Sue Marty, Ph.D., D.A.B.T. The Dow Chemical Company TERA Endocrine Workshop April 23, 2013

Transcription

1 Sue Marty, Ph.D., D.A.B.T. The Dow Chemical Company TERA Endocrine Workshop April 23, 2013

2 Agenda Value of WoE/MoA determinations in EDSP Information used to examine potential endocrine activity and/or MoA: EDSP Tier 1 screening assay data Previous toxicity data (CFR Part 158 studies) ToxCast Published literature An example with an antiandrogenic response

3 Value of WoE and MoA EDSP Tier 1 was designed to minimize false negatives WoE ensures a more scientific approach to data evaluation Pattern of effects shows reproducible biological activity Confidence in decision for Tier 2 testing Understanding the MoA Ensures that Tier 1 results are not non-specific findings Can be useful to tailor Tier 2 testing

4 EDSP Tier 1: Assay Redundancy and WoE E Anti-E A Anti-A Steroidogenesis HPG HPT In vitro E A Estrogen Receptor Binding X X Estrogen Receptor Transactivation X Androgen Receptor Binding X X Aromatase X Steroidogenesis X X In vivo Uterotrophic (rat) X Hershberger (rat) X X X Female Pubertal (rat) X X X X X Male Pubertal (rat) X X X X X Amphibian Metamorphosis (frog) X Fish Short Term Reproduction X X X X X X X E = estrogen; A = androgen

5 WoE Methods 1) Gather and organize all relevant information; 2) Determine quality and reliability of data; 3) Integrate data from different sources to facilitate evaluation and data comparisons; 4) Evaluate data for factors such as consistency, coherence, adequacy, plausibility, etc; and 5) Apply a weighting or hypothesis-based evaluation system to draw conclusions on the existing dataset

6 A Case Study with Compound X: Decreased Androgen Response seen in EDSP Tier 1

7 Compound X Previous toxicity studies for compound X: Target organs: liver and kidney Kidneys: Minimal-to-slight cortical tubule nephrosis ( ) Liver: Minimal-to-slight hepatocellular hypertrophy Mild-to-moderate induction of hepatic microsomal metabolism No histopathological changes in reproductive organs, AST, or mammary tissues

8 EDSP Tier 1: Results for Compound X Compound X Anti-A Steroidogenesis HPG In vitro E A Estrogen Receptor Binding --- Estrogen Receptor Transactivation (weak) Androgen Receptor Binding --- X Aromatase --- X Steroidogenesis --- X X In vivo Uterotrophic (rat) --- Hershberger (rat) X X Female Pubertal (rat) X X Male Pubertal (rat) X X X Amphibian Metamorphosis (frog) --- Fish Short Term Reproduction --- X X X X E = estrogen; A = androgen

9 Androgen Pathway In vitro Results Data Set AR binding ToxCast (5 ARrelated assays) Steroido -genesis Compound X Negative 5 of 5 AR assays negative Negative General Assay Notes Examines parent (not metabolites) Useful to confirm positive/negatives, but may be difficult to use if results differ from EDSP Tier 1 in vitro assays; Predictiveness of ToxCast for androgen pathway unclear (examines parent): 1. ATG_AR_TRANS 2. NCGC_AR_Agonist 3. NVS_NR_rAR 4. NVS_NR_hAR 5. NCGC_AR_Antagonist Isolated positive results either very specific, cell context-dependent result or non-specific effect. Not statistically significant; induction may not be biologically meaningful (1.5-fold identified in validation program)

10 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Compound X AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay FSTRA

11 Hershberger Assay Detects interactions with AR and 5α-reductase inhibitors Castrated animals no evaluation of broad-based steroidogenesis inhibitors or HPG axis interactions RESULTS FOR COMPOUND X: Androgenic portion: No organ weight changes Antiandrogenic portion: Increase liver weights Decreased LABC and seminal vesicles No significant effects on glans penis, ventral prostate and Cowper s glands Pattern does not support 5 α -reductase inhibition

12 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Compound X AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay FSTRA

13 Androgen Pathway In vivo Results Data Set Compound X General Assay Notes Hershberger assay Positive Antiandrogenicity Antiandrogenicity due to enhanced testosterone clearance.is cmpd an enzyme inducer? Examine liver wts and serum testosterone (not definitive), save liver tissue; Examine ToxCast for active enzymes [e.g., rat CYP 2A1, human CYP 2B6 (corresponds with rat 2B1), human PXR, PXRE associated with CYP3A4 (rat CYP 3A2), PPARα, phase II UDPGT]

14 Testosterone Metabolism at Multiple Sites CH 3 CH 3 O CH 3 OH O CH 3 H 3 C Dihydrosestosterone (DHT) O 17-beta-dehydrogenase OH 5-alpha-reductase O Androstenedione (AS) CH 3 CH 3 OH Testosterone (TES) CYP HO aromatase 17-beta-dehydrogenase estradiol (E2) HO estrone (E) CH 3 O HO O H 3 C CH 3 OH 2-hydroxytestosterone (2-OHTES ) O CH OH 3 H 3 C OH 6-hydroxytestosterone (6-OHTES) H 3 C CH 3 OH O OH (7-hydroxytestosterone (7-OHTES) O CH OH 3 H 3 C 17-hydroxytetosterone (17-OHTES) OH

15 Plasma Radioactivity ( g-eq/g plasma) Time-Course of 14 C-Testosterone Radioactivity in Hershberger Rats Figure 5. Plasma Time-course of 14 C Testosterone Derived Radioactivity from Control and Treated Hershberger Rats on TD Control Treated Time (min)

16 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Compound X AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay metabolism FSTRA

17 Cmpd X: Male Pubertal Assay Results No significant differences in body weight Increased liver weight (similar to Hershberger) Decreased serum testosterone No change in the age or body weight at preputial separation * # Decreased ventral prostate, seminal vesicles, dorsolateral prostate, LABC and epididymides weights No effect on testis weights* No effect on adrenal weights* No testicular histopathology * # No change in pituitary weight or histopathology #

18 Cmpd X: Female Pubertal Results On PND 35, high-dose females weighed less than controls Increased liver weights Increased age at vaginal opening, but no change in body weight at VO Delay in age at first estrus No effect on estrous cycle (% cycling and regularly cycling)* # Decreased ovarian and uterine weights No effect on adrenal weights* Decreased pituitary weight, but no associate histopathology # No ovarian or uterine histopathology * #

19 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Test Compound AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay metabolism (adrenal wt) (adrenal wt) - (Not full pattern) (Not full pattern) FSTRA

20 Androgen Pathway In vivo Results and OSRI Data Set FSTRA Compound X Negative General Assay Notes Antiandrogens: decreased fecundity, gonadal histopathology (oocyte atresia. increased spermatogenia), decreased GSI and tubercles in males; Steroidogenesis inhibitors: Responses may vary with target but generally gonadal histopathology (oocyte atresia/decreased maturation. increased sperm and/or Leydig cells), decreased E2 and vitellogenin in females, increased GSI in males

21 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Test Compound AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay metabolism (adrenal wt) (adrenal wt) - (Not full pattern) (Not full pattern) FSTRA ---

22 Androgen Pathway In vivo Results and OSRI Data Set Part 158 Studies Compound X Negative for Endocrine Effects General Assay Notes OECD 421/422, multi-gen: mating and fertility indices, gestation index/duration, implantation nos., corpora lutea nos., pup nos., estrous cyclicity, reproductive and AST organ weights and histopathology, mammary histopathology, pituitary histopathology (hypertrophy in the adenohypophysis?); Subchronic toxicity: reproductive and AST organ weights and histopathology, mammary histopathology, pituitary histopathology (hypertrophy in the adenohypophysis?) Chronic study may be helpful (e.g., Leydig cell tumors), but must consider strain and senescence Devt Tox: litter no., corpora lutea no., resorptions, sex ratios, malformations

23 Antiandrogenic Responses: Choosing an MoA Assay Example Cmpd Results Test Compound AR Antagonist Flutamide Steroidogenesis Inhibitor Ketoconazole Disruption HPG Axis Atrazine AR binding Finasteride (5αreductase) Steroidogenesis Hershberger assay Pubertal male assay Pubertal female assay metabolism (adrenal wt) (adrenal wt) - (Not full pattern) (Not full pattern) FSTRA --- Part

24 Endocrine Pathways and Published Literature Published Literature: Consider using ToxRTool to evaluate data reliability (i.e., quality of a test report) Does not address: Study relevance appropriate? Fit for purpose? Adequacy usefulness of the data For Chemical X, 2 published studies given a Klimisch rating of 3

25 Conclusions Goal of EDSP Tier 1 assays is to ID potential interactions with estrogen, androgen and thyroid pathways (limit false negatives) Profile of effects predicted for a specific MoA may not occur May be difficult to determine MoA based on Tier 1 results Potential for endocrine activity was detected even if MoA is unclear Apical endpoints also detect indirect endocrine effects; thus, not all cmpds positive in Tier 1 will directly affect estrogen, androgen or thyroid pathways