Document ID: EPA-HQ-OPP-2002-0233-0010
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2002-09-13T04:00Z

[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
1
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
Reviewer:
Esther
Seto
,
Date
January
21,
2002
Peer
Review:
Ibrahim
Barsoum,
PhD
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
U.
S.
Environmental
Protection
Agency
_______________

STUDY
TYPE:
Acute
Pulmonary
Toxicity
/
Pathogenicity
PMRA
DATA
CODE
M4.
2.
3
USEPA
OPPTS
885.3150
TEST
MATERIAL
(PURITY):
Sporothrix
flocculosa,
pure
culture
(MPCA)
in
sterile
water
1.15
x
10
8
CFU/
mL
SYNONYMS:
Pseudozyma
flocculosa,
STF,
Stephanoascus
flocculosa
CITATION:
Harrington,
Kelly,
A.
(June
19,
1997).
"Acute
Intratracheal
Infectivity
Testing
of
Sporothrix
flocculosa,
a
Fungal
Pesticide"
IIT
Research
Institute,
Chicago,
Illinois,
USA.
IITRI
Project
No.
L08641,
Study
No.
3
In­
Life
Study
Dates:
October
9,
1996
­
October
14,
1996
(toxicity
range­
finding
assay)
and
October
15.
1996
­
November
5,
1996
(infectivity
assay).
Unpublished.

SPONSOR:
Université
Laval
Québec,
Canada
G1K
7P4
EXECUTIVE
SUMMARY:
In
an
acute
pulmonary
toxicity/
infectivity
study,
groups
of
young
adult
CD
rats
(4/
sex/
scheduled
sacrifice
date)
were
exposed
by
the
intratracheal
route
to
an
undiluted
suspension
of
Sporothrix
flocculosa
(TS)
at
a
dose
of
3.
2
x
10
7
cfu/
animal
(in
0.
1
mL).
Animals
were
then
observed
for
up
to
14
days.
An
equal
number
of
young
adult
CD
rats
were
similarly
injected
with
heat­
killed
test
substance
(KTS).
An
undosed
naive
control
(NC)
group
consisting
of
4
rats/
sex
was
also
included
in
the
study.
Cage
side
observations
for
clinical
symptoms
was
performed
daily
and
animal
body
weights
and
food
consumption
were
monitored.

Designated
test
animals
from
the
TS
and
KTS
groups
were
sacrificed
on
days
0,
7
and
14
and
gross
necropsies
were
performed.
During
the
course
of
the
study,
5
TS­
dosed
and
10
KTS­
dosed
animals
died
prior
to
their
scheduled
sacrifice
dates.
These
animals
were
also
necropsied.
The
NC
group
of
animals
was
sacrificed
and
necropsied
at
the
end
of
the
14
day
study.
Infectivity
and
clearance
were
assessed
by
quantitatively
recovering
the
MPCA
from
the
blood,
lungs
and
lymph
nodes,
spleen,
kidneys,
liver,
heart,
stomach
and
small
intestine,
caecum
and
brain.

Laboured
respiration
was
observed
in
3/
12
TS­
dosed
male
rats,
1/
12
TS­
dosed
female
rats,
1/
12
KTSdosed
male
rats
and
4/
12
KTS­
dosed
female
rats.
The
posture
of
one
female
TS­
dosed
animal
was
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
2
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
hunched.
Rough
hair
coat
was
observed
in
3/
12
TS­
dosed
male
rats,
1/
12
KTS­
dosed
male
rats
and
4/
8
KTS­
dosed
female
rats.
One
rat
from
each
of
the
male
TS­
dosed,
male
KTS­
dosed
and
female
KTS­
dosed
groups
had
ocular
discharge.
Nasal
discharge
was
noted
in
2/
12
male
TS­
dosed
rats,
1/
12
KTS­
dosed
rat
and
2/
12
female
KTS­
dosed
rats.
One
male
TS­
dosed
rat
appeared
lethargic
on
day
4.
The
presence
or
absence
of
clinical
symptoms
were
not
indicative
of
spontaneous
deaths.

Due
to
the
large
number
of
spontaneous
deaths
(3/
12
male
TS­
dosed
rats,
6/
12
male
KTS­
dosed
rats,
2/
12
female
TS­
dosed
rats
and
4/
12
female
KTS­
dosed
rats)
and
a
number
of
missed
data
collections,
data
for
evaluating
body
weights,
food
consumption
and
relative
organ
weight
were
limited.
At
the
end
of
the
14day
long
study,
administration
of
S.
flocculosa
did
not
have
a
statistically
significant
effect
on
body
weight.
Analysis
of
daily
food
consumption
and
relative
organ
weights
was
skewed
as
it
was
not
determined
or
did
not
include
animals
that
died
prior
to
their
scheduled
sacrifice
dates.

At
necropsy,
lesions
and
enlargement
of
the
lung
were
observed
in
6/
12
male
TS­
dosed
rats,
5/
12
male
KTS­
dosed
rats,
4/
12
female
TS­
dosed
rats
and
6/
12
female
KTS­
dosed
rats.
Confluent
dark
areas
were
seen
in
the
kidneys
of
one
male
TS­
dosed
rat.
Lesions
and
enlargement
of
the
spleen
were
noted
in
1/
12
male
TS­
and
KTS­
dosed
rats
and
2/
12
female
KTS­
dosed
rats.
Liver
lesions
were
observed
in
one
animal
from
each
of
the
male
TS­
dosed,
male
KTS­
dosed
and
female
KTS­
dosed
groups.
These
necropsy
findings
were
considered
consistent
with
the
method
of
dosing
and
the
body's
normal
immunological
response
to
a
foreign
substance.

Sporothrix
flocculosa
was
detected
in
the
lungs
and
lymph
nodes
and
the
stomach
and
small
intestine
of
TS­
dosed
animals
only.
Counts
in
these
tissues
were
below
the
limit
of
detection
by
day
7.

Based
on
this
study,
S.
flocculosa
is
toxic,
but
not
infective
or
pathogenic,
at
the
dose
administered
when
introduced
by
the
intratracheal
route
to
male
and
female
CD
rats.
This
acute
pulmonary
study,
however,
is
classified
as
UNACCEPTABLE
due
to
major
deficiencies
in
the
collected
data
and
a
possible
dosing
error,
as
indicated
by
the
presence
of
the
MPCA
in
the
stomach
and
small
intestines
on
the
day
of
dosing.
A
subsequent
range­
finding
acute
pulmonary
toxicity
study
was
conducted
and
reviewed.

COMPLIANCE:
Signed
and
dated
GLP
compliance
and
Quality
Assurance
statements
were
provided.
An
unsigned
Data
Confidentiality
statement
was
included.

I.
MATERIALS
AND
METHODS
A.
MATERIALS:

1.
Test
Material:
Sporothrix
flocculosa
(produced
by
test
facility)
Description:
MPCA
Lot/
Batch
#:
UK34­
35
Purity:
toxicity
range­
finding
asay:
7.
5
x
10
8
cells/
mL
or
1.
15
x
10
8
cfu/
mL
infecitivity
assay:
6.
0
x
10
8
cells/
mL
or
3.
2
x
10
8
cfu/
mL
CAS
#:
n/
a
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
3
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
Test
substance
homogeneity
was
determined
by
withdrawing
aliquots
from
three
locations
(top,
centre
and
bottom)
of
a
10
mL
tube
containing
test
substance
in
ASTM
type
1
water.
Samples
from
the
three
locations
were
plated
in
triplicate
on
YM
plates.
Colonies
were
counted
after
incubation
for
three
days
at
25

C.
The
average
plate
count
of
the
triplicate
samples
taken
from
each
location
were
comparable,
indicating
that
the
test
substance
was
homogeneous
throughout
the
volume
of
the
tube.

2.
Sample
Preparation:
Sporothrix
flocculosa
(batch
number
UK34­
45)
was
provided
to
the
test
facility
in
the
form
of
two
petri
dishes
(P1
and
P2)
of
yeast
malt
agar
with
white
mould.
IITRI
then
inoculated
two
malt
agar
plates
(P3
and
P4)
with
a
colony
from
P1.
One
week
prior
to
dosing
in
the
toxicity
range­
finding
component
of
the
study,
300
mL
of
yeast
malt
broth
was
inoculated
with
test
substance
from
P4
and
incubated
at
room
temperature
for
seven
days.
On
the
day
of
dosing,
the
broth
culture
was
centrifuged
and
the
pellet
was
resuspended
in
5
mL
of
ASTM
type
1
water.
The
titre
of
the
resuspended
solution
was
determined
to
be
7.
5
x
10
8
cells/
mL
by
hemacytometer
count
(corresponding
to
1.
15
x
10
8
cfu/
mL
as
determined
by
plate
count).
This
solution
was
used
undiluted
for
dosing.
One
week
before
the
start
of
the
infectivity
assay,
300
mL
of
yeast
malt
broth
was
inoculated
with
test
substance
from
P4
and
incubated
at
room
temperature
for
seven
days.
On
the
day
of
dosing,
the
broth
culture
was
centrifuged
and
resuspended
in
7
mL
of
ASTM
type
1
water.
The
titre
of
the
resuspended
solution
was
determined
to
be
6.
0
x
10
8
cells/
mL
by
hemacytometer
count.
Half
of
the
solution
was
heat­
treated
for
20
minutes
at
approximately
82

C
to
render
the
active
ingredient
non­
viable
and
was
used
as
KTS
(killed
test
substance).
The
rest
of
the
solution
was
used
as
TS
(test
substance).
Both
KTS
and
TS
were
plated
to
verify
titre
and
inactivation,
respectively.
The
titre
of
the
TS
solution
was
3.2
x
10
8
cfu/
mL
and
the
titre
of
the
KTS
solution
was
0
cfu/
mL.

3.
Test
animals:
Species
/
Strain:
CD
Rat
Age/
weight
at
dosing:
approximately
6
weeks
of
age
males:
222.93
­
274.76
g
females:
160.07
­
214.38
g
Source:
Charles
River
Laboratories
Portage,
MI
Housing:
The
animals
were
housed
2/
cage
in
polypropylene
cages
with
hardwood
chip
bedding.
Animal
rooms
and
cages
were
cleaned
and
sanitized
prior
to
initiation
of
the
study
and
cages
were
cleaned
twice
weekly
thereafter.
Control
group
animals
(naive
control
and
killed
test
substance­
dosed
groups)
were
housed
in
one
room
while
test
substance­
dosed
groups
were
housed
in
a
second
room.
Diet:
Certified
Purina
Rodent
Chow
5002
(PMI
Feeds
Inc.,
St.
Louis,
MO)
was
provided
ad
libitum.
Water:
City
of
Chicago
tap
water
was
provided
ad
libitum.
Environmental
conditions:
Temperature:
Humidity:
Air
changes:
Photoperiod:
18
­
25

C
27
­
70
%
Not
reported
12
hrs
dark
/
12
hrs
light
Acclimation
period:
One
week
acclimation
period
for
animals
used
in
toxicity
range­
finding
assay.
Two
week
acclimation
period
for
animals
used
in
infectivity
assay.
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
4
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
B.
STUDY
DESIGN
and
METHODS:

1.
In
life
dates
­
October
9,
1996
­
October
14,
1996
(toxicity
range­
finding
assay)
October
15.
1996
­
November
5,
1996
(infectivity
assay)

2.
Animal
assignment
and
treatment
­
After
one
week
of
quarantine,
three
rats
per
sex
were
selected
randomly,
weighed
and
used
in
the
5­
day
preliminary
range­
finding
toxicity
assay.
Rats
were
anesthetized
with
ether
and
dosed
intratracheally
with
0.
1
mL
of
the
test
substance,
containing
1.
15
x
10
7
cfu,
prepared
for
the
toxicity
range­
finding
assay.
The
rats
were
observed
for
five
days
post
dosing.

For
the
infectivity
assay,
the
remaining
animals
were
weighed
one
week
later
and
assigned
to
treatment
groups
such
that
no
animal's
body
weight
varied
from
the
group
mean
body
weight
by
more
than
20%.
Treatment
groups
included
TS­
dosed
rats,
KTS­
dosed
rats
and
a
naive
control
(NC)
group
of
rats.
The
number
of
animals
and
the
sacrifice
time
for
each
of
the
test
groups
is
shown
in
Table
1.
The
rats
were
anesthetized
with
ether,
dosed
intratracheally
and
observed
for
14
days.

TABLE
1.
Treatment,
mortality/
animals
treated
Group
Number
Treatment
Scheduled
Sacrifice
Mortality
(#
dead/
total)

Males
Females
Combined
1
TS
b
0
0/
4
0/
4
0/
8
2
KTS
c
0
0/
4
0/
4
0/
8
3
TS
b
7
3/
4
a
2/
4
5/
8
4
KTS
c
7
4/
4
4/
4
8/
8
5
TS
b
14
0/
4
a
0/
4
0/
8
6
KTS
c
14
2/
4
0/
4
2/
8
7
NC
d
14
0/
4
0/
4
0/
8
a
one
male
rat
in
the
group
was
inadvertently
dosed
twice
b
dosed
with
0.
1
mL
of
TS
containing
3.
2
x
10
7
cfu
c
dosed
with
0.1
mL
of
KTS
containing
the
equivalent
of
3.2
x
10
7
cfu
of
heat­
killed
active
ingredient
d
naive
control
group
3.
Body
Weight
­
Animal
body
weights
were
determined
upon
receipt,
prior
to
randomization,
at
the
time
of
test
substance
dosing
(day
0)
and
weekly
thereafter.

4.
Food
Consumption
­
Food
consumption
was
monitored
when
food
was
replenished.
Average
daily
food
consumption
per
rat
was
calculated.

5.
Clinical
Observations
­
Cageside
observations
were
made
daily
during
the
toxicity
range­
finding
and
infectivity
assays.

6.
Necropsies
­
Necropsies
were
performed
upon
death
or
at
scheduled
sacrifice
times.
Organ
weights
[2000­
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2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
5
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
(relative
to
body
weights)
were
recorded.
Gross
observations
of
external
features
and
internal
organs
and
cavities
of
each
animal
were
made.

7.
Microbial
Enumeration
­
Samples
of
blood
(from
surviving
animals
only),
brain,
caecum,
kidney,
liver,
heart,
lung
and
lymph
nodes,
spleen,
stomach
and
small
intestines
were
aseptically
removed
and
placed
in
sterile
blending
bags
containing
0.
1%
peptone.
The
tissues
were
blended
and
aliquots
were
serially
diluted
in
0.
1%
peptone
and
plated
onto
YM
or
MA
(see
study
for
determination
sensitivity
of
detection).
The
plates
were
incubated
for
at
least
72
hours
at
approximately
25

C.
Counts
were
reported
as
viable
cfu/
mL
of
blood
or
cfu/
gram
of
tissue.

8.
Statistics
­
Mean
body
weights,
cumulative
body
weight
gains,
average
daily
food
consumption
and
organ
weights
(as
a
percentage
of
total
body
weight)
were
calculated
for
each
combination
of
sex
and
treatment
along
with
corresponding
standard
deviations.
Treatment
groups
were
compared
using
analysis
of
variance
(ANOVA)
followed,
where
appropriate,
by
Dunnett's
test.
A
p

0.05
was
considered
significant
for
all
statistical
analyses.

II.
RESULTS
AND
DISCUSSION:

A.
Mortality
­
None
of
the
three
male
and
three
female
rats
dosed
in
the
range­
finding
assay
died
during
the
5­
day
observation
period.

A
total
of
15
rats
(3/
8
male
TS­
dosed
rats,
6/
8
male
KTS­
dosed
rats,
2/
8
female
TS­
dosed
rats
and
4/
8
female
KTS­
dosed
rats)
died
on
days
2
and
3
of
the
infectivity
assay.
These
mortalities
are
summarized
in
Table
1.
The
male
rat
in
group
3
that
was
inadvertently
dosed
twice
was
one
of
the
rats
that
died.

The
LC50
of
S.
flocculosa
formalesis>
3.2
x
10
7
cfu/
animal.
for
females
is
>
3.
2
x
10
7
cfu/
animal
combined
is
>
3.
2
x
10
7
cfu/
animal.

Thehigh
spontaneousdeath
ratein
TS­
and
KTS­
dosedratsmay
havebeenpartiallydueto
thelargesize
of
S.
flocculosa.
The
higher
death
rate
of
KTS­
dosed
versus
TS­
dosed
rats
may
have
been
due
to
toxins
which
were
activated
by
the
autoclaving
step
used
to
produce
the
KTS.
Regardless
of
the
cause
of
deaths,
the
large
number
of
deaths
made
it
difficult
to
properly
assess
the
data
collected
in
this
study.

B.
Clinical
Observations
­
During
the
toxicity
range­
finding
assay,
one
female
exhibited
laboured
respiration
on
the
day
of
dosing.
No
other
adverse
clinical
signs
were
observed
in
the
other
animals
used
for
the
toxicity
range­
finding
assay.

Results
of
clinical
observations
of
the
test
animals
over
the
course
of
the
14­
day
infectivity
assay
are
summarized
in
Table2.
Theseresultsincludethoseofanimalswhich
weresacrificed
on
day0(
4
animals/
sex/
treatment
group).

TABLE
2.
Clinical
Observations
TS
(12
animals
/
sex)
KTS
(12
animals
/
sex)
NC
(12
animals
/
sex)

Observation
M
F
M
F
M
F
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
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Infectivity
and
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/
6
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flocculosa
/
STF]
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M4.
2.
3
/
USEPA
OPPTS
885.3150
founddead3
2
6
40
0
labouredrespiration3
1
1
40
0
hunchedposture0
1
0
00
0
roughhaircoat3
0
1
40
0
oculardischarge1
0
1
10
0
nasaldischarge2
0
1
20
0
lethargic1
0
0
00
0
no
observed
signs
7
11
11
7
4
4
Rough
hair
coat
and
laboured
respiration
was
first
observed
between
days
0
­
2
and
persisted
in
some
animals
up
until
day
11.
The
onset
of
hunched
posture,
nasal
discharge,
ocular
discharge
and
lethargy
occurred
between
days
2
­
4
and
persisted
for
only
one
to
two
days.

Of
the
15
rats
that
died,
only
6
exhibited
adverse
clinical
symptoms
prior
to
their
death,
and
not
all
of
the
rats
displaying
symptoms
died.

C.
Body
Weight
­
The
body
weights
of
a
number
of
animals
that
died
and
the
body
weights
of
whole
groups
of
animals
on
particular
days
were
inadvertently
not
measured.
These
omissions
in
body
weight
measurements
were
considered
a
major
deficiency.
Based
on
the
available
data,
mean
body
weights
and
body
weight
gains
are
summarized
in
Tables
3
and
4.
Analysis
of
the
limited
data
indicated
no
statistically
significant
differences
in
mean
body
weight
or
mean
body
weight
gain
between
groups
at
the
end
of
the
14day
observation
period.

Individual
body
weight
data
revealed
that
all
animals
that
died
prior
to
their
scheduled
sacrifice
dates
had
lost
a
significant
amount
of
weight
(35.
74
­
63.
05
g
for
TS­
dosed
rats;
30.
09
­
50.
26
g
for
KTS­
dosed
rats)
within
two
to
three
days
after
dosing.

Two
female
rats
in
the
NC
group
were
deprived
of
water
during
the
second
week
of
the
study
leading
to
a
loss
in
weight.
The
duration
of
time
for
which
these
animals
were
deprived
was
not
available.

TABLE
3.
Mean
Body
Weights
Body
Weight
(g)

Treatment
Group
Sex
Parameter
Day
0
Day
2
Day
3
Day
7
Day
14
TS
M
Mean
SD
N
261.63
12.79
12
200.62
7.67
2
191.21

1
267.1

1
317.96
48.17
4
KTS
M
Mean
SD
N
253.73
11.52
12
210.81
15.49
5

297.45
17.65
2
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
7
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
NC
M
Mean
SD
N
254.20
13.88
4

338.24
27.08
4
TS
F
Mean
SD
N
192.39
10.80
12
145.75

1

242.71
32.94
2
233.01
9.74
4
KTS
F
Mean
SD
N
192.13
13.74
12
157.03
2.01
2

225.32
21.93
4
NC
F
Mean
SD
N
191.33
9.72
4

190.96
50.39
4
­
data
not
available
due
to
spontaneous
deaths,
death
of
all
animals
in
group
or
missed
body
weight
measurements
TABLE
4.
Mean
Body
Weight
Gains
Total
Gain
(g)

Treatment
Group
Sex
Parameter
Day
0
­
14
TS
M
Mean
SD
N
68.13
29.52
4
KTS
M
Mean
SD
N
56.88
23.01
2
NC
M
Mean
SD
N
84.04
17.16
4
TS
F
Mean
SD
N
41.35
2.86
4
KTS
F
Mean
SD
N
30.18
15.27
4
NC
F
Mean
SD
N
­0.38
53.19
4
D.
Food
Consumption
­
Food
consumption
data
are
presented
in
Table
5.
Food
consumption
was
not
determined
for
animals
which
died
prior
to
their
scheduled
sacrifice
dates.
Analysis
of
the
limited
data
indicated
no
statistically
significant
differences
between
treatment
groups.

TABLE
5.
Average
Daily
Food
Consumption
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
8
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
Treatment
Group
Sex
Parameter
Average
Daily
Consumption
(g)

TS
M
Mean
SD
N
23.0
4.6
5
KTS
M
Mean
SD
N
20.6
1.6
4
NC
M
Mean
SD
N
25.9
1.6
4
TABLE
5.
Average
Daily
Food
Consumption
contd.

Treatment
Group
Sex
Parameter
Average
Daily
Consumption
(g)

TS
F
Mean
SD
N
16.3
2.1
6
KTS
F
Mean
SD
N
15.1
3.3
4
NC
F
Mean
SD
N
15.6
3.6
4
E.
Necropsy
Results
­
Lung
lesions
were
observed
in
TS­
and
KTS­
dosed
animals
and
in
both
animals
that
died
prior
to
their
scheduled
sacrifice
dates
and
those
that
survived
until
sacrifice.
Other
lesions
in
the
spleen,
liver
and
kidney
were
observed
primarily
in
the
animals
that
died.
Three
of
the
15
animals
that
died
exhibited
no
gross
lesions.
No
lesions
were
found
in
any
of
the
NC
animals
or
in
any
of
the
4/
sex
TS­
and
KTS­
dosed
animals
sacrificed
post­
dosing
on
day
0.
Necropsy
findings
are
summarized
in
Table
6.

TABLE
6.
Necropsy
Findings
TS(
12
animals/
sex)
KTS(
12
animals/
sex)
NC(4
animals/
sex)

Organ
­observation
M
FMFMF
Kidneys
­confluent
dark
1–––––
Liver
­confluent
dark
­diffuse
dark
red
­foci,
diffuse
dark
red
1
–
–
–
–
–
–
–
1
–
1
–
–
–
–
–
–
–
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
9
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
Lung
­cyst
­diffuse
red
lesion
­enlarged
­focus,
multiple
dark
red
­granular,
diffuse
dark
red
­mass
­pigmentation,
mottled
­single
red
lesion
­focus,
multiple
diffuse
1
1
2
–
–
1
–
1
–
–
1
1
–
–
2
–
–
–
1
–
1
–
1
–
1
–
1
2
–
3
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Spleen
­confluent
dark
­enlarged
­nodule,
multiple
1
–
–
–
–
–
–
1
–
–
1
1
–
–
–
–
–
–
No
gross
lesions
78
7
4
44
F.
Organ
Weights
­
The
effectsoftestsubstance
onrelative
organweightsaresummarizedinTables7
and
8.
Relative
organ
weights
were
not
calculated
for
1
KTS­
dosed
male,
1
TS­
dosed
female
and
2
KTSdosed
females
were
not
calculated
since
their
body
weights
were
inadvertently
not
taken
at
the
time
of
their
spontaneous
deaths.

Statistical
analysis
was
conducted
only
on
Day
14
data
as
relative
organ
weights
of
NC
rats
were
only
available
for
Day
14.
The
relative
weights
of
the
lungs
and
lymph
nodes
of
TS­
and
KTS­
dosed
male
rats
and
KTS­
dosed
female
rats
were
significantly
increased
compared
to
their
NC
counterparts.
The
KTSdosed
rats
also
had
significantly
increased
relative
spleen
weights
as
compared
to
NC
animals.

TABLE
7.
Relative
Organ
Weights
of
Male
Rats
Relative
Organ
Weight
(%)
a
Treatment
Group
Day
Parameter
Lungs
and
Lymph
Nodes
Spleen
Kidneys
Liver
Heart
Stomach
and
Small
Intestine
Caecum
Brain
TS
0
Mean
SD
N
0.588
0.0399
4
0.234
0.0231
4
1.041
0.0432
4
4.781
0.1789
4
0.497
0.0995
4
5.812
1.0454
4
2.246
0.2904
4
0.788
0.0495
4
KTS
0
Mean
SD
N
0.783
0.1431
4
0.268
0.0303
4
1.019
0.0695
4
5.217
0.1936
4
0.448
0.0297
4
5.954
1.4792
4
2.188
0.3856
4
0.768
0.0304
4
TS
2
Mean
SD
N
0.3288
0.3103
2
0.313
0.0568
2
1.298
0.1407
2
6.476
0.0102
2
0.555
0.2100
2
5.835
1.7558
2
0.861
0.0411
2
1.004
0.0183
2
KTS
b
2
Mean
SD
N
2.978
0.2469
5
0.323
0.0434
5
1.252
0.1407
5
6.085
0.6457
5
0.414
0.0360
5
3.136
1.3865
5
1.128
0.2886
5
0.910
0.0263
5
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
10
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
TS
3
Mean
SD
N
3.962
–
1
0.538
–
1
1.311
–
1
7.137
–
1
0.467
–
1
1.545
–
1
0.882
–
1
0.966
–
1
TS
7
Mean
SD
N
1.201
–
1
0.352
–
1
0.999
–
1
4.444
–
1
0.638
–
1
5.560
–
1
1.962
–
1
0.776
–
1
TS
14
Mean
SD
N
1.378*
0.4414
4
0.281
0.0324
4
0.903
0.0534
4
4.407
0.2441
4
0.396
0.0406
4
2.783
0.7500
4
2.637
0.4895
4
0.646
0.1204
4
KTS
14
Mean
SD
N
1.524*
0.5578
2
0.617*
0.3987
2
0.994
0.0628
2
4.678
0.7775
2
0.364
0.0047
2
3.544
0.4449
2
2.333
0.1315
2
0.698
0.0024
2
NC
14
Mean
SD
N
0.738
0.2227
4
0.199
0.0172
4
0.920
0.0448
4
4.778
0.3979
4
0.401
0.0345
4
3.339
0.1582
4
2.411
0.4032
4
0.578
0.0543
4
a
Relative
organ
weight
=
[absolute
organ
weight
(g)
/
body
weight
(g)]
x
100%
b
Mean
and
standard
deviation
calculated
for
only
5/
6
rats
which
died
on
day
2.
The
body
weight
of
the
6
th
animal
was
inadvertently
not
taken.
*statistically
significant
from
NC,
p

0.
05
(ANOVA
and
Dunnett's
test
performed
on
Day
14
data
only)

TABLE
8.
Relative
Organ
Weights
of
Female
Rats
Relative
Organ
Weight
(%)
a
Treatment
Group
Day
Parameter
Lungs
and
Lymph
Nodes
Spleen
Kidneys
Liver
Heart
Stomach
and
Small
Intestine
Caecum
Brain
TS
0
Mean
SD
N
0.686
0.0858
4
0.228
0.0329
4
0.960
0.0438
4
0.4271
0.5159
4
0.485
0.0486
4
5.218
0.5456
4
2.309
0.1835
4
0.994
0.0427
4
KTS
0
Mean
SD
N
0.854
0.0835
4
0.242
0.0339
4
0.870
0.0834
4
4.355
0.3931
4
0.416
0.0174
4
4.588
1.3688
4
2.293
0.4117
4
0.932
0.0908
4
TS
2
Mean
SD
N
3.862
–
1
0.297
–
1
1.260
–
1
5.777
–
1
0.428
–
1
3.683
–
1
1.164
–
1
1.215
–
1
KTS
2
Mean
SD
N
3.207
0.3977
2
0.274
0.0094
2
1.250
0.0822
2
5.959
0.1208
2
0.425
0.0355
2
4.916
3.2068
2
0.896
0.1083
2
1.154
0.0613
2
TS
3
Mean
SD
N
ND
b
ND
1
ND
ND
1
ND
ND
1
ND
ND
1
ND
ND
1
ND
ND
1
ND
ND
1
ND
ND
1
KTS
3
Mean
SD
N
ND
ND
2
ND
ND
2
ND
ND
2
ND
ND
2
ND
ND
2
ND
ND
2
ND
ND
2
ND
ND
2
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
11
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
TS
7
Mean
SD
N
1.247
0.2855
2
0.312
0.0065
2
0.825
0.2751
2
3.763
1.1773
2
0.380
0.0169
2
4.850
1.4324
2
1.661
0.0163
2
0.795
0.0829
2
TS
14
Mean
SD
N
1.271
0.1771
4
0.360
0.0802
4
0.896
0.0570
4
4.197
0.2994
4
0.441
0.1002
4
3.203
0.1360
4
2.345
0.2067
4
0.799
0.0947
4
KTS
14
Mean
SD
N
1.645*
0.4418
4
0.761*
0.3774
4
0.976
0.1646
4
4.865
0.7747
4
0.415
0.0561
4
3.284
0.4018
4
2.265
0.6521
4
0.832
0.1178
4
NC
14
Mean
SD
N
0.891
0.1105
4
0.218
0.0296
4
1.127
0.1010
4
4.370
0.3211
4
0.449
0.0684
4
5.175
2.2565
4
1.739
0.5391
4
1.006
0.2046
4
a
Relative
organ
weight
=
[absolute
organ
weight
(g)
/
body
weight
(g)]
x
100%
b
ND
­
not
determined;
body
weight
not
determined
*statistically
significant
from
NC,
p

0.
05
(ANOVA
and
Dunnett's
test
performed
on
Day
14
data
only)

G.
Microbial
Enumeration
­
Enumeration
of
the
test
substance
from
various
organs
and
blood
is
summarized
in
Tables
9
and
10.
No
test
substance
was
recovered
from
any
NC
or
KTS­
dosed
animals.
Viable
S.
flocculosa
was
recovered
only
from
the
lungs
and
lymph
nodes
(day
0)
and
the
stomach
and
small
intestines
(day
0
and
day
2)
from
TS­
dosed
animals.

TABLE
9.
Recovery
of
Sporothrix
flocculosa
from
Selected
Tissues
and
Blood
of
Male
Rats
Log
[(
cfu/
g
of
tissue)
+
1]
or
Log
[(
cfu/
mL
of
blood)
+
1]

Group
Day
Parameter
Blood
Lung
&
Lymph
Nodes
Spleen
Kidneys
Liver
Heart
Stomach
&
Small
Intestine
Caecum
Brain
TS
0
Mean
SD
GeoMn
b
N
BDL
a
BDL
BDL
4
7.239
0.322
1.73
x
10
7
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
4.320
0.667
2.09
x
10
4
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
KTS
0
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
TS
2
Mean
SD
GeoMn
N
ND
c
ND
ND
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
1.791
2.533
6.18
x
10
1
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
KTS
2
Mean
SD
GeoMn
N
ND
ND
ND
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
BDL
BDL
BDL
6
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
12
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
TS
3
Mean
SD
ND
ND
ND
1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL
GeoMn
N
1
TS
7
Mean
SD
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL
GeoMn
N
1
TS
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
KTS
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
NC
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
a
BDL
­
below
detection
limit
b
GeoMn
­
geometric
mean
of
cfu/
tissue
or
cfu/
mL
c
ND
­
not
done
TABLE
10.
Recovery
of
Sporothrix
flocculosa
from
Selected
Tissues
and
Blood
of
Female
Rats
Log
[(
cfu/
g
of
tissue)
+
1]
or
Log
[(
cfu/
mL
of
blood)
+
1]

Group
Day
Parameter
Blood
Lung
&
Lymph
Nodes
Spleen
Kidneys
Liver
Heart
Stomach
&
Small
Intestine
Caecum
Brain
TS
0
Mean
SD
GeoMn
b
N
BDL
a
BDL
BDL
4
7.014
0.354
1.03
x
10
7
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
3.867
0.553
7.35
x
10
3
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
KTS
0
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
TS
2
Mean
SD
ND
c
ND
ND
1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
3.202

1.59
x
10
3
1
BDL

1
BDL
GeoMn
N
1
KTS
2
Mean
SD
GeoMn
N
ND
ND
ND
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
13
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
TS
3
Mean
SD
ND
ND
ND
1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL

1
BDL
GeoMn
N
1
KTS
3
Mean
SD
GeoMn
N
ND
ND
ND
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
TS
7
Mean
SD
GeoMn
N
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
BDL
BDL
BDL
2
TS
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
KTS
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
NC
14
Mean
SD
GeoMn
N
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
BDL
BDL
BDL
4
a
BDL
­
below
detection
limit
b
GeoMn
­
geometric
mean
of
cfu/
tissue
or
cfu/
mL
c
ND
­
not
done
H.
Reviewer's
Conclusions
­
Among
the
animals
whose
scheduled
sacrifice
dates
were
on
days
7
or
14,
laboured
respiration
was
observed
in
3/
8
TS­
dosed
male
rats,
1/
8
TS­
dosed
female
rats,
1/
8
KTS­
dosed
male
rats
and
4/
8
KTS­
dosed
female
rats.
Laboured
respiration
was
first
noted
in
some
animals
on
day
2
and,
in
some
cases,
would
re­
occur
sporadically
up
to
day
11.
The
posture
of
one
female
TS­
dosed
animal
was
hunched
on
day
2.
Rough
hair
coat
was
observed
as
early
as
on
day
0
and
up
until
day
11
in
3/
8
TSdosed
male
rats,
1/
4
KTS­
dosed
male
rats
and
4/
8
KTS­
dosed
female
rats.
One
rat
from
each
of
the
male
TS­
dosed,
male
KTS­
dosed
and
female
KTS­
dosed
groups
had
ocular
discharge.
Nasal
discharge
was
noted
in
2/
8
male
TS­
dosed
rats,
1/
8
KTS­
dosed
rat
and
2/
8
female
KTS­
dosed
rats.
Ocular
and
nasal
discharge
occurred
between
days
2
­
3.
One
male
TS­
dosed
rat
appeared
lethargic
on
day
4.
The
presence
or
absence
of
clinical
symptoms
were
not
indicative
of
spontaneous
deaths.

Due
to
the
large
number
of
spontaneous
deaths
(3/
8
male
TS­
dosed
rats,
6/
8
male
KTS­
dosed
rats,
2/
8
female
TS­
dosed
rats
and
4/
8
female
KTS­
dosed
rats)
and
a
number
of
missed
data
collections,
data
for
evaluating
body
weights,
food
consumption
and
relative
organ
weight
were
limited.
At
the
end
of
the
14day
long
study,
administration
of
S.
flocculosa
did
not
have
a
statistically
significant
effect
on
body
weight.
Analysis
of
daily
food
consumption
and
relative
organ
weights
was
skewed
as
it
was
not
determined
or
did
not
include
animals
that
died
prior
to
their
scheduled
sacrifice
dates.

At
necropsy,
lesions
and
enlargement
of
the
lung
were
observed
in
6/
12
male
TS­
dosed
rats,
5/
12
male
KTS­
dosed
rats,
4/
12
female
TS­
dosed
rats
and
6/
12
female
KTS­
dosed
rats.
Confluent
dark
areas
were
seen
in
the
kidneys
of
one
male
TS­
dosed
rat.
Lesions
and
enlargement
of
the
spleen
were
noted
in
1/
12
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
14
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
male
TS­
and
KTS­
dosed
rats
and
2/
12
female
KTS­
dosed
rats.
Lung
lesions
were
observed
in
one
animal
from
each
of
the
male
TS­
dosed,
male
KTS­
dosed
and
female
KTS­
dosed
groups.
These
necropsy
findings
were
considered
consistent
with
the
method
of
dosing
and
the
body's
normal
immunological
response
to
a
foreign
substance.

Sporothrix
flocculosa
was
detected
in
the
lungs
and
lymph
nodes
and
the
stomach
and
small
intestine
of
TS­
dosed
animals
only.
Counts
in
these
tissues
were
below
the
limit
of
detection
by
day
7.

A
number
of
explanations
could
be
provided
for
the
significant
number
of
deaths
of
both
TS­
and
KTSdosed
Both
viable
and
heat­
killed
test
substance
may
have
been
toxic.
Clinical
observations
(i.
e.,
laboured
breathing)
and
gross
necropsy
findings
(i.
e.,
lung
abnormalities)
may
have
been
the
result
of
pulmonary
blockage
following
intratracheal
administration
of
either
TS
or
KTS.
The
higher
death
rate
of
KTS­
dosed
versus
TS­
dosed
rats
may
have
been
due
to
toxins
which
were
activated
by
the
autoclaving
step
used
to
produce
the
KTS.
The
relatively
large
size
of
the
test
substance
may
have
contributed
to
the
deaths.
An
alternative
explanation
may
be
a
possible
problem
with
the
dosing
technique,
as
indicated
by
the
presence
of
S.
flocculosa
in
the
stomach
and
small
intestines
on
day
0,
since
none
of
the
undosed
NC
group
animals
died
prior
to
their
scheduled
sacrifice
dates.
The
probability
of
a
dosing
error
was
reinforced
by
a
subsequent
acute
pulmonary
range­
finding
study
(see
review
for
separate
Acute
Pulmonary
­
Range
Finding
Study)
in
which
no
deaths
were
observed
despite
a
higher
dose
of
TS.

Based
on
this
study,
S.
flocculosa
is
toxic,
but
not
infective
or
pathogenic,
at
the
dose
administered
when
introduced
by
the
intratracheal
route
to
male
and
female
CD
rats.
This
study,
however,
is
classified
as
unacceptable
due
to
major
deficiencies
in
the
collected
data.
According
to
USEPA
OPPTS
885.3150,
the
minimum
dose
level
is
10
8
cfu
per
animal.
Animals
in
this
study
were
only
dosed
with
3.
2
x
10
7
cfu.
Furthermore,
the
test
substance
used
in
this
study
was
not
the
technical
grade
active
ingredient
(as
recommended
by
USEPA
OPPTS
885.3150)
but
was
instead
prepared
by
the
test
facility
using
a
method
different
from
the
proposed
manufacturing
method.
The
applicant
has
agreed,
however,
to
upgraded
label
statements
requiring
a
respirator
for
all
workers
and
bystanders
during
times
of
potential
exposure.

I.
Deficiencies
­
In
addition
to
the
deficiencies
already
noted
above,
the
number
of
air
changes
was
not
reported.
This
additional
deficiency
is
not
considered
to
have
any
impact
on
the
interpretation
of
this
study.