64
www.huntingdon.com Endocrine Disruptor Screening Program Webinar week 20-23 January 2014
www.huntingdon.com
Endocrine Disruptor
Screening Program
Webinar week
20-23 January 2014
www.huntingdon.com
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
Leslie Akhurst & Yen-Ling Cheung
IN VITRO TESTS FOR
ENDOCRINE DISRUPTION
3
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IN VITRO TESTS FOR ENDOCRINE
DISRUPTION
Leslie
ER transcriptional activation assay
Steroidogenesis assay
Yen
Aromatase
ER & AR binding assays
Leslie
Summary
4
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EPA Tier 1 EDSP Tests
5
Assays
HPG HPT
E Anti-E A Anti-A T E
In vitro
ERα Transcriptional Activation X
ER Binding X X
AR Binding X X
Steroidogenesis X X
Aromatase X
In vivo
Uterotrophic X
Hershberger X X
Pubertal Male X X X X X
Pubertal Female X X X X X
Fish short tem reproduction X X X X X X X
Amphibian metamorphosis X
Modes of Action Covered by Assay
Receptor binding Steroidogenesis
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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
6
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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
7
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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
8
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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
9
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ER Transcriptional Activation Assay
Acceptance Criteria
10
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
17α-
Methyltestosterone -6.0 to -5.1 -8.0 to -6.2 - - 10-11 to 10-5 M
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OECD Validated Spreadsheet Lab name: Date: Worker name: Plate name: Performance standard
ID1 1.00E-05 M ID2 1.00E-04 M ID3 1.00E-06 M ID4 1.00E-08 M fold-induction (FI) 4.7 Pass
FI VC_Mean + 2SD 1.3
RawData 1 2 3 4 5 6 7 8 9 10 11 12 FI PC10 1.4
A 1312 1854 1308 315 326 389 2176 1925 2085 2397 1825 1974
B 654 665 551 327 252 213 2333 2166 2162 2113 2299 2078
C 515 606 467 400 336 261 2021 1908 1626 1933 1732 1807
D 523 462 498 364 273 242 841 988 802 659 1310 749
E 515 530 518 363 342 244 524 486 516 494 538 620
F 385 426 441 454 364 416 380 400 306 377 509 514
G 401 377 452 408 373 405 334 441 375 413 340 348
H 396 477 438 391 588 388 2280 1875 1835 1970 2311 2195
VC_mean→ 446.3 (PC (1 nM E2) + VC)_mean→ 2077.7
fold-induction→ 4.7
Raw - VC_mean→ 865.9 1407.5 862.0 -131.1 -120.5 -56.9 1729.2 1479.0 1639.2 1950.3 1378.3 1528.0
207.9 218.5 104.6 -119.2 -194.6 -233.0 1886.8 1720.0 1716.0 1667.0 1852.4 1631.2
68.9 160.2 21.2 -46.3 -109.9 -185.4 1574.3 1461.8 1179.7 1486.9 1285.7 1361.1
76.8 15.9 51.6 -82.1 -173.5 -203.9 394.6 541.5 356.2 213.2 863.3 303.2
68.9 83.4 71.5 -83.4 -104.6 -202.6 78.1 39.7 70.2 47.7 91.4 173.5
-60.9 -19.9 -5.3 7.9 -82.1 -30.5 -66.2 -46.3 -140.4 -68.9 62.2 67.5
-45.0 -68.9 5.3 -38.4 -72.8 -41.0 -112.5 -5.3 -71.5 -33.1 -105.9 -98.0
-50.3 30.5 -7.9 -55.6 141.7 -58.3 1833.8 1428.7 1388.9 1524.0 1864.3 1749.1
PC (1 nM E2)_mean→ 1631.5
(Raw - VC_mean) / PC_mean→ 53.1% 86.3% 52.8% -8.0% -7.4% -3.5% 106.0% 90.7% 100.5% 119.5% 84.5% 93.7%
12.7% 13.4% 6.4% -7.3% -11.9% -14.3% 115.6% 105.4% 105.2% 102.2% 113.5% 100.0%
4.2% 9.8% 1.3% -2.8% -6.7% -11.4% 96.5% 89.6% 72.3% 91.1% 78.8% 83.4%
4.7% 1.0% 3.2% -5.0% -10.6% -12.5% 24.2% 33.2% 21.8% 13.1% 52.9% 18.6%
4.2% 5.1% 4.4% -5.1% -6.4% -12.4% 4.8% 2.4% 4.3% 2.9% 5.6% 10.6%
-3.7% -1.2% -0.3% 0.5% -5.0% -1.9% -4.1% -2.8% -8.6% -4.2% 3.8% 4.1%
-2.8% -4.2% 0.3% -2.4% -4.5% -2.5% -6.9% -0.3% -4.4% -2.0% -6.5% -6.0%
-3.1% 1.9% -0.5% -3.4% 8.7% -3.6% 112.4% 87.6% 85.1% 93.4% 114.3% 107.2%
ID1 ID2 ID3 ID4
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
Testosterone Corticosterone 17α estradiol 17βestradiol
Pass
HLS 14/09/2010 Leslie Akhurst 570 nm
17βestradiol17α estradiolCorticosteroneTestosterone
0%20%40%60%80%
100%120%140%160%180%200%
-14 -12 -10 -8 -6 -4
log[(M)]ID1
0%20%40%60%80%
100%120%140%160%180%200%
-14 -12 -10 -8 -6 -4
log[(M)]ID2
0%20%40%60%80%
100%120%140%160%180%200%
-14 -12 -10 -8 -6 -4
log[(M)]ID3
0%20%40%60%80%
100%120%140%160%180%200%
-14 -12 -10 -8 -6 -4
log[(M)]ID4
11
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OECD Validated Spreadsheet
12
For PC50 calculation ID1 ID2 ID3 ID4
0.5
log [(M)] log [(M)] log [(M)] log [(M)]
-5 119.0% 1.70E-06 -4 -9.7% x -6 93.5% x -8 123.1% x
-6 29.3% x -5 -10.9% x -7 84.8% x -9 101.1% x
-7 6.8% x -6 -9.4% x -8 89.1% 1.46E-09 -10 92.4% 1.63E-11
-8 1.9% x -7 -9.5% x -9 42.4% x -11 38.7% x
-9 -0.9% x -8 -3.9% x -10 6.1% x -12 3.0% x
-10 -0.2% x -9 -6.4% x -11 -3.0% x -13 -3.3% x
-11 5.5% x -10 -0.2% x -12 -6.2% x -14 -5.1% x
1.70E-06 0.00E+00 1.46E-09 1.63E-11
For PC10 calculation ID1 ID2 ID3 ID4
0.1
log [(M)] log [(M)] log [(M)] log [(M)]
-5 119.0% x -4 -9.7% x -6 93.5% x -8 123.1% x
-6 29.3% 1.39E-07 -5 -10.9% x -7 84.8% x -9 101.1% x
-7 6.8% x -6 -9.4% x -8 89.1% x -10 92.4% x
-8 1.9% x -7 -9.5% x -9 42.4% 1.28E-10 -11 38.7% 1.57E-12
-9 -0.9% x -8 -3.9% x -10 6.1% x -12 3.0% x
-10 -0.2% x -9 -6.4% x -11 -3.0% x -13 -3.3% x
-11 5.5% x -10 -0.2% x -12 -6.2% x -14 -5.1% x
1.39E-07 0.00E+00 1.28E-10 1.57E-12
Raw / VC_mean→ 5.45 7.68 4.67 0.82 0.52 0.45 4.82 4.47 5.34 7.13 5.28 5.90
(fold-induction) 2.20 2.42 2.02 0.39 0.66 0.60 4.96 4.46 4.13 4.90 5.45 5.23
1.51 1.11 1.23 0.57 0.68 0.58 4.86 4.60 4.62 4.43 4.80 5.25
1.19 0.99 1.06 0.58 0.84 0.41 2.90 2.70 2.67 2.67 2.66 2.47
1.08 1.02 0.80 1.18 0.85 0.49 1.12 1.23 1.40 1.21 1.01 1.15
1.00 1.09 0.89 0.81 0.77 0.63 0.90 0.84 0.89 0.80 0.80 1.00
1.29 1.43 0.97 0.60 1.53 0.85 0.74 0.69 0.81 0.80 0.69 0.87
0.99 1.01 0.87 1.18 0.88 1.08 5.13 4.89 5.72 5.47 4.94 4.72
fold-induction of VC_mean→ 1.00 fold-induction of PC_mean→ 5.15
fold-induction of VC_SD→ 0.12 fold-induction of PC_SD→ 0.38
fold-induction of VC_mean + 2SD→ 1.23 fold-induction of corresponding to the PC10→ 1.41
ID1 ID2 ID3 ID4
log [(M)] mean SD log [(M)] mean SD log [(M)] mean SD log [(M)] mean SD
-5 5.94 1.56 -4 0.60 0.20 -6 4.88 0.44 -8 6.10 0.94
-6 2.21 0.20 -5 0.55 0.14 -7 4.52 0.42 -9 5.19 0.27
-7 1.28 0.21 -6 0.61 0.06 -8 4.69 0.15 -10 4.83 0.41
-8 1.08 0.10 -7 0.61 0.22 -9 2.76 0.13 -11 2.60 0.11
-9 0.96 0.15 -8 0.84 0.35 -10 1.25 0.14 -12 1.13 0.10
-10 0.99 0.10 -9 0.73 0.10 -11 0.88 0.03 -13 0.86 0.11
-11 1.23 0.24 -10 0.99 0.48 -12 0.74 0.06 -14 0.79 0.09
17α estradiol 17βestradiol
Testosterone Corticosterone 17α estradiol 17βestradiol
Testosterone Corticosterone 17α estradiol 17βestradiol
Testosterone Corticosterone
0.0
1.0
2.0
3.0
4.0
5.0
6.0
-12 -11 -10 -9 -8 -7 -6
log[(M)]
VC ID3 PC
0.01.02.03.04.05.06.07.08.0
-11 -10 -9 -8 -7 -6 -5
log[(M)]
VC ID1 PC
0.0
1.0
2.0
3.0
4.0
5.0
6.0
-10 -9 -8 -7 -6 -5 -4
log[(M)]
VC ID2 PC
0.01.02.03.04.05.06.07.08.0
-14 -13 -12 -11 -10 -9 -8
log[(M)]
VC ID4 PC
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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
13
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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?
14
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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.
15
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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 (17β-
estradiol) were met for 10 of the 19 chemicals. Nevertheless, out-of-
range values were mostly very close to the test guideline ranges
Response curve parameters for the weak estrogen agonist (17α-
estradiol) were met for only 5 of the 19 chemicals. Nevertheless, out-
of-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.
16
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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
17
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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
18
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Steroidogenesis Pathways
19
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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%
20
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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)
≥ 2 times SC (amended to ≥ 1.5 times in
OECD guideline and SEP
document)
≥ 7.5 times SC
Inhibition (1 µM
prochloraz) ≤ 0.5 times SC ≤ 0.5 times SC
21
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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%
22
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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.
23
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Aromatase (CYP19) Assay
US-EPA OCSPP 890.1200
Human recombinant microsomes
Human CYP19 and human P450 reductase
24
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Aromatase (CYP19) Assay
Measures conversion of [3H]androstendione
(ASDN) to estrone by release of 3H2O
25
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Aromatase (CYP19) assay
Assay
Conducted in polypropylene test tubes
Buffer
[3H]androstendione
NADPH
Propylene glycol
Test article (or controls)
Microsomal protein
Incubation time – 15 minutes
Stop – methylene chloride (DCM)
26
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Aromatase (CYP19) assay
8 concentrations of test article
Ideally log M -3 to log M -10 in triplicate
8 concentrations of positive control
log M -5 to log M -10 in duplicate
Full activity control - quadruplicate
Background control - quadruplicate
27
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Aromatase (CYP19) Assay
Controls:
Positive control – formestane
Full activity control – solvent instead of test article
Background activity controls – solvent with buffer
instead of NADPH
28
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Aromatase (CYP19 assay)
Analysts best friend
29
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Aromatase (CYP19 assay)
Analyst specific – full proficiency assessment is
required for each analyst
Proficency chemicals:
Formestane, econazole, fenarimol, nitrofen and
atrazine
30
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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
31
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Aromatase (CYP19) Assay –
guideline criteria
32
Performance Criteria for Aromatase
Assay Parameter
Recommended Values
Minimum Aromatase Activity 0.1 nmol/mg-protein/min
Mean Background Control
Activity
≤ 10% of Full Activity
Coefficient of Variation (CV) for
replicates within each sample type
and concentration of 4-OH ASDN
<15%
Criteria for Positive Control Response Curves
Parameter Lower
Limit
Upper Limit
Slope -1.2 -0.8
Top (%) 90 110
Bottom (%) -5 +6
Log IC50 -7.3 -7.0
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Aromatase (CYP19) Assay
Performance criteria from positive control (n=12)
33
Parameter Lower limit
criteria
Upper limit
criteria
Actual lower
limit
Actual upper
limit
Slope -1.2 -0.8 -1.010 -0.812
Top (%) 90 110 90.1 107.2
Bottom (%) -5 +6 0.1 1.1
Log IC50 (M) -7.3 -7.0 -7.74 -7.30
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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
34
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Estrogen receptor (ER) binding
assay
US-EPA OCSPP 890.1250
Rat uterine cytosol preparation
Saturation binding experiments
Competitive binding experiments
35
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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)
36
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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)
37
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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.
38
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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
39
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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
40
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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 half-
log 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”
41
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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)
42
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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)
43
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Estrogen (ER) receptor binding
assay
Analyst specific – full proficiency assessment is
required for each analyst
44
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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)
(Hill) Slope (log10(M)-1) -1.1 to -0.7 (-1.2 to -0.6)
Solvent concentration
Ethanol ≤3%
DMSO ≤10%
Negative control (octyltriethoxysilane)
does not displace more than 25% of
[3H]-17β-estradiol from the ER on
average across all concentrations
≤25%
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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
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Androgen (AR) receptor binding
assay
US-EPA OCSPP 890.1150
Rat ventral prostate cytosol preparation
Saturation binding experiments
Competitive binding experiments
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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)
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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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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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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
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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
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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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Androgen (AR) receptor binding
assay
Analyst specific – full proficiency assessment is
required for each analyst
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Androgen (AR) receptor binding
assay – guideline criteria and our
experience
Parameter Unit R1881 Dexamethasone
Lower limit Upper Limit Lower limit Upper Limit
Bottom
plateau
level
% binding -2.0
(1.104)
2.0
(1.794)
-12
(7.369)
12
(15.65)
Top plateau
level
% binding 82
(99.1)
114
(105.9)
87
(97.3)
106
(101.9)
Hill Slope Log10 (M)-1 -1.2
(-1.174)
-0.8
(-0.890)
-1.4
(-8.947)
-0.6
(-1.179)
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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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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
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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
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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!
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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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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
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In Vitro Endocrine Disruptor Assays
Thank you for listening!
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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
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
+44 (0) 1480 892031
