Document ID: EPA-HQ-OPP-2002-0233-0013
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2002-09-13T04:00Z

[Sporodex
L
/
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Oral
Infectivity
and
Toxicity
Study
/1
[Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
2.
2
/
USEPA
OPPTS
885.3050
Reviewer:
Esther
Seto
,
Date
January
8,
2002
Peer
Review:
Ibrahim
Barsoum,
PhD
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
U.
S.
Environmental
Protection
Agency
_______________

STUDY
TYPE:
Acute
Oral
Toxicity
­
Rat
PMRA
DATA
CODE
M4.2.
2
/
USEPA
OPPTS
885.3050
TEST
MATERIAL
(PURITY):
Sporodex®
(WP
formulation)
­
1997
Formulation
5.6­
5.8
x
10
8
Pseudozyma
flocculosa
colony
forming
units
/
mL
SYNONYMS:
Pseudozyma
flocculosa
[STF],
Sporothrix
flocculosa,
Stephanoascus
flocculosa
CITATION:
Descôteaux,
Jean­
Paul.
(September
9,
1996).
"Report
of
an
Acute
Oral
Toxicity
/
Infectivity
Study
of
Sporodex®
Administered
by
Gavage
to
Fisher
344
Rats."
Institut
Armand­
Frappier,
Laval,
Québec,
Canada.
Study
Number
951890.
In­
Life
Study
Dates:
February
6­
7,
1996
­
February
27­
28,
1996.
Unpublished.

SPONSOR:
Plant
Products
Co.,
Ltd.
314
Orenda
Road
Brampton,
Ontario
Canada
EXECUTIVE
SUMMARY:
In
an
acute
oral
toxicity
study,
groups
of
fasted,
6
­
7
week
old
Fisher
344
rats
(12/
sex)
were
given
a
single
oral
dose
of
Sporodex
WP
in
USP
sterile
water
for
injection
at
doses
of
5.8
x
10
8
colony­
forming
units
(CFU)
per
animal
for
males
and
5.
6
x
10
8
CFU
per
animal
for
females.
The
animals
were
then
observed
for
a
period
of
up
to
21
days
with
interim
scheduled
sacrifices.

Oral
LD50
Males
>
5.8
x
10
8
CFU
per
animal.
Females
>
5.6
x
10
8
CFU
per
animal.

No
mortalities
occurred.
Limit
test.

Pseudozyma
flocculosa
is
of
LOW
Toxicity
based
on
theLD50
in
female
Fisher
344
rats.
There
were
no
clinical
signs
of
toxicity
and
all
of
the
animals
gained
weight
during
the
study.
No
gross
findings
were
observed
at
necropsy.
No
label
comments
are
required.

This
acute
oral
study
is
classified
acceptable.
This
study
satisfies
the
guideline
requirement
for
an
acute
oral
study
(OPPTS
885.3050)
in
the
rat.

COMPLIANCE:
Signed
and
dated
GLP,
Quality
Assurance,
and
Data
Confidentiality
statements
were
[Sporodex
L
/
2001­
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/
PLG]
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PROTECTED
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Acute
Oral
Infectivity
and
Toxicity
Study
/2
[Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
2.
2
/
USEPA
OPPTS
885.3050
provided.
The
study
meets
GLP
standards
with
the
following
exceptions:
1)
The
purity
and
the
characterization
of
the
active
ingredient
and
the
stability
of
the
test
material
was
established
by
the
sponsor's
representative's
laboratory
and
was
not
included
in
the
compliance
statement.
2)
Some
SOPs
were
used
as
drafts.

I.
MATERIALS
AND
METHODS
A.
MATERIALS:

1.
Test
Material:
Sporodex®
Description:
pinkish
fine
powder
Lot/
Batch
#:
UK34­
35
Purity:
3.5
x
10
8
colony
forming
units
/
g
CAS
#:
n/
a
The
purity
and
the
stability
of
the
test
substance
was
established
and
documented
by
the
sponsor's
representative
(Dr.
Richard
Bélanger;
Université
Laval).

2.
Sample
Preparation:
A
solution
of
the
test
substance
was
prepared
by
dissolving
0.
5
g
of
Sporodex®
in
100
mL
of
USP­
grade
sterile
water
for
injection
(Abbott
Laboratories,
Montréal,
Québec)
and
mixing
in
a
blender
for
approximately
2
minutes.
The
solution
was
adjusted
by
weight
per
volume
to
establish
the
recommended
dose
of
1
x
10
8
CFU/
mL
by
the
addition
of
sterile
water
and
mixing
with
a
stir
bar.

Half
of
the
solution
was
heat­
treated
for
approximately
20
minutes
at
82
±
2

C
to
render
the
active
ingredient
non­
viable
and
was
used
as
killed
test
substance
[TS(
K)]
to
dose
control
group
animals.
The
rest
of
the
solution
was
used
as
viable
test
substance
[TS(
V)]
to
dose
animals
in
the
treatment
groups.

Immediately
before
and
after
dosing,
various
dilutions
of
TS(
K)
and
TS(
V)
were
plated
on
yeast
malt
peptone
dextrose
agar
(YMA)
media
(Quélab,
Montréal,
Québec),
containing
0.
05
g/
L
of
chloramphenicol,
to
confirm
their
titres.
The
titre
of
the
TS(
V)
preparation
was
5.
6
­
5.
8
x
10
8
CFU/
g
and
the
titre
of
the
TS(
K)
preparation
was
0
CFU/
g.

USP­
grade
sterile
water
for
injection
(Abbott
Laboratories,
Montréal,
Québec)
was
also
used
as
a
control.

3.
Test
animals:
Species:
Rat
Strain:
Fisher
344,
substrain
NHsd
Age/
weight:
animal
receipt
4
­5
weeksold
males:
67.3
­
80.5
g
(based
on
ten
animals)
females:
68.3
­
82.1
g
(based
on
ten
animals)
at
time
of
dosing
6
­7
weeksold
males:
111
­
129
g
females:
100
­
109
g
Source:
Harlan
Sprague
Dawley
Inc.
Indianapolis,
Indiana,
USA
[Sporodex
L
/
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Oral
Infectivity
and
Toxicity
Study
/3
[Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
2.
2
/
USEPA
OPPTS
885.3050
Housing:
Rats
were
individually
housed,
in
wire­
bar
covered
cages
with
beta
chip
bedding,
throughout
the
acclimation
and
treatment
periods.
Diet:
Prolab
Rodent
Chow
4018
(Charles
River
Laboratories,
St­
Constant,
Québec)
was
provided
ad
libitum
for
the
first
week
of
the
acclimation
period.
For
the
remainder
of
the
study,
the
rats
received
a
diet
of
certified
Rodent
Diet
5002
(PMI
Feeds
Inc.)
ad
libitum.
Water:
Municipal
tap
water
was
provided
ad
libitum
Environmental
conditions:
Temperature:
Humidity:
Air
changes:
Photoperiod:
21
­
24

C
19
­
54
%
not
reported
12
hrs
dark
/
12
hrs
light
Acclimation
period:
approximately
two
weeks
B.
STUDY
DESIGN
and
METHODS:

1.
In
life
dates
­
Start:
February
6­
7,
1996
End:
February
27­
28,
1996
2.
Animal
assignment
and
treatment
­
Animals
were
assigned,
on
the
basis
of
body
weight,
to
the
test
groups
noted
in
Table
1.
Individual
body
weights
did
not
vary
from
the
group
mean
body
weight
(by
sex)
by
more
than
20%.
Following
a
16
hour
fast,
rats
were
given
a
single
dose
of
TS(
V),
TS(
K)
or
USP­
grade
sterile
water
by
gavage
then
observed
daily
and
weighed
weekly
until
sacrifice.
Necropsies
were
performed
following
sacrifice
on
the
scheduled
days.

TABLE
1.
Doses,
mortality/
animals
treated
Test
Group
Test
Substance
Dose
Level
Sacrifice
Day
Males
Females
Combined
1
TS(
V)

:5.8
x
10
8
CFUin
1
mL

:5.6
x
10
8
CFUin
1
mL
0
0/
4
0/
4
0/
8
2
TS(
K)

:
heat­
killed
active
ingredient
equal
to
5.
8
x
10
8
CFUin
1
mL

:
heat­
killed
active
ingredient
equal
to
5.
6
x
10
8
CFUin
1
mL
0
0/
4
0/
4
0/
8
3
TS(
V)

:5.8
x
10
8
CFUin
1
mL

:5.6
x
10
8
CFUin
1
mL
7
0/
4
0/
4
0/
8
4
TS(
K)

:
heat­
killed
active
ingredient
equal
to
5.
8
x
10
8
CFUin
1
mL

:
heat­
killed
active
ingredient
equal
to
5.
6
x
10
8
CFUin
1
mL
7
0/
4
0/
4
0/
8
5
TS(
V)

:5.8
x
10
8
CFUin
1
mL

:5.6
x
10
8
CFUin
1
mL
21
0/
4
0/
4
0/
8
6
TS(
K)

:
heat­
killed
active
ingredient
equal
to
5.
8
x
10
8
CFUin
1
mL

:
heat­
killed
active
ingredient
equal
to
5.
6
x
10
8
CFUin
1
mL
21
0/
4
0/
4
0/
8
[Sporodex
L
/
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Oral
Infectivity
and
Toxicity
Study
/4
[Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
2.
2
/
USEPA
OPPTS
885.3050
7
Control
1
mL
of
USP
sterile
water
for
injection
21
0/
4
0/
4
0/
8
3.
Clinical
Observations
­
Cage­
side
observations
for
mortality
and
moribundity
were
made
at
least
once
daily.
Cage­
side
observations
also
included
examination
of
the
general
condition,
appearance
and
demeanor
of
each
rat,
and
observation
of
the
animal's
movement
within
the
cage.

4.
Body
Weights
­
Body
weights
were
measured
upon
receipt
of
the
animals,
on
the
day
of
randomization,
prior
to
gavage
and
weekly
thereafter.
All
body
weight
measurements
were
taken
following
an
overnight
fasting
period.

5.
Feed
Consumption
­
Measurement
of
feed
consumption
began
on
the
day
of
dosing
and
continued
weekly
thereafter.

6.
Necropsy
­
Animals
were
sacrificed
according
to
the
schedule
in
Table
1.
All
test
animals
were
fasted
overnight
prior
to
sacrifice.
On
the
day
of
scheduled
sacrifice,
animals
were
weighed
and
anaesthetized
using
Ketamine/
Xylazine
prior
to
blood
collection
by
cardiac
puncture.
The
necropsy
included
an
examination
of
the
external
surface
of
the
body,
all
orifices,
cranial
cavity,
external
surface
of
the
brain,
the
thoracic,
abdominal
and
pelvic
cavities
and
the
viscera.
The
following
tissues
were
collected
from
each
test
animal
for
enumeration
of
the
test
substance:
brain,
lungs,
liver,
kidneys,
stomach
and
small
intestine
(duodenum,
jejunum,
ileum),
caecum,
mesenteric
lymph
nodes,
and
spleen.

7.
Sensitivity
of
Detection
­
Aliquots
containing
the
viable
test
substance
diluted
to
approximately
10
4
,
10
5
,and10
6
viable
units/
mL
were
placed
into
sterile
bags
(Seward
Medical,
England)
and
homogenized
with
either
the
lungs
or
caecums
removed
from
3
male
and
3
female
rats.
Additional
samples
containing
the
test
substance
without
the
addition
of
animal
tissues
served
as
the
untreated
controls.
The
homogenized
samples
and
the
untreated
controls
were
standardized
for
volume
and
plated
on
duplicate
plates
of
YMA
media
supplemented
with
0.
05
g/
L
of
chloramphenicol.
After
incubation
at
approximately
27

C
for
36
±
12
hours,
the
substance
titres
were
determined
by
counting
colonies.
The
sensitivity
of
detection
was
expressed
as
a
factor
of
the
percent
recovery
and
the
limit
of
detection
observed.

8.
Enumeration
of
Test
Substance
­
Each
organ,
aseptically
collected
at
the
time
of
necropsy,
was
weighed
and
homogenized.
Organ
homogenates
and
blood
samples
were
diluted,
as
necessary,
with
peptone
water.
A
100
µl
aliquot
was
then
plated
on
duplicate
YMA
agar
supplemented
with
0.
05
g/
L
of
chloramphenicol
and
incubated
at
approximately
27

C
for
36
±
12
hours.
Only
plates
with
counts
of
between
10
­
120
colonies
were
included
for
the
determination
of
the
number
of
CFU/
g
of
tissue
or
CFU/
mL
of
blood.
The
number
of
CFU/
g
of
organ
was
calculated
as
follows:

(mean
plate
count
/
0.
1
g
of
organ
homogenate)*
weight
of
organ
homogenate
weight
of
organ.

9.
Statistics
­
The
analysis
of
variance
(ANOVA)
method
was
used
to
determine
whether
statistically
significant
differences
in
body
weight
and
feed
consumption
existed
between
groups.

II.
RESULTS
AND
DISCUSSION:

A.
Mortality
is
given
in
Table
1.
[Sporodex
L
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/
PLG]
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Toxicity
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/5
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M4.
2.
2
/
USEPA
OPPTS
885.3050
The
oral
LD50
for
males
is
greater
than
5.
8
x
10
8
CFU
per
animal.
females
is
greaterthan5.6
x
10
8
CFU
per
animal.

B.
Clinical
observations
­
There
were
no
clinical
signs
of
toxicity
at
any
point
of
the
study.
All
animals
appeared
normal
in
general
condition,
demeanor
and
movement.

C.
Body
Weight
­
All
animals
gained
weight
(see
Table
2
for
summary).
No
statistically
significant
difference
in
body
weight
or
weight
gain
between
TS(
K),
TS(
V)
and
USP
sterile
water
control
groups
was
found.
A
significant
statistical
difference
(P
<0.001),
however,
was
found
between
sexes
for
all
groups
including
control
groups
and
was,
therefore,
not
considered
treatment­
related.

TABLE
2.
Rat
body
weight
summary
Test
Substance
Sex
Body
Weight
(g)

Day
0
a
Day
7
b
Day
14
c
Day
21
c
TS(
V)
M
120
±
9
150
±
14
173
±
8
197
±
7
TS(
K)
M
121
±
8
148
±
12
165
±
23
190
±
26
USP­
sterile
water
M
122
±
7
149
±
9
175
±
11
200
±
11
TS(
V)
F
104
±
4
121
±
6
132
±
4
143
±
4
TS(
K)
F
105
±
4
123
±
4
129
±
5
141
±
5
USP­
sterile
water
F
105
±
4
122
±
7
131
±
9
143
±
10
a
:
each
value
represents
the
mean
and
standard
deviation
calculated
on
the
basis
of
12
animals
for
TS(
V)
and
TS(
K)
treatment
groups
and
on
the
basis
of
4
animals
for
the
USP­
sterile
water
group
b
:
each
value
represents
the
mean
and
standard
deviation
calculated
on
the
basis
of
8
animlas
for
TS(
V)
and
TS(
K)
treatment
groups
and
on
the
basis
of
4
animals
for
the
UPS­
sterile
water
group
c
:
each
value
represents
the
mean
and
standard
deviation
calculated
on
the
basis
of
4
animals
for
TS(
V)
and
TS(
K)
treatment
groups
and
on
the
basis
of
4
animals
for
the
UPS­
sterile
water
group
D.
Feed
Consumption
­
No
statistically
significant
difference
in
weekly
feed
consumption
between
TS(
K),
TS(
V)
and
USP
sterile
water
control
groups
was
found.
A
significant
statistical
difference
(P
<0.001),
however,
was
found
between
sexes
for
all
groups
including
control
groups
and
was,
therefore,
not
considered
treatment­
related.

E.
Necropsy
­
There
were
no
gross
necropsy
findings
for
any
of
the
animals
in
the
study.

F.
Sensitivity
of
Detection
­
In
the
presence
of
lung
and
caecum
tissue,
the
percentage
recovery
of
the
active
ingredient
was
61­
91%
and
54­
83%,
respectively.
The
sensitivity
of
detection
in
the
lungs
was
established
as
132
CFU/
mL.
The
sensitivity
of
detection
in
the
caecum
was
established
as
142
CFU/
mL.
Assuming
that
the
density
of
the
organ
homogenate
was
1
g/
mL,
the
sensitivities
of
detection
in
the
lungs
and
in
the
caecum
could
also
be
expressed
as
132
CFU/
g
and
142
CFU/
g,
respectively.

G.
Enumeration
of
the
Test
Substance
­
Pseudozyma
flocculosa,
at
levels
of
1000
and
160
(estimated
[Sporodex
L
/
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Oral
Infectivity
and
Toxicity
Study
/6
[Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
2.
2
/
USEPA
OPPTS
885.3050
value)
CFU/
g
of
tissue,
was
recovered
from
the
stomach
and
small
intestines
of
2/
4
male
rats
which
were
sacrificed
one
hour
after
administration
of
TS(
V).
Pseudozyma
flocculosa
was
also
recovered
from
the
stomach
and
small
intestines
of
1/
4
female
rats
sacrificed
one
hour
after
administration
of
TS(
V)
at
a
level
of
780
CFU/
g
of
tissue.
No
Pseudozyma
flocculosa
was
recovered
by
the
seventh
day
post
dosing
in
any
of
the
animals
or
in
any
of
the
other
collected
organs
or
fluids.

H.
Reviewer's
Conclusions:
According
to
U.
S.
EPA
OPPTS
885.3050,
the
recommended
test
substance
for
an
acute
oral
toxicity/
pathogenicity
study
is
the
TGAI.
The
test
substance
used
in
this
study
was
a
1997
formulation
of
Sporodex
WP
(wettable
powder).
PMRA
and
EPA
previously
accepted
the
wettable
powder
end­
use
product
as
the
test
substance.
A
change
in
the
intended
formulation
of
the
end­
use
product
from
a
wettable
powder
to
a
liquid
formulation
(Sporodex
L),
however,
triggered
the
need
for
a
rationale
for
the
test
substance.
The
applicant
requested
a
waiver
from
submitting
a
replacement
acute
oral
study
using
the
TGAI
or
the
liquid
formulation
based
on
the
fact
that
the
new
formulants
found
in
Sporodex
L
are
of
food
grade
quality
and
that
the
levels
of
other
formulants
have
been
significantly
reduced.
The
toxicity
of
the
liquid
formulation
is,
therefore,
expected
to
be
less
than
that
of
the
wettable
powder
formulation
that
was
tested.
The
waiver
rationale
is
accepted.

The
acute
oral
study
is
classified
as
acceptable.
Based
on
the
results
of
this
study,
Sporodex
L
and
its
active
ingredient,
P.
flocculosa,
is
not
considered
toxic
or
pathogenic
to
male
or
female
Fisher
344
rats.
The
detection
of
P.
flocculosa
in
the
stomachs
and
small
intestines
of
some
of
the
treated
rats
is
consistent
with
the
route
of
administration.
Clearance
of
P.
flocculosa
appears
to
occur
within
seven
days
of
dosing.

I.
Deficiencies
­
The
number
of
air
changes
and
the
organ
weights
were
not
reported.
These
deficiencies
are
not
considered
to
have
a
significant
impact
on
the
interpretation
of
the
study.
Clearance
was
not
established
by
examining
the
feces
for
the
presence
of
P.
flocculosa.
This
is
not
considered
a
deficiency
since
clearance,
instead,
was
estimated
by
enumerating
P.
flocculosa
from
collected
tissues,
organs
and
fluids.