1
July
19,
2002
MEMORANDUM
SUBJECT:
Transmittal
of
Meeting
Minutes
of
the
FIFRA
Scientific
Advisory
Panel
Meeting
Held
June
26­
27,
2002
TO:
Marcia
E.
Mulkey,
Director
Office
of
Pesticide
Programs
FROM:
Paul
I.
Lewis,
Designated
Federal
Official
FIFRA
Scientific
Advisory
Panel
Office
of
Science
Coordination
and
Policy
THRU:
Larry
C.
Dorsey,
Executive
Secretary
FIFRA
Scientific
Advisory
Panel
Office
of
Science
Coordination
and
Policy
Sherell
A.
Sterling,
Acting
Director
Office
of
Science
Coordination
and
Policy
Please
find
attached
the
meeting
minutes
of
the
FIFRA
Scientific
Advisory
Panel
open
meeting
held
in
Arlington,
Virginia
from
June
26­
27,
2002.
This
report
addresses
a
set
of
scientific
issues
being
considered
by
the
Environmental
Protection
Agency
regarding
determination
of
the
appropriate
FQPA
Safety
Factor(
s)
in
the
organophosphorous
pesticide
cumulative
risk
assessment:
susceptibility
and
sensitivity
to
the
common
mechanism,
acetylcholinesterase
inhibition.

Attachment
2
cc:

Stephen
Johnson
Susan
Hazen
Adam
Sharp
James
Jones
Janet
Andersen
Debbie
Edwards
Anne
Lindsay
Steve
Bradbury
Denise
Keehner
Linda
Moos
Lois
Rossi
Frank
Sanders
Margaret
Stasikowski
William
Jordan
Antonio
Bravo
Douglas
Parsons
David
Deegan
Vanessa
Vu
(SAB)
OPP
Docket
FIFRA
Scientific
Advisory
Panel
Members
Stephen
M.
Roberts,
Ph.
D.
Fumio
Matsumura,
Ph.
D.
Herbert
Needleman,
M.
D.
Christopher
J.
Portier,
Ph.
D.
Mary
Anna
Thrall,
D.
V.
M.

FQPA
Science
Review
Board
Members
John
Bigbee,
Ph.
D.
William
Brimijoin,
Ph.
D.
Amira
T.
Eldefrawi,
Ph.
D.
Jean
Harry,
Ph.
D.
Dale
Hattis,
Ph.
D.
George
Lambert,
M.
D.
Michael
McClain,
Ph.
D.
Carey
Pope,
Ph.
D.
Nu­
May
Ruby
Reed,
Ph.
D.
Lester
Sultatos,
Ph.
D.
3
SAP
Meeting
Minutes
No.
2002­
03
June
26­
27,
2002
FIFRA
Scientific
Advisory
Panel
Meeting,
held
at
the
Sheraton
Crystal
City
Hotel,
Arlington,
Virginia
A
Set
of
Scientific
Issues
Being
Considered
by
the
Environmental
Protection
Agency
Regarding:

Determination
of
the
Appropriate
FQPA
Safety
Factor(
s)
in
the
Organophosphorous
Pesticide
Cumulative
Risk
Assessment:
Susceptibility
and
Sensitivity
to
the
Common
Mechanism,
Acetylcholinesterase
Inhibition
4
NOTICE
This
report
has
been
written
as
part
of
the
activities
of
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(FIFRA),
Scientific
Advisory
Panel
(SAP).
This
report
has
not
been
reviewed
for
approval
by
the
United
States
Environmental
Protection
Agency
(Agency)
and,
hence,
the
contents
of
this
report
do
not
necessarily
represent
the
views
and
policies
of
the
Agency,
nor
of
other
agencies
in
the
Executive
Branch
of
the
Federal
government,
nor
does
mention
of
trade
names
or
commercial
products
constitute
a
recommendation
for
use.

The
FIFRA
SAP
was
established
under
the
provisions
of
FIFRA,
as
amended
by
the
Food
Quality
Protection
Act
(FQPA)
of
1996,
to
provide
advice,
information,
and
recommendations
to
the
Agency
Administrator
on
pesticides
and
pesticide­
related
issues
regarding
the
impact
of
regulatory
actions
on
health
and
the
environment.
The
Panel
serves
as
the
primary
scientific
peer
review
mechanism
of
the
EPA,
Office
of
Pesticide
Programs
(OPP),
and
is
structured
to
provide
balanced
expert
assessment
of
pesticide
and
pesticide­
related
matters
facing
the
Agency.
Food
Quality
Protection
Act
Science
Review
Board
members
serve
the
FIFRA
SAP
on
an
ad­
hoc
basis
to
assist
in
reviews
conducted
by
the
FIFRA
SAP.
Further
information
about
FIFRA
SAP
reports
and
activities
can
be
obtained
from
its
website
at
http://
www.
epa.
gov/
scipoly/
sap/
or
the
OPP
Docket
at
(703)
305­
5805.
Interested
persons
are
invited
to
contact
Larry
Dorsey,
SAP
Executive
Secretary,
via
e­
mail
at
dorsey.
larry@.
epa.
gov.
5
SAP
Meeting
Minutes
No.
2002­
03
FIFRA
Scientific
Advisory
Panel
Meeting,
June
26­
27,
2002,
held
at
the
Sheraton
Crystal
City
Hotel,
Arlington,
Virginia
A
Set
of
Scientific
Issues
Being
Considered
by
the
Environmental
Protection
Agency
Regarding:

Determination
of
the
Appropriate
FQPA
Safety
Factor(
s)
in
the
Organophosphorous
Pesticide
Cumulative
Risk
Assessment:
Susceptibility
and
Sensitivity
to
the
Common
Mechanism,
Acetylcholinesterase
Inhibition
Mr.
Paul
Lewis
Stephen
M.
Roberts,
Ph.
D.
Designated
Federal
Official
FIFRA
SAP
Session
Chair
FIFRA
Scientific
Advisory
Panel
FIFRA
Scientific
Advisory
Panel
Date:
July
19,
2002
Date:
July
19,
2002
6
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
Scientific
Advisory
Panel
Meeting
June
26­
27,
2002
Determination
of
the
Appropriate
FQPA
Safety
Factor(
s)
in
the
Organophosphorous
Pesticide
Cumulative
Risk
Assessment:
Susceptibility
and
Sensitivity
to
the
Common
Mechanism,
Acetylcholinesterase
Inhibition
PARTICIPANTS
FIFRA
SAP
Session
Chair
Stephen
M.
Roberts,
Ph.
D.,
University
of
Florida,
Gainesville,
FL
Designated
Federal
Official
Mr.
Paul
Lewis,
FIFRA
Scientific
Advisory
Panel
Staff,
Office
of
Science
Coordination
and
Policy
FIFRA
Scientific
Advisory
Panel
Fumio
Matsumura,
Ph.
D.,
University
of
California
at
Davis,
Davis,
CA
Herbert
Needleman,
M.
D.,
University
of
Pittsburgh,
Pittsburgh,
PA
Christopher
J.
Portier,
Ph.
D.,
National
Institute
of
Environmental
Health
Science
Research
Triangle
Park,
NC
Mary
Anna
Thrall,
D.
V.
M.,
Colorado
State
University,
Fort
Collins,
CO
FQPA
Science
Review
Board
Members
John
Bigbee,
Ph.
D.,
Virginia
Commonwealth
University,
Richmond,
VA
William
Brimijoin,
Ph.
D.,
Mayo
Clinic
and
Medical
School,
Rochester,
MN
Amira
T.
Eldefrawi,
Ph.
D.,
University
of
Maryland
School
of
Medicine,
Baltimore,
MD
Jean
Harry,
Ph.
D.,
National
Institute
of
Environmental
Health
Science,
Research
Triangle
Park,
NC
Dale
Hattis,
Ph.
D.,
Clark
University,
Worcester,
MA
George
Lambert,
M.
D.,
Environmental
and
Occupational
Health
Sciences
Institute,
UMDNJ,
Piscataway,
NJ
Michael
McClain,
Ph.
D.,
McClain
and
Associates,
Randolph,
NJ
Carey
Pope,
Ph.
D.,
Oklahoma
State
University,
Stillwater,
OK
Nu­
May
Ruby
Reed,
Ph.
D.,
California
Environmental
Protection
Agency,
Sacramento,
CA
Lester
Sultatos,
Ph.
D.,
New
Jersey
Medical
School,
Newark,
NJ
INTRODUCTION
The
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(FIFRA),
Scientific
Advisory
Panel
(SAP)
has
completed
its
review
of
the
set
of
scientific
issues
being
considered
by
the
7
Agency
pertaining
to
determination
of
the
appropriate
FQPA
Safety
Factor(
s)
in
the
organophosphorous
pesticide
cumulative
risk
assessment:
susceptibility
and
sensitivity
to
the
common
mechanism,
acetylcholinesterase
inhibition.

Advance
notice
of
the
meeting
was
published
in
the
Federal
Register
on
May
31,
2002.
The
review
was
conducted
in
an
open
Panel
meeting
held
in
Arlington,
Virginia,
on
June
26­
27,
2002.
The
meeting
was
chaired
by
Dr.
Stephen
Roberts.
Mr.
Paul
Lewis
served
as
the
Designated
Federal
Official.

Before
the
Agency
presentation
on
issues
pertaining
to
determination
of
the
appropriate
FQPA
safety
factor,
Mr.
Francis
B.
Suhre
(Office
of
Pesticide
Programs,
EPA)
provided
the
Panel
a
status
report
on
organophosporus
pesticide
cumulative
risk
estimates:
comparison
of
outputs
from
different
models.

Vicki
Dellarco,
Ph.
D.
(Office
of
Pesticide
Programs,
EPA),
began
the
Agency
presentations
by
providing
an
introduction
and
overview
of
the
approach
to
evaluating
susceptibility/
sensitivity
of
children
in
cumulative
risk
assessments
and
review
of
available
animal
studies.
Stephanie
Padilla,
Ph.
D.
(Office
of
Research
and
Development,
EPA)
summarized
age
dependent
sensitivity
and
susceptibility.
Vicki
Dellarco,
Ph.
D.
(Office
of
Pesticide
Programs,
EPA)
ended
the
Agency
presentation
by
discussing
the
risk
characterization
of
sensitivity
and
susceptibility.
Other
EPA
participants
were
Randy
Perfetti,
Ph.
D.
(Office
of
Pesticide
Programs,
EPA)
and
Karl
Baetcke,
Ph.
D.
(Office
of
Pesticide
Programs,
EPA).

In
preparing
these
meeting
minutes,
the
Panel
carefully
considered
all
information
provided
and
presented
by
the
Agency
presenters,
as
well
as
information
presented
by
public
commenters.
These
meeting
minutes
address
the
information
provided
and
presented
at
the
meeting,
especially
the
response
to
the
charge
by
the
Agency.

PUBLIC
COMMENTERS
Oral
statements
were
made
by:

Jennifer
Sass,
Ph.
D.,
on
behalf
of
the
Natural
Resources
Defense
Council
Angelina
Duggan,
Ph.
D.,
on
behalf
of
Sound
Science
Policy
Alliance
Larry
Sheets,
Ph.
D.,
Bayer
Crop
Science,
on
behalf
of
CropLife
America
James
Gibson,
Ph.
D.,
The
Brody
School
of
Medicine
of
Eastern
Carolina
University,
on
behalf
of
Sound
Science
Policy
Alliance
Jack
M.
Zabik,
Ph.
D.,
Dow
AgroSciences,
on
behalf
on
CropLife
America
8
Mr.
Ed
Gray,
McDermott
Will
and
Emery,
on
behalf
of
FQPA­
Implementation
Working
Group
Mr.
Art
Beltrone,
private
citizen
Judith
Schreiber,
Ph.
D.
State
of
New
York,
Office
of
the
Attorney
General
Written
statements
were
received
as
follows:

No
written
comments
were
received.

CHARGE
Issue
1.
Role
of
Cholinesterases
and
Acetylcholine
As
discussed
in
the
EPA
report,
inhibition
of
acetylcholinesterase
(AChE)
in
the
young
can
result
in
cholinergic
toxicity
as
in
the
adult,
but
evidence
has
also
been
emerging
over
the
last
several
years
that
indicates
that
AChE
and
acetylcholine
may
serve
as
neuromodulators
in
development.

Question
1.1
Please
comment
on
the
extent
to
which
the
report
adequately
summarizes
the
current
state
of
knowledge.
Does
the
scientific
evidence
support
the
conclusion
that
perturbation
of
the
cholinergic
nervous
system
during
development
by
inhibiting
AChE
can
potentially
lead
to
deficits
in
the
structure
and
function
of
the
central
and
peripheral
nervous
systems?

Issue
2.
Age­
Dependent
Sensitivity
to
Cholinesterase
inhibition
in
Animal
Studies
Age­
dependent
sensitivity
(i.
e.,
young
animals
can
exhibit
higher
levels
of
cholinesterase
(ChE)
inhibition
at
the
same
dose
or
inhibition
at
lower
doses
compared
to
adults)
has
been
observed
in
several
laboratory
studies
following
treatment
(acute
and/
or
repeated
oral
gavage
doses)
of
neonatal,
juvenile,
and
adult
rats
with
organophosphorus
(OP)
pesticides.
The
exact
mechanisms
of
this
age­
dependent
sensitivity
are
not
known,
but
several
studies
have
demonstrated
that
toxicokinetic
factors
may
be
responsible.
Most
notably,
the
more
limited
ability
of
the
young
to
detoxify
OP
pesticides
by
A­
esterases
and
carboxylesterases
appears
to
be
an
important
factor
underlying
the
increased
sensitivity
of
the
immature
rat
to
ChE
inhibition.
There
appears
to
be
more
rapid
recovery
of
inhibited
AChE
(synthesis
of
new
ChE
enzyme)
in
postnatal
(and
fetal)
rat
tissues,
but
information
on
comparative
recovery
in
children
and
human
adults
is
lacking.

Question
2.1
Please
comment
on
the
extent
to
which
the
report
adequately
discussed
and
summarized
9
the
current
understanding
of
age­
dependent
sensitivity
to
ChE
inhibition,
the
prevailing
views
in
the
scientific
community
concerning
the
biological
factors
involved,
and
the
role
esterases
may
play
as
a
major
factor
accounting
for
potential
increased
sensitivity
of
the
immature
rat.

Question
2.2
Please
comment
on
the
timing
of
administration
(i.
e.,
the
developmental
stage
treated)
and
the
differential
found
between
adults
and
the
young
animal.

Question
2.3
Please
comment
on
the
extent
to
which
comparative
ChE
data
on
six
OP
pesticides
(chlorpyrifos,
diazinon,
dimethoate,
methamidophos,
malathion,
methyl
parathion)
may
represent
a
reasonable
subset
of
different
structural
and
pharmacokinetic
characteristics
of
the
cumulative
group
of
OP
pesticides
to
define
an
upper
bound
on
the
differential
sensitivity
that
may
be
expected
at
different
life
stages
of
the
immature
animal.
As
an
example,
there
are
no
chemicalspecific
comparative
cholinesterase
data
on
azinphos­
methyl
(AZM),
an
important
contributor
of
risk
for
the
food
pathway.
Pesticide­
specific
comparative
cholinesterase
data
on
the
other
six
pesticides
from
the
OP
class
(including
data
on
malathion,
a
member
of
the
same
chemical
subgroup
as
AZM)
show
a
limited
range
of
differential
sensitivities
­­
from
one­
fold
(no
increased
sensitivity)
up
to
three­
fold
­­
between
the
young
and
adults.
EPA
regards
these
data
on
other
OPs
as
providing
sufficient
evidence
to
assess
the
potential
for
AZM
to
show
age­
dependent
sensitivity,
and
to
reasonably
predict
the
degree
of
potential
difference
in
sensitivity
between
the
young
and
adults.
Given
the
results
of
the
other
OPs,
EPA
concludes
that
it
is
unlikely
that
AZM
would
exceed
a
magnitude
of
difference
greater
than
approximately
3­
fold
following
treatment
of
PND
11
through
21
pups
versus
adult
animals.

Issue
3.
Relevance
of
the
Animal
Findings
to
Children
Age
dependent
sensitivity
to
cholinesterase
inhibition
has
been
associated
with
the
limited
ability
of
the
immature
rat
to
detoxify
OP
pesticides
by
esterases.
In
rats,
A­
esterase
activity
increases
from
birth
to
reach
adult
levels
around
postnatal
day
21.
Fetal
rats
possess
very
little
carboxylesterase
activity
with
increasing
activity
as
the
postnatal
rat
matures,
reaching
adult
values
after
puberty
(50
days
of
age).
Data
showing
increased
sensitivity
of
the
young
animal
to
cholinesterase
inhibition
compared
to
adults
has
generally
been
derived
from
acute
dosing
of
PND
7
or
PND
11
pups,
or
repeated
dosing
of
PND
11
to
PND
21
pups.
The
available
data
also
show
as
the
young
rat
rapidly
matures
in
its
ability
to
detoxify
by
esterases,
the
differential
in
cholinesterase
inhibition
becomes
smaller.
Thus,
the
relative
sensitivities
of
immature
rats
found
in
the
studies
of
dosing
pups
through
PND
11
to
21
are
smaller
compared
to
studies
of
dosing
a
PND
11
pup.
The
dosing
studies
of
PND
11
through
21
pups
are
considered
to
better
approximate
the
maturation
profile
of
the
A­
esterases
of
the
highly
exposed
children's
age
group
in
the
OP
cumulative
risk
assessment,
the
one
and
two
year
olds,
compared
to
a
study
of
a
PND
11
pup
which
is
similar
to
a
newborn.
Thus,
the
repeated
rat
dosing
studies
more
closely
mimic
10
the
maturation
or
developmental
profile
of
A­
esterase
appearance
in
children
around
the
one
and
two
year
olds
where
children
are
reaching
adult
levels
of
A­
esterase
activity.
The
use
of
dosing
studies
of
PND
11
through
21
is
consistent
with
the
exposure
patterns
of
children.
Humans
generally
do
not
begin
to
consume
fresh
(uncooked)
fruits
and
vegetables
until
after
six
months
of
age
or
more.
Furthermore,
repeated
dosing
studies
were
used
to
determine
relative
sensitivity
because
people
are
exposed
every
day
to
an
OP
pesticide
through
food,
and
thus
an
animal
study
using
repeat
exposures
is
considered
appropriate.
Finally,
following
exposure
to
an
OP,
regeneration
of
cholinesterase
to
pre­
exposure
levels
does
not
occur
for
days
or
weeks,
making
the
exposed
individual
potentially
more
vulnerable
to
subsequent
exposures
during
that
period.

Question
3.1
Please
comment
on
the
maturation
profile
of
A­
esterase
and
the
uncertainties
surrounding
these
data
in
young
children.
Because
no
human
data
are
available
on
the
maturation
profile
of
carboxylesterases,
please
comment
on
what
should
be
assumed
in
humans,
especially
children
age
1
to
2
years,
given
the
animal
data
and
what
science
understands
in
general
about
detoxification
maturation
profiles.

Question
3.2
Please
comment
on
the
extent
to
which
the
biological
understanding
of
observed
agedependent
sensitivity
to
cholinesterase
inhibition
in
laboratory
animal
studies
informs
our
understanding
about
the
likelihood
of
similar
effects
occurring
in
children;
in
particular,
what
can
be
inferred
from
animal
and
human
information
regarding
the
potential
for
different
age
groups
to
show
increased
sensitivity
if
exposed
to
cholinesterase­
inhibiting
pesticides.
Does
the
scientific
evidence
support
the
conclusion
that
infants
and
children
are
potentially
more
sensitive
to
organophosphorus
cholinesterase
inhibitors?

Question
3.3
Please
comment
on
the
conclusions
regarding
the
faster
recovery
in
the
young
animal
of
AChE
activity.
Because
there
is
no
human
information
on
the
recovery
of
AChE
in
children
compared
to
adults,
please
comment
on
the
extent
to
which
recovery
of
AChE
in
children
should
be
factored
into
conclusions
regarding
potential
risk
to
children.

PANEL
CONSIDERATION
OF
AGENCY
APPLICATION
OF
THE
FQPA
SAFETY
FACTOR
The
Panel
was
not
explicitly
charged
with
making
a
determination
as
to
whether
the
EPA,
in
the
Agency
background
document
developed
for
this
meeting,
had
made
an
appropriate
choice
of
a
3x
versus
the
presumptive
10x
FQPA
safety
factor,
and
the
rational
11
for
its
decision.
Nonetheless,
discussion
of
this
point
arose
several
times
during
the
two­
day
Panel
session.
Given
the
importance
of
the
issue,
an
attempt
is
made
here
to
summarize
the
views
expressed
by
Panel
members
along
with
the
logic
behind
these
views.
The
Panel
recognizes
that
it
is
constituted
as
a
technical
advisory
body,
not
a
group
intended
to
provide
legal/
policy
advice.
However
the
choice
to
apply
particular
FQPA
safety
factors
in
EPA's
cumulative
risk
analysis
clearly
involves
both
policy
and
science.
A
legal/
policy
interpretation
is
needed
to
define
the
standard
of
evidence
required
to
depart
from
the
mandated
default
10­
fold
factor
in
any
ultimate
risk
management
decisions
that
might
be
made
on
the
basis
of
the
cumulative
risk
analysis.
Technical
judgments
are
also
needed
in
assessing
whether
any
particular
standard
of
evidence
has
been
met
by
the
data
available
for
individual
organophosphate
pesticides
or
AChE
inhibitors
as
a
common
mechanism
group.
The
discussion
below
summarizes
the
Panel's
assessment
of
the
scientific
evidence
pertaining
to
the
FQPA
safety
factor.

A
majority
of
the
Panel
members
who
commented
on
the
Agency
decision
of
an
appropriate
FQPA
safety
factor
disagreed
with
the
Agency's
proposal
to
deal
with
the
FQPA
requirements
to
ensure
protection
of
infants
and
children
by
selective
application
of
a
3X
safety
factor.
These
Panel
members
concluded
that
the
confidence
with
the
available
data
was
not
sufficient
to
assure
adequate
protection
with
less
than
the
10x
FQPA
safety
factor.
Other
Panel
members
were
prepared
to
accept
the
EPA
proposal,
some
with
certain
reservations.

The
Agency
has
proposed
not
to
apply
the
full
10x
FQPA
safety
factor
in
cases
where
animal
studies
have
indicated
that
younger
animals
(rats)
are
no
more
sensitive
than
adults
to
AChE
inhibition
by
repeated
(as
opposed
to
single
dose)
exposure
to
OPs.
Where
there
are
data
that
indicate
no
greater
sensitivity
for
cholinergic
inhibition
in
weanling
animals
than
in
adults,
the
Agency
would
apply
no
special,
additional
safety
factor.
The
Agency
proposes
to
apply
a
3X
safety
factor
(described
as
a
database
uncertainty
factor)
in
cases
of
chemicals
that
have
been
shown
to
be
about
three­
fold
more
potent
as
AChE
inhibitors
in
weanling
rats
than
in
adults.
The
Agency
also
proposes
to
apply
the
same
safety
factor
to
the
24
remaining
chemicals
currently
under
review,
while
awaiting
receipt
of
new
data
from
ongoing
studies
of
developmental
neurotoxicity
in
rats.

Various
reasons
were
cited
by
the
Panel
members
who
recommended
instead
that
the
EPA
apply
across
the
board
a
uniform
10X
FQPA
safety
factor.
The
most
widely
cited
reason
for
this
recommendation
was
a
concern
that
the
existing
animal
database
does
not
provide
sufficient
assurance
that
young
children
are
not
at
substantially
greater
risk
than
adults
from
exposures
to
OPs.
This
concern
was
based
on
the
uncertainties
arising
from
several
deficiencies
in
the
EPA's
cumulative
risk
analysis.
These
deficiencies
include
the
following:

1.
Extrapolation
from
data
on
a
limited
set
of
compounds.

The
EPA's
proposal
to
use
a
3­
fold
factor
for
the
cumulative
risk
assessment
is
based
on
relative
sensitivity
to
cholinesterase
inhibition
from
a
set
of
six
organophosphorus
toxicants.
At
12
most,
an
approximate
3­
fold
difference
in
sensitivity
to
cholinesterase
inhibition
was
noted
in
younger
animals
following
repeated
dosing.
The
EPA
considered
that
this
subset
of
compounds
represents
the
range
of
variability
of
differential
responses
for
all
30
compounds
under
consideration.
The
age­
dependence
of
differences
in
sensitivity
to
cholinesterase
inhibition
by
the
other
24
OP
toxicants
is
unknown.
This
data
gap
alone
was
felt
to
make
it
prudent
to
accept
the
10x
default.

2.
Uncertainties
about
the
mechanisms
of
age­
dependent
sensitivity
in
young
rats
and
their
applicability
to
human
beings.

Even
with
the
six
compounds
known
to
have
relatively
small
age­
dependent
sensitivity
in
young
rats,
the
extrapolation
to
humans
is
problematic.
First,
the
mechanism
of
age­
dependent
sensitivity
in
rats
has
not
yet
been
fully
elucidated.
More
important,
we
lack
comprehensive
information
about
the
relative
biotransformation
capacities
for
OPs
in
young
and
adult
humans,
and
about
the
relative
rates
of
enzyme
recovery
by
de
novo
synthesis
and
other
mechanisms.
Without
detailed
information
of
this
sort
(admittedly
difficult
to
obtain)
we
cannot
be
sure
that
the
relatively
rapid
decrease
in
OP
sensitivity
in
weanling
rats
will
also
apply
to
children
in
the
critical
1­
2
year
age
group.

3.
Limitations
of
animal
models
to
identify
effects
of
cholinesterase
inhibition
in
children
While
the
Agency
noted
that
the
OP
cumulative
risk
assessment
is
based
on
AChE
inhibition
and
cholinergic
toxicity,
more
relevant
indications
of
whether
an
exposure
to
OPs
are
"
safe
for
children''
are
needed,
specifically
behavioral
and
cognitive
measures
such
as
IQ,
attention,
language
function,
etc.
Much
uncertainty
is
introduced
by
using
AChE
inhibition
as
a
surrogate
for
these
endpoints.
For
example,
as
was
pointed
out
at
the
meeting,
it
is
not
known
whether
a
given
level
of
AChE
inhibition
has
the
same
consequences
for
a
young
child
as
for
an
adult.
Information
is
largely
lacking
about
the
sensitivity,
specificity
and
predictive
power
of
AChE
inhibition
as
a
marker
for
neurobehavioral
effects
of
OPs
based
on
current
animal
models.
In
addition,
such
information
is
also
lacking
in
terms
of
high
quality
epidemiological
studies
of
exposure
to
pesticides
to
infants
and
children.
Particularly,
the
lack
of
long
term
neurobehavioral
studies
at
any
stage
of
development
creates
a
great
deal
on
uncertainty
in
trying
to
identify
the
risks
of
the
OPs
to
children.

4.
Uncertainties
about
the
potential
frequency
of
"high­
level
exposure".

Another
consideration
in
the
application
of
the
FQPA
safety
factor
is
confidence
in
the
extent
to
which
the
exposure
assessment
truly
captures
high­
end
exposures,
particularly
in
children.
One
Panel
member
pointed
out
that
although
the
Agency
proposes
to
consider
upper
percentile
estimates
of
exposure
in
the
cumulative
risk
assessment,
these
estimates
may
not
be
as
high
as
the
percentiles
imply.
As
evidence
for
this,
an
example
was
cited
in
which
consumption
of
small
amounts
of
a
single
food
item
(e.
g.,
apple
or
pear)
containing
a
single
OP
at
the
upper
end
of
its
PDP
range
could
result
in
exposure
above
the
95
th
percentile
for
cumulative
dietary
13
exposure
calculated
by
the
Agency.
In
view
of
this,
an
argument
could
be
made
for
an
additional
FQPA
safety
factor
if
the
benchmark
for
risk
management
decision
is
a
percentile
of
exposure
that
does
not
adequately
address
infrequent,
but
not
truly
rare,
exposure
events.

While
aware
of
all
these
issues,
other
Panel
members
nonetheless
considered
that
the
Agency's
proposal
for
a
3X
safety
factor
was
reasonable,
with
certain
provisions.
The
major
provision
asked
for
by
some
of
these
panel
members
was
to
use
3X
safety
factors
even
for
agents
that
showed
no
age­
dependent
sensitivity
in
rats
and
an
increase
to
10X
in
the
case
of
agents
that
have
not
yet
been
evaluated
for
potential
age­
dependent
sensitivity.
This
position
was
based
on
a
reasonable
level
of
confidence
in
the
existing
animal
database
for
the
six
different
OP
anticholinesterases
so
far
evaluated
for
age­
dependent
sensitivity.
This
database
showed
no
compounds
with
more
than
3X
greater
potency
in
weanling
than
in
adult
rats,
and
several
that
show
identical
potency
in
these
two
age
groups
(e.
g.,
methamidophos).
Reasonable
confidence
was
expressed
that
the
animal
data
can
be
extrapolated
to
humans
in
light
of
the
recent
data
that
illuminate
the
mechanisms
underlying
age­
dependent
sensitivity
to
OP
anticholinesterases
in
the
rat.
These
data
demonstrate
that
at
least
a
large
portion
of
the
age
dependent
sensitivity
reflects
the
maturation
profiles
of
enzymes
involved
in
metabolism
and
elimination
of
such
agents.
Although
comparative
information
on
humans
is
not
complete,
the
species
extrapolation
is
strengthened
by
information
on
A­
esterase
maturation
indicating
similarly
rapid
maturation
during
the
period
equivalent
to
early
infancy,
with
near
adult
levels
reached
by
the
time
of
weaning
in
rats
in
humans.
Finally,
one
Panel
member
noted
that
many
of
the
agents
in
question
have
been
in
use
for
decades
and
yet,
despite
isolated
cases
of
acute
toxicity,
no
clear
evidence
of
developmental
abnormalities
has
emerged.

DETAILED
RESPONSE
TO
THE
AGENCY'S
CHARGE
The
specific
issues
to
be
addressed
by
the
Panel
are
keyed
to
the
Agency's
background
document,
dated
June
3,
2002,
and
are
presented
as
follows:

Issue
1.
Role
of
Cholinesterases
and
Acetylcholine
As
discussed
in
the
EPA
report,
inhibition
of
acetylcholinesterase
(AChE)
in
the
young
can
result
in
cholinergic
toxicity
as
in
the
adult,
but
evidence
has
also
been
emerging
over
the
last
several
years
that
indicates
that
AChE
and
acetylcholine
may
serve
as
neuromodulators
in
development.

Question
1.1
Please
comment
on
the
extent
to
which
the
report
adequately
summarizes
the
current
state
of
knowledge.
Does
the
scientific
evidence
support
the
conclusion
that
perturbation
of
the
cholinergic
nervous
system
during
development
by
inhibiting
AChE
can
potentially
lead
to
deficits
in
the
structure
and
function
of
the
central
and
peripheral
nervous
systems?
14
As
discussed
in
the
EPA
report,
inhibition
of
acetylcholinesterase
(AChE)
in
the
young
can
result
in
cholinergic
toxicity
as
in
the
adult,
but
evidence
has
also
been
emerging
over
the
last
several
years
to
indicate
that
AChE
and
acetylcholine
(ACh)
may
serve
as
neuromodulators
in
development.

The
Panel
concluded
that
there
is
a
significant
potential
that
brain
development
could
be
affected
by
any
agent
that
blocks
the
activity
of
AChE
and
raises
the
level
of
synaptic
(or
nonsynaptic
acetylcholine.
Thus
the
Panel
agreed
that
the
scientific
evidence
support
the
conclusion
that
perturbation
of
the
cholinergic
nervous
system
during
development,
by
inhibiting
AChE,
could
potentially
lead
to
deficits
in
the
structure
and
function
of
the
central
and
peripheral
nervous
systems.

Overview
The
Panel
commends
the
Agency
on
the
preparation
of
the
section
of
the
report
dealing
with
the
potential
role(
s)
of
organophosphate
(OP)
inhibitors
on
the
structure
and
function
of
the
developing
nervous
system.
Section
II
A
of
the
report
presents
information
regarding
the
roles
of
acetylcholine
and
AChE
in
neurodevelopment.
It
is
well
known
that
inhibition
of
AChE
catalytic
function
leads
to
the
accumulation
of
acetylcholine,
which
in
addition
to
its
role
in
cholinergic
transmission,
also
participates
in
the
structural
development
of
neurons.
Compelling
evidence
demonstrates
that
AChE
is
a
multifunctional
protein
with
a
catalytic
domain
and
a
surface
adhesive
domain
that
may
be
important
for
morphogenesis
in
the
nervous
system.
In
vitro
studies
in
which
the
adhesive
site
is
perturbed
have
clearly
demonstrated
a
direct
developmental
role
for
this
domain
in
both
the
central
and
peripheral
nervous
systems.
Finally,
inhibition
of
AChE
in
the
adult
leads
to
the
expression
of
a
novel
AChE
isoform
(AChE­
R)
which
has
a
different
tissue
distribution
from
the
normal
synaptic
form
(AChE­
S)
and
may
serve
different
functions.
Thus,
the
potential
effect(
s)
of
OP
inhibitors
on
the
developing
nervous
system
are
complex.
An
elaboration
of
the
Panel's
position
is
provided
below.

Elevated
acetylcholine
levels
and
neuronal
development
The
cumulative
risk
assessment
of
OP
anti­
AChEs
is
based
on
their
common
mechanism
of
toxicity,
i.
e.,
phosphorylation
of
AChE
leading
to
accumulation
of
acetylcholine
and
consequent
cholinergic
signs
of
toxicity.
Importantly,
acetylcholine
is
itself
a
neuromodulator.
Thus,
the
elevated
levels
of
acetylcholine,
subsequent
to
AChE
inhibition,
might
disrupt
neurodevelopment
by
affecting
axonal
outgrowth
and
guidance
(Coronas,
et
al.,
2000;
Wessler,
et
al.,
1998).
The
published
data
provide
ample
evidence
that
acetylcholine
modulates
neural
growth
and
plasticity
in
addition
to
its
well­
known
role
in
interneuronal,
neuromuscular
and
neuroglandular
signal
transmission.
Acetylcholine
effects
are
mediated
by
diverse
subtypes
of
ionotropic
and
metabotropic
receptors.
Inhibition
of
synaptic
AChE
by
OPs
causes
excessive
activation
of
nicotinic
and
muscarinic
receptors.
The
former
responds
by
rapid
conformational
change
to
an
inactive,
desensitized
state.
On
the
other
hand,
muscarinic
receptors
respond
by
15
down
regulation
(i.
e.
their
numbers
are
reduced).
The
functional
impact
of
such
changes
on
the
developing
brain
would
be
very
serious
if
they
were
prolonged,
for
example,
if
AChE
activity
does
not
recover.

Eskenazi
and
co­
workers
(1999)
recently
reviewed
the
evidence
that
repeated
low­
level
exposure
of
animals
to
OP
pesticides
might
affect
neurodevelopment
and
growth
in
developing
animals.
For
example,
animal
studies
have
reported
neurobehavioral
effects
such
as
impairment
on
maze
performance,
locomotion,
and
balance
in
neonates
exposed
in
utero
and
during
early
postnatal
periods.
Possible
mechanisms
for
these
effects
include
inhibition
of
brain
AChE,
downregulation
of
muscarinic
receptors,
decreased
brain
DNA
synthesis,
and
reduced
brain
weight
in
the
offspring.
Research
findings
also
suggest
that
it
is
biologically
plausible
that
OP
exposure
may
cause
dysregulation
of
the
autonomic
nervous
system.
Downstream
effects
at
multiple
sites,
including
the
lungs,
could
predispose
children
to
a
variety
of
disabilities.
All
such
changes
can
be
considered
endpoints
elicited
by
the
common
mechanism
of
toxicity
and
must
be
anticipated
from
exposure
to
any
OP
anticholinesterase.

Another
downstream
effect
that
could
potentially
result
from
the
common
mechanism
of
OP
toxicity
is
the
compensatory
upregulation
of
novel
forms
of
AChE
that
do
not
function
quite
like
the
normal
forms.
Some
recent
research
suggests
that
inhibition
of
AChE
in
adults
stimulates
production
of
an
AChE
variant
known
as
"read­
through"
AChE
(because
the
normal
transcriptional
splicing
at
the
C­
terminus
is
omitted).
The
major
AChE
expressed
in
nervous
tissue
is
the
so­
called
"synaptic"
form.
Chronic
inhibition
of
AChE
activity
can
lead
to
the
expression
of
the
unique
"read­
through"
product,
which
is
secreted
as
a
monomer
(Grisaru,
et
al.,
1999;
Soreq,
H.
and
S.
Seidman,
2001).
This
protein
has
the
same
enzyme
kinetics
as
the
synaptic
form
and
thus
would
behave
like
other
forms
of
AChE
in
a
typical
assay.
However,
because
read­
through
AChE
has
a
different
distribution
within
the
cell,
and
from
tissue
to
tissue,
it
may
not
have
the
same
functional
impact
as
normal
AChE.
The
presence
of
read­
through
AChE
has
not
yet
been
described
in
the
fetus
or
the
neonate,
nor
has
there
been
any
study
of
the
potential
for
developmentally
significant
modulation
of
this
form
after
OP
exposure.
Nonetheless,
the
possibility
of
such
effects,
or
additional
changes
in
protein
expression
that
may
eventually
be
revealed
by
proteomic
studies,
reinforces
concerns
that
OPs
might
exert
developmental
neurotoxicity
through
their
common
mode
of
action.
These
observations
also
give
rise
to
a
concern
that
apparent
recovery
of
assayed
total
brain
cholinesterase
following
OP
inhibition
might
not
indicate
a
return
to
a
completely
normal
state
in
a
developing
nervous
system.

Direct
role
for
AChE
in
development
There
is
also
evidence
that
AChE
inhibitors
could
disturb
neuronal
development
by
mechanisms
in
addition
to
the
common
mode
of
action.
AChE
is
developmentally
expressed
by
neurons
during
axonal
outgrowth
and
migration,
periods
when
its
role
in
terminating
cholinergic
transmission
would
be
unnecessary
(Drews,
U.
1975;
Grisaru,
et
al.,
1999;
Layer,
P.
G.
and
E.
Willbold.
1995;
Soreq,
H.
and
S.
Seidman.
2001).
Experimental
studies
in
vitro,
involving
perturbation
of
AChE
either
by
certain
non­
OP
AChE
inhibitors
or
AChE­
specific
antibodies,
16
confirm
a
specific
developmental
role
for
AChE
(Bigbee,
et
al.,
1999;
Dupree
and
Bigbee.
1994;
Layer,
P.
G.,
et
al.,
1993).
In
addition,
observations
by
Slotkin
and
collaborators
have
demonstrated
persistent
neurobehavioral
and
DNA/
protein
abnormalities
in
rats
subjected
to
moderate
or
low
dose
AChE
inhibitor
treatment
in
utero
or
in
an
early
postnatal
period.
A
direct
role
for
AChE
in
the
process
of
neural
development
has
also
been
demonstrated
by
genetic
manipulation
of
AChE
expression,
either
by
stable
transfection
or
by
antisense
treatment
(Bigbee,
et
al.,
2000;
Brimijoin,
S.
and
C.
Koenigsberger.
1999;
Grisaru,
et
al.,
1999;
Sternfeld,
et
al.,
1998).

In
tissue
culture,
AChE
that
is
catalytically
inactivated
by
point
mutation
of
the
active
site
serine
can
still
support
some
morphogenic
phenomena
(Sternfeld,
et
al.,
1998).
Such
findings
indicate
that
the
morphogenic
potential
of
this
enzyme
is
at
least
partially
independent
of
its
esterase
activity,
possibly
because
of
morphogenic
properties
in
the
adhesive
domain
surrounding
the
opening
of
the
active
site
gorge.
Results
from
studies
using
transgenic
mice,
however,
have
produced
results
that
raise
questions
about
the
significance
of
adhesion­
based
functions
of
AChE
in
brain
development.
In
one
study,
it
was
shown
that
neuronal
development
and
structure
of
the
brain
are
apparently
normal
in
AChE
knockout
heterozygote
mice
that
have
only
50%
of
normal
AChE
expression
levels
(Xie
et
al.
2000).
Even
complete
AChE
knockout
causes
no
profound
changes
in
the
structure
of
cholinergic
pathways
in
the
brain
as
revealed
by
histochemistry
and
immunohistochemistry
(Mesulam
et
al.,
2002).

It
can
also
be
questioned
whether
OPs
are
likely
to
influence
the
adhesive
functions
of
AChE,
in
contrast
to
certain
long­
chain,
bis­
quaternary
AChE
inhibitors
that
bind
reversibly
to
catalytic
and
peripheral
sites.
No
evidence
exists
to
indicate
that
OPs
bind
to
the
adhesive
domain.
On
the
other
hand,
as
pointed
out
in
the
EPA
report,
the
possibility
exists
that
an
OP
could
alter
the
three­
dimensional
structure
of
AChE
by
binding
to
the
active
site,
thereby
subtly
altering
the
surface
adhesive
domain.
For
this
reason,
as
was
pointed
out
by
one
Panel
member,
there
is
need
for
additional
pharmacodynamic
studies
to
better
define
the
different
OPs
and
their
structural
interactions
with
AChE.

At
present,
it
would
be
prudent
to
recognize
the
potential
for
developmental
toxicity
stemming
from
mechanisms
that
operate
in
addition
to
the
common
mode
of
OP
toxicity.
Thus
we
must
recognize
that
the
degree
of
AChE
inhibition
may
not
fully
capture
the
ability
of
an
OP
to
perturb
the
development
of
the
nervous
system.
AChE
is
uniquely
high
during
critical
periods
of
development
and
thus
may
be
especially
vulnerable
for
short
periods.
Furthermore,
a
given
degree
of
inhibition
of
AChE
in
the
fetus
or
neonate
may
have
a
greater
effect
than
the
same
level
of
inhibition
in
the
adult.
However,
the
current
review
is
almost
completely
qualitative.
There
is
no
quantitative
analysis
relating
either
the
presence
or
the
extent
of
developmental
effects
and
duration
of
measured
or
estimated
cholinesterase
inhibition
in
the
developing
brain.
This
quantitative
component
is
a
key
missing
link
in
the
chain
of
analysis
that
is
needed
to
assess
whether
the
degree
of
cholinesterase
inhibition
that
has
been
judged
statistically
detectable
for
adults
should
also
be
expected
to
be
without
appreciable
consequence
during
development.
17
The
Panel
generally
agreed
that
the
existing
evidence
falls
short
of
what
would
be
needed
to
prove
that
AChE
inhibition
during
development
will
cause
later
deficits
in
nervous
system
structure
and
function.
However,
while
definitive
evidence
is
lacking,
the
potential
nevertheless
exists.
Of
particular
importance
to
the
risk
assessment
of
OP
toxicants,
more
recent
information
suggests
that
some
OP
inhibitors
of
AChE
can
modify
neuronal
growth
in
vitro.
It
should
be
stressed,
however
(as
noted
in
the
Report)
that
some
anticholinesterases
have
no
apparent
effect
on
neurite
outgrowth.
Some
studies
suggest
that
neurodevelopment
may
be
affected
in
vivo
by
some
OP
toxicants,
but
most
of
these
studies
utilize
unrealistic
exposure
conditions
and
thus
have
uncertain
relevance
for
risk
assessment.
In
addition,
there
could
be
very
subtle
changes
not
disclosed
by
standard
behavioral
tasks.
The
two
best­
described
systems
are
both
sensory
in
nature
and
are
difficult
to
assess.
The
report
did
not
summarize
the
studies
for
neuro­
behavioral
effects
from
fetal
exposure
to
the
OPs.
Thus
it
is
difficult
to
determine
whether
there
is
a
common
potential
for
neurological
and
behavioral
effects.
This
issue
needs
greater
clarification
in
the
document,
especially
as
it
relates
to
registrant­
provided
developmental
neurotoxicity
(DNT)
studies.
Without
this
comprehensive
review
and
evaluation
of
a
larger
number
of
DNT
studies,
it
is
difficult
to
assess
whether
the
existing
data
support
or
refute
a
common,
additional
developmental
risk
above
the
adult
risk,
for
a
common
level
of
AChE
inhibition.

Issue
2.
Age­
Dependent
Sensitivity
to
Cholinesterase
inhibition
in
Animal
Studies
Age­
dependent
sensitivity
(i.
e.,
young
animals
can
exhibit
higher
levels
of
cholinesterase
(ChE)
inhibition
at
the
same
dose
or
inhibition
at
lower
doses
compared
to
adults)
has
been
observed
in
several
laboratory
studies
following
treatment
(acute
and/
or
repeated
oral
gavage
doses)
of
neonatal,
juvenile,
and
adult
rats
with
organophosphorus
(OP)
pesticides.
The
exact
mechanisms
of
this
age­
dependent
sensitivity
are
not
known,
but
several
studies
have
demonstrated
that
toxicokinetic
factors
may
be
responsible.
Most
notably,
the
more
limited
ability
of
the
young
to
detoxify
OP
pesticides
by
A­
esterases
and
carboxylesterases
appears
to
be
an
important
factor
underlying
the
increased
sensitivity
of
the
immature
rat
to
ChE
inhibition.
There
appears
to
be
more
rapid
recovery
of
inhibited
AChE
(synthesis
of
new
ChE
enzyme)
in
postnatal
(and
fetal)
rat
tissues,
but
information
on
comparative
recovery
in
children
and
human
adults
is
lacking.

Question
2.1
Please
comment
on
the
extent
to
which
the
report
adequately
discussed
and
summarized
the
current
understanding
of
age­
dependent
sensitivity
to
ChE
inhibition,
the
prevailing
views
in
the
scientific
community
concerning
the
biological
factors
involved,
and
the
role
esterases
may
play
as
a
major
factor
accounting
for
potential
increased
sensitivity
of
the
immature
rat.

Age­
dependent
sensitivity
(i.
e.,
young
animals
can
exhibit
higher
levels
of
cholinesterase
(ChE)
inhibition
at
the
same
dose,
or
inhibition
at
lower
doses
compared
to
adults)
has
been
observed
in
several
laboratory
studies
following
treatment
(acute
and/
or
repeated
oral
gavage
18
doses)
of
neonatal,
juvenile,
and
adult
rats
with
organophosphorus
(OP)
pesticides.
The
exact
mechanisms
of
this
age­
dependent
sensitivity
are
not
known,
but
several
studies
have
demonstrated
that
toxicokinetic
factors
may
be
responsible.
Most
notably,
the
more
limited
ability
of
the
young
to
detoxify
OP
pesticides
by
A­
esterases
and
carboxylesterases
appears
to
be
an
important
factor
underlying
the
increased
sensitivity
of
the
immature
rat
to
ChE
inhibition.
There
also
appears
to
be
more
rapid
recovery
of
inhibited
AChE
(synthesis
of
new
ChE
enzyme)
in
postnatal
(and
fetal)
rat
tissues,
but
information
on
comparative
recovery
in
children
and
human
adults
is
lacking.

The
Panel
considered
the
Agency's
summarization
of
the
current
literature
to
be
adequate
in
some
areas
and
deficient
in
others.
The
discussion
and
summation
of
the
age­
dependent
toxicity
of
the
six
OP
insecticides
for
which
data
are
available
was
concise
and
complete,
and
Tables
1
and
2
were
helpful
and
informative.
Toxicokinetic
factors
were
proposed
to
be
critically
important
in
age­
related
sensitivity
and
limited
discussion
was
provided
in
the
document
on
the
detoxifying
esterases
(carboxylesterase
and
A­
esterase)
and
their
differential
expression
during
maturation.
However,
little
is
mentioned
regarding
differences
in
oxidative
metabolism
and
its
potential
role
in
differential
sensitivity
via
differential
rates
of
metabolism
of
some
OPs
to
more
active
forms.
The
report
documented
the
role
of
A
and
B
esterases
in
the
limitation
of
AChE
inhibitor
action
and
the
importance
of
AChE
resynthesis
as
a
means
of
differential
recovery
from
enzyme
inhibition.
However,
the
discussion
of
the
biological
factors
[specifically,
A­
esterases
and
carboxylesterases]
that
might
result
in
age­
dependent
susceptibility
to
toxicity
of
certain
OPs
could
be
significantly
improved
by
presenting
a
more
balanced
interpretation
of
the
available
data.

Some
anticholinesterases
show
distinct
age­
related
differences
in
effects,
while
other
OP
agents
appear
to
express
little
age­
related
differences.
Differences
in
sensitivity
tend
to
be
smaller
with
repeated
dosing
and
may
also
be
a
function
of
age
of
the
developing
animal.
Several
factors
may
contribute
to
this
finding,
including
faster
recovery
of
acetylcholinesterase
in
tissues
of
young
animals
and
increasing
levels
of
detoxifying
esterases
with
increasing
maturity
of
metabolic
systems.
Several
studies
have
shown
that
the
sensitivity
of
the
target
enzyme
in
tissues
from
different
age
groups
does
not
differ.
Thus,
sensitivity
of
acetylcholinesterase
molecules
themselves
probably
does
not
contribute
to
age­
related
sensitivity.
Differences
in
cholinergic
receptor
adaptation
were
also
considered.
Cholinergic
receptors
often
downregulate
following
cholinesterase
inhibition,
but
differences
in
receptor
adaptation
do
not
appear
responsible
for
agerelated
sensitivity.
The
Agency's
background
document
also
mentions
the
presence
of
muscarinic
autoreceptors,
capable
of
inhibiting
acetylcholine
release
presynaptically.
In
fact,
the
postnatal
maturation
of
the
muscarinic
autoreceptor
correlates
roughly
with
decreasing
acute
sensitivity
to
OP
toxicants
and
may
therefore
play
a
role
in
age­
related
sensitivity.

The
Agency's
background
document
summarized
evidence
that
supports
important
roles
for
A­
esterases
and
carboxylesterases
in
the
increased
sensitivity
of
the
immature
rat,
but
ignores
observations
or
interpretations
that
might
suggest
other
possibilities.
Consequently
the
document
tends
to
overstate
the
degree
to
which
the
mechanisms
of
age­
dependent
toxicity
of
OPs
are
understood.
This
is
most
apparent
with
regard
to
three
issues:
19
1.
The
document
summarizes
several
studies
that
have
reported
correlation
between
the
temporal
patterns
of
development
of
A­
esterase
and
carboxylesterase
activities
and
OP
sensitivity.
However,
the
document
does
not
mention
that
some
of
those
studies
also
have
reported
a
decreased
capacity
of
immature
rats
to
oxidatively
activate
these
same
insecticides.
Immature
rats
have
reduced
A­
esterase
and
carboxylesterase
activities,
but
they
also
have
a
similarly
reduced
capacity
to
produce
the
oxygen
analogs
from
the
parent
insecticides.
This
is
an
important
potentially
offsetting
observation
that
should
be
discussed
in
the
report.
It
should
be
noted
that
no
targeted
mechanistic
studies
have
evaluated
the
role
of
these
esterases
in
age­
related
sensitivity.
Thus,
only
a
correlation
between
inherent
esterase
activity
levels
and
sensitivity
to
the
anticholinesterases
support
the
concept
of
esterase­
mediated
differential
sensitivity.

2.
The
report
presents
evidence
in
support
of
a
role
for
A­
esterase
in
detoxification
of
certain
OPs
and
in
age­
dependent
sensitivity,
but
does
not
discuss
evidence
that
might
be
contrary
to
this
view.
There
are
only
three
oxons
that
have
been
identified
that
are
substrates
for
A­
esterase
in
vitro
–
paraoxon,
chlorpyrifos
oxon,
and
diazoxon.
Studies
with
knockout
mice
have
indicated
that
paraoxon
metabolism
by
A­
esterase
is
probably
insignificant
in
vivo.
And
as
indicated
in
the
document,
knockout
mice
were
much
more
sensitive
to
chlorpyrifos
oxon
or
diazoxon.
However,
not
mentioned
in
the
document
was
the
observation
that
knockout
mice
were
only
slightly
more
sensitive
to
the
parent
compounds
chlorpyrifos
and
diazinon,
and
even
then
only
at
high
doses,
suggesting
that
A­
esterase
may
not
be
an
important
detoxification
pathway
upon
exposure
to
the
parent
insecticides.
In
addition,
some
reports
in
the
literature
have
suggested
that
A­
esterase
in
the
rat
probably
only
plays
a
role
in
detoxification
when
the
chlorpyrifos
or
diazinon
doses
are
very
large.
At
small
to
moderate
doses,
detoxification
by
A­
esterase
is
probably
insignificant
compared
to
detoxification
through
carboxylesterase.
The
report
should
include
some
discussion
of
this
issue.

3.
The
document,
referring
to
Table
2
on
p.
22,
states
that
the
temporal
pattern
of
A­
esterase
and
carboxylesterase
activities
correlate
reasonably
well
with
studies
on
OP
sensitivity.
But
it
does
not
discuss
possible
exceptions
to
this
correlation.
For
example,
methyl
paraoxon
is
not
a
substrate
for
A­
esterase,
and
has
limited
interactions
with
carboxylesterase.
Therefore,
one
should
expect
limited
age­
dependent
sensitivity
yet
its
acute
age­
dependent
sensitivity
(from
Table
1)
is
almost
the
same
as
that
of
chlorpyrifos,
and
its
age­
dependent
toxicity
after
repeated
administration
might
even
exceed
that
of
chlorpyrifos
(again
from
Table
1).
These
observations
could
suggest
involvement
of
other
factors
in
the
age­
dependent
sensitivity
of
at
least
methyl
parathion.

The
discussion
focused
on
developmental
profiles
of
esterases
exclusively
and
ignores
changes
in
cytochrome
P450
activities
with
age
as
a
potential
contributing
toxicokinetic
factor
in
agerelated
sensitivity
to
OPs.
The
discussion
should
be
expanded
to
include
a
description
of
the
state
of
knowledge
on
P450
development
in
the
rat,
focusing
primarily
on
isoforms
known
or
suspected
20
to
be
involved
with
OP
bioactivation
and
detoxification.

The
Agency
needs
to
include
a
discussion
in
the
background
document
on
the
implications
of
different
possible
dose
metrics
in
explaining
age­
related
sensitivities
through
metabolism.
There
should
be
a
clear
articulation
of
reasonable
alternative
hypotheses
about
which
dose
metric(
s)
for
ChE
could
be
important
for
the
developmental
pharmacodynamic
actions
of
anti­
cholinesterase
agents.
For
example,
it
is
possible
that
the
best
dose
metric
for
predicting
effects
could
be
"peak"
levels
of
cholinesterase
inhibition
on
one
day
or
several
days
of
successive
exposure.
Alternatively,
an
"AUC"
measure
of
the
integral
of
%
inhibition
X
time
could
prove
to
be
the
closest
causally
relevant
predictor
of
developmental
effects.
There
are
also
a
few
more
complex
hypotheses.
In
any
event,
given
each
of
these
and/
or
other
plausible
measures
of
internal
delivered
"dose",
a
discussion
should
be
included
on
the
roles
of
activating
vs.
detoxifying
enzyme
activities
and
other
factors
in
this
context.
As
an
example,
for
measures
of
acute
peak
cholinesterase
inhibition
by
OPs
requiring
activation
for
biological
activity,
activating
enzyme
activities
will
be
important
and
detoxifying
enzymes
such
as
the
esterases
will
tend
to
be
less
important.
The
opposite
would
tend
to
be
the
case
if
AUC
(integrated
%
inhibition
X
time)
over
an
extended
period
of
dosing
is
more
important
for
causing
developmental
effects—
in
that
case,
activating
activity
would
be
somewhat
less
important
and
detoxifying
enzyme
activities
for
both
the
parent
chemical
and
the
activated
intermediate
would
tend
to
be
more
important.

Question
2.2
Please
comment
on
the
timing
of
administration
(i.
e.,
the
developmental
stage
treated)
and
the
differential
found
between
adults
and
the
young
animal.

The
Panel
interpreted
the
question
as
a
query
on
the
impact
of
dosing
parameters
on
relative
sensitivity
of
different
age
groups.
The
Panel
concluded
that
the
timing
of
exposures
is
critically
important
in
evaluation
of
age­
related
differences
in
sensitivity
to
anticholinesterases.
The
Agency's
background
document
describes
a
number
of
studies,
some
with
prenatal,
some
with
postnatal,
and
some
with
combined
prenatal/
postnatal
exposures.
Based
on
cholinesterase
inhibition,
the
studies
utilizing
exclusively
prenatal
dosing
appear
to
report
consistently
equal
or
lesser
effects
in
the
developing
organism
than
in
the
dam.
This
may
in
some
cases
be
due
to
the
timing
of
biochemical
measurements
relative
to
exposure,
but
the
findings
generally
suggest
no
higher
sensitivity
to
cholinesterase
inhibition
in
prenatally­
exposed
animals.
The
reverse
is
often
found
when
animals
are
exclusively
treated
postnatally.
In
essence,
higher
toxicity
and
more
extensive
cholinesterase
inhibition
are
often
noted
in
neonatal
animals
compared
to
older
immature
animals,
and
even
greater
differences
in
sensitivity
arise
when
comparing
very
young
animals
to
adults
dosed
similarly
with
a
number
of
OP
toxicants.
With
acute,
relatively
high
exposures,
several
OP
insecticides
are
markedly
more
toxic
to
very
young
individuals.
This
kind
of
stage­
related
sensitivity
is
compound
specific
and
it
appears
to
be
directly
related
to
the
maturational
state
of
A­
esterases
and
carboxylesterases.
Compounds
that
are
not
substrates
for
one
or
both
of
these
developmentally
regulated
enzymes
appear
generally
not
to
show
differential
inhibition
based
on
timing
of
bolus
injection.
In
some
cases,
however,
age­
related
sensitivity
may
21
occur
with
OP
toxicants
that
are
not
well
detoxified
by
either
carboxylesterase
or
A­
esterase
(e.
g.,
young
animals
are
markedly
more
sensitive
to
methyl
parathion).
Other
toxicokinetic
or
toxicodynamic
factors
may
therefore
play
an
important
role
in
age­
related
sensitivity.

In
contrast,
when
immature
and
adult
rats
were
repeatedly
exposed
to
some
OP
insecticides
(e.
g.,
chlorpyrifos),
relatively
little
age­
related
differences
in
cholinergic
toxicity
were
noted.
The
ability
to
recover
between
exposures
in
tissues
from
younger
animals
may
be
important
in
this
regard,
i.
e.,
if
AChE
molecules
are
being
synthesized
faster
in
immature
animals,
overall
enzymatic
activity
will
recover
faster
following
each
cholinesterase
inhibitor
exposure,
thereby
reducing
accumulation
of
insult.
Because
of
the
relatively
short
maturation
period
in
rodents,
however,
repeated
dosing
studies
can
change
the
baseline,
i.
e.,
the
animal
is
becoming
less
sensitive
to
the
pesticide
throughout
the
dosing
period.
Thus,
lesser
age­
related
differences
in
sensitivity
with
repeated,
compared
to
acute
exposures,
may
be
due
both
to
inherent
differences
in
recovery
potential
and
to
decreased
sensitivity
as
the
dosing
period
progresses.
One
could
question
if
changes
in
enzyme
recovery
may
even
cause
a
reversal
of
age­
related
sensitivity
in
repeated
dosing
paradigms;
that
is
a
situation
in
which
adults
are
more
sensitive
than
younger
animals.
In
fact,
several
studies
(Chakraborti
et
al.,
1993;
Pope
and
Liu,
1997;
Zheng
et
al.,
2000)
suggest
that
while
neonatal
rats
are
markedly
more
sensitive
to
acute
exposures
to
chlorpyrifos,
fewer
differences
are
noted
with
daily
dosing,
and
if
intermittent
dosing
(every
four
days)
is
used,
more
extensive
neurochemical
changes
(i.
e.,
AChE
inhibition,
muscarinic
receptor
downregulation)
may
occur
in
adults.
These
findings
imply
that
more
rapid
recovery
of
AChE
activity
noted
in
the
immature
animal's
brain
following
OP
exposure
can
in
fact
contribute
to
more
rapid
functional
recovery
of
neurotransmission.

Question
2.3
Please
comment
on
the
extent
to
which
comparative
ChE
data
on
six
OP
pesticides
(chlorpyrifos,
diazinon,
dimethoate,
methamidophos,
malathion,
methyl
parathion)
may
represent
a
reasonable
subset
of
different
structural
and
pharmacokinetic
characteristics
of
the
cumulative
group
of
OP
pesticides
to
define
an
upper
bound
on
the
differential
sensitivity
that
may
be
expected
at
different
life
stages
of
the
immature
animal.
As
an
example,
there
are
no
chemical­
specific
comparative
cholinesterase
data
on
azinphosmethyl
(AZM),
an
important
contributor
of
risk
for
the
food
pathway.
Pesticide­
specific
comparative
cholinesterase
data
on
the
other
six
pesticides
from
the
OP
class
(including
data
on
malathion,
a
member
of
the
same
chemical
subgroup
as
AZM)
show
a
limited
range
of
differential
sensitivities
­­
from
one­
fold
(no
increased
sensitivity)
up
to
three­
fold

between
the
young
and
adults.
EPA
regards
these
data
on
other
OPs
as
providing
sufficient
evidence
to
assess
the
potential
for
AZM
to
show
age­
dependent
sensitivity,
and
to
reasonably
predict
the
degree
of
potential
difference
in
sensitivity
between
the
young
and
adults.
Given
the
results
of
the
other
OPs,
EPA
concludes
that
it
is
unlikely
that
AZM
would
exceed
a
magnitude
of
difference
greater
than
approximately
3­
fold
following
treatment
of
PND
11
through
21
pups
versus
adult
animals.
22
The
majority
of
the
Panel
members
concluded
that
the
comparative
data
on
six
OP
pesticides
(chlorpyrifos,
diazinon,
dimethoate,
methamidophos,
malathion,
and
methyl
parathion)
should
not
be
considered
to
represent
a
reasonable
subset
of
different
structural
and
pharmacokinetic
characteristics
of
the
cumulative
group
of
OP
pesticides
to
define
an
upper
bound
on
the
differential
sensitivity
that
may
be
expected
at
different
life
stages
of
the
immature
animal.
However
one
Panelist
dissented
from
this
view,
and
agreed
with
the
report
that
these
six
pesticides
could
be
used
to
define
an
upper
bound
on
the
differential
sensitivity
for
the
cumulative
group.

Specific
comments
by
Panelists
against
the
use
of
the
6
OPs
as
a
representative
subset
of
the
cumulative
group
were
as
follows:

The
currently
available
data
on
direct
postnatal
exposure
of
six
OP
pesticides
shed
some
light
on
the
potential
differential
sensitivity
of
OPs
during
stages
of
development.
The
Agency
is
to
be
commended
for
the
extensive
effort
in
addressing
these
rather
complicated
issues.
However,
the
complex
interplay
of
many
factors
(e.
g.,
pharmacokinetics
and
pharmacodynamics
that
are
chemical­
and
developmental
stage­
specific)
leading
up
to
the
inhibition
of
brain
ChE
inhibition
is
the
source
of
substantial
uncertainty
for
predicting
the
upper
bound
of
the
differential
sensitivity
for
all
the
OPs
under
evaluation.

The
document
suggests
that
the
age­
related
change
in
sensitivity
to
certain
OPs
is
largely
a
function
of
toxicokinetic
factors
since
age­
related
changes
in
acetylcholinesterase
catalysis
and
sensitivity
to
inhibitors
do
not
occur.
If
this
is
the
case,
one
must
consider
whether
or
not
the
toxicokinetic
characteristics
of
any
remaining
members
of
the
cumulative
assessment
group
are
sufficiently
different
from
the
six
indicated
in
the
document,
so
as
to
lead
to
a
juvenile/
adult
differential
toxicity
greater
than
a
3­
fold
uncertainty
factor.
Based
on
the
lack
of
information
in
the
open
literature
regarding
the
toxicokinetic
characteristics
of
the
remaining
pesticides
(most
importantly
their
metabolism
and
volumes
of
distribution),
one
must
conclude
that
there
simply
is
not
enough
information
available
to
know
whether
or
not
the
six
insecticides
indicated
in
the
document
are
representative
toxicokinetically
of
the
cumulative
group.
Consequently,
we
do
not
know
if
those
six
OPs
can
define
an
upper
bound
for
the
possible
differential
age­
dependent
sensitivity
of
other
OPs.

One
Panelist
offered
differences
in
potency
among
ChE
agents
as
an
illustration
of
the
uncertainties
involved
in
extrapolating
biological
properties
between
agents.
A
more
that
10­
fold
difference
in
the
relative
potency
factor
(RPF)
is
observed
between
the
metabolic
activation
pair
of
acephate
and
methamidophos,
just
within
adult
female
rats.
For
these
two
chemicals,
and
with
the
rich
database
available
for
methamidophos,
the
Agency's
document
stated
that
it
is
not
possible
to
determine
"whether
acephate
would
show
comparable
responses
in
adult
and
young
rats"
(page
13,
Determination
of
the
Appropriate
FQPA
Safety
Factor(
s)
in
the
Organophosphorus
Pesticide
Cumulative
Risk
Assessment;
June
10,
2002).
Other
than
obtaining
chemical­
specific
data,
much
more
information
is
needed
for
a
reliable
estimate
of
a
range
of
agerelated
sensitivity
of
OPs.
There
are
insufficient
data
to
fully
support
a
3­
fold
uncertainty
factor
23
based
on
an
estimated
upper
bound
of
3­
fold
age­
related
differential
sensitivity.

It
should
also
be
noted
that
dose­
response
modeling
would
give
a
more
consistent
comparison
for
the
age­
related
sensitivity
among
chemicals,
and
the
Agency's
analysis
showed
that
the
upper
bound
would
be
4­
fold
based
on
data
for
methyl
parathion.
Presumably
this
is
only
based
on
the
data
from
repeated
dosing,
and
not
including
the
single
dosing
study
that
showed
up
to
7­
fold
differences.
Thus,
given
the
current
data,
it
may
be
prudent
to
consider
an
upper
bound
of
greater
than
3
just
for
the
toxicity
side
of
the
uncertainty
factor
consideration.

Overall,
it
is
ill­
advised
to
speak
of
an
"upper
bound"
from
the
six
available
observations
in
this
case.
"Upper
bound"
conveys
the
impression
of
a
firm,
known
upper
limit
and
the
existing
data
cannot
support
a
conclusion
of
this
sort
with
any
reasonable
degree
of
confidence.
It
is
even
challenging
to
attempt
a
distributional
treatment
from
such
a
small
number
of
chemicals
but
this
is
the
best
treatment
that
can
be
made.
A
first
step
should
be
to
apply
either
the
Agency's
exponential
model
as
presented
at
the
February
2002
SAP
meeting,
or,
where
the
data
are
insufficient
for
this,
a
simplified
version
of
it
to
express
the
apparent
relative
potency
based
on
estimated
ED10's
of
the
chemicals
for
either
acute
or
repeated
dosing
exposures
for
animals
of
various
young
age
groups
versus
adults.
The
simplified
exponential
model
is
needed
because
some
of
the
current
calculations
distort
the
relative
potency
of
the
cholinesterase
inhibition
results
in
young
versus
adult
animals
by
failing
to
take
into
account
the
fact
that
no
more
than
100%
of
the
enzyme
can
be
inhibited.
For
example,
the
calculation
from
the
Moser
et
al
acute
dosing
data
for
male
animals
is
based
on
a
simple
ratio
of
89%
inhibition
in
pups
versus
39%
inhibition
in
adults.
Clearly,
with
a
simple
ratio,
even
if
the
true
potency
ratio
in
the
two
groups
were
100
or
1000,
the
calculation
could
not
produce
a
result
larger
than
100/
39
or
approximately
2.5.
The
2.
3
in
the
document
becomes
about
5
when
one
applies
a
simple
one­
parameter
version
of
the
exponential
model.
One
Panelist
suggested
a
revised
experimental
model,
as
presented
by
the
Agency,
that
uses
a
basic
exponential
form,
but
omits
the
high
dose
saturation
level
of
inhibition
and
the
expanded
model's
low
dose
nonlinearity
feature:

Fraction
inhibited
=
1
–
e
­kd
(1)

Where
d
is
the
dose
and
k
is
the
measure
of
potency
(inhibition
units
per
dose
at
low
doses).
This
model
at
least
corrects
for
the
fact
that
one
cannot
get
more
than
100%
inhibition
while
calculating
apparent
potency
in
each
group.

Using
this
simplified
exponential
model,
the
relative
potency
for
two
comparable
experiments
in
animals
of
different
age
is
just
the
ratio
of
k1
for
the
younger
age
group
to
k2
for
the
older/
adult
age
group,
or:

Potency
in
young
age
group
relative
to
adults
(k1/
k2)=
24
d
2
(adult
animals)
ln
(1
­
Fraction
inhibited
in
young)

d
1
(young
animals)
ln(
1
­
Fraction
inhibited
in
adults)

(Alternatively,
one
could
use
equation
1
to
estimate
ED10's
for
each
group
and
take
a
ratio
of
the
ED10's
as
the
measure
of
relative
potency.
The
results
of
this
would
be
very
similar
to
the
ratio
of
"k"
potency
factors
described
above).

This
equation
for
relative
potency
in
adult
and
young
animals
incorporates
a
saturation
at
100%
cholinesterase
inhibition
and
also
corrects
for
the
situation
where
the
inhibition
findings
are
from
different
doses.
Putting
in
an
upper
limit
of
inhibition
short
of
100%
(as
is
found
necessary
in
some
cases
in
the
Agency's
modeling)
would
tend
to
increase
the
pup/
adult
sensitivity
ratios
in
cases
where
the
pup
shows
greater
inhibition
than
the
adult.

A
particular
challenge
for
this
proposed
analysis
applies
to
cases
such
as
malathion
where
in
some
cases
there
is
no
detectable
cholinesterase
inhibition
in
adult
animals
at
rather
high
doses,
but
there
is
appreciable
inhibition
at
comparable
and
lower
doses
in
younger
animals.
Simply
excluding
these
cases
risks
biasing
the
analysis,
so
some
truncated
distributional
analysis
is
needed
here.

Other
specific
comments
offered
by
one
Panelist
in
support
of
the
use
of
the
6
OPs
as
a
representative
subgroup
of
the
cumulative
risk
were
as
follows:

First,
there
is
no
inherent
difference
in
the
ChE
enzymes
or
its
binding
to
an
OP
between
young
and
adult
animals.
Second,
the
difference
between
inhibition
of
ChE
between
newborn,
pups
and
adult
animals
is
primarily
due
to
two
factors,
which
are
the
rate
of
regeneration
of
the
enzyme
and
the
level
of
various
enzymes,
such
as
the
esterases
and
others,
that
detoxify
the
OP,
neither
of
which
will
be
different
among
the
compounds
that
are
tested.
The
main
difference
among
the
test
compounds
is
going
to
be
the
relative
rate
of
detoxification.

In
general,
the
6
OPs
for
which
data
are
available
for
ChE
inhibition
of
young
and
adult
animals
are
qualitatively
similar
with
respect
to
ChE
inhibition.
For
these
compounds,
the
ratio
of
ChE
inhibition
of
adult
to
pup
sensitivity
ranged
from
no
difference
to
three
fold.
Based
on
this
information,
the
Agency
has
included
a
3­
fold
uncertainty
factor.
The
3­
fold
factor
is
reasonable
since
the
range
of
1
to
3
fold
is
based
on
dosing
of
large
amounts
of
OPs
directly
to
the
pup
and
adult
animals,
which
represents
exaggerated
exposure
conditions.
Under
more
realistic
conditions
of
exposure
to
pregnant
or
lactating
dams,
the
degree
of
inhibition
in
the
neonates
and
the
pups
was
generally
less
than
the
dam.

Overall,
the
prediction
of
the
range
of
enzyme
inhibition
is
more
limited
than
the
prediction
of
toxicity
and
the
lack
of
information
for
the
other
OPs
and
the
uncertainty
in
making
25
this
estimate
is
taken
into
account
by
the
incorporation
of
a
3­
fold
uncertainty
factor.

Issue
3.
Relevance
of
the
Animal
Findings
to
Children
Age
dependent
sensitivity
to
cholinesterase
inhibition
has
been
associated
with
the
limited
ability
of
the
immature
rat
to
detoxify
OP
pesticides
by
esterases.
In
rats,
Aesterase
activity
increases
from
birth
to
reach
adult
levels
around
postnatal
day
21.
Fetal
rats
possess
very
little
carboxylesterase
activity
with
increasing
activity
as
the
postnatal
rat
matures,
reaching
adult
values
after
puberty
(50
days
of
age).
Data
showing
increased
sensitivity
of
the
young
animal
to
cholinesterase
inhibition
compared
to
adults
has
generally
been
derived
from
acute
dosing
of
PND
7
or
PND
11
pups,
or
repeated
dosing
of
PND
11
to
PND
21
pups.
The
available
data
also
show
as
the
young
rat
rapidly
matures
in
its
ability
to
detoxify
by
esterases,
the
differential
in
cholinesterase
inhibition
becomes
smaller.
Thus,
the
relative
sensitivities
of
immature
rats
found
in
the
studies
of
dosing
pups
through
PND
11
to
21
are
smaller
compared
to
studies
of
dosing
a
PND
11
pup.
The
dosing
studies
of
PND
11
through
21
pups
are
considered
to
better
approximate
the
maturation
profile
of
the
A­
esterases
of
the
highly
exposed
children's
age
group
in
the
OP
cumulative
risk
assessment,
the
one
and
two
year
olds,
compared
to
a
study
of
a
PND
11
pup
which
is
similar
to
a
newborn.
Thus,
the
repeated
rat
dosing
studies
more
closely
mimic
the
maturation
or
developmental
profile
of
A­
esterase
appearance
in
children
around
the
one
and
two
year
olds
where
children
are
reaching
adult
levels
of
A­
esterase
activity.
The
use
of
dosing
studies
of
PND
11
through
21
is
consistent
with
the
exposure
patterns
of
children.
Humans
generally
do
not
begin
to
consume
fresh
(uncooked)
fruits
and
vegetables
until
after
six
months
of
age
or
more.
Furthermore,
repeated
dosing
studies
were
used
to
determine
relative
sensitivity
because
people
are
exposed
every
day
to
an
OP
pesticide
through
food,
and
thus
an
animal
study
using
repeat
exposures
is
considered
appropriate.
Finally,
following
exposure
to
an
OP,
regeneration
of
cholinesterase
to
preexposure
levels
does
not
occur
for
days
or
weeks,
making
the
exposed
individual
potentially
more
vulnerable
to
subsequent
exposures
during
that
period.

Question
3.1
Please
comment
on
the
maturation
profile
of
A­
esterase
and
the
uncertainties
surrounding
these
data
in
young
children.
Because
no
human
data
are
available
on
the
maturation
profile
of
carboxylesterases,
please
comment
on
what
should
be
assumed
in
humans,
especially
children
age
1
to
2
years,
given
the
animal
data
and
what
science
understands
in
general
about
detoxification
maturation
profiles.

The
Panel
concluded
that
there
is
appreciable
residual
uncertainty
about
the
differences
in
activity
at
early
versus
adult
life
stages
in
relevant
activation
and
detoxification
pathways
in
animals
and
humans,
especially
for
detoxification
by
carboxylesterases.

Many
Panel
members
provided
generally
similar
perspectives.
The
discussion
below
26
begins
with
evaluations
of
the
specific
data
cited
in
the
Agency's
background
document
for
the
changes
in
A­
esterase
levels
during
development.
With
this
as
background,
the
Panel
responded
to
the
last
part
of
the
question
with
a
review
of
the
general
profile
of
changes
in
whole­
body
elimination
half
lives
for
drugs
in
general,
and
drugs
eliminated
by
various
specific
pathways.
In
the
absence
of
more
direct
evidence
for
developmental
changes
in
carboxylesterases
and
other
even
less
well
characterized
routes
of
elimination,
these
data
provide
the
most
applicable
starting
point
for
defining
baseline
expectations
and
associated
uncertainties.

Specific
Data
on
Changes
in
A­
esterases
and
P450
Activating
and
Detoxifying
Enzymes
During
Development
It
would
be
useful
to
include
more
information
in
the
Agency's
background
document
on
metabolic
enzymes
and
metabolism
since
the
rate
of
detoxification
appears
to
contribute
to
the
differences
in
the
relative
inhibition
of
ChE
at
various
ages
as
compared
to
the
adult
in
rats.
Carboxylesterases
and
A­
esterases
have
been
shown
to
be
important
in
the
detoxification
of
some
OP
toxicants
in
rats,
and
may
contribute
to
age­
related
differences
in
sensitivity
in
humans.
However,
some
studies
suggest
that
other
metabolic
factors
may
also
be
important
contributors
to
age­
related
sensitivity
for
other
OP
agents.
The
entire
spectrum
of
enzymes
responsible
for
activation/
detoxification
of
the
OP
toxicants
should
be
evaluated
for
potential
changes
in
enzyme
expression
and
function
during
human
development
and
their
potential
contributions
to
relative
sensitivity.
Determination
of
activities
of
all
processes
in
human
tissues
would
be
ideal,
but
difficult
to
accomplish.
Additionally,
while
the
relative
contributions
of
blood
and
tissue
detoxification
can
be
estimated
in
animal
models,
this
information
is
unknown
in
humans
for
most
if
not
all
OP
toxicants.
This
subject
therefore
represents
a
potentially
significant
uncertainty
in
how
young
children
may
respond
to
OP
toxicants
relative
to
adults
based
on
differential
metabolism.

Both
the
carboxyesterases
and
A­
esterase
are
non­
specific
esterases.
Data
are
available
concerning
changes
in
the
levels
of
A­
esterase
in
blood
with
age
in
humans,
which
are
about
20
%
of
adult
levels
at
birth
and
near
adult
levels
by
6
months
of
age;
however,
there
are
fewer
data
for
the
carboxylesterases
during
development.
Several
Panel
members
felt
that
data
should
be
collected
at
least
with
blood
carboxylesterases
to
limit
the
uncertainty
associated
with
that
missing
information.
There
is
a
complete
lack
of
data
about
the
activity
levels
of
these
esterases
in
the
liver
and
other
tissues
where
the
bulk
of
the
detoxification
is
likely
to
occur.

At
birth,
the
esterases
in
general,
like
many
other
enzymes
responsible
for
metabolism,
are
at
a
low
level—
approximately
20%
of
adult
values.
These
enzymes
increase
rapidly
during
the
first
few
months
and
although
variable,
are
near
the
adult
level
(60­
70%)
at
six
months.
The
fact
that
the
OP
exposure
of
very
young
infants
is
estimated
to
be
smaller
than
that
of
other
age
groups
tends
to
reduce
concerns
arising
from
neonatal
deficiencies
in
esterases
that
detoxify
OPs.
One
Panel
member
noted
that
the
development
of
the
various
esterases
appears
to
be
generally
similar
and
the
carboxyesterases
are
likely
to
be
similar
to
the
A­
esterases
in
this
regard.

Some
Panel
members
felt
strongly
that
EPA
should
not
accept
the
remaining
data
gaps
on
the
relative
importance
of
different
esterases
for
detoxification
of
different
OPs
for
any
length
of
27
time.
Now
that
EPA
research
scientists
have
developed
an
in­
house
assay
that
at
least
approximately
tracks
the
age
dependent
shift
in
blood
samples'
ability
to
alter
OP
availability
in
vitro
based
on
A­
esterase
and
carboxylesterase
activities,
these
assays
should
be
performed
with
human
blood
samples
at
all
ages
of
interest
and
with
all
environmentally
relevant
OPs.
The
problem
with
carboxylesterase
is
that
human
blood
contains
very
little
of
this
enzyme,
which
is
largely
confined
to
liver.
Therefore,
for
the
foreseeable
future,
the
Agency
must
continue
to
reason
by
analogy
with
animal
data
and
with
the
developmental
profile
of
other
liver
drug
metabolizing
enzymes.
In
this
context,
however,
it
does
appear
reasonable
to
assume
that
the
youngest
infants
will
indeed
be
deficient
in
carboxylesterase
expression,
and
that
expression
of
this
enzyme
will
approach
adult
levels
sometime
in
early
childhood—
possibly
in
the
1­
2
year
bracket.

Some
OPs
are
initially
metabolized
by
cytochrome
P450s
to
oxon
intermediates.
It
appears
that
the
P450s
involved
are
P450
3a
and
2D6
families.
Cytochrome
P4502
D6
expression
is
decreased
in
the
newborn's
liver
and
then
approaches
the
adult
level
within
a
few
weeks.
Family
3
enzyme
overall
activity
is
generally
thought
to
be
increased
during
the
newborn,
infancy
and
early
childhood
stages
of
life.
Family
3a
during
development
is
primarily
composed
of
P4503a4
and
3a7.
P4503a7
is
the
fetal
form
of
family
3a
and
is
expressed
in
high
levels
in
the
fetal,
newborn
and
infant
liver
as
compared
to
the
adult.
The
P450
3a4
is
expressed
at
higher
activity
levels
during
these
periods
than
in
adulthood.
These
findings
are
somewhat
substrate
dependent
and
to
the
Panel's
knowledge,
studies
of
the
capacity
of
3a7
to
metabolize
OPs
have
not
been
conducted.
The
changing
expression
of
these
P450
forms
may
add
to
the
overall
toxicities
of
the
OPs
to
the
human
during
development.
The
expression
of
these
enzymes
in
the
human
brain
during
development
has
not
yet
been
extensively
studied.

Detoxification
Maturation
Profiles
Overall,
the
pattern
of
age­
related
change
in
the
A­
esterase
bears
a
close
resemblance
to
general
patterns
of
change
for
elimination
inferred
from
human
observations
of
age­
related
changes
in
the
pharmacokinetics
of
therapeutic
drugs.
Table
1
reproduces
the
results
of
an
analysis
by
Hattis
et
al.
(2002,
in
press)
and
Ginsberg
et
al.
(2002).
The
table
shows
geometric
means
±
1
standard
error
range
of
the
ratios
of
the
half
lives
of
drugs
eliminated
by
a
variety
of
pathways
in
children
of
various
age
groups
relative
to
adult
half
lives.
Overall,
premature
infants
show
on
average
about
a
four­
fold
prolongation
of
elimination
half
life
for
the
typical
drug;
and
infants
under
2
months
of
age
have
about
double
the
half
life
of
adults.
The
6
month
to
2
year
age
group
shows,
if
anything,
a
slightly
shorter
geometric
mean
half
life
than
in
comparable
adult
studies.
If
these
patterns
hold
for
activation
and
inactivation
pathways
for
OPs,
then
agents
that
do
not
require
metabolic
activation
would
be
expected
to
pose
greater
risks
in
very
young
full
term
infants
(achieving
comparable
blood
levels
at
about
half
the
long
term
internal
dose
per
mg/
kg
of
external
dose)
but
children
in
other
age
groups
would,
on
average
show
no
greater
pharmacokinetic
sensitivity
than
adults.
Other
things
being
equal,
it
seems
most
likely
that
the
unmeasured
carboxylesterase
will
behave
similarly,
but
how
confident
one
should
be
about
this
is
open
to
question.
28
A
further
topic
where
data
are
available
is
the
extent
of
human
inter­
individual
variability
in
half
lives
as
a
function
of
age.
Variability
is
much
larger
than
adults
in
the
age
groups
up
until
about
six
months,
but
reverts
approximately
to
adult
levels
of
pharmacokinetic
variability
thereafter.
29
Table
I.
Geometric
Mean
Ratios
of
Child/
Adult
Elimination
Half­
Lives.
Data
Represent
Regression
Results
from
135
Data
Groups
for
41
Drugs,
Log(
Arithmetic
Mean
Half­
Life)
Data
Major
Elimination
Pathway
Premature
neonates
Full
term
neonates
1
wk­
2
mo
2­6
mo
6
mo­2
yr
2­
12
yr
12
­18yr
All
pathways
3.
89
(2.8­
5.4)
a
1.96
(1.7­
2.3)
1.93
(1.7­
2.2)
1.17
(1.0­
1.3)
0.79
(0.66­
0.94)
0.98
(0.89­
1.1)
1.11
(0.86­
1.4)

All
CYP
(P450
metabolism)
4.52
(2.5­
8.0)
1.83
(1.4­
2.3)
3.51
(3.1­
4.0)
1.22
(0.96­
1.6)
0.51
(0.41­
0.65)
0.61
(0.52­
0.72)
0.73
(0.26­
2.0)

All
Non­
CYP
3.
43
(2.4­
4.8)
1.80
(1.5­
2.1)
1.46
(1.3­
1.7)
1.06
(0.91­
1.2)
0.98
(0.78­
1.2)
0.92
(0.81­
1.03)
1.11
(0.87­
1.4)

Unclassified
1.
00
(0.83­
1.2)
0.94
(0.94­
1.06)
more
detailed
classification:
CYP1A2
2.74
(0.9­
7.6)
9.45
(2.9­
31)
4.29
(3.8­
4.9)
1.24
(1.0­
1.5)
0.57
(0.44­
0.72)
0.54
(0.45­
0.64)

Renal
2.
78
(1.4­
5.4)
2.75
(1.8­
4.1)
1.15
(0.86­
1.6)
0.81
(0.60­
1.1)
0.60
(0.48­
0.74)
1.13
(0.73­
1.7)

Glucuronidation
4.
40
(4.1­
4.7)
2.98
(2.8­
3.2)
2.15
(1.7­
2.7)
0.98
(0.84­
1.1)
1.19
(1.0­
1.4)
1.36
(1.2­
1.5)
1.47
(1.3­
1.7)

CYP3A
5.
28
(2.7­
10)
2.08
(1.4­
3.2)
1.91
(1.5­
2.5)
0.41
(0.27­
0.63)
0.61
(0.45­
0.84)
0.73
(0.25­
2.1)

CYP2C9
2.
19
(1.7­
2.8)
0.55
(0.39­
0.79)
0.77
(0.51­
1.2)

Other,
mixed
CYP's
1.
27
(0.7­
2.3)
1.08
(0.58­
2.0)

Other
Non­
CYP's
(not
renal,
glucuronidation)
0.41
(.
03­
5)
1.22
(0.94­
1.6)
1.05
(0.80­
1.4)
0.77
(0.58­
1.0)
1.24
(0.94­
1.6)
1.41
(0.82­
2.4)
a
Parentheses
show
the
±
1
standard
error
range.
30
Question
3.2
Please
comment
on
the
extent
to
which
the
biological
understanding
of
observed
age­
dependent
sensitivity
to
cholinesterase
inhibition
in
laboratory
animal
studies
informs
our
understanding
about
the
likelihood
of
similar
effects
occurring
in
children;
in
particular,
what
can
be
inferred
from
animal
and
human
information
regarding
the
potential
for
different
age
groups
to
show
increased
sensitivity
if
exposed
to
cholinesteraseinhibiting
pesticides.
Does
the
scientific
evidence
support
the
conclusion
that
infants
and
children
are
potentially
more
sensitive
to
organophosphorus
cholinesterase
inhibitors?

The
scientific
evidence
supports
the
conclusion
that
infants
and
children
are
potentially
more
sensitive
to
OP
cholinesterase
inhibitors
than
are
adults.
There
are
still
important
unresolved
questions
including:
1)
What
is
the
extent
of
age­
dependency
in
human
fetuses,
children,
juveniles,
adults
(and
the
elderly)
and
is
it
larger
or
smaller
than
in
rats?
2)
What
are
the
ages
at
which
higher
sensitivity
is
present
in
humans
as
compared
with
rats
(e.
g.,
are
1­
2
yr.
humans
best
modeled
by
the
PND
21
rat)?
3)
Are
underlying
mechanisms
contributing
to
agerelated
sensitivity
fundamentally
similar?
and
4)
Does
a
certain
degree
of
acetylcholinesterase
inhibition
in
the
immature
system
leads
to
equivalent
neurochemical
consequences
as
those
observed
in
adults
­
or,
by
contrast,
are
there
likely
to
be
some
adverse
neurodevelopmental
consequences
for
amounts
of
brain
cholinesterase
inhibition
that
are
considered
reasonably
tolerable
by
adults?

The
understanding
of
differential
age­
related
toxicity
in
experimental
animals
exposed
to
OP
toxicants
suggests
that,
with
acute
high
exposures,
young
children
may
be
markedly
more
sensitive
to
some
agents.
This
is
likely
based
on
both
toxicodynamic
and
toxicokinetic
factors
including
differences
in
expression
of
detoxifying
esterases,
possible
differences
in
activation
of
some
agents,
and
in
maturation
of
adaptive
processes
that
limit
or
modulate
anticholinesterase
toxicity.
With
other
OP
toxicants
(e.
g.,
methamidophos),
lesser
or
even
no
age­
related
differences
in
acute
sensitivity
may
exist.
Some
studies
suggest,
however,
that
differences
in
sensitivity
are
less
pronounced
or
non­
existent
with
repeated
dosing.
Both
kinetic
(e.
g.,
detoxification)
and
dynamic
(e.
g.,
feedback
inhibition
of
acetylcholine
release)
pathways
are
most
likely
important
in
contributing
to
age­
related
differences
in
sensitivity
to
high
dose
exposures,
i.
e.,
these
processes
are
likely
challenged
only
when
high
levels
of
the
toxicant
occur
in
the
system.
Thus,
with
repeated,
lower
exposures,
lesser
differences
in
sensitivity
would
be
expected.
As
noted
above,
however,
it
is
likely
that
the
reduced
age­
related
differences
with
repeated
exposures
in
rodents
is
due
to
rapid
maturation
of
the
animal
with
consequent
decreased
sensitivity
over
the
course
of
exposure.
Therefore,
the
Panel
agreed
that
the
scientific
evidence
supports
the
conclusion
that
infants
and
children
are
potentially
more
sensitive
to
OP
cholinesterase
inhibitors
to
acute
high
dose
exposures.
With
lower
and
repeated
exposures,
the
evidence
for
higher
sensitivity
in
young
individuals
is
not
as
convincing.

In
the
absence
of
directly
applicable
data,
it
was
felt
that
humans
might
differ
from
rats
in
31
the
extent
and
nature
of
age­
dependent
sensitivity
for
enzyme
inhibition.
All
the
animal
data
were
generated
using
either
direct
exposure
to
neonates,
juvenile
and
adult
animals
at
very
high
doses
or
the
treatment
of
pregnant
or
lactating
animals,
also
at
relatively
high
dose
levels.
The
data
from
the
repeated
direct
dosing
experiments
yielded
ChE
inhibition
sensitivity
ratios
of
1
to
3­
fold
for
pups
versus
the
adults.
This
could
become
as
much
as
10­
fold
following
acute
dosing.
Whether
this
makes
a
substantial
difference
in
humans
likely
depends
on
the
exposure
level.

As
stated
above,
the
remaining
data
gap
regarding
human
blood
A­
esterase­
mediated
detoxification
of
the
different
OP
anticholinesterases
should
be
addressed
by
further
research.
A
number
of
experimental
approaches
were
proposed
by
Panel
members.
One
was
the
use
of
an
in
vitro
model
recently
developed
(Padilla
et
al.,
2002).
Now
that
EPA
scientists
have
developed
an
in­
house
assay
that
at
least
approximately
(and
perhaps
quite
accurately)
tracks
the
age
dependent
shift
in
blood
esterase
abilities
to
alter
OP
availability
in
vitro,
as
a
direct
comparison
between
species,
these
assays
should
be
run
with
human
and
rat
blood
samples
at
all
ages
of
interest
and
with
all
environmentally
relevant
OPs.
For
carboxylesterase,
a
complication
exists
for
projection
between
species,
i.
e.,
in
humans
(in
contrast
to
rodents),
very
little
of
this
enzyme
is
found
in
the
blood.
One
Panel
member
recommended
a
set
of
studies
on
the
age­
related
variation
in
sensitivity
of
blood
cholinesterases
to
inhibition
by
OP
inhibitors
in
a
primate
model,
preferably
a
higher
primate.
An
advantage
of
a
primate
model
is
the
similarity
in
plasma
carboxylesterase
activity,
i.
e.,
primates
are
deficient
in
this
pathway.
Such
studies
would
provide
the
most
relevant
possible
animal
data
on
several
fronts,
including
the
difficult
question
of
whether
AChE
and
BChE
resynthesis
is
indeed
faster
in
young
children
than
in
adults,
and
at
what
developmental
stage.
They
would
also
provide
information
on
the
potential
importance
of
carboxylesterases
and
Aesterases
at
different
ages.
In
the
foreseeable
future,
however,
we
must
continue
to
reason
by
analogy
with
rodent
data
and
with
the
developmental
profile
of
other
liver
drug
metabolizing
enzymes.
In
this
context,
it
does
appear
reasonable
to
assume
that
the
youngest
infants
will
indeed
be
deficient
in
tissue
carboxylesterase
expression,
and
that
expression
of
this
enzyme
will
approach
adult
levels
sometime
in
early
childhood—
probably
in
the
1­
2
year
bracket
or
sooner.

Two
Panel
members
felt
strongly
that
the
studies
presented
by
the
Agency
have
limited
application
to
understanding
the
effects
of
OP
insecticides,
specifically
in
children.
While
adverse
effects
related
to
mechanisms
other
than
acetylcholinesterase
inhibition
are
considered
in
the
risk
assessment
for
individual
OP
agents,
there
is
concern
that
such
possible
effects
could
be
"hidden"
in
the
process
of
cumulative
risk
assessment.
The
evaluation
of
OP
toxicity
can
be
considered
to
belong
in
the
realms
of
behavioral
teratology
and
toxicology.
James
Wilson,
who
opened
this
field,
delineated
dose­
response
relationships
of
prenatal
toxicants.
At
highest
exposures,
the
outcome
is
fetal
death;
at
somewhat
lower
doses,
congenital
defects;
at
lesser
doses,
growth
retardation
is
seen;
and
finally,
at
the
lowest
exposures,
functional
deficits,
most
notably
behavior,
become
visible.
It
is
in
this
lowest
exposure
stratum
that
examination
of
OP
toxicity
should
continue.
The
concern
with
the
effects
of
OPs
prenatally
and
postnatally
is
associated
with
the
brain.
This
relates
to
the
impact
of
OPs
on
children's
function,
and
among
their
most
critical
functions
is
their
ability
to
think,
talk
and
pay
attention.
Not
to
include
data
on
these
outcomes
excludes
important
variables
in
the
assessment
and
therefore
introduces
important
specification
32
error.
Wilson's
work
and
the
work
of
many
others
have
shown
that
systematically
measured
behavior
may
demonstrate
toxicological
effects
at
lower
doses
than
those
that
yield
phenotypic
or
biochemical
alterations.

These
same
Panel
members
further
stated
that
EPA­
listed
studies
of
animal
behavioral
effects,
some
of
which
were
not
associated
with
cholinergic
alterations,
were
conducted
at
doses
of
OP
pesticides
previously
thought
to
be
without
effect.
Levin
and
colleagues
reported
long
term
behavioral
changes
in
offspring
following
maternal
chlorpyrifos
exposure.
The
nature
of
the
changes
(loss
of
sensitivity
to
cholinergic
muscarinic
antagonist)
suggested
that
the
behavioral
effects
were
not
cholinergic
in
origin.
These
and
other
data
point
to
mechanisms
besides
AChE
inhibition
that
may
also
be
at
work
in
OP
toxicity.
Thus,
reliance
on
a
single
biochemical
assay
to
measure
brain
damage
may
become
problematic.

Expanding
on
this
issue,
the
Panel
members
pointed
out
that
when
using
a
marker,
in
this
case
brain
AChE
levels
as
a
marker
for
more
proximate
effects
of
OPs,
one
is
required
to
calibrate
it
and
determine
its
validity
in
estimating
the
process
or
event
that
it
stands
for.
To
determine
this,
it
is
necessary
to
measure
both
the
marker
and
the
process
of
interest
(e.
g.,
synaptogenesis,
behavioral
outcome)
and
determine
the
correlation
between
the
two
variates,
the
coefficient
of
determination,
the
sensitivity,
specificity,
and
predictive
power,
both
positive
and
negative,
of
the
marker.
These
factors
have
precise
meanings
in
science.
Sensitivity
is
the
probability
that
an
outcome
(e.
g.,
impaired
learning)
will
be
identified
by
the
marker.
Specificity
is
the
probability
that
the
absence
of
such
an
outcome
will
be
correctly
identified.
Predictive
power
positive
is
the
probability
that
a
positive
test
will
identify
a
specified
outcome.
EPA
has
not
indicated
anywhere
in
its
report
that
these
important
determinations
have
been
accomplished.
As
a
consequence,
the
amount
of
measurement
error
in
the
cumulative
risk
assessment
is
unknown.
Since
this
measurement
error
is
nonsystematic
(neither
systematically
higher
or
lower
AChE
levels
than
the
true
values)
and
non­
differential
(not
increased
in
subjects
with
higher
brain
AChE,
etc.,
than
with
lower
levels),
the
direction
of
the
bias
introduced
by
measurement
error
is
toward
the
null.
That
is,
it
would
tend
to
underestimate
the
size
of
the
effect
under
study,
in
this
case
the
sensitivity
of
children
to
OPs.
From
these
points,
these
Panel
members
concluded
that
the
EPA
report
contains
substantial
measurement
and
specification
errors,
and
as
a
consequence,
underestimates
the
risk
of
OPs
for
child
health.

In
general,
however,
it
should
be
stressed
that
the
cumulative
risk
assessment
for
the
OP
insecticides
is
indeed
based
on
acetylcholinesterase
inhibition
and
cholinergic
toxicity.
While
noncholinergic
endpoints
may
weigh
on
the
risk
assessment
of
individual
agents,
the
cumulative
risk
assessment
is
driven
by
cholinergic
mechanisms
initiated
by
acetylcholinesterase
inhibition
and
related
to
consequent
increases
in
acetylcholine,
if
the
common
mechanism
for
OP
insecticides
is
acetylcholinesterase
inhibition
and
cholinergic
toxicity.
Based
on
this
endpoint,
there
is
compelling
evidence
to
support
the
conclusion
of
potentially
higher
sensitivity
in
infants
and
children.

Question
3.3
33
Please
comment
on
the
conclusions
regarding
the
faster
recovery
in
the
young
animal
of
AChE
activity.
Because
there
is
no
human
information
on
the
recovery
of
AChE
in
children
compared
to
adults,
please
comment
on
the
extent
to
which
recovery
of
AChE
in
children
should
be
factored
into
conclusions
regarding
potential
risk
to
children.

The
Panel
agreed
that
given
the
conservation
of
neurodevelopmental
processes
across
species,
all
aspects
of
this
biological
process
identified
to
be
critical
in
the
rodent
model
should
be
taken
into
consideration
when
evaluating
these
compounds
for
their
potential
risk
to
children.
The
Panel
raised
some
issues
regarding
the
interpretation
of
the
biological
consequences
of
the
apparent
faster
recovery
of
AChE
activity
in
the
young
animals
–
that
is
the
Panel
had
reservations
about
whether
the
faster
recovery
could
be
regarded
as
indicating
a
return
to
a
completely
normal
state
that
is
free
of
further
neurodevelopmental
consequences.

The
Agency's
background
document
provides
information
regarding
what
appears
to
be
a
faster
recovery
of
AChE
in
young
animals
as
compared
to
the
adult.
The
available
data
are
quite
limited,
however,
and
it
is
not
possible
to
reach
a
conclusion
regarding
the
dynamics
of
the
underlying
mechanisms
of
how
this
phenomenon
occurs
and
its
biological
impact.
Given
the
species
conservation
of
many
such
biological
processes,
as
well
as
the
high
degree
of
structural
and
functional
homology
between
AChEs
and
ACh
receptors
in
rats
and
humans,
differential
recovery
rates
should
ultimately
be
factored
into
conclusions
regarding
possible
risk
to
children.
How
this
will
be
done
in
the
absence
of
biological
data
is
a
question.

The
general
mechanism
proposed
for
differential
recovery
rates
deals
with
higher
on­
going
macromolecular
synthesis
in
immature
tissues
than
adult
tissue.
There
may
also
be
differences
in
the
ability
of
tissues
to
respond
to
AChE
inhibition
by
inducing
the
synthesis
of
AChE.
For
example,
some
studies
suggest
that
anticholinesterases
can
activate
the
transcription
of
AChE
(Soreq
and
Seidman,
2001).
These
phenomena
however,
have
not
been
adequately
evaluated
in
animal
models
following
OP
exposure.

In
order
to
fully
appreciate
the
importance
compensatory
mechanisms
in
the
younger
animal,
information
is
needed
on
relevant
transmitter
systems
including
synthesis
rates,
turnover
rates,
and
equilibrium
levels
of
the
transmitters,
as
well
as
the
pharmacology,
numbers
and
binding
capacities
of
the
transmitter
receptors.
Finally,
we
need
to
know
much
more
about
the
down
stream
effects
of
increased
acetylcholine
levels
resulting
from
an
inhibition
of
AChE.
Once
this
is
known,
we
will
have
a
better
idea
of
exactly
what
the
inhibition
of
AChE
activity
and
its
time
to
recovery
may
mean
in
the
young
animal.

The
compensatory
ability
of
the
developing
animal
also
shows
itself
in
the
relatively
normal
phenotypes
seen
with
certain
knockout
animals
and
genetic
mutants.
One
might
take
comfort
in
reasoning
that
adaptive
mechanisms
seen
in
experimental
animal
models
are
also
likely
to
operate
in
humans.
A
strong
caution
needs
to
be
raised,
however,
because
compensatory
and
adaptive
mechanisms
can
still
lead
to
permanently
abnormal
outcomes.
Recovery
of
whole
brain
34
AChE
does
not
necessarily
imply
return
to
a
normal
state,
especially
in
the
developing
nervous
system.
That
is
because
the
formation
of
brain
architecture
and
the
elaboration
and
stabilization
of
synapses
must
continue
during
the
period
of
neurochemical
disruption.
The
possible
result
is
a
permanent
alteration
in
the
characteristics
of
synapses
formed
in
the
interval
prior
to,
during,
and
following
exposure.
In
addition,
the
replenishment
of
AChE
may
merely
reflect
synthesis
of
catalytically
active
but
functionally
deficient
molecules,
with
regard
to
cholinergic
neurotransmission.

As
noted
previously,
however,
when
exposure
periods
are
separated
in
time
(4
day
intervals
between
exposures),
adult
rats
show
more
cumulative
AChE
inhibition
and
downregulation
of
receptors
(Chakraborti
et
al.,
1993).
These
findings
suggest
that
the
more
robust
recovery
of
AChE
in
immature
animals
indeed
represents
enhanced
functional
recovery.
The
major
AChE
expressed
in
nervous
tissue
is
the
so­
called
"synaptic"
form
(AChE­
S).
Chronic
inhibition
of
AChE
activity
can
lead
to
the
expression
of
a
unique
transcript,
referred
to
as
the
"read­
through"
form
(AChE­
R)
that
is
secreted
as
a
monomer
(Grisaru,
et
al.,
1999;
Soreq
and
Seidman,
2001).
This
protein
has
the
same
enzyme
kinetics
as
the
synaptic
form,
and
thus
would
appear
in
an
enzyme
assay
as
normal
AChE.
However,
because
the
enzyme
has
a
different
distribution,
it
may
not
have
the
same
functional
impact
as
the
normal
AChE­
S.
The
relevance
of
these
findings
to
the
issue
currently
under
review
remains
to
be
determined,
yet
they
raise
concerns
regarding
the
dynamics
of
the
overall
process
of
cholinesterase
inhibition
during
development.
With
all
of
these
biological
processes,
the
consequences
of
such
inhibition
and
replenishment
would
depend
upon
the
stage
of
brain
development
occurring
during
this
period.

ADDITIONAL
COMMENTS
One
Panel
member
provided
comments
on
the
exposure
assessment
for
consideration
in
the
selection
of
an
appropriate
FQPA
uncertainty
factor.
This
Panel
member
analyzed
the
EPA's
treatment
of
exposures
(e.
g.
dietary
exposure).
References
in
the
document
to
95
th
,99
th
,
99.5th,
99.
9th
percentiles
imply
a
view
that
such
numbers
bracket
the
high
end
exposures.
A
simple
calculation
of
the
corresponding
consumption
would
show
otherwise.
This
point
can
be
illustrated
by
using
the
Agency's
cumulative
exposure
for
individuals
1­
2
years
old
and
assuming
thatthe
entire
amountofexposure
comesfroma
single
chemicalina
single
foodformofa
commodity.
For
example,
let's
assume
that
the
entire
exposure
is
from
azinphos
methyl
(AZM)
in
fresh
apple
or
pear.
The
1999
PDP
single
serving
monitoring
data
showed
that
AZM
was
detected
in
76.2%
(1088
of
1427
samples)
of
apples
at
0.01­
0.55
ppm,
and
43.2%
(152
of
352
samples)
of
pears
at
0.
013­
0.87
ppm.
Taking
into
account
the
0.1
of
RPF
for
AZM,
and
using
the
highest
detected
residue
(0.
55
ppm
for
apple
or
0.
87
ppm
for
pears),
the
cumulative
dietary
exposure
of
0.0002
mg/
kg/
day
at
the
95
th
percentile
is
equivalent
to
the
consumption
of
either
1.3­
1.9
oz.
of
apple
or
0.
8­
1.
2
oz.
of
pears.
These
levels
of
consumption
do
not
appear
to
represent
the
high
end
of
consumption
even
from
just
fresh
apple
or
pears.
Only
as
the
cumulative
exposure
moves
toward
the
higher
distributional
percentiles
does
it
begin
to
appear
more
unlikely
to
be
contributed
from
a
single
source.
35
This
type
of
analysis
is
helpful
to
provide
a
context
for
exposure
estimates
in
a
cumulative
risk
assessment.
Obviously,
to
choose
an
uncertainty
factor
to
account
for
the
exposure
component
we
must
know
what
percentile
captures
the
reasonably
expected
high
end.
In
this
illustration,
an
argument
can
be
made
for
an
additional
FQPA
uncertainty
factor
if
the
benchmark
for
risk
management
decision
is
based
on
the
95
th
percentile
of
dietary
exposure.
Fortunately,
for
the
exposure
assessment,
especially
the
dietary
route,
sufficient
data
are
available
for
a
much
more
informed
decision.
The
Agency
is
encouraged
to
provide
documentation
that
goes
beyond
the
numerical
exposure
values
and
percentiles
present
in
the
Agency's
background
document.

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