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2001­
0304
/
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Sensitivity
of
Detection
Study
/1
[Pseudozyma
flocculosa
/STF]

Reviewer:
Esther
Seto
,
Date
Jan.
11,
2001
Peer
Review:
Ibrahim
Barsoum,
PhD
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
U.
S.
Environmental
Protection
Agency
_______________

STUDY
TYPE:
Sensitivity
of
Detection
of
Sporothrix
flocculosa
for
Toxicity/
Pathogenicity
Testing
in
Rats
TEST
MATERIAL
(PURITY):
Sporothrix
flocculosa
(pure
culture)
8.95
x
10
5
Colony
Forming
Units
(CFU)
per
millilitre
SYNONYMS:
Pseudozyma
flocculosa
[STF],
Stephanoascus
flocculosa
CITATION:
Harrington,
K.
(1997)
Sensitivity
of
Detection
of
Sporothrix
flocculosa
for
Toxicity/
Pathogenicity
Testing
in
Rats.
IIT
Research
Institute
(Chicago,
Illinois).
Laboratory
report
number
L08641,
August
23,
1996
­
Sept.
18,
1996.
Unpublished.

SPONSOR:
Dr.
Richard
Bélanger.
Université
Laval
Québec,
Canada
G1K7P4
EXECUTIVE
SUMMARY:
The
objective
of
this
study
was
to
determine
a
method
to
reproducibly
recover
Sporothrix
flocculosa
from
the
tissues
of
male
and
female
CD
rats.
A
total
of
four
male
and
four
female
CD
rats
were
used
in
the
study.
Assays
were
carried
out
to
determine
the
levels
of
recovery
from
CD
rat
lung
and
caecum
tissues
inoculated
with
nominal
doses
of
10
2
and
10
4
CFU/
mL
(corresponding
to
measured
doses
of
4.
2
x
10
2
and4.2
x
10
4
CFU/
mL
respectively)
of
the
test
substance.
The
caecum
represented
tissues
in
which
normal
flora
microorganisms
could
compete
with
the
test
substance
for
isolation
on
recovery
media.
The
lung
represented
tissues
which
could
reasonably
be
expected
to
be
devoid
of
competing
organisms.

After
removal
from
the
uninoculated
animals,
lung
and
caecum
tissues
were
blended
and
aliquots
of
the
test
substance
were
added
to
the
tissue
and
peptone
control
samples.
The
suspensions
were
then
either
mixed
and
plated
(pre­
blended
samples)
or
re­
blended
prior
to
plating
(post­
blended
samples)
onto
yeast
malt
agar
(YM),
YM
supplemented
with
chloramphenicol
(YM/
Chl)
or
Martin's
agar
(MA)
for
enumeration.

YM
provided
acceptable
levels
of
recovery
of
the
test
substance
from
the
lung
tissues
of
male
and
female
CD
rats,
whereas
the
selective
properties
of
MA
were
required
to
provide
recovery
from
the
heavily
contaminated
caecum
tissues.
Mechanical
processing
such
as
vortexing
or
blending,
resulted
in
increased
recoveries
(relative
to
the
inoculation
dose).
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The
minimum
detection
limit
for
recovery
of
microorganisms
from
lung
and
caecum
tissues
was
defined
as
30
CFU/
mL.
The
sensitivity
of
detection
(i.
e.
ratio
of
CFU
recovered:
CFU
of
inoculum)
ranged
from
1:
1.
2
(YM
recovery
from
caecum
tissue
inoculated
with
10
2
CFU/
mL)
and
2.
4:
1
(MA
recovery
from
caecum
tissue
inoculated
with
10
4
CFU/
mL).
Based
on
the
results
of
this
study,
YM
will
be
used
as
the
nonselective
recovery
medium
for
rat
tissues
with
the
exception
of
the
stomach
and
intestinal
tract.
MA
will
be
employed
as
the
selective
recovery
medium
for
the
stomach
and
intestinal
tract
in
subsequent
pathogenicity/
toxicity
studies.

This
sensitivity
of
detection
study
for
toxicity
and
pathogenicity
testing
of
Sporothrix
flocculosa
is
classified
as
acceptable.
The
high
standard
deviation
values
and
extremely
high
percentage
recovery
values
for
lung
and
caecum
tissues
at
the
inoculated
dose
of
10
4
CFU/
mL
were
noted
in
the
review.
These
observations,
however,
do
not
impact
on
the
proposal
to
use
MA
as
selective
recovery
medium
for
stomach
and
intestinal
tract
tissues
and
YM
as
recovery
medium
for
all
other
rat
tissues
in
subsequent
toxicity/
pathogenicity
studies.

COMPLIANCE:
Signed
and
dated
GLP
and
Quality
Assurance
statements
were
provided
stating
that
the
study
was
conducted
in
accordance
with
U.
S.
EPA
Good
Laboratory
Practice
Standards
(40
CFR
160).
The
Data
Confidentiality
statement
was
not
dated
or
signed.

I.
MATERIALS
AND
METHODS
A.
MATERIALS:

1.
Test
Material:
Sporothrix
flocculosa
Description:
fungal
culture
Lot/
Batch
#:
produced
Sept.
6,
1996
Purity:
pure
culture
of
Sporothrix
flocculosa
in
yeast
malt
broth
8.9
x
10
5
CFU/
mL
CAS
#:
n/
a
Verification
of
homogeneity
was
conducted
(not
in
this
study
but
in
subsequent
toxicity/
pathogenicity
studies)
by
removing
samples
from
three
locations
(top,
centre,
bottom)
within
a
container
of
the
test
substance
suspension
and
performing
triplicate
plate
counts
using
Yeast
Malt
Agar
(YM).

Sporothrix
flocculosa
batch
number
UK34­
35,
was
provided
to
the
test
facility
in
the
form
of
two
petri
dishes
(P1
and
P2)
of
yeast
malt
agar
with
white
mold.
Yeast
malt
broth
was
inoculated
with
a
colony
picked
from
P1
and
incubated
for
7
days
at
room
temperature.
This
culture
was
used
as
the
inoculum
for
the
sensitivity
of
detection
assay.

2.
Vehicle
and/
or
positive
control:
All
dilutions
of
the
test
substance
suspension
were
prepared
in
sterile
ASTM
Type
1
H2
O
(purified
water)
as
the
carrier.

3.
Test
animals:
Species/
Strain:
Male
and
female
Sprague
Dawley
CD
Rats.
Age/
weight
at
dosing:
The
animals
were
41
(males)
and
43
(females)
days
of
age
upon
receipt.
The
body
weight
range
on
the
day
of
the
assay
was
242
­
262
g
for
the
male
rats
and
162
­
192
g
for
the
female
rats.
Source:
Charles
River
Breeding
Laboratories
(Portage,
MI)
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Housing:
The
animals
were
housed
up
to
two
per
cage
in
plastic
cages
(10.5"
wide
x
18"
long
x
8"
high)
with
hardwood
chip
bedding.
Diet:
Ceritified
Purina
Rodent
Chow
5002
(PMI
Feeds,
Inc.,
St.
Louis,
MO),
lot
June
12
96
1B
was
provided
ad
libitum,
except
during
an
overnight
fasting
period.
Water:
City
of
Chicago
tap
water
was
provided
ad
libitum.
Fresh
water
was
supplied
twice
weekly.
Environmental
conditions:
Temperature:
Humidity:
Air
changes:
Photoperiod:
22
­
24

C
38
­
50%
n/
a
12
hrs
dark/
12
hrs
light
with
fluorescent
lights
Acclimation
period:
The
rats
were
quarantined
from
August
28,
1996
to
September
13,
1996
prior
to
being
released
for
testing.

B.
STUDY
DESIGN
and
METHODS:

1.
Test
Substance
Titre
­
The
test
substance
was
prepared
in
purified
water,
mixed
by
vortexing
for
at
least
one
minute
and
allowed
to
settle
for
approximately
one
minute.
An
aliquot,
from
the
centre
of
the
suspension,
was
withdrawn
and
plated
on
triplicate
YM
plates.
Four
hours
later,
a
second
aliquot
was
withdrawn
and
plated
on
triplicate
YM
plates.
The
plates
were
incubated
for
112
hours
before
being
examined
for
colonies.

2.
Experimental
Design
and
Test
Procedure
­
The
detection
of
the
test
substance
on
selective
and
nonselective
bacterial
recovery
media
was
tested
quantitatively.
The
sensitivity
of
detection
in
the
presence
of
lung
and
caecal
tissues,
as
well
as
the
effect
of
blending
on
the
recovery
of
the
test
substance,
was
also
tested.
Four
rats
per
sex
were
sacrificed
by
CO2
asphyxiation
and
the
lungs
and
caecum
were
aspetically
removed
from
each
uninoculated
animal.
Each
tissue
was
placed
in
a
pre­
weighed,
sterile
blending
bag
containing
0.
1
%
peptone
(Difco),
blended
manually
or
using
a
Stomacher
blendinger
(Seward
Medical,
London,
England)
and
the
gross
weight
determined
(bag,
peptone
plus
tissue).
Appropriate
dilutions
of
the
test
substance
suspension
were
made
in
order
to
add
a
target
dose
of
2
x
10
2
and2
x
10
4
CFU/
mL
(actual
dose
8.
4
x
10
2
and8.4
x
10
4
)
to
the
blending
bags
containing
an
equal
volume
of
peptone
(control
containing
no
tissue),
blended
lungs
or
blended
caeca
such
that
the
final
concentration
of
the
active
ingredient
was
1
x
10
2
and1
x
10
4
CFU/
mL
(actual
concentration
4.
2
x
10
2
and4.2
x
10
4
CFU/
mL).
The
samples
were
mixed
by
vortexing
and
enumerated
by
removing
a
0.
1
mL
aliquot
and
spreading
on
duplicate
plates
of
Yeast
Malt
Agar
(YM),
YM
supplemented
with
chloramphenicol
(YM/
Chl)
and
Martin's
Agar
(MA)
media.
These
are
referred
to
as
Pre­
blended
(Pre)
samples.
The
suspensions
were
then
re­
blended
and
re­
plated.
These
are
referred
to
as
Post­
blended
(Post)
samples.
The
MPCA
titre
in
the
tissues
was
determined
by
colony
counts
after
incubation
at
room
temperature
in
the
dark
for
112
hours.

3.
Calculation
of
Data
­
Microbial
Enumeration
­
Mean
microbial
numbers
from
duplicate
plate
counts
for
each
tissue
(Pre
and
Post)
were
obtained
and
expressed
as
CFU/
mL.
The
sensitivity
of
detection
and
the
limit
of
detection
were
expressed
as
factors
of
the
percent
recovery.

II.
RESULTS
AND
DISCUSSION:

A.
Test
Substance
Titre
­
The
titre
of
the
test
substance
was
determined
to
be
8.9
x
10
5
CFU/
mL
by
plate
count.
The
titre
of
the
dosing
materials
were
determined
to
be
8.
4
x
10
2
and8.4
x
10
4
CFU/
mL.

B.
Sensitivity
of
Detection
of
Test
Substance
­
Results
of
the
study
showing
the
%
recovery
in
the
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flocculosa
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different
sample
suspensions
(generated
from
mean
plate
counts
from
replicate
plates)
are
summarized
in
Table
1.
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Table
1.
Percentage
of
Recovery
of
Sporothrix
flocculosa
in
Test
Sample
Suspensions
nominal
dose
of
10
2
CFU/
mL
(measured
dose
of
4.
2
x
10
2
CFU/
mL)
nominal
dose
of
10
4
CFU/
mL
(measured
dose
of
4.
2
x
10
4
CFU/
mL)

YM
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
Control
c
141.67
(10.10)
150.60
(26.10)
171.43
(16.84)
141.07
(12.63)

Lung
d
170.54
(27.21)
166.37
(24.44)
199.70
(21.92)
382.74
(247.46)

Caecum
d
101.19
(53.75)
80.36
(33.37)
267.26
(219.15)
278.27
(212.62)

YM/
Chl
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
Control
c
167.26
(5.89)
156.55
(10.94)
153.57
(18.52)
152.98
(26.10)

Lung
d
195.54
(19.59)
195.24
(20.11)
288.99
(204.35)
386.01
(242.19)

Caecum
d
171.13
(29.84)
149.40
(8.99)
180.06
(45.81)
266.96
(220.06)

MA
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
%
Recovery
Pre
blended
a
%
Recovery
Post
blended
b
Control
c
114.29
(26.94)
189.29
(33.67)
175.00
(3.37)
182.74
(5.89)

Lung
d
211.61
(12.31)
208.63
(7.98)
402.38
(447.10)
568.45
(436.03)

Caecum
d
232.14
(8.80)
194.05
(14.09)
148.81
(49.88)
289.58
(236.25)

a
Pre:
sample
plated
after
vortexing
b
Post:
sample
plated
after
re­
blending
c
Two
samples
tested
(mean
and
standard
deviation)
d
Four
samples
tested
(mean
and
standard
deviation)
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Table
2.
Sensitivity
of
Detection
of
P.
flocculosa
*

Media
Tissue
Dose
(CFU/
mL)
Limit
of
Detection
a
Post
Blending
(CFU/
mL)
Sensitivity
of
Detection
b
Post
Blending
YM
Lung
4.2
x
10
2
30
1.2:
1
4.2
x
10
4
30
2.2:
1
Caecum
4.2
x
10
2
53
0.57:
1
4.2
x
10
4
30
1.6:
1
YM/
Chl
Lung
4.2
x
10
2
30
1.4:
1
4.2
x
10
4
30
2.3:
1
Caecum
4.2
x
10
2
30
1.1:
1
4.2
x
10
4
30
1.6:
1
MA
Lung
4.2
x
10
2
30
1.5:
1
4.2
x
10
4
30
3.32:
1
Caecum
4.2
x
10
2
30
1.4:
1
4.2
x
10
4
30
1.7:
1
*
The
study
report
made
an
error
in
calculating
limit
of
detection
and
sensitivity
of
detection.
The
values
in
the
above
table
are
the
corrected
values.
a
The
minimum
limit
of
detection
is
30
CFU/
mL.
The
limit
of
detection
is
based
on
the
sensitivity
of
detection
(i.
e.
if
the
ratio
of
CFU
recovered
:
CFU
dosed
is
>
1,
the
limit
of
detection
is
30;
if
the
ratio
is
<
1,
the
minimum
limit
of
detection
is
divided
by
the
value
of
the
ratio).
b
The
sensitivity
of
detection
is
defined
as:

CFU
recovered
from
post­
blended
samples
combined
with
tissue
CFU
recovered
from
pre­
blended
samples
combined
with
peptone
only
plated
on
YM.

C.
Pre
vs.
Post
­
Re­
blending
the
samples
did
not
result
in
significant
changes
in
recovery
from
the
lung
at
the
10
2
CFU/
mL
dose
level.
At
the
same
dose
level,
recovery
from
the
post­
blended
caecum
samples,
however,
was
lower
than
pre­
blended
samples
on
all
three
media.
At
the
10
4
CFU/
mL
dose
level,
blending
resulted
in
increased
recoveries
from
both
the
lung
and
the
caecum
on
all
three
media.

D.
YM
vs.
YM/
Chl
vs.
MA
­
YM,
YM/
Chl
and
MA
all
provided
acceptable
levels
of
recovery
of
the
test
substance
from
the
lung
and
caecum
of
male
and
female
CD
rats,
with
yields
from
the
caecum
maximum
on
MA
selective
medium.
Based
on
the
results
of
this
study,
YM
will
be
employed
as
the
non­
selective
recovery
medium
for
"sterile"
tissues
as
represented
by
the
lung.
MA
selective
medium
will
be
employed
for
recovery
from
non­
sterile
tissues
(e.
g.,
stomach
and
intestinal
tract)
as
represented
by
the
caecum.
[2000­
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/
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PROTECTED
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Sensitivity
of
Detection
Study
/7
[Pseudozyma
flocculosa
/STF]

REVIEWER'S
CONCLUSIONS
This
sensitivity
of
detection
study
is
acceptable.
The
high
standard
deviation
values
and
extremely
high
percentage
recovery
values
for
lung
and
caecum
tissues
at
the
inoculated
dose
of
10
4
CFU/
mL
were
noted
in
the
review.
These
observations,
however,
do
not
impact
on
the
acceptability
of
using
MA
as
selective
recovery
medium
for
stomach
and
intestinal
tract
tissues
and
YM
as
recovery
medium
for
all
other
rat
tissues
in
subsequent
toxicity/
pathogenicity
studies.

Recoveries
were
generally
higher
than
the
amount
dosed.
Mechanical
processing
(e.
g.,
vortexing,
blending)
appeared
to
fragment
mycelial
segments
or
disaggregate
clumped
cells
resulting
in
a
higher
recovery
compared
to
the
amount
dosed.
Re­
blending
of
the
samples,
however,
did
not
consistently
increase
or
decrease
recovery.

DEFICIENCIES
The
number
of
air
changes
per
hour
was
not
reported.
This
omission
is
considered
to
have
no
impact
on
the
interpretation
of
the
study
results.
