1
UNITED
STATES
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
D.
C.
20460
OFFICE
OF
PREVENTION
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
TXR#:
0051477
DATE:
February
19,
2003
SUBJECT:
Imazalil:
Report
of
the
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTRAC)

FROM:
Abdallah
Khasawinah,
Ph.
D.,
Toxicologist
Reregistration
Branch
4
Health
Effects
Division
(
7509C)

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

TO:
Ray
Kent,
Branch
Chief
Reregistration
Branch
4
Health
Effects
Division
(
7509C)

PC
Code:
111901
cc:
Anna
Lowit
(
RRB2),
Susan
Hummel
(
RRB4),
Cecelia
Watson
(
SRRD)

On
January
21,
2003,
the
Health
Effects
Division
(
HED)
Mechanism
of
Toxicity
Science
Assessment
Review
Committee
(
MTRAC)
reviewed
the
recommendations
of
the
toxicology
reviewer
for
imazalil
with
regard
to
the
proposed
mechanism
of
action
in
causing
liver
tumors
in
male
mice.
The
conclusions
drawn
at
this
meeting
are
presented
in
this
report.
2
Action:
The
Mechanism
of
Toxicity
Assessment
Review
Committee
(
MTRAC)
met
on
January
21,
2003
to
evaluate
the
mechanistic
and
other
relevant
data
to
determine
whether
the
liver
tumorigenic
effects
of
imazalil
in
the
mouse
liver
are
related
to
its
effects
on
hepatic
cell
proliferation.
The
mechanistic
data
included
a
cell
proliferation
study
in
male
CD­
1
mice
submitted
by
the
Registrant
(
MRID
45713701
and
its
addendums).

Conclusions:
Based
on
the
weight
of
evidence
and
MRID
45713701
findings,
the
committee
concluded
that
the
new
data
does
not
support
a
mechanism
of
action
to
explain
the
tumorigenic
effects
of
imazalil
in
the
rodent
liver.
The
committee
explained
its
decision
on
the
following:

(
1)
No
hepatic
cell
proliferation
was
demonstrated
in
rat
or
the
new
mouse
study.
In
the
mouse,
there
was
a
decline
of
cell
proliferation
as
measured
by
Bromo­
deoxyuridine
(
BrdU)
or
Proliferating
Cell
Nuclear
Antigen
(
PCNA)
staining.

(
2)
BrdU
is
the
preferred
method
for
measuring
cell
proliferation.
The
mouse
study
findings
were
variable.
BrdU
staining
occurred
only
in
the
control
and
the
high
dose
animals
but
none
in
the
intermediate
doses.

(
3)
PCNA
staining
(
not
the
best
method
for
measuring
cell
proliferation)
yielded
also
variable
results
with
a
declining
trend
suggesting
inhibition
of
cell
proliferation.

(
4)
Even
if
hepatic
cell
proliferation
was
demonstrated,
there
is
currently
no
scientific
support
to
link
it
to
tumorigenic
effects.

The
Committee
concluded,
therefore,
that
the
new
data
in
the
mouse
does
not
demonstrate
a
mechanism
of
action
to
explain
the
tumoregeinc
effects
of
imazalil
in
rodents.
3
Committee
Members
in
Attendance
Members
who
were
present
and
gave
concurrence
to
this
report
were
Karl
Baetcke,
Pauline
Wagner,
William
Burnam
and
Anna
Lowit.

Members
in
Absentia:
Vicki
Dellarco
Also
in
Attendance
were:
Ray
Kent
Data
Evaluation
/
Report
Presentation
Abdallah
Khasawinah,
Ph.
D.
Toxicologist
Reregistration
Branch
4
Health
Effects
Division
(
HED)
4
Cl
Cl
O
N
N
CH
2
I.
BACKGROUND
INFORMATION
A.
Introduction:
Imazalil
[
1­(
2­(
2,4­
dichlorophenyl)­
2­(
2­
propenyloxy)
ethyl)­
1H­
imidazole]
is
a
systemic
fungicide
registered
for
post­
harvest
treatment
of
citrus
fruits
and
bananas
(
import
tolearance
only)
and
for
seed
treatment
of
barley
and
wheat
prior
to
planting.
Imazalil
is
also
used
in
egg
handling
facilities.
Imazalil
is
known
as
Enilconazole
in
veterinary
medicine.
The
reregistration
of
imazalil
is
being
supported
by
Janssen
Pharmaceutica
and
Makhteshim­
Agan
of
North
America
Inc.

Imazalil
is
used
to
prevent,
treat
and
control
diseases
caused
by
a
variety
of
pathogenic
organisms
(
fungi),
which
include
(
but
are
not
limited
to)
Aspergillus
in
egg
handling
facilities
and
equipment,
blue
mold
in
citrus
fruits
and
Fusarium
in
wheat
and
barley
seeds.
imazalil
inhibits
cytochrome
P­
450
dependent
biosynthesis
of
ergosterol,
an
essential
component
of
the
cell
membrane
of
the
fungi
cells.

B.
Chemical
Identification
Chemical
Structure:

Molecular
formula:
C
14
H
14
Cl
2
N
2
O
Molecular
weight:
297.18
Synonyms:
(
R23979),
Enilconazole,
Fungaflor,
Fecundal,
Fungazil
Melting
point:
52.7

C
Vapor
pressure
(
20

C):
1.2x10­
6
mm
Hg
Water
solubility
(
20

C):
180
­
293
ppm
Log
K
ow:
3.82
Solubility:
very
soluble
in
methanol,
ethanol,
2­
propanol,
xylene,
benzene,
toluene,
and
solutions
of
mineral
and
organic
acids.
Soluble
in
n­
heptane,
hexane,
and
petroleum
ether
CAS
Registry
No.:
35554­
44­
0
PC
Code:
111901
5
C.
Toxicity
Summary:

Imazalil
is
placed
in
Category
II,
III,
and
IV
for
oral,
dermal
and
inhalation
toxicity,
respectively.
It
is
highly
irritating
to
rabbit
eye
(
Category
I),
but
is
not
a
skin
irritant
(
Category
IV)
or
a
dermal
sensitizer.
Imazalil
(
EC
formulation)
is
readily
absorbed
by
the
rat
skin
with
a
41
%
absorption
of
the
applied
dose
within
10
hours
of
application.

The
thyroid
and
the
liver
are
primary
target
organ
for
Imazalil
toxicity.
Enlarged
livers
were
seen
in
rabbits
after
6
days
of
dermal
application
at
250
mg/
kg/
day
(
MRID
42085201),
increased
liver
weights
and
liver
to
body
weight
ratios,
increased
centrilobular
swollen
hepatocytes
and
increased
vacuolization
in
hepatocytes
after
one
month
of
dietary
treatment
at
32.1
mg/
kg/
day
in
rats
(
MRID
43965704),
and
similar
histopathologic
effects
in
mice
(
at
38.6
mg/
kg/
day
in
the
diet
(
MRID
43222601).
In
a
chronic
dietary
rat
study,
there
was
an
increased
incidence
of
intra
cytoplasmic
inclusion
bodies
of
hepatocytes,
increased
severity
of
hepatocyte
vacuolization
as
well
as
bile
duct
proliferation
at
15.5
mg/
kg/
day
(
MRID
47026101).
Liver
histopathologic
lesions
were
also
seen
in
a
23­
month
study
in
mice
at
28.0
mg/
kg/
day
(
42972001).
Increased
liver
vacuolization
was
also
seen
in
male
rats
in
a
2­
generation
reproduction
study
at
80
mg/
kg/
day
(
MRID
42570701
&
42949402).
Increased
liver
weights
were
seen
in
dogs
treated
for
one
year
at
20
mg/
kg/
day
(
41328802).
The
absolute
and
relative
weight
of
thyroid
glands
was
increased
in
male
rats
fed
Imazalil
for
two
years
at

65.8
mg/
kg/
day
(
MRID
44858001).
Microscopic
changes
were
also
seen
in
the
affected
thyroids.

Carcinogenicity
studies
in
rodents
indicate
that
Imazalil
was
carcinogenic
to
male
Swiss
albino
mice
and
Wistar
rats,
based
on
significant
increase
in
liver
adenomas
and
combined
adenomas/
carcinomas.
In
rats
there
was
also
increased
incidence
of
combined
thyroid
follicular
cell
adenomas/
carcinomas.
The
HED
CPRC
(
1994)
and
CARC
(
1998)
classified
Imazalil
a
Group
C­
carcinogen
and
recommended
a
linear
low
dose
approach
(
Q
1
*)
for
quantification
of
human
cancer
risk.
The
CARC
(
1999)
reclassified
imazalil
under
the
July
1999
Draft
Guidelines
for
Carcinogenic
Assessment
into
the
category
"
Likely
to
be
carcinogenic
in
humans".
The
Committee
reaffirmed
its
earlier
decision
by
recommending
a
linear
low­
dose
(
Q
1
*)
extrapolation
for
quantification
of
human
cancer
risk.
This
extrapolation
is
supported
by
the
lack
of
confirmation
of
the
mode
of
action.
The
most
potent
unit
risk
,
Q
1
*(
mg/
kg/
day)­
1
for
imazalil
based
on
male
mouse
liver
adenoma
and/
or
carcinoma
combined
tumor
rates
is
6.2
x
10­
2
in
human
equivalents
(
HED
Doc
013842).

Imazalil
was
non
mutagenic
both
in
vivo
and
in
vitro
mutagenicity
assays
(
Salmonella
test,
chromosomal
aberration
using
hyman
lymphocytes
or
Chinese
hamster
V79
cells,
micronucleus
test,
Unscheduled
DVA
synthesis
assay).

The
registrant
has
conducted
several
non
guideline
studies
on
the
enzyme
induction
properties
of
imazalil
and
its
BrdU
immunostaining
and
PCNA
staining
of
hepatocytes.
These
studies
are
summarized
in
the
following
table.
6
MRID
Study
Summary
45160101
;
male
rats
0,
400,
1200
or
3200
ppm
dietary;
(
0,
40.8,
123
or
328
mg/
kg/
day);
phenobarbital
1200
ppm
(
126
mg/
kg/
day),
in
the
diet
for
4
weeks
followed
by
a
4
or
9
week
recovery.
The
study
authors
contend
that
the
primary
effect
of
imazalil
is
on
the
liver
causing
microsomal
enzyme
induction
resulting
in
hepatocellular
hypertrophy.
Enhanced
hepatic
metabolism
of
the
thyroid
hormone
(
T4)
was
demonstrated
in
this
study
by
increased
hepatic
UDP­
glucuronyl
transferase
activity
with
Lthyroxine
which
caused
decreased
serum
levels.
The
decreased
serum
levels
of
the
T4
hormone
causes
an
increased
release
of
TSH.
The
higher
serum
TSH
levels,
in
turn
may
lead
to
thyroid
follicular
cell
hypertrophy,
hyperplasia
and
neoplasia.

45376801
Supplement
to
45160101
Same
conditions
Imazalil
affected
the
thyroid
peroxidase
and
liver
enzyme
activities
in
a
similar
way
as
phenobarbital.
Liver
aniline
hydroxylase,
N­
ethylmorphine
N­
demethylase,
7­
pentoxyresorufin
O­
dealkylase,
and
UDP­
glucuronyltransferase,
and
thyroid
peroxidase
activities
were
all
induced
during
the
imazalil
treatment.
The
largest
inductive
effect
of
both
imazalil
and
phenobarbital
was
observed
on
the
7­
Pentoxyresorufin
O­
dealkylase
activity
(
1,600
­
2,700%
of
the
control
for
imazalil
and
43,500%
of
the
control
for
phenobarbital
at
week
4
of
treatment).
The
5

monodeiodinase
was
not
induced
and
was
slightly
depressed
by
either
the
imazalil
or
the
phenobarbital
treatment.

The
supplementary
information
demonstrated
that
imazalil
had
a
clear
inductive
effect
on
7­
ethoxyresorufin
O­
dealkylase
activity
in
a
dose
related
manner
(
157%,
296%
400%
relative
to
the
control
at
400,
1200
and
3200
ppm
dietary
feeding
levels
of
imazalil
for
4
weeks,
respectively)
in
a
fashion
similar
to
phenobarbital
(
positive
control
used
in
the
study).
However,
the
lauric
acid
hydroxylase
activity
was
only
slightly
induced
by
both
imazalil
and
phenobarbital.

The
enzyme
activities
in
the
imazalil
and
phenobarbital
treated
rats
returned
to
normal
during
the
4
­
9
week
recovery
periods.

45362602
supplement
to
45160101
same
conditions
evaluated
for
the
percentage
of
labeled
cells
stained
immunocytochemically
for
Proliferating
Cell
Nuclear
Antigen
(
PCNA).
Liver
sections
from
ten
rats
per
group
were
examined
at
the
end
of
the
treatment
period
and
from
ten
per
group
after
a
4­
week
recovery
period.
No
treatment­
effect
was
observed
at
either
time
point;
therefore,
administration
of
imazalil
had
no
effect
on
cell
proliferating
potential
of
the
rat
liver.

43222601,
Swiss
mice
0,
50,
200
or
600
ppm
for
1
or
3
months
The
LOAEL
in
this
study
is
200
ppm
(
38.6
and
45.6
mg/
kg/
day
in
males
and
females
respectively)
and
is
based
on
increased
incidence
and
severity
of
histopathologic
effects,
increased
microsomal
protein
and
increased
microsomal
cytochrome
P450
content
in
the
livers
of
both
males
and
females.
The
NOAEL
in
this
study
is
50
ppm
(
9.5
and
11.3
mg/
kg/
day
in
males
and
females,
respectively).

45362601
Supplement
to
43222601,
Hepatocytes
were
evaluated
for
the
percentage
of
labeled
cells
stained
immunochemically
for
Proliferating
Cell
Nuclear
Antigen
(
PCNA).
At
the
end
of
the
13­
week
treatment
period,
the
labeling
index
for
all
treated
groups
was
similar
to
that
of
controls.
These
results
show
that
treatment
with
imazalil
for
up
to
13
weeks
had
no
effect
on
cell
proliferation
in
the
liver
as
measured
by
PCNA.
MRID
Study
Summary
7
45713701;
45708401;
45783801;
45822503;
non­
guideline
hepatocellular
proliferation
study;
male
mice;
0,
100,
200,
400,
600
or
1200
ppm
(
0,
18.7,
38.3,
73.4,
110.4,
or
233.2
mg/
kg/
day)
in
the
diet,
for
2
or
13
weeks
followed
by
2
week
recovery
or
4
week
recovery.
At
sacrifice,
liver
sections
prepared
for
BrdU
immunohistochemical
and
PCNA
staining
of
hepatocytes.
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.
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.
"
There
was
a
decreased
proportion
of
PCNA­
labeled
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)

The
Registrant,
in
an
Overview
Summary
(
MRID
45822502)
suggests
that
imazalil
might
be
acting
as
a
mitogen
inhibiting
cell
proliferation.
However,
mitogens
stimulate
cell
proliferation
in
the
absence
of
cytotoxicity/
necrosis/
regeneration.
The
Registrant
also
argues
that
the
collective
evidence
from
various
studies
on
imazalil
(
previously
reviewed
by
HED)
suggests
that
imazalil
effects
on
the
liver,
including
tumorigenic
effects,
are
threshold
based
and
reversible.

II.
DATA
PRESENTATION
A.
Available
Data:

1.
1999
CARC
Report:
EXECUTIVE
SUMMARY
Previously,
the
Cancer
Peer
Review
Committee
(
CPRC;
August
24,
1994)
classified
Imazalil
as
a
Group
C­
possible
human
carcinogen
and
recommended
the
low
dose
linear
extrapolation
(
Q
1*)
approach
for
quantification
of
human
cancer
risk.
This
decision
was
based
on
an
increased
incidence
of
hepatocellular
adenomas
and
combined
adenomas
and
carcinomas
in
male
Swiss
albino
mice
at
the
high
dose.
The
increase
was
statistically
significant
by
pair­
wise
comparison,
with
a
statistically
significant
trend.
Females
had
a
significant
dose­
related
trend
for
hepatocellular
carcinomas
and
combined
adenomas/
carcinomas.
No
apparent
increase
in
tumors
was
noted
in
Wistar
rats.
The
Committee
determined
that
the
dosing
was
not
adequate
for
assessing
the
carcinogenic
potential
in
the
rat
and
therefore,
recommended
that
a
new
2­
year
chronic
feeding/
carcinogenicity
study
in
rats
using
higher
dose
levels
be
performed.

At
the
July
22,
1998
CARC
meeting,
the
Committee
(
September,
4,
1998)
reaffirmed
the
CPRC's
previous
decision
in
classifying
Imazalil
as
a
Group
C
carcinogen
and
the
use
of
a
linear
low­
dose
extrapolation
approach
for
quantification
of
human
cancer
risk
based
on
liver
tumors
in
male
mice.
8
On
October
27,
1999,
the
CARC
met
to
evaluate
the
newly
completed
rat
chronic/
carcinogenicity
study.
The
CARC
concluded
that
Imazalil
was
carcinogenic
to
male
rats
because:
1)
There
was
a
significant
increase
by
pair­
wise
comparison
of
the
2400
ppm
(
134.8
mg/
kg/
day)
group
with
the
controls
for
hepatocellular
adenoma
and
combined
adenomas/
carcinomas.
2)
The
incidence
of
hepatocellular
adenomas
was
outside
the
range
of
the
historical
control
data
and
the
combined
incidence
was
driven
by
the
adenomas.
3)
There
were
also
significant
increasing
trends
for
hepatocellular
adenomas
and
combined
adenomas/
carcinomas
;
and
4)
There
was
a
significant
increase
by
pair­
wise
comparisons
of
the
1200
and
2400
ppm
(
65.8
and
134.8
mg/
kg/
day,
respectively)
groups
with
the
controls
for
thyroid
follicular
cell
combined
adenomas/
carcinomas.
There
was
also
a
significant
increasing
trend
for
thyroid
follicular
cell
combined
adenomas/
carcinomas.
The
incidence
of
combined
thyroid
tumors
was
outside
the
historical
control.
No
increases
in
liver
and
thyroid
tumors
were
noted
in
females.
The
dosing
at
the
highest
dose
in
both
sexes
was
considered
to
be
adequate
and
not
excessive
based
on
decreases
in
body
weight
gains
and
non­
neoplastic
changes
in
the
liver
(
both
sexes)
and
thyroid
(
males
only).
The
CARC
considered
the
liver
and
thyroid
tumors
in
males
to
be
treatment­
related.

Under
the
Draft
Guidelines
for
Carcinogen
Risk
Assessment
(
July,
1999),
Imazalil
is
classified
in
the
category
"
Likely
to
be
carcinogenic
in
humans".
The
CARC's
decision
was
based
on
the
following:

1.
There
were
increases
in
hepatocellular
adenomas
and
combined
liver
adenomas/
carcinomas
in
male
Swiss
albino
mice
and
Wistar
rats.
In
male
rats,
there
was
also
an
increased
incidence
of
combined
thyroid
follicular
cell
adenomas/
carcinomas.
2.
Imazalil
was
non
mutagenic
in
in
vitro
and
in
vivo
mutagenicity
assays.

3.
It
is
structurally
related
to
triazole
compounds,
which
are
hepatocarcinogens
in
mice.

The
CARC
recommended
a
linear
low­
dose
(
Q
1*)
extrapolation
approach
for
the
quantification
of
human
cancer
risk
based
on
the
most
potent
liver
tumors
in
mice.
This
approach
is
supported
by
the
lack
of
confirmation
of
the
mode
of
action.

2.
Thyroid
Effects
Study
in
Rats:
In
a
one­
month
oral
toxicity
study
(
MRID
45160101),
imazalil
(
50%
a.
i.
premix)
was
administered
in
the
diet
to
groups
of
50
male
SPF
Wistar
rats,
substrain
Hannover,
at
concentrations
of
0
(
vehicle
control),
400,
1200,
or
3200
ppm
(
0,
40.8,
123,
or
328
mg/
kg/
day)
for
up
to
4
weeks
followed
by
a
recovery
period
of
4
or
9
weeks
(
13
weeks
total
study
duration).
A
total
of
50
rats
fed
1200
ppm
(
126
mg/
kg/
day)
phenobarbital
under
the
same
conditions
as
the
test
animals
served
as
a
positive
control.
Ten
rats
per
group
were
sacrificed
at
weeks
1,
2,
4,
8,
and
13
for
evaluation
of
all
parameters.
9
The
only
clinical
sign
noted
was
food
wastage
by
a
few
high­
dose
rats
during
weeks
1
to
8
of
the
study;
the
positive
controls
showed
signs
of
sedation
during
weeks
1
and
2.
Midand
high­
dose
male
rats,
respectively,
weighed
up
to
8%
(
p<
0.01)
and
19%
(
p<
0.01)
less
than
vehicle
controls,
gained
15%
(
p<
0.01)
and
36%
(
p<
0.01)
less
weight,
and
consumed
7%
(
p<
0.01)
and
15%
(
p<
0.01)
less
food
during
the
4­
week
treatment
period.
Consequently,
the
food
conversion
ratio
was
not
significantly
affected
by
treatment
with
the
test
material.
Compensatory
weight
gain
was
observed
during
the
recovery
period.
Positive
controls
showed
no
effects
on
weight
gain
or
food
consumption
during
the
4­
week
treatment
period.

Both
imazalil
and
phenobarbital
(
positive
control)
induced
P­
450
dependent
activities
and
UDP
glucuronyltransferase
activity.
Imazalil
affected
the
thyroid
peroxidase
and
5'­
monodeiodinase
activities
in
a
similar
way
as
phenobarbital.
Most
enzyme
activities
in
the
imazalil
and
phenobarbital
treated
rats
returned
to
normal
during
recovery
period.

Absolute
and/
or
relative
liver
weights
were
significantly
(
p<
0.01
or
<
0.05)
increased
by
7­
27%
at
all
doses
after
treatment
for
1
or
4
weeks
and
at
the
mid­
and
high­
dose
levels
after
2
weeks.
Absolute
and
relative
liver
weights
in
positive
controls
were
30%
to
39%
(
p<
0.01)
greater
than
that
of
controls
throughout
the
treatment
period.
Relative
thyroid
weight
in
high­
dose
rats
was
significantly
increased
(
19%,
p<
0.01)
only
at
week
2,
whereas
absolute
and
relative
thyroid
weights
in
positive
controls
were
increased
by
18­
21%
(
p<
0.01)
at
weeks
2
and
4.
Associated
gross
findings
included
swollen
liver
in
50­
70%
(
p<
0.01
or
<
0.05)
of
high­
dose
rats
at
weeks
1,
2,
and
4
and
in
40%
(
N.
S.)
of
middose
rats
at
week
4
and
a
dark
liver
in
70%
(
p<
0.01)
of
high­
dose
rats
at
week
4.
The
liver
was
swollen
in
70­
100%
of
positive
controls
during
the
treatment
period.
No
effect
was
observed
on
labeling
index
in
the
liver
or
thyroid
gland
at
any
dose
of
imazalil.

Treatment­
related
microscopic
findings
occurring
at
incidences
significantly
greater
(
p<
0.01
or
<
0.05
unless
otherwise
noted)
than
those
of
controls
included
centrilobular
and
periportal
hepatocyte
hypertrophy
and
hepatocyte
vacuolation.
The
incidence
of
centrilobular
hypertrophy
was
20%
(
N.
S.),
50%,
and
50%
in
low­
dose
rats;
60,
90,
and
100%
in
mid­
dose
rats;
and
90­
100%
in
high­
dose
rats
at
weeks
1,
2,
and
4,
respectively,
compared
with
0%
in
controls.
Periportal
hypertrophy
did
not
occur
in
controls
or
lowdose
rats,
but
the
incidence
was
10%
(
N.
S.),
60%,
and
60%
in
mid­
dose
rats
and
90,
80,
and
100%
in
high­
dose
rats
at
weeks
1,
2,
and
4,
respectively.
Hepatocyte
vacuolation
occurred
in
0,
0,
and
10%
(
N.
S.)
of
low­
dose
rats,
40%
(
N.
S.),
20%
(
N.
S.),
and
10%
(
N.
S.)
of
mid­
dose
rats,
and
80­
90%
of
high­
dose
rats
at
weeks
1,
2,
and
4,
respectively,
compared
with
0%
of
controls.
The
severity
of
the
liver
lesions
also
increased
with
dose
and
duration
of
treatment.
Except
for
one
mid­
dose
rat
at
week
8,
none
of
these
liver
lesions
were
observed
in
any
rat
during
the
recovery
period.
The
incidences
of
all
three
liver
lesions
and
thyroid
follicular
hypertrophy
were
significantly
increased
in
positive
control
rats
at
two
or
all
three
time
points
during
treatment.
10
Serum
thyroxine
(
T4)
and
thyroid
stimulating
hormone
(
TSH)
levels
were
generally
correlated
with
each
other
at
weeks
1,
2,
4,
and
8,
particularly
in
mid­
and
high­
dose
rats.
Although
T4
and
TSH
levels
in
low­
dose
rats
fluctuated
during
treatment
and
recovery,
the
pattern
did
not
show
the
consistency
as
observed
for
mid­
and
high­
dose
rats.
One
week
after
treatment
started,
serum
T4
levels
were
decreased
by
12%
(
p<
0.05)
and
17%
(
p<
0.01)
in
mid­
and
high­
dose
rats,
respectively,
compared
with
controls,
whereas
TSH
levels
were
increased
by
15%
(
N.
S.)
and
19%
(
N.
S.).
With
continued
treatment,
at
week
4,
serum
T4
levels
in
mid­
and
high­
dose
rats,
respectively,
rose
dramatically
to
levels
88%
and
52%
greater
than
that
of
controls
and
TSH
levels
decreased
to
10%
(
N.
S.)
and
20%
(
N.
S.)
below
that
of
controls.
After
treatment
was
terminated,
T4
levels
decreased
to
levels
that
were
only
11%
(
p<
0.05)
and
5%
(
N.
S.)
higher
than
that
of
controls
in
midand
high­
dose
rats,
respectively,
at
week
8,
and
serum
TSH
levels
again
increased
to
53%
(
p<
0.01)
and
42%
(
p<
0.05),
respectively,
greater
than
that
of
controls.
At
week
13,
T4
levels
in
mid­
and
high­
dose
remained
comparable
to
those
observed
at
week
8,
but
TSH
levels
decreased
relative
to
those
observed
at
week
8.
A
statistically
significant
serum
triiodothyronine
(
T3)
level
was
noted
in
the
high
dose
group
at
week
4
only.

In
conclusion,
this
study
was
not
conducted
to
determine
a
no­
observed­
adverse­
effect
level
(
NOAEL)
and
none
was
established,
because
liver
lesions
were
observed
at
all
dose
levels.
The
study
authors
contend
that
the
primary
effect
of
imazalil
is
on
the
liver
causing
microsomal
enzyme
induction
resulting
in
hepatocellular
hypertrophy.
Enhanced
hepatic
metabolism
of
the
thyroid
hormone
(
T4)
was
demonstrated
in
this
study
by
increased
hepatic
UDP­
glucuronyl
transferase
activity
with
L­
thyroxine,
which
caused
decreased
serum
levels.

3.
Hepatic
Enzyme
Induction/
Inhibition
Study
in
Rats:
In
a
one­
month
oral
toxicity
study
(
MRID
45160101),
imazalil
(
50%
a.
i.
premix)
was
administered
in
the
diet
to
groups
of
50
male
SPF
Wistar
rats,
substrain
Hannover,
at
concentrations
of
0
(
vehicle
control),
400,
1200,
or
3200
ppm
(
0,
40.8,
123,
or
328
mg/
kg/
day)
for
up
to
4
weeks
followed
by
a
recovery
period
of
4
or
9
weeks
(
13
weeks
total
study
duration).
A
total
of
50
rats
fed
1200
ppm
(
126
mg/
kg/
day)
phenobarbital
under
the
same
conditions
as
the
test
animals
served
as
a
positive
control.
Ten
rats
per
group
were
sacrificed
at
weeks
1,
2,
4,
8,
and
13
for
evaluation
of
all
parameters.

In
the
study
supplement
(
MRID
45376801),
a
final
report
of
the
hepatic
drug
metabolizing
and
thyroid
enzymes
is
presented.
Liver
and
thyroid
microsomes
were
prepared
from
liver
and
thyroid
pieces
and
assayed
for
protein
and
cytochrome
P­
450
contents
(
liver
microsomes).
Liver
aniline
hydroxylase,
N­
ethylmorphine
N­
demethylase,
7­
ethoxyresorufin
O­
deethylase,
7­
pentoxyresorufin
O­
dealkylase,
lauric
acid
hydroxylase,
UDP­
glucuronyltransferase,
and
5

monodeiodinase
and
thyroid
peroxidase
activities
were
analyzed.
With
the
exception
of
the
7­
ethoxyresorufin
O­
deethylase
and
lauric
acid
hydroxylase,
these
enzyme
activities
were
reported
in
MRID
45160101.
11
Both
imazalil
and
phenobarbital
induced
P­
450
dependent
activities
and
UDP
glucuronyltransferase
activity.
Imazalil
affected
the
thyroid
peroxidase
and
liver
enzyme
activities
in
a
similar
way
as
phenobarbital.
Liver
aniline
hydroxylase,
N­
ethylmorphine
N­
demethylase,
7­
pentoxyresorufin
O­
dealkylase,
and
UDP­
glucuronyltransferase,
and
thyroid
peroxidase
activities
were
all
induced
during
the
imazalil
treatment.
The
largest
inductive
effect
of
both
imazalil
and
phenobarbital
was
observed
on
the
7­
Pentoxyresorufin
O­
dealkylase
activity
(
1,600
­
2,700%
of
the
control
for
imazalil
and
43,500%
of
the
control
for
phenobarbital
at
week
4
of
treatment).
The
5

monodeiodinase
was
not
induced
and
was
slightly
depressed
by
either
the
imazalil
or
the
phenobarbital
treatment.

The
supplementary
information
demonstrated
that
imazalil
had
a
clear
inductive
effect
on
7­
ethoxyresorufin
O­
dealkylase
activity
in
a
dose
related
manner
(
157%,
296%
400%
relative
to
the
control
at
400,
1200
and
3200
ppm
dietary
feeding
levels
of
imazalil
for
4
weeks,
respectively)
in
a
fashion
similar
to
phenobarbital
(
positive
control
used
in
the
study).
However,
the
lauric
acid
hydroxylase
activity
was
only
slightly
induced
by
both
imazalil
and
phenobarbital.

The
enzyme
activities
in
the
imazalil
and
phenobarbital
treated
rats
returned
to
normal
during
the
4
­
9
week
recovery
periods.

This
additional
information
supplements
the
original
report
does
not
affect
the
conclusions
stated
in
the
DER
of
MRID
#
45160101.

This
study
was
conducted
to
determine
the
effects
of
imazalil
administration
to
male
rats
on
the
induction
of
the
hepatic
drug
metabolizing
enzymes
and
the
related
thyroid
enzymes
to
explain
the
hepatocellular
hypertrophy
and
the
thyroid
follicular
cell
hypertrophy
seen
in
long­
term
studies
with
imazalil.
As
stated
previously
in
the
DER
of
MRID
45160101,
these
findings
may
provide
the
mechanism
by
which
imazalil
induced
thyroid
neoplasms
in
long­
term
studies.

4.
PCNA
Quantitative
Analysis
of
Livers
­
Rats:
This
study
(
MRID
45362602)
is
a
supplement
to
MRID
45160101.
Livers
from
20
male
rats
administered
imazalil
base,
technical
(
96.9%)
at
dietary
concentrations
of
0,
400,
1200,
or
3200
ppm
of
imazalil
(
0,
40.8,
123,
or
328
mg/
kg/
day)
or
1200
phenobarbital
(
126
mg/
kg/
day;
positive
control)
for
4
weeks
were
evaluated
for
the
percentage
of
labeled
cells
stained
immunocytochemically
for
Proliferating
Cell
Nuclear
Antigen
(
PCNA).
Liver
sections
from
ten
rats
per
group
were
examined
at
the
end
of
the
treatment
period
and
from
ten
per
group
after
a
4­
week
recovery
period.
No
treatment­
effect
was
observed
at
either
time
point;
therefore,
administration
of
imazalil
had
no
effect
on
cell
proliferating
potential
of
the
rat
liver.

5.
Subchronic
Oral
Hepatotoxicity
Study
in
Mice:
In
a
3­
month
oral
mechanistic
toxicity
study
(
MRID
43222601,
43292402)
designed
to
study
effects
on
the
liver,
12
Imazalil
Base
(
96.9%)
was
administered
in
the
diet
to
25
male
and
25
female
Swiss
mice
for
3
months
at
nominal
dosage
levels
of
0,
50,
200
or
600
ppm
(
approximate
doses
of
0,
9.5,
38.6
or
115
mg/
kg/
day
for
males
and
0,
11.3,
45.6
or
138
mg/
kg/
day
for
females,
as
adjusted
for
actual
achieved
concentrations
of
about
83%
of
nominal).
Additional
groups
of
15
mice/
sex/
group
were
also
given
Imazalil
base
in
the
diet
at
0,
50,
200
or
600
ppm,
but
were
sacrificed
at
1
month.
The
following
parameters
were
evaluated:
clinical
signs,
body
weights,
food
consumption,
clinical
chemistries
(
3
liver
enzymes
only),
gross
necropsy,
organ­,
weights,
histopathology
(
gall
bladder
and
liver­
only),
electron
microscopy
of
liver,
liver
microsomal
protein
and
cytochrome
P450
content,
liver
enzymatic
activities
of
7
P450
isoenzymes,
liver
testosterone
metabolism
and
serum
concentrations
of
Imazalil.

No
treatment­
related
effects
on
mortality,
clinical
signs,
body
weights,
body
weight
gains
or
food
consumption
were
observed.
At
200
ppm,
the
following
treatment­
related
effects
were
observed:
increased
incidence
of
dark
liver
at
gross
necropsy
in
males,
increased
incidence
and
severity
of
"
centrilobular
clearer
aspect"
and
of
large
and/
or
small
vacuoles
in
the
hepatocytes
of
males
and
females,
increased
liver
microsomal
protein
in
males
and
females,
and
increased
microsomal
cytochrome
P450
content
in
males
and
females.
At
600
ppm,
the
following
treatment­
related
effects
were
observed:
increased
incidence
of
dark
livers
at
gross
necropsy
in
males
and
females,
increased
absolute
liver
weights
and
relative
liver/
body
weight
ratios
in
males
and
females,
increased
incidence
and
severity
of
"
centrilobular
clearer
aspect"
and
of
large
and/
or
small
vacuoles
in
the
hepatocytes
of
males
and
females,
increased
individual
cell
necrosis
in
hepatocytes
of
males,
increased
diffuse
swelling
of
hepatocytes
in
females,
increased
liver
microsomal
protein
in
males
and
females,
and
increased
microsomal
cytochrome
P450
content
in
males
and
females.
Electron
microscopy
revealed
increased
numbers
of
lipid
droplets
in
hepatocytes,
which
corresponded
with
the
increased
vacuolization
observed
by
light
microscopy,
and
a
morphologically
changed
rough
endoplasmic
reticulum
(
RER)
in
the
hepatocytes
of
600
ppm
males
and
females.
Regarding
liver
enzymatic
activities
of
7
P450
isoenzymes.,
dosing
with
Imazalil
at
200
ppm
and
600
ppm
significantly
induced
certain
enzymatic
activities
but
also
had
an
inhibitory
effect
on
other
metabolic
activities.
At
600
ppm,
the
total
activity
of
testosterone
hydroxylases
was
increased
in
both
males
and
females.
Low
levels
of
Imazalil
were
detected
in
the
serum
of
some
males
and
females
at
600
ppm
only.

The
LOEL
in
this
study
is
200
ppm
(
38.6
and
45.6
mg/
kg/
day
in
males
and
females
respectively)
and
is
based
on
increased
incidence
and
severity
of
histopathologic
effects,
increased
microsomal
protein
and
increased
microsomal
cytochrome
P450
content
in
the
livers
of
both
males
and
females.
The
NOEL
in
this
study
is
50
ppm
(
9.5
and
11.3
mg/
kg/
day
in
males
and
females,
respectively).
13
6.
PCNA
Quantitative
Analysis
of
Livers
­
Mouse:
This
study
(
MRID
45362601)
is
a
supplement
to
MRID
43222601.
Liver
sections
from
20
male
and
20
female
mice
administered
Imazalil
base,
technical
(
96.9%
purity)
at
dietary
concentrations
of
0,
50,
200,
or
600
ppm
(
approximate
doses
of
0,
9.5,
38.6
or
115
mg/
kg/
day
for
males
and
0,
11.3,
45.6
or
138
mg/
kg/
day
for
females,
as
adjusted
for
actual
achieved
concentrations
of
about
83%
of
nominal)
for
13
weeks
were
evaluated
for
the
percentage
of
labeled
cells
stained
immunochemically
for
Proliferating
Cell
Nuclear
Antigen
(
PCNA).
No
treatmenteffect
was
observed;
therefore,
oral
administration
of
imazalil
had
no
effect
on
cell
proliferating
potential
of
the
rat
liver.

7.
Hepatocellular
Proliferation
Study
­
Mouse:
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
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
BrdU­
immunohistochemical
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.
14
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
PCNA­
labeled
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).

8.
Overview
of
Liver
Effects
and
Cell
Cycle
Changes
in
Mice:
In
this
overview
prepared
by
the
Registrant
(
MRID
45822502),
concludes
that
the
effects
of
imazalil
on
the
liver
are
nongenotoxic,
threshold
based
and
reversible.
Cell
proliferation
data
indicate
that
imazalil
inhibits
the
cell
proliferation
at
dietary
concentrations
of
200
ppm
and
greater.
According
to
this
review,
the
tumoregenic
response
may
be
related
to
the
inhibition
of
cell
proliferation
which
permits
altered
liver
cell
population
that
escape
the
cell
cycle
to
have
a
selective
growth
advantage.

III.
CONCLUSIONS
Based
on
the
weight
of
evidence
and
MRID
45713701
findings,
the
committee
concluded
that
the
new
data
does
not
support
a
mechanism
of
action
to
explain
the
tumorigenic
effects
of
imazalil
in
the
rodent
liver.
The
committee
explained
its
decision
on
the
following:

(
1)
NO
hepatic
cell
proliferation
was
demonstrated
in
rat
or
the
new
mouse
study.
In
the
mouse,
there
was
a
decline
of
cell
proliferation
as
measured
by
Bromo­
deoxyuridine
(
BrdU)
or
Proliferating
Cell
Nuclear
Antigen
(
PCNA)
staining.

(
2)
BrdU
is
the
preferred
method
for
measuring
cell
proliferation.
The
mouse
study
findings
were
variable.
BrdU
staining
occurred
only
in
the
control
and
the
high
dose
animals
but
none
in
the
intermediate
doses.

(
3)
PCNA
staining
(
not
the
best
method
for
measuring
cell
proliferation)
yielded
also
variable
results
with
a
declining
trend
suggesting
inhibition
of
cell
proliferation.

(
4)
Even
if
hepatic
cell
proliferation
was
demonstrated,
there
is
currently
no
scientific
support
to
link
it
to
tumorigenic
effects.

The
Committee
concluded,
therefore,
that
the
new
data
in
the
mouse
does
not
demonstrate
a
mechanism
of
action
to
explain
the
tumorigenic
effects
of
imazalil
in
rodents.
