OECD
phase
1A
work
on
a
non
OECD
phase
1A
work
on
a
non­
spawning
fish
assay
spawning
fish
assay
­
draft
results
draft
results
­

Hirofumi
Yokota
Chemicals
Evaluation
and
Research
Institute,
Japan
(
CERI)
Non
Non­
spawning
fish
assay
spawning
fish
assay

To
detect
chemicals
that
affect
androgenic
or
estrogenic
activity
in
fish
exposed
during
a
limited
part
of
their
lifecycle
not
including
reproduction.


Three
core
biological
endpoints:

­
Gross
morphology
2nd
sex
characters
and
gonado­
somatic
index
(
GSI)

­
Plasma
or
liver
vitellogenin
(
VTG)
levels
­
Histopathology
of
excised
gonads.


Test
species
­
Fathead
minnow
(
Pimephales
promelas)

­
Medaka
(
Oryzias
latipes)

­
Zebrafish
(
Danio
renio)
Outline
of
non­
spawning
fish
assay
14
21
Days
after
initiation
of
the
exposure
0
Daily
observation
on
mortality
and
abnormal
response
Separate
with
mesh
barrier
Male
×
5
Female
×
5
Male
×
5
Female
×
5
Male
×
5
Female
×
5
­
Sexual
characters
­
GSI
­
VTG
level
­
Gonad
histology
Endpoints

To
obtain
initial
information
on:

i)
the
relevance
of
the
endpoints
used
in
terms
of
their
sensitivity
and
(
species)
specificity
ii)
the
reproducibility
of
test
results.


Participating
laboratories
­
CERI
(
Lead
laboratory)

­
Bayer
CropScience
AG.
(
Germany)

­
METOCEAN
ENVIRONMENT
INC.
(
METO)
(
Japan)

­
National
Institute
for
Environmental
Studies
(
NIES)
(
Japan)


Test
substance
­
17 ­
Estradiol
(
E2)
(
estrogen)

­
17 ­
Trenbolone
(
TB)
(
androgen)

OECD
phase
1A
validation
study
Main
endpoint
Gross
morphology
VTG
induction
Gonad
histology
Test
species
Sex
GSI
2nd
sex
characters
Male
 
 
 
Fathead
minnow
Female
×
×
 
Male
×
 
 
Medaka
Female
×
×
 
Male
×
 
Zebrafish
Female
×
ND
 
NA
 
:
Cleary
detected
in
all
studies,
 
:
Detected
in
all
studies,
 
:
Detected
in
a
few
studies,

×
:
Not
detected
ND:
Not
determined,
NA:
Not
available
yet
Draft
results
of
the
studies
with
E2
E2
exposure
with
fathead
minnow

Laboratory
in
charge:
CERI,
NIES,
Bayer

Nominal
conc.:
100,
32,
10
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
8
to
9
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
10
or
15
L
/
vessel

Dilution
water:
dechlorinated
tap
water
or
reconstituted
water

Test
temperature:
25
±
2
 

Food:
brine
shrimp

Chemical
analysis:
LC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
EnBio
or
Biosense)
GSI
of
male
fathead
minnow
:
CERI
:
NIES
:
Bayer
0
0.5
1
1.5
2
2.5
3
3.5
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)
CERI
NIES
Bayer
**
0
0.5
1
1.5
2
2.5
3
3.5
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)
CERI
NIES
Bayer
**

14­
day
21­
day
0
5
10
15
20
25
30
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

Number
of
nuptial
tubercles
CERI
NIES
0
5
10
15
20
25
30
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

Number
of
nuptial
tubercles
CERI
NIES
*
*

**

Secondary
sexual
characters
of
male
fathead
minnow
:
CERI
:
NIES
14­
day
21­
day
0.01
0.1
1
10
100
1000
10000
100000
1000000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*

*
*
*

*
*
*
*
*
*

VTG
conc.

(

µ
g/

mL)

0.01
0.1
1
10
100
1000
10000
100000
1000000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*
*

*
*
*
*
*
*

VTG
conc.

(

µ
g/

mL)
VTG
of
male
fathead
minnow
14­
day
21­
day
:
CERI/
EnBio
:
NIES/
EnBio
:
Bayer/
Biosense
:
NIES/
Biosense
(
Blood)
(
Blood)
(
Blood)
(
Plasma)

The
plasma
sample
from
several
fish
was
not
obtained
due
to
the
technical
problem.
E2
exposure
with
medaka

Laboratory
in
charge
:
CERI,
NIES,
METO

Nominal
conc.:
100,
32,
10
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
4
to
5
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
1.8
to
5
L
/
vessel

Dilution
water:
dechlorinated
tap
water

Test
temperature:
24
±
2
 

Food:
brine
shrimp
(
newly
hatched
larvae)


Chemical
analysis:
LC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
Transgenic,
EnBio
or
Biosense)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)

GSI
of
male
medaka
14­
day
21­
day
:
CERI
:
NIES
:
METO
0
50
100
150
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

Number
of
papillary
processes
CERI
NIES
METO
0
50
100
150
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

Number
of
papillary
processes
CERI
NIES
METO
*
*
*

Secondary
sexual
characters
of
male
medaka
14­
day
21­
day
:
CERI
:
NIES
:
METO
Male
Female
VTG
of
male
medaka
0.1
1
10
100
1000
10000
100000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

ng/

mg
liver)
CERI/
Trans
Genic
NIES/
Biosense
NIES/
Enbio
METO/
Trans
Genic
*
*
*

*
*
*

*
*
*

*
*
0.1
1
10
100
1000
10000
100000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

ng/

mg
liver)
CERI/
Trans
Genic
NIES/
Biosense
NIES/
Enbio
METO/
Trans
Genic
*
*
*
*
*
*

*
*

*
*
*

:
CERI/
Transgenic
:
NIES/
Biosense
:
METO/
Transgenic
:
NIES/
EnBio
14­
day
21­
day
E2
exposure
with
zebrafish

Laboratory
in
charge:
CERI,
NIES,
METO,
Bayer

Nominal
conc.:
100,
32,
10
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
4
to
6
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
4.5
to
15
L
/
vessel

Dilution
water:
dechlorinated
tap
water
or
reconstituted
water

Test
temperature:
26
±
2
 

Food:
brine
shrimp

Chemical
analysis:
LC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
EnBio
or
Biosense)
0
0.5
1
1.5
2
2.5
3
3.5
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)
CERI
NIES
METO
Bayer
0
0.5
1
1.5
2
2.5
3
3.5
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

GSI
(%)
CERI
NIES
METO
Bayer
*
*

*

GSI
of
male
zebrafish
14­
day
21­
day
:
CERI
:
NIES
:
Bayer
:
METO
One
male
fish
in
Bayer
was
identified
as
female
fish
after
excision
of
gonads.
0.01
0.1
1
10
100
1000
10000
100000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
CERI/
Enbio
NIES/
Enbio
NIES/
Biosense
METO/
Enbio
Bayer/
Biosense
*
*

*
*
*

*
*

VTG
conc.

(

µ
g/

mL)
0.01
0.1
1
10
100
1000
10000
100000
Control
10
32
100
E2
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
CERI/
Enbio
NIES/
Enbio
NIES/
Biosense
METO/
Enbio
Bayer/
Biosense
*

*
*

*
*

VTG
conc.

(

µ
g/

mL)

VTG
of
male
zebrafish
The
data
from
Bayer
is
not
submitted
yet.

(
Blood)
(
Blood)
(
Blood)
(
Blood)
(
Plasma)

:
CERI/
EnBio
:
NIES/
EnBio
:
Bayer/
Biosense
:
NIES/
Biosense
:
METO/
EnBio
14­
day
21­
day
Draft
results
of
the
studies
with
TB
Main
endpoint
Gross
morphology
VTG
reduction
Gonad
histology
Test
species
Sex
GSI
2nd
sex
characters
Male
×
×
×
Fathead
minnow
Female
 
 
 
Male
×
×
×
Medaka
Female
 
 
 
Male
×
×
Zebrafish
Female
 
ND
 
NA
 
:
Cleary
detected
in
all
studies,
 
:
Detected
in
all
studies,
 
:
Detected
in
a
few
studies,

×
:
Not
detected
ND:
Not
determined,
NA:
Not
available
yet
TB
exposure
with
fathead
minnow

Laboratory
in
charge:
CERI,
NIES,
Bayer

Nominal
conc.:
5000,
500,
50
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
8
to
9
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
10
or
15
L
/
vessel

Dilution
water:
dechlorinated
tap
water
or
reconstituted
water

Test
temperature:
25
±
2
 

Food:
brine
shrimp

Chemical
analysis:
GC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
EnBio
or
Biosense)
:
CERI
:
NIES
:
Bayer
GSI
of
female
fathead
minnow
0
5
10
15
20
25
30
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
0
5
10
15
20
25
30
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
*

14­
day
21­
day
0
2
4
6
8
10
12
14
16
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

Number
of
nuptial
tubercles
0
2
4
6
8
10
12
14
16
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

Number
of
nuptial
tubercles
Secondary
sexual
characters
of
female
fathead
minnow
:
CERI
:
NIES
14­
day
21­
day
VTG
of
female
fathead
minnow
:
CERI/
EnBio
:
NIES/
EnBio
:
Bayer/
Biosense
:
NIES/
Biosense
(
Blood)
(
Blood)
(
Blood)
(
Plasma)

14­
day
21­
day
The
plasma
sample
from
one
fish
was
not
obtained
due
to
the
technical
problem.

1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*
*
*

*
*
*
*

VTG
conc.

(

µ
g/

mL)
1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*

*
*
*

*
*
*

*
*

VTG
conc.

(

µ
g/

mL)
TB
exposure
with
medaka

Laboratory
in
charge
:
CERI,
NIES,
METO

Nominal
conc.:
5000,
500,
50
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
4
to
5
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
1.8
to
5
L
/
vessel

Dilution
water:
dechlorinated
tap
water

Test
temperature:
24
±
2
 

Food:
brine
shrimp
(
newly
hatched
larvae)


Chemical
analysis:
GC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
Transgenic,
EnBio
or
Biosense)
0
5
10
15
20
25
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
0
5
10
15
20
25
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
*
*

:
CERI
:
NIES
:
METO
GSI
of
female
medaka
14­
day
21­
day
Some
females
in
CERI
and
METO
spawned
during
the
exposure
period.
Secondary
sexual
characters
of
female
medaka
0
50
100
150
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

Number
of
papillary
processes
*
*

*
*
*
*
0
50
100
150
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

Number
of
papillary
processes
*
*
*
*
*
*

:
CERI
:
NIES
:
METO
14­
day
21­
day
1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

ng/

mg
liver)
*
*

*
*
1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

ng/

mg
liver)
*
*
*

*
*

*
*

*
*
*

VTG
of
female
medaka
14­
day
21­
day
:
CERI/
Transgenic
:
NIES/
Biosense
:
METO/
Transgenic
:
NIES/
EnBio
TB
exposure
with
zebrafish

Laboratory
in
charge:
CERI,
NIES,
METO,
Bayer

Nominal
conc.:
5000,
500,
50
ng/
L
and
control

Number
of
fish:
10
 
and
10
 
/
treatment

Age
of
fish:
4
to
6
month
old

Exposure
system:
flow­
through
exposure

Stock
solution:
an
aqueous
solution

Test
solution
vol.:
4.5
to
15
L
/
vessel

Dilution
water:
dechlorinated
tap
water
or
reconstituted
water

Test
temperature:
26
±
2
 

Food:
brine
shrimp

Chemical
analysis:
LC­
MC
(
once
a
week)


VTG
analysis:
ELISA
(
EnBio
or
Biosense)
GSI
of
female
zebrafish
0
5
10
15
20
25
30
35
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
0
5
10
15
20
25
30
35
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

GSI
(%)
*

14­
day
21­
day
:
CERI
:
NIES
:
Bayer
:
METO
Each
one
male
fish
in
CERI
and
Bayer
was
identified
as
female
fish
after
excision
of
gonads.
0.01
0.1
1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*
*
*
*

*
*

VTG
conc.

(

µ
g/

mL)

0.01
0.1
1
10
100
1000
10000
100000
Control
50
500
5000
TB
nominal
concentration
(
ng/
L)

VTG
conc.

(

 g/

mL
blood
or
plasma)
*
*

*
*

*
*

*
*

VTG
conc.

(

µ
g/

mL)
VTG
of
female
zebrafish
14­
day
21­
day
The
data
from
Bayer
is
not
submitted
yet.

(
Blood)
(
Blood)
(
Blood)
(
Blood)
(
Plasma)

:
CERI/
EnBio
:
NIES/
EnBio
:
Bayer/
Biosense
:
NIES/
Biosense
:
METO/
EnBio
Draft
summary

GSI
­
Significant
differences
were
not
always
observed
in
all
studies.

­
The
values
in
both
intra­
and
inter­
laboratory
varied
considerably
possibly
due
to
difference
in
sexual
maturity.

­
Some
female
medaka
and
zebrafish
spawned
during
exposure.


Secondary
sexual
characters
(
fathead
minnow
and
medaka)

­
Significant
differences
were
not
always
observed
in
all
studies.

­
The
quantification
method
was
not
standardized
between
laboratory.


VTG
­
Significant
differences
were
observed
in
all
studies.

­
The
values
in
both
the
control
and
treatment
groups
differed
with
the
ELISA
kits.

­
The
preparation
method
of
the
samples
was
not
standardized.
Future
plan

All
data
except
for
gonadal
histology
will
be
submitted
to
the
lead
laboratory
by
August
18th.


The
round­
robin
evaluation
of
gonadal
histology
will
be
conducted
by
several
fish
pathologists
in
September.


The
draft
report
will
be
submitted
to
OECD
by
September
1st
and
then
circulated
to
the
members
of
the
OECD
FDG.


The
final
results
will
be
discussed
by
the
OECD
FDG
meeting
which
will
be
held
on
October
21st
and
22nd.
Acknowledgement

Bayer
CropScience
AG.


Chemicals
Evaluation
and
Research
Institute,
Japan

METOCEAN
ENVIRONMENT
INC.


National
Institute
for
Environmental
Studies

Ms.
Anne
GOURMELON
(
OECD)
