CANCER
ASSESSMENT
DOCUMENT
EVALUATION
OF
THE
CARCINOGENIC
POTENTIAL
OF
PHMB
PC
CODE
111801
TXR
No.
0052040
FINAL
July
16,
2003
CANCER
ASSESSMENT
REVIEW
COMMITTEE
HEALTH
EFFECTS
DIVISION
OFFICE
OF
PESTICIDE
PROGRAMS
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
1200
PENNSYLVANIA
AVENUE,
NW
WASHINGTON,
DC
20460
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
DATA
PRESENTATION:
Jonathan
Chen
DOCUMENT
PREPARATION:
Jessica
Kidwell,
Executive
Secretary
COMMITTEE
MEMBERS
IN
ATTENDANCE:
(
Signature
indicates
concurrence
with
the
assessment
unless
otherwise
stated).

William
Burnam
Marion
Copley
Vicki
Dellarco
Kit
Farwell
Abdallah
Khasawinah
Nancy
McCarroll
Tim
McMahon
Linda
Taylor
Yintak
Woo
NON­
COMMITTEE
MEMBERS
IN
ATTENDANCE
(
Signature
indicates
concurrence
with
the
pathology
report
and
statistical
analysis
of
data,
respectively)

John
Pletcher,
Consulting
Pathologist
Lori
Brunsman,
Statistician
OTHER
ATTENDEES:
None
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
ii
TABLE
OF
CONTENTS
EXECUTIVE
SUMMARY
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1
I.
INTRODUCTION
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5
II.
BACKGROUND
INFORMATION
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5
III.
EVALUATION
OF
CARCINOGENICITY
STUDIES
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1.
2­
Year
Chronic
Toxicity
and
Carcinogenicity
Study
in
Rats
(
1996)
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6
2.
2­
Year
Oncogenicity
Study
in
Mice
(
1996)
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15
3.
Dermal
Carcinogenicity
Study
in
Mice
(
1977)
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26
IV.
TOXICOLOGY
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31
1.
Metabolism
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31
2.
Mutagenicity
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32
3.
Structure­
Activity
Relationship
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33
4.
Subchronic
and
Chronic
Toxicity
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34
5.
Mode
of
Action
Studies
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36
V.
COMMITTEE'S
ASSESSMENT
OF
THE
WEIGHT­
OF­
THE­
EVIDENCE
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36
VI.
CLASSIFICATION
OF
CARCINOGENIC
POTENTIAL
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39
VII.
QUANTIFICATION
OF
CARCINOGENIC
POTENTIAL
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40
VIII.
BIBLIOGRAPHY
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41
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
1
EXECUTIVE
SUMMARY
On
April
9,
2003,
the
Cancer
Assessment
Review
Committee
of
the
Health
Effects
Division
of
the
Office
of
Pesticide
Programs
met
to
evaluate
the
carcinogenic
potential
of
PHMB.

Dr.
Jonathan
Chen
of
the
Antimicrobials
Division
presented
the
chronic
toxicity/
carcinogenicity
studies
in
Wistar
rats
and
C57B1/
10JfCD­
1/
Alpk
and
Alderley
Park
mice
by
describing
the
experimental
design;
reporting
on
survival
and
body
weight
effects
treatment­
related
non­
neoplastic
and
neoplastic
lesions,
statistical
analysis
of
the
tumor
data,
and
the
adequacy
of
the
dose
levels
tested;
and
presenting
the
weight
of
the
evidence
for
the
carcinogenicity
of
PHMB.

PHMB
was
administered
in
the
diet:
1)
to
male
and
female
Wistar
rats
(
52
rats/
sex)
at
dose
levels
of
0,
200,
600,
or
2000
ppm
(
0,
12.2,
36.3,
or
126.1
mg/
kg/
day
for
males;
0,
14.9,
45.3,
or
162.3
mg/
kg/
day
for
females)
of
PHMB
for
105
weeks;
and
2)
to
C57B1/
10J
f
CD­
1/
Alpk
mice
(
55/
sex/
group)
at
0,
400,
1,200
or
4,000
ppm
(
equivalent
to
55,
167,
or
715
mg/
kg/
day
for
males
and
69,
217,
or
856
mg/
kg/
day
for
females)
for
2
years.

In
addition,
four
groups
of
specific
pathogen
free
(
50M
+
50F)
Alderley
Park
Mice
received
dermally
0.3
mL
of
test
material
at
doses
of
0
(
solvent
in
methanol),
0.6
mg
(
0.2%
PHMB
in
ethanol),
6.0
mg
(
2%
PHMB
in
ethanol),
or
30.0
mg
(
10%
PHMB
in
ethanol)
per
mouse
per
day
for
five
days
a
week
for
80
weeks.
The
treatment
dosages
are
approximately
equivalent
to
0,
15,
150
and
750
mg/
kg/
day
of
20%
PHMB
solution.

The
CARC
concluded
that
PHMB
showed
evidence
of
carcinogenicity
based
on
the
following:

Rat
­
Oral
=
There
was
no
treatment­
related
increase
in
any
tumors
in
male
Wistar
rats.

=
Vascular
Tumors:
Female
Wistar
rats
from
the
Pathology
Working
Group
(
PWG)
diagnoses
had
significant
increasing
trends
in
liver
hemangiomas
and
liver
hemangiosarcomas,
both
at
p<
0.05.
There
was
also
a
significant
increasing
trend
for
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined
at
p<
0.01.
There
were
significant
differences
in
the
pair­
wise
comparisons
of
the
2000
ppm
dose
group
with
the
controls
for
liver
hemangiomas
and
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined,
both
at
p<
0.05.
The
incidence
of
hemangiomas
and
hemangiosarcomas
for
all
sites
combined
was
2/
42
(
5%),
1/
42
(
2%),
3/
40
(
8%),
and
6/
35
(
17%)
for
the
0,
200,
600,
and
2000
ppm
dose
groups,
respectively.
Historical
control
data
regarding
hemangiosarcomas
shows
the
range
of
hemangiosarcomas
by
tissue
in
rats
was
0­
1.9%.
The
CARC
considered
the
tumor
response
at
the
high
dose
(
17%
for
the
combined
re­
read
of
liver
and
original
diagnoses
at
other
sites)
to
be
treatment­
related
and
driven
by
the
increase
in
hemangiomas
when
all
sites
are
combined.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
2
=
Adequacy
of
Dosing:
The
CARC
concluded
that
dosing
at
the
highest
dose
(
2000
ppm)
was
considered
to
be
adequate,
but
not
excessive,
for
both
male
and
female
rats.
In
high
dose
females,
this
was
based
on
increased
mortality
and
a
reduction
in
body
weight
of
5­
8%
throughout
the
study.
In
high­
dose
males,
body
weights
were
significantly
reduced
(
4­
6%)
through
week
79.
Food
utilization
(
g
growth/
100
g
feed)
for
the
first
12
weeks
decreased
significantly
(
97­
8%)
in
both
sexes
at
2000
ppm.

Mouse
­
Oral
=
Vascular
Tumors:
Male
C57B1/
10J
f
CD­
1/
Alpk
mice
from
the
PWG
diagnoses
had
significant
increasing
trends
in
hemangiomas,
hemangiosarcomas,
and
combined
hemangiomas
and
hemangiosarcomas,
all
at
p<
0.01.
There
were
also
significant
differences
in
the
pair­
wise
comparisons
of
the
4000
ppm
dose
group
with
the
controls,
for
hemangiomas
(
p<
0.01),
hemangiosarcomas
(
p<
0.05),
and
combined
hemangiomas
and/
or
hemangiosarcomas
(
p<
0.01).
The
incidence
of
hemangiomas
was
2/
55
(
4%),
3/
55
(
5%),
4/
55
(
7%),
11/
53
(
21%),
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
The
incidence
of
hemangiosarcomas
was
5/
55
(
9%),
4/
55
(
7%),
6/
55
(
11%),
and
12/
53
(
23%)
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
The
incidence
of
combined
hemagiomas
and
hemangiosarcomas
was
6/
55
(
11%),
6/
55
(
11%),
9/
55
(
16%),
and
20/
53
(
38%)
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
For
hemangiosarcomas,
the
tumor
incidence
in
the
4000
ppm
dose
group
(
23%)
exceeded
the
historical
control
range
(
1.8­
18.3%).
The
CARC
considered
the
increase
in
vascular
tumors
(
hemangiomas,
hemangiosarcomas,
and
combined)
at
the
high
dose
(
which
is
considered
to
be
excessive)
to
be
treatment­
related.
Although
not
statistically
significant,
the
tumor
response
(
9/
55
[
16%]
for
the
combined
hemangiomas
and
hemangiosarcomas)
in
mice
at
the
mid­
dose
of
1200
ppm
is
considered
treatment­
related
since
this
tumor
type
was
also
seen
in
female
mice
(
orally
and
dermally)
and
female
rats.

=
Vascular
Tumors:
Female
C57B1/
10J
f
CD­
1/
Alpk
mice
from
the
PWG
diagnoses
had
significant
increasing
trends
in
hemangiomas
(
p<
0.05),
hemangiosarcomas
(
p<
0.05),
and
combined
hemangiomas
and
hemangiosarcomas
(
p<
0.01).
There
was
also
a
significant
difference
(
p<
0.05)
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls,
for
combined
hemangiomas
and/
or
hemangiosarcomas.
The
incidence
of
combined
hemangiomas
and/
or
hemangiosarcomas
was
8/
54
(
15%),
5/
53
(
9%),
7/
54
(
13%),
and
15/
49
(
31%)
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
For
hemangiosarcomas,
the
tumor
incidence
in
the
4000
ppm
dose
group
(
31%)
exceeded
the
historical
control
range
(
0­
10.9%).
There
are
no
historical
control
data
for
hemangiomas
by
tissue.
The
CARC
considered
the
combined
vascular
tumors
at
the
high
dose
(
which
is
considered
to
be
excessive)
to
be
treatmentrelated
There
were
no
increases
in
these
tumors
at
doses
which
were
not
considered
excessive.

=
Rectal­
Anal
Junction
Tumors:
In
male
mice
there
was
a
significant
increasing
trend
at
p
<
0.01,
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
3
controls
at
p
<
0.05,
for
rectal­
anal
junction
squamous
cell
carcinomas.
The
incidence
of
rectalanal
junction
tumors
was
0/
45,
0/
45,
0/
45,
and
5/
48
for
the
0,
400,
1200
and
4000
ppm
dose
groups,
respectively.
In
female
mice
there
was
a
significant
increasing
trend
at
p
<
0.01
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls
at
p
<
0.01,
for
rectal­
anal
junction
squamous
cell
carcinomas.
The
incidence
of
rectal
anal
junction
tumors
was
0/
42,
0/
43,
0/
43,
8/
28
for
the
0,
400,
1200
and
4000
ppm
dose
groups,
respectively.
The
CARC
considered
the
carcinomas
at
the
high
dose
(
which
is
considered
to
be
excessive)
in
both
males
and
females
to
be
treatment­
related
but
suggestive
of
a
local
irritant
effect.
These
tumors
did
not
contribute
to
the
CARC's
weight­
of­
the­
evidence
analyses.

=
Adequacy
of
Dosing:
Dosing
was
considered
to
be
excessive
at
the
high
dose
of
4000
ppm
based
on
decreased
overall
body
weight
gains
of
50%
in
males
and
32%
in
females
at
termination.
In
addition,
decreased
body
weight
gains
of
33%
in
males
and
19%
in
females
were
seen
at
13
weeks.
The
study
indicated
that
the
animals
at
the
4000
ppm
dose
group
had
increased
food
consumption
(
p<
0.05
or
0.01)
from
approximately
week
12
through
termination
in
males
(
87­
29%)
and
females
(
87­
26%).

Dosing
was
considered
to
be
adequate,
but
not
excessive,
in
both
males
and
females
at
the
middose
of
1200
ppm
based
on
decreased
overall
body
weight
gains
(
males,
7%;
females,
2%),
increases
in
hematology
parameters,
and
non­
neoplastic
histopathological
changes
in
the
gall
bladder,
liver,
and
rectal­
anal
junction.

Mouse
­
Dermal
=
Vascular
Tumors:
Female
Alderley
Park
mice
had
significant
increasing
trends
in
liver
angiosarcomas
(
p
<
0.01)
and
vascular
tumors
from
all
sites
combined
(
p
<
0.05).
There
was
also
a
significant
difference
in
the
pair­
wise
comparison
of
the
30
mg
dose
group
with
the
controls
for
liver
angiosarcomas
at
p
<
0.05.
The
incidence
of
liver
angiosarcomas
was
0/
41,
0/
45,
0/
40,
and
2/
35
for
the
0,
0.6,
6.0,
and
30
mg
dose
groups,
respectively.
The
CARC
considered
the
tumor
increase
at
the
high
dose
to
be
equivocal
since
the
tumors
were
only
seen
at
an
excessive
dose.
Vascular
tumors
were
not
seen
at
lower
doses.

There
was
no
treatment­
related
increase
in
any
tumors
in
male
Alderley
Park
mice.

=
Adequacy
of
Dosing:
Dosing
was
considered
to
be
excessive
at
the
high
dose
(
750
mg/
kg/
day
of
20%
PHMB
equivalent)
in
both
sexes
due
to
increased
mortality
(
78%,
high
dose,
versus
33%
and
28%
in
the
male
and
female
controls,
respectively)
and
decreased
body
weight
gain.
Overall
body
weight
gain
decreases
of
45%
and
17%
were
seen
in
males
and
females,
respectively.
No
treatment­
related
effects
were
noted
at
the
mid­
dose
level
of
150
mg/
kg/
day
of
20%
PHMB
equivalent.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
4
Mutagenicity
=
There
is
no
mutagenicity
concern.

Structure
Activity
Relationship
=
No
appropriate
structural
analogs
could
be
located
for
comparison
purposes.
In
general,
with
the
exception
of
exposure
via
the
inhalation
route,
high
molecular
weight
(
MW)
polymers
are
not
expected
to
be
of
significant
carcinogenic
concern
because
of
poor
bioavailability.
PHMB
has
indeed
been
shown
to
have
low
bioavailability.
However,
SAR
consideration
suggests
that
there
is
a
likelihood
that
the
lower
MW
fractions
of
PHMB
may
be
metabolized
to
polyamines
in
analogy
to
the
metabolism
of
arginine
(
a
guanidine­
containing
amino
acid)
to
form
biogenic
polyamines
such
as
putrescine,
spermidine
and
spermine.
There
is
strong
evidence
that
these
polyamines
are
readily
taken
up
by
cells
and
may
play
an
important
role
in
promoting
skin
and
colon
carcinogenesis
by
serving
as
ingredients
to
support
neoplastic
growth.
A
metabolism
study
submitted
by
the
company
showed
that
low
MW
fractions
of
PHMB
can
indeed
be
metabolized
in
the
rat;
however,
the
metabolites
have
not
yet
been
identified.

Mode
of
Action
=
No
mode
of
action
studies
were
available.

In
accordance
with
the
EPA
Draft
Guidelines
for
Carcinogen
Risk
Assessment
(
July
1999),
the
CARC
classified
PHMB
into
the
category
"
Suggestive
Evidence
of
Carcinogenicity,
but
Not
Sufficient
to
Assess
Human
Carcinogenic
Potential"
by
the
oral
and
dermal
routes.
The
quantification
of
human
cancer
risk
is
not
required.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
5
Figure
1:
Chemical
Structure
of
PHMB
I.
INTRODUCTION
On
April
9,
2003,
the
Cancer
Assessment
Review
Committee
(
CARC)
of
the
Health
Effects
Division
of
the
Office
of
Pesticide
Program
met
to
evaluate
the
carcinogenic
potential
of
PHMB.
This
was
the
first
time
this
compound
was
assessed
for
carcinogenicity
by
the
CARC.

II.
BACKGROUND
INFORMATION
PHMB,
also
known
as
Baquacil;
Baquacil
SB;
Cosmoquil
CQ;
polihexanide;
polyhexamethylbiguanide;
poly(
hexamethylenebiguanide)
hydrochloride;
Vantocil
1B;
or
polyhexamethylene
biguanide.
The
CAS
Number
of
this
chemical
is
32289­
58­
0.
The
PC
Code
of
PHMB
is
111801.
PHMB
is
a
group
of
polymers
(
see
Figure
1)
that
has
been
used
as
an
antimicrobial
agent
in
a
wide
variety
of
applications
including
oil­
in­
water
and
water­
in­
oil
emulsions,
industrial
reagents,
silicone
systems,
cellulose
solutions
and
oil
recovery
systems.
Now,
PHMB
is
primarily
used
as
a
non­
chlorinated
antimicrobial
agent
in
swimming
pool
and
spa
facilities.

On
12/
18/
2000,
01/
25/
2001,
01/
30/
2003
and
02/
06/
2003,
the
Health
Effects
Division's
Hazard
Identification
Assessment
Review
Committee
(
HIARC)
evaluated
the
toxicology
data
base
of
PHMB,
established
the
Reference
Doses
(
RfDs)
and
selected
the
toxicological
endpoints
for
dietary
as
well
as
occupational
exposure
risk
assessments.

III.
EVALUATION
OF
CARCINOGENICITY
STUDIES
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
6
1.
2­
Year
Chronic
Toxicity
and
Carcinogenicity
Study
in
Rats
(
1996)

Reference:

Horner,
S.
A.
(
1996)
Polyhexamethylene
Biguanide:
Two
Year
Feeding
Study
in
Rats.
Zeneca
Central
Toxicology
Laboratory,
Alderley
Park,
Macclesfield,
Cheshire,
UK.
Laboratory
Project
ID:
Report
No.
CTL/
P/
4663,
Study
No.
PR0936,
June
5,
1996.
MRID
44059301.
Unpublished.

Busey,
W.
M.
(
1996)
Polyhexamethylene
Biguanide
(
PHMB):
Two
Year
Feeding
Study
in
Rats.
Pathology
Working
Group
Peer
Review
of
Proliferative
Vascular
Lesions
in
Male
and
Female
Rats.
Zeneca
CTL,
Alderley
Park,
Macclesfield,
Cheshire,
UK.
Study
No.
PR0936,
Report
No.
CTL/
C/
3172,
May
13,
1996.
MRID
44042801.
Unpublished.

A.
Experimental
Design
A
combined
chronic
toxicity/
oncogenicity
study
in
Alpk:
APfSD
Wistar
rats
was
conducted
by
Central
Toxicology
Laboratory,
Alderley
Park,
Macclesfield,
Cheshire,
England,
for
Zeneca
Specialities,
Wilmington,
Delaware
and
dated
June
5,
1996
(
Report
No.
CTL/
P/
4663,
Study
No.
PR0936,
MRID
No.
44059301).
A
Pathology
Working
Group
(
PWG)
peer
review
reevaluated
the
vascular
lesions
of
this
study
in
a
report
dated
May
13,
1996
(
Report
No.
CTL/
C/
3172,
MRID
No.
44042801).
Both
the
original
and
the
reread
diagnoses
are
presented
in
this
document.

The
study
design
allocated
groups
of
52
rats
per
sex
to
dose
levels
of
0,
200,
600,
or
2000
ppm
(
0,
12.2,
36.3,
or
126.1
mg/
kg/
day
for
males;
0,
14.9,
45.3,
or
162.3
mg/
kg/
day
for
females)
of
PHMB
for
105
weeks.
An
additional
12
rats
per
sex
per
dose
were
designated
for
interim
sacrifice
at
week
52.

B.
Discussion
of
Tumor
Data
Survival
Analysis
The
statistical
evaluation
of
mortality
indicated
no
significant
incremental
changes
with
increasing
doses
of
PHMB
in
male
Alpk:
APfSD
Wistar
rats.
Female
Alpk:
APfSD
Wistar
rats
had
a
significant
increasing
trend
in
mortality
with
increasing
doses
of
PHMB
(
p<
0.01)
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
2000
ppm
dose
group
(
p
<
0.05)
with
the
control
group
(
Memo,
L.
Brunsman,
12/
03/
02,
TXR
No.
0051368).
Tables
1
and
2
show
the
mortality
results.
Table
1.
PHMB
­
1996
Alpk:
APfSD
Wistar
Rat
Study
Male
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
26
27­
52
53i
53­
78
79­
105f
Total
(
ppm)
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
7
0
0/
64
0/
64
12/
64
5/
52
27/
47
32/
52
(
0)
(
0)
(
10)
(
57)
(
62)

200
0/
64
3/
64
12/
61
7/
49
26/
42
36/
52
(
0)
(
5)
(
13)
(
58)
(
69)

600
0/
64
2/
64
12/
62
10/
50
20/
40
32/
52
(
0)
(
3)
(
19)
(
48)
(
62)

2000
3/
64
2/
61
10/
59
5/
49
22/
44
32/
54
(
5)
(
3)
(
10)
(
48)
(
59)

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

iInterim
sacrifice
at
week
53.

fFinal
sacrifice
at
week
105.

(
)
Percent.
Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
8
Table
2.
PHMB
­
1996
Alpk:
APfSD
Wistar
Rat
Study
Female
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
26
27­
52
53I
53­
78
79­
105f
Total
(
ppm)

0
0/
64
0/
64
12/
64
6/
52
19/
46
25/
52
(
0)
(
0)
(
12)
(
41)
(
48)**

200
0/
64
0/
64
12/
64
5/
52
15/
47
20/
52
(
0)
(
0)
(
10)
(
32)
(
38)

600
1/
64
1/
63
12/
62
7/
50
14/
43
23/
52
(
2)
(
2)
(
14)
(
32)
(
44)

2000
1/
64
4/
63
12/
59
6/
47
21/
41
32/
52
(
2)
(
6)
(
12)
(
47)
(
62)*

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

iInterim
sacrifice
at
week
53.

fFinal
sacrifice
at
week
105.

(
)
Percent.

Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
9
Tumor
Analysis
The
study
pathologist
and
study
peer
reviewer
determined
that
there
were
3
hemangiosarcomas
of
the
liver
in
2000
ppm
females.
Benign
hemangiomas
were
not
observed.
The
increased
incidence
of
hemangiosarcomas
in
females
gave
positive
results
in
trend
analyses
(
p<
0.05).
A
single
observation
of
benign
hemangioma
was
made
in
each
of
the
control
and
high
dose
males;
no
hemangiosarcomas
were
observed
in
males.
A
Pathology
Working
Group
(
PWG)
was
convened
in
1996
to
confirm
the
diagnoses
of
the
vascular
neoplasms
(
MRID
44042801).
The
PWG
determined
that
there
were
2
hemangiomas
and
1
hemangiosarcoma
in
2000
ppm
females
and
2
hemangiomas
in
2000
ppm
males.
The
PWG
concluded
that
the
findings
of
vascular
neoplasms
in
high
dose
females
were
incidental.
However,
the
report
of
the
PWG
consensus
indicated
that
no
hemangiosarcoma
or
hemangioma
had
been
observed
in
female
controls
in
18
studies
with
the
same
strain
of
rat.
Dr.
J.
M.
Pletcher,
EPA's
consulting
pathologist,
confirmed
the
validity
of
the
PWG
report
(
TXR
No.
0052000).

There
were
no
compound­
related
tumors
observed
in
male
rats.
Female
rats
from
the
original
study
diagnoses
had
significant
increasing
trends
in
liver
hemangiosarcomas
and
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined,
both
at
p
<
0.05.
There
was
also
a
significant
difference
in
the
pair­
wise
comparison
of
the
2000
ppm
dose
group
with
the
controls
for
liver
hemangiosarcomas
at
p
<
0.05.

Female
rats
from
the
PWG
diagnoses
had
significant
increasing
trends
in
liver
hemangiomas
and
liver
hemangiosarcomas,
both
at
p
<
0.05.
There
was
also
a
significant
increasing
trend
for
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined
at
p
<
0.01.
There
were
significant
differences
in
the
pair­
wise
comparisons
of
the
2000
ppm
dose
group
with
the
controls
for
liver
hemangiomas
and
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined,
both
at
p
<
0.05
(
Memo,
L.
Brunsman,
12/
03/
02,
TXR
No.
0051368).

The
statistical
analyses
of
the
female
rats
were
based
upon
Peto's
prevalence
test.
Tables
3
and
4
show
the
tumor
analyses
results.

Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
Alpk:
ApfSD
Wistar
rats
and
conducted
by
the
Syngenta
Central
Toxicology
Laboratory
(
Alderley
Park,
UK)
are
provided
(
MRID
457108­
08).
The
total
number
of
rats
with
hemangiosarcomas
recorded,
irrespective
of
site,
are
summarized
in
Table
5.
The
occurrence
of
hemangiosarcomas
by
tissue
in
rats
is
shown
in
Table
6.
The
historical
control
range
for
hemangiosarcomas
was
0­
1.9%
for
both
male
and
female
rats.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
10
Table
3.
PHMB
­
1996
Alpk:
APfSD
Wistar
Rat
Study
ORIGINAL
STUDY
DIAGNOSES
Female
Vascular
Tumor
Rates+
and
Peto's
Prevalence
Test
Results
(
p
values)
Dose
(
ppm)

0
200
600
2000
Liver
Hemangiosarcomas
0/
42
1/
42
0/
40
3a/
35
(%)
(
0)
(
2)
(
0)
(
9)
p
=
0.0107*
0.1284
­
0.0338*
Other
Sites
(
NOT
LIVER)
Hemangiomas
1/
42
1/
41
3/
39
3b/
34
(%)
(
2)
(
2)
(
8)
(
9)
p
=
0.0733
0.4208
0.1430
0.0748
Other
Sites
(
NOT
LIVER)

Hemangiosarcomas
1c/
40
0/
40
0/
35
0/
28
(%)
(
2)
(
0)
(
0)
(
0)
p
=
­
­
­
­
Combined
2/
42
2/
42
3/
40
6/
35
(%)
(
5)
(
5)
(
8)
(
17)
p
=
0.0234*
0.3094
0.2804
0.0575
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
observation
of
the
first
tumor.

aFirst
liver
hemangiosarcoma
observed
at
week
91,
dose
2000
ppm.

bFirst
other
sites
(
NOT
LIVER)
hemangioma
observed
at
week
92,
dose
2000
ppm.

cFirst
other
sites
(
NOT
LIVER)
hemangiosarcoma
observed
at
week
95,
dose
0
ppm.

Note:
Significance
of
trend
denoted
at
control.
Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.
If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
11
Table
4.
PHMB
­
1996
Alpk:
APfSD
Wistar
Rat
Study
Female
Vascular
Tumor
Rates+
and
Peto's
Prevalence
Test
Results
(
p
values)
Dose
(
ppm)
0
200
600
2000
PWG
RE­
READ
OF
LIVER
VASCULAR
TUMORS
Hemangiomas
0/
42
0/
42
0/
40
2a/
35
(%)
(
0)
(
0)
(
0)
(
6)
p
=
0.0107*
­
­
0.0808*

Hemangiosarcomas
0/
27
0/
32
0/
29
1b/
20
(%)
(
0)
(
0)
(
0)
(
5)
p
=
0.0232*
­
­
0.1226
ORIGINAL
DIAGNOSES
OF
VASCULAR
TUMORS
AT
OTHER
SITES
Hemangiomas
1/
42
1/
41
3/
39
3c/
34
(%)
(
2)
(
2)
(
8)
(
9)
p
=
0.0733
0.4208
0.1430
0.0748
Hemangiosarcomas
1d/
40
0/
40
0/
35
0/
28
(%)
(
2)
(
0)
(
0)
(
0)
p
=
­
­
­
­
COMBINED
PWG
RE­
READ
OF
LIVER
AND
ORIGINAL
DIAGNOSES
AT
OTHER
SITES
Combined
2/
42
1/
42
3/
40
6/
35
(%)
(
5)
(
2)
(
8)
(
17)
p
=
0.0079**
­
0.2869
0.0344*
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
observation
of
the
first
tumor.
a
First
liver
hemangioma
observed
at
week
91,
dose
2000
ppm.
b
First
liver
hemangiosarcoma
observed
at
week
105,
dose
2000
ppm.
c
First
other
sites
(
NOT
LIVER)
hemangioma
observed
at
week
92,
dose
2000
ppm.
d
First
other
sites
(
NOT
LIVER)
hemangiosarcoma
observed
at
week
95,
dose
0
ppm.

Note:
Significance
of
trend
denoted
at
control.
Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.
If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
12
Table
5.
Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
Alpk:
ApfSD
Wistar
rats
and
conducted
by
the
Syngenta
Central
Toxicology
Laboratory
(
Alderley
Park,
UK)
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
13
Table
6.
Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
Alpk:
ApfSD
Wistar
rats
and
conducted
by
the
Syngenta
Central
Toxicology
Laboratory
(
Alderley
Park
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
14
C.
Non­
Neoplastic
Lesions
In
addition
to
liver
hemangiomas
and
hemangiosarcomas,
the
liver
is
also
the
target
organ
in
males
and
females.
Plasma
alkaline
phosphatase
activity
was
elevated
significantly
over
controls
(
p<
0.01)
in
females
from
the
main
study
dosed
at
2000
ppm
(
843­
74%).
In
2000
ppm
males
the
enzyme
was
significantly
increased
by
36%
and
27%
at
weeks
14
and
27,
respectively.
In
females
at
2000
ppm,
absolute
liver
weight
was
reduced
significantly
(
p<
0.05)
by
11%.
In
2000
ppm
males,
microscopic
observations
of
liver
hepatocyte
fat
and
spongiosis
were
increased
44%
and
22%
over
controls,
respectively.
Corresponding
increases
in
these
lesions
were
not
seen
in
females.
There
were
no
corroborating
gross
pathology
findings
of
the
liver
abnormalities.

D.
Adequacy
of
the
Dosing
for
Assessment
of
Carcinogenicity
The
dosing
was
considered
to
be
adequate,
but
not
excessive,
for
both
male
and
female
rats.
In
the
rat
study
at
the
high
dose
(
2000
ppm)
there
was
significantly
increased
mortality
in
females
(
62%
vs.
48%
in
controls).
Body
weights
were
significantly
reduced
by
5­
8%
in
high­
dose
females
throughout
the
study.
In
high­
dose
males,
body
weights
were
significantly
(
p<
0.01
or
0.05)
lower
than
controls
(
94­
6%)
through
week
79.
Food
utilization
(
g
growth/
100
g
feed)
for
the
first
12
weeks
decreased
significantly
(
p<
0.01)
vs
controls
(
97­
8%
in
both
sexes
at
2000
ppm).
Plasma
alkaline
phosphatase
activity
was
significantly
elevated
43­
74%
over
controls
in
2000
ppm
females
from
the
main
study
at
the
27,
53,
79,
and
105­
week
intervals.
In
males
at
2000
ppm,
alkaline
phosphatase
activity
was
increased
significantly
by
36%
and
27%
during
weeks
14
and
27,
respectively.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
15
2.
2­
Year
Oncogenicity
Study
in
Mice
(
1996)

Reference:

Milburn
G.
M.
(
1996)
Polyhexamethylene
Biguanide:
Two
Year
Oncogenicity
Study
in
Mice.
Central
Toxicology
Laboratory,
Alderley
Park,
Macclesfield,
Cheshire,
UK
SK10
4TJ.
Laboratory
Project
ID:
PM0937.
June
21,
1996.
MRID
44074201.
Unpublished.

Mann,
P.
(
2002)
Polyhexamethylene
Biguanide
(
PHMB):
Two
Year
Oncogenic
Study
in
Mice.
Pathology
Working
Group.
Peer
Review
of
Proliferative
Vascular
Lesions
in
Male
and
Female
Mice.
Report
Identification
CTL
Study
No.
PM0937.
EPL
Project
Number.
698­
001.
MRID
45710802.
Unpublished.

A.
Experimental
Design
In
this
mouse
oncogenicity
study
(
MRID
44074201),
poly(
hexamethylenebiguanide)
hydrochloride
(
PHMB,
20.2%
a.
i.)
was
administered
in
the
diet
to
C57B1/
10J
f
CD­
1/
Alpk
mice
(
55/
sex/
group)
at
0,
400,
1,200
or
4,000
ppm
(
equivalent
to
55,
167,
or
715
mg/
kg/
day
for
males
and
69,
217,
or
856
mg/
kg/
day
for
females)
for
2
years.
A
Pathology
Working
Group
(
PWG)
peer
review
reevaluated
the
vascular
lesions
of
this
study
in
a
report
dated
June
27,
2002
(
MRID
45710802).
Only
the
PWG
re­
read
diagnoses
of
the
vascular
tumors
are
presented
in
this
document.

B.
Discussion
of
Tumor
Data
Survival
Analysis
The
statistical
evaluation
of
mortality
indicated
a
significant
increasing
trend
with
increasing
doses
of
PHMB
in
female
C57B1/
10J
f
CD­
1/
Alpk
mice.
There
was
no
statistically
significant
incremental
change
in
mortality
with
increasing
doses
of
PHMB
in
male
C57B1/
10J
f
CD­
1/
Alpk
mice
(
Memo,
L.
Brunsman,
12/
03/
02,
TXR
No.
0051368).
Tables
7
and
8
show
the
mortality
results.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
16
Table
7.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
Male
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
26
27­
52
53­
78
79­
105
f
Total
(
ppm)

0
0/
55
0/
55
5/
55
24/
50
29/
55
(
0)
(
0)
(
9)
(
48)
(
53)

400
0/
55
2/
55
6/
53
18/
47
26/
55
(
0)
(
4)
(
11)
(
38)
(
47)

1200
0/
55
0/
55
6/
55
11/
49
17/
55
(
0)
(
0)
(
11)
(
22)
(
31)*
N
4000
0/
55
5/
55
7/
50
17/
43
29/
55
(
0)
(
9)
(
14)
(
40)
(
53)

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

f
Final
sacrifice
at
week
105.

(
)
Percent.

N:
Negative
change
from
control.

Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
17
Table
8.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
Female
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
26
27­
52
53­
78
79­
105
f
Total
(
ppm)

0
1/
55
0/
54
7/
54
17/
47
25/
55
(
2)
(
0)
(
13)
(
36)
(
45)**

400
1/
55
1/
54
0/
53
17/
53
19/
55
(
2)
(
2)
(
0)
(
32)
(
35)

1200
1/
55
0/
54
5/
54
14/
49
20/
55
(
2)
(
0)
(
9)
(
29)
(
36)

4000
3/
55
3/
52
10/
49
23/
39
39/
55
(
5)
(
6)
(
20)
(
59)
(
71)**

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

f
Final
sacrifice
at
week
105.

(
)
Percent.

Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
18
Tumor
Analysis
The
study
pathologist
and
study
peer
reviewer
determined
that
there
was
an
increase
in
vascular
tumors,
mainly
hemangiosarcoma,
in
both
sexes
at
the
high
dose
(
4000
ppm)
when
compared
to
controls.
The
reviewing
pathologist
agreed
with
the
increase
in
vascular
tumors,
but
not
always
with
the
diagnosis
(
hemangioma
vs.
hemangiosarcoma).
A
pathologist
working
group
(
PWG)
was
conducted
in
2002
to
confirm
the
diagnoses
of
the
vascular
neoplasms
(
MRID
45710802).
The
PWG
confirmed
the
study
pathologist
and
peer
reviewer's
conclusion,
that
there
was
clear
evidence
of
a
treatment­
related
increase
in
incidence
of
animals
with
either
hemangioma
or
hemangiosarcoma
in
the
high
dose
of
both
sexes.
This
increase
was
largely
due
to
the
increase
in
the
number
of
vascular
tumors
in
the
liver.
PWG
suggested
that
it
is
more
appropriate
to
consider
the
total
number
of
animals
with
vascular
neoplasms,
rather
than
the
individual
organs
with
either
primary
or
metastatic
lesions.
Dr.
J.
M.
Pletcher,
EPA's
consulting
pathologist,
confirmed
the
validity
of
the
PWG
report
(
TXR
No.
0052033).

As
shown
in
Table
9,
male
mice
had
significant
increasing
trends
at
p<
0.01,
and
significant
differences
in
the
pair­
wise
comparisons
of
the
4000
ppm
dose
group
with
the
controls,
for
hemangiomas
(
p<
0.01),
hemangiosarcomas
(
p<
0.05)
and
combined
hemangiomas
and/
or
hemangiosarcomas
(
p<
0.01).
There
was
also
a
significant
increasing
trend
at
p
<
0.01,
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls
at
p
<
0.05,
for
rectal­
anal
junction
squamous
cell
carcinomas.

Female
mice
had
significant
increasing
trends
for
hemangiomas
(
p<
0.05),
hemangiosarcomas
(
p<
0.05),
and
combined
hemangiomas
and
hemangiosarcomas
(
p<
0.01).
There
was
also
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls,
for
combined
hemangiomas
and/
or
hemangiosarcomas,
at
p<
0.05.
There
was
also
a
significant
increasing
trend
at
p<
0.01,
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls
at
p<
0.01
for
rectal­
anal
junction
squamous
cell
carcinomas.

The
statistical
analyses
of
the
male
mice
were
based
upon
the
Exact
trend
test
and
the
Fisher's
Exact
test
for
pair­
wise
comparisons.
The
statistical
analyses
of
the
female
mice
were
based
upon
Peto's
prevalence
test
(
Memo,
L.
Brunsman,
12/
03/
02,
TXR
No.
0051368).
See
Tables
9
through
12
for
tumor
analyses
results.

Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
C57BL/
10J/
CD­
1
Alpk
mouse
and
conducted
by
the
Syngenta
Central
Toxicology
Laboratory
(
Alderley
Park,
UK)
are
provided
(
MRID
457108­
04).
The
total
number
of
rats
with
hemangiosarcoma
recorded,
irrespective
of
site
are
summarized
in
Table
13.
There
are
no
historical
control
data
for
hemangiosarcoma
by
tissue.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
19
Table
9.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
2002
PWG
Re­
Read
Male
Vascular
Tumor
Rates+
(
All
Sites)
and
Fisher's
Exact
Test
and
Exact
Trend
Test
Results
(
p
values)
Dose
(
ppm)

0
400
1200
4000
Hemangiomas
2/
55
3/
55
4/
55
11a/
53
(%)
(
4)
(
5)
(
7)
(
21)

p
=
0.0007**
0.5000
0.3393
0.0063**

Hemangiosarcomas
5/
55
4/
55
6b/
55
12/
53
(%)
(
9)
(
7)
(
11)
(
23)

p
=
0.0067**
0.5000
0.5000
0.0468*

Combined
6c/
55
6c/
55
9c/
55
20d/
53
(%)
(
11)
(
11)
(
16)
(
38)

p
=
0.0000**
0.6195
0.2899
0.0010**
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
week
39.

a
First
hemangioma
observed
at
week
39,
dose
4000
ppm.
b
First
hemangiosarcoma
observed
at
week
59,
dose
1200
ppm.
c
One
animal
in
each
of
the
0,
400
and
1200
ppm
dose
groups
had
both
an
hemangioma
and
an
hemangiosarcoma.
d
Three
animals
in
the
4000
ppm
dose
group
had
both
an
hemangioma
and
an
hemangiosarcoma
Note:
Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
20
Table
10.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
Male
Rectal­
Anal
Junction
Tumor
Rates+
and
Fisher's
Exact
Test
and
Exact
Trend
Test
Results
(
p
values)

Dose
(
ppm)

0
400
1200
4000
Squamous
Cell
Carcinomas
0/
45
0/
45
0/
45
5a/
48
(%)
(
0)
(
0)
(
0)
(
10)

p
=
0.0011**
1.0000
1.0000
0.0329*
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
week
36.

a
First
squamous
cell
carcinoma
observed
at
week
36,
dose
4000
ppm.

Note:
Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
21
Table
11.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
2002
PWG
Re­
Read
Female
Vascular
Tumor
Rates+
(
All
Sites)
and
Peto's
Prevalence
Test
Results
(
p
values)
Dose
(
ppm)
0
400
1200
4000
Hemangiomas
6/
51
2/
53
5a/
52
8/
44
(%)
(
12)
(
4)
(
10)
(
18)

p
=
0.0294*
­
­
0.1884
Hemangiosarcomas
6/
54
4/
53
4/
54
10b/
49
(%)
(
11)
(
8)
(
7)
(
20)

p
=
0.0300*
­
­
0.1262
Combined
8c/
54
5d/
53
7e/
54
15f/
49
(%)
(
15)
(
9)
(
13)
(
31)

p
=
0.0023**
­
­
0.0288*
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
observation
of
the
first
tumor.

a
First
hemangioma
observed
at
week
69,
dose
1200
ppm.
b
First
hemangiosarcoma
observed
at
week
54,
dose
4000
ppm.
c
Four
animals
in
the
0
ppm
dose
group
had
both
an
hemangioma
and
an
hemangiosarcoma.
d
One
animal
in
the
400
ppm
dose
group
had
both
an
hemangioma
and
an
hemangiosarcoma.
e
Two
animals
in
the
1200
ppm
dose
group
had
both
an
hemangioma
and
an
hemangiosarcoma.
f
Three
animals
in
the
4000
ppm
dose
group
had
both
an
hemangioma
and
an
hemangiosarcoma.

Note:
Significance
of
trend
denoted
at
control.
Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.
If
*,
then
p
<
0.05.
If
**,
then
p
<
0.0
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
22
Table
12.
PHMB
­
1996
C57B1/
10J
f
CD­
1/
Alpk
Mouse
Study
Female
Rectal­
Anal
Junction
Tumor
Rates+
and
Peto's
Prevalence
Test
Results
(
p
values)

Dose
(
ppm)

0
400
1200
4000
Squamous
Cell
Carcinomas
0/
42
0/
42
0/
43
8a/
28
(%)
(
0)
(
0)
(
0)
(
29)

p
=
0.0000**
­
­
0.0002**
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
observation
of
the
first
tumor.

a
First
squamous
cell
carcinoma
observed
at
week
86,
dose
4000
ppm.

Note:
Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
23
Table
13.
Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
C57BL/
10J/
CD­
1
Alpk
mouse
and
conducted
by
the
Syngenta
Central
Toxicology
Laboratory
(
Alderley
Park,
UK).
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
24
C.
Non­
Neoplastic
Lesions
At
the
1,200
ppm
level,
mice
were
observed
with
decreased
overall
body
weight
gains
(
males
97%;
females
92%);
increases
in
hematology
parameters
(
p<
0.05)
including
hemoglobin
in
females
(
86%),
hematocrit
in
females
(
86%),
and
RBCs
in
females
(
85%);
decreased
absolute
weight
of
the
liver
in
males
(
915%)
and
females
(
921%);
increased
incidences
of
gross
pathological
changes
including
distended
caeca
in
females
(
4%
treated
vs.
0%
in
controls);
traumatized
pinnae
of
ears
in
males
(
36%
treated
vs.
4%
controls)
and
females
(
44%
treated
vs.
4%
controls);
increased
incidences
of
nonneoplastic
lesions
including
luminal
dilatation
of
the
gall
bladder
in
females
(
9%
treated
vs.
2%
controls),
hepatocyte
hypertrophy
of
the
liver
in
males
(
13%
treated
vs.
0%
controls)
and
females
(
35%
treated
vs.
0%
controls),
increased
ploidy
of
the
liver
in
males
(
13%
treated
vs.
0%
controls)
and
females
(
36%
treated
vs.
0%
controls),
pigmentation
of
the
liver
in
females
(
11%
treated
vs.
0%
controls),
and
inflammation
of
the
rectal­
anal
junction
in
males
(
44%
treated
vs.
2%
controls)
and
females
(
47%
treated
vs.
21%
controls).

At
the
4,000
ppm
dose
level,
decreased
overall
body
weight
gain
was
observed
in
males
(
950%)
and
females
(
932%);
increased
food
consumption
(
p<
0.05
or
0.01)
from
approximately
week
12
through
termination
in
males
(
87­
29%)
and
females
(
87­
26%);
decreased
food
utilization
(
p<
0.01)
during
weeks
1­
12
in
males
(
940%)
and
females
(
920%);
increased
hemoglobin
in
males
(
87%)
and
females
(
817%),
hematocrit
in
males
(
85%)
and
females
(
816%),
and
RBCs
in
males
(
810%)
and
females
(
817%);
decreased
weight
of
the
liver
in
males
(
20%)
and
females
(
30%);
decreased
weight
of
the
testes
of
males
(
915%)
and
in
adrenals
of
females
(
922%);
and
increased
incidences
of
gross
pathological
changes
including
swollen
anuses
in
males
(
18%
treated
vs.
0%
controls)
and
females
(
7%
treated
vs.
0%
controls),
distended
caeca
in
males
(
9%
treated
vs.
0%
controls)
and
females
(
13%
treated
vs.
0%
controls),
pinnae
of
ears
traumatized
in
males
(
27%
treated
vs.
4%
controls)
and
females
(
22%
treated
vs.
4%
controls),
distention
of
the
gall
bladder
in
males
(
49%
treated
vs.
11%
controls)
and
females
(
47%
treated
vs.
9%
controls),
and
liver
mass
in
males
(
23%
treated
vs.
9%
controls)
and
females
(
15%
treated
vs.
8%
controls).
Increased
incidences
of
non­
neoplastic
microscopic
lesions
were
observed,
including
luminal
dilatation
of
the
gall
bladder
in
males
(
48%
treated
vs.
20%
controls)
and
females
(
39%
treated
vs.
2%
controls),
epithelial
hyperplasia
of
the
gall
bladder
in
males
(
25%
treated
vs.
0%
controls)
and
females
(
13%
treated
vs.
0%
controls),
hepatocyte
hypertrophy
of
the
liver
in
males
(
53%
treated
vs.
0%
controls)
and
females
(
49%
treated
vs.
0%
controls),
ploidy
of
the
liver
in
males
(
53%
treated
vs.
0%
controls)
and
females
(
38%
treated
vs.
0%
controls),
pigmentation
of
the
liver
in
males
(
36%
treated
vs.
0%
controls)
and
females
(
42%
treated
vs.
0%
controls),
and
inflammation
of
the
rectal­
anal
junction
in
males
(
82%
treated
vs.
2%
controls)
and
females
(
74%
treated
vs.
21%
controls).

Toxicity
observed
common
to
both
sexes
of
the
1,200
and
4,000
ppm
treatment
groups
included
decreased
overall
weight
gains,
pinnae
of
ears
traumatized,
hepatocyte
hypertrophy
of
the
liver,
increased
ploidy
of
the
liver,
and
inflammation
of
the
rectal­
anal
junction.
There
was
also
epithelial
hyperplasia
of
the
gallbladder
common
to
females
of
both
treatment
groups,
but
occurring
in
males
only
at
4,000
ppm.
No
treatment­
related
effects
were
seen
in
mice
in
the
400
ppm
dose
group.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
25
D.
Adequacy
of
Dosing
for
Assessment
of
Carcinogenicity
Dosing
was
considered
to
be
excessive
at
the
high
dose
of
4000
ppm
based
on
decreased
overall
body
weight
gains
of
50%
in
males
and
32%
in
females
at
termination.
In
addition,
decreased
body
weight
gains
of
33%
in
males
and
19%
in
females
were
observed
at
13
weeks.
The
study
indicated
that
the
animals
at
the
4000
ppm
dose
group
had
increased
food
consumption
(
p<
0.05
or
0.01)
from
approximately
week
12
through
termination
in
males
(
87­
29%)
and
females
(
87­
26%).

Dosing
was
considered
to
be
adequate,
but
not
excessive,
in
both
males
and
females
at
the
mid­
dose
of
1200
ppm
based
on
decreased
overall
body
weight
gains
(
males,
7%;
females,
2%),
and
increases
in
hematology
parameters,
and
non­
neoplastic
histopathological
changes
in
the
gall
bladder,
liver,
and
rectal­
anal
junction.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
26
3.
Dermal
Carcinogenicity
Study
in
Mice
(
1977)

Reference:

Clapp,
MJ.
L.;
Iswarn,
T.
J.;
Major,
P.
(
1977).
Polyhexamethylene
Biguanide:
80
Week
Skin
Painting
Study
in
Mice:
Report
No.
CTL/
P/
331
(
Amended).
ICI
Americas,
Inc.,
Washington,
Del;
CDL:
233269.
MRID
00066475.
Unpublished
Data.

ICI
Americas,
Inc.
(
1975).
Polyhexamethylene
Biguanide:
80
Week
Painting
Study
in
Mice:
Appendix
B:
Pathology
Individual
Animal
Data:
Report
No.
CTL/
P/
331.
CDL:
235604­
A.
MRID
00104796.

A.
Experimental
Design
Four
groups
of
specific
pathogen
free
(
50M
+
50F)
Alderley
Park
Mice
received
dermally
0.3
mL
of
test
material
at
doses
of
0
(
solvent
in
ethanol),
0.6
mg
(
0.2%
PHMB
in
ethanol),
6.0
mg
(
2%
PHMB
in
ethanol)
and
30.0
mg
(
10%
PHMB
in
ethanol)
per
day
for
five
days
a
week
for
80
weeks.
The
treatment
dosages
are
approximately
equivalent
to
0,
15,
150
and
750
mg/
kg/
day
of
a
20%
PHMB
solution.

B.
Discussion
of
Tumor
Data
Survival
Analysis
The
statistical
evaluation
of
mortality
indicated
a
significant
increasing
trend
with
increasing
doses
of
PHMB
in
male
and
female
SPF
Alderley
Park
mice
(
Memo,
L.
Brunsman,
12/
03/
02,
TXR
No.
0051368).
Tables
14
and
15
show
the
mortality
results.

Tumor
Analysis
There
were
no
compound­
related
tumors
observed
in
male
mice.
Female
mice
had
significant
increasing
trends
in
liver
angiosarcomas
(
p
<
0.01)
and
vascular
tumors
from
all
sites
combined
(
p
<
0.05).
There
was
a
significant
difference
in
the
pair­
wise
comparison
of
the
30
mg
dose
group
with
the
controls
for
liver
angiosarcomas
at
p
<
0.05.
The
statistical
analyses
of
the
female
mice
were
based
upon
Peto's
prevalence
test.
See
Table
16
for
tumor
analyses
results.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
27
Table
14.
PHMB
­
1977
SPF
Alderley
Park
Mouse
Study
Male
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
20
21­
40
41­
60
61­
81
f
Total
(
mg/
animal)
(
mg/
kg/
day
of
20%
PHMB
equivalent)

0.0
0/
49#
1/
49
6/
48
9/
42
16/
49
(
0.0)
(
0)
(
2)
(
12)
(
21)
(
33)**

0.6
1/
50
0/
49
1/
49
7/
48
9/
50
(
15)
(
2)
(
0)
(
2)
(
15)
(
18)

6.0
1/
50
0/
49
6/
49
6/
43
13/
50
(
150)
(
2)
(
0)
(
12)
(
14)
(
26)

30.0
3/
50
4/
47
7/
43
25/
36
39/
50
(
750)
(
6)
(
9)
(
16)
(
69)
(
78)**

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

f
Final
sacrifice
at
week
80.

#:
One
animal
missexed
and
removed
from
study
at
week
4.

(
)
Percent.

Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
28
Table
15.
PHMB
­
1977
SPF
Alderley
Park
Mouse
Study
Female
Mortality
Rates+
and
Cox
or
Generalized
K/
W
Test
Results
Weeks
Dose
1­
20
21­
40
41­
60
61­
81f
Total
(
mg/
animal)
(
mg/
kg/
day
of
20%
PHMB
equivalent)

0.0
2/
50
2/
48
4/
46
6/
42
14/
50
(
0.0)
(
4)
(
4)
(
9)
(
14)
(
28)**

0.6
1/
50
1/
49
3/
48
11/
45
16/
50
(
15)
(
2)
(
2)
(
6)
(
24)
(
32)

6.0
1/
50
2/
49
7/
47
8/
40
18/
50
(
150)
(
2)
(
4)
(
15)
(
20)
(
36)

30.0
3/
50
2/
47
7/
45
27/
38
39/
50
(
750)
(
6)
(
4)
(
16)
(
71)
(
78)**

+
Number
of
animals
that
died
during
interval/
Number
of
animals
alive
at
the
beginning
of
the
interval.

f
Final
sacrifice
at
week
80.

(
)
Percent.

Note:
Time
intervals
were
selected
for
display
purposes
only.

Significance
of
trend
denoted
at
control.

Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.

If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
29
Table
16.
PHMB
­
1977
SPF
Alderley
Park
Mouse
Study
Female
Vascular
Tumor
Rates+
and
Peto's
Prevalence
Test
Results
(
p
values)
Dose
­
mg/
animal
(
mg/
kg/
day
of
20%
PHMB
equivalent)
0
0.6(
15)
6.0(
150)
30.0(
750)
Liver
Hemangioendotheliomas
0/
37
0/
40
0/
37
1a/
31
(%)
(
0)
(
0)
(
0)
(
3)
p
=
0.1059
­
­
­

Liver
Angiosarcomas
0/
41
0/
45
0/
40
2b/
35
(%)
(
0)
(
0)
(
0)
(
6)
p
=
0.0025**
­
­
0.0401*

Other
Sites
(
NOT
LIVER)

Hemangioendotheliomas
1c/
41
0/
44
0/
40
0/
33
(%)
(
2)
(
0)
(
0)
(
0)
p
=
­
­
­
­

Combined
1/
41
0/
45
0/
40
3/
35
(%)
(
2)
(
0)
(
0)
(
9)
p
=
0.0129*
­
­
0.1606
+
Number
of
tumor
bearing
animals/
Number
of
animals
examined,
excluding
those
that
died
before
observation
of
the
first
tumor.

a
First
liver
hemangioendothelioma
observed
at
week
68,
dose
30.0
mg/
kg/
day.

b
First
liver
angiosarcoma
observed
at
week
60,
dose
30.0
mg/
kg/
day.

c
First
other
sites
(
NOT
LIVER)
hemangioendothelioma
observed
at
week
62,
dose
0.0
mg/
kg/
day.

Note:
Significance
of
trend
denoted
at
control.
Significance
of
pair­
wise
comparison
with
control
denoted
at
dose
level.
If
*,
then
p
<
0.05.
If
**,
then
p
<
0.01.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
30
Historical
control
data
regarding
hemangiosarcomas
from
studies
using
the
Alderley
Park
mice
and
conducted
by
the
ICI
Central
Toxicology
Laboratory
are
provided
(
MRID
225025201)
and
are
summarized
in
Table
17.

Table
17.
Historical
Control
Data
Regarding
the
Incidence
of
Liver
Tumors
in
Control
Alderley
Park
Mice
in
80
Week
Studies
at
the
ICI
Central
Laboratory
(
MRID
225025201)

1
2
3
4
5
6
1965
1065
1969
1969
1970
1972
M
F
M
F
M
F
M
F
M
F
M
F
Hepatosarcoma
4
1
3
0
1
0
3
0
0
0
0
0
Hepatoma
2
0
5
0
5
0
9
4
5
1
4
0
Hemangioendothel
ioma
0
0
0
0
0
1
0
0
0
0
0
0
Total
no.
of
tumors
6
1
8
0
6
0
12
4
5
1
4
0
No.
of
animal
examined
41
39
35
39
30
31
45
50
32
39
45
49
%
each
sex
with
tumors
14.6
2.6
22.9
0
20
0
26.7
8
15.6
2.6
8.9
0
Combined
%
with
tumors
8.75
12.5
9.8
16.8
8.6
4.3
Overall
Incidence
of
Liver
Tumors
=
9.9%
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
31
C.
Non­
Neoplastic
Lesions
The
treatment
dosages
are
equivalent
to
0,
15,
150
and
750
mg/
kg/
day
of
20%
PHMB
solutions.
Mice
that
received
the
highest
dose
level
showed
a
poorer
condition,
being
very
thin
throughout
the
experiment.
Death
in
both
males
and
females
in
the
highest
dose
group
were
slightly
higher
than
in
other
groups
during
the
first
year.
This
pattern
continued
throughout
the
remainder
of
the
study,
resulting
in
a
high
mortality
rate
(
75%
in
males
and
females)
in
the
highest
dose
animals
at
termination,
compared
with
approximately
30%
in
the
other
groups.
The
highest
dose
level
of
PHMB
resulted
in
noticeable
irritation
to
the
skin
of
both
males
and
females
immediately
after
application.
Erythema
and
some
clumping
of
the
growing
fur
were
noticed
during
the
first
few
weeks
and
after
the
4th
week;
hyperkeratosis
became
evident,
especially
in
males.
No
differences
were
apparent
between
the
controls
and
those
mice
receiving
0,
0.6
or
6.0
mg
PHMB
per
mouse
per
application.
A
significant
reduction
in
mean
body
weight
was
observed
for
both
male
and
female
animals
that
received
the
highest
dose
level.
There
were
no
overall
differences
in
food
consumption
between
the
control
and
treatment
groups.

D.
Adequacy
of
Dosing
for
Assessment
of
Carcinogenicity
Dosing
was
considered
to
be
excessive
at
the
high
dose
(
750
mg/
kg/
day
of
20%
PHMB
equivalent)
in
both
sexes
due
to
increased
mortality
(
78%,
high
dose,
versus
33%
and
28%
in
the
male
and
female
controls,
respectively)
and
decreased
body
weight
gain.
Overall
body
weight
gain
decreases
of
45%
and
17%
were
seen
in
males
and
females,
respectively.
No
treatment­
related
effects
were
noted
at
the
mid­
dose
level
of
150
mg/
kg/
day
of
20%
PHMB
equivalent
(
NOAEL).

IV.
TOXICOLOGY
1.
Metabolism
Bioavailability
of
PHMB
was
investigated
in
male
and
female
Sprague­
Dawley
rats,
fed
diets
of
200ppm
or
2000ppm
(
10
and
100
mg/
kg
nominal
dose)
for
fourteen
days
followed
by
a
single
radiolabelled
dose
of
either
0.08
mg/
kg
or
0.8
mg/
kg
(
MRID
#'
s
43567001
and
43599901),
or
after
a
single
100
mg/
kg
dose
(
MRID
#'
s
00077926
and
00086363).
In
both
studies,
feces
represented
the
major
route
of
excretion
at
all
dose
levels,
comprising
greater
than
90%
of
the
administered
dose.
A
similar
excretion
in
feces
was
observed
in
bile­
cannulated
rats
after
a
single
radiolabelled
dose
of
20
mg/
kg
(
MRID
#
43567001).
Thus,
fecal
excretion
of
PHMB­
derived
radioactivity
represents
unabsorbed
test
material.
The
excretion
pattern
of
low,
mid,
and
high
molecular
weight
fractions
of
PHMB
was
similar.
Bioavailability
was
4.7%
and
3.9%
for
males
and
females,
respectively,
at
the
10
mg/
kg
dose,
and
3.0%
and
2.6%
for
males
and
females,
respectively,
at
the
100
mg/
kg
dose.
Tissue
distribution
in
rats
given
10
mg/
kg
PHMB
showed
concentrations
in
the
liver
and
kidney
of
male
rats
to
be
0.568
g/
g
and
0.499
g/
g,
respectively.
In
female
rats,
liver
and
kidney
concentrations
were
0.752
g/
g
and
0.807
g/
g
respectively.
As
a
percentage
of
the
dose,
liver
of
male
and
female
rats
contained
0.18%
and
0.19%
of
the
dose
respectively,
while
kidneys
contained
0.03%
and
0.04%
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
32
of
the
dose
respectively.
Metabolite
analysis
of
pooled
urine
from
rats
administered
a
low
molecular
weight
fraction
of
PHMB
at
20
mg/
kg
(
the
fraction
showing
the
greatest
absorption)
revealed
the
presence
of
more
than
one
metabolite,
but
identification
was
not
performed
due
to
the
small
amount
of
sample
available
for
analysis.

2.
Mutagenicity
In
a
battery
of
mutagenicity
assays,
PHMB
was
not
mutagenic
in
bacteria
or
clastogenic
in
cultured
human
lymphocytes.
There
was
also
no
evidence
of
in
vivo
clastogenicity
or
aneugenicity
in
mouse
bone
marrow
or
unscheduled
DNA
synthesis
in
rat
liver.
No
mutagenic
studies
were
found
in
the
open
literature.
The
following
guideline
studies
were
acceptable
and
satisfy
the
pre­
1991
requirements
for
mutagenicity
testing.

In
two
independently
performed
microbial
gene
mutation
assays
(
MRID
No.
41687004),
Salmonella
typhimurium
strains
TA1535,
TA1537,
TA1538,
TA98,
and
TA100
were
exposed
to
0.32
­
500
µ
g/
plate
Vantocil
IB
(
19.6%
a.
i.)
in
the
absence
or
presence
of
S9
activation.
Additional
testing
was
carried
out
using
comparable
doses
with
and
without
S9
in
TA1537
and
TA98.
The
S9
fraction
was
derived
from
Aroclor
1254­
induced
rat
livers
and
the
test
material
was
delivered
to
the
test
system
in
dimethyl
sulfoxide.
The
utility
of
testing
a
bactericidal
agent
in
a
microbial
mutation
assay
is
questionable.
Nevertheless,
cytotoxicity
was
observed
for
the
majority
of
strains
at
$
200
:
g/
plate
+/­
S9.
All
strains
responded
in
the
expected
manner
to
the
nonactivated
and
S9­
activated
positive
controls.
There
was,
however,
no
evidence
that
Ventocil
IB
induced
a
mutagenic
response
in
any
strain
at
any
nonactivated
or
S9­
activated
dose.

In
an
in
vitro
mammalian
cell
cytogenetic
assay
(
MRID
No.
41404501/
42149905),
human
lymphocytes
derived
from
male
and
female
donors
were
exposed
to
Vantocil
IB
(
19.6%
a.
i.
in
water)
doses
of
5,
25,
or
50
µ
g/
mL
without
S9
activation
(
both
donors)
and
levels
of
25,
100
or
187.5
µ
g/
mL
+
S9
(
male
donor)
or
at
25,
100
or
250
µ
g/
mL
(
female
donor)
with
S9
activation
for
approximately
2.5
­
3.5
hours.
The
S9
liver
homogenate
was
derived
from
Aroclor
1254
induced
Sprague­
Dawley
rat
livers.
The
test
material
was
delivered
to
the
test
system
in
physiological
saline.
A
50%
reduction
in
mitotic
index
occurred
at
50
µ
g/
mL
­
S9
(
both
donors)
and
at
100
µ
g/
mL
+
S0
(
male
donor)
or
at
250
µ
g/
mL
+
S9
(
female
donor).
The
positive
controls
induced
the
expected
high
yield
of
chromosome
aberrations
in
the
lymphocytes
derived
from
the
male
and
female
donors.
There
was,
however,
no
evidence
that
Vantocil
IB
induced
a
clastogenic
effect.
This
study
is
classified
as
Acceptable
and
satisfies
the
guideline
requirement
for
an
in
vitro
cytogenetic
assay.

In
a
mouse
micronucleus
assay
(
MRID
No.
41096901/
41404503),
groups
of
five
male
and
five
female
C57BL/
6JfCD­
1/
Alpk
mice
received
single
oral
gavage
administrations
of
250
or
400
mg/
kg
Vantocil
IB
(
19.6%
a.
i.)
prepared
in
deionized
water.
Bone
marrow
cells
from
mice
in
the
highdose
group
were
examined
for
the
incidence
of
micronucleated
polychromatic
erythrocytes
(
MPEs).
Low­
dose
animals
were
sacrificed
at
24
hours.
Two
animals
receiving
400
mg/
kg
died
prior
to
the
scheduled
sacrifice.
There
was
also
clear
evidence
of
target
cell
cytotoxicity
in
the
high­
dose
males
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
33
and
females
at
all
sacrifice
intervals.
The
positive
control
induced
the
expected
high
yield
of
MPEs
in
males
and
females.
Vantocil
IB
did
not,
however,
induce
a
clastogenic
or
aneugenic
effect
in
either
sex
at
any
dose
or
sacrifice
time.
The
study
is
classified
as
Acceptable
and
satisfies
the
requirements
for
FIFRA
Test
Guideline
84­
2
for
a
micronucleus
assay.

In
two
independently
performed
in
vivo/
in
vitro
unscheduled
DNA
synthesis
(
UDS)
assays
(
MRID
No.
41404502/
42149903),
groups
of
two
to
three
male
rats
were
administered
single
oral
gavage
doses
of
750
or
1500
mg/
kg
Vantocil
IB
(
19.6%)
prepared
in
deionized
water.
Animals
were
sacrificed
at
4
and
12
hours
post
treatment
and
recovered
hepatocytes
were
scored
for
UDS.
Clinical
toxicity
(
i.
e.,
excessive
salivation
and
subdued
nature)
was
observed
at
1500
mg/
kg;
higher
levels
were
lethal.
No
cytotoxicity
for
the
target
organ
was
seen
at
either
level.
The
positive
control
induced
the
expected
high
yield
of
hepatocytes
with
net
nuclear
grains.
There
was,
however,
no
evidence
that
the
Vantocil
IB
induced
a
genotoxic
response
at
either
dose
or
sacrifice
time.
This
study
is
classified
as
Acceptable
and
satisfies
the
guideline
requirement
for
a
UDS
assay
(
84­
4).

3.
Structure­
Activity
Relationship
No
appropriate
structural
analogs
could
be
located
for
comparison
purposes.
In
general,
with
the
exception
of
exposure
via
the
inhalation
route,
high
MW
polymers
are
not
expected
to
be
of
significant
carcinogenic
concern
because
of
poor
bioavailability.
PHMB
has
indeed
been
shown
to
have
low
bioavailability.
However,
SAR
consideration
suggests
that
there
is
a
likelihood
that
the
lower
MW
fractions
of
PHMB
may
be
metabolized
to
polyamines
in
analogy
to
the
metabolism
of
arginine
(
a
guanidine­
containing
amino
acid)
to
form
biogenic
polyamines
such
as
putrescine,
spermidine
and
spermine.
There
is
strong
evidence
that
these
polyamines
are
readily
taken
up
by
cells
and
may
play
an
important
role
in
promoting
skin
and
colon
carcinogenesis
by
serving
as
ingredients
to
support
neoplastic
growth.
The
metabolism
study
submitted
by
the
company
showed
that
low
MW
fractions
of
PHMB
can
indeed
be
metabolized
in
the
rat;
however,
the
metabolites
have
not
yet
been
identified.
It
would
be
important
to
carry
out
the
identification
study.
If
the
metabolites
can
be
identified
to
be
polyamines,
they
could
provide
a
potential
mode
of
action
to
explain
the
carcinogenicity
of
PHMB.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
34
4.
Subchronic
and
Chronic
Toxicity
a)
Subchronic
Toxicity
Rats
In
a
90­
day
rat
oral
toxicity
study,
young
adult
specific
pathogen
free
(
S.
P.
F)
Wister
rats
(
25M
+
25
F)
received
levels
of
0,
2500,
and
5000
ppm
(
0,
310,
620
mg/
kg/
day)
of
poly(
hexamethylenebiguanide)
hydrochloride
(
25%)
for
90
days
in
the
diet.
At
the
end
of
the
90­
day
test
period
all
animals
were
killed
with
chloroform,
and
an
immediate
postmortem
examination
was
made.
All
animals
survived
the
90­
day
test
period.
There
were
no
specific
adverse
effects
of
the
compound.
Food
consumption
was
comparable
for
the
test
groups
and
controls.
Body
weight
was
moderately
reduced
in
males
(
13.2%
less
than
controls),
and
females
(
16.6%
less
than
controls)
fed
the
compounds
at
the
highest
dietary
level
(
5000
ppm).
No
abnormalities
in
hematological
parameters
were
observed.
No
gross
abnormalities
were
observed.
No
remarkable
change
in
organ/
body
weight
ratios
was
detected.
Microscopic
evaluation
revealed
that
the
liver
of
some
females
given
the
compound
at
a
level
of
5000
ppm
showed
an
unusual
degree
of
iron
pigment
both
within
the
liver
cells
and
kupffer
cells.
Although
the
report
states
that
no
iron
pigments
were
seen
in
animals
fed
2500
ppm
test
material
in
the
diet,
the
study
does
not
include
detailed
histopathological
results
of
the
2500
ppm
animals.
Therefore,
the
NOAEL
cannot
be
determined
in
this
study.

Dogs
In
a
90­
day
dog
oral
toxicity
study,
three
groups
of
Beagle
dogs
(
4M
+
4F
per
group),
12.4
­
14.6
kg
initial
body
weight,
received
poly(
hexamethylenebiguanide)
hydrochloride
(
25%)
at
dietary
levels
of
0,
5500
and
11,000
ppm
(
0,
137
or
275
mg/
kg/
day)
ad
libitum
for
90
days.
General
observation,
food
consumption,
body
weight
and
the
following
clinico­
pathological
studies
were
performed:
hemoglobin,
packed
cell
volume
(
hematocrit),
leucocyte
count
(
total),
leucocyte
count
(
differential),
blood
urea,
serum
alkaline
phosphatase,
BSP
(
liver
function
test)
and
urine
analyses
(
pH,
specific
gravity,
glucose,
protein,
bilirubin,
and
microscopy
of
centrifuge
deposits).
At
the
end
of
the
test
period
the
animals
were
killed
with
an
overdose
of
pentobarbitone
administered
intravenously.
A
full
postmortem
examination
and
microscopic
examination
were
taken
from
the
following:
brain
(
cerebrum,
cerebellum
and
pons),
spinal
cord,
pituitary,
submaxillary
gland,
thyroid,
thymus,
heart,
lung,
aorta,
stomach,
duodenum,
ileum,
colon,
liver,
spleen,
kidney,
bladder,
adrenal,
ovary
and
uterus
or
testis
and
epididymis,
and
sciatic
nerves.

Both
treated
and
control
animals
maintained
an
excellent
condition
throughout
and
no
adverse
effects
were
noted.
No
food
consumption
data
was
submitted.
Mean
body
weights
were
comparable
between
control
and
treated
animals
except
for
the
11,000
ppm
female
dogs
which
gained
significantly
less
total
weight
than
the
control
female
dogs.
Results
of
the
hematological
parameters
were
unremarkable.
Clinical
function
tests
showed
no
difference
in
retention
of
BSP
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
35
attributable
to
test
material.
Urine
analyses
did
not
appear
to
be
influenced
by
treatment.
Organ/
body
weight
ratios
showed
no
significant
variation
from
the
normal
as
a
result
of
treatment.
No
gross
pathology
attributable
to
test
material
was
detected.
Microscopic
examination
revealed
slight
hemosiderons
in
2
out
of
4
males
at
11,000
ppm.
No
other
microscopic
abnormalities
attributable
to
treatment
were
present
at
either
dose
level.

The
NOAEL
for
this
90­
day
dog
feeding
study
appears
to
be
5500
ppm
(
low
dose).
The
high
dose
treatment
caused
less
total
weight
gain
in
female
dogs
and
slight
hemosiderons
in
2
out
of
4
male
dogs.

b)
Chronic
Toxicity
Dogs
In
a
chronic
toxicity
study
(
MRID
#
43620501),
poly(
hexamethylenebiguanide)
hydrochloride
(
25%)
was
administered
to
groups
of
4
male
and
female
Beagle
dogs
in
the
diet
initially
at
dose
levels
of
0,
300,
1500,
and
4500
ppm
(
0,
7.5,
37.5,
and
112.5
mg/
kg/
day
nominal
dose)
for
one
year.
Following
an
unexpectedly
severe
reaction
in
3
of
4
males
at
4500
ppm
(
scrotal
skin
lesions),
the
high
dose
was
discontinued
on
week
9
or
10,
reduced
to
3000
ppm
(
75
mg/
kg/
day),
and
then
recommenced
on
week
11
or
12.
Up
to
and
including
the
3000
ppm
dose,
there
were
no
consistent
effects
of
PHMB
on
body
weight,
weight
gain,
food
consumption,
or
hematological
parameters.
Plasma
alanine
aminotransferase
(
ALT)
activity
was
significantly
increased
in
male
and
female
dogs
at
the
3000
ppm
dose
level
beginning
at
week
8,
but
there
was
variability
in
the
response,
and
only
one
male
dog
was
available
for
measurement
after
week
10.
Testes
weight
was
decreased
29%
and
32%
for
the
left
and
right
testis
of
high
dose
male
dogs,
and
testicular
tubular
degeneration
was
observed
in
the
surviving
male
dog
as
well
as
in
one
dog
sacrificed
intercurrently.
Liver
weight
in
high
dose
male
dogs
was
decreased
14%,
and
microscopic
changes
of
the
liver
were
also
observed
in
male
dogs
at
the
high
dose.
In
one
female
dog
at
the
high
dose,
significant
clinical
signs
(
decreased
activity,
stiff/
splayed
gait,
slight
tremors)
were
observed
which
were
not
reversible.
In
addition,
plasma
alanine
aminotransferase
was
increased
almost
10­
fold
over
the
pre­
treatment
level
by
week
35
of
treatment.
Plasma
aspartate
aminotransferase
in
this
dog
was
almost
doubled
by
week
35
of
treatment.
Marked
dermatitis
of
the
limbs
was
also
observed
in
this
dog.
The
LOAEL
is
3000
ppm
(
75
mg/
kg/
day)
for
male
and
female
dogs,
based
on
changes
in
testis
and
liver
weight
and
microscopic
observations
in
male
dogs,
and
based
on
clinical
signs
of
toxicity
and
clinical
chemistry
alterations
in
the
female
dog.
The
NOAEL
is
1500
ppm
(
37.5
mg/
kg/
day)
for
male
and
female
dogs.

This
study
is
classified
as
acceptable
and
satisfies
the
guideline
requirement
[
OPPTS
870.4100;
OPP
§
83­
1]
for
a
chronic
oral
toxicity
study
in
dogs.
5.
Mode
of
Action
Studies
There
are
no
mode
of
action
studies
available
at
this
time.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
36
V.
COMMITTEE'S
ASSESSMENT
OF
THE
WEIGHT­
OF­
THE­
EVIDENCE
1.
Carcinogenicity
The
CARC
concluded
that
PHMB
showed
evidence
of
carcinogenicity
based
on
the
following:

Rat
­
Oral
There
was
no
treatment­
related
increase
in
any
tumors
in
male
Wistar
rats.

Vascular
Tumors:
Female
Wistar
rats
from
the
PWG
diagnoses
had
significant
increasing
trends
in
liver
hemangiomas
and
liver
hemangiosarcomas,
both
at
p<
0.05.
There
was
also
a
significant
increasing
trend
for
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined,
at
p
<
0.01.
There
were
significant
differences
in
the
pair­
wise
comparisons
of
the
2000
ppm
dose
group
with
the
controls
for
liver
hemangiomas
and
hemangiomas
and/
or
hemangiosarcomas
at
all
sites
combined,
both
at
p
<
0.05.
The
incidence
of
hemangiomas
and
hemangiosarcomas
for
all
sites
combined
was
2/
42
(
5%),
1/
42
(
2%),
3/
40
(
8%),
and
6/
35
(
17%)
for
the
0,
200,
600,
and
2000
ppm
dose
groups,
respectively.
Historical
control
data
regarding
hemangiosarcomas
shows
the
range
of
hemangiosarcomas
by
tissue
in
rats
was
0­
1.9%.
The
CARC
considered
the
tumor
response
at
the
high
dose
(
17%
for
the
combined
re­
read
of
liver
and
original
diagnoses
at
other
sites)
to
be
treatment­
related
and
driven
by
the
increase
in
hemangiomas
when
all
sites
are
combined.

Adequacy
of
Dosing:
The
CARC
concluded
that
dosing
at
the
highest
dose
(
2000
ppm)
was
considered
to
be
adequate,
but
not
excessive,
for
both
male
and
female
rats.
In
high
dose
females,
this
was
based
on
increased
mortality
and
a
reduction
in
body
weight
of
5­
8%
throughout
the
study.
In
high­
dose
males,
body
weights
were
significantly
reduced
(
4­
6%)
through
week
79.
Food
utilization
(
g
growth/
100
g
feed)
for
the
first
12
weeks
decreased
significantly
(
97­
8%)
in
both
sexes
at
2000
ppm.

Mouse
­
Oral
Vascular
Tumors:
Male
C57B1/
10J
f
CD­
1/
Alpk
mice
from
the
PWG
diagnoses
had
significant
increasing
trends
in
hemangiomas,
hemangiosarcomas,
and
combined
hemangiomas
and
hemangiosarcomas,
all
at
p<
0.01.
There
were
also
significant
differences
in
the
pair­
wise
comparisons
of
the
4000
ppm
dose
group
with
the
controls,
for
hemangiomas
(
p<
0.01),
hemangiosarcomas
(
p<
0.05),
and
combined
hemangiomas
and/
or
hemangiosarcomas
(
p<
0.01).
The
incidence
of
hemangiomas
was
2/
55,
3/
55,
4/
55,
11/
53,
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
The
incidence
of
hemangiosarcomas
was
5/
55,
4/
55,
6/
55,
and
12/
53
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
The
incidence
of
combined
hemagiomas
and
hemangiosarcomas
was
6/
55,
6/
55,
9/
55,
and
20/
53
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
For
hemangiosarcomas,
the
tumor
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
37
incidence
in
the
4000
ppm
dose
group
(
23%)
exceeded
the
historical
control
range
(
1.8­
18.3%).
The
CARC
considered
the
increase
in
vascular
tumors
(
hemangiomas,
hemangiosarcomas,
and
combined)
at
the
high
dose
(
which
is
considered
to
be
excessive)
to
be
treatment­
related.
Although
not
statistically
significant,
the
tumor
response
(
9/
55
[
16%]
for
the
combined
hemangiomas
and
hemangiosarcomas)
in
mice
at
the
mid­
dose
of
1200
ppm
is
considered
treatment­
related
since
this
tumor
type
was
also
seen
in
female
mice
(
orally
and
dermally)
and
female
rats.

Vascular
Tumors:
Female
C57B1/
10J
f
CD­
1/
Alpk
mice
from
the
PWG
diagnoses
had
significant
increasing
trends
in
hemangiomas
(
p<
0.05),
hemangiosarcomas
(
p<
0.05),
and
combined
hemangiomas
and
hemangiosarcomas
(
p<
0.01).
There
was
also
a
significant
difference
(
p<
0.05)
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls,
for
combined
hemangiomas
and/
or
hemangiosarcomas.
The
incidence
of
combined
hemangiomas
and/
or
hemangiosarcomas
was
8/
54
(
15%),
5/
53
(
9%),
7/
54
(
13%),
and
15/
49
(
31%)
for
the
0,
400,
1200,
and
4000
ppm
dose
groups,
respectively.
For
hemangiosarcomas,
the
tumor
incidence
in
the
4000
ppm
dose
group
(
31%)
exceeded
the
historical
control
range
(
0­
10.9%).
There
are
no
historical
control
data
for
hemangiomas
by
tissue.
The
CARC
considered
the
combined
vascular
tumors
at
the
high
dose
(
which
is
considered
to
be
excessive)
to
be
treatment­
related.
There
were
no
increases
in
these
tumors
at
doses
which
were
not
considered
excessive.

Rectal­
Anal
Junction
Tumors:
In
male
mice
there
was
a
significant
increasing
trend
at
p
<
0.01
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls
at
p
<
0.05,
for
rectal­
anal
junction
squamous
cell
carcinomas.
The
incidence
of
rectal­
anal
junction
tumors
was
0/
45,
0/
45,
0/
45,
and
5/
48
for
the
0,
400,
1200
and
4000
ppm
dose
groups,
respectively.
In
female
mice
there
was
a
significant
increasing
trend
at
p<
0.01,
and
a
significant
difference
in
the
pair­
wise
comparison
of
the
4000
ppm
dose
group
with
the
controls
at
p<
0.01,
for
rectal­
anal
junction
squamous
cell
carcinomas.
The
incidence
of
rectal
anal
junction
tumors
was
0/
42,
0/
43,
0/
43,
8/
28
for
the
0,
400,
1200
and
4000
ppm
dose
groups,
respectively.
The
CARC
considered
the
carcinomas
at
the
high
dose
(
which
is
considered
to
be
excessive)
in
both
males
and
females
to
be
treatment­
related
but
suggestive
of
a
local
irritant
effect.
These
tumors
did
not
contribute
to
the
CARC's
weight­
of­
the­
evidence
analyses.

Adequacy
of
Dosing
Dosing
was
considered
to
be
excessive
at
the
high
dose
of
4000
ppm
based
on
decreased
overall
body
weight
gains
of
50%
in
males
and
32%
in
females
at
termination.
In
addition,
decreased
body
weight
gains
of
33%
in
males
and
19%
in
females
were
seen
at
13
weeks.
The
study
indicated
that
the
animals
at
the
4000
ppm
dose
group
had
increased
food
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
38
consumption
(
p<
0.05
or
0.01)
from
approximately
week
12
through
termination
in
males
(
87­
29%)
and
females
(
87­
26%).

Dosing
was
considered
to
be
adequate,
but
not
excessive,
in
both
males
and
females
at
the
mid­
dose
of
1200
ppm
based
on
decreased
overall
body
weight
gains
(
males,
7%;
females,
2%),
increases
in
hematology
parameters,
and
non­
neoplastic
histopathological
changes
in
the
gall
bladder,
liver,
and
rectal­
anal
junction.

Mouse
­
Dermal
Vascular
Tumors:
Female
Alderley
Park
mice
had
significant
increasing
trends
in
liver
angiosarcomas
(
p
<
0.01)
and
vascular
tumors
from
all
sites
combined
(
p
<
0.05).
There
was
also
a
significant
difference
in
the
pair­
wise
comparison
of
the
30
mg
dose
group
with
the
controls
for
liver
angiosarcomas
at
p
<
0.05.
The
incidence
of
liver
angiosarcomas
was
0/
41,
0/
45,
0/
40,
and
2/
35
for
the
0,
0.6,
6.0,
and
30
mg
dose
groups,
respectively.
The
CARC
considered
the
tumor
increase
at
the
high
dose
to
be
equivocal
since
the
tumors
were
only
seen
at
an
excessive
dose.
Vascular
tumors
were
not
seen
at
lower
doses.

There
was
no
treatment­
related
increase
in
any
tumors
in
male
Alderley
Park
mice.

Adequacy
of
Dosing
Dosing
was
considered
to
be
excessive
at
the
high
dose
(
750
mg/
kg/
day
of
20%
PHMB
equivalent)
in
both
sexes
due
to
increased
mortality
(
78%,
high
dose,
versus
33%
and
28%
in
the
male
and
female
controls,
respectively)
and
decreased
body
weight
gain.
Overall
body
weight
gain
decreases
of
45%
and
17%
were
seen
in
males
and
females,
respectively.
No
treatment­
related
effects
were
noted
at
the
mid­
dose
level
of
150
mg/
kg/
day
of
20%
PHMB
equivalent
(
NOAEL).

2.
Mutagenicity
There
is
no
mutagenicity
concern.

3.
Structure
Activity
Relationship
No
appropriate
structural
analogs
could
be
located
for
comparison
purposes.
In
general,
with
the
exception
of
exposure
via
the
inhalation
route,
high
molecular
weight
(
MW)
polymers
are
not
expected
to
be
of
significant
carcinogenic
concern
because
of
poor
bioavailability.
PHMB
has
indeed
been
shown
to
have
low
bioavailability.
However,
SAR
consideration
suggests
that
there
is
a
likelihood
that
the
lower
MW
fractions
of
PHMB
may
be
metabolized
to
polyamines
in
analogy
to
the
metabolism
of
arginine
(
a
guanidine­
containing
amino
acid)
to
form
biogenic
polyamines
such
as
putrescine,
spermidine
and
spermine.
There
is
strong
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
39
evidence
that
these
polyamines
are
readily
taken
up
by
cells
and
may
play
an
important
role
in
promoting
skin
and
colon
carcinogenesis
by
serving
as
ingredients
to
support
neoplastic
growth.
A
metabolism
study
submitted
by
the
company
showed
that
low
MW
fractions
of
PHMB
can
indeed
be
metabolized
in
the
rat;
however,
the
metabolites
have
not
yet
been
identified.

4.
Mode
of
Action
No
mode
of
action
studies
were
available.

VI.
CLASSIFICATION
OF
CARCINOGENIC
POTENTIAL
In
accordance
with
the
EPA
Draft
Guidelines
for
Carcinogen
Risk
Assessment
(
July
1999),
the
CARC
classified
PHMB
into
the
category
"
Suggestive
Evidence
of
Carcinogenicity,
but
Not
Sufficient
to
Assess
Human
Carcinogenic
Potential"
by
the
oral
and
dermal
routes.
The
weightof
the­
evidence
considerations
for
this
classification
are
as
follows:

(
i)
A
treatment­
related
statistically
significant
increase
(
trend
and
pair­
wise)
in
vascular
tumors
(
mainly
benign)
was
seen
in
female
rats
at
an
oral
dose
that
was
considered
to
be
adequate,
but
not
excessive.
This
was
considered
as
the
strongest
evidence
in
the
CARC's
evaluation
of
PHMB.

(
ii)
Oral
exposure
to
male
and
female
mice
also
resulted
in
treatment­
related
vascular
tumors
seen
at
an
excessive
dose.
However,
at
the
next
highest
dose
level,
which
was
considered
adequate
but
not
excessive,
there
was
a
slight
but
not
statistically
significant
increase
in
this
same
tumor
which
added
to
the
CARC's
concern
for
this
tumor
type.

(
iii)
It
is
noted
that
dermal
exposure
to
female
mice
resulted
in
an
equivocal
increase
in
vascular
tumors
seen
at
only
an
excessive
dose.

(
iv)
No
treatment­
related
increase
in
any
tumors
was
seen
in
male
rats
via
the
oral
route
or
in
male
mice
via
the
dermal
route
of
exposure.

VII.
QUANTIFICATION
OF
CARCINOGENIC
POTENTIAL
The
quantification
of
human
cancer
risk
is
not
required.
PHMB
CANCER
ASSESSMENT
DOCUMENT
FINAL
REPORT
40
VIII.
BIBLIOGRAPHY
MRID
No.
CITATION
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MJ.
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­­­­­­­­­­­­
Brunsman,
L.
L.
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Qualitative
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