Overview
of
Amphibian
Workshop
International
Workshop
on
the
Use
of
Anuran
Models
for
Endocrine
Disruption
and
Reproductive
Toxicology
June
2003,
Duluth,
MN
Leslie
Touart
International
Workshop
on
the
Use
of
Anuran
Models
for
Endocrine
Disruption
and
Reproductive
Toxicology
°
Experts
from
11
countries
participated
°
Day
1
­
presentations
 
general
endocrinology
 
reproductive
biology
 
test
methods
for
screening
and
testing
°
Day
2
 
breakout
group
discussions
 
screening
assays
and
testing
methods
for
thyroid
axis
disruption
 
diagnostic
indicators
of
amphibian
endocrine
disruption
 
reproductive
endocrinology
and
testing
°
Workshop
proceedings
to
be
submitted
for
publication
Fall
2003
Workshop
Summary
°
Discussion
that
screening
assay
is
intended
as
general
vertebrate
model
for
effects
to
thyroid
hormone
axis
°
Choice
of
test
species?
X.
laevis
immediately
practical,
X.
tropicalis
desirable
when
more
information
available
°
Many
endpoints
and
important
MOAs
to
consider
were
discussed
°
Mechanistic/
diagnostic
endpoints
desirable,
but
should
be
balanced
with
integrative
(
apical)

measures
that
are
simple,
unambiguous
Workshop
Summary
(
cont.)

°
Proposal
to
start
with
NF
51
to
continue
up
to
NF60,
duration
minimum
14
days
°
Important
considerations
and
issues
were
listed
(
i.
e.,
food/
feeding,
water
quality,
mixed
MOAs,

etc.)

°
Flow­
through
design
preferred,
but
semi­
static
ok
°
Some
chemicals
identified
for
key
MOAs
Workshop
Summary
(
cont.)

°
Tier
2
not
focused
on
general
vertebrate
screening
but
for
assessing
ecological
risk
in
amphibians
°
Emphasis
for
Tier
2
on
Xenopus
sp.,
extrapolation
critical
°
Apical
endpoints
 
fecundity,
fertility,

hatchability,
sexual
development
&

differentiation,
and
viable
offspring
°
Full
life­
cycle
or
multi­
generation
desirable,
but
partial
life­
cycle
more
practical
OECD
Amphibian
Expert
Group
The
main
objectives
of
the
meeting
were
to:

°
discuss
and
agree
on
the
preferred
approach
for
a
frog
metamorphosis
assay
for
the
detection
of
thyroid
disruptors
°
discuss
and
agree
on
the
main
parameters
of
a
protocol
°
agree
on
a
timeframe
and
action
plan
for
further
work
in
the
coming
months.
Thyroid
related
modes
of
action
and
possible
endpoints.

+
1
+
1
+
1
+
1
?
1
+

?
1
?
1
?
?

+
1
+
1
+

Mol.
Biology:

°
TR 
mRNA
°
TSH 
mRNA
°
CRF
°
UDPGT
++
2
+?

+
2
+
2
Biochemistry
:

°
T3/
T4
°
Enzymes
(
D2/
D3)
+
1
++
1
+
2
+?
1
?
1
?
2
++
1
+
2
Histology:

 
Thyroid
 
Pituitary
+
1
+
1
+?
1
?
1
?
2
+
1
+
2
Morphology:

°
Dev.
Stage
°
Hind
limb
TR
­
agonism
­
antagonism
Neuroendocrine
HP
axis
Elimination
­
deiodination
­
conjugation
(
UDPGT)

Transport
­
TTR
displacement
Synthesis
­
I­
uptake
­
TPO
inhibition
MOA
Possible
endpoints
+
endpoints
able
to
measure
effects
on
the
associated
thyroid
related
mode
of
action;

1
core
endpoints
of
the
frog
metamorphosis
assay
proposed
by
the
Amphibian
Expert
Group;

2
optional
endpoints
of
the
frog
metamorphosis
assay
proposed
by
the
Amphibian
Expert
Group.
Thyroid
pathways
and
relevant
chemicals.

IOP
Transformation
Corticosteriod
(
research
need)

Neuroendocrine
NH3,
T4,
T3,
brominated
flame
retardants
(
e.
g.
tetrachlorobisphenol)

Thyroid
Receptor
Research
need
 
not
known
Elimination
Research
need
 
not
known
Transport
PTU,
Methimazole,
Perchlorate
Synthesis
Chemicals
which
have
an
action
Mode
of
action
Chemicals
and
countries
which
conducted
the
Amphibian
metamorphosis
assay.

­

TBBA
­

 ­
trenbolone
­

Estradiol
+

Perchlorate
+

Methimazole
+

+

IOP
+

T3
+

+

+

T4

+

Amitrole
­

Zineb
+

+

ETU
+

+

+

PTU

United
States
Japan
Germany
Chemicals
Shaded
cell:
country
used
the
chemical;

+
chemical
came
up
positive;

­
chemical
showed
no
thyroid
related
effect;


chemicals
preferred
for
the
optimization
phase
Need
to
establish
the
expression
profiles
of
the
biomarkers
throughout
metamorphosis
Optional
Core
spinoff
Core
spinoff
Semi­
quantve
PCR
Semi­
quantve
PCR
Semi­
quantve
PCR
Molecular
biology:

°
TR ­
mRNA
°
TR ­
mRNA
°
TSH 
sub­
unit­
mRNA
­­­­­­­­­­­­­

­­

No
assay/
method
available
°
Deiodinase
activity
(
D2/
D3)
°
Develop
a
protocol
for
extraction
from
whole
body
homogenates
or
specific
tissues,

°
Develop
a
standardized
RIA
protocol
for
T3/
T4
Optional
RIA
using
whole
body/
brain
Biochemistry:

°
T3/
T4
Optional
Pituitary
histology
°
Define
whether
quantitative
evaluation
is
possible
/
needed;

°
Define
histological
endpoints
to
be
looked
at
(
eg.
epithelial
cell
height)

Core
Core/
Opti
onal
Qualitative
Quantitative
Thyroid
histology
Core
Optional
NF
scale
Analo/
digital
calipers/
videoframe
grab
and
image
analysis
software
Developmental
morphology:

°
Staging
(
includes
hind
limb
development)

°
hindlimb
length
Research
needs
Priority
level
Measurement
technique
Endpoints
Additional
Work
 
Protocol
optimization
underway
 
complete
Dec
2003
 
Compare
protocols
beginning
NF
51
continue
for
21
days
and
beginning
NF
54
continue
for
14
days
 
US,
Germany,
Japan
participating
 
Draft
protocol
for
validation
work
Jan
2004
 
Interlaboratory
validation
exercise
Summer
2004
