Document ID: EPA-HQ-OPP-2003-0293-0006
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-09-04T04:00Z

­
1­
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
DATE:
July
9,
2003
MEMORANDUM
TXR
No.
0052006
SUBJECT:
Mechanism
of
Toxicity
SARC
Second
Report:
Acifluorfen
(
PC
Code:
114402)

FROM:
Paul
Chin
Reregistration
Branch
I
Health
Effects
Division
(
7509C)

TO:
Pauline
Wagner,
Co­
Chair
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
Health
Effects
Division
(
7509C)
and
Karl
Baetcke,
Co­
Chair
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
Health
Effects
Division
(
7509C)

cc:
Anna
Lowit
(
RRB2),
Kit
Farwell
(
RRB1),
Christina
Scheltema
(
SRRD)

The
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
reviewed
the
toxicological
data
supporting
peroxisome
proliferation
as
a
proposed
mode
of
action
for
the
carcinogenic
effects
of
acifluorfen
in
mice
on
April
17,
2003.
The
first
report
contains
the
conclusions
from
this
meeting.

At
the
recent
Cancer
Assessment
Review
Committee
(
CARC)
meeting
(
May
21,
2003),
the
CARC
concluded
that
the
increased
levels
of
enzymes
(
ALP
and
SGPT)
seen
at
40.5
mg/
kg/
day
in
the
carcinogenicity
study
in
mice
(
CD­
1)
were
considered
equivocal
because
of
large
standard
deviations.
Therefore,
the
first
MTARC
report
was
revised
to
reflect
this
conclusion.

THIS
DOCUMENT
SUPERSEDES
THE
PREVIOUS
MTARC
REPORT
DATED
MAY
14,
2003
(
TXR
No.
0050227).
­
2­
Committee
Members
in
Attendance
Members
who
were
present
and
gave
electronic
concurrence
to
this
report
were:
Vicki
Dellarco,
Alberto
Protzel,
Anna
Lowit,
Ayaad
Assaad,
Karen
Hamernik,
Karl
Baetcke,
Pauline
Wagner,
Elizabeth
Mendez,
and
John
Doherty.

Also
in
attendance
were:
Whang
Phang
(
RRB1),
Michael
Metzger
(
RRB1),
Kit
Farwell
(
RRB1),
Tawanda
Spears
(
SRRD),
Brian
Dementi
(
TOX)

Data
Evaluation
/
Report
Preparation
Byong­
Han
Chin,
Ph.
D
Toxicologist
­
3­
I.
Conclusion
The
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC)
reviewed
and
debated
the
toxicological
data
supporting
peroxisome
proliferation
as
a
proposed
mode
of
action
for
the
carcinogenic
effects
of
acifluorfen
in
mice.
The
MTARC
concluded
that
the
currently
available
data
are
considered
to
be
sufficient
to
support
peroxisome
proliferation
as
the
mode
of
action
of
acifluorfen­
induced
liver
tumors
in
mice
according
to
the
criteria
recommended
by
International
Life
Sciences
Institute
(
ILSI);
these
include:

1.
Changes
in
liver
morphology
were
observed
in
both
rats
and
mice
treated
with
acifluorfen.
These
effects
include:
dose­
dependent
increase
in
relative
liver
weights,
and
increased
incidence
of
cellular
hypertrophy,
and
increased
number
of
peroxisomes.
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.
Dose­
dependent
increase
in
the
levels
of
CN­
insensitive
acyl
(
palmitoyl)
CoA
oxidase
activities
involved
in
peroxisomal
fatty
acid
metabolism.

Previously,
the
MTARC
concluded
that
the
peroxisome
proliferation
is
the
mode
of
action
of
lactofen
in
inducing
liver
tumors
in
rodents.
Since
acifluorfen
is
the
major
metabolite
of
lactofen
in
the
rodents,
acifluorfen
is
considered
likely
to
contribute
to
the
peroxisome
proliferation
induced
by
lactofen.
Therefore,
the
data
on
lactofen
provide
some
support
for
acifluorfen.

II.
Background
Information
ACIFLUORFEN
(
Sodium
5­[
2­
chloro­
4­(
trifluoromethyl)
phenoxy]­
2­
nitrobenzoate)
is
the
active
ingredient
of
two
herbicides
formulations,
Tackle
and
Blazer,
which
were
originally
manufactured
by
two
separate
companies.
Acifluorfen
is
a
contact
herbicide
used
for
selective
pre­
and
post­
emergence
control
of
annual
broad­
leaf
weeds
and
some
grasses
in
rice,
peanut,
and
soybean
production.
Acifluorfen
is
also
a
major
metabolite
of
another
registered
herbicide
lactofen.

Although
toxicological
data
are
available
on
both
acifluorfen
products,
the
data
provided
on
Blazer
are
incomplete,
whereas,
the
data
on
Tackle
are
more
current
and
complete.
Therefore,
discussions
of
the
toxicology
of
acifluorfen
are
predominantly
based
on
the
data
derived
from
the
more
recent
studies
with
Tackle,
and
where
appropriate
the
data
on
Blazer
are
also
utilized.
It
should
be
noted
that
Tackle
contains
about
20%
to
24%
of
acifluorfen
as
the
active
ingredient,
whereas
Blazer
contains
approximately
40%
of
acifluorfen.
­
4­
O
Cl
NO2
COONa
F3C
In
rats,
acifluorfen
was
rapidly
absorbed
following
oral
administration
and
eliminated
mainly
in
the
urine
(
46­
58%)
and
feces
(
21­
41%).
The
major
component
present
in
urine
was
unchanged
acifluorfen
and
the
major
component
in
feces
was
unchanged
acifluorfen
and
an
amine
metabolite.
No
tissue
accumulation
was
observed.

III.
Chemical
and
Physical
Properties
of
Acifluorfen
Chemical
Structure:

Common
Synonyms:
Blazer,
Tackle
Physical
Properties:
Melting
point:
>
212o
F
Water
Solubility:
>
25%

CAS
No.:
62476­
59­
9
IV.
Regulatory
History
The
registrant
(
BASF)
submitted
a
petition
(
MRID
No.
45323500)
requesting
that
risk
assessment
for
acifluorfen
be
based
on
the
MOE
approach
rather
than
using
a
Q
1*
in
human
equivalents
(
from
K.
Blundell,
BASF
to
Ms.
Christina
Scheltema,
Chemical
Review
Manager,
SRRD,
MRID
No.
45323500
dated
Feb.
2,
2001).
The
petition
reviewed
and
summarized
earlier
data
submissions
which
supported
peroxisome
proliferation
as
the
mode
of
action
of
acifluorfen
for
induction
of
liver
tumors.
The
current
unit
risk
[
Q
1*]
is
1.27
x
10­
2
(
mg/
kg/
day)­
1
(
memorandum
from
L.
Brunsman
to
P.
Chin,
dated
November
8,
2001).

The
data
supporting
the
proposed
mode
of
action
as
presented
by
the
registrants
were
considered
at
a
preliminary
meeting
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTARC),
HED
on
Aug.
23,
2001.
The
Committee
evaluated
the
available
data
according
to
criteria
recommended
by
the
International
Life
Sciences
Institute
(
ILSI)
workshop
on
peroxisome
proliferation
for
determining
whether
or
not
liver
tumors
were
induced
by
acifluorfen
via
a
peroxisomal
proliferation.
The
ILSI
criteria
are
as
follows:

1.
Changes
in
liver
morphology
indicating
hepatomegaly
as
measured
by
increased
relative
liver
weights
and
an
increased
number
of
peroxisomes
as
measured
by
morphometric
analysis.
­
5­
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
CN­
insensitive
acyl
(
palmitoyl)
CoA
oxidase
activities.

The
MTARC
concluded
that
the
available
data
were
insufficient
to
support
the
proposed
mode
of
action
of
peroxisome
proliferation
for
acifluorfen.
BASF
was
informed
of
this
decision
(
e­
mail
to
R.
Hawks
from
B.
H.
Chin,
dated
9/
12/
01).
Subsequently
the
registrant
proceeded
to
develop
the
necessary
data
and
submitted
the
following
new
mechanism
studies
with
acifluorfen:

1)
The
induction
of
the
number
and
size
of
hepatic
peroxisomes
in
mice
following
4
week
dietary
feeding
with
acifluorfen
(
MRID
45693401),

2)
S­
phase
response
study
in
the
liver
of
mice
following
3
days,
1
week,
and
2
weeks
feeding
with
acifluorfen
(
MRID45803601)

3)
Enzyme
induction
study
in
the
liver
of
mice
following
4
weeks
feeding
with
acifluorfen
(
MRID
45793901).

V.
Evaluation
of
the
Toxicology
Data
Base
for
Acifluorfen
Subchronic
Toxicity
Studies
with
acifluorfen
Available
studies
are
adequate
to
satisfy
subchronic
testing
requirements
for
acifluorfen.
The
subchronic
feeding
studies
in
rats
and
mice
showed
decrease
in
body
weight
and
liver
toxicity
such
as
increased
liver
weight
and
increased
incidence
of
cellular
hypertrophy.
There
is
no
subchronic
toxicity
in
dogs
on
acifluorfen.
However,
there
was
an
acceptable
chronic
feeding
toxicity
study
in
dogs.

Chronic
Toxicity
and
Carcinogenicity
Studies
with
acifluorfen
and
Classification
of
Carcinogenic
Potential
The
chronic
feeding
toxicity
studies
in
rats,
mice
and
dogs
demonstrated
that
acifluorfen
induced
liver
toxicity
(
acidophilic
cells
in
the
liver
and
increased
liver
weight)
and
kidney
toxicity
(
nephritis/
pyelonephritis
and
increased
kidney
weight).
Acifluorfen
induces
an
increase
in
combined
malignant
and
benign
liver
tumor
incidence
in
two
mouse
carcinogenicity
studies
employing
different
strains
of
mice
(
B6C3F1
and
CR­
CD­
1)
[
HED
Doc.
No.
003410;
001099;
003963;
003409;
003556].
The
data
were
evaluated
by
the
HED
Cancer
Peer
Review
Committee
(
HED
Doc.
No.
007698
dated
March
17,
1988).
Acifluorfen
was
classified
as
a
Group
B,
probable
human
carcinogen,
and
the
unit
risk
[
q*
1]
was
calculated
to
be
1.27
x
10­
2
(
mg/
kg/
day)­
1
(
memorandum
from
L.
Brunsman
to
P.
Chin,
dated
November
8,
2001).
A
two­
year
bioassay
in
the
F­
344
rat
administering
Tackle
(
approximately
20­
24%
acifluorfen)
in
the
diet
for
24
months
did
not
show
any
treatment­
related
increase
in
tumor
incidence.
­
6­
Mutagenicity
The
genetic
toxicology
studies
indicate
that
acifluorfen
was
weakly
mutagenic
in
Salmonella
typhimurium
TA100
and
in
Saccharomyces
cerevisiae
in
the
presence
of
S9
metabolic
activation.
However,
in
a
repeat
study,
acifluorfen
was
negative
in
Salmonella
typhimurium
strains
TA98,
TA100,
TA1535,
and
TA1537
in
the
presence
and
absence
of
metabolic
activation.
Also,
the
test
material
was
negative
for
gene
mutations
in
Chinese
hamster
ovary
(
CHO)
cells
and
for
clastogenic
effects
in
vivo.
In
addition,
acifluorfen
was
not
mutagenic
in
DNA
synthesis
assay
in
primary
rat
hepatocytes
and
in
in
vivo
dominant
lethal
assay
in
rats.
The
mutagenicity
studies
satisfy
the
pre­
1991
mutagenicity
guideline
requirements.
Summaries
of
the
submitted
acceptable
mutagenicity
studies
are
presented
below
(
Table
1).

Mechanism
Studies
with
acifluorfen
The
relevant
data
concerning
possible
acifluorfen
induced
peroxisome
proliferation
are
presented
as
follows:

A.
Liver
Toxicity
(
Table
2)

Pertinent
results
from
various
guideline
studies
that
are
relevant
to
the
proposed
mechanism
of
action
of
acifluorfen
are
summarized
in
Table
2.
There
are
several
studies
that
show
acifluorfen
induced
liver
toxicity.
The
subchronic
feeding
toxicity
studies
in
rats
and
mice,
chronic
feeding
toxicity
studies
in
rats
and
dogs,
and
carcinogenicity
study
in
mice
(
CD­
1
and
B6C3F1)
showed
that
acifluorfen
caused
liver
toxicity
as
characterized
by
increased
absolute
and
relative
liver
weights.
In
addition,
acifluorfen
caused
increased
microscopic
change
in
the
liver
in
the
chronic
rat
study
at
125
mg/
kg/
day
(
increased
incidence
of
acidophilic
cells)
and
in
the
chronic
dog
study
at
112.5
mg/
kg/
day
(
congestion,
brown
pigment,
fatty
vacuoles
and
inflammation).
In
the
90­
day
study
in
B6C3F1
mice,
fatty
infiltration
of
the
liver
was
observed
at
187.5
mg/
kg/
day
or
higher
and
increased
incidence
of
histopathological
changes
in
the
liver
(
hypertrophy,
increased
mitotic
activity,
and
oval
cell
proliferation)
and
increased
levels
of
enzymes
(
ALP
and
SGPT)
were
seen
at
375
mg/
kg/
day
or
higher.
These
levels
were
above
the
dose
levels
which
induce
peroxisome
proliferation.
It
is
noted
that
increased
levels
of
enzymes
(
ALP
and
SGPT)
seen
at
40.5
mg/
kg/
day
in
the
carcinogenicity
study
in
mice
(
CD­
1)
were
considered
equivocal
because
of
large
standard
deviations.

B.
Peroxisome
Proliferation
(
Table
3)

A
study
designed
to
determine
whether
or
not
acifluorfen
induces
an
increase
in
peroxisome
proliferation
is
available
and
the
executive
summary
of
the
study
follows:

The
effects
of
4­
week
dietary
administration
of
Blazer
Technical
(
Lot
#
01501L300,
purity
46%
acifluorfen)
on
the
induction
of
the
number
and
size
of
hepatic
peroxisomes
in
male
and
female
B6C3F1
mice
(
5/
sex/
dose)
was
investigated
(
MRID
45693401).
The
­
7­
administered
concentrations
were
0,
350,
1735,
or
5210
ppm
(
equivalent
to
0,
92,
484,
and
1346
mg/
kg/
day
in
males
and
0,
139,
644,
and
1939
mg/
kg/
day
in
females,
respectively).
Mean
daily
intakes
of
the
a.
i.
were
42/
64,
223/
296
and
619/
892
mg/
kg/
day,
[
males/
females],
respectively.
Blazer
Technical
induced
a
slight
to
moderate
increase
in
the
number
of
peroxisomes
within
the
centrilobular
region
of
the
high­
dose
group
of
both
sexes
observable
by
light
microscopy.
No
changes
were
found
in
male
and
female
mice
of
lower
dose
groups.
By
electron
microscopy,
treatment
with
Blazer
Technical
induced
a
dose­
related
increase
in
the
number,
size,
and
area
of
hepatic
peroxisomes
in
mice
treated
with

350
ppm
test
material,
thereby
suggesting
that
Blazer
Technical
is
a
weak
peroxisome
proliferator.
At
350
ppm,
only
slight
increase
of
size
1
peroxisome
(
up
to
1.5
fold;
p<
0.05)
in
males
and
females
were
observed.

At
1735
ppm,
significant
increase
in
number
of
peroxisomes
(
2­
6
fold;
p<
0.01)
in
both
sexes
were
observed.
In
both
sexes
of
mice,
dose­
related
significant
increase
(
p<
0.01)
in
total
peroxisomal
area
was
apparent
(
2.7
fold
increases
in
males
and
2.2
fold
increases
in
females).

At
5210
ppm,
significant
increase
in
number
of
peroxisomes
in
males
(
3­
23
fold;
p<
0.01)
and
females
(
1.5­
35
fold;
p<
0.01)
were
observed.
In
both
sexes
of
mice,
dose­
related
significant
increase
(
p<
0.01)
in
total
peroxisomal
area
was
apparent
(
7.2
fold
increases
in
males
and
8.3
fold
increases
in
females).

By
electron
microscopy,
the
positive
control
diisononyl
phthalate
(
DINP)
treatment
for
4
weeks
in
mice
significantly
increased
the
size,
number,
and
cytoplasmic
area
of
peroxisomes
in
male
mice
treated
with

500
ppm
and
female
mice
treated
with

1500
ppm.
Particularly
increased
were
the
number
of
microperoxisomes
in
mice
treated
with

4000
ppm
DINP.
There
were
also
a
dose­
related
increase
in
cytoplasmic
volume
of
male
and
female
mice
treated
with

1500
ppm
positive
control
(
MRID
45686501).

C.
Evidence
of
Cell
Proliferation
(
Table
3)

A
study
designed
to
determine
whether
or
not
acifluorfen
induces
cell
proliferation
as
measured
by
increased
relative
liver
weights
and
increased
replicative
DNA
synthesis
as
measured
by
increased
hepatocellular
BrdU
nuclear
labeling
is
available.
The
executive
summary
of
the
study
follows:

In
a
DNA
synthesis
(
S­
phase
response)/
cell
proliferation
study
(
MRID
45803601),
Blazer
Technical
(
46.1%
sodium
acifluorfen
in
acetone)
was
administered
to
groups
of
8
male
and
8
female
B6C3F1
mice
at
dietary
concentrations
of
0,
350,
1735
and
5210
ppm
(
i.
e.:
160,
800
and
2400
ppm
of
the
active
ingredient),
mean
daily
intakes
of
the
a.
i.
for
males/
females
of
40/
54,
227/
287
and
714/
845
mg/
kg/
day,
respectively,
for
3
days,
1
week
or
2
weeks.
Food
consumption
and
body
weights
were
determined
weekly.
The
animals
were
examined
at
least
once
a
day;
and
additionally,
comprehensive
clinical
examination
was
performed
weekly.
One
week
prior
to
necropsy,
osmotic
mini­
pumps
containing
bromodeoxyuridine
(
BrdU)
were
implanted
subcutaneously.
Cell
proliferation
(
S­
phase
response)
and
apoptosis
were
determined
in
the
liver.
­
8­
No
animals
died
during
the
study
and
no
clinical
signs
were
observed.
The
test
material
had
no
effect
on
food
or
water
consumption,
and
there
were
no
consistent
effects
on
male
or
female
body
weight.

Dose­
related
increases
in
liver
weights
(
generally
both
absolute
and
relative)
and
findings
of
moderate
or
moderate
to
severe
increases
in
panlobular
hypertrophy
of
hepatocytes
were
seen
in
males
and
females
exposed
to
714/
845
a.
i.
mg/
kg/
day,
respectively,
on
all
treatment
schedules
(
3­
day,
1­
and
2­
week).
Liver
hypertrophy
was
most
pronounced
in
the
males
and
peaked
after
1
week
of
treatment.
By
2
weeks,
an
increase
in
single
cell
necrosis
and
apoptotic
cells
was
observed
primarily
in
high­
dose
males.
This
finding,
suggesting
cytotoxicity
to
the
target
organ,
indicates
that
dosing
was
adequate
to
assess
cell
proliferation
in
the
liver.
The
oral
administration
of
Blazer
Technical
(
46.1%
acifluorfen­
sodium)
to
mice
produced
a
dose­
dependent
and
significant
induction
of
BrdU
labeling
in
the
liver,
which
is
indicative
of
cell
proliferation
(
S­
phase
response),
in
all
dose
groups.
The
most
pronounced
effect
for
each
of
these
three
parameters
(

liver
weight,

liver
hypertrophy
and

BrdU
labeling)
for
both
sexes
was
seen
after
1
week
of
treatment.

This
study
is
classified
as
acceptable
(
non­
guideline).
Although
this
study
does
not
satisfy
the
requirement
for
any
current
FIFRA
Test
Guideline
84­
2,
the
results
may
be
used
in
a
possible
mode
of
action
analysis
of
the
test
substance.

D.
Evidence
of
Increased
Enzymatic
Activity
(
Table
3)

A
study
designed
to
determine
whether
or
not
acifluorfen
induces
enzymes
involved
in
peroxisomal
fatty
acid
metabolism,
especially
CN­
insensitive
acyl
(
palmitoyl)
CoA
oxidase
activities
is
available.
The
executive
summary
of
the
study
follows:

In
an
enzyme
induction
study
(
MRID
45793901),
Blazer
technical
(
46.1%
acifluorfen­
Na
a.
i.
in
acetone)
was
administered
to
groups
of
10
male
and
10
female
B6C3F1
mice
at
dietary
concentrations
of
0,
350,
1735
and
5210
ppm
(
160,
800
and
2400
ppm
of
active
ingredient)
for
4
weeks.
Mean
daily
intakes
of
the
a.
i.
were
36.9/
54.6,
180/
255
and
709/
933
mg/
kg/
day,
[
males/
females],
respectively.
Food
consumption
and
body
weights
were
determined
weekly.
Animals
were
examined
once
a
day,
and
a
comprehensive
clinical
examination
carried
out
weekly.
In
the
first
5
animals
of
each
group,
glutathione
concentration
(
GSH)
in
the
liver
was
determined;
in
the
second
5
animals,
the
amount
of
cyanide­
insensitive
palmitoyl­
CoA­
oxidation
(
PALCoA)
in
total
protein
was
measured.

At
the
high­
dose,
significant
(
p

0.01)
decreases
in
male
body
weights
were
recorded
on
days
14,
21
and
28;
the
greatest
decrease
in
male
body
weights
(
9.2%)
occurred
on
Day
28.
Body
weight
gain
for
this
group
was
also
significantly
(
p

0.01)
reduced
compared
to
control
at
the
same
time
intervals.
No
clear
adverse
effects
were
found
on
female
body
weight.
There
was
a
significant
(
p

0.01)
and
dose­
dependent
increase
in
PALCoA,
which
is
involved
in
peroxisomal
fatty
acid
metabolism,
ranging
from
a
58
to
576%
increase
for
males
and
a
3
to
707%
increase
for
females
at
350
to
5210
ppm,
respectively.
Increased
­
9­
PALCoA
was
accompanied
by
decreased
GSH
concentrations
in
high­
dose
males
(
9%

)
and
females
(
15%

)
;
the
response
was
dose
related
for
both
sexes
but
only
reached
statistical
significance
in
the
high­
dose
females.
Based
on
these
considerations,
it
was
concluded
that
Blazer
technical,
containing
46.1%
NA
acifluorfen
as
the
a.
i.,
showed
clear
evidence
of
the
induction
of
the
peroxisomal
enzyme
system.
Thus,
the
demonstration
of
peroxisomal
enzyme
system
induction
in
the
liver
satisfies
one
of
the
necessary
criteria
to
classify
a
nongenotoxic
substance
as
a
peroxisome
proliferator.

The
following
special
study
also
supports
the
findings
of
the
new
mechanism
study
described
above.
In
this
special
study,
hepatocytes
isolated
from
male
Sprague­
Dawley
rats
were
exposed
to
acifluorfen
or
lactofen
(
MRID
No.:
45283902)
to
examine
the
induction
of
palmitoyl
CoA
oxidase
activity
following
in
vitro
exposure.
This
study
showed
that
both
acifluorfen
and
lactofen
displayed
increased
activity
of
palmitoyl
CoA
oxidase
activity
following
in
vitro
exposure
(
Table
3).

VI
Data
from
Lactofen
Studies
The
relevance
of
peroxisome
proliferation
(
PP)
to
hepatocarcinogenesis
has
been
previously
discussed
for
lactofen
(
see
HED
Memorandum:
Lactofen:
Report
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
from
Robert
F.
Fricke
to
Christine
Olinger,
dated
March
12,
2001).

A.
Rationale
for
Using
Lactofen
Data
1.
Lactofen
is
a
diphenyl
ether
herbicide
and
the
structure
of
lactofen
is
very
similar
to
that
of
acifluorfen
(
see
below).

2.
The
metabolism
study
of
lactofen
in
rats
showed
that
acifluorfen
is
a
major
metabolite
of
lactofen
(
Accession
No.
071222).
Seventy
two
hours
after
administration
of
lactofen
to
rats,

97%
of
the
radiolabel
was
recovered
in
the
excreta
(
urine
and
feces).
Urinary
excretion
comprised
39
­
56%
of
the
dose
while
the
fecal
output
totaled

43
­
67%
of
the
dose.
While
the
parent
compound,
lactofen,
was
the
major
component
in
the
feces,
the
major
metabolite
in
urine
was
acifluorfen
which
accounted
for
>
90%
of
the
radioactivity
recovered
in
this
fraction.
­
10­
C
H
3
O
O
CH
3
O
Cl
F
F
F
O
O
N
+

O
O
O
Cl
F
F
F
OH
O
N+
O
O
Figure
1:
Structures
of
Lactofen
and
Acifluorfen
Lactofen
(
PPG­
844)

Acifluorfen
(
PPG­
847
)

3.
Following
a
single
oral
dose
of
radiolabeled
lactofen
at
50
mg/
kg
to
the
SD
rats
and
mice
(
strain
not
identified),
acifluorfen
was
the
primary
metabolite
of
lactofen
in
the
plasma.
The
plasma
level
of
acifluorfen
at
peak
ranged
61­
71%
of
the
administered
radioactivity
[
Hawks,
(
2001).
p22
of
the
BASF
Report
dated
January
31,
2001
entitled
Sodium
Acifluorfen:
Request
for
Reassessment
of
Current
Cancer
Classification
Considering
Peroxisome
Proliferation
 
Associated
Mode
of
Action].

B.
The
Lactofen
Data
in
Supporting
That
the
Peroxisome
Proliferation
is
the
Mode
of
Action
of
Lactofen
The
submitted
lactofen
data
in
supporting
that
peroxisome
proliferation
is
the
mode
of
action
of
lactofen
are
as
follows:

1.
The
mutagenicity
studies
showed
that
lactofen
is
neither
mutagenic
nor
genotoxic.
A
non­
guideline
mutagenicity
study
showed
equivocal
(
probably
negative)
binding
of
lactofen
to
DNA.
­
11­
2.
Changes
in
liver
morphology
were
observed
in
both
rats
and
mice
treated
with
lactofen
in
the
diet
for
8
and
7
weeks,
respectively
(
MRID
No.
45283904).
These
effects
include:
dose­
dependent
increase
in
relative
liver
weights
and
increased
number
of
peroxisomes
as
measured
by
electron
microscopic
analysis.
Further,
in
both
rats
and
mice
there
was
a
dose­
dependent
increase
in
nuclear
enlargement,
cytoplasmic
eosinophilia,
hypertrophy
and
peroxisomal
staining.
In
addition,
treatment
with
lactofen
resulted
in
dose­
dependent
increases
(
particularly
in
female
mice)
in
the
activities
of
hepatic
CN­
insensitive
palmitoyl
CoA
oxidase
and
carnitine
acetyl
transferase.

3.
Dose­
dependent
increase
in
CN­
insensitive
palmitoyl
CoA
oxidase
was
also
observed
in
primary
rat
hepatocytes
treated
with
lactofen
(
MRID
No.
45283902).

4.
The
doses
at
which
carcinogenicity
were
observed
[
mouse,
LOAEL
=
50
ppm
(
7.1
mg/
kg/
day);
rat,
LOAEL
=
2000
ppm
(
76
mg/
kg/
day)]
were
consistently
higher
than
the
doses
which
caused
peroxisome
proliferation
(
for
details
see
HED
Memorandum:
Lactofen:
Report
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
from
Robert
F.
Fricke
to
Christine
Olinger,
dated
March
12,
2001).

The
major
metabolite
of
lactofen
in
the
rodent
is
acifluorfen
(
see
Table
4.
Comparative
Metabolism
of
acifluorfen
and
lactofen
in
Rats).
Since
the
MTARC
concluded
that
the
peroxisome
proliferation
is
the
mode
of
action
of
lactofen,
peroxisome
proliferation
data
generated
using
lactofen
can
be
considered
in
evaluating
the
hypothesis
that
acifluorfen
is
a
peroxisome
proliferation.

VII.
Other
Modes
of
Action
As
stated
previously,
the
weight
of
the
evidence
indicates
that
acifluorfen
does
not
possess
mutagenic
activity.
Currently,
no
other
mode
of
action
has
been
hypothesized
for
acifluorfen.

VIII.
Structural
Activity
Relationships
(
SAR)

As
a
member
of
the
diphenyl
ether
chemical
family,
acifluorfen
(
lactofen's
major
metabolite)
is
structurally
related
to
four
other
chemicals,
namely
lactofen,
nitrofen,
oxyfluorfen,
and
fomesafen.
Lactofen
produces
hepatocellular
adenomas
and
carcinomas
in
mice
and
increases
in
liver
neoplastic
nodules
and
foci
of
cellular
alteration
(
possible
precursor
of
tumors)
in
rats.
Nitrofen
produces
hepatocellular
carcinomas
in
mice
and
pancreatic
carcinomas
in
rats,
oxyfluorfen
produces
marginally
positive
liver
tumors
in
mice
but
is
negative
in
rats,
and
fomesafen
produces
hepatocellular
adenomas
and
carcinomas
in
mice.

Acifluorfen
contains
the
following
basic
structural
features
in
common
with
other
compounds
causing
peroxisome
proliferation:

S
a
chlorinated
benzene
ring
to
which
an
ether
linkage
is
attached,

S
a
terminal
carboxyl
group
(
Eacho,
1996),
and
S
a
lipophillic
backbone
(
Kozuka,
1991).
­
12­
VII.
SUMMARY
Based
on
the
available
toxicity
data,
there
is
evidence
to
support
that
acifluorfen
is
a
non­
genotoxic
hepatocarcinogen.
Tables
5
and
7
show
"
Summary
of
Peroxisomal
Effects
and
Liver
Tumor
Induction
in
Male
and
Female
Mice
Administered
Acifluorfen".
Tables
6
and
8
show
"
Summary
of
Liver
Toxicity
and
Tumor
Induction
in
Male
and
Female
Mice
Administered
Acifluorfen".
Table
9
provides
quantitative
evaluation
of
peroxisomes
and
cytoplasmic
volume
(%)
in
male
and
female
mice
fed
acifluorfen
(
Blazer
Technical)
or
positive
control
(
DINP)
for
4
weeks.
These
data
(
Tables
5­
9)
provide
supporting
evidence
of
peroxisome
proliferation
as
a
mode
of
action
for
liver
tumors
induction
by
acifluorfen
according
to
the
criteria
recommended
by
ILSI;
these
include:

1.
Changes
in
liver
morphology
were
observed
in
both
rats
and
mice
treated
with
acifluorfen.
These
effects
include:
dose­
dependent
increase
in
relative
liver
weights,
and
increased
incidence
of
cellular
hypertrophy,
and
increased
number
of
peroxisomes
as
measured
by
electron
microscopic
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.
Dosed­
dependent
increase
in
the
levels
of
enzymes
involved
in
peroxisomal
fatty
acid
metabolism,
especially
CN­
insensitive
acyl
(
palmitoyl)
CoA
oxidase
activities.

4.
Previously,
the
MTARC
concluded
that
the
peroxisome
proliferation
is
the
mode
of
action
of
lactofen
in
inducing
liver
tumors
in
rodents.
Since
acifluorfen
is
the
major
metabolite
of
lactofen
in
the
rodents,
it
is
probable
that
acifluorfen
contributes
to
the
peroxisome
proliferation
and
liver
tumors
induced
by
lactofen.
Therefore,
the
mode
of
action
data
on
lactofen
provide
support
for
acifluorfen.
­
13­
VIII.
References
1.
Eacho,
P.
I.,
Foxworthy,
P.
S.,
Lawrence,
J.
W.,
Herron,
D.
K.,
and
Noonan,
D.
J.
(
1996)
Common
structural
requirements
for
peroxisome
proliferation
by
tetrazole
and
carboxylic
acidcontaining
compounds.
Ann.
NY
Acad.
Sci.
804:
387­
402.

2.
Hawks,
R.
(
2001).
Sodium
Acifluorfen:
Request
for
reassessment
of
current
cancer
classification
considering
peroxisome
proliferation­
associated
mode
of
action.
BASF
Registration
Doc.
No.
2001/
5000878.
BASF
Corp.
January
31,
2001.
MRID
No.
45323500.

3.
IARC
Technical
Report
No.
24:
Peroxisome
proliferation
and
its
role
in
carcinogenesis.
December,
1994.

4.
Kozuka,
H.,
Yamada,
J.,
Horie,
S,
Watanabe,
T.,
Suga,
T.
and
Ikeda,
T.
(
1991)
Characteristics
of
induction
of
peroxisomal
fatty
acid
oxidation­
related
enzymes
in
rat
liver
by
drugs.
Biochem.
Pharm.
41(
4):
617­
623.

5.
Lactofen:
Report
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
from
Robert
F.
Fricke
to
Christine
Olinger,
dated
March
12,
2001.

6.
Reevaluation
of
Classification
of
Carcinogenicity
of
Acifluorfen
Following
Science
Advisory
Panel
(
SAP)
Review
of
Data.
The
HED
Cancer
Peer
Review
Committee
(
HED
Doc.
No.
007698
dated
March
17,
1988).

7.
Goldenthal,
E.
I.;
Jessup,
D.
C.;
Geil,
R.
G.;
et
al.
(
1979)
Lifetime
Dietary
Feeding
Study
in
Mice:
285­
013a.
(
Unpublished
study
received
Mar
29,
1979
under
707­
149;
prepared
by
International
Research
and
Development
Corp.,
submitted
by
Rohm
&
Haas
Co.,
Philadelphia,
Pa.;
CDL:
098024­
A;
098025).
MRID
00082897.

8.
Barnett,
J.;
Jenkins,
L.;
Parent,
R.
(
1983)
Evaluation
of
the
Potential
Oncogenic
and
Toxicological
Effects
of
Long­
term
Dietary
Administration
of
Tackle
to
Fischer
344
Rats:
GSRI
Project
No.
413­
985­
41.
Final
rept.
(
Unpublished
study
received
May
16,
1983
under
359­
708;
prepared
by
Gulf
South
Research
Institute,
submitted
by
Rhone­
Poulenc,
Inc.,
Monmouth
Junction,
NJ;
CDL:
250289­
A;
250290;
250291).
MRID
00128353.
­
14­

Table
1.
Genotoxicity/
Mutagenicity
Profile
of
Acifluorfen
ASSAY
TYPE
RESULT
MRID
Ames
assay
B
standard
plate
assay
(
Blazer,
42.8%)
Gene
mutation
"
Weakly"
Positive
in
TA
100
only
41480101
Ames
assay
B
standard
plate
and
standard
plate
with
preincubation
using
manufactoring
use
product
(
46%)
Gene
mutation
Negative
in
TA
100
45393902
Ames
assay
B
standard
plate
assay
using
highly
pure
material
(
99.5%)
Gene
mutation
Negative
45393901
Ames
assay
B
standard
plate
assay
and
standard
plate
with
preincubation
both
using
end­
use
product
(
25.5%)
Gene
mutation
Negative
both
with
and
without
preincubation
45323501
Forward
gene
mutation
in
CHO
cells
Gene
mutation
Negative
41480103
Forward
mutation
in
TK
+/­
locus
in
mouse
lymphoma
cells
Gene
mutation
Negative
00122739
Drosophila
melanogaster­
(
Tackle
2S)

White­
ivory
mutation
Sex­
linked
recessive
lethal
Y
chromosome
loss
Dominant
lethal
mutations
Bithorax
test
Gene
mutation
Gene
mutation
Gene
mutation
Chromosome
Aberrations
Chromosome
Aberrations
Negative
Negative
Positive
Positive
Negative
00122737
In
vivo
rat
cytogenetic
assay
Chromosome
Aberrations
Negative
00122741
Dominant
lethal
assay
in
rats
Chromosome
Aberrations
Negative
00122738
In
vitro
unscheduled
DNA
synthesis
Other
mechanisms
Negative
00122742
Saccharomyces
cerevisiae
D5
mitotic
recombination
(
Tackle
2S,
29.7%)
Other
mechanisms
"
Weak"
positive
00148272
TOTAL
11
negative
4
positive
­
15­

Table
2.
Acifluorfen
Toxicity
Profile
(
Appendix
1
presents
the
Executive
Summaries
for
critical
studies)

STUDY
TYPE
 
DOSE
LEVELS
NOAEL/

LOAEL
(
mg/
kg/
day)
Liver
weight
Liver
enzymes
Histopathology
Chronic/
Carcinogenicity
­
Rat
Fischer
344
(
1983)
0,
25,

150,
500,
2500,
or
5000
ppm.
(
0,
1.25,
7.50,
25.0,
125,

or
250
mg/
kg/
day
based
on
1
ppm=
0.05
mg/
kg/
day)

TEST
material
=
19.2­
25.6%
a.
i.

(
MRID
No.
00128353;
Accession
No's.
071315
through
071317
and
250289
through
250792)
25/
125
increased
absolute
and
relative
liver
weights
equivocal
increased
level
of
ALP
at
250
mg/
kg/
day
increased
incidence
of
acidophilic
cells
in
the
liver
No
treatment­
related
increase
in
tumors
Carcinogenicity
in
Mice
B6C3F1
(
1982).
0,
625,
1250,

or
2500
ppm
(
males:
0,
119,
259,
655
mg/
kg/
day;

females:
0,
143,
313,
711
mg/
kg/
day)
TEST
material
=

20.4­
23.2%
a.
i.

(
MRID
No.
00122732;
Accession
No's.
071312,
071313,

071314,
250463,
and
250464)
<
119
(
LDT)
increased
absolute
and
relative
liver
weights
Not
observed
at
LDT
p<
0.05­
0.01
liver
adenomas;

liver
carcinomas;
combined
liver
adenomas
&
carcinomas
p<
0.01
trend
for
adenomas
(


)
;
carcinomas
(

)
;

adenomas
&
carcinomas
combined
(


)

Carcinogenicity
in
Mice
CD­
1
(
1979).
0,
7.5,
45,
or
270
ppm
(
1.125,
6.75,
or
40.5
mg/
kg/
day
based
on
1
ppm
=

0.15
mg/
kg/
day)
for
24
months.
TEST
material
=
39.4­

40.5%
a.
i.

(
MRID
No.
00082897)
6.75/
40.5
increased
absolute
and
relative
liver
weights
equivocal
increased
levels
of
ALP
and
SGPT.
p<
0.05
combined
liver
adenomas
&
carcinomas
(
270
ppm

)

Chronic
Feeding
Study
in
Dogs
(
1983).
Tackle
"
2S"

(
Acifluorfen,
purity
was
unspecified).
0,
20,
300,
or
4500
ppm
(
0,
0.5,
7.5
or
112.5
mg/
kg/
day
based
on
1
ppm
=
0.025
mg/
kg/
day)
for
2
years
(
MRID
No.
00131162;
Accession
No's.
251297
and
251298)
7.5/
112.5
increased
absolute
and
relative
liver
weights
increased
level
of
LDH
increased
incidence
of
microscopic
changes
in
the
liver
(
congestion,
brown
pigment,
fatty
vacuoles
and
inflammation).
STUDY
TYPE
 
DOSE
LEVELS
NOAEL/

LOAEL
(
mg/
kg/
day)
Liver
weight
Liver
enzymes
Histopathology
­
16­

Subchronic
feeding
study
in
rats
Fischer
344
(
1982).
0,

20,
80,
320,
1250,
2500
or
5000
ppm
(
0,
2,
8,
32,
125,

250,
or
500
mg/
kg/
day
based
on
1
ppm
=
0.1
mg/
kg/
day)

TEST
material
=
20.4­
23.6%
a.
i.

(
MRID
No.
00122730;
Accession
No.
071308)
32/
125
increases
in
liver
weights
at
250
or
higher
increased
levels
of
SGPT
and
ALP
increased
incidence
of
hypertrophy
of
liver
(
including
increased
cellular
hypertrophy,

mitotic
activity,
individual
cell
death,
and
proliferation
of
oval
or
bile
duct
cells)

Subchronic
feeding
study
in
mice
B6C3F1
(
1982)
0,
20,

80,
320,
1250,
2500
or
5000
ppm
(
0,
3,
12,
48,
187.5,

375,
or
750
mg/
kg/
day
based
on
1
ppm
=
0.15
mg/
kg/
day)
for
3
months
TEST
material
=
20.4­
23.2%
a.
i.

(
MRID
No.
00252826;
Accession
No.
071308)
48/
187.5
At
375
mg/
kg/
day
as
test
material
intake:

Increases
in
absolute
and
relative
liver
weights
At
375
mg/
kg/
day
and
above:
Increased
levels
of
SGPT
and
ALP
At
187.5
mg/
kg/
day:

histopathologic
changes
(
fatty
infiltration)
of
the
liver
At
375
mg/
kg/
day
and
above:

histopathological
changes
in
the
liver
(
hypertrophy,
increased
mitotic
activity,
oval
cell
proliferation)
­
17­

Table
3.
Summary
of
Mechanistic
Studies
with
Acifluorfen
STUDY
TYPE
B
DOSE
LEVELS
Liver
weight
Liver
enzymes
Electron
microscopy
Induction
of
the
number
and
size
of
hepatic
peroxisomes
in
mice
following
4
week
dietary
feeding
(
MRID
45693401).

0,
350,
1735,
or
5210
ppm
Blazer
(
46%
a.
i.),
test
material
(
equivalent
to
0,
92,
484,
and
1346
mg/
kg/
day
in
males
and
0,
139,
644,
and
1939
mg/
kg/
day
in
females)

mean
daily
intakes
of
the
a.
i.
were
42/
64,
223/
296
and
619/
892
mg/
kg,
[
males/
females]
NA
NA
Induced
dose­
related
increase
in
the
number,
size,

and
area
of
hepatic
peroxisomes
in
mice
treated
with

350
ppm
Dose­
related
increase
in
total
peroxisomal
area
in
high
dose
groups
(
7.2
fold
increases
in
males
and
8.3
fold
increases
in
females).

S­
phase
response
study
in
the
liver
of
mice
following
3
days,
1
week,
and
2
weeks
feeding
(
MRID45803601)

0,
350,
1735
and
5210
ppm
Blazer
(
46
%
a.
i.)
(
i.
e.:

160,
800
and
2400
ppm
of
the
active
ingredient),

mean
daily
intakes
of
a.
i.
for
males/
females
of
40/
54,
227/
287
and
714/
845
mg/
kg/
day
Induced
doserelated
increases
in
absolute
and
relative
liver
weights
NA
At
high
dose,
Induced
a
doserelated
increases
in
panlobular
hypertrophy
of
hepatocytes
on
all
treatment
schedules.

Liver
hypertrophy
was
most
pronounced
in
the
males
and
peaked
after
1
week
of
treatment.

By
2
weeks,
an
increase
in
single
cell
necrosis
and
apoptotic
cells
was
observed
primarily
in
high­
dose
males.

Inducted
BrdU
labeling
in
the
liver,
which
is
indicative
of
cell
proliferation
(
S­
phase
response).
STUDY
TYPE
B
DOSE
LEVELS
Liver
weight
Liver
enzymes
Electron
microscopy
­
18­

Enzyme
induction
study
in
the
liver
of
mice
following
4
weeks
feeding
(
MRID
45793901).

0,
350,
1735
and
5210
ppm
Blazer
(
46
%
a.
i.)
(
160,

800
and
2400
ppm
of
active
ingredient)
for
4
weeks.

Mean
daily
intakes
of
a.
i.
were
36.9/
54.6,
180/
255
and
709/
933
mg/
kg/
d,
[
males/
females]
NA
Induced
dosedependent
increase
in
cyanideinsensitive
palmitoyl­

CoAoxidase
5
­
7
fold
in
high
dose
groups
(
males
and
females)

Males:
158,
458,

and
676%
increase
at
37,
180,
and
709
mg/
kg/
day
Females:
3,
316,

and
807%
increase
at
55,
255,
and
933
mg/
kg/
day
NA
Measurement
of
Peroxisome
Proliferation
in
Primary
Rat
Hepatocytes
Induced
by
Lactofen
and
Five
of
its
Metabolites
including
acifluorfen
(
45283902)

0.003,
0.01,
0.03,
and
0.1
mM
NA
Acifluorfen
or
lactofen
induced
CN­
insensitive
palmitoyl
CoA
oxidase
activities
at
0.01
mM
and
above.
EM
analysis
did
not
indicate
an
increased
peroxisome
number.
­
19­

Table
4.
Comparative
Metabolism
of
acifluorfen
and
lactofen
in
Rats
Dose
(
mg/
kg)
Sex
Cumulative
%
Radioactivity
Excreted
Urine
Feces
24
hrs
48
hrs
72
hrs
96
hrs
24
hrs
48
hrs
72
hrs
96
hrs
Acifluorfen
(
1)
116
Males
48
41
117
Females
60
23
Lactofen
(
2)
125
Males
29
36
44
34
62
67
125
Females
19
53
56
18
35
43
Urinary
and
Fecal
Metabolites
Major
component
in
Urine
%
of
Total
Urinary
radioactivity
found
(%
of
administered
dose)
Major
component
in
feces
%
of
Total
Fecal
radioactivity
found
(%
of
administered
dose)

Acifluorfen
(
1)
116/
117
Males/
Females
Acifluorfen
93
(
50)
Acifluorfen
amine
Acifluorfen
60­
80
(
19­
26)

13
(
4)

Lactofen
(
2)
125/
125
Males/
Females
Acifluorfen
91
(
46)
Lactofen
Acifluorfen
amine
Acifluorfen
30­
37
(
17­
20)

7
(
4)

6
(
3)

(
1)
Excerpted
from
MRID
00122746;
Accession
No.
071321.
The
elimination
half­
life
of
acifluorfen
was
3.7
hours
in
females
versus
8.8
hours
in
males.

(
2)
Excerpted
from
Accession
No.
071222;
HED
Doc.
No.
014148
­
20­

Table
5.
Summary
of
Peroxisomal
Effects
and
Liver
Tumor
Induction
in
Male
Mice
Administered
Acifluorfen
Dose
(
mg/
kg/
day)

as
active
ingredient
Mean
Number
of
Peroxisome
according
to
size
classification
(
relative
to
controls,
%)

MRID
45693401
(
a)
Induction
of
Peroxisomal
Enzyme
Activities;

Palmitoyl
CoA
oxidase
MRID
45793901
(
b)
Cell
proliferation;

BrdU
labeling
in
the
liver
(
S­
phase
response)

MRID
45803601
(
c)
Tumor
incidence
(%
incidence)

18­
month
carcinogenicity
study
in
mice
MRID
00122732
(
d)

(
Note
2)

1
(<
0.1

m2)
2
(<
0.3

m2)
3
(<
0.5

m2)
Total
area
of
peroxisomes
(
note
1)
Adenomas
Carcinomas
Adenomas/
Carcinomas
Combined
0
7.8
±
1.5
(
SD)
8.0
±
2.0
0.5
±
0.4
1.25
±
0.18
5.24
±
0.5
1.01
±
0.3
8/
58
(
14
%)
1/
48
(
2
%)
9/
58
(
16
%)

37­
42
11.8*

(
51%)

±
4.1
9.6
(
20%)

±
1.7
0.3
±
0.2
1.49
(
19%)

±
0.29
8.29**

(
58%)

±
0.57
4.07
**(
304%)

±
1.86
NT
NT
NT
119
NT
NT
NT
NT
NT
NT
18/
60
(
30
%)
*
3/
50
(
6
%)
21/
60
(
35
%)
*

180­
227
15.4**

(
97%)

±
3.1
18.7**

(
134%)

±
2.3
2.9**

(
480%)

±
1.6
3.43**

(
174%)

±
0.63
25.29**

(
383%)

±
2.93
14.09
**(
1300%)

±
2.69
NT
NT
NT
259
NT
NT
NT
NT
NT
NT
12/
56
(
21
%)
4/
46
(
9
%)
16/
56
(
29
%)

619­
714
22.3**

(
185%)

±
9.4
35.7**

(
346%)

±
10.1
11.4**

(
2180%)

±
3.4
9.03**

(
622%)

±
2.54
35.41**

(
576%)

±
2.49
20.61**(
1947%)

±
3.57
25/
59
(
42
%)
**
15/
44
(
34
%)
**
40/
59
(
68
%)

**

NT:
Not
tested
*
Significantly
different
than
control
(
p<
0.05)
**
Significantly
different
than
control
(
p<
0.01)

Note
1:
The
area
occupied
by
peroxisomes
was
expressed
in
relation
to
cytoplasmic
region
as
%.
Size
classes
1­
5
(<
0.1
­
>
0.75

m2).

Note
2:
Significance
of
pairwise
comparison
with
control
(
Fisher's
Exact
Test)
denoted
at
Dose
level.
Tumor
bearing
animals/
animal
at
risk
(
i.
e.,
the
animals,

which
died
prior
to
the
week
of
the
first
tumor
occurrence
for
each
tumor
type,
are
removed
from
the
animals
at
risk).

a)
4­
week
treatment
at
42,
223,
and
619
mg/
kg/
day
b)
4
week
treatment
at
37,
180,
and
709
mg/
kg/
day
c)
1
week
treatment
at
40,
227,
and
714
mg/
kg/
day
d)
18
month
study.
Overall
mean
doses
were
119,
259,
and
655
mg/
kg/
day.
SD:
Standard
deviation
­
21­

Table
6.
Summary
of
Liver
Toxicity
and
Tumor
Induction
in
Male
Mice
Administered
Acifluorfen
Dose
(
mg/
kg/
day)

as
active
ingredient
Subchronic
study
(
a)

(
MRID
00252826)
Cell
Proliferation
Study
(
b)

(
MRID
45803601)
18­
month
carcinogenicity
study
in
mice
(
MRID
00122732)

Fatty
infiltration
of
liver
hypertrop
hy,
mitotic
activity
and
increased
levels
of
SGPT
and
ALP
Hypert
rophy
Single
cell
necrosis
and
apoptotic
cells
Body
weight,
g
(%
of
controls)
Absolute
Liver
weight,
g
(%
of
controls)
Relative
Liver
weight
(%
of
controls)
Body
weight,

%
of
controls
Liver
weight,
g
(%
of
controls)
Adenomas/
Carcinomas
Combined
0
30.6
1.51
4.94
2.03
9/
58
(
16
%)

40­
48
NF
NF
NF
NF
30.5
(
99.7%)
1.63
(
108%)**
5.34
(
108)**
NT
NT
NT
119
NT
NT
NT
NT
NT
NT
NT
90**
2.41
(
119%)
21/
60
(
35
%)
*

188
+++
(
d)
NF
NT
NT
NT
NT
NT
NT
NT
NT
227
NT
NT
NF
NF
31.3
(
102%)
2.13
(
141%)**
6.80
(
137%)

**
NT
NT
NT
259
NT
NT
NT
NT
NT
NT
NT
87**
2.95
**

(
145%)
16/
56
(
29
%)

375
+++
+++
NT
NT
NT
NT
NT
NT
NT
NT
655­
750
+++
+++
+++
+++
27.3
(
89%)
**
1.90
(
125%)**
6.94
(
140%)

**
75**
3.75
**

(
185%)
40/
59
(
68
%)
**

NT:
Not
tested
NF:
Not
found
*
Significantly
different
than
control
(
p<
0.05)
**
Significantly
different
than
control
(
p<
0.01)

a)
Treated
for
90­
days
at
3,
12,
48,
188,
375,
and
750
mg/
kg/
day.
­
22­

b)
Treated
for
2
weeks
at
40,
227,
and
714
mg/
kg/
day
c)
18
month
study.
Overall
mean
doses
were
119,
259,
and
655
mg/
kg/
day.
d)
+++
means
positive
responses.
­
23­

Table
7.
Summary
of
Peroxisomal
Effects
and
Liver
Tumor
Induction
in
Female
Mice
Administered
Acifluorfen
Dose
(
mg/
kg/
day)

as
active
ingredient
Mean
Number
of
Peroxisome
according
to
size
classification
(
relative
to
controls,
%)

MRID
45693401
(
a)
Induction
of
Peroxisomal
Enzyme
Activities;

Palmitoyl
CoA
oxidase
MRID
45793901
(
b)
Cell
proliferation;

BrdU
labeling
in
the
liver
(
S­
phase
response)

MRID
45803601
(
c)
Tumor
incidence
(%
incidence)

18­
month
carcinogenicity
study
in
mice
MRID
00122732
(
d)

(
Note
2)

1
(<
0.1

m2)
2
(<
0.3

m2)
3
(<
0.5

m2)
Total
area
of
peroxisomes
(
note
1)
Adenomas
Carcinomas
Adenomas/
Carcinomas
Combined
0
9.5
±
2.8
(
SD)
8.2
±
0.7
0.4
±
0.3
1.27
±
0.13
5.65
±
0.94
1.02
±
0.24
1/
55
(
1
%)
0/
45
(
0
%)
1/
55
(
2
%)

54­
55
64
13.2*

(
39%)

±
1.4
7.5
±
0.8
0.2
±
0.2
1.25
±
0.18
5.81
(+
3%)

±
0.54
1.31*
(
28%)

±
0.31
NT
NT
NT
143
NT
NT
NT
NT
NT
NT
5
/
59
(
5
%)
1/
47
(
2
%)
6/
59
(
10
%)

255­
313
12.1
(
27%)

±
2.1
15.9**

(
94%)

±
1.5
2.2**

(
450%
)

±
1.0
2.81**

(
121%)

±
0.37
17.84**

(+
216%)

±
1.79
3.68**
(
259%)

±
0.70
4/
57
(
4
%)
1/
44
(
2
%)
5/
57
(
9
%)

711
NT
NT
NT
NT
NT
NT
19/
58
(
19
%)
**
5/
46
(
11
%)
*
24/
58
(
41
%)
**

845­
933
14.6
(
54%)

±
5.9
35.7**

(
335%)

±
11.4
14.0**

(
3400%)

±
3.9
10.6**

(
735%
)

±
2.95
45.57**

(+
707%)

±
3.65
6.19**
(
505%)

±
1.37
NT
NT
NT
NT:
Not
tested
*
Significantly
different
than
control
(
p<
0.05)
**
Significantly
different
than
control
(
p<
0.01)
SD:
Standard
deviation
Note
1:
The
area
occupied
by
peroxisomes
was
expressed
in
relation
to
cytoplasmic
region
as
%.
Size
classes
1­
5
(<
0.1
­
>
0.75

m2).

Note
2:
Significance
of
pairwise
comparison
with
control
(
Fisher's
Exact
Test)
denoted
at
Dose
level.
Tumor
bearing
animals/
animal
at
risk
(
i.
e.,
the
animals,
which
died
prior
to
the
week
of
the
first
tumor
occurrence
for
each
tumor
type,
are
removed
from
the
animals
at
risk).

a)
4
week
treatment
at
64,
296,
and
892
mg/
kg/
day.
b)
4
week
treatment
at
55,
255,
and
933
mg/
kg/
day
c)
1
week
treatment
at
54,
287,
and
845
mg/
kg/
day
d)
18
month
study.
Overall
mean
doses
were143,
313,
and
711
mg/
kg/
day.
­
24­

Table
8.
Summary
of
Liver
Toxicity
and
Tumor
Induction
in
Female
Mice
Administered
Acifluorfen
Dose
(
mg/
kg/
d)

as
active
ingredient
Subchronic
study
(
a)

MRID
00252826
Cell
Proliferation
Study
(
b)

(
MRID
45803601)
18­
month
carcinogenicity
study
in
mice
[
c]

(
MRID
00122732)

Fatty
infiltration
of
liver
hypertrophy,

mitotic
activity
and
increased
levels
of
SGPT
and
ALP
Hypertroph
y
Single
cell
necrosis
and
apoptotic
cells
Body
weight,
g
(%
of
controls)
Absolute
Liver
weight,

g
(%
of
controls)
Relative
Liver
weight
(%
of
controls)
Body
weight,

%
of
controls
Liver
weight,
g
(%
of
controls)
Adenomas/
Carcinomas
Combined
0
25.6
1.3
5.07
1.70
1/
55
(
2
%)

48­
54
NF
NF
NF
NF
25.6
1.31
5.14
(
101%)
NT
NT
NT
143
NT
NT
NT
NT
NT
NT
NT
89**
1.59
(
96%)
6/
59
(
10
%)

188
+++(
d)
NF
NT
NT
NT
NT
NT
NT
NT
NT
287­
313
NT
NT
NF
NF
25.5
1.62
(
125%)

**
6.35
(
125%)**
78**
1.90
(
112%)*
5/
57
(
9
%)

375
+++
+++
NT
NT
NT
NT
NT
NT
NT
NT
711­
845
+++
+++
+++
NF
24.8
(
97%)
2.19
(
168%)

**
8.81
(
174%)

**
66**
2.32
(
136%)

**
24/
58
(
41
%)
**

NT:
Not
tested
NF:
Not
found
*
Significantly
different
than
control
(
p<
0.05)
**
Significantly
different
than
control
(
p<
0.01)

a)
Treated
for
90­
days
at
3,
12,
48,
188,
375,
and
750
mg/
kg/
day.

b)
Treated
for
2
weeks
at
54,
287,
and
845
mg/
kg/
day
c)
18
month
study.
Overall
mean
doses
were143,
313,
and
711
mg/
kg/
day.

d)
+++
means
positive
responses.
­
25­
TABLE
9.
Quantitative
evaluation
of
peroxisomes
and
cytoplasmic
volume
(%)
in
male
and
female
mice
fed
Blazer
Technical
(
acifluorfen)
or
DINP
(
positive
control)
for
4
weeks.

Test
Material
Diet
conc.
(
ppm)
Size
Classification
(
males)
Size
Classification
(
females)

1
2
3
4
5
Total
Area
of
peroxi
somes
(
in
%)
1
2
3
4
5
Total
Area
of
peroxi
somes
(
in
%)

Blazer
Technicala
0
7.8
8.0
0.5
0.0
0.0
1.25
9.5
8.2
0.4
0.0
0.0
1.27
350
11.8*
9.6
0.3
0.1
0.0
1.49
13.2*
7.5
0.21
0.0
0.0
1.25
1735
15.4**
18.7**
2.9**
0.2*
0.0
3.43**
12.1
15.9**
2.2**
0.2
0.0
2.81**

5210
22.3**
35.7**
11.4**
2.8**
0.3**
9.03**
14.6
35.7**
14.0**
4.3**
1.0*
10.6**

DINPb
0
12.1
10.7
1.0
0.1
0.1
1.93
24.6
12.1
1.4
0.1
0.0
2.48
500
18.2*
11.0
1.3
0.0
0.0
2.16
22.3
9.3
0.5
0.0
0.0
1.88
1500
55.0**
15.5
0.8
0.1
0.0
3.56**
32.4
19.9**
1.2
0.0
0.0
3.63*

4000
49.6**
47.3**
10.9**
1.1**
0.1
10.6**
31.3
37.2**
11.1**
2.1**
0.1
9.52**

8000
18.3**
35.5**
27.9**
13.9**
2.1**
18.7**
26.4
35.5**
18.6**
9.7**
1.8
14.8**

aData
from
pages
28­
29
MRID
45693401
bData
from
page
29
of
MRID
45686501
*
p

0.05;
**
p

0.01
­
26­
APPENDIX
I
Supporting
Executive
Summaries
Subchronic
toxicity
in
rats
In
a
90­
day
feeding
study
(
MRID
No.
00122730;
Accession
No.
071308),
Tackle
(
20.4­
23.6%
a.
i.)
was
administered
to
Fischer
344
rats
(
30/
sex/
dose)
in
the
diet
at
levels
of
0,
20,
80,
320,
1250,
2500
or
5000
ppm
(
0,
2,
8,
32,
125,
250
or
500
mg/
kg/
day
based
on
a
conversion
factor
of
1
ppm
=
0.1
mg/
kg/
day)
for
3
months.
Body
weights
for
2500
and
5000
ppm
males
and
5000
ppm
females
were
decreased
(
92.3­
95.5%,
65.3%­
71.2%
and
83.3­
90.6%
of
the
controls,
respectively).
Food
consumption
for
the
5000
ppm
males
and
females
was
decreased
throughout
most
of
the
study
period.
At
2500
ppm
or
above
in
one
or
both
sexes,
there
were
significant
(
p<
0.05)
changes
in
hematology
parameters
(
erythrocyte
counts,
hematocrit,
and
hemoglobin
concentration),
clinical
chemistry
values
(
serum
electrolytes,
calcium,
phosphorus,
glutamic
pyruvic
transaminase
and
alkaline
phosphatase
activities,
BUN,
creatinine,
serum
protein,
albumin
and
globulin
concentrations)
and
urinalysis
parameters
(
nitrate
and
urobilinogen
content).
Liver
and
kidney
weights
(
both
absolute
and
relative)
were
increased.
Histopathological
changes
were
seen
in
the
liver
of
2500
ppm
or
above
males
and
females
(
including
increased
cellular
hypertrophy,
mitotic
activity,
individual
cell
death,
and
proliferation
of
oval
or
bile
duct
cells).

In
the
1250
ppm
group
(
in
one
or
both
sexes),
there
were
decreases
in
hematology
parameters
(
erythrocyte
counts
and
hematocrit
values),
increase
in
absolute
and
relative
liver
weights
(
20­
21%
and
22­
74%,
respectively),
increase
in
absolute
and
relative
kidney
weights
(
11­
13%
and
10­
12%,
respectively),
and
increased
incidence
of
hypertrophy
of
liver
cells
when
compared
to
the
controls.

No
significant
treatment­
related
effects
were
seen
in
males
or
females
of
320
ppm
or
below.
The
LOAEL
for
subchronic
toxicity
is
1250
ppm
(
125
mg/
kg/
day)
based
on
decreases
in
hematology
parameters,
increases
in
liver
and
kidney
weights,
and
increased
incidence
of
hypertrophy
of
liver
cells
when
compared
to
the
controls.
The
NOAEL
for
systemic
toxicity
is
320
ppm
(
32
mg/
kg/
day).

NOTE:
In
the
original
DER
[
TXR
No.
003409
(
same
as
TXR
No.
003556)],
this
study
was
classified
as
supplementary.
The
registrant
was
requested
to
submit
the
following:
a)
Individual
animal
histopathology
on
the
animals
of
the
20­
2500
ppm
groups;
b)
An
explanation
of
why
in
the
"
author's
report
the
least
frequent
clinical
observation
was
"
loss
of
hair"
while
the
submitted
daily
record
of
clinical
signs
did
not
mention
this
event
[
pages
98­
102
of
the
report].
­
27­
In
the
Agency's
response
to
registrant's
comment
on
previous
review,
this
study
was
classified
as
supplementary
(
TXR
No.
003963).
The
explanation
of
regarding
"
loss
of
hair"
was
satisfactory
but
the
individual
animal
histopathology
on
the
animals
of
the
20­
2500
ppm
groups
was
not
submitted.
This
study
is
classified
as
Unacceptable/
guideline
but
upgradable.
The
data
in
this
study
was
used
to
set
the
doses
in
the
rat
chronic/
carcinogenicity
study.

Subchronic
toxicity
in
mice
In
a
90­
day
feeding
study
(
MRID
No.
00252826;
Accession
No.
071308),
Tackle
(
20.4­
23.2%
a.
i.)
was
administered
to
B6C3F1
mice
(
30/
sex/
dose)
in
the
diet
at
levels
of
0,
20,
80,
320,
1250
or
2500
ppm
(
0,
3,
12,
48,
187.5
or
375mg/
kg/
day
based
on
a
conversion
factor
of
1
ppm
=
0.15
mg/
kg/
day)
for
3
months.
Changes
in
mean
body
weights,
hematology
(
total
white
blood
cell
numbers,
MCV,
reticulocyte
counts),
clinical
chemistry
(
SGPT,
alkaline
phosphatase,
serum
glucose),
liver
weights
(
absolute
and
relative),
and
histopathological
changes
in
the
liver
(
hypertrophy,
increased
mitotic
activity,
individual
cell
death
and
focal
necrosis)
were
noted
in
males
and
females
at
the
2500
and
5000
ppm
doses
at
30
and
90
days.
Fatty
infiltration
of
the
liver
was
observed
in
males
and
females
at
the
1250,
2500
and
5000
ppm
doses
at
30
days
and
at
the
1250
and
2500
doses
at
90
days.

The
NOAEL
for
systemic
toxicity
is
320
ppm
(
48
mg/
kg/
day)
and
the
LOAEL
is
1250
ppm
(
187.5
mg/
kg/
day)
based
on
histopathologic
changes
(
fatty
infiltration)
of
the
liver.
This
study
is
classified
as
unacceptable/
guideline
but
upgradable.
The
data
in
this
study
were
used
to
select
the
doses
in
the
mouse
chronic/
carcinogenicity
study.

Chronic
toxicity
in
dogs
In
a
chronic
toxicity
study
(
MRID
No.
00131162;
Accession
No's.
251297
and
251298),
Tackle
"
2S"
(
Acifluorfen,
sodium
salt;
purity
was
unspecified)
was
administered
to
Beagle
dogs
(
8/
sex/
dose)
in
the
diet
at
levels
of
0,
20,
300
or
4500
ppm
(
0,
0.5,
7.5
or
112.5
mg/
kg/
day
based
on
a
conversion
factor
of
1
ppm
=
0.025
mg/
kg/
day)
for
2
years.

Body
weights
were
lower
than
respective
controls
in
males
(­
8
to
­
20%)
and
females
(­
9
to
­
10%)
at
4500
ppm
(
high­
dose)
throughout
the
study
(
statistical
analysis
was
not
performed).
In
addition,
increases
in
liver
and
kidney
weights
(
both
absolute
and
relative)
were
seen
in
high
dose
males
and
females.
Additionally,
in
both
sexes
at
high
dose,
red
blood
cell
counts,
hemoglobin,
hematocrit
and
cholesterol
values
were
significantly
lower
and
leukocyte
counts
and
urinary
volume
were
significantly
higher
than
those
of
the
controls.
In
males
at
high
dose,
serum
level
of
creatinine
was
lower
than
that
of
the
controls.
Platelet
counts,
lactate
dehydrogenase
activity
and
specific
gravity
of
urine
were
higher
than
respective
controls.
In
females
at
high
dose,
serum
levels
of
calcium
and
albumin
were
lower
when
compared
to
the
controls.
Histologically,
there
were
increased
­
28­
incidence
of
microscopic
changes
in
the
liver
(
congestion,
brown
pigment,
fatty
vacuoles
and
inflammation)
when
compared
to
the
controls.

The
LOAEL
for
systemic
toxicity
is
4500
ppm
(
112.5
mg/
kg/
day)
based
on
decreased
body
weight
gain,
increased
absolute
and
relative
liver
and
kidney
weights,
changes
in
hematology,
biochemistry,
and
urinalysis
parameters
and
increased
incidence
of
microscopic
changes
in
the
liver
(
congestion,
brown
pigment,
fatty
vacuoles
and
inflammation).
The
NOAEL
for
systemic
toxicity
is
300
ppm
(
7.5
mg/
kg/
day).

This
chronic
feeding
study
in
dogs
is
classified
as
Unacceptable/
guideline
and
does
not
satisfies
the
guideline
data
requirement
for
a
chronic
toxicity
study
(
83­
1)
in
dogs.
However,
it
can
be
upgraded.
Reasons
for
this
classification
are
the
purity
of
the
test
substance
was
not
adequately
identified
in
the
report
and
stability
of
the
test
substance
in
dog
chow
was
not
reported.
However,
a
new
study
is
not
required
because
when
this
study
and
the
data
of
another
chronic
feeding
study
in
dog
(
MRID
No.
00107484)
with
Blazer
are
analyzed
together,
they
provide
a
reasonable
understanding
of
the
chronic
toxicity
of
acifluorfen
in
dogs.
Another
chronic
feeding
study
in
dog
(
MRID
No.
00107484)
with
Blazer
is
an
old
study
(
reported
in
1978)
which
was
classified
as
core
guideline
data,
however,
the
DER
for
this
study
was
not
available.
In
the
old
study,
there
were
changes
in
hematological
and
biochemical
parameters
related
to
the
effects
in
the
liver
of
the
treated
dogs
at
high
dose
(
1800
to
5400
ppm)
when
compared
to
the
controls.
Histopathologically,
treatment
related
alterations
were
observed
in
the
liver,
kidney,
gall
bladder
and
eyes
when
compared
to
the
controls.
The
NOAEL
for
systemic
toxicity
was
50
ppm
and
LOAEL
was
300
ppm
(
mid
dose)
based
on
a
coagulation
effects
when
compared
to
the
controls
(
HED
Doc.
No.
001099;
extracted
from
the
earlier
review
of
A.
Arce
dated
May
2,
1979).

Chronic
toxicity/
carcinogenicity
in
mice
[
feeding]

(
1)
In
a
24­
month
carcinogenicity
study
(
MRID
No.
00082897),
Blazer
(
39.4­
40.5%
a.
i.)
was
administered
to
CR
CD­
1
mice
(
80/
sex/
dose)
in
the
diet
at
levels
of
0,
7.5,
45
or
270
ppm
for
24
months
(
0,
1.125,
6.75
or
40.5
mg/
kg/
day
based
on
a
conversion
factor
of
1
ppm
=
0.15
mg/
kg/
day).
The
highest
dose
level
was
initially
administered
to
mice
at
a
dose
of
1.25
ppm
on
study
weeks
1
to
16
before
being
increased
to
270
ppm.
Blazer
produced
a
statistically
significant
(
p<
0.05)
increase
in
the
total
number
of
liver
tumors
(
primarily
adenomas)
in
high
dose
(
270
ppm)
female
mice.
No
significant
increase
in
liver
tumors
occurred
in
male
mice.

In
high
dose
males,
there
was
a
dose­
related
increase
in
absolute
and
relative
liver
weights
and
relative
kidney
weights.
In
addition,
there
was
dose
related
elevation
of
alkaline
phosphatase
and
serum
glutamic
pyruvic
transaminase
activities
in
mid
and
high
dose
male
mice.
However,
due
to
the
variability
of
the
standard
deviation
for
these
parameters,
statistical
analysis
was
not
performed.
­
29­
The
NOAEL
for
systemic
toxicity
is
45
ppm
(
6.75
mg/
kg/
day)
and
the
LOAEL
is
270
ppm
(
40.5
mg/
kg/
day)
based
on
increased
absolute
and
relative
liver
weights,
and
increased
relative
kidney
weights.
It
is
noted
that
increased
levels
of
enzymes
(
ALP
and
SGPT)
seen
at
40.5
mg/
kg/
day
in
this
study
were
considered
equivocal
because
of
large
standard
deviations.

This
study
by
itself
does
not
satisfy
the
guideline
requirement
for
a
carcinogenicity
study
(
83­
2b)
in
mice.
The
deficiencies
of
the
study
report
are
as
follows:
resolution
of
histopathology
questions;
explanation
of
why
the
270
ppm
diet
was
analyzed
only
once;
provide
stability
of
the
compound
in
feed
data;
explanation
of
Table
4
(
Food
Consumption).
However,
the
requirement
for
carcinogenicity
study
for
mice
is
satisfied
when
considered
together
with
the
other
mouse
carcinogenicity
study
(
MRID
No.
00122732;
Accession
No.
071312,
071313,
071314,
250463,
and
250464).
When
the
data
of
these
two
studies
are
analyzed
together,
they
provide
a
reasonable
understanding
of
the
carcinogenicity
of
acifluorfen
on
mice.

(
2)
In
a
18­
month
carcinogenicity
study
(
MRID
No.
00122732;
Accession
No's.
071312,
071313,
071314,
250463,
and
250464),
Tackle
(
20.4­
23.2%
a.
i.)
was
administered
to
B6C3F1
mice
(
60/
sex/
dose)
in
the
diet
at
levels
of
0,
625,
1250
or
2500
ppm
(
0,
119,
259
or
655
mg/
kg/
day
for
males
and
0,
143,
313
or
711
mg/
kg/
day
for
females)
for
18
months.

An
increase
in
mortality
was
seen
in
high
dose
males
(
control,
1/
60;
high­
dose,
10/
60).
Body
weights
in
all
treated
mice
were
reduced
when
compared
to
controls.
Beginning
at
week
2
for
mid­
and
high­
dose
males
and
week
6
for
low­
dose
males,
body
weights
were
significantly
reduced
(
p<
0.01)
relative
to
controls.
The
body
weight
decrease
for
low­,
mid­,
and
high­
dose
males
was
8%,
10%
and
23%
of
the
controls,
respectively
at
week
13
and
10%,
13%
and
25%
of
the
controls,
respectively
at
week
79.
Similar
results
were
obtained
with
females,
except
that
reduced
body
weights
of
low­
and
mid­
dose
females
were
not
significant
until
week
13.
The
body
weight
decrease
for
low­,
mid­,
and
highdose
females
was
6%,
5%
and
14%
of
the
controls,
respectively
at
week
13
and
11%,
22%
and
34%
of
the
controls,
respectively
at
week
79.
Mean
food
consumption
in
highdose
males
was
higher
relative
to
the
controls
most
of
the
study
period.

Mean
corpuscular
volume
(
MCV)
and
segmented
neutrophil
counts
were
decreased
and
lymphocyte
and
RBC
counts
were
increased
compared
to
controls
in
all
treated
males
at
final
sacrifice.
Segmented
neutrophil
counts
was
decreased
and
lymphocyte
counts
were
increased
in
all
treated
females
(
at
interim
sacrifice)
and
in
mid­
and
high­
dose
females
(
at
final
sacrifice)
when
compared
to
controls.

Mean
absolute
and
relative
liver
weights
of
treated
males
and
females
were
greater
than
the
controls
at
interim
and
final
sacrifice.
Gross
pathology
showed
an
apparent
doserelated
increase
in
incidence
of
liver
masses
in
treated
males
when
compared
to
the
controls
at
final
sacrifice.
In
high
dose
females,
an
increase
in
incidence
(
37%)
of
liver
masses
was
observed.
The
incidence
of
white
foci
(
1
mm)
on
the
nonglandular
portion
of
­
30­
the
stomach
were
seen
in
high­
dose
males
and
mid­
and
high­
dose
females.
In
addition,
one
high­
dose
male
and
one
high­
dose
female
each
had
an
ulcer
of
the
stomach.

Acifluorfen
was
associated
with
statistically
significant
positive
trends
for
liver
tumors
(
adenomas,
carcinomas,
and
adenomas/
carcinomas
combined)
and
stomach
tumors
(
papillomas)
in
both
sexes.
The
liver
tumors
were
significantly
increased
above
the
controls
at
the
lowest
dose
level
tested
(
625
ppm)
in
male
mice
and
at
the
highest
dose
level
tested
(
2500
ppm)
in
both
sexes.
In
addition,
the
stomach
tumors
were
significantly
increased
above
the
controls
at
the
highest
dose
level
tested
(
2500
ppm)
in
both
sexes.
The
highest
dose
tested
was
considered
adequate
in
evaluation
of
carcinogenic
potential
of
the
test
chemical.

The
NOAEL
for
systemic
toxicity
is
not
established.
The
LOAEL
for
systemic
toxicity
is
equal
or
lower
than
625
ppm
(
119
and
143
mg/
kg/
day
for
males
and
females,
respectively)
based
on
reduced
body
weight,
increased
absolute
and
relative
liver
weights,
and
changes
in
hematologic
parameters
(
decreased
MCV
counts,
decreased
segmented
neutrophil
counts,
increased
RBC
counts,
and
increased
lymphocyte
counts).
This
study
is
classified
as
Acceptable/
Guideline
and
satisfies
the
Subdivision
F
guideline
requirement
for
a
carcinogenicity
study
(
83­
2b)
in
mice.

Chronic
toxicity/
carcinogenicity
in
rats
[
feeding]

In
a
two­
year
feeding/
carcinogenicity
study
(
MRID
No.
00128353;
Accession
No's.
071315
through
071317
and
250289
through
250792),
Tackle
(
19.1­
25.6%
a.
i.)
was
administered
to
Fischer
344
rats
(
73/
sex/
dose)
in
the
diet
at
levels
of
0,
25,
150,
500,
2500
or
5000
ppm
for
2
years
(
0,
1.25,
7.50,
25.0,
125
or
250
mg/
kg/
day
based
on
a
conversion
factor
of
1
ppm=
0.05
mg/
kg/
day).

All
males
and
61/
65
females
in
5000
ppm
group
died
before
termination
of
the
study.
Mean
body
weight
was
significantly
decreased
in
males
at
the
2500
(
7­
9%)
and
5000
ppm
(
18­
35%)
throughout
the
study
relative
to
the
controls.
In
females,
the
body
weight
was
also
decreased
in
2500
(
6­
16%)
and
5000
ppm
(
11­
28%)
groups
relative
to
the
controls.

Red
cell
counts,
hematocrit
and
hemoglobin
values
were
significantly
lower
in
the
5000
ppm
males
when
compared
to
the
controls.

In
the
5000
ppm
males
and
females,
blood
glucose,
triglyceride,
and
serum
globulin
and
total
protein
were
significantly
lower
than
those
of
the
control
animals;
BUN,
creatinine
and
alkaline
phosphatase
were
significantly
higher
than
those
of
the
control
animals.
In
the
2500
ppm
males,
blood
glucose,
triglyceride
and
globulin
were
significantly
lower
and
BUN
was
significantly
higher
when
compared
to
the
control
animals.
In
the
2500
ppm
females,
triglyceride
and
globulin
were
significantly
lower
and
alkaline
phosphatase
was
significantly
higher
when
compared
to
the
control
animals.

Gross
necropsy
showed
that
there
was
an
increased
incidence
of
kidney
and
liver
discoloration,
stomach
ulcers
and
reduced
testes
size
for
the
5000
ppm
males
when
­
31­
compared
to
the
controls.
In
females,
an
increased
incidence
of
kidney
lesions
(
distended
pelvis/
calculi)
was
observed
in
the
2500
and
5000
ppm
animals
and
stomach
ulceration
was
observed
in
the
5000
ppm
animals
when
compared
to
the
controls.

In
2500
ppm
males,
there
were
increases
in
absolute
and
relative
liver
weights,
decreases
in
spleen
weights,
and
increases
in
relative
kidney
weights.
In
5000
ppm
males,
there
were
increases
in
liver
weights
and
decreases
in
spleen
weights.
In
females
at
2500
and
5000
ppm,
there
were
increases
in
relative
liver
weights
and
decreases
in
heart
weights.

Histologically,
there
were
increased
incidences
of
acidophilic
cells
in
the
liver
of
the
5000
ppm
group
in
both
sexes
and
in
the
2500
ppm
females.
In
addition,
there
were
increased
incidences
of
nephritis/
pyelonephritis
in
2500
and
5000
ppm
group
females.
An
increase
in
the
incidence
of
stomach
ulcer
was
seen
in
5000
ppm
males
and
females.
Testicular
atrophy
was
also
seen
in
5000
ppm
males.

Under
the
condition
of
the
study,
no
treatment­
related
increase
in
tumor
incidence
was
found
in
the
acifluorfen
treated
rats.

The
NOAEL
for
systemic
toxicity
is
500
ppm
(
25
mg/
kg/
day);
the
LOAEL
is
2500
ppm
(
125
mg/
kg/
day)
based
on
reduced
body
weight,
increased
absolute
and
relative
liver
weights
and
increased
kidney
weights,
increased
incidence
of
nephritis/
pyelonephritis,
increased
incidence
of
acidophilic
cells
in
the
liver,
and
related
changes
in
clinical
chemistry
parameters
The
highest
dose
(
5000
ppm)
tested
was
considered
excessively
toxic
in
evaluation
of
carcinogenic
potential
of
the
test
chemical
based
on
findings
of
reduced
body
weights,
increased
mortality,
increased
liver
weights
and
liver
enzyme
changes
(
alkaline
phosphatase),
renal
changes
(
nephritis
and
pyelonephritis),
stomach
ulcers
and
decreased
testes
size.
This
study
is
classified
as
Acceptable/
guideline
and
satisfies
the
guideline
data
requirement
for
a
combined
chronic/
carcinogenicity
study
(
83­
5)
in
rats.

Another
rat
chronic
feeding
study
(
MRID
No.
00087478)
with
Blazer
was
also
conducted
by
Rohm
and
Haas.
The
DER
(
HED
Doc.
No.
001099
and
001251)
for
this
study
indicated
no
increase
in
any
tumor
incidence
in
treated
animals
relative
to
the
controls.
However,
the
experimental
design
of
the
study
used
multiple
shifts
in
dose
levels
at
different
durations
of
the
study.
It
is
difficult
to
establish
at
what
dose
levels
or
times
that
changes
in
biological
parameters
occurred.
However,
when
the
data
of
these
two
studies
are
analyzed
together,
they
provide
a
reasonable
understanding
of
the
chronic
toxicity
of
acifluorfen
in
rats.