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The Uterotrophic Assay

The Uterotrophic Assay. The OECD Validation Program and More … A Case Study in Validation. J. William ‘Willie’ Owens The Procter & Gamble Co. Overview of Presentation. Illustrate All the Parts of a Validation Program Illustrate Standardization of Protocol

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The Uterotrophic Assay

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  1. The Uterotrophic Assay The OECD Validation Program and More … A Case Study in Validation J. William ‘Willie’ Owens The Procter & Gamble Co.

  2. Overview of Presentation Illustrate All the Parts of a Validation Program Illustrate Standardization of Protocol Illustrate Conduct and Logistics of Study Illustrate Variety of Issues that Arise Illustrate that Nothing is Perfect

  3. The Scientific Rationale • Estrogen naturally regulates the growth of • several female tissues: uterus, vagina, and • mammary gland • Estrogen stimulated growth of uterus is cyclic, • rapid, and dramatic – in laboratory rodents: • 4-4.5 days total cycle, estrogen phase ~ 2 days • uterus does not reach min or max in cycle, dynamic range increases to 5-6 fold without cycle and minimal uterine weight

  4. The Scientific Rationale • Rodent uterus presents opportunity for rapid • assay for estrogen agonists and antagonists • The conditions and data: • Low endogenous levels of estrogens (immature or ovariectomized female) • 3 days administration, sacrifice on 4th day (twice the time of natural estrogen growth) • Quantitative data – continuous gravimetric weight (not qualitative score or quantal categories, e.g., vaginal cornification assay)

  5. The Uterotrophic Assay The History – early 1930s Need to identify ‘estrogen’ – follow biological activity through chemical fractionation Original pharmaceutical needs – assay possible natural and synthetic estrogens Vaginal assay subjective and quantal – but castrated male assay, used weights of responsive tissues in the reproductive tract

  6. The Uterotrophic Assay The History – early 1930s At least 6 laboratories published basic protocols Rat and mouse Immature & intact, immature & OVX, adult OVX Consecutive administration for 3-4 days Sometimes with imbibed fluid, sometimes blotted weights Other variations Primary focus was POTENT estrogens

  7. The Uterotrophic Assay The History – 1970s Estrogen receptor discovered Antagonists discovered Compound discovery uses receptor assay first Promising agents (still potent) proceed to the uterotrophic assay (conserve animals/time) Antagonist assay – coadminister test agent with potent reference, look for decrease in weight versus reference only group Research on complementary molecular /cellular markers

  8. The Uterotrophic Assay The History – 1990s Estrogen ‘mimics’ emerge as issue Typically, weak agonists 2-5 orders of magnitude lower in binding affinity than 17-beta estradiol Development of in vitro assays (reporter genes) Development of new molecular and cellular markers Reapplication of pharmaceutical paradigm Addition of in vitro techniques to tiered scheme Attempts to relate markers to uterine weight increase Begin application of assay to very weak agonists

  9. Types of Data Mechanistic Data The endpoint or measurement is linked mechanistically through a mode of action to an effect or condition Empirical Data An association is demonstrated between the endpoint to an effect or condition Adverse Effect Endpoint used for risk assessment

  10. The Uterotrophic Assay What is its model and prediction? The growth of the uterus is a culmination of an estrogen mode of action A cascade of molecular and cellular events that begins with a ligand binding the estrogen receptor (Mechanistic data) Good ‘in principle’ concordance with binding data Antiestrogens block response Response absent in alpha-estrogen receptor knock out mice

  11. The Uterotrophic Assay What is its model and prediction? Basic prediction: Positive uterine growth indicates that substance may trigger estrogen mediated effects Testing for adverse affects may be warranted If a relevant route of administration is used, the LOEL may provide helpful information on the expected potency

  12. Scientific Rationale & Regulatory Use In principle, we have a match up where the uterotrophic assay can deliver the information that is needed by the regulators.

  13. The OECD Program Phase I Standardize common protocols Demonstrate it works with reference estrogen Refine as necessary Note: Validation works with a given protocol With several versions – OECD accepted the need to work with several protocols to demonstrate their characteristics

  14. The OECD Program • Phase I • Rat as species of choice (mouse later?) • EE (ethinyl estradiol – not 17beta-estradiol as it binds avidly to alpha-fetoprotein) • Two basic versions: intact, immature ♀ & OVX ♀ • Two routes: oral gavage and sc injection • Also test extension of dosing to 7 days (from 3) • Both wet (imbibed fluid) and blotted (no fluid) uterine weights • Record body weights (toxicity, statistics) • Voluntary expts. (evaluate histology, BrdU)

  15. The OECD Program • Immature, Intact Female • - Start pnd 17-20 (birth = pnd 0) • Once per day, 3 consecutive days, ~ same time each day • Sacrifice, 24 hours after last dose • Both oral gavage and sc injection expts • n = 6

  16. The OECD Program Uterine Weight Long Evans Rats Showing ‘Window’ for Stable Uterine Weights 100 Entering puberty E2 levels rising Low E2 levels Stable weight & low SD’s 80 60 Mean Uterine Weight (mg) 40 20 0 PND 20 21 22 23 24 25 26 27 28 29 30 31 32 n= 10 9 10 10 9 13 10 9 10 10 10 10 13 Postnatal Day Example from Lichtensteiger’s lab Univ of Zurich

  17. The OECD Program • OVX Female • - OVX 6-8 weeks of age (42-56 days)- Allow 7 days to regress • Once per day, 3 consecutive days, ~ same time each day • Satellite protocol using 7 day dosing • Sacrifice, 24 hours after last dose • Sc injection only • n = 6

  18. Protocol B – immature model, subcutaneous injection Protocol A – immature model, oral gavage Lab 1 Lab 2 Lab 3 450 160 180 500 Lab 1 Lab 4 Lab 2 450 400 160 140 Lab 1 Lab 5 Lab 3 400 Lab 2 350 140 120 Lab 6 Lab 7 350 Lab 3 300 120 Lab 7 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 100 Lab 1 Lab 8 300 Lab 7 250 100 Lab 8 Lab 3 Lab 9 Lab 8 250 80 Lab 9 200 Lab 7 80 Lab 10 Lab 9 200 Protocol C’ – adult OVX model, subcutaneous injection, doses extended to 7 days 60 Protocol C – adult OVX model, subcutaneous injection Lab 11 Lab 10 150 Lab 11 60 Lab 11 150 40 Lab 11 Lab 12 Lab 18 100 40 100 Lab 12 Lab 15 Lab 19 20 50 20 50 Lab 17 Lab 13 0 0 0 0 Lab 18 Lab 14 Lab 15 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 Lab 16 0.0 0.01 0.03 0.1 0.3 1.0 10.0 3.0 Ethinyl Estradiol Administered Dose (µg/kg bw/day) Uterine Weight (mg)

  19. The OECD Program • Phase 1 Conclusions • Basically, success! • No evident difference in versions (none superior) • Expected difference for EE in po vs sc • Blotted weight slightly more sensitive (less variable) • Some animal husbandry issues with immature • Extend OVX regression to 14 days • No impact from strain, diet, etc. • Overall consistency • Adequate power with n = 6

  20. 140 Phase 1 – labs 1 - 4, 10-14 days, rest 7 days regression 120 100 80 Uterine Weight (mg) Phase 2 –14 days or more regression 60 40 20 0 0 5 10 20 25 30 35 Number The OECD Program OVX Control Blotted Weights

  21. The OECD Program • Phase 1 Observations • Lab 19 possibly less sensitive, no evident reason • Antagonists (ZM 189,154) demonstration was successful • Slightly greater induction of blotted weight with immature animals 5-fold vs 3.5 fold • Imbibed fluid possibly greater in OVX @ 3 days (but far less imbibed fluid @ 7 days) • Model dose curve with Hill equation ED10 supports statistical analysis

  22. The OECD Program • Phase II • Two segments: Dose response Coded (blind) Multichemical • Demonstrate capability to detect weak agonists • Demonstrate reproducibility (both EE and weak) • Continue testing of protocol capabilities • Continue wet and blotted uterine weights • Continue body weights (toxicity, statistics) • Voluntary experiments (histology, BrdU)

  23. The OECD Program Chemical Selection • Weak Agonists • Bisphenol A • Genistein • Methoxychlor • Nonylphenol • o,p’-DDTNegative Chemical • Dibutylphthalate

  24. Chemical Name (Abbreviation) Mean IC50 (M)  S.E.M. RBA (%) Log RBA 17-Estradiol (E2) 8.99 x 10-10 0.27 x 10-10 100.00 2.00 Ethinyl Estradiol (EE) 4.73 x 10-10 0.60 x 10-10 190.06 2.28 Genistein (GN) 2.00 x 10-7 0.21 x 10-7 0.443 -0.35 Dihydroxymethoxychlor (HPTE) 3.55 x 10-7 0.15 x 10-7 0.253 -0.60 Methoxychlor (MX) 1.44 x 10-4 0.66 x 10-4 0.001 -3.20 4-Nonylphenol (NP) 3.05 x 10-6 0.15 x 10-6 0.029 -1.53 Bisphenol A (BPA) 1.17 x 10-5 0.64 x 10-5 0.008 -2.11 o,p’-DDT 6.43 x 10-5 0.89 x 10-5 0.001 -2.85 Chemical Selection ER Binding Data

  25. Lab 5 Lab 3 5 4 Lab 3 Lab 14 Protocol A - immature, oral gavage ProtocolB – immature, s.c. Lab 7 Lab 13 4 Lab 13 Lab 7 3 Lab 16 Lab 11 3 Lab 15 2 Lab 16 2 1 1 0 0 Ratio of uterine wt (test/control) 0 200 400 600 800 1000 0 200 400 600 800 1000 1200 5 5 Protocol C – OVX, s.c. 4 4 Lab 5 Lab 7 3 3 Lab 7 Lab 13 Lab 14 Lab 13 2 2 Lab 16 Protocol CX - OVX, s.c., 7 days 1 1 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 mg/kg/day mg/kg/day The OECD Program Bisphenol A

  26. 4 4 Lab 4 Lab 4 3 3 Lab 9 Lab 9 Lab 14 Lab 14 Lab 16 Lab 16 2 2 1 1 Protocol B - immature, s.c. Protocol A - immature, oral gavage 0 0 Ratio of uterine wt (test/control) 600 0 20 40 60 80 100 0 100 200 300 400 500 4 4 Protocol C - OVX, s.c. 3 3 Lab 4 Lab 4 2 2 Lab 9 Lab 9 Lab 16 1 1 Protocol D - OVX, s.c., 7 days 0 0 0 20 40 60 80 100 0 20 40 60 80 100 mg/kg/day mg/kg/day The OECD Program Genistein

  27. 4 4 Lab 1 Lab 1 Lab 9 Lab 9 3 3 Lab 10 Lab 10 Lab 16 Lab 16 2 2 1 1 Protocol A - immature, oral gavage Protocol B - immature, s.c. 0 0 Ratio of uterine wt (test/control) 0 100 200 300 400 500 600 0 200 400 600 800 1000 4 4 Protocol C - OVX, s.c. Protocol CX - OVX, s.c., 7 days 3 3 Lab 8 Lab 8 Lab 10 2 2 Lab 10 Lab 16 1 1 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 mg/kg/day mg/kg/day The OECD Program Methoxychlor

  28. Lab 2 Lab 4 4 4 Lab 5 Protocol A - immature, oral gavage Protocol B - immature, s.c. Lab 2 Lab 7 Lab 4 Lab 11 3 3 Lab 7 Lab 16 Lab 15 Lab 16 Lab 14 2 2 1 1 0 0 0 50 100 150 200 250 300 350 400 Ratio of uterine wt (test/control) 0 20 40 60 80 100 120 4 4 Protocol C - OVX, s.c. Protocol CX - OVX, s.c., 7 days 3 3 Lab 5 Lab 14 Lab 4 2 2 Lab 4 Lab 7 Lab 7 Lab 16 1 1 0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 mg/kg/day mg/kg/day The OECD Program Nonylphenol

  29. 4 4 Lab 6 Protocol B - immature, s.c. Lab 6 Lab 8 Lab 8 3 3 Lab 10 Lab 10 Lab 16 Lab 16 2 2 1 1 Protocol A - immature, oral gavage 0 0 0 100 200 300 400 500 600 700 0 50 100 150 200 250 Ratio of uterine wt (test/control) 4 4 Protocol C - OVX, s.c. Protocol CX - OVX, s.c., 7 days 3 3 Lab 8 Lab 8 2 2 Lab 10 Lab 10 Lab 16 1 1 0 0 0 50 100 150 200 250 5 25 50 100 200 mg/kg/day mg/kg/day The OECD Program o,p’-DDT

  30. The OECD Program Dose Response Summary All weak agonists were detected Protocols were qualitatively similar Includes one po study with OVX Route of administration influence substance-specific for lowest dose that is statistically significantBPA & GN / NP /MX & o,p’-DDT Blotted weights again slightly less variable Magnitude of response varied (partial agonists)

  31. The OECD Program Dose Response Summary • However, all was not perfection: • - One lab did not record terminal body weights Therefore, not included in analysis • Several instances statistical significance was not recorded • One discordant data set

  32. The OECD Program Dose Response Summary 5 out of 83, no statistical significance BPA, NP, o,p’-DDT – the weakest agonists 4 cases – labs did only 3 intermediate doses - highest dose missing - uterine weights were rising

  33. 5 Bisphenol A - immature, s.c. Lab 3 Lab 5 4 Lab 7 Lab 11 3 Lab 12 Ratio of uterine weight (test/control) Lab 13 Lab 14 2 Lab 15 Lab 16 1 Lab 17 0 0 100 200 300 400 500 600 700 800 900 mg/kg/d The OECD Program Dose Response Summary

  34. 4 Lab 2 Nonylphenol - immature, s.c. Lab 4 Lab 5 3 Lab 7 Lab 11 Lab 12 Lab 14 2 Ratio of uterine weight (test/control) Lab 15 Lab 16 Lab 17 1 0 0 20 40 60 80 100 120 The OECD Program Dose Response Summary mg/kg/d

  35. 70 Lab 12 (2) 60 3 Labs appear high and would decrease sensitivity Lab 5 (1) 50 Lab 17 (2) 40 Blotted Uterine Wt (mg) 30 20 Control Group Uterine Wts by Ranked Number – Multichemical and Dose Response 10 0 0 10 20 30 40 50 60 The OECD Program Immature Vehicle Control Weights

  36. The OECD Program Coded Multichemical - Design All Samples are Coded and Prepared at Repository so Dilutions at Laboratories are Same Two EE Doses Included - Compare to same doses in Phase 1 (Reproducibility over time) - Same EE doses used in Dose Response (Reproducibility over time – not simultaneous) One Dose = to One from Dose Response for Each Weak Agonist - 3rd or 4th dose from dose response (Reproducibility over time) One Dose of Negative Chemical

  37. Protocol B – immature model, subcutaneous injection Protocol A – immature model, oral gavage Lab 1 Lab 2 Lab 3 450 160 180 500 Lab 1 Lab 4 Lab 2 450 400 160 140 Lab 1 Lab 5 Lab 3 400 Lab 2 350 140 120 Lab 6 Lab 7 350 Lab 3 300 120 Lab 7 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 100 Lab 1 Lab 8 300 Lab 7 Uterine Weight (mg) 250 100 Lab 8 Lab 3 Lab 9 Lab 8 250 80 Lab 9 200 Lab 7 80 Lab 10 Lab 9 200 Protocol C’ – adult OVX model, subcutaneous injection, doses extended to 7 days 60 Protocol C – adult OVX model, subcutaneous injection Lab 11 Lab 10 150 Lab 11 60 Lab 11 150 40 Lab 11 Lab 12 Lab 18 100 40 100 Lab 12 Lab 15 Lab 19 20 50 20 50 Lab 17 Lab 13 0 0 0 0 Lab 18 Lab 14 Lab 15 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0 Lab 16 0.0 0.01 0.03 0.1 0.3 1.0 10.0 3.0 Ethinyl Estradiol Administered Dose (µg/kg bw/day)

  38. Lab 6 6.00 6.00 Lab 14 Lab 13 Protocol A - immature, oral gavage Protocol B - immature, s.c. Lab 4 Lab 8 5.00 5.00 Lab 7 #1 Lab 10 #1 Lab 7 #2 Lab 10 #2 4.00 4.00 Lab 8 Lab 3 Lab 9 #1 Lab 2 3.00 3.00 Lab 9 #2 Lab 1 Lab 11 Lab 4 Lab 10 #1 Lab 9 #1 2.00 2.00 Lab 10 #2 Lab 9 #2 Lab 13 Lab 16 1.00 1.00 Lab 15 Lab 14 Lab 16 Lab 7 #1 0.00 0.00 Lab 7 #2 Phase 2 Phase 2 Phase 1 Phase 1 Phase 2 Phase 2 Phase 1 Phase 1 EE=0.3 EE=1 EE=0.3 EE=1 EE = 1 EE = 1 EE = 3 EE = 3 Ratio of uterine wt (test/control) 6.00 6.00 Protocol CX - OVX, s.c., 7 days Protocol C - OVX, s.c. injection 5.00 5.00 Lab 14 Lab 7 #1 Lab 7 #1 4.00 4.00 Lab 7 #2 Lab 7 #2 Lab 8 Lab 8 3.00 3.00 Lab 9 #1 Lab 9 #1 Lab 9 #2 Lab 9 #2 Lab 10 #1 Lab 10 #1 2.00 2.00 Lab 10 #2 Lab 10 #2 Lab 13 1.00 1.00 0.00 0.00 Phase 2 Phase 2 Phase 1 Phase 1 Phase 2 Phase 2 Phase 1 Phase 1 EE=0.3 EE=1 EE=0.3 EE=1 EE=0.3 EE=1 EE=0.3 EE=1 The OECD Program Coded Multichemical

  39. The OECD Program Coded Multichemical Laboratories replicated their EE results: - Between Phase 1 and Phase 2 (where laboratories were in both phases) - Within Phase 2 where duplicates were done

  40. The OECD Program Coded Multichemical • Two sets of EE problems: • Some ambiguity in directions • “High and low EE doses” • - The high EE concentration was the low dilution • - The low EE concentration was the high dilution • Apparently, some labs misreported the doses. • Four discovered this when requested to review records • One could not resolve the issue in reviewing records • A second discovered a dilution error, where low EE • concentration was several fold lower than intended

  41. The OECD Program Coded Multichemical 2. One laboratory repeated its ‘insensitivity’ with EE in Phase 1 in Phase 2. Protocol C – adult OVX model, subcutaneous injection This one lab did not observe statistical difference with coded EE in Phase 2 0.0 0.01 0.03 0.1 0.3 1.0 3.0 10.0

  42. Lab 1 Lab 2 Lab 3 Lab 18 Lab 6 Lab 19 4.50 4.50 Lab 20 Protocol B – immature, s.c. Protocol A - immature, oral gavage 4.00 Lab 8 Lab 1 4.00 Lab 9 Lab 2 3.50 3.50 Lab 11 Lab 3 3.00 3.00 Lab 10 Lab 6 2.50 Lab 12 2.50 Lab 8 Lab 13 Lab 9 2.00 2.00 Lab 17 Lab 10 1.50 1.50 Lab 16 Lab 13 1.00 1.00 Lab 14 Lab 16 0.50 Lab 14 0.50 0.00 0.00 Ratio of uterine wt (test/control) NP GN MX NP BPA MX DDT DBP BPA DDT DBP GN 4.50 4.50 Protocol C prime - OVX, s.c., 7 days Protocol C - OVX, s.c. injection 4.00 4.00 3.50 3.50 Lab 20 3.00 3.00 Lab 8 Lab 8 2.50 2.50 Lab 9 Lab 9 Lab 10 2.00 Lab 10 2.00 Lab 13 Lab 13 1.50 1.50 Lab 16 1.00 1.00 Lab 14 0.50 0.50 0.00 0.00 GN NP NP MX MX GN BPA BPA DDT DBP DDT DBP The OECD Program Coded Multichemical Results Compound

  43. The OECD Program Coded Multichemical Multichemical doses were either #3 or #4 out of five from dose response expts Several in lower portion of dose response curve Thus, in both dose response and multichemical, several laboratories did not achieve statistical significance (but this pattern ‘repeated’ in both sets of experiments)

  44. The OECD Program Coded Multichemical Bisphenol A – Protocol A 600 mg/kg/d Dose Response 3 pos/1 neg Multichemical 6 pos/4 neg 1 negative: 3 animals died n=3 Nonylphenol – Protocol B 80 mg/kg/d Dose Response 6 pos/3 neg (4 pos @ 80) Multichemical 11 pos/4 neg 1 negative: Lab 12 with high control uterine wts

  45. The OECD Program Coded Multichemical Results with negative chemical – dibutylphthalate 36 total experiments in 4 protocols 3 events with statistically significant increase 2 events with statistically significant decrease All within +/- 40% weight of controls Implication – there will be possible instances of both false positives and false negatives due to group to group variability

  46. The OECD Program Coded Multichemical Other observations: Dose preparation issues Hydrophobic substances difficult to prepare Heating and mechanical techniques were needed to achieve dispersion (not complete dissolution) Two laboratories reported inability to prepare Toxicity observed in some laboratories Deaths observed and euthanasia required with some chemicals

  47. The OECD Program Phytoestrogen Dietary Analysis Concerns over phytoestrogens in lab diets Rare instance of high control uterine wts leading to insensitivity of bioassay Linked to high phytoestrogen content of diet All laboratories retained sample of diet Diets analyzed (genistein, daidzein, coumestrol)

  48. 70 Lab 5 60 Lab 12 (2 points) Lab 17 50 40 Control Uterine Weights - blotted (mg) Lab 20 30 20 10 0 0 100 200 300 400 500 Genistein equivalents (micrograms/gram diet) The OECD Program Phytoestrogen Dietary Analysis Control Uterine Weights and Dietary Phytoestrogen

  49. 70 Lab 12 60 Lab 5 Lab 17 50 40 Lab 20 Uterine Weight - blotted (mgs) 30 20 10 0 20 30 40 50 60 70 Body Weight (grams) The OECD Program Phytoestrogen Dietary Analysis Control Uterine Weights and Body Weight

  50. The OECD Program Laboratory Strain Concern over sensitivity of rodent strain In this study, laboratories reported using Sprague Dawley & and Wistar rats (both supply houses and in-house sources) No difference in uterine weights of control animals attributable to strain No difference in responses to EE or any of the weak agonists attributable to strain

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