II.
B.
5
­
Page
1
of
12
Appendix
II.
B.
5
Prepared
by:
U.
S.
Environmental
Protection
Agency
II.
B.
5
­
Page
2
of
12
Table
of
Contents
a.
Introduction
............................................................................................................
3
b.
Comparison
of
Submitted
Protocols
used
for
the
Measurement
of
Tissue
and
Blood
ChE
Activity
in
Rats
...........................................................................................
3
c.
Investigation
of
Reactivation
in
Sampling
Handling
and
AChE
Analysis
.........
5
d.
Summary
of
Motor
Activity
Experiments
Performed
in
EPA's
Dose­
Response
Experiments
...................................................................................................................
7
e.
Summary
of
Protocol
and
Preliminary
Results
of
Seven
Chemical
(
Equipotent)
Mixture
Experiment
.................................................................................
9
f.
References
............................................................................................................
12
List
of
Tables
Table
II.
B.
5­
1.
Comparison
of
the
submitted
protocols
for
the
N­
methyl
carbamates
used
in
measuring
ChE
activity
in
tissue
and
blood..................................................
4
Table
II.
B.
5­
2.
Composition
of
seven
chemical
mixture
with
the
NMCs..........................
9
List
of
Figures
Figure
II.
B.
5­
1.
Reactivation
experiment
with
oxamyl
.....................................................
6
Figure
II.
B.
5­
2.
Plots
of
motor
activity
data
from
NHEERL
experiments
for
seven
Nmethyl
carbamates
...................................................................................................
8
Figure
II.
B.
5­
3.
Brain
cholinesterase
results
from
seven
chemical
equipotent
mixture.
10
Figure
II.
B.
5­
4.
Comparison
of
brain
cholinesterase
from
dose­
response
and
single
dosing
in
the
mixture
study.....................................................................................
11
II.
B.
5
­
Page
3
of
12
a.
Introduction
The
following
text
provides
the
information
mentioned
in
the
Hazard/
RPF
chapter
of
the
Preliminary
Cumulative
Risk
Assessment
of
the
NMCs.

The
following
topics
are
covered
in
this
Appendix:


information
provided
in
standard
operating
procedures
for
the
measurement
of
tissue
and
blood
ChE
activity;


investigation
of
reactivation
in
sampling
handling
and
AChE
analysis
during
EPA's
time
course
and
dose­
response
experiments;


summary
of
motor
activity
experiments
performed
in
EPA's
dose­
response
experiments
and;


summary
of
protocol
and
preliminary
results
of
seven
chemical
(
equipotent)
mixture
experiment.

b.
Comparison
of
Submitted
Protocols
used
for
the
Measurement
of
Tissue
and
Blood
ChE
Activity
in
Rats
Table
III.
B.
5­
1
provides
a
summary
of
the
ChE
measurement
protocols
submitted
for
registration
studies
to
EPA1.
The
protocols
available
at
present
time
indicate
that
the
experimental
conditions
among
laboratories
vary
but
that
dilutions
are
generally
limited
to
approximately
1:
20
and
that
samples
are
frozen
immediately.
A
reagent
test
kit
from
Boehringer
Mannheim
was
indicated
in
three
of
the
protocols.
The
Hitachi
717
was
indicated
in
three
protocols.
Although
information
regarding
the
time
of
sample
handling
is
more
limited,
the
available
information
suggests
that
reasonable
precautions
were
taken
in
these
studies
to
reduce
reactivation
prior
to
analysis.

1
SOPs
for
pirimicarb
were
provided
to
EPA
on
7/
25/
2005.
The
evaluation
of
pirimicarb
SOPs
will
be
included
in
the
revised
risk
assessment.
II.
B.
5
­
Page
4
of
12
Table
II.
B.
5­
1.
Comparison
of
the
submitted
protocols
for
the
N­
methyl
carbamates
used
in
measuring
ChE
activity
in
tissue
and
blood.

SOP
Details
Methomyl
Oxamyl
Formetanate
Carbofuran
Mrids
44487501,
44472001
44254401,
44420301,

44472001
not
indicated
45675701
Tissue
Rat
RBC,
tissue
Rat
RBC,
tissue
plasma,
whole
blood,

brain
whole
blood,
plasma
or
serum,
RBC,
brain
Test
kit
BMD
ChE
Reagent
Set
#
124117
BMD
ChE
Reagent
Set
#
124117
BMC
ChE
kit
#
124117
None,
Reagent
Recipe
given
Instrument
Hitachi
717
Hitachi
717
Hitachi
717
Gilford
Instruments
System
2600
Anticoagulant
EDTA
EDTA
Heparin
EDTA
Centrifugation
speed
speed,
time,
temp
not
indicated
speed,
time,
temp
not
indicated
plasma:
800
g
10
minutes
1600
g
for
10
min
@
0­

5
°
C
Centrifuge
refrigerated?
not
indicated
not
indicated
Yes
0­
5
°
C
Dilution
1:
20
Triton
1:
20
Triton
not
specified
Sorenson's
buffer
1:
20
Triton
X
(
ice­
cold)

Temp
tissue
frozen
­
70C,
RBC
samples
not
indicated
tissue
frozen
­
70C,
RBC
samples
not
indicated
not
specified
stored
at
­
72C
until
assay;
assay
at
30C
Sample
on
ice
tissue
sample
snap
frozen
if
not
analyzed
immediately;

RBC
not
indicated
tissue
sample
snap
frozen
if
not
analyzed
immediately;

RBC
not
indicated
yes,
record
time
of
sample
receipt
in
lab
on
ice
until
delivered
to
lab;
processed
samples
frozen
until
analysis
Time
limitation
for
sample
life
not
indicated
not
indicated
2
hrs,
time
of
sample
and
analysis
thawed
RBC
sample
within
30
minutes.

Time
of
receipt
of
sample?
not
indicated
not
indicated
yes
record
of
time
at
each
step
in
processing
#
and
Time
of
a
sample
batch
for
processing
not
indicated
not
indicated
30
max
at
a
time,
record
of
time
of
steps
in
process
24
samples
in
a
batch,

~
1
hr
till
frozen
Time
from
sample
drawn
to
sample
assayed
not
indicated
not
indicated
max
time
not
indicated
RBC:
1
to
7
days
II.
B.
5
­
Page
5
of
12
c.
Investigation
of
Reactivation
in
Sampling
Handling
and
AChE
Analysis
In
response
to
questions
raised
by
the
SAP
in
February,
2005
concerning
the
in
vitro
reactivation
of
the
cholinesterase
activity
in
the
samples
from
the
N­
methyl
carbamate­
treated
animals
in
NHEERL
experiments,
the
Agency
conducted
a
series
of
experiments.

On
March
27,
2004,
we
conducted
the
initial
assessment
of
the
level
of
cholinesterase
activity
in
brains
from
animals
treated
with
different
dosages
of
oxamyl.
We
used
only
½
of
the
brain
(
sectioned
down
the
middle,
sagitally)
for
this
assessment.
The
other
½
of
the
brain
was
kept
in
the
­
80

C
freezer
frozen
(
without
homogenization).
About
one
year
later
on
April
22,
2005,
we
assessed
the
activity
in
the
other
half
of
those
brains
under
various
conditions:


Is
reactivation
caused
by
storage
in
a
freezer
for
one
year?
The
same
conditions
were
used
for
this
assay
as
were
originally
used
for
cholinesterase
activity
assessment
to
determine
if
storage
for
a
year
had
caused
reactivation
of
any
activity.
(
Diluted
1:
3
during
homogenization
and
then
immediately
analyzed
for
cholinesterase
activity
using
the
radiometric
assay).


Is
reactivation
promoted
by
dilution
of
the
homogenate?
The
homogenate
(
1:
3
dilution)
was
immediately
diluted
again
to
create
a
total
dilution
of
1:
9
and
the
cholinesterase
activity
was
assessed
using
the
radiometric
assay.
This
was
to
determine
the
effect
of
dilution
on
the
reactivation
of
carbamate­
inhibited
cholinesterase
activity.


Is
reactivation
promoted
by
allowing
the
tissues
sit
on
ice
for
1.5
hours?
The
homogenate
1:
3
was
allowed
to
sit
on
ice
for
1.5
hours
before
assessment
of
activity.
This
was
to
determine
the
effect
of
time
on
the
reactivation
of
carbamateinhibited
cholinesterase
activity.


Is
reactivation
possible
in
these
tissues?
The
homogenate
(
1:
3)
was
subjected
to
a
marked
dilution
(
1:
40)
and
increase
in
temperature
(
37

C)
to
measure
the
amount
of
reactivation
that
was
possible
in
these
samples
over
a
period
of
12
minutes.

The
results
of
all
four
of
these
experiments
are
presented
in
Figure
II.
B.
5­
1
below.
Storage
of
the
tissue
in
the
freezer
for
one
year
before
analysis
did
not
appear
to
differ
from
the
original
dose­
response
assessment.
Allowing
the
homogenized
tissue
to
sit
for
1.5
hours
before
analysis
produced
slight
(

5%)
reactivation
in
the
0.5
and
1.0
mg/
kg
dosage
groups,
but
no
reactivation
in
the
highest
(
1.5
mg/
kg)
dosage
group.
Dilution
of
the
tissue,
however,
had
much
more
of
an
effect
on
the
level
of
cholinesterase
reactivation
with
a
7­
10%
increase
in
activity,
especially
at
the
dosage
groups
with
more
inhibition).
Therefore
it
has
to
be
assumed
that
this
level
of
reactivation
does
occur
during
the
initial
1:
3
homogenization,
but
is
unavoidable
as
the
homogenization
process
is
necessary
for
further
handling
of
the
tissue.
Finally,
if
the
carbamate­
inhibited
tissue
is
encouraged
to
reactivate
by
substantial
dilution
of
the
II.
B.
5
­
Page
6
of
12
tissue
and
increases
in
temperature,
one
can
see
a
progressive
gaining
of
cholinesterase
activity
in
all
dosage
groups
that
exhibited
cholinesterase
inhibition.

Figure
II.
B.
5­
1.
Reactivation
experiment
with
oxamyl
OXAMYL
REACTIVATION
Reactivation
Time
(
minutes)
2
4
6
8
10
12
Brain
Cholinesterase
Activity
%
CONTROL
0
10
20
30
40
50
60
70
80
90
100
110
120
Control
0.0666
mg/
kg
0.1
mg/
kg
0.5
mg/
kg
1.0
mg/
kg
1.5
mg/
kg
Original
3/
27/
04
1:
3
immediately
(
4/
22/
05)
1:
3,
1.5
hr
wait
(
4/
22/
05)
1:
9
immediately
(
4/
22/
05)
II.
B.
5
­
Page
7
of
12
d.
Summary
of
Motor
Activity
Experiments
Performed
in
EPA's
Dose­
Response
Experiments
As
part
of
EPA's
dose­
response
studies,
the
acute
neurobehavioral
effects
of
N­
methyl
carbamate
insecticides
have
been
systematically
compared.
The
results
of
neurobehavioral
effects
have
been
compared
graphically
to
the
results
of
AChE
measurements.
The
use
of
an
automated
assessment
of
motor
activity
along
with
ChE
activity
measurements
allows
a
functional
outcome
that
can
be
directly
related
to
the
ChE
inhibition
in
RBC
and
brain.
The
results
presented
below
were
presented
at
the
March,
2005
meeting
of
the
Society
of
Toxicology
(
Moser
et
al,
2005).

We
evaluated
all
seven
N­
methyl
carbamates
as
described
in
USEPA,
2005.
Adult
male
Long­
Evans
rats
(
n=
10/
dose)
were
dosed
by
oral
gavage
with
either
carbaryl
(
3­
50
mg/
kg),
propoxur
(
0.3­
20
mg/
kg),
oxamyl
(
0.07­
1.5
mg/
kg),
methomyl
(
0.1­
2.5
mg/
kg),
methiocarb
(
0.5­
25
mg/
kg),
formetanate
(
0.1­
5
mg/
kg),
or
carbofuran
(
0.1­
1.5
mg/
kg).
Ten
minutes
after
dosing,
rats
were
scored
for
obvious
signs
of
cholinergic
toxicity
("
tox
score").
Horizontal
and
vertical
activity
were
tabulated
during
subsequent
20­
minute
sessions
in
a
figure­
eight
chamber.
Brain
and
blood
tissues
were
taken
immediately
thereafter
to
determine
ChE
activity
as
described
above.

As
shown
in
Figure
II.
B.
5­
2,
exposure
to
each
of
the
NMCs
resulted
in
decreased
motor
activity.
The
vertical
activity
dose­
response
curves
were
significantly
different
from
horizontal
activity,
with
greater
decreases
in
vertical
activity
at
the
higher
doses.
Nonetheless,
the
correlation
between
horizontal
and
vertical
activity
was
good,
indicating
a
high
concordance
of
effect
on
both
measures.
Within­
subject
regressions
indicated
a
better
correlation
between
motor
activity
and
brain
ChE
inhibition
than
with
blood
ChE.
Brain
ChE
is
equally
or
more
sensitive
at
the
low
end
of
the
dose­
response.

The
ranking
of
toxicity
as
measured
by
the
"
tox
score"
ranged
from
no
effect
(
carbaryl,
formetanate)
to
a
dose­
related
increase
in
the
incidence
and
severity
(
propoxur,
carbofuran).
Thus,
some
NMCs
showed
pronounced
effects
on
motor
activity
at
doses
that
produced
little
or
no
obvious
toxicity
as
evaluated
by
this
clinical
observation.
This
illustrates
that
observable
signs
of
cholinergic
toxicity
are
not
always
predictive
of
the
magnitude
of
behavioral
neurotoxicity.
In
contrast,
motor
activity
is
a
sensitive
endpoint
that
reveals
the
biological
relevance
of
ChE
inhibition
produced
by
these
N­
methyl
carbamates.

For
some
NMCs,
data
are
available
for
motor
activity
and
clinical
signs
in
studies
submitted
for
pesticide
registration.
The
Agency
has
not
yet
extracted
the
motor
activity
data
from
registration
studies
for
comparison
with
EPA's
behavioral
data
or
for
formal
comparison
with
ChE
data.
However,
evaluation
of
summary
information
provided
in
EPA's
study
reviews
(
ie,
Data
Evaluation
Records,
not
provided
here)
indicates
that
ChE
is
equally
or
more
sensitive
than
behavioral
effects.
II.
B.
5
­
Page
8
of
12
Figure
II.
B.
5­
2.
Plots
of
motor
activity
data
from
NHEERL
experiments
for
seven
N­
methyl
carbamates
Propoxur
Dose
mg/
kg
0.00.31.0
3.0
10.0
20.0
%
Control
±
SEM
0
20
40
60
80
100
120
horizontal
vertical
*

*

**

*
*
Tox
Score
0.0
0.3
1.0
3.0
10.0
20.0
%
Showing
Signs
0
20
40
60
80
100
Methiocarb
Dose
mg/
kg
0.000.50
2.00
5.00
12.00
25.00
%
Control
±
SEM
0
20
40
60
80
100
120
140
horizontal
vertical
**

*
*
*
*
*
*
Tox
Score
0.0
0.5
2.0
5.0
12.0
25.0
%
Showing
Signs
0
20
40
60
80
100
Oxamyl
Dose
mg/
kg
0.00
0.07
0.10
0.50
1.00
1.50
%
Control
±
SEM
0
20
40
60
80
100
120
140
horizontal
vertical
*
*
*
*
Tox
Score
0.0
0.1
0.1
0.5
1.0
1.5
%
Showing
Signs
0
20
40
60
80
100
*
Methomyl
Dose
mg/
kg
0.
000.10
0.25
0.60
1.25
2.5
0
%
Control
±
SEM
0
20
40
60
80
100
120
140
horizontal
vertical
*
*
*
*
Tox
Score
0.00
0.10
0.25
0.60
1.25
2.50
%
Showing
Signs
0
20
40
60
80
100
Carbofuran
Dose
(
mg/
kg)
0.00
0.10
0.30
0.50
0.75
1.50
Percent
control
activity
0
20
40
60
80
100
120
140
horizontal
vertical
*
*
**
*

*

*
*
Tox
Score
0.00
0.10
0.30
0.50
0.75
1.50
%
Showing
Signs
0
20
40
60
80
100
Formetanate
Dose
(
mg/
kg)
0.00.10.3
0.6
1.5
5.0
Percent
control
activity
0
20
40
60
80
100
120
140
horizontal
vertical
*
*

*
*
Tox
Score
0.0
0.1
0.3
0.6
1.5
5.0
%
Showing
Signs
0
20
40
60
80
100
*
*

Carbaryl
Dose
mg/
kg
0.0
3.0
7.5
15.0
30.0
50.0
%
Control
±
SEM
0
20
40
60
80
100
120
horizontal
vertical
*

*
*
*
*
*

*
Tox
Score
all
normal
*
II.
B.
5
­
Page
9
of
12
e.
Summary
of
Protocol
and
Preliminary
Results
of
Seven
Chemical
(
Equipotent)
Mixture
Experiment
A
mixture
study
was
conducted
using
7
N­
methyl
carbamates
(
carbaryl,
carbofuran,
formetanate,
methiocarb,
methomyl,
oxamyl,
and
propoxur).
A
doseadditive
experimental
design
was
used
and
the
proportion
of
the
carbamates
in
the
mixture
was
based
on
their
potency:
they
were
combined
in
an
equipotent
manner
using
the
individual­
chemical
benchmark
values
as
the
point
of
comparison.
The
composition
of
the
mixture
is
shown
in
Table
II.
B.
5­
2.

Table
II.
B.
5­
2.
Composition
of
a
seven
chemical
mixture
with
the
NMCs
N­
Methyl
Carbamate
Pesticide
Percentage
of
Mixture
Carbaryl
41.60%

Carbofuran
1.46%

Formetanate
1.63%

Methiocarb
19.60%

Methomyl
5.05%

Oxamyl
1.49%

Propoxur
29.10%

The
mixture
study
consisted
of
13
dosage
groups
with
10
male,
Long­
Evans
rats
(
90
days
old)
in
each
group.
The
N­
methyl
carbamates
were
given
in
two
vehicles:
either
corn
oil
(
carbaryl,
carbofuran,
methiocarb
and
propoxur)
or
water
(
formetanate,
oxamyl,
and
methomyl).
Every
animal
received
both
vehicles,
one
dose
immediately
after
the
other.
Five
different
dosage
levels
of
the
mixture
were
given,
predicted
to
produce
<
5%,
10%,
25%,
45%
or
60%
brain
cholinesterase
inhibition,
and
also
each
N­
methyl
carbamate
was
given
alone
at
a
previously
tested
dosage
to
confirm
the
original
dose­
response
data
(
7
singlechemical
experimental
groups).
The
control
group
received
the
vehicles
only.
The
study
was
staggered
over
four
consecutive
days
with
all
dosage
levels
being
tested
each
day.
All
dosing
solutions
were
made
fresh
each
day.
The
animals
were
dosed,
clinical
observation
of
the
degree
of
cholinergic
toxicity
was
recorded
10
minutes
after
dosing,
and
then
motor
activity
was
assessed
for
the
next
20
minutes.
Immediately
after
removal
from
the
activity
chambers,
the
animals
were
briefly
anesthesized
with
CO2
and
killed
by
40
minutes
after
dosing.
Brains
were
taken
and
frozen
at
­
80

C
until
analysis;
RBCs
were
prepared
from
trunk
blood
samples.
Cholinesterase
activity
was
determined
using
the
radiometric
assay
as
previously
described.

As
can
be
seen
from
Figure
II.
B.
5­
3
below,
increasing
dosages
of
the
mixture
produced
increasing
decrements
in
brain
cholinesterase
activity.
II.
B.
5
­
Page
10
of
12
Moreover,
the
dose­
additive
model
predicted
the
degree
of
cholinesterase
inhibition
within
the
95%
confidence
limits
of
each
predicted
value.

It
is
important
to
note
that
the
single
dose
repeat
of
each
N­
methyl
carbamate
(
open
symbols,
Figure
II.
B.
5­
4)
overlapped
the
original
doseresponse
curve
values
for
each
methyl
carbamate,
except
for
methomyl
and
propoxur
which
showed
5­
10%
less
activity.

Figure
II.
B.
5­
3.
Brain
cholinesterase
results
from
seven
chemical
equipotent
mixture
Dose
of
Mixture
(
mg/
kg)
0
2
4
6
8
10
12
14
16
Cholinesterase
activity
(%
control)

20
30
40
50
60
70
80
90
100
110
120
Expected
Values
(+
95%
confidence
limits)
Acutal
Data
(+
sem)

Dose­
Additive
Design
Equipotent
Mixture
Control
II.
B.
5
­
Page
11
of
12
Figure
II.
B.
5­
4.
Comparison
of
brain
cholinesterase
from
dose­
response
and
single
dosing
in
the
mixture
study
Single
Dosing
in
the
Mixture
Study
Dose
(
mg/
kg)
0.1
1
10
100
Cholinesterase
(%
control)

0
10
20
30
40
50
60
70
80
90
100
110
120
Propoxur
Carbaryl
Oxamyl
Methomyl
Methiocarb
Folrmetanate
Carbofuran
Open
Symbols
are
Single
Dose
Repeats
in
Mixture
Study
II.
B.
5
­
Page
12
of
12
f.
References
Moser,
V.
C.,
Phillips,
P.
M.,
McDaniel,
K.
L.,
Marshall,
R.
S.,
Padilla,
S.,
Lowit,
A.
2005.
Comparison
of
acute
neurobehavioral
effects
of
N­
methyl
carbamate
insecticides.
Presented
at
the
44th
Annual
Meeting
of
the
Society
of
Toxicology;
New
Orleans,
Louisiana.
