The Legacy of the F344 Rat at the National Toxicology Program

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1. The Legacy of the F344 Rat at the National Toxicology Program IATP Lecture The 33r JSTP Annual Meeting, Osaka January 26-27, 2017
2. Legacy of the F344 Rat at the National Toxicology Program (NTP) • NCI/NTP (National Cancer Institute/National Toxicology Program) • NCI in 1960s & NTP from 1970s to present • NTP levels of evidence of carcinogenicity • Origin and history of NCI/NTP use of F344 rat for cancer bioassays • Origin & selection of rat and mouse strains • F344 rat used by NTP from 1960s to 2006 • Over 570 NCI/NTP 2-year carcinogenicity bioassays using F344 rats • NTP switch from F344 to Wistar Han (briefly) and then to Harlan Sprague Dawley • F344-specific tumor responses : Mononuclear cell leukemia, Leydig cell tumors, Tunica vaginalis mesothelioma • Concerns about other F344 selected tumor responses: Kidney and Forestomach
3. Origin of the F344 Rat • Originally produced at Columbia University in September 1920 • 344th brother-sister mating of rats from the Fischer commercial breeding colony • F344 rat rapidly became favorite strain for tumor transplantation studies in the 1950’s • Selected in 1970 by the National Cancer Institute (NCI) for their cancer bioassay program • F344 rat has been used for over 50 years for cancer bioassays
4. National Cancer Institute (NCI) & National Toxicology Program (NTP) • NCI established 1937 as independent research institute • 1944 – NCI placed under the National Institute of Health (NIH) • Early history was to identify anti-cancer drugs • 1961 – focus on basic research & hazard identification • Rodent cancer bioassay established for hazard identification • 1970 – F344 selected as choice for rat cancer bioassay (B6C3F1 selected as choice for mouse cancer bioassay) • NCI cancer bioassay program transferred to NTP • 1978 – formal creation of NTP • Continued use of F344 and B6C3F1 mouse for rodent cancer bioassay
5. NTP F344 Rat Cancer Bioassay • Basic design has been 50 rats per dose per sex with exposures starting at 5 to 6 weeks of age • 3 or more doses used • Interim sacrifices for selected bioassays • (Current default design now starts with in utero exposures) • Cancer responses classified as “levels of evidence of carcinogenicity” • Clear evidence of carcinogenicity • Some evidence of carcinogenicity • Equivocal evidence of carcinogenicity • No evidence of carcinogenicity • Inadequate study for carcinogenic activity
6. Reasons for 2006 NTP Switch from the F344 Rat for Toxicity & Carcinogenicity Testing – Part 1 • High and variable background incidence of mononuclear cell leukemia (MNCL) & associated early mortality • Currently classified as large granular lymphocytic (LGL) leukemia • High incidence of Leydig cell tumors (LCT) • Tunica vaginalis mesotheliomas (TVM) associated with LCT F344 rat has high sensitivity to develop these 3 tumors
7. Reasons for 2006 NTP Switch from the F344 Rat for Toxicity & Carcinogenicity Testing – Part 2 • Poor reproductive performance & therefore not useful for reproductive toxicity studies or bioassays starting with in utero exposure • Sporadic seizures and idiopathic chylothorax • Other F344 rat concerns (not restricted to F344 rats) • Renal tumors secondary to chronic progressive nephropathy • Forestomach neoplasia secondary to irritation in gavage studies Clearly, there was no single reason for switching from the F344 rat
8. Disadvantages of Switch from F344 Rat • Loss of a robust historical control database • Need to become familiar with a new rat model • Initial switch to Wistar Han rat was short-lived • Wistar Han rat low litter size was not sufficient for NTP reproductive toxicity studies • Thus, NTP switched from Wistar Han to Harlan Sprague Dawley • Need to develop a new historical control database
9. Legacy of the F344 Rat at the National Toxicology Program (NTP) • NCI/NTP (National Cancer Institute/National Toxicology Program) • NCI in 1960s & NTP from 1970s to present • NTP levels of evidence of carcinogenicity • Origin and history of NCI/NTP use of F344 rat for cancer bioassays • Origin & selection of rat and mouse strains • F344 rat used by NTP from 1960s to 2006 • Over 570 NCI/NTP 2-year carcinogenicity bioassays using F344 rats • NTP switch from F344 to Wistar Han (briefly) and then to Harlan Sprague Dawley • F344-specific tumor responses : Mononuclear cell leukemia, Leydig cell tumors, Tunica vaginalis mesothelioma • Concerns about other F344 selected tumor responses: Kidney, Thyroid, Forestomach
10. Mononuclear Cell Leukemia (MNC) (LGL Leukemia) • Species and strain-specific • High prevalence & highly variable background incidence in male and female F344 rats • Male overall control range – 2% to 72% • Female overall control range – 6% to 66% • Progressive increased incidence since 1970s • High incidence of MNCL – major cause of early mortality • Sporadic exacerbation of MNCL by various treatment • Unexplained dramatic decrease associated with splenic toxicity and after splenectomy
11. Effect of Housing on Control Incidences of MNCL and Leydig Cell Tumors
12. Factors Affecting Incidence of F344 MNCL • Initially seen during mammary gland transplantation studies in 1950s • MNCL quickly outgrew the mammary adenocarcinoma • Leading to death in 14 to 25 days after 115th transplant generation • Easily transplanted – this allowed for extensive study of MNCL • Variables, factors & events influence background incidence • Higher in males than in females • Affected by diet • Decreased incidence in corn oil gavaged males • Increased incidence in inhalation studies • Decreased incidence associated with splenic toxicity & with splenectomy • Decreased incidence associated with liver tumor response
13. Features of MNCL Disease in F344 Rats • Macroscopic – Splenic enlargement & liver infiltration • Anemia and death in 2 to 6 weeks • Arises in spleen marginal zone & spreads to splenic red pulp • Splenic lymphoid depletion & erythrophagocytosis; secondary involvement of liver • Subsequently many organs involved but bone marrow involvement is late • Onset at 18 months with death typically by 22 months of age • Main cause of death in F344 rats 20 months and older • Behavior, immunopositive features, phenotype & NK activity similar to LGL cells
14. MNCL in Spleen Splenomegay (control spleen at top) Leukemia infiltrates & lymphoid depletion
15. MNCL in Liver
16. MNCL versus Human Leukemia F344 MNCL (LGL Leukemia) • Has LGL features • Common with high & variable incidence • F344 strain-specific • Bone marrow involvement only late in disease • No oncogenic transformation • No viral etiology Human Aggressive NK Cell Leukemia • Morphologically similar to F344 MNCL • Has LCL features • Extremely rare (only 98 cases reported worldwide) • Extensive bone marrow involvement • Dominant oncogenic transformation • Viral etiology (Ebstein-Barr virus)
17. NTP Studies with Potential MNCL Responses (Treatment-Related Increases or Decreases) • 64 Studies between 1978 & 2006 (the switch from the F344 was in 2006) • 38 Studies with MNCL positive response according to NTP • 26 Studies with dramatic decreased MNCL response (often to 0%) according to NTP • 62 of these 64 studies had primary tumor responses in other tissues • Such as kidney tumors, liver tumors, endocrine tumors, etc.
18. Examples of Non-Controversial MNCL Responses in NTP Studies • Non-controversial positive response = robust dose-response in both sexes • Because of the high and variable background incidence of MNCL, many other responses considered positive by NTP are potentially debatable • Two non-controversial MNCL responses • Furan • Male – 16%, 22%, 34%, 50% (Historical mean 21%; range = 4% to 38%) • Female – 16%, 18%, 34%, 42% (Historical mean 27%; range = 16% to 38%) • Tetrafluoroethylene • Male – 68%, 86%, 76%, 62% (Historical mean 56%; range = 38% to 66%) • Female – 32%, 62%, 46%, 72% (Historical mean 42%; range = 30% to 54%)
19. NTP Studies with Potential MNCL Responses • 2/64 had only MNCL tumor responses in both sexes in the main study • o-Nitroanisole (NTP says clear evidence of carcinogenicity) & dimethyl morpholinophosphoramidate (DMMPA) (NTP says some evidence of carcinogenicity) • o-Nitroanisole • Male – 52%, 50%, 84%, 68% (Historical mean 45%; range = 32% to 62%) • Female – 28%, 22%, 28%, 52% (Historical mean 27%; range = 14% to 36%) • DMMPA • Male – 28%, 42%, 38%, 50% (Historical mean 17%; range = 2% to 26%) • Female – 18%, 26%, 24%, 36% (Historical mean 13%; range = 2% to 42%)
20. NTP Positive MNCL Responses in One or Both Sexes • Potentially positive studies prior to NTP establishing levels of evidence of carcinogenicity – 6 very old NCI studies • Clear evidence of carcinogenicity – 9 NTP studies • Some evidence of carcinogenicity – 7 NTP studies • Equivocal evidence of carcinogenicity – 16 NTP studies • No evidence of carcinogenicity – >550 NTP F344 rat studies
21. NTP Positive MNCL Responses in One or Both Sexes • Potentially positive studies prior to NTP establishing levels of evidence of carcinogenicity – 6 very old NCI studies • Pathology findings questionable; leukemia diagnoses not defined • Clear evidence of carcinogenicity – 9 NTP studies • 2/9 had robust dose response in both sexes • Some evidence of carcinogenicity – 7 NTP studies • 2/7 had response in both sexes • Equivocal evidence of carcinogenicity – 16 NTP studies • 2/16 had questionable MNCL response in both sexes • No evidence of carcinogenicity – >550 NTP F344 rat studies
22. NTP Positive MNCL Responses in One or Both Sexes • Potentially positive studies prior to NTP establishing levels of evidence of carcinogenicity – 6 very old NCI studies • Pathology findings questionable; leukemia diagnoses not defined • Clear evidence of carcinogenicity – 9 NTP studies • 2/9 had robust dose response in both sexes • 7/9 single sex response • 3/7 statistical findings of low stringency • Some evidence of carcinogenicity – 7 NTP studies • 2/7 had response in both sexes • 7/7 positivity challenged during peer review because of variable historical control data • 7/7 remained classified as some evidence of carcinogenicity by NTP • Equivocal evidence of carcinogenicity – 16 NTP studies • 16/16 had questionable MNCL response in one sex • 2/16 had questionable MNCL response in both sexes • No evidence of carcinogenicity – >550 NTP F344 rat studies
23. Summary Conclusions on MNCL Responses in NTP F344 Rat Studies • The NTP decided to no longer use the F344 rat for their bioassay program • Due to the high and variable background incidence of MNCL in males and females • Due to unique sensitivity of F344 rat to spontaneous MNCL • Due to early mortality from MNCL in rats 20 months • Due to difficulty to make definitive identification of a treatment-related increased in MNCL • A higher stringency statistical test with use of historical control data should be used in assessing MNCL responses • There is no definitive human counterpart the F344 MNCL • F344 MNCL responses not of significant concern for human health
24. Leydig Cell Hyperplasia and Leydig Cell Tumors (LCT) • Age-related common spontaneous testes lesions in F344 (Incidences as high as 100%) • Typically benign; focal or multifocal; usually bilateral • Morphological features similar between hyperplasia and tumors • Distinction between hyperplasia and tumor based on size • Leydig cell adenoma exceeds diameter of 3 seminiferous tubules • May contain cystic areas and areas of basophilic & spindleoid cells • Associated with hormone imbalance between testicular LH receptor & serum testosterone
25. Leydig Cell Hyperplasia
26. Leydig Cell Adenomas
27. Variables Influencing Spontaneous Incidence of LCTs • Strain – F344 has highest spontaneous incidence of LCTs • NTP historical control rate = 54% to 98% • Breeder effect • Breeder 1 F344 rats – 76% • Breeder 2 F344 rats – 90% • Some evidence that increased body weight associated with decreased incidence • Age-dependent with LCTs common in second year of life • Decreased incidence in single housed rats possibly due to stress
28. Comparison with Human LCTs F344 LCT • Common with spontaneous incidences as high as 100% • Age-related increase incidence • Morphology similar to human • Typically benign Human LCT • Extremely rare (~0.1% of all human cancer) • 2 age peaks • 5-10 years & 20-60 years old • Morphology similar to F344 rat • Typically benign
29. NTP Studies with Increased LCTs • Difficult to identify an increase because of high background incidence • NTP historical control incidence = 54% to 98% • 7 studies identified by NTP with increased LCTs • 2 studies with clear evidence of LCT carcinogenicity according to NTP • 5 studies with equivocal evidence of LCT carcinogenicity according to NTP • 5/7 were inhalation studies with single housing and increased stress • Decreased incidence in inhalation controls allowed identification of marginal potentially treatment-related increase in LCTs • For 1 study (Kava kava extract) LCT was the only tumor response
30. Studies with Treatment-Related Increase in LCTs According to NTP • Clear evidence of LCT carcinogenicity • Ethyl benzene (inhalation study)* • Isoprene (inhalation study) • Equivocal evidence of LCT carcinogenicity • Cumene (inhalation study) • Kava kava extract (gavage study) • Leucomalachite green (dietary study) • Tetrafluoroethylene (inhalation study) • Tetralin (inhalation study) * LCT data is more consistent with equivocal evidence of carcinogenicity
31. Summary Conclusions on LCT Responses in NTP F344 Rat Studies • There is a very high background incidence of LCT in male F344 rats • This makes the F344 rat not useful for reliably identifying testicular carcinogens • The high incidence of LCT was a major reason for the NTP decision to no longer use the F344 rat in their cancer bioassay program • The clinical presentation of LCT in F344 rats is unique and different from the clinical presentation of LCTs in humans • Considering all facts together, a treatment-related LCT response in the F344 rat is not of significant concern to human health
32. Tunica Vaginalis Mesothelioma (TVM) • Age-associated male tumor response • Spontaneous incidence 0.2% to 5% in NTP F344 rats • F344 rat is uniquely sensitive • Associated with presence of Leydig cell tumors • Histomorphology • Similar to pleural mesothelioma • Starts as single layer of hyperplastic mesothelial cells in tunica vaginalis and may become papillary and multilayered • Can have glandular, tubular or sarcomatous phenotype • May spread into abdominal cavity, particularly when treatment-related • All considered malignant by NTP
33. Tunical Vaginalis Mesothelioma
34. TVMs Arise in the Tunica Vaginalis
35. TVMs Associated with Leydig Cell Tumors
36. Hypothetical Factors Associated with Treatment-Related Increas