Document ID: EPA-HQ-ORD-2006-0187-0004
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2006-03-14T05:00Z

Fax­
on­
Demand
Telephone:
(
202)
401­
0527
Item
No.:
6065
Office
of
Pesticide
Programs
Science
Policy
on
The
Use
of
Data
on
Cholinesterase
Inhibition
for
Risk
Assessments
of
Organophosphorous
and
Carbamate
Pesticides
August
18,
2000
Office
of
Pesticide
Programs
US
Environmental
Protection
Agency
Washington
DC
20460
TABLE
OF
CONTENTS
EXECUTIVE
SUMMARY
1
LIST
OF
ABBREVIATIONS
4
ORGANIZATION
OF
POLICY
DOCUMENT
5
1.
INTRODUCTION
7
1.1
RISK
ASSESSMENT
FRAMEWORK
7
1.2
BIOLOGY
AND
TOXICOLOGY
OF
CHOLINESTERASE
INHIBITION
......
9
2.
HISTORICAL
BACKGROUND
14
2.1
OPP
=

s
HISTORICAL
APPROACH
14
2.2
REVIEWS
OF
PROPOSED
AGENCY/
OPP
SCIENCE
POLICY
POSITIONS..............................................................................................
15
3.
IDENTIFICATION
OF
THE
TOXICOLOGICAL
ENDPOINTS
FOR
ASSESSMENT
OF
CHOLINESTERASE
INHIBITORS
18
3.1
EVALUATION
OF
EFFECTS
ON
CHOLINERGIC
FUNCTIONS
19
3.1.1
CONCLUSIONS
19
3.1.2
RATIONALE..................................................................................
20
3.2
NERVOUS
SYSTEM
CHOLINESTERASE
INHIBITION
25
3.2.1
CONCLUSIONS
25
3.2.2
RATIONALE..................................................................................
26
3.3
BLOOD
CHOLINESTERASE
INHIBITION
28
3.3.1
CONCLUSIONS
28
3.3.2
RATIONALE..................................................................................
30
4.
WEIGHT­
OF­
THE­
EVIDENCE
ANALYSIS
FOR
SELECTION
OF
CRITICAL
EFFECTS
34
4.1
ANALYSIS
OF
INDIVIDUAL
STUDIES
37
4.2
INTEGRATIVE
ANALYSIS
OF
THE
DATA
BASE....................................
38
4.3
COLLECTION
OF
PERIPHERAL
NERVOUS
SYSTEM
CHOLINESTERASE
INHIBITION
DATA..................................................
41
5.
CONCLUSION
43
6.
REFERENCES
45
EXECUTIVE
SUMMARY
The
Office
of
Pesticide
Programs
(
OPP)
published
a
policy
statement
on
the
use
of
data
on
cholinesterase
inhibition
(
and
other
events
associated
with
cholinergic
effects
related
to
nervous
system
function)
in
human
health
risk
assessment
of
certain
classes
of
pesticide
chemicals
for
review
by
the
FIFRA
Scientific
Advisory
Panel
(
SAP)

in
1997
and
for
public
comment
in
1997
and
1998
(
US
EPA,
1997b).
The
1997
science
policy
document
described
the
approaches
OPP
would
employ
in
assessing
the
potential
for
human
health
hazard
from
the
cholinergic
effects
on
nervous
system
function
following
exposure
to
cholinesterase­
inhibiting
pesticides.

The
1997
policy
document
has
been
reorganized
and
revised,
taking
into
consideration,
as
appropriate,
comments
offered
by
the
public,
the
SAP,
other
EPA
offices
and
other
government
agencies.
As
did
the
1997
policy,
this
revised
science
policy
emphasizes
the
weighing
of
all
relevant
evidence
when
selecting
endpoints
for
the
hazard
assessment
of
anticholinesterase
pesticides.
This
"
weight­
of­
the­
evidence"

review,
conducted
on
a
case­
by­
case,
chemical­
by­
chemical
basis,
is
accomplished
by
performing
an
integrative
analysis
after
assessing
all
the
individual
lines
of
evidence
(
including
all
available
data
on
cholinesterase
inhibition
in
all
compartments
­­
central
nervous
system,
peripheral
nervous
system,
red
blood
cells,
and
plasma
­­
as
well
as
data
on
clinical
signs,
symptoms
and
other
physiological
or
behavioral
effects).

Weighing
of
the
evidence
must
include
considerations
of
many
factors,
including
the
adequacy
of
study
protocols;
quality
of
data;
number
of
studies
on
each
endpoint;

dose­
dependency
of
responses;
time
course
and
duration
of
effects;
and
similarities
or
differences
of
responses
observed
in
all
the
species,
strains,
and
sexes
tested
for
each
duration
and
route
of
exposure
evaluated.

In
a
weight­
of­
the­
evidence
assessment
of
cholinesterase­
inhibiting
substances,

acetylcholinesterase
inhibition
in
the
nervous
system
is
viewed
as
a
key
event
in
the
mechanism
of
toxicity
of
these
compounds
and
an
important
critical
effect
to
consider
in
the
hazard
assessment.
Evaluations
of
the
cholinergic
effects
(
i.
e.,
physiological
and
behavioral
changes
and
measures
of
cholinesterase
inhibition
in
the
central
and
peripheral
nervous
systems)
caused
by
exposure
to
the
cholinesterase­
inhibiting
organophosphorous
and
carbamate
pesticides
provide
direct
evidence
for
characterizing
potential
human
health
hazard.
Because
of
likely
differences
in
both
the
chemicals
=

and
the
cholinesterases
=

pharmacodynamic
properties,
measures
of
cholinesterase
inhibition
in
both
the
central
and
peripheral
nervous
systems
are
important
for
a
thorough
evaluation
of
potential
hazard.
However,
direct
measurement
of
cholinesterase
activity
in
peripheral
nervous
system
tissues
are
rarely
available
at
the
present
time.
When
these
data
are
not
available,
as
a
matter
of
prudent
science
policy
protective
of
human
health,
EPA
will
treat
cholinesterase
inhibition
in
the
blood
as
a
surrogate
measure
for
the
peripheral
nervous
system
in
animals
and
for
both
the
peripheral
and
central
nervous
systems
in
humans.
Information
from
blood
cholinesterase
inhibition
data
is
considered
to
provide
important
insights
into
potential
hazard.
Red
blood
cell
(
RBC)
measures
of
acetylcholinesterase
(
AChE)
are
generally
preferred
over
plasma
measures
of
cholinesterase
activity
because
data
on
red
blood
cells
may
provide
a
better
representation
of
the
inhibition
of
the
neural
target
enzyme,

acetylcholinesterase.
OPP,
however,
may
use
plasma
cholinesterase
inhibition
data
under
certain
circumstances,
such
as
if
red
blood
cell
data
are
insufficient,
of
poor
quality,
or
unavailable;
if
there
is
a
lack
of
dose­
dependency
for
the
red
blood
cell
acetylcholinesterase
inhibition;
or,
if
the
dose
responses
for
inhibition
of
plasma
cholinesterase
more
closely
approximate
those
for
AChE
inhibition
in
the
nervous
system
than
do
the
dose
responses
for
RBC
acetylcholinesterase
inhibition.

It
should
be
noted
that
the
present
policy
provides
guidance
only
on
how
to
deal
with
data
as
they
relate
to
the
cholinergic
endpoints
associated
with
nervous
system
function
following
exposure
to
organophosphorous
and
carbamate
pesticides.
This
scope
is
consistent
with
all
earlier
descriptions
of
Agency
assessment
approaches
as
well
as
that
of
other
organizations
with
regard
to
the
evaluation
of
cholinesteraseinhibiting
substances
(
e.
g.,
WHO
JMPR
(
1990,1999),
DPR­
CalEPA
(
1997)
and
other
national
authorities
such
as
Canada's
PMRA
(
Franklin,
1999).
When
applying
the
weight­
of­
the­
evidence
approach
for
selecting
critical
effect(
s)
for
derivation
of
a
reference
dose
(
RfD)
or
concentration
(
RfC),
however,
the
entire
toxicological
data
base
on
a
pesticide
must
be
evaluated
(
i.
e.,
there
also
must
be
consideration
of
endpoints
not
related
to
the
cholinergic
consequences
of
anticholinesterase
activity,
for
instance,
liver
or
developmental
toxicity
or
carcinogenicity).
It
is
possible
that,
for
one
or
more
of
the
exposure
scenarios
being
evaluated,
the
non­
cholinergic
effects
will
be
identified
as
critical
or
co­
critical,
and
they
may
become
a
more
appropriate
basis
for
deriving
RfDs
or
RfCs.

Finally,
OPP
policy
documents
are
meant
to
be
A
living
documents,@
that
is,
they
are
open
to
periodic
updating
and
revision
to
reflect
advances
in
the
science.
Thus,

this
policy,
too,
will
be
updated
to
incorporate
important
new
scientific
knowledge
as
it
becomes
available.
For
example,
the
routine
availability
of
data
on
acetylcholinesterase
activity
in
the
peripheral
nervous
system
may
allow
for
refinements
in
the
hazard
assessment
approach
for
anticholinesterase
chemicals.

Also,
as
knowledge
increases
about
the
potential
roles
of
the
different
cholinesterases
in
the
developing
organism,
particularly
as
they
impact
the
development
of
the
nervous
system,
it
may
allow
for
refinements
in
evaluating
the
potential
differential
sensitivity
and
susceptibility
of
the
young
versus
adults.
In
fact,
a
substantial
research
effort
has
been,
and
continues
to
be,
made
to
determine
what
roles
acetylcholine­,

butyrylcholineand
other
esterases
may
play
in
the
development
of
the
nervous
system
and
in
cell
growth,
proliferation
and
death
in
other
tissues.
OPP
encourages
further
discussion
of
the
possible
implications
of
the
research
findings,
both
for
future
research
planning
and
for
the
Agency
=

s
regulation
of
cholinesterase­
inhibiting
pesticides.
LIST
OF
ABBREVIATIONS
Scientific
Terms:

ACHE
Acetylcholinesterase
BMD
Benchmark
Dose
BuChE
Butyrylcholinesterase
LOAEL
Lowest­
Observed­
Adverse­
Effect
Level
MF
Modifying
factor
NOAEL
No­
Observed­
Adverse­
Effect
Level
PoD
Point
of
Departure
RBC
Red
Blood
Cell
(
or
erythrocyte)

RfC
Reference
Concentration
RfD
Reference
Dose
UF
Uncertainty
Factor
Organizational
Terms:

DPR­
CalEPA
Department
of
Pesticide
Regulation­
California
Environmental
Protection
Agency
FIFRA
SAP
EPA
=

s
FIFRA
Scientific
Advisory
Panel
ILSI
International
Life
Sciences
Institute
PMRA
Canada
Pesticide
Management
Regulatory
Agency­
Canada
NRC/
NAS
National
Research
Council­
National
Academy
of
Sciences
OPP
Office
of
Pesticide
Programs
SAB
EPA
=

s
Science
Advisory
Board
SAP
EPA
=

s
FIFRA
Scientific
Advisory
Panel
TRAC
Tolerance
Reassessment
Advisory
Committee
WHO/
FAO
JMPR
World
Health
Organization/
Food
and
Agricultural
Organization
Joint
Meeting
on
Pesticide
Residues
ORGANIZATION
OF
POLICY
DOCUMENT
This
science
policy
document
describes
the
approaches
that
the
Office
of
Pesticide
Programs
(
OPP)
employs
when
evaluating
data
on
cholinesterase
inhibition
and
other
cholinergic
effects
related
to
nervous
system
function
that
are
the
consequences
of
acetycholinesterase
inhibition
(
i.
e.,
physiological
or
behavioral
changes)
in
assessing
the
potential
hazard
following
exposure
to
organophosphorous
or
carbamate
pesticides
that
inhibit
cholinesterase.

 
Section
1(
Introduction)
presents
a
very
brief
description
of
the
Agency
=

s
general
approach
to
non­
cancer
risk
assessment.
This
chapter
also
includes
a
brief
introduction
to
the
biology
of
the
cholinergic
nervous
system,
description
of
cholinesterase
enzymes
and
their
distribution,
the
inhibition
of
acetylcholinesterase
as
a
mechanism
of
toxicity
for
cholinesterase­
inhibiting
pesticides,
and
the
consequences
of
acetylcholinesterase
inhibition
in
the
body.

 
Section
2
(
Historical
Background)
presents
the
history
that
has
led
to
the
development
of
this
policy
document.

 
Section
3
(
Identification
of
the
Toxicological
Endpoints
for
Assessment
of
Cholinesterase
Inhibitors)
describes
the
scientific
rationale
for
selection
of
endpoints
for
anticholinesterase
pesticides,
their
toxicological
significance
and
relevance
to
hazard
assessment.

 
Section
4
(
Weight­
of­
the­
Evidence
Analysis
for
Selection
of
Critical
Effects)

describes
the
weight­
of­
the­
evidence
approach
that
is
used
to
select
the
critical
effect(
s)
for
the
risk
assessment
of
anticholinesterase
pesticides.
 
Section
5
(
Conclusions)
provides
a
summary
of
the
key
elements
of
the
weightof
the­
evidence
approach
that
OPP
uses
when
evaluating
anticholinesterase
organophosphorous
and
carbamate
pesticides.

 
Section
6
(
References)
contains
the
literature
citations
and
other
references
used
as
source
material
for
the
policy
document.
1.
INTRODUCTION
The
purpose
of
this
document
is
to
set
forth
the
principles
and
procedures,

including
a
weight­
of­
the­
evidence
approach,
used
by
OPP
for
the
selection
of
appropriate
endpoints
for
assessing
potential
hazards
to
humans
exposed
to
anticholinesterase
pesticides.
In
addition,
this
science
policy
document
also
describes
science
policy
approaches
specific
to
effects
related
to
cholinesterase
inhibition
that
will
be
used
to
address
inadequacies
in
data
or
lack
of
knowledge.
The
Agency=
s
policy
which
addresses
the
potential
for
pre­
and
postnatal
effects
and
the
completeness
of
databases
with
respect
to
toxicity
and
exposure
as
they
relate
to
infants
and
children
when
conducting
risk
assessments
and
making
regulatory
decisions
regarding
the
setting
of
tolerances
(
residues
in
food)
under
the
1996
Food
Quality
Protection
Act
can
be
found
in
the
draft
guidance
document
entitled
AThe
Office
of
Pesticide
Programs=
Policy
on
Determination
of
the
Appropriate
FQPA
Safety
Factor(
s)
for
Use
in
the
Tolerance­
setting
Process@
(
US
EPA,
1999b).

1.1
RISK
ASSESSMENT
FRAMEWORK
Regulatory
decision
making
in
EPA
is
described
as
consisting
of
two
major
steps­­
risk
assessment
and
risk
managementBwhich
are
closely
related
but
different
processes.
Risk
assessment
defines
the
potential
for
adverse
effects
1
to
occur
in
individuals
or
populations,
while
risk
management
weighs
risk
reduction
alternatives
and
integrates
the
risk
assessment
with
social,
economic,
and
other
factors,
as
appropriate.
The
Agency
uses
the
paradigm
put
forward
by
the
National
Research
Council
of
the
National
Academy
of
Sciences
in
1983
and
modified
in
1994
(
NRC/
NAS,

1983;
1994)
that
defines
and
organizes
risk
assessment
into
four
phases:
hazard
identification,
dose­
response
assessment,
exposure
assessment,
and
risk
characterization.
Risk
assessment
for
noncancer
effects
including
those
addressed
in
1
Adverse
effects
include
alterations
from
the
baseline
that
diminish
an
organism's
ability
to
survive,
reproduce,
or
adapt
to
the
environment.
Neurotoxicity
is
defined
as
an
adverse
change
in
the
structure
or
function
of
the
central
and/
or
peripheral
nervous
system
following
exposure
to
an
agent
(
US
EPA
1998a).
this
policy
is
generally
based
on
identifying
a
no­
observed­
adverse­
effect­
level
(
NOAEL)
or
calculating
a
benchmark
dose
(
BMD)
for
a
critical
effect
2
,
which
is
usually
determined
from
laboratory
animal
studies,
for
use
as
a
Point
of
Departure
(
PoD)
when
deriving
a
reference
dose
(
RfD)
or
reference
concentration
(
RfC).
The
point
of
departure
(
PoD)
is
generally
defined
as
a
point
estimate
of
an
empirically­
measured
or
modeled
dose
or
exposure
level
that
is
used
as
the
Ajumping­
off@
point
for
extrapolation
to
exposure
levels
below
those
tested,
where
actual
human
exposures
are
actually
likely
to
be
occurring.
The
PoD
can
be
a
dose
at
which
no
effects
are
found
or
a
dose
level
which
is
associated
with
some
percent
of
response
relative
to
the
control
or
baseline
level
of
response.
The
PoD
is
divided
by
one
or
more
uncertainty
factors
(
UF)

or
modifying
factors
(
MF).
The
UFs
(
typically
3­
or
10­
fold
in
magnitude)
reflect
uncertainties
inherent
in
the
extrapolation
from
laboratory
animal
species
to
humans
(
the
interspecies
UF),
in
the
variations
in
sensitivity
among
members
of
the
human
population
(
the
intraspecies
UF),
for
the
use
of
subchronic
rather
than
chronic
data
(
the
subchronic
to
chronic
UF),
the
use
of
a
lowest­
observed­
adverse­
effect
level
(
LOAEL)

rather
than
a
NOAEL
(
the
LOAEL
to
NOAEL
UF),
and
the
comprehensiveness
and
quality
of
the
database
available,
i.
e.,
whether
or
not
all
potential
endpoints
of
concern
are
identified
and
evaluated
in
acceptable
studies
(
the
database
UF).
A
modifying
factor
may
be
used
to
address
scientific
uncertainties
in
the
principal
study
used
for
RfD/
C
derivation
which
are
not
explicitly
addressed
by
the
other
standard
Ufs.

2
ACritical
effect@
is
defined
in
EPA=
s
Integrative
Risk
Information
System
(
IRIS)
as
Athe
first
adverse
effect,
or
its
known
precursor,
that
occurs
as
the
dose
rate
increases.@
Information
on
the
derivation
of
reference
doses
or
reference
concentrations
can
be
found
at
the
IRIS
web
site
http://
www.
epa.
gov/
ngispgm3/
iris/
rfd.
htm.
The
result
of
dividing
a
PoD
by
the
appropriate
uncertainty
factors
and/
or
modifying
factor
is
a
reference
dose
(
RfD)
for
oral
or
dermal
exposuresBor
reference
concentration
(
RfC)
for
inhalation
exposure(
s).
The
RfD
or
RfC
is
defined
as
an
estimate,
within
an
order
of
magnitude,
of
exposure
assumed
to
be
without
appreciable
risk
for
adverse
noncancer
health
effects.
In
the
risk
characterization
step,
the
RfD
and
RfC
values
are
compared
to
potential
or
known
exposure
levels.
Risk
characterization
also
fully
describes
the
nature
and
extent
of
the
risks
posed,
and
how
well
the
data
support
the
conclusions,
including
a
discussion
of
the
limitations
and
uncertainties
involved.
Sometimes,
because
of
these
limitations
and
uncertainties,
further
data
may
be
collected
to
reduce
the
uncertainties
and
refine
the
risk
assessment.

The
Agency
has
acknowledged
that
the
historical
approach
to
defining
a
NOAEL
and
calculating
RFDs
and
RfCs
has
limitations
(
see
USEPA,
1994;
1995;
1996).
In
response,
the
Agency
has
developed
draft
guidance
on
an
alternative
methodBthe
Benchmark
Dose
(
BMD)
Approach
(
USEPA,
1996).
The
BMD
is
defined
as
the
statistical
lower
confidence
limit
on
the
dose
producing
a
predetermined
level
of
change
in
response
compared
with
the
background
response.
A
BMD
is
derived
by
fitting
a
mathematical
model
to
the
dose­
response
data.
3
The
Agency
is
still
gaining
experience
with
BMD
analyses
and
has
not
yet
formally
finalized
standard
operating
procedures.
OPP,
however,
will
use
the
BMD
approach
for
derivation
of
RfDs
and
RfCs
to
the
extent
possible.

1.2
BIOLOGY
AND
TOXICOLOGY
OF
CHOLINESTERASE
INHIBITION
Acetylcholine
plays
an
important
role
in
the
functioning
of
the
nervous
system.
4
3
Draft
benchmark
software
for
hazard
endpoints
is
currently
available
at
http:\
www.
epa.
gov\
ncea\
bmds.
4
Summary
reviews
of
the
cholinergic
components
of
the
nervous
system
and
of
the
toxicity
of
anticholinesterase
chemicals
can
be
found
in
several
chapters
of
two
widely­
available
textbooks
(
Ecobichon
(
1996),
Hoffman,
et
al.,
(
1996),
and
Taylor
(
1996a,
1996b))
and
in
Dementi
(
1997)
which
served
as
a
technical
support
document
to
the
1997
policy
document.
Acetylcholine
is
a
neurotransmitter
which
enables
chemical
communication
to
occur
between
a
nerve
cell
and
a
target
cell.
This
target
cell
may
be
another
nerve
cell,

muscle
fiber
or
gland.
Upon
stimulation,
the
nerve
cell
releases
acetylcholine
into
the
synapse
(
or
space)
between
the
two
cells.
This
released
acetylcholine
binds
to
receptors
on
a
target
cell,
thereby
passing
the
signal
on
to
that
nerve
cell,
muscle
or
gland.
The
end
result
of
the
stimulation
of
cholinergic
pathway(
s)
includes,
for
example,
the
contraction
of
smooth
(
e.
g.,
in
the
gastrointestinal
tract)
or
skeletal
muscle,
changes
in
heart
rate
or
glandular
secretion
(
e.
g.,
sweat
glands)
or
communication
between
nerve
cells
in
the
brain
or
in
the
autonomic
ganglia
of
the
peripheral
nervous
system.
Cholinergic
pathways
innervate
virtually
every
organ
in
the
body,
including
the
brain
and
peripheral
nervous
system.

There
are
two
major
divisions
of
the
nervous
system,
both
of
which
contain
cholinergic
pathways
that
may
be
affected
by
cholinesterase­
inhibiting
chemicals:

 
the
peripheral
nervous
system,
consisting
of
neuromuscular
junctions
in
skeletal
muscle,
and
tissues
of
the
autonomic
nervous
system,
consisting
of
ganglia
of
the
sympathetic
and
parasympathetic
nervous
systems,
smooth
muscles,
cardiac
muscle,
and
glands;
and
 
the
central
nervous
system,
consisting
of
brain
and
spinal
cord.

The
distribution
of
cholinergic
receptors
in
the
central
nervous
system
and
the
peripheral
nervous
system
is
not
uniform
(
Brimijoin,
1992).
For
example,
certain
brain
regions
of
the
mature
organism
are
rich
in
cholinergic
neurons
(
e.
g.,
the
striatum,

hippocampus,
cerebral
cortex),
while
other
regions
are
less
well
innervated
and
have
less
cholinesterase
activity
(
e.
g.,
cerebellum).
There
are
two
major
types
of
cholinergic
receptors
­­
muscarinic
and
nicotinic
­­
and
there
are
several
subtypes
of
each.
These
receptor
types
also
are
differentially
distributed
in
different
regions
of
the
central
and
peripheral
nervous
systems,
thus
contributing
to
the
complexity
of
effects
that
may
occur.

Acetylcholinesterase
(
AChE)
is
found
in
cholinergic
neurons,
in
the
vicinity
of
synapses,
and
in
other,
non­
neural
tissues.
It
is
highly
concentrated
at
the
neuromuscular
and
other
neuroeffector
junctions.
It
is
the
enzyme
that
breaks
down
acetylcholine
and
terminates
its
action
in
the
synapses
between
neurons
and
between
neurons
and
muscle
fibers
or
glands.
Inhibition
of
AChE
leads
to
an
accumulation
of
acetylcholine
and
a
prolongation
of
the
action
of
acetylcholine
at
the
nerve­
nerve,

nerve­
muscle
or
nerve­
gland
interface.
Peripherally,
the
accumulation
of
acetylcholine
can
result
in
cholinergic
responses
such
as
smooth
muscle
contractions
(
e.
g.,

abdominal
cramps),
glandular
secretions
(
e.
g.,
sweating),
skeletal
muscle
twitching,

and,
at
higher
concentrations,
flaccid
paralysis.
In
addition,
there
may
be
centrallymediated
effects
on
learning,
memory
and
other
behavioral
parameters.
Thus,
the
inhibition
of
AChE
potentially
results
in
a
broad
range
of
adverse
effects,
having
an
impact
on
most
bodily
functions,
and
depending
on
the
magnitude
and
half­
life
of
an
exposure
dose,
these
effects
can
be
serious,
even
fatal.

Effects
caused
by
AChE
inhibition
may
be
a
result
of
action
on
neurons
in
the
central
nervous
system
and/
or
the
peripheral
nervous
system.
Access
of
chemicals
to
the
central
nervous
system
and
the
peripheral
nervous
system
may
be
different
because
of
differences
in
pharmacokinetic
properties
of
these
two
compartments
(
e.
g.,

differences
in
absorption,
distribution,
metabolism,
elimination).
These
differences
may
be
due
to
chemical
specific
characteristics
as
well
as
the
characteristics
of
the
exposed
organism
(
e.
g.,
degree
of
maturation
of
the
blood­
brain
barrier).
The
pattern
of
effects
seen
may
also
depend
upon
factors
such
as
the
pharmacodynamic
characteristics
(
i.
e.,

binding
potency,
rate
of
reversal)
of
the
cholinesterase­
inhibiting
chemical
and
the
molecular
form
of
cholinesterase
with
which
it
is
interacting
(
e.
g.,
see
Scarsella,
et
al.,

1979).

Butyrylcholinesterase
(
BuChE)
is
similar
in
structure
to
AChE,
but
it
is
encoded
by
a
separate
gene.
BuChE,
which
is
synthesized
primarily
in
the
liver
and
found
in
plasma
and
other
tissues,
is
generally
distinguished
from
AChE
by
BuChE=
s
slower
rate
of
hydrolysis
of
acetylcholine,
by
function
and
by
localization
using
histochemical
techniques
after
subjecting
the
experimental
model
to
inhibitors
which
selectively
block
the
activity
of
one
but
not
the
other
enzyme
(
Taylor
and
Radic,
1994).
Furthermore,
the
binding
affinity
of
anticholinesterase
chemicals
for
each
enzyme
can
differ
among
these
substances
(
Silver,
1974;
Taylor
and
Radic,
1994).
Both
enzymes
are
present
during
development
of
the
nervous
system,
with
the
ratios
of
one
to
the
other
changing
substantially
over
time
and
with
location
(
Hoffman,
et
al.,
1996).
While
no
neurological
function
has
been
shown
definitively
for
BuChE
in
the
developing
or
mature
nervous
system,
the
BuChE
present
in
the
plasma
will
catalyze
the
hydrolysis
and
inactivation
of
ingested
esters
from
plant
sources
(
e.
g.,
cocaine
and
related
synthetic
local
anesthetics
(
Hoffman,
et
al.,
1996)
and
neuromuscular
blocking
agents
such
as
succinylcholine
(
Taylor,
1996b).
Likewise,
there
is
no
known
physiological
or
biochemical
function
for
erythrocyte
acetylcholinesterase
(
Brimijoin,
1992;
Dementi,

1997).

As
discussed
later,
the
blood
cholinesterase
enzymes
are
regarded,
as
a
matter
of
policy,
as
surrogate
measures
of
neuronal
cholinesterase
activity.
Of
the
two
common
blood
elements
measured,
red
blood
cells
(
RBC)
contain
AChE
exclusively,

while
the
ratio
of
AChE
to
BuChE
in
plasma
varies
widely
among
humans,
dogs,
and
rats,
the
species
in
which
these
measures
are
most
typically
made
for
risk
assessment
and
regulatory
purposes.
While
human
plasma
is
overwhelmingly
BuChE,
the
plasma
of
dogs
and
rats
contains
both
AChE
and
BuChE
(
Scarsella,
1979;
Edwards
and
Brimijoin,
1983).

The
question
of
whether,
and,
if
so,
how,
BuChE
plays
a
role
in
the
development
and/
or
functioning
of
the
nervous
system
still
awaits
resolution.
Research
has
been,

and
continues
to
be,
conducted
to
determine
if
butyrylcholinesterase
plays
a
role
in
nervous
system
morphogenesis
(
development)
and
function,
and
whether,
and
if
so,

how,
butyryl­
and/
or
acetylcholinesterase
and
other
esterases
play
a
more
general
role
in
cell
growth
and
death,
including
in
carcinogenesis.
In
addition,
the
dose
response
relationships
attendant
to
acetylcholinesterase=
s
function(
s)
in
the
development
of
the
nervous
system
remain
to
be
described
and
compared
with
those
of
the
endpoints
currently
used
in
the
evaluation
of
nervous
system
function.
OPP
is
preparing
a
brief
summary
of
the
available
literature
on
the
role
of
the
cholinesterases
(
and,
perhaps,

other
esterases)
in
these
areas.
OPP
also
is
preparing
a
series
of
questions
to
serve
as
a
starting
point
for
discussion
for
addressing
whether
or
not
this
information
may
justify
further
revisions
to
the
present
policy.
This
effort
is
being
conducted
separately
from
the
revision
of
the
current
policy
document.
2.
HISTORICAL
BACKGROUND
2.1
OPP
=

s
HISTORICAL
APPROACH
Cholinesterase
inhibition
and
the
cholinergic
effects
(
i.
e.,
the
physiological
or
behavioral
changes)
caused
by
organophosphorous
and
carbamate
pesticides
have
long
been
endpoints
that
OPP
has
used
in
assessing
potential
human
health
hazards.

For
well
over
a
decade,
OPP
has
regarded
data
showing
cholinesterase
inhibition
in
brain,
RBC,
or
plasma,
and
data
on
physiological
or
behavioral
changes
as
critical
effects
(
i.
e.,
effects
that
should
be
considered
for
use
in
the
derivation
of
an
RfD
or
RfC).
OPP
has
used
statistical
significance,
rather
than
a
fixed
percentage
of
response
from
baseline,
as
the
primary,
but
not
exclusive,
determinant
of
toxicological
and
biological
significance
in
selecting
Points
of
Departure
(
e.
g.,
NOAELs
or
LOAELs
or
Benchmark
Doses).
This
approach
treats
cholinesterase
activity
data
like
most
continuous
endpoints
(
i.
e.,
graded
measures
of
response
such
as
changes
in
organ
weight,
hormone
levels
or
enzyme
activity),
where
no
fixed
generic
percentage
of
change
from
the
baseline
is
considered
to
separate
adverse
from
non­
adverse
effects
(
US
EPA,
1995).
OPP
believes
that
a
fixed
percentage
for
describing
adversity
that
would
apply
to
all
cholinesterase­
inhibiting
pesticides
or
to
all
compartments
(
i.
e.,

blood,
central
nervous
system,
peripheral
nervous
system)
cannot
be
determined
realistically
or
scientifically
justified.
Each
data
set
must
be
judged
on
its
own
merits,

consistent
with
the
weight­
of­
the­
evidence
approach
that
OPP
is
implementing.
The
use
of
uncertainty
factors
and
the
use
of
statistical
significance
are
consistent
with
Agency
practice
for
all
non­
cancer,
systemic
toxicity
endpoints.

OPP=
s
Reference
Dose
Tracking
Report
(
US
EPA,
1997a)
lists
chronic
Reference
Doses
for
over
50
chemicals
based
in
whole,
or
in
part,
on
cholinesterase
inhibition.
There
are,
however,
many
more
than
50
risk
assessments
that
make
use
of
this
endpoint
in
acute
and
chronic
dietary
exposure/
risk
assessments
and
in
other,

nondietary
scenarios
representing
both
short­
term
and
intermediate­
term
exposure(
s).
2.2
REVIEWS
OF
PROPOSED
AGENCY/
OPP
SCIENCE
POLICY
POSITIONS
Prior
to
1997,
one
internal
Agency
colloquium
(
US
EPA,
1988)
and
two
public
Science
Advisory
Board
(
SAB)/
Scientific
Advisory
Panel
(
SAP)
meetings
(
SAB/
SAP,

1990;
1993)
considered
draft
Agency
guidance
on
the
use
of
cholinesterase
data
in
risk
assessment.
An
additional
SAP/
SAB
review
in
1992
of
a
proposed
reference
dose
for
aldicarb
also
addressed
the
issue
of
cholinesterase
inhibition
as
an
endpoint
in
risk
assessment
(
SAB/
SAP,
1992).
Each
of
these
reviews
yielded
somewhat
different
perspectives
and
recommendations,
based
in
part
on
somewhat
differing
proposed
policies,
but
primarily
on
differing
points
of
view
of
each
peer
review
group.
The
area
of
greatest
divergence
among
these
reports
and
in
their
recommendations
involved
the
interpretation
and
use
of
blood
measures
of
cholinesterase
inhibition,
particularly
in
plasma,
for
deriving
reference
doses.
Some
reviewers
and
panels
placed
less
(
or
no)

reliance
on
plasma
measures
of
cholinesterase
inhibition
and/
or
less
reliance
on
red
blood
cell
measures
of
AChE
inhibition
as
a
critical
effect
than
OPP
traditionally
has
placed
on
each.
EPA
has
never
finalized
guidance
on
this
topic
for
use
Agency­
wide.

In
1997,
OPP
published
its
own
policy
statement
on
the
use
of
data
on
cholinesterase
inhibition
for
risk
assessments,
accompanied
by
case
studies
illustrating
the
application
of
this
policy
and
a
review
of
pertinent
literature
on
cholinesterase
inhibition
prepared
by
OPP
staff
for
public
comment
and
SAP
review
(
US
EPA,
1997b;

Dementi,
1997).
In
1998,
as
part
of
the
OPP
review
process
for
science
policy
issues
agreed
upon
in
conjunction
with
the
Tolerance
Reassessment
Advisory
Committee
(
TRAC),
OPP
again
made
the
1997
policy
paper
available
for
broader
public
comment
(
US
EPA,
1998b).

The
1997
OPP
policy
statement
described
a
weight­
of­
the­
evidence
approach
for
use
when
evaluating
the
data
on
cholinesterase
inhibition
and
its
consequent
potential
adverse
cholinergic
effects.
The
SAP
expressed
support
for
the
use
of
such
an
approach
(
SAP,
1997).
Briefly,
the
SAP
stated
that:
...
the
weight
of
evidence
approach
is
indeed
reasonable
and
justified
on
the
basis
of
the
available
scientific
data
so
long
as
these
data
are
derived
from
rigorous
experiments
with
standardized
methods
and
proper
controls.
In
particular,
this
approach
allows
flexibility
to
weight
heavily
inhibition
in
non­
target
tissue
when
the
overall
toxicologic
context
suggests
that
other
approaches
pose
danger
of
serious
risk
from
overexposure.
(
Emphasis
added
in
original.)

The
1997
policy
paper
also
proposed
that
the
differences
of
opinion
with
respect
to
the
use
of
blood
measures
in
risk
assessment
could
be
reduced
or
resolved
by
the
collection
of
peripheral
nervous
system
tissue
measurements
of
AChE
inhibition
in
animal
studies
which
might
serve
instead
of
the
blood
measures
as
critical
effects
for
use
in
hazard
assessment.
The
1997
SAP
concluded
that
the
use
of
blood
measures
Ais
readily
justified
if
the
discrepancy
between
blood
cholinesterase
and
functional
endpoints
is
not
too
great@
and
recommended
that
data
on
AChE
inhibition
in
the
peripheral
nervous
system
be
collected.
On
these
points,
the
SAP
also
noted
that:

 
There
was
unanimous
support
for
the
notion
that,
under
SOME
circumstances,

measurement
of
SOME
blood­
borne
cholinesterases
would
be
appropriate
to
consider
in
establishing
RfDs
for
anticholinesterases....(
Emphasis
added
in
original.)

and
 
Measured
inhibition
of
cholinesterase
activities
in
any
of
the
blood
fractions
is
best
regarded
as
an
imperfect
mirror
of
enzyme
inhibition
in
the
true
target
tissues...
OPP
subsequently
asked
the
International
Life
Sciences
Institute
(
ILSI)/
Risk
Science
Institute
to
convene
a
workgroup
to
help
further
define
the
feasibility
and
details
for
collecting
these
data.
This
workgroup's
report
concluded
that
it
was
currently
feasible
to
measure
AChE
inhibition
in
the
peripheral
nervous
system
(
Mileson,
et.
al.,
1999b).
The
ILSI
workgroup
further
concluded,
"
Methods
and
techniques
currently
available
are
adequate
to
characterize
the
AChE
activity
in
the
peripheral
nervous
system,
but
additional
studies
would
help
to
improve
these
methods."
3.
IDENTIFICATION
OF
THE
TOXICOLOGICAL
ENDPOINTS
FOR
ASSESSMENT
OF
CHOLINESTERASE
INHIBITORS
This
Section
explains
the
science
policy
decisions
and
rationale
specific
to
the
evaluation
of
the
various
cholinergic
effects
on
nervous
system
function
caused
by
anticholinesterase
pesticides.
This
rationale
forms
the
basis
of
the
weight­
of­

theevidence
approach
described
later
in
Section
4.
The
general
principles,
including
definitions
of
key
terms,
and
approaches
used
by
OPP
and
EPA
for
evaluating
the
neurotoxic
potential
of
environmental
agents
can
be
found
in
the
Agency's
Guidelines
for
Neurotoxicity
Risk
Assessment
(
US
EPA,
1998a).

This
Section
is
organized
around
conclusions
followed
by
a
rationale
addressing
three
key
types
of
endpoints
generally
assessed
currently
for
cholinesterase­
inhibiting
pesticides:

1)
evaluations
of
physiological
and
behavioral/
functional
effects;

2)
measures
of
acetylcholinesterase
inhibition
in
the
neural
tissues
(
i.
e.,
brain
and
peripheral
nervous
system);
and
3)
measures
of
cholinesterase
inhibition
in
the
blood
(
i.
e.,
red
blood
cells
and
plasma).
3.1
EVALUATION
OF
EFFECTS
ON
CHOLINERGIC
FUNCTIONS
3.1.1
CONCLUSIONS
!
Clinical
signs/
symptoms
5
in
humans
and
behavioral
or
physiological
effects
in
humans
and
animals
provide
the
most
direct
evidence
of
the
potential
adverse
consequences
of
human
exposure
to
anticholinesterase
pesticides.

!
Effects
observable
in
humans
6
can
cover
a
broader
range
than
those
that
can
be
observed
in
animal
studies,
including
psychological
complaints,
cognitive
complaints
and
other
subjective
effects,
and
performance
measures
of
learning
and
memory.
Human
studies
following
either
deliberate
or
inadvertent
exposure,
nevertheless,
are
currently
quite
limited
in
the
scope
of
the
evaluations
made
and
scale
of
the
measurements
used.
As
for
animal
studies,
it
is
possible
that
one
or
more
effects
of
concern
may
be
occurring
but
measures
for
their
evaluation
were
not
or
could
not
be
incorporated
in
the
study
design.
Also,
the
generally
small
numbers
of
subjects
may
limit
the
power
of
the
study
to
detect
effects
of
concern.
!

5
A
"
symptom"
is
defined
as
a
condition
which
is
a
departure
from
normal
function
reported
by
the
person
experiencing
and
reporting
that
condition
(
e.
g.,
headache,
nausea).
A
"
clinical
sign"
is
an
objectively­
measured
effect
(
e.
g.,
heart
rate,
blood
pressure)
indicative
or
suggestive
of
a
condition
for
an
individual
(
human
or
animal)
observed
and
recorded
by
another
such
as
a
physician.
6
EPA
is
currently
reviewing
its
policy
concerning
human
studies
with
respect
to
ethical
and
scientific
standards
for
their
acceptability
and
use
in
risk
assessments,
particularly
with
respect
to
decisions
under
FQPA.
The
Agency
neither
requires
nor
encourages
the
conduct
of
human
hazard
identification
studies
to
detect
potential
adverse
effects
of
pesticides.
EPA
has
held
two
meetings
of
a
joint
SAP/
SAB
(
Science
Advisory
Board)
panel
(
December
1998
and
November,
1999
(
US
EPA,
1998c,
1999d))
on
the
ethical
elements
of
this
issue.
The
Panel
is
expected
to
issue
its
report
in
the
Summer
of
2000.
Evaluation
of
physiological
and
behavioral
changes
(
i.
e.,
functional
data)
in
animal
studies
also
are
limited
in
terms
of
the
scope
of
effects
assessed
and
the
measurements
employed.
As
with
human
studies,
it
is
possible
that
one
or
more
effects
of
concern
may
be
occurring
but
procedures
for
measuring
these
effects
were
not
or
could
not
be
incorporated
in
the
study
design,
leaving
the
possibility
of
a
false
negative.

!
Because
of
the
limited
range
of
measures
of
behavioral
and
physiological
effects
evaluated
historically,
functional
data
obtained
from
human
and
animal
studies
should
not
be
relied
on
solely,
to
the
exclusion
of
other
kinds
of
pertinent
information,
when
weighing
the
evidence
for
selection
of
the
critical
effect(
s)
that
will
be
used
as
the
basis
of
the
RfD
or
RfC.

3.1.2
RATIONALE
Many
of
the
adverse
acute
and
longer­
term
effects
of
anticholinesterase
organophosphorous
pesticides
that
have
been
observed
in
humans
were
described
by
Morgan
(
1989)
and
updated
by
Reigart
and
Roberts
(
1999):

Most
commonly
reported
in
humans
are
headache,
nausea,
and
dizziness.
Anxiety
and
restlessness
are
prominent.
Worsening
may
result
in
muscle
twitching,
weakness,
tremor,
incoordination,
vomiting,
abdominal
cramps,
diarrhea.
Often
prominent
are
sweating,
salivation,
tearing,
rhinorrhea,
and
bronchorrhea.
Blurred
and/
or
dark
vision,
and
excessive
contraction
of
the
pupil
of
the
eye
(
miosis)
may
also
be
seen.
Tightness
in
the
chest,
wheezing
and
productive
cough
may
progress
to
frank
pulmonary
edema.
Bradycardia
may
progress
to
sinus
arrest,
or
tachycardia
and
hypertension.
Confusion,
bizarre
behavior,
and
toxic
psychosis
may
occur.
In
severe
poisonings,
toxic
myocardiopathy,
unconsciousness,
incontinence,
convulsions,
respiratory
depression
and
death
may
be
seen.
Repeated
absorption,
but
not
enough
to
cause
acute
poisoning
may
result
in
persistent
anorexia,
weakness,
and
malaise.

As
noted,
many
of
the
effects
described
above
may
be
seen
after
acute
exposures
of
humans
to
anticholinesterase
pesticides.
There
also
are
case
reports
describing
long(
er)­
term
effects
following
acute
high­
level
exposures.
Little
information
exists
describing
effects
following
long(
er)­
term,
low­
level
exposures
to
humans.
Several
cholinesterase
inhibiting
chemicals
from
both
the
organophosphorous
and
carbamate
classes
have
been
used
and/
or
are
being
explored
for
usefulness
in
the
treatment
of
certain
neuromuscular
diseases
and
neurocognitive
disorders
such
as
Alzheimer=
s
disease
(
Taylor,
1996a;
Standaert
and
Young,
1996).
The
literature
describing
the
results
of
clinical
trials
and
imposition
of
treatment
regimens
for
these
drugs
may
be
useful
in
providing
insights
into
the
nature
(
both
therapeutic
and
adverse)
of
human
responsiveness
to
these
substances.

Increasing
levels
of
exposure
generally
result
in
progressively
more
serious
effects,
although
the
exact
pattern
of
effects
differs
among
anticholinesterase
chemicals
and
may
be
influenced
by
the
age
of
the
patient,
genetic
differences,
drug
interactions,
and
other
factors.
Different
cholinesterase­
inhibiting
chemicals
may,
and
generally
do,
produce
different
spectra
of
clinical
signs
and
behavioral
effects.
This
complexity,
in
part,
may
arise
from
differences
between
the
absorbed
chemicals
in
distribution
between
the
central
and
peripheral
nervous
systems
and
differential
binding
in
those
nervous
system
compartments,
or
differential
interactions
with
the
two
major
types
of
cholinergic
receptors
(
i.
e.,
muscarinic
and
nicotinic
receptors).
The
nature
and
temporal
pattern
of
effects
also
depends
on
the
magnitude,
duration,
and
frequency
of
exposure,
as
well
as
whether
metabolic
activation
is
needed.
Perhaps
one
third
of
the
effects
caused
by
anticholinesterase
chemicals
(
e.
g.,
headache,
confusion,
tremor,
and
convulsions)
can
be
attributed
primarily
to
effects
on
the
central
nervous
system
(
Minton
and
Murray,
1988).
For
many
effects,
however,
it
is
difficult
to
distinguish
whether
they
are
centrally
or
peripherally
mediated
or
both.

For
any
pesticide,
including
cholinesterase
inhibitors,
OPP
may
require
different
toxicology
studies
in
laboratory
animals,
depending
upon
the
use
and
exposure
patterns
of
the
substance.
Not
all
of
these
studies
include
the
requirement
for
measurement
of
cholinesterase
activity
or
the
effects
occurring
as
a
consequence
of
its
inhibition.
Those
that
do
or
could
are
noted
with
an
asterisk
(*).
The
key
studies
required
for
a
food­
use
pesticide
of
conventional
chemistry
are:

!
Acute
oral,
dermal,
and
inhalation
lethality
tests
in
mammals;

!
Acute
or
subchronic
(
90­
day)
delayed
neurotoxicity
study
in
hens;

!
Acute
and
subchronic
(
90­
day)
neurotoxicity
screening
battery
in
rats*,
which
includes:

<
Functional
observational
battery,
which
is
a
set
of
structured
observations
outside
the
home
cage,
including
assessments
of
autonomic
signs,
pupillary
response
to
light
or
pupil
size,
arousal,
reactivity,
posture
and
gait,
grip
strength,
limb
splay,
and
simple
sensory
reflexes
(
e.
g.,
tail
pinch
and
a
sudden
sound);

<
Automated
motor
activity;

<
Histopathology
of
neural
tissue
from
animals
prepared
by
in
situ
perfusion;

<

Responses
to
visual
or
proprioceptive
(
i.
e.,
sense
of
body
position
or
awareness
of
pressure)
stimuli
are
optional,
but
not
commonly
done.

!
21­
Day
or
subchronic
(
90­
day)
dermal
toxicity
study
in
mammals*;

!
Subchronic
(
90­
day)
inhalation
study
in
mammals*
(
if
appropriate
on
basis
of
anticipated
human
route
of
exposure);

!
Two
chronic
toxicity
studies*,
one
in
the
rat
and
one
in
the
dog;
!
Two
prenatal
developmental
toxicity
studies*,
one
in
a
rodent
and
one
in
a
nonrodent
species;
and
!
Two­
generation
reproduction
study
in
rodents*;
and
!
Developmental
neurotoxicity
study
in
rats*,
which
includes,
in
pups:

<
detailed
observations,
developmental
landmarks,
motor
activity,
auditory
startle
reflexes,
learning
and
memory
test,
and
neuropathology
on
postnatal
days
11
and
60
<
detailed
observations
for
neurological
effects
also
are
made
in
dams
While
they
never
have
been
a
part
of
EPA's
data
requirements
and,
thus,
there
are
no
EPA
testing
guidelines
for
them,
human
hazard
identification
studies
on
some
pesticides
have
been
submitted
by
the
chemical=
s
sponsor(
s)
and,
in
the
past,
prior
to
the
passage
of
FQPA,
considered
for
use
in
risk
assessments.
These
hazard
identification
studies
typically
are
designed
to
identify
no­
effect
levels
for
ChEIassociated
enzyme
activity
and,
sometimes,
for
some
clinical
effects.
Although
many
of
these
human
hazard
identification
studies
with
cholinesterase
inhibitors
are
acute
(
i.
e.,
single
dose)
in
their
exposure
duration,
some
have
incorporated
short­
term
(
e.
g.,
4­
10
day)
or
longer
(
e.
g.,
21­
28
day)
repeat
dosing.
Measures
of
cholinesterase
inhibition
in
either
whole
blood
(
which
is
a
mixture
of
plasma
and
RBCs),
or
separately
in
RBCs
and
plasma
are
usually
included.
Sometimes,
reporting
of
some
clinical
symptoms
and
signs
are
included;
in
a
few
cases,
objective
physiological
measures,
such
as
blood
pressure,
pulse
rate
or
temperature,
have
been
reported.

Human
hazard
identification
studies
can
be
designed
to
detect
more
effects
in
addition
to
blood
enzyme
inhibition
(
e.
g.,
mild
sweating
and
nausea)
compared
to
animal
studies,
due
to
self­
reporting
of
complaints,
including
sensory,
cognitive,
and
psychological
effects.
Formal
evaluations
(
by
interview
or
test),
however,
are
very
uncommon
as
are
measurements
of
physiological
parameters
like
heart
function
(
e.
g.,
heart
rate
and
blood
pressure)
and
breathing
rate.
More
sophisticated
neurobehavioral
test
batteries,
such
as
intelligence
tests
or
simple
memory
tests,
used
in
epidemiological
studies
(
for
example,
Anger,
et
al.,
1996),
are
rarely,
if
ever,
used
in
human
hazard
identification
studies
of
cholinesterase­
inhibiting
organophosphorous
and
carbamate
pesticides.
The
reports
of
certain
kinds
of
animal
studies
include
detections
of
overt
clinical
signs,
including
many
of
the
autonomic
signs,
and
motor
effects,
such
as
tremors.
In
the
rodent
neurotoxicity
screening
battery
studies,
the
data
are
gathered
systematically
by
observers
unaware
of
treatment.
The
measurements
of
effects
are
defined
quantitatively,
albeit,
usually
on
an
ordinal
scale
(
e.
g.,
+
1,
+
2).
Valid
screening
studies
also
include
automated
and
quantitative
measures
of
motor
activity,
grip
strength,
and
limb
splay,
though
changes
in
these
measures
are
not
a
distinguishing
characteristic
of
cholinesterase
inhibition.
EPA=
s
test
guidelines
for
the
neurotoxicity
screening
batteries
were
published
in
1991.
OPP
has
received
data
from
these
neurotoxicity
screening
studies
on
many
of
the
anticholinesterase
organophosphorous
and
carbamate
pesticides.
Of
the
roughly
30
effects
that
may
occur
following
acute
exposures
as
listed
by
Morgan
(
1989)
and
updated
by
Reigart
and
Roberts
(
1999),
perhaps
one
third
would
not
be
seen
in
routine
animal
studies,
or
even
in
the
neurotoxicity
screening
battery,
as
they
are
currently
designed,
especially
the
sensory,
cognitive,
and
psychological
effects.
Thus,
because
of
the
limitations
in
the
study
design
and
conduct
of
both
human
and
animal
studies,
OPP
may
not
understand
fully
the
profile
of
effects
of
concern
that
may
result
from
exposure
to
the
cholinesteraseinhibiting
pesticides.

3.2
NERVOUS
SYSTEM
CHOLINESTERASE
INHIBITION
3.2.1
CONCLUSIONS
!
Inhibition
of
acetylcholinesterase
in
the
nervous
system
(
both
central
and
peripheral)
is
generally
accepted
as
a
key
component
of
the
mechanism
of
toxicity
leading
to
adverse
cholinergic
effects.
The
inhibition
of
this
enzyme
provides
direct
evidence
of
potential
adverse
effects.
Interference
with
the
timely
deactivation
of
neuronal
or
neuroeffector
acetylcholine
results
in
the
protraction
of
the
actions
of
acetylcholine
at
these
sites,
which
in
turn
results
in
adverse
cholinergic
effects.
Because
the
inhibition
of
acetylcholinesterase
is
a
key
event
that
can
lead
to
adverse
effects,
data
showing
this
response
provide
valuable
information
in
assessing
potential
hazards
posed
by
anticholinesterase
pesticides.

!
Measures
of
acetylcholinesterase
activity
in
both
central
and
peripheral
nervous
tissues
are
important
for
a
full
assessment
of
the
potential
for
hazard
because
the
enzyme
and
each
chemical
may
have
different
pharmacokinetic
and
pharmacodynamic
properties
in
each
compartment
of
the
nervous
system.
!
The
relationships
between
the
functional
effects
and
changes
in
acetylcholinesterase
activity
in
both
nervous
system
compartments
often
are
difficult
to
characterize
with
existing
data
for
a
variety
of
reasons
(
e.
g.,
development
of
tolerance,
heterogeneity
of
cholinergic
pathways
including
the
molecular
form(
s)
of
AChE
present
at
each
location,
limited
data
on
the
regional
distribution
of
acetylcholinesterase,
the
time
course
of
inhibition
in
each
region,
and
limited
evaluation
of
functional
effects).

3.2.2
RATIONALE
The
inhibition
of
acetylcholinesterase
is
a
key
step
in
the
mechanism
of
toxicity
of
certain
organophosphorous
and
carbamate
pesticides
(
Mileson,
et
al,
1998;
Mileson,
1999a;
US
EPA,
1999a,
1999c),
and,
therefore,
measures
of
cholinesterase
inhibition
represent
a
critical
biochemical
biomarker
of
potential
adverse
effects.
Inhibition
of
acetylcholinesterase
in
the
central
nervous
system
is
considered
to
be
an
indicator
of
an
adverse
effect.
Nonetheless,
reductions
in
neural
AChE
activity
may
not
always
be
accompanied
by
overt
clinical
signs
or
symptoms
because,
for
example,
the
critical
functions
of
those
specific
neurons
may
not
be
sufficiently
evaluated
to
detect
related
changes
or
tolerance
may
have
developed.
The
time
at
which
potential
functional
effects
are
evaluated
may
also
contribute
to
an
apparent
lack
of
concordance
between
functional
effects
and
the
neurochemical
effects
(
i.
e.,
cholinesterase
inhibition).
Based
on
these,
and,
perhaps,
other
factors,
it
is
difficult
to
determine,
with
accuracy
or
consistently,
the
degree
of
cholinesterase
inhibition
that
will
cause
specific
physiological
or
behavioral
changes.
Thus,
OPP
considers
a
treatment­
related
decrease
in
brain
or
peripheral
tissue
AChE
activity,
in
itself,
toxicologically
important.
Data
showing
such
a
decrease
are
appropriate
for
use
as
a
critical
effect
for
the
derivation
of
RfDs
and
RfCs,
as
well
as
for
characterizing
potential
human
hazards.

Historically,
data
on
central
nervous
system
AChE
inhibition
have
come
from
single
or
repeated
exposure
animal
studies,
in
which
whole
brain
homogenates
are
assayed
at
one
or
two
time
points.
For
the
past
several
years,
more
detailed
measurements
of
brain
AChE
inhibition
have
been
required.
These
requirements,
as
part
of
the
neurotoxicity
screening
battery,
or
as
separate
studies,
have
sought
to
characterize
the
time
course
of
inhibition
in
plasma,
RBCs,
and
brain,
including
in
specific
brain
regions,
after
acute
and
90­
day
exposures.
Even
so,
most
of
the
existing
data
sets
will
generally
contain
measures
only
of
whole
brain
AChE
activity,
but
not
usually
regional
brain
measurements,
or
time­
course
data,
particularly
following
acute
exposures.
The
lack
of
regional
brain
measures
appears
to
be
a
limitation,
given
that
the
distribution
of
cholinergic
pathways
and
the
concentration
and
molecular
form
of
AChE
in
different
brain
regions
is
not
homogenous.
Thus,
whole
brain
measurements
of
AChE
inhibition
may
reveal
little
or
no
change
in
activity
while
masking
significant
changes
in
specific
brain
regions
associated
with
particular
cholinergically­
mediated
functions
(
e.
g.,
the
hippocampus
and
memory).

Unfortunately,
measures
of
AChE
inhibition
in
peripheral
neural
tissues
or
neuroeffector
junctions
are
rare.
AChE
inhibition
data
from
the
peripheral
nervous
system
potentially
have
unique
value
because
many
of
the
adverse
signs
and
symptoms
associated
with
exposure
to
anticholinesterase
pesticides
(
e.
g.,
diarrhea,
excess
salivation)
are
a
result
of
effects
on
the
peripheral
nervous
system.
Because
of
the
potential
pharmacokinetic
differences
between
the
central
and
peripheral
nervous
compartments,
measures
of
AChE
activity
in
both
of
these
systems
are
important
for
the
full
assessment
of
chemicals
on
the
nervous
system.
Certain
chemicals
may
have
equivalent
access
to
a
specific
compartment,
in
both
degree
and
rate
of
interaction.
On
the
other
hand,
there
are
others
for
which
the
rate
of
access
to,
and
concentration
in,
peripheral
tissues
is
far
greater
than
in
the
central
nervous
system.
These
patterns
could
shift
with
longer
term
exposures.

Although
AChE
inhibition
data
in
peripheral
nervous
system
tissues
have
not
been
required
in
toxicological
studies
submitted
to
EPA
and,
at
the
moment,
no
standard
protocol
exists
for
the
generation
of
such
data,
OPP
indicated
in
1997
that
the
collection
of
these
measures
potentially
could
become
an
alternative
to
the
use
of
blood
cholinesterase
inhibition
measures
in
animal
studies
in
the
hazard
and
risk
assessment
process.
As
discussed
earlier,
the
SAP
(
SAP,
1997)
and
an
expert
panel
of
ILSI
(
Mileson,
et
al.
1999b)
have
stated
that
it
is
feasible
to
measure
AChE
inhibition
in
peripheral
nervous
system
tissues.
The
1997
SAP
report
asserted,
"
it
is
important
that
joint
efforts
be
mounted
to
evaluate
AChE
inhibition
in
the
peripheral
neural
tissues
per
se
and
in
the
neuroeffector
junctions."
The
SAP
expressed
the
view
that
it
is
technically
feasible
to
routinely
conduct
AChE
assays
on
the
peripheral
nervous
system,
while
recognizing
the
difficulties
involved.
The
SAP
further
suggested
that
skeletal
muscles,
heart,
lung,
salivary
glands,
diaphragm,
and
autonomic
ganglia
(
e.
g.,
superior
cervical
ganglia)
be
considered
as
appropriate
tissues
to
examine.
The
SAP
considered
that
standardized
and
reproducible
dissection
and
homogenization
of
tissue,
assays
with
minimal
tissue
dilution,
selection
of
the
most
relevant
tissue
targets,
and
standardization
of
tissue
storage
conditions
were
the
most
important
technical
issues
to
resolve
when
measuring
AChE
activity
in
the
peripheral
nervous
system.
Work
is
underway
in
EPA=
s
National
Health
and
Environmental
Effects
Laboratory
(
NHEERL)
to
develop
and
standardize
protocols
for
assaying
enzyme
activity
in
various
peripheral
tissues
(
e.
g.,
see
Marshall,
et
al.,
1999).

3.3
BLOOD
CHOLINESTERASE
INHIBITION
3.3.1
CONCLUSIONS
!
Inhibition
of
blood
cholinesterases
(
i.
e.,
plasma
and
red
blood
cell)
is
not
itself
an
adverse
effect,
but
may
indicate
a
potential
for
adverse
effects
on
the
nervous
system.
As
a
matter
of
science
policy,
blood
cholinesterase
data
are
considered
appropriate
surrogate
measures
of
potential
effects
on
peripheral
nervous
system
acetylcholinesterase
activity
in
animals,
for
CNS
acetylcholinesterase
activity
in
animals
when
CNS
data
are
lacking
and
for
both
peripheral
and
central
nervous
system
acetylcholinesterase
in
humans.

!
As
such,
blood
cholinesterase
inhibition
data
are
considered
appropriate
endpoints
for
derivation
of
reference
doses
or
concentrations
when
considered
in
a
weight­
of­
the­
evidence
analysis
of
the
entire
database
on
a
single
pesticide
or
on
two
or
more
pesticides
assigned
to
a
common
mechanism
of
toxicity
group,
where
acetylcholinesterase
inhibition
is
the
common
mechanism
of
toxicity.
!
Red
blood
cell
measures
of
acetylcholinesterase
inhibition,
if
reliable,
generally
are
preferred
over
plasma
data.
Since
the
red
cell
contains
only
acetylcholinesterase,
the
potential
for
exerting
effects
on
neural
or
neuroeffector
acetylcholinesterase
may
be
better
reflected
by
changes
in
red
blood
cell
acetylcholinesterase
than
by
changes
in
plasma
cholinesterases
which
contain
both
butyrylcholinesterase
and
acetylcholinesterase
in
varying
ratios
depending
upon
the
species.
This
conclusion
rests
on
data
showing
that
chemicals
may
have
significantly
different
interactions
with
AChE
and
BuChE,
including
their
affinities
for
binding
with
the
enzymes.

!
Although
RBC
acetylcholinesterase
data
are
generally
preferred,
in
some
cases,
reliance
on
measures
of
RBC
may
not
be
appropriate
because
of
methodological
issues
concerning
blood
measures
of
cholinesterase
activity.
When
making
weight­
of­
the­
evidence
judgments
concerning
the
selection
of
RBC
versus
plasma
measures
of
cholinesterase
inhibition
as
endpoints
for
derivation
of
reference
doses
or
concentrations,
it
is
critical
to
consider
all
aspects
of
the
information
database,
including
the
adequacy
of
the
study
protocol,
quality
of
the
data,
dose­
dependency
of
the
responses,
as
well
as
available
data
on
measures
of
brain
acetylcholinesterase
inhibition
and
functional
effects.

!
Plasma
contains
both
butyrylcholinesterase
and
acetylcholinesterase
in
varying
ratios
depending
upon
the
species.
The
separate
characterization
of
RBC
and
plasma
measures
of
cholinesterase
inhibition
provides
an
additional
means
of
measuring
effects.
Additionally,
having
separate
RBC
and
plasma
data
allow
for
more
informative
animal­
to­
human
comparisons.
3.3.2
RATIONALE
As
a
biomarker
of
exposure,
blood
cholinesterase
inhibition
can
be
correlated
with
the
extent
of
exposure.
As
discussed
earlier,
there
is
often
a
direct
relationship
between
a
greater
magnitude
of
exposure
and
an
increase
in
incidence
and
severity
of
clinical
signs
and
symptoms
as
well
as
blood
cholinesterase
inhibition.
In
other
words,
the
greater
the
exposure,
the
greater
the
cholinesterase
inhibition
in
the
blood
and
the
greater
the
potential
for
an
adverse
effect
to
occur.
Both
plasma
and
RBC
measures
of
cholinesterase
inhibition
also
provide:

C
pharmacokinetic
evidence
of
absorption
of
the
pesticide
and/
or
its
active
metabolite(
s)
into
the
bloodstream
and
systemic
circulation;
and
C
pharmacodynamic
evidence
of
binding
to
AChE,
the
neural
form
of
the
target
enzyme,
or
to
plasma
BuChE,
an
enzyme
similar
in
structure
to
AChE
Because
the
interaction
with
AChE
is
widely
accepted
as
a
key
event
of
the
mechanism
of
toxicity
for
anticholinesterase
pesticides,
inhibition
of
this
cholinesterase
in
the
blood
creates
the
presumption
that
a
chemical
also
is
causing
inhibition
of
neural
AChE.
Chemicals
are
absorbed
into
the
blood
and
transported
to
the
peripheral
nervous
system.
Pharmacokinetically,
the
blood
compartment
and
the
peripheral
nervous
system
are
"
outside
of"
the
central
nervous
system,
i.
e.,
separated
from
the
CNS
by
the
blood­
brain
barrier.
Thus,
blood
measures
of
cholinesterase
activity
are
viewed
as
a
better
surrogate
for
the
effects
on
AChE
in
the
peripheral
nervous
system
than
are
enzyme
changes
in
the
central
nervous
system.
Because
data
on
AChE
inhibition
in
the
peripheral
nervous
system
have
rarely
been
gathered
in
animals,
blood
cholinesterase
inhibition
measures
are
generally
the
only
information
available
to
assess
the
potential
of
chemicals
to
inhibit
AChE
in
the
peripheral
nervous
system.
In
human
studies,
blood
cholinesterase
inhibition
measures
serve
as
surrogates
for
effects
in
both
the
central
and
peripheral
nervous
systems
because
neither
of
these
neural
tissues
is
available
for
evaluation
directly.
As
discussed
earlier,
evaluations
of
clinical
signs
and
symptoms
have
limitations,
and
thus
should
not
be
relied
on
solely,
to
the
exclusion
of
other
data.
Therefore,
blood
cholinesterase
inhibition
data
are
considered
appropriate
endpoints
for
derivation
of
reference
doses
or
concentrations
when
considered
in
a
weight­
of­
the­
evidence
analysis
of
the
entire
database
on
a
single
pesticide
or
on
two
or
more
pesticides
assigned
to
a
common
mechanism
of
toxicity
group,
where
acetyl­
cholinesterase
inhibition
is
the
common
mechanism
of
toxicity.

The
usefulness
of
collecting
blood
cholinesterase
inhibition
data
(
both
RBC
and
plasma)
is
illustrated
further
by
its
use
in
monitoring
workers
for
occupational
exposures
(
even
in
the
absence
of
signs,
symptoms,
or
other
behavioral
effects).
Blood
cholinesterase
inhibition
(
RBC
and/
or
plasma)
is
considered
as
providing
a
sufficient
basis
for
removing
workers
from
the
exposure
environment,
given
the
assumption
that
if
one
were
to
protect
against
enzyme
inhibition,
one
would
protect
against
effects
of
graver
concern.
For
example,
the
California
Department
of
Health
Services
(
CDHS)
requires
monitoring
of
agricultural
workers
who
have
contact
with
highly
toxic
organophosphorous
or
carbamate
compounds
(
EPA
Toxicity
Category
I
or
II
pesticides;
LD50
#
500
mg/
kg
in
rats)(
CDHS,
1988).
CDHS
removes
workers
from
the
workplace
whose
plasma
levels
show
40%
or
greater
cholinesterase
inhibition
from
baseline,
or
whose
red
blood
cell
cholinesterase
levels
show
30%
or
greater
inhibition.
Workers
may
not
return
until
their
cholinesterase
values
return
to
within
80%
of
baseline.
The
World
Health
Organization
(
WHO)
also
has
guidelines
with
the
same
RBC
action
levels
(
i.
e.,
30%
or
greater
inhibition),
and
considers
plasma
inhibition
of
50%
of
baseline
to
indicate
a
"
toxic"
decrease
(
Fillmore
and
Lessinger,
1993).
Fillmore
and
Lessinger
also
reviewed
the
California
program
and
found
that
"
The
relative
risk
of
pesticide
poisoning
was
increased
in
workers
whose
initial
baseline
plasma
levels
were
low,
or
if
their
levels
had
already
dropped
to
60­
80%
of
their
baseline
previously
in
the
season."

Although
a
pesticide=
s
effect(
s)
on
either
RBC
and
plasma
cholinesterase
activity
is
considered
to
provide
information
on
its
potential
to
inhibit
AChE
in
the
nervous
system,
data
from
RBCs,
which
contain
AChE
exclusively,
may
better
reflect
neuronal
AChE
inhibition
than
data
from
the
plasma,
which
is
a
variable
mixture
of
butyrylcholinesterase
and
acetylcholinesterase.
As
discussed
earlier,
acetylcholinesterase
is
the
enzyme
involved
in
the
mechanism
of
toxicity
for
the
cholinergic
effects
of
anticholinesterase
pesticides.
Although
BuChE
is
somewhat
similar
in
structure
to
AChE,
BuChE
is
nevertheless
sufficiently
different
in
important
ways
which
often
result
in
it
having
binding
affinities
to
anticholinesterase
agents
as
well
as
other
characteristics
that
are
quite
different
from
those
of
acetylcholinesterase
(
Silver,
1974;
Taylor
and
Radic,
1994).
The
composition
of
plasma
cholinesterases
varies
widely
among
humans,
dogs,
and
rats,
the
species
for
which
these
measures
are
most
typically
made.
Human
plasma
is
overwhelmingly
BuChE
with
a
ratio
of
BuChE
to
AChE
of
1,000:
1
(
Edwards
and
Brimijoin,
1983).
In
dogs,
there
is
a
little
more
than
10%
acetylcholinesterase
in
plasma
with
a
ratio
of
BuChE
to
AChE
of
7:
1
(
Scarsella
et
al.,
1979).
In
rats,
plasma
contains
approximately
50%
or
more
of
AChE
with
a
BuChE
to
AChE
ratio
of
1:
3
in
males
and
2:
1
in
females
(
Edwards
and
Brimijoin,
1983).
While
it
is
technically
possible
to
ascertain
the
contribution
of
each
ChE
to
the
level
of
inhibition
in
plasma,
this
type
of
data
is
rarely
available.
Thus,
the
relationship
between
blood
measures
of
AChE
and
BuChE
or
other
factors
is
usually
not
known.
For
these
reasons,
a
treatment­
related
decrease
in
plasma
cholinesterase
activity,
viewed
in
isolation,
provides
less
insight
into
the
potential
of
a
chemical
to
cause
neural
AChE
inhibition
than
do
data
on
RBC
AChE
inhibition.

Historically,
there
have
been
technical
difficulties
with
the
measurement
of
the
inhibition
of
plasma
and
RBC
cholinesterase(
s),
particularly
for
the
latter
(
see
Wilson,
et
al.,
1996).
Although
in
recent
years
there
have
been
improvements
in
blood
measures
of
cholinesterase
activity,
it
is
important
to
consider
carefully
the
methodological
issues
that
may
affect
the
accuracy
and
variability
of
the
data
when
assessing
the
effects
of
pesticides
on
cholinesterase
activity
in
blood.
There
are
many
methods
available
for
measuring
blood
cholinesterase
activity.
The
colorimetric
method,
based
on
the
Ellman
reaction,
is
considered
a
reliable
method
when
performed
properly,
and
is
commonly
used
for
measuring
plasma
and
RBC
cholinesterase
activity
(
Ellman,
et
al.,
1961;
US
EPA,
1992;
ASCP,
1994).
While
well
suited
to
the
measurement
of
cholinesterase
inhibition
induced
by
organophosphorous
pesticides,
the
Ellman
method
may
underestimate
cholinesterase
activity
in
both
plasma
and
RBC
following
carbamate
exposure
because
of
the
relatively
unstable
binding
of
the
carbamate
esters
to
the
acetylcholinesterase.
The
radiometric
method
may
be
better
suited
for
measuring
carbamate­
inhibited
cholinesterase
(
Johnson
and
Russell,
1975;
Wilson,
et
al.,
1996).
The
refinement
of
measurement
methods
continues
in
NHEERL.
If,
and
when,
the
conduct
of
future
animal
studies
on
pesticides
includes
the
measurement
of
peripheral
nervous
system
acetylcholinesterase
activity,
in
addition
to
central
nervous
system
measures,
then
less
reliance
can
be
placed
on
the
use
of
blood
measures
as
critical
effects
for
the
derivation
of
RfDs/
RfCs
in
the
risk
assessment
process.
However,
as
noted
above,
blood
measures
will,
in
any
case,
continue
to
provide
important
information
in
characterizing
human
hazard
because
they
still
will
provide
a
means
of
animal­
to­
human
comparison
of
cholinesterase
inhibition.
As
noted
above,
work
on
standardizing
methods
for
measuring
acetycholinesterase
activity
in
the
peripheral
nervous
system
is
underway.
OPP/
EPA
expects
that
the
standardization
and
use
of
these
methods
will
result
in
a
database
that
improves
the
scientific
understanding
of
the
risks
of
cholinesterase­
inhibiting
compounds.
4.
WEIGHT­
OF­
THE­
EVIDENCE
ANALYSIS
FOR
SELECTION
OF
CRITICAL
EFFECTS
The
present
science
policy
has
been
prepared
considering
the
comments
received
from
the
SAP
and
the
public
in
1997
and
during
the
public
comment
period
in
1998.
This
revised
policy
continues
to
embrace
the
weight­
of­
the­
evidence
approach
of
considering
all
relevant
data
in
an
integrative
manner
that
was
described
in
the
1997
OPP
document
(
US
EPA,
1997).
This
revised
policy
expands
the
discussion
of
the
approach
and
clarifies
the
weight­
of­
the­
evidence
approach
by
describing
more
explicitly
under
what
conditions
and
how
plasma
and/
or
RBC
cholinesterase
data
would
be
considered.
The
policy
also
re­
emphasizes
the
potential
usefulness
of
collection
of
peripheral
neural
data
on
AChE
inhibition
to
reduce
reliance
on
the
surrogate
blood
measures.

OPP
is
using
the
weight­
of­
the­
evidence
approach
described
here
to
analyze
individual
studies
as
well
as
the
complete
database
on
a
pesticide
when
selecting
critical
effects
for
hazard
assessment.
The
primary
objective
of
the
weight­
of­
theevidence
analysis
for
anticholinesterase
pesticides
is
to
select
Points
of
Departure
(
PoDs)
(
i.
e.,
NOAELs,
LOAELs,
or
benchmark
doses)
for
critical
effects
to
be
used
in
the
calculation
of
RfDs,
RfCs
or
margins
of
exposure
(
MOE)
for
all
of
the
routes
and
durations
of
exposure
appropriate
for
a
pesticide
given
its
use
and
exposure
patterns
when,
after
review
of
the
entire
toxicological
database,
it
is
concluded
that
the
cholinergic
effect(
s)
induced
by
the
substance
being
evaluated
do,
in
fact,
represent
the
critical
effect(
s).
Briefly,
the
weight­
of­
the­
evidence
approach
includes
consideration
of
all
available
data
on:

!
clinical
signs
and
other
physiological
and
behavorial
effects
in
humans
and
animals;

!
symptoms
in
humans;

!
central
nervous
system
acetylcholinesterase
inhibition;

!
peripheral
nervous
system
acetylcholinesterase
inhibition;

!
red
blood
cell
acetylcholinesterase
inhibition;
and
!
plasma
cholinesterase
inhibition
(
BuChE
in
humans;
mixed
AChE/
BuChE
in
animals).

A
comparison
of
the
pattern
of
doses
required
to
produce
physiological
and
behavioral
effects
and
cholinesterase
inhibition
in
different
compartments
is
conducted.
In
addition
to
these
parallel
analyses
of
the
dose­
response
information,
comparisons
of
the
temporal
aspects
(
e.
g.,
time
of
onset
and
peak
effects
and
duration
of
effects)
of
each
relevant
endpoint
are
made.
This
analysis
should
be
done
for
each
relevant
route
and
duration
of
exposure
(
e.
g.,
acute,
intermediate
and/
or
chronic
exposures)
for
each
available
species/
strain/
sex
of
animals.
Furthermore,
the
potential
for
differential
sensitivity/
susceptibility
of
adult
versus
young
animals
(
i.
e.,
effects
following
perinatal
or
postnatal
exposures)
to
anticholinesterase
chemicals
should
be
assessed.
These
analyses
should
be
conducted
in
the
context
of
the
adequacy
of
the
protocols
used
and
the
quality
of
the
available
data.
Based
on
this
weight­
of­
the­
evidence
analysis
for
an
anticholinesterase
pesticide,
OPP
may
select
as
the
critical
effects
any
one
or
more
of
the
behavioral
and
physiological
changes
or
enzyme
measures
listed
above.

Although
clinical
signs/
symptoms,
physiological
and
behavioral
changes
are
considered
very
important
for
characterizing
an
adverse
effect
in
humans,
these
endpoints
are
not
given
disproportionate
emphasis
or
relied
on
solely,
or
even
always
necessarily
preferred,
in
selecting
critical
effects
for
risk
assessment
because
the
evaluations
of
such
endpoints
have
limitations.
Comprehensive
measures
of
AChE
activity
in
nervous
system
tissues,
particularly
as
they
may
reflect
age­
related
differences,
are
considered
very
important
and
are
given
considerable
prominence
in
the
weight­
of­
the­
evidence
analysis
for
selection
of
critical
effects
because,
as
discussed
earlier,
acetylcholinesterase
inhibition
is
considered
a
key
event
in
the
mechanism
of
toxicity
for
the
cholinesterase­
inhibiting
organophosphorous
and
carbamate
pesticides
and
a
substantial
body
of
literature
exists
which
links
enzyme
inhibition
with
a
broad
range
of
adverse
effects.
Thus,
data
on
cholinesterase
inhibition
may
be
viewed
as
predictors
of
potential
adverse
responses
mediated
via
cholinergic
pathways
and
may
be
used
instead
of,
or
in
the
absence
of,
data
on
clinical
signs
and
symptoms,
and
other
physiological
and
behavioral
effects.
Direct
measures
of
AChE
inhibition
in
the
neural
target
tissues,
(
i.
e.,
central
and
peripheral
nervous
systems)
are
preferred.
However,
when
such
data
are
missing
or
inadequate,
they
would
obviously
receive
less
weight
in
the
analysis.
In
these
circumstances,
measures
of
cholinesterase
inhibition
in
the
blood
(
plasma
and/
or
RBC)
are
viewed
as
reasonable
surrogates
for
the
peripheral
nervous
system
given
that
the
blood
is
the
pharmacokinetic
compartment
into
which
chemicals
are
absorbed
and
transported
to
the
peripheral
nervous
system.
In
animals,
data
on
blood
cholinesterase
inhibition
also
are
considered
important
companion
data
for
central
nervous
system
AChE
inhibition
data,
even
though
the
brain
constitutes
a
different
pharmacokinetic
compartment.
As
noted
earlier,
blood
measures
(
both
plasma
and
RBC)
of
cholinesterase
activity
in
human
studies
must
serve
as
surrogates
for
enzyme
activity
in
both
central
and
peripheral
nervous
systems,
in
light
of
the
lack
of
availability
of
data
on
these
parameters.
As
discussed
in
Section
3.3,
within
the
blood
compartment,
RBC
AChE
data,
if
reliable,
are
generally
preferred
over
plasma
data.
Even
though
plasma
contains
a
mixture
of
AChE
and
BuChE,
plasma
cholinesterase
data
should
be
evaluated
and
considered
in
the
parallel
analyses
as
described
below.
Also,
there
may
be
certain
situations
where
plasma
cholinesterase
inhibition
may
be
selected
as
the
critical
effect
for
the
risk
assessment.

Evaluation
of
the
statistical
and
toxicological
significance
of
the
study
results
and
application
of
uncertainty
factors
follows
the
Agency's
established
procedures
for
derivation
of
an
RfD
or
RfC
and
the
principles
articulated
in
the
FQPA
10X
Safety
Factor
policy.
A
description
of
the
strengths,
weaknesses,
and
limitations
of
the
database
is
included;
this
evaluation
may
lead
to
the
identification
of
data
needed
to
refine
the
data
base
and
the
risk
assessment.
Any
residual
concerns
(
i.
e.,
significant
uncertainties)
are
accommodated
for
when
making
the
FQPA
10X
Safety
Factor
determination.

Practically,
the
weight­
of­
the­
evidence
analysis
may
be
viewed
as
having
several
steps:
first,
the
individual
studies
are
evaluated;
second,
all
studies
in
the
database
and
their
relationship
to
one
another
are
examined
in
an
integrated
manner;
and
lastly,
the
critical
effects
are
selected
for
risk
assessment
and
additional
data
needs
identified.
Below
is
a
more
detailed
discussion
of
these
steps.

4.1
ANALYSIS
OF
INDIVIDUAL
STUDIES
For
a
full
evaluation
of
an
anticholinesterase
pesticide,
the
important
elements
of
a
study
should
include:

!
Evaluations
of
physiological
and
behavioral
effects;

!
Measures
of
central
nervous
system
acetylcholinesterase
activity
(
in
animal
studies)
(
often
these
will
be
whole
brain
measures
rather
than
measures
in
specific
brain
regions)

!
Measures
of
peripheral
nervous
system
acetylcholinesterase
inhibition
(
in
animal
studies)
(
rarely
available
at
the
present
time);

!
Measures
of
RBC
and
plasma
cholinesterase
inhibition.

First,
each
study
is
critically
evaluated.
Results
should
be
assessed
in
the
context
of
both
statistical
and
biological
significance.
No
fixed
percentage
of
change
(
e.
g.
20%
for
cholinesterase
enzyme
inhibition)
is
predetermined
to
separate
adverse
from
non­
adverse
effects.
OPP's
experience
with
the
review
of
toxicity
studies
with
cholinesterase­
inhibiting
substances
shows
that
differences
between
pre­
and
postexposure
of
20%
or
more
in
enzyme
levels
is
nearly
always
statistically
significant
and
would
generally
be
viewed
as
biologically
significant.
The
biological
significance
of
statistically­
significant
changes
of
less
than
20%
would
have
to
be
judged
on
a
caseby
case
basis,
noting,
in
particular,
the
pattern
of
changes
in
the
enzyme
levels
and
the
presence
or
absence
of
accompanying
clinical
signs
and/
or
symptoms.
The
study
evaluation
involves
consideration
of,
among
other
factors:
the
adequacy
of
study
protocol
and
design
(
e.
g.,
experimental
group
size
and
characteristics
(
e.
g.,
single
gender
or
both),
dose
spacing,
methods
used
for
neurochemical
and
functional
evaluations);
whether
pre­
exposure
data
were
obtained
in
the
subsequently­
exposed
individuals
(
i.
e.
measures
are
taken
before
and
after
treatment
in
the
same
individual,
then
statistically
analyzed
as
such)
or
a
separate
control
group
was
used,
and
the
general
conduct
of
the
study.
The
consistency
of
the
findings
within
the
study
when
repeated
measures
are
taken,
the
dose­
dependency
of
the
responses,
as
well
as
the
temporal
aspects
of
effects
(
e.
g.,
the
time­
of­
onset,
steady
state,
time­
to­
peak
effects
and
the
time
until
complete
recovery)
and
the
statistical
significance
of
any
differences
measured
between
unexposed
and
exposed
groups
are
to
be
examined
before
reaching
any
conclusions
regarding
biological
significance.
The
relationship
of
the
different
effects
seen
to
one
another
also
should
be
considered
in
interpreting
the
findings.
Following
critical
evaluation
of
the
validity
of
the
study,
candidate
points
of
departure
(
i.
e.,
NOAELs,
LOAELs,
or
benchmark
doses)
are
identified
or
calculated.
4.2
INTEGRATIVE
ANALYSIS
OF
THE
DATA
BASE
When
evaluating
the
entire
database
and
selecting
an
endpoint(
s)
as
the
critical
effect(
s)
to
serve
as
the
PoD
in
the
derivation
of
a
RfD
or
RfC,
parallel
analyses
of
the
dose­
response
(
i.
e.,
changes
in
magnitude
of
enzyme
inhibition
or
of
a
different
effect
with
increasing
dose)
and
the
temporal
pattern
of
all
relevant
effects
will
be
compared
across
all
of
the
different
compartments
affected
(
e.
g.,

plasma,
RBC,
peripheral
nervous
system,
brain),
and
for
the
functional
changes
to
the
extent
the
database
permits.
The
overall
adequacy
of
the
test
protocols
and
the
quality
of
the
data
also
are
important
elements
of
the
analysis.

The
consistency
(
or,
the
lack
thereof)
of
LOAELs,
NOAELs,
or
BMDs
for
each
category
of
effects
(
e.
g.,
clinical
signs,
cholinesterase
inhibition
in
the
various
compartments,
etc.)
for
the
test
species/
strains/
sex
available
and
for
each
duration
and
route
of
exposure
should
be
noted.
If
scientifically
valid,
reliable,
and
ethically
appropriate
to
use,
human
data
may
be
preferable
to
animal
data
because
they
preclude
the
need
for
extrapolation
of
results
across
species,
avoiding
the
uncertainties
attendant
to
this
aspect
of
the
risk
assessment
process.
Confidence
in
the
selection
of
an
endpoint(
s)
for
derivation
of
an
RfD
or
RfC
is
enhanced
by
the
factors
described
in
Table
1.
The
findings
for
anticholinesterase
pesticides
will
span
a
broad
continuum,

and
their
databases
will
range
from
those
which
are
comprehensive
and
robust
to
those
Table
1.
Factors
to
consider
for
selection
of
Points
of
Departure
for
anticholinesterase
chemicals
Higher
confidence:

$
Methodologies
employed
are
valid
and
reliable
$
Sample
processing
is
reliable
and
appropriate
$
Reliable
data
are
available
for
RBC,
plasma,
and
brain
cholinesterase
inhibition
as
well
as
functional
data
in
individual
studies
$
Responses
are
dose
dependent,
and
NOAELs
are
apparent
$
Time­
to­
peak
effects
and
steady
state
are
demonstrated
$
Responses
for
each
end
point
evaluated
are
consistent
within
multiple
tests
in
the
same
species/
strain/
sex
which
are
limited
and
of
poor
quality.
Thus,
end
point
selection
and
weight­
of­

theevidence
judgments
must
be
made
on
a
case­
by­
case
basis.
For
example,
often
cholinesterase
inhibition
data
in
a
single
compartment
may
be
inconsistent
across
studies
involving
the
same
species
or
strain.
In
some
cases,
large
differences
may
be
noted
in
the
magnitude
of
cholinesterase
inhibition
in
one
compartment
in
comparison
to
all
the
other
compartments.
In
other
cases,
there
is
no
dose­
effect
relationship
for
cholinesterase
inhibition
in
one
or
more
compartments.
Time
course
data
for
cholinesterase
inhibition
also
are
often
limited.
Brain
measures
of
AChE
activity
are
often
limited
to
whole
brain
at
termination
of
an
animal
study.
So,
a
typical
database
for
an
anticholinesterase
pesticide
will
likely
contain
a
number
of
inadequacies
that
can
have
a
broad
spectrum
of
influence
from
none
to
substantial
on
the
selection
of
critical
effects.
It
should
be
emphasized,
however,
that
the
lack
of,
or
deficiency
in,
any
one
factor
listed
in
Table
1
would
not
necessarily
discount
the
usefulness
of
a
study
in
selecting
an
endpoint
for
calculation
of
an
RfD
or
RfC.
Functional
evaluations
are
limited
in
both
human
and
animal
studies.
Therefore,
as
described
earlier,
measures
of
cholinesterase
inhibition
are
included
in
the
weight­
of­
the­
evidence
evaluation;
the
reliance
on
all
relevant
data
is
considered
to
be
both
scientifically
sound
and
public
health
protective.
Cholinesterase
inhibition
in
the
blood
may
occur
at
lower
doses
than
other
cholinergic
effects
(
e.
g.,
brain
AChE
inhibition,
functional
effects).
The
NOAEL
or
equivalent
benchmark
dose
for
RBC
AChE
inhibition
and
that
for
plasma
and/
or
brain
may
not
be
the
same.
This
could
be
due
to
methodological
problems
or
to
the
different
binding
affinities
of
a
pesticide
to
AChE
compared
to
those
for
BuChE
or
to
a
number
of
other
factors.
As
explained
in
Section
3.3,
if
the
measurements
of
AChE
inhibition
in
RBCs
are
considered
methodologically
sound,
these
data
generally
are
preferred
over
plasma
cholinesterase
activity
data
as
predictors
of
neural
AChE
activity,
even
if
the
plasma
NOAEL/
BMD
is
lower.
However,
if
the
RBC
data
are
unreliable
(
e.
g.,
questions
exist
about
the
methodology
or
there
is
no
dose­
dependency)
or
the
dose
response
for
inhibition
of
plasma
cholinesterase
more
closely
approximates
that
for
AChE
inhibition
in
the
nervous
system
than
does
the
dose
response
for
RBC
acetylcholinesterase
inhibition,
plasma
cholinesterase
inhibition
may
be
the
more
prudent
endpoint
to
use
to
represent
the
critical
effect.
Occasionally,
because
of
methodological
difficulties
or
for
other,
poorly­
understood
reasons,
empirical
correlations
between
the
doses
that
cause
plasma
and
brain
cholinesterase
inhibition
(
in
the
same
or
other
studies)
may
be
stronger
than
those
between
the
doses
for
RBC
and
brain
enzyme
inhibition.

The
weight­
of­
the­
evidence
approach
emphasizes
the
determination
of
the
quality
of
the
cholinesterase
data,
especially
the
RBC
measures.
Standard
operating
procedures
for
measuring
cholinesterase
activity
have
continued
to
evolve
over
the
last
decade
(
Wilson,
et.
al.,
1996;
Hunter,
et.
al.,
1997);
detailed
information
on
the
method(
s)
and
procedures
used
for
measurements
of
cholinesterase
activity
following
treatment
is
important.
The
method
used
for
carbamate
pesticides
is
particularly
important
because
the
reliability
of
data
on
cholinesterase
effects
depends
not
only
on
the
specific
methodology
used,
but
to
a
great
extent
on
sample
processing
(
given
the
readily
reversible
nature
of
the
carbamylated
AchE).

4.3
COLLECTION
OF
PERIPHERAL
NERVOUS
SYSTEM
CHOLINESTERASE
INHIBITION
DATA
As
discussed
earlier,
the
1997
FIFRA
Scientific
Advisory
Panel
endorsed
collection
of
peripheral
nervous
system
AChE
data
as
being
technically
feasible
and
advised
OPP
that
these
data
may
be
a
better
indicator
of
cholinergic
effects
than
are
blood
cholinesterase
measures.
The
ILSI
Panel
provided
more
technical
guidance
along
with
a
number
of
recommendations
for
further
studies
to
improve
the
methodologies,
while,
nonetheless
concluding
that
such
measures
could
be
taken
now
(
Mileson,
et
al.,
1999b).
OPP
agrees
that
peripheral
nervous
system
measurements
from
a
suitable
set
of
tissues
could
provide
an
alternative
to
blood
measures.
OPP,

with
ongoing
technical
and
research
support
from
NHEERL,
will
continue
to
support
the
development
and
validation
of
methodologies
for
measuring
peripheral
neural
AChE
inhibition.
This
was
a
major
aspect
of
OPP=
s
policy
in
1997
and
was
endorsed
by
the
1997
SAP.
Once
a
methodology
is
validated,
data
from
such
studies
will
be
sought
on
a
regular
basis
and
used
to
supplement
or
replace
blood
measures
which
now
serve
as
a
surrogate
for
the
peripheral
nervous
system.
In
the
interim,
any
data
on
peripheral
tissues
will
be
evaluated
and
incorporated
into
the
risk
assessment
on
a
case­
by­
case
basis.
OPP
strongly
encourages
the
development
of
any
data
aimed
at
refining
risk
assessments
based
upon
blood
measures
to
be
focused
on
peripheral
nervous
system
measures
of
AChE.
Additional
data
to
differentiate
between
the
acetylcholinesterase
and
butyrylcholinesterase
in
plasma,
a
procedure
recommended
by
the
SAP
in
1997,

would
also
be
useful.

Additional
studies
to
provide
data
on
metabolism,
pharmacokinetics
and
pharmacodynamics
also
may
be
useful
to
aid
in
the
characterization
of
the
cholinesterase
inhibiting
properties
and
potential
hazard
of
organophosphorous
and
carbamate
pesticides.
To
lessen
the
uncertainties
inherent
in
route­
to­
route
extrapolation,
endpoint
specific
data
could
be
collected
on
exposure
routes
of
interest,

such
as
cholinesterase
inhibition
following
dermal
exposure.
5.
CONCLUSION
The
elements
of
the
weight­
of­
the­
evidence
evaluations
used
for
selecting
toxicity
endpoint(
s)
are
summarized
in
Table
2.
Weight­
of­
the­
evidence
judgments
must
be
sound
and
supported
by
the
data
on
the
individual
pesticides.
The
risk
assessor
should
provide
a
hazard
characterization
that
summarizes
the
endpoint
data
that
are
available
for
consideration,
discusses
the
strengths,
limitations
and
uncertainties
of
the
data,
and
describes
how
well
the
data
supports
the
conclusions.

The
rationale
for
selection
of
the
critical
effect(
s)
must
be
clearly
articulated
in
this
characterization.
Table
2.
Elements
of
the
Weight­
of­
the­
Evidence
Approach
OPP
is
using
a
weight­
of­
the­
evidence
approach
to
analyze
individual
studies
and
the
overall
database
on
an
organophosphorous
or
carbamate
cholinesteraseinhibiting
pesticide
to
select
the
appropriate
critical
effect(
s)
for
hazard
assessment.

Based
on
this
weight­
of­
the­
evidence
analysis,
OPP
may
select
as
critical
effects
any
one
or
more
of
the
following
functional
or
biochemical
measures:

!
physiological
and
behavioral
effects
in
humans
and
animals;

!
central
or
peripheral
nervous
system
tissue
acetylcholinesterase
inhibition;

!
red
blood
cell
acetylcholinesterase
inhibition;

!
plasma
cholinesterase
inhibition
(
butyrylcholinesterase
in
humans;
a
mixture
of
acetylcholinesterase
and
butyrlcholinesterase
in
animals).

Because
functional
evaluations
have
limitations,
measures
of
acetylcholinesterase
inhibition
are
considered
in
the
weight­
of­
the­
evidence
for
selection
of
critical
effects.
Although
data
on
acetylcholinesterase
inhibition
in
neural
tissues
are
preferred,
peripheral
nervous
system
data
are
rarely
available
in
animals,
and
in
humans
neither
peripheral
nor
central
nervous
system
data
are
typically
available.
Blood
measures
can
serve
as
surrogate
information
for
projecting
potential
hazards,
as
a
matter
of
science
policy
in
protecting
human
health.
Although
plasma
cholinesterase
data
may
be
used
under
certain
circumstances,
in
general,
red
blood
cell
data,
if
available
and
reliable,
are
preferred
over
plasma
data.

In
selecting
the
appropriate
endpoint(
s)
for
hazard
assessment,
all
available
response
data
(
i.
e.,
all
available
cholinesterase
enzyme
activity
and
functional
data)
are
evaluated
for
data
quality,
and
analyzed
for
comparisons
of
dose­
dependency,
time
to
onset,
steady
state,
time
to
peak
effects,
duration
of
effects
within
and
across
all
test
species/
sexes
for
all
durations
and
routes
of
exposure.
Evaluation
of
statistical
and
toxicological
significance,
and
application
of
uncertainty
factors,
follows
established
Agency
procedures
for
the
derivation
of
reference
doses
or
reference
concentrations.
If
limitations
are
identified
in
the
database,
additional
data
may
be
required
to
reduce
any
uncertainties
and
refine
the
hazard
assessment
and/
or
accommodated
for
during
the
FQPA
10X
Safety
Factor
decision
process.
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