OFFICE
OF
PREVENTION,
PESTICIDES,
AND
TOXIC
SUBSTANCES
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
May
22,
2002
MEMORANDUM
SUBJECT:
Lactofen
­
Report
of
the
Cancer
Assessment
Review
Committee
FROM:
Sanjivani
Diwan
Executive
Secretary
Cancer
Assessment
Review
Committee
Health
Effects
Division
(
7509C)

TO:
Robert
Fricke,
Toxicologist
Reregistration
Branch
2
Health
Effects
Division
(
7509C)

The
Cancer
Assessment
Review
Committee
met
on
January
23,
2002
to
evaluate
the
carcinogenic
potential
of
Lactofen.
Attached
please
find
the
Final
Cancer
Assessment
Document.

cc:
K.
Dearfield
R.
Hill
Y.
Woo
J.
Pletcher
CANCER
ASSESSMENT
DOCUMENT
EVALUATION
OF
THE
CARCINOGENIC
POTENTIAL
OF
LACTOFEN
(
SECOND
REVIEW)
P.
C.
CODE:
128888
FINAL
REPORT
21­
May­
2002
TXR
#
0050184
CANCER
ASSESSMENT
REVIEW
COMMITTEE
HEALTH
EFFECTS
DIVISION
OFFICE
OF
PESTICIDE
PROGRAMS
______________________________________________________________________________
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
DATA
PRESENTATION:
Robert
F.
Fricke
DOCUMENT
PREPARATION:
Robert
F.
Fricke
COMMITTEE
MEMBERS
IN
ATTENDANCE:
(
Signature
indicates
concurrence
with
the
assessment
unless
otherwise
stated).
List
the
Committee
members
Karl
Baetcke
William
Burnam
Kerry
Dearfield
Vicki
Dellarco
Richard
Hill
Nancy
McCarroll
Esther
Rinde
Joycelyn
Stewart
Clark
Swentzel
Linda
Taylor
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Duplicate
Signature
Page
(
FAX
Copy)
contains
Yin­
Tak
Woo's
signature
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
CONTENTS
Executive
Summary
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iv
I.
Introduction
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1
II.
Background
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1
III.
Studies
Reviewed
by
the
CARC
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1
IV.
Conclusions
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2
V.
Cancer
Classification
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3
Attachments
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5
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
EXECUTIVE
SUMMARY
On
January
23,
2002,
the
Cancer
Assessment
Review
Committee
(
CARC)
met
to
reevaluate
the
cancer
classification
of
lactofen
in
light
of
the
conclusions
[
TXR
No.
014590]
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC).
The
MTARC
concluded
that
lactofen
acts
via
a
mechanism
involving
peroxisome
proliferation
as
a
result
of
activation
of
peroxisome
proliferatoractivated
receptor
 
(
PPAR
 )
activation.

On
January
17,
2001,
the
MTARC
reviewed
the
merits
of
the
toxicological
data
supporting
peroxisome
proliferation
as
the
proposed
mode
of
action
for
lactofen.
Based
on
the
weight­
ofevidence
from
guideline,
as
well
as
non­
guideline
mechanistic
studies,
the
MTARC
concluded
that
there
are
sufficient
data
to
classify
lactofen
as
a
non­
genotoxic
hepatocarcinogen
in
rodents
with
peroxisome
proliferation
being
a
plausible
mode
of
action.

This
conclusion
is
based
on
the
results
of
a
special
study
in
rodents
which
evaluated
the
biochemical
and
histopathological
markers
for
peroxisome
proliferation
in
livers
(
MRID
No.:
45283904).
In
mice
there
was
a
dose­
dependent
increase
in
relative
liver
weights
and
liver
enzyme
activities
used
as
biomarkers
for
peroxisome
proliferation.
Histological
evaluations
of
the
livers
also
revealed
dosedependent
increases
in
nuclear
enlargement,
cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomal
staining.
These
results
not
only
show
dose­
dependent
increases
in
the
parameters
measured,
but
more
importantly,
a
non­
linear
dose­
response
curve.

The
NOAEL
for
this
study
was
established
at
2
ppm
(
0.3
mg/
kg/
day),
based
on
increased
activities
of
liver
enzymes
and
increased
incidence
of
liver
histopathological
findings
at
the
LOAEL
of
10
ppm
(
1.5
mg/
kg/
day).

Based
on
the
liver
tumors
seen
in
both
sexes
of
the
mouse,
and
in
consideration
of
the
mechanistic
data
provided,
the
CARC
concluded
that
lactofen
should
be
classified
as
likely
to
be
carcinogenic
to
humans
at
high
enough
doses
to
cause
these
biochemical
and
histopathological
effects
in
livers
of
rodents
but
unlikely
to
be
carcinogenic
at
doses
below
those
causing
these
changes.
Further
the
margin
of
exposure
(
MOE)
approach
should
be
used
for
estimating
human
cancer
risk,
using
a
NOAEL
of
2
ppm
(
0.3
mg/
kg/
day).
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
I.
INTRODUCTION
On
January
23,
2002,
the
Cancer
Assessment
Review
Committee
(
CARC)
met
to
evaluate
the
conclusions
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
and
to
reevaluate
the
cancer
classification
of
lactofen.

The
data
were
presented
by
Dr.
Robert
F.
Fricke
of
Reregistration
Branch
2
of
the
Health
Effects
Division.

II.
BACKGROUND
INFORMATION
Initial
evaluation
of
the
carcinogenic
potential
of
lactofen
took
place
on
February
12,
1987
by
the
Carcinogenicity
Peer
Review
Committee
(
CPRC).
Based
on
liver
tumors
seen
in
both
sexes
of
mice,
the
CPRC
concluded
that
lactofen
should
be
classified
as
a
B2
(
Probable
Human
Carcinogen)
[
Attachment
1,
Peer
Review
of
Lactofen,
Memo
from
J.
Hauswirth
to
R.
Mountfort,
dated
April
08,
1987,
TXR
No.
:
none].
Subsequent
quantitative
evaluation
of
the
tumor
data
resulted
in
a
Q
1*
of
0.119
(
mg/
kg/
day)­
1
in
human
equivalents
for
lactofen.

The
Registrant
(
Valent
U.
S.
A.)
submitted
a
petition
(
MRID
No.
45160301)
requesting
that
risk
assessment
for
lactofen
be
based
on
the
MOE
approach
rather
than
using
a
Q
1*.
The
petition
reviewed
and
summarized
earlier
data
submissions,
which
supported
peroxisome
proliferation
as
the
mechanism
of
action
of
lactofen.

On
January
17,
2001,
the
MTARC
reviewed
the
merits
of
the
toxicological
data
supporting
peroxisome
proliferation
as
the
proposed
mode
of
action
for
lactofen.
Based
on
the
weight­
ofevidence
from
guideline,
as
well
as
non­
guideline
mechanistic
studies,
the
MTARC
concluded
that
peroxisome
proliferation
is
the
mode
of
activation
of
lactofen
[
Attachment
2,
TXR
No.
014590].
The
MTARC
also
recommended
that
the
CARC
reevaluate
the
carcinogenicity
classification
of
lactofen
in
light
of
the
mode
of
action
of
lactofen
as
it
pertains
to
the
formation
of
liver
tumors
in
mice.

III.
STUDIES
REVIEWED
BY
THE
CARC
The
CARC
reviewed
summaries
of
the
mechanistic
studies
as
presented
in
the
report
of
the
MTARC
.
These
studies
included
several
non­
guideline
in
vitro
and
in
vivo
mechanism
studies
that
characterized
lactofen­
induced
peroxisome
proliferation.

Of
the
studies
evaluated
by
the
CARC,
a
non­
guideline
study,
in
which
biochemical
and
histopathological
markers
for
peroxisome
proliferation
were
measured
in
both
rat
and
mouse
livers
(
MRID
No.:
45283904),
was
considered
most
pertinent.

In
this
study,
male
and
female
mice
were
exposed
to
technical
lactofen
at
0,
2,
10,
50,
or
250
ppm
or
to
nafenopin
(
positive
control
for
peroxisome
proliferation)
at
500
ppm.
After
7
weeks
of
treatment,
the
mice
were
sacrificed
and
the
livers
examined
biochemically
and
pathologically.
Dose­
dependent
increases
were
observed
(
Table
1)
in
relative
liver
weights,
catalase
and
acyl
CoA
oxidase,
and
carnitine
acetyl
transferase
(
females
only)
was
observed.
Histological
evaluations
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
(
Table
2)
also
revealed
dose­
dependent
increases
in
nuclear
enlargement,
cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomal
staining
in
livers.
Nafenopin­
treated
mice
showed
significant
increases
in
all
of
the
parameters
measured.

These
results
not
only
show
dose­
dependent
increases
in
the
parameters
measured
(
see
above),
but
more
importantly,
a
non­
linear
dose­
response
curve
with
a
NOAEL.
A
NOAEL
of
2
ppm
(
0.3
mg/
kg/
day
using
a
conversion
factor
of
1
ppm
=
0.15
mg/
kg/
day)
was
based
on
increases
in
relative
liver
weight
(
male
mice),
carnitine
acetyl
transferase
and
palmitoyl
CoA
oxidase
(
female
mice)
at
a
LOAEL
of
10
ppm
(
1.5
mg/
kg/
day).

The
NOAEL
for
this
study
was
established
at
2
ppm
(
0.3
mg/
kg/
day),
based
on
increased
activities
of
liver
enzymes
and
increased
incidence
of
liver
histopathological
findings
at
the
LOAEL
of
10
ppm
(
1.5
mg/
kg/
day).

IV.
CONCLUSIONS:

The
CARC
concluded
that
a
NOAEL
of
0.3
mg/
kg/
day
should
be
used
to
calculate
the
MOE.
The
MOE
approach
should
be
used
for
quantification
of
risk
and
the
acceptable
MOEs
will
be
risk
management
decisions.

The
CARC
also
concluded
that
a
NOAEL
of
0.3
mg/
kg/
day
can
be
used
for
chronic
and
reproductive
NOAELs.
The
NOAEL
of
0.3
mg/
kg/
day
is
also
protective
of
carcinogenic
effects,
which
has
a
NOAEL
of
10
ppm
(
1.5
mg/
kg/
day).

Chronic
Toxicological
Effects:
From
the
results
of
a
chronic
feeding
study
in
the
dog,
the
NOAEL
was
established
at
40
ppm
(
0.8
mg/
kg/
day),
based
on
increased
incidence
of
proteinaceous
casts
in
the
kidneys
and
statistically
significant
decreases
in
the
absolute
weight
of
thyroid
and
adrenal
glands
in
males
at
the
LOAEL
of
200
ppm
(
3.96
mg/
kg/
day).
The
chronic
RfD
is
established
at
0.008
mg/
kg/
day
(
100x
uncertainty
factor).

Reproductive
Toxicity:
Based
on
the
results
of
a
two­
generation
reproduction
study
in
the
rat,
the
NOAELs
for
parental,
reproductive
and
offspring
toxicity
were
all
established
at
50
ppm
(
2.6
mg/
kg/
day).
Effects
observed
at
the
LOAEL
of
500
ppm
(
26
mg/
kg/
day)
included
parental
toxicity
consisting
of
deaths,
decreased
body
weight/
gain
and
increased
incidence
of
histopathological
findings,
reproductive
toxicity
consisting
of
decreased
male
fertility,
and
offspring
toxicity
consisting
of
decreased
pup
body
weight,
decreased
liver
weights,
and
decreased
spleen
weights.

Carcinogenicity:
Based
on
the
results
of
a
carcinogenicity
study
in
the
mouse,
the
carcinogenic
effects
were
seen
at
doses
greater
than
10
ppm
(
1.5
mg/
kg/
day)
and
there
was
an
increased
incidence
of
non­
neoplastic
and
neoplastic
liver
masses
seen
in
both
sexes
at
the
LOAEL
of
50
ppm
(
7.5
mg/
kg/
day).
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
V.
CANCER
CLASSIFICATION
In
accordance
with
the
EPA
Draft
Guidelines
for
Carcinogen
Risk
Assessment
(
July
1999),
based
on
the
liver
tumors
seen
in
both
sexes
of
the
mouse,
and
in
consideration
of
the
mechanistic
data
provided,
the
CARC
concluded
that
lactofen
should
be
classified
as
"
likely
to
be
carcinogenic
to
humans
at
high
enough
doses
to
cause
these
biochemical
and
histopathological
changes
in
the
liver
of
rodents
but
unlikely
to
be
carcinogenic
to
humans
below
those
doses
causing
these
changes."
Furthermore,
the
CARC
recommended
using
an
MOE
approach
for
estimating
human
cancer
risk.
Therefore,
a
NOAEL
of
2
ppm
(
0.3
mg/
kg/
day)
should
be
used
for
calculating
the
MOE.

Table
1:
Evaluation
of
Liver
Parameters
in
Male
and
Female
Mice
After
7
Weeks
of
Treatment
with
Lactofen
(
Technical,
78.2%;
pure,
99.8%)
or
Nafenopin
(
NAF)

Sex
Dose
(
ppm)
Relative
Liver
Wt
(
g/
100
g)
Catalase
Carnitine
Acetyl
Transferase
Palmitoyl
CoA
Oxidase
Male
0
3.9
0.34
2.92
3.1
2
(
Tech)
4.2
0.49
2.96
5.2
10
(
Tech)
5.0***
0.41
3.87***
6.1
50
(
Tech)
4.5**
0.49
3.27
7.2
250
(
Tech)
7.7***
0.94***
3.1
40***

250
(
pure)
6.2***
0.93***
6.77***
29***

Female
0
4.0
0.22
3.21
2.1
2
(
Tech)
4.1
0.26
3.71
4.3
10
(
Tech)
4.4
0.21
4.27**
11***

50
(
Tech)
4.7**
0.30*
4.68***
13***

250
(
Tech)
6.7***
0.69***
4.53**
35***

250
(
pure)
7.3***
00.44***
7.77***
27***

Male
500
NAF
12.8***
0.70***
8.31***
49***

Female
500
NAF
11.9***
0.76***
9.94***
38***

**
p

0.01;
***
p

0.001
1
Data
summarized
from
Tables
5
and
6
of
MRID
No.
452383904.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Table
2:
Evaluation
of
Histopathological
Parameters
in
Male
and
Female
Mice
After
7
Weeks
Treatment
with
Lactofen
(
Technical,
78.2%;
Pure,
99.8%)
or
Nafenopin
(
NAF)
1
Sex
Dose
(
ppm)
Nuclear
Enlargement
Cytoplasmic
Eosinophilia
Hypertrophy
Peroxisomal
Staining
Male
0



+

2
(
Tech)



+

10
(
Tech)
±
±
±
+

50
(
Tech)
+
+
+
++

250
(
Tech)
+
++
++
++

250
(
Pure)
++
+++
++
++

Female
0



+

2
(
Tech)



+

10
(
Tech)
±


+

50
(
Tech)
+
+
+
++

250
(
Tech)
++
++
++
+++

250
(
Pure)
++
+++
+++
++

Male
500
NAF
++
+++
+++
+++

Female
500
NAF
+++
+++
+++
+++

1
Data
summarized
from
Table
7
and
8
of
MRID
No.
452383904.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Attachment
1
Peer
Review
of
Lactofen,
September
12,
1987
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
I(

APR
8,
1987
[
TXR
NO.
0050971]
Subject:
Peer
Review
of
Lactofen
From:
Judith
W.
Hauswirth,
Ph.
D.
Acting
Section
Head,
Section
VI
Toxicology
Branch/
HED
(
TS­
769C)

To:
Richard
Mountfort
Product
Manger
#
23
Registration
Division
(
TS­
767C)

The
Toxicology
Branch
Peer
Review
Ccumittee
met
on
February
12,
1987
to
discuss
and
evaluate
the
weight
ot
the
evidence
on
Lactofen,
with
particular
reference
to
its
oncogenic
potential.

.
Individuals
in
Attendance:

1.
Peer
Review
Committee:
(
Signatures
indicate
concurrence
with
peer
review
unless
otherwise
stated).

Theodore
M.
Farber
William
L.
Burnan
Reto
Engler
John
A.
Quest
Esther
Rinde
Judith
W.
Hauswirth
Richard
Levy
Louis
Kasza
Richard
Hill
Robert
Beliles
2.
Reviewers:(
Non­
panel
members
responsible
for
data
presentation,­
signatures
indicate
technical
accuracy
of
panel
report).

Joycelyn
E.
Stewart
(
Reviewer)

Albin
B.
Kocialski
(
Section
Head)
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Bernice
Fisher
(
Reviewer)

Allen
Katz
(
Reviewer)

3.
Peer
Review
Members
in
Absentia:
(
Committee
members
who
were
not
able
to
attend
the
discussion;
signatures
indicate
concurrence
with
the
overall
conclusions
of
the
Committee.)

Anne
Barton
Stephen
Johnson
Diane
Beal
B.
Material
Reviewed:

The
material
available
tor
review
consisted
of
DER's
for
mouse
oncogenicity
and
lifetime
rat
feeding
studies
on
Lactofen,
summary
tables
on
incidence
of
neoplastic
and
non­
neoplastic
lesions
tor
both
studies,
historical
control
data
on
pertinent
tumors
and
Toxicology
branch
"
One­
Liners".

C.
Background
Information:

Registration
for
Cobra
.
Herbicide
(
a.
i.,
Lactofen)
is
being
sought
by
PPG
Industries.
The
herbicide
is
to
be
used
pre
and/
or
post
emergence
for
control
of
susceptible
broadleaf
weeds
affecting
soybeans,
cotton
and
peanuts.
There
are
no
present
or
previous
tolerances
in
the
USA.
Lactofen
is
supplied
as
a
manufacturing
use
concentrate
containing
60%
a.
i.
and
as
an
emulsifiable
concentrate
containing
2
pounds
per
gallon
a.
i.

The
chemical
name
of
Lactofen
is
1­(
carboethyoxy)
ethyl
5­(
2­
chloro­
4­
(
tri­
fluoromethyl)
phenoxy)­
2­
nitrobenzoate.
Lactofen
is
also
referred
to
as
PPG­
844
in
this
report.

D.
Evaluation
of
Oncogenicity
Studies:

Oncogenicity
study
in
mice
PPG­
844
Technical.
Hazleton
Laboratories
America,
Vienna,
VA.
Study
No.
250­
152,
August
29,
1985.
Accession
No.
073848.

1.
Mouse
Oncogenicity
Study:

a.
Discussion
of
Study:

Lactofen
(
78.26%
pure)
was
administered
in
the
diet
to
groups
of
60
male
and
60
female
Crl:
CD­
1
(
CP,)(
SR)
mice
at
levels
of
10,
50
and
2­
50
ppm
for
78
weeks.
The
study
was
conducted
by
Hazleton
Laboratories,
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
America
and
was
dated
(
3
August
29,
1985.
The
incidence
of
hepatocellular­­
neoplasm
is
summarized
in
the
following
table.

Incidence
of
Hepatocellular
Neoplasms
Dose
(
ppm)

Tumor
Type
0
10
50
250
Males
hepatocellular
adenoma
2/
50(
4)
2/
50(
4)
5/
50
(
10)
6/
50
(
12)
carcinoma
7/
50(
15)**
7/
50(
15)
1O/
50
(
20)
15/
50(
30)*
total
tumor
9/
50(
18)**
9/
50(
18)
15/
50(
31)
21/
50(
42)**
bearing
animals
females
Hepatocellular
adenoma.
0/
5(
7(
0)
2/
50(
4)
4/
50(
8)
3/
50
(
6)
carcinoma
2/
50(
4)**
1/
50(
2)
0/
50(
0)
5/
50(
10)
total
tumor
2/
50(
4)**
3/
50(
6)
4/
50(
8)
8/
50(
16)*
bearing
animals
The
number
in
parentheses
is
the
percentage
incidence.
Cochran­
Armitage
Trend
test
is
indicated
on
Control
and
Fisher
Exact
test
is
indicated
on
Dose
group
as:
**
p<
0.01
and
*
p<
O.
05
No
other
tumor
response
related
to
Lactofen
administration
was
seen.

Historical
control
data
was
provided
on
liver
tumors
in
CD­
1
mice
sacrificed
after
78
weeks.
The
data
was
derived
from
three
studies
conducted
from
1979­
1980
by
the
performing
laboratory.

Historical
Control
Data
Hepatocellular
Tumors
Total
incidence
Range(%)

males
adenoma
3/
140
0­
4
carcinoma
19/
140
3­
17
females
adenoma
3/
156
0­
7
carcinoma
5/
156
0­
14
The
incidence
of
hepatocellular
adenomas
and
carcinomas
was
well
outside
the
historical
control
range
tor
male
mice
at
the
highest
dose
tested
(
250
ppm).
In
females,
carcinomas
and
adenomas,
considered
alone,
were
within
the
historical
control
range.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
b.
MTD
Considerations:

Body
weights
and
food
consumption
were
unaffected
by
treatment.
Survival
was
slightly
affected
at
the
high
dose
for
males
and
mid
and
high
dose
for
females
but
the
differences
were
not
statistically
significant.
Hemoglobin,
hematocrit
and
RBC
values
were
decreased
in
the
high
dose
females
when
compared
to
controls
at
58
weeks
by
13,
16
and
11%,
respectively.
This
effect
was
not
seen
at
78
weeks
with
the
values
being
only
5,
3
and
2%
lower,
respectively
than
corresponding
controls.
Significantly
increased
liver/
body
weight
ratios
were
observed
in
all
treated
males
an
in
mid
and
high
dose
females.

Non­
neoplastic
liver
pathology
in
treated
groups
consisted
of
the
following:

1.
Hepatocytomegaly
was
increased
in
all
treated
male
groups
and
in
the
mid
and
high
dose
females;
2.
necrosis
of
individual
hepatocytes
was
increased
in
the
high
dose
males;
3.
Foci/
areas
of
cellular
alteration
were
increased
in
mid
and
high
dose
males
and
high
dose
females;
and
4.
sinusoidal
cell
pigmentation
was
increased
in
all
male
and
female
treated
groups
(
Note:
the
control
rate
was
22%
for
males
and
42%
for
females).

In
a
90­
day
CD­
1
mouse
feeding
study,
all
animals
treated
with
5000
and
10,000
ppm
Lactofen
were
found
dead
by
week
three
and
week
two,
respectively.
At
2000
ppm
statistically
significant
lower
body
weights
were
seen.
Also
at
20OO
ppm
an
increased
incidence
of
hepatocytic
vacuolization,
necrosis
of
individual
hepatocytes,
hyperplasia
of
biliary
epithelium,
hepatocellular
swelling
and
extramedullary
hematopoiesis
was
seen
in
both
sexes.

The
Committee
concurred
that
based
upon
the
toxicity,
as
seen
in
both
the
90­
day
and
oncogenicity
studies,
that
an
MTD
has
not
been
reached
in
the
latter
study
and
that
higher
dosages
(>
250
ppm)
would
have
been
tolerated
chronically.

2.
Two­
Year
Rat
feeding
study:

Two­
Year
feeding
study
of
the
oncogenicity
and
Chronic
Toxicity
of
PPG­
844
in
Rats.
Hazleton
Laboratories,
America,
Madison,
WI.
Study
N0.
6100­
100,
July
30,
1995.
Accession
Nos.
073837­
073840.

a.
Discussion
of
Study:

Lactofen
(
73.5
­
78.3%
a.
i.)
was
administered
in
the
diet
to
Charles
River
CD
Sprague­
Dawley
derived
rats
at
dosages
of
O,
50,
500,1000
and
200b.
ppm
for
two
years.
A
total
of
84
animals
per
sex
were
started
in
each
group.
Clinical
chemistry,
blood
chemistry
and
urinalysis
were
performed
at
6,
12,
18
and
24
months
on
8
rats/
sex/
group.
At
6,
12,
and
18
months
8
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
rats/
sex/
group
were
sacrificed
and
liver
tissues
were
examined
microscopically.

An
increased
incidence
of
"
neoplastic"
nodules
(
sometimes
referred
to
as
proliferative
nodules
in
the
report
but
listed
under
neoplastic
lesions)
was
seen
in
male
and
female
rats
at
the
highest
dose
tested.

Incidence
of
Proliferative
Nodules
(
Neoplastic)
Dosages
(
ppm)
0
50
500
1000
2000
Males
5+/
63(
8)
1/
59(
2)
3/
60(
5)
5/
61(
8)
15/
59(
25)**
Females
1/
60(
2)**
0/
59(
0)
2/
60(
3)
2/
58(
3)
7/
60(
12)*

+
includes
one
hepatocellular
carcinoma
The
number
in
parentheses
indicates
the
percentage
incidence.
Cochran­
Armitage
Trend
test
is
indicated
on
Control
and
Fisher
Exact
test
is
indicated
on
dose
group
as:
**
p
<
0.01
and
*
p<
0.05.

Survival
analysis
of
rat
data
in
males
indicated
a
survival
disparity.
Although
the
Cochran­
Armitage
Trend
analysis
of
tumor
rates
does
not
adjust
for
this
fact,
a
more
sensitive
method
of
Trend
analysis
(
Peto's)
would
have
resulted
in
a
smaller
p
value,
i.
e.
a
more
significant
result.
This
is
true
because
of
the
earlier
occurrence
or
greater
mortality
in
the
highest
dose
group.

No
evidence
for
early
development
of
neoplastic
lesions
was
apparent
from
the
6,
12
or
18­
month
interim
sacrifices.
The
only
hepatocellular
carcinoma
seen
was
one
in
the
male
control
group.

Historical
control
data
provided
by
the
performing
laboratory
in
untreated
Sprague­
Dawley
rats
from
1980
to
1983
indicated
that
in
males
the
total
incidence
of
neoplastic
nodules
from
four
studies
was
7/
342
with
a
range
of
1­
2%
and
in
females
the
total
incidence
of
neoplastic
nodules
was
1/
347
and
the
range
was
0­
1%.

An
increased
incidence
of
endometrial
stromal
polyps
was
observed
in
treated
females
(
p<
0.05
for
all
groups
by
Fisher's
exact
test)
when
compared
to
the
controls;
however,
the
incidence
was
within
the
historical
control
range
of
the
performing
laboratory
and
was
not
considered
to
be
treatment
related.

b.
MTD
Considerations:

The
Committee
concurred
that
the
high
dose
in
this
study
approached
an
MTD.
This
conclusion
was
based
upon:

!
a
statistically
significant
decrease
in
body
weight
in
both
sexes
when
compared
to
control
values
(
10­
15%
in
males
and
10­
18%
in
females
during
the
study);
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
!
SGOT,
SGPT
and
alanine
phosphatase
levels
were
increased
in
all
high
dose
animals
throughout
the
study;
and
!
basophilic
and
eosinophilic
foci
of
cellular
alteration
were
statistically
significantly
(
p<
O.
05)
increased
in
high
close
animals
of
both
sexes.

E.
Additional
Toxicology
Information:

1.
Metabolism:

Lactofen
is
rapidly
excreted
after
oral
administration
with
95%
of
the
radiolabel
excreted
within
72
hours
in
rats.
Male
rats
excreted
the
majority
of
the
radioactivity
in
feces
(
56%),
while
female
rats
excreted
most
in
the
urine
(
52%).

In
mice
65%
to
69%
of
the
orally
administered
radioactivity
is
excreted
within
4
days;
male
mice
excreted
50%
and
female
mice
excreted
34%
of
the
absorbed
dose
in
the
feces.
A
small
portion
remained
bound
to
mouse
liver.
In
male
rhesus
monkeys
fecal
and
urinary
excretions
of
14C
Lactofen
were
each
approximately
40%
of
the
administered
dose
and
were
complete
in
144
and
72
hours,
respectively
after
oral
administration.

Two
types
of
transformations
were
observed
from
the
metabolism
of
PPG844.
One
route
involved
hydrolysis
of
the
ethyl
ester
to
form
PPG­
947.
This
reaction
was
followed
by
further
hydrolysis
to
PPG­
84;
(
Acifluorfen,
see
#
4
below).
The
second
route
of
metabolism
involved
reduction
of
the
nitro
group
or
PPG­
844
to
form
PPG­
1576,
which
was
further
hydrolyzed
to
PPG­
2838
and
finally
to
PPG­
2053.
The
proposed
metabolic
pathway
can
be
found
in
Figure
1
(
pg.
7).

2.
Mutagenicity:

Lactofen
did
not
cause
an
increase
in
chromosomal
aberrations
when
tested
with
and
without
metabolic
activation
in
CHU
cells,
was
negative
both
with
and
without
metabolic
activation
in
the
CHQ/
HGPRT
mammalian
cell
forward
mutation
assay
and
did
not
induce
unscheduled
DNA
synthesis
in
isolated
rat
hepatocytes.
Lactofen
did
have
a
low
covalent
binding
index
to
mouse
liver
DNA
in
vivo
and
was
positive
in
the
Ames
Salmonella/
microsome
plate
test
to
tester
strain
TA
1538
without
metabolic
activation
but
at
dosages
that
precipitated
out
in
the
medium
(
50O0
and
7500
ug/
plate).

3.
Reproduction
and
Teratology:

Lactofen
was
not
embryo/
fetotoxic
when
evaluated
in
rats
and
rabbits
at
dosages
up
to
400
mq/
kg
and
20
M/
kg,
respectively.
In
a
2­
generation
reproduction
study
at
500
ppm
Lactofen
administration
resulted
in
decreased
pup
weight
and
increased
pup
heart
and
liver
weights.
The
NOEL
for
these
effects
was
50
ppm.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
METABOLISM
OF
PPG­
844
4.
Structure
Activity
Relationship:

Lactofen
and
its
metabolites
are
structurally
related
to
four
chemicals
that
are
oncogenic
in
rodents:


Acifluorfen
(
the
major
metabolite
of
Lactofen;
see
PPG­
647
Fig.
1)
induces
hepatocellular
adenomas
and
carcinomas
in
mice
but
is
negative
for
oncogenicity
in
rat;


Nitrofen
produces
hepatocellular
carcinomas
in
mice
and
pancreatic
carcinomas
in
rats;


Oxyfluorfen
produces
marginally
positive
liver
tumors
in
mice
but
is
negative
in
the
rat;
and

Fomesafen
produces
hepatocellular
adenomas
and
carcinomas
in
mice
(
Category
C
oncogen).

5.
Special
Studies:

Lactofen
is
not
an
inducer
of
microsomal
enzyme
activity.
Lactofen
(
technical
PPG­
844,
but
not
pure
PPG­
844)
and
Nafenopin,
a
known
peroxisome
proliferator,
induce
catalase
activity
and
increased
liver
peroxisome
and
alkaline
phosphatase
staining
in
rats
and
mice.
However,
liver
samples
from
Chimpanzees
treated
with
Lactofen
(
technical
PPU­
844,
5.
and
75
mg/
kg/
day
for
90
days)
showed
no
increase
in
the
activity
of
acyl
Coenzyme
A
oxidase
or
catalase
the
increased
levels
of
which
are
indicative
of
peroxisome
proliferation.

e.
Weight
of
Evidence
Considerations:

The
Committee,
considered
the
following
facts,
regarding
the
toxicology
data
on
Lactofen
to
be
of
importance
in
a
weight
of
the
evidence
determination
of
oncogenic
potential.

1.
Administration
of
Lactofen
in
the
diet
of
male
and
tamale
Crl:
CD­
1(
CR)(
BR)
mice
was
associated
with
an
increased
incidence
of
hepatocellular
tumors
(
adenomas,
carcinomas
and
total
hepatic
tumor
bearing
animals
in
males
and
adenomas
and
total
hepatic
tumor
bearing
animals
in
females).

2.
Historical
control
information
from
the
performing
laboratory
for
the
incidence
of
hepatocellular.
tumors
in
CD­
1
mice
at
78
weeks
indicated
that
the
incidence
of
hepatocellular
adenomas
and
carcinomas
was
well
outside
the
historical
control
range
for
male
mice
at
the
highest
dose
tested.
In
mice,
carcinomas
and
adenomas
at
the
highest
dose
tested
were
within
the
historical
control
range.

3.
The
MTD
was
not
reached
in
the
mouse
oncogenicity
study
and
doses
higher
than
250
ppm
could
have
been
tolerated
chronically
in
CD­
1
mice.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
4.
An
increased
incidence
of
"
neoplastic"
nodules
(
sometimes
referred
to
as
proliferative
nodules
in
the
study
report
but
listed
under
neoplastic
lesions)
was
seen
in
male
and
female
CD
sprague­
Dawley
derived
rats
at
the
highest
dose
tested
(
2000
ppm).

5.
No
evidence
tor
early
development
of
neoplastic
lesions
in
the
liver
was
apparent
from
the
6,12
or
l8­
month
interim
sacrificers
in
the
chronic
rat
feeding
study.

6.
The
MTD
was
approached
at
the
high
dose
in
the
rat
study
based
upon
increased
body
weight
gain
in
both
sexes
(>
10%),.
increased
levels
of
SGOT,
SGPT
and
alkaline
phosphatase
in
both
sexes
and
increased
basophilic
and
eosinophilic
foci
of
cellular
alteration
in
the
livers
of
both
sexes.

7.
Lactofen
is
rapidly
excreted
in
the
rat
(
95%
in
three
days).
Excretion
in
the
mouse
is
slower
since
only
b5­
69%
is
excreted
within
four
days.
Some
radioactivity
remains
bound
to
mouse
liver.

8.
Lactofen
was
negative
in
three
short
term
assays
for
mutagenicity
but
was
positive
at
high
doses
that
precipitated
in
the
medium
in
the
Ames
Salmonella
assay
(
strain
TA
1538)
and
had,
a
low
covalent
binding
index
to
mouse
liver
DNA
in
vivo.
The
latter
result
corroborates
the
finding
in
the
mouse
metabolism
study
showing
binding
radioactivity
from
14C­
Lactofen
in
the
liver.

9.
Lactofen
was
not
embryo/
fetotoxic
when
evaluated
in
rats
andrabbits
In
a
2­
generation
reproduction
study
at
500
ppm
Lactofen
administration
resulted
in­
decreased
pup
weight
and
increased
pup
heart
and
liver
weights.
The
NOEL
for
these
effects
was
50
ppm.

10.
Lactofen
and
its
metabolites
are
structurally
related
to
four
chemicals
that
are
oncogenic
in
rodents.
Acifluorfen,
Nitrofen,
Oxyfluorfen
and
Fomesafen
all
induce
hepatocellular
tumors
in
mice.

G.
Classification
of
Oncogenic
Potential:

The
Committee
concluded
based
upon
the
available
evidence
that
Lactofen
meets
the
criteria
of
a
category
B2
oncogen
(
probable
human
carcinogen).
Lactofen
met
criteria
a
for
this
category
in
that
it
induced
"
an
increased
incidence
of
malignant
tumors
or
combined
malignant
and
benign
tumors
a)
in
multiple
species
or
strains..."
In
CD­
1
mice
an
increased
incidence
of
hepatocellular
carcinomas
in
males
and
an
increased
incidence
of
combined
hepatocellular
adenomas
and
carcinomas
in
both
males
and
females
was
associated
with
administration
of
Lactofen.
In
Charles
River
CD
Sprague­
Dawley
rats
an
increased
incidence
of
liver
neoplastic
nodules
was
seen
in
both
sexes.
Although
an
increase
in
malignant
tumors
was
not
seen
in
rats,
the
Committee
felt
that
a
B2
classification
was
appropriate
since
a
tumor
response
was
seen
in
two
species
at
the
same
site.
Supportive
information
was
provided
by
structure
activity
correlations.
Four
structurally
similar
chemicals,
Acifluorfen,(
the
major
metabolite
of
Lactofen),
Nitrofen,
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
1
See
for
example
the
third
edition
of
Casarett
and
Doull's
Toxicology:
The
Basic
Science
of
Poisons
(
edited
by
C.
D.
Klaassen,
M.
0.
Amdur
and
J.
Doull),
3rd
edition
Macmillan
Publishing
Company,
New
York,
1986.
p.
136.
Oxyfluorfen
and
Fomesafen,
all
produce
hepatocellular
tumors
in
rodents.
Lactofen
was
negative
in
three
short
term
assays
for
mutagenicity,
but
did
have
a
low
binding
index,
to
mouse
liver
DNA
in
vivo.
Results
of
the
Ames/
Salmonella
plate
assay
were
positive
in
one
strain,
but
only
at
doses
that
precipitated
in
the
medium
and,
therefore,
could
not
be
interpreted
as
a
definitive
positive
result.

Since
an
MTD
was
not
reached
in
the
mouse
oncogenicity
study
on
Lactofen,
the
Committee
felt
that
the
tumor
response
seen
at,
the
highest
could
not
be
considered
to
be
a
high
dose
effect
only
and
that
it
Lactofen
had
been
tested
at
higher
dosage
levels,
as
well,
induction
of
liver
tumors
would
have
been
seen
at
multiple
dosage
levels.

The
Committee
discussed
the
registrant's
contention
that
the
mechanism
of
tumor
induction
by
Lactofen
is
through
peroxisome
proliferation
and
that
such
a
mechanism
has
a
threshold
in
contrast
to
a
non­
threshold
mechanism
attributed
to
direct
acting
carcinogens.
The
Committee
felt
that,
although
this
could
be
part
of
the
mechanism
for
liver
tumor
formation
by
Lactofen,
such
a
mechanism
has
not
been
proven
to
be
directly,
but
only
indirectly,
associated
with
liver
tumor
formation
by
this
and
other
chemicals1.

1
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Attachment
2
Lactofen
­
Report
of
the
Mechanism
of
Toxicity
Assessment
Committee
January
17,
2001
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
OFFICE
OF
PREVENTION,
PESTICIDES,
AND
TOXIC
SUBSTANCES
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
TXR
No.:
014590
March
12,
2001
MEMORANDUM
SUBJECT:
LACTOFEN:
Report
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
FROM:
Robert
F.
Fricke
Reregistration
Branch
II
Health
Effects
Division
(
7509C)

THROUGH:
Karl
Baetcke,
MTARC,
Co­
chair,
Health
Effects
Division
(
7509C)

and
Pauline
Wagner
MTARC,
Co­
chair,
Health
Effects
Division
(
7509C)

TO:
Christine
Olinger,
Risk
Assessor
Reregistration
Branch
I
Health
Effects
Division
(
7509C)

EPA
Identification
Nos
PC
Code:
128888
DP
Barcode:
D267472
Submission:
S582396
Case:
819544
Action
Requested:
The
Registrant
(
Valent
U.
S.
A.)
has
submitted
a
petition
(
MRID
No.
45160301)
requesting
that
risk
assessment
for
lactofen
be
based
on
the
MOE
approach
rather
than
using
a
Q
1*
of
0.119
(
mg/
kg/
day)­
1
in
human
equivalents
(
HED
Doc
No.:
014237,
July
12,
2000).
The
petition
reviewed
and
summarized
earlier
data
submissions,
which
supported
peroxisome
proliferation
as
the
mechanism
of
action
of
lactofen.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
Conclusions:
On
January
17,
2001,
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
reviewed
the
merits
of
the
toxicological
data
supporting
peroxisome
proliferation
as
the
proposed
mode
of
action
for
lactofen.
Based
on
the
weight­
of­
evidence
from
guideline,
as
well
as
mechanistic
studies
,
the
MTARC
concluded
that
the
evidence
presented
on
peroxisome
proliferation
indicates
that
lactofen
operates
via
a
mode
of
action
involving
PPAR
activation.

Evaluation
Criteria:
Based
on
the
International
Life
Sciences
Institute
(
ILSI)
recommendations),
the
following
criteria
were
used
to
assess
the
mode
of
action
of
lactofen:

1.)
Changes
in
liver
morphology
indicating
hepatomegaly
as
measured
by
increased
relative
liver
weights
and
increased
number
of
peroxisomes
as
measured
by
morphometric
analysis.

2.)
Evidence
of
cell
proliferation
as
measured
by
increased
relative
liver
weights
and
increased
replicative
DNA
synthesis
as
measured
by
increased
hepatocellular
BrdU
nuclear
labeling
in
light
microscopy.

3.)
Increased
levels
of
enzymes
involved
in
peroxisomal
fatty
acid
metabolism,
especially
CNinsensitive
acyl
(
palmitoyl)
CoA
oxidase
activities.

Weakness
of
Submitted
Data
Supporting
Mode
of
Action
on
Peroxisome
Proliferation:

The
only
weakness
in
the
submitted
data
was
that
BrdU
labeling
was
not
performed
to
evaluate
replicative
DNA
synthesis.
The
MTARC
concluded
that
this
shortcoming
did
not
impact
on
their
conclusions;
ample
evidence
was
submitted
which
demonstrated
hepatic
hyperplasia
after
treatment
with
lactofen.

Strengths
of
the
Submitted
Data
in
Supporting
Mode
of
Action
on
Peroxisome
Proliferation:

1.)
The
results
guideline
studies
showed
that
lactofen
is
neither
mutagenic
nor
genotoxic.
A
non­
guideline
study
showed
equivocal
(
probably
negative)
binding
of
lactofen
to
DNA.

2.)
Changes
in
liver
morphology
were
observed
in
both
rats
and
mice
treated
with
lactofen.
These
effects
include:
dose­
dependent
increase
in
relative
liver
weights
and
increased
number
of
peroxisomes
as
measured
by
electron
microscopic
analysis.
Further,
in
mice
there
was
a
dose­
dependent
increase
in
nuclear
enlargement,
cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomal
staining.

3.)
There
was
evidence
of
cell
proliferation
as
measured
by
increased
relative
liver
weights
and
histological
evidence
indicating
hepatic
hyperplasia.
These
effects
were
dose­
related
with
a
clearly
defined
threshold.

4.)
Dose­
dependent
increases
(
particularly
in
female
mice)
in
the
activities
of
hepatic
CNinsensitive
palmitoyl
CoA
oxidase
and
carnitine
acetyl
transferase.
Dose­
dependent
increase
in
CN­
insensitive
palmitoyl
CoA
oxidase
was
also
observed
in
primary
rat
hepatocytes
treated
with
lactofen.
LACTOFEN
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
5.)
The
doses
at
which
carcinogenicity
was
observed
(
mouse,
LOAEL
=
50
ppm;
rat,
LOAEL
=
2000
ppm)
were
consistent
or
higher
than
the
doses
which
caused
peroxisome
proliferation.

Other
Issues
Discussed:

The
MTARC
recommended
that
the
cancer
classification
be
reviewed
by
the
Cancer
Assessment
Review
Committee.
Since
the
toxicological
database
for
lactofen
are
relatively
extensive,
the
MTARC
further
recommended
that
the
Registrant
submit
the
lactofen
studies
to
ILSI
as
a
case
study.
1
Assessment
of
the
Mode
of
Action
for
Lactofen
I.
Background
A
joint
meeting
(
March
29,
2000)
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
and
Cancer
Assessment
Review
Committee
(
CARC)
evaluated
the
adequacy
of
the
toxicological
database
for
diclofop­
methyl
in
support
of
peroxisome
proliferation
as
the
mechanism
of
action
for
liver
carcinogenicity.
The
basis
for
the
evaluation
was
two
detailed
literature
reviews
on
the
role
of
peroxisome
proliferation
in
hepatocarcinogenesis.
One
review
1
by
the
International
Life
Sciences
Institute
(
ILSI)
evaluated
the
human
cancer
risk
of
peroxisome
proliferation,
while
the
other
2
evaluated
the
human
cancer
risk
of
the
plasticiser
di(
2­
ethylhexyl)
phthalate
(
DEHP).

As
a
result
of
this
joint
meeting
of
the
MTARC
and
CARC,
criteria
(
based
on
the
ILSI
recommendations)
were
established
which
should
be
met
before
a
non­
genotoxic
hepatocarcinogenic
substance
can
be
classified
as
a
peroxisome
proliferator
(
PP).
These
criteria
are:

1.)
Changes
in
liver
morphology
indicating
hepatomegaly
as
measured
by
increased
relative
liver
weights
and
increased
number
of
peroxisomes
as
measured
by
morphometric
analysis.

2.)
Evidence
of
cell
proliferation
as
measured
by
increased
relative
liver
weights
and
increased
replicative
DNA
synthesis
as
measured
by
increased
hepatocellular
BrdU
nuclear
labeling
in
light
microscopy.

3.)
Increased
activity
of
enzymes
involved
in
peroxisomal
fatty
acid
metabolism,
especially
acyl
(
palmitoyl)
CoA
oxidase
activities.

II.
Mechanism
of
Action
of
Peroxisome
Proliferators
PPs
are
a
diverse
group
of
chemicals
and
include
synthetic
(
industrial
plasticisers
and
solvents
and
hypolipidemic
drugs),
as
well
as
the
naturally
occurring
compounds
(
certain
fatty
acids,
prostaglandins,
and
steroids).
Although
the
synthetic
PPs
are
structurally
dissimilar,
they
share
a
similarity
with
fatty
acids
­
a
large
hydrophobic
region
and
an
aliphatic
chain
with
a
terminal
acidic
group,
usually
carboxylic
acid
(
Figure
1).

Research
over
the
past
decade
showed
that
the
rodent
is
highly
responsive
to
PPs
and
that
the
liver
is
the
primary
target
organ.
The
action
of
PPs
on
rodent
liver
leads
to
specific
and
well
characterized
toxicological
events.
Short­
term
effects
(
as
early
as
one
week)
on
the
liver
include
hepatomegaly
due
to
hypertrophy
and
hyperplasia,
increase
in
the
number
and
size
of
peroxisomes,
and
the
transcriptional
induction
of
peroxisomal
enzymes
(
acyl
CoA
oxidase),
endoplasmic
reticulum
(
cytochrome
P450)
and
cytosol
(
fatty
acid
binding
protein).
While
these
short­
term
effects
on
the
liver
are
reversible,
long­
term
exposure
leads
to
the
development
of
hepatic
cancer.

Figure
1:
Chemical
Structures
of
Some
Peroxisome
Proliferators
Lactofen
Assessment
of
Mode
of
Action
2
C
H
3
CH
3
NH
N
N
Cl
S
OH
O
O
OH
O
CH
3
C
H
3
O
O
CH
3
CH
3
O
O
CH
3
CH
3
O
O
CH
3
O
CH
3
CH
3
Wy­
14,643
Nafenopin
DEHP
Clofibrate
A
receptor­
based
mechanism
for
the
proliferation
of
peroxisomes
was
established
with
the
identification
of
peroxisome
proliferator­
activated
receptor
 
(
PPAR )
3,
which
is
a
member
of
the
nuclear
receptor
superfamily.
The
binding
of
PPAR 
to
the
retinol
X
receptor
(
RXR)
produces
a
heterodimer
which
becomes
fully
active
with
the
binding
of
a
PP
to
the
active
site.
The
active
PPAR ­
RXR­
PP
complex
is
capable
of
regulating
gene
expression
through
interaction
with
the
peroxisome
proliferator
responsive
elements
(
PPRE)
of
target
genes.

In
a
recent
review4,
the
large
number
of
PPs
preferentially
activate
PPAR ;
several
hypolilidemic
drugs
(
clofibrate)
and
Wy­
14,643
have
been
shown
to
directly
bind
to
PPAR .
They
further
point
out
that
the
ability
of
a
PP
to
bind
to
and/
or
activate
PPAR 
correlated
well
with
the
hepatocarcinogenic
potency
of
the
PP.
Further
definitive
evidence
for
the
involvement
of
PPAR 
in
the
development
of
hepatocellular
tumors
was
demonstrated
using
transgenic
mice
which
lacked
expression
of
PPAR 
mRNA.

The
introduction
of
PPAR 
null
mice(­/­)
by
Lee
et
al.
5,
the
obligatory
role
of
PPAR 
in
mediating
the
cellular
effects
of
PP
was
established.
Short­
term
exposures
to
Wy­
14,643
or
clofibrate
showed
that
while
wild­
type
(+/+)
mice
developed
the
classic
hepatic
effects,
the
effects
in
treated
null
(­/­)
mice
were
comparable
to
the
(­/­)
controls.
In
a
chronic
toxicity
study
6
(+/+)
and
(­/­)
mice
were
fed
either
basal
diet
or
diet
containing
0.1%
Wy­
14,643.
After
one
week
of
treatment,
the
(+/+)
mice
had
a
significant
increase
(
93%)
in
relative
liver
weight;
further
increases
in
relative
liver
weights
were
observed
at
5
weeks
(
245%)
and
11
months
(
355%)
of
treatment.
By
contrast,
the
relative
liver
weights
of
treated
(­/­)
mice
were
comparable
to
control
(­/­)
mice
at
all
timepoints.
To
evaluate
replicative
DNA
synthesis,
the
BrdU
labeling
index
was
measured
in
both
(+/+)
and
(­/­)
mice.
While
the
BrdU
labeling
index
was
significantly
increased
in
(+/+)
mice
after
1
and
5
weeks
of
treatment
compared
to
(+/+)
controls,
the
BrdU
labeling
indexes
of
treated
(­/­)
mice
were
comparable
to
(­/­)
controls
at
these
same
timepoints.
Gross
examination
revealed
multiple
visible
nodules
in
the
livers
of
treated
(+/+)
mice;
microscopic
examination
showed
a
100%
incidence
of
hepatocellular
neoplasms.
The
livers
of
the
treated
(­/­)
mice
were
grossly
and
microscopically
comparable
to
(­/­)
control
mice.
Lactofen
Assessment
of
Mode
of
Action
3
These
data
firmly
established
the
central,
and
necessary,
role
of
PPAR 
in
nongenotoxic
hepatocarcinogenesis.

III.
Evaluation
of
Toxicity
Database
for
Lactofen
Both
guideline
and
non­
guideline
(
mechanistic)
studies
have
been
reviewed.
Pertinent
results
of
guideline
studies
relevant
to
the
proposed
mechanism
of
action
of
lactofen
are
summarized
in
Table
1.

A.
Subchronic
Toxicity
Studies
with
Lactofen
1)
4­
Week
Feeding
Study
in
the
Rat
(
MRID
No:
00117563):
In
a
4­
week
rangefinding
study,
rats
were
fed
diets
at
0,
200,100,
5000
or
10000
ppm
lactofen
(
76%
a.
i.).
Animals
in
the
high­
dose
group
had
100%
mortality
by
day
7.
Increased
liver
and
kidney
weights
were
observed
at
doses
of
1000
ppm
and
higher.

2)
Subchronic
Feeding
Study
in
the
Rat
(
MRID
No:
00117564):
In
a
90­
day
subchronic
toxicity
study,
rats
were
fed
diets
containing
0,
40,
200
or
1000
ppm
lactofen
(
males:
0,
2.9,
14,
74
mg/
kg/
day;
females:
0,
3.5,
17,
or
85
mg/
kg/
day).
At
the
high­
dose
level,
increased
relative
(
to
body
weight)
liver
weights
in
males
(
3.4%,
control
2.8%)
and
females
(
3.1%,
control
2.7%)
and
absolute
liver
weights
in
males
(
18.4
g,
control
15.0
g)
were
observed.
Gross
findings
were
observed
only
in
high­
dose
animals
and
included
dark
livers
in
high­
dose
males
(
15/
19)
and
females
4/
21);
darkened
renal
cortex
was
also
observed
in
highdose
males
(
15/
19)
and
females
(
3/
21).
Histopathological
evaluation
of
high­
dose
animals
revealed
brown
pigmentation
in
hepatocytes
and/
or
Kupffer
cells
(
males,
17/
19;
females,
7/
21;
none
in
controls),
and
acidophilic
hepatocellular
degeneration
in
males
(
10/
19,
none
in
control)
and
females
(
1/
21,
none
in
controls)
and
hyperplasia
of
bile
ducts
in
males
(
6/
19;
controls,
1/
20).
Other
microscopic
lesions
included
brown
pigment
in
tubular
epithelium
in
the
kidneys
of
high­
dose
animals
(
males,
12/
19;
females,
3/
21;
none
in
controls).

3)
Subchronic
Feeding
Study
in
the
Mouse
(
MRID
No:
00132882):
In
a
90­
day
subchronic
toxicity
study,
mice
were
fed
diets
containing
0,
40/
2000,
200,
1000,
5000,
or
10000
ppm
lactofen
(
calculated
doses:
0,
5.7,
29/
286,
143,
714
or
1429
mg/
kg/
day).
Further
as
a
result
of
100%
mortality
in
5000
and
10000
ppm
males
and
females,
the
40
ppm
dose
was
increased
to
2000
ppm
at
week
5.
With
the
exception
of
one
female,
all
of
the
2000
ppm
animals
died
between
5
and
10
weeks
after
the
increase
in
the
dose.
Dose­
related
increases
in
serum
cholesterol
and
total
protein
levels,
as
well
as
liver
enzymes
(
ALP,
ALT
and
AST)
were
observed
in
200
and
1000
ppm
males
and
females.
Comparable
increases
in
absolute
and
relative
liver
weights
were
seen
at
200
ppm
(
males,
43
­
48%;
females,
45
­
53%)
and
1000
ppm
(
males,
198
­
221%;
females,
189
­
190%).
Gross
examination
revealed
enlarged
livers
in
200,
1000
and
2000
ppm
mice
and
enlarged
spleens
in
1000
and
2000
ppm
mice.
At
2000
and/
or
1000
ppm,
microscopic
examination
of
the
liver
showed
hepatocytic
vacuolization,
necrosis
of
individual
hepatocytes,
bile
retention,
coagulative
necrosis,
hyperplasia
of
biliary
epithelium
and
increased
extramedullary
hematopoiesis;
the
kidney
showed
nephrosis
and
cortical
fibrosis/
scarring.
Lactofen
Assessment
of
Mode
of
Action
4
B.
Chronic
Toxicity,
Carcinogenicity
and
Reproductive
Toxicity
Studies
with
Lactofen
1)
Combined
2­
Year
Chronic
Feeding/
Carcinogenicity
Study
in
the
Rat
(
MRID
No.:
00150329):
In
this
study,
rats
were
fed
diets
containing
0,
50,
500,
1000,
or
2000
ppm
(
0,
2,
19,
38,
and
76
mg/
kg/
day,
based
on
20
ppm
=
1
mg/
kg)
lactofen
for
104
weeks.
Effects
seen
at
1000
ppm
included
increased
incidence
of
mottled
diffusely
dark
livers
and
kidneys,
increased
aspartate
aminotransferase,
alanine
aminotransferase
and
alkaline
phosphatase
activities,
decreased
cholesterol,
blood
urea
nitrogen,
and
total
protein
globulin
levels,
and
increased
incidence
in
the
pigmentation
of
hepatocytes,
Kupffer
cells
and
renal
cortical
tubule
cells.
Effects
seen
at
the
2000
ppm
were
similar
to
those
seen
at
1000
ppm,
but
more
severe.
Other
effects
at
2000
ppm
included
increased
incidence
in
basophilic
or
eosinophilic
foci
of
cellular
alteration
and
increased
incidence
of
neoplastic
liver
nodules.

2)
18­
Month
Carcinogenicity
Study
in
the
Mouse
(
MRID
No.
00150343):
In
this
study,
mice
were
fed
diets
containing
lactofen
at
0,
10,
50,
or
250
ppm
(
0,
1.4,
7.1,
or
36
mg/
kg/
day,
based
on
1
ppm
=
0.143
mg/
kg)
for
78
weeks.
Effects
seen
at
50
ppm
included
increased
liver
weight,
increased
incidence
of
dark
colored
and/
or
enlarged
livers,
hepatocytomegaly
(
also
observed
in
males
at
10
ppm);
increased
incidences
of
focal
cell
alteration
(
females
only),
and
hepatocellular
adenomas
;
sinusoidal
cell
pigmentation
in
the
liver
was
observed
in
all
dose
groups.
At
the
highest­
dose
tested
(
250
ppm),
the
severity
of
these
signs
of
toxicity
was
increased.
Other
effects
noted
at
250
ppm
included
increased
incidence
of
non­
neoplastic
and
neoplastic
liver
masses
and
increased
kidney
pigmentation.

3)
Chronic
Feeding
Study
in
the
Dog
(
MRID
No:
41967901):
In
a
one­
year
toxicity
study,
dogs
were
fed
diets
containing
0,
40,
200,
or
1000/
3000
(
0,
0.79,
4.0,
20/
59
mg/
kg/
day
based
on
1
mg/
kg
=
40
ppm)
lactofen;
because
of
lack
of
significant
toxicity
at
1000
ppm,
the
dose
was
increased
to
3000
ppm
after
4­
months
of
treatment.
Dogs
fed
the
1000/
3000
ppm
diet
showed
a
slight
increase
in
peroxisomal
staining
(
based
on
the
intensity
of
brown
stippling
of
D.
A.
B.
stained
slides)
in
the
livers.
Relative
liver
weight
was
increased
in
high­
dose
females.
There
was
no
evidence
of
nuclear
enlargement,
increased
mitotic
activity,
inflammation,
or
focal
necrosis.
Lactofen
Assessment
of
Mode
of
Action
5
Table
1:
Summary
of
Guideline
Toxicity
Studies
with
Lactofen
Study
(
MRID
No.)
Liver
Weight
Liver
Enzymes
Liver
Histopathology
4­
Week
Feeding
­
Rat
(
00117563)

0,
200,
1000,
5000,
10000
ppm
100%
lethality
at
10000
ppm
5000
ppm


and


:

Increased
relative
wt
Not
measured
Not
measured
13­
Week
Feeding
­
Rat
(
00117564)

0,
40,
200,
1000
ppm


:
0,
2.9,
14,
74
mg/
kg/
day


:
0,
3.5,
17,
85
mg/
kg/
day
1000
ppm:
Increased
abs
and
rel
in


and
rel
in


1000
ppm


:
increased
ALT,
AST
and
ALK
1000
ppm,


and


:
Brown
pigmentation
in
hepatocytes
and/
or
Kupfer
cells
and
acidophilic
hepatocellular
degeneration.

13­
Week
Feeding
­
Mice
(
00132882)

0,
40/
2000,
200,
1000,
5000,
10000
ppm
0,
5.7/
286,
29,
143,
714
mg/
kg/
day
40
ppm
increased
to
2000
ppm
at
wk
7
100%
lethality
at
HDT

200
ppm


and


:

Increased
relative
and
absolute
wt

200
ppm:
Increased
ALK
in


and


,
increased
ALT
and
AST
in



1000
ppm:
Increased
ALT
and
AST
in


,

increased
total
cholesterol
in


and



200
ppm:
Enlarged
liver
in


and


,
extramedullary
hematopoiesis
in


,


1000
ppm


and


:
Hepatocellular
vacuolation,

bile
retention,
hepatocellular
swelling,
and
extramedullary
hematopoiesis

1000
ppm


:
Necrosis
of
individual
hepatocytes,

Carcinogenicity
­
Mouse
(
00150343)

0,
10,
50
,
250
ppm
0,
1.4,
7.1,
36
mg/
kg/
day

10
ppm


,

50
ppm


:
Increased
abs
and
rel
wts
Not
measured
Hepatomegaly:

10
ppm


and

50
ppm


Focus/
Area
of
Cell
Alteration:
50
ppm


and
250
ppm


Sinusoidal
cell
pigmentation.:

10
ppm
in


and


Necrosis
of
individual
hepatocytes:
250
ppm


Hepatocellular
Adenoma:

50
ppm


and


Carcinoma:
250
ppm


and


Combined:
250
ppm


and


Lactofen
Assessment
of
Mode
of
Action
Study
(
MRID
No.)
Liver
Weight
Liver
Enzymes
Liver
Histopathology
6
2­
year
Combined
Chronic/
Onco
­
Rat
(
00150329)

0,
50,
500,
1000,
2000
ppm
0,
2,
19,
38,
76
mg/
kg/
day

1000
ppm,


,

500
ppm


:
Increased
relative
wt

500
ppm,


and


:

Increased
AST,
ALT
and
ALK
at
18
mo.

1000
ppm,:
Pigmentation
in
hepatocytes(


only)
and
Kupfer
cells
(


and


)

2000
ppm:
Increased
basophilic
and
eosinophylic
(


only)
foci
of
cellular
alteration
and
proliferative
nodules
1­
Year
Chronic
Feeding
Study
­
Dog
(
41967901)

0,
40,
200,
and
1000/
3000
ppm
0,
0.8,
4,
20/
59
mg/
kg/
day
1000/
3000
ppm


:

Statistically
significant
increases
in
the
relative
liver
weight;
absolute
liver
weight
not
affected.
Not
measured
No
histopathological
findings
observed
in
the
liver
2­
Generation
Reproduction
­
Rat
(
00132885)

0,
50
500,
2000
ppm
F1
Adults

500
ppm


and
2000
ppm


:
Increased
relative
wt
2000
ppm


:

Increased
absolute
wt.

F1
Pups

500
ppm


:

Increased
absolute
wt
2000
ppm


:

Increased
relative
wt.
Not
measured
F1
Adults
2000
ppm


and


:
Centrilobular
necrosis/
degeneration,
brown
pigmentation
[
hemosiderin]
in
hepatocytes
and
reticuloendothelial
cells
Lactofen
Assessment
of
Mode
of
Action
7
4)
Reproductive
Toxicity
Study
in
the
Rat
(
MRID
No:
00132885):
In
this
study,
rats
were
fed
diets
containing
lactofen
at
0,
50,
500,
or
2000
ppm
(
F0
males/
females:
0,
2.6/
3.1,
26.2/
31.8,
103.5/
121.3
mg/
kg/
day;
F1
males/
females:
0,
2.7/
3.3,
26.732.9,
or
115.4/
138.9
mg/
kg/
day).
For
parental
groups
at
the
high­
dose
level,
there
was
increases
in
spleen
and
liver
weights;
increased
incidence
of
liver
[
hepatocytic
centrilobular
degeneration
and
necrosis]
and
spleen
[
extramedullary
hematopoiesis]
microscopic
lesions.
For
offspring
groups
no
liver
toxicity
was
noted
C.
Mutagenicity
and
Genotoxicity
Studies
with
Lactofen
The
guideline
and
non­
guideline
studies
indicate
that
lactofen
is
neither
genotoxic
nor
mutagenic.
Equivocal
(
probably
negative)
results
were
observed
in
an
in
vivo
DNA
binding
assay
with
radiolabeled
lactofen.
Results
are
summarized
in
Table
2.

Table
2:
Summary
of
Mutagenic
and
Genotoxic
Effects
of
Lactofen
Assay
Results
Salmonella
typhimurium/
mammalian
microsome
mutagenicity
assay
(
MRID
No.
00150346,
00150346)
Negative:
±
S9
including
Salmonella
typhimurium/
mammalian
microsome
mutagenicity
assay
(
MRID
No.
00150347)
Negative
±
S9
In
vitro
cytogenetic
assay
with
Chinese
hamster
ovary
(
CHO)
cells
(
MRID
No.
00150348)
Negative
for
clastogenic
effects
±
S9
In
vitro
cytogenetic
assay
with
Chinese
hamster
ovary
(
CHO)
cells
(
MRID
No.
00150626)
Negative
for
clastogenic
effects
±
S9
In
vitro
unscheduled
DNA
synthesis
in
primary
mouse
hepatocytes
(
MRID
No.
00150349
and
00162141)
Negative
In
vitro
unscheduled
DNA
synthesis
in
primary
rat
hepatocytes
(
MRID
No.
00150627)
Negative
In
vivo
DNA
covalent
binding
in
mouse
liver
(
MRID
No.
00150350)
Equivocal
(
probably
negative)
Low
level
of
binding
D.
Mechanistic
Studies
with
Lactofen
Several
non­
guideline
in
vitro
and
in
vivo
mechanism
studies
have
been
submitted
which
further
characterized
lactofen­
induced
peroxisome
proliferation.
These
studies
include
the
measurement
of
biochemical
markers
for
peroxisome
proliferation
in
the
mouse
and
rat,
as
well
as
in
primary
rat
hepatocytes,
and
a
DNA
binding
study
in
the
mouse.
A
summary
of
the
results
of
these
mechanistic
studies
is
summarized
in
the
Table
3.
Lactofen
Assessment
of
Mode
of
Action
8
Table
3:
Summary
of
Results
for
Mechanistic
Studies
with
Lactofen
Study
(
MRID
No)
Liver
Weights
Enzyme
Pathology/
histopathology
Electron
Microscopy
Analysis
of
biochemical
and
microscopic
parameters
in
Chimpanzee
liver
(
45283901,
45283905)

5
and
75
mg/
kg/
day
Not
measured
Aryl
CoA
oxidase,
catalase
and
carnitine
acetlytransferase
activities
not
affected
by
treatment
No
nuclear
enlargement,
cytoplasmic
eosinophilia
or
hepertrophy
observed
in
liver
biopsies
after
0,
1,
and
3
months
of
treatment
Slight
+
response
for
peroxisomal
staining
(
brown
stippling)

Results
of
the
Analysis
of
Biochemical
Parameters
in
Mouse
and
Rat
Liver
Following
Exposure
to
PPG­
844
(
45283904)

Mouse:
0,
2,
10,
50,
250
technical
250
ppm
pure
Rat:
0
and
2000
ppm
technical
Mice
at
7­
Weeks:


10
ppm


and

50
ppm


Increased
rel
wt
Catalase
and
CN­
insensitive
palmiloyl
CoA
oxidase
increased
in


at
250
ppm
and


at

50
ppm.
Pathology:
Rats
(
2000
ppm)
and
mice
(

50
ppm)
showed
increased
nuclear
enlargement,

cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomes
in
number
of
peroxisomes.

EM:
Rats
(
2000
ppm)
and
mice
(
250
ppm)

showed
quantitative
increase
in
the
number
of
peroxisomes
Measurement
of
Peroxisome
Proliferation
in
Primary
Rat
Hepatocytes
Induced
by
PPG­
844
and
Five
of
its
Metabolites
(
45283902)
Not
applicable
Concentration­
dependent
increase
in
CN­
insensitive
palmitoyl
CoA
oxidase
activities
with
each
of
the
metabolites.
EM:
Lactofen
(
0.01
mM)
increased
number
of
peroxisomes
and
glycogen
aggregates.

Other
metabolites
showed
occasional
peroxisomes
Lactofen
Assessment
of
Mode
of
Action
9
1)
Subchronic
Toxicity
Study
in
Male
Chimpanzees
(
MRID
No.:
45283901,
45283905):
In
this
study,
three
male
chimpanzees
per
dose
group
were
orally
dosed
with
lactofen
at
5
and
75
mg/
kg/
day
for
three
months,
followed
by
a
two­
month
recovery
period.
Whole
blood
was
collected
before
and
during
treatment
for
clinical
pathological
evaluations;
liver
biopsies
were
obtained
before
and
after
30
and
90
days
of
treatment.
Compared
to
pretreatment
values,
the
activities
of
acetyl
CoA
oxidase,
catalase
and
carnitine
acetyl
transferase
in
the
liver
were
not
affected
by
treatment.
Histopathological
evaluation
of
the
liver
biopsies
did
not
show
any
evidence
of
nuclear
enlargement,
cytoplasmic
eosinophilia,
or
hypertrophy;
peroxisome
content
in
the
liver
biopsies
did
not
show
any
change
in
the
pre­
and
posttreatment
evaluations.
Further,
electron
microscopic
evaluation
of
liver
did
not
reveal
any
evidence
of
peroxisome
proliferation.
Based
on
the
results
of
this
study
lactofen
was
not
a
peroxisome
proliferator
in
chimpanzees.

2)
Measurement
of
Biochemical
and
Histopathological
Markers
for
Peroxisome
Proliferation
in
Rat
and
Mouse
Livers
(
MRID
No.:
45283904):
In
a
dietary
study
male
and
female
Crl:
CD(
S)
Br
rats
and
Crl:
CD1
mice
were
fed
diets
containing
lactofen
or
nafenopin,
a
positive
control
for
peroxisome
proliferation.
Biochemical
markers
of
peroxisomal
proliferation
included
measurement
of
hepatic
acyl
CoA
oxidase,
catalase
and
carnitine
acetyl
transferase
activities.
Light
and
electron
microscopic
were
used
to
evaluate
livers
for
evidence
of
peroxisome
proliferation
Male
and
female
mice
were
exposed
to
technical
lactofen
at
0,
2,
10,
50,
or
250
ppm,
pure
lactofen
at
250
ppm,
or
nafenopin,
at
500
ppm.
After
7
weeks
of
treatment,
male
and
female
mice
showed
significant
biochemical
and
pathological
effects
on
the
liver.
Dosedependent
increases
in
relative
liver
weights,
catalase
and
acyl
CoA
oxidase
were
observed
in
males
and
females;
females
also
showed
a
significant
increase
in
carnitine
acetyl
transferase
(
Table
4).
Liver
histology
(
Table
5)
revealed
significant,
dose­
dependent
increases
in
nuclear
enlargement,
cytoplasmic
eosinphilia,
hypertrophy
and
peroxisomal
staining.
Nafenopintreated
mice
showed
significant
increases
in
all
of
the
parameters
measured.

Similar
findings
were
also
observed
in
an
8­
week
study,
in
which
rats
were
fed
diets
containing
lactofen
at
0
or
2000
ppm
or
nafenopin
at
500
ppm.
Lactofen­
treated
rats
had
significantly
increased
relative
liver
weights,
carnitine
acetyl
transferase
and
acyl
CoA
oxidase
(
Table
6).
Histological
examination
revealed
increased
incidence
of
nuclear
enlargement,
cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomal
staining
(
Table
7);
catalase
activity
was
not
affected
by
treatment.
Nafenopin­
treated
rats
showed
significant
increases
in
all
of
the
parameters
indicative
of
peroxisome
proliferation
Electron
microscopic
analysis
was
performed
on
the
livers
of
male
rats
and
mice.
As
a
quantitative
measurement
of
peroxisome
proliferation,
the
ratio
of
peroxisomes
to
mitochondria
were
determined.
The
ratios
were
1/
4.7
(
21%)
and
1/
3.0
(
33%)
for
control
and
treated
(
2000
ppm,
8
weeks)
male
rats,
respectively,
and
1/
4.8
(
21%)
and
1/
1.5
(
66%)
for
control
and
treated
(
250
ppm,
7
weeks)
mice,
respectively.

Table
4:
Evaluation
of
Liver
Parameters
in
Male
and
Female
Mice
After
7
Weeks
of
Treatment
with
Lactofen
(
Technical,
78.2%;
pure,
99.8%)
or
Nafenopin
(
NAF)
Lactofen
Assessment
of
Mode
of
Action
10
Sex
Dose
(
ppm)
Relative
Liver
Wt
(
g/
100
g)
Catalase
Carnitine
Acetyl
Transferase
Palmitoyl
CoA
Oxidase
Male
0
3.9
0.34
2.92
3.1
2
(
Tech)
4.2
0.49
2.96
5.2
10
(
Tech)
5.0***
0.41
3.87***
6.1
50
(
Tech)
4.5**
0.49
3.27
7.2
250
(
Tech)
7.7***
0.94***
3.1
40***

250
(
pure)
6.2***
0.93***
6.77***
29***

Female
0
4.0
0.22
3.21
2.1
2
(
Tech)
4.1
0.26
3.71
4.3
10
(
Tech)
4.4
0.21
4.27**
11***

50
(
Tech)
4.7**
0.30*
4.68***
13***

250
(
Tech)
6.7***
0.69***
4.53**
35***

250
(
pure)
7.3***
00.44***
7.77***
27***

Male
500
NAF
12.8***
0.70***
8.31***
49***

Female
500
NAF
11.9***
0.76***
9.94***
38***

**
p

0.01;
***
p

0.001
1
Data
summarized
from
Tables
5
and
6
of
MRID
No.
452383904.

Table
5:
Evaluation
of
Histopathological
Parameters
in
Male
and
Female
Mice
After
7
Weeks
Treatment
with
Lactofen
(
Technical,
78.2%;
Pure,
99.8%)
or
Nafenopin
(
NAF)
1
Sex
Dose
(
ppm)
Nuclear
Enlargement
Cytoplasmic
Eosinphilia
Hypertrophy
Peroxisomal
Staining
Male
0



+

2
(
Tech)



+

10
(
Tech)
±
±
±
+

50
(
Tech)
+
+
+
++

250
(
Tech)
+
++
++
++

250
(
Pure)
++
+++
++
++

Female
0



+

2
(
Tech)



+

10
(
Tech)
±


+

50
(
Tech)
+
+
+
++

250
(
Tech)
++
++
++
+++

250
(
Pure)
++
+++
+++
++

Male
500
NAF
++
+++
+++
+++

Female
500
NAF
+++
+++
+++
+++

1
Data
summarized
from
Table
7
and
8
of
MRID
No.
452383904.
Lactofen
Assessment
of
Mode
of
Action
11
Table
6:
Evaluation
of
Liver
Parameters
in
Male
and
Female
Rats
After
8
Weeks
of
Dietary
Treatment
with
Lactofen
(
Technical,
78.2%)
or
Nafenopin
(
NAF)

Sex
Dose
(
ppm)
Relative
Liver
Wt
(
g/
100
g)
Catalase
Carnitine
Acetyl
Transferase
Palmitoyl
CoA
Oxidase
Male
0
3.0
0.37
3.5
3.4
2000
4.3***
0.36
6.4***
8.6**

Female
0
2.8
0.20
7.3
5.3
2000
3.8***
0.19
2.4***
18.3***

Male
500
NAF
5.4***
0.60***
2.9
13.3***

Female
500
NAF
3.7***
0.30***
10.2***
12.5**

**
p

0.01;
***
p

0.001
1
Data
summarized
from
Table
11
of
MRID
No.
452383904.

Table7:
Evaluation
of
Histopathological
Parameters
in
Male
and
Female
Rats
After
8
Weeks
Treatment
with
Lactofen
(
Technical,
78.2%)
or
Nafenopin
(
NAF)
1
Sex
Dose
(
ppm)
Nuclear
Enlargement
Cytoplasmic
Eosinphilia
Hypertrophy
Peroxisomal
Staining
Male/
0



+

2000
++
++
++
++

Female
0



+

2000
++
++
++
++

Male
500
NAF
++
++
++
+++

Female
500
NAF
++
++
++
++

1
Data
summarized
from
Table
12
of
MRID
No.
452383904.

3.)
Measurement
of
Biochemical
Markers
for
Peroxisome
Proliferation
in
Primary
Rat
Hepatocytes
(
MRID
No.:
45283902):
In
this
study,
primary
cultures
of
rat
hepatocytes
were
exposed
to
PPG­
844
(
lactofen,
99.8%)
or
five
of
its
principal
animal
metabolites
(
structures
Figure
2):
PPG­
947
(
desethyl
lactofen
98.9%
purity),
PPG­
847
(
acrifluorfen,
100%
purity),
PPG­
1576
(
amino
lactofen,
95.6%
purity),
and
PPG­
2053
(
amino
acifluorfen,
94.6%
purity),
PPG­
2838
(
desethyl
amino
lactofen,
70.6%
purity).
Because
of
the
low
purity
of
PPG
2838,
it
was
excluded
from
the
definitive
study.
CN­
insensitive
palmitoyl
CoA
oxidase
activity,
a
biomarker
for
peroxisome
proliferation
was
measured
after
68
hr
of
exposure.
Solvent
(
DMSO)
and
positive
(
clofibrate)
were
also
evaluated.

Additionally,
control,
lactofen,
metabolite
and
clofibrate­
treated
hepatocyte
were
examined
using
an
electron
microscope.

CN­
insensitive
palmitoyl
CoA
oxidase
data
are
summarized
in
Table
8.
Of
the
test
chemicals
evaluated,
treatment
with
lactofen
resulted
in
greater
than
5­
fold,
concentrationdependent
increase
in
palmitoyl
CoA
oxidase
activity
compared
to
the
solvent
control.
the
highest
increase
for
the
metabolites
was
approximately
3­
fold.
Decending
order
of
potency,
based
on
palmitoyl
CoA
oxidase
activity,
was
PPG­
844,
­
947,
­
847,
­
1576
and
­
2053.
Lactofen
Assessment
of
Mode
of
Action
12
C
H
3
O
O
CH
3
O
Cl
F
F
F
O
O
N
+

O
O
C
H
3
OH
O
O
C
l
F
F
F
O
O
NH
3
+

C
H
3
OH
O
O
Cl
F
F
F
O
O
N+
O
O
C
H
3
O
O
CH
3
O
Cl
F
F
F
O
O
NH
3
+

O
Cl
F
F
F
OH
O
N+
O
O
O
Cl
F
F
F
OH
O
NH
3
+
Clofibrate
produced
significant
increases
(
6.9
to
7.7­
fold)
in
enzyme
activity
at
both
of
the
concentrations
test.

Electron
micrographs
of
lactofen
and
clofibrate­
treated
hepatocytes
revealed
an
increased
numbers
of
dense,
anucleoide
peroxisomes
compared
to
the
solvent
control.
Additionally,
lactofen­
treated
hepatocytes
showed
increased
amounts
of
cytoplasmic
glycogen
aggregates.
Treatment
with
the
metabolites
did
not
cause
any
increase
in
the
number
of
peroxisomes
relative
to
the
control.

Figure
2:
Structures
of
Lactofen
and
Primary
Metabolites
Lactofen
(
PPG­
844)
Desethyl
amino
lactofen
(
PPG­
2838)

Desethyl
Lactofen
(
PPG­
947)
Amino
Lactofen
(
PPG­
2838
)

Acifluorfen
(
PPG­
847
)
Amino
Acifluorfen
(
PPG­
2053)
Lactofen
Assessment
of
Mode
of
Action
13
Table
8:
Effect
of
Lactofen
and
Metabolites
on
CN­
Insensitive
Palmitoyl
CoA
Oxidase
Activity
(
nmol/
min/
mg
protein)
in
Primary
Rat
Hepatocytes
Addition
Concentration,
mM
0.003
0.01
0.03
0.10
PPG­
844
(
Lactofen)
1.55*
2.47*
NT
2.63*

PPG­
947
1.61*
1.68*
NT
1.74*

PPG­
847
NT
2
1.35*
1.35*
1.75*

PPG­
1576
NT
1.26*
0.743
1.573
PPG­
2053
NT
0.86
0.983
1.46*

Controls
Solvent
Control
(
DMSO)
0.50
Positive
Control
Clofibrate,
0.16
mM
3.46*

Clofibrate,
0.50
mM
3.86*

1
Data
summarized
from
Table
2
of
MRID
No.
45283902.
2
NT
=
not
tested
3
Mean
of
two
observations,
significance
not
determined.
*
Significantly
different
from
solvent
control,
p

0.05.

IV.
Other
Modes
of
Action
Studies
with
transgenic
mouse
confirmed
that
essentially
all
of
the
of
the
effects
of
PPs
in
rodent
liver
are
mediated
by
PPAR .

V.
Recommendations
to
the
MTARC
Based
on
the
weight­
of­
the­
evidence
of
the
toxicity
database,
there
are
sufficient
data
to
classify
lactofen
as
a
non­
genotoxic
hepatocarcinogen
in
rodents
with
peroxisome
proliferation
being
a
plausible
mode
of
action.

VI.
Relevancy
to
Humans
The
human
relevancy
of
the
role
of
peroxisome
proliferators
was
not
addressed,
and
will
be
deferred
pending
a
proposed
ILSI
evaluation
of
pertinent
data.

VI.
References
Cattly,
R.
C.,
DeLuca,
J.,
Elcombe,
C.,
Fenner­
Crisp,
P.,
Lake,
B.
G.,
Marsman,
D.
S.,
Pastoor,
T.
A.,
Popp,
J.
A.,
Robinson,
D.
E.,
Schwetz,
B.,
Tugwood,
J.,
and
Wahli,
W.,
Do
peroxisome
proliferating
compounds
pose
a
hepatocarcinogenic
hazard
to
humans?
Regulatory
Toxicology
and
Pharmacology
27:
47­
60
(
1998).

Doull,
J.,
Cattley,
R.,
Elcombe,
C.,
Lake,
B.
G.,
Swenberg,
J.,
Wilkinson,
C.,
Wiliams,
G.,
and
van
Gemert,
M.
A
cancer
risk
assessment
of
di(
2­
ethylhexyl)
phthaltae:
Application
of
the
new
U.
S.
EPA
Risk
Assessment
Guidelines,
Regulatory
Toxicology
and
Pharmacology
29:
327­
357
(
1999).
Lactofen
Assessment
of
Mode
of
Action
14
1.
Cattly,
R.
C.,
DeLuca,
J.,
Elcombe,
C.,
Fenner­
Crisp,
P.,
Lake,
B.
G.,
Marsman,
D.
S.,
Pastoor,
T.
A.,
Popp,
J.
A.,
Robinson,
D.
E.,
Schwetz,
B.,
Tugwood,
J.,
and
Wahli,
W.,
Do
peroxisome
proliferating
compounds
pose
a
hepatocarcinogenic
hazard
to
humans?
Regulatory
Toxicology
and
Pharmacology
27:
47­
60
(
1998).

2.
Doull,
J.,
Cattley,
R.,
Elcombe,
C.,
Lake,
B.
G.,
Swenberg,
J.,
Wilkinson,
C.,
Wiliams,
G.,
and
van
Gemert,
M.
A
cancer
risk
assessment
of
di(
2­
ethylhexyl)
phthaltae:
Application
of
the
new
U.
S.
EPA
Risk
Assessment
Guidelines,
Regulatory
Toxicology
and
Pharmacology
29:
327­
357
(
1999).

3.
Issemann,
I.
And
Green,
S.,
Activation
of
a
member
of
the
steroid
hormone
receptor
superfamily
by
peroxisome
proliferators.
Nature
(
London)
347:
645­
650
(
1990).

4.
Corton,
J.
C.,
Lapinskas,
P.
J.
and
Gonzalex,
F.
J.,
Central
role
of
PPAR 
in
the
mechanism
of
actions
of
hepatocrcinogenic
peroxide
proliferators,
Mutation
Research,
448:
139­
151
(
2000).

5.
Lee,
S.
S.,
Pineau,
T.,
Drago,
J.,
Lee,
E.
J.,
Owens,
J.
W.,
Kroetz,
D.
L.,
Fernandez­
Saluguero,
P.
M.,
Westphal,
H.,
and
Gonzalez,
F.
J.,
Targeted
disruption
of
the
 
isoform
of
the
peroxisome
proliferator­
activated
receptor
gene
in
mice
results
in
abolishment
of
the
pleiotropic
effects
of
peroxisome
proliferators,
Mol.
Cell.
Biol.,
15:
3012­
3022
(
1995).

6.
Peters,
J.
M.,
Cattley,
R.
C.
and
Gonzalez,
F.
J.,
Role
of
PPAR

in
the
mechanism
of
action
on
nongenotoxic
carcinogen
and
peroxisome
proliferator
Wy­
14,643,
Carcinogenesis,
18,
2029­
2033
(
1997).
Issemann,
I.
And
Green,
S.,
Activation
of
a
member
of
the
steroid
hormone
receptor
superfamily
by
peroxisome
proliferators.
Nature
(
London)
347:
645­
650
(
1990).

Corton,
J.
C.,
Lapinskas,
P.
J.
and
Gonzalex,
F.
J.,
Central
role
of
PPAR 
in
the
mechanism
of
actions
of
hepatocrcinogenic
peroxide
proliferators,
Mutation
Research,
448:
139­
151
(
2000).

Lee,
S.
S.,
Pineau,
T.,
Drago,
J.,
Lee,
E.
J.,
Owens,
J.
W.,
Kroetz,
D.
L.,
Fernandez­
Saluguero,
P.
M.,
Westphal,
H.,
and
Gonzalez,
F.
J.,
Targeted
disruption
of
the
 
isoform
of
the
peroxisome
proliferatoractivated
receptor
gene
in
mice
results
in
abolishment
of
the
pleiotropic
effects
of
peroxisome
proliferators,
Mol.
Cell.
Biol.,
15:
3012­
3022
(
1995).

Peters,
J.
M.,
Cattley,
R.
C.
and
Gonzalez,
F.
J.,
Role
of
PPAR

in
the
mechanism
of
action
on
nongenotoxic
carcinogen
and
peroxisome
proliferator
Wy­
14,643,
Carcinogenesis,
18,
2029­
2033
(
1997).
