OECD validation program of the H295R
Steroidogenesis Assay to screen for
inhibitors and inducers of sex steroid
hormone production.
Markus Hecker &
G. Timm, A.-M. Vinggaard, C. Nelleman, Y.
Akahori, R. Cooper, H. Hollert, S-Y. Han, M. B.
Murphy, J. L. Newsted, J. P. Giesy
www.entrix.com
U.S. Endocrine Disruptor Screening Program
Universe of chemicals - 87,000 chemicals
900 pesticide active ingredients
2,500 other pesticide formulate ingredients
75,500 industrial chemicals
8,000 cosmetics, food additives and nutritional
supplements
Initial focus on:
Pesticide actives
High production volume inerts & high throughput
U.S. Endocrine Disruptor Screening Program
Tier 1
 In vitro and in vivo screens
 Detect potential to interact with endocrine
system
Tier 2
 Multi-generation studies covering a broad range
of taxa
 Provide data for hazard assessment
OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals
Level 1
Sorting & prioritization based
upon existing information
Level 2
In vitro assays providing
mechanistic data
Level 3
In vivo assays providing data about single
endocrine Mechanisms and effects
Level 4
In vivo assays providing data
about multiple endocrine
mechanisms and effects
Level 5
In vivo assays providing data on effecst
from endocrine & other mechanisms
•physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability
•human & environmental exposure, e.g., production volume, release, use patterns
•hazard, e.g., available toxicological data
•ER, AR, TR receptor binding affinity
•High Through Put Prescreens
•Transcriptional activation
•Thyroid function
•Aromatase & Steroidogenesis
in vitro in vitro •Fish hepatocyte VTG assay
•Aromatase
& Steroidogenesis
•Aryl hydrocarbon receptor recognition/binding •QSARs
• Others (as appropriate)
•Uterotrophic Assay (estrogenic related)
•Hershberger Assay (androgenic related)
•Non-receptor mediated hormone function
•Fish VTG assay
(estrogenic related)
•Others (e.g. thyroid)
•Enhanced OECD 407 (endpoints based
on endocrine mechanisms)
•Male and female pubertal assays
•Adult intact male assay
•Fish gonadal histopathology
assay
•Frog metamorphosis assay
•1-generation assay (TG415 enhanced)
•Partial and full life cycle assays in
fish, birds, amphibians &
•2-generation assay (TG416 enhanced)
invertebrates (develop. & reprod.)
•Reproductive screening (TG421 enhanced)
•Combined 28 day/reproduction screening test (TG 422 enhanced)
Note: Document prepared by the Secretariat of the Test Guidelines Programme based on the agreement reached at the 6th Meeting of the EDTA Task Force
Level 2 (Tier 1) — In Vitro, Mechanistic Assays
Assay Type
OECD Japan U.S. Status
Rat cytosol
ER Binding
hr Recombinant
√
√
ERTA CERI
√
√
rr Recombinant
√
√
Aromatase
√
KGN Cell
Steroidogenesis
H295R
√
√
Ф Pre-validation completed ● Validation completed
●
√
√
Microsomal
Ф
Ф
●
√
Rat cytosol
AR Binding
√
√
●
●
√
Ф
H295R Cell Line
CYP11A
Cholesterol
CYP17
CYP17
Pregnenolone
3β-HSD
3β-HSD
CYP17
Progesterone
CYP21
11-Deoxycorticosterone
CYP11B2
Corticosterone
CYP11B2
Aldosterone
DHEA
17α-OH Pregnenolone
3β-HSD
CYP17
17α-OH Progesterone
CYP21
Deoxycortisol
CYP11B1
Cortisol
Androstenedione
17β-HSD
CYP19
Testosterone Estrone
CYP19
17β-HSD
17β-estradiol
H295R Cell Line
The H295R Cell Line has been shown to detect
effects of chemicals on the production of
steroid hormones in a manner that was
consistent with the mode of action of the
model chemicals. a
The observed changes were also consistent
with observed effects of these chemicals in
various animal models. a
a Hecker
et al. 2006
H295R Steroidogenesis Assay
Seed plate at 300,000 cells/mL &
incubate at 37°C for 24 h
Hormone Analysis
Dose cells after 24 h at 37oC
& incubate at 37°C for 48 h
Remove medium &
extract in preparation
for hormone analysis
Objectives
 Test the revised H295R Steroidogenesis Assay
protocol at seven independent laboratories;
 Compare the specific changes in the production
of T and E2 in response to 12 model compounds
with different modes of interaction with
steroidogenic pathways (weak, medium and
strong inhibitors and inducers; negative
chemicals);
 Validate the quality control criteria to be met
when conducting the H295R Steroidogenesis
Assay;
Objectives
 Assess the transferability, reproducibility,
sensitivity and applicability of the assay;
 Identify and validate appropriate data evaluation
procedures;
 Apply the developed protocols and evaluation
procedures to 16 supplemental chemicals with
largely unknown types of interaction with the
production of T and E2.
Participating Laboratories
 US Environmental Protection Agency
Endocrinology Laboratory, U.S.A.
 Chemicals Assessment Center
Chemical Evaluation and Research Institute, Japan
 National Intitute of Toxicological Research
Korea Food and Drug Administration, Korea
 Danish Institute for Food and Veterinary Research
Department of Toxicology and Risk Assessment, Denmark
 Institute of Zoology, University of Heidelberg, Germany
 Center of Coastal Pollution and Conservation, City
University of Hong Kong, SAR, China
 University of Saskatchewan, Saskatoon, SK, Canada
QC-Plates – Inter-Lab Comparison
All Experiments (Testosterone)
4
Lab 1
Fold-Change
Lab 2
3
Lab 3
Lab 4
2
Lab 6
1
0
SC
For1
For10
Pro0.3
All Experiments (Estradiol)
All Experiments (Estradiol)
1000
1.4
1.2
Fold-Change
100
Fold-Change
Pro3
10
1
0.1
1
0.8
0.6
0.4
0.2
0
0.01
SC
For1
For10
SC
Pro0.3
Pro3
Testosterone – Core Chemical Examples
Prochloraz
Forskolin
2
6
Lab3
Lab4
Lab1
Lab6
T fold-change (SC=1)
Lab2
4
2
Lab2
Lab3
Lab4
Lab6
1.5
1
0.5
0
0
0.0001
0.001
0.01
0.1
µM
1
10
0.0001
100
0.001
Nonoxynol-9
Lab1
Lab2
Lab3
Lab4
0.01
µM
0.1
Lab6
2
1
0
0.0001
0.001
0.01
0.1
µM
3
T fold-change (SC=1)
T fold-change (SC=1)
Lab1
1
1
10
Estradiol – Core Chemical Examples
Forskolin
Letrozole
1000
Lab1
100
E2 fold-change (SC=1)
Lab2
Lab3
Lab4
10
Lab6
1
0.1
1.2
Lab2
Lab3
0.9
Lab4
Lab6
0.6
0.3
0
0.0001
0.001
0.01
0.1
µM
1
10
100
0.0001
0.001
0.01
EDS
4
Lab1
E2 fold-change (SC=1)
E2 fold-change (SC=1)
Lab1
Lab2
Lab3
Lab4
Lab6
3
2
1
0
0.0001
0.001
0.01
0.1
µM
1
10
100
0.1
µM
1
10
100
Fold-Change/LOEC - Testosterone
Fold-Change (Testosterone)
a
Prochloraz
Aminoglutethimide
Letrozole
Nonoxynol-9
Molinate
Benomyl
EDS
HCG
Paraben
Atrazine
Forskolin
Trilostane
a
Lab 1
LOEC
0.0001
100
100
10
nd
nd
nd
nd
10
100
10
mu
0.1
Max Change









Lab 2
LOEC
0.1
100
100
10
nd
nd
nd
nd
nd
1
1
mu
0.01
Max Change









Lab 3
LOEC
0.0001
10
a
100
c
nd
100
nd
nd
nd
1
100
1
mu
1
Max Change








only one experiment was conducted or considered for data evaluation;
Not statistically significant; p = 0.051;
c
Cytotoxicity observed at concentration at which effects occurred at other laboratories = 10µ
b
Lab 4
LOEC
0.001
100
100
10
nd
mu
nd
nd
nd
nd
nd
1
mu
1
Max Change









Lab 6
LOEC
0.01
100
100
10
nd
nd
nd
nd
nd
nd
1
mu
0.01
Max Change









Fold-Change/LOEC - Estradiol
Fold-Change (Estradiol)
a
Lab 1
LOEC
Letrozole
Prochloraz
Aminoglutethimide
Benomyl
EDS
Nonoxynol-9
HCG
Paraben
Molinate
Atrazine
Forskolin
Trilostane
a
0.001
0.1
100
nd
nd
nd
nd
mu
nd
100
10
mu
0.01
mu
1
Max Change






mu




Lab 2
LOEC
0.001
1
mu
10
nd
nd
nd
nd
10
100
0.001
mu
0.1
100
Lab 3
Max Change
LOEC
Max Change
mu

 0.0001


0.1


10
a

nd
nd

nd
a

nd
mu


10
mu


100
mu


1

 0.1 mu
mu


0.1
only one experiment was conducted or considered for data evaluation;
Not statistically significant; p = 0.051;
c
Cytotoxicity observed at concentration at which effects occurred at other laboratories = 10µ
b
Lab 4
LOEC
0.01
1
b
100
a
nd
nd
nd
a
nd
nd
mu
100
mu
10
mu
0.1
mu
1
Max Change








Lab 6
LOEC
0.01
0.1
100
nd
nd
nd
nd
nd
100
0.1
mu
0.01
mu
1
Max Change








Testosterone – Supplemental Chemicals
Testosterone
LOEC
st
1 Lab
Ketoconazole
Bisphenol A
Piperonyl butoxide
Genistein
Finasteride
Dinitrophenol
Spironolactone
Fenarimol
Danazol
DEHP
Dimethoate
Flutamide
Glyphosate
Prometon
Tricrecyl phosphate
Mifepristone
a
1
10
10
10
10
0.0001
1
2
nd
Lab
1
10
10
10
100
100
st
1 Lab
Max Change
nd
2 Lab














10

10
0.1





considered because clear concentration-response at all but the greatest concentration
Inducers
Inhibitors
≤ 1 µM or 
≤10 µM or 
≤100 µM or
Max concentration (>20%) or
Max concentration (<20%)
No effect
≤ 0.01 µM or 
≤ 1 µM or 
≤ 10 µM or 
≤ 100 µM or 
Max concentration (> 20%)
Estradiol – Supplemental Chemicals
Estradiol
st
1 Lab
Danazol
Ketoconazole
Fenarimol
Finasteride
Glyphosate
Dinitrophenol
Spironolactone
Piperonyl butoxide
Dimethoate
Tricrecyl phosphate
Flutamide
Prometon
Bisphenol A
DEHP
Mifepristone
Genistein
a
1
10
10
10
10
100
10
1a
0.1
10
LOEC
nd
2 Lab
10
10
1
100
100
1
1
1
10
Max Change
nd
1 Lab
2 Lab
st



















considered because clear concentration-response at all but the greatest concentration
Inducers
Inhibitors
≤ 1 µM or 
≤10 µM or 
≤100 µM or
Max concentration (>20%) or
Max concentration (<20%)
No effect
≤ 0.01 µM or 
≤ 1 µM or 
≤ 10 µM or 
≤ 100 µM or 
Max concentration (> 20%)
Conclusions
 Relative dose-response profiles are comparable for
all model compounds tested across laboratories
 The assay can discern between strong, medium and
weak inducers and inhibitors of T and E2
production as well as negative responses
Some variation between laboratory data
 Method detection limits vary -> difficulties to directly
compare changes (decreases) that are close or below MDL
 Some lab specific technical issues with cell viability assays
Overall Conclusions
The H295R Steroidogenesis Assay is a rapid,
economic, reliable, and cost effective
screen of chemicals for their potential to
alter Steroidogenesis (priority setting, Tier
1/Level 2 screening)
Data suggest good transferability of the
assay
Overall Conclusions – Comparison to In Vivo Data
 The H295R Steroidogenesis Assay was predictive of in
vivo effects reported for changes in hormone
concentrations or other related endpoints related to
reproduction (e.g. Hershberger)
 The H295R Assay always detected an effect for
chemicals that were reported to interfere with
hormone production/concentrations in vivo
 Some of the trends in the responses were not
comparable (e.g. increase vs. decrease in T production
after exposure to aromatase inhibitors)
 There was only one chemical for which both in vivo
and in vitro data were available that would have been
characterized as false positives by the H295R Assay.
Overall Conclusions – Comparison to In Vivo Data
Chemical
Testosterone
In Vivo
H295R
Estradiol
H295R
In Vivo



Aminoglutethimide

Atrazine
/Benomyl


Bisphenol A
n.d.

Butyl paraben

Danazol
n.d.

DEHP
Dimethoate
n.d.
Dinitrophenol
n.d.
EDS
n.d.



Fenarimol
Finasteride

Flutamide


Forskolin
n.d.



Genistein
Glyophosate
-d
HCG



Ketoconazole



Letrozole



Mifepristone

Molinate
n.d. c
Nonoxynol-9
n.d.

Piperonyl butoxide
n.d.



Prochloraz

Prometon


Spironolactone

Tricrescyl phosphate
/n.d.
a
-a
Trilostane
/a
Corrected for cross-reactivity
b
Identified as reproductive toxicant in vivo
c
Data not considered because inconclusive results
d
H295R Assay does only capture effects downstream of LH/FS


n.d.
n.d.


n.d.
n.d.

n.d.
n.d.
n.d.

-d



n.d.
n.d.

-b
n.d. b

-
Species
Reference
Fish; Rat
Fish; Rat
Rat
Rat
Human
Rat
Sheep
Fish
Amphibian
Rat
Monkey; Rat
Fish
-d
Fish; Rat
Rat
Human
Rat
Amphibian; Rat
Fish
Amphibian; Rat
Rat
Fish; Human; Rat
Bergman and Laskey 1993; Monteiro et al. 2000
Spano et al. 2004; Wetzel et al 1994
Carter and Laskey 1982; Spencer et al. 1998
Taxvig et al. 2008
Murakami et al.1993; Peters et al. 1980
Davis et al. 1994; Noriega et al. 2009
Rawlings et al. 1998
Ankley et al. 2005
Canosa and Ceballos 2001
Adamson et al. 2008; Mikkilä et al. 2006
Harrison et al. 1999;Ohno et al. 2003
Soso et al. 2006
-d
Monteiro et al. 2000; O’Connor et al. 2002
Kumru et al. 2007
Fassett et al. 2008; Wang et al. 1994
Ellis et al. 1998
Vinggaard et al. 2005; Brande-Lavritsen et al. 2008
Villeneuve et al. 2006
Canosa and Ceballos 2001; Yamasaki et al. 2004
Latendresse et al. 1995
Jungmann et al. 1983; Villeneuve et al. 2008
Thanks To
 Soon Young Han (Korea Food and Drug Administration, Seoul, Korea)
 Ralph Cooper, John Laskey and Angela Buckalew (US-EPA)
 Anne-Marie Vinggaard and Christine Nellemann (Danish Institute for
Food and Veterinary Research, Denmark)
 Yumi Akahori and Makoto Nakai (Chemicals Assessment Center, Japan)
 Stefanie Grund and Henner Hollert (University of Heidelberg, Germany)
 Margaret Murphy and Rudolph Wu (City University of Hong Kong, Hong
Kong, China)
This work was facilitated through a grant from the U.S. Environmental
Protection Agency.
Thank You!
Dr. Markus Hecker
ENTRIX, Inc.
Toxicology Centre
University of Saskatchewan
44 Campus Drive, Saskatoon
SK S7N 5B3, Canada
Tel: (306) 966-5233
Cell: (517) 899-0594
Email: mhecker@entrix.com