Research
Triangle
Park,
North
Carolina
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
L.
Earl
Gray,
Jr.,
Ph.
D.
(
EPA)

Paul
M.
D.
Foster,
Ph.
D.
(
NIEHS)

Eric
A.
Crecelius,
Ph.
D.
(
Battelle)

Rochelle
W.
Tyl,
Ph.
D
(
RTI
International)

Julia
D.
George,
Ph.
D.
(
RTI
International)
BACKGROUND
This
study
was
performed
under
a
subcontract
to
Battelle
Laboratories,

Columbus,
Ohio
for
prime
contract
No.
68­
W­
01­
023
for
the
U.
S.
Environmental
Protection
Agency
One
of
the
tests
being
considered
for
inclusion
in
this
screening
program
is
a
mammalian,
two­
generation
reproductive
toxicity
test
EPA
OPPTS
Health
Effects
Test
Guideline
870.3800:
Reproduction
and
Fertility
Effects
(
U.
S.
EPA
1998).

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.

In
the
standard
two­
generation
test,
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.
These
animals
are
used
to
breed
the
F2
generation.

The
study
design
being
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.
OBJECTIVES
1.
Whether
some
of
the
effects
from
perinatal
exposure
to
Vinclozolin
(
VIN)
or
to
Di­
n­
butyl
phthalate
(
DBP)
that
can
be
easily
detected
after
puberty
are
missed
in
weanling
animals
of
the
F1
generation.

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.
HYPOTHESIS

The
"
Standard
2­
Generation
Protocol"
cursory
examination
of
up
to
three
F1
males
per
litter
at
weaning
and
only
one
F1
male
at
adulthood
allows
adverse
reproductive
effects
that
appear
at
and
after
puberty
to
be
missed

Examination
of
three
or
more
F1
males
at
or
after
puberty,
in
addition
to
the
F1
males
examined
at
weaning,
will
detect
additional
reproductive
effects,
and
provide
a
more
complete
and
accurate
characterization
of
the
effects
of
the
test
compound
APPROACH

Vinclozolin
(
VIN)
and
dibutyl
phthalate
(
DBP),
two
known
and
wellcharacterized
anti­
androgens,
were
used,
each
at
two
doses.


The
high
dose
of
each
compound
was
a
known
effect
level.


The
low
dose
of
VIN
was
expected
to
produce
hypospadias
and
vaginal
pouches
that
would
be
hard
to
detect
in
weanlings,
but
easier
to
detect
in
adults.


The
low
dose
of
DBP
was
the
LOAEL
(
lowest
observable
adverse
effect
level)
for
this
compound.


These
compounds
and
the
selected
doses
were
identified
by
basic
research
protocols,
and
were
used
to
test
this
hypothesis
in
rats.
Vinclozolin
(
VIN)

Chemical
Name:
3­(
3,5­
Dichlorophenyl)­
5­
ethenyl­
5­
methyl­
2,4­
oxazolidinedione
CAS
Number:
5­
0471­
44­
8
Supplier:
Chem
Services,
Inc.

Manufacturer's
Batch
No.:
270­
71B
Appearance:
colorless,
crystalline
solid
Molecular
Formula:
C
12
H
9
Cl
2
NO
3
Molecular
Weight:
286.114
Structure:

Common
use:
systemic
dicarboximide
fungicide
used
on
grapes,
other
fruit,

vegetables,
hops,
ornamental
plants
and
turf
(
Kelce
et
al.,
1997).

O
N
O
O
Cl
Cl
Endocrine­
Disrupting
Properties
of
Vinclozolin

Multigenerational
studies
in
rats
indicate
that
in
utero/
lactational
exposure
results
in
demasculinized
male
offspring
(
van
Ravenzwaay,
1992).


Gray
et
al.
(
1994)
confirmed
and
extended
these
findings
by
administering
vinclozolin
in
corn
oil
by
gavage
once
daily
on
gd
14
through
pnd
3
in
rats
at
0,
100,
or
200
mg/
kg/
day.
Male
offspring
exhibited
dose­
related
incidences
and
severities
of
male
reproductive
tract
malformations
(
see
below)
and
renal
system
malformations,
including
hydroureter,

hydronephrosis,
and
urinary
bladder
stones.


In
vitro
studies
indicate
that
the
two
vinclozolin
metabolites
bind
to
the
androgen
receptor
(
Kelce
et
al.,
1994a)
and,
acting
as
antiandrogens,
inhibit
subsequent
androgen
receptordependent
transcriptional
activation
(
Wong
et
al.,
1995).


This
mechanism
has
been
confirmed
in
vivo
(
Kelce
et
al.,
1997)
with
exposure
to
200
mg/
kg/
day
vinclozolin,
resulting
in
alteration
of
the
expression
of
androgen­
dependent
genes.


The
adverse
effects
of
vinclozolin
are
mediated
by
its
metabolites
(
Kelce
et
al.,
1994b).


The
adult
rat
is
also
responsive
to
exposure
to
vinclozolin,
but
reproductive
tract
malformations
are,
as
expected,
not
produced
(
Anderson
et
al.,
1995).

Wistar
and
Long­
Evans
rats
were
orally
dosed
with
vinclozolin
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
anogenital
distance;
retained
nipples/
areolas;
hypospadias;
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
(
Hellwig
et
al.,
2000).


At
12
mg/
kg/
day,
retained
nipples/
areolas
were
present
in
both
strains
in
preweanling
males
but
persisted
only
in
Long­
Evans
rats
(
Hellwig
et
al.,
2000).


Long­
Evans
rats
(
but
not
Wistar)
also
exhibited
a
low
incidence
of
hypoplasia
of
accessory
sex
organs.
The
NOAELs
(
no
observable
adverse
effect
level)
were
therefore
12
mg/
kg/
day
in
Wistar
rats
and
6
mg/
kg/
day
in
Long­

Evans
rats
(
there
were
no
effects
in
either
strain
at
3
or
1
mg/
kg/
day)
(
Hellwig
et
al.,
2000).


Wistar
and
Long­
Evans
rats
were
orally
dosed
with
vinclozolin
from
gd
14
to
pnd
3
at
200,
12,
6,
3,
1,
or
0
mg/
kg/
day
(
Hellwig
et
al.,
2000).
The
high
dose
(
200
mg/
kg/
day)
was
maternally
toxic,
and
male
offspring
from
both
strains
exhibited
reduced
anogenital
distance;
retained
nipples/
areolas;
hypospadias;
penile
hypoplasia;
development
of
a
vaginal
pouch;
hypoplasia
and
chronic
inflammation
of
the
epididymides,

prostate,
seminal
vesicles.


Maternal
rats
were
dosed
by
gavage
on
gd
14
through
pnd
3
with
vinclozolin
at
0,
100,
and
200
mg/
kg/
day
(
first
study),
with
expected
male
offspring
reproductive
malformations
observed
at
both
doses
(
Gray
et
al.,

1994).


In
a
second
study,
the
doses
were
0,
3.125,
6.25,
12.5,
25,
50,
and
100
mg/
kg/
day
(
Gray
et
al.,
1999).
In
male
offspring,
reduced
anogenital
distance
was
observed
at

3.125
mg/
kg/
day,
retained
areolas
were
observed
at

6.25
mg/
kg/
day,
and
ventral
prostate
weight
was
reduced
and
hypospadia
observed
at
50
mg/
kg/
day.
Ectopic
testes
were
only
observed
at
100
and
200
mg/
kg/
day.
Di­
butylphthalate
(
DBP)

CAS
Number:
84­
74­
2
Supplier:
Sigma­
Aldrich,
Inc.

Manufacturer's
Batch
No.:
080K1023
Appearance:
clear,
colorless
liquid
Molecular
Formula:
C
16
H
22
O
4
Molecular
Weight:
278.35
Chemical
Structure:

Common
use:
a
coalescing
aid
in
latex
adhesives,
as
a
plasticizer
in
cellulose
plastics,
and
as
a
solvent
in
dyes.
O
O
O
O
Dibutylphthalate
(
DBP)
cont'd.


Exposure
during
gestation
results
in
developmental
toxicity,
and
DBP
crosses
the
placenta
in
rats
(
Saillenfait
et
al.,
1998;
Ema
et
al.,
1993,
1994,
1995a,
1998).
The
intestinal
metabolite,
mono­
n­
butyl
phthalate,
also
causes
developmental
toxicity
in
rats;
this
is
most
likely
the
proximate
toxicant
(
Ema
et
al.,
1995b).


Although
DBP
acts
as
an
antiandrogen,
it
does
not
bind
to
the
androgen
receptor
(
Foster
et
al.,
2000)


DBP
apparently
acts
by
inhibiting
fetal
testicular
testosterone
biosynthesis
in
vitro
and
in
vivo
(
Mylchreest
et
al.,
1999).
In
adult
rats,
it
also
causes
testicular
toxicity
but,
as
expected,
no
malformations
(
Cater
et
al.,

1977).
Daily
oral
(
gavage)
administration
of
DBP
to
dams,
during
gestation
and
lactation
of
100
mg/
kg/
day
through
750
mg/
kg/
day,
results
in
dose­
related
reproductive
malformations
in
male
offspring,
with
approximately
75%
of
the
male
offspring
affected
at
750
mg/
kg/
day.
The
male
malformations
include
shortened
anogenital
distance,
small
flaccid
testes,
agenesis
of
portions
(
caput,
corpus,
cauda)
of
or
the
entire
epididymis,
delayed
puberty,
retained
nipples
and
areolae,
etc.
(
Gray
et
al.,
1998;
2000).


An
oral
dose
of
50
mg/
kg/
day
has
been
defined
as
the
NOAEL
by
Mylchreest
et
al.
(
1998a,
b,
1999).

Therefore,
for
this
study,
DBP
in
corn
oil
will
be
administered
by
oral
gavage
once
daily
on
gd
6
through
pnd
21
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
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
One­
Generation
Extension
Study
Design
and
Target
Doses
One­
Generation
Extension
Target
Doses
Group
No.
No.
Animals
Dosed
No.
Days
Exposure
Dosing
Period
(
gd­
pnd)
Dose
(
mg/
kg/
day)
Dosing
Concentration
(
mg/
kg/
day)
Dose
Volume
(
ml/
kg)

1
25
36­
38
6­
21
0
0
5
2
25
36­
38
6­
21
50
VIN
10
VIN
5
3
25
36­
38
6­
21
100
VIN
20
VIN
5
4
25
36­
38
6­
21
100
DBP
20
DBP
5
5
25
36­
38
6­
21
500
DBP
100
DBP
5
Summary
of
F0
Maternal
Data

Mean
periodic
maternal
body
weights
and
weight
gains

Feed
consumption
(
expressed
as
g/
animal/
day
and
g/
kg
body
weight/
day)
during
gestation
and
lactation

Survival
indices

Gestational
length

Mean
litter
size

Mean
number
of
live
and
dead
offspring

Prenatal
(
postimplantation)
loss
(%)
=


Number
and
percent
of
mothers
showing
treatment­
related
behavioral
abnormalities
in
nesting
and
nursing

Gestational
index
(%)
=


Gross
necropsy

Number
of
uterine
nidation
scars
at
necropsy
No.
pregnant
females
with
live
litters
No.
pregnant
females
x
100
No.
implantation
scars
No.
live
pups
at
birth
No.
implantation
scars
x
100
 
Summary
of
F1
Litter
Lactational
Data

Total
litter
size

Number
and
percent
of
stillborn

Number
and
percent
of
live
births

Anogenital
distance
and
body
weight
on
pnd
0
and
21

Periodic
viability
counts

Periodic
body
weights
by
sex
per
litter
from
birth
to
weaning
(
taken
on
pnd
0,
4,
7,
14,
and
21
by
individual
pup)


Sex
ratio
(%
males
per
litter)
Summary
of
F1
Litter
Lactational
Data
Cont.

Live
Birth/
Survival
Indices:

Live
birth
index:
=

4­
day
survival
index
=

7­
day
survival
index
=

14­
day
survival
index
=

21­
day
survival
index
=

Lactation
index
=

Number
of
male
pups
per
litter
with
(
and
number
per
male
of)
areolae
and/
or
nipples
on
pnd
11­
13
and
21
No
live
pupsat
birth
Total
no
pupsat
born
x
.
.
100
No
pups
surviving
days
precull
Total
no
live
pupsat
birth
x
.
(
)

.
4
100
No
pups
surviving
days
Total
no
live
pupsat
days
postcull
x
.

.
(
)

7
4
100
No
pups
surviving
days
Total
no
live
pupsat
days
x
.
.
14
7
100
No
pups
surviving
days
Total
no
live
pupsat
days
postcull
x
.

.
(
)

21
4
100
No
pups
surviving
days
Total
no
live
pupsat
days
x
.
.
21
14
100
Summary
of
Data
From
Retained
Male
F1
Offspring

Mean
periodic
body
weights
and
weight
gains

Age
and
body
weight
at
acquisition
of
preputial
separation

Organ
weights

Reproductive
system
external
and/
or
gross
abnormalities

Presence
of
areolae
and/
or
nipples
at
adult
necropsy

Anogenital
distance
at
adult
necropsy
External
and
Internal
Examination
of
F1
Males
at
Necropsy
(
pnd
21
or
95)


Each
male
selected
for
pnd
21
necropsy
or
pnd
95
adult
necropsy
was
examined:


Externally

Nipples
and
areolae
were
counted
and
position
recorded

hypospadias,
epispadias,
and
cleft
phallus

AGD
was
measured

Undescended
testes

Preputial
separation

Location
of
each
testis
(
scrotal,
abdominal,
gubernaculum
attached
to
abdominal
wall)


Gubernacular
cords,
present
or
absent
and
length
in
mm

Presence
of
cranial
suspensory
ligaments

Testes
which
were
small,
absent,
fluid
filled,
enlarged,
appeared
infected,
or
other

Epididymides
which
were
small,
absent,
or
infected
(
including
region
of
effects)


Ventral
prostate
which
was
small,
absent,
or
infected

Dorsolateral
prostate
which
was
small,
absent,
or
infected

Seminal
vesicles
which
were
small,
absent,
infected,
or
one
side
larger
than
the
other

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:


Kidneys
with
hydronephrosis
or
calcium
deposits

Hydroureter(
s)


Urinary
bladder
stones
or
blood
in
urinary
bladder
The
following
organs
were
weighed:


Each
testis
individually

Each
corpus
plus
caput
epididymides

Each
cauda
epididymides

Entire
seminal
vesicle,
plus
coagulating
glands
with
fluid
as
a
unit,
if
possible

The
prostate
ventral
and
dorsolateral
lobes
separately

Paired
adrenals

Liver

Levator
ani
plus
bulbocavernosus
muscle
complex

Cowper's
(
bulbourethral)
glands
as
a
pair

Glans
penis
(
only
if
preputial
separation
has
occurred)
Pnd
21
Necropsy
Guideline
Two­
Gen
Study
Weights:
Body
X
Brain
X
Spleen
X
Thymus
X
Liver
Adrenal
Testis
Corpus/
Caput
Epidiymis
Cauda
Epididymis
Seminal
Vesicles/
Coagulating
Gland
Prostate
LABC
Cowper's
Glands
Extended
One­
Gen
X
X
X
X
X
X
X
X
X
X
Pnd
21
Necropsy
Guideline
Two­
Gen
Study
AGD
Areolae
Nipples
Hypospadias
Epispadias
Cleft
Phallus
Soiled
Inguinal
Region
Gubernacular
Cord
a
Cranial
Suspensory
Ligaments
a
a
Present/
Absent;
Length
Extended
One­
Gen
X
X
X
X
X
X
X
X
X
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
in
preweanling
males
by
pnd
13,
based
on
retention
of
areolae
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*
*

Percent
Males
with
One
or
More
Areolae
(
postnatal
day
13)

(%)
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*

Percent
Males
with
One
or
More
Areolae
(
postnatal
day
21)

(%)
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*

Percent
Males
with
One
or
More
Areolae
(
postnatal
day
95)

(%)
Both
doses
of
VIN
and
the
high
dose
of
DBP
were
detected
as
effect
levels
from
data
on
AGD
at
birth.
This
effect
persisted
throughout
the
study
to
pnd
95.
0.0
0.5
1.0
1.5
2.0
2.5
0
mg/
kg/
day
Vinclozolin
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
0
mg/
kg/
day
Di­
n­
Butyl
Phthalate
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
Adjusted
Anogenital
Distance
(
postnatal
day
0)

(

mm)
*
*
*
*
p<
0.05
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
0
mg/
kg/
day
Vinclozolin
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
0
mg/
kg/
day
Di­
n­
Butyl
Phthalate
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0/
05
*
*
*

Adjusted
Anogenital
Distance
(
postnatal
day
21)

(

mm)
0
10
20
30
40
0
mg/
kg/
day
Vinclozolin
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
0
mg/
kg/
day
Di­
n­
Butyl
Phthalate
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
*
*

Adjusted
Anogenital
Distance
(
postnatal
day
95)

(

mm)
*
p<
0.05
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
antiandrogenic
activity
than
retained
areolae.
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*

Percent
Males
with
One
or
More
Nipples
(
postnatal
day
13)

(%)
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*

Percent
Males
with
One
or
More
Nipples
(
postnatal
day
21)

(%)
0
10
20
30
40
50
60
70
80
90
100
0
mg/
kg/
day
50
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Vinclozolin
100
mg/
kg/
day
Di­
n­
Butyl
Phthalate
500
mg/
kg/
day
Di­
n­
Butyl
Phthalate
*
p<
0.05
*
*
*

Percent
Males
with
One
or
More
Nipples
(
postnatal
day
95)

(%)
Were
male
reproductive
system
malformations
detected
at
the
pnd
21
necropsy
for
both
test
chemicals
at
both
doses?
F
1
M
a
le
O
f
fs
p
rin
g
R
e
p
r
o
d
u
c
t
iv
e
S
y
s
te
m
M
a
lf
o
rm
a
t
io
n
s
a
t
th
e
P
n
d
2
1
N
e
c
ro
p
s
y
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
k
g
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
/
d
a
y
)

P
a
ra
m
e
te
r
0
5
0
1
0
0
1
0
0
5
0
0
N
o
.
p
u
p
s
7
4
8
2
6
5
7
1
6
5
C
o
w
p
e
r
'
s
g
la
n
d
m
is
s
in
g
:

L
e
f
t
0
(
0
.0
)
a
1
(
1
.
2
)
2
(
3
.1
)
0
(
0
.
0
)
1
(
1
.
5
)

R
ig
h
t
0
(
0
.0
)
3
(
3
.
7
)
2
(
3
.1
)
0
(
0
.
0
)
0
(
0
.
0
)

B
ila
t
e
ra
l
0
(
0
.0
)
5
(
6
.
1
)
4
3
(
6
6
.2
)
1
(
1
.
4
)
6
(
9
.
2
)

T
o
ta
l
0
(
0
.0
)
9
(
1
1
.0
)
4
7
(
7
2
.3
)
1
(
1
.
4
)
7
(
1
0
.8
)

E
p
id
i
d
ym
i
s
(
m
id
e
s
)

m
is
s
in
g
,
re
d
u
c
e
d
in
s
iz
e
:

L
e
f
t
0
(
0
.0
)
0
(
0
.
0
)
1
(
1
.5
)
0
(
0
.
0
)
8
(
1
2
.
3
)

R
ig
h
t
0
(
0
.0
)
0
(
0
.
0
)
1
(
1
.5
)
0
(
0
.
0
)
8
(
1
2
.
3
)

B
ila
t
e
ra
l
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.0
)
0
(
0
.
0
)
5
(
7
.
7
)

T
o
ta
l
0
(
0
.0
)
0
(
0
.
0
)
2
(
3
.1
)
0
(
0
.
0
)
2
1
(
3
2
.
3
)

E
p
is
p
a
d
ia
s
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.
0
)

H
y
p
o
s
p
a
d
ia
s
0
(
0
.0
)
8
.0
(
9
.
7
)
5
2
(
8
0
.0
)
0
(
0
.
0
)
2
(
3
.
1
)

M
a
le
s
w
it
h
>
1
g
u
b
e
r
n
a
c
u
la
r
c
o
rd
7
4
(
1
0
0
.
0
)
8
2
(
1
0
0
.0
)
6
5
(
1
0
0
.
0
)
6
9
(
9
7
.
2
)
5
9
(
9
0
.
8
)

M
a
le
s
w
it
h
>
1
c
ra
n
ia
l
s
u
s
p
e
n
s
o
ry
lig
a
m
e
n
t
0
(
0
.0
)
1
(
1
.
3
)
0
(
0
.0
)
2
.
2
(
3
.1
)
2
.2
(
3
.
4
)

L
e
va
to
r
a
n
i
b
u
lb
o
­

c
a
v
e
rn
o
s
u
s
c
o
m
p
le
x
:

M
is
s
in
g
0
(
0
.0
)
0
(
0
.
0
)
1
(
1
.5
)
0
(
0
.
0
)
0
(
0
.
0
)

S
p
o
n
g
y
0
(
0
.0
)
0
(
0
.
0
)
1
(
1
.5
)
0
(
0
.
0
)
0
(
0
.
0
)

P
e
n
is
r
e
d
u
c
e
d
in
s
iz
e
0
(
0
.0
)
0
(
0
.
0
)
3
(
4
.6
)
0
(
0
.
0
)
0
(
0
.
0
)

P
h
a
llu
s
,
c
le
ft
0
(
0
.0
)
4
(
4
.
9
)
2
5
(
3
8
.5
)
0
(
0
.
0
)
2
(
3
.
1
)

P
ro
s
t
a
t
e
m
is
s
in
g
:

D
o
rs
a
l
0
0
2
1
(
3
2
.3
)
0
1
(
1
.
5
)

V
e
n
t
ra
l
0
0
4
(
6
.2
)
0
1
(
1
.
5
)

V
le
ft
lo
b
e
0
0
1
(
1
.5
)
0
1
(
1
.
5
)

T
o
t
a
l
0
(
0
.0
)
0
(
0
.
0
)
2
6
(
4
0
.0
)
0
3
(
4
.
6
)

S
e
m
in
a
l
v
e
s
ic
le
s
:

Missing
0
(
0.0)
0
(
0.0)
0
(
0.0)
0
4
(
6.2)

M
is
s
h
a
p
e
n
0
(
0
.0
)
0
(
0
.
0
)
7
(
1
0
.
8
)
0
3
(
4
.
6
)

T
o
t
a
l
0
(
0
.0
)
0
(
0
.
0
)
7
(
1
0
.
8
)
0
7
(
1
0
.
8
)

T
e
s
te
s
u
n
d
e
s
c
e
n
d
e
d
0
(
0
.0
)
0
(
0
.
0
)
3
(
4
.6
)
0
4
(
6
.
1
)

H
y
d
r
o
n
e
p
h
ro
s
is
:
b
L
e
f
t
0
(
0
.0
)
1
(
1
.
2
)
0
(
0
.0
)
0
0
(
0
.
0
)

R
ig
h
t
3
(
4
.0
)
4
(
4
.
9
)
0
(
0
.0
)
2
(
2
.
8
)
6
(
9
.
2
)

B
ila
t
e
ra
l
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.0
)
1
(
1
.
4
)
0
(
0
.
0
)

T
o
t
a
l
3
(
4
.0
)
5
(
6
.
1
)
0
(
0
.0
)
3
(
4
.
2
)
6
(
9
.
2
)

a
N
um
b
e
r
(
a
n
d
%
)
w
i
th
th
e
in
d
ic
a
te
d
fin
d
in
g
.
A
m
a
le
m
a
y
b
e
c
o
u
n
te
d
m
o
re
th
a
n
o
n
c
e
i
f
h
e
e
xh
ib
ite
d
m
o
re
th
a
n
o
n
e
m
a
lfo
rm
a
tion
.

b
T
h
e
in
cid
e
n
c
e
o
f
h
yd
r
o
n
e
p
h
ro
s
is
,
a
c
om
m
o
n
fin
d
in
g
in
m
a
le
C
D
®
(
S
p
r
a
g
u
e
­
D
a
w
le
y)
r
a
ts
is
p
ro
vid
e
d
fo
r
in
te
rn
a
l
q
u
a
li
ty
c
o
n
tro
l
.
T
h
e
re
w
a
s
n
o
c
h
em
ic
a
l­
o
r
d
o
s
e
­
re
la
te
d
in
c
id
e
n
ce
.
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).
Were
male
reproductive
system
malformations
detected
at
pnd
95
for
both
test
chemicals
at
both
doses?
a
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
kg
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
/
d
a
y
)

P
a
ram
e
te
r
0
5
0
1
0
0
1
0
0
5
0
0
No.
males
82
95
74
81
74
C
o
w
p
e
r's
g
la
n
d
s
:

M
is
s
in
g
0
(
0
.0
)
6
(
6
.3
2
)
5
6
(
7
5
.
7
)
0
(
0
.
0
)
5
(
6
.8
)

R
e
d
u
c
e
d
in
s
iz
e
0
(
0
.0
)
1
(
1
.0
)
8
(
1
0
.
8
)
0
(
0
.
0
)
3
(
4
.0
)

E
p
id
id
ym
is
m
is
s
in
g
0
(
0
.
0
)
0
(
0
.0
)
4
(
5
.
4
)
0
(
0
.
0
)
3
3
(
4
4
.6
)

R
e
d
u
c
e
d
in
s
iz
e
1
(
1
.2
)
0
(
0
.0
)
1
2
(
1
6
.
2
)
0
(
0
.
0
)
5
2
(
7
0
.
3
)

E
p
is
p
a
d
ia
s
0
(
0
.0
)
4
(
4
.3
)
1
1
(
1
4
.
9
)
0
(
0
.
0
)
0
(
0
.0
)

G
la
n
s
p
e
n
is
n
o
t
c
om
p
le
t
e
ly
d
e
ta
c
h
e
d
b
0
(
0
.0
)
3
(
3
.2
)
2
0
(
2
7
.
0
)
0
(
0
.
0
)
7
(
9
.5
)

H
yp
o
s
p
a
d
ia
s
0
(
0
.0
)
1
5
(
1
5
.8
)
7
3
(
9
8
.
6
)
0
(
0
.
0
)
1
2
(
1
6
.
2
)

L
A
B
C
:
b
M
is
s
in
g
0
(
0
.
0
)
0
(
0
.0
)
2
(
2
.
7
)
0
(
0
.
0
)
0
(
0
.0
)

R
e
d
u
c
e
d
in
s
iz
e
0
(
0
.0
)
2
(
2
.1
)
3
8
(
5
1
.
4
)
0
(
0
.
0
)
4
(
5
.4
)

M
a
l
fo
rm
e
d
0
(
0
.
0
)
1
(
1
.0
)
5
(
6
.
7
)
0
(
0
.
0
)
0
(
0
.0
)

M
ale
s
w
ith
>
1
g
u
b
e
r
n
a
c
u
la
r
c
o
rd
5
.5
(
6
.2
)
1
1
(
1
1
.6
)
1
3
(
1
7
.
6
)
1
.0
(
1
.2
)
6
(
8
.1
)

M
ale
s
w
ith
>
1
c
ra
n
ia
l
s
u
s
p
e
n
s
o
ry
lig
a
m
e
n
t
0
(
0
.
0
)
0
(
0
.0
)
1
.0
(
1
.
4
)
0
(
0
.
0
)
6
.2
(
8
.
4
)

P
h
a
llu
s
,
c
le
ft
1
(
1
.2
0
)
4
1
(
4
3
.2
)
7
4
(
1
0
0
.0
)
2
.0
(
2
.5
)
2
6
(
3
5
.
1
)

P
re
p
u
c
e
p
a
rt
ia
l
ly
o
r
f
u
l
ly
d
e
ta
c
h
e
d
8
1
(
9
8
.
8
)
9
4
(
9
9
.0
)
7
4
(
1
0
0
.0
)
8
1
(
1
0
0
.0
)
7
4
(
1
0
0
.0
)

P
re
p
u
tia
l
g
la
n
d
s
,
p
u
s
fi
l
le
d
b
0
(
0
.
0
)
1
(
1
.2
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.0
)

P
ro
s
ta
te
,
d
o
rs
a
l:

M
is
s
in
g
0
(
0
.0
)
0
(
0
.0
)
1
7
(
2
3
.
0
)
0
(
0
.
0
)
3
(
4
.0
)

R
e
d
u
c
e
d
in
s
ize
0
(
0
.0
)
2
(
2
.4
)
2
0
(
2
7
.
0
)
2
(
2
.
5
)
7
(
9
.5
)

A
b
n
o
rm
a
l
/
in
f
e
c
t
e
d
0
(
0
.
0
)
2
(
2
.4
)
3
(
4
.
0
)
0
(
0
.
0
)
1
(
1
.4
)

(
C
o
n
tin
u
e
d
)

T
e
x
t
T
a
b
le
4
.
F
1
M
a
le
O
f
fs
p
rin
g
R
e
p
r
o
d
u
ct
iv
e
S
ys
te
m
M
a
lf
o
rm
a
t
io
n
s
a
t
th
e
P
n
d
9
5
N
e
c
ro
p
s
y
Control
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.

Text
Table
4
(
continued)
Vehicle
(
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.
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.
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.
Text
Table
5.
Gross
Findings
at
Unscheduled
Necropsy
a
Vehicle
Control
(
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.

mg/
kg/
day)
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?
T
e
x
t
T
a
b
le
6
.
C
o
m
p
a
ris
o
n
o
f
t
h
e
I
n
c
id
e
n
c
e
o
f
M
a
le
R
e
p
r
o
d
u
c
t
iv
e
S
y
s
te
m
M
a
l
f
o
rm
a
t
io
n
s
o
n
p
n
d
2
1
V
e
rs
u
s
p
n
d
9
5
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
k
g
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
/
d
a
y
)

P
a
ra
m
e
t
e
r
0
5
0
1
0
0
1
0
0
5
0
0
N
o
.
m
a
le
s
,
p
n
d
2
1
7
4
8
2
6
5
7
1
6
5
N
o
.
m
a
le
s
,
p
n
d
9
5
8
2
9
5
7
4
8
1
7
4
C
o
w
p
e
r
'
s
g
la
n
d
m
is
s
in
g
/

re
d
u
c
e
d
in
s
iz
e
:

P
n
d
2
1
0
(
0
.
0
)
a
9
(
1
1
.0
)
4
7
(
7
2
.
3
)
1
(
1
.
4
)
7
(
1
0
.
8
)

P
n
d
9
5
1
(
1
.
2
)
7
(
7
.
4
)
6
3
(
8
5
.
1
)
0
(
0
.
0
)
8
(
1
0
.
8
)

E
p
id
i
d
y
m
i
d
e
s
m
is
s
i
n
g
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
2
(
3
.
1
)
0
(
0
.
0
)
1
4
(
2
1
.
5
)

P
n
d
9
5
0
(
0
.
0
)
0
(
0
.
0
)
4
(
5
.
4
)
0
(
0
.
0
)
3
3
(
4
4
.
6
)

E
p
id
id
y
m
id
e
s
r
e
d
u
c
e
d
in
s
iz
e
/
a
b
n
o
r
m
a
l
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
4
(
6
.
2
)

P
n
d
9
5
1
(
1
.
2
)
0
(
0
.
0
)
1
9
(
2
5
.
7
)
0
(
0
.
0
)
5
2
(
7
1
.
6
)

E
p
is
p
a
d
ia
s
:

P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
0
(
0
.
0
)
4
(
4
.
3
)
1
1
(
1
4
.
9
)
0
(
0
.
0
)
0
(
0
.
0
)

H
y
p
o
s
p
a
d
ia
s
:

P
n
d
2
1
0
(
0
.
0
)
8
.0
(
9
.
7
)
5
2
(
8
0
.
0
)
0
(
0
.
0
)
2
(
3
.
1
)

P
n
d
9
5
0
(
0
.
0
)
1
5
(
1
5
.
8
)
7
3
(
9
8
.
6
)
0
(
0
.
0
)
1
2
(
1
6
.
2
)

L
A
B
C
m
is
s
in
g
/
re
d
u
c
e
d
in
s
iz
e
:
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
1
(
1
.
5
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
0
(
0
.
0
)
2
(
2
.
1
)
4
0
(
5
4
.
0
)
0
(
0
.
0
)
4
(
5
.
4
)

P
a
ra
m
e
t
e
r
0
5
0
1
0
0
1
0
0
5
0
0
P
h
a
llu
s
,
c
le
ft
:

P
n
d
2
1
0
(
0
.
0
)
4
(
4
.
9
)
2
5
(
3
8
.
5
)
0
(
0
.
0
)
2
(
3
.
1
)

P
n
d
9
5
1
(
1
.
2
)
4
1
(
4
3
.1
5
)
7
4
(
1
0
0
.0
)
2
.0
(
2
.
4
7
)
2
6
(
3
5
.
1
4
)

P
ro
s
t
a
t
e
d
o
rs
a
l
lo
b
e
m
is
s
in
g
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
2
1
(
3
2
.
3
)
0
(
0
.
0
)
1
(
1
.
5
)

P
n
d
9
5
0
(
0
.
0
)
0
(
0
.
0
)
1
7
(
2
3
.
0
)
0
(
0
.
0
)
3
(
4
.
0
)

P
ro
s
t
a
t
e
d
o
rs
a
l
lo
b
e
re
d
u
c
e
d
in
s
iz
e
/
a
b
n
o
r
m
a
l
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
0
(
0
.
0
)
3
(
3
.
2
)
2
0
(
2
7
.
0
)
2
(
2
.
5
)
8
(
1
0
.
8
)

P
ro
s
t
a
t
e
v
e
n
t
ra
l
lo
b
e
m
is
s
in
g
P
n
d
2
1
0
(
0
.
0
)
0
(
0
.
0
)
5
(
7
.
7
)
0
(
0
.
0
)
2
(
3
.
1
)

P
n
d
9
5
0
(
0
.
0
)
0
(
0
.
0
)
1
2
(
1
6
.
2
)
0
(
0
.
0
)
3
(
4
.
0
)

(
C
o
n
tin
u
e
d
)
T
e
x
t
T
a
b
le
6
(
c
o
n
tin
u
e
d
)
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
k
g
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
kg
/
d
a
y
)

P
a
ra
m
e
te
r
0
5
0
1
0
0
1
0
0
5
0
0
P
ro
s
ta
te
v
e
n
t
ra
l
lo
b
e
re
d
u
c
e
d
in
s
iz
e
/
a
b
n
o
r
m
a
l
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
1
(
1
.2
)
7
(
7
.4
)
4
5
(
6
0
.
8
)
2
(
2
.5
)
8
(
1
0
.8
)

S
e
m
in
a
l
v
e
s
ic
le
s
m
is
s
in
g
/

re
d
u
c
e
d
in
s
iz
e
/
a
b
n
o
r
m
a
l
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
8
(
1
2
.
3
)
0
(
0
.0
)
7
(
1
0
.8
)

P
n
d
9
5
0
(
0
.0
)
5
(
6
.1
)
6
3
(
8
5
.
1
)
0
(
0
.0
)
3
9
(
5
2
.
7
)

T
e
s
te
s
u
n
d
e
s
c
e
n
d
e
d
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
3
(
4
.6
)
0
(
0
.
0
)
4
(
6
.
1
)

P
n
d
9
5
0
(
0
.0
)
1
(
1
.0
)
1
5
(
2
0
.
3
)
0
(
0
.0
)
1
0
(
1
3
.
3
)

T
e
s
te
s
re
d
u
c
e
d
in
s
iz
e
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
1
(
1
.2
)
1
(
1
.0
)
1
7
(
2
3
.
0
)
0
(
0
.0
)
4
5
(
6
0
.
8
)

T
e
s
te
s
f
la
c
c
id
/
s
o
ft
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
0
(
0
.0
)
0
(
0
.0
)
1
(
1
.4
)
0
(
0
.0
)
3
2
(
4
3
.
2
)

M
a
le
s
w
ith
>
1
g
u
b
e
rn
u
c
le
a
r
w
t.
b
P
n
d
2
1
7
4
(
1
0
0
.
0
)
8
2
(
1
0
0
.
0
)
6
5
(
1
0
0
.
0
)
6
9
(
9
7
.2
)
5
9
(
9
0
.
8
)

P
n
d
9
5
5
.
5
(
6
.2
)
1
1
(
1
1
.6
)
1
3
(
1
7
.
6
)
1
.
0
(
1
.2
)
6
(
8
.1
)

M
a
le
s
w
ith
>
1
c
ra
n
ia
l
s
u
s
p
e
n
s
o
ry
lig
a
m
e
n
t
b
P
n
d
2
1
0
(
0
.0
)
1
(
1
.
3
2
)
0
(
0
.0
)
2
.2
(
3
.1
)
2
.2
(
3
.4
)

P
n
d
9
5
0
(
0
.0
)
0
(
0
.0
)
1
(
1
.4
)
0
(
0
.
0
)
6
.
2
(
8
.
1
)

V
a
g
in
a
l
p
o
u
c
h
P
n
d
2
1
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.0
)
0
(
0
.
0
)
0
(
0
.
0
)

P
n
d
9
5
0
(
0
.0
)
2
(
2
.4
)
4
3
(
5
8
.
1
)
0
(
0
.0
)
1
(
1
.4
)

a
D
a
ta
p
re
s
e
n
te
d
a
s
n
u
m
b
e
r
(%
)
o
f
m
a
l
e
s
e
x
h
ib
i
t
i
n
g
th
e
f
i
n
d
i
n
g
.
M
a
l
e
s
m
a
y
b
e
c
o
u
n
te
d
m
o
r
e
th
a
n
o
n
c
e
i
f
th
e
y
e
xh
ib
ite
d
m
o
re
tha
n
o
n
e
fin
d
in
g
.

b
T
h
e
in
cid
e
nc
e
s
o
f
th
e
s
e
fin
din
g
s
a
r
e
tak
e
n
f
rom
S
um
m
a
r
y
T
a
b
le
1
3
(
an
d
T
e
xt
T
a
b
le
3
)
fo
r
p
n
d
2
1
,
a
n
d
from
S
um
m
a
r
y
T
a
b
l
e
2
0
(
a
n
d
T
e
xt
T
a
b
l
e
4
)
fo
r
p
n
d
9
5
.
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:

°
epididimides
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
phallus
(
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)

°
vaginal
pouch
(
not
detected
in
any
group
on
pnd
21)
Were
there
differences
in
the
effects
on
weights
of
various
male
reproductive
system
organs
between
the
two
chemicals
at
the
same
time
point?

VIN:
Seminal
vesicle
and
coagulating
gland
prostate
LABC
Cowper's
gland
DBP:
testes
epididymides
Were
there
differences
in
the
effects
on
weights
of
various
male
reproductive
system
organs
within
each
chemical
at
the
two
different
time
points?
C
o
m
p
a
ris
o
n
o
f
I
n
c
id
e
n
c
e
s
o
f
M
a
le
R
e
p
ro
d
u
c
tiv
e
S
y
s
te
m
M
a
l
fo
rm
a
t
io
n
s
o
n
P
n
d
2
1
a
n
d
P
n
d
9
5
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
k
g
/

d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
P
a
ra
m
e
t
e
r
0
5
0
1
0
0
1
0
0
P
n
d
2
1
a
N
o
.
m
a
le
s
e
x
a
m
in
e
d
7
4
8
2
6
5
7
1
N
o
.
l
i
tt
e
rs
e
x
a
m
in
e
d
2
3
2
5
2
2
2
3
P
n
d
9
5
b
N
o
.
m
a
le
s
e
x
a
m
in
e
d
8
2
9
5
7
4
8
1
N
o
.
l
i
tt
e
rs
e
x
a
m
in
e
d
2
3
2
5
2
2
2
3
I
n
c
id
e
n
c
e
o
f
to
ta
l
n
o
.
o
f
m
a
lfo
rm
a
tio
n
s
b
a
s
e
d
o
n
n
o
.
F
1
m
a
le
s
e
x
a
m
in
e
d
P
n
d
2
1
c
,
d
0
(
0
.0
)
2
1
(
2
5
.
6
)
1
6
9
(
2
6
0
.0
)
1
(
1
.
4
)
4
3
P
n
d
9
5
c
,
e
8
(
9
.8
)
f
9
5
(
1
0
0
.0
)
5
3
2
(
7
1
8
.9
)
1
1
(
1
3
.
6
)
2
3
7
T
o
ta
l
n
u
m
b
e
r
o
f
F
1
m
a
le
s
w
ith
>
1
m
a
lfo
rm
a
tio
n
s
P
n
d
2
1
c
,
d
,
g
0
(
0
.0
)
9
(
1
1
.
0
)
5
4
(
8
3
.
1
)
1
(
1
.
4
)
2
9
P
n
d
9
5
c
,
e
7
(
8
.5
)
f
4
7
(
4
9
.
5
)
7
4
(
1
0
0
.
0
)
7
(
8
.
6
)
6
8
T
o
t
a
l
n
o
.
o
f
F
1
l
i
t
t
e
r
s
w
i
th
>
1
m
a
le
w
ith
>
1
m
a
lfo
rm
a
tio
n
P
n
d
2
1
c
,
d
,
g
0
(
0
.0
)
7
(
2
8
.
0
)
2
1
(
9
5
.
5
)
1
(
4
.
4
)
2
1
P
n
d
9
5
c
,
e
7
(
3
0
.
4
)
f
2
1
(
8
4
.
0
)
2
2
(
1
0
0
.
0
)
7
(
3
0
.4
)
2
2
N
o
.
m
a
lfo
rm
e
d
m
a
le
s
p
e
r
a
f
fe
c
te
d
l
i
t
t
e
r
s
P
n
d
2
1
c
,
d
,
g
0
.0
0
1
.2
9
2
.
5
7
1
.
0
0
P
n
d
9
5
1
.0
0
f
2
.2
4
3
.
3
6
1
.
0
0
a
T
h
e
n
u
m
b
e
r
o
f
F
1
m
a
le
w
e
a
n
l
in
g
s
/
l
i
tte
r
e
xam
in
e
d
in
th
is
p
r
o
to
c
o
l
v
e
rs
u
s
th
e
n
um
b
e
rs
from
th
e
1
9
9
8
O
P
P
T
S
te
s
tin
g
g
u
id
e
l
in
e
a
re
a
p
p
r
o
xim
a
te
ly
e
q
u
iva
le
n
t
(
3
.2
2
­
3
.5
4
vs
.

3
)
.

b
T
h
e
O
P
P
T
S
te
s
tin
g
g
u
id
e
l
in
e
s
p
e
c
i
fi
e
s
o
n
e
m
a
le
/
l
i
t
te
r
r
e
ta
i
n
e
d
to
a
d
u
l
th
o
o
d
.

T
h
e
r
e
fo
r
e
,
th
is
p
r
o
to
c
o
l
p
ro
v
i
d
e
s
a
t
l
e
a
s
t
th
r
e
e
t
im
e
s
th
e
s
e
n
s
i
ti
v
i
ty
to
d
e
te
c
t
a
d
u
l
t
m
a
le
m
a
l
fo
rm
a
t
io
n
s
(
3
.2
2
­
3
.8
0
v
s
.
1
)
.

c
D
a
ta
p
r
e
s
e
n
te
d
a
s
n
u
m
b
e
r
(%
)
.

d
P
n
d
2
1
d
a
ta
ta
k
e
n
f
r
o
m
S
u
m
m
a
r
y
T
a
b
l
e
1
4
a
n
d
In
d
i
vi
d
u
a
l
A
n
im
a
l
T
a
b
l
e
s
A
­
1
5
a
n
d
A
­
1
7
.

e
P
n
d
9
5
d
a
ta
ta
k
e
n
f
r
o
m
S
u
m
m
a
r
y
T
a
b
l
e
2
1
a
n
d
In
d
i
vi
d
u
a
l
A
n
im
a
l
T
a
b
l
e
s
A
­
2
6
a
n
d
A
­
2
8
.

f
P
re
d
o
m
i
n
a
n
t
ly
m
in
o
r
e
ffe
c
ts
(
e
.
g
.,
e
p
id
id
ym
is
a
n
d
te
s
ti
s
r
e
d
u
c
e
d
i
n
s
i
ze
,
p
r
o
s
ta
te
a
b
n
o
rm
a
l
[
h
a
rd
a
n
d
b
ro
w
n
],
e
tc
.)
.

g
P
re
d
om
in
a
n
tly
m
is
s
in
g
C
o
w
p
e
r
'
s
g
la
n
d
(
s
)
.
a
V
e
h
ic
le
C
o
n
t
ro
l
(
m
g
/
k
g
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
/
d
a
y
)

P
a
ra
m
e
t
e
r
0
5
0
1
0
0
1
0
0
5
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Text
Table
8
(
continued)

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

,


,



,
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).


,
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.
In
addition
to
addressing
the
specific
objectives
stated
above,

the
data
in
this
study
invite
comparison
of
the
effects
of
these
antiandrogens
that
work
through
different
mechanisms
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
#
##
 
#
#

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
Would
effects
observed
on
pnd
95
have
been
observed
if
we
only
examined
one
adult
male
per
litter
in
each
group?
CONCLUSIONS
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
dose
VIN).
CONCLUSIONS
(
CONTINUED)

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.
ACKNOWLEDGEMENTS
This
study
was
performed
under
a
subcontract
to
Battelle
Laboratories,
Columbus,
Ohio
for
prime
contract
No.
68­
W­
01­
023
for
the
U.
S.
Environmental
Protection
Agency
Laboratory
Staff
(
RTI
International)


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

Ms.
D.
B.
Bynum
Chemistry

Dr.
E.
A.
Crecelius,
PNNL,
Battelle
Marine
Sciences
Laboratory,
Sequim,
WA.


Mr.
M.
M.
Veselica,
Supervisor,
RTI
Materials
Handling
Facility

Mr.
D.
L.
Hubbard

Mr.
R.
A.
Price
Animal
Care
(
RTI
International)


Dr.
D.
B.
Feldman,
DVM,
ACLAM,

Veterinarian

Mr.
F.
N.
Ali,
Manager
of
RTI
Animal
Research
Facility
Quality
Assurance

Ms.
D.
J.
Smith
(
RTI)


Ms.
M.
D.
Phillips
(
RTI)


Ms.
T.
M.
Kenney
(
RTI)


Ms.
C.
Sexsmith,
QA
Consultant

Ms.
K.
Cummings
(
ILS)


Ms.
T.
Pollock
(
Battelle)
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Long­
Evans
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(
1994a).
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vitro/
in
vivo
evidence
that
vinclozolin
(
V)
is
an
environmental
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Suppl.
1),
189.

Kelce,
W
.
R.
E.
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P.
Gamcik,
S.
C.
Laws,
and
L.
E.
Gray,
Jr.
(
1994b).
Environmental
hormone
disruptors:
evidence
that
vinclozolin
developmental
toxicity
is
mediated
by
antiandrogenic
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Appl.
Pharmacol.
126,
275­
285.

Kelce,
W.
R.,
C.
Lambright,
L.
E.
Gray,
Jr.,
and
K.
Roberts
(
1997).
Vinclozolin
and
pp'­
DDE
alter
androgen­
dependent
gene
expression:
in
vivo
confirmation
of
an
androgen
receptor
mediated
mechanism.

Toxicol.
Appl.
Pharmacol.
142,
192­
200.
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E.,
R.
C.
Cattley,
M.
Sar,
and
P.
M.
Foster
(
1998a).
The
effects
of
di(
n­
butyl)
phthalate
on
prenatal
androgen­
regulated
male
sexual
differentiation
are
not
mediated
by
direct
interaction
with
the
androgen
receptor.
Teratology
57(
4/
5),
199
(
Abstract
No.
25).

Mylchreest,
E.,
R.
C.
Cattley,
and
P.
M.
Foster
(
1998b).
Male
reproductive
tract
malformations
in
rats
following
gestational
and
lactational
exposure
to
di(
n­
butyl)
phthalate:
an
antiandrogen
mechanism?
Toxicol.
Sci.
43(
1),

47­
60.
Mylchreest,
E.,
M.
Sar,
R.
C.
Cattley,
and
P.
M.
Foster
(
1999).
Disruption
of
androgen­
regulated
male
reproductive
development
by
di(
n­
butyl)
phthalate
during
late
gestation
in
rats
is
different
from
flutamide.

Toxicol.
Appl.
Pharmacol.
156,
81­
95.

Saillenfait,
A.
M.,
J.
P.
Payan,
J.
P.
Fabry,
D.
Beydon,
I.
Langonne,
F.
Gallissot,
and
J.
P.
Sabate
(
1998).

Assessment
of
the
developmental
toxicity,
metabolism,
and
placental
transfer
of
di­
n­
butyl
phthalate
administered
to
pregnant
rats.
Toxicol.
Sci.
45,
212­
224.

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S.
EPA
(
1998).
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S.
Environmental
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Pesticides
and
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Guidelines,
OPPTS
870.3700,
Prenatal
Developmental
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1998.

van
Ravenzwaay,
B.
(
1992).
Discussion
of
prenatal
and
reproductive
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Reg.
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83­
258
(
vinclozolin).

Data
submission
to
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from
BASF
Corporation,
MRID
425813­
02.

Wong,
C.­
I.,
W.
R.
Kelce,
M.
Sar,
and
E.
M.
Wilson
(
1995).
Androgen
receptor
antagonist
versus
agonist
activities
of
the
fungicide
vinclozolin
relative
to
hydroxyflutamide.
J.
Biol.
Chem.
270,
19998­
20003.5.
T
e
x
t
T
a
b
le
7
.
E
v
a
lu
a
ti
o
n
o
f
D
e
te
c
tio
n
o
f
M
a
le
R
e
p
ro
d
u
c
tiv
e
S
y
s
te
m
M
a
lfo
rm
a
ti
o
n
s
in
th
is
P
r
o
to
c
o
l
V
e
rs
u
s
in
th
e
O
P
P
T
S
T
e
s
tin
g
G
u
id
e
li
n
e
s
V
e
h
ic
le
C
o
n
tr
o
l
(
m
g
/
k
g
/
d
a
y
)
V
IN
(
m
g
/
k
g
/
d
a
y
)
D
B
P
(
m
g
/
k
g
/
d
a
y
)

P
a
r
a
m
e
te
r
0
5
0
1
0
0
1
0
0
5
0
0
P
n
d
2
1
a
N
o
.
m
a
le
s
e
x
a
m
in
e
d
7
4
8
2
6
5
7
1
6
5
N
o
.
l
i
t
t
e
r
s
e
x
a
m
in
e
d
2
3
2
5
2
2
2
3
2
3
M
e
a
n
n
u
m
b
e
r
o
f
F
1
m
a
l
e
o
f
f
s
p
r
i
n
g
e
x
a
m
in
e
d
p
e
r
l
i
tte
r
3
.2
2
3
.2
8
3
.3
8
3
.2
4
3
.5
4
P
n
d
9
5
b
N
o
.
m
a
le
s
e
x
a
m
in
e
d
8
2
9
5
7
4
8
1
7
4
N
o
.
l
i
t
t
e
r
s
e
x
a
m
in
e
d
2
3
2
5
2
2
2
3
2
3
M
e
a
n
n
u
m
b
e
r
o
f
F
1
m
a
l
e
o
f
f
s
p
r
i
n
g
e
x
a
m
in
e
d
p
e
r
l
i
tte
r
3
.5
6
3
.8
0
3
.3
6
3
.5
2
3
.2
2
In
c
id
e
n
c
e
o
f
to
ta
l
n
o
.
o
f
m
a
lfo
rm
a
tio
n
s
b
a
s
e
d
o
n
n
o
.
F
1
m
a
l
e
s
e
x
a
m
i
n
e
d
P
n
d
2
1
c
,
d
0
(
0
.0
)
2
1
(
2
5
6
)
1
6
9
(
2
6
0
.0
)
1
(
1
.4
)
4
3
(
6
6
.2
)

P
n
d
9
5
c
,
e
8
(
9
.8
)
f
9
5
(
1
0
0
.0
)
5
3
2
(
7
1
8
.9
)
1
1
(
1
3
.6
)
2
3
7
(
3
2
0
.3
)

T
o
ta
l
n
u
m
b
e
r
o
f
F
1
m
a
le
s
w
i
th
>
1
m
a
lfo
rm
a
tio
n
o
n
p
n
d
9
5
c
7
(
8
.5
)
f
4
7
(
4
9
.5
)
7
4
(
1
0
0
.0
)
7
(
8
.6
)
6
8
(
9
1
.9
)

T
o
t
a
l
n
o
.
o
f
F
1
l
it
t
e
r
s
w
i
t
h
>
1
m
a
le
w
i
t
h
>
1
m
a
lfo
rm
a
tio
n
s
o
n
p
n
d
9
5
c
7
(
3
0
.4
)
f
2
1
(
8
4
.0
)
2
2
(
1
0
0
.0
)
7
(
3
0
.4
)
2
2
(
9
5
.7
)

N
o
.
m
a
l
fo
rm
e
d
m
a
le
s
p
e
r
a
ffe
c
te
d
l
itte
r
s
o
n
p
n
d
9
5
1
.0
f
2
.2
4
3
.3
6
1
.0
3
.0
9
a
T
h
e
n
u
m
b
e
r
o
f
F
1
m
a
le
w
e
a
n
l
in
g
s
/
l
i
tte
r
e
x
a
m
in
e
d
i
n
th
i
s
p
r
o
to
c
o
l
v
e
rs
u
s
th
e
n
u
m
b
e
rs
fr
o
m
th
e
1
9
9
8
O
P
P
T
S
te
s
t
in
g
g
u
i
d
e
l
in
e
a
r
e
a
p
p
r
o
x
im
a
te
ly
e
q
u
i
v
a
le
n
t.

b
T
h
e
O
P
P
T
S
t
e
s
t
in
g
g
u
id
e
l
in
e
s
p
e
c
i
f
i
e
s
o
n
e
m
a
le
/
l
i
t
t
e
r
r
e
t
a
i
n
e
d
t
o
a
d
u
l
t
h
o
o
d
.
T
h
e
r
e
f
o
r
e
,
t
h
is
p
r
o
t
o
c
o
l
p
r
o
v
i
d
e
s
a
t
l
e
a
s
t
t
h
r
e
e
t
i
m
e
s
t
h
e
p
o
w
e
r
t
o
d
e
t
e
c
t
a
d
u
l
t
m
a
le
m
a
l
f
o
r
m
a
t
io
n
s
.

c
D
a
ta
p
r
e
s
e
n
te
d
a
s
n
u
m
b
e
r
(%
)
.

d
P
n
d
2
1
d
a
ta
ta
k
e
n
fro
m
S
u
m
m
a
r
y
T
a
b
l
e
1
4
a
n
d
In
d
iv
id
u
a
l
A
n
im
a
l
T
a
b
le
s
A
­
1
5
a
n
d
A
­
1
7
.

e
P
n
d
9
5
d
a
ta
ta
k
e
n
fro
m
S
u
m
m
a
r
y
T
a
b
l
e
2
1
a
n
d
In
d
iv
id
u
a
l
A
n
im
a
l
T
a
b
le
s
A
­
2
6
a
n
d
A
­
2
8
.

f
P
re
d
o
m
i
n
a
n
t
l
y
m
i
n
o
r
e
f
fe
c
ts
(
e
.
g
.
,
e
p
id
id
y
m
i
s
a
n
d
te
s
t
i
s
r
e
d
u
c
e
d
i
n
s
i
z
e
,
p
r
o
s
ta
te
a
b
n
o
rm
a
l
[
h
a
r
d
a
n
d
b
ro
w
n
],
e
tc
.)
