7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience

http://slidepdf.com/reader/full/scientific-insights-endocrine-disruption-uterotrophic-and-hershberger-assay 1/5

You can read more of our 

articles, posters and casestudies on our website

www.huntingdon.com

Crop protection

Chemical

Pharmaceuticals

Biologics

Endocrine disruption:

Uterotrophic and

Hershberger assay

validation and

experience

Scientifc insights

Bringing you articles full of in-depth

scientic insight on subjects ranging from

pharmaceutical development through to crop

protection registrations.

UK 

sales@ukorg.huntingdon.com

USA

sales@priceton.huntingdon.com

 Japan

+81 (0) 3 3238 6381-7

7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience

http://slidepdf.com/reader/full/scientific-insights-endocrine-disruption-uterotrophic-and-hershberger-assay 2/5

2

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

7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience

http://slidepdf.com/reader/full/scientific-insights-endocrine-disruption-uterotrophic-and-hershberger-assay 3/5

3

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).

7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience

http://slidepdf.com/reader/full/scientific-insights-endocrine-disruption-uterotrophic-and-hershberger-assay 4/5

4

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.

7/29/2019 Scientific insights: Endocrine disruption: Uterotrophic and Hershberger assay validation and experience

http://slidepdf.com/reader/full/scientific-insights-endocrine-disruption-uterotrophic-and-hershberger-assay 5/5

5

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.