The Legacy of the F344 Rat at the National Toxicology Program

The Legacy of the F344 Rat at the National Toxicology Program

IATP Lecture The 33r JSTP Annual Meeting, Osaka January 26-27, 2017

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

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

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

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

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

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

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




• F344-specific tumor responses : Mononuclear cell leukemia, Leydig cell tumors, Tunica vaginalis mesothelioma• Concerns about other F344 selected tumor responses: Kidney, Thyroid,

Forestomach

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

Effect of Housing on Control Incidences of MNCL and Leydig Cell

Tumors

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

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

MNCL in Spleen

Splenomegay (control spleen at top)

Leukemia infiltrates & lymphoid depletion

MNCL in Liver

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)

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.

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%)

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%)

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


• 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


• 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

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

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

Leydig Cell Hyperplasia

Leydig Cell Adenomas

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

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

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

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

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

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

Tunical Vaginalis Mesothelioma

TVMs Arise in the Tunica Vaginalis

TVMs Associated with Leydig Cell Tumors

Hypothetical Factors Associated with

Treatment-Related Increase in F344 Rat TVMs• Hormone imbalance

• Secondary to LCT with local release of mitogenic growth factors• Mechanical pressure & shearing forces• Expression of autocrine mitogenic growth factors

• Direct or indirect chemical effect• Increased mitogenic effect from chemicals or metabolites• Oxidative stress

Comparison with Human TVMs

F344 TVM• Arise in tunica vaginalis• Morphology similar to human

TVM• Likely mechanistically secondary

to Leydig cell tumors• Specificity to F344 rat

Human TVM• Arise from testis scrotal sac• Morphology similar to F344 TVM• More invasive & metastatic than

F344 TVM• Not secondary to Leydig cell

tumors• Alternative mechanism for

induction compared to F344 TVM

NTP Studies with Increased TVMs• 17 NTP studies with a treatment-related increase in TVMs• 4/17 Positive TVM response • (these studies done before NTP had levels of evidence categories for

carcinogenicity)

• 9/17 Clear evidence of TVM carcinogenicity• 1/17 Some evidence of TVM carcinogenicity• 3/17 Equivocal evidence of TVM carcinogenicity

Studies with Treatment-Related Increases in TVMs According to NTP• 17 NTP studies with treatment-related increased TVMs• 14/17 had clear evidence of carcinogenicity or were judged positive• 13/17 were multisite carcinogens with other tumor responses• 3/17 with TVM as the only treatment-related tumor response• Methyleugenol• O-Nitrotoluene• O-Toluidine hydrochloride

• 1/17 with some evidence of TVM carcinogenicity plus a questionable treatment-relate nasal tumor response• Pentachlorophenol

Acrylamide TVM Response• NTP Drinking water study in F344 rats• 4% (control), 4%, 1%, 10%, 18% (treated)

• Johnson et al., 1986 drinking water study in F344 rats• 5% (control), 0%, 12%, 18%, 17% (treated)

• Friedman et al., 1995 drinking water study in F344 rats• 4% (control), 4%, 4%, 8%, 17% (treated)

• Maronpot et al., 2015 drinking water study in Wistar rats• 0% (control), 0%, 0%, 0% (treated)

Results reflect the unique sensitivity of F344 rat to development of TVMs

Summary Conclusions on TVM Responses in NTP F344 Rat Studies• The F344 rat is uniquely sensitive to development of TVM• This sensitivity is not seen in other rat strains and stocks

• Development of TVM is closely associated with presence of Leydig cell tumors• Treatment-related increases likely reflect hormonal imbalance and/or

mechanical pressure from adjacent Leydig cell tumors

• Since TVMs are F344 strain-specific & only seen in the presence of Leydig cell tumors, they are of questionable significance to human health




• F344-specific tumor responses : Mononuclear cell leukemia, Leydig cell tumors, Tunica vaginalis mesothelioma• Concerns about other F344 selected tumor responses: Kidney and Forestomach

Kidney Tumor Response in the NTP Quercetin and Coumarin Rat Cancer

Bioassays

Kidney Tumor Responses in F344 NTP Studies• A low but statistically significant increase in renal tubular neoplasms• Seen in several NTP studies; more common in males• 49/>550 for males; 13/>550 for females

• Associated with chronic progressive nephropathy (CPN)• Associated with severity of CPN• CPN is a degenerative & regenerative age-related lesion in rats• CPN results in a 10-fold increase in renal tubule cell proliferation• CPN not restricted to F344 rats; also seen in Sprague Dawley rats

•Marginal responses are often followed up by step sections of remaining fixed kidney tissue

Male F344 Rat NTP Quercetin Data from

the 2-Year Study• Renal tubular adenoma• 1/50 (control), 2/50, 7/50, 8/50 (treated)

• Renal tubular carcinoma• 0/50 (control), 0/50, 0/50, 1/50 (treated)

• Chronic progressive nephropathy (CPN) severity score (0 to 4 maximum)• 2.7 (control), 2.7, 3.0, 3.2 (treated)

Renal Lesions in NTP Quercetin Study

Chronic progressive nephropathy (CPN) CPN tubule profile with thickened basement membrane

Renal Lesions in NTP Quercetin Study

Atypical tubule hyperplasia Tubular adenoma

Distinctive Morphology of Hereditary Type Eosinophilic Renal Tubular

Carcinoma

Well delineated lobules with central necrosis Higher magnification showing eosinophilic cytoplasm and variably sized nuclei

Crissman et al., 1992; Hard et al., 1994; Thurman et al., 1995

NTP Evaluation of F344 Quercetin Study Data• NTP considered the kidney tumor response as some evidence of

carcinogenicity• NTP bioassay considered a hazard identification study and does not

consider other factors such as mode of action and human relevance• Consequently, the renal tissue was re-examined by an expert in rat

kidney lesions

Re-Evaluation of Histopathology Slides by Rat Kidney Tumor Expert (Dr. Gordon Hard)• Tumor response was associated with CNP and with the severity of CPN• There was increased severity of CPN in high-dose versus control at 6 & 15

months• There was dose-related increased severity of CPN in the 2-year study• No evidence of direct renal toxicity from quercetin at any study

• 13-week, 6-month, 15-month, and 2-year studies• Number of atypical tubular hyperplasia (ATH) foci and adenomas in 2-year

study was reduced using published diagnostic criteria• Downgraded some adenomas to ATH and some ATH to tubule profiles• 5 lesions, including the tubular carcinoma, have phenotype of familial renal

tumor of spontaneous origin

Male F344 NTP Quercetin Data from the NTP 2-Year Study & Expert Re-

Evaluation• Renal tubular adenoma• 1/50 (control), 2/50, 7/50, 8/50 (treated)• 0/50 (control), 0/50, 5*/50, 5/50 (treated)

• Renal tubular carcinoma• 0/50 (control), 0/50, 0/50, 1/50 (treated)• 0/50 (control), 0/50, 0/50, 1*/50 (treated)

• Chronic progressive nephropathy (CPN) severity score (0 to 4 maximum)• 2.7 (control), 2.7, 3.0, 3.2 (treated) - severity score 0 to 4• 5.7 (control), 5.9, 6.2, 6.5 (treated) – severity score 0 to 8 *Familial

renal tumor

Expert re-evaluation

Weight of Evidence Indicates the Quercetin Renal Tumor Response is Not of Significant Concern to

Human Health• Marginal kidney tumor response secondary to exacerbation of CPN• There is no strict human counterpart of CPN• CPN associated with 10X increase in tubular cell proliferation• Absence of chemical toxicity of quercetin on kidney• Other studies of quercetin in rats, mice, and hamsters are without

renal toxicity or renal tumors• 1 carcinoma and 1 adenoma in rats given quercetin are spontaneous

tumors of familial/hereditary origin

Male F344 Rat NTP Coumarin Data from the 2-Year Study

• Chemically related increased severity of chronic progressive nephropathy (CPN) & associated tubular hyperplasia in treated rats• Hyperplasia• 6% (control), 30%, 20%, 12% (treated)

• Renal tubular adenoma• 2% (control), 12%, 14%, 10% (treated)

• Renal tubular carcinoma• 0% (control), 1%, 0%, 0% (treated)

Renal Lesions in NTP Coumarin Study

CPN Tubule Hyperplasia in Coumarin-Treated Rat Renal Tubular Adenoma in Coumarin-Treated Rat

• Kidney tumor response secondary to exacerbation of CPN• Dose-related increase in chronic progressive nephropathy (CPN)• Dose-related CPN also present at 15-month interim evaluation• No pathological evidence of direct renal toxicity at bioassay doses• Exacerbation of CPN in treated rats was the principal cause of

decreased survival• CPN is associated with 10X increase in tubular cell proliferation• There is no strict human counterpart of CPN

Weight of Evidence Indicates the Coumarin Renal Tumor Response is Not of Significant Concern to

Human Health

Forestomach Tumor Responses in NTP F344 Rat Studies

• Forestomach tumor responses have been seen in NTP gavage studies• 20/>550 in male and 16/>550 in female bioassays

• Forestomach tumors are associated with hyperplasia of the forestomach mucosa• Inflammation is a component of forestomach hyperplasia•Mucosal ulceration may be present• Cell proliferation is markedly increased

Male F344 Rat NTP 2,4-Hexadienal & Ethyl Acrylate Data from 2-Year

Studies•2,4-Hexadienal •Hyperplasia 6% (control), 38%, 84%, 100% (treated)•Papilloma 0% (control), 6%, 20%, 58% (treated)•Carcinoma 0% (control), 0%, 2%, 4% (treated)

• Ethyl acrylate •Hyperplasia 2% (control), 82%, 92% (treated)•Papilloma 2% (control), 30%, 58% (treated)•Carcinoma 2% (control), 10%, 24% (treated)

Forestomach Hyperplasia in a F344 Rat Treated with Ethyl Acrylate

Representative Example of Markedly Increased Cell Proliferation in Forestomach Hyperplasia

PCNA stain for cell proliferation

Forestomach Hyperplasia with Inflammation in a F344 Rat Treated

with 2,4-Hexadienal

Representative Example of Forestomach Papilloma and

Carcinoma

2,4-Hexadienal Study Papilloma Representative Example of Forestomach Squamous Cell Carcinoma

NTP Evaluation of F344 2,4-Hexadienal & Ethyl Acrylate Studies• NTP considered the forestomach tumor response in the 2,4-

Hexadienal study as clear evidence of carcinogenicity• NTP considered the forestomach tumor response in the ethyl

acrylate study as positive for carcinogenicity• NTP bioassay is a hazard identification study and does not

consider other factors such as likely mode of action and human relevance

Weight of Evidence of Male F344 2,4-Hexadienal & Ethyl Acrylate Forestomach Tumor Responses• Both compounds were irritating to the forestomach causing

hyperplasia and associated inflammation• Similar hyperplasia and tumors seen in female rats and in male and female mice• Markedly enhanced cell proliferation associated with development of benign and malignant

tumors

• This mode of action & human relevance has been described previously for similar tumor responses to forestomach irritation commonly associated with gavage studies• Proctor et al., (2007) Toxicol Sci 98:313-326

• No definitive anatomic counterpart in humans

Therefore, forestomach tumors secondary to irritation are not relevant to human health.

The Legacy of the NTP F344 Rat• The F344 rat has been the NTP cancer bioassay workhorse for over 5

decades• Its use has resulted in an extensive historical database and much

useful information on factors associated with development of cancer• A highly variable background incidence and increase in background

MNCL and LCTs, low fecundity, and other factors led to NTP switching to a different rat for cancer bioassays• While the utility of the F344 rat for short term toxicity and research

studies remains, the F344 rat is not recommended for chronic toxicity and carcinogenicity hazard identification studies

The Legacy of the F344 Rat at the National Toxicology Program

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