Published on February 4, 2014
Endocrine Disruptor Screening Program Webinar week 20-23 January 2014 www.huntingdon.com
Science and methodologies behind performance and interpretation EDSP in vivo mammalian assays Bob Parker PhD, Diplomate ABT Director, Safety Assessment Reproductive and Developmental Toxicology www.huntingdon.com
Science and Methodologies behind the Performance and Interpretation of Endocrine Disruptor Screening Program’s Tier 1 In Vivo Mammalian Assays: Hershberger, Uterotropic, Male Pubertal and Female Pubertal Studies – A Study Director’s Perspective Robert M. Parker, PhD, DABT Colin Williams Huntingdon Life Sciences www.huntingdon.com
EDSP Tier 1 Assays In vitro Estrogen Receptor Binding Estrogenic Non-estrogenic ER Activation Androgen Receptor Binding Androgenic Non-androgenic Aromatase Steroidogenesis Testosterone Uterotrophic Female Pubertal HPG/HPT EcoTox In vivo Estrogen www.huntingdon.com Hershberger Male Pubertal HPG/HPT Thyroid Function Fish Short-term Reproduction (HPG) Amphibian Metamorphosis (HPT) From: Marty MS, Carney EW and Rowlands JC. Toxicol Sci 120(S1), S93–S108 , 2011.
The EDSP Tier 1 screening assays encompass key endpoints within a Mode of Action (e.g., receptor binding) and along endocrine pathways (e.g., effects on HPG and HPT axes, steroidogenesis) through which a chemical has the potential to interact with the Estrogen, Androgen, or Thyroid hormonal pathways.* Steroidogenesis Inhibitor g Male Pubertal Study g g g HPG Axis HPT Axis g g g g g1 Androgen g Estrogen Androgen Antagonist Hershberger Estrogen Agonist Androgen Agonist Mode of Action Estrogen Antagonist Receptor Binding Study Design Uterotrophic Female Pubertal Study g g g g *Complementary endpoints across assays are indicated (solid red box) within each column. 1 5-reductase inhibition only www.huntingdon.com U.S. EPA 2011a. EPA-HQ-OPPT-2010-0877-0021
Examples of Neuro-Endocrine Pathways that are affected by Endocrine Disrupting Compounds Hypothalamic-Pituitary -Adrenal Axis Somatotropin Axis Hypothalamic-PituitaryGonadal Axis Hypothalamic-PituitaryThyroid Axis RETINOID SIGNALING PATHWAY VITAMIN D SIGNALING PATHWAY Black arrows denote contiguous pathways. Red arrows highlight examples of cross-talk between pathways www.huntingdon.com DETAILED REVIEW PAPER ON THE STATE OF THE SCIENCE ON NOVEL IN VITRO AND IN VIVO SCREENING AND TESTING METHODS AND ENDPOINTS FOR EVALUATING ENDOCRINE DISRUPTORS. Series on Testing & Assessment, No. 178; ENV/JM/MONO(2012)23
Reproductive Physiology: Rat and Human Similarities • Steroid hormone control of reproductive function relies on testosterone, estradiol, dihydrotestosterone and progesterone. • CNS-hypothalamic secretion of gonatropin-releasing hormone controls pituitary synthesis and release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that regulate germ cell development after puberty. LH surges induce spontaneous ovulation in the female, LH regulates testis Leydig cell testosterone production. • Hormonal regulation of uterine function and onset of delivery. • Androgens are required to maintain male spermatogenesis and secondary sex characteristics. • Dramatic endocrine changes resulting from CNS-hypothalamic-pituitarygonadal maturation responsible for puberty in males and females. Females generally attain puberty at an earlier age than males of the same species. www.huntingdon.com Gray et al, 2004 ILAR Journal, 45:4, 425
Reproductive Physiology: Rat and Human Differences • The rat placenta lacks aromatase; estrogen is produced during pregnancy by the ovary. Human placental tissue expresses high levels of aromatase. • Rat sexual differentiation is perinatal, whereas CNS sexual differentiation is postnatal, regulated to a great degree by aromatization of testosterone to estradiol. In nonhuman primates and presumably humans, more CNS events are prenatal, and androgens are more important than in rats . • The rat has a 4- to 5-day estrous cycle, with no functional corpora luteum. The estrous cycle can be monitored easily by examining daily cytology. The female rat displays sexual receptivity only during estrus after ―lights out‖ after a proestrus vaginal smear. This behavior is exquisitely dependent on estrogen followed by progesterone. Humans have a menstrual cycle approximately 28 days in duration and do not display periods of peak behavioral ―estrus‖ during the cycle. • Puberty in the rat (as measured by the age at vaginal opening and the onset of estrous cyclicity) occurs at about 32 days of age in females and 42 days of age (as measured by preputial separation an androgen-dependent event) in male Sprague-Dawley and Long-Evans rat strains. In humans, puberty occurs at 9 to 12 years of age in girls, and 10 to 14 years of age in boys. www.huntingdon.com Gray et al, 2004 ILAR Journal, 45:4, 425
OPPTS 890.1500: PUBERTAL DEVELOPMENT AND THYROID FUNCTION IN INTACT JUVENILE/PERIPUBERTAL MALE RATS 4 21 23 25 30 Postnatal Day 35 40 45 50 53 Body Weights checked daily; Dose daily adjusting for body weight Daily examination for PS Cull to 8 – 10 pups Wean & Group Assign Dosing Period BW Blood Collection Necropsy www.huntingdon.com
Thyroid Antagonist Thyroid Agonist Estrogen Antagonist * Androgen Antagonist Androgen Agonist Growth Age and Weight at Preputial Separation Hormones Thyroxine (T 4) Testosterone Thyroid Stimulating Hormone (TSH) Organ weights A Testis (separately) Epididymides (separately) Ventral Prostate Dorsolateral Prostate SV (with CG) with Fluid SV (with CG) without Fluid Levator ani/Bulbocavernosus muscles Thyroid Liver Pituitary Histopathology: Testis Epididymus Thyroid follicular epithelial height Thyroid colloid area Estrogen Agonist * MALE PUBERTAL ASSAY ENDPOINTS B B B B B C C ` D G E C F * Not designed to detect this modality however effects do occur A. Androgen agonist - A statistically significant increase in any two or more of the five required androgen-dependent tissue weights is considered positive; B. Paradoxical weight decrease; C. Atrophy; D. Aspermia and ductal atrophy; E. Variable progression to tubular atrophy; F. Hyperplasia/ hypertrophy of the interstitial cells of the testis; G. Hypospermatogenesis and interstitial cellular atrophy www.huntingdon.com
OPPTS 890.1450: PUBERTAL DEVELOPMENT AND THYROID FUNCTION IN INTACT JUVENILE/PERIPUBERTAL FEMALE RATS Postnatal Day 4 21 22 25 30 35 40 42 Body Weights checked daily; Dose daily adjusting for body weight Daily examination for VO; then daily vaginal lavage for cyclicity Cull to 8 – 10 pups Wean & Group Assign Dosing Period BW Blood Collection Necropsy www.huntingdon.com
Steroidogenesis Inhibition Thyroid Antagonist Thyroid Agonist Androgen Antagonist * Androgen Agonist * Estrogen Antagonist Growth Age and Weight at Vaginal Opening Hormones Thyroxine (T4) Thyroid Stimulating Hormone (TSH) Estrous Cyclicity Age at first estrus Organ weights: Ovaries Uterus Thyroid Liver Adrenals (paired) Histopathology: Ovarian Uterus Thyroid follicular epithelial height Thyroid colloid area Estrogen Agonist FEMALE PUBERTAL ASSAY ENDPOINTS A A B B A A * Not designed to detect this modality however effects do occur A. Androgen agonist - A statistically significant increase in any two or more of the five required androgen-dependent tissue weights is considered positive; B. Paradoxical weight decrease; C. Atrophy; D. Aspermia and ductal atrophy; E. Variable progression to tubular atrophy; F. Hyperplasia/ hypertrophy of the interstitial cells of the testis; G. Hypospermatogenesis and interstitial cellular atrophy www.huntingdon.com
Combined Male and Female Pubertal Assay Schematic Postnatal Day 4 23 25 30 35 40 42 45 50 53 Female Body Weights checked daily; Dose daily adjusting for body weight Daily examination for VO; vaginal lavage for cyclicity Cull to 8 – 10 pups Wean & Group Assign Male Body Weights checked daily; Dose daily adjusting for body weight Daily examination for PS Female Dosing Period Male Dosing Period •Female •BW •Blood Collection •Necropsy www.huntingdon.com •Male •BW •Blood Collection •Necropsy
Combined Pubertal Study versus Individual Male and Female Pubertal Studies It is acceptable to run a combined male and female pubertal study. (OPPTS 890.1500 and 890.1450). The combined study duration is the same as the Male Pubertal Assay. Separately conducted studies require 50 time-mated F0 animals. In contrast, a combined Male and Female Pubertal Assay design requires 26 timemated F0 animals. This 48% reduction in the number of F0 animals meets the spirit of the three Rs espoused by ECVAM. The other major advantages of a combined pubertal study design are: 1) most pups are evaluated rather than having one sex discarded per single gender study designs; 2) reduced reporting time; and 3) decreased costs (e.g., fewer animals, rooms; cages, technical evaluations, technician time). The only minor disadvantage is concentration of technician time during the VO and PPS evaluations. www.huntingdon.com
Concerns and Potential Pitfalls for Pubertal Studies Scheduling Parturition is often over two days causing a staggered start Stagger may be required based on necropsy technical staff Litter sizes may be unbalanced Method of animal allocation Very detailed (performed on PND 21; tight schedule) Ranked bodyweight and distributing litters across groups No placing of same-sex litter mates in the same group (controls Litter Effect) EPA Required Spreadsheets and Statistical Analysis Requires manual entry and therefore another QC/QA effort www.huntingdon.com
Concerns and Potential Pitfalls for Pubertal Studies Caging and Bedding Requirements Feed and Water Requirements Treatment (between 7 and 9 am) Oral gavage recommended Stainless steel catheter with ball Dose administration (Day of Necropsy) Transfer from dosing room to holding room Transfer from holding room to Necropsy Lab to minimize stress effects Initiation of necropsy two hours after dosing Euthanasia – decapitation (alternatives now acceptable) Completion of Necropsy by 1:00 pm www.huntingdon.com
Problems with Interpreting Pubertal Assays Inherent variability in (apical endpoints) Significant inherent biological variability in the endpoints (puberty onset (age and weight at VO and PPS), estrous cycle, organ weights) complicates interpretation. Endpoints can be altered by either endocrine or non-endocrine modes of action or by non-specific, systemic toxicity, or by impairment in growth (such as reduced food consumption). Assay Specificity Female pubertal assay: Insufficient monitoring period for estrous cycling Ovarian and uterine weights complicated by estrous cycling Male pubertal assay: False negative: Phenobarbital (< 100 mg/kg/day) thyroid effects not detected Male and Female Pubertal Assays: ―SAP: “…that a negative control substance has not been identified (in the pubertal assays)…is a major limitation to the Tier I battery. Lacking demonstration of expected negative results remains an issue for the validity of these assays”. EPA is in the process of conducting negative control studies www.huntingdon.com
Using Body Weight for MTD and Pubertal Assay Specificity A 10% decrement in final body weight has been established as a criterion for establishment of a Maximum Tolerated Dose, but subsequent studies (Marty et al., 2003; Laws, et al., 2007) suggest more than 6% decrement in final body weight of males may result in thyroid perturbations. The possibility of body weight-associated changes rather than direct endocrine disruption should be considered when there is >6% change in body weight in male rats. Feed restriction studies performed with the pubertal assay designs: 9-12% change in terminal body weight Decreased absolute Adrenal, Pituitary (♀♂) and Ovarian weights Decreased absolute Epididymal, Ventral Prostate and Seminal Vesicles weights T3 and T4 are sensitive to body weight changes 9% body weight change altered thyroid endpoints (♂) SAP: ―Body weight reductions were closely associated with perturbations in the onset of puberty and/or normal cycling. Therefore the specificity of the pubertal assays for detecting alterations in the HPG axis due to purely endocrine-related disruption is currently unclear‖. www.huntingdon.com
OPPTS 890.1400: HERSHBERGER BIOASSAY STUDY DESIGN Postnatal Day 0 42 49 59 60 Post-surgical care continued acclimatisation Acclimatisation Castration Dosing period Necropsy Related timings must remain the same but the plan may be shifted provided dosing commences no earlier than Day 49 and no later than Day 60 www.huntingdon.com
Steroidogenesis Inhibition Thyroid Antagonist Thyroid Agonist Androgen Antagonist Estrogen Antagonist Androgen Agonist Growth Weight at Necropsy Clinical Signs Hormones (Optional) Testosterone Follicle Stimulating Hormone Leutinizing Hormone Organ weights A,B Cowper's gland Glans Penis Ventral Prostate SV (with CG) with Fluid Levator ani/Bulbocavernosus Liver - optional Kidneys (paired) - optional Adrenals (paired) - optional Estrogen Agonist HERSHBERGER ASSAY ENDPOINTS A. Androgen agonist - A statistically significant increase in all five androgen-dependent organs is a clear indication of potential androgenic activity while any two or more of the five required androgen-dependent tissue weights should be considered a positive androgen agonist result. B. Androgen antagonist - A statistically significant decrease in all five androgen-dependent organs is a clear indication of potential androgenic activity while any two or more of the five required androgen-dependent tissue weights should be considered a positive androgen agonist result. www.huntingdon.com
Issues with Hershberger Assay 1.EPA and OECD test guidelines require a castrated male model. 2.Weights of the target tissues may be altered by agents other than androgen agonists or antagonists, therefore significant alterations in two or more target organ weights are required for a positive assay outcome. 3.Glans penis weights can only be collected from animals that have completed preputial separation, yet there is wide inter-laboratory variation in the mean age at which preputial separation occurs. 4.For optional endpoints, ease of measurement and ability to interpret results need to be fully considered to avoid potential ambiguities. If optional measurements are included, it is advisable to develop a priori a set of internal interpretive criteria specified in the study protocol before conducting the assays. 5.If optional steroid hormone levels are measured, the anesthetic agent and euthanizing method should be chosen carefully to avoid artifacts. www.huntingdon.com Borgert et al. 2011. Regulatory Toxicology and Pharmacology 59: 397–411
OPPTS 890.1600: UTEROTROPHIC ASSAY STUDY DESIGN: OVERIECTOMIZED RAT Postnatal Day 35 42 Acclimatisation 49 56 Post-surgical care continued acclimatisation Vaginal Swabbing Animal arrival 59 Ovariectomy Dosing Necropsy Related timings must remain the same but the plan may be shifted provided ovariectory is conducted in animals between 6 and 8 weeks of age www.huntingdon.com
Steroidogenesis Inhibition Steroidogenesis Induction Thyroid Antagonist Thyroid Agonist Androgen Antagonist Androgen Agonist Estrogen Antagonist UTEROTROPHIC ASSAY ASSAY ENDPOINTS Growth Uterine Weights A With Fluid Without Fluid Estrogen Agonist Uterotrophic Assay Endpoints A. Positive response Estrogen Agonist: Statistically significant increase of the mean uterus weight (wet and or blotted) www.huntingdon.com
Issues with Uterotrophic Assay 1. EPA has stated a preference for the ovariectomized female rat model while OECD favors the immature rat model (based on animal welfare concerns). 2. Immature model appears more sensitive to dietary phytoestrogen content, to body weight influences on uterine weight, and to systemic toxicity. 3. Ovariectomy increases animal manipulation/stress, introduces an artificial loss of organ function to the assay, and requires additional acclimatization after surgery as well as the requirement to confirm complete ovariectomy prior to the assay and at necropsy; small ovarian remnants can alter assay outcome. 4. EPA favors subcutaneous route while OECD states most relevant route of exposure should be used with consideration given to first pass metabolism. 5. For substances rapidly deactivated by first-pass hepatic metabolism, the subcutaneous route may produce positive results that are irrelevant for the route of administration used in Tier 2 testing and for actual environmental exposures. www.huntingdon.com Borgert et al. 2011. Regulatory Toxicology and Pharmacology 59: 397–411
Weight-of-Evidence Considerations Tier 1 screening tests are intended to minimise false negatives – focus is on sensitivity, not specificity In vitro tests indicate mode of action, not endocrine activity In vivo tests include influence of kinetics / metabolism / endocrine feedback control systems – but also nonendocrine responses Tests are complementary and ―redundant‖ – each mode of action is assessed in more than one test Look for consistent / inconsistent results Consider the range, nature and magnitude of effects www.huntingdon.com
Flowchart for assessment of endocrine disrupting properties for human health Multi-endpoint studies (apical, in vivo) Supporting studies Targeted endpoint studies (mechanistic, in vitro & in vivo) (non apical, in vivo) No adverse health effects giving concern for endocrine activity A Endocrine activity giving concern for endocrine toxicity B No ED concern per Weybridge? Adverse effects giving concern for endocrine toxicity Endocrine activity giving concern for endocrine toxicity C Adverse health effect in apical study supported by mechanistic evidence of endocrine mediated effect Endocrine activity giving concern for endocrine toxicity D No evidence of Endocrine activity E No or insufficient evidence of ED MoA per Weybridge C. When adverse effects on endocrine relevant endpoints in apical or supporting non-apical in vivo studies are supported by mechanistic data from in vitro and in vivo studies, (i.e. the sequence of the biochemical and cellular events that www.huntingdon.com
Flowchart for Assessment of Endocrine Disrupting Properties for Human Health Adverse health effect in apical study supported by mechanistic evidence of endocrine mediated effect Yes Sufficient evidence of ED as per Weybridge Are adverse effects specific? No Yes Relevance of ED mechanism to humans? (unless exposure is negligible) No Risk assessment based on non-endocrine endpoint Yes Determine potency of ED according to proposed criteria www.huntingdon.com Risk assessment based on endocrine endpoint with assessment factors according to potency Modified from ECETOC Technical Report 106, June 2009 Bars R et al. 2011. Reg Toxicol Pharmaco 37–46.
Relevance and strength Weighting of Tier 1 Endocrine Screening Endpoints Borgert et al. (2011a) proposed a framework were the relevance of each endpoint is assigned a weight according to its importance for evaluating a specific hypothesis (described above). ―Weight‖ implies that all data do not contribute equally to answering the question posed. Thus, ―weighting‖ involves a careful consideration of the specific hypothesis to be evaluated and how each particular measurement (data) informs that hypothesis. Ideally, weight would be assigned quantitatively based on objective measurements of predictive power, false positive and negative detection rates, and potency or strength of the response. This value is deemed a relevance weight, designated ―WREL.‖ The strength of response produced by the test chemical in a particular assay or endpoint is also given weight, a value deemed the response weight, ―WRES‖. www.huntingdon.com
ANY BURNING QUESTIONS? www.huntingdon.com
References Bars R, Broeckaert F, Fegert I, Gross M, et al. 2011. Science based guidance for the assessment of endocrine disrupting properties of chemicals. Regulatory Toxicology and Pharmacology 59 (2011) 37–46. Borgert CJ, Mihaich EM, Ortego LS, et al. 2011a. Hypothesis-driven weight of evidence framework for evaluating data within the US EPA's Endocrine Disruptor Screening Program. Regul Toxicol Pharmacol. 61:185-191. Borgert CJ, Mihaich EM, Quill TF, Marty MS et al. 2011b. Evaluation of EPA's Tier 1 Endocrine Screening Battery and recommendations for improving the interpretation of screening results. Regul Toxicol Pharmacol. 59:397-411. Borgert CJ, Baker SP, Matthews JC. 2013. Potency matters: Thresholds govern endocrine activity. Regul. Toxicol.Pharmacol., 67(1):83–88. Detailed Review Paper on the State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors. Series on Testing & Assessment, No. 178; ENV/JM/MONO(2012)23. ECETOC Guidance on Identifying Endocrine Disrupting Effects .Technical Report 106 (June 2009) http://www.ecetoc.org/technical-reports Gray, LE, Wilson V, Noriega N et al. 2004. Use of the Laboratory Rat as a Model in Endocrine Disruptor Screening and Testing. ILAR, Journal 45(4): 425-437. Kortenkamp A, Martin O, Faust M, Evans R, McKinlay R, Orton F and Ro E. 2011. State of rhe Art Assessment of Endocrine Disrupters: Final Report . European Commission, DG Environment , Section 7.2.2., p.127. www.huntingdon.com
References Laws SC, Stoker TE, Ferrell JM, Hotchkiss MG and Cooper RL. 2007. Effects of altered food intake during pubertal development in male and female wistar rats. Toxicol Sci 100:194-202. Marty MS, Johnson KA and Carney EW. 2003. Effect of feed restriction on Hershberger and pubertal male assay endpoints. Birth Defects Research Part B: Developmental and Reproductive Toxicology 68(4), 363374. Marty MS, Carney EW and Rowlands JC. 2011 Endocrine Disruption: Historical Perspectives and Its Impact on the Future of Toxicology Testing. Toxicol Sci 120(S1), S93–S108 . OECD (Organization for Economic Cooperation and Development). 2012. Guidance Document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption no 150. Organisation for Economic Cooperation and Development, Paris, 24-Aug-2012. Picut CA, Remick AK, Asakawa MG, Simons ML and Parker GA. 2013) Histologic Features of Prepubertal and Pubertal Reproductive Development in Female Sprague-Dawley Rats. Toxicol Pathol U.S. EPA (Enviromental Protection Agency) 2011a. Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. EPA-HQ-OPPT-2010-0877-0021. US EPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013. Weybridge, 1996. European Workshop on the impact of endocrine disrupters on human health and wildlife. 2–4 December 1996, Weybridge, UK. In: Report of Proceedings EUR 17549 Copenhagen, Denmark: European Commission DG XII, April 16, 1997). Available from: European Environment Agency, Kongens Nytorv 6, DK-1050 Copenhagen K, Denmark. Zoeller RT, Brown TR,L. Doan LL, Gore AC et al. 2012. Endocrine-Disrupting Chemicals and Public Health Protection: A Statement of Principles from the Endocrine Society. Endocrinology 153: 4097–4110. www.huntingdon.com
Other webinars this week 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 firstname.lastname@example.org +44 (0) 1480 892031 www.huntingdon.com
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