White
Paper
on
Species/
Strain/
Stock
in
Endocrine
Assays
Research
Triangle
Park,
North
Carolina
White
Paper
on
Species/
Strain/
Stock
in
Endocrine
Assays
Prepared
by:

Sherry
P.
Parker,
Rochelle
W.
Tyl
For:

Battelle
Memorial
Institute
as
a
part
of
EPA
Prime
Contract
68­
W­
01­
023,
James
Kariya,
US
EPA,

Work
Assignment
Manager.

Reviewed
but
not
in
complete
concurrence:

J.
Spearow
Background

There
is
evidence
that
different
species
and
strains
within
species
exhibit
differing
sensitivities
to
endocrine­
active
compounds

Selection
of
appropriate
species
and
strain(
s),
or
at
least
understanding
their
differential
responsivity,
is
important
in
EDSP
assays

EPA
testing
guidelines
recommend
using
the
rat
but
not
strains
with
low
fecundity.
The
most
commonly
used
rat
strain
for
these
guideline
studies
is
the
Sprague­
Dawley
rat

In
the
December
2001
meeting
of
the
EDMVS,
committee
members
discussed
strains
and
stocks
and
concluded
that
the
EPA
should
prepare
a
white
paper
summarizing
what
is
known
about
interspecies
and
intraspecies
strain/
stock
similarities
and
differences
in
responses
to
EACs,
and
provide
the
rationale
for
strain/
stock
selection
Concern
Animal
models
used
in
assays
to
detect
endocrine
disruption
have
been
chosen
on
the
basis
of
convenience
and
familiarity,
and
species/
strains/
stocks
which
are
more
frequently
used
are
those
which
are
bred
specifically
for
robust
fecundity
and
likely
reduced
sensitivity
to
endocrine
perturbations
(
NTP's
Report
of
the
Endocrine
Disruptors
Low
Dose
Peer
Review,
2000).
Purpose
To
summarize
the
interspecies
and
intraspecies
similarities
and
differences
in
response
to
endocrine
endpoints,
in
order
to
determine
whether
specific
species/
strains
should
be
preferred
or
avoided
when
screening
for
endocrine
activity.
Literature
Search
Strategy

Databases
searched
included
MedLine,
PubMed,

Biological
Abstracts,
Chemical
Abstracts,
Toxline
including
DART
(
Developmental
and
Reproductive
Toxicology)
for
published
articles/
abstracts

For
intraspecies
comparisons,
the
focus
was
on
"
rat
strain."
When
there
was
a
paucity
of
references
pertaining
to
a
general
endocrine
endpoint,
"
mouse
strain"
was
added
to
the
search.
For
interspecies
comparisons,
the
focus
was
on
rats
and
mice.


Search
Terms:
"
rat
strain"
and
keywords
from
EDSP
protocols,
in
addition
to
specific
strains
and
specific
authors
Scope

Endocrine
endpoints
in
assays
under
consideration
by
EDSP

Intraspecies
and
interspecies
studies
conducted
in
a
single
laboratory
(
to
minimize
confounders);
since
these
studies
were
few,

multiple
laboratory
comparisons
were
also
used
when
necessary
and/
or
appropriate.


Published
data

Focus
on
rat
strains
Inbred
Versus
Outbred
Strains
Inbred

>
20
generations
of
inbreeding

Known
genetic
background

Less
variable
response
to
EACs

Small
litter
size

Less
historical
data
Outbred

<
1%
inbreeding/
generation

Variable
genetic
background

More
diverse
responses
to
EACs

Large
litter
size
(
due
to
selection
for
high
fecundity)


More
historical
data
Confounders
Affecting
Comparisons
of
Reproductive
Toxicity
Data
Same
laboratory,
different
times
or
different
laboratories.


Animals

Source/
supplier
(
the
same
strain
from
different
suppliers
will
most
likely
be
genetically
different)


Age,
weight,
and
health
status

Husbandry

Housing

Caging/
water
bottles

Feed
and
Water

Temperature
and
relative
humidity
and
Light
cycle

Technician
skills
and
experience

Source
of
the
test
material

Study
Design

Number
of
animals/
dose
groups,
dose
levels,
vehicle,

route

Data
(
how
collected
and
analyzed)
Endocrine
Endpoints
in
EDSP
Assays

Fertility
and
Gestational
Indices

Survival
and
Growth
Indices

Reproductive
Tract
Development

Urethral
Vaginal
Distance
(
UVD)


Vaginal
Patency
in
Females

Age
of
First
Estrus
in
Females

Estrous
Cyclicity

Uterine
Weight

Anogenital
Distance
(
AGD)


Retention
of
Nipples/
Areolae
in
Preweanling
Males

Preputial
Separation
in
Males

Sex
Accessory
Structures

Andrology

Behavioral
Assessments
(
Clinical
Observations)


Hormonal
Controls

Gross
Examinations

Organ
Weights
and
Histopathology
Summary
of
Agent­
and
Endpoint­
Specific
Intraspecies
Differences
14
LE
>
Wistar
vinclozolin
7
SD,
LE
flutamide
7
LE
SD
p,
p'­
DDE
Nipple
retention
7
SD,
LE
flutamide
7
SD
LE
p,
p'­
DDE
AGD
6
F344
SD
tamoxifen
6
SD,
F344
E2
4
F344
SD
D4
3
SD,
F344
EE,
DES
2
AP>
SD
NP
1
Wistar,
SD
Da/
Han
BPA
1
SD
Wistar,
Da/
Han
EE
Uterine
Weight
References
(
from
Table
2
of
the
white
paper)

Less
Sensitive/
Insensitive
Strains
Sensitive
Strains
Chemical
Endocrine
Endpoint
37
Jcl:
ICR
mouse
CD­
1
mouse
DEHP
39
ICR,
CD­
1,
S15
mouse
B6,
C17/
Jls
mouse
E2
38
C57BL/
6N
mouse
E2
38
C57BL/
6N,
ICR
mouse
BPA
14
Wistar
LE
vinclozolin
8
F344
SD
low
dose
E2
8
F344,
SD
E2
7
LE,
SD
flutamide
Male
reproductive
organ
wts.
9
SD
AP
BPA
7
SD,
LE
p,
p'­
DDE
VO
7
SD,
LE
p,
p'­
DDE
8
F344,
SD
E2
PPS
References
(
from
Table
2
of
the
white
paper)

Less
Sensitive/
Insensitive
Strains
Sensitive*

Strains
Chemical
Endocrine
Endpoint
Summary
of
Agent­
and
Endpoint­
Specific
Intraspecies
Differences
(
Continued)
13
LE
(
FSH,
Prl,

LH)

SD
(
FSH,
E2,
T4)

p,
p'DDE
Hormone
Levels
39
CD­
1,
S15
mouse
B6,
C17/
Jls
mouse
E2
17
SD
lead
15
SD
AP
BPA
Andrology
31
SD
F344
BDCM
30
SD,
LE
F344
atrazine
29
F344,
SD,
LE
Holtzman
atrazine
Fertility/
gestational
effects
22
F344
SD
atrazine
21
SD
LE
atrazine
18
SD,
LE
F344,
BN
feed
restriction
Estrous
cycle/
ovulation
References
(
from
Table
2
of
the
white
paper)

Less
Sensitive/

Insensitive
Strains
Sensitive*

Strains
Chemical
Endocrine
Endpoint
Summary
of
Agent­
and
Endpoint­
Specific
Intraspecies
Differences
(
Continued)
28
F344
(
T3)

TSH,
TRH
28
SD
(
T3)

SD,
F344
(
T4)

TSH,
TRH
27
LE
(
T4)

TCDD
25
SD
(
Prl)

F344
(
Prl)

BPA
25
SD
(
Prl)

F344
(
Prl)

E2
26
SD
(
E2,
P)

Holtzman
(
P)

atrazine
24
SD
(
LH,
Prl)

LE
(
LH,
Prl)

atrazine
23
LE
(
T,
LH)

Han/
Wistar
(
T,
LH)

TCDD
22
F344
(
Prl)

SD
(
Prl)

E2
13
SD
(
Prl,
LH,
T,

DHT,
TSH)

LE
(
E2,
T4,
T,
DHT,

TSH)

p,
p'DDE
13
LE
(
FSH,
Prl,
LH)

SD
(
FSH,
E2,
T4)

p,
p'DDE
Hormone
Levels
References
(
from
Table
2
of
the
white
paper)

Less
Sensitive/
Insensitive
Strains
Sensitive*
Strains
Chemical
Endocrine
Endpoint
Summary
of
Agent­
and
Endpoint­
Specific
Intraspecies
Differences
(
Continued)
22
F344
(
females)

SD
(
females)

atrazine
40
WF
(
cadmium)

F344
(
females)

cadmium
14
Wistar
(
males)

LE
(
males)

vinclozolin
12
Wistar
(
males)

F344>
ACI>
Lewis>
CD
(
males)

DMAB
10
SD
(
females)

F344
(
females)

BPA
Histopathology
(
reproductive
organs)
35
SD,
BN
F344
DES
34
Wistar,
Donryu
F344>
BN
E2
33
SD
F344
E2
Pituitary
Weights
References
(
from
Table
2
of
the
white
paper)

Less
Sensitive/

Insensitive
Strains
Sensitive*
Strains
Chemical
Endocrine
Endpoint
Summary
of
Agent­
and
Endpoint­
Specific
Intraspecies
Differences
(
Continued)
1.
Diel
et
al.,
2001
2.
Odum
et
a.,
1999a
3.
Steinmetz
et
al.,
1998
4.
McKim
et
al.,
2001
5.
Christian
et
al.,
1998
6.
Bailey
et
al.,
2002
7.
You
et
al.,
1998
8.
Putz
et
al.,
2001
9.
Tinwell
et
al.,
2002
10.
Long
et
al.,
2000
11.
Gray
and
Ostby,
1995
12.
Shirai
et
al.,
1990
13.
O'Connor
et
al.,
1999
14.
Hellwig
et
al.,
2000
15.
Tinwell
et
al.,
2000
16.
Wilkinson
et
al.,
2000
17.
Apostoli
et
al.,
1998
18.
Putz
et
al.,
2001
19.
Tropp
et
al.,
2001
20.
Ando­
Lu
et
al.,
1998
21.
Cooper
et
al.,
2000
22.
Eldridge
et
al.
1994;
Smith
et
al.,
1994
23.
Haavisto
et
al.,
2001
24.
Cooper
et
al.,
2000
25.
Steinmetz
et
al.,
1997
26.
Cummings
et
al,
2000
27.
Pohjanvirta
et
al.,
1989
28.
Fail
et
al.,
1999
29.
Cummings
et
al.,
2000
30.
Narotsky
et
al.,
2001
31.
Bielmeier
et
al.,
2001
32.
Liberati
et
al.,
2002
33.
Schechter
et
al.,
1987
34.
Yin
et
al.,
2001
35.
Wendell
et
al.,
1996,
1997,
1998,
2000;

Chun
et
al.,
1998
36.
Rehm
and
Waalkes,
1988
37.
Oishi
et
al.,
1993
38.
Nagao
et
al.,
2002
39.
Spearow
et
al.,
1999;
2001
40.
Rehm
and
Waalkes,
1988
References
for
Summary
Tables
Rat
Interstrain
Comparisons
(
based
on
current
data)

Outbred

Uterine
weight
affected
by
many
chemicals

AGD
and
nipple
retention,

sensitive
in
some
(
depending
on
chemical)


Male
reproductive
organs
affected
by
variety
of
chemicals

Effects
on
hormone
levels
are
dependent
on
the
hormone
measured
and
chemical
Inbred

Greater
effects
of
chemicals
on
pituitary
weight

Uterine
weight
less
affected

More
sensitive
to
gestation/
fertility
effects

Effects
on
hormone
levels
are
dependent
on
the
hormone
measured
and
chemical

Comparisons
based
mostly
on
F344
strain
(
little
data
in
other
inbred
strains)
Interspecies
Similarities
and
Differences

Few
studies
have
been
conducted
in
a
single
laboratory
comparing
the
effects
of
endocrine­
disrupting
chemicals
in
more
than
one
strain
within
a
species,
and
even
fewer
studies
have
been
conducted
in
a
single
laboratory
comparing
the
effects
of
endocrine­
disrupting
chemicals
in
more
than
one
species.


Difficult
to
compare
species
when
variability
across
strains
within
a
species
is
high

NTP
studies
of
the
effects
of
24
different
chemicals
on
male
reproductive
parameters
in
B6
mice
and
F344
rats
across
7
labs
show
a
58%
correlation
in
response
to
reproductive
toxicants
(
even
with
the
same
rat
and
mouse
strains).
Genetic
Differences
in
Response
to
Endocrine­
Active
Chemicals
Chemical
Genotype
Endpoint
Effect
There
are
strain
(
genotype)
by
environmental
agent
by
endpoint
interactions.
These
need
to
be
considered
in
selecting
the
appropriate
species/
strains
for
EDSP
assays.
Conclusions

Comparisons
revealed
variability
in
effects
produced
by
endocrinedisrupting
chemicals
on
endocrine
endpoints
from
strain
to
strain.

Endocrine
effects
were
chemical
specific,
strain
specific,
endpoint
specific,
and,
in
some
cases,
laboratory
specific.
There
were
more
sensitive
and
less
sensitive
strains
to
endocrine­
active
compounds
among
both
outbred
and
inbred
strains,
depending
on
the
chemical
used
and
the
endpoints
evaluated.


Inbred
strains
are
homogeneous
at
all
loci,
and
have
a
limited
range
of
responses
(
less
variability,
but
an
effect
may
be
missed),
so
using
several
genetically­
defined
inbred
strains
in
endocrine
screens
may
be
the
only
way
to
provide
a
broad
spectrum
of
responsivity.
If
selecting
a
single
strain
for
endocrine
screens,
outbred
strains
have
more
genetic
variability,
exhibit
a
broader
range
of
responsivity
(
with
a
greater
likelihood
of
detecting
an
effect),
and
may
be
more
appropriate.
Outbred
strains,
which
are
heterogeneous
like
humans
and
other
species
of
interest,
may
provide
a
more
appropriate
animal
model
for
determining
the
effects
of
EACs.
Conclusions
(
continued)


Since
the
actions
of
EACs
were
generally
observed
for
more
than
one
endpoint,
there
is
a
greater
likelihood
of
detecting
an
endocrine
disruptor
in
a
study
with
many
endpoints.


In
current
OECD
and
EPA
validation
efforts
for
the
Uterotrophic
and
Hershberger
Assays
(
looking
at
many
of
the
same
endpoints),
there
was
no
effect
on
responsivity
of
different
strains
(
housing,
feed,
bedding,
etc.)
with
potent
androgens
and
estrogens.
Acknowledgements
Shelley
Tyl
(
RTI)

Bonnie
Hamby
(
RTI)

Carol
Sloan
(
RTI)

Jim
Kariya
(
EPA)

Jimmy
Spearow
(
UC
Davis)

Dave
Houchens
(
Battelle)

Jerry
Johnson
(
Battelle)
