Avian
Two­
Generation
Toxicity
Test
Detailed
Review
Paper
NACEPT
Endocrine
Disruptor
Methods
Validation
Subcommittee
August
2003
Leslie
Touart
Detailed
Review
Paper:

AVIAN
TWO­
GENERATION
TOXICITY
TEST
WORK
PERFORMED
BY
On
behalf
of
the
United
States
Environmental
Protection
Agency
EPA
CONTRACT
NUMBER
68­
W­
01­
023
OVERVIEW
AND
SCIENTIFIC
BASIS
OF
AVIAN
TWO­
GENERATION
TESTS
°
Hormonal
control
of
sexual
differentiation
in
birds
differs
from
that
of
mammals
°
Birds
lack
fetoprotein
°
Oviparity
in
birds
allows
retention
of
compounds
°
Current
protocols
 
are
not
sufficiently
robust
to
differentiate
endocrine
disrupting
chemicals
from
other
reproductive
or
developmental
toxins
 
are
not
designed
to
determine
long­
term
effects
of
in
ovo
exposure
 
Do
not
assess
effects
at
the
4
critical
life
stages
that
could
be
sensitive
to
endocrine
disruption
Test
Species
°
Japanese
Quail
(
Coturnix
japonica)

°
Bobwhite
(
Colinus
virginianus)

 
terrestrial
habit
 
accepted
model
for
toxicity
tests
 
adaptable
to
laboratory
conditions
 
indeterminate
layer
 
precocial
young
Japanese
Quail
Family
Phasianidae
(
Pheasants/
Partridges)

°
Old
World
quail
°
Rapid
incubation
and
maturation
 
Reproductively
mature
in
6
to
8
weeks
°
Sexually
dimorphic
°
Can
be
kept
in
breeding
condition
all
year
(
optimal
5
to
6
months)

°
High
rate
of
egg
production
(
300
eggs
per
year)

°
Peak
production
within
2­
3
weeks
of
onset
of
lay
°
Highly
adaptable
to
battery
cages
°
Endocrine
and
behavioral
patterns
are
well
characterized
°
Large
number
of
cultivated
strains
Bobwhite
Family
Odontophoridae
(
New
World
Quails)

°
Indigenous
to
North
and
South
America
°
Reproductively
mature
in
24
weeks
°
sexually
dimorphic
°
Adapts
well
to
laboratory
°
Highly
photsensitive
°
females
produce
about
1
egg
per
day
°
Peak
egg
production
at
6
weeks
after
onset
of
lay
°
Little
deliberate
selective
breeding
Advantages
Japanese
Quail
°
Endocrine,
behavior
patterns
known
°
Small
bird;
occupies
230
cm2
per
bird
°
Reach
sexual
maturity
by
6
weeks
°
Prolific
layer
°
Early
maturity
(
36
days
male,
42
days
female)

°
Short
incubation
period
(
16­
17
days)

°
Males
are
aggressive
breeders
°
Males
maintain
high
fertility
(
90%)

°
Adapts
well
to
breeder
cages
°
Produce
large
egg
(
8%
of
body
wt)

°
Naturally
hardy
in
laboratory
°
Highly
photosensitive
°
Dimorphism
of
plumage
by
3
weeks
°
Male
cloacal
gland
°
History
of
toxicity
testin
°
Spermatogenesis
is
well
characterized
Bobwhite
°
Less
domesticated,
wild
type
°
Small
bird;
occupies
400­
900
cm2
per
bird
°
Prolific
layer
(
somewhat
less
than
Japanese
quail)

°
Males
are
aggressive
breeders
°
Males
maintain
high
fertility
(
95%)

°
Adapts
well
to
laboratory
°
Produces
large
egg
(
8%
­
10.5%
of
body
wt)

°
Highly
photosensitive
°
Populations
not
prone
to
photperiodic
drift
°
Dimorphism
of
plumage
color
°
History
of
use
in
toxicity
testing
°
More
yolk
per
egg
(
39.8%)
by
weight)
compared
with
Japanese
quail
(
31.9%)
Disadvantages
Japanese
Quail
°
Inbreeding
not
tolerated
(
impaired
fertility)

°
Strains
differ
in
body
weight,

maturation
rate,
egg
production,

lipid
deposition
in
body
and
egg
°
Populations
can
show
marked
photoperiodic
drift
with
large
variability
in
reproductive
response
°
Food
wastage
making
food
consumption
measurement
difficult
°
Most
strains
have
colored
eggshells
that
are
difficult
to
candle
(
some
white
egg
strains
available)

°
Less
yolk
per
egg
compared
to
bobwhite
°
Little
used
in
U.
S.
for
toxicity
tests
Bobwhite
°
Long
incubation
period
(
23
days)

°
Long
maturation
period
(
24
months)

°
Food
wastage
making
food
consumption
measurement
difficult
°
Sex
cannot
be
distinguished
by
plumage
until
12
weeks
of
age
°
Lack
cloacal
gland
°
Spermatogenesis
not
characterized
Exposure
Considerations
°
Exposure
of
parental
(
P1)
generation
 
Pre­
egg
laying
 
Risk
of
loss
of
statistical
power
due
to
infertile
pairs
 
Effects
on
sexual
maturation
possible
 
Bioaccumulation
of
test
substance
 
post­
initiation
of
laying
(
proven
breeders)

 
Statistical
advantages
 
removing
nonproductive
birds
before
exposure
 
Use
pretreatment
measures
as
covariates
 
Reduction
in
cost
from
reduced
exposure
period
 
Rapidly
of
manifestation
of
reproductive
effects
observable
Exposure
Considerations
(
cont.)

°
Exposure
of
offspring
(
F1)
of
parents
 
No
exposure
(
in
ovo
exposure
only)

 
Avoids
masking
of
endocrine­
mediated
effects
by
high
mortality
of
chicks
from
direct
toxicity
 
Exposure
from
hatch
through
egg­
laying
 
Worse
case
scenario
 
Allows
observation
of
effects
at
all
life
stages
 
Mortality
of
chicks
could
mask
endocrine­
mediated
effects
°
F2
chicks
not
exposed
Exposure
Considerations
(
cont.)

°
Combined
exposure
scenarios
for
P1
and
F1
 
Maximize
attributes,
minimize
disadvantages
 
Expose
maximum
number
of
reproductive
processes
 
Expose
the
maximum
number
of
life
stages
 
Increase
power
of
test
 
Not
mask
endocrine­
related
effects/
not
confound
interpretation
of
results
 
Worst­
case
environmental
exposure
 
Time
and
cost­
effectiveness
 
Dosing
Study
Avian
Dosing
Study
Schematic
P
F1
F2
Egg
laying
begins
P1A
P1B
9
weeks
12
weeks
4
weeks
Treatment
begins
Egg
laying
begins
Routes
of
Administration
of
Chemical
°
Food
 
More
natural
exposure,
but
greater
than
in
wild
 
Avoids
intermittent
high
body
loading
 
Detect
food
avoidance/
anorexia
consequences
 
Low
labor
cost
for
administration
 
Dose
estimation
not
precise
 
Costs
verifying
concentration
and
stability
high
 
Effect
of
natural
endocrine
active
substances
in
feed
unknown
°
Water
 
Ecologically
relevant
route
 
Dispersion
of
chemical
more
easily
achieved
than
in
diet
 
Not
used
for
chemicals
of
low
solubility
in
water
 
Evaporation
may
concentrate
chemical
 
Water
spillage
more
serious
than
feed
spillage
Route
of
Administration
(
cont.)

°
Bolus
 
Most
accurate
dose
estimate
 
Best
for
unstable
or
volatile
chemicals
 
Low
analytical
costs
to
verify
dose
 
Handling
stress
 
Greater
absorption
rate,
saturation
of
hepatic
enzymes
 
Can
result
in
lower
tolerated
test
concentrations
 
Intubation
trauma
 
Regurgitation
of
emetic
compounds
 
High
labor
cost
of
dosing
Dosing
Options
°
Constant
dietary
concentration
 
More
directly
comparable
to
environmental
concentrations
 
Young
can
receive
higher
doses
than
parents
°
Constant
daily
dosage
 
Can
compare
effects
between
life
stages
and
species
of
different
body
size
°
Both
methods
are
highly
artificial
 
Determine
by
data
needs
of
risk
assessment
process
Statistical
Approaches
°
Multiple
comparison
methods­
NOAEC
 
Robust
to
non­
normal
errors
 
Sensitive
to
loss
of
replicates
 
Requires
greater
number
of
test
animals
 
NOEC/
LOEC
limited
to
concentrations
on
test
°
Regression
methods­
ECx,
BMD
 
Less
affected
by
loss
of
replicates
 
Estimate
a
dose­
response
curve
 
Estimation
of
ECx
not
limited
to
test
concentraions
 
Assess
time­
series,
time
lag
between
exposure
and
response
Fitness
Endpoints
°
Growth
Rate
°
Food
Consumption
°
Measures
of
Reproductive
Performance
 
Fecundity
 
maximum
production
period
 
Fertilization
Success
 
Separation
of
gender­
specific
effects
 
Fertility
trials
 
Gamete
Viability
 
Sperm
motility
and
morphology
 
Sperm
mobility
 
Interaction
of
sperm
with
egg
perivitelline
layers
Fitness
Endpoints
(
cont.)

 
Egg
quality
 
Candling­
distribution
of
background
cracking
 
Eggshell
thickness
 
Eggshell
Strength
 
Puncture
test
 
Compression
test
 
Eggshell
matrix
proteins
as
biomarkers
 
Hatching
Success
 
F1
and
F2
post­
hatch
suvivorship
 
Primary,
integrating
production
endpoint
 
High
CVs
Fitness
Endpoints
(
cont.)

°
Changes
in
breeding
behavior
 
Sensitive
endpoint
 
Ecologically
relevant
°
Neurological/
CNS
impairment
tests
 
Integrated
response
to
toxin
 
Open­
field
tests
Physiological
Endpoints
°
Organ
growth
and
morphological
changes
 
Gonadosomatic
index
 
Organ:
brain
weight
index(
brain,
thyroid,
adrenal
gland)

 
Oocyte
diameter
 
Oviduct
differentiation
 
Developmental
landmarks
 
Gross
landmarks
 
Age
at
first
egg
 
Cloacal
gland
size,
age
at
foam
production
 
Age
of
sexually
dimorphic
plumage
development
 
Age
at
first
crowing
 
Sternotracheal
(
syringeal)
muscles
 
Medualary
bone
 
Histopathology
of
juvenile
and
adult
tissues
°
Sex
Ratio
Biochemical
Measures
°
Biomarkers
of
hepatic
metabolic
changes
 
Vitellogenin
 
Alkaline­
labile
Phosphate
Assay
 
RIA,
ELISA
 
No
universal
VTG
antibody
available
 
Quail
VTG
ELISA
assay
recently
developed
 
Circulating
very
low­
density
lipoprotein
 
Enzymatic
method
in
development
 
Cytochrome
P450
1A
induction
 
EROD
activity
 
Aromatase
immunocytochemical
assay
Biochemical
Measures
(
cont.)

°
Plasma,
egg
and
fecal/
urate
hormone
levels
 
Handling
stress
can
alter
circulating
steroid
levels
 
Fecal/
Urate
sampling
 
Long
history
of
use
in
conservation
biology
(
field
samples)
for
steroid
hormones
 
Recent
application
in
avian
studies
 
Rapid
extraction
methods
available
 
May
be
useful
for
thyroid
hormones,
but
not
tested
 
RIA,
ELISA
assays
 
Commercial
kits
for
steroid
and
thyroid
hormones
applicable
to
birds
CANDIDATE
PROTOCOLS
Short­
Term
Life
Cycle
Test
(
Proven
Breeders):

 
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
(
OECD
April
2000)

Life
Cycle
Tests:

 
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species
(
ASTM
E1062­
86)

 
OECD
Test
Guideline
206:
Avian
Reproduction
Test(
OECD
1993)

 
EPA
OPPTS
850.2300:
Avian
Reproductive
Test
(
USEPA
1996);
suggested
adaptations
for
two­
generation
test
added
by
EDSTAC
(
EPA
1998)

Two­
Generation
Test:

 
Proposal
for
a
Avian
Two­
Generation
Toxicity
Test
in
Japanese
Quail
(
OECD
1999)
Recommended
Protocol
°
Preferred
species:
Japanese
quail
°
Exposure
protocol:
TBD
based
on
Avian
Dosing
Study
°
Fitness
Endpoints:

°
Egg
production
°
Fertility
°
Eggshell
integrity
°
Embryo
viability
°
Hatchability
°
Sex
ratio
°
Chick
health
°
Signs
of
toxicity
Recommended
Protocol
(
cont.)

°
Endocrine
Endpoints
Gross
morphology
&
histopathology
 
Organ/
gland
weights/
bone
length(
chicks)

 
Organ/
gland
histology
 
Spermatid
counts
&
morphology
 
Gross
anomalies
Developmental
landmarks
 
Feather
dimorphism
 
Cloacal
gland
size
 
Sexual
maturation/
behavior
Plasma
and
fecal/
urate
hormones
 
Steroid
hormones
 
Thyroid
hormones
Significant
Data
Gaps
Lack
of
clear
information
on
source
of
and
metabolic
fate
of
xenobiotics
in
ovo.

The
effects
of
anti­
estrogens
in
juvenile
and
sexually
mature
test
species.

The
effects
of
anti­
androgens
in
the
developing
embryo
or
hatchling.

The
effects
of
thyroid
hormone
agonists
(
or
thyroid
stimulation)
on
reproduction.

Interactive
effects
of
endocrine­
active
substances,

especially
natural
phytosteroids
in
diets.
Data
Gaps
(
cont.)

The
effects
of
strain
differences
on
test
outcome
and
interpretation:
what
traits
are
co­
selected
with
high
body
weight
or
high
fecundity;
what
qualities
should
be
selected
for
toxicity
testing
in
randombred
lines.

A
statistical
approach
for
delayed
effects.

Specific
information
on
husbandry
requirements
of
the
Japanese
quail
that
will
result
in
consistent
results
in
laboratory
toxicity
tests.
Specifically
information
for
dealing
with
fear,
social
stress,
injurious
pecking,
etc.
Implementation
Considerations
and
Future
Research
°
Pre­
validation
studies
 
To
select
appropriate
exposure
regimen,
recommend
a
direct
performance
comparison
of
proven­
breeder
and
pre­
breeding
exposure
regimens
combined
with
nontreatment
and
worst­
case
hatch­
through
egglaying
F1
exposure
scenarios
 
Do
endocrine­
mediated
effects
occur
during
maturation
that
are
overlooked
when
only
in
ovo
exposure
is
considered?

 
Do
compounds
that
are
directly
toxic
to
chicks
mask
detection
of
endocrine­
mediated
effects?
Implementation/
Research
(
cont.)

 
Recommend
verifying
the
relative
sensitivity
of
the
Japanese
quail
and
Bobwhite
in
a
side­

byside
comparison
study
 
Recommend
determining
the
effects
of
strain
selection
in
Japanese
quail
on
test
outcome
to
minimize
nontreatment
variability
across
laboratories
 
If
ANOVA
methods
are
to
be
applied
to
the
test,
recommend
investigation
of
statistical
approach
for
delayed
effects
Implementation/
Research
(
cont.)

 
Recommend
development
of
T4/
T3
assays
in
fecal/
urate
samples
for
noninvasive
monitoring
of
thyroid
function
 
Recommend
PCR
methods
for
genetic
sex
determination
be
optimized
for
Japanese
quail
 
Recommend
determining
the
interactive
effects
of
phytosteroids
in
feed
on
test
outcome
 
Recommend
evaluating/
standardizing
husbandry
practices
 
Recommend
guidelines
for
histology
preparation
and
examination
of
tissues
°
Validation
of
the
study
design
through
interlaboratory
comparisons
Issues/
Concerns
°
Animal
usage
°
Value
added
of
2
gen
v.
1
gen
°
Japanese
quail
sensitivity
°
Lack
of
avian
assay
in
Tier
1
°
Time
delay
in
validation
(&
costs)

°
Linkage
with
existing
avian
testing
framework
