Document ID: EPA-HQ-OPP-2003-0024-0082
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-05T04:00Z

August
4,
2003
MEMORANDUM
SUBJECT:
Transmittal
of
Meeting
Minutes
of
the
FIFRA
Scientific
Advisory
Panel
Meeting
Held
June
17­
20,
2003
TO:
James
J.
Jones,
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
Joseph
J.
Merenda,
Jr.,
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
17
to
20,
2003.
This
report
addresses
a
set
of
scientific
issues
being
considered
by
the
Environmental
Protection
Agency
regarding
the
potential
developmental
effects
of
atrazine
on
amphibians.

Attachment
2
cc:

Susan
Hazen
Adam
Sharp
Anne
Lindsay
Janet
Andersen
Debbie
Edwards
Steven
Bradbury
Jay
Ellenberger
Arnold
Layne
Tina
Levine
Frank
Sanders
Betty
Shackleford
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.
Steven
Heeringa,
Ph.
D.
Gary
E.
Isom,
Ph.
D.
Fumio
Matsumura,
Ph.
D.
Mary
Anna
Thrall,
DVM
FQPA
Science
Review
Board
Members
Joel
Coats,
Ph.
D.
Peter
Delorme,
Ph.
D.
Robert
J.
Denver,
Ph.
D.
James
Gibbs,
Ph.
D.
Sherril
L.
Green,
DVM
Darcy
B.
Kelley,
Ph.
D.
Werner
Kloas,
Ph.
D.
Gerald
A.
LeBlanc,
Ph.
D.
Carl
Richards,
Ph.
D.
David
Skelly,
Ph.
D.
3
SAP
Report
No.
2003­
01
REPORT
FIFRA
Scientific
Advisory
Panel
Meeting,
June
17­
20,
2003,
held
at
the
Crowne
Plaza
Hotel
Arlington,
Virginia
A
Set
of
Scientific
Issues
Being
Considered
by
the
Environmental
Protection
Agency
Regarding:

POTENTIAL
DEVELOPMENTAL
EFFECTS
OF
ATRAZINE
ON
AMPHIBIANS
4
NOTICE
These
meeting
minutes
have
been
written
as
part
of
the
activities
of
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA),
Scientific
Advisory
Panel
(
SAP).
These
meeting
minutes
represent
the
views
and
recommendations
of
the
FIFRA
SAP,
not
the
United
States
Environmental
Protection
Agency
(
Agency).
The
content
of
these
meeting
minutes
do
not
represent
information
approved
or
disseminated
by
the
Agency.
They
have
not
been
reviewed
for
approval
by
the
Agency
and,
hence,
the
contents
of
these
meeting
minutes
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
is
a
Federal
advisory
committee
operating
in
accordance
with
the
Federal
Advisory
Committee
Act
and
was
established
under
the
provisions
of
FIFRA,
as
amended
by
the
Food
Quality
Protection
Act
(
FQPA)
of
1996.
The
FIFRA
SAP
provides
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
Report
No.
2003­
01
REPORT:
FIFRA
Scientific
Advisory
Panel
Meeting,
June
17­
20,
2003,
held
at
the
Crowne
Plaza
Hotel,
Arlington,
Virginia
A
Set
of
Scientific
Issues
Being
Considered
by
the
Environmental
Protection
Agency
Regarding:

POTENTIAL
DEVELOPMENTAL
EFFECTS
OF
ATRAZINE
ON
AMPHIBIANS
Mr.
Paul
Lewis
Stephen
Roberts,
Ph.
D.
Designated
Federal
Official
FIFRA
SAP
Session
Chair
FIFRA
Scientific
Advisory
Panel
FIFRA
Scientific
Advisory
Panel
Date:
August
4,
2003
Date:
August
4,
2003
6
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
Scientific
Advisory
Panel
Meeting
June
17­
20,
2003
POTENTIAL
DEVELOPMENTAL
EFFECTS
OF
ATRAZINE
ON
AMPHIBIANS
PARTICIPANTS
FIFRA
SAP
Session
Chair
Stephen
M.
Roberts,
Ph.
D.,
Professor
and
Program
Director,
University
of
Florida
Center
for
Environmental
&
Human
Toxicology,
Gainesville,
Florida
FIFRA
Scientific
Advisory
Panel
Members
Steven
Heeringa,
Ph.
D.,
Research
Scientist
and
Director
for
Statistical
Design
Institute
for
Social
Research,
University
of
Michigan,
Ann
Arbor,
MI
Gary
E.
Isom,
Ph.
D.,
Professor
of
Toxicology,
School
of
Pharmacy
and
Pharmacal
Sciences,
Purdue
University,
West
Lafayette,
IN
Fumio
Matsumura,
Ph.
D.,
Professor
of
Environmental
Toxicology
and
Director
of
the
Center
for
Environmental
Health
Sciences
University
of
California
at
Davis,
Davis,
CA
Mary
Anna
Thrall,
DVM.,
Professor,
Department
of
Microbiology,
Immunology
and
Pathology,
Colorado
State
University,
Fort
Collins,
CO
FQPA
Science
Review
Board
Members
Joel
Coats,
Ph.
D.,
Professor
and
Chair,
Department
of
Entomology,
Iowa
State
University,
Ames,
Iowa
Peter
Delorme,
Ph.
D.,
Senior
Evaluation
Officer,
Environmental
Assessment
Division
Pest
Management
Regulatory
Agency,
Health
Canada,
Ottawa,
Ontario,
Canada
Robert
J.
Denver,
Ph.
D.,
Associate
Professor
and
Associate
Chair
for
Undergraduate
Studies,
Department
of
Molecular,
Cellular
and
Developmental
Biology;
Associate
Professor,
Department
of
Ecology
and
Evolutionary
Biology,
The
University
of
Michigan,
Ann
Arbor,
MI
James
Gibbs,
Ph.
D.,
Associate
Professor,
Faculty
of
Environmental
and
Forest
Biology,
SUNY­
ESF,
Syracuse,
NY
Sherril
L.
Green,
DVM,
Associate
Professor,
Department
of
Comparative
Medicine,
Stanford
University
School
of
Medicine,
Stanford,
CA
7
Darcy
B.
Kelley,
Ph.
D.,
Professor
of
Biological
Sciences,
Columbia
University,
New
York,
NY
Werner
Kloas,
Ph.
D.,
Professor
of
Endocrinology,
Department
of
Endocrinology,
Institute
of
Biology,
Hymboldt
University,
Berlin;
Head
of
Department
of
Inland
Fisheries,
Leibniz­
Institute
of
Freshwater
Ecology
and
Inland
Fisheries,
Berlin,
Germany
Gerald
A.
LeBlanc,
Ph.
D.,
Professor
of
Toxicology,
Department
of
Environmental
&
Molecular
Toxicology,
North
Carolina
State
University,
Raleigh,
NC
Carl
Richards,
Ph.
D.,
Director
and
Professor,
Minnesota
Sea
Grant
College
Program,
University
of
Minnesota
Duluth,
Duluth,
MN
David
Skelly,
Ph.
D.,
Associate
Professor
of
Ecology,
Yale
School
of
Forestry
and
Environmental
Studies,
New
Haven,
CT
PUBLIC
COMMENTERS
Oral
statements
were
made
by:

Ronald
Kendall,
Ph.
D.,
Texas
Tech
University,
Glen
Van
Der
Kraak,
Ph.
D.,
University
of
Guelph,
Canada,
Ernest
Smith,
Ph.
D.,
Texas
Tech
University,
John
Giesy,
Ph.
D.,
Michigan
State
University,
Louis
Du
Preez,
Ph.
D.,
Potchefstroom
University,
South
Africa,
K.
R.
Solomon,
Ph.
D.,
University
of
Guelph,
Canada,
Timothy
Gross,
Ph.
D.,
United
States
Geological
Survey
and
the
University
of
Florida,
Robert
Sielken,
Jr.,
Ph.
D.
Sielken
and
Associates,
Inc.,
James
Carr,
Ph.
D.,
Texas
Tech
University,
and
Ms.
Catherine
Bens,
United
States
Department
of
Agriculture,
on
behalf
of
Ecorisk,
Inc.

John
Ashby,
Ph.
D.
and
Charles
Breckenridge,
Ph.
D.
on
behalf
of
Syngenta
Tyrone
Hayes,
Ph.
D.,
the
University
of
California,
Berkeley,
as
a
private
citizen
Nigel
Noriega,
Ph.
D.,
USEPA
,
as
a
private
citizen
Janis
McFarland,
Ph.
D.
on
behalf
of
Syngenta
Angelina
Duggan,
Ph.
D.
on
behalf
of
CropLife
America
Mr.
Scott
Slaughter
on
behalf
of
the
Center
for
Regulatory
Effectiveness
Mr.
Jere
White,
Kansas
Corn
Growers
Association
and
Kansas
Grain
Sorghum
Producers
Association,
Ms.
Stephanie
Whalen,
Hawaii
Agriculture
Research
Center,
Bill
Kubecka,
DVM,
Kubecka
Farms,
and
Mr.
Gary
Marshall,
Missouri
Corn
Merchandising
Council
and
Missouri
8
Corn
Growers
Association,
on
behalf
of
the
Triazine
Network
Richard
Fawcett,
Ph.
D.,
on
behalf
of
the
Iowa
Corn
Growers
Association
Mr.
Robert
Hedberg
on
behalf
of
the
Weed
Science
Society
of
America
Jennifer
Sass,
Ph.
D.,
on
behalf
of
the
Natural
Resources
Defense
Council
Steven
Sheffield,
Ph.
D.,
George
Mason
University,
as
a
private
citizen
Mr.
James
Tozzi
on
behalf
of
Multinational
Business
Services
Mr.
John
Hall
on
behalf
of
the
Kentucky
Corn
Growers
Association
Mr.
Daniel
Botts
on
behalf
of
the
Florida
Fruit
and
Vegetable
Association
Written
statements
were
received
from:

Center
for
Regulatory
Effectiveness
Ecorisk,
Inc.

Natural
Resources
Defense
Council
Rachel
Carson
Council
Rachel
Jordan,
as
a
private
citizen
Sielken
&
Associates
Consulting,
Inc.

State
of
New
York,
Office
of
the
Attorney
General
Steven
Sheffield,
Ph.
D.,
George
Mason
University,
as
a
private
citizen
Syngenta
Tyrone
Hayes,
University
of
California,
Berkeley,
as
a
private
citizen
Triazine
Network
Weed
Science
Society
of
America
INTRODUCTION
9
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
Agency
pertaining
to
the
potential
developmental
effects
of
atrazine
on
amphibians.
Advance
notice
of
the
meeting
was
published
in
the
Federal
Register
on
February
24,
2003
and
May
8,
2003.
The
review
was
conducted
in
an
open
Panel
meeting
held
in
Arlington,
Virginia,
from
June
17­
20,
2003.
The
meeting
was
chaired
by
Stephen
Roberts,
Ph.
D.
Mr.
Paul
Lewis
served
as
the
Designated
Federal
Official.
Steven
Bradbury,
Ph.
D.
(
Office
of
Pesticide
Programs,
EPA)
summarized
the
goals
and
objectives
of
the
Agency's
presentation.
Thomas
Steeger,
Ph.
D.
(
Office
of
Pesticide
Programs,
EPA)
and
Mr.
Joseph
Tietge
(
Office
of
Research
and
Development,
EPA)
provided
an
overview
of
the
atrazine
studies
and
conceptual
model
for
potential
studies,
respectively.
Thomas
Steeger,
Ph.
D.
(
Office
of
Pesticide
Programs,
EPA)
completed
the
Agency's
presentation
by
providing
concluding
remarks.

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.
This
document
addresses
the
information
provided
and
presented
within
the
structure
of
the
charge
by
the
Agency.

CHARGE
1.
In
reviewing
the
available
laboratory
and
field
studies,
the
Agency
used
a
number
of
criteria
to
evaluate
individual
investigations.
Criteria
such
as
experimental
design,
test
protocols,
and
quality
assurance
information
were
used
to
ascertain
the
reliability
of
the
generated
data
in
terms
of
its
ability
to
adequately
assess
a
hypothesis
that
atrazine
elicits
developmental
effects
in
amphibians,
and
if
so,
the
nature
and
strength
of
associated
dose­
response
relationships.

a)
Does
the
SAP
have
any
comments
and
recommendations
regarding
the
EPA's
approach
and
criteria
used
to
evaluate
the
studies?

b)
Given
the
evaluation
criteria
employed
by
the
Agency,
please
comment
on
EPA's
overall
characterization
of
the
currently
available
studies.

c)
Please
comment
on
the
availability,
as
of
February
28,
2003,
of
additional,
relevant
studies
in
the
open
literature
that
were
not
addressed
in
the
white
paper.

d)
Since
February
28,
2003,
is
the
Panel
aware
of
any
studies
that
would
be
relevant?

2.
In
its
evaluation
of
existing
field
studies,
the
Agency
has
concluded
that
these
investigations
are
of
limited
value.
The
reasons
include:
(
1)
the
high
variability
in
environmental
conditions
and
uncertainties
in
the
pre­
existing
status
and
condition
of
field­
collected
animals,
(
2)
the
spatial
and
temporal
aspects
of
atrazine
exposure
(
i.
e.,
spatial
and
temporal
variability
over
the
course
of
the
studies
and
the
extent
to
which
such
aspects
of
atrazine
exposure
were
empirically
measured
or
otherwise
accounted
for),
and
(
3)
the
possible
co­
occurrence
of
additional
chemical
and/
or
nonchemical
stressors.
10
a)
To
the
extent
that
the
field
studies
appear
to
indicate
that
atrazine
may
not
adversely
affect
development,
please
comment
on
EPA's
conclusion
that
the
body
of
data
from
field
studies
does
not
provide
the
means
to
ascertain
whether
the
lack
of
a
relationship
between
atrazine
exposure
and
developmental
effects
is
due
to
the
absence
of
a
causal
relationship
or
limitation
in
study
methodologies.

b)
To
the
extent
that
any
field
studies
appear
to
indicate
that
atrazine
may
adversely
affect
development,
please
comment
on
EPA's
conclusion
that
these
field
studies
do
not
provide
sufficient
information
to
resolve
the
potential
role
of
additional
co­
occurring
stressors.

3.
In
an
evaluation
of
the
existing
laboratory­
based
studies,
the
Agency
concluded
that
there
was
sufficient
information
to
establish
a
hypothesis
that
atrazine
could
cause
adverse
gonadal
developmental
effects.
However,
due
to
different
experimental
designs
and
variability
in
the
nature
and
extent
of
experimental
conditions
(
e.
g.,
level
of
excessive
mortality,
delayed
development
in
untreated
organisms,
lack
of
response
to
positive
controls)
it
was
not
possible
to
adequately
assess
the
hypothesis
that
atrazine
causes
developmental
effects.
It
was
further
concluded
that
the
current
body
of
information
did
not
provide
the
means
to
characterize
the
nature
of
any
associated
dose­
response
relationships.

a)
Please
comment
on
EPA's
determination
that
the
laboratory
studies
provide
a
plausible
basis
for
the
means
to
establish
a
hypothesis
concerning
the
potential
for
atrazine
to
cause
developmental
effects.
Also,
please
comment
on
whether
the
overall
body
of
available
data
is
adequate
to
demonstrate
whether
or
not
atrazine
causes
developmental
effects
under
the
conditions
described
in
these
studies.

b)
Please
comment
on
EPA's
conclusion
that
given
the
variability
in
the
available
dose­
response
data
across
the
studies
(
e.
g.,
an
approximately
250­
fold
difference
in
reported
thresholds
for
observed
developmental
effects
as
well
as
reports
of
monotonic
and
non­
monotonic
doseresponse
curves),
it
is
not
possible
to
ascertain
the
relationship,
if
any,
of
atrazine
exposure
to
developmental
effects
in
amphibians.

4.
Many
of
the
available
studies
proposed
that
aromatase
induction
results
in
elevated
estrogen
levels
that
lead
to
feminization
(
ovotestes/
intersex/
hermaphroditism)
in
genetically
male
amphibians.

a)
Please
comment
on
EPA's
conclusion
that,
to
date,
aromatase
induction
by
atrazine
has
not
been
demonstrated
in
any
anuran
in
controlled
laboratory
investigations.

b)
The
variability
associated
with
plasma
sex
steroid
concentrations
and
aromatase
activities
is
high.
Is
this
variability
normal?
Please
comment
on
any
readily
apparent
or
available
methodological
improvements
(
e.
g.,
changes
in
sampling
design,
analytical
techniques)
that
could
efficiently
address
this
variability
in
future
studies.
11
c)
Please
comment
on
whether
there
are
additional
data,
other
than
those
summarized
in
the
white
paper,
that
suggest
late
exposure
of
amphibians
(
i.
e.,
juveniles
or
adults)
to
estrogens
or
estrogenic
chemicals
can
induce
ovotestes
formation.

d)
Please
comment
on
whether
there
are
additional
data,
other
than
those
summarized
in
the
white
paper,
that
suggest
alternative
mechanisms
that
could
explain
the
apparent
feminization
of
genetically­
male
amphibians.

5.
With
regard
to
specific
endpoints,
the
Agency
does
not
currently
have
sufficient
information
to
quantitatively
relate
gonadal/
laryngeal
effects
to
reproductive
outcomes.
A
major
underlying
uncertainty
is
the
ecological
relevance
of
ovotestes
occurrence
to
the
maintenance
of
anuran
populations.

a)
Can
the
Panel
provide
sources
of
data
on
background
rates
of
ovotestes
occurrence
in
amphibian
species
and
any
associated
considerations
for
interpreting
this
information
in
the
context
of
the
reviewed
studies?

b)
Can
the
Panel
characterize
any
evidence
that
suggests
that
the
presence
of
ovotestes
in
male
anurans
results
in
reproductive
impairment
via
reductions
in
fertility?

c)
The
reduction
of
laryngeal
muscle
area
suggests
diminished
testosterone
in
males.
If
this
is
found
to
be
a
valid
observation
and
if
estrogen
concentrations
do
increase
as
testosterone
concentrations
decrease,
what
other
endpoints
(
e.
g.,
secondary
sexual
characteristics
and
reproductive
behavior)
would
likely
be
affected?

6.
While
some
of
the
available
data
indicate
there
may
be
an
association
between
atrazine
exposure
and
developmental
effects
in
amphibians,
the
Agency's
evaluation
of
the
existing
body
of
laboratory
and
field
studies
has
determined
that
there
is
not
sufficient
scientific
evidence
to
indicate
that
atrazine
consistently
produces
effects
across
the
range
of
amphibian
species
examined.
However,
the
current
body
of
knowledge
has
deficiencies
and
uncertainties
that
limit
its
usefulness
in
assessing
potential
developmental
atrazine
effects
and
the
extent
of
any
associated
cause­
effect
and
dose­
response
relationships.
Consequently,
the
Agency
has
determined
that
there
are
not
sufficient
data
to
reject
the
hypothesis
that
atrazine
can
cause
adverse
developmental
effects
in
amphibians.

Does
the
SAP
concur
with
these
conclusions?
If
not,
what
lines­
of­
evidence
would
lead
to
an
alternative
conclusion?

7.
Assuming
the
Agency
determined
an
ecological
risk
assessment
with
a
greater
degree
of
certainty
concerning
developmental
effects
of
atrazine
on
amphibians
were
needed,
please
comment
on
EPA's
conclusion
that
additional
information
is
required
to
evaluate
potential
causal
relationships
between
atrazine
exposure
and
gonadal
development.
Please
also
comment
on
the
added
utility,
if
any,
of
additional
information
to
interpret
the
shape
of
dose­
response
curves
for
potential
developmental
endpoints
and
the
extent
to
which
threshold
or
non­
threshold
response
12
relationships
can
be
quantified.

8.
The
Agency
has
developed
a
conceptual
model
from
which
to
develop
a
set
of
study
protocols
for
evaluating
the
potential
effects
of
atrazine
on
gonadal
development
in
amphibians.
The
Agency
has
proposed
a
research
approach
using
focused,
empirical,
laboratory
studies
based
on
initial
investigations
with
X.
laevis
followed
by
selective,
confirmatory
studies
with
frog
species
native
to
North
America.

a)
Please
comment
on
the
proposed
sequence
of
study
objectives.

b)
Please
comment
on
whether
the
Agency's
first
set
of
proposed
studies
has
accounted
for
the
major
sources
of
uncertainty
associated
with
the
potential
effects
of
atrazine
on
anuran
sexual
differentiation.
In
addition
to
time
to
metamorphosis,
gonadal
abnormalities,
and
sex
ratios
in
the
proposed
Phase
I
assays,
please
comment
on
any
other
endpoints
that
should
be
considered
in
this
initial
phase.

c)
Please
also
comment
on
the
range,
spacing
and
number
of
atrazine
concentrations
that
should
be
employed
in
the
proposed
testing
sequence
to
resolve
uncertainties
in
the
shape
and
nature
of
dose­
response
relationships
for
any
observed
developmental
effects.

d)
Please
comment
on
the
Agency's
recommendation
that
X.
laevis
be
used
as
the
primary
biological
model
in
the
proposed
studies
and
whether
or
not
the
mechanisms
involved
in
sexual
differentiation
of
the
ranid
and
pipid
species
are
sufficiently
similar
to
predict
effects
and
associated
dose­
response
curves
for
Rana
and/
or
to
efficiently
design
Rana
studies.

e)
In
this
regard,
are
there
important
differences
between
the
species
to
conclude
that
any
affected
developmental
processes
observed
in
X.
laevis
would
not
occur
in
Rana?

f)
Alternatively,
are
there
developmental
pathways
in
Rana,
but
not
in
X.
laevis,
that
raise
concerns
about
using
X.
laevis
as
the
primary
biological
model
in
any
future
atrazine
studies?

g)
Assuming
X.
laevis
and
Rana
are
sufficiently
concordant
from
a
toxicodynamic
perspective
with
regard
to
potential
developmental
effects
of
atrazine,
what
critical
toxicokinetic
processes
should
be
considered
for
extrapolating
X.
laevis
dose­
response
relationships
to
Rana
and/
or
for
designing
subsequent
studies
with
Rana?

DETAILED
RESPONSE
TO
THE
CHARGE
The
specific
issues
to
be
addressed
by
the
Panel
are
keyed
to
the
Agency's
background
documents
"
White
Paper
on
Potential
Developmental
Effects
of
Atrazine
on
Amphibians",
dated
May
29,
2003,
and
are
presented
as
follows:

1.
In
reviewing
the
available
laboratory
and
field
studies,
the
Agency
used
a
number
of
criteria
to
evaluate
individual
investigations.
Criteria
such
as
experimental
design,
test
13
protocols,
and
quality
assurance
information
were
used
to
ascertain
the
reliability
of
the
generated
data
in
terms
of
its
ability
to
adequately
assess
a
hypothesis
that
atrazine
elicits
developmental
effects
in
amphibians,
and
if
so,
the
nature
and
strength
of
associated
doseresponse
relationships.

a)
Does
the
SAP
have
any
comments
and
recommendations
regarding
the
EPA's
approach
and
criteria
used
to
evaluate
the
studies?

b)
Given
the
evaluation
criteria
employed
by
the
Agency,
please
comment
on
EPA's
overall
characterization
of
the
currently
available
studies.

The
Panel
considered
parts
A
and
B
of
question
1
together.
The
Agency's
criteria
in
evaluating
the
available
studies
included
experimental
design,
protocols
and
quality
assurance,
strength
of
cause/
effect
and/
or
concentration/
response,
mechanistic
plausibility
and
ecological
relevance.
These
criteria
reflect
requirements
for
sound
and
consistent
science
in
ecological
risk
assessments.

The
Panel
concluded
that
the
review
was
thorough,
the
approaches
and
criteria
were
appropriate
and
that
the
conclusions
were
valid,
given
the
data
reviewed.
The
Panel
raised
a
few
minor
concerns
relating
to
the
Agency's
approach.
One
experiment
(
Syngenta
#
2233­
02)
categorized
as
a
field
study
is
actually
a
mesocosm
experiment.
Mesocosms,
while
potentially
powerful,
are
not
capable
of
providing
inferences
regarding
the
fate
of
natural
populations.
With
regard
to
the
analysis
of
laboratory
studies,
the
Panel
expressed
minor
concerns
about
the
reanalysis
of
the
data
but
agreed
that
the
overall
characterization
of
the
studies
was
reasonable.

The
Panel
agreed
that
additional
studies
are
warranted.
Studies
from
several
research
laboratories
provided
evidence
that
atrazine
can
cause
developmental
abnormalities
in
amphibians.
Although
not
considered
in
the
Agency's
White
Paper,
the
findings
are
consistent
with
studies
on
effects
of
atrazine
exposure
to
other
vertebrates,
both
aquatic
(
e.
g.
fish)
[
Moore,
2003]
and
terrestrial
(
some
rodents).
Given
the
conservation
of
many
basic
pathways
for
endocrine
regulation,
these
studies
in
other
species
are
relevant
to
the
issues
addressed
in
the
Agency's
White
Paper.
However,
a
range
of
abnormalities
have
been
reported
in
amphibians
and
these
are
not
consistent
from
study
to
study
(
bearing
in
mind
that
the
literature
includes
studies
in
several
anuran
species).

c)
Please
comment
on
the
availability,
as
of
February
28,
2003,
of
additional,
relevant
studies
in
the
open
literature
that
were
not
addressed
in
the
white
paper.
Since
February
28,
2003,
is
the
Panel
aware
of
any
studies
that
would
be
relevant?

The
Agency
appropriately
identified
the
extant
published
literature
relevant
to
atrazine
effects
on
the
development
of
the
amphibian
gonad.
The
Panel
recommended
adding
mortality
studies
to
the
Agency's
assessment
because
they
offer
a
toxicological
perspective
regarding
the
atrazine
concentrations
used
in
developmental
studies.
The
Panel
also
identified
selected
additional
published
studies
("
open
literature")
that
do
not
need
to
be
included
in
the
Agency's
14
White
Paper,
but
are
relevant
to
ongoing
deliberations
and
may
be
useful
for
the
Agency's
risk
assessment.
These
studies
encompass
several
topics
including:

1)
Effects
of
atrazine
on
survival
and
early
development
of
anurans
Allran
JW
and
Karasov
WH.
2001.
Effects
of
atrazine
on
embryos,
larvae,
and
adults
of
anuran
amphibians.
Environ
Toxicol
Chem.
Apr;
20(
4):
769­
775.

Bevan
CL,
Porter
DM,
Prasad
A,
Howard
MJ,
and
Henderson
LP.
2003.
Environmental
estrogens
alter
early
development
in
Xenopus
laevis.
Environ
Health
Perspect.
Apr;
111(
4):
488­
496.

Morgan
MK,
Scheuerman
PR,
Bishop
CS,
and
Pyles
RA.
1996.
Teratogenic
potential
of
atrazine
and
2,4­
D
using
FETAX.
J
Toxicol
Environ
Health.
Jun
7;
48(
2):
151­
168.

2)
Hormone
effects
on
survival
and
early
development
of
anurans
Antila
E.
1977.
Early
steroid
metabolism
in
Xenopus
laevis,
Rana
temporaria
and
Triturus
vulgaris
embryos.
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Aug
11;
8(
2):
71­
77.

Fort
DJ,
McLaughlin
DW,
Rogers
RL,
and
Buzzard
BO.
2003.
Evaluation
of
the
developmental
toxicities
of
ethanol,
acetaldehyde,
and
thioacetamide
using
FETAX.
Drug
Chem
Toxicol.
Feb;
26(
1):
23­
34.

Nishimura
N,
Fukazawa
Y,
Uchiyama
H,
and
Iguchi
T.
1977.
Effects
of
estrogenic
hormones
on
early
development
of
Xenopus
laevis.
J
Exp
Zool.
Jul
1;
278(
4):
221­
233.

3)
Effects
of
atrazine
on
vulnerability
to
infection
and
immune
function
Christin
MS,
Gendron
AD,
Brousseau
P,
Menard
L,
Marcogliese
DJ,
Cyr
D,
Ruby
S,
and
Fournier
M.
2003.
Effects
of
agricultural
pesticides
on
the
immune
system
of
Rana
pipiens
and
on
its
resistance
to
parasitic
infection.
Environ
Toxicol
Chem.
May;
22(
5):
1127­
1133.

Gendron
AD,
Marcogliese
DJ,
Barbeau
S,
Christin
MS,
Brousseau
P,
Ruby
S,
Cyr
D,
and
Fournier
M.
2003.
Exposure
of
leopard
frogs
to
a
pesticide
mixture
affects
life
history
characteristics
of
the
lungworm
Rhabdias
ranae.
Oecologia.
May;
135(
3):
469­
476.

4)
Normative
and
experimental
data
on
hormones,
sex
determination
and
sexual
differentiation
in
anurans;
hormone
sensitive
biomarkers
Bögi
C,
Schwaiger
J,
Ferling
H,
Mallow
U,
Steineck
C,
Kalbfus
W,
Negele
RD,
Lutz
I,
and
Kloas
W.
2003.
Endocrine
effects
of
environmental
pollution
on
Xenopus
laevis
and
Rana
temporaria.
Environ
Res.
(
in
press).
15
Bögi
C,
Schwaiger
J,
Ferling
H,
Mallow
U,
Steineck
C,
Kalbfus
W,
Negele
RD,
Lutz
I,
and
Kloas
W.
2002.
Endocrine
effects
of
environmental
pollution
on
amphibians.
Proceedings
of
2nd
Status­
Seminar
Endocrine
Disrupters,
2nd
­
4th
April
2001,
Berlin,
Germany,
http://
www.
statusumwelthormone
de,
59­
62.

Bögi
C,
Levy
G,
Lutz
I,
and
Kloas
W.
2002.
Functional
genomics
and
sexual
differentiation
in
amphibians.
Comp
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559­
570.

Catz
D,
Fischer
L,
and
Kelley
D.
1995.
Androgen
regulation
of
a
laryngeal­
specific
myosin
heavy
chain
isoform
whose
expression
is
sexually
differentiated.
Dev
Biol.
171:
448­
457.

Kang
L,
Marin
M,
and
Kelley
D.
1995.
Androgen
biosynthesis
and
secretion
in
developing
Xenopus
laevis.
Gen
Comp
Endocrinol.
100:
293
­
307.

Kelley
DB.
1982.
Female
sex
behaviors
in
the
South
African
clawed
frog,
Xenopus
laevis:
gonadotropin­
releasing,
gonadotropic,
and
steroid
hormones.
Horm
Behav.
Jun;
16(
2):
158­
174.

Kelley
DB
and
Pfaff
DW.
1976.
Hormone
effects
on
male
sex
behavior
in
adult
South
African
clawed
frogs,
Xenopus
laevis.
Horm
Behav.
Jun;
7(
2):
159­
182.

Kloas
W.
2002.
Amphibians
as
model
for
the
study
of
endocrine
disruptors.
Int
Rev
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216,
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57.

Kloas
W,
Levy
G,
Bögi
C,
Opitz
R,
and
Lutz
I.
2002.
Effects
of
environmental
chemicals
on
reproductive
biology
of
amphibians.
Proceedings
of
2nd
Status
Seminar
Endocrine
Disrupters,
2nd
­
4th
April
2001,
Berlin,
Germany,
http://
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status­
umwelthormone.
de,
55­
58.

Kloas
W,
Bögi
C,
Levy
G,
Würtz
S,
and
Lutz
I.
2002.
Sexual
differentiation
in
amphibians.
Proceedings
of
the
21st
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of
the
European
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(
Bonn,
Germany,
26­
30
August,
2002),
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Kloas
W,
Lutz
I,
and
Einspanier
R.
1999.
Amphibians
as
model
to
study
endocrine
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II.
Estrogenic
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of
environmental
chemicals
in
vitro
and
in
vivo.
Drugs
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the
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I,
Opitz
R,
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Kloas
W.
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Amphibians
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model
to
study
endocrine
disruptors:
I.
In
vivo
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on
reproductive
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Proceedings
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Seminar
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­
4th
April
2001,
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Levy
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I,
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R,
Krüger
A,
and
Kloas
W.
2002.
Bisphenol
A
induces
feminization
in
Xenopus
laevis
tadpoles
via
estrogen­
response
systems.
Proceedings
of
the
21st
Conference
of
the
European
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Endocrinologists
(
Bonn,
Germany,
26­
30
August,
2002),
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91­
94.
16
Levy
G,
Lutz
I,
Opitz
R,
Krüger
A,
and
Kloas
W.
2003.
Bisphenol
A
induces
feminization
in
Xenopus
laevis
tadpoles.
Environ
Res.
(
in
press).

Lutz
I,
Würtz
S,
Schulz
A,
Levy
G,
Bögi
C,
Opitz
R,
and
Kloas
W.
2002.
Establishment
of
estrogen
receptor­
mRNA
as
estrogenic
biomarker
in
the
amphibian
Rana
temporaria.
Proceedings
of
the
21st
Conference
of
the
European
Comparative
Endocrinologists
(
Bonn,
Germany,
26­
30
August,
2002),
Monduzzi
Editore,
95­
98.

Lutz
I,
and
Kloas
W.
1999.
Amphibians
as
model
to
study
endocrine
disruptors:
I.
Environmental
pollution
and
estrogen
receptor
binding.
Drugs
and
Hormones
as
Pollutants
of
the
Aquatic
Environment
 
Determination
and
Ecotoxicological
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Sci
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225:
49­
57.

Marin
ML,
Tobias
ML,
and
Kelley
DB.
1990.
Hormone­
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in
the
sexual
differentiation
of
laryngeal
muscle
fiber
number
in
Xenopus
laevis.
Development.
110:
703­
711.

Miyashita
K,
Shimizu
N,
Osanai
S,
and
Miyata
S.
2000.
Sequence
analysis
and
expression
of
the
P450
aromatase
and
estrogen
receptor
genes
in
the
Xenopus
ovary.
J
Steroid
Biochem
Mol
Biol.
75:
101­
107.

Miyata
S,
Koike
S,
and
Kubo
T.
1999.
Hormonal
reversal
and
the
genetic
control
of
sex
differentiation
in
Xenopus.
Zool
Sci.
15:
335­
340.

Mosconi
G,
Carnevali
O,
Franzoni
MF,
Cottone
E,
Kloas
W,
Lutz
I,
Yamamoto
K,
Kikuyama
S,
and
Polzonetti­
Magni
AM.
2002.
Environmental
estrogens
and
reproductive
biology
in
amphibians.
Gen
Comp
Endocrinol.
126:
125­
129.

Opitz
R,
Levy
G,
Bögi
C,
Lutz
I,
and
Kloas
W.
2002.
Endocrine
disruption
in
fishes
and
amphibians.
Transworld
Research
Network,
Recent
Res
Devel
Endocrinol.
3:
127­
170.

Robertson
J
and
Kelley
D.
1996.
Thyroid
hormone
controls
the
onset
of
androgen
sensitivity
in
the
developing
larynx
of
Xenopus
laevis.
Dev
Biol.
176:
108­
123.

Tobias
M,
Tomasson
J,
and
Kelley
DB.
1998.
Attaining
and
maintaining
strong
vocal
synapses
in
female
Xenopus
laevis.
J
Neurobiol.
37:
441­
448.

Tobias
ML,
Marin
ML,
and
Kelley
DB.
1991.
Temporal
constraints
on
androgen
directed
laryngeal
masculinization
in
Xenopus
laevis.
Dev
Biol.
147:
260­
270.

Tobias
ML,
Marin
ML
and
Kelley
DB.
1991.
Development
of
functional
sex
differences
in
the
larynx
of
Xenopus
laevis.
Dev
Biol.
147:
251­
259.

van
Wyk
JH,
Pool
EJ,
and
Leslie
AJ.
2003.
The
effects
of
anti­
androgenic
and
estrogenic
17
disrupting
contaminants
on
breeding
gland
(
nuptial
pad)
morphology,
plasma
testosterone
levels,
and
plasma
vitellogenin
levels
in
male
Xenopus
laevis
(
African
clawed
frog).
Arch
Environ
Contam
Toxicol.
44:
247­
256.

Varriale
B
and
Chieffi
P.
1997.
Oestrogen
control
of
the
sexual
dimorphism
in
the
Harderian
gland
of
Xenopus
laevis.
J
Steroid
Biochem
Mol
Biol.
Aug;
62(
5­
6):
455­
60.

Wetzel
DM
and
Kelley
DB.
1983.
Androgen
and
gonadotropin
effects
on
male
mate
calls
in
South
African
clawed
frogs,
Xenopus
laevis.
Horm
Behav.
17:
388­
404.

5)
Anuran
husbandry
Bögi
C,
Schwaiger
J,
Ferling
H,
Mallow
U,
Steineck
C,
Kalbfus
W,
Negele
RD,
Lutz
I,
and
Kloas
W.
2003.
Endocrine
effects
of
environmental
pollution
on
Xenopus
laevis
and
Rana
temporaria.
Environ
Res.
(
in
press).

Major
N
and
Wassersug
RJ.
1998.
Survey
of
current
techniques
in
the
care
and
maintenance
of
the
African
clawed
frog
(
Xenopus
laevis).
Contemp
Top
Lab
Anim
Sci.
Sep;
37(
5):
57­
60.

Moore
A,
Scott
AP,
Lower
N,
Katsiadaki
I,
and
Greenwood
L.
2003.
The
effects
of
4­
nonylphenol
and
atrazine
on
Atlantic
salmon
(
Salmo
salar
L)
smolts.
Aquaculture
222:
253­
263.

Wright
KM.
2001.
Diets
for
captive
amphibians.
In
K.
M.
Wright
and
R.
B.
Whittaker
(
ed.)
Amphibian
Medicine
and
Captive
Husbandry.
Krieger
Publishing
Company,
Malabar,
FL.
pp.
63­
72.

2.
In
its
evaluation
of
existing
field
studies,
the
Agency
has
concluded
that
these
investigations
are
of
limited
value.
The
reasons
include:
(
1)
the
high
variability
in
environmental
conditions
and
uncertainties
in
the
pre­
existing
status
and
condition
of
fieldcollected
animals,
(
2)
the
spatial
and
temporal
aspects
of
atrazine
exposure
(
i.
e.,
spatial
and
temporal
variability
over
the
course
of
the
studies
and
the
extent
to
which
such
aspects
of
atrazine
exposure
were
empirically
measured
or
otherwise
accounted
for),
and
(
3)
the
possible
co­
occurrence
of
additional
chemical
and/
or
non­
chemical
stressors.

a)
To
the
extent
that
the
field
studies
appear
to
indicate
that
atrazine
may
not
adversely
affect
development,
please
comment
on
EPA's
conclusion
that
the
body
of
data
from
field
studies
does
not
provide
the
means
to
ascertain
whether
the
lack
of
a
relationship
between
atrazine
exposure
and
developmental
effects
is
due
to
the
absence
of
a
causal
relationship
or
limitation
in
study
methodologies.
18
The
Panel
concluded
that
the
absence
of
an
established
causal
relationship
derived
from
laboratory
studies
was
not
critical
in
limiting
the
interpretation
of
the
field
studies.
Ecological
field
studies
are
routinely,
and
successfully,
conducted
in
the
absence
of
such
information.
However,
the
Panel
believed
strongly
that
all
of
the
field
studies
reviewed
had
serious
design
or
methodological
flaws
that
limit
their
usefulness
in
evaluating
hypotheses
related
to
the
effects
of
atrazine
exposure
on
anuran
developmental
responses.
Common,
important
problems
in
the
field
studies
considered
included
inappropriate
site
selection
practices
(
e.
g.,
designation
of
control
sites
with
concentrations
of
atrazine
that
exceeded
some
exposure
sites)
and
failure
to
identify
a
sampling
frame
and
to
choose
sampling
sites
randomly
from
within
it,
as
well
as
insufficient
statistical
power
associated
with
too
few
sampling
sites
to
evaluate
study
hypotheses.
These
problems
render
interpretation
of
results
problematic,
if
not
impossible.

It
also
was
noted
that
the
field
studies
focused
on
measurement
of
endpoints
identified
in
laboratory
studies.
None
of
the
field
studies
measured
responses
for
which
field
studies
are
most
revealing.
Specifically,
whereas
abundance
and
age
structure
were
measured
occasionally,
highly
relevant
endpoints
related
to
reproduction,
recruitment
and
population
viability
were
entirely
absent.
It
should
also
be
noted
that,
aside
from
one
mesocosm
experiment,
all
of
the
field
studies
were
observational.
While
observational
field
studies
are
necessary
and
potentially
yield
strong
inference,
carefully
designed
field
experiments
offer
opportunities
to
manipulate
the
natural
environment,
thereby
controlling
for
some
potentially
confounding
factors
and
allowing
direct
interpretation
of
responses.
Such
an
opportunity
was
unexploited
in
the
pool
of
field
studies
considered
by
the
Panel.

b)
To
the
extent
that
any
field
studies
appear
to
indicate
that
atrazine
may
adversely
affect
development,
please
comment
on
EPA's
conclusion
that
these
field
studies
do
not
provide
sufficient
information
to
resolve
the
potential
role
of
additional
co­
occurring
stressors.

The
Panel
determined
that
the
field
studies
provided
important
information.
Most
notably,
multiple
studies
conducted
by
different
labs
have
uncovered
gonadal
abnormalities
in
wild
populations
of
anurans.
Since
the
Panel
concluded
that
atrazine
exposure
is
a
plausible
hypothesis
explaining
gonadal
abnormalities
and
comparable
abnormalities
have
been
described
in
natural
populations
of
native
amphibians,
further
field
studies
are
warranted
and
can
be
conducted
in
the
absence
of
knowledge
concerning
a
specific
mechanism(
s)
that
cause(
s)
deformities.
The
Panel
concluded
that
the
field
studies
conducted
to
date
do
not,
however,
provide
sufficient
information
to
resolve
the
potential
role
of
additional
co­
occurring
stressors,
for
reasons
cited
in
the
response
to
Question
2(
a).

3.
In
an
evaluation
of
the
existing
laboratory­
based
studies,
the
Agency
concluded
that
there
was
sufficient
information
to
establish
a
hypothesis
that
atrazine
could
cause
adverse
gonadal
developmental
effects.
However,
due
to
different
experimental
designs
and
variability
in
the
nature
and
extent
of
experimental
conditions
(
e.
g.,
level
of
excessive
mortality,
delayed
development
in
untreated
organisms,
lack
of
response
to
positive
controls)
it
was
not
possible
to
adequately
assess
the
hypothesis
that
atrazine
causes
developmental
effects.
It
was
further
concluded
that
the
current
body
of
information
did
19
not
provide
the
means
to
characterize
the
nature
of
any
associated
dose­
response
relationships.

a)
Please
comment
on
EPA's
determination
that
the
laboratory
studies
provide
a
plausible
basis
for
the
means
to
establish
a
hypothesis
concerning
the
potential
for
atrazine
to
cause
developmental
effects.
Also,
please
comment
on
whether
the
overall
body
of
available
data
is
adequate
to
demonstrate
whether
or
not
atrazine
causes
developmental
effects
under
the
conditions
described
in
these
studies.

The
Panel
concurred
with
the
Agency's
determination
that
the
laboratory
studies
on
the
effects
of
atrazine
on
anuran
gonadal
development
are
sufficient
to
hypothesize
that
atrazine
interferes
with
normal
development.
Available
data
on
the
effects
of
atrazine
on
gonadal
development
of
frogs
are
limited;
however,
existing
lines
of
evidence
support
the
hypothesis.
Seven
laboratory
investigations
were
designed
to
evaluate
the
effects
of
atrazine
exposure
on
larval
frog
gonadal
development
and
are
described
in
the
Agency's
White
Paper.
Five
studies
detected
abnormalities
of
gonadal
development,
including
the
development
of
ovotestis
(
Hayes
et
al.,
2003;
Carr
et
al.,
2003)
and
reductions
in
primary
spermatogonia
(
males)
and
oogonia
(
females)
(
Tavera­
Mendoza
et
al.,
2001a,
2001b).
Differences
among
the
types
of
effects
observed
in
these
studies
could
be
due
to
species
differences,
differences
in
exposure,
and
specific
endpoints
selected
for
evaluation.
The
two
studies
that
reported
no
effects
of
atrazine
on
gonadal
development
were
unpublished
reports.
One
study
suffered
from
very
high
mortality
among
organisms
in
all
treatments
(
Hecker
et
al.,
2003a).
Both
studies
(
Hecker
et
al.,
2003a
and
Hecker
et
al.,
2003b),
indicated
problems
relating
to
poor
water
quality.
The
inability
to
detect
gonadal
abnormalities
with
atrazine
exposure
in
these
studies
should
not
detract
from
the
positive
results
noted
in
the
majority
of
the
studies.
Deficiencies
in
all
laboratory
studies
were
noted
as
related
to
experimental
design,
data
analyses,
or
performance
standards.

Significant
data
gaps
exist
in
our
understanding
of
the
effects
of
atrazine
on
anuran
development.
These
gaps
include
a
lack
of
understanding
of
the
mechanism
by
which
atrazine
might
elicit
developmental
toxicity,
the
nature
of
the
concentration­
response
relationship,
definition
of
susceptible
windows
of
exposure,
variable
terminology
used
to
describe
effects,
and
identification
of
a
threshold
concentration.
Panel
members
agreed
that
sufficient
data
were
available
to
establish
the
hypothesis
that
atrazine
interferes
with
normal
gonadal
development
in
anurans
but
were
hesitant
to
accept
the
hypothesis
with
the
limited
available
data.
It
was
agreed
that
more
data
are
necessary
to
properly
test
the
hypothesis.
These
data
should
be
generated
under
standardized
conditions
and
must
be
subject
to
independent
verification.

Clarification
was
requested
of
the
Agency
as
to
whether
agreement
with
the
charge
statement:
" 
data
is
adequate
to
demonstrate
 (
that) 
atrazine
causes
developmental
effects
under
the
conditions
described
in
these
studies"
implies
acceptance
of
the
hypothesis
or
implies
that
sufficient
data
exist
to
warrant
concern.
During
the
Panel
deliberations,
the
Agency
expressed
the
latter
interpretation.
All
Panel
members
agreed
that
sufficient
data
existed
to
warrant
concern.
20
b)
Please
comment
on
EPA's
conclusion
that
given
the
variability
in
the
available
doseresponse
data
across
the
studies
(
e.
g.,
an
approximately
250­
fold
difference
in
reported
thresholds
for
observed
developmental
effects
as
well
as
reports
of
monotonic
and
nonmonotonic
dose­
response
curves),
it
is
not
possible
to
ascertain
the
relationship,
if
any,
of
atrazine
exposure
to
developmental
effects
in
amphibians.

A
major
deficiency
that
exists
among
laboratory
studies
of
the
effects
of
atrazine
on
anuran
gonadal
development
has
been
the
difficulty
in
defining
the
concentration­
response
relationship,
and
accordingly,
a
threshold
concentration.
The
magnitude
of
effects
reported
in
the
peer­
reviewed
scientific
literature
are
summarized
in
Table
1
below.

Table
1.
Effects
of
Atrazine
on
General
Gonadal
Development
or
Germ
Cell
Content
of
Gonads
Percent
Animals
with
Effects
Atrazine
Concentration
(
mcg/
L)

0.01
0.1
1.0
10
20­
25
200
Hayes
et
al.
(
2002a)
0
~
18
~
18
~
18
~
18
~
18
Carr
et
al.
(
2003)
 
 
4.5
7
13
 
Hayes
et
al.
(
2003)
 
65
 
 
20
 
Tavera­
Mendoza
et
al.
(
2001a)
 
 
 
 
~
70
 
Tavera­
Mendoza
et
al.
(
2001b)
 
 
 
 
~
30
 
 
not
tested
From
these
data,
the
Panel
could
draw
no
conclusions
regarding
a
concentration­
response
relationship.
More
data
are
required
to
define
the
concentration­
response
relationship
between
atrazine
and
gonadal
development
of
anuran
larvae.
However,
the
Panel
believes
that
the
data
supports
the
hypothesis
that
the
effect
of
atrazine
on
amphibian
general
gonadal
development
occurs
with
a
threshold
concentration
between
0.01
and
25
ug/
L.

It
was
noted
that,
in
general,
maximum
effects
on
gonadal
development
at
atrazine
exposure
concentrations
(
when
observed)
averaged
~
20%,
irrespective
of
exposure
concentration.
One
Panel
member
noted
that
this
may
reflect
a
plateau
of
the
concentrationresponse
curve
at
this
level
of
effect
and
that
perhaps
effects
of
greater
magnitude
should
not
be
expected
in
future
studies.
Another
Panel
member
disagreed,
suggesting
that
there
might
be
a
possibility
of
monotonic
as
well
as
non­
monotonic
responses.

Precedence
was
discussed
for
chemicals
having
the
ability
to
cause
intersex
conditions
in
only
a
small
percentage
of
the
exposed
population
in
laboratory­
controlled
experiments.
21
Tributyltin
is
a
marine
biocide
known
to
cause
intersex
conditions
in
some
snail
species.
Incidents
of
100%
intersex
females
have
been
noted
in
field
populations
inhabiting
tin­
contaminated
environments
(
Curtis
and
Barse,
1990;
Gooding
et
al.,
1999;
Gooding,
2003;
Morcillo
and
Porte,
1999;
and
Smith,
1981).
During
many
laboratory
experiments
(
Gooding
et
al.,
2003;
Oberdorster
et
al.,
1998;
and
Smith,
1981),
a
maximum
incidence
of
intersex
of
only
~
30%
has
been
observed.
This
discrepancy
has
been
attributed
to
limitations
of
laboratory
experiments.
For
example,
exposure
of
maternal
organisms
during
oocyte
maturation
or
of
embryos
may
be
required
to
maximally
induce
intersex
among
the
resulting
organisms.
In
the
absence
of
such
exposure
during
laboratory
experiments,
perhaps
only
the
most
susceptible
individuals
develop
intersex
conditions.
The
limited
susceptibility
of
anuran
larvae
to
the
developmental
effects
of
atrazine,
coupled
with
variations
in
experimental
design,
could
result
in
low
and
variable
responses
that
may
mask
any
suggestion
of
a
concentration­
response
relationship.

4.
Many
of
the
available
studies
proposed
that
aromatase
induction
results
in
elevated
estrogen
levels
that
lead
to
feminization
(
ovotestes/
intersex/
hermaphroditism)
in
genetically
male
amphibians.

a)
Please
comment
on
EPA's
conclusion
that,
to
date,
aromatase
induction
by
atrazine
has
not
been
demonstrated
in
any
anuran
in
controlled
laboratory
investigations.

The
Panel
agreed
with
the
Agency's
conclusion
that,
to
date,
aromatase
induction
by
atrazine
has
not
been
demonstrated
in
anurans
by
controlled
laboratory
studies.
The
experimental
designs
used
by
several
investigators
in
order
to
demonstrate
effects
on
aromatase
induction
using
long
term
exposures
are
inappropriate
to
demonstrate
any
influence
of
atrazine,
if
there
might
be
one.

The
aromatase
hypothesis,
as
applied
to
the
partial
feminizing
effects
of
atrazine
on
male
Xenopus
laevis,
originated
with
the
well­
established
feminizing
effects
of
estrogen
in
this
species.
Exposure
of
Xenopus
laevis
tadpoles
to
sufficient
estradiol
during
the
critical
period
resulted
in
100%
phenotypic
females.
When
backcrossed
to
genetic
males
(
zz
genotype),
half
of
these
females
produce
only
male
offspring.
The
results
indicated
that
half
of
the
phenotypic
females
were
genotypic
males.

Indirect
support
for
the
aromatase
hypothesis
comes
from
findings
in
a
human
carcinoma
cell
line
(
H295R)
in
which
atrazine
(
and
two
other
triazines)
increased
aromatase
(
CYP19)
enzyme
activity
and
mRNA
levels
(
Sanderson
et
al.,
2000,
2001).
In
addition,
Hayes
et
al.
(
2002a)
reported
that
exposure
of
adult
male
Xenopus
laevis
to
atrazine
decreased
plasma
testosterone,
and
these
investigators
hypothesized
that
this
decrease
was
due
to
the
induction
of
aromatase.
The
Hayes
et
al.
(
2002a)
study
did
not
directly
address
the
hypothesis
that
atrazine
induces
aromatase,
and
the
apparent
decrease
in
plasma
testosterone
might
be
explained
by
other
mechanisms
(
e.
g.,
changes
in
hormone
clearance).
Studies
submitted
to
the
Agency
by
Ecorisk
(
Giesy
et
al.,
2003;
Hecker
et
al.,
2003b)
were
purportedly
designed
to
evaluate
the
hypothesis
that
atrazine
induces
aromatase
in
Xenopus
laevis.
However,
the
Panel
identified
at
least
two
flaws
in
the
design
of
these
studies
that
limit
their
usefulness.
First,
the
investigators
exposed
22
adult
frogs
to
atrazine
for
long
periods
(
26,
43
or
47
days).
In
such
long
term
exposure
experiments,
compensatory
physiological
adjustments
could
have
obscured
any
effects
that
atrazine
may
have
had
on
aromatase
activity/
expression
in
the
short
term.
Second,
the
deleterious
effects
of
atrazine
on
amphibians
are
hypothesized
to
occur
during
gonadal
development
(
during
a
sensitive
premetamorphic
tadpole
stage).
Thus,
the
appropriate
developmental
stages
were
not
tested,
and
developmental
changes
in
sensitivity
to
atrazine
could
confound
interpretation.

Possible
feminizing/
demasculinizing
effects
of
atrazine
could
be
produced
by
modes
of
action
other
than
induction
of
aromatase
(
enhanced
estrogen
synthesis).
For
example,
atrazine
might
exert
an
antiandrogenic
effect.
Feminization
effects
caused
by
estrogen
administration
to
Xenopus
tadpoles
are
dose
dependent
(
Kloas
et
al.,
1999;
and
unpublished
data)
and
can
also
be
obtained
at
least
in
part
by
antiandrogens
such
as
cyproterone
acetate,
p,
p'­
DDE,
and
vinclozolin
(
Kloas,
2002;
Bogi
et
al.,
2002).

Several
potential
modes
of
action
should
be
considered
in
addition
to
the
aromatase
hypothesis.
While
the
available
data
do
not
support
any
one
mode
of
action,
the
following
hypotheses
could
be
proposed:

(
1)
Estrogenic
effects
of
atrazine:
a)
the
induction
of
aromatase
resulting
in
the
elevation
in
plasma
and/
or
intragonadal
estradiol
levels
b)
agonist
action
mediated
by
binding
to
the
estrogen
receptor
(
no
data
available
in
amphibians)

(
2)
Antiandrogenic
effects:
a)
inhibition
of
5 ­
reductase
resulting
in
a
decrease
in
the
dihydrotestrosterone
(
DHT)/
testosterone(
T)
ratio
b)
interference
with
androgen
receptor
mediated
actions
(
no
data
available
in
amphibians)

(
3)
Generalized
effects
on
steroid
metabolism
(
4)
Interference
via
the
hypothalamus­
pituitary­
gonad
axis
(
i.
e.,
Cooper
et
al.,
2000)

Furthermore,
atrazine
could
exert
feminizing
effects
through
multiple
modes
of
action.
While
focus
has
been
placed
on
a
causal
endocrine
disruptive
effect
of
atrazine,
one
cannot
currently
rule
out
the
hypothesis
that
the
feminizing
effects
of
atrazine
are
due
to
a
generalized
(
or
even
specific)
interference
with
genetic
pathways
responsible
for
gonadal
development.
Atrazine
could
act
upstream
of
the
endocrine
system
(
endocrine
changes
have
not
been
demonstrated
in
tadpoles
in
any
study)
and
endocrine
(
epigenetic)
changes
caused
by
atrazine
could
be
a
byproduct
and
not
the
cause.
In
addition,
atrazine
could
affect
the
thyroid
system,
which
is
known
to
influence
gonadal
development
and
steroid
hormone
action
in
developing
amphibians.

b)
The
variability
associated
with
plasma
sex
steroid
concentrations
and
aromatase
activities
is
high.
Is
this
variability
normal?
Please
comment
on
any
readily
apparent
or
available
methodological
improvements
(
e.
g.,
changes
in
sampling
design,
analytical
23
techniques)
that
could
efficiently
address
this
variability
in
future
studies.

Inter­
individual
variability
in
plasma
sex
steroid
concentrations
and
steroid
converting
enzyme
activities
in
lower
vertebrates
such
as
fishes,
amphibians
and
reptiles
is
often
high.
This
variability
may
be
attributed
to
sex
differences,
age,
stage
of
reproductive
development,
dietary
influences,
and
social
dominance,
among
others.
However,
the
Panel
was
unable
to
determine
whether
the
variability
reported
in
the
studies
submitted
to
the
EPA
by
ECORISK
was
due
to
biological
variation
or
technical
limitations.
The
most
common
technique
used
to
analyze
steroid
hormones
in
animal
tissues
and
plasma
is
radioimmunoassay
(
RIA).
Although
less
common,
enzyme­
linked
immunosorbant
assay
(
ELISA)
is
also
used
to
measure
plasma
steroids,
and
this
technique
was
used
in
the
ECORISK
studies.
It
is
noteworthy
that
the
estradiol
measurements
in
male
Xenopus
laevis
obtained
by
the
ECORISK
group
by
ELISA
are
one
to
two
orders
of
magnitude
higher
than
those
reported
in
the
open
literature
in
which
RIA
was
used.
Also,
in
the
ECORISK
studies,
there
is
no
mention
of
attempts
to
validate
the
ELISAs
for
use
with
Xenopus
laevis
plasma
(
also,
appropriate
validations
were
not
conducted
in
the
original
reports
that
described
the
ELISA
techniques
which
are
cited
in
the
ECORISK
reports
(
Hecker
et
al.,
2003b).
Recovery
analyses
(
using
radioactive
hormone)
following
organic
extraction
were
not
conducted
in
these
studies,
but
are
essential
to
control
for
variation
in
extraction
efficiency
among
samples.
This
alone
could
account
for
significant
variation
in
the
reported
values.
No
other
standard
assay
validations
were
conducted
(
described
below)
nor
were
intra­
or
interassay
coefficients
of
variation
reported.
These
omissions
make
it
difficult
to
evaluate
the
validity
of
the
findings
reported
by
ECORISK.

In
the
studies
published
by
Hayes
(
2002a),
and
as
indicated
during
his
presentation
to
the
FIFRA
SAP,
Hayes
had
been
unable
to
measure
plasma
estradiol
in
Xenopus
using
RIA.
However,
members
of
the
Panel
noted
that
other
researchers
have
successfully
measured
estradiol
in
male
Xenopus
laevis
(
Tobias
et
al.,
1998;
Bögi
et
al.,
2003).
In
addition,
in
the
Hayes
et
al.
(
2002a)
study,
measures
of
plasma
testosterone
are
based
on
a
sample
size
of
only
four
per
treatment.
The
Hayes
et
al.
(
2002a)
study
is
the
only
direct
evidence
for
an
endocrine
disruptive
effect
of
atrazine
in
an
amphibian,
and
as
such
should
be
replicated
and
extended
from
adults
to
the
appropriate
developmental
stages.

It
is
essential
that
the
assays
used
for
plasma
sex
steroid
measurements
be
validated
for
use
in
the
amphibian
species
under
study.
Validations
should
include,
but
are
not
limited
to:
1)
parallelism
between
the
standard
curve
and
dilutions
of
plasma/
serum;
2)
recovery
of
labeled
(
i.
e.,
radioactive)
hormone
added
to
the
plasma/
serum
sample
before
extraction;
3)
verification
that
residual
plasma
sex
hormone
binding
globulins
and
other
plasma
proteins
do
not
interfere
in
the
assay;
4)
verification
of
the
specificity
of
the
antiserum;
and
5)
corroboration
of
results
by
an
independent
method
(
e.
g.,
comparison
of
ELISA
and
RIA).
As
mentioned
above,
an
important
quality
control
is
to
conduct
recovery
estimates
(
using
a
radiolabeled
tracer)
for
each
extracted
sample
to
account
for
differences
in
extraction
efficiencies.

Regarding
the
sampling
technique
for
wild­
caught
Xenopus
laevis,
the
ECORISK
group
trapped
frogs,
transported
them
to
the
laboratory,
and
then
maintained
them
for
48
hours
in
the
24
laboratory,
purportedly
to
allow
them
to
recover
from
capture
stress.
No
attempt
was
made
to
evaluate
whether
animals
had
indeed
recovered
from
capture
stress
(
e.
g.
by
measuring
plasma
corticosterone)
or
if
the
animals
continued
to
suffer
from
confinement
stress.
Stress
can
have
profound
effects
on
plasma
sex
steroid
concentrations,
and
individuals
respond
differently
to
stress.
Thus,
a
major
component
of
the
variability
in
measurements
could
be
due
to
the
method
of
sampling.
Future
studies
of
adults
(
if,
in
fact,
this
life
history
stage
is
studied)
should
strive
to
collect
plasma
from
animals
immediately
after
capture
(
in
field
experiments).
Also,
the
time
of
day
when
plasma
is
collected
in
both
field
and
laboratory
studies
should
be
considered
in
the
design
of
experiments
and
reported.
There
are
distinct
circadian
rhythms
in
plasma
hormones
in
frogs
and
thus
the
choice
of
time
of
day
or
night
to
sample
(
it
should
be
noted
that
Xenopus
laevis
are
nocturnal)
could
impact
the
results.

In
relation
to
biological
variability,
every
effort
should
be
made
to
record
and
correlate
physiological
parameters
that
might
account
for
variability
in
plasma
sex
steroid
concentrations
independent
of
exposure
to
atrazine
or
other
toxicants.
These
parameters
should
include,
but
are
not
limited
to,
body
weight
(
lean
vs.
fat
wet
and
dry
weights),
stage
of
reproductive
development
(
e.
g.,
calculation
of
gonadosomatic
index,
plasma
vitellogenin
concentration
in
females),
and
stress
(
e.
g.,
plasma
corticosterone).

Future
mechanistic
studies
should
focus
on
short
term
exposures
in
tadpoles/
juveniles/
adults
(
e.
g.
6
hr,
12
hr,
1
d,
3
d,
7
d,
and
14
d).
Measures
should
include
estradiol,
the
androgens
T
and
DHT
and
activities
of
aromatase
as
well
as
5 
­
reductase.
In
testing
the
aromatase
hypothesis,
biochemical
assays
could
be
included
that
test
for
direct
interference
of
atrazine
with
aromatase
(
or
5 ­
reductase)
enzyme
activity.
Possible
effects
of
atrazine
on
aromatase
gene
expression
should
be
tested
using
reverse
transcriptase­
polymerase
chain
reaction
(
RT­
PCR)
techniques
such
as
those
described
by
Miyata
et
al.
(
1999)
and
Miyashita
et
al.
(
2000).
Particular
emphasis
should
be
placed
on
using
these
techniques
to
analyze
effects
of
atrazine
on
aromatase
gene
expression
in
larval
amphibians.
A
complementary
approach
for
testing
for
estrogenic
actions
would
be
to
determine
effects
of
atrazine
exposure
on
estrogenic
biomarkers
now
available
for
Xenopus
such
as
plasma
vitellogenin
(
ELISA­
kit,
BIOSENSE,
Norway
or
TOWA­
KAGAKU,
Japan)
or
vitellogenin
mRNA
(
RT­
PCR,
Kloas
et
al.,
1999).

c)
Please
comment
on
whether
there
are
additional
data,
other
than
those
summarized
in
the
white
paper,
that
suggest
late
exposure
of
amphibians
(
i.
e.,
juveniles
or
adults)
to
estrogens
or
estrogenic
chemicals
can
induce
ovotestes
formation.

The
Panel
is
unaware
of
reports
that
address
the
possibility
of
ovotestis
formation
in
Xenopus
at
late
exposures.
For
other
amphibians,
it
is
known
in
bufonids
(
Ponse,
1945)
and
some
ranid
species
(
Browder,
1975)
that
there
may
arise
sex
reversal
at
later
stages.

d)
Please
comment
on
whether
there
are
additional
data,
other
than
those
summarized
in
the
white
paper,
that
suggest
alternative
mechanisms
that
could
explain
the
apparent
feminization
of
genetically­
male
amphibians.
25
As
mentioned
in
response
to
question
4a,
a
mode
of
action
other
than
aromatase
induction
might
be
responsible
for
feminizing/
demasculinizing
effects
on
gonadal
development
of
genetically
male
amphibians.
One
experimental
approach
to
address
specifically
such
effects
could
be
to
produce
genetic
male
(
ZZ)
female
phenotypes
and
use
their
progeny
(
all
ZZ
males)
specifically
for
such
experiments.
In
addition,
slight
effects
on
the
thyroid
system
cannot
be
excluded
by
the
experiments
presented
up
to
now.

5.
With
regard
to
specific
endpoints,
the
Agency
does
not
currently
have
sufficient
information
to
quantitatively
relate
gonadal/
laryngeal
effects
to
reproductive
outcomes.
A
major
underlying
uncertainty
is
the
ecological
relevance
of
ovotestes
occurrence
to
the
maintenance
of
anuran
populations.

a)
Can
the
Panel
provide
sources
of
data
on
background
rates
of
ovotestes
occurrence
in
amphibian
species
and
any
associated
considerations
for
interpreting
this
information
in
the
context
of
the
reviewed
studies?

The
Panel
began
its
response
to
this
question
by
defining
ovotestes
as
follows:
the
occurrence
of
frank
testicular
tissue
and
ovarian
segments
within
a
recognizable
gonad.
Witschi
(
1956)
reported
1
case
of
ovotestes.
There
is
at
least
one
study
in
which
the
prevalence
of
ovotestes
in
a
control
population
of
laboratory
frogs
has
been
described
(
Bögi
et
al.,
2003;
Levy
et
al.,
2003).
To
the
Panel's
knowledge,
the
background
rates
of
ovotestes
in
wild
amphibian
populations
have
not
been
reported.
The
Panel
believed
the
frequency
of
occurrence
of
ovotestes
in
normal
healthy
populations
of
amphibians
is
probably
very
low
and
likely
varies
among
species.
This
is
based
on
the
relatively
rare
occurrence
of
ovotestes
in
Panel
members'
laboratory
amphibians.
Without
objective
analysis
and
surveys
on
the
background
rates
of
ovotestes
in
wild
populations,
it
is
not
possible
to
assess
the
impact,
if
any,
that
the
presence
of
ovotestes
in
male
frogs
may
have
on
anuran
populations.

b)
Can
the
Panel
characterize
any
evidence
that
suggests
that
the
presence
of
ovotestes
in
male
anurans
results
in
reproductive
impairment
via
reductions
in
fertility?

To
the
Panel's
knowledge,
there
are
no
published
reports
that
indicate
that
ovotestes
in
male
anurans
either
do
or
do
not
result
in
reproductive
impairment.
In
amphibians
and
other
species,
decreases
in
sperm
production
are
correlated
with
a
decrease
in
testicular
size.
The
consequences
of
gonadal
abnormalities
for
reproductive
impairment
of
individuals
or
for
wild
populations
of
anurans
are
entirely
unknown.
Three
broad
hypotheses
exist:

1.
Gonadal
abnormalities
among
genetic
males
may
have
no
effect
on
local
population
dynamics.
This
could
occur
if
abnormal
males
do
not
breed
or
participate
in
breeding
congresses.
The
breeding
system
of
many
anurans
leads
to
a
small
fraction
of
males
achieving
matings
with
a
disproportionate
fraction
of
females.

2.
Gonadal
abnormalities
among
genetic
males
may
lead
to
declines
in
wild
amphibian
26
populations.
This
could
occur
if
abnormal
males
participate
in
breeding
congregations
and
achieve
matings
with
females
that
lead
to
incomplete
or
entire
failure
of
fertilization.

3.
Gonadal
abnormalities
in
genetic
male
frogs
may
lead
to
increases
in
wild
amphibian
populations.
This
could
occur
if
abnormal
genetic
males
act
as
females
in
the
breeding
dynamics
of
natural
populations.

Thus,
it
will
be
impossible
to
evaluate
the
ecological
relevancy
of
reported
gonadal
abnormalities
without
measuring
endpoints
related
to
reproductive
behavior.
The
Panel
therefore
recommended
that
feminized
phenotypic
males
be
included
in
"
grow­
out"
studies
for
the
purpose
of
using
them
in
breeding
experiments
to
test
the
hypothesis
that
ovotestes
impair
reproductive
function.

c)
The
reduction
of
laryngeal
muscle
area
suggests
diminished
testosterone
in
males.
If
this
is
found
to
be
a
valid
observation
and
if
estrogen
concentrations
do
increase
as
testosterone
concentrations
decrease,
what
other
endpoints
(
e.
g.,
secondary
sexual
characteristics
and
reproductive
behavior)
would
likely
be
affected?

The
Panel
concluded
that
the
following
endpoints
could
be
used.
All
are
endocrine
biomarkers.
The
first
six
can
be
evaluated
using
noninvasive
methods
and
would
not
require
sacrificing
the
animal:

(
1)
Snout
to
vent
length,
body
weight
(
feminized
males
should
be
bigger)
(
2)
Nuptial
pads
(
3)
Enlargement
of
the
ventral
folds
of
the
cloacae
(
4)
Strength
and
pattern
of
the
male
calling
signal
(
5)
Clasping
(
6)
Seminal
fluid
analysis
(
sperm
count,
motility
evaluation,
morphology)
(
7)
Time
course
examining
synthesis
of
vitellogenin
by
the
liver
in
response
to
estrogen
challenge
(
8)
Oviduct
development
(
9)
Proteins
expressed
in
Harderian
glands
around
the
eye
(
three
proteins
expressed
by
females,
1
in
males)
(
10)
Number
and
size
of
muscle
fibers
in
larynx,
myosin
expression
in
larynx
muscle
Continuous
studies
(
e.
g.,
studies
that
follow
atrazine­
exposed
animals
through
sexual
maturity)
would
be
of
great
value
to
field
studies.
The
animal's
gonadal
development
could
be
assessed
using
minimally
invasive
techniques
such
as
ultrasonography,
endoscopy,
or
magnetic
resonance
imaging.

6.
While
some
of
the
available
data
indicate
there
may
be
an
association
between
atrazine
exposure
and
developmental
effects
in
amphibians,
the
Agency's
evaluation
of
the
existing
body
of
laboratory
and
field
studies
has
determined
that
there
is
not
sufficient
scientific
evidence
to
indicate
that
atrazine
consistently
produces
effects
across
the
range
of
amphibian
species
examined.
However,
the
current
body
of
knowledge
has
deficiencies
and
27
uncertainties
that
limit
its
usefulness
in
assessing
potential
developmental
atrazine
effects
and
the
extent
of
any
associated
cause­
effect
and
dose­
response
relationships.
Consequently,
the
Agency
has
determined
that
there
are
not
sufficient
data
to
reject
the
hypothesis
that
atrazine
can
cause
adverse
developmental
effects
in
amphibians.
Does
the
SAP
concur
with
these
conclusions?
If
not,
what
lines­
of­
evidence
would
lead
to
an
alternative
conclusion?

The
EPA
expressed
several
conclusions
in
this
question.
The
Panel
examined
each
conclusion
separately.

The
first
conclusion
put
forth
was:
"
there
is
not
sufficient
scientific
evidence
to
indicate
that
atrazine
consistently
produces
effects
across
the
range
of
amphibian
species
examined."
The
Panel
agreed
with
this
conclusion.
The
Panel
previously
noted
in
response
to
question
#
3
that
there
was
sufficient
information
to
establish
a
hypothesis
that
atrazine
could
cause
adverse
gonadal
developmental
effects
in
amphibians.
Studies
were
available
that
reported
results
from
eight
species
of
frog,
including,
Xenopus
laevis
(
African
clawed
frog),
Rana
pipiens
(
northern
leopard
frog),
and
Rana
clamitans
(
green
frogs)
in
laboratory
or
controlled
exposure
studies
(
mesocosm
studies).
Field
observational
studies
reported
on
effects
in
X.
laevis,
R.
pipiens,
Acris
crepitans
(
cricket
frogs),
Bufo
maninus
(
cane
toad)
and
Bufo
terrestris
(
southern
toad).
The
response
of
the
species,
both
in
terms
of
the
endpoints
considered
and
the
magnitude
of
response,
was
inconsistent
across
the
species
studied
and
among
studies
which
used
the
same
species.
Comparison
among
the
studies
was
difficult
because
of
the
problems
identified
with
respect
to
the
design
and
conduct
of
both
the
laboratory
and
field
studies,
which
confound
their
interpretation.

The
second
conclusion
put
forth
was:
"
the
current
body
of
knowledge
has
deficiencies
and
uncertainties
that
limits
its
usefulness
in
assessing
potential
developmental
atrazine
effects ".
The
Panel
agreed
with
the
conclusions
in
the
Agency's
White
Paper
that
there
were
deficiencies
and
uncertainties
with
respect
to
the
methods,
conduct,
and
results
of
the
studies
submitted,
as
were
identified
in
responses
to
questions
2
and
3.
Among
the
major
factors
identified
were
difficulties
with
the
husbandry
in
laboratory
studies,
presence
of
atrazine
in
control
exposures
and
reference
sites
and
a
lack
of
consideration
of
and/
or
information
on
the
presence
or
potential
impact
of
other
stressors
in
observational
field
studies.
Given
these
deficiencies
and
limitations,
the
Panel
concluded
that
the
current
data
would
not
be
suitable
for
ecological
risk
assessment.
Further,
it
was
recognized
by
the
Panel
that
in
order
to
conduct
a
scientifically
sound
ecological
risk
assessment,
the
Agency
needs
to
have
results
from
studies
where
other
factors
can
be
ruled
out
as
a
cause
in
either
the
presence
or
the
absence
of
effects.

The
third
conclusion
put
forth
was
that
the
uncertainties
and
deficiencies
limited
"
the
extent
of
(
the
identification
of)
any
associated
cause­
effect
and
concentration­
response
relationships."
The
Panel
concluded
that
although
they
agreed
that
a
causal
relationship
can
be
hypothesized
between
atrazine
and
effects
on
gonadal
development,
the
uncertainties
and
deficiencies
in
existing
studies
precluded
acceptance
of
the
hypothesis
(
see
response
to
question
3).
Further,
the
exact
nature
of
the
response
in
gonadal
development
in
amphibians
(
shape
of
the
concentration/
response
function,
presence
of
a
threshold)
cannot
be
characterized
at
this
point
for
28
the
species
tested.
The
Panel
also
addressed
this
issue
in
its
response
to
questions
3(
b)
and
7.
Finally,
the
Panel
noted
that
knowledge
of
the
concentration/
response
function
is
a
necessary
element
to
conduct
an
ecological
risk
assessment.

The
final
conclusion
put
forth
was:
"
the
Agency
has
determined
that
there
are
not
sufficient
data
to
reject
the
hypothesis
that
atrazine
can
cause
adverse
developmental
effects
in
amphibians."
The
Panel
agreed
that
the
available
data
suggest
that
atrazine
can
affect
gonadal
development
in
amphibians.
However,
the
available
data
do
not
allow
a
proper
characterization
of
the
nature
and
magnitude
of
the
response
at
either
the
organism
or
population
level,
nor
do
they
offer
sufficient
support
for
the
identification
of
a
plausible
mechanism.

The
Panel
further
agreed
that
information
gained
from
the
available
studies
contributes
to
our
knowledge
and
will
be
useful
in
the
design
and
conduct
of
future
studies.
It
was
further
concluded
by
the
Panel
that
the
adverse
effects,
in
this
case
the
presence
of
abnormalities
in
gonadal
development
described,
need
to
be
connected
to
the
assessment
endpoints
of
reproductive
competence
(
i.
e.,
fertilization
success
and
subsequent
potential
effects
at
the
population
level).

7.
Assuming
the
Agency
determined
an
ecological
risk
assessment
with
a
greater
degree
of
certainty
concerning
developmental
effects
of
atrazine
on
amphibians
were
needed,
please
comment
on
EPA's
conclusion
that
additional
information
is
required
to
evaluate
potential
causal
relationships
between
atrazine
exposure
and
gonadal
development.
Please
also
comment
on
the
added
utility,
if
any,
of
additional
information
to
interpret
the
shape
of
dose­
response
curves
for
potential
developmental
endpoints
and
the
extent
to
which
threshold
or
non­
threshold
response
relationships
can
be
quantified.

The
Panel
agreed
with
the
conclusion
that
additional
information
is
required
to
evaluate
potential
causal
relationships
between
atrazine
exposure
and
gonadal
development.
Several
points
were
made
in
regard
to
this
conclusion.
There
is
a
need
to
confirm
the
causal
relationship
that
is
suggested
by
the
existing
data,
and
some
similarity
of
data,
or
patterns
or
trends,
from
different
labs
needs
to
be
presented
to
show
repeatability
of
the
effects.
One
of
the
tenets
of
the
scientific
method
is
the
repeatability
of
experiments.
Further,
as
previously
noted,
it
is
necessary
to
characterize
the
nature
of
the
dose­
response
(
or
more
correctly,
concentration­
response)
function.
Finally,
there
is
a
need
to
identify
a
plausible
mechanism,
supported
by
data.
The
characterization
of
a
mechanism
can,
in
part,
aid
in
the
extrapolation
of
results
from
surrogate
test
species
to
species
of
concern
in
the
environment.

With
respect
to
concentration­
response
curves,
the
Panel
emphasized
that
these
are
extremely
important
to
the
question
of
any
detrimental
effects
of
a
toxicant
to
an
organism
and
necessary
for
risk
assessment.
Regardless
of
whether
behavior
demonstrates
either
a
monotonic
or
atypical
concentration­
response
relationships
for
a
given
endpoint,
it
should
be
possible
to
ascertain
the
shapes
of
the
curves,
given
enough
concentrations,
replications,
and
controlled
conditions.
Repeatability
in
other
laboratories
should
be
feasible,
if
the
same
species,
stage,
water,
concentrations
and
timing
are
utilized.
29
It
was
further
put
forth
that
studies
on
the
quantitative
structure­
activity
relationships
(
QSAR)
can
often
provide
information
about
a
mechanism
of
action
or
provide
a
rationale
for
the
data
that
are
generated
from
comparative
testing.
Experiments
using
a
series
of
closely
related
compounds
(
cyanazine,
propazine,
simazine,
terbuthylazine,
etc.)
could
elucidate
patterns
that
would
help
explain
the
interaction
between
the
molecules
and
the
putative
receptor,
addressing
the
causal
relationship.
This
approach
seems
to
be
lacking
so
far.
It
could
be
valuable
in
the
in
vivo
tests
for
gonadal
development
as
well
as
for
induction
of
aromatase
or
expression
of
mRNA
for
aromatase.

There
are
few
studies
among
those
reviewed
in
the
Agency's
White
Paper
that
acknowledge
the
existence
of
metabolites
that
may
be
biologically
significant.
Three
chlorinated
metabolites
are
potentially
bioactive
in
the
same
mode
as
atrazine
­­
deethyl
atrazine,
deisopropyl
atrazine,
and
didealkyl
atrazine,
which
is
also
called
diamino
chlorotriazine.
There
also
is
one
major
dechlorinated
metabolite
(
hydroxy
atrazine)
that
should
be
evaluated
as
well,
based
on
its
presence
as
a
primary
degradate
of
atrazine.
These
have
been
addressed
in
the
context
of
water
quality
and
mammalian
toxicology,
but
scarcely
in
the
amphibian
development
studies.
They
should
be
evaluated
because
they
are
commonly
occurring
transformation
products,
and
they
also
could
be
interesting
if
included
in
the
QSAR
studies
suggested
above.

8.
The
Agency
has
developed
a
conceptual
model
from
which
to
develop
a
set
of
study
protocols
for
evaluating
the
potential
effects
of
atrazine
on
gonadal
development
in
amphibians.
The
Agency
has
proposed
a
research
approach
using
focused,
empirical,
laboratory
studies
based
on
initial
investigations
with
X.
laevis
followed
by
selective,
confirmatory
studies
with
frog
species
native
to
North
America.

a)
Please
comment
on
the
proposed
sequence
of
study
objectives.

The
Panel
was
in
agreement
that
a
logical
progression
of
studies
as
proposed
by
the
Agency
would
elucidate
effects
and
document
mechanisms
associated
with
any
gonadal
abnormalities
due
to
atrazine
exposure.
The
Panel
had
a
number
of
suggestions
as
to
how
this
model
could
be
improved
and
which
of
the
studies
provided
the
most
immediate
and
useful
tests
of
the
atrazine
hypothesis.
There
was
consensus
that
laboratory
experiments
(
Phase
1
of
the
proposed
approach)
should
proceed
immediately.
These
studies
would
confirm
whether
gonadal
deformities
occur
with
exposure
to
atrazine
and
bracket
concentrations
at
which
effects
are
observed.
One
Panel
member
suggested
that
Phase
1
testing
include
characterization
of
a
concentration­
response
relationship
using
some
cellular/
molecular
marker
of
atrazine's
effect.
Such
an
endpoint
would
likely
be
sensitive
and
detectable
in
a
timely
fashion.

Panel
members
concurred
that
the
studies
identified
under
Phase
5
should
be
initiated
as
early
as
possible
within
the
framework
of
the
study
plan.
These
studies
would
require
grow­
out
of
individuals
with
gonadal
deformities
to
appropriate
life
stages
to
examine
the
effects
of
the
reported
gonadal
deformities
on
fecundity
and
fertility.
The
Panel
concluded
that
these
studies
30
were
essential
since
true
ecological
effects
are
dependent
on
the
hypothesis
that
reproduction
of
populations
is
impaired
by
individual
reproductive
impairment.

The
Panel
believed
that,
subject
to
study
design
constraints
(
i.
e.,
both
logistical
and
the
necessity
for
a
sound
statistical
design),
the
ideal
study
would
address
the
effect
of
atrazine
on
gonadal
development/
morphology
and
have
sufficient
individuals
to
allow
for
the
continuation
of
the
study
for
assessment
of
the
effects
on
fertility/
reproduction.
If
such
a
study
were
not
feasible,
grow­
out
studies
under
Phase
5
should
begin
as
soon
as
possible
as
noted
above.

There
was
consensus
among
the
Panel
that
the
Agency
should
proceed
with
confirmatory
studies
with
a
North
American
Rana
species
due
to
potential
differences
in
response
with
those
species
compared
to
Xenopus.

Some
Panel
members
believed
that
the
importance
of
field
studies
should
be
more
prominent
in
the
Agency's
approach.
Field
studies
often
alter,
sometimes
drastically,
conclusions
derived
from
laboratory
studies.
The
same
study
organisms
under
multiple,
interacting
stressors
in
a
field
situation
often
exhibit
a
dramatically
elevated
sensitivity
to
a
particular
compound
in
comparison
to
exposure
to
it
in
isolation
in
a
lab
situation.
These
Panel
members
contended
that
field
studies
are
vital
for
addressing
population­
level
effects
of
atrazine
and
generating
insights
about
ecologically
relevant
endpoints
and
therefore
they
should
not
be
consigned
to
a
second
priority
to
Phase
1
lab
studies.

Experimental,
field­
based
studies
could
be
quite
incisive.
More
specifically,
a
highly
revealing
experimental
system
in
the
field
would
be
a
small,
temporary
wetland
with
established,
seasonal
breeding
populations
of
amphibians.
Multiple
wetlands
can
be
surrounded
with
drift
fences
such
that
all
individuals
entering
(
pre­
breeding
adults)
and
leaving
(
post­
breeding
adults
and
metamorphs)
are
captured,
marked,
measured,
and
released.
Many
such
wetlands
could
be
included
in
a
given
study
with
subsets
left
as
controls
and
others
experimentally
treated
with
various
levels
of
atrazine.
A
wealth
of
demographic
information
could
be
gleaned
from
such
an
experimental
study
(
studies
by
KA
Berven
[
1990]
provide
a
useful
example
of
this
approach
and
the
information
that
can
be
gained
from
it).
Moreover,
high
densities
of
such
wetlands
with
little
or
no
previous
exposure
to
atrazine
are
readily
available,
thereby
limiting
many
of
the
complications
associated
with
contaminated
controls
that
have
arisen
in
many
of
the
studies
conducted
on
the
topic
to
date
in
agricultural
areas.
Additional
benefits
would
accrue
if
laboratory
studies
were
able
to
identify
a
suite
of
external
morphological
characteristics
that
were
associated
with
feminization/
demasculinization
and
were
indices
of
internal
gonadal
abnormalities
noted
during
laboratory
studies.
Use
of
such
indices
would
potentially
obviate
the
need
to
sacrifice
animals
in
the
field
for
time­
consuming
histopathological
analysis
and
thereby
increase
efficacy
of
field
surveys.

b)
Please
comment
on
whether
the
Agency's
first
set
of
proposed
studies
has
accounted
for
the
major
sources
of
uncertainty
associated
with
the
potential
effects
of
atrazine
on
anuran
sexual
differentiation.
In
addition
to
time
to
metamorphosis,
gonadal
abnormalities,
and
sex
ratios
in
the
proposed
Phase
I
assays,
please
comment
on
any
other
endpoints
that
31
should
be
considered
in
this
initial
phase.

There
was
Panel
consensus
that
the
Agency's
first
set
of
proposed
studies
have
accounted
for
the
major
sources
of
uncertainty
associated
with
the
potential
effects
of
atrazine
on
anuran
sexual
differentiation.

The
Panel
had
a
number
of
suggestions
on
potential
endpoints
that
could
be
measured
in
Phase
1.
Many
of
these
endpoints
have
been
presented
in
response
to
question
5c.
The
Panel
was
in
consensus
that
a
clear
set
of
definitions
concerning
the
terminology
for
classifying
gonadal
deformities
should
be
developed
by
the
Agency.
This
is
essential
for
quantifying
results
of
past
and
future
studies.

Regarding
the
major
sources
of
uncertainty
associated
with
the
potential
effects
of
atrazine
on
anuran
sexual
differentiation,
the
Panel
agreed
with
the
Agency
that
the
lack
of
standardization
of
husbandry
protocols
for
laboratory
Xenopus
laevis
and
Rana
pipiens
likely
played
a
significant
role.
The
Panel
is
aware
that
the
Agency
has
expertise
in
these
areas.
The
Panel
concurred
that
ASTM
guidelines
for
water
quality
should
be
followed.
For
example,
pH,
conductivity,
ammonia
(
total,
ionized
and
unionized
forms),
nitrate,
nitrite,
dissolved
oxygen,
chlorine
or
chloramine
levels,
copper
and
iron
levels
should
be
standardized
among
experiments.
Alterations
in
any
of
these
parameters
may
alter
experimental
results,
particularly
those
involving
growth
rates
and
development.
With
FETAX,
Holtfreter's
solutions
with
adequate
calcium
are
adequate
for
raising
tadpoles.

Animals
should
be
loaded
in
flow­
through
tanks
at
a
density
according
to
ASTM
guidelines.
Flow
through
tanks
are
preferable,
but
data
collected
from
static
renewal
tanks
would
be
acceptable
provided
animals
are
loaded
according
to
ASTM
guidelines
and
water
quality
is
assessed
on
a
daily
basis
(
and
maintained
within
the
ASTM
guidelines).

Xenopus
laevis
are
carnivores.
Therefore,
diet
should
contain
at
least
14%
protein.
Diets
formulated
for
herbivores
or
for
omnivorous
fish
or
turtles
are
not
suitable.
Diets
formulated
especially
for
Xenopus
are
commercially
available
for
both
tadpoles
and
adults.
The
quantity
of
feed
(
g/
animal)
should
be
based
on
the
manufacturer's
recommendation
and
adjusted
as
the
animal
grows.

The
following
reference
is
recommended
as
a
guide
for
housing
and
husbandry
of
anurans:
Amphibian
Medicine
and
Captive
Husbandry.
(
Eds.):
KM
Wright
and
BR
Whitaker,
Krieger
Publishing
Co.,
Malabar,
FL.

One
Panel
member
felt
that
a
larger
number
of
male/
female
pairs
should
be
used
to
develop
tadpole
treatment
groups.
The
current
use
of
three
pairs
is
minimal
and
may
contribute
to
variation
in
results
among
experiments
reported
to
date.

One
Panel
member
believed
that
a
stock
colony
of
Xenopus
animals
should
be
developed
that
researchers
could
draw
upon.
This
would
minimize
potential
variation
among
populations
32
studied
in
different
laboratories.
This
stock
colony
should
include
phenotypic
females
with
a
ZZ
genotype
so
that
sex
ratios
can
be
accurately
determined.

c)
Please
also
comment
on
the
range,
spacing
and
number
of
atrazine
concentrations
that
should
be
employed
in
the
proposed
testing
sequence
to
resolve
uncertainties
in
the
shape
and
nature
of
dose­
response
relationships
for
any
observed
developmental
effects.

The
Panel
recognized
that
it
must
answer
this
question
in
the
context
of
realistic
constraints
on
the
cost
and
effort
that
can
be
devoted
to
any
single
study
to
determine
if
aqueous
concentration
of
atrazine
bears
a
relationship
to
gonadal
irregularities
in
amphibians.
The
scope
of
any
new
experimental
study
will
be
determined
by
the
number
of
concentrations
and
controls
tested,
the
number
of
intra­
laboratory
replications
(
e.
g.
tanks)
for
each
concentration
level,
and
the
number
of
test
animals
per
experimental
replication.
Range
and
spacing
of
the
experimental
concentration
levels
are
related
to
the
number
of
feasible
experimental
points
and
may
also
be
governed
by
the
hypothesized
functional
form
of
any
underlying
relationship
of
concentration
levels
to
response.

The
Panel
considered
the
components
of
the
experimental
design
for
a
new
study
in
the
following
order:
1)
selection
of
controls;
2)
range
of
observations
for
experimental
concentrations;
3)
number
of
independent
replicates
per
treatment;
4)
number
of
test
animals
per
experimental
replicate;
and
5)
number
and
spacing
of
experimental
concentrations.

1)
Selection
of
controls.
The
experiment
should
include
untreated
control
replicates
and
a
positive
control
treatment
in
which
test
animals
are
exposed
to
a
concentration
of
17 ­
estradiol.
The
Panel
supported
the
Agency's
statement
that
a
positive
androgen
control
group
is
not
needed.

2)
Range
of
observations:
The
experimental
concentrations
used
in
the
experiment
should
include
an
untreated
control
and
span
the
range
of
ecologically
relevant
atrazine
concentrations
to
include
at
least
one
concentration
exceeding
the
upper
percentile
bounds
that
have
been
measured
in
natural,
aquatic
environments.

3)
Number
of
replications
per
treatment:
From
the
Xenopus
Hayes
et
al.,
(
2002a)
and
Carr
et
al.
(
2003)
studies
and
Rana
study
(
Hayes
et
al.,
2002b),
estimates
of
the
empirical
intra­
replication
(
intra­
tank)
correlation
can
be
calculated.
The
estimated
intra­
class
correlation
should
be
used
to
determine
the
number
of
replicates
per
treatment
arm
and
the
allocation
of
total
sample
size
to
replications
and
test
animals
per
replication.
Since
there
will
be
uncertainty
in
the
estimation
of
intra­
class
correlation
for
a
future
study,
the
Panel
advised
the
Agency
to
err
in
selecting
values
that
are
near
the
high
end
of
the
range
of
values
observed
in
the
previous
studies.
Underestimation
of
the
intra­
replicate
correlation
in
planning
the
sample
size
allocation
can
seriously
attenuate
the
true
power
of
the
tests
of
the
hypothesis
for
the
chosen
endpoints.
In
contrast,
overestimation
of
the
intra­
class
correlation
will
produce
some
cost
inefficiency
in
the
design,
but
will
not
generally
endanger
the
power
to
detect
significant
true
effects
in
the
experimental
study.
33
4)
Number
of
test
animals
subjects
per
experimental
replicate:
Having
established
a
working
value
for
the
intra­
replicate
correlation
for
the
class
of
outcomes
of
interest
and
the
desired
levels
of
statistical
power
for
the
specific
hypothesis
tests,
the
optimal
number
of
animal
subjects
per
replicate
can
be
determined
using
standard
sample
size
allocation
formulas.
The
allocation
is
obviously
constrained
by
bio­
loading
and
water
quality
considerations
that
are
discussed
in
the
Agency's
White
Paper.

5)
Number
and
spacing
of
experimental
concentrations:
Determination
of
the
optimal
number
and
spacing
of
treatments
is
governed
by
the
hypothesized
shape
of
any
underlying
concentrationresponse
relationship.
The
Panel
had
determined
that
data
from
existing
studies
support
testing
the
hypothesis
that
there
is
a
relationship
between
atrazine
concentrations
and
gonadal
abnormalities
in
amphibians.
Hayes,
in
his
presentation
to
the
Panel,
provided
data
and
arguments
that
the
relationship
is
not
monotonic
­­
potentially
an
"
inverted"
response.
However,
Hayes'
conclusion
has
not
been
replicated
in
other
studies.
The
Panel
believes
there
is
insufficient
data
for
an
understanding
of
the
shape
of
the
concentration­
response
curve.
Data
are
needed
to
evaluate,
at
the
low
end
of
the
curve,
whether
a
non
monotonic
relationship
exists.
A
robust
design
approach
would
be
to
follow
the
pattern
of
previous
studies
and
use
multiple
concentration
points
to
accommodate
the
possibility
that
any
effect
is
monotonic,
or
alternatively
that
there
is
a
simple
non­
monotonic
relationship
between
atrazine
concentration
and
rates
of
gonadal
abnormalities
in
the
amphibian
test
subjects.
There
is
an
advantage
to
retaining
concentration
points
that
have
been
used
in
the
prior
research
by
Hayes,
Carr,
Hecker
and
others:
0,0.01,0.1,
1,
10
and
25
ug/
l.
The
Panel
also
recommended
adding
an
upper
concentration
level
that
exceeds
the
25
ug/
l
value
at
which
Hayes'
and
Carr's
studies
have
detected
increases
in
the
number
of
abnormalities.
Such
a
spacing
of
concentration
treatments
would
be
sufficient
to
test
the
hypothesis
of
an
effect
of
atrazine
concentration
on
gonadal
abnormalities
and
to
secondarily
test
whether
any
real
effect
is
monotonic
or
non­
monotonic
across
atrazine
concentration
levels.

d)
Please
comment
on
the
Agency's
recommendation
that
X.
laevis
be
used
as
the
primary
biological
model
in
the
proposed
studies
and
whether
or
not
the
mechanisms
involved
in
sexual
differentiation
of
the
ranid
and
pipid
species
are
sufficiently
similar
to
predict
effects
and
associated
dose­
response
curves
for
Rana
and/
or
to
efficiently
design
Rana
studies.

The
Panel
concluded
that
Rana
should
be
used
as
a
collaborating
species.
The
choice
of
X.
laevis
as
a
primary
biological
model
is
well
justified
since
developmental
and
sexual
differentiation
of
Xenopus
laevis
have
been
extensively
studied,
the
published
literature
is
extensive,
and
this
is
not
the
case
for
any
North
American
species.
However,
the
primary
ecological
concern
for
the
Agency
remains
effects,
if
any,
on
North
American
anurans.
Rana
is
the
best
studied
North
American
genus
and
is
the
best
choice
for
study.
Sexual
differentiation
in
ranids
has
not
been
as
extensively
studied
as
in
Xenopus
but,
given
strong
conservation
of
basic
developmental
mechanisms
and
no
known
dissimilarities,
there
is
no
reason
at
this
time
to
believe
that
mechanisms
will
differ
substantively.

e)
In
this
regard,
are
there
important
differences
between
the
species
to
conclude
that
any
34
affected
developmental
processes
observed
in
X.
laevis
would
not
occur
in
Rana?

Several
Panel
members
stated
that
there
is
little
or
no
evidence
to
demonstrate
that
there
are
significant
differences
between
Rana
or
Xenopus
developmental
pathways
that
would
preclude
the
Agency
from
using
Xenopus
as
a
model
in
future
studies.
However,
some
Panel
members
noted
that
there
are
significant
differences
between
the
two
groups
of
species
in
timing
of
life
cycle
events
such
that
concerns
about
differences
in
developmental
pathways
cannot
be
eliminated.

f)
Alternatively,
are
there
developmental
pathways
in
Rana,
but
not
in
X.
laevis,
that
raise
concerns
about
using
X.
laevis
as
the
primary
biological
model
in
any
future
atrazine
studies?

No
differences
have
been
identified
to
date
that
would
raise
concerns
about
differences
in
developmental
pathways.

g)
Assuming
X.
laevis
and
Rana
are
sufficiently
concordant
from
a
toxicodynamic
perspective
with
regard
to
potential
developmental
effects
of
atrazine,
what
critical
toxicokinetic
processes
should
be
considered
for
extrapolating
X.
laevis
dose­
response
relationships
to
Rana
and/
or
for
designing
subsequent
studies
with
Rana?

To
the
Panel's
knowledge,
there
are
no
pharmacokinetic
studies
on
Xenopus
laevis
or
Rana
pipiens
that
would
allow
direct
comparisons
between
uptake,
metabolism,
or
depuration.
However,
given
that
Xenopus
is
a
fully
aquatic
species
and
Rana
is
semiterrestrial
there
are
bound
to
be
differences.
The
significance
of
such
differences,
if
any,
is
uncertain.
Measuring
whole
body
burden
and/
or
residues
in
specific
tissues
will
yield
information
on
this
topic.
35
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E.
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Laboratory
Study
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DL,
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PD,
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Giesy
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