Document ID: EPA-HQ-OPP-2002-0233-0011
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
17,
2002
Peer
Review:
Ibrahim
Barsoum,
PhD
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
U.
S.
Environmental
Protection
Agency
_______________

STUDY
TYPE:
Acute
Pulmonary
­
Range
Finding
Study
PMRA
DATA
CODE
M4.
2.
3
USEPA
OPPTS
885.3150
TEST
MATERIAL
(PURITY):
Sporothrix
flocculosa
[STF],
pure
culture
(MPCA)
in
sterile
water
SYNONYMS:
Pseudozyma
flocculosa,
STF,
Stephanoascus
flocculosa
CITATION:
Harrington,
Kelly,
A.
(July,
1997).
"Acute
Pulmonary
Range
Finding
Toxicity
Test
of
Sporothrix
flocculosa,
a
Fungal
Pesticide,
in
Rats."
IIT
Research
Institute,
Chicago,
Illinois,
USA.
IITRI
Project
No.
L08641,
Study
No.
5
In­
Life
Study
Dates:
March
21,
1997
­
April
4,
1997.
Unpublished.

SPONSOR:
Université
Laval
Québec,
Canada
G1K
7P4
EXECUTIVE
SUMMARY:
A
large
number
of
spontaneous
deaths
were
observed
in
both
treatment
and
control
groups
in
a
prior
acute
pulmonary
toxicity
/
infectivity
assay
(see
separate
review).
In
order
to
determine
whether
the
test
substance
(in
both
its
viable
and
non­
viable
forms),
Sporothrix
flocculosa,
was
the
cause
of
the
deaths,
a
subsequent
acute
pulmonary
range­
finding
toxicity
study
was
conducted.
In
this
range­
finding
study,
groups
of
young
adult
CD
rats
(5/
sex/
dose
level)
were
exposed
by
the
intratracheal
route
to
Sporothrix
flocculosa
(4.2
x
10
8
cfu/
mL)
in
ASTM
Type
1
water
at
doses
of
4.
2
x
10
7
,3.4
x
10
7
,
6.8
x
10
6
and3.4
x
10
6
cfu/
animal.
Animals
were
then
observed
for
14
days.
There
were
no
mortalities
and
all
animals
gained
weight
during
the
study.

LD50
Males
>
4.2
x
10
7
cfu/
animal
Females
>
4.2
x
10
7
cfu/
animal
Combined
>
4.
2
x
10
7
cfu/
animal
No
mortalities
occurred.

Sporothrix
flocuclosa
is
classified
as
being
of
SLIGHT
Toxicity
(EPA
Toxicity
Category
IV)
based
on
adverse
effects
observed
in
some
test
animals.
Rough
hair
coat
occurred
in
a
dose­
dependent
manner
with
all
5
animals/
sex
exhibiting
this
symptom
at
the
highest
dose
of
4.
2
x
10
7
CFU/
animal.
One
female
dosed
with4.2
x
10
7
CFU
experienced
tremors,
closed
eyes
and
rough
hair
coat.
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
2
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
This
acute
pulmonary
study
is
considered
supplemental.
This
study
does
not
satisfy
the
guideline
requirement
for
an
acute
pulmonary
study
(OPPTS
885.3150)
in
the
rat
(see
section
II­
F).

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:
6.2
x
10
8
cells/
mL
as
determined
by
hemacytometer
count
4.2
x
10
8
cfi/
mL
as
determined
by
plate
count
CAS
#:
n/
a
Verification
of
stability
and
homogeneity
conducted
as
part
of
toxicity
and
pathogenicity
studies.

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.
The
test
substance
was
subcultured
approximately
every
three
months
on
malt
agar
to
maintain
viability.
One
week
before
the
start
of
the
in­
life
phase,
300
mL
of
yeast
malt
broth
was
inoculated
with
a
subculture
of
the
test
substance
and
incubated
at
room
temperature
for
seven
days.
On
the
day
of
dosing,
the
broth
culture
was
centrifuged
and
resuspended
in
4
mL
of
ASTM
type
1
water.
The
titre
of
the
resuspended
solution
was
determined
to
be
6.2
x
10
8
cells/
mL
by
hemacytometer
count
(corresponding
to
4.
2
x
10
8
colony
forming
units
(cfu)/
mL
as
determined
by
plate
count).
The
solution
was
diluted
to
5
x
10
8
,1
x
10
8
and5
x
10
7
cells/
mL
(corresponding
to
3.
4
x
10
8
,6.8
x
10
7
and
3.
4
x
10
7
cfu/
mL,
respectively).

3.
Test
animals:
Species
/
Strain:
CD
Rat
Age/
weight
at
dosing:
approximately
6
weeks
of
age
males:
195.11
­
224.48
g
females:
155.88
­
188.64
g
Source:
Charles
River
Laboratories
Portage,
MI
Housing:
During
quarantine,
the
rats
were
housed
up
to
two
per
cage
in
polypropylene
cages
with
hardwood
chip
bedding.
Upon
assignment
to
test
groups
,
and
for
the
remainder
of
the
study,
the
rats
were
singly
housed.
Diet:
Certified
Purina
Rodent
Chow
5002
(PMI
Feeds
Inc.,
St.
Louis,
MO)
ad
libitum
Water:
City
of
Chicago
water
ad
libitum
Environmental
conditions:
Temperature:
Humidity:
Air
changes:
Photoperiod:
20
­
24

C
8
­
35
%
Not
reported
12
hrs
dark
/
12
hrs
light
Acclimation
period:
one
week
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
3
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
B.
STUDY
DESIGN
and
METHODS:

1.
In
life
dates
­
Start:
March
21,
1997
End:
April
4,
1997
3.
Animal
assignment
and
treatment
­
Animals
were
assigned
to
the
test
groups
noted
in
Table
1.
Rats
were
anesthetized
with
ether
and
administered
a
dose
of
0.
1
ml
test
substance
intratracheally.
They
were
observed
twice
daily
(only
once
daily
on
weekends)
and
weighed
weekly
for
14
days
after
dosing.
Survivors
were
sacrificed
and
necropsies
were
not
performed.

TABLE
1.
Concentrations,
exposure
conditions,
mortality/
animals
treated
Group
Dose
(cfu)
Mortality
(#
dead/
total)

Males
Females
Combined
1
4.2
x
10
7
0/
5
0/
5
0/
10
2
3.4
x
10
7
0/
5
0/
5
0/
10
3
6.8
x
10
6
0/
5
0/
5
0/
10
4
3.4
x
10
6
0/
5
0/
5
0/
10
5.
Statistics
­TheLD50
was
set
at
greater
than
4.
2
x
10
7
cfu/
animal.
No
calculations
were
necessary.

II.
RESULTS
AND
DISCUSSION:

A.
Mortality
is
given
in
Table
1.

The
LD50
(C.
I.)
for
males
is
>
4.
2
x
10
7
cfu/
animal.
for
females
is
>
4.
2
x
10
7
cfu/
animal
combined
is
>
4.
2
x
10
7
cfu/
animal.

B.
Clinical
observations
­
A
summary
of
the
incidence
of
clinical
observations
is
presented
in
Table
2.

TABLE
2.
Clinical
Observations
Observations
Dose
(CFU/
animal)

4.2
x
10
7
3.4
x
10
7
6.8
x
10
6
3.4
x
10
6
males
females
males
females
males
females
males
females
rough
hair
coat
555211
0
0
tremors
010000
0
0
closed
eye(
s)
010000
0
0
no
signs
observed
000344
5
5
[2000­
0680
and
2001­
0304
/
PLG]
~
PROTECTED
~
Acute
Pulmonary
Infectivity
and
Toxicity
/
4
[Pseudozyma
flocculosa
/
STF]
DACO
M4.
2.
3
/
USEPA
OPPTS
885.3150
Rough
hair
coat
occurred
in
a
dose­
dependent
manner
beginning,
in
some
animals,
on
day
3
with
sporadic
re­
occurrence
until
day
11.
One
female,
dosed
at
4.
2
x
10
7
CFU,
presented
with
tremors,
closed
eyesand
rough
hair
coat.
The
tremors
were
noted
only
on
day
3
and
the
closed
eyes
were
noted
on
days
3
and
5.

C.
Body
Weight
­
All
animals
gained
weight
during
the
study.
One
female,
dosed
at
4.
2
x
10
7
CFU,
lost
weight
between
days
0
and
7
but
gained
weight
thereafter.
Statistical
analysis
was
not
conducted
to
compare
body
weight
data
between
groups.

E.
Reviewer's
Conclusions
­
The
recommended
test
substance
for
an
acute
pulmonary
study
is
the
technical
grade
active
ingredient
(TGAI).
Instead,
the
test
substance
used
in
this
study
was
the
MPCA
and
was
prepared
by
the
test
facility
using
a
method
different
from
the
proposed
manufacturing
methods.
The
applicant
has
agreed
to
upgraded
label
statements
requiring
respirators
for
all
users.

According
to
USEPA
OPPTS
885.3150,
Acute
Pulmonary
Toxicity/
Pathogenicity,
the
minimum
dose
is
10
8
units
of
the
MPCA
per
test
animal.
The
maximum
dose
level
used
in
this
study,
however,
was
only
4.
2
x
10
7
CFU/
animal.
Furthermore
the
purpose
of
this
study
was
to
determine
the
toxicity
range
of
S.
flocculosa
in
order
to
aid
in
the
interpretation
of
the
large
number
of
spontaneous
deaths
from
the
acute
pulmonary
toxicity
/
infectivity
study
conducted
previously.
Infectivity,
as
required
by
USEPA
OPPTS
885.3150,
was
not
addressed
by
this
study.
This
study
does
not
comply
with
guideline
requirements
and
is,
therefore,
considered
supplemental.

The
range­
finding
study
suggests
that
the
LD50
of
S.
flocculosa
is
greater
than
4.
2
x
10
7
CFU/
animal
in
male
and
female
rats.
Sporothrix
flocculosa
is
classified
as
slightly
toxic
based
on
rough
hair
coat
(observed
in
55%
of
treated
male
rats
and
40%
of
treated
female
rats)
which
occurred
in
a
dose­
dependent
manner.

F.
Deficiencies
­
In
addition
to
the
deficiencies
already
noted
above,
the
number
of
air
changes
was
not
reported
and
the
relative
humidity
of
the
room
in
which
the
animals
were
housed
fell
below
the
target
of
3070
These
additional
deficiencies
are
not
considered
to
have
any
impact
on
the
interpretation
of
this
study.