Rodent liver tumors
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1. Rodent Liver Tumors Bob Maronpot, Raleigh, NC IET Established 1970 50+ Year Anniversary
2. Rodent Liver Tumors Bob Maronpot, Raleigh, NC • A little bit of National Cancer Institute (NCI) and National Toxicology Program (NTP) rodent cancer bioassay history • NTP liver tumor data • Liver tumor images • Current safety assessment perspective IET Established 1970 50+ Year Anniversary
3. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers 1775 – Scrotal cancer in chimney sweeps. Cancer was attributed to the tar and soot in the chimneys. This is an early and famous example of occupational cancer in humans. Rodent Bioassay Timeline
4. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers • Yamagiwa & Ichikawa – 1915-1924 Rodent Bioassay Timeline Ichikawa Koichi 1888-1948 Katsusaburo Yamagiwa (1863-1930) Tar and soot painted on rabbit ears produced cancer • Murphy & Sturm – 1925 • Lung tumors in tar-painted mice • Cook et al. – 1932 • Cancer induction by PAHs
5. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Murphy & Sturm – 1925 • Lung tumors in tar-painted mice • Cook et al. – 1932 • Cancer induction by PAHs • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers • Yamagiwa & Ichikawa – 1918 • Tar & soot on rabbit ears Rodent Bioassay Timeline Takaoki Sasaki 1878-1966 Tomizo Yoshida 1903-1973 o-Amidoazotoluene diet and liver cancer. Effects of dose on latency. Use of stop studies. Sasaki and Yoshida – 1935 • Sasaki and Yoshida – 1935 • o-amidoazotoluene diet and liver cancer • Berenblum – 1941 • Concept of co-carcinogenesis • Initiation, promotion, progression • Magee & Barnes – 1956 • Nitrosamines & liver cancer in rats
6. 1700’s 1950 1960 1970 1980 1990 2000 2010 • Bernardino Ramazzini – 1713 • John Hill – 1761 • Percival Pott – 1775 • Elmslie -1866 • Jonathon Hutchinson – 1888 • Rehn – 1895 • Yamagiwa & Ichikawa – 1918 • Murphy & Sturm – 1925 • Cook et al. – 1932 • Sasaki & Yoshida – 1935 • Berenblum – 1941 • Magee & Barnes – 1956 • Realization that chemicals, environmental factors, and aspects of lifestyle cause cancer Realization that chemicals, environmental factors, and aspects of lifestyle cause cancer
7. Concept of the Rodent Bioassay & Its Establishment by the National Cancer Insititute (NCI) •1962 – First contracted cancer bioassay •1969 – Innes et al*., study published •20,000 mice; 127 different chemicals; 18-mo studies •Selection of B6C3F1 mouse •1971 – U.S. National Cancer Act •Decision made to standardize bioassay testing •~1975 – Inbred F344 rat selected •Small size, vigor & survival, disease resistance *Innes et al., JNCI 42(6): 1104-1114 (1969)
8. Concept of the Rodent Bioassay & Its Establishment by the National Cancer Insititute (NCI) •1962 – First contracted cancer bioassay •1969 – Innes et al*., study published •20,000 mice; 127 different chemicals; 18-mo studies •Selection of B6C3F1 mouse •1971 – U.S. National Cancer Act •Decision made to standardize bioassay testing •~1975 – Inbred F344 rat selected •Small size, vigor & survival, disease resistance *Innes et al., JNCI 42(6): 1104-1114 (1969)
9. Thou shalt use standardize tests
10. Thou shalt use two species
11. Thou shalt use the MTD & 1/2 MTD
12. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP CANCER BIOASSAY TIMELINE The NCI cancer bioassay 50 Male and 50 female F344 rats 50 Male and 50 female B6C3F1 mice Maximum tolerated dose (MTD) & 1/2 MTD Test duration of 18 months or 2 years Pathology evaluation
13. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP Input from National and International Organizations Refining of the Bioassay • Standardization of bioassay – Originally designed for screening • Extensive pathology with peer review* – Standardization of diagnostic nomenclature • Statistical evaluation standardized • Historical control database • Search for alternative models *Maronpot & Boorman (1982) Toxicol Pathol 10(2): 71-78 Standardization of bioassay Originally designed for screening Extensive pathology with peer review* Standardization of diagnostic nomenclature Statistical evaluation standardized Historical control database Search for alternative models
14. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP Limitations of the bioassay • Resource intensive • Bioassay not validated • Inherent insensitivity for detecting weak or moderate carcinogens • Single chemical exposure vs “real world” • Not sure if an agent has carcinogenic potential under actual human exposure conditions • Debate regarding relevance • Rodent-specific mechanisms • High doses • Resource intensive • Bioassay not validated • Inherent insensitivity for detecting weak or moderate carcinogens • Single chemical exposure vs “real world” • Not sure if an agent has carcinogenic potential under actual human exposure conditions • Debate regarding relevance • Rodent-specific mechanisms • High doses
15. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP Alternative models & ancillary approaches • Strain A mouse • Two-stage & neonatal models • Humanized mice • Ito medium-term model • Transgenic models • Local subcutaneous injection • Medaka & guppy models • Genotoxicity batteries Strain A mouse model Two-stage & neonatal rodent models Use of humanized mice Ito medium-term model Transgenic mouse models Subcutaneous injection Medaka & guppy models Genotoxicity studies
16. # Mouse (%), n=490* Rat (%), n=490* 1 Liver (27.1) Liver (10.6) 2 Lung (8.8) Kidney, tubular cell (9.2) 3 Forestomach (4.7) Mammary gland (5.9) 4 Hematopoietic system (4.5) Lung (4.6) 5 Harderian gland Thyroid gland, follicular cell (2.7) Thyroid gland, follicular cell (4.5) 6 Kidney, tubular cell (2.5) Forestomach (4.3) 7 Vascular System (Unspecified) (2.3) Urinary bladder (4.1) 8 Mammary gland (2.2) Skin (3.8) 9 Ovary (2) Hematopoietic system (3.7) 10 Skin (1.6) Adrenal medulla Oral cavity Zymbal gland (3.5) Target Organs of Chemical-induced Carcinogenicity *n=490 studies where the same chemical was tested in both F344 rats and B6C3F1 mice Courtesy of A. Pandiri 2020
17. Historical control incidences of liver tumors in rats (F344/N) and mice (B6C3F1) Background Liver Tumor Incidence Tumor type Male Mouse % (Range%) Female Mouse % (Range%) Male Rat % (Range%) Female Rat % (Range%) Hepatocellular Adenoma 54.91 (34-78) 25.68 (10-67) 1.43 (0-6) 0.86 (0-4) Hepatocellular Carcinoma 30 (16-50) 12.93 (4-20) 0.57 (0-4) 0.14 (0-2) Hepatoblastoma 3.27 (0-8) 0.55 (0-2) 0 0 Combined 71.82 (62-84) 34.43 (16-73) 2 (0-6) 1 (0-4) Mouse, n=550; Rat, n=700 Courtesy of A. Pandiri 2020
18. Liver 57 % Lung 22 % Kidney 22 % Mammary gland 14 % Hematopoietic 13 % Forestomach 12 % Thyroid 10 % Vascular System 9 % Frequency of Tissue Response in 290 Cancer- Positive NTP Mouse and/or Rat Studies Data courtesy of D. Malarkey 2022
19. • 30% (146) of 490 NTP studies had an hepatocellular tumor response in rats and/or mice* • Species dependence: mouse – 95/146 (65%), rat – 14/146 (9.6%), or both species 37/146 (25.3%) Liver Tumor Incidences Based on 490 Studies Liver tumors N=146/490* Mouse Male n (%) Mouse Female n (%) Rat Male n (%) Rat Female n (%) Nodule 0 1 (0.6) 10 (6.8) 6 (4.1) Hepatocellular Adenoma 6 (4.1) 14 (9.5) 1 (0.6) 5 (3.4) Hepatocellular carcinoma 64 (43.8) 88 (60.2) 31 (21.2) 30 (20.5) Hepatoblastoma 20 (13.7) 13 (8.9) 0 (0) 1 (0.6) Combined 90 (61.6) 116 (79.5) 42 (28.8) 42 (28.8) * 490 studies with same chemical tested in both rats and mice Data courtesy of A. Pandiri 2022
20. Hepatocellular Adenomas and Carcinomas
21. Hepatic Foci of Cellular Alteration
22. Hepatocellular Adenoma Untreated Male B6C3F1 Mouse
23. Hepatocellular Adenoma
24. Hepatocellular Carcinoma
25. Hepatocellular Carcinoma
26. Progression of Proliferative Liver Lesions Basophilic Focus Hepatocellular adenoma Metastatic carcinoma Hepatocellular carcinoma
27. Hepatoblastoma Turusov et al., Tox Path 30(5):580-591 (2002) (63/140 studies had hepatoblastoma) (Evaluated 500 hepatoblastomas)
28. Carcinoma Arising in Adenoma
29. Cholangioma Sprague Dawley Male
30. Cystic Cholangioma Male F344 Rat Control
31. Cholangiocarcinoma B6C3F1 Untreated male
32. Hepatocholangiocarcinoma Hepatocholangioma Treated Female Sprague Dawley Treated Male F344
33. Hepatocholangiocarcinoma with intestinal metaplasia
34. Other types of liver tumors Hemangiosarcoma Histiocytic sarcoma Stellate cell tumor Lymphoma
35. What Have We Learned from the Conventional Cancer Studoes with Respect to Liver Tumors?
36. Malarkey, DE, Hoenerhoff, MJ, and Maronpot, RR. 2018. Carcinogenesis: Manifestations and Mechanisms in Fundamentals of Toxicologic Pathology, 3rd Edition, Wallig, MA, Haschek, WM, Rousseaux, CG, Bolon, B, and Mahler, BW, Editors, Academic Press, San Diego. Pp 83-104.
37. Multistage hepatocarcinogenesis normal focus of altered hepatocytes hepatocellular adenoma hepatocellular carcinoma H-ras activation altered Brca1 altered TGFa Cathepsins Osteopontin Goliath MIG MHC class II B-catenin apoptosis c-fos cyr61
38. There were and still are some strong opinions about the significance & relevance of rodent bioassays. There were and still are some strong opinions about the significance & relevance of rodent bioassays
40. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP 2022 • Nuclear receptor activation • CAR/PXR, AhR, PPAR-a • Cytotoxicity and regenerative hyperplasia • Endocrine modifiers • Epigenetic modifiers • Mitogen/tumor promoter • Inflammation • Oxidative stress • Hormonal perturbation • Immunosuppression • Suppression of apoptosis Mechanisms associated with bioassay tumor responses Mechanisms associated with bioassay tumor responses
41. Contemporary efforts to identify carcinogens • Core set of mechanistic assays • DNA repair & reactivity • Receptor-mediated assays • Intercellular communication • Enzyme induction • Cell cycle perturbations • Endocrine disruption • Effects on methylation • Oxidative stress • Immunosuppression • Other contemporary investigative approaches • NEGCARC (Genotoxicity, endocrine, histopathology) for pharmaceuticals • Tox 21 & high throughput screening assays • Genomics, proteomics, metabonomics • Mutations in cancer genes • Structure activity relationships • Epigenetic changes • Adverse outcome pathway/MOA Throughput QSAR Relationships Cell culture and Genetox assays Organoids, metabolically competent Lower order model organisms Rodent models Human relevance
42. Considerations in Safety Assessment Studies for IET • Contemporary regulatory requirements for conventional animal safety assessment studies are still required nationally and internationally. • IET should maintain expertise in conduct of conventional safety assessment studies • Contemporary regulatory requirements for conventional animal safety assessment studies are still required nationally and internationally. • IET should maintain expertise in conduct of conventional safety assessment studies
43. Considerations in Safety Assessment Studies for IET • At the present time there are no regulatory approved mechanistic assays or investigative studies to replace contemporary animal safety assessment studies. • At the present time there are no regulatory approved mechanistic assays or investigative studies to replace contemporary animal safety assessment studies • Experience with alternative short duration carcinogenicity studies will be important for IET to maintain expertise in safety assessment. •Experience with alternative short duration carcinogenicity studies will be important for IET to maintain expertise in safety assessment.
44. Considerations in Safety Assessment Studies for IET • Depending on the test agent and anticipated extent and duration of human exposure, there is some consideration for reducing a strict regulatory requirement for 2 traditional two-year carcinogenicity studies. • Depending on the test agent and anticipated extent and duration of human exposure, there is some consideration for reducing a strict regulatory requirement for 2 traditional two-year carcinogenicity studies.
45. Considerations in Safety Assessment Studies for IET • Contemporary development of laboratory expertise with alternative cellular based and other mechanistic studies to potentially replace conventional in vivo animal studies is strongly recommended for the future of IET. • Contemporary development of laboratory expertise with alternative cellular based and other mechanistic studies to potentially replace conventional in vivo animal studies is strongly recommended for the future of IET.