scientific insights: endocrine disruption: uterotrophic and hershberger assay validation and...
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7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience
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Endocrine disruption: Uterotrophic and
Hershberger assay validation and
experience
The endocrine system regulates all biological
processes throughout life including the function of
the reproductive systems. Endocrine disruptors are
chemicals that interfere with the endocrine (or hormone
system) in animals, including humans. Although there
has been much debate in the scientic community
since the term endocrine disruptor was rst coined in1991, the devastating consequences of exposure to
chemicals that interfere with the reproductive systems
cannot be ignored.
Up until its ban in the 1970s, diethylstilboestrol (DES)
was being widely prescribed to pregnant women to
block spontaneous abortion and to promote foetal
growth. Only after the offspring reached puberty did
the devastating consequences become clear as DES
affected the reproductive systems and also caused
vaginal cancer. Although this was an extreme case
whereby a potent synthetic oestrogen was deliberately
administered, evidence has been accruing of
effects caused by low-level exposure to both wildlife
and humans. This includes strong evidence that
chemical exposure has been associated with adverse
developmental and reproductive effects on sh and
wildlife in specic locations.
As a consequence, the USA, Japan, EU and OECD
have been looking at establishing testing approaches
with the aim of assessing the risks associated with
chemicals that potentially have endocrine disrupting
properties. One of the leading and rst nationally,
legally binding programs was the endocrine disruptor screening program (EDSP) of the USA.
Endocrine disruptor screening
program (EDSP)The US Environmental Protection Agency (EPA) adopted
a two tier approach to testing endocrine disruptors. Tier
1 consists of a battery of eleven in vitro and in vivo tests
which, as a whole, allow the detection of oestrogen
and androgen-mediated effects by various modes of
action (see Table 1). Once a chemical is identied as
a potential endocrine disruptor during Tier 1 screening,the EPA will request additional testing in order to further
assess the compound and it’s interaction with the
oestrogen, androgen or thyroid hormonal systems. To
date no denitive designs have been decided upon.
HLS is able to complete the full battery of Tier 1 tests
and specically the Department of Pharmacology runs
the Uterotrophic and Hershberger assays. Although the
assays had been run many times previous to the EPA
requirements, the EPA also published test guidelineswith very specic methodologies and it was therefore
decided to re-validate both assays in accordance with
the specic guidance.
Tier 1 in vivo Studies
Hershberger Bioassay
The Hershberger Bioassay serves as an in vivo
screening assay for both androgen agonists and
antagonists by assessing changes in the weight of ve
androgen-dependent tissues – the ventral prostate (VP),seminal vesicles (SV), levator ani-bulbo cavernosus
(LABC) muscle, paired Cowper’s glands (COW) and
the glans penis (GP). These ve tissues all respond to
androgen agonists with an increase in absolute weight,
whilst antagonists cause a decrease in weight (following
pre-treatment with a potent reference androgen). A
positive androgen agonist result, at any dose level,
is considered to have occurred when a statistically
signicant increase, in two or more of the ve androgen
dependent tissue weights compared to the vehicle
control, is observed and all the target tissues display
some degree of increased growth. A positive androgenantagonist result is considered to have occurred when
a statistically signicant reduction, in two or more of the
ve androgen dependent tissue weights compared to
the vehicle control group, is observed and all the target
tissues displayed some degree of reduced growth.
The primary model for the Hershberger Bioassay is
the surgically castrated male rat, this ensures males
with minimal levels of circulating androgens. The
hypothalamic-pituitary-gonad (HPG) axis is rendered
unable to compensate via feedback mechanisms and
thus the ability of the tissues to respond is maximised
and starting tissue weight variability is minimized. Theintact (uncastrated) stimulated male is an alternative
model, to avoid the castration step; however this
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Table 1: US-EPA’s Tier 1 Battery and modes of action detected
Screening assays Receptor Binding Steroidogenesis HPG-axis3 HPT-axis3
O2 Anti-O2 A2 Anti-A2 O-production2 A-production2
In vivo
Uterotrophic x
Hershberger x x x
Pubertal Male x x x x x
Pubertal Female x x4 x x x
Amphibian Metamoprhosis x
Fish Short-term
Reproduction
(male and female)
x x4 x x x x x
In vitro
ER binding1 x x4
ERα Transcriptional
Activation
x
AR Binding1 x x
Steroidogenesis H295R x x
Aromatase Recombinant x
1 Oestrogen and Androgen Receptor binding2 Oestrogen and Androgen3 Hypothalamic-pituitary-gonadal or -thyroidal axis4 Assays are expected to detect anti-oestrogens, but this was not established during the validation process since no oestrogen receptor
antagonists were tested
model is not as sensitive, starting tissue weight is morevariable and it is not able to consistently detect effects
from weak anti androgens. The validation study for the
Hershberger Bioassay at Huntingdon Life Sciences
(HLS) was therefore performed in the surgically
castrated male rat.
For both the androgenic and anti-androgenic phases,
Sprague-Dawley rats were obtained from an approved
supplier, allowed an initial acclimatisation period of
7 days to ensure the animals were healthy and then
the rats were castrated, at ages of 42-48 days, using
approved anaesthetics and aseptic techniques. The ratswere then allowed a further 9-10 days acclimatisation to
allow for recovery from surgery and regression in the
target tissue weights.
In Phase 1 (androgenic phase) testosterone propionate
(0.1 - 1 mg/kg/day) was administered daily by sub-
cutaneous injection for 10 consecutive days. In Phase 2
(anti-androgenic phase) utamide (0.3 - 10 mg/kg/day)
was administered daily by oral gavage in combination
with daily subcutaneous injections of testosterone
propionate (0.4 mg/kg/day) for 10 consecutive days.
Doses were selected on the basis of EPA guidelines,
previous experience and the scientic literature. In both
phases animals were aged 54-58 days at the start of dosing. Approximately 24 hours after the last dose,
animals were killed and the ve androgen-dependent
tissues excised, carefully trimmed of excess adhering
tissues and fat and weighed without blotting to the
nearest 0.1 mg. Animals were aged 64-68 days at
necropsy.
Testosterone propionate, at subcutaneous doses of
0.1 - 1 mg/kg/day, induced dose-dependent positive
androgen agonist activity, with statistically signicant
increases in all ve androgen-dependent tissues
recorded compared with vehicle-treated control animals
(see Figure 1).
In general, the Hershberger Bioassay is able to detect
statistically signicant effects following changes in sex
organ weights of around 20% to 50% (depending upon
the tissue) and above.
Flutamide, at oral doses of 0.3 - 10 mg/kg/day, induced
dose-dependent positive androgen antagonist activity
in castrated rats. There were statistically signicant
decreases in three out of the ve androgen-dependent
tissues at 0.3 mg/kg/day, in four out of the ve tissues at
1, 3 and 5 mg/kg/day and in all ve androgen-dependent
tissues at 10 mg/kg/day (see Figure 2).
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Figure 1: Effects of subcutaneous administration of testosterone
propionate on seminal vesicle (SV), ventral prostate (VP),
paired Cowper’s glands (COW), levator ani-bulbocavernosus
LABC) and glans penis (GP) weights in castrated rats
Statistical signicance: p<0.05 for GP at 0.1 and p<0.001 at 0.2-1 mg/kg/day.Statistical signicance: p<0.01 for LABC and COW at 0.1 and p<0.001 at
.2-1 mg/kg/day.
Statistical signicance: p<0.001 for SV and VP at 0.1-1 mg/kg/day.
Figure 2: Effects of oral administration of utamide on seminal
vesicle (SV), ventral prostate (VP), paired Cowper’s glands
COW), levator ani-bulbocavernosus (LABC) and glans penis
GP) weights in castrated rats
Statistical signicance: p<0.05 for VP, LABC and COW at 0.3 and p<0.001 at
-10 mg/kg/day.
Statistical signicance: p<0.001 for SV at 1-10 mg/kg/day.
Statistical signicance: p<0.01 for GP at 10 mg/kg/day.
Sprague-Dawley rats were obtained from an approved
supplier, allowed an initial acclimatisation period of 14
days to ensure the animals were healthy and then the
rats were ovariectomised, at ages of 43-50 days, using
approved anaesthetics and aseptic techniques. The rats
were then acclimatised for a further 14 days to allow
for recovery from surgery and regression in the uterine
tissues. Completeness of ovariectomy was conrmed byswabbing epithelial cells of the vagina for 5 consecutive
days (starting on Day 9 or 10 and nishing on Day 14
after ovariectomy).
17 alpha-ethynyl oestradiol (0.01 - 10 mg/kg/day)
was administered daily by subcutaneous injection for
4 consecutive days. Approximately 24 hours after the
last dose, animals were killed and the uterine tissue
carefully dissected and examined for the presence of
ovarian tissue and the wet and blotted uterine weights
were recorded to the nearest 0.1 mg.
17 alpha-ethynyl oestradiol, at subcutaneous doses of
0.01 - 10 mg/kg, induced positive oestrogen agonistactivity in ovariectomised rats in this study. There were
marked, statistically signicant increases in both wet
and blotted uterine weights compared with vehicle-
treated control animals (see Figures 3 - 4).
In general, the uterotrophic assay is able to detect
statistically signicant effects following changes in
uterus weights of approximately 25% and above.
Figure 3: Effects of subcutaneous administration of
17 alpha-ethynyl oestradiol on wet uterine weights in
ovariectomised rats
Figure 4: Effects of subcutaneous administration of 17 alpha-ethynyl oestradiol on dry uterine weights in
ovariectomised rats
0.0
50.0
100.0
150.0
200.0
250.0
0
300
600
900
1200
1500
1800
0.03 0.3 3
P e r c e n t a g e c h a n g e f r o m v e h i c l e
( L A B C a n d G P )
P e r c e n t a g e c h a n g e f r o m v e h i c l e
(
S V , V
P a n d C O W )
Dose (mg/kg/day)
SV
VP
COW
LABC
GP
SV
VPCOW
LABC
GP
-100
-80
-60
-40
-20
0
20
0.1 1.0 10.0
Percentagechangefromvehicle
Dose (mg/kg/day)
SV
VP
LABC
COW
GP
0
200
400
600
800
0.01 0.1 1 10
P e r c e n t a g e c h a n g e f r o m v
e h i c l e
Dose (mg/kg/day)
Wet uterus weight
0
50
100
150
200
0.01 0.1 1 10
P e r c e n t a g e c h a n g e f r o m v
e h i c l e
Dose (mg/kg/day)
Dry uterus weightUterotrophic AssayThe Uterotrophic assay serves as an in vivo screening
assay for oestrogen agonists by assessing effects on
wet and dry (blotted) uterus weights. The validation study
for the Uterotrophic assay at Huntingdon Life Sciences
was performed in the surgically ovariectomised female
rat. The Uterotrophic assay relies for its sensitivity on an
animal test system in which the hypothalamic-pituitary-
gonad (HPG) axis is not functional, leading to low levels
of circulating oestrogen, ensuring low baseline uterine
weights and a maximum response to administered
oestrogens. Although the immature female after
weaning could be used rather than the ovariectomisedrat, it is not as sensitive and is not able to consistently
detect effects from weak oestrogen agonists.
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Conclusion At HLS, from validation studies and sponsor studies
conducted so far, the Department of Pharmacology
has demonstrated that we are able to performed two
of the Tier 1 tests as dened by the EPA’S endocrine
disruptor screening programme (Hershberger and
Uterotrophic assays). These assays have been shown
to be relatively sensitive, being able to pick up changesin tissue weights of around 20-30% as being statistically
signicant. The data produced by these assays give an
indication of direct receptor mediated androgenic, anti-
androgenic and steroidogenesis activity (Hershberger
assay) and direct receptor mediated oestrogenic activity
(uterotrophic assay).
Any positive results would need to be considered in
conjunction with other results from the Tier 1 program,
as the battery of tests as a whole is designed to
allow detection of oestrogen and androgen-mediated
effects, by various modes of action, including receptor
binding, transcriptional activation, steroidogenesis andhypothalamic-pituitary-gonadal feedback. In September
2011, the EPA issued its nal guidance on the weight of
evidence (WoE) analysis that it will use to evaluate the
data submitted in response to test orders issued for Tier
1 screening. This WoE analysis, in turn, will be used
Key Points
• The US EPA’s Tier 1 endocrine disruption screeningprogram consists of eleven assays, six in vivo and ve in vitro, designed to screen substancesfor interactions with the oestrogen, androgen andthyroid hormone systems.
• The Department of Pharmacology at HLS havedeveloped two of the in vivo tests and have
sensitive assays for the Hershberger Bioassay andUterotrophic assay.
• Any positive results will need to be assessed inconjunction with the other tests in the program usingthe EPA’s weight of evidence approach.
to determine what substances merit further evaluation
under Tier 2 as substances that have the potential to
interact with oestrogen, androgen, or thyroid hormonal
pathways.