Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
EPA
Reviewer:
Abdallah
Khasawinah,
Ph.
D.
Signature:
Reregistration
Branch
4,
HED
(
7509C)
Date:
EPA
Secondary
Reviewer:
William
Dykstra,
Ph.
D.
Signature:
Reregistration
Branch
4,
HED
(
7509C)
Date:

TXR#:
0051041
DATA
EVALUATION
RECORD
STUDY
TYPE:
Hepatocellular
Proliferation
­
Mouse
(
Non­
guideline)

DP
BARCODE:
D284584,
D286951,
D287541
SUBMISSION
CODE:
S619276,
S625382,
S627058
P.
C.
CODE:
111901
TOX.
CHEM.
NO.:
497AB
TEST
MATERIAL
(
PURITY):
Imazalil
Technical
(
96.2%
purity)

SYNONYMS:
R023979;
(
±
)
­
2­
ethoxy­
2,3­
dihydro­
3,3­
dimethylbenzofuran­
5­
ylmethanesulfonate
(
IUPAC)

CITATION:
O'Neill,
T.
P.
(
2002)
Cell
Proliferation
Study
in
Male
CD­
1
Mice
After
Administered
Imazalil
in
the
Diet
for
2
or
13
Weeks
­
Final
Report:
Lab
Study
Number:
WIL­
436001.
WIL
Research
Laboratories,
Inc.
(
Ashland,
OH)
574
pages.
July
9,
2002.
MRID
45713701.
Unpublished.

O'Neill,
T.
P.
(
2002)
Cell
Proliferation
Study
in
Male
CD­
1
Mice
After
Administered
Imazalil
in
the
Diet
for
2
or
13
Weeks
­
Interim
Final
Report:
Lab
Study
Number:
WIL­
436001.
WIL
Research
Laboratories,
Inc.
(
Ashland,
OH)
573
pages.
June
27,
2002.
MRID
45708401.
Unpublished.

O'Neill,
T.
P.
(
2002)
Cell
Proliferation
Study
in
Male
CD­
1
Mice
After
Administered
Imazalil
in
the
Diet
for
2
or
13
Weeks
­
Addendum
to
the
Final
Report:
Supplement
to
MRID
45713701,
Lab
Study
Number:
WIL­
436001.
WIL
Research
Laboratories,
Inc.
(
Ashland,
OH)
40
pages.
October
11,
2002.
MRID
45783801.
Unpublished.

O'Neill,
T.
P.
(
2002)
Cell
Proliferation
Study
in
Male
CD­
1
Mice
After
Administered
Imazalil
in
the
Diet
for
2
or
13
Weeks
­
Addendum
II
to
the
Final
Report:
Supplement
to
MRID
45713701,
Lab
Study
Number:
WIL­
436001.
WIL
Research
Laboratories,
Inc.
(
Ashland,
OH)
55
pages.
December
17,
2002.
MRID
45822503.
Unpublished
SPONSOR:
Janssen
Pharmaceutica
N.
V.,
B­
2340,
Beerse,
Belgium
EXECUTIVE
SUMMARY:
In
a
nonguideline
hepatocellular
proliferation
study
(
MRID
45713701,
45708401,
45783801
&
45822503)
conducted
to
investigate
the
mechanism
of
liver
tumor
induction
observed
at
same
dosage
levels
in
oncogenicity
studies,
groups
of
male
Crl:
CD­
1R(
ICR)
BR
mice
(
10/
dose/
sacrifice
time)
were
given
imazalil
(
purity
96.2%,
Lot.
No.
ZR023979G3H771)
in
the
diet
ad
libitum
at
concentrations
of
0.0,
100,
200,
400,
600
or
1200
ppm
(
0,
18.7
±
1.1,
38.3
±
2.7,
73.4
±
5.13,
110.4
±
11.7,
233.2
±
24.5
mg/
kg/
day)
for
2
or
13
weeks.
Bromo­
deoxyuridine
(
BrdU)
in
sterile
phosphate­
buffered
saline
(
30
mg/
mL)
was
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
2
of
14
administered
subcutaneously
(
through
an
implanted
osmotic
minipump)
seven
days
prior
to
the
appropriate
mice
sacrifice
period.
Animals
were
sacrificed
after
2
week
interim
period;
4
week
recovery
period
(
the
0
and
1200
ppm
group
only);
13
week
primary
sacrifice;
17
week
recovery
period
(
the
0
and
1200
ppm
group
only).
At
sacrifice,
the
animals
were
weighed
and
the
liver
removed,
weighed,
and
sections
prepared
for
serial
review
by
Hematoxylin
and
Eosin
or
BrdUimmunohistochemical
and
Proliferating
Cell
Nuclear
Antigen
(
PCNA)
staining
of
hepatocytes.

Administration
of
imazalil
to
male
mice
in
their
diets
for
up
to
13
weeks
affected
survival
and
body
weight
only
at
the
1200
ppm
level.
Clinical
findings,
food
consumption
and
macroscopic
changes
were
not
affected
by
the
imazalil
treatment.
The
primary
organ
of
imazalil
toxicity
in
this
test
was
the
liver.
Imazalil
produced
microscopic
changes
(
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation,
and
hepatocellualr
necrosis)
in
a
dose
related
manner.
Liver
enzymes
levels
ALD
and
SDH
were
elevated
but
not
LDH.
With
the
exception
of
a
single
hepatocellular
nucleus
in
a
200
ppm
animal
positive
for
BrdU
labeling,
none
was
positive
in
all
the
100,
200,
400,
and
600
ppm
groups.
BrdU
labeling
in
the
high
dose
group
of
1200
ppm
was
lower
than
in
the
control
group
at
both
the
interim
sacrifice
of
two
weeks
or
the
13
week
primary
sacrifice.
The
4
week
and
17
week
recovery
sacrifices
were
negative
for
any
BrdU
labeling
in
both
the
control
and
1200
ppm
groups.
This
was
confirmed
by
repeating
the
staining
procedure
for
the
control
and
high
dose
groups
at
the
interim
and
13
week
sacrifices.
The
BrdU
staining
procedure
was
also
verified
by
producing
positive
results
in
cells
taken
from
duodenum
sections.
It
was
observed
that
control
animals
had
a
higher
labeling
percentage
than
the
highest
imazalil
dose
tested.

PCNA
labeling
index
as
an
indicator
of
hepatic
cell
proliferation
was
(
statistically
significant)
lower
in
the
imazalil
tested
dose
groups
than
in
the
control
group.
This
demonstrates
the
lack
of
induction
of
hepatic
cell
proliferation
by
imazalil.
"
There
was
a
decreased
proportion
of
PCNAlabeled
hepatocyte
nuclei
and
fewer
hepatocyte
nuclei
overall
in
like­
sized
total
areas
of
the
livers
of
the
200,
400,
600
and
1200
ppm
group
mice
compared
to
mice
in
the
control
group",
suggesting
an
inhibition
of
cell
proliferation
in
the
imazalil
treated
mice.

The
LOAEL
for
imazalil
in
the
male
mice
is
200
ppm
(
38.3
±
2.7
mg/
kg/
day)
based
on
its
hepatotoxicity
(
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation,
and
hepatocellualr
necrosis
and
elevated
sorbitol
dehydrogenase
levels).
The
NOAEL
is
100
ppm
(
18.7
±
1.1
mg/
kg/
day).

This
study
is
considered
Acceptable/
Non
Guideline
for
the
intended
purpose.

COMPLIANCE:
Signed
and
dated
GLP,
Quality
Assurance,
Data
Confidentiality,
and
Flagging
statements
were
provided.
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
3
of
14
I.
MATERIALS
AND
METHODS
A.
MATERIALS:

1.
Test
Material:
Imazalil
technical
(
R023979)
Description:
Clear,
viscous,
orange­
yellow
liquid
Lot
#:
ZR023979G3H771
Purity:
96.2%
a.
i.
Stability
of
compound:
considered
stable
at
room
temperature
CAS
#:
35554­
44­
0
Structure:

2.
Vehicle:
PMI
Certified
Rodent
LabDiet
®
(
No.
5002)
was
used
for
preparing
the
test
diet
mixtures.

3.
Test
animals:
Species:
Male
mice
Strain:
Crl:
CD­
1R(
ICR)
BR
Age
at
study
initiation:
60
Days
Weight
at
study
initiation:
21.5
­
35.4
g
Source:
Charles
River
Laboratories,
Inc.,
Kingston,
NY
Housing:
Individually
housed
in
clean
cages
suspended
above
cage­
board
or
other
suitable
material
Diet:
PMI
Certified
Rodent
LabDiet
®
(
No.
5002),
ad
libitum
Water:
Tap
water
filtered
by
reverse
osmosis,
ad
libitum
Environmental
conditions:
Temperature:
21.7
22.8oC
(
recorded
daily)
Humidity:
20.7
­
67.4%
(
recorded
daily)
Air
changes:
Not
reported
Photo
period:
12­
Hour
light/
dark
cycle
Acclimation
period:
13
Days
B.
STUDY
DESIGN
1.
In­
life
dates
­
Start:
09/
14/
2001
End:
04/
24/
2002
2.
Animal
assignment
Prior
to
initiation
of
treatment,
the
mice
were
weighed
and
examined
in
detail
for
physical
abnormalities.
Mice
judged
suitable
for
assignment
to
the
study
were
selected
by
the
study
director
and
assigned
to
the
test
groups
shown
in
Table
1
using
a
computer­
based
randomization
procedure
that
ensured
the
homogeneity
of
group
means
for
body
weight.
Individual
body
weights
at
randomization
were
within
±
20
%
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
4
of
14
of
the
mean.

Table
1.
Study
Design.

Group
#
Treatment
Dose
(
ppm)
Number
of
Mice
2
Week
Necropsy
4
Weeka
Necropsy
13
Week
Necropsy
17
Weeka
Necropsy
1
Untreated
0
40
10
10
10
10
2
Imazalil
100
20
10
10
3
Imazalil
200
20
10
10
4
Imazalil
400
20
10
10
5
Imazalil
600
20
10
10
6
Imazalil
1200
40
10
10
10
10
a
Recovery
Groups
3.
Test
Diet
Preparation
and
Analysis
A
premix
of
imazalil
and
diet
was
prepared
by
homogenizing
appropriate
amounts
of
imazalil
and
rodent
feed.
Desired
dietary
concentrations
were
then
prepared
by
homogenizing
premix
amounts
with
known
quantities
of
the
rodent
feed.
Samples
for
homogeneity
analysis
were
taken
from
the
top,
middle
and
bottom
of
each
diet.
Samples
for
stability
analysis
were
taken
from
the
middle
portion
of
each
diet
and
stored
under
laboratory
conditions
for
8
and
10
days.
Samples
for
concentration
analysis
were
collected
weekly
from
each
dosing
formulation
for
study
weeks
0,
1,
2,
3,
8
and
12.
The
test
diets
were
prepared
weekly
and
stored
at
room
temperature
in
properly
labeled
storage
bags.

The
analytical
data
indicated
that
the
variance
between
nominal
and
actual
dose
to
the
animals
was
acceptable.

Results
 
Homogeneity:
The
concentrations
of
imazalil
were
within
­
3
to
7.5%
of
target
concentrations
in
samples
from
the
top,
middle
and
bottom
of
the
batch
indicating
they
were
homogeneously
distributed
throughout
the
diet.

Stability:
Imazalil
was
considered
to
be
stable
within
the
diet
for
a
period
of
10
days.
The
mean
concentration
of
the
stored
formulations
ranged
from
86.0
to
97.5%
of
the
time
zero
concentrations.

Dose
concentration:
­
All
diets
were
found
to
be
within
90.3­
106%
of
nominal
concentrations
except
for
one
time
reading
of
116%
in
group
4.

4.
Dose
selection
rationale
The
objective
of
the
study
was
to
identify
a
mode
of
action
and
dose­
response
characteristics
for
imazalil­
induced
liver
tumor
response
in
the
same
strain
of
mice
used
in
the
previous
bioassay.
Increased
incidences
of
hepatocellular
neoplasms
were
found
in
a
carcinogenicity
study
with
male
mice
fed
200
or
600
ppm
imazalil
(
MRID
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
5
of
14
42972001).
The
doses
for
the
present
study
were
chosen
to
correspond
with
the
doses
in
the
bioassay
study.
The
doses
of
100,
400
and
1200
ppm
were
selected
to
provide
additional
dose
response
information.

4.
Statistics
The
body
weight,
body
weight
change,
food
consumption,
clinical
pathology
and
liver
weights
were
analyzed
by
ANOVA
followed
by
Dunnett's
test
(
p

0.05)
to
detect
significant
differences.
PCNA
data
was
analyzed
using
two­
tailed
tests
for
minimum
significance
levels
of
1%
and
5%,
comparing
each
test
article­
treated
group
to
the
control
group.
The
total
number
of
labeled
nuclei
and
labeling
index
data
were
subjected
to
a
prametric
one­
way
analysis
of
variance
to
determine
intergroup
differences.

C.
METHODS
1.
Animal
observations
All
mice
were
observed
twice
daily
for
moribundity
and
death
examined
and
once
daily
for
clinical
observations.
Detailed
physical
examinations
were
done
weekly
on
all
mice.

2.
Body
weight
and
food
consumption
Body
weights
and
food
consumption
were
recorded
weekly
starting
two
weeks
prior
to
dietary
administration
of
imazalil.
In
addition,
daily
food
consumption
and
daily
intake
of
test
material
were
calculated.

3.
Animal
preparation
Animals
were
anesthetized
using
isoflurane.
A
30
mg/
mL
solution
of
bromodeoxyuridine
(
BrdU)
in
sterile
phosphate­
buffered
saline
was
loaded
in
osmotic
minipumps
which
were
then
implanted
subcutaneously
seven
days
prior
to
the
appropriate
mice
sacrifice
period.

4.
Sacrifice
and
pathology
At
sacrifice,
mice
anesthetized
by
carbon
dioxide
and
blood
collected
from
the
vena
cava
for
evaluation
of
alanine
aminotransferase,
lactate
dehydrogenase
and
sorbitol
dehydrogenase.
The
liver
from
scheduled
and
unscheduled
sacrifices
was
removed
and
weighed.
Portions
(~
5
mm
in
thickness)
from
the
left
lateral,
right
lateral,
median
and
caudate
portions
of
the
liver,
as
well
as
a
portion
of
the
small
intestine,
were
excised,
fixed
in
10%
neutral­
buffered
formalin,
and
embedded
in
paraffin.
For
unscheduled
deaths,
the
remaining
liver
was
minced,
placed
in
a
cryovial,
flash
frozen
in
liquid
nitrogen
and
stored
at
­
70oC
for
possible
future
analysis.

Serial
sections
of
each
liver
portion
were
prepared
according
to
standard
operating
procedures
and
stained
with
Hematoxylin
and
Eosin
or
immunohistochemically
stained
for
BrdU­
labeled
nuclear
DNA,
both
with
(
test)
and
without
(
negative
control)
the
presence
of
anti­
BrdU
antibody.
"
Established
immunohistochemical
methods
were
used
to
identify
cells
in
the
S
phase
using
a
monoclonal
antibody
raised
against
a
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
6
of
14
purified
form
of
BrdU".
Labeling
index
(
LI)
was
used
to
measure
the
percentage
of
cells
in
the
DNNA
replicative
or
S
phase
thet
had
incorporated
BedU,
an
exogenous
DNA
precursor.
The
duodenum
was
processed
similarly
and
served
as
a
BrdU
positive
control.
Slides
of
the
liver
and
duodenum
were
scanned
by
light
microscopy
to
verify
BrdU
incorporation.

Liver
sections
were
scanned
for
uniform
BrdU
labeling
in
hepatocytes
of
the
different
lobes
of
the
liver.
Once
the
BrdU
labeling
was
qualitatively
determined
to
be
uniform
between
the
three
lobes
of
the
liver,
only
the
left
lobe
was
scored
for
BrdU
incorporation.
At
least
2000
hepatocellular
nuclei
per
animal
were
counted
and
scored
with
the
investigator
blind
to
the
animal
treatment.

Because
of
insufficient
staining
techniques
utilized
for
the
BrdU
labeling,
all
liver
and
duodenum
samples
were
restained
using
an
improved
staining
procedure
(
not
described
in
the
report).
BrdU
labeling
was
re­
evaluated
for
the
control
and
high
dose
groups
at
the
2
and
13
week
sacrifice.

Proliferating
Cell
Nuclear
Antigen
(
PCNA)
immunostaining
of
hepatocytes
to
determine
the
effects
of
imazalil
on
hepatocyte
proliferation
was
conducted
after
the
finding
that
imazalil
did
not
increase
(
induce)
BrdU
incorporation.
Tissue
blocks
containing
samples
of
liver
from
four
sections
and
small
intestine
(
as
positive
control)
from
the
control
group
and
the
high
dose
group
(
all
necropsies)
were
submitted
by
the
sponsor
to
Experimental
Pathology
Laboratories,
Inc.
(
EPL
®
)
for
PCNA
immunostaining
using
a
commercial
kit
(
Zymed
®
PCNA
staining
kit)
(
MRID
45783801.
Hepatocyte
nuclei
(
labeled
and
unlabeled)
were
counted
microscopically
in
a
grid
fashion.
Subsequent
to
the
finding
that
the
high
dose
group
animals
had
decreased
PCNA
labeling
compared
to
the
control
group
animals,
PCNA
immnonstaining
was
performed
on
the
other
test
groups
for
week
2
and
week
13
necropsies
(
MRID
45822503).

II.
RESULTS
A.
OBSERVATIONS
One
animal
in
the
1200
ppm
group
was
sacrificed
in
extremis
on
day
5
of
treatment
exhibiting
hypo­
activity,
tremors,
dermal
atonia,
shallow
respiration,
emaciation,
body
cool
to
touch,
prostration
and
unkempt
appearance
attributable
to
the
imazalil
treatment.
One
animal
in
the
200
ppm
group
was
found
dead
on
day
65
and
was
not
treatment
related.
No
other
mortalities
or
clinical
symptoms
were
reported
that
would
be
related
to
treatment.

B.
BODY
WEIGHT
AND
FOOD
CONSUMPTION
There
were
sporadic
and
inconsistent
body
weight
changes
among
groups
reaching
statistical
significance
(
p<
0.05­
0.01)
in
a
non
dose­
related
manner.
The
body
weight
gain
in
the
1200
ppm
group
was
18.3%
­
26.7%
lower
than
the
untreated
group
during
the
first
2
weeks
of
imazalil
administration
then
became
comparable
throughout
the
remainder
of
the
study.
With
the
exception
of
statistically
significant
(
p<
0.05
or
p<
0.01)
increases
in
food
consumption
in
the
200
­
1200
ppm
groups
during
study
weeks
6
and
7,
there
were
no
treatment­
related
effects
on
food
consumption
by
the
mice.
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
7
of
14
C.
COMPOUND
CONSUMPTION
The
calculated
mean
compound
consumption
over
the
13
week
feeding
period
(
calculated
by
the
reviewer
from
table
17A
of
the
study
report)
for
the
100,
200,
400,
600
and
1200
ppm
groups
is
18.7
±
1.1,
38.3
±
2.7,
73.4
±
5.13,
110.4
±
11.7,
233.2
±
24.5
mg/
kg/
day,
respectively.

D.
CLINICAL
CHEMISTRY
The
serum
chemistry
parameters
are
presented
in
Table
2.
A
statistically
significant
(
p<
0.01)
increase
(
97%
over
the
control)
in
alanine
transferase
(
ALT)
levels
were
reported
in
the
1200
ppm
imazalil
group
at
the
13
week
sacrifice
and
returned
to
normal
levels
after
4
weeks
of
recovery
period.
Mean
sorbitol
dehydrogenase
levels
were
elevated
(
114
­
380%
of
the
controls)
in
all
imazalil
groups
at
both
sacrifice
times
but
were
statistically
significant
only
at
the
600
(<
0.05)
for
the
2
week
sacrifice
and
1200
(<
0.01)
ppm
for
the
2
and
13
week
sacrifices.
Their
levels
returned
to
normal
during
the
recovery
periods.
Mean
lactate
dehydrogenase
levels
were
statistically
and
significantly
lower
in
the
600
and
1200
ppm
imazalil
groups
compared
to
the
control,
but
were
comparable
to
the
control
group
values
at
the
13
week
sacrifice
suggesting
that
this
decrease
was
an
anomaly
rather
than
treatment­
related.
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
8
of
14
Table
2.
Summary
of
serum
chemistry
values
in
male
mice
fed
diets
containing
imazalil
up
to
13
weeksa
Week
of
Sacrifice
Alanine
Transferase
(
U/
L)

0
100
ppm
200
ppm
400
ppm
600
ppm
1200
ppm
2
43
±
6.9
(
7)
39
±
6.1
(
10)
36
±
7.8
(
10)
29
±
8.2
(
10)
41
±
13.3
(
10)
44
±
16.8
(
10)

4
(
recovery)
32
±
7.2
(
10)
NA
NA
NA
NA
35
±
7.5
(
10)

13
43
±
6.5
(
10)
39
±
7.2
(
10)
45
±
23.3
(
9)
43
±
9.3
(
10)
40
±
18.0
(
10)
77
±
31.2**
(
10)

17
(
recovery)
39
±
10.9
(
10)
NA
NA
NA
NA
42
±
11.6
(
9)

Sorbitol
Dehydrogenase
(
U/
L)

2
5.6
±
1.78
(
3)
12.1
±
5.96
(
6)
11.9
±
3.17
(
4)
13.3
±
6.51
(
10)
18.2
±
6.70*
(
10)
21.3
±
9.22**
(
10)

4
(
recovery)
10.2
±
4.62
(
10)
NA
NA
NA
NA
9.8
±
6.30
(
9)

13
36.4
±
7.92
(
10)
41.7
±
8.86
(
10)
53.2
±
25.64
(
10)
51.8
±
14.43
(
10)
58.2
±
12.27
(
10)
96.4
±
36.48**
(
10)

17
(
recovery)
26.8
±
1.54
NA
NA
NA
NA
29.4
±
5.20
Lactate
Dehydrogenase
(
U/
L)

2
1573
±
925
(
7)
1407
±
702
(
10)
1683
±
452
(
10)
682
±
412*
(
10)
418
±
136**
(
10)
365
±
88**
(
10)

4
(
recovery)
166
±
97
(
10)
NA
NA
NA
NA
425
±
139
(
10)

13
423
±
95
(
10)
590
±
266
(
10)
484
±
168
(
9)
608
±
177
(
10)
579
±
246
(
10)
643
±
409
(
10)

17
(
recovery)
491
±
192
(
10)
NA
NA
NA
NA
432
±
115
(
9)

Reproduced
from
Table
20,
MRID
45713701
*
p

0.05,
**
p

0.01
NA
Not
Applicable
D.
ORGAN
WEIGHT
AND
PATHOLOGY
1.
Liver
weight
As
shown
in
Table
3,
there
was
a
statistically
significant
increase
in
the
absolute
liver
weight
(
36.7%,
13.6%
and
48.6%
increase)
and
relative
to
final
body
weight
(
40.6%,
12.6%
and
31.7%
increase)
at
the
2
week
interim
sacrifice,
4
week
recovery
sacrifice
and
13
week
primary
sacrifice,
respectively,
in
the
1200
ppm
treated
mice
compared
to
the
control
animals.
Liver
weights
(
absolute
and
relative)
were
comparable
to
the
control
group
at
the
17
week
sacrifice
time.
Increased
liver
weights
relative
to
body
weight
were
noted
in
the
600
ppm
group
at
study
weeks
2
and
13
necropsies
(
14.6%
and
13.5%
increase,
respectively).
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
9
of
14
Table
3.
Absolute
and
relative
liver
weights
of
male
mice
fed
diets
containing
imazalil
up
to
13
weeksa
Week
of
Sacrifice
Absolute
(
g)
0
100
ppm
200
ppm
400
ppm
600
ppm
1200
ppm
2
2.066
2.093
2.053
2.164
2.290
2.824**

4
(
recovery)
2.035
NA
NA
NA
NA
2.312*

13
2.301
2.243
2.310
2.562
2.566
3.289*

17
(
recovery)
2.104
NA
NA
NA
NA
2.096
Relative
to
final
body
weight
g%

2
5.844
5.923
5.938
6.234
6.699**
8.216**

4
(
recovery)
5.599
NA
NA
NA
NA
6.294**

13
5.725
5.474
5.548
6.135
6.499**
8.380**

17
(
recovery)
5.026
NA
NA
NA
NA
5.141
Data
from
Tables
26­
33,
MRID
45713701
*
p

0.05,
**
p

0.01
a
Based
on
10
mice/
group
except
for
the
200
ppm
group
13
week
sacrifice
was
9
mice
2.
Liver
pathology
Macroscopic
examination
did
not
reveal
any
liver
lesions
in
the
imazalil­
treated
animals.

3.
Histopathology
Histopathological
findings
of
the
liver
are
summarized
in
Table
4.
All
mice
in
the
200,
400,
600
and
1200
ppm
groups
developed
treatment­
related
centrilobular
hepatocellular
hypertrophy
with
increasing
incidence
and
severity
at
the
2
week
interim
sacrifice.
Hepatocellular
vacuolation
with
increasing
incidence
and
severity
were
also
noted
in
mice
treated
with
400,
600
or
1200
ppm
of
imazalil.
Minimal
hepatocellular
necrosis
was
observed
at
low
incidence
in
the
600
and
1200
ppm
groups.
At
the
4
week
recovery
necropsy
minimal
to
moderate
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation
was
seen
in
all
the
1200
ppm
animals,
while
hepatocellular
necrosis
incidence
was
similar
to
the
control
group.
At
the
13
week
necropsy
examination
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation
occurred
with
increasing
incidence
and
severity
in
all
animals
treated
with
200
ppm
of
imazalil
and
above
and
a
slightly
higher
incidence
of
minimal
hepatocellular
necrosis
in
the
1200
ppm
group.
Hepatocellular
vacuolation
with
lower
incidence
and
severity
persisted
during
the
recovery
period
at
17
weeks
sacrifice.
Minimal
hepatocellular
hypertrophy
was
note
only
in
one
animal
at
the
17
weeks
recovery
necropsy
with
an
absence
of
hepatocellualr
necrosis.
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
10
of
14
Table
4.
Summary
of
Microscopic
Findings
(
Liver)

Week
2
of
Sacrifice
0
100
ppm
200
ppm
400
ppm
600
ppm
1200
ppm
No.
examined
Hypertrophy,
hepatocellular
Minimal
Mild
Moderate
Necrosis,
Minimal
Inflamation,
Subacute,
Minimal
Vacoulation,
Hepatocellular
Minimal
Mild
10
0
None
None
None
000
None
None
10
0
None
None
None
000
None
None
10
33
None
None
0
None
0
None
None
10
10
82
None
0155
None
10
10
37
None
22
10
55
10
10
None
3711844
Week
4
of
Sacrifice
(
Recovery)

No.
examined
Hypertrophy,
hepatocellular
Minimal
Mild
Moderate
Necrosis,
Minimal
Inflamation,
Subacute,
Minimal
Vacoulation,
Hepatocellular
Minimal
Mild
Inflamation,
Chronic,
Mild
Mineralization,
Minimal
10
0
None
None
None
120
None
None
00
0
0
0
0
10
10
3521132111
Week
13
of
Sacrifice
No.
examined
Hypertrophy,
hepatocellular
Minimal
Mild
Moderate
Severe
Necrosis,
Minimal
Inflamation,
Subacute,
Minimal
Vacoulation,
Hepatocellular
Minimal
Mild
Moderate
Severe
Inflamation,
Chronic,
Mild
10
0
None
None
None
None
120
None
None
None
None
0
10
0
None
None
None
None
120
None
None
None
None
0
922
None
None
None
0
None
3210
None
2
10
972
None
None
139351
None
0
10
10
253
None
13
10
154
None
0
10
10
None
None
2834
10
None
3430
Week
17
of
Sacrifice
(
Recovery)

No.
examined
Hypertrophy,
hepatocellular
Minimal
Vacoulation,
Hepatocellular
Minimal
Mild
Moderate
10
0
None
0
None
None
None
0
0
0
0
9
117223
Reproduced
from
Tables
34
­
37,
MRID
45713701
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
11
of
14
5.
Hepatocellular
Proliferation
The
labeling
indices
for
imazalil
shown
in
Table
5.
With
the
exception
of
a
single
hepatocellular
nucleus
in
a
200
ppm
animal
positive
for
BrdU
labeling,
none
was
positive
in
all
the
100,
200,
400,
and
600
ppm
groups.
BrdU
labeling
in
the
high
dose
group
of
1200
ppm
was
similar
or
lower
than
in
the
control
group
at
both
the
interim
sacrifice
of
two
weeks
or
the
13
week
primary
sacrifice.
The
4
week
and
17
week
recovery
sacrifices
were
negative
for
any
BrdU
labeling
in
both
the
control
and
1200
ppm
groups.
According
to
the
study
report,
nuclear
labeling
with
BrdU
was
demonstrated
in
the
crypts
of
the
duodenum
of
all
but
one
animal,
indicating
both
exposure
of
the
animals
to
BrdU
and
the
adequacy
of
the
staining
method.

As
a
result
of
the
negative
results
regarding
BrdU
labeling
of
hepatocytes,
livers
from
the
control
and
1200
ppm
mice
at
the
interim
and
13
week
sacrifice
were
restained
using
an
improved
procedure
(
was
not
described).
Following
re­
staining,
the
mean
labeling
index
for
the
control
animals
and
the
1200
ppm
animals
at
the
2
week
sacrifice
was
10.79
and
7.10
BrdU
positive
cells
per
1000
hepatocytes,
respectively.
At
the
13
week
primary
sacrifice,
the
labeling
index
was
31.12
and
13.96
BrdU
positive
cells
per
1000
hepatocytes
for
the
control
and
1200
ppm
animals,
respectively.

TABLE
5.
Percent
of
BrdU­
labeled
nuclei
(
±
standard
deviation)
in
hepatocytes
of
mice
fed
diets
containing
imazalil
up
to
13
weeksa
Week
of
Sacrifice
Control
100
ppm
200
ppm
400
ppm
600
ppm
1200
ppm
2
0.99
±
1.1994
0.00
0.00
0.00
0.00
0.71
±
0.3823
4
(
recovery)
0.00
­
­
­
­
0.00
13
3.11
±
2.5509
0.00
0.00
0.00
0.00
1.394
±
1.1497
17
(
recovery)
0.00
­
­
­
­
0.00
*
Data
from
Tables
53­
56,
MRID
45713701
calculated
by
the
reviewer
Based
on
10
mice/
group
except
for
the
200
ppm
group
13
week
sacrifice
was
9
mice
To
further
demonstrate
the
potential
effects
of
imazalil
on
hepatocytes
cell
proliferation,
the
sponsor
subjected
liver
sections
from
the
control
and
imazalil
test
groups
to
PCNA
immunostaining
using
readily
commercial
staining
kits.
The
results
of
this
demonstrated
that
the
PCNA
labeling
index
in
the
high
dose
group
was
lower
than
in
the
control
group
demonstrating
the
lack
of
hepatic
cell
proliferation
by
imazalil.
The
results
are
summarized
in
Table
6.
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
12
of
14
TABLE
6.
Summary
of
Hepatocyte
Nuclei
Counts
and
PCNA
Labeling
Indices
Treatment
Group
Sacrifice
Week
Unlabeled
plus
Labeled
Nuclei
PCNA
labeled
Nuclei
Labeling
Index
%
Number
of
Grid
areas
Counted
for
PCNA
Labeled
Nuclei
Control
Group
2
2811
9
±
11.2
0.3
±
0.40
32
4
2592
9
±
9.2
0.4
±
0.37
32
13
2496
20
±
15.0
0.8
±
0.72
32
17
2613
4
±
3.6
0.15
±
0.18
32
100
ppm
2
2817
9
±
13.8
0.3
±
0.54
32
13
2679
12
±
6.8
0.4
±
0.28
32
200
ppm
2
2702
7
±
8.5
0.2
±
0.27
32
13
2457
9*
±
5.7
0.3*
±
0.23
33
400
ppm
2
2435
7
±
8.8
0.3
±
0.40
32
13
2216
4**
±
3.6
0.2**
±
0.18
34
600
ppm
2
2243
1
±
0.8
0.0
±
0.04
33
13
2151
3**
±
2.4
0.1**
±
0.12
36
1200
ppm
Group
2
2454
1
±
0.7
0.0
±
0.03
32
4
2383
1*
±
2.8
0.0*
±
0.13
33
13
2124
7**
±
12.4
0.3*
±
0.56
40
17
2152
1
±
0.8
0.1
±
0.05
34
Reproduced
from
Tables
65
&
66
on
pages
15
&
16
and
Table
on
page
41
of
Study
Report
MRID
45822503.
Labeling
Index
=
Total
Number
of
PCNA­
Labeled
nuclei
X
100%
Total
Number
of
Unlabeled
and
PCNA­
Labeled
Nuclei
*
=
Significantly
different
from
the
control
group
at
0.05
using
Dunnett's
test.
**
=
Significantly
different
from
the
control
group
at
0.01
using
Dunnett's
test.

III.
DISCUSSION
and
CONCLUSIONS
A.
INVESTIGATORS'
CONCLUSIONS:

Administration
of
imazalil
to
mice
in
their
diets
for
up
to
13
weeks
affected
survival
and
body
weights
only
at
the
1200
ppm
level.
Clinical
findings,
food
consumption,
mean
lactate
dehydrogenase
levels,
macroscopic
changes
and
BrdU
labeling
were
not
affected
by
the
imazalil
treatment.
Higher
SDH
levels
(
compared
to
controls)
were
noted
at
100
ppm
and
above.
However,
at
the
100
ppm
the
increased
SDH
levels
were
not
accompanied
with
microscopic
findings
or
other
increases
in
liver
enzymes.
Alanine
transferase
was
statistically
significantly
increased
at
the
1200
ppm
after
13
weeks
of
exposure
and
returned
to
normal
levels
during
the
recovery
period.
There
was
a
statistically
significant
increase
in
the
absolute
liver
weight
(
36.7%,
13.6%
and
48.6%
increase)
and
relative
to
final
body
weight
(
40.6%,
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
13
of
14
12.6%
and
31.7%
increase)
at
the
2
week
interim
sacrifice,
4
week
recovery
sacrifice
and
13
week
primary
sacrifice,
respectively
in
the
1200
ppm
treated
mice
compared
to
the
control
animals.
Increased
liver
weights
relative
to
body
weight
were
noted
in
the
600
ppm
group
at
study
weeks
2
and
13
necropsies
(
14.6%
and
13.5%
increase,
respectively).
Microscopically,
all
mice
in
the
200,
400,
600
and
120
ppm
groups
developed
treatment­
related
centrilobular
hepatocellular
hypertrophy
with
increasing
incidence
and
severity
at
the
2
week
interim
sacrifice.
Hepatocellular
vacuolation
with
increasing
incidence
and
severity
were
also
noted
in
mice
treated
with
400,
600
or
120
ppm
of
imazalil.
Minimal
hepatocellular
necrosis
was
observed
at
low
incidence
in
the
600
and
1200
ppm
groups.
At
the
4
week
recovery
necropsy
minimal
to
moderate
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation
was
seen
in
all
the
1200
pm
animals,
while
hepatocellular
necrosis
incidence
was
similar
to
the
control
group.
At
the
13
week
necropsy
examination
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation
occurred
with
increasing
incidence
and
severity
in
all
animals
treated
with
200
ppm
of
imazalil
and
above
and
a
slightly
higher
incidence
of
minimal
hepatocellular
necrosis
in
the
1200
ppm
group.
Hepatocellular
vacuolation
with
lower
incidence
and
severity
persisted
during
the
recovery
period
at
17
weeks
sacrifice.
Minimal
hepatocellular
hypertrophy
was
noted
only
in
one
animal
at
the
17
weeks
recovery
necropsy
with
an
absence
of
hepatocellualr
necrosis.
The
investigators
concluded
that
the
NOAEL
for
the
dietary
oral
administration
of
imazalil
to
male
Crl:
CD­
1
(
ICR)
mice
for
2
or
13
weeks
is
100
ppm
(
mg/
kg/
day).
Imazalil
did
not
increase
BrdU
nuclear
staining
of
hepatocytes
in
the
liver
of
any
treatment
group,
nor
it
increased
PCNA
labeling
in
liver
cells.
Contrary
to
expectations,
there
was
a
decrease
of
BrdU
staining
of
the
hepatocytes
in
the
high
dose
group
compared
to
the
control
group.
The
statistically
significant
decrease
in
proportion
of
hepatocyte­
labeled
hepatocyte
nuclei
was
considered
"
paradoxical"
suggesting
an
inhibition
of
cell
proliferation
in
the
imazalil
treated
male
mice.

B.
REVIEWER
COMMENTS:

The
reviewer
finds
the
investigator's
conclusions
acceptable
and
supported
by
the
study
data.
The
primary
organ
of
imazalil
toxicity
in
this
test
was
the
liver.
Imazalil
produced
microscopic
changes
(
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation,
and
hepatocellualr
necrosis)
in
a
dose
related
manner.
Liver
enzymes
levels
ALT
and
SDH
were
elevated
but
not
LDH.
Imazalil
did
not
induce
any
BrdU
nuclear
labeling
of
hepatocytes
at
any
of
the
doses
tested.
This
was
confirmed
by
repeating
the
staining
procedure.
The
BrdU
staining
procedure
was
also
verified
by
producing
positive
results
in
cells
taken
from
duodenum
sections.
It
was
observed
that
control
animals
had
a
higher
labeling
percentage
than
the
highest
dose
tested.

PCNA
labeling
index
as
an
indicator
of
hepatic
cell
proliferation
was
lower
in
the
imazalil
treated
groups
at
all
dose
levels
than
in
the
control
group
at
all
sacrifice
times.
This
demonstrates
the
lack
of
induction
of
hepatic
cell
proliferation
by
imazalil.

The
LOAEL
for
imazalil
in
the
male
mice
is
200
ppm
(
38.3
±
2.7
mg/
kg/
day)
based
on
its
hepatoxicity
(
centrilobular
hepatocellular
hypertrophy
and
hepatocellular
vacuolation,
and
hepatocellualr
necrosis
and
elevated
sorbitol
dehydrogenase
levels).
The
NOAEL
is
100
ppm
(
18.7
±
1.1
mg/
kg/
day).

C.
STUDY
DEFICIENCIES:
Imazalil
/
PC
111901
Hepatic
Cell
Proliferation
(
Male
Mice)
(
2002)
Non­
guideline
Page
14
of
14
No
deficiencies
were
found
that
would
affect
the
interpretation
of
the
study.
The
improved
staining
BrdU
staining
procedure
was
not
described.

This
study
is
considered
Acceptable/
Non
Guideline
for
the
intended
purpose.
