This webinar discusses the in vitro studies required for the EPA’s Endocrine Disruptor Screening Program; we will take a closer look at each of studies, their challenges, their endpoints, their acceptance criteria and discuss our experience of the validation of the assays. We will also look at some ways in which you can ensure that your studies run smoothly and efficiently. We will close out by discussing the EDSP’s Data Evaluation Record (DER) and the Standard Evaluation Procedures (SEP). More info at http://www.huntingdon.com/Chemical/Endocrinedisruptorscreeningprogram/Webinars

1. Endocrine Disruptor
Screening Program
Webinar week
20-23 January 2014
www.huntingdon.com

2. In vitro tests for endocrine
disruption

Leslie Akhurst MSc, BSc (Hons)


Head of In vitro Safety Assessment
Yen-Ling Cheung PhD, MSc

Scientific Manager, Metabolism
www.huntingdon.com

3. IN VITRO TESTS FOR
ENDOCRINE DISRUPTION
Leslie Akhurst & Yen-Ling Cheung
www.huntingdon.com
3

4. IN VITRO TESTS FOR ENDOCRINE
DISRUPTION

Leslie
ER transcriptional activation assay
 Steroidogenesis assay


Yen
Aromatase
 ER & AR binding assays


Leslie

Summary
www.huntingdon.com
4

5. EPA Tier 1 EDSP Tests
Assays
Modes of Action Covered by Assay
Receptor binding
Steroidogenesis HPG
E Anti-E A Anti-A
T
E
HPT
In vitro
ERα Transcriptional Activation
X
ER Binding
X
X
AR Binding
X
X
Steroidogenesis
X
Aromatase
X
X
In vivo
Uterotrophic
X
Hershberger
X
X
Pubertal Male
X
X
Pubertal Female
Fish short tem reproduction
Amphibian metamorphosis
www.huntingdon.com
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
5

6. IN VITRO ED ASSAYS

Tier 1 in vitro assays are intended to provide
some mechanistic data for single known
pathways, whereas in vivo assays capture
multiple modes of action
www.huntingdon.com
6

7. ER Transcriptional Activation
Assay





OCSPP 890.1300 (Oct 2009)
OECD 455 (7 September 2009, revised 2 October 2012)
hERα-HeLa-9903 cell line
 Expresses human estrogen receptor α
 Contains firefly luciferase reporter gene
Validated by the Japanese Chemicals Evaluation and
Research Institute (CERI)
Assay designed to investigate agonist interactions, not
antagonist. Therefore ER binding assay is also needed
before concluding that the chemical does not bind to the
receptor
www.huntingdon.com
7

8. ER Transcriptional Activation Assay






96 well plate assay
Expose cells to 8 conc of test/ref substance in triplicate. Test up to 1 mM
or solubility limit of test substance.
Cytotoxicity of test materials assessed using MTT/MTS. Wells with <80%
viability not included in data analysis
A reference chemical plate is run alongside each test to monitor stability of
cell line response
 17β-estradiol (strong estrogen)
 17α-estradiol (weak estrogen)
 17α-methyltestosterone (very weak agonist)
 Corticosterone (negative)
Ligand binds to hERα → activates reporter gene → luciferase expression
measured using luminometer
The test must be conducted on at least two occasions
www.huntingdon.com
8

9. ER Transcriptional Activation Assay
Acceptability Criteria






EC50, PC50, PC10 and Hill slopes measured for each reference
chemical and should fall within the acceptability range
The mean luciferase activity of the PC should be ≥ 4-fold VC
The fold-induction corresponding to the PC10 of the PC should
be > 1±2 SD of the VC
Consistent responses must be observed on two assay
occasions
Solubility should not be exceeded and cytotoxicity should not
be ≤80% of VC
Test substance is positive if the maximum response (RPCmax)
≥ 10% of response of PC in two runs
www.huntingdon.com
9

10. ER Transcriptional Activation Assay
Acceptance Criteria
Name
logPC50
logPC10
logEC50
Hill slope
Test range
17β-Estradiol (E2)
-11.4 to -10.1
<-11
-11.3 to -10.1
0.7 to 1.5
10-14 to 10-8 M
17α-Estradiol
-9.6 to -8.1
-10.7 to -9.3
-9.6 to -8.4
0.9 to 2.0
10-12 to 10-6 M
Corticosterone
-
-
-
-
10-10 to 10-4 M
17αMethyltestosterone
-6.0 to -5.1
-8.0 to -6.2
-
-
10-11 to 10-5 M
www.huntingdon.com
10

11. OECD Validated Spreadsheet
Lab name: HLS
ID1
1.00E-05 M
Date:
ID2
14/09/2010
1.00E-04 M
Testosterone
RawData
A
B
C
D
E
F
G
H
VC_mean→
1
1312
654
515
523
515
385
401
396
2
1854
665
606
462
530
426
377
477
3
1308
551
467
498
518
441
452
438
446.3
4
ID3
6
315
326
389
327
252
213
400
336
261
364
273
242
363
342
244
454
364
416
408
373
405
391
588
388
(PC (1 nM E2) + VC)_mean→
fold-induction→
865.9
207.9
68.9
76.8
68.9
-60.9
-45.0
-50.3
1407.5
218.5
160.2
15.9
83.4
-19.9
-68.9
30.5
862.0
104.6
21.2
51.6
71.5
-5.3
5.3
-7.9
-131.1
-119.2
-46.3
-82.1
-83.4
7.9
-38.4
-55.6
(Raw - VC_mean) / PC_mean→
53.1%
12.7%
4.2%
4.7%
4.2%
-3.7%
-2.8%
-3.1%
86.3%
13.4%
9.8%
1.0%
5.1%
-1.2%
-4.2%
1.9%
52.8%
6.4%
1.3%
3.2%
4.4%
-0.3%
0.3%
-0.5%
-8.0%
-7.3%
-2.8%
-5.0%
-5.1%
0.5%
-2.4%
-3.4%
Plate name:
ID4
17α estradiol
5
Raw - VC_mean→
Leslie Akhurst
1.00E-06 M
Worker name:
Corticosterone
-120.5
-56.9
-194.6
-233.0
-109.9
-185.4
-173.5
-203.9
-104.6
-202.6
-82.1
-30.5
-72.8
-41.0
141.7
-58.3
PC (1 nM E2)_mean→
-7.4%
-11.9%
-6.7%
-10.6%
-6.4%
-5.0%
-4.5%
8.7%
-3.5%
-14.3%
-11.4%
-12.5%
-12.4%
-1.9%
-2.5%
-3.6%
570 nm
1.00E-08 M
Performance standard
fold-induction (FI)
FI VC_Mean + 2SD
17βestradiol
8
1925
2166
1908
988
486
400
441
1875
9
2085
2162
1626
802
516
306
375
1835
10
2397
2113
1933
659
494
377
413
1970
11
1825
2299
1732
1310
538
509
340
2311
FI PC10
12
1974
2078
1807
749
620
514
348
2195
4.7
1729.2
1886.8
1574.3
394.6
78.1
-66.2
-112.5
1833.8
1631.5
1479.0
1720.0
1461.8
541.5
39.7
-46.3
-5.3
1428.7
1639.2
1716.0
1179.7
356.2
70.2
-140.4
-71.5
1388.9
1950.3
1667.0
1486.9
213.2
47.7
-68.9
-33.1
1524.0
1378.3
1852.4
1285.7
863.3
91.4
62.2
-105.9
1864.3
106.0%
115.6%
96.5%
24.2%
4.8%
-4.1%
-6.9%
112.4%
90.7%
105.4%
89.6%
33.2%
2.4%
-2.8%
-0.3%
87.6%
100.5%
105.2%
72.3%
21.8%
4.3%
-8.6%
-4.4%
85.1%
119.5%
102.2%
91.1%
13.1%
2.9%
-4.2%
-2.0%
93.4%
84.5%
113.5%
78.8%
52.9%
5.6%
3.8%
-6.5%
114.3%
4.7
1.3
1.4
Pass
Pass
1528.0
1631.2
1361.1
303.2
173.5
67.5
-98.0
1749.1
93.7%
100.0%
83.4%
18.6%
10.6%
4.1%
-6.0%
107.2%
7
2176
2333
2021
841
524
380
334
2280
2077.7
ID1
ID2
ID3
ID4
Testosterone
Corticosterone
17α estradiol
17βestradiol
log [(M)]
mean
SD
log [(M)]
mean
SD
log [(M)]
mean
SD
log [(M)]
mean
SD
-5
64.1%
19.2%
-4
-6.3%
2.5%
-6
99.0%
7.8%
-8
99.2%
18.2%
-6
10.8%
3.9%
-5
-11.2%
3.6%
-7
108.8%
6.0%
-9
105.2%
7.3%
-7
5.1%
4.3%
-6
-7.0%
4.3%
-8
86.1%
12.5%
-10
84.5%
6.2%
-8
2.9%
1.9%
-7
-9.4%
3.9%
-9
26.4%
6.0%
-11
28.2%
21.6%
-9
4.6%
0.5%
-8
-8.0%
3.9%
-10
3.8%
1.2%
-12
6.4%
3.9%
-10
-1.8%
1.8%
-9
-2.1%
2.8%
-11
-5.2%
3.0%
-13
1.2%
4.7%
-11
-2.2%
2.3%
-10
-3.1%
1.2%
-12
-3.9%
3.3%
-14
-4.8%
2.4%
log[PCMax (M)], RPCMax, PCMax (M)
-5
64.1% 1.00E-05
-9
-2.1% 1.00E-09
-7
108.8% 1.00E-07
-9
105.2% 1.00E-09
log[PC50 (M)], PC50 (M)
-5.26
5.44E-06
-8.60
2.48E-09
-10.61
2.44E-11
log[PC10 (M)], PC10 (M)
-6.15
7.11E-07
-9.73
1.87E-10
-11.83
1.46E-12
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
-14 -12 -10
ID1
www.huntingdon.com
-8
log[(M)]
-6
-4
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
-14 -12 -10
ID2
-8
log[(M)]
-6
-4
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
-14 -12 -10
ID3
-8
log[(M)]
-6
-4
-14 -12 -10
ID4
-8
-6
-4
log[(M)]
11

12. OECD Validated Spreadsheet
ID2
ID3
For PC50 calculation ID1
Testosterone
Corticosterone
17α estradiol
0.5
log [(M)]
log [(M)]
log [(M)]
-5
119.0% 1.70E-06
-4
-9.7% x
-6
93.5% x
-6
29.3% x
-5
-10.9% x
-7
84.8% x
-7
6.8% x
-6
-9.4% x
-8
89.1%
-8
1.9% x
-7
-9.5% x
-9
42.4% x
-9
-0.9% x
-8
-3.9% x
-10
6.1% x
-10
-0.2% x
-9
-6.4% x
-11
-3.0% x
-11
5.5% x
-10
-0.2% x
-12
-6.2% x
1.70E-06
0.00E+00
ID2
ID3
For PC10 calculation ID1
Testosterone
Corticosterone
17α estradiol
0.1
log [(M)]
log [(M)]
log [(M)]
-5
119.0% x
-4
-9.7% x
-6
93.5% x
-6
29.3% 1.39E-07
-5
-10.9% x
-7
84.8% x
-7
6.8% x
-6
-9.4% x
-8
89.1% x
-8
1.9% x
-7
-9.5% x
-9
42.4%
-9
-0.9% x
-8
-3.9% x
-10
6.1% x
-10
-0.2% x
-9
-6.4% x
-11
-3.0% x
-11
5.5% x
-10
-0.2% x
-12
-6.2% x
1.39E-07
0.00E+00
Raw / VC_mean→
(fold-induction)
5.45
2.20
1.51
1.19
1.08
1.00
1.29
0.99
1.00
0.12
1.23
7.68
2.42
1.11
0.99
1.02
1.09
1.43
1.01
ID4
1.46E-09
1.46E-09
1.28E-10
1.28E-10
0.82
0.52
0.45
4.82
4.47
0.39
0.66
0.60
4.96
4.46
0.57
0.68
0.58
4.86
4.60
0.58
0.84
0.41
2.90
2.70
1.18
0.85
0.49
1.12
1.23
0.81
0.77
0.63
0.90
0.84
0.60
1.53
0.85
0.74
0.69
1.18
0.88
1.08
5.13
4.89
fold-induction of VC_mean→
fold-induction of PC_mean→
5.15
fold-induction of VC_SD→
fold-induction of PC_SD→
0.38
fold-induction of VC_mean + 2SD→
fold-induction of corresponding to the PC10→
1.41
ID1
ID2
ID3
Testosterone
Corticosterone
17α estradiol
log [(M)]
mean
SD
log [(M)]
mean
SD
log [(M)]
mean
-5
5.94
1.56
-4
0.60
0.20
-6
4.88
-6
2.21
0.20
-5
0.55
0.14
-7
4.52
-7
1.28
0.21
-6
0.61
0.06
-8
4.69
-8
1.08
0.10
-7
0.61
0.22
-9
2.76
-9
0.96
0.15
-8
0.84
0.35
-10
1.25
-10
0.99
0.10
-9
0.73
0.10
-11
0.88
-11
1.23
0.24
-10
0.99
0.48
-12
0.74
VC
4.67
2.02
1.23
1.06
0.80
0.89
0.97
0.87
ID1
PC
VC
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-11 -10 -9
-8
log[(M)]
www.huntingdon.com
ID2
PC
VC
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-7
-6
-5
ID3
5.34
4.13
4.62
2.67
1.40
0.89
0.81
5.72
-8
-7
log[(M)]
-6
-5
-4
7.13
4.90
4.43
2.67
1.21
0.80
0.80
5.47
5.28
5.45
4.80
2.66
1.01
0.80
0.69
4.94
5.90
5.23
5.25
2.47
1.15
1.00
0.87
4.72
ID4
SD
0.44
0.42
0.15
0.13
0.14
0.03
0.06
17βestradiol
log [(M)]
mean
-8
6.10
-9
5.19
-10
4.83
-11
2.60
-12
1.13
-13
0.86
-14
0.79
PC
VC
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-10 -9
17βestradiol
log [(M)]
-8
123.1% x
-9
101.1% x
-10
92.4% 1.63E-11
-11
38.7% x
-12
3.0% x
-13
-3.3% x
-14
-5.1% x
1.63E-11
ID4
17βestradiol
log [(M)]
-8
123.1% x
-9
101.1% x
-10
92.4% x
-11
38.7% 1.57E-12
-12
3.0% x
-13
-3.3% x
-14
-5.1% x
1.57E-12
ID4
SD
0.94
0.27
0.41
0.11
0.10
0.11
0.09
PC
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-12 -11 -10 -9
log[(M)]
-8
-7
-6
-14 -13 -12 -11 -10
-9
-8
log[(M)]
12

13. ER Transcriptional Activation Assay –
our experience





All 10 proficiency chemicals listed in 2009 guideline tested (2012
guideline now lists 14 chemicals – only 4 from 2009 guideline)
7 positive chemicals all confirmed as positive
Corticosterone and atrazine confirmed as negative
Dibutyl phthalate was negative in 2 tests and weakly positive in 2
tests. Other labs have also experienced this. On examination of the
CERI draft validation report, a positive response was observed for
DBT based on logPC10 but not logPC50 values, which is what we
also observed.
Use of an antagonist (4-hydroxytamoxifen) eliminated the response
shown by dibutyl phthalate - therefore true ER binding
www.huntingdon.com
13

14. ER Transcriptional Activation
Assay – our experience


Edge effects - if suspected the plate layout should be altered.
Overall we concluded that there were no edge effects but we
did observe row and column effects for untreated plates.
However, the levels of statistical significance using Tukey’s
test were lower than observed in the CERI validation report
No test substances found positive to date, but when we do,
should we also test using an ERα antagonist to confirm that
the response is ERα-specific?
www.huntingdon.com
14

15. ER Transcriptional Activation Assay –
Challenges


Acceptance criteria for reference chemicals are not met on
every occasion. Often values (PC10, EC50 etc) lie just outside
the ranges. Gary Timm at the EPA e-mailed me: “we regard
the performance criteria as "acceptance" criteria, not
"rejection" criteria, so missing one by a small margin will not
invalidate your results during EPA review”.
CVs are sometimes >20% for triplicate cultures. However,
data sent to me by CERI has shown their CVs to be >20% as
well! No mention of CV in validation report.
www.huntingdon.com
15

16. SAP Review

The EPA’s 2013 SAP review of the Tier 1 tests included 19
chemicals for the ER TA assay (one from HLS):




All response curve parameters for the strong estrogen agonist (17βestradiol) were met for 10 of the 19 chemicals. Nevertheless, out-ofrange values were mostly very close to the test guideline ranges
Response curve parameters for the weak estrogen agonist (17αestradiol) were met for only 5 of the 19 chemicals. Nevertheless, outof-range values were mostly very close to the guideline ranges
All of the test facilities had difficulties meeting the Guideline ranges for
the very weak estrogen agonist, 17α-methyltestosterone
Conclusion: while performance criteria were generally not met for the
majority of the chemicals, out-of-range values were often close to the
Guideline ranges. All but one chemical were classified as either
negative or positive in the assay, however inability to fulfil the
performance criteria may affect the interpretation of at least 8 of 19
chemicals.
www.huntingdon.com
16

17. Steroidogenesis Assay






OCSPP 890.1550 (Oct 2009)
OECD 456 (July 2011)
Human H295R adreno-carcinoma cell line
Expresses genes that encode all key enzymes in
steroidogenesis pathway
Cells produce all steroid hormones found in adult adrenal
cortex and gonads
Assay validated by Hecker et al for detection of testosterone
and 17β-estradiol (E2) - for agonists and antagonists
www.huntingdon.com
17

18. Steroidogenesis Assay








Establish cells in 24 well plate for 24 h
Use only cells between 4 and 10 passages from frozen
Expose cells to 6 conc. of test chemical in triplicate.
Concentrations should not exceed solubility limit; 100 µM is
max concentration.
Cell viability assessed using MTT; wells with <80% viability
not included in data analysis
Include known inhibitor (prochloraz) and inducer (forskolin) on
a separate QC plate
48 h incubation then remove supernatants for hormone
analysis
We use ELISA (mass spec is an alternative)
Test conducted on three independent occasions
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18

19. Steroidogenesis Pathways
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19

20. Steroidogenesis Assay – Acceptance
Criteria




All measurements given as fold
increase/decrease relative to solvent control
QC plate must meet acceptability ranges for
induction and inhibition
Chemical positive if fold induction/inhibition is
statistically significant at two adjacent
concentrations in at least 2 of the 3 tests
Within plate CVs should be ≤ 30%
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20

21. Steroidogenesis Assay – QC Plate
Acceptance Criteria
Testosterone
Estradiol
Minimal basal production
500 pg/mL
40 pg/mL
Basal production
≥ 5 times MDL
≥ 2.5 times MDL
Induction (10 µM
forskolin)
Inhibition (1 µM
prochloraz)
www.huntingdon.com
≥ 2 times SC
(amended to ≥ 1.5 times in
OECD guideline and SEP
document)
≥ 7.5 times SC
≤ 0.5 times SC
≤ 0.5 times SC
21

22. Steroidogenesis Assay- Challenges





Narrow window for passage number, but should wait for results
from 1st test in order to set concentrations for 2nd and 3rd tests, so
not always possible to use one frozen vial of cells for all 3 tests
Need to measure potential interference of test chemical with ELISA
QC plate - not always possible to achieve a 2 fold induction in
testosterone over negative control for forskolin (often values around
1.7-1.8 fold). Communication with EPA reassured us that their lab
also experienced this. EPA stated that these were acceptance
criteria not rejection criteria, so the report will not automatically be
disqualified if acceptance criteria are not met. SEP document and
OECD TG has since lowered acceptance limit to 1.5 fold.
Occasionally other criteria not met but usually very close to
acceptance range
Interplate CV (between tests) for solvent controls sometimes >30%
www.huntingdon.com
22

23. Steroidogenesis Assay – SAP Review



Four of the five performing laboratories (16 of 18 compounds) did not
provide data and results for the laboratory proficiency test which is
recommended to demonstrate laboratory proficiency with running the
assay (HLS provided summary data).
All intraplate coefficients of variation (CVs) were within acceptable limits
(≤30%), but interplate CVs for some studies were ≥30%.
Conclusion: the laboratories performance of the assay was generally
consistent across all 18 test compounds, and the performance criteria
were generally met for all compounds. In most cases where the
performance criteria were not met, the values only slightly exceeded the
expected values and did not impact the interpretation or reliability of the
study. The results demonstrate that the Steroidogenesis Assay as
performed by the testing laboratories can distinguish between chemicals
that alter or do not alter testosterone and/or estrogen levels in vitro.
www.huntingdon.com
23

24. Aromatase (CYP19) Assay
US-EPA OCSPP 890.1200
 Human recombinant microsomes
 Human CYP19 and human P450 reductase

www.huntingdon.com
24

25. Aromatase (CYP19) Assay

Measures conversion of [3H]androstendione
(ASDN) to estrone by release of 3H2O
www.huntingdon.com
25

26. Aromatase (CYP19) assay

Assay
Conducted in polypropylene test tubes
 Buffer
3
 [ H]androstendione
 NADPH
 Propylene glycol
 Test article (or controls)
 Microsomal protein
 Incubation time – 15 minutes
 Stop – methylene chloride (DCM)

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26

27. Aromatase (CYP19) assay

8 concentrations of test article


8 concentrations of positive control



Ideally log M -3 to log M -10 in triplicate
log M -5 to log M -10 in duplicate
Full activity control - quadruplicate
Background control - quadruplicate
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27

28. Aromatase (CYP19) Assay

Controls:
Positive control – formestane
 Full activity control – solvent instead of test article
 Background activity controls – solvent with buffer
instead of NADPH

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28

29. Aromatase (CYP19 assay)

Analysts best friend
www.huntingdon.com
29

30. Aromatase (CYP19 assay)

Analyst specific – full proficiency assessment is
required for each analyst
Proficency chemicals:
Formestane, econazole, fenarimol, nitrofen and
atrazine

www.huntingdon.com
30

31. Aromatase (CYP19 assay)
Our experience
Generally straight forward
 Improved full control activities by using a smaller
volume of chilled recombinant microsomes
 Improved reproducibility using the Hamilton Robot
for liquid handling steps

www.huntingdon.com
31

32. Aromatase (CYP19) Assay –
guideline criteria
Performance Criteria for Aromatase
Assay Parameter
Recommended Values
Minimum Aromatase Activity
0.1 nmol/mg-protein/min
≤ 10% of Full Activity
Mean Background Control
Activity
<15%
Coefficient of Variation (CV) for
replicates within each sample type
and concentration of 4-OH ASDN
Criteria for Positive Control Response Curves
Parameter
Upper Limit
Lower
Limit
Slope
-1.2
-0.8
Top (%)
90
110
Bottom (%)
-5
+6
Log IC50
-7.3
-7.0
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32

33. Aromatase (CYP19) Assay

Performance criteria from positive control (n=12)
Parameter
Slope
Top (%)
Bottom (%)
Log IC50 (M)
Lower limit
criteria
-1.2
90
-5
-7.3
www.huntingdon.com
Upper limit
criteria
-0.8
110
+6
-7.0
Actual lower Actual upper
limit
limit
-1.010
-0.812
90.1
107.2
0.1
1.1
-7.74
-7.30
33

34. Aromatase (CYP19) Assay – SAP
review

SAP – review of 18 compounds
•
•
•
•
Performance criteria was generally met in each study
Some studies lacked mid-log concentrations
Deviations from performance criteria were minor
Overall the assays from the labs were able to distinguish
between inhibitors and non-inhibitors of this activity
www.huntingdon.com
34

35. Estrogen receptor (ER) binding
assay




US-EPA OCSPP 890.1250
Rat uterine cytosol preparation
Saturation binding experiments
Competitive binding experiments
www.huntingdon.com
35

36. Estrogen (ER) receptor binding
assay

Saturation binding
Optimal protein concentration determination for saturation
binding
(0.03 nM [3H]-17-estradiol ; 25-35% binding)
Saturation binding: estradiol (8 conc)

Competitive binding
Optimal protein concentration determination for
competitive binding
(1.0 nM [3H]-17-estradiol ; 10-15% binding)
www.huntingdon.com
36

37. Estrogen (ER) receptor binding
assay

Saturation assay:
Day 1
 TEDG
+ PMSF buffer
 [3H]-17-estradiol (8 conc: 0.3 nM – 3 nM)
 Cold 17 -estradiol (100  label)
 Uterine cytosol
 Incubation (4ºC for 16 – 20 hours)
www.huntingdon.com
37

38. Estrogen (ER) receptor binding
assay
Saturation assay:
Day 2
 60%
(v/v) Hydroxyapatite (HAP) in TEDG + PMSF
added to assay tubes
 Incubated at 4ºC for 5 minutes with 10 sec vortex in
between (3 times)
 After 3rd vortex 2 ml cold TEDG + PMSF buffer
added then vortex
 Centrifuge at 1000  g for 10 min @ 4ºC. SNT
decanted.
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38

39. Estrogen (ER) receptor binding
assay
Saturation assay continued:
Day 2
 Repeated
twice more
 After 3rd centrifugation, drain tube, 1.5 mL ethanol
added, incubate with ethanol and 3 vortex at 5 min
intervals, centrifuged again
 Supernatant (1 mL) added to 14 mL scintillation
fluid for liquid scintillation counting
www.huntingdon.com
39

40. Estrogen (ER) receptor binding
assay

Saturation binding requirements:
3 Runs
 Specific binding curve
 Linear Scatchard plot
 Kd = 0.03 to 1.5 nM
 Bmax = 10 to 150 fmol ER/100 µg protein

www.huntingdon.com
40

41. Estrogen (ER) receptor binding
assay

Competitive binding assay
• Solvents: absolute ethanol (max 3%), water and DMSO (max 10%)
• Solubility test undertaken to ascertain whether final concentration of
1 mM is achievable
• If required lower concentration is prepared at log = -3.5 M then by halflog molar decrease until solubility can be achieved
• Not soluble at 1 µM or above in ETOH, DMSO or H2O and is not
interactive at 1 µM, the chemical is classified as “equivocal” or
“equivocal” up to conc tested rather than “not interactive”
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41

42. Estrogen (ER) receptor binding
assay

Competitive binding assay continued
 Negative
control: Octyltriethoxysilane
(8 concentrations between: log Molar -3 to -10)
 Weak
positive control: 19-Norethindone (or
norethynodrel)
(8 concentrations between: log Molar -4 to -8.5)
www.huntingdon.com
42

43. Estrogen (ER) receptor binding
assay

Competitive binding assay continued
 Reference
chemical: 17-Estradiol
(7 concentrations between: log Molar -8 to -11)
 Test
chemical:
(8 concentrations between : log Molar -3 to -10)
www.huntingdon.com
43

44. Estrogen (ER) receptor binding
assay

Analyst specific – full proficiency assessment is
required for each analyst
www.huntingdon.com
44

45. Estrogen (ER) receptor binding
assay – guideline criteria and our
experience
Competitive Binding Assay Performance Criteria
Criterion
Tolerance Limit(s)
Radioinert 17β-estradiol fitted curve parameters
Loge(residual Std. Dev.)
≤2.35 (0.93 to 2.17)
Top (% binding)
94 to 111 (81 to 141)
Bottom (% binding)
-4 to 1 (-3.6 to 2.2)
(Hill) Slope (log10(M)-1)
-1.1 to -0.7 (-1.1 to -0.6)
Weak Positive control (norethynodrel) fitted curve parameters (19-norethindrone)
Loge(residual Std. Dev.)
≤2.60 (1.46 to 2.21)
Top (% binding)
90 to 110 (87 to 128)
Bottom (% binding)
-5 to 1 (-15.4 to 2.7)
-1)
(Hill) Slope (log10(M)
-1.1 to -0.7 (-1.2 to -0.6)
Solvent concentration
Ethanol
≤3%
DMSO
≤10%
Negative control (octyltriethoxysilane)
≤25%
does not displace more than 25% of
[3H]-17β-estradiol from the ER on
average across all concentrations
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45

46. Estrogen (ER) receptor binding
assay – SAP review

SAP – review of 18 compounds
• Performance was generally consistent across the compounds
• Not all assays met the performance criteria
• Overall the laboratories performance was generally acceptable
www.huntingdon.com
46

47. Androgen (AR) receptor binding
assay




US-EPA OCSPP 890.1150
Rat ventral prostate cytosol preparation
Saturation binding experiments
Competitive binding experiments
www.huntingdon.com
47

48. Androgen (AR) receptor binding
assay

Saturation binding
Optimal protein concentration determination for saturation
binding
(0.25 nM [3H]-R1881 ; 25-35% binding)
Saturation binding: [3H]-R1881 (8 conc)

Competitive binding
Optimal protein concentration determination for
competitive binding
(1.0 nM [3H]-R1881 ; 10-15% binding)
www.huntingdon.com
48

49. Androgen (AR) receptor binding
assay

Saturation assay:
Day 1
 Low
salt TEDG buffer
 [3H]-R1881 (8 conc: 0.25 nM – 10 nM)
 Triamcinolone acetonide
 Cold R1881 (100  label)
 Rat prostate cytosol
 Incubation (4ºC for 20 hours)
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49

50. Androgen (AR) receptor binding
assay
Saturation assay:
Day 2
 60%
(v/v) Hydroxyapatite (BIO-RAD HT-GEL) in
50 mM Tris buffer
 Added 100 µl of incubation mixture to HAP assay
tubes
 Incubated at 4ºC for 20 minutes with vortex-mixing
every 5 minutes for 10 sec
 Centrifuge for 3 minutes at 4ºC at 600  g
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50

51. Androgen (AR) receptor binding
assay
Saturation assay:
Day 2 continued
 Wash
pellet with 50 mM Tris, pH 7.4 (4ºC); 4 times
in all with centrifugation in between
 After 4th wash add 2 mL ethanol, incubate for 10
mins, vortex at 5 min intervals, centrifuge for 10 min
 Supernatant (1 mL) added to 14 mL scintillation
fluid for liquid scintillation counting
www.huntingdon.com
51

52. Androgen (AR) receptor binding
assay

Saturation binding requirements:
3 Runs
 Specific binding curve
 Linear Scatchard plot
 Kd = 0.685 to 1.57 nM
 Bmax = 10 to 150 fmol AR/100 µg protein

www.huntingdon.com
52

53. Androgen (AR) receptor binding
assay

Competitive binding assay
 Receptor
conc optimised for protein which binds 10
to 15% at 1 nM [3H]-R1881
 Weak positive control: Dexamethasone
(8 concentrations between : log Molar -3 to -10)
 Reference chemical: R-1881
(5 concentrations between: log Molar -7 to -11)
 Test
chemical
(8 concentrations between: log Molar -3 to -10)
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53

54. Androgen (AR) receptor binding
assay

Analyst specific – full proficiency assessment is
required for each analyst
www.huntingdon.com
54

55. Androgen (AR) receptor binding
assay – guideline criteria and our
experience
Parameter
Bottom
plateau
level
Unit
R1881
Dexamethasone
Lower limit Upper Limit Lower limit Upper Limit
% binding
-2.0
(1.104)
2.0
(1.794)
-12
(7.369)
12
(15.65)
Top plateau % binding
level
82
(99.1)
114
(105.9)
87
(97.3)
106
(101.9)
-1.2
(-1.174)
-0.8
(-0.890)
-1.4
(-8.947)
-0.6
(-1.179)
Hill Slope
Log10 (M)-1
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55

56. Androgen (AR) receptor binding
assay – SAP review

SAP review of 18 compounds
Saturation binding performance criteria were not
meet for all cytosol preparations
 Competitive binding

 cytosol
still could identify test chemicals which were
“ AR binders”
 Control chemicals either met or slightly exceeded
the performance criteria
 Assays submitted were generally acceptable and
the data was considered reliable.
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56

57. SEP




Standard Evaluation Protocol
Issued by EPA in September 2011 for each study
type
Provides guidance to EPA staff reviewing data
SEP and DER documents make it clear that EPA
expects to see data from lab proficiency tests
www.huntingdon.com
57

58. DER





Data Evaluation Record
To help EPA review of data, the DER
standardises how the data are reported for each
study type
Lists deviations from protocol or information
missing from report
Additional to CROs own report
HLS has completed many DERs which have
been submitted and reviewed by the EPA. No
comments received on the DERs to date
www.huntingdon.com
58

59. Challenges to labs performing ED tests







Time consuming to set up
Many proficiency chemicals to test, on at least one occasion
Proficiency chemicals should be tested for each person
conducting test, and should be repeated if lab personnel
change
Acceptability criteria not always met - if close to range you
must judge whether EPA will accept study
EPA expect to see proficiency data in the study report (but
proficiency is a separate study……)
DER document adds to time taken to report data
HLS has experienced and overcome all these challenges!
www.huntingdon.com
59

60. How You Can Help Your Studies Run
Smoothly



These tests can be lengthy to conduct, especially as
they have to be conducted on 2 or 3 occasions, and
may be repeated if acceptance criteria are not met.
Allow sufficient months for testing to be conducted in
advance of your deadline
Provide relevant data on the test substance e.g.
solubility in organic (DMSO, ethanol) and aqueous
solvents
Instruct CRO in advance whether you need them to
prepare your DER
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60

61. Summary






HLS offers all 5 in vitro assays (and all 6 in vivo)
Experience gained since 2009
When setting up assays, HLS had contact with
EPA for advice
Many studies completed
Thorough understanding of EDSP program
Experienced at preparing DER documents in
addition to study reports
www.huntingdon.com
61

62. In Vitro Endocrine Disruptor Assays

Thank you for listening!
www.huntingdon.com
62

63. Other webinars this week

Wednesday 22nd

EDSP Tier 1 In Vivo Mammalian Assays
 Bob

Parker
Thursday 23rd

Amphibian metamorphosis assay for the
EPA’s EDSP
 Carole
Jenkins
www.huntingdon.com

64. HLS EDSP expert team





Ephi Gur – Team lead and Regulatory
Bob Parker – Toxicology
Will Davies – Toxicology
John Carter – In vitro technologies
Carole Jenkins – Aquatic toxicology

Contact via me
 reganj@ukorg.huntingdon.com
 +44
(0) 1480 892031
www.huntingdon.com