EDSP webinar 2: In vitro assays for the EDSP

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Information about EDSP webinar 2: In vitro assays for the EDSP
Education

Published on February 4, 2014

Author: JimBRegan

Source: slideshare.net

Description

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 www.huntingdon.com 18

Steroidogenesis Pathways www.huntingdon.com 19

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% www.huntingdon.com 20

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

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

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

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

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

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)  www.huntingdon.com 26

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 www.huntingdon.com 27

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

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

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

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

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 www.huntingdon.com 32

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

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

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

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

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

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. www.huntingdon.com 38

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

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

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” www.huntingdon.com 41

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

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

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

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 www.huntingdon.com 45

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

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

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

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) www.huntingdon.com 49

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 www.huntingdon.com 50

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

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

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) www.huntingdon.com 53

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

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 www.huntingdon.com 55

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. www.huntingdon.com 56

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

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

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

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 www.huntingdon.com 60

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

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

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  reganj@ukorg.huntingdon.com  +44 (0) 1480 892031 www.huntingdon.com

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