Proposed
OPPTS
Science
Policy:
PPAR ­
Mediated
Hepatocarcinogenesis
in
Rodents
and
Relevance
to
Human
Health
Risk
Assessments
November
5,
2003
Office
of
Prevention,
Pesticides
&
Toxic
Substances
U.
S.
Environmental
Protection
Agency
Washington,
D.
C.
20460
Page
2
of
39
PREFACE
The
U.
S.
Environmental
Protection
Agency
(
EPA)
uses
two
assumptions
concerning
the
assessment
of
laboratory
animal
tumors
(
USEPA,
1999;
2003).
The
first
is
that
the
tumor
findings
in
the
experimental
animals
are
relevant
to
the
assessment
of
potential
cancer
hazards
and
risks
in
humans.
The
second
is
that,
if
the
animal
evidence
is
sufficient
to
support
a
conclusion
that
a
carcinogenic
response
has
occurred
in
the
test
species,
and
the
mode
of
action
(
mechanistic)
information
on
the
way(
s)
a
chemical
may
induce
tumors
either
is
absent
or
fails
to
support
a
non­
linear
dose
response,
a
linear
dose­
response
extrapolation
is
used
to
estimate
risks
at
environmental
exposure
levels.
Each
of
these
assumptions
is
rebuttable
in
the
face
of
convincing
scientific
information.

EPA
has
issued
general
guidance
on
the
use
of
animal
and
other
data
to
assess
the
human
carcinogenic
potential
of
environmental
agents
(
USEPA,
1986)
and
has
proposed
updates
to
that
guidance
more
recently
(
USEPA,
1996;
USEPA,
1999;
USEPA,
2003).
To
date,
EPA
has
also
developed
science
policies
on
the
interpretation
for
three
specific
animal
tumor
responses.
These
science
policies
addressed
proliferative
lesions
in
the
rat
liver
(
Rinde
et
al.
1987),
male
rat­
limited
kidney
tumors
associated
with
accumulation
of
alpha
2u­
globulin
(
USEPA,
1991),
and
thyroid
tumors
resulting
from
disruption
of
thyroid­
pituitary
homeostasis
(
USEPA,
1997).
Accumulated
information
on
rodent
liver
tumors
that
are
induced
through
the
activation
of
the
peroxisome
proliferator­
activated
receptor
 
(
PPAR )
led
the
International
Life
Sciences
Institute
Risk
Science
Institute
(
ILSI
RSI)
to
establish
an
expert
working
group
to
update
the
state
of
the
science
on
PPAR 
agonist­
induced
carcinogenesis
in
rodents
and
the
human
relevance
of
such
animal
tumors
(
ILSI,
in
press).
The
report
of
this
ILSI
RSI
workgroup
provides
the
current
scientific
understanding
of
the
mode(
s)
of
action
of
three
of
the
PPAR 
agonist­
induced
tumors
observed
in
rodent
bioassays;
liver
tumors
in
rats
and
mice
and
Leydig
cell
and
pancreatic
acinar
cell
tumors
in
rats.

There
are
a
number
of
pesticides
and
industrial
chemicals
that
increase
peroxisomes
(
number
and
size)
and
liver
tumors
in
rodents
via
activation
of
PPAR .
This
proposed
guidance
document
is
intended
to
provide
direction
to
the
scientists
in
the
Office
of
Pollution,
Prevention
and
Toxic
Substances
in
evaluating
liver
tumor
data
observed
following
exposure
to
this
subgroup
of
carcinogens.
This
document
also
responds
to
the
EPA
policy
concerning
risks
to
infants
and
children
(
USEPA,
1995;
USEPA,
2003)
which
requires
that
each
risk
assessment
present
findings
explicit
for
these
lifestages.
OPPTS
will
depart
from
the
science
policy
within
this
document
where
the
facts
or
circumstances
warrant.
In
such
cases,
OPPTS
will
explain
why
a
different
course
was
taken.
Page
3
of
39
TABLE
OF
CONTENTS
PREFACE
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Page
2
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39
I.
Introduction
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Page
4
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39
II.
Overview
of
Peroxisome
Proliferation
and
PPAR 
Agonism
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Page
5
of
39
III.
Establishing
PPAR ­
Agonism
as
a
Mode
of
Action
for
Rodent
Hepatocarcinogenicity
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Page
6
of
39
IV.
Human
and
Non­
human
Primate
Response
to
PPAR 
Agonists
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Page
8
of
39
V.
Development
of
PPAR 
Activity
and
Responses
to
PPAR 
Agonists
in
the
Fetus
and
Neonate
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Page
12
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39
Ontogeny
of
PPAR
 
Expression,
Peroxisomal
Assemblage,
Peroxisomal
Numerical
or
Volume
Density,
and
Peroxisomal
Enzyme
Activities
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Page
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39
PPAR 
Agonism
in
the
Fetus
and
Neonate
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Page
13
of
39
Conclusions
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Page
14
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39
VI.
Proposed
Science
Guidance
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Page
14
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39
Proposed
Science
Policy
Statements
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Page
14
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39
Framework
to
Establish
the
PPAR 
Agonist
MOA
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Page
15
of
39
Data
Needs
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Page
16
of
39
REFERENCES
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Page
18
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39
APPENDIX
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25
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39
A­
1.
PPAR 
Activity
and
Peroxisome
Assemblage/
Content
in
Fetal
Liver
Tissue
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25
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39
A­
2.
Peroxisome
Assemblage/
Content
and
Peroxisomal
Enzyme
Activity
in
Neonatal
Liver
Tissue
.
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Page
27
of
39
A­
3.
Response
of
the
fetal
liver
to
the
PPAR 
agonist
clofibrate
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Page
28
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39
A­
4.
Response
of
Neonates
Following
Lactational
Exposures
to
PPAR 
Agonists
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Page
32
of
39
A­
5.
Response
of
Neonates
Following
Direct
Exposures
to
PPAR 
Agonists
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Page
37
of
39
Page
4
of
39
I.
Introduction
There
has
been
substantial
scientific
interest
regarding
the
role
of
peroxisome
proliferation
in
rodent
hepatocarcinogenesis
and
its
relevance
for
human
carcinogenesis
at
this
and
other
potential
sites.
Several
scientific
groups
have
examined
the
state
of
the
science
on
PPAR 
agonist
­
induced
rodent
liver
tumors
over
the
years.
A
workgroup
convened
under
the
auspices
of
the
International
Agency
for
Research
on
Cancer
concluded
that
the
mode
of
action
(
MOA)
for
liver
tumors
induced
in
rodents
by
PPAR 
agonists
is
unlikely
to
be
operative
in
humans
(
IARC,
1995).
The
participants
of
a
workshop
held
under
the
auspices
of
the
International
Life
Sciences
Institute
Health
and
Environmental
Sciences
Institute
concluded
that
although
it
appeared
unlikely
that
PPAR 
agonists
could
induce
liver
tumorigenesis
in
humans,
the
possibility
could
not
be
ruled
out
(
Cattley
et
al.
1998).

Recent
scientific
developments
have
led
to
a
reevaluation
of
the
state
of
the
science
to
characterize
the
mode(
s)
of
action
(
e.
g.,
PPAR ­
agonism)
and
human
relevance
of
rodent
tumors
induced
by
certain
peroxisome
proliferating
agents.
To
that
end,
the
ILSI
Risk
Science
Institute
convened
a
workgroup
to
upgrade
the
state
of
the
science
for
PPAR 
agonist­
induced
rodent
liver
tumors,
as
well
as
to
evaluate
the
mode(
s)
of
action
for
Leydig
cell
and
pancreatic
acinar
cell
tumors,
which
also
are
observed
frequently
in
rats
with
PPAR 
agonists.
The
workgroup
provided
a
detailed
analysis
of
the
key
events
in
the
mode
of
action
of
PPAR ­
agonist
induced
liver
tumors
in
rodents,
and
then
proceeded
with
a
concordance
analysis
of
the
evidence
that
these
key
events
can
occur
in
humans.
The
workgroup
concluded
that
while
the
PPAR 
receptor
can
be
activated
in
humans,
there
are
substantial
species
differences
in
toxicodynamics
that
make
it
very
unlikely
that
the
downstream
key
events
and
therefore
hepatocarcinogenesis
would
occur
in
humans.
Finally,
the
workgroup
concluded
that
there
is
insufficient
information
at
this
time
to
firmly
establish
a
mode
of
action
for
the
Leydig
cell
and
pancreatic
acinar
cell
tumors
in
rats
(
ILSI,
in
press).
It
should
be
noted
that
the
ILSI
expert
panel's
report
on
"
PPAR 
Agonist­
Induced
Rodent
Tumors:
Mode(
s)
of
Action
and
Human
Relevance"
was
peer
reviewed
by
an
independent
group
of
17
scientists
from
academia,
government
and
industry.

The
purpose
of
this
OPPTS
guidance
document
is
to
describe
the
approach
the
Office
will
proposes
to
use
to
evaluate
the
scientific
information
regarding
the
mode
of
action
of
PPAR 
agonists
in
rodent
hepatocarcinogenesis
and
the
relevance
of
this
mode
of
action
for
human
hepatocarcinogenesis.
Other
tumor
types
(
e.
g.
Leydig
cell
and
pancreatic
acinar
cell
tumors)
that
may
be
associated
with
PPAR ­
agonists
are
briefly
described.
The
document
provides
an
overview
of
the
evidence
for
a
PPAR ­
agonist
mode
of
action
for
liver
tumors
in
rodents,
and
an
overview
of
all
known
age
and
species
differences
in
the
key
events.
Finally,
the
document
will
provide
guidance
in:
1)
using
a
framework
to
describe
and
to
present
the
proposed
PPAR ­
mediated
mode
of
action;
2)
data
needed
to
demonstrate
that
rodent
liver
tumors
have
arisen
as
a
result
of
a
PPAR 
agonist
mode
of
action;
and
3)
the
relevance
of
this
mode
of
action
for
hepatocarcinogenesis
in
humans.
OPPTS
will
depart
from
this
proposed
science
policy
where
the
facts
and
circumstances
warrant.
Page
5
of
39
II.
Overview
of
Peroxisome
Proliferation
and
PPAR 
Agonism
Events
leading
to
carcinogenic
response
following
exposure
to
an
environmental
agent
are
varied
and
may
range
from
mutations
of
the
genome
to
the
activation/
deactivation
of
genes
and/
or
receptors.
An
example
of
this
latter
category
is
PPAR 
agonism.
This
event
is
key
in
rodent
liver
carcinogenesis
triggered
by
administration
of
certain
peroxisome
proliferating
agents.
An
increase
in
the
number
and
volume
of
peroxisomes
present
in
liver
cells
is
a
key
characteristic
of
PPAR 
agonism.
It
is
generally
accepted
that
peroxisome
proliferation
is
associated
with
liver
tumor
formation
in
rodents
although
this
phenomenon
has
not
been
established
as
a
causal
effect.

In
addition
to
inducing
hepatocarcinogenesis
in
rodents,
PPAR 
agonists
have
also
been
observed
to
induce
pancreatic
acinar
cell
and
Leydig
cell
tumors
in
rats.
Of
15
PPAR 
agonists
tested
to
date,
nine
have
been
shown
to
induce
all
three
tumors
in
non­
F344
rat
strains
but
not
in
mice.
In
the
case
of
Leydig
cell
tumor
formation,
two
potential
MOAs
based
on
activation
of
PPAR 
have
been
proposed.
One
MOA
invokes
the
induction
of
hepatic
aromatase
activity
leading
to
an
increase
in
serum
estradiol
level.
The
second
MOA
purports
that
PPAR 
agonists
inhibit
testosterone
biosynthesis.
Although
agonism
of
PPAR 
may
lead
to
the
induction
of
aromatase
or
inhibition
of
testosterone
biosynthesis,
the
data
available
to
date
are
insufficient
to
support
which,
if
either,
of
these
two
proposed
MOAs
is
operative.
For
pancreatic
acinar
cell
tumor
(
PACT)
formation,
a
MOA
has
been
proposed
in
which
PPAR ­
agonists
cause
a
decrease
in
bile
acid
synthesis
and/
or
change
the
composition
of
the
bile
acid
resulting
in
cholestasis.
These
steps
increase
the
level
of
the
growth
factor
cholecystokinin
(
CCK)
which
then
binds
to
its
receptor,
CCK
A,
leading
to
acinar
cell
proliferation.
Some
evidence
exists
to
support
this
proposed
MOA
and
there
does
not
appear
to
be
evidence
of
any
other
MOA
operating
in
the
formation
of
PACTs
after
exposure
to
PPAR 
agonists.
However,
the
data
are
not
considered
sufficient
to
establish
a
MOA
with
confidence,
because
it
has
only
been
described
for
two
chemicals,
PFOA
and
WY14643,
in
one
laboratory.
As
a
result,
the
evidence
is
considered
insufficient
to
infer
that
this
MOA
may
be
generalized
to
all
PACT­
inducing
PPAR 
agonists.
Page
6
of
39
III.
Establishing
PPAR ­
Agonism
as
a
Mode
of
Action
for
Rodent
Hepatocarcinogenicity
Although
the
precise
mechanistic
steps
leading
to
hepatic
carcinogenesis
in
rodents
after
PPAR 
agonist
exposure
are
not
completely
understood,
knowledge
of
the
mode
of
action
has
been
characterized
(
ILSI,
in
press).
As
shown
in
Figure
1,
it
has
been
proposed
that
PPAR 
agonists
activate
PPAR 
(
which
regulates
the
transcription
of
genes
involved
in
peroxisome
proliferation,
cell
cycle
control,
apoptosis,
and
lipid
metabolism).
Activation
of
PPAR 
leads
to
an
increase
in
cell
proliferation
and
a
decrease
in
apoptosis,
which
in
turn
leads
to
preneoplastic
cells
and
further
clonal
expansion
and
formation
of
liver
tumors.
As
depicted
in
Figure
1,
the
biological
events
in
PPAR ­
induced
hepatocarcinogenesis
may
be
classified
as
either
causal
(
i.
e.,
required
for
this
MOA)
or
associative
(
i.
e.,
markers
of
PPAR 
agonism
but
not
shown
to
be
directly
involved
in
the
etiology
of
liver
tumors).
PPAR 
activation,
changes
in
rates
of
liver
cell
proliferation/
apoptosis,
and
selective
clonal
expansion
are
causally
related
to
PPAR ­
mediated
liver
tumor
formation.
Of
these,
only
PPAR 
activation
is
highly
specific
for
this
MOA
while
cell
proliferation/
apoptosis
and
clonal
expansion
are
common
to
other
modes
of
action.
Among
the
associative
events
are
peroxisome
proliferation
(
a
highly
specific
indicator
that
this
MOA
is
operative)
and
peroxisomal
gene
expression.
Peroxisomal
proliferation
may
also
result
in
hepatocyte
oxidative
stress
which
may
contribute
to
the
mode
of
action
by
causing
indirect
DNA
damage
and
leading
to
mutations,
or
by
stimulating
cell
proliferation.
However,
increases
in
oxidative
damage
to
DNA
have
not
been
unambiguously
demonstrated
for
PPAR 
agonists.
Oxidative
stress
is
a
general
phenomenom,
and
thus
does
not
represent
a
highly
specific
marker
for
PPAR ­
agonist
induced
liver
carcinogenesis.
For
a
more
detailed
description
of
the
PPAR 
agonist
mode
of
carcinogenic
action
see
ILSI
(
in
press).
Page
7
of
39
Causative
Events
PPAR 
Agonist
Activation
of
PPAR
 
Cell
Proliferation
Decreased
Apoptosis
Preneoplastic
Foci
Clonal
Expansion
Liver
Tumors
°
Expression
of
Peroxisomal
Genes
°
Increase
in
Peroxisomes
(
number
&
size)
Associative
Events*

*
Although
there
are
other
biological
events
(
e.
g.,
Kupffer
cell
mediated
events,
inhibition
of
gap
junctions),
the
measurements
of
peroxisome
proliferation
and
peroxisomal
enzyme
activity
(
in
particular
acyl­
CoA)
are
widely
used
as
reliable
markers
of
PPAR 
activation.
Figure
1.
Key
Events
in
the
Mode
of
Action
for
PPAR 
Agonist
Induced
Rodent
Liver
Tumors
When
postulating
a
PPAR ­
agonist
mediated
MOA
for
rodent
liver
tumorigenesis,
one
of
the
first
indications
that
this
MOA
may
be
operative
is
the
increase
in
the
number
and
size
of
peroxisomes
in
the
cytoplasm
of
hepatocytes.
It
should
be
noted
that
although
peroxisome
proliferation
itself
has
not
been
shown
to
be
a
causal
event
in
liver
tumorigenesis
,
it
is
a
highly
specific
biomarker
of
the
PPAR 
agonist
MOA.
Evidence
of
peroxisome
proliferation
(
and
by
extension
PPAR 
agonism)
has
also
been
obtained
through
biochemical
assays
­
conducted
as
part
of
long
term
cancer
studies
or
as
independent
mechanistic
studies
­
that
evaluate
the
activity
of
peroxisome­
specific
enzymes
involved
in
the
metabolism
of
fatty
acids.
These
assays
include
activity
level
measurements
for
acyl­
CoA
oxidase,
palmitoyl
CoA
oxidase,
carnitine
acetyl
transferase,
and
CYP4A.
Increases
in
palmitoyl
CoA,
cartinine
acetyl
transferase,
and
CYP4A
activity
have
been
noted
in
conjunction
with
liver
tumorigenesis
after
exposure
to
certain
peroxisome
proliferators
(
PP)
such
as
clofibrate,
lactofen,
trichloroacetic
acid,
oxidiazon,
and
di­(
2­
ethylhexyl)
phthalate
(
DEHP)(
ILSI,
in
press).
Furthermore,
the
observation
that
acyl
CoA
oxidase
induction
after
PPAR 
agonist
exposure
is
not
seen
in
PPAR ­
null
mice,
has
served
to
identify
changes
in
this
enzyme
activity
as
a
key
event
in
the
induction
of
rodent
liver
tumors
by
PPAR ­
agonists
and
a
biomarker
for
the
MOA
(
Ward
et
al.
1998).
In
addition
to
biochemical
alterations,
other
liver
changes
noted
after
exposure
to
PPAR 
agonists
Page
8
of
39
include:
hepatocyte
hypertrophy,
increased
liver
weights,
and
increased
liver
cell
mitotic
activity
(
reviewed
in
Cattley
et
al.
1998).

The
discovery
and
cloning
of
the
PPAR 
in
1990
by
Isseman
and
Green
provided
insight
into
the
role
of
PPAR 
in
the
processes
leading
to
hepatocarcinogenesis
in
rodents
(
Isseman
and
Green,
1990).
Conclusive
evidence
of
the
pivotal/
causal
role
of
PPAR 
in
the
induction
of
liver
tumors
by
certain
PPAR 
agonists
was
obtained
from
studies
on
PPAR ­
null
knockout
mice.
In
a
series
of
seminal
experiments,
Lee
et
al.
(
1995)
and
Peters
et
al.
(
1997)
exposed
wild
type
(+/+)
and
PPAR ­
null
(­/­)
mice
to
either
WY14643
or
clofibrate
­
two
known
PPAR 
agonists
­
at
doses
known
to
induce
hepatocarcinogenesis
in
rodents.
Short­
term
exposure
(
2
weeks)
to
either
one
of
these
PPAR 
agonists
failed
to
elicit
in
PPAR ­
null
mice
the
early
hepatic
effects
characteristic
of
liver
tumor
induction
such
as
increases
in
liver
weights,
hepatic
peroxisome
proliferation,
CYP4A
induction
as
well
as
induction
of
other
peroxisomal
enzymes.
In
contrast,
wild
type
(+/+)
mice
displayed
all
these
indicators
of
hepatocellular
alteration.
These
findings
were
corroborated
by
experiments
using
longer
periods
of
exposure
(
5
weeks
to
11
months)
in
which
WY14643
exposure
failed
to
elicit
in
PPAR ­
null
mice
the
increases
in
acyl
CoA
oxidase,
CDK­
1,
CDK­
4,
and
c­
myc
seen
in
wild
type
mice.
WY14643
treatment
also
failed
to
induce
replicative
DNA
synthesis
(
as
measured
through
BrDU
labeling
indices)
in
PPAR ­
null
mice
under
conditions
that
elicited
this
effect
in
the
wild
type
mice.
Moreover,
WY14643
exposure
for
11
months
led
to
a
100%
incidence
of
hepatic
neoplasms
in
wild
type
mice
while
the
PPAR ­
null
mice
showed
none.
PPAR ­
null
mice
exposed
to
DEHP
for
24
weeks
did
not
manifest
any
of
the
changes
associated
with
PPAR ­
mediated
liver
tumors
such
as
peroxisome
proliferation,
increased
peroxisomal
enzyme
activity,
or
increased
CYP4A
mRNA
levels
(
Ward
et
al.
1998).
Experiments
on
null
mice
after
WY14643,
clofibrate,
or
DEHP
exposure
provide
persuasive
evidence
that
a
PPAR ­
mediated
MOA
is
operating
in
the
induction
of
hepatocellular
neoplasms.

IV.
Human
and
Non­
human
Primate
Response
to
PPAR 
Agonists
Studies
conducted
in
numerous
test
species
indicate
that
while
rodents
(
mice
and
rats)
are
highly
responsive
to
PPAR 
agonist­
induced
hepatocarcinogenicity,
other
species
(
e.
g.,
hamster,
dogs,
guinea
pigs,
New
and
Old
World
primates,
and
humans)
appear
to
be
refractory
(
Cattley
et
al.
1998;
Doull
et
al.
1999).
For
example,
a
wide
range
of
PPAR 
agonists
which
produce
peroxisome
proliferation
in
rodent
hepatocytes,
have
little
or
no
effect
in
guinea
pig
or
monkeys
when
the
test
compounds
were
evaluated
by
changes
in
peroxisome
proliferation
and/
or
enzyme
activities
(
e.
g.,
palmitoyl­
CoA)
(
Doull
et
al.
1999).
Furthermore,
liver
tumors
are
not
found
in
Syrian
hamsters
after
40­
60
weeks
administration
of
WY14643
and
nafenopin
(
Lake
et
al.
1993).
A
variety
of
studies
have
been
conducted
to
characterize
the
response
of
humans
and
non­
human
primates
to
PPAR 
agonists,
and
to
determine
the
cause(
s)
of
the
apparent
refractory
nature
of
the
response.
These
studies
are
summarized
below.
Page
9
of
39
Two
major
epidemiology
studies
have
been
conducted
to
assess
the
effect
of
prolonged
exposure
to
the
hypolipidemic
drugs
gemfibrozil
or
clofibrate.
In
the
Helsinki
Heart
Study
over
4000
men
with
high
cholesterol
were
treated
with
gemfibrozil
or
a
placebo
for
five
years.
Although
substantial
decreases
in
the
serum
lipid
levels
were
noted
in
the
gemfibrozil
treated
group
compared
to
the
placebo
group,
the
death
rate
and
liver
cancer
rates
in
both
groups
were
comparable.
It
is
noteworthy,
however,
that
in
this
study
the
liver
cancer
incidence
was
reported
in
conjunction
with
gall
bladder
and
intestinal
cancers
(
Frick
et
al.,
1987;
Huttunen
et
al.,
1994).

In
the
second
study,
over
15,000
men
with
ischemic
heart
disease
received
clofibrate
or
a
placebo
for
five
years,
and
their
health
status
was
followed
for
eight
years
after
cessation
of
treatment
(
CPI,
1984).
During
the
follow
up
conducted
4
years
after
treatment
stopped,
the
group
treated
with
clofibrate
had
a
24%
increase
in
mortality
relative
to
the
high
cholesterol
patients
treated
with
placebo.
These
mortalities
were
attributed
to
diseases
(
including
malignancies)
of
the
gall
bladder,
intestines,
liver
and,
pancreas.
Eight
years
after
treatment
was
stopped,
however,
the
cancer
death
rate
between
the
two
groups
was
comparable.
As
was
the
case
in
the
Helsinki
Heart
Study,
cancer
data
were
not
provided
specifically
for
the
liver
but
rather
they
were
presented
in
combination
with
the
gall
bladder,
pancreatic,
and
intestinal
cancer
incidences.
Thus,
neither
of
these
epidemiological
studies
has
provided
evidence
of
PPAR ­
mediated
hepatocarcinogenesis;
however,
these
data
are
inconclusive
because
of
the
limited
durations
of
exposure
The
apparent
species
differences
between
rodents
and
human
and
nonhuman
primates
in
the
response
to
PPAR 
agonists
is
also
supported
by
a
long­
term
study
in
monkeys.
Marmosets
exposed
for
6.5
years
to
clofibrate
at
relatively
high
doses
(
94
mg/
kg/
day
or
higher)
did
not
develop
liver
tumors
(
Tucker
and
Orton,
1995).
Although
the
duration
of
this
study
did
not
represent
a
lifetime
exposure,
the
results
strongly
suggest
that
primates
appear
to
be
refractory
to
PPAR 
agonists
(
Doull
et
al.,
1999
and
ILSI,
in
press).

Examination
of
liver
biopsies
from
patients
receiving
hypolipidemic
drugs
(
clofibrate
or
ciprofibrate)
to
treat
hyperlipidemia
provide
another
line
of
evidence
for
human
response
to
PPAR 
agonists.
Clofibrate
exposure
­
at
clinically
relevant
doses
­
has
been
shown
only
to
induce
a
1.5­
fold
increase
in
the
number
of
hepatic
peroxisomes
and
a
23%
increase
in
the
volume
density
of
these
organelles.
Similar
findings
have
been
reported
for
ciprofibrate
where
liver
biopsies
from
patients
treated
with
the
drug
for
6
months
to
two
years
exhibited
a
30%
increase
in
the
volume
density
of
hepatic
peroxisomes
(
Bentley
et
al.,
1993;
Hinton
et
al.,
1983).
In
addition,
there
was
no
evidence
of
liver
tumors
from
liver
biopsies
that
were
conducted
on
patients
treated
with
gemfibrozil,
clofibrate
or
fenofibrate
for
periods
ranging
for
up
to
5.3
years
and
followed
up
for
an
additional
7.9
years
(
Blumcke
et
al.,
1983;
De
La
Iglesia
et
al.,
1982;
Gariot
et
al.,
1987).

Further
evidence
that
humans
appear
to
be
refractory
to
the
hepatocarcinogenic
properties
of
PPAR 
agonists
is
provided
by
experiments
in
which
exposure
to
Page
10
of
39
ciprofibrate,
clofibrate,
gemfibrozil,
and
fenofibrate
(
hypolipidemic
drugs
known
to
be
PPAR 
agonists)
failed
to
elicit
a
response
in
human
hepatocytes
that
is
observed
in
rodent
hepatocytes.
Human
hepatocytes
cultured
in
the
presence
of
these
compounds
did
not
exhibit
increases
in
replicative
DNA
synthesis
or
acyl
CoA
oxidase
activity
and/
or
expression
(
i.
e.
mRNA
levels),
suppression
of
apoptosis,
or
increased
peroxisome
proliferation.
These
findings
are
consistent
with
the
results
obtained
after
exposure
to
other
PPAR 
agonists
that
are
not
hypolipidemic
drugs
such
as
mono­
2­
ethylhexyl
phthalate
(
MEHP)
(
Baker
et
al.,
1996;
Kamendulis
et
al.,
2002;
Hasmall
et
al.
1999
and
2000).

In
vitro
and
in
vivo
data
on
monkeys
(
cynomolgus,
marmoset,
and
Rhesus)
support
the
findings
from
human
in
vitro
and
liver
biopsy
studies.
Palmitoyl
CoA
oxidase
activity
was
evaluated
in
monkeys
after
in
vivo
exposure
to
a
variety
of
PPAR 
agonists
(
e.
g.,
bezafibrate,
clofibrate,
di­
2­
ethylhexyl
phthalate
(
DEHP),
MEHP,
fenofibrate,
nafenopin,
and
LY171883).
The
changes
noted
in
enzymatic
activity
after
PPAR 
agonist
exposure
were
minimal
or
non­
existent
relative
to
control
(
ILSI,
in
press).
Moreover,
cynomolgus
monkeys
exposed
in
vivo
to
DEHP,
di­
isononyl
phthalate
and
clofibrate
also
failed
to
exhibit
an
increase
in
DNA
synthesis
(
Pugh
et
al.,
2000).
It
should
be
noted,
however,
that
cynomolgus
monkeys
treated
for
two
weeks
with
clinically
relevant
doses
of
fenofibrate
or
ciprofibrate
exhibited
a
3­
fold
increase
in
the
number
of
hepatic
peroxisomes,
a
2­
fold
increase
in
liver
size,
and
hepatocellular
hypertrophy
(
Qualls
et
al.,
2003).
Although
these
changes
appear
to
be
similar
to
the
response
in
rodents
to
PPAR 
agonist
exposure,
there
was
no
evidence
of
increases
in
peroxisomal
enzyme
activity
(
e.
g.,
acyl
CoA
oxidase,
catalase,
or
carnitine
acetyltransferase).

Research
on
several
aspects
of
PPAR 
agonism
has
been
undertaken
to
attempt
to
ascertain
the
basis
for
the
differential
response
between
rodents
and
humans
after
exposure
to
PPAR 
agonists.
These
investigations
have
focused
on
PPAR 
gene
expression,
PPAR 
gene
structure,
and
peroxisome
proliferator
response
element
(
PPRE)
structure.
After
the
discovery
and
cloning
of
the
gene
encoding
the
human
PPAR ,
Tugwood
et
al.
(
1996)
and
Palmer
et
al.
(
1998)
demonstrated
that
PPAR 
expression
in
humans
is
10­
fold
lower
than
that
seen
in
rodents.
While
this
may
account
in
part
for
the
unresponsiveness
of
humans
to
the
hepatocarcinogenic
properties
of
PPAR 
agonists,
it
is
unknown
how
much
it
contributes
to
the
differences
between
the
species.

Transfection
experiments
have
demonstrated
that
human
PPAR 
(
hPPAR )
is
very
similar
to
the
rodent
PPAR 
and
capable
of
transactivating
reporter
genes
(
containing
a
rodent
PPRE
in
their
promoter
region)
following
clofibrate
exposure,
thereby
establishing
that
hPPAR"
is
functional
(
Sher
et
al.,
1993;
Mukherjee
et
al.,
1994;
Pineau
et
al.,
1996).
This
finding
was
confirmed
by
in
vivo
experiments
in
which
PPAR ­
null
mice
were
infected
by
an
adenovirus
carrying
the
hPPAR 
gene.
The
hPPAR 
gene
was
able
to
"
rescue"
these
knockout
mice
as
evidenced
by
the
increases
in
peroxisome
proliferation
and
peroxisomal
enzyme
gene
expression
(
Yu
et
al.,
2001).
Page
11
of
39
Other
studies
have
focused
on
the
potential
variability
in
the
structure
of
the
PPAR 
gene
in
human
populations.
The
hPPAR 
8/
14
variant
identified
by
Tugwood
et
al.
(
1996)
results
in
a
truncated
hPPAR ,
while
hPPAR 
6/
29,
identified
by
Roberts
et
al.
(
1998),
results
in
a
four
amino
acid
substitution.
The
hPPAR 
8/
14
variant
has
been
detected
in
every
sample
examined
to
date
in
two
laboratories
while
hPPAR 
6/
29
is
extremely
rare
having
been
isolated
only
once
(
Roberts
et
al.,
2000).
Two
point
mutations
(
PPAR *
3
and
PPAR *
2)
were
identified
by
Sapone
et
al.
(
2000).
PPAR *
3
is
relatively
common
in
Northern
India
populations
while
PPAR *
2
is
relatively
rare.
Relative
to
wild
type,
PPAR *
3
results
in
unresponsiveness
to
low
concentrations
of
ligand
as
well
as
a
lower
non­
ligand
dependent
trans­
activating
(
i.
e.
constitutive)
activity.
In
contrast,
PPAR *
2
exhibits
a
slightly
higher
constitutive
activity
than
wild
type.
However,
PPAR *
2
activity
after
WY14643
exposure
is
comparable
to
wild
type.
Precisely
how
these
mutations
may
render
humans
less
sensitive
to
PPAR 
agonists
is
not
known.

Another
factor
that
may
contribute
to
the
apparent
lack
of
response
in
humans
to
the
hepatocarcinogenic
inducing
properties
of
PPAR 
agonists
may
be
related
to
a
difference
in
the
hPPREs
response
to
hPPAR 
induction.
This
theory
stems
from
transfection
studies
in
which
Sher
et
al.
(
1993)
demonstrated
that
hPPAR 
can
transactivate
reporter
genes
containing
a
murine
PPRE.
However,
the
gene
and
protein
expression
of
various
biomarkers
indicative
of
PPAR ­
mediated
liver
tumorigenesis
and
regulated
by
hPPRE,
including
acyl
CoA
oxidase,
were
not
increased
in
human
hepatocytes
following
exposure
to
several
PPAR 
agonists.
These
findings
were
corroborated
by
data
collected
after
patients
were
exposed
at
clinically
relevant
doses
to
various
fibrates
(
e.
g.,
bezafibrate,
fenofibrate,
and
gemfibrozil)
which
indicate
that
acyl
CoA
oxidase
mRNA
levels
were
unaffected
(
Roglans
et
al.,
2002).
In
contrast,
other
human
genes
regulated
by
PPAR 
agonists
­
but
not
implicated
in
hepatic
carcinogenesis
­
(
e.
g.
,
lipoprotein
kinase,
carnitine
palmitoyl
transferase,
etc.)
are
effectively
regulated
by
PPAR 
agonists
(
discussed
in
ILSI,
in
press).
Moreover,
in
experiments
in
which
HepG2
cells
were
transfected
with
hPPAR 
(
at
the
levels
of
PPAR 
seen
in
rodents)
and
treated
with
fibrate
did
not
lead
to
increases
in
acyl
CoA
oxidase
mRNA
levels
(
Hsu
et
al.,
2001;
Lawrence
et
al.,
2001a).
The
possibility
that
the
hPPRE
for
acyl
CoA
oxidase
may
differ
from
the
rodent
in
its
ability
to
bind
hPPAR 
has
been
further
suggested
by
experiments
showing
that
this
peroxisomal
enzyme
cannot
be
activated
in
cells
transfected
with
a
PPAR 
expression
vector
and
that
it
may
require
high
concentrations
of
PPAR 
agonists
to
be
induced
(
Woodyatt
et
al.,
1999;
Varanasi
et
al.,
1998;
Rodriguez
et
al.,
2000).
Furthermore,
Lambe
et
al.
(
1999)
have
shown
that
when
rat
acyl
CoA
oxidase
PPRE
is
disrupted
by
site
directed
mutagenesis,
the
hPPRE
for
this
enzyme
could
not
rescue
the
null
phenotype
in
reporter
gene
assays.
Although
it
appears
that
the
downstream
effects
of
PPAR 
agonism
such
as
induction
of
acyl
CoA
oxidase,
CYP4A1,
DNA
replication,
or
apoptosis
suppression
do
not
occur
in
humans,
hPPAR 
in
COS­
1
or
NIH
3T3
cells
can
be
activated
by
exposure
to
MEHP
as
indicated
by
the
increases
in
luciferase
reporter
gene
activity
(
Dirven
et
al.,
1993;
Maloney
&
Waxman,
1999;
Hasmall
et
al.,
2000).
Page
12
of
39
In
summary,
although
humans
possess
a
functional
PPAR ,
and
the
human
receptor
can
be
activated
by
peroxisome
proliferators,
humans
(
and
non­
human
primates)
appear
to
be
refractory
to
the
key
events
associated
with
the
induction
of
liver
tumors
by
PPAR 
agonists.
It
is
the
totality
of
evidence
that
provides
a
strong
argument
that
PPAR ­
induced
liver
carcinogenesis
is
not
likely
to
occur
in
humans.
Human
and
non­
human
primate
hepatocytes
treated
with
a
range
of
different
PPAR 
agonists
do
not
exhibit
increased
replicative
DNA
synthesis,
suppression
of
apoptosis,
increased
expression
of
marker
mRNAs
and
proteins
including
acyl
CoA
oxidase
or
peroxisome
proliferation.
In
vivo
studies
with
monkeys
showed
that
treatment
with
the
hypolipdemic
drugs
fenofibrate
or
ciprofibrate
induced
an
increase
in
liver
weights
(
up
to
2­
fold)
and
peroxisome
numbers
(
up
to
about
3­
fold),
but
only
slight
or
no
increases
in
peroxisomal
enzyme
activities.
Similarly,
analyses
of
biopsies
from
liver
tissue
of
patients
treated
with
hypolipodemic
drugs
showed
only
slight
increases
or
no
increases
in
peroxisome
numbers
or
volume
density.
Although
the
epidemiological
literature
and
cancer
studies
in
nonhuman
primates
are
inconclusive,
they
do
not
provide
evidence
for
the
potential
of
PPAR 
agonists
to
induce
liver
tumors
in
humans.

V.
Development
of
PPAR 
Activity
and
Responses
to
PPAR 
Agonists
in
the
Fetus
and
Neonate
Previous
sections
of
this
document
provided
an
overview
of
the
evidence
and
basis
for
the
establishment
of
PPAR ­
agonism
as
a
mode
of
action
for
the
induction
of
liver
tumors
observed
in
adult
rodents.
This
section
focuses
on
the
ontogeny
of
the
response
to
PPAR 
agonists
during
fetal
and
postnatal
development
and
the
sensitivity
of
the
developing
organism
relative
to
the
adult.
No
data
on
the
ontogeny
of
the
response
during
human
development
were
found
in
the
literature.
Thus,
conclusions
concerning
the
ontogeny
of
the
response
to
PPAR 
agonists
and
the
relative
sensitivity
of
developing
humans
must
rely
on
evidence
provided
by
laboratory
studies
in
animals.

Ontogeny
of
PPAR
 
Expression,
Peroxisomal
Assemblage,
Peroxisomal
Numerical
or
Volume
Density,
and
Peroxisomal
Enzyme
Activities
A
detailed
review
of
studies
available
on
the
ontogeny
of
peroxisomes
and
peroxisomal
enzyme
activities
during
development
in
the
rodent
liver
is
provided
in
Appendices
A­
1
and
A­
2.
These
studies
indicate
that
expression
of
the
PPAR 
gene,
assemblage
of
peroxisomal
proteins
into
plasmids,
the
content
of
peroxisomes,
and
peroxisomal
enzyme
activity
appears
to
occur
late
in
rodent
fetal
development
(
i.
e.,
gestational
day
15
or
later)
(
Braissant
and
Wahli,
1998;
Wilson
et
al,
1991;
Tsukada,
et
al.,
1968;
Stefanini
et
al.,
1989;
Stefanini,
et
al.,
1985;
Cibelli,
et
al.,
1988).
Thus,
it
appears
that
the
direct
effects
of
a
PPAR 
agonist
on
PPAR 
and
secondary
effects
on
peroxisomal
proliferation
and
enzyme
activites
would
not
be
operative
prior
to
gestational
day
15
or
later.

In
the
neonatal
rat,
peroxisomal
assemblage,
peroxisomal
numerical
density
and
the
volume
of
peroxisomes
have
been
reported
to
be
similar
among
neonatal
and
adult
rats,
as
have
peroxisomal
enzyme
activities
(
Stefanini
et
al.,
1999;
Stefanini
et
al.,
Page
13
of
39
1995;
Cimini
et
al.,
1994;
Singh
and
Lazo,
1992;
Dostal
et
al.,
1987;
Staubli
et
al.,
1977;
Weibel
et
al.,
1969).

Based
on
the
available
evidence,
it
appears
that
PPAR 
expression
is
low
or
absent
in
the
rodent
fetus
until
just
before
birth.
In
neonatal
rodents,
the
expression
of
PPAR 
mRNA
is
comparable
to
or
less
than
that
seen
in
the
adult
rodent.
Thus,
it
is
plausible
that
rodent
embryos
would
not
respond
to
the
effects
of
a
PPAR 
agonist
until
late
in
development
whereas
the
neonate
might
be
expected
to
respond
to
a
PPAR 
agonist
like
adult
rodents.

PPAR 
Agonism
in
the
Fetus
and
Neonate
Several
studies
have
been
conducted
to
examine
the
effects
of
exposure
to
PPAR 
agonists
during
fetal
and
postnatal
development
in
rats
and
mice.
These
studies
are
reviewed
in
detail
in
Appendices
A­
3
­
A­
5.
Administration
of
the
PPAR 
agonist
clofibrate
to
rats
or
mice
during
pregnancy
can
accentuate
peroxisome
assemblage
or
peroxisomal
proliferation
in
the
liver
of
their
fetuses
late
in
development
(
i.
e.,
during
gestational
days
17­
21)(
Wilson
et
al.,
1991;
Stefanini
et
al.,
1989;
Cibelli,
et
al.,
1988).
Markers
of
peroxisomal
assemblage,
the
proteins
PMP­
70
and
DHAP­
AT,
are
increased,
relative
to
controls,
to
an
equal
or
greater
extent
in
the
mouse
fetus
compared
to
clofibrate­
treated
dams.
However,
the
total
levels
of
these
assemblage
proteins
(
i.
e.,
on
a
µ
g/
mg
or
units/
mg
protein
basis)
are
lower
in
the
fetus
than
in
treated
dams.
Peroxisomal
numbers
also
increased
in
rat
or
mouse
fetuses
from
dams
treated
with
clofibrate,
particularly
in
gestational
day
19
fetuses.
The
activity
of
catalase,
a
peroxisomal
enzyme
which
is
also
found
in
the
cytoplasm,
increases
somewhat
in
gestational
day
19
to
21
fetuses
and
neonates
from
mothers
treated
with
clofibrate
but
the
specific
activity
of
the
enzyme
does
not
exceed
the
level
of
activity
observed
in
clofibrate­
treated
rat
or
mouse
dams.
Similarly,
palmitoyl
CoA
oxidase
activity
was
enhanced
as
much
as
8­
fold
in
gestational
day
21
rat
fetuses
but,
again,
the
enhanced
specific
activity
of
the
enzyme
was
about
equal
to
that
seen
in
the
clofibrate­
treated
dams.

Although
limited,
these
data
when
considered
together
suggest
that:
1)
the
rodent
fetus
responds
to
a
PPAR 
agonist
like
the
adult
rodent
dam
(
i.
e.,
there
are
increases
in
peroxisomal
assemblage/
proliferation
and
peroxisomal
enzyme
activities;
2)
effects
of
a
PPAR 
agonist
on
the
fetus
occur
late
in
development;
and
3)
although
the
enhancement,
relative
to
controls,
of
peroxisomal
numbers
and
peroxisomal
enzyme
activities
during
fetal
development
may
exceed
that
observed
in
the
PPAR 
agonist­
treated
pregnant
rat
or
mouse,
the
increased
levels
of
peroxisomal
protein
or
peroxisomal
enzyme
activities
do
not
exceed
the
levels
found
in
the
pregnant
dam.
Thus,
it
does
not
appear
that
the
fetal
rodent
is
more
sensitive
than
the
adult
rodent
dam
to
the
effects
of
a
PPAR 
agonist.

Treatment
of
rat
dams
during
lactation
also
leads
to
an
enhancement
of
peroxisomal
assemblage,
peroxisomal
enzyme
activities,
and
the
numerical
density
of
peroxisomes
in
their
nursing
pups
(
Stefanini
et
al.,
1999;
Stefanini
et
al.,
1995;
Cimini
Page
14
of
39
et
al.,
1994;
Singh
and
Lazo,
1992;
Fahl
et
al.,
1983).
Generally,
nursing
neonates
appear
no
more
sensitive
than
their
PPAR 
agonist
treated
dams
because
increases
in
these
parameters
are
similar
to
the
increases
observed
in
the
treated
dams.
Furthermore,
increases
in
liver
weights,
an
effect
characteristic
of
PPAR 
agonism,
in
nursing
neonates
exposed
to
a
PPAR 
agonist
were
no
greater
or
less
than
that
reported
in
treated
dams
(
Singh
and
Lazo,
1992;
Cimini
et
al.,
1994;
Osterburg
et
al.,
1992;
Schroeder,
1983).
At
this
time,
however,
it
is
unknown
how
much
of
the
compound
is
metabolized
by
the
dams
and/
or
transferred
to
the
neonates
via
the
milk;
hence
it
is
possible
that
a
difference
in
the
internal
dose
may
play
a
role
in
the
differential
responses
noted
between
nursing
neonates
and
their
dams.

Direct
exposure
of
neonates
to
PPAR 
agonists
results
in
an
increase
in
peroxisomal
enzyme
activities
and
an
increase
in
the
numerical
density
or
volume
of
peroxisomes;
the
increases
in
these
parameters
are
comparable
to
those
observed
in
young
adults
or
adult
rats
(
Yu
et
al.,
2001;
Yamoto,
1996;
Dostal
et
al.,
1987;
Staubli
et
al.,
1977;).
At
a
dose
of
a
PPAR 
agonist
that
affects
peroxisome
enzyme
activity
or
peroxisome
numbers,
no
effect
on
liver
weights
was
observed
(
Yu
et
al.,
2001)
or
the
increase
in
liver
weights
observed
in
neonates
was
no
greater
than
that
observed
in
treated
adults
(
Dostal
et
al.,
1997;
Yamoto,
1996)

Conclusions
The
data
available
on
the
effects
of
PPAR 
agonist
in
the
rodent
fetus
or
neonate
(
e.
g.,
increases
in
peroxisome
numbers
and
size,
peroxisome
enzyme
activities,
and
liver
weights)
provides
support
that
there
is
not
an
increased
sensitivity
relative
to
an
adult
to
hepatocarcinogenicity
during
fetal
or
neonatal
development.
Any
conclusions
regarding
this
hepatocarcinogenic
mode
of
action
in
adult
rodents
would
also
appear
to
apply
to
young
rodents,
and
similarly
any
conclusions
regarding
the
relevance
of
this
mode
of
action
for
human
hepatocarcinogenesis
would
apply
to
the
young,
as
well
as
the
adults.

VI.
Proposed
Science
Guidance
Proposed
Science
Policy
Statements
Although
the
precise
mechanism
for
the
formation
of
liver
tumors
by
a
PPAR 
agonist
has
not
been
established,
key
events
for
the
mode
of
action
leading
to
hepatocarcinogenesis
have
been
identified.
Key
events
for
the
mode
of
action
that
have
been
causally
related
to
liver
tumor
formation
include:
activation
of
PPAR ,
perturbation
of
cell
proliferation
and
apoptosis,
and
selective
clonal
expansion.
Key
events
that
are
associated
with
PPAR 
agonism
and
liver
tumor
formation
and
that
are
reliable
markers
that
a
chemical
has
induced
PPAR 
include
expression
of
peroxisomal
genes
(
e.
g.,
palmitoyl
CoA
oxidase,
acyl
CoA
oxidase)
and
peroxisome
proliferation
(
i.
e.,
an
increase
in
the
number
and
size
of
peroxisomes).
Page
15
of
39
It
has
been
well
established
that
chemicals
that
are
PPAR 
agonists
can
induce
liver
tumors,
and
perhaps
other
tumors,
in
rats
and
mice
but
the
potential
for
PPAR 
agonists
to
induce
liver
tumors
in
other
species,
including
humans,
appears
to
be
unlikely.
This
is
because
evidence
obtained
from
in
vivo
and
in
vitro
studies
with
hamsters,
guinea
pigs,
non­
human
primates,
and
humans
(
i.
e.,
cells
in
culture
or
biopsies)
shows
that,
quantitatively,
these
other
species
are
apparently
refractory
to
the
effects
of
a
PPAR 
agonist.
Lines
of
evidence
supporting
this
presumption
include
minimal
or
no
effects
on
peroxisome
proliferation,
peroxisomal
enzyme
activity,
or
hepatocellular
proliferation
in
species
other
than
rats
or
mice.
Moreover,
epidemiological
studies
have
not
provided
evidence
of
increased
incidence
of
liver
neoplasms
in
humans;
however,
these
data
are
inconclusive
because
of
the
limited
durations
of
exposure.

Recognizing
the
mode
of
action
data
that
show
a
linkage
between
PPAR 
agonism
and
liver
tumor
formation,
OPPTS
proposes
to
adopt
the
following
science
policy:

°
When
liver
tumors
are
observed
in
long
term
studies
in
rats
and
mice,
and
1)
the
data
are
sufficient
to
establish
that
the
liver
tumors
are
a
result
of
a
PPAR 
agonist
MOA
and
2)
other
potential
MOAs
have
been
evaluated
and
found
not
operative,
the
evidence
of
liver
tumor
formation
in
rodents
should
not
be
used
to
characterize
potential
human
hazard.

°
There
is
limited
evidence
that
a
chemical
may
induce
pancreatic
and
Leydig
cell
tumors
through
a
PPAR 
agonist
mode
of
action.
However,
the
evidence
is
inadequate
at
this
time
to
support
a
linkage
between
PPAR 
agonism
and
formation
of
these
tumor
types.
Thus,
it
is
presumed
that
chemicals
in
this
subclass
that
induce
pancreatic
or
Leydig
cell
tumors
may
pose
a
carcinogenic
hazard
for
humans.

Different
types
of
data
on
a
chemical
may
be
provided
that
indicate
that
a
chemical
induces
liver
tumors
via
a
PPAR 
agonist
mode
of
action.
The
approach
to
establishing
that
a
PPAR 
agonist
mode
of
action
is
operative
and
the
data
needed
to
support
this
presumption
are
discussed
below.

Framework
to
Establish
the
PPAR 
Agonist
MOA
The
Agency
uses
an
analytical
framework
for
judging
whether
available
evidence
for
an
agent
supports
a
mode
of
action
for
tumor
induction
in
animals
(
USEPA,
1999,
Sonich­
Mullin
et
al.,
2000).
This
framework
was
considered
in
evaluating
the
postulated
mode
of
action
for
PPAR 
agonists.

The
framework
for
analyzing
mode
of
action
begins
with
a
summary
description
of
the
postulated
mode(
s)
of
action.
This
is
followed
by
questions
to
be
addressed
to
the
available
empirical
data
and
experimental
observations
anticipated
to
be
pertinent.
The
areas
of
inquiry
in
the
framework
are:
Page
16
of
39
(
i)
identification
of
key
event(
s).
A
"
key
event"
is
defined
as
an
empirically
observable,
precursor
step
that
is
a
necessary
element
of
the
mode
of
action,
or
is
a
marker
for
such
an
element
(
e.
g.,
increased
cell
growth
and
organ
weight,
hyperplasia,
cellular
proliferation,
hormone
or
other
protein
perturbations,
receptor­
ligand
changes,
DNA
or
chromosome
effects,
cell
cycle
effects).,

(
ii)
strength,
consistency,
specificity
of
association
(
e.
g.,
causality
is
supported
by
a
significant
statistical
and
biological
association
between
key
events
and
a
tumor
response
in
well
conducted
studies
and
with
consistent
observations
in
a
number
of
such
studies,
with
differing
experimental
designs),

(
iii)
dose­
response
relationships
(
i.
e.,
key
event(
s)
and
tumor
response
increase
correlatively
with
dose),

(
iv)
temporal
relationships
(
i.
e.,
if
an
event(
s)
is
an
essential
element
of
tumorigenesis,
it
should
precede
tumor
appearance),

(
v)
biological
plausibility
and
coherence
(
i.
e.,
is
the
mode
of
action
consistent
with
what
is
known
about
carcinogenesis
in
general
and
for
the
case
specifically?),

(
vi)
other
modes
of
action
(
i.
e.,
have
alternative
modes
of
action
for
the
tumor
response
been
considered
and
are
they
supported
by
the
data?).

It
should
be
emphasized
that
the
topics
described
above
for
analysis
should
not
be
regarded
as
a
checklist
of
necessary
"
proofs".
The
judgment
whether
a
postulated
mode
of
action
is
supported
by
available
data
takes
into
account
the
weight
of
the
evidence
and
the
analysis
as
a
whole.

Data
Needs
Chemicals
can
produce
tumors
at
a
given
site
by
more
than
one
mode
of
action.
Thus,
before
a
PPAR 
agonist
MOA
can
be
defined
as
a
cause
of
the
liver
tumors,
it
is
also
critical
to
ensure
that
other
MOAs
do
not
contribute
significantly
to
the
development
of
the
tumors.
For
instance,
it
is
important
to
ensure
that
direct
DNA
reactivity
is
not
the
source
of
the
carcinogenic
findings.
The
results
of
in
vitro
and
in
vivo
short
term
tests
for
mutagenicity
and
the
evaluation
of
the
presence
of
structural
alerts
and
structure­
activity
relationships
are
helpful.
Likewise,
chemicals
producing
rodents
liver
tumors
exclusively
through
PPAR 
activation
do
not
cause
cytotoxicity;
such
findings
need
to
be
evaluated
at
doses
that
have
produced
PPAR 
agonist
precursor
effects
and
liver
tumors
to
ensure
that
cytotoxicity
is
not
prominent.

Parameters
chosen
to
demonstrate
that
activation
of
PPAR 
is
the
mode
of
action
for
the
induction
of
rodent
liver
tumors
must
be
both
sensitive
and
specific.
In
other
words,
precursor
events,
whether
causally
related
to
or
associated
with
liver
tumor
formation,
must
clearly
show
that
tumors
are
due
to
a
PPAR 
agonist
MOA
and
Page
17
of
39
exclude
other
potential
MOAs.
Valuable
data
to
address
specificity
can
also
be
obtained
from
PPAR 
knockout
mouse
bioassays.
Demonstration
of
the
absence
of
hepatocarcinogenicity
and
related
liver
toxicity
in
a
PPAR 
knockout
mouse
but
the
presence
of
liver
tumors
and
toxic
responses
in
the
wild
type
mouse
not
only
provides
evidence
that
a
PPAR 
agonist
MOA
is
operating
in
the
induction
of
the
liver
tumors
but
also
demonstrates
that
other
MOAs
(
e.
g.,
direct
mutagenic
effects,
cytotoxicity)
are
not
major
contributors
to
the
onset
of
hepatocarcinogenesis.
However,
it
is
recognized
that
a
PPAR 
knockout
mouse
is
not
generally
used
to
demonstrate
that
a
chemical
induces
liver
tumors
via
a
PPAR
 
agonist
MOA.
Thus,
other
data
may
be
used
to
demonstrate
that
a
PPAR
 
agonist
MOA
for
liver
tumor
formation
is
operative.

Demonstration
that
a
PPAR 
agonist
MOA
is
operative
can
be
shown
by
a
data
set
that
includes
in
vitro
evidence
of
PPAR 
agonism
(
i.
e.,
evidence
from
an
in
vitro
receptor
assay),
in
vivo
evidence
of
an
increase
in
number
and
size
of
peroxisomes,
increases
in
the
activity
of
acyl
CoA
oxidase,
and
hepatic
cell
proliferation.
The
in
vivo
evidence
should
be
collected
from
studies
designed
to
provide
the
data
needed
to
show
dose­
response
and
temporal
concordance
between
precursor
events
and
liver
tumor
formation.

Because
some
chemicals
have
been
shown
to
induce
little
or
no
effect
on
PPAR 
activity
but
produce
other
effects
associated
with
PPAR 
agonisms
(
ILSI,
in
press),
a
receptor
assay
should
be
used
to
demonstrate
that
a
compound
is
a
PPAR 
agonist
and
is
not
inducing
effects
via
other
PPAR
receptors.
Evidence
of
peroxisome
proliferation
is
a
fundamental
aspect
of
PPAR 
agonists
and
along
with
evidence
from
an
in
vitro
reporter
assay
provides
definitive
evidence
that
a
PPAR 
agonist
MOA
is
operative.
Increases
in
peroxisomal
enzyme
activities
are
commonly
used
markers
for
peroxisome
proliferation
and
data
from
measurements
of
peroxisomal
enzyme
activity,
when
combined
with
direct
evidence
of
peroxisome
proliferation,
enhance
the
ability
to
establish
temporal
and
dose­
response
concordance
between
key
events
and
liver
tumor
formation.
Finally,
hepatic
cell
proliferation
is
a
key,
causal
event
leading
to
the
formation
of
liver
tumors
by
PPAR 
agonists
and
evidence
of
hepatic
cell
proliferation,
when
combined
with
other
evidence,
may
also
provide
important
information
on
the
temporal
aspect
of
tumor
development
and
the
dose­
response
concordance
of
precursor
events
and
tumor
formation.

Other
information
that
is
desirable
and
may
strengthen
the
weight
of
evidence
for
demonstrating
that
a
PPAR 
agonist
MOA
is
operative
includes
data
on
hepatic
CYP4A1
induction,
palmitoyl
CoA
activity,
hepatocyte
hypertrophy,
increase
in
liver
weights,
decrease
in
the
incidence
of
apoptosis,
increase
in
microsomal
fatty
acid
oxidation,
and
enhanced
formation
of
hydrogen
peroxide.
Page
18
of
39
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APPENDIX
A­
1.
PPAR 
Activity
and
Peroxisome
Assemblage/
Content
in
Fetal
Liver
Tissue
PPAR 
activity
is
expressed
late
in
rodent
embryonic
development.
In
a
study
using
in
situ
hybridization
with
antisense
and
sense
riboprobes,
expression
of
PPAR­
 
in
E8.5
(
gestation
day
8.5),
E11.5,
E13.5,
E15.5,
and
E18.5
Sprague­
Dawley
rat
embryos
was
determined
(
Braissant
and
Wahli
1998).
Low
levels
of
transcripts
of
the
PPAR 
gene
were
first
detected
in
the
livers
of
E13.5
embryos
and
became
highly
expressed
in
E18.5
embryos.

Wilson
et
al.,
(
1991)
assayed
liver
tissue
of
gestational
day
13
to
19
Swiss­
Webster
fetal
mice
for
peroxisomal
matrix,
membrane­
associated,
and
integral
membrane
proteins.
Markers
for
these
proteins
used
in
the
study
were:
matrix
­
catalase
(
not
specific
to
peroxisomes
but
also
found
in
the
cytoplasm);
membraneassociated
protein
­
dihydroxyacetone
phosphate
acyltransferase
(
DHAP­
AT);
and
integral
membrane
protein
­
peroxisomal
membrane
protein
(
PMP
70).
DHAP­
AT,
which
provides
evidence
of
peroxisome
assemblage
during
ontogeny,
increased
about
2­
fold
in
fetuses
from
gestational
day
13
to
19.
There
were
low
levels
of
PMP
70
in
13
­
and
15­
day
fetuses;
levels
of
this
peroxisomal
membrane
protein
were
increased
somewhat
in
15­
and
19­
day
fetuses.
These
data,
and
the
fact
that
the
numerical
density
of
peroxisomes
could
not
be
quantified
in
fetal
liver
tissue
due
to
weak
staining
of
the
peroxisomes
indicate
that
the
assemblage
of
peroxisomal
proteins
to
form
plasmids
has
not
been
completed
in
the
gestational
day
19
mouse
fetus.

Catalase
and
palmitoyl
CoA
oxidase
activities
were
first
detected
in
the
gestational
day
15
Wistar
rat
fetus
(
Cibelli,
et
al.,
1988).
In
the
liver,
there
was
about
a
2­
3
fold
increase
in
catalase
activity
and
palmitoyl
CoA
oxidase
activity
between
gestational
days
19
and
birth.
Though
these
enzymes
are
not
the
products
of
PPAR 
target
genes,
they
are
indicative
of
peroxisome
proliferation
and
therefore
useful
in
establishing
the
"
competency"
of
these
test
animals
to
respond
to
PPAR 
agonist
exposure.

Peroxisomes
are
present
(
few
to
many)
in
gestational
day
19
rat
fetuses,
few
are
present
in
gestational
day
15
fetuses,
and
peroxisomes
are
not
evident
in
fetal
liver
tissue
before
gestational
days
14­
15
(
Tsukada,
et
al.,
1968;
Stefanini
et
al.,
1985;
Stefanini
et
al.,,
1989;
Cibelli
et
al.,
1988).
In
mice,
few
peroxisomes
are
found
in
15­
day
fetal
livers
(
Wilson
et
al.,
1991).
Page
26
of
39
Table
1.
Summary
of
Data
on
Peroxisome
Assemblage/
Content
and
PPAR 
Activity
in
Fetal
Liver
Tissue
Parameter
Indicator
Reference
Assemblage
(
membrane
proteins)
Swiss­
Webster
mice
­
DHAP­
AT
­
levels
increased
from
gestational
days
13­
19;

PMP
70
­
low
levels
in
gestational
day
13
and
15
fetuses
and
increased
slightly
in
gestational
day
17
to
19
fetuses.
Wilson
et
al.,
1991.

Peroxisomal
content
Few
to
many
in
gestational
day
19
rat
embryos
but
not
evident
before
gestational
days
14­
15.

Mean
number
in
thin
section
of
cell
increases
from
2.5
to
7.8
between
16
and
21
days
of
development;
7.8
in
newborn
and
30.1
in
adult.

Few
found
in
gestational
day
15
mouse
embryos;
inability
to
quantify
the
numerical
density
in
gestational
day
19
mouse
embryos
suggests
assemblage
of
peroxisomal
proteins
into
plasmids
has
not
been
completed.
Tsukada,
et
al.,
1968;
Stefanini
et
al.,

1989;
Cibelli
et
al.,
1988
Stefanini
et.
al.,
1985
Wilson,
et
al.,
1991
PPAR 
gene
or
peroxisomal
activity
Low
levels
of
PPAR 
gene
transcripts
first
detected
in
gestational
day
13.5
SD­
rat
embyros;
highly
expressed
in
gestational
day
18.5
embryos
Catalase
and
palmitoyl
CoA
oxidase
activities
first
detected
in
gestational
day
15
embryos
and
increase
2­
to
3­
fold
at
birth.
Braissant
and
Wahli,
1998
Cibelli,
et
al.,
1988
Page
27
of
39
A­
2.
Peroxisome
Assemblage/
Content
and
Peroxisomal
Enzyme
Activity
in
Neonatal
Liver
Tissue
The
activities
of
DHAP­
AT
and
alkyl
DHAP
synthetase,
enzymes
involved
in
the
synthesis
of
plasmalogens
and
that
are
found
in
peroxisomes,
were
reported
to
be
similar
in
19­
day
Sprague­
Dawley
neonates
and
adult
rats
(
Singh
and
Lazo,
1992).

Stefanini
et
al.,
(
1995)
found
no
statistically
significant
differences
in
numerical
density
or
volume
density
of
peroxisomes
in
livers
of
14,
21,
or
35­
day
F344
rat
neonates
and
no
differences
between
neonate
groups
and
adult
females.
In
a
more
recent
report
(
Stefanini
et.
al.,
1999)
no
differences
were
found
between
14
or
21­
day
neonates
or
between
these
groups
and
adult
F344
rats
in
peroxisomal
 ­
oxidation;
the
numerical
density
and
volume
density
of
liver
peroxisomes
were
also
comparable
among
groups.
However,
from
a
qualitative
standpoint,
hepatic
peroxisomes
were
reported
to
be
more
numerous
and
larger
in
adult
females
than
in
neonates.
There
were
gradual
increases
in
catalase
and
palmitoyl
CoA
oxidase
activities
among
the
age
groups
(
14­
day
neonate
<
21­
day
neonate
<
adult).
No
differences
were
reported
for
the
specific
volume
density
among
7­
8.5­
10­
13­
or
17­
day
Wistar­
derived
rat
neonates
or
among
these
neonates
or
when
compared
with
adult
data
from
a
separate
report
(
Staubli,
et
al.,
1977;
Weibel,
et
al.,
1969).

There
is
evidence
from
other
reports
that
peroxisomal
enzyme
activities
that
are
markers
of
PPAR 
expression
(
e.
g.,
palmitoyl
CoA
oxidase
and
carnitine
acetyl
transferase)
are
similar
among
control
neonatal
rats
of
different
ages
and
neonatal
and
adult
rats.
As
shown
by
Dostal
et
al.(
1987),
untreated
Sprague­
Dawley
neonatal
or
young
adult
rats
(
10,
18,
25
or
46
days
of
age)
were
found
to
have
levels
of
palmitoyl
CoA
oxidase
activity
(
range
­
3.61
­
5.31
nmol/
min/
mg
protein
in
neonates
and
young
adults)
comparable
to
adult
rats
90
days
of
age
(
4.8
nmol/
min/
mg
protein).
Carnitine
acetyl
transferase
activity
was
also
shown
to
be
comparable
among
neonates
of
different
ages
and
similar
to
adults;
carnitine
acetyl
transferase
activity
was:
6.97
+
0.06,
5.85
+
0.41,
11.4
+
1.0,
8.10
+
0.55,
6.18
+
0.41,
and
4.98
+
0.63
nmol/
min/
mg
protein
for
10,
18,
20,
25,
46,
and
90
day
rats,
respectively.
Stefanini
et.
al.,(
1999)
found
no
differences
between
14
or
21­
day
neonatal
F344
rats
or
between
these
age
groups
and
adult
rats
in
peroxisomal
 ­
oxidation
but
during
development
there
were
increases
in
catalase
(
14­
day
neonate
about
5­
fold
less
than
the
adult)
and
palmitoyl
CoA
oxidase
(
14­
day
neonate
about
2­
fold
less
than
the
adult)
activities.
In
another
study
using
F344
rats
(
Cimini
et
al.,
1994),
palmitoyl
CoA
oxidase
activity
was
found
to
be
minimal
in
14­
day
neonates
(
0.2
+
0.02
mU/
mg
protein)
when
compared
with
enzyme
activity
in
adults
(
3.7
+
0.30
mU/
mg
protein.
DHAP­
AT
was
reported
to
be
similar
in
14­
day
neonates
and
adults
and
catalase
enzyme
activity
increased
about
3­
fold
between
14
days
and
adulthood.
Page
28
of
39
Table
2.
Ontogeny
of
Peroxisome
Assemblage/
Content
and
PPAR 
Activity
in
Neonatal
Liver
Tissue
Parameter
Indicator
Reference
Assemblage
DHAP­
AT
and
alkyl
DHAP
synthetase
activities
similar
in
day
19
neonatal
and
adult
rats.

No
difference
in
DHAP­
AT
activity
between
14­
day
neonates
and
adults
Singh
and
Lazo,
1992
Cimini
et
al.,
1994
Peroxisomal
content
No
differences
in
numerical
density
or
volume
of
peroxisomes
among
14­,
21­,
or
35­
day
or
among
these
age
groups
and
adult
F344
rats.
Qualitatively,
peroxisomes
more
numerous
and
larger
in
adults.

No
differences
in
specific
volume
density
of
peroxisomes
among
Wistar­
derived
7­,
8.5­,
10­,
13­,
or
17­
day
rat
neonates
or
between
these
age­
groups
and
adults
Stefanini,
et
al.,
1995;
Stefanini,
et
al.,
1999
Staubli,
et
al.,
1977;
Weibel,
et
al.,
1969
Peroxisomal
activity
No
differences
in
peroxisomal
 ­
oxidation
among
14
day,
21
day,
and
adult
F344
rats;
palmitoyl
CoA
oxidase
2­
fold
less
in
14
day
neonates
than
in
adults.

Palmitoyl
CoA
oxidase
activity
comparable
among
10
day,
18
day,
25
day,
46
day
and
adult
SD
rats;
carnitine
acetyl
transferase
activity
similar
among
different
age
groups.

Palmitoyl
CoA
oxidase
activity
minimal
(
about
19­
30­
fold
less
than
adult
activity)
in
14­
day
F344
neonatal
rats.
Catalase
activity
(
not
specific
to
peroxisomes)
3­
fold
less
in
14­
day
neonates
than
in
adults.
Stefanini,
et
al.,
1999
Dostal,
et
al.,
1987
Cimini,
et
al.,
1994
A­
3.
Response
of
the
fetal
liver
to
the
PPAR 
agonist
clofibrate
Several
studies
have
shown
that
treatment
of
pregnant
rats
and
mice
with
the
PPAR 
agonist,
clofibrate,
can
induce
peroxisome
proliferation
in
fetal
liver
tissue.

Cibelli
et
al
(
1988)
administered
clofibrate
(
0.8%
in
the
diet)
to
Wistar
rats
at
various
stages
of
pregnancy
for
7
days.
On
the
eighth
day,
dams
were
sacrificed,
and
the
maternal
and
fetal
livers
were
removed;
some
dams
were
allowed
to
deliver
for
examination
of
newborn
livers.
Liver
weights
were
comparable
among
treated
and
control
dams
and
among
treated
and
control
fetuses.
There
was
a
qualitative
increase,
relative
to
unexposed
fetuses,
in
the
size
of
peroxisomes
in
15­
day
fetuses
from
treated
dams
and
many
peroxisomes
were
observed
in
19­
day
fetuses
and
the
newborn.
There
was
no
effect
on
catalase
activity
in
15­
or
17­
day
embryos
but
there
was
a
3­
fold
increase
in
catalase
activity
in
the
newborn.
Palmitoyl
CoA
oxidase
activity
was
increased
4­
fold
in
15­
day
embryos
and
6­
8
fold
in
19­
to
21­
day
embryos.
By
birth,
palmitoyl
CoA
oxidase
activity
was
similar
in
the
livers
of
the
treated
pups
and
dams.
In
the
dams,
palmitoyl
CoA
oxidase
activity
increased
3­
to
4­
fold
and
catalase
activity
increased
1.6­
to
1.8­
fold
during
days
15­
17
of
gestation,
and
during
gestation
days
17
to
21,
palmitoyl
CoA
oxidase
activity
increased
4­
to
5­
fold
and
catalase
activity
increased
1.4­
to
1.6
fold.
Page
29
of
39
Stefanini
et.
al.
(
1989)
treated
pregnant
Wistar
rats
with
a
diet
containing
0.8%
clofibrate
for
7
days,
and
dams
at
13,
15,
17,
19
and
21
days
of
pregnancy
were
sacrificed.
Delivery
was
induced
in
dams
close
to
term.
Livers
were
removed
from
the
fetuses
and
newborn
rats
and
morphometric
analyses
were
conducted
on
hepatocyte
peroxisomes.
The
volume
density
of
peroxisomes
and
the
peroxisomal
numerical
density
were
significantly
increased
in
all
test
animals
but
particularly
in
fetuses
over
19
days
and
the
newborn.
Increases
in
the
numerical
densities
of
15­,
17­,
19­,
21­
day
fetuses
and
the
newborn,
relative
to
controls,
were
1.5­,
1.3­,
1.8­,
2.5­,
and
2.4­
fold,
respectively.
Increases
in
the
volume
density
of
peroxisomes
for
these
same
age
groups
were
3.4­,
3.3­,
3.9­,
4.3­,
and
4.6­
fold,
respectively.

Wilson
et
al.
(
1991)
treated
pregnant
Swiss­
Webster
mice
with
400
mg/
kg/
day
clofibrate
by
gavage
from
gestation
days
6­
19.
Peroxisomal
density
was
increased
2­
fold
in
maternal
mice
after
7
days
of
treatment.
There
was
also
an
increase
in
the
number
of
peroxisomes
in
15­
day
fetuses
from
treated
dams
versus
untreated
dams
but
because
of
poor
staining
the
peroxisomal
density
could
not
be
quantified
in
the
fetuses
from
treated
dams.
Maximal
increases
in
peroxisomal
membrane
protein
70
were
also
observed
7
days
after
initiation
of
treatment
in
maternal
liver
tissue
and
general
increases
in
peroxisomal
proteins
were
observed
in
fetal
liver
tissues
from
clofibrate
treated
dams
between
13
and
19
days
of
gestation.
At
gestation
day
19,
DHAP­
AT
and
PMP
70
were
each
increased
about
2­
fold
in
clofibrate
treated
dams;
DHAP­
AT
was
also
increased
about
2­
fold
in
gestation
day
19
fetuses
but
PMP­
70
was
increased
about
5­
fold.
Catalase
activity
in
liver
tissue
of
gestational
day
19
fetuses
was
increased
somewhat
(
1.2­
fold
to
1.8­
fold)
relative
to
catalase
activity
in
fetuses
from
untreated
dams
but
there
was
no
effect
on
catalase
activity
in
treated
dams.
Because
catalase
is
found
in
the
cytoplasm
as
well
in
peroxisomes,
changes
in
catalase
activity
can
not
be
ascribed
solely
to
the
effects
on
peroxisomes.
Page
30
of
39
Table
3.
Fetal
Responses
to
the
PPAR 
Agonist,
Clofibrate.

Treatment
Fetal
Response
(
compared
to
control)
Dams
Response
(
compared
to
control)
Reference
Pregnant
Wistar
rats
treated
with
0.8%
dietary
supplement
for
1
week
prior
to
sacrifice
gestational
day
15
­

palmitoyl
CoA
oxidase
activity
increased
4­

fold;
catalase
activity
not
affected;

gestational
days
19­
21
­

palmitoyl
CoA
oxidase
activity
increased
6­

to
8­
fold;
at
birth,
specific
activity
about
equal
to
treated
dams
catalase
activity
increased
3­
fold
in
the
newborn;
specific
activity
less
than
treated
dams
peroxisomes
­
few
in
gestational
day
15
fetuses,
many
in
gestational
day
19
fetuses
and
newborn,
relative
to
controls
liver
weights
­
no
increases
during
any
stage
of
development
gestational
day
15
­

palmitoyl
CoA
oxidase
activity
increased
3.1­
fold;

catalase
activity
increased
1.8­
fold
gestational
days
19­
21
­

palmitoyl
CoA
oxidase
activity
increased
5­
fold;

catalase
activity
increased
1.4­
to
1.6­
fold
peroxisomes
­
not
examined
in
dams
liver
weights
­
no
statistically
significant
increases
during
gestation
Cibelli,
et
al.,
1988
Pregnant
Wistar
rats
treated
with
0.8%
dietary
supplement
for
1
week
prior
to
sacrifice.
Numerical
density
of
peroxisomes
­

increased
1.3­
to
2.5­
fold
in
gestational
day
15,

17,
19,
21
fetuses
or
newborn;

Volume
density
of
peroxisomes
­
increased
3.4­
to
4.6­
fold
in
gestational
day
15,
17,
19,
21
fetuses
or
newborn
Not
examined
Stefanini
et
al.,
1989
Treatment
Fetal
Response
(
compared
to
control)
Dams
Response
(
compared
to
control)
Reference
Page
31
of
39
400
mg/
kg
administered
by
gavage
to
pregnant
Swiss­

Webster
mice
from
gestation
day
6
to
gestation
day
19.
Dams
were
sacrificed
at
13,
15,
17
and
19
days
of
gestation,
and
maternal
and
fetal
livers
were
removed.
Peroxisomal
density
could
not
be
quantified
in
fetuses;
general
increases
in
peroxisomal
proteins
in
gestational
days
14­
19
fetuses;

PMP
70
increased
>
5­
fold
in
gestation
days
14­

19
fetuses;
no
effect
in
gestational
day
13
or
15
fetuses;
specific
activity
in
17­
or
19­
day
fetuses
about
4­
fold
less
than
that
in
treated
dams
DHAP­
AT
increased
about
2­
fold
in
gestational
day
14­
19
fetuses;
minimal
effect
on
gestational
day
15
fetuses;
no
effect
on
gestational
day
13
fetuses;
specific
activity
about
50­
fold
less
in
gestational
day
17
or
19
fetuses
than
in
treated
dams
Catalase
­
1.2­
to
1.8­
fold
increase
in
gestational
day
19
fetuses;
no
effect
in
gestational
day
13,
15,
or
17
gestational
day
fetsuses
Peroxisomal
density
increased
2­

fold
after
7
days
treatment;

PMP­
70
and
DHAP­
AT
each
increased
about
2­
fold
Catalase
­
unaffected
Wilson
et
al.,
1991
Page
32
of
39
A­
4.
Response
of
Neonates
Following
Lactational
Exposures
to
PPAR 
Agonists
Peroxisomal
enzyme
activities
were
increased
in
nursing
pups
from
Sprague­
Dawley
rat
dams
that
were
treated
with
0.025%
ciprofibrate
in
the
diet
from
postnatal
days
3­
19
(
Singh
and
Lazo,
1992).
DHAP­
AT
and
alkyl­
DHAP
synthetase
activities
were
increased
in
the
livers
of
19­
day
neonates
by
3.9
and
2.6­
fold,
respectively;
corresponding
increases
in
activities
of
these
two
enzymes
were
also
seen
in
the
ciprofibrate­
treated
dams
(
4.2
and
3.2­
fold,
respectively).
Liver
weights
of
treated
dams
were
increased
1.8­
fold
and
in
19­
day
pups
liver
weights
were
increased
1.5­
fold.

Cimini
et
al.
(
1994)
treated
F344
damswith
1g/
kg/
day
DEHP
by
gavage
for
up
to
21
days
from
day
of
delivery
through
lactation.
Pups
were
sacrificed
on
day
14,
day
21
or
day
35
following
14
days
of
recovery.
Relative
liver
weights
increased
1.65­
fold
in
the
dams
at
weaning,
and
1.47­
fold
in
14­
and
21­
day
pups.
At
day
21,
palmitoyl
CoA
oxidase
increased
9.3
fold
in
dams,
while
it
increased
6­
fold
in
the
nursing
pups
at
14
days
and
4.85­
fold
at
21
days.
However,
palmitoyl
CoA
oxidase
activity
was
substantially
less
in
the
pups
than
in
the
dams
treated
with
DEHP
(
pups,
1.2
mU/
mg
protein
at
14
days;
dams,
34.4
mU/
mg
protein
at
21
days).
DHAP­
AT
was
increased
about
2­
fold
in
14­
and
21­
day
neonates,
but
DHAP
AT
levels
were
unaffected
in
DEHP­
treated
dams.
Catalase
activity
was
increased
about
2­
fold
in
14­
day
and
21­
day
neonates
and
adults.
Following
14
days
of
recovery,
most
enzyme
levels
returned
to
normal
in
the
dams
and
pups,
although
catalase
activity
remained
slightly
higher
in
both
the
dams
and
pups.

In
a
separate
study,
pregnant
lactating
F344
dams
were
administered
by
gavage
1
g/
kg/
day
DEHP
for
21
days
beginning
at
the
day
of
delivery
and
the
nursed
pups
were
sacrificed
after
2
or
3
weeks,
or
following
a
14
day
recovery
period
(
Stefanini,
et
al.,
1995).
The
numerical
density
or
volume
density
of
peroxisomes
was
increased
marginally
(<
2­
fold),
relative
to
controls,
in
both
pup
groups.
Dams
treated
for
21
days
with
DEHP
showed
a
more
pronounced
increase
in
the
volume
density
of
peroxisomes
(
about
2­
fold),
but
the
numerical
density
of
peroxisomes
was
increased
in
the
dams
to
the
same
degree
as
the
2­
or
3­
week
pups.
The
increases
in
volume
density
or
numerical
density
of
peroxisomes
did
not
decline
to
control
levels
in
the
3­
week
pups
after
a
14
day
recovery
period.
Volume
density
of
peroxisomes
apparently
declined
to
close
to
control
levels
after
a
recovery
period
of
8
days
in
dams
treated
for
threeweeks
but
there
was
no
apparent
decline
in
the
numerical
density
of
peroxisomes.
Relative
liver
weights
were
increased
about
equally
in
2
and
3
week­
old
neonates
and
adults
(
1.5
to
1.6­
fold).

Neonatal
F344
rats
exposed
for
14
days
following
birth
to
nafenopin
(
NF)
or
Wy­
14,643
(
WY)
through
milk
showed
increases
in
peroxisomes
and
peroxisomal
enzyme
activities
(
Fahl,
1983).
The
dams
were
treated
twice
daily
by
gavage
with
100
mg/
kg
NF
or
WY.
Increases
in
peroxisome
numbers
were
comparable
in
lactating
dams
treated
with
nafenopin
and
their
suckling
offspring.
In
pups
exposed
lactationally
to
NF
for14
days,
there
was
a
3­
fold,
35­
fold,
29­
fold
and
14­
fold
increase
in
the
activities
of
Page
33
of
39
catalase.
carnitine
acetyl
transferase,
peroxisomal
enoyl
CoA
hydratase,
and
palmitoyl
CoA
oxidase,
respectively;
exposure
to
WY
for
14
days
resulted
in
a
3­
fold,
15­
fold,
46­
fold,
and
12­
fold
increase
in
the
activities
of
catalase.
carnitine
acetyl
transferase,
peroxisomal
enoyl
CoA
hydratase,
and
palmitoyl
CoA
oxidase,
respectively.
The
increases
in
peroxisomal
enzyme
activities
catalase
and
peroxisomal
enoyl­
CoA
hydratase
were
similar
to
those
seen
in
the
dams
treated
with
the
PPAR 
agonists
for
14
days.

Effects
of
treatment
with
ciprofibrate
on
PPAR 
expression
were
also
investigated
in
lactating
F344
rats
and
their
pups
(
Stefanini,
et
al.,
1999).
Dams
were
treated
with
ciprofibrate
(
0.025%
in
the
diet)
beginning
on
the
day
of
delivery
for
21
days
or
a
week
after
delivery
for
14
days
(
i.
e.,
days
7­
21
after
delivery).
Pups
from
dams
treated
with
ciprofibrate
for
21
days
were
sacrificed
at
14
or
21
days;
pups
from
dams
treated
with
ciprofibrate
from
day
7
to
day
21
were
sacrificed
at
21
days.
Palmitoy
CoA
oxidase
activity
was
increased
12­
14­
fold,
relative
to
controls,
in
pups
from
dams
treated
with
ciprofibrate
from
either
day
7
to
day
21
(
14
days)
or
for
21
days;
the
levels
of
activity
(
approximately
4
U/
g
protein)
were
comparable
to
adults
treated
with
ciprofibrate
for
21
days
.
However,
in
the
7­
21­
day
pups,
greater
increases
in
enzyme
activity,
relative
to
controls,
were
seen
than
in
adults
treated
with
ciprofibrate
for
14
days
and
the
level
of
enzyme
activity
in
the
pups
exposed
lactationally
to
ciprofibrate
was
also
greater
(
about
2X)
than
the
treated
dams
(
7­
21­
day
pups
­
about
4
U/
g
protein
or
a
12­
fold
increase
relative
to
controls;
adults
­
about
2
U/
mg
protein
or
a
5­
fold
increase
relative
to
controls).
Increases
in
cyanide
insensitive
peroxisomal
 ­
oxidation
activity,
up
to
10­
fold,
relative
to
controls,
were
seen
in
14­
day
pups
from
treated
dams
and
in
day
7
to
day
21
pups.
However,
the
induced
levels
of
 ­
oxidation
(
U/
g
of
liver
tissue)
in
day
1­
14
pups
and
in
day
7­
21
pups
were
less
than
the
induced
levels
of
 ­
oxidation
seen
in
dams
treated
with
ciprofibrate
for
14
days.
The
levels
of
induced
 ­
oxidation
in
21­
day
pups
from
dams
treated
with
ciprofibrate
for
21
days
were
comparable.
There
was
about
a
3
to
4­
fold
increase,
relative
to
controls,
in
numerical
density
of
liver
peroxisomes
in
day
1­
14
pups
and
in
day
7­
21
pups.
The
increase
in
this
parameter
in
dams
treated
with
ciprofibrate
for
either
14
days
or
for
21
days
was
about
2­
fold.
Finally,
increases
in
liver
weights
were
somewhat
more
pronounced
in
the
pups
lactationally
exposed
to
DEHP
than
in
treated
dams
(
relative
liver
weight
increases
­
day
1­
14
pups,
2.25X
and
day
7­
21
adults,
1.47X;
day
7­
21
pups,
2.63X
and
day
7­
21
adults,
1.47X;
day
1­
21
pups,
2.63X
and
day
1­
21
adults,
1.94X.
In
a
two­
generation
rat
reproduction
study
with
diclofop­
methyl
submitted
to
the
Agency
(
Osterburg,
1992),
increased
liver
weights
were
reported
at
100
ppm
(
the
highest
dose
tested)
in
F0
and
F1
adults
(
males
12%
and
31%;
females
26%
and
13%),
no
increases
in
liver
weights
were
reported
for
F1
male
or
female
day
4
offspring
but
increased
liver
weights
were
reported
for
F2
male
day
4
offspring
(
24%)
and
F2
female
day
4
offspring(
23%).
At
the
highest
dose
tested,
histologic
examination
showed
that
there
was
nuclear
swelling
of
hepatocytes
and
hepatocyte
hypertrophy
in
both
F0
and
F1
adults.
No
hepatic
effects
were
observed
in
F1
offspring
but
cellular
hypertrophy
and
nuclear
swelling
was
observed
in
F2
offspring.
Thus,
the
results
of
the
study
with
diclofop­
methyl
show
that,
qualitatively,
exposure
of
dams
to
a
PPAR 
Page
34
of
39
agonist
can
lead
to
effects
on
liver
weights
and
liver
histology
in
neonates
that
are
consistent
with
PPAR 
agonism.

In
contrast,
a
two­
generation
reproduction
study
with
lactofen
submitted
to
the
Agency
showed
no
effects
on
liver
weights
or
liver
histology
in
neonates(
Schroeder,
1983).
In
this
study,
liver
weights
were
increased
somewhat
(
113%)
in
F1
adult
females
treated
with
2000ppm
but
not
in
F1
or
F2
weanlings.
No
liver
histopathology
was
observed
in
F0
adult
males
(
histopathology
was
not
conducted
on
female
F0
adults)
but
intrahepatic
bile
duct
proliferation,
centrolobular
degeneration/
necrosis
was
observed
in
male
and
female
rats
dying
on
test.
Histopathology
examination
of
liver
tissue
from
F1
or
F2
weanlings
revealed
no
histopathologic
effects.

The
results
of
these
two
studies
show
that
when
dams
are
treated
with
a
PPAR 
agonist
(
i.
e.,
diclofop­
methyl)
during
gestation
and
lactation,
liver
hypertrophy
may
be
induced
in
the
nursing
neonate.
However,
the
liver
effects
of
the
PPAR 
agonist
in
neonates
are
no
more
pronounced
than
those
observed
in
adults
(
i.
e.,
diclofop­
methyl)
or
they
are
absent
in
the
neonate
but
present
in
the
adult
(
i.
e.,
lactofen).
Page
35
of
39
Table
4.
PPAR 
Agonism
in
Dams
and
in
Neonates
Exposed
by
Lactation
to
Peroxisome
Proliferators
Chemical
Treatment
Neonatal
Response
Adult
Response
Reference
Ciprofibrate
0.025%
in
the
diet
of
SD
rat
dams
from
postnatal
days
3­
19
DHAP­
AT
increased
3.9­
fold
and
alkyl­
DHAP
synthetase
increased
2.6­
fold
in
19­

day
nursing
neonates;

Liver
weights
increased
1.8­
fold
DHAP­
AT
increased
4.2­
fold
and
alkyl­

DHAP
synthetase
3.2­
fold
at
postnatal
day
19
Liver
weights
increased
1.5­
fold
Singh
and
Lazo,
1992
Ciprofibrate
F344
rat
dams
treated
with
0.025%
ciprofibrate
in
the
diet
from
day
of
delivery
to
day
21
or
a
week
after
delivery
for
14
days
palmitoyl
CoA
oxidase
activity
increased
12­
14­

fold
in
21­
day
pups
from
dams
treated
for
21
days
or
21­
day
pups
from
dams
treated
from
postnatal
days
7­
21.

peroxisomal
 ­
oxidation
increased
up
to
10­
fold
in
day­
14
pups
and
in
day
21
pups
from
dams
treated
from
day
7­
21.

numerical
density
of
peroxisomes
increased
in
3­

to
4­
fold
in
14­
day
pups
and
in
day
21
pups
from
dams
treated
from
day
7
­
21;
palmitoyl
CoA
oxidase
activity
increased
5­
fold
in
dams
treated
for
14
days
(
day
7­

21);
peroxisomal
 ­
oxidation
increased
6­
8­

fold
(
estimated
from
fig.
1)
in
adults
treated
for
14
days
or
21
days;

numerical
density
of
peroxisomes
increased
2­
fold
in
dams
treated
for
14
or
21
days.
Stefanini
et
al.,
1999
DEHP
F344
rat
dams
gavaged
with
1g/
kg/
day
from
parturition
to
day
21.
at
14
or
21
days,

palmitoyl
CoA
oxidase
increased
4.85
to
6­
fold;

DHAP­
AT
increased
about
2­
fold;
at
21
days
and
14
days
recovery,
DHAP­
AT
remained
increased
(
about
2­
fold).

catalase
increased
2­
fold.

relative
liver
weights
increased
1.47­
fold
in
14­

day
neonates
and
about
the
same
amount
in
21­

day
neonates
at
21
days:

palmitoyl
CoA
oxidase
increased
9­
fold;

DHAP­
AT
unchanged;
at
21
days
and
14
days
recovery,
DHAP­
AT
returned
to
control
levels.

catalase
increased
2­
fold.

relative
liver
weights
increased
1.65­
fold
Cimini
et
al.,

1994
DEHP
F344
rat
dams
gavage
with
1g/
kg/
day
from
parturition
to
day
21
numerical
density
and
volume
density
of
peroxisomes
increased
marginally
(<
2­
fold)
in
2­

or
3­
week
nursing
pups;
volume
or
numerical
density
of
peroxisomes
in
3­
week
pups
did
not
decline
following
a
14­
day
recovery
period.
a
more
pronounced
increase
in
volume
density
of
peroxisomes
(
about
2­
fold)
than
2­
or
3­
week
nursing
pups;
volume
density,
but
not
numerical
density,
of
peroxisomes
declined
to
control
levels
following
a
8­
day
recovery
in
dams
treated
for
21
days.
Stefanini
et
al.,
1995
Chemical
Treatment
Neonatal
Response
Adult
Response
Reference
Page
36
of
39
Nafenopin
(
NF)
or
Wy­

14,643
(
Wy)
F344
rat
dams
gavaged
with
100
mg/
kg
NF
or
Wy
twice
daily
from
delivery
to
postnatal
day
14
catalase,
NF
and
Wy
­
3­
fold
increase
at
14
days;

carnitine
acetyl
transferase,
NF
35­
fold
increase
and
Wy
15­
fold
increase;

peroxisomal
enoyl
CoA
hydratase,
NF
29­
fold
increase
and
Wy
46­
fold
increase;

palmitoyl­
CoA
oxidase,
NF
14­
fold
increase
and
Wy
12­
fold
increase.
catalase
and
peroxisomal
enoyl­
CoA
hydratase
were
similar
to
those
seen
in
the
day
14
neonates;

peroxisome
numbers
were
comparable,

qualitatively,
in
lactating
dams
treated
with
nafenopin
and
their
suckling
offspring
Fahl
et
al.,

1983
Diclofop
methyl
Sprague­
Dawley
male
and
female
rats
administered
diclofopmethyl
via
the
diet
for
two
consecutive
generations;

dose
levels
­
females
0,

0.9,
2.5,
or
8.5
mg/
kg/
day
liver
weights
­

no
increases
in
F1
male
or
female
day
4
offspring
F2
males
­
increased
24%;
F2
females
­

increased
23%

no
hepatic
histopathology
liver
weights
­

F0
males
increased
12%;
F1
males
increased
31%;

F0
females
increased
26%;
F1
females
increased
13%

nuclear
swelling
and
hepatocyte
hypertrophy
in
both
F0
and
F1
adults
Osterburg,

1992
Lactofen
Sprague­
Dawley
male
and
female
rats
administered
lactofen
via
the
diet
for
two
generations;
dose­
levels
­

F0
females
0,
3.1,
31.8,

or
121.3
mg/
kg/
day;
F1
females
3.3,
32.9,
or
121.3
mg/
kg/
day
liver
weights
­
no
increases
in
F0
or
F1
pups;
no
hepatic
histopathology
observed
in
F0
or
F1
offspring
liver
weights
­
increased
13%
in
F1
adult
females;
centrolobular
degeneration/
necrosis
was
observed
in
male
and
female
rats
dying
on
test
Schroeder,

1983
Page
37
of
39
A­
5.
Response
of
Neonates
Following
Direct
Exposures
to
PPAR 
Agonists
A
study
designed
to
investigate
the
effects
of
a
PPAR 
agonist
on
neonatal
rats
of
different
ages
was
conducted
by
Dostal
et
al.
(
1987).
Male
Sprague­
Dawley
rats
6,
14,
16,
21,
42,
or
86
days
of
age
were
administered
(
by
gavage)
daily
doses
of
DEHP
for
5
days,
and
24
hours
after
sacrifice
activities
of
hepatic
peroxisomal
enzymes,
palmitoyl
CoA
oxidase
and
carnitine
acetyltranferase
were
determined.
The
doses
administered
were
0,
10,
100,
1000,
or
2000
mg/
kg/
day.
Administration
of
1000
mg/
kg/
day
caused
significant
decreases
in
body
weight
and
mortality
(
66­
70%)
in
pups
14­
18
days
of
age,
and
administration
of
2000
mg/
kg/
day
caused
mortality
in
virtually
all
pups
of
these
ages
(
the
authors
concluded
that
body
weight
decrements
and
mortality
were
not
associated
with
effects
on
peroxisome
proliferation
activity).
At
a
non­
lethal
dose
(
100
mg/
kg/
day),
absolute
liver
weight
increases
relative
to
those
in
the
controls
were
0,
17,
3,
10,
and
14%
(
6­
10,
14­
18,
21­
25,
42­
46,
and
86­
89­
day
old
pups
and
adults,
respectively).
At
100
mg/
kg/
day,
measurements
of
palmitoyl
CoA
activity
showed
that
there
was
a
greater
increase
only
in
the
14­
18­
day
pups
when
compared
with
86­
90­
day
adults
(
6.9­
fold
increase
versus
a
3.98­
fold
increase).
A
greater
increase
in
carnitine
acetyl
transferase
also
was
shown
at
this
dose
level
only
for
14­
18
day
pups
when
compared
with
86­
90­
day
adults
(
7.8­
fold
increase
versus
a
4.4­
fold
increase).
The
data
on
increased
liver
weights
and
peroxisomal
enzyme
activities
from
this
study
indicate
that
there
is
little
difference
in
the
response
of
neonatal
or
young
adult
rats
compared
with
adult
rats
to
treatment
with
DEHP.

Administration
of
clofibrate
to
4­,
8­,
or
12­
week
old
male
or
female
F344
rats
for
7
days
(
200
mg/
kg/
day)
induced
increases
in
liver
weights,
peroxisomal
 ­
oxidation,
and
the
percentage
of
peroxisomal
area
relative
to
hepatocellular
cytoplasm
(
Yamoto,
1996).
Increases
in
these
parameters
among
the
4­,
8,
and
12­
week
old
rats,
respectively,
were:
relative
liver
weights
­
males
­
108,
161,
and
168%;
females
­
108,
119,
and
117%;
palmitoyl
CoA
oxidation
­
males
­
206,
589,
and
1072%
and
females
­
145,
152,
and
312%;
percentage
of
peroxisomal
area
to
hepatocellular
cytoplasm
­
males
­
134,
479,
and
657%
and
females
­
168,
236,
169%.
All
increases
were
statistically
significant
(
p
<
0.05).
The
results
of
this
study
indicate
that
the
effects
of
the
PPAR 
agonist
clofibrate
are
weak
in
the
immature
rat
and
that
susceptibility
to
the
effects
of
the
chemical
increase
as
rats
approach
adulthood.

Peroxisome
volume
density
was
increased
6­
fold
and
peroxisome
number
was
increased
2­
fold
following
treatment
by
intubation
of
neonatal
Wistar­
derived
rats
with
100
mg/
kg/
day
nafenopin
from
lactation
days
5
through
9
(
Staubli,
et
al.,
1977).
Following
a
recovery
period
of
7
days,
peroxisome
volume
density
and
number
closely
approached,
but
did
not
attain,
control
values.
Page
38
of
39
Table
5.
Data
on
liver
effects
in
Neonates
or
Weanlings
Exposed
Directly
to
PPAR 
agonists
Chemical
Treatment
Neonatal/
Weanling
Response
Adult
Response
Reference
DEHP
male
Sprague­
Dawley
rats
6,
14,
16,
21,
42,
or
86
days
of
age
gavaged
with
0,
100,

1000,
or
2000
mg/
kg/
day
for
5
days
(
NOTE:
1000
and
2000
mg/
kg/
day
were
lethal
doses)
at
dose
=
100
mg/
kg/
day:

Palmitoyl
CoA
oxidase
increase
6­
10
day­
3X
14­
18
day­
7X
21­
25day­
2X
carnitine
acetyl
transferase
increase
6­
10
day­
2.7X
14­
18
day­
7.8X
21­
25
day­
2.4X
increases
in
peroxisomes
­
similar
at
all
ages
(
qualitatively)

liver
weight
increases
14­
18
day
­
1.2X
at
dose
=
100
mg/
kg/
day:

Palmitoyl
CoA
oxidase
increase
42­
46
day­
2.5X
86­
90
day­
4X
carnitine
acetyl
transferase
increase
42­
46
day­
young
adult
­
3.6X
86­
90
day
adult
­
4.4X
increases
in
peroxisomes
­
similar
at
all
ages
(
qualitatively)

liver
weight
increases
42­
46
day
young
adult
­
1.1X
86­
90
day
adult
­
1.1X
Dostal
et
al.,

1987
Nafenopin
5­
day
Wistar­
derived
rat
pups
intubated
with
100
mg/
kg/
day
from
day
5
through
day
9
peroxisome
volume
density
increase
­

6­
fold
in
day
9
neonates
peroxisome
number
increase
­
2­
fold
in
9
day
neonates
no
data
Staubli,
et
al.,

1977
Chemical
Treatment
Neonatal/
Weanling
Response
Adult
Response
Reference
Page
39
of
39
Clofibrate
4­,
8­,
or
12­
week
male
and
female
F344
rats
gavaged
with
200
mg/
kg/
day
for
7
days.
palmitoyl
CoA
oxidase
increase
males
4­
week
weanling­
206
%

8­
week
young
adult
­
589%
females
4­
week
weanling
­
145%

8­
week
young
adult
­
152%

percentage
of
peroxisomal
area
to
cytoplasm
­
increase
males
4­
week
weanling­
134%

8­
week
young
adult­
479%
females
4­
week
weanling­
168%

8­
week
young
adult­
236%

increased
liver
weights
males
4­
week
weanling
­
108%

8­
week
young
adult
­
161%
females
4­
week
weanling
­
108%

8­
week
young
adult
­
119%
palmitoyl
CoA
oxidase
increase
males
12­
week
adult­
1072%
females
12­
week
adult
­
312%

percentage
of
peroxisomal
area
to
cytoplasm
­
increase
males
12­
week
adult­
657%
females
12­
week
adult
­
169%

increased
liver
weights
males
12­
week
adult
­
168%
females
12­
week
adult
­
117%
Yamoto,
1996
