Document ID: EPA-HQ-OPP-2002-0233-0014
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2002-09-13T04:00Z

[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
1
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
Reviewer:
Esther
Seto
,
Date
February
1,
2002
Peer
Review:
Ibrahim
Barsoum,
PhD
Microbial
Pesticides
Branch
Biopesticides
and
Pollution
Prevention
Division
U.
S.
Environmental
Protection
Agency
_______________

STUDY
TYPE:
Acute
Dermal
Toxicity
­
Rabbit
PMRA
DATA
Code
M4.4
/
USEPA
OPPTS
885.3100
Acute
Dermal
Irritation
­
Rabbit
PMRA
DATA
Code
M4.5.
2
/
USEPA
OPPTS
870.2500
TEST
MATERIAL
(PURITY):
Sporothrix
flocculosa
(MPCA)

SYNONYMS:
Pseudozyma
flocculosa
[STF]

CITATION:
Johnson,
William
D.
(July
29,
1997).
"Acute
Dermal
Toxicity
/
Irritation
Study
of
Sporothrix
flocculosa,
a
Fungal
Pesticide,
in
Rabbits."
IIT
Research
Institute,
Life
Sciences
Department,
Chicago,
IL,
USA.
IITRI
Project
No.
L08641,
Study
No.
4.
InLife
Study
Dates:
November
13,
1996
­
November
27,
1996.
Unpublished.

SPONSOR:
Dr.
Richard
Bélanger
Université
Laval
Québec,
Canada
EXECUTIVE
SUMMARY:
In
an
acute
dermal
toxicity
study,
a
single
group
of
New
Zealand
White
rabbits
(5/
sex)
was
dermally
exposed
to
Sporothrix
flocculosa,
for
24
hours
to
an
area
of
approximately
10%
of
the
dorsal
skin
surface.
Following
exposure,
the
animals
were
observed
for
a
period
of
14
days.

Dermal
LD50
Males
>
1.2
x
10
7
CFU/
animal
(equivalent
to
approximately
0.82­
0.90
g/
kg
bw)
Females
>
1.2
x
10
7
CFU/
animal
(equivalent
to
approximately
0.80­
0.91
g/
kg
bw)
Combined
>
1.
2
x
10
7
CFU/
animal
(equivalent
to
approximately
0.
80­
0.91
g/
kg
bw)
Limit
Dose.
No
mortalities
observed.

No
treatment­
related
signs
of
toxicity
or
skin
irritation
were
observed
in
any
animal
during
the
14­
day
observation
period.
At
the
dose
administered,
Sporothrix
flocculosa
is
not
considered
toxic
or
irritating
to
the
skin.

The
recommended
test
substance
for
acute
dermal
toxicity
and
acute
dermal
irritation
studies
is
the
end­
use
product.
Instead,
the
test
substance
was
produced
by
the
test
facility
using
a
method
different
from
the
proposed
manufacturing
method.
A
waiver
rationale
was
submitted
to
address
the
toxicity
and/
or
irritation
potential
of
the
formulation
ingredients.
[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
2
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
This
acute
dermal
study
is
classified
as
acceptable
for
assessing
the
dermal
toxicity
and
dermal
irritation
potential
of
Sporothrix
flocculosa,
the
MPCA
found
in
Sporodex
L.

COMPLIANCE:
Signed
and
dated
GLP
and
Quality
Assurance
statements
were
included.
A
Statement
of
No
Data
Confidentiality
was
provided
but
was
not
signed
or
dated.

I.
MATERIALS
AND
METHODS
A.
MATERIALS:

1.
Test
Material:
Sporothrix
flocculosa
Description:
MPCA
Lot/
Batch
#:
UK34­
35
Purity:
1.1
x
10
7
cells/
mL
by
hemacytometer
count
6.2
x
10
6
CFU/
mL
by
plate
count
CAS
#
TGAI:
N/
A
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
was
comparable,
indicating
that
the
test
substance
was
homogeneous
throughout
the
volume
of
the
tube.

2.
Sample
Preparation:
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
mould.
IITRI
then
inoculated
two
malt
agar
plates
(P3
and
P4)
with
a
colony
from
P1.
A
scrape
of
P4
culture
was
added
to
50
mL
of
YM
broth
and
incubated
at
room
temperature.
On
the
day
of
dosing,
the
YM
broth
containing
Sporothrix
flocculosa
was
filtered
through
sterile
gauze
pads
into
100
mL
of
sterile
ASTM
type
1
purified
water,
vortexed
and
administered
to
the
animals.
The
titre
of
the
dosing
suspension
was
determined
to
be
6.2
x
10
6
CFU/
mL
by
plate
count
(corresponding
to
1.
1
x
10
7
cells/
mL
by
hemactyometer
count).

3.
Test
animals:
Species:
Rabbit
Strain:
New
Zealand
White
Age/
weight
at
dosing:
approximately
2.
5
months
of
age
males:
2.22
­
2.
46
kg
females:
2.39
­
2.
50
kg
Source:
Kuiper
Rabbit
Ranch,
Gary,
IN,
USA
Housing:
The
rabbits
were
housed
individually
in
stainless
steel
cages
(61
x
45.5
x
41
cm)
with
absorbent
liners
placed
under
the
stainless
steel
mesh
floor
to
absorb
liquids.
Diet:
Each
animal
was
provided
with
150
g
of
Certified
Purina
Lab
Rabbit
Chow
HF
#
5326
(PMI
Feeds
Inc.,
St.
Louis,
MO)
daily.
Water:
City
of
Chicago
water
was
supplied
ad
libitum.
[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
3
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
Environmental
conditions:
Temperature:
Humidity:
Air
changes:
Photoperiod:
22.5
­
23.0

C
40
­
56%
Not
Reported
12
hrs
dark
/
12
hrs
light
Acclimation
period:
The
animals
were
held
in
quarantine
for
1
week.

B.
STUDY
DESIGN
and
METHODS:

1.
In
life
dates
­
November
13,
1996
­
November
27,
1996.

2.
Animal
assignment
and
treatment
Five
rabbits
per
sex
were
randomly
selected
and
assigned
to
groups
as
summarized
in
Table
1.
Twentyfour
hours
prior
to
treatment,
fur
from
approximately
10%
of
the
dorsal
skin
surface
of
each
rabbit
was
clipped.
A
total
of
2
mL
of
the
test
substance
was
applied
to
the
prepared
skin
of
each
rabbit.
The
test
sites
were
covered
with
12.
8
x
11.
5
cm
surgical
dressing
(Surgipad,
J&
J
Products,
New
Brunswick,
NJ),
plastic
film,
lint­
free
cloth
and
elastic
adhesive
bandage
(Elastoplast,
Beiersdorf
Inc.
k
Nowalk,
CT).
The
wrappings
were
removed
after
24
hours
and
the
application
sites
were
rinsed
with
water
and
towel­
dried.

TABLE
1.
Doses,
mortality/
animals
treated
Dose
Males
Females
Combined
1.2
x
10
7
CFU/
animal
0/
5
0/
5
0/
10
3.
Clinical
Observations
All
rabbits
were
observed
frequently
immediately
following
dosing
and
once
per
day
for
13
days
after
removal
of
the
wrappings.
Clinical
observations
included
the
examination
of
the
application
site
for
signs
of
dermal
irritation
approximately
30­
60
minutes
after
unwrapping
and
daily
thereafter.
Any
skin
reactions
observed
were
graded
according
to
the
Draize
method
(see
Appendix
1).

4.
Body
Weights
All
rabbits
were
weighed
prior
to
dosing
and
weekly
thereafter.

5.
Necropsies
At
the
end
of
the
study,
all
rabbits
were
euthanized
and
discarded
without
necropsy.

6.
Statistics
The
dermal
LD50
was
set
at
greater
than
1.
2
x
10
7
CFU/
animal.
No
calculations
were
required.

II.
RESULTS
AND
DISCUSSION:

A.
Mortality
[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
4
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
None
of
the
rabbits
died
during
the
course
of
the
study.
The
dermal
LD50
is
greater
than
1.
2
x
10
7
CFU/
animal
for
both
male
and
female
rabbits.

B.
Clinical
observations
One
male
rabbit
exhibited
slight
diarrhea
7
days
following
unwrapping.
No
other
adverse
clinical
symptoms
were
observed
during
the
treatment
and
observation
period.
No
signs
of
dermal
irritation
(i.
e.,
erythema
or
edema)
were
observed
in
any
rabbit
upon
removal
of
the
wrappings
or
during
the
subsequent
observation
period.

C.
Body
Weight
Individual
body
weight
data
are
summarized
in
Table
2.
One
male
rabbit
lost
weight
within
the
first
week
but
experienced
a
slight
weight
gain
thereafter.

TABLE
2.
Body
Weight
Males
Body
Weight
(kg)

Animal
Number
Week
0
Week
1
Week
2
Cumulative
Body
Weight
Change
(kg)
(week
2
­
week
0)

981
2.45
2.61
2.71
0.26
982
2.38
2.49
2.72
0.34
983
2.22
2.40
2.58
0.36
985
2.34
2.32
2.35
0.01
Mean
2.36
2.49
2.62
0.27
±S.
D.
a
0.09
0.14
0.17
0.15
Females
Body
Weight
(kg)

Animal
Number
Week
0
Week
1
Week
2
Cumulative
Body
Weight
Change
(kg)
(week
2
­
week
0)

986
2.50
2.64
2.78
0.28
987
2.20
2.46
2.62
0.42
988
2.39
2.60
2.69
0.28
989
2.41
2.60
2.69
0.28
990
2.46
2.60
2.79
0.33
Mean
2.39
2.58
2.71
0.32
[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
5
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
±
S.
D.
0.
12
0.
07
0.
07
0.
06
a
S.
D.
­
standard
deviation
E.
Reviewer's
Conclusions
The
recommended
test
substance
for
acute
dermal
toxicity
and
acute
dermal
irritation
studies
is
the
end­
use
product.
Instead,
the
test
substance
was
produced
by
the
test
facility
using
a
method
different
from
the
proposed
manufacturing
method.
A
waiver
rationale
was
submitted
to
address
the
toxicity
and/
or
irritation
potential
of
the
formulation
ingredients.
All
formulation
ingredients
are
either
food­
grade
or
relatively
nontoxic
One
formulation
ingredient
may
cause
irritation
of
the
skin
with
prolonged
contact.

The
LD50
for
Sporothrix
flocculosa,
the
MPCA
in
Sporodex
L,
in
male
and
female
rats
is
greater
than
1.
2
x
10
7
CFU/
animal.
No
treatment­
related
signs
of
toxicity
or
skin
irritation
were
observed
in
any
animal
during
the
14­
day
observation
period.

F.
Deficiencies
The
number
of
air
changes
per
hour
was
not
reported.
However,
given
the
data
presented,
this
omission
is
considered
to
have
no
impact
on
the
interpretation
of
the
study
results.
[
Sporodex
L
/
2001­
0304
/
PLG
]
~
PROTECTED
~
Acute
Dermal
Toxicity
/
Acute
Dermal
Irritation
/
6
[
Pseudozyma
flocculosa
/
2000­
0680
/
STF]
DACO
M4.
4
and
M4.
5.
2
/
USEPA
OPPTS
885.3100
and
870.2500
Appendix
1:
Description
of
Skin
Reactions
Evaluation
of
Skin
Reactions
Score
Erythema
and
eschar
formation
No
erythema
0
Very
slight
erythema
(barely
perceptible)
1
Well­
defined
erythema
2
Moderate
to
severe
erythema
3
Severe
erythema
(beet
redness)
to
slight
4
eschar
formation
(injuries
in
depth)

Edema
Formation
No
edema
0
Very
slight
edema
(barely
perceptible)
1
Slight
edema
(edges
of
area
well­
defined
2
by
definite
raising)
Moderate
edema
(raised
approximately
3
1.
0
mm)
Severe
edema
(raised
more
than
1.
0
mm
4
beyond
the
area
of
exposure)

Draize,
J.
H.,
Appraisal
of
the
Safety
of
Chemicals
in
Foods,
Drugs,
and
Cosmetics,
Assoc.
Food
and
Drug
Officials
of
the
U.
S.,
Austin,
Texas,
1959.