Endocrine Mechanisms in Carcinogenesis

Endocrine mechanisms in carcinogenesis

Charles Wood Boehringer Ingelheim Inc. Ridgefield, CT

26 Oct 2020

3.1 Scope and objectives

Scope: Carcinogenicity testing/risk assessment, not translational models Objectives: • Provide brief overview of the mode of action (MOA) and adverse outcome pathway (AOP) frameworks • Review example MOAs/AOPs and common key events for common endocrine-related tumor outcomes in rodents • Discuss considerations for risk assessment and emerging applications

3.2 Case study (environmental chemical x)

2-year chronic/carcinogenicity study in male Wistar rats Dose (ppm) 0 40 200 1000 2000

Thyroid follicular cell adenomas 3/59 2/60 4/60 9/60 10/59 Thyroid follicular cell carcinomas 0/59 0/60 1/60 1/60 4/59 Combined adenomas and carcinomas 3/59 2/60 5/60 10/60 13/59

Bold = pairwise p<0.05 vs controls; dose trend p<0.01 for all Historical control mean incidence for combined tumors = 14% Other tumor outcomes: ↑ incidence of liver adenomas+carcinomas in female mice at ≥ 1000ppm

3.3 Case study (environmental chemical x)

Is this finding human relevant? What type of quantitative risk assessment should be used? Are there susceptible populations? Are other toxicity studies warranted?

3.4 A few definitions

• Mechanism of action = “detailed understanding and description of events, often at the molecular level” • Mode of action (MOA) = “sequence of key events and processes… proceeding through operational and anatomical changes, and resulting in cancer formation;” chemical- or drug-specific; may include metabolism and dose considerations • Adverse outcome pathway (AOP) = series of measurable and causally-linked key events, generally starting with a molecular initiating event and leading to an adverse health outcome; biology-focused, chemical- or drug-agnostic

2005 U.S. EPA Cancer Guidelines Ankley and Edwards. Curr Opin Toxicol. 2018 Jun 1;9:1-7.

3.5 Broad goals of the MOA/AOP frameworks

• Organize mechanistic information into discrete events leading to a cancer outcome, enabling regulatory review • Inform risk assessment process (e.g. human relevance, dose response, susceptible populations) • Facilitate understanding/study of complex mixtures and cumulative risk • Enable use of novel alternative methods (e.g. cell-based testing strategies) and predictive modeling strategies

https://www.epa.gov/sites/production/files/2013-09/documents/cancer_guidelines_final_3-25-05.pdf OECD Guidance Document on Developing and Assessing Adverse Outcome Pathways, https://one.oecd.org/document/ENV/JM/MONO(2013)6/en/pdf

3.6 Generic AOP for cancer outcome

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Tumor (↓ organ Altered cell ↑ Cellular function or Receptor/ligand signaling, gene proliferation, capacity to interaction activation hyperplasia respond)

Increasing level of biological complexity

Ankley, et al. Environ Toxicol Chem. 2010 Mar;29(3):730-741. OECD Guidance Document on Developing and Assessing Adverse Outcome Pathways, https://one.oecd.org/document/ENV/JM/MONO(2013)6/en/pdf

3.7 Case study: AOP for thyroid FC tumors

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↑ Follicular adenomas ± carcinomas

3.8 Case study: AOP for thyroid FC tumors

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↑ Follicular cell ↑ Follicular “Antithyroid” ↓ Serum T3/T4, hypertrophy & adenomas ± effect ↑ Serum TSH proliferation carcinomas

www.epa.gov -- EPA/630/R-97/002

3.9 Case study: AOP/MOA for thyroid FC tumors

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↑ Follicular cell ↑ Follicular “Antithyroid” ↓ Serum T3/T4, hypertrophy & adenomas ± effect ↑ Serum TSH proliferation carcinomas

↑ liver weight, ↓ serum T3/T4, ↑ Ki67 labeling ↑ PROD activity, ↑ serum TSH, index, ↑ follicular Example data ↑ Cyp2b expression, ↑ pituitary TSH cell hypertrophy/ supporting key ↑ T4-UDP-GT activity or β-Tsh hyperplasia events

3.10 Evaluating MOAs/AOPs

Strength, consistency, & specificity Dose-response relationship MOAs: Health organizations/ Temporal relationship regulatory agencies Biological plausibility, coherence AOPs: OECD (Advisory Group Alternative MOAs on Molecular Screening and Conclusion Toxicogenomics) Human Relevance

MOA: Boobis et al. Crit Rev Toxicol. 2006;36(10):781-792. Meek et al. J Appl Toxicol. 2014;34(1):1-18. AOP: Becker et al. Regul Toxicol Pharmacol. 2015;72(3):514-537.

3.11 Case study: Risk assessment of thyroid tumors (1)

• Thyroid FC tumors were considered to be treatment-related at doses ≥1000 ppm. • There was no evidence of direct mutagenic activity in genotoxicity studies. • Other alternative MOAs for thyroid tumors were excluded. • The proposed liver-mediated thyroid MOA was accepted based on KE data provided. • The MOA data provide justification for a threshold-based approach for human cancer risk assessment: "Not likely to be carcinogenic to humans at doses that do not alter rat thyroid hormone homeostasis.”

Dellarco et al. Crit Rev Toxicol. 2006;36(10):793-801. www.epa.gov -- EPA/630/R-97/002 https://archive.epa.gov/raf/web/pdf/thy_appx.pdf

3.12 Case study: Risk assessment of thyroid tumors (2)

• Human relevance is still the default assumption, but thyroid hormone changes in the rat should protect against any potential cancer risk in human. • “[I]f humans develop cancer through thyroid-pituitary disruption, it appears that humans are less sensitive to the carcinogenic effects than are rodents. Rodents show significant increases in cancer with thyroid-pituitary disruption; humans show little, if any.” • The Reference Dose (RfD) approach is “generally based on thyroid-pituitary disruptive effects themselves, in lieu of tumor effects, when data permit.” • Quantification of human cancer risk would not be required if overall NOAEL for non- cancer risk assessment was below dose levels that alter thyroid hormone homeostasis.

EPA/630/R-97/002, 1998, https://www.epa.gov/osa/assessment-thyroid-follicular-cell-tumors https://archive.epa.gov/raf/web/pdf/thy_appx.pdf

3.13 Case study: Risk assessment of thyroid tumors (3)

• However… this MOA may be considered relevant for developmental neurotoxicity. • The Food Quality Protection Act of 1996 directs EPA to provide enhanced protections for infants and children by using an additional tenfold margin of safety in setting tolerances for certain chemicals. • “Chemicals that perturb thyroid homeostasis and result in hypothyroidism are known to be associated with neurological disorders and alterations in neurological development, both in animals and humans.” • If a neurodevelopmental concern is raised by existing data, studies to address developmental neurotoxicity may be requested.

https://archive.epa.gov/raf/web/pdf/thy_appx.pdf

3.14 Case study: Risk assessment of thyroid tumors (4)

Finally, does this MOA indicate “endocrine disruption”? • U.S. EPA – no specific guidance • Joint Research Centre (EC, 2016) – no (indirect effect) • “Histopathological findings in rat thyroid and increased thyroid weight in presence of liver histopathology (including liver enzyme induction) were attributed to a liver-mediated mechanism not considered to be ED-mediated. Since… metabolism and excretion of thyroid hormones by the liver was not considered as an endocrine MoA, such effects were not considered relevant to conclude on ED.” • ANSES Workshop (EC, 2017) – yes (in developmental setting) • “[I]t would be unusual and problematic to discard hepatic effects leading to increased TH clearance as ‘secondary’ or not relevant for designating a chemical as a thyroid disruptor… effects on target tissues (e.g. foetal brain) of thyroid hormone insufficiency during development caused by activation of liver enzymes in a pregnant animal would not be reversible and would not be considered ‘non-adverse’.”

https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-research-reports/ https://orbit.dtu.dk/files/162452424/Thyroid_workshop_final_report.pdf

3.15 Case study (environmental chemical x)

Is this finding human relevant? What type of quantitative risk assessment should be used? Are there susceptible populations? Are other toxicity studies warranted?

3.16 Rodent carcinogenicity study outcomes (EPA)

Mr = male rat; Fr = female rat; n=288 cpds in mouse (38%+) Mm = male mouse, Fm = female mouse n=307 cpds in rat (38%+) * P < 0.05 for rat vs mouse Hill et al. Toxicol Sci. 2017;155(1):157-169.

3.17 Rodent carcinogenicity study outcomes (EPA)

Mr = male rat; Fr = female rat; Endocrine-related sites accounted for 38% (155/411) Mm = male mouse, Fm = female mouse of all treatment-related tumor outcomes. Hill et al. Toxicol Sci. 2017;155(1):157-169.

3.18 Common MOA/AOP categories

• Mutagenic • Non-mutagenic + mitogen = o Cytotoxic o Mitogenic • Non-hormonal • Hormonal

Boobis et al. Crit Rev Toxicol. 2006;36(10):781-792. Wood et al. Toxicol Pathol. 2015;43(6):760-775.

3.19 Mitogenicity model of carcinogenesis (1)

• Cancer can be defined as poorly controlled cell growth arising from uncorrected DNA errors in critical parts of oncogenes or tumor suppressor genes. • Spontaneous errors occur every time DNA is copied, due to oxidative damage, deamination, adduct formation, etc. • When errors occur, they are permanently fixed during DNA replication in the S-phase of the cell cycle. • Based on these concepts, an agent can increase risk of cancer by causing direct DNA damage, promoting errors through increased DNA replication, or inducing a combination of these effects.

Cohen and Ellwein. Science. 1990;249(4972):1007-1011. Preston-Martin et al. Cancer Res. 1990;50(23):7415-7421.

3.20 Mitogenicity model of carcinogenesis (2)

• Hormones often induce mitogenic responses in endocrine target tissues, increasing cell proliferation via classical ligand/receptor (or alternative) pathways. • This enhanced cell division may increase clonal expansion of cells with prior DNA damage, the number of target cells for DNA-damaging agents, and/or the probability of spontaneous genetic errors. • Mitogenicity does not necessarily lead to carcinogenicity… Co-factors include time/age (for key mutations to accumulate), differentiation state (e.g. higher numbers of susceptible progenitor/stem cells), genetics (e.g. poor DNA repair), and concurrent genotoxicity, cytotoxicity, or inflammation.

Tomasetti and Vogelstein. Science. 2015;347(6217):78-81. Wood et al. Toxicol Pathol. 2015;43(6):760-775.

3.21 Endocrine-related MOAs/AOPs for cancer

Tumor site Appendix Disclaimers: Thyroid (follicular cell) 1-4 • Not intended to be Thyroid (C-cell) 5 comprehensive or “definitive” – Testis (Leydig cell) 6-12 some may have data gaps Ovary 13-16 • Presented as described in the Uterus 17-18 peer-reviewed literature or other Mammary gland 19-20 publicly available review Pituitary gland 21 documents -- does not necessarily Adrenal gland 22-24 indicate prior acceptance by Neuroendocrine (ECL cell) 25 health regulatory agency or OECD

3.22 Example MOAs/AOPs for thyroid FC tumors

↑ induction of UGTs ± CAR activity in liver ↑ metabolism & clearance of T4/T3  thyroid peroxidase (TPO) Index agents: phenobarbital, thiazopyr ↓ incorporation of active iodide into iodotyrosines Index agent: 6-propylthiouracil  iodide pump activity ↓ uptake of inorganic iodide Index agent: perchlorate

Follicular cell cytotoxicity Index agents: PCBs

Hill et al. Environ Health Perspect. 1998;106(8):447-457; Hurley. Environ Health Perspect. 1998;106(8):437-445; Byrne et al. Endocrinology. 1987;121(2):520-527. MOA/AOP 1-4 EPA/630/R-97/002, https://www.epa.gov/sites/production/files/2014-11/documents/thyroid.pdf

3.23 Key event: Serum hormones

In general, measurement of reproductive hormones as “add-on” endpoints in standard toxicity studies should be avoided or used with caution.

Biological factors Power calculation for hormones in female rats • Cycle stage • Pulsatility P4 E2 LH PRL • Puberty CV (%) 97 32 25 275 • Senescence Power (%) 80 80 80 80 50% Δ 65 11 7 207 # animals Intra- and inter- 25% Δ 211 32 21 678 required individual variability

Stanislaus et al. Toxicol Pathol. 2012;40(6):943-950.

3.24 Key event: Serum hormones

• Anticipate normal variability across individuals. • Detecting hormone disturbances can be complicated by many factors, including estrous/menstrual cyclicity, diurnal variation, age/maturation, and stress. • Timing of test article effect may be critical due to compensatory changes. • Know your model and assay. • Always interpret in context of other information related to endocrine effects (histopathology, organ weights, biochemical and in vitro data, etc.).

Stanislaus et al. Toxicol Pathol. 2012;40(6):943-950; Anderson et al. Toxicol Pathol.2103;41(6):921-934. Chapin and Creasy. Toxicol Pathol. 2012;40(7):1063-1078; Weinbauer et al. Toxicol Pathol. 2008;36(7S):7S-23S.

3.25 Surrogate/complementary biomarkers to serum hormones

• Histopathology (ex. accessory sex glands, vaginal epithelium) • Immunohistochemistry (ex. tyrosine hydroxylase in hypothalamus) • Gene expression (ex. Tshb in pituitary gland) • Toxicity biomarkers (ex. antimuellerian hormone for follicular toxicity) • Enzyme activity assays (ex. T4-UDP-GT activity) • In vitro assays (ex. altered steroidogenesis)

Rouquié et al. Regul Toxicol Pharmacol. 2014;70(3):673-680. Ortiga-Carvalho et al. J Clin Invest. 2005;115(9):2517-2523.

3.26 Key event: Cell proliferation

• Central event in many endocrine-mediated MOAs/AOPs for cancer • Quantifiable by standard IHC markers: BrdU, Ki67, ±PCNA • Design considerations: power, time points, burst vs plateau effect • Enhanced coverage with digital image analysis but must know your target cells Ki67 in mouse uterus

Wood et al. Toxicol Pathol. 2015;43(6):760-775.

3.27 Use of archival samples in cancer MOA/AOP studies

• FFPE samples can provide an important resource for AOP-building and expedited MOA studies. • Newer methods (NG Sequencing, IHC/ISH, mass spec, etc.) can now overcome many of the technical issues introduced by formalin fixation. • Key research needs include better quality metrics, demodification methods, and tailored bioinformatic approaches.

Hester et al. Toxicol Sci. 2016;154(2):202-213; Wehmas et al. Toxicol Sci. 2018;162(2):535-547; Lin and Chen. Arch Pathol Lab Med. 2014;138(12):1564-1577.

3.28 Case study: AOP/MOA for thyroid FC tumors

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↑ Follicular cell ↑ Follicular (“Antithyroid” ↓ Serum T3/T4, hypertrophy & adenomas ± effect) ↑ Serum TSH proliferation carcinomas

CAR activity: hepatocyte ↑ TSH or β- FC hypertrophy, Supporting data Tsh hypertrophy, expression ↑ Ki67 LI in from archival ↑ Cyp2b in liver in pituitary thyroid FFPE samples

3.29 Novel alternative methods and AOP networks

• Current initiatives (including EDSP) aim to shift regulatory endpoints from “apical” effects to early molecular changes in endocrine pathways. • A major focus of this effort is to map early effects (e.g. receptor binding/activity, altered steroidogenesis, cell- based responses) to MIEs and KEs in AOP networks. • AOP-based endpoints would inform tiered testing approaches, cumulative assessments, and waiver models (including carci studies).

e.g. U.S. EPA 2017, https://www.regulations.gov/docket?D=EPA-HQ-OPP-2017-0214

3.30 “The EPA will reduce its requests for, and our funding of, mammal studies by 30 percent by 2025 and eliminate all mammal study requests and funding by 2035. Any mammal studies requested or funded by the EPA after 2035 will require Administrator approval on a case-by-case basis.”

-- Andrew R. Wheeler, U.S. EPA Administrator Memorandum on 10 September 2019

https://www.epa.gov/sites/production/files/2019-09/image2019-09-09-231249.txt

3.31 Summary notes

• Endocrine-related tumors are common outcomes in chronic rodent bioassays (>60% of rat tumors). • Most endocrine-related tumors in rodents occur by indirect pituitary-mediated MOAs/AOPs (see appendix). • Endocrine-related tumor outcomes with an accepted MOA are generally assessed using threshold methods based on early key events. • Newer alternative methods are being increasingly used to screen for and investigate endocrine-related MOAs/AOPs.

3.32 Thanks for your attention!

3.33 Endocrine-related MOAs/AOPs for cancer Appendix

Thyroid (1-5) Pituitary (21) Key for appendix slides

Uterus (18-19) Adrenal gland (22-24)

Testis (6-12)

Mammary gland (20-21)

Ovary (13-16) Neuroendocrine (GIT) (25)

3.34 Appendix MOAs/AOPs for thyroid FC tumors

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↑ Follicular cell ↑ Follicular “Antithyroid” ↓ Serum T3/T4, hypertrophy & adenomas and effect ↑ Serum TSH proliferation carcinomas

MOA/AOP 1-4 EPA/630/R-97/002, https://www.epa.gov/sites/production/files/2014-11/documents/thyroid.pdf

3.35 Appendix MOAs/AOPs for thyroid FC tumors

Follicular cell cytotoxicity Index agents: PCBs

3.36 Appendix MOAs/AOPs for thyroid C-cell tumors

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GLP-1R ↑ calcitonin ↑ C-cell ↑ C-cell cell activation release/synthesis proliferation/ adenomas ± & serum levels hyperplasia carcinomas

Index agents: GLP-1R agonists

Rosol. Toxicol Pathol. 2013;41(2):303-309. Knudsen et al. Endocrinology. 2010;151(4):1473-1486. MOA/AOP 5

3.37 Appendix MOAs/AOPs for Leydig cell tumors

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↑ Leydig cell ↑ Leydig cell “Antiandrogen” ↑ Serum LH proliferation/ adenomas ± effect hyperplasia carcinomas

Cook et al. Crit Rev Toxicol. 1999;29(2):169-261. MOA/AOP 6-11

3.38 Appendix MOAs/AOPs for Leydig cell tumors

 androgen receptor activity ↑ induction of UDPGTs in liver ↓ negative feedback of testosterone in hypothalamus ↑ metabolism & clearance of testosterone Index agents: vinclozalin, flutamide ↓ serum testosterone Index agent: various pesticides

 testosterone synthesis ↓ serum testosterone Index agent: lansoprazole

Cook et al. Crit Rev Toxicol. 1999;29(2):169-261. Fort et al. Fundam Appl Toxicol.1995;26(2):191-202. MOA/AOP 6-8

3.39 Appendix MOAs/AOPs for Leydig cell tumors

↑ dopaminergic activity ↓ serum prolactin ↑ PPAR-α agonist activity ↓ LH receptors on Leydig cells ± ↑ aromatase and serum estrogen ± transient ↓ in serum testosterone ± ↓ serum testosterone ± ↑ GnRH Index agents: fibrates, PFOA Index agents: sulfoxaflor, oxolinic acid

 5α-reductase activity ↓ DHT from testosterone Index agents: finasteride

Cook et al. Crit Rev Toxicol. 1999;29(2):169-261; Rasalpour et al. Crit Rev Toxicol. 2014;44 Suppl 2:25-44. Klaunig et al. Crit Rev Toxicol. 2003;33(6):655-780; Yamada et al. Toxicol Appl Pharmacol. 1995;134(1):35-42. MOA/AOP 9-11

3.40 Appendix MOA/AOP for Leydig cell tumors

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↑ GnRH agonist ↑ activation of ↑ Leydig cell ↑ Leydig cell activity1 LHRH receptors proliferation/ adenomas ± (± ↑ serum LH) on Leydig cells2 hyperplasia carcinomas

1 Induces Leydig cell proliferation at low levels; at higher levels, negative feedback inhibition may decrease serum LH and reduce Leydig cell proliferation/tumors 2 rat only; other species do not appear to have LHRH Index agent: leuprolide

Cook et al. Crit Rev Toxicol. 1999;29(2):169-261. MOA/AOP 12 Prentice and Meikle. Hum Exp Toxicol. 1995;14(7):562-572.

3.41 Appendix MOAs/AOPs for ovarian tumors

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↑ stromal or “Antiestrogen” ↑ Serum LH ± ↑ ovarian adenomas, epithelial cell effect FSH granulosa cell tumors hyperplasia

Index agents: Selective ER modulators Capen. Toxicol Pathol. 2004;32 Suppl 2:1-5. Long et al. Toxicol Pathol. 2001;29(4):403-410. Cohen et al. Reprod Toxicol. 2000;14(1):37-44. MOA/AOP 13-16

3.42 Appendix MOAs/AOPs for ovarian tumors

↑ ER degradation/↓ ER expression in hypothalamus ↓ Ø feedback from endogenous E2, ↑ GnRH Estrogen receptor (ER) blockade in hypothalamus ↓ Ø feedback from Follicular arrest or oocyte toxicity in ovary endogenous E2, ↑ GnRH ↓ endogenous E2 synthesis ↓ Ø feedback from endogenous E2, ↑ GnRH

 aromatase in ovary ↓ endogenous E2 synthesis ↓ Ø feedback from endogenous E2, ↑ GnRH

Capen. Toxicol Pathol. 2004;32 Suppl 2:1-5. Long et al. Toxicol Pathol. 2001;29(4):403-410. MOA/AOP 13-16 Cohen et al. Reprod Toxicol. 2000;14(1):37-44.

3.43 Appendix MOA/AOP for uterine tumors

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↓ serum prolactin ↑ endometrial ↑ dopaminergic ↑ endometrial ↓ luteotropism, proliferation, activity* ↑ serum E2:P4 carcinomas ↑ luteolysis, hyperplasia ↓ P4 release

*Note: In female rats, prolactin acts as the luteotrophic hormone rather Index agent: bromocriptine than LH. Dopamine agonists bind to dopamine D2 receptors in the pituitary and inhibit pituitary prolactin release. Harleman et al. Toxicol Pathol. 2012;40(6):926-930. Klaunig et al. Regul Toxicol Pharmacol. 2016;74 Suppl:S44-S56. MOA/AOP 17

3.44 Appendix MOA/AOP for uterine tumors

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↑ ERα activity altered ↑ atypical ± anovulation ↑ endometrial (perinatal differentiation, endometrial ↑E2:P4 exposure carcinomas period) ↑ progenitor cells hyperplasia

Index agent: diethylstilbestrol, genistein Newbold et al. Cancer Res. 2001;61(11):4325-4328. Suen et al. Toxicol Pathol. 2018;46(5):574-596. Suen et al. Mol Cancer Res. 2019;17(12):2369-2382. MOA/AOP 18

3.45 Appendix MOA/AOP for mammary gland tumors

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“anti-LH effect”: anovulation, ↓ latency ± ↓ GnRH pulse in ↑ mammary ↓ LH surge from ↑ time in estrus, gland ↑incidence of hypothalamus ↑ serum E2 ± mammary anterior pituitary hyperplasia prolactin tumors

Index agents: chloro-S-triazines

Simpkins et al. Toxicol Sci. 2011;123(2):441-459. O’Connor et al. Drug Chem Toxicol. 2000;23(4):575-601. https://archive.epa.gov/scipoly/sap/meetings/web/pdf/finalpartb_atz.pdf MOA/AOP 19

3.46 Appendix MOA/AOP for mammary gland tumors

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↓ - feedback, ↑ mammary ↓ latency ± ↓ dopaminergic ↑ serum gland ↑incidence of activity prolactin hyperplasia mammary tumors

Index agent: haloperidol, reserpine Simpkins et al. Toxicol Sci. 2011;123(2):441-459. O’Connor et al. Drug Chem Toxicol. 2000;23(4):575-601. MOA/AOP 20

3.47 Appendix MOA/AOP for pituitary tumors

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“anti-LH effect”: anovulation, ↑ lactotroph ↓ GnRH pulse in ↑ pituitary ↓ LH surge from ↑ time in estrus, hypertrophy & hypothalamus adenomas anterior pituitary ↑ serum E2 hyperplasia

Index agents: chloro-S-triazines

O’Connor et al. Drug Chem Toxicol. 2000;23(4):575-601. https://archive.epa.gov/scipoly/sap/meetings/web/pdf/finalpartb_atz.pdf MOA/AOP 21

3.48 Appendix MOA/AOP for adrenal medullary tumors

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↑ blood ↑ catecholamine ↑ chromaffin cell ↑ nodular ↑ pheochromo- calcium synthesis proliferation hyperplasia cytomas

Index agents: vitamin D3, sugar alcohols

Tischler et al. Toxicol Sci. 1999;51(1):9-18; Rosol et al. Toxicol Sci. 2001;29(1):41-48. Greim et al. 2016 MAK Value Documentation MOA/AOP 22

3.49 Appendix MOA/AOP for adrenal medullary tumors

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uncoupling of ↑ noradrenaline ↑ chromaffin cell ↑ nodular ↑ pheochromo- oxidative synthesis proliferation hyperplasia cytomas phosphorylation

Index agents: acrylamide, anilines

Greim et al. 2016 MAK Value Documentation MOA/AOP 23

3.50 Appendix MOA/AOP for adrenal medullary tumors

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↑ neural ↓ blood ↑ chromaffin cell ↑ nodular ↑ pheochromo- stimulation of pressure and/or proliferation hyperplasia cytomas chronic medulla hypoxemia

Index agents: reserpine

Tischler et al. Toxicol Sci. 1999;51(1):9-18. Tischler et al. Fundam Appl Toxicol. 1997;35(2):216-220. MOA/AOP 24

3.51 Appendix MOA/AOP for gastric neuroendocrine tumors

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 ↑ ECL cell gastric H+/K+ ↑ gastrin release ↑ gastric ECL-like proliferation and ATPase, ↓HCl by G cells (carcinoid) secretion hyperplasia tumors

Index agents: omeprazole

Betton et al. Toxicol Pathol. 1988;16(2):288-298. MOA/AOP 25

3.52 Appendix References

AOP/MOA frameworks • Ankley et al. 2010. Environ Toxicol Chem 29, 730–41. https://pubmed.ncbi.nlm.nih.gov/20821501/ • Ankley and Edwards. 2018. Curr Opin Toxicol 9, 1-7. https://pubmed.ncbi.nlm.nih.gov/29682628 • Boobis et al. 2006. Crit Rev Toxicol 36, 781–92. https://pubmed.ncbi.nlm.nih.gov/17118728/ • Vinken et al. 2017. Arch Toxicol 91, 3697-707. https://pubmed.ncbi.nlm.nih.gov/28660287/ • Meek et al. 2003. Crit Rev Toxicol 33, 591–653. https://pubmed.ncbi.nlm.nih.gov/14727733/ • Meek et al. 2014. J Appl Toxicol 44, 1-49. https://pubmed.ncbi.nlm.nih.gov/24777878/ • AOP: OECD Guidance Document, https://one.oecd.org/document/ENV/JM/MONO(2013)6/en/pdf • Simon et al. 2014. Crit Rev Toxicol 44, 17–43. https://pubmed.ncbi.nlm.nih.gov/25070415/ • U.S. EPA. 2005. EPA/630/P-03/001F. http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-25-05.PDF • U.S. EPA. 2017. EPA-HQ-OPP-2017-0214. https://www.regulations.gov/docket?D=EPA-HQ-OPP-2017-0214

3.53 Appendix References

Mitogenicity and cancer • Cohen and Ellwein. 1990. Science. 249, 1007-11. https://pubmed.ncbi.nlm.nih.gov/2204108/ • Preston-Martin et al. 1990. Cancer Res. 50, 7415-21. https://pubmed.ncbi.nlm.nih.gov/2174724/ • Tomasetti and Vogelstein. 2015. Science. 347, 78-81. https://pubmed.ncbi.nlm.nih.gov/25554788/ • U.S. EPA. 2005. EPA/630/P-03/001F. http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-25-05.PDF • Wood et al. 2015. Toxicol Pathol 43, 760-75. https://pubmed.ncbi.nlm.nih.gov/25903269/

Serum hormones • Anderson et al. 2013. Toxicol Pathol 41, 921-34. https://pubmed.ncbi.nlm.nih.gov/23334695/ • Chapin and Creasy. 2012. Toxicol Pathol 40, 1063-78. https://pubmed.ncbi.nlm.nih.gov/22552397/ • Ortiga-Carvalho et al 2005. J Clin Invest 115, 2517-23. https://pubmed.ncbi.nlm.nih.gov/16100573/ • Stanislaus et al. 2012. Toxicol Pathol 40, 943-50. https://pubmed.ncbi.nlm.nih.gov/22569585/

3.54 Appendix References

Thyroid (follicular cell) • Byrne et al. 1987. Endocrinology. 121, 520-7. https://pubmed.ncbi.nlm.nih.gov/3036477/ • Dellarco et al. 2006. Crit Rev Toxicol. 36, 793-801. https://pubmed.ncbi.nlm.nih.gov/17118729/ • European Commission, Joint Research Centre. 2016. https://ec.europa.eu/jrc/en/publication/eur-scientific- and-technical-research-reports/ • European Commission, ANSES Workshop. 2017. https://orbit.dtu.dk/files/162452424/Thyroid_workshop_final_report.pdf • Hill et al. 1998. Environ Health Perspect 106, 447-57. https://pubmed.ncbi.nlm.nih.gov/9681971/ • Hurley. 1998. Environ Health Perspect 106, 437-45. https://pubmed.ncbi.nlm.nih.gov/9681970/ • Rouquié et al. 2014. Regul Toxicol Pharmacol 70, 673-80. https://pubmed.ncbi.nlm.nih.gov/25455223/ • U.S. EPA. 1998. EPA/630/R-97/002. https://www.epa.gov/osa/assessment-thyroid-follicular-cell-tumors • U.S. EPA. 2005. IRIS Assessment of Perchlorate (ClO4-) and Perchlorate Salts. https://cfpub.epa.gov/ncea/iris/iris_documents/documents/subst/1007_summary.pdf

3.55 Appendix References

Thyroid (c-cell) • Rosol et al. 2013. Toxicol Pathol 41, 303-9. https://pubmed.ncbi.nlm.nih.gov/23471186/ • Knudsen et al. 2010. Endocrinology 151, 1473-86. https://pubmed.ncbi.nlm.nih.gov/20203154/

Testis (Leydig cell) • Cook et al 1999 Crit Rev Toxicol 29, 169-261. https://pubmed.ncbi.nlm.nih.gov/10213111/ • Fort et al. 1995 Fundam Appl Toxicol 26, 191-202. https://pubmed.ncbi.nlm.nih.gov/7589908/ • Klaunig et al. 2003 Crit Rev Toxicol 33, 655-780. https://pubmed.ncbi.nlm.nih.gov/14727734/ • Prentice and Meikle 1995 Hum Exp Toxicol 14, 562-72. https://pubmed.ncbi.nlm.nih.gov/7576816/ • Rasoulpour et al 2014 Crit Rev Toxicol 44 Suppl 2, 25-44. https://pubmed.ncbi.nlm.nih.gov/24832552/ • Yamada et al. 1995 Toxicol Appl Pharmacol 134, 35-42. https://pubmed.ncbi.nlm.nih.gov/7676456/

3.56 Appendix References

Ovary • Capen. 2004. Toxicol Pathol 32 Suppl 2, 1-5. https://pubmed.ncbi.nlm.nih.gov/15503657/ • Cohen et al. 2000. Reprod Toxicol 14, 37-44. https://pubmed.ncbi.nlm.nih.gov/10689201/ • Laws et al. 2014. PLoS Genet 10, e1004230. https://pubmed.ncbi.nlm.nih.gov/24603706/ • Long et al. 2001. Toxicol Pathol 29, 719-26. https://pubmed.ncbi.nlm.nih.gov/11794385/

Uterus • Harleman et al. 2012. Toxicol Pathol 40, 926-30. https://pubmed.ncbi.nlm.nih.gov/22585942/ • Klaunig et al. 2016. Regul Toxicol Pharmacol 74 Suppl, S44-56. https://pubmed.ncbi.nlm.nih.gov/26148665/ • Newbold et al. 2001. Cancer Res 61, 4325-8. https://pubmed.ncbi.nlm.nih.gov/11389053/ • Suen et al. 2018. Toxicol Pathol 46, 574-596. https://pubmed.ncbi.nlm.nih.gov/29895210/ • Suen et al. 2019. Mol Cancer Res 17, 2369-2382. https://pubmed.ncbi.nlm.nih.gov/31597742/

3.57 Appendix References

Mammary gland and pituitary • Cooper et al. 2000. Toxicol Sci 53, 297-307. https://pubmed.ncbi.nlm.nih.gov/10696778/ • O’Connor et al. 2000. Drug Chem Toxicol 23, 575-601. https://pubmed.ncbi.nlm.nih.gov/11071396/ • Simpkins et al. 2011. Toxicol Sci 123, 441-59. https://pubmed.ncbi.nlm.nih.gov/21768606/ • U.S. EPA. 2000. SAP Report No. 2000-05. https://archive.epa.gov/scipoly/sap/meetings/web/pdf/finalatrazine.pdf

3.58 Appendix References

Adrenal gland • Greim et al. 2016. MAK Value Documentation. https://onlinelibrary.wiley.com/doi/full/10.1002/3527600418.mbphaeoe4916 • Rosol et al. 2001. Toxicol Sci 29, 41-8. https://pubmed.ncbi.nlm.nih.gov/11215683/ • Tischler et al. 1997. Fundam Appl Toxicol 35, 216-20. https://pubmed.ncbi.nlm.nih.gov/9038243/ • Tischler et al. 1999. Toxicol Sci 51, 9-18. https://pubmed.ncbi.nlm.nih.gov/10496673/

Neuroendocrine • Betton et al. 1988. Toxicol Pathol 16, 288-98. https://pubmed.ncbi.nlm.nih.gov/2903544/

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