP
(
Tables
5
and
A4).

F0
Lactation
(
DBP)

F0
maternal
lactational
body
weights
in
the
DBP­
treated
animals
exhibited
a
dose­
related
decreasing
trend
on
pnd
4
and
7,
with
a
significant
depression
in
maternal
body
weight
at
500
mg/
kg/
day
DBP
on
pnd
7
(
Tables
6
and
A5).
Maternal
lactational
body
weight
was
not
significantly
affected
by
DBP
treatment
on
pnd
0,
14,
or
21.
Maternal
lactational
body
weight
change
did
not
exhibit
any
consistent
treatment
effect
for
any
of
the
intervals
measured
(
Table
6).
However,
significant
pairwise
differences
from
the
control
group
were
noted
at
100
mg/
kg/
day
DBP
for
pnd
0
to
4
(
decrease)
and
7
to
14
(
increase).
F0
maternal
lactational
feed
consumption,
expressed
as
g/
day
and
g/
kg/
day,
was
largely
unaffected
by
DBP
treatment,
with
feed
consumption
from
pnd
0
to
4
and
7
to
14
(
g/
day)
exhibiting
a
decreasing
dose­
related
trend
(
Tables
7
and
A6).
Overall
treatment
effects
were
observed
for
maternal
feed
consumption
on
pnd
7
to
14
(
g/
day
and
g/
kg/
day),
but
there
were
no
pairwise
differences
between
the
values
in
the
DBP­
treated
groups
and
in
the
control
group
for
these
periods
(
Table
7).
Maternal
clinical
observations
during
lactation
included
alopecia
in
six
females,
rust­
colored
fur
in
four
females,
and
piloerection,
weight
loss,
and
reddish
vaginal
discharge
in
one
female
each
at
0
mg/
kg/
day,
alopecia
in
11
females,
rust­
colored
fur
in
nine
females,
efflux
of
dosing
solution
or
piloerection
in
four
females,
rooting
postdosing
in
two
females,
and
salivation
prior
to
dosing
or
sore(
s)
in
one
female
each
at
100
mg/
kg/
day;
and
alopecia
in
eight
females,
piloerection
in
four
females,
rust­
colored
fur
in
three
females,
rooting
08055.001.012
31
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03
31
postdosing
in
two
females,
and
chromodacryorrhea,
red
ear(
s),
efflux
of
dosing
solution,
swollen
genital
area,
salivation
prior
to
dosing,
and
sore(
s)
in
one
female
each
at
500
mg/
kg/
day
(
Tables
8
and
A7).

F0
Female
Reproductive
Indices
(
DBP)

Pregnancy
was
confirmed
in
24,
24,
and
25
F0
females
in
the
0,
100,
and
500
mg/
kg/
day
DBP
groups,
respectively
(
Tables
9
and
A8).
F0
control
and
DBP­
treated
groups
were
similar
with
respect
to
fertility
index,
gestational
index,
and
gestational
length
(
Table
9).
There
were
also
no
differences
across
groups
for
the
number
of
total
implantation
sites
per
litter,
percent
postimplantation
loss
per
litter,
or
number
of
total,
live,
or
dead
pups
per
litter
at
birth
(
pnd
0)
(
Table
9).

Unscheduled
F0
Female
Necropsy
(
DBP)

There
were
no
unscheduled
necropsies
for
the
F0
females
treated
with
DBP
(
Tables
8
and
A­
7).

Scheduled
F0
Female
Necropsy
(
DBP)

Necropsy
findings
for
F0
females
at
scheduled
sacrifice
were
minimal
and
consisted
of
alopecia
on
different
parts
of
the
body,
occurring
in
four
to
eight
animals
in
the
control
and
treated
groups
(
Tables
8
and
A­
7).
Rust­
colored
fur
was
noted
in
two
control
animals,
eight
low
dose
animals,
and
three
high
dose
animals.
One
retained
pup
in
uterine
horn
and
one
pup
lodged
in
the
vagina
were
noted
in
one
low
dose
F0
female
(
No.
4),
and
a
mammary
mass
was
noted
in
one
low
dose
female
(
No.
123)
(
Table
8).

F1
Observations
(
DBP)
Fate
of
F1
Animals
During
Lactation
(
DBP)

There
were
24,
23,
and
24
live
litters
on
pnd
0
at
0,
100,
and
500
mg/
kg/
day,
respectively
(
Tables
9
and
A­
8).
F1
pup
mortality
for
pnd
0­
21
was
14,
16,
and
36
pups
at
0,
100,
and
500
mg/
kg/
day,
respectively.
By
weaning
on
pnd
21,
there
were
23
F1
litters
each
at
0,
100,
and
500
mg/
kg/
day
(
Tables
10
and
A­
8).
08055.001.012
32
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32
Observations
of
F1
Pups
During
Lactation
(
DBP)

Live
birth
and
stillbirth
indices
were
unaffected
across
all
groups,
as
were
the
survival
indices
for
pnd
7,
14,
and
21,
and
the
lactational
index
for
pnd
4
to
21
(
Tables
9
and
A­
8).
The
survival
index
for
pnd
4
exhibited
a
decreasing
trend
and
overall
treatment
effect
but
no
significant
difference
between
the
control
and
the
100
or
500
mg/
kg/
day
DBP
groups
(
Table
9).
The
mean
number
of
live
pups
per
litter
for
pnd
0,
7,
14,
and
21
was
unaffected
across
all
groups
(
Tables
10
and
A­
8).
A
decreasing
linear
trend
was
observed
for
the
number
of
live
pups
per
litter
on
pnd
4,
but
there
was
no
overall
treatment
effect.
Absolute
average
F1
female
AGDs
per
litter
on
pnd
0
exhibited
a
slight
increasing
trend,
with
the
mean
AGD
in
the
control
group
measuring
1.07
mm,
compared
to
1.13
mm
and
1.15
mm
for
the
100
and
500
mg/
kg/
day
groups,
respectively
(
Tables
10
and
A­
9).
When
F1
female
AGDs
were
adjusted
for
body
weight,
an
increasing
linear
trend
was
observed,
with
the
high
dose
exhibiting
significantly
longer
AGDs
than
the
control
group
(
1.19
mm
vs.
1.06
for
controls;
Table
10).
However,
mean
F1
male
AGDs
(
absolute
or
adjusted
for
body
weight)
exhibited
a
significant
dose­
related
decrease,
with
AGDs
in
the
500
mg/
kg/
day
DBP
group
significantly
shorter
than
the
control
group
(
Table
10).
Average
absolute
F1
male
AGD
was
2.18
mm
in
the
control
group,
compared
to
2.16
and
1.91
mm
in
the
100
and
500
mg/
kg/
day
DBP
groups,
respectively.
Average
adjusted
F1
male
AGD
was
2.15
mm
in
the
control
group,
compared
to
2.14
and
1.95
mm
in
the
100
and
500
mg/
kg/
day
DBP
groups,
respectively.
Mean
F1
pup
body
weights
per
litter
(
sexes
combined
or
separately)
were
significantly
depressed
in
the
500
mg/
kg/
day
dose
group
on
pnd
0,
4,
7,
14,
and
21
(
Tables
10
and
A­
10).
The
sex
ratio
(
percent
male
pups/
litter)
was
unaffected
by
DBP
treatment
on
both
pnd
0
and
4
(
Table
10).
The
average
number
of
nipples
per
male
pup
and
the
percent
male
pups
with
one
or
more
nipples
were
significantly
increased
in
the
500
mg/
kg/
day
DBP
group
compared
to
the
control
group
(
Tables
10
and
A­
11).
With
respect
to
areolae,
the
number
per
pup
and
percent
pups
with
one
or
more
were
increased
at
both
100
and
500
mg/
kg/
day
DBP.
Clinical
signs
observed
for
pnd
0
through
21,
other
than
dead
or
missing
(
presumed
dead)
pups,
were
minimal
and
included
a
white
area
on
the
abdomen
(
1)
in
the
vehicle
control
group;
hematoma
(
2),
sore
(
s),
scabs,
and/
or
scars
at
the
umbilicus
(
11),
and
small,
pale,
and
dehydrated
(
1)
at
100
mg/
kg/
day;
and
missing
tail
(
1),
clubbed
left
hindlimb
(
1),
and
pale
in
color
(
1)
at
500
mg/
kg/
day
DBP
(
Tables
11
and
A­
12).

Unscheduled
Necropsy
of
F1
Pups
During
Lactation
(
DBP)

Necropsy
findings
of
F1
pups
found
dead
or
euthanized
moribund
on
pnd
0­
21
included
the
usual
findings:
pups
that
died
on
pnd
0
exhibited
open
(
fetal
state)
or
closed
ductus
arteriosus
(
postnatal
state),
no
air
(
fetal
state)
or
air
(
postnatal
state)
in
lungs,
no
or
little
milk
in
stomach,
and
autolysis
of
abdominal
organs
(
Tables
12
and
A­
13).
Pups
that
died
later
during
lactation
primarily
exhibited
no
or
little
milk
in
stomach
and
autolysis
of
abdominal
organs.
Necropsy
findings
for
F1
pups
culled
on
pnd
4
indicated
that
all
pups
in
the
control
and
100
and
500
mg/
kg/
day
DBP
groups
were
sexed
correctly
(
Table
12).
08055.001.012
33
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03
33
Necropsy
of
F1
Pups
at
Pnd
21
(
DBP)

After
selection
on
pnd
21,
there
were
74,
71,
and
65
F1
male
offspring
evaluated
at
scheduled
sacrifice
on
pnd
21
at
0,
100,
and
500
mg/
kg/
day
DBP,
respectively
(
Tables
13,
A­
15,
and
A­
16).
F1
male
body
weight
at
sacrifice
on
pnd
21
exhibited
a
dose­
related
decreasing
trend
and
was
significantly
reduced
at
500
mg/
kg/
day
DBP
(
Tables
13
and
A­
16).
AGD
(
absolute
or
adjusted
for
body
weight)
was
significantly
reduced
at
the
high
dose
of
DBP.
Similarly,
the
average
number
of
areolae
or
nipples
per
pup
and
the
percent
male
pups
with
one
or
more
nipples
or
areolae
were
also
increased
at
the
high
dose
of
DBP.
The
percent
male
pups
necropsied
on
pnd
21
with
cleft
phallus
was
increased
at
the
high
dose
at
3%,
compared
to
0%
for
the
control
group
(
Tables
13
and
A­
15).
DBP
treatment
resulted
in
an
increasing
trend
for
the
incidence
of
hypospadias,
but
no
effect
on
the
incidence
of
epispadias,
or
soiled
inguinal
region.
No
pnd
21
F1
males
were
observed
to
have
a
partially
or
entirely
detached
prepuce,
which
is
an
appropriate
observation
at
this
age
(
Table
13),
nor
was
there
a
treatment
effect
for
the
percent
males
with
at
least
cranial
suspensory
ligament
or
the
length
of
the
right
or
left
gubernacular
cord
(
Table
13).
The
percent
males
with
at
least
one
gubernacular
cord
exhibited
a
dose­
related
decreasing
trend,
with
the
500
mg/
kg/
day
DBP
group
significantly
less
than
the
control
group
(
91%
vs
100%).

Mean
organ
weights
(
absolute
or
adjusted
with
respect
to
body
weight)
for
paired
adrenal,
absolute
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
levator
ani
plus
bulbocavernosus
complex
(
LABC),
and
paired
Cowper's
gland
weights
were
significantly
decreased
at
500
mg/
kg/
day
DBP,
whereas
adjusted
liver
weight
was
increased.
At
100
mg/
kg/
day
DBP,
adjusted
liver
weight
was
increased,
whereas
absolute
right
or
left
corpus
plus
caput
epididymis,
and
paired
Cowper's
gland
weights
were
decreased,
as
were
adjusted
right
or
left
testis,
right
or
left
corpus
plus
caput
epdidymis,
and
paired
Cowper's
gland
weight
(
Tables
13
and
A­
16).

Necropsy
findings
for
F1
pups
on
pnd
21
at
scheduled
necropsy
included,
most
notably,
missing
Cowper's
glands
(
bilateral)
in
0,
1,
and
6
pups
in
the
0,
100,
and
500
mg/
kg/
day
DBP
groups
(
Tables
14
and
A­
17).
Hydronephrosis
(
right
kidney)
was
observed
in
2,
3,
or
5
animals
in
the
0,
100,
or
500
mg/
kg/
day
DBP.
High
dose
males
exhibited
an
array
of
epididymal
anomalies,
including
missing
caput
(
right
or
bilateral,
1
each),
missing
corpus
and
caput
(
right,
left,
or
bilateral,
1
each),
missing
corpus
and
caput
on
one
side
and
whole
epididymis
on
the
other
side
(
4),
missing
whole
epididymis
(
right
or
bilateral,
2
each),
epididymis
partially
developed
or
reduced
in
size
(
5).
Other
findings
observed
only
in
the
high
dose
animals
included
missing
left
Cowper's
gland
(
1),
missing
prostate
(
dorsal,
ventral,
ventral
left
lobe;
1
each),
missing
or
misshapen
seminal
vesicles
(
7
animals),
testis
undescended
or
in
abdominal
cavity
(
4),
and
testis
reduced
and
brown
in
color
(
1).
Necropsy
of
F1
females
on
pnd
21
indicated
that
all
females
were
correctly
sexed
(
Tables
14
and
A­
18).
08055.001.012
34
5/
5/
03
34
Fate
of
Pnd
95
F1
Males
(
DBP)

During
the
postwean
period,
one
high
dose
F1
male
was
euthanized
moribund
on
pnd
72,
and
another
high
dose
F1
male
was
euthanized
moribund
on
pnd
79
(
Tables
15
and
A­
19).
There
were
82,
81,
and
74
F1
males
at
0,
100,
and
500
mg/
kg/
day,
respectively,
evaluated
at
scheduled
necropsy
on
pnd
95
(
Table
15).

F1
Male
Postwean
Observations
(
DBP)

Absolute
F1
male
age
at
acquisition
of
preputial
separation
exhibited
a
dose­
related
increasing
trend,
with
both
the
100
and
500
mg/
kg/
day
DBP­
treated
males
achieving
preputial
separation
later
than
the
control
males
(
Tables
16
and
A­
20).
The
absolute
mean
postnatal
day
of
preputial
separation
was
40.7
days
for
the
100
mg/
kg/
day
group
and
42.2
days
for
the
500
mg/
kg/
day
dose
group,
compared
to
40.0
days
for
the
vehicle
control
group.
When
postnatal
day
of
preputial
separation
was
adjusted
for
body
weight,
both
DBP­
treated
groups
were
still
significantly
delayed
compared
to
the
control
group
in
a
dose­
related
manner
(
40.6
and
42.3
at
100
and
500
mg/
kg/
day,
respectively,
vs.
40.1
days
for
the
control
group;
Table
16).
F1
male
body
weight
on
the
day
of
acquisition
was
not
affected
by
DBP
treatment
(
Table
16).

F1
male
body
weight
exhibited
a
dose­
related
decreasing
trend
and
was
significantly
depressed
at
500
mg/
kg/
day
DBP
during
the
postwean
period
from
pnd
21
to
81,
but
not
from
pnd
84
to
pnd
95
(
Tables
17,
A­
21,
and
A­
22).
F1
male
body
weight
at
100
mg/
kg/
day
DBP
was
significantly
decreased
only
on
pnd
32.
Differences
in
body
weight
change
(
pnd
21
to
95)
were
less
pronounced,
with
the
high
dose
group
gaining
significantly
less
weight
than
the
control
group
for
the
periods
of
pnd
21
to
25,
25
to
28,
28
to
32,
32
to
35,
and
42
to
46.
F1
male
body
weight
change
was
also
significantly
less
than
the
control
group
at
100
mg/
kg/
day
for
pnd
25
to
28.
A
dose­
related
decreasing
trend
and
overall
treatment
effect,
with
no
significant
pairwise
differences
between
the
treated
groups
and
the
control
group,
was
observed
for
body
weight
change
on
pnd
35
to
39.
Body
weight
change
for
pnd
74
to
77
exhibited
an
overall
treatment
effect
but
no
dose­
related
pattern
or
pairwise
differences
between
the
treated
and
the
control
groups
(
Table
17).
Finally,
F1
male
body
weight
change
exhibited
an
overall
treatment
effect,
with
the
100
mg/
kg/
day
group
significantly
greater
than
the
control
group
on
pnd
81
to
84
and
21
to
95
(
Table
17).

Feed
consumption
(
expressed
as
g/
day)
exhibited
a
decreasing
trend
and
overall
treatment
effect
on
pnd
21­
25,
25
to
28,
28
to
32,
32
to
35,
35
to
39,
39
to
42,
and
42
to
46,
with
the
high
dose
group
significantly
lower
than
the
controls
(
Tables
18,
A­
23,
and
A­
24).
Feed
consumption
(
g/
day)
was
increased
at
100
mg/
kg/
day
on
pnd
39
to
42.
Feed
consumption
(
g/
day)
exhibited
a
treatment
effect
that
was
determined
by
the
100
mg/
kg/
day
DBP­
treated
group
for
all
intervals
from
pnd
49
to
pnd
95
and
for
pnd
21
to
95
(
postweaning
period)
(
Table
18).
The
100
mg/
kg/
day
DBP
group
was
significantly
increased
for
feed
consumption
(
g/
day)
over
the
control
group
for
all
intervals
from
pnd
49
to
pnd
95
and
pnd
21
to
95.
Significant
linear
trends
were
noted
for
feed
consumption
on
pnd
46
to
49
and
49
to
53.
Feed
consumption
at
500
mg/
kg/
day
was
increased
for
pnd
74
to
77
(
Table
18).
When
feed
consumption
was
expressed
as
g/
kg/
day,
an
increasing
08055.001.012
35
5/
5/
03
35
trend
and
overall
dose
effect
was
noted
for
all
intervals
except
pnd
21
to
25,
35
to
39,
63
to
67,
and
91
to
95.
For
those
intervals
exhibiting
both
a
significant
trend
and
overall
effect
of
treatment,
feed
consumption
(
g/
kg/
day)
was
increased
compared
to
the
control
group
in
both
the
100
and
500
mg/
kg/
day
DBP
groups,
with
the
exception
of
pnd
25
to
28
and
28
to
32,
for
which
only
the
higher
dose
was
significantly
increased.
For
pnd
21
to
25,
a
treatment
effect
was
noted
but
no
pairwise
differences
between
the
treated
groups
and
the
control
group.
Feed
consumption
(
g/
kg/
day)
on
pnd
35
to
39,
63
to
67,
and
91
to
95
exhibited
a
treatment
effect,
with
both
DBP
groups
significantly
greater
than
the
controls,
although
the
pattern
was
not
strictly
dose
related
(
Table
18).

Clinical
observations
included,
most
notably,
cleft
phallus
in
1,
1,
and
31
males
in
the
0,
100,
and
500
mg/
kg/
day
dose
groups,
and
hypospadias
in
0,
0,
and
12
males
at
0,
100,
and
500
mg/
kg/
day
DBP
(
Tables
19
and
A­
25).
Other
clinical
observations
included
sore(
s)
in
four
males,
dehydration
or
rust­
colored
fur
in
two
males
each,
and
alopecia,
chromodacryorrhea,
torn
ear,
and
weight
loss
due
to
a
sipper
tube
malfunction
in
one
male
each
at
0
mg/
kg/
day;
chromodacryorrhea
or
sore(
s),
in
three
males
each,
scar
at
the
umbilicus
in
two
males,
and
alopecia
or
penis
pointed
anteriorly
in
one
male
each
at
100
mg/
kg/
day;
and
undescended
testes
in
four
males,
vaginal
pouch
or
dehydration
in
three
males,
ataxia,
chromodacryorrhea
or
piloerection
in
two
males,
and
scruffy
appearance,
red
ear,
dragging
hind
limb(
s),
skinny
apearance,
labored
breathing,
rust­
colored
fur,
sore(
s),
or
dark
testes
(
external
appearance)
in
one
each
of
the
males
at
500
mg/
kg/
day
DBP
(
Table
19).

Unscheduled
Necropsy
of
Postwean
F1
Males
(
DBP)

Necropsy
of
the
two
males
sacrificed
moribund
indicated
that
they
both
had
reduced
epididymides
(
left
or
bilateral)
and
testes
that
were
undescended
and/
or
reduced
in
size
(
left
or
bilateral;
Table
21).
One
male
had
additional
urogenital
anomalies,
including
cleft
phallus,
bilaterally
missing
Cowper's
glands,
hypospadias,
missing
LABC,
missing
dorsal
and
ventral
prostate,
and
reduced
seminal
vesicles
(
Tables
21
and
A­
28).
Other
observations
are
noted
in
Table
21.

Scheduled
Necropsy
of
F1
Males
on
Pnd
95
(
DBP)

At
necropsy
on
pnd
95,
the
average
body
weight
of
the
F1
males
did
not
exhibit
any
doserelated
changes
(
Tables
20
and
A­
27).
Average
AGD
(
absolute)
exhibited
a
dose­
related
decreasing
trend
and
overall
treatment
effect,
and
was
significantly
decreased
at
500
mg/
kg/
day
DBP.
The
average
absolute
AGD
was
37.0
and
33.0
mm
for
the
100
and
500
mg/
kg/
day
groups,
compared
to
37.4
mm
for
the
control
group
(
Tables
20
and
A­
26).
When
AGD
was
adjusted
for
body
weight,
only
the
high
dose
was
significantly
less
than
the
control
group
(
33.1
mm
vs.
37.4),
although
AGD
for
the
low
dose
group
was
also
slightly
decreased
at
36.8
mm
(
98.4%
of
the
control
value).
The
incidence
of
control
males
with
one
or
more
nipples
or
areolae,
or
hypospadias,
was
0%.
However,
the
incidence
of
these
parameters
and
cleft
phallus
(
control
=
1.2%)
was
clearly
increased
after
treatment
with
500
mg/
kg/
day
DBP.
The
number
of
males
with
one
or
more
nipples
08055.001.012
36
5/
5/
03
36
or
one
or
more
areolae
exhibited
a
dose­
related
increasing
trend
and
overall
treatment
effect,
with
significant
increases
at
500
mg/
kg/
day.
Approximately
32
and
36%
of
the
high
dose
animals
had
one
or
more
nipples
or
areolae
on
pnd
95,
compared
to
0%
for
the
control
group
(
Table
20).
The
percent
males
with
hypospadias
or
cleft
phallus
also
increased
in
a
dose­
related
manner.
A
significant
increase
in
the
percent
affected
males
at
500
mg/
kg/
day
DBP,
compared
to
the
control
group,
was
observed
for
hypospadias
and
cleft
phallus,
reaching
16
and
35%,
respectively,
compared
to
0
and
1.2
%
in
the
control
animals
(
Table
20).
Epispadias
or
soiled
inguinal
region
were
not
observed
in
any
animals
in
the
control
or
DBP
treated
groups.
The
percent
males
with
a
partially
or
entirely
attached
prepuce
was
not
affected
by
DBP
treatment,
nor
was
there
a
treatment
effect
for
the
percent
males
with
at
least
one
gubernacular
cord.
The
average
length
of
the
right
or
left
gubernacular
cord
exhibited
a
significant
trend
and
overall
treatment
effect,
respectively,
but
these
effects
were
not
clearly
dose­
related
(
Table
20).
The
percent
males
with
at
least
one
cranial
suspensory
ligament
was
0,
0,
and
8.4
for
the
0,
100,
and
500
mg/
kg/
day
DBP
groups,
which
was
a
significant
increase
at
the
high
dose
(
Table
20).
Mean
absolute
weights
for
paired
adrenal
glands
or
Cowper's
glands
were
equivalent
across
treatment
groups
for
the
F1
males
on
pnd
95
(
Tables
20
and
A­
27).
Absolute
liver
weight
was
significantly
increased
above
the
control
group
at
100
(
but
not
500)
mg/
kg/
day.
Absolute
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
and
LABC
weights
exhibited
a
decreasing
linear
trend
and
overall
treatment
effect,
and
were
significantly
decreased
at
500
mg/
kg/
day
DBP.
Mean
adjusted
weights
(
adjusted
with
respect
to
terminal
body
weight)
for
liver
was
significantly
increased
at
the
high
dose
compared
to
the
control
animals,
and
adjusted
paired
adrenal
gland
and
Cowper's
gland
weights
were
unaffected
by
DBP
treatment.
However,
weights
of
all
other
tissues,
including
right
or
left
testis,
right
or
left
corpus
plus
caput
epididymis,
right
or
left
cauda
epididymis,
seminal
vesicle
with
coagulating
glands,
prostate
(
dorsal,
ventral,
or
whole),
and
LABC
exhibited
decreasing
trends
and
overall
treatment
effects
and
were
significantly
decreased
at
the
higher
dose.
With
the
exception
of
adjusted
right
cauda
epididymis
weight,
no
significant
differences
from
controls
were
observed
for
adjusted
organ
weights
at
100
mg/
kg/
day,
and
all
of
the
values
(
except
liver
weight)
at
this
dose
were
similar
to
those
seen
in
control
animals
(
Table
20).
Adjusted
right
cauda
epididymis
weight
was
significantly
lower
than
the
control
value.

Necropsy
findings
for
F1
pups
at
scheduled
necropsy
on
pnd
95
were
more
varied
than
those
observed
at
pnd
21
and
were
primarily
observed
at
the
high
dose
(
Tables
21
and
A­
28).
They
included
anomalies
of
the
Cowper's
glands
(
12
males),
epididymides
(
61
males),
dorsal
prostate
(
11
males),
ventral
prostate
(
11
males),
seminal
vesicles
(
32
males),
and
testis
(
56
males).
Additional
observations
are
presented
in
Tables
21
and
A­
28.
080555.001.012
37
5/
5/
03
37
DISCUSSION
This
study
was
designed
to
test
the
hypothesis
that
the
current
U.
S.
EPA
OPPTS
testing
guideline
for
Reproduction
and
Fertility
Effects
(
OPPTS
870.3800;
U.
S.
EPA,
1998)
is
relatively
insensitive
for
detection
of
male
reproductive
system
malformations
since
it
mandates
necropsy
of
three
animals/
sex/
litter
on
pnd
21
(
at
weaning),
and
only
one
animal/
sex/
litter
retained
through
acquisition
of
puberty
to
adulthood,
with
functional
and
structural
reproductive
system
evaluations
and
andrological
endpoints
assessed
on
these
F1
animals
(
one
per
litter)
as
adults.
A
secondary
hypothesis
was
whether
specific
effects
and
incidences
of
effects
identified
in
the
adults
would
be
missed
in
the
weanling
sacrifice.
These
hypotheses
was
tested
by
employing
two
known
potent
antiandrogens
 
VIN
and
DBP
 
each
at
a
high
effect
level
and
at
a
low
effect
level,
based
on
U.
S.
EPA
in­
house
data,
with
the
F0
dams
(
25/
group)
dosed
from
gd
6
through
pnd
20.
The
F1
litters
were
standardized
on
pnd
4
to
ten
pups
with
retention
of
all
male
F1
pups.
AGD
was
recorded
at
birth,
retained
nipples/
areolae
were
recorded
on
pnd
11­
13,
and
AGD
and
retained
nipples/
areolae
recorded
at
weaning
and
adult
sacrifices.
On
pnd
21,
approximately
one­
half
of
the
F1
males
per
litter
were
carefully
necropsied.
On
pnd
95,
the
remaining
F1
males
per
litter
were
carefully
necropsied.

The
hypotheses
can
be
broken
down
into
a
series
of
questions
(
and
the
answers
from
this
study)
as
follows:

1.
Can
this
study
design
and
the
performing
laboratory
detect
both
doses
of
both
test
compounds
(
VIN
and
DBP)
as
effect
levels?
Both
doses
of
both
test
chemicals
were
detected
as
effect
levels
by
pnd
13
from
data
on
AGD
at
birth
(
both
doses
of
VIN
and
the
high
dose
of
DBP)
and
on
retention
of
areolae
in
preweanling
males
on
pnd
11­
13
(
both
doses
of
both
test
compounds).
Interestingly,
the
incidence
of
nipples
on
pnd
11­
13
males
was
significantly
increased
for
both
doses
of
VIN
and
for
the
high
dose
(
but
not
the
low
dose)
of
DBP.
Since
retained
nipples
are
not
observed
in
control
males,
and
retained
areolae
are
(
at
0­
3.5%
incidence
in
the
performing
laboratory),
the
anticipation
was
that
retained
nipples
would
be
a
more
sensitive
indicator
of
anti­
androgenic
activity
than
retained
areolae.
See
Text
Table
2
for
AGDs
and
retained
nipples
and/
or
areolae
from
birth
to
pnd
95.

2.
Were
male
reproductive
system
malformations
detected
at
the
pnd
21
necropsy
for
both
test
chemicals
at
both
doses?
In
utero/
lactational
exposure
to
both
doses
of
both
test
chemicals
resulted
in
male
reproductive
system
malformations
in
a
dose­
related
incidence
and
severity.
The
male
reproductive
malformations
at
the
low
dose
of
DBP
were
biologically
significant
(
never
observed
in
controls)
but
clearly
not
statistically
significant.
They
included
missing
Cowper's
glands
and
presence
of
cranial
suspensory
ligaments
(
normally
observed
only
in
females).
Text
Table
3
presents
the
data
in
support
of
this
answer.
080555.001.012
38
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38
Text
Table
2.
Anogenital
Distance
and
Retention
of
Nipples
and
Areolae
in
F1
Malesa
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Male
absolute
AGD
(
mm):
pnd
0
2.18
1.96***
1.56***
2.16
1.91***

pnd
21
15.31
14.52*
11.01***
15.47
12.55***

pnd
95
37.44
35.81**
29.95***
37.00
32.96***

Female
AGD
(
mm)

pnd
0:

absolute
1.07
1.10
1.09
1.13
1.15
adjusted
1.06
1.11
1.11
1.12
1.19**

Pnd
11­
13
No.
nipples/
male
0.00
0.43*
5.97***
0.02
0.99***

Males
with
>
1
nipplesb
0
(
0.00)
11
(
13.14)**
56
(
86.76)***
0.9
(
1.31)
c
15
(
22.86)***

No.
areolae/
male
0.23
6.43***
7.66***
0.94**
4.01***

Males
with
>
1
areolaeb
6
(
7.69)
d
77
(
93.71)***
64
(
97.79)***
21
(
29.41)**
49
(
75.71)***

Pnd
21
No.
nipples/
male
0.0
0.46
4.36***
0.01
0.63***

Males
with
>
1
nippleb
0
(
0.0)
15
(
18.29)**
59
(
90.63)***
1
(
1.43)
23
(
35.38)***

No.
areolae/
male
0.0
0.73*
4.02***
0.0
0.71***

Males
with
>
1
areolaeb
0
(
0.0)
25
(
30.49)***
58
(
89.06)***
0
(
0.0)
16
(
24.62)***

Pnd
95
No.
nipples/
male
0.0
1.60***
5.86***
0.01
1.04***

Males
with
>
1
nippleb
0
(
0.0)
48
(
50.53)***
70
(
94.59)***
1
(
1.23)
24
(
32.43)***

No.
areolae/
male
0.0
2.14***
6.57***
0.04
1.14***

Males
with
>
1
areolaeb
0
(
0.0)
56
(
58.95)***
72
(
97.30)***
2
(
2.47)
27
(
36.49)***

a
Data
taken
from
summary
tables
10,
13,
and
20.

b
Data
presented
as
number
(%)

c
Not
statistically
significantly
increased
d
The
performing
laboratory's
historical
control
incidence
range
of
%
males
with
>
areola(
e)
is
0.0­
3.7%

*,
**,
***
=
p
<
0.05,
0.01,
0.001
relative
to
concurrent
control
group
value
080555.001.012
39
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39
Text
Table
3.
F1
Male
Offspring
Reproductive
System
Malformations
at
the
Pnd
21
Necropsy
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
No.
pups
74
82
65
71
65
Cowper's
gland
missing:

Left
0
(
0.0)
a
1
(
1.2)
2
(
3.1)
0
(
0.0)
1
(
1.5)

Right
0
(
0.0)
3
(
3.7)
2
(
3.1)
0
(
0.0)
0
(
0.0)

Bilateral
0
(
0.0)
5
(
6.1)
43
(
66.2)
1
(
1.4)
6
(
9.2)

Total
0
(
0.0)
9
(
11.0)
47
(
72.3)
1
(
1.4)
7
(
10.8)

Epididymis(
mides)
missing,
reduced
in
size:

Left
0
(
0.0)
0
(
0.0)
1
(
1.5)
0
(
0.0)
8
(
12.3)

Right
0
(
0.0)
0
(
0.0)
1
(
1.5)
0
(
0.0)
8
(
12.3)

Bilateral
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
5
(
7.7)

Total
0
(
0.0)
0
(
0.0)
2
(
3.1)
0
(
0.0)
21
(
32.3)

Epispadias
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Hypospadias
0
(
0.0)
8.0
(
9.7)
52
(
80.0)
0
(
0.0)
2
(
3.1)

Males
with
>
1
gubernacular
cord
74
(
100.0)
82
(
100.0)
65
(
100.0)
69
(
97.2)
59
(
90.8)

Males
with
>
1
cranial
suspensory
ligament
0
(
0.0)
1
(
1.3)
0
(
0.0)
2.2
(
3.1)
2.2
(
3.4)

Levator
ani
bulbocavernosus
complex:

Missing
0
(
0.0)
0
(
0.0)
1
(
1.5)
0
(
0.0)
0
(
0.0)

Spongy
0
(
0.0)
0
(
0.0)
1
(
1.5)
0
(
0.0)
0
(
0.0)

Penis
reduced
in
size
0
(
0.0)
0
(
0.0)
3
(
4.6)
0
(
0.0)
0
(
0.0)

Phallus,
cleft
0
(
0.0)
4
(
4.9)
25
(
38.5)
0
(
0.0)
2
(
3.1)

Prostate
missing:

Dorsal
0
0
21
(
32.3)
0
1
(
1.5)

Ventral
0
0
4
(
6.2)
0
1
(
1.5)

V
left
lobe
0
0
1
(
1.5)
0
1
(
1.5)

Total
0
(
0.0)
0
(
0.0)
26
(
40.0)
0
3
(
4.6)

Seminal
vesicles:

Missing
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
4
(
6.2)
080555.001.012
40
5/
5/
03
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
40
Misshapen
0
(
0.0)
0
(
0.0)
7
(
10.8)
0
3
(
4.6)

Total
0
(
0.0)
0
(
0.0)
7
(
10.8)
0
7
(
10.8)

Testes
undescended
0
(
0.0)
0
(
0.0)
3
(
4.6)
0
4
(
6.1)

Hydronephrosis:
b
Left
0
(
0.0)
1
(
1.2)
0
(
0.0)
0
0
(
0.0)

Right
3
(
4.0)
4
(
4.9)
0
(
0.0)
2
(
2.8)
6
(
9.2)

Bilateral
0
(
0.0)
0
(
0.0)
0
(
0.0)
1
(
1.4)
0
(
0.0)

Total
3
(
4.0)
5
(
6.1)
0
(
0.0)
3
(
4.2)
6
(
9.2)

a
Number
(
and
%)
with
the
indicated
finding.
A
male
may
be
counted
more
than
once
if
he
exhibited
more
than
one
malformation.

b
The
incidence
of
hydronephrosis,
a
common
finding
in
male
CD
®
(
Sprague­
Dawley)
rats
is
provided
for
internal
quality
control.
There
was
no
chemical­
or
dose­
related
incidence.

3.
Were
male
reproductive
system
malformations
detected
at
pnd
95
for
both
test
chemicals
at
both
doses?
In
utero/
lactational
exposure
to
both
test
chemicals
at
both
doses
resulted
in
male
reproductive
system
malformations
in
a
dose­
and
chemical­
related
incidence
and
severity
on
pnd
95.
For
the
low
dose
DBP,
findings
included
cleft
phallus,
dorsal
and
ventral
prostate
lobes
reduced
in
size,
and
enlarged
testes.
Admittedly,
these
were
observed
at
a
low
incidence
at
this
dose,
but
they
were
biologically
significant
although
not
likely
statistically
significant.
Text
Table
4
presents
the
data
in
support
of
this
answer.

4.
Did
the
F1
males
that
died
or
were
sacrificed
moribund
also
exhibit
male
reproductive
malformations?
There
were
two
F1
males
each
at
the
VIN
high
dose
and
the
DBP
high
dose
that
died
or
were
sacrificed
moribund.
They
did
exhibit
the
same
male
reproductive
system
malformations
as
those
observed
in
the
adult
males
at
scheduled
necropsy
(
see
Text
Table
5
below).

5.
Were
the
incidences
of
findings
present
at
both
pnd
21
and
95
necropsies
different?
Were
they
greater
on
pnd
95?
Would
they
have
been
detected
on
pnd
95
with
only
one
male
evaluated
per
litter?
As
is
obvious
from
Text
Table
6,
in
almost
every
case
for
findings
present
at
both
pnd
21
and
95,
the
incidence
is
higher
on
pnd
95.
In
addition,
some
observations
were
present
only
on
pnd
95
(
such
as
vaginal
pouch
on
Text
Table
6
and
findings
in
Text
Table
3
marked
with
footnote
"
b").
080555.001.012
41
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03
41
Text
Table
4.
F1
Male
Offspring
Reproductive
System
Malformations
at
the
Pnd
95
Necropsya
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
No.
males
82
95
74
81
74
Cowper's
glands:

Missing
0
(
0.0)
6
(
6.32)
56
(
75.7)
0
(
0.0)
5
(
6.8)

Reduced
in
size
0
(
0.0)
1
(
1.0)
8
(
10.8)
0
(
0.0)
3
(
4.0)

Epididymis
missing
0
(
0.0)
0
(
0.0)
4
(
5.4)
0
(
0.0)
33
(
44.6)

Reduced
in
size
1
(
1.2)
0
(
0.0)
12
(
16.2)
0
(
0.0)
52
(
70.3)

Epispadias
0
(
0.0)
4
(
4.3)
11
(
14.9)
0
(
0.0)
0
(
0.0)

Glans
penis
not
completely
detached
b
0
(
0.0)
3
(
3.2)
20
(
27.0)
0
(
0.0)
7
(
9.5)

Hypospadias
0
(
0.0)
15
(
15.8)
73
(
98.6)
0
(
0.0)
12
(
16.2)

LABC:
b
Missing
0
(
0.0)
0
(
0.0)
2
(
2.7)
0
(
0.0)
0
(
0.0)

Reduced
in
size
0
(
0.0)
2
(
2.1)
38
(
51.4)
0
(
0.0)
4
(
5.4)

Malformed
0
(
0.0)
1
(
1.0)
5
(
6.7)
0
(
0.0)
0
(
0.0)

Males
with
>
1
gubernacular
cord
5.5
(
6.2)
11
(
11.6)
13
(
17.6)
1.0
(
1.2)
6
(
8.1)

Males
with
>
1
cranial
suspensory
ligament
0
(
0.0)
0
(
0.0)
1.0
(
1.4)
0
(
0.0)
6.2
(
8.4)

Phallus,
cleft
1
(
1.20)
41
(
43.2)
74
(
100.0)
2.0
(
2.5)
26
(
35.1)

Prepuce
partially
or
fully
detached
81
(
98.8)
94
(
99.0)
74
(
100.0)
81
(
100.0)
74
(
100.0)

Preputial
glands,
pus
filled
b
0
(
0.0)
1
(
1.2)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Prostate,
dorsal:

Missing
0
(
0.0)
0
(
0.0)
17
(
23.0)
0
(
0.0)
3
(
4.0)

Reduced
in
size
0
(
0.0)
2
(
2.4)
20
(
27.0)
2
(
2.5)
7
(
9.5)

Abnormal/
infected
0
(
0.0)
2
(
2.4)
3
(
4.0)
0
(
0.0)
1
(
1.4)

(
Continued)
080555.001.012
42
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03
42
Text
Table
4
(
continued)

Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Prostate,
ventral:

Missing
0
(
0.0)
0
(
0.0)
12
(
16.2)
0
(
0.0)
3
(
4.0)

Reduced
in
size
0
(
0.0)
4
(
4.9)
43
(
58.1)
1
(
1.2)
4
(
5.4)

Abnormal/
infected
1
(
1.2)
3
(
3.1)
5
(
6.7)
1
(
1.2)
6
(
8.1)

Seminal
vesicles:

Missing
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
8
(
10.8)

Misshapen/
infected
0
(
0.0)
0
(
0.0)
6
(
8.1)
0
(
0.0)
5
(
6.8)

Reduced
in
size
0
(
0.0)
5
(
6.1)
58
(
78.4)
0
(
0.0)
27
(
36.5)

Testes
Undescended
0
(
0.0)
1
(
1.0)
15
(
20.3)
0
(
0.0)
10
(
13.5)

sc
in
abdom.
wallc
0
(
0.0)
0
(
0.0)
8
(
10.8)
0
(
0.0)
2
(
2.7)

Reduced
in
size
1
(
1.2)
1
(
1.0)
17
(
23.0)
0
(
0.0)
45
(
60.8)

Flaccid/
soft
0
(
0.0)
0
(
0.0)
1
(
1.4)
0
(
0.0)
32
(
43.2)

Enlarged
0
(
0.0)
0
(
0.0)
0
(
0.0)
1
(
1.2)
2
(
2.7)

Abnormal/
infected
0
(
0.0)
0
(
0.0)
3
(
4.0)
0
(
0.0)
1
(
1.4)

Urinary
bladder:
b
Adhered
to
prostate
0
(
0.0)
0
(
0.0)
1
(
1.4)
0
(
0.0)
0
(
0.0)

Calculi
present
1
(
1.2)
0
(
0.0)
3
(
4.0)
0
(
0.0)
1
(
1.4)

Vaginal
pouch
b
0
(
0.0)
2
(
2.4)
43
(
58.1)
0
(
0.0)
1
(
1.4)

a
Data
are
presented
as
number
(%)
with
the
indicated
finding;
data
from
summary
tables
20
and
21.
On
this
table,
a
male
may
be
counted
more
than
once
if
he
exhibited
more
than
one
malformation.

b
Findings
not
reported
at
the
pnd
21
necropsy.

c
Undescended
testes
imbedded
subcutaneously
(
sc)
in
the
abdominal
wall.
080555.001.012
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43
Text
Table
5.
Gross
Findings
at
Unscheduled
Necropsy
a
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
No.
males
0
0
2
b
0
2
c
Cleft
phallus
 
 
2
 
1
Cowper's
gland,
missing
 
 
1
 
1
Epididymis,
reduced
in
size
 
 
0
 
2
Hypospadias
 
 
2
 
1
LABC,
missing
 
 
1
 
1
Penis,
pus
 
 
1
 
0
Prostate:

Dorsal
missing
 
 
0
 
1
Ventral
missing
 
 
0
 
1
Whole
gland
missing
 
 
1
 
0
Whole
gland
reduced
in
size
 
 
1
 
0
Seminal
vesicles,
reduced
in
size
 
 
2
 
1
Testis:

Undescended
 
 
1
 
2
sc
in
abdomen
 
 
1
 
0
Reduced
in
size
 
 
1
 
2
Urinary
bladder
calculi
 
 
1
 
0
Vaginal
pouch
 
 
1
 
0
a
Data
from
summary
table
21.
On
this
table,
a
male
may
be
counted
more
than
once
if
he
exhibited
more
than
one
malformation.

b
One
high
dose
VIN
male
was
euthanized
moribund
on
pnd
64,
and
one
high
dose
VIN
male
was
found
dead
on
pnd
80.

c
Both
high
dose
DBP
males
were
euthanized
moribund,
one
each
on
pnd
72
and
79.
080555.001.012
44
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44
The
incidences
of
the
findings
for
the
epididymides
are
presented
separately
for
missing
(
agenesis
of
caput,
corpus
and/
or
cauda
or
of
the
entire
organ)
and
for
reduced
in
size/
abnormal.
The
description
"
abnormal"
i.
e.,
changes
in
color,
appearance,
texture
or
presence
of
infection
is
almost
exclusively
documented
on
pnd
95.
Missing
parts
or
whole
epididymis(
des)
were
noted
at
the
high
dose
of
both
VIN
and
DBP
on
both
pnd
21
and
95,
with
incidences
for
both
time
points
higher
in
DBP
than
in
VIN
and
with
higher
incidences
on
pnd
95
than
on
pnd
21
for
both
chemicals.
Epididymis(
des),
reduced
in
size/
abnormal,
were
not
noted
in
any
VIN
group
nor
in
the
low
dose
of
DBP
on
pnd
21.
At
the
high
dose
of
DBP
on
pnd
21,
the
finding
was
present
but
the
incidence
was
low
(
6.2%).
On
pnd
95,
over
25%
of
the
males
at
the
high
dose
VIN
exhibited
the
finding,
while
the
incidence
for
the
high
dose
DBP
males
was
over
70%
(
Text
Table
6).
Therefore,
for
both
time
points,
the
incidence
was
higher
at
500
mg/
kg
DBP
than
at
100
mg/
kg
VIN.

The
incidence
of
the
findings
for
the
prostate
were
presented
separately
for
dorsal
and
ventral
lobes
and
for
missing
(
agenesis
of
part[
s]
or
the
entire
lobe),
versus
reduced
in
size/
abnormal.
"
Abnormal",
i.
e.,
change
in
color,
appearance,
texture,
or
presence
of
infection
is
almost
exclusively
documented
on
pnd
95.

For
the
prostate,
missing
dorsal
lobes
were
documented
at
a
relatively
high
incidence
at
100
mg/
kg/
day
on
pnd
21
(
32.3%)
and
on
pnd
95
(
23.0%)
for
VIN,
and
at
a
much
lower
incidence
for
DBP
at
500
mg/
kg/
day
on
pnd
21
(
1.5%)
and
on
pnd
95
(
4.0%).
Prostate
dorsal
lobe,
reduced
in
size/
abnormal,
was
not
present
on
pnd
21
for
any
VIN
or
DBP
doses.
On
pnd
95,
this
finding
was
present
at
50
mg/
kg
(
33.2%)
and
at
100
mg/
kg
/
day
(
27.0%)
VIN,
and
at
100
mg/
kg/
day
(
2.5%)
and
at
500
mg/
kg
(
10.8%)
DBP.
The
agenesis
of
the
dorsal
lobe
of
the
prostate
was
much
more
common
in
VIN
than
in
DBP
at
their
high
doses
on
both
pnd
21
and
95.
Small/
abnormal
dorsal
prostate
lobes
were
present
only
on
pnd
95
with
higher
incidences
in
the
VIN
low
and
high
doses
than
in
the
DBP
low
and
high
doses.

Prostate
,
ventral
lobe
missing,
was
not
present
in
controls
or
at
either
VIN
or
DBP
low
doses,
with
a
7.7%
incidence
at
100
mg/
kg/
day
VIN
and
a
3.1%
incidence
at
500
mg/
kg/
day
DBP
on
pnd
21.
Agenesis
of
the
ventral
lobe
was
present
at
both
high
doses
of
VIN
and
DBP
on
pnd
95.
Ventral
lobe,
reduced
in
size/
abnormal,
was
not
present
in
any
group
on
pnd
21
and
was
present
on
pnd
95
at
0
mg/
kg
(
1.2%;
one
male
with
ventral
lobe
"
brown
and
hard"),
at
50
mg/
kg/
day
(
7.4%)
and
100
mg/
kg/
day
(
60.8%)
VIN,
and
at
100
mg/
kg/
day
(
2.5%)
and
500
mg/
kg/
day
(
10.8%)
DBP.
The
ventral
lobe
of
the
prostate
was
detected
as
missing
in
the
high
doses
of
both
chemicals
at
a
low
incidence
on
pnd
21.
On
pnd
95,
the
incidence
at
100
mg/
kg/
day
VIN
was
increased
over
twofold
(
to
16.2%)
relative
to
the
incidence
on
pnd
21.
The
incidence
was
similar
for
both
time
points
(
3­
4.0%)
for
500
mg/
kg/
day
DBP.

Hypospadias
was
not
detected
in
the
vehicle
control
group
or
in
the
low
dose
DBP
group
on
pnd
21
or
pnd
95.
At
the
low
dose
VIN,
the
incidence
on
pnd
95
(
15.8%)
was
almost
twice
that
of
the
incidence
on
pnd
21
(
9.7%).
At
the
high
dose
VIN,
the
incidences
were
very
high
at
both
pnd
21
(
80.0%)
and
at
pnd
95
(
98.6%).
At
the
high
dose
DBP,
the
incidence
on
pnd
95
(
16.2%)
was
over
fivefold
greater
than
at
pnd
21
(
3.1%).
The
incidence
of
hypospadias
was
higher
for
VIN
than
080555.001.012
45
5/
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03
45
for
DBP
and
higher
on
pnd
95
than
on
pnd
21
for
both
chemicals.
Epispadias
was
not
detected
in
any
group
on
pnd
21,
and
was
not
detected
in
the
vehicle
control
group
or
in
either
DBP
group
on
pnd
95.
Epispadias
was
detected
in
the
low
dose
VIN
(
4.3%)
and
in
the
high
dose
VIN
(
14.9%)
on
pnd
95.
Thus,
the
incidence
of
epispadias
was
also
greater
for
VIN
than
for
DBP
(
it
was
undetected
in
DBP
groups)
and
was
higher
on
pnd
95
than
on
pnd
21
for
VIN
(
it
was
undetected
in
VIN
groups
on
pnd
21).

Effects
on
seminal
vesicles
(
missing/
reduced
in
size/
abnormal)
were
not
present
in
the
control
group
or
in
the
low
dose
VIN
or
DBP
groups
on
pnd
21,
and
not
in
the
controls
or
the
low
dose
DBP
on
pnd
95.
These
effects
were
observed
in
approximately
equal
incidences
in
the
high
dose
VIN
(
12.3%)
and
the
high
dose
DBP
(
10.8%)
on
pnd
21.
On
pnd
95,
the
incidence
in
the
low
dose
VIN
was
6.1%,
with
a
very
high
incidence
in
the
high
dose
VIN
(
85.1%,
sevenfold
higher
than
on
pnd
21);
at
the
high
dose
DBP,
on
pnd
95,
the
incidence
was
52.7%
(
a
fivefold
increase
over
the
incidence
in
this
group
on
pnd
21).

The
presence
of
gubernaculum
and
cranial
suspensory
ligaments
in
the
F1
males
on
pnd
21
and
95
and
their
relationship
with
undescended
testes
requires
further
discussion.

Imajima
et
al.
(
1997)
and
Shono
et
al.
(
2000)
reported
postnatal
cryptorchidism,
preceded
by
delays
in
the
transabdominal
descent
of
testes
in
rat
fetuses,
after
gestational
exposure
to
a
very
high
oral
dose
(
1000
mg/
kg/
day)
of
monobutyl
phthalate
(
the
major
intestinal
metabolite
of
DBP)
on
gd
15­
18
(
Imajima
et
al.,
1997)
or
on
gd
7­
10,
11­
14,
or
15­
18
(
Shono
et
al.,
2000).
Testes
normally
descend
to
the
inguinal
ring
by
term
and
into
the
scrotal
sacs
during
late
lactation
(
typically
pnd
16­
20
in
the
performing
laboratory).
Cryptorchidism
(
undescended
testes)
was
observed
in
the
present
study
in
both
the
weanling
(
pnd
21)
and
adult
(
pnd
95)
necropsies
for
both
VIN
and
DBP
high
dose
groups.

Very
recent
data
have
indicated
that
male
perinatal
reproductive
development
is
regulated
not
just
by
testosterone
(
and
DHT;
made
by
the
Leydig
cells)
and
Mdllerian­
inhibiting
substance
(
MIS;
made
by
the
Sertoli
cells)
which
cause
the
Mdllerian
ducts,
which
form
female
reproductive
structures
to
regress
in
males,
but
also
by
a
peptide
hormone,
now
designated
as
insulin­
like
factor
3
(
ISNL3)
produced
by
the
Leydig
cells
(
Kubota
et
al.,
2002).
ISNL13
was
discovered
in
the
1980s,
but
its
function
in
regulating
the
development
of
the
gubernaculum
(
which
attaches
to
the
caudal
portion
of
the
testis
and
epididymis
and
is
responsible,
in
whole
or
part,
for
testis
descent
into
the
lower
abdomen
to
the
inguinal
ring
in
utero
and
into
the
scrotal
sacs
during
late
lactation
in
rodents)
was
not
identified
until
ISNL13­
knockout
mice
were
constructed
(
with
normal
T
and
MIS),
which
exhibited
bilateral
cryptorchidism
(
Nef
and
Parada,
1999;
Zimmermann
et
al.,
1999).
ISNL13
has
also
been
shown
to
play
a
role
in
DES­
induced
cryptorchidism
in
mice
(
Emmen
et
al.,
2000a),
which
implies
estrogen
involvement.
The
role
of
ISNL13
in
human
cryptorchidism
is
not
clear,
with
evidence
both
for
its
role
(
Tomboc
et
al.,
2000)
and
against
any
role
(
Baker
et
al.,
2002).
Emmen
et
al.
(
2000b)
have
shown
that
both
androgen
and
Ins13
are
required
for
rodent
gubernuclear
outgrowth
in
vitro.
080555.001.012
46
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46
Dr.
L.
Early
Gray's
laboratory
at
U.
S.
EPA
(
NHEERL)
(
Gray
et
al.,
2000;
Gray
and
Foster,
2002;
Wilson
et
al.,
2003)
has
reported
that
gavage
administration
to
rats
of
DBP
(
or
DEHP
or
BBP)
at
750
mg/
kg/
day
on
gd
14
to
pnd
3
results
in
male
reproductive
system
malformations,
including
cryptorchidism
associated
with
abnormalities
of
the
gubernaculum.
In
addition,
examination
of
fetal
testes
from
dams
exposed
to
DEHP
at
750
mg/
kg/
day
on
gd
14­
18
indicated
that
not
only
was
testosterone
production
reduced,
but
also
that
Ins13
mRNA
was
inhibited
by
approximately
80%
(
Wilson
et
al.,
2003).
When
both
DBP
and
BBP,
each
by
gavage
at
500
mg/
kg/
day,
on
gd
14­
18
(
Gray
et
al.,
in
preparation)
were
administered
to
rat
dams,
no
offspring
males
in
the
combination
group
exhibited
normal
gubernacula,
and
a
few
males
exhibited
retained
cranial
suspensory
ligaments,
which
normally
maintain
the
ovaries
in
the
upper
abdomen
in
females.
In
some
cases,
only
the
gubernaculum
was
affected,
with
no
other
male
reproductive
system
malformations
or
vice
versa,
indicating
an
effect
of
the
phthalates
at
high
oral
doses
on
both
testosterone
and
ISNL13
synthesis.
Consistent
with
this
interpretation,
DBP
administered
by
gavage
at
a
high
oral
dose
(
500
mg/
kg/
day)
to
dams
in
the
present
study
during
gestation
and
lactation,
produced
a
small
but
biologically
significant
incidence
of
undescended
testes,
as
well
as
other
male
reproductive
system
malformations
in
male
offspring
on
pnd
21
and
95.

Therefore,
the
cryptorchidism
observed
by
Imagima
et
al.
(
1997),
Shono
et
al.
(
2000),
Gray
and
Foster
(
2002),
Wilson
et
al.
(
2003),
and
in
this
study
from
administration
of
various
phthalates
(
monobutyl
phthalate,
DBP,
DEHP,
BBP,
etc.)
resulted
only
from
very
high
oral
"
bolus"
doses
during
the
sensitive
period.

In
the
present
study,
the
presence
of
cranial
suspensory
ligaments
in
males
and
the
reduced
number
of
males
with
at
least
one
gubernaculum
on
pnd
21,
were
observed
at
the
low
and
high
doses
of
DBP
(
and
in
one
male
at
the
low
dose
VIN
on
pnd
21).
Cranial
suspensory
ligaments
were
also
observed
on
pnd
95
at
the
high
dose
of
VIN
(
one
male)
and
at
the
high
dose
of
DBP
(
six
males);
cranial
suspensory
ligaments
are
normally
observed
only
in
females,
attached
to
the
ovaries
and
the
under
side
of
the
diaphragm.
Undescended
testes
were
observed
on
pnd
21
at
the
high
dose
VIN
(
three)
and
at
the
high
dose
DBP
(
four);
and
on
pnd
95
at
the
low
dose
VIN
(
one),
the
high
dose
VIN
(
15),
and
at
the
high
dose
DBP
(
10)
but
not
in
the
low
dose
DBP.
Of
the
males
with
undescended
testes
at
the
high
VIN
and
the
high
DBP
doses,
eight
of
15
at
100
mg/
kg/
day
VIN
and
two
at
500
mg/
kg/
day
DBP
exhibited
testes
imbedded
subcutaneously
in
the
abdominal
wall,
strong
evidence
for
abnormal/
absent
gubernacula.

The
diagnostic
offspring
male
reproductive
malformations
observed
after
in
utero
exposure
to
VIN
are
hypospadias
and
agenesis
(
absence)
of
the
ventral
prostate.
In
Text
Table
6,
which
compares
the
percentage
and
incidence
of
malformations
at
pnd
21
and
95,
the
incidence
of
hypospadias
was
increased
on
pnd
95
over
the
incidence
on
pnd
21
for
both
doses
of
VIN
(
and
for
the
high
dose
of
DBP).
Agenesis
of
the
ventral
prostate
exhibited
a
higher
incidence
on
pnd
95
than
on
pnd
21
for
the
high
dose
of
VIN;
it
was
not
detected
at
either
time
point
for
the
low
dose
of
VIN.
It
was
also
detected
at
the
high
(
but
not
the
low)
dose
of
DBP
at
both
time
points
at
approximately
equal
incidences.
When
one
separates
the
data
on
missing
ventral
prostate
lobe
080555.001.012
47
5/
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03
47
from
data
on
ventral
prostate
lobe
"
reduced
in
size/
abnormal,"
the
sensitivity
of
the
pnd
95
necropsy
(
over
the
pnd
21
necropsy)
becomes
very
clear
for
the
latter
finding.
In
Text
Table
6
on
pnd
21,
there
were
no
incidences
of
ventral
prostate
lobe
reduced
in
size/
abnormal.
However,
on
pnd
95,
the
incidences
were
increased
at
the
low
(
7.4%)
and
at
the
high
dose
(
60.8%)
on
VIN
and
at
the
low
(
2.5%
and
high
(
10.8%)
doses
of
DBP.
Therefore,
this
effect
was
missed
entirely
on
pnd
21.

Also
for
Text
Table
6,
those
male
malformations
observed
on
pnd
95
but
not
on
pnd
21
in
either
or
both
test
chemicals
or
in
both
dose
groups
per
chemical
include
the
following:

°
epididimydes
reduced
in
size/
abnormal
(
not
observed
in
either
VIN
groups
on
pnd
21)

°
epispadias
(
not
detected
in
any
group
on
pnd
21)

°
levator
ani
bulbocavernosus
(
LABC)
complex,
missing/
reduced
in
size
(
not
observed
in
the
low
dose
of
VIN
or
either
dose
of
DBP
on
pnd
21)

°
cleft
phalllus
(
not
observed
in
the
low
dose
of
DBP
on
pnd
21)

°
dorsal
and/
or
ventral
lobes
of
prostate
reduced
in
size/
abnormal
(
not
detected
in
any
group
on
pnd
21)

°
seminal
vesicles
missing/
reduced
in
size/
abnormal
(
not
observed
in
the
low
dose
of
VIN
on
pnd
21)

°
undescended
testes
(
not
observed
in
the
low
dose
of
VIN
on
pnd
21)

°
testes
reduced
in
size
or
flaccid/
soft
(
not
detected
in
any
group
on
pnd
21)

°
and
vaginal
pouch
(
not
detected
in
any
group
on
pnd
21)

An
approach
to
determining
whether
effects
observed
on
pnd
95
would
have
been
observed
if
we
only
examined
one
adult
male
per
litter
in
each
group
is
presented
in
Text
Table
7.
In
this
table,
the
number
(
and
%)
of
male
reproductive
system
malformations
and
the
number(
and
%)
of
males
with
one
or
more
reproductive
system
malformations
are
presented
for
pnd
21
and
95.
Then,
the
incidence,
based
on
the
number
of
males
with
one
or
more
reproductive
system
malformations
(
taken
from
the
individual
animal
data
tables;
Appendix
I,
Tables
A­
26
and
A­
28)
and
on
the
number
of
litters
in
each
group,
with
>
1
male
with
>
1
reproductive
malformations,
is
presented.
These
numbers
are
used
to
detemine
whether
one
male
randomly
selected
to
represent
his
litter
on
pnd
95
would
have
been
malformed.
080555.001.012
48
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48
Text
Table
6.
Comparison
of
the
Incidence
of
Male
Reproductive
System
Malformations
on
pnd
21
Versus
pnd
95
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
No.
males,
pnd
21
74
82
65
71
65
No.
males,
pnd
95
82
95
74
81
74
Cowper's
gland
missing/
reduced
in
size:

Pnd
21
0
(
0.0)
a
9
(
11.0)
47
(
72.3)
1
(
1.4)
7
(
10.8)

Pnd
95
1
(
1.2)
7
(
7.4)
63
(
85.1)
0
(
0.0)
8
(
10.8)

Epididymides
missing
Pnd
21
0
(
0.0)
0
(
0.0)
2
(
3.1)
0
(
0.0)
14
(
21.5)

Pnd
95
0
(
0.0)
0
(
0.0)
4
(
5.4)
0
(
0.0)
33
(
44.6)

Epididymides
reduced
in
size/
abnormal
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
4
(
6.2)

Pnd
95
1
(
1.2)
0
(
0.0)
19
(
25.7)
0
(
0.0)
52
(
71.6)

Epispadias:

Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
0
(
0.0)
4
(
4.3)
11
(
14.9)
0
(
0.0)
0
(
0.0)

Hypospadias:

Pnd
21
0
(
0.0)
8.0
(
9.7)
52
(
80.0)
0
(
0.0)
2
(
3.1)

Pnd
95
0
(
0.0)
15
(
15.8)
73
(
98.6)
0
(
0.0)
12
(
16.2)

LABC
missing/
reduced
in
size:

Pnd
21
0
(
0.0)
0
(
0.0)
1
(
1.5)
0
(
0.0)
0
(
0.0)

Pnd
95
0
(
0.0)
2
(
2.1)
40
(
54.0)
0
(
0.0)
4
(
5.4)

(
Continued)
080555.001.012
49
5/
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03
49
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Phallus,
cleft:

Pnd
21
0
(
0.0)
4
(
4.9)
25
(
38.5)
0
(
0.0)
2
(
3.1)

Pnd
95
1
(
1.2)
41
(
43.15)
74
(
100.0)
2.0
(
2.47)
26
(
35.14)

Prostate
dorsal
lobe
missing
Pnd
21
0
(
0.0)
0
(
0.0)
21
(
32.3)
0
(
0.0)
1
(
1.5)

Pnd
95
0
(
0.0)
0
(
0.0)
17
(
23.0)
0
(
0.0)
3
(
4.0)

Prostate
dorsal
lobe
reduced
in
size/
abnormal
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
0
(
0.0)
3
(
3.2)
20
(
27.0)
2
(
2.5)
8
(
10.8)

Prostate
ventral
lobe
missing
Pnd
21
0
(
0.0)
0
(
0.0)
5
(
7.7)
0
(
0.0)
2
(
3.1)

Pnd
95
0
(
0.0)
0
(
0.0)
12
(
16.2)
0
(
0.0)
3
(
4.0)

Prostate
ventral
lobe
reduced
in
size/
abnormal
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
1
(
1.2)
7
(
7.4)
45
(
60.8)
2
(
2.5)
8
(
10.8)

Seminal
vesicles
missing/
reduced
in
size/
abnormal
Pnd
21
0
(
0.0)
0
(
0.0)
8
(
12.3)
0
(
0.0)
7
(
10.8)

Pnd
95
0
(
0.0)
5
(
6.1)
63
(
85.1)
0
(
0.0)
39
(
52.7)

Testes
undescended
Pnd
21
0
(
0.0)
0
(
0.0)
3
(
4.6)
0
(
0.0)
4
(
6.1)

Pnd
95
0
(
0.0)
1
(
1.0)
15
(
20.3)
0
(
0.0)
10
(
13.3)

Testes
reduced
in
size
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
1
(
1.2)
1
(
1.0)
17
(
23.0)
0
(
0.0)
45
(
60.8)

(
Continued)
080555.001.012
50
5/
5/
03
50
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Testes
flaccid/
soft
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
0
(
0.0)
0
(
0.0)
1
(
1.4)
0
(
0.0)
32
(
43.2)

Males
with
>
1
gubernuclear
wt.
b
Pnd
21
74
(
100.0)
82
(
100.0)
65
(
100.0)
69
(
97.2)
59
(
90.8)

Pnd
95
5.5
(
6.2)
11
(
11.6)
13
(
17.6)
1.0
(
1.2)
6
(
8.1)

Males
with
>
1
cranial
suspensory
ligamentb
Pnd
21
0
(
0.0)
1
(
1.32)
0
(
0.0)
2.2
(
3.1)
2.2
(
3.4)

Pnd
95
0
(
0.0)
0
(
0.0)
1
(
1.4)
0
(
0.0)
6.2
(
8.1)

Vaginal
pouch
Pnd
21
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
(
0.0)

Pnd
95
0
(
0.0)
2
(
2.4)
43
(
58.1)
0
(
0.0)
1
(
1.4)

a
Data
presented
as
number
(%)
of
males
exhibiting
the
finding.
Males
may
be
counted
more
than
once
if
they
exhibited
more
than
one
finding.

b
The
incidences
of
these
findings
are
taken
from
Summary
Task
13
(
and
Text
Table
3)
for
pnd
21,
and
from
Summary
Task
20
(
and
Text
Table
4)
for
pnd
95.

For
the
pnd
95
necropsy,
approximately
50%
of
the
males
and
84%
of
the
litters
at
the
low
dose
of
VIN
and
all
of
the
males
in
all
of
the
litters
at
the
high
dose
of
VIN
exhibited
at
least
one
reproductive
malformation.
Approximately
90%
of
the
males
and
30%
of
the
litters
at
the
low
dose
of
DBP
and
92%
of
the
males
and
96%
of
the
litters
at
the
high
dose
of
DBP
also
exhibited
at
least
one
reproductive
malformation.
These
values
indicate
that
the
number
of
affected
males
per
affected
litter
would
be
1.0,
2.2,
3.4,
1.0,
and
3.1
at
0,
50,
and
100
VIN
and
at
100
and
500
DBP,
respectively
(
Text
Table
7).
This
information
is
interpreted
to
mean
that
affected
males
in
the
low
and
high
dose
VIN
and
the
high
dose
DBP
would
have
been
detected
at
either
the
pnd
21
or
95
necropsies
(
under
the
current
testing
guidelines).
The
incidence
at
the
low
dose
DBP
(
1.0
affected
males
per
affected
litter)
implies
that
detection
of
males
with
reproductive
system
malformations
on
pnd
21
(
three
males
examined
out
of
five
males
present
at
weaning
in
the
current
guidelines)
and
at
pnd
95
(
one
male
examined
out
of
two
males
remaining
after
the
pnd
21
necropsy
in
the
080555.001.012
51
5/
5/
03
51
current
guidelines)
would
likely
be
detected
but
at
an
erroneously
lower
incidence.
Since
approximately
three
males/
litter
were
examined
on
pnd
21
(
as
specified
by
the
1998
OPPTS
testing
guideline),
the
likelihood
of
detecting
effects
on
pnd
21
is
not
changed,
and
the
calculation
for
results
on
pnd
21
for
this
protocol,
versus
the
current
testing
guidelines,
was
not
performed.

The
best
approach
to
determine
whether
examination
of
more
male
pups
at
weaning,
and,
more
importantly,
retention
of
more
male
offspring
to
adulthood
(
versus
the
one/
sex/
litter
in
the
standard
two­
generation
study
design)
would
allow
detection
of
more
male
reproductive
malformations
and
higher
incidences
of
these
malformations,
would
be
to
start
with
the
full
offspring
data
set
from
this
study.
Then,
repeated
random
samples
of
three
pups/
litter
on
pnd
21
and
one
male/
litter
on
pnd
95
would
be
selected
(
using
mathematical
random
number
generators
from
SAS
®
)
.
Approximately
1000
such
random
samples
would
be
obtained
and
then
analyzed
to
determine
whether
retention
of
more
pups/
litter
would
result
in
smaller
variances
(
increased
precision)
for
estimated
differences
and
therefore
more
significant
p­
values.
This
repeated
analysis
is
basically
a
Monte­
Carlo
simulations
assessment
(
and
use
of
a
one­
tailed
t­
test).
The
results
should
provide
the
probability
of
detecting
significant
effects
(
for
the
parameters
selected)
with
the
randomly
selected
three
male
pups/
litter
on
pnd
21
and
with
the
randomly
selected
one
male/
litter
on
pnd
95
versus
the
probability
of
detection
with
the
entire
data
set
from
this
study.

6.
Were
there
differences
in
the
effects
on
weights
of
various
male
reproductive
system
organs
between
the
two
chemicals
at
the
same
time
point,
and
within
each
chemical
at
the
two
different
time
points?
Text
Table
8
presents
the
absolute
and
adjusted
(
adjusted
for
body
weight
as
covariate)
weights
of
all
the
reproductive
organ
weights
on
pnd
21
and
95
for
all
groups.

For
testis
weights,
effects
were
more
profound
from
DBP
with
significant
reductions
at
the
low
dose
DBP
(
but
not
at
the
low
dose
VIN)
on
pnd
21
and
greater
reductions
at
the
high
dose
DBP
than
at
the
high
dose
VIN
for
both
pnd
21
and
pnd
95.

For
epididymal
corpus
plus
caput
together
and
for
cauda
epididymis,
reductions
were
greater
at
the
high
dose
DBP
than
at
the
high
dose
VIN
for
both
pnd
21
and
pnd
95.
Interestingly,
the
weight
of
the
corpus
plus
caput
was
reduced
for
both
the
low
dose
VIN
and
DBP
for
pnd
21
but
not
for
pnd
95.
The
cauda
epididymis
weight
was
reduced
for
the
right
(
but
not
left)
cauda
for
the
low
dose
VIN
only
on
pnd
95.
The
adjusted
(
but
not
the
absolute)
weight
of
the
right
(
but
not
the
left)
cauda
was
also
reduced
for
the
low
dose
DBP
only
for
pnd
95.

Seminal
vesicle
plus
coagulating
gland
weights
were
more
profoundly
reduced
at
the
high
dose
VIN
than
at
the
high
dose
DBP
for
both
pnd
21
and
95.
In
addition,
the
absolute
weights
were
significantly
reduced
at
the
low
dose
VIN
(
but
not
DBP)
for
pnd
21,
but
not
for
pnd
95.
080555.001.012
52
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52
Text
Table
7.
Evaluation
of
Detection
of
Male
Reproductive
System
Malformations
in
this
Protocol
Versus
in
the
OPPTS
Testing
Guidelines
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Pnd
21a
No.
males
examined
74
82
65
71
65
No.
litters
examined
23
25
22
23
23
Mean
number
of
F1
male
offspring
examined
per
litter
3.22
3.28
3.38
3.24
3.54
Pnd
95b
No.
males
examined
82
95
74
81
74
No.
litters
examined
23
25
22
23
23
Mean
number
of
F1
male
offspring
examined
per
litter
3.56
3.80
3.36
3.52
3.22
Incidence
of
total
no.
of
malformations
based
on
no.
F1
males
examined
Pnd
21
c,
d
0
(
0.0)
21
(
256)
169
(
260.0)
1
(
1.4)
43
(
66.2)

Pnd
95
c,
e
8
(
9.8)
f
95
(
100.0)
532
(
718.9)
11
(
13.6)
237
(
320.3)

Total
number
of
F1
males
with
>
1
malformation
on
pnd
95
c
7
(
8.5)
f
47
(
49.5)
74
(
100.0)
7
(
8.6)
68
(
91.9)

Total
no.
of
F1
litters
with
>
1
male
with
>
1
malformations
on
pnd
95
c
7
(
30.4)
f
21
(
84.0)
22
(
100.0)
7
(
30.4)
22
(
95.7)

No.
malformed
males
per
affected
litters
on
pnd
95
1.0f
2.24
3.36
1.0
3.09
a
The
number
of
F1
male
weanlings/
litter
examined
in
this
protocol
versus
the
numbers
from
the
1998
OPPTS
testing
guideline
are
approximately
equivalent.

b
The
OPPTS
testing
guideline
specifies
one
male/
litter
retained
to
adulthood.
Therefore,
this
protocol
provides
at
least
three
times
the
power
to
detect
adult
male
malformations.

c
Data
presented
as
number
(%).

d
Pnd
21
data
taken
from
Summary
Table
14
and
Individual
Animal
Tables
A­
15
and
A­
17.

e
Pnd
95
data
taken
from
Summary
Table
21
and
Individual
Animal
Tables
A­
26
and
A­
28.

f
Predominantly
minor
effects
(
e.
g.,
epididymis
and
testis
reduced
in
size,
prostate
abnormal
[
hard
and
brown],
etc.)
080555.001.012
53
5/
5/
03
53
The
whole
prostate
weight
was
more
profoundly
reduced
for
the
high
dose
VIN
than
for
the
high
dose
DBP
for
both
pnd
21
and
95;
it
was
also
significantly
reduced
as
absolute
and
adjusted
weights
at
the
low
dose
VIN
on
pnd
21
but
not
on
pnd
95.
The
ventral
lobe
weights
paralleled
the
intact
gland
weights
exactly.
The
dorsal
lobe
weight
was
significantly
reduced
only
at
the
high
dose
of
both
VIN
and
DBP,
with
the
weights
at
the
high
dose
VIN
more
reduced
than
the
weights
at
the
high
dose
DBP
on
both
pnd
21
and
95.

The
levator
ani
bulbocavernosus
(
LABC)
weights
(
absolute
and
adjusted)
were
significantly
reduced
at
the
low
and
high
dose
VIN,
and
at
the
high
dose
(
but
not
the
low
dose)
DBP,
with
the
reductions
greater
for
VIN
than
for
DBP
at
both
pnd
21
and
95.

Cowper's
gland
weights
were
unaffected
at
the
low
dose
VIN
and
significantly
reduced
for
absolute
weights
on
both
pnd
21
and
pnd
95,
and
for
adjusted
weights
only
for
pnd
95
at
the
high
dose
VIN.
For
DBP
groups,
absolute
and
adjusted
Cowper's
gland
weights
were
reduced
at
the
high
and
low
doses
only
for
pnd
21
(
and
not
for
pnd
95).

In
summary,
testes
and
epididymides
weights
were
more
affected
(
reduced)
by
DBP.
Seminal
vesicle
and
coagulating
gland,
prostate,
LABC,
and
Cowper's
gland
weights
were
more
affected
(
reduced)
by
VIN
(
Text
Table
8).

In
addition
to
addressing
the
specific
objectives
stated
above,
the
data
in
this
study
invite
comparison
of
the
effects
of
these
two
dissimilar
antiandrogens.

As
an
example
of
this,
the
kinds
and
incidences
of
F1
male
reproductive
malformations
observed
at
pnd
95
in
the
low
and
high
dose
groups
of
VIN
versus
DBP
are
presented
in
Text
Table
9.
The
enhanced
pnd
95
data
resulting
from
the
present
study
design
provide
an
opportunity
to
compare
male
reproductive
anomalies
that
are
most
likely
persistent.
There
were
dose­
related
incidences
in
both
chemicals,
with
the
low
dose
of
VIN
more
effective
(
more
malformations
observed)
than
the
low
dose
of
DBP
(
incidence
of
specific
malformations
was
less
or
none).
This
assessment
is
obviously
related
to
the
doses
selected
for
each
chemical
and
the
different
mechanisms
of
action
of
the
two
chemicals.
VIN
is
a
dicarboximide
fungicide,
considered
an
androgen­
receptor
antagonist
(
Gray
et
al.,
1994).
DBP
is
an
industrial
chemical
used
as
a
coalescing
agent
in
latex
adhesives,
as
a
plasticizer
in
cellulose
plastics,
and
as
a
solvent
for
dyes
(
Kavlock
et
al.,
2002).
DBP
or
its
monoester,
mBuP,
does
not
bind
to
the
androgen
receptor
(
Kavlock
et
al.,
2002),
and
it
does
not
act
on
reproductive
development
via
its
peroxisomal
proliferating
activity
(
Ward
et
al.,
1998).
Its
developmental
anti­
androgenic
mechanism
is
via
inhibition
of
fetal
testicular
testosterone
biosynthesis
in
the
fetal
interstital
Leydig
cells,
perhaps
by
delaying
Leydig
cell
differentiation
and
resulting
in
Leydig
cell
hyperplasia
in
utero
(
Parks
et
al.,
2000).
DBP
may
also
act
by
its
inhibition
of
the
transcription
of
the
Insl3
gene
also
in
the
Leydig
cells
(
Kubota
et
al.,
2002).
The
peptide
hormone
Insl3
is
responsible
for
the
normal
development
and
function
of
the
gubernaculum
in
fetal
male
rats.
Knockout
mice
with
normal
T
and
MIS
080555.001.012
54
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54
Text
Table
8.
Comparison
of
Male
Reproductive
System
Organ
Weights
on
pnd
21
versus
pnd
95a
Vehicle
Control
(
mg/
kg/
day)
VIN
(
mg/
kg/
day)
DBP
(
mg/
kg/
day)

Parameter
0
50
100
100
500
Testis
Right
Ab
pnd
21
 
(
0.1323)
 
99
(
0.1198)
 
999
(
0.1017)

Right
Ab
pnd
95
 
(
1.7702)
 
999
(
1.5284)
 
999
(
1.2383)

Right
Aj
pnd
21
 
(
0.1295)
8
(
0.1334)
 
9
(
0.1221)
999
(
0.1105)

Right
Aj
pnd
95
 
(
1.7680)
 
999
(
1.5335)
 
999
(
1.2416)

Left
Ab
pnd
21
 
(
0.1305)
 
99
(
0.1186)
 
999
(
0.0983)

Left
Ab
pnd
95
 
(
1.7688)
 
999
(
1.4970)
 
999
(
1.3982)

Left
Aj
pnd
21
 
(
0.1281)
 
 
99
(
0.1189)
999
(
0.1078)

Left
Aj
pnd
95
 
(
1.7649)
 
999
(
1.5056)
 
999
(
1.4089)

Corpus
and
Caput
Epididymis
Right
Ab
pnd
21
 
(
0.0139)
9
(
0.0125)
999
(
0.0100)
9
(
0.0123)
999
(
0.0088)

Right
Ab
pnd
95
 
(
0.3623)
 
999
(
0.3267)
 
999
(
0.2405)

Right
Aj
pnd
21
 
(
0.0137)
9
(
0.0123)
999
(
0.0105)
9
(
0.0121)
999
(
0.0094)

Right
Aj
pnd
95
 
(
0.3617)
 
999
(
0.3282)
 
999
(
0.2402)

Left
Ab
pnd
21
 
(
0.0137)
 
999
(
0.0099)
999
(
0.0111)
999
(
0.0084)

Left
Ab
pnd
95
 
(
0.3563)
 
99
(
0.3329)
 
999
(
0.2548)

Left
Aj
pnd
21
 
(
0.0130)
 
999
(
0.0103)
999
(
0.0108)
999
(
0.0092)

Left
Aj
pnd
95
 
(
0.3617)
 
9
(
0.3282)
 
999
(
0.2543)

(
Continued)
080555.001.012
55
5/
5/
03
55
Cauda
Epididymis
Right
Ab
pnd
21
 
(
0.0099)
 
999
(
0.0069)
 
999
(
0.0062)

Right
Ab
pnd
95
 
(
0.2857)
9
(
0.2719)
999
(
0.2309)
 
999
(
0.1128)

Right
Aj
pnd
21
 
(
0.0097)
 
999
(
0.0072)
 
999
(
0.0069)

Right
Aj
pnd
95
 
(
0.2854)
9
(
0.2716)
999
(
0.2316)
9
(
0.2735)
999
(
0.1143)

Left
Ab
pnd
21
 
(
0.0096)
 
999
(
0.0076)
 
999
(
0.0064)

Left
Ab
pnd
95
 
(
0.2720)
 
999
(
0.2263)
 
999
(
0.1362)

Left
Aj
pnd
21
 
(
0.0095)
 
999
(
0.0079)
 
999(
0.0070)

Left
Aj
pnd
95
 
(
0.2714)
 
999
(
0.2277)
 
999
(
0.1363)

Seminal
vesicles
plus
coagulating
glands
Ab
pnd
21
 
(
0.0180)
999
(
0.0145)
999
(
0.0082)
 
999
(
0.0097)

Ab
pnd
95
 
(
1.4707)
 
999
(
0.7097)
 
999
(
1.1111)

Aj
pnd
21
 
(
0.0178)
999
(
0.0143)
999
(
0.0085)
 
999
(
0.0102)

Aj
pnd
95
 
(
1.4585)
 
999
(
0.7378)
 
999
(
1.1186)

Prostate
Whole
Gland
Ab
pnd
21
 
(
0.0483)
9
(
0.0441)
999
(
0.0235)
 
999
(
0.0292)

Ab
pnd
95
 
(
1.0810)
 
999
(
0.4226)
 
999
(
0.8601)

Aj
pnd
21
 
(
0.0478)
9
(
0.0436)
999
(
0.0249)
 
999
(
0.0322)

Aj
pnd
95
 
(
1.0799)
 
999
(
0.4261)
 
999
(
0.8689)

Ventral
lobe
Ab
pnd
21
 
(
0.0251)
9
(
0.0225)
999
(
0.0117)
 
999
(
0.0163)

Ab
pnd
95
 
(
0.6418)
 
999
(
0.1897)
 
999
(
0.4762)

(
Continued)

Aj
pnd
21
 
(
0.0246)
9
(
0.0220)
999
(
0.0127)
 
999
(
0.0178)

Aj
pnd
95
 
(
0.6417)
 
999
(
0.1899)
 
999
(
0.4812)
080555.001.012
56
5/
5/
03
56
Dorsal
lobe
Ab
pnd
21
 
(
0.0232)
 
999
(
0.0118)
 
999
(
0.0126)

Ab
pnd
95
 
(
0.4393)
 
999
(
0.2303)
 
99
(
0.3791)

Aj
pnd
21
 
(
0.0229)
 
999
(
0.0126)
 
999
(
0.0139)

Aj
pnd
95
 
(
0.4381)
 
999
(
0.2337)
 
9
(
0.3829)

LABC
Ab
pnd
21
 
(
0.0562)
99
(
0.0474)
999
(
0.0313)
 
999
(
0.0419)

Ab
pnd
95
 
(
1.3577)
999
(
1.1463)
999
(
0.5539)
 
999
(
0.9411)

Aj
pnd
21
 
(
0.0551)
99
(
0.0459)
999
(
0.0337)
 
99
(
0.0456)

Aj
pnd
95
 
(
1.3508)
999
(
1.1400)
999
(
0.5703)
 
999
(
0.9547)

Cowper's
glands
Ab
pnd
21
 
(
0.0036)
 
99
(
0.0021)
9
(
0.0030)
99
(
0.0027)

Ab
pnd
95
 
(
0.1480)
 
999
(
0.0688)
 
 
Aj
pnd
21
 
(
0.0035)
 
 
9
(
0.0030)
9
(
0.0029)

Aj
pnd
95
 
(
0.1479)
 
999
(
0.0697)
 
 
a
The
control
values
are
presented
(
in
parentheses)
for
comparison
with
the
values
from
the
treated
groups;
if
there
is
a
statistically
significant
change,
the
degree
of
significance
is
presented
by
up/
down
arrows
and
the
value
is
in
parentheses.
pnd
21
organ
weights
in
grams
from
Summary
Table
13
pnd
95
organ
weights
in
grams
from
Summary
Table
20
Ab
=
Absolute
organ
weight
in
grams
Aj
=
Adjusted
organ
weight
in
grams
(
adjusted
for
body
weight
as
covariate).
LABC
=
Levator
ani
bulbocavernosus
(
LABC)
muscle
9,
99,
999,
statistically
significantly
reduced
at
p
<
0.05,
p<
0.01
and
p<
0.001,
respectively,
by
appropriate
statistical
tests
(
see
summary
tables
and
text
for
details).

8,
statistically
significantly
increased
at
p<
0.05
by
appropriate
statistical
tests
(
see
summary
tables
and
text
for
details).
 
,
no
statistically
significant
difference
from
the
control
group
value.
080555.001.012
57
5/
5/
03
57
exhibited
bilateral
cryptorchidism
(
Nef
and
Parada,
1999;
Zimmerman
et
al.,
1999).
The
gubernaculum
attaches
to
the
caudal
portion
of
the
testis
and
the
caudal
epididymis
and
to
the
inguinal
ring
(
and
scrotal
sac).
It
is
responsible
for
the
descent
of
the
testis
to
the
inguinal
ring
in
utero
and,
with
androgen,
for
testicular
descent
into
the
scrotal
sac
during
later
lactation.
When
it
is
absent
or
abnormal
(
e.
g.,
threadlike),
the
testis(
es)
do
not
descend
to
the
inguinal
ring
during
gestation
and
do
not
normally
descend
into
the
scrotal
sacs
postnatally.
DBP
at
high
oral
doses
(
750
mg/
kg/
day,
gd
14­
pnd
3;
Gray
et
al.,
2000)
has
been
shown
to
cause
cryptorchidism
with
abnormal
gubernacula
(
Gray
and
Foster,
2002)
in
rats.
Both
VIN
(
at
both
doses)
and
DBP
(
at
the
high
dose)
in
the
present
study
caused
undescended
testes,
with
some
of
these
males
exhibiting
testes
embedded
in
the
abdominal
wall
at
the
high
doses
of
VIN
(
eight
males)
and
DBP
(
two
males).

Additionally,
differences
between
the
range
and
severity
of
effects
caused
by
the
two
test
chemicals
are
evident
(
Text
Table
9).
VIN
causes
a
low
incidence
of
flaccid/
soft
testes
or
retention
of
cranial
suspensory
ligaments
in
males,
while
DBP
causes
a
midrange
incidence.
In
contrast,
VIN
causes
a
higher
incidence
of
epispadias,
hypospadias,
missing
or
small
ventral
prostate,
or
vaginal
pouch
than
DBP
does.
It
appears
as
if
VIN
more
strongly
affects
those
structures
under
DHT
control
(
lower
reproductive
tract,
prostate,
seminal
vesicles,
external
genitalia).
The
other
findings
on
Text
Table
9
occurred
with
approximately
equal
incidences
in
the
high
dose
groups
of
both
chemicals
(
and
with
a
slightly
less
concordance
in
the
low
dose
groups
of
both
chemicals).

Text
Table
9.
Comparison
of
the
Kinds
and
Incidences
of
F1
Male
Reproductive
Malformations
by
Chemical
and
by
Dose
Observed
on
pnd
95a
VIN
Dose
DBP
Dose
Finding
Low
High
Low
High
Cowpers
gland
missing/
reduced
in
size
##
##
 
#
#

Epididymides,
missing/
reduced
in
size
 
#
#
 
#
#

LABCb
missing/
reduced
in
size/
malformed
 
#
 
#

Hypospadias
##
###
 
#
#

Cleft
phallus
##
###
#
##

Prostate:
Dorsal
#
##
#
##

Ventral
#
###
 
#
#

Seminal
vesicles,
missing/
misshapen
#
###
 
#
#

Epispadias
#
##
 
 
Vaginal
pouch
#
##
­­
 
Testes
undescended
#
##
 
#
#

Testes
embedded
in
abdominal
wall
 
#
#
 
#

Testes
reduced
in
size
#
##
 
#
#
080555.001.012
58
5/
5/
03
VIN
Dose
DBP
Dose
Finding
Low
High
Low
High
58
Testes
flaccid/
soft
 
#
 
#
#

Glans
penis
not
completely
detached
#
##
 
#
#

Males
with
>
1
cranial
suspensory
ligament
 
#
 
#
#

 
=
no
incidence
#
=
small
incidence
(
1­
5%),
##
=
mid
range
incidence
(
6­
74%),
###
=
high
incidence
(>
75%)

a
PND
95
data
taken
from
Individual
Animal
Tables
20
and
21.

b
LABC
=
Levator
Ani
plus
Bulbocavernosus
Complex
080555.001.012
59
5/
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03
59
CONCLUSIONS
1.
Specific
male
offspring
malformations
were
detected
on
pnd
95
but
not
on
pnd
21.
Examples
include
prostate
dorsal
lobe
abnormal/
reduced
in
size
(
VIN,
both
doses;
DBP,
high
dose),
prostate
ventral
lobe
abnormal/
reduced
in
size
(
both
compounds,
both
doses),
and
epispadias
(
VIN,
both
doses).

2.
The
incidence
of
specific
male
offspring
malformations
detected
on
pnd
95
was
higher
than
the
incidence
of
the
same
malformation
observed
on
pnd
21.
Examples
include
agenesis
of
all
or
parts
of
the
epididymis(
des)
(
high
dose
of
both
VIN
and
DBP),
hypospadias
(
low
dose
VIN),
and
missing/
reduced
in
size/
abnormal
seminal
vesicles
(
high
dose
of
both
VIN
and
DBP).

3.
The
effects
of
VIN
on
the
incidence
of
hypospadias
and
ventral
prostate
agenesis
were
more
obvious
at
pnd
95
than
at
pnd
21.
This
effect
was
more
apparent
at
the
low
dose
than
at
the
high
dose.
Specifically,
hypospadias
was
observed
in
9.7%
vs
15.8%
of
the
animals
on
pnd
21
and
95,
respectively,
whereas
high
dose
animals
exhibited
hypospadias
at
80.0%
vs
98.6%
on
pnd
21
and
95,
respectively.

4.
The
effects
of
DBP
(
high
dose)
on
the
incidence
of
epididymal
agenesis
on
pnd
95
was
approximately
twice
that
observed
on
pnd
21,
and
thus
were
more
obvious
on
pnd
95
than
on
pnd
21.

5.
Adverse
effects
on
the
weight
of
some
male
reproductive
tissues
were
more
apparent
at
pnd
95
than
on
pnd
21.
Examples
include
adjusted
right
or
left
testis
weight
(
high
dose
VIN),
absolute
right
cauda
epididymis
weight
(
low
dose
VIN),
adjusted
right
cauda
epididymis
weight
(
low
dose
VIN
and
DBP),
absolute
LABC
weight
(
low
dose
VIN),
adjusted
LABC
weight
(
high
dose
VIN
and
DBP),
and
absolute
and
adjusted
Cowper's
gland
weight
(
high
dose
VIN).

6.
Adverse
reproductive
system
effects
in
toto
(
structural
malformations
and
other
abnormalities)
of
the
low
and
high
doses
of
VIN
and
the
high
dose
of
DBP
on
F1
adult
male
offspring
would
most
likely
be
statistically
significant
with
either
one
or
three
adult
males/
litter,
and
would
have
been
detected
with
either
study
design.

7.
Adverse
reproductive
system
structural
effects
in
toto
at
the
low
dose
of
DBP
on
F1
adult
male
offspring
were
clearly
biologically
significant
but
not
necessarily
or
likely
statistically
significant,
with
either
one
or
three
adult
males/
litter,
and
provide
an
example
of
effects
that
would
not
be
detected
with
either
study
design.

8.
The
more
males
examined
per
litter,
the
better
the
characterization
of
the
litter
as
responding
or
not
responding
adversely
to
exposure,
and
the
smaller
the
variance
term
for
pooled
litters
within
each
treatment
group.
The
enhanced
sensitivity
with
more
males
examined
per
litter
would
increase
the
likelihood
of
detection
of
effects
as
statistically
and
biologically
significant.
Also,
for
effects
with
low
incidence,
such
as
in
the
low
dose
DBP
group
in
this
study,
the
risk
with
fewer
males
examined
per
litter
is
that
the
effect
might
be
missed,
i.
e.,
the
litter
would
be
designated
as
not
responding,
on
the
basis
of
the
one
male
examined,
if
that
male
did
not
exhibit
the
effect.
080555.001.012
60
5/
5/
03
60
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64
PROTOCOL
DEVIATIONS
1.
Nine
instances
of
deviations
from
the
protocol
were
noted,
all
involving
relative
humidity
levels
in
the
animal
rooms,
as
follows:

Room
Date
%
RH
Duration
303
8/
15/
02
71.0
1
hour
303
9/
17/
02
85.5
1
hour
403
9/
17/
02
80.4
1
hour
403
10/
9/
02
85.0
1
hour
404
9/
17/
02
85.3
1
hour
404
10/
9/
02
78.9
1
hour
503
9/
17/
02
78.4
1
hour
503
10/
9/
02
76.1
1
hour
407
10/
9/
02
79.9
1
hour
2.
On
8/
2/
02,
the
lights
in
the
ARF
did
not
go
off
at
8
pm
as
scheduled.
The
lights
were
turned
off
manually
at
9:
30
pm.
Thus,
the
lights
were
on
15.5
hours
instead
of
14.
This
deviation
occurred
during
the
gestational
period.
Based
on
the
pregnancy
rates,
successful
deliveries,
and
subsequent
rearing
of
the
young,
this
deviation
in
the
light
cycle
did
not
appear
to
have
an
adverse
effect
on
the
study.

3.
Samples
of
the
dose
formulations
were
scheduled
to
be
taken
on
the
first
day
of
dosing
on
gd
6,
pnd
0,
7,
14,
and
21.
Due
to
an
oversight
by
the
laboratory
staff,
the
samples
for
pnd
0
and
20
were
not
taken
until
the
second
day
of
dosing
for
those
time
points.
Results
of
the
analysis
(
see
Appendix
II)
suggest
that
the
dosing
formulation
had
not
been
compromised
by
this
oversight,
and
that
it
did
not
adversely
affect
the
results
of
the
analysis
or
the
study.

4.
The
post­
dosing
time
for
Animal
#
98,
Rx
Code
31036
was
inadvertently
not
recorded
on
8/
16/
02.

5.
The
post­
dosing
observation
time
for
4
females
in
Rx
01822
was
from
1­
5
minutes
over
the
2
hours
post­
dosing
time
on
8/
5/
03.

6.
On
8/
15/
02,
two
female
pups
were
found
dead
and
one
male
pup.
External
and
visceral
findings
were
recorded
for
one
female
and
one
male.
Documentation
for
external
and
visceral
exam
for
one
female
was
inadvertently
not
recorded.

7.
Presence
or
absence
of
cranial
suspensory
ligament
was
inadvertently
not
recorded
at
necropsy
for
Male
1348,
Rx
Code
77491,
and
Male
1038,
Rx
Code
31038.

8.
Weight
of
Glans
Penis
inadvertently
not
recorded
at
necropsy
for
Male
#
1044,
Rx
Code
31038.

9.
The
presence
or
absence
of
gubernacular
cords
was
not
recorded
at
necropsy
for
Male
#
1158,
Rx
Code
31038.

10.
The
weight
of
the
Glans
Penis
was
taken
at
necropsy
even
though
the
Glans
Penis
was
noted
as
partially
detached
for
Male
1156,
Rx
Code
31038.
The
weight
was
not
entered.
080555.001.012
65
5/
5/
03
65
11.
The
presence
or
absence
of
cranial
suspensory
ligaments
was
not
recorded
at
pnd
21
necropsy
for
6
pups
in
Rx
Code
77491,
for
6
pups
in
Rx
Code
31036,
for
9
pups
in
Rx
Code
01822,
for
6
pups
in
Rx
Code
24038,
and
for
7
pups
in
Rx
Code
17225.

12.
Animals
were
ordered
at
10­
11
weeks
of
age
with
a
weight
range
of
200­
300
g
and
should
have
been
ordered
at
age
of
9­
10
weeks
with
a
weight
range
of
200­
225
g.

13.
Presence
or
absence
of
hypospadias
not
recorded
for:
dam
10
pup
2,
dam
21
pup
2
in
Rx
77491;
dam
12
pup
5,
dam
41
pup
2
in
Rx
31036;
dam
42
pup
1,
dam
23
pup
5
in
Rx
01822;
dam
7
pup
2,
dam
84,
pup
4
in
Rx
24038;
dam
6
pup
1,
dam
16
pup
2
in
Rx
17725.

14.
Presence
or
absence
of
epispadias
not
recorded
for:
dam
10
pup
2,
dam
21
pup
2
in
Rx
77491;
dam
12
pup
5,
dam
41
pup
2
in
Rx
31036;
dam
3
pup
1,
dam
8
pup
3,
dam
8
pup
4,
dam
13
pup
3,
dam
23
pup
3,
dam
23
pup
5,
dam
42
pup
1,
dam
51
pup
3
in
Rx
01822;
dam
7
pup
2
dam
84
pup
4
in
Rx
24038;
dam
6
pup
1,
dam
16
pup
2,
dam
114
pup
2
in
Rx
17725.

15.
Presence
or
absence
of
cleft
phallus
not
recorded
for:
dam
10
pup
2,
dam
21
pup
2
in
Rx
77491;
dam
12
pup
5,
dam
41
pup
2
in
Rx
31036;
dam
3
pup
1,
dam
8
pup
3,
dam
8
pup
4,
dam
13
pup
3,
dam
23
pup
3,
dam
23
pup
5,
dam
42
pup
1,
dam
42
pup
2
in
Rx
01822;
dam
7
pup
2,
dam
84
pup
4
in
Rx
24038;
dam
6
pup
1,
dam
16
pup
2
in
Rx
17725.

16.
Inguinal
regions
soiled
with
urine
not
recorded
for:
dam
10
pup
2
in
Rx
77491;
dam
41
pup
2
in
Rx
31036;
dam
13
pup
2,
dam
13
pup
3,
dam
23
pup
5,
dam
42
pup
1,
dam
51
pup
3
in
Rx
01822;
dam
7
pup
2,
dam
84
pup
4
in
Rx
24038;
dam
16
pup
2,
dam
77
pup
1
in
Rx
17725.

17.
The
length
of
the
right
gubernacular
cord
was
not
recorded
because
the
cord
was
inadvertently
cut
for
dam
23
pup
2
in
Rx
01822.
The
length
of
neither
gubernacular
cord
was
not
recorded
because
both
were
inadvertently
cut
for
dam
115
pup
2
in
Rx
17725.

18.
F1
males
1266
through
1323
were
weighed
on
9/
15/
02
instead
of
9/
14/
02
as
required.
Note:
neither
body
or
feed
weights
were
done
on
9/
15/
02.

19.
Presence
or
absence
of
gross
necropsy
findings
not
recorded
for:
dam
3
pup
1,
dam
42
pup
1
in
Rx
01822;
dam
24
pup
1
in
Rx
24038;
dam
6
pup
1,
dam
15
pup
2
in
Rx
17725.

20.
Seminal
vesicles
were
lost
prior
to
weighing
for
dam
52
pup
1
in
Rx
31036.

21.
Number
of
nipples
and
number
of
areola
not
recorded
for:
dam
23
pup
5
in
Rx
01822;
dam
34
pup
3
in
Rx
24038.

22.
Levator
ani
plus
bulbocavernosus
lost
prior
to
weighing
for
dam
109
pup
1
in
Rx
24038.

23.
One
cowpers
gland
lost
prior
to
weighing
for
dam
16
pup
1
in
Rx
17725.

In
the
Study
Director's
professional
opinion,
these
deviations
did
not
affect
the
study
design,
performance,
or
interpretation
and
are
presented
for
completeness.

____________________________________
___________________

Julia
D.
George,
Ph.
D.
Date
Study
Director
080555.001.012
66
5/
5/
03
66
Table
1.
Analyses
of
Dose
Formulationsa
Test
chemical
RTI
Rx
Code
RTI
Color
Code
Battelle
Sample
Code
Sample
Typeb
Nominal
Concentration
(
mg/
ml)
Analytical
Concentration
(
mg/
ml)
c
Mean
%
of
Nominal
+
RSDd
vehicle
NA
NA
2­
10­
A
preship
0
NDe
­­

DBP
NA
NA
2­
10­
b
preship
20
18.8
94.1
±
2.52
2­
10­
c
preship
100
94.2
94.3
±
2.09
Vinclozolin
NA
NA
2­
10­
d
preship
10
9.77
97.7
±
2.28
NA
NA
2­
10­
e
preship
20
19.5
97.5
±
1.89
DBP
24038­
GD6
purple
1778­
12­
1
first
dosing
day
20
19.0
94.4
±
2.01
20438­
PND0
purple
1778­
12­
2
first
dosing
day
20
18.3
20438­
PND7
purple
1778­
12­
3
first
dosing
day
20
18.7
20438­
PND14
purple
1778­
12­
4
first
dosing
day
20
19.0
20438­
PND21
purple
1778­
12­
5
first
dosing
day
20
19.3
17725­
GD6
red
1778­
13­
1
first
dosing
day
100
93.8
94.5
±
2.16
17725­
PND0
red
1778­
13­
2
first
dosing
day
100
93.6
17725­
PND7
red
1778­
13­
3
first
dosing
day
100
97.7
17725­
PND14
red
1778­
13­
4
first
dosing
day
100
92.3
17725­
PND21
red
1778­
13­
5
first
dosing
day
100
95.0
Vinclozolin
31036­
GD6
green
1779­
86­
1
first
dosing
day
10
9.48
94.6
±
1.26
31036­
PND0
green
1779­
86­
2
first
dosing
day
10
9.27
31036­
PND7
green
1779­
86­
3
first
dosing
day
10
9.58
31036­
PND14
green
1779­
86­
4
first
dosing
day
10
9.47
31036­
PND21
green
1779­
86­
5
first
dosing
day
10
9.51
01822­
GD6
orange
1779­
87­
1
first
dosing
day
20
18.7
93.6
±
1.76
01822­
PND0
orange
1779­
87­
2
first
dosing
day
20
18.9
01822­
PND7
orange
1779­
87­
3
first
dosing
day
20
18.8
(
Continued)
080555.001.012
67
5/
5/
03
67
Test
Chemical
RTI
Rx
Code
RTI
Color
Code
Battelle
Sample
Code
Sample
Typeb
Nominal
Concentration
(
mg/
ml)
Analytical
Concentration
(
mg/
ml)
c
Mean
%
of
Nominal
+
RSDd
01822­
PND14
orange
1779­
87­
4
first
dosing
day
20
18.1
01822­
PND21
orange
1779­
87­
5
first
dosing
day
20
19.0
DBP
10303­
05­
01
NA
1778­
10­
1
postdose
20
18.8
94.3
±
0.49
10303­
05­
01
NA
1778­
10­
2
postdose
20
18.9
10303­
06­
01
NA
1778­
11­
1
postdose
100
83.5
94.1
±
1.36
10303­
06­
01
NA
1778­
11­
1
R­
1f
postdose
100
93.8
10303­
06­
01
NA
1778­
11­
1
R­
2
postdose
100
95.2
10303­
06­
01
NA
1778­
11­
1
R­
3
postdose
100
95.9
10303­
06­
01
NA
1778­
11­
2
postdose
100
93.2
Vinclozolin
10303­
07­
01
NA
1779­
84­
1
postdose
10
8.79
95.8
±
3.34
10303­
07­
01
NA
1779­
84­
1
R­
1
postdose
10
9.75
10303­
07­
01
NA
1779­
84­
1
R­
2
postdose
10
9.95
10303­
07­
01
NA
1779­
84­
1
R­
3
postdose
10
9.71
10303­
07­
01
NA
1779­
84­
2
postdose
10
9.35
10303­
08­
01
NA
1779­
85­
1
postdose
20
18.2
94.8
±
5.79
10303­
08­
01
NA
1779­
85­
2
postdose
20
17.7
10303­
08­
01
NA
1779­
85­
2
R­
1
postdose
20
19.5
10303­
08­
01
NA
1779­
85­
2
R­
2
postdose
20
19.5
10303­
08­
01
NA
1779­
85­
2
R­
3
postdose
20
20.1
(
Continued)
080555.001.012
68
5/
5/
03
68
a
Dosing
solutions
were
formulated
on
7/
9/
02
in
corn
oil
vehicle
for
administration
at
5.0
ml/
kg.
The
doses
were
therefore
0,
50,
and
100
mg/
kg/
day
vinclozolin,
and
100,
and
500
mg/
kg/
day
DBP.

b
Samples
were
taken
prior
to
shipping
from
Battelle
to
RTI
(
preship),
on
the
first
day
dosing
for
gd
6
and
pnd
7
and
14,
on
the
second
day
of
dosing
on
pnd
0
and
20,
and
after
dosing
was
completed
(
postdose).

c
n=
3
for
individual
determinations.

d
Data
are
presented
as
mean
%
(%
relative
standard
deviation).

e
ND
=
not
detected;
estimated
limit
of
detection
is
0.57
microg/
ml
for
DBP
and
0.11
microg/
ml
for
vinclozolin.

f
Analyses
labeled
"
R"
were
reanalyses
of
samples
for
which
less
than
90%
of
the
nominal
concentration
was
recovered.