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Robert R. Maronpot1, Abraham Nyska2, Jennifer E. Foreman3, Yuval Ramot4
1Maronpot Consulting LLC, Raleigh, North Carolina, USA, 2Sackler School of Medicine, Tel Aviv University and Timrat, Israel, 3ExxonMobil Biomedical Sciences, Inc., Annadale, New Jersey, USA, 4Hadassah-Hebrew University Medical Center, Jerusalem, Israel
Keywords: Mononuclear cell leukemia, LGL leukemia, Leydig cell tumor, tunica vaginalis mesothelioma, cancer bioassay, carcinogenesis bioassay, National Toxicology Program, staging leukemia
The Fischer 344 (F344) rat was used by the National Toxicology Program (NTP) for over 5 decades for toxicity and carcinogenicity studies. However, in 2006, the NTP decided to switch to a different rat stock due largely to high background control incidences of Leydig cell tumors (LCTs) and mononuclear cell leukemia (MNCL), also known as large granular lymphocytic (LGL) leukemia. In the current review, we aim (1) to provide a summary of NTP bioassays with treatment-associated effects involving MNCL and LCTs in addition to male F344-specific tunica vaginalis mesothelioma (TVM); (2) to describe important pathobiological differences between these F344 rat tumor responses and similar target tissue-tumor response in humans; and (3) to present the NTP reasons for switching away from the F344 rat. We show that because of the highly variable background incidence of F344 MNCL, more reliance on historical control data than is usual for most tumor responses is warranted to evaluate potential effect of any chemical treatment in this rat strain. The high spontaneous incidence of LCTs in the testes of male F344 rats has made this tumor endpoint of little practical use in identifying potential testicular carcinogenic responses. TVM responses in F344 rats have a biological plausible relationship to LCTs unlike TVM in humans. Given their high spontaneous background incidence and species-specific biology, we contend that MNCL and LCT, along with TVM responses, in F344 rat carcinogenicity studies are inappropriate tumor types for human health risk assessment and lack relevance in predicting human carcinogenicity.
Table of Contents
- A Brief History of the NCI/NTP Carcinogenesis Bioassay
- NTP Switch from the F344 Rat
- Mononuclear Cell Leukemia (MNCL)
5.1. Early History of MNCL
5.2. Natural History of Spontaneous and Transplanted MNCL
5.3. The MNCL Transplant Model
5.4. Staging MNCL
5.5. Cytological, Immunophenotypic, and Functional Features of F344 MNCL Cells
5.6. F344 MNCL and Human LGL Leukemia
5.7. NTP Studies with Potential MNCL Responses
5.7.1. Early studies evaluated before use of levels of evidence of carcinogenicity.
5.7.2. Studies with clear evidence of carcinogenicity for MNCL.
5.7.3. Studies with some evidence of carcinogenicity for MNCL.
5.7.4. Studies with equivocal evidence of carcinogenicity for MNCL.
- Leydig Cell Tumors (LCT)
6.1. Features of Leydig Cell (LC) Proliferative Lesions
6.2. Chemically Induced Proliferative LC Lesions
6.3. Factors Influencing the Spontaneous Incidence of Leydig Cell Tumors (LCTs)
6.3.1. Strain and breeder
6.3.2. Dependence on body weight
6.3.3. Age dependence
6.3.4. Dependence on administration route
6.3.5. Individual caging vs. group caging
6.3.6. Effect of sexual activity
6.4. Human Leydig Cell Tumors (LCTs)
6.5. NTP Studies with an LCT Tumor Response
- Tunica Vaginalis Mesothelioma (TVM)
7.1. Features and Pathogenesis of Tunica Vaginalis Mesotheliomas in F344 Rats
7.2. TVM in Humans
7.3. NTP Studies with Increased Incidences of TVMs
- Perspective on the legacy of the F344/N rat
The Fischer 344 rat was originally produced by Dr. Maynie Rose Curtis at Columbia University in September 1920 from the 344th brother-sister mating of rats from the Fischer commercial breeder colony (Rao & Boorman 1990). This inbred rat became a favorite strain for use in tumor transplantation studies in the 1950’s (Dunning & Curtis 1957). Because of its small size, what was considered at the time to be favorable fertility, and consistent response to a number of chemical carcinogens, it was selected as the rat of choice for National Cancer Institute (NCI) cancer bioassays in 1970 (Cameron et al., 1985; Goodman et al., 1985; Weisburger 1983). Use of the F344 rat by the NCI and the National Toxicology Program (NTP) in carcinogenicity studies over 5 decades has led to the creation of the largest rat cancer bioassay database in the world. In 2006 the NTP made a decision to switch from the F344, first to the Wistar rat and subsequently to the Sprague Dawley rat for their toxicity and carcinogenicity studies (King-Herbert and Thayer 2006; King-Herbert et al., 2010). Because NTP toxicity and carcinogenicity testing practices have tended to create a testing paradigm followed by other investigators, it is unlikely that the F344 rat will see much use in carcinogenesis bioassays in the future.
There are multiple objectives to our review:
First, to provide background on NTP and discuss its reasons for switching away from use of the F344 rat. Second, to provide a retrospective summary and evaluation of mononuclear cell leukemia (MNCL)
Based on their high spontaneous background incidence and species/strain- specific biology, our conclusion is that these tumor responses in F344 rat carcinogenicity studies differ from and/or are due to different mechanism from those in humans. Thus, increased frequencies of these tumors in F344 rats do not predict human carcinogenicity.
This is not intended to be a comprehensive review of all existing literature on MNCL, LCTs, and TVMs, and it is not our intention to challenge the final NTP conclusions from the corresponding cancer bioassays. It is important to note that NTP conclusions are made in regards to the strength of evidence that a chemical exposure is responsible for increased incidence of neoplasms in rodents. The conclusions are not intended to evaluate human relevance. The intent of this review is to examine the large database and to specifically comment on the significance of these responses with respect to human health risk.
Source material for general information on the NTP carcinogenesis bioassays plus data and commentary on MNCL, LC tumors, and TVMs is derived from the publically available NTP database and published NTP toxicity/carcinogenicity technical reports (http://ntp.niehs.nih.gov).
Background incidence distributions for Figures 1 and 2 were modeled using the mean incidences and range of incidences of spontaneous neoplasms in F344 rats as reported by Haseman et al., 1998 for MNCL, LCT, and combined adenoma and carcinoma liver tumors. Liver tumors were chosen for comparative purposes to illustrate incidence and variability of a common tumor type. The modeled distributions were generated in an Excel spreadsheet using random numbers that were based on the mean incidence and a rough standard deviation for each tumor type. The rough standard deviation was generated by taking the reported range (Incidencemax – Incidencemin) divided by four, which is an accepted statistical practice (Triola 2010). The random number generation was repeated 27 times to produce a modeled distribution for the feeding studies and 18 times to produce a modeled distribution for the inhalation studies. These values were selected based on the number of studies used to compile the descriptive statistics taken from Haseman et al., 1998. (27 feeding studies and 18 inhalation studies)
3. A Brief History of the NCI/NTP Carcinogenesis Bioassay
As mentioned previously the F344 rat was derived in the 1920’s to fill a need for a reproducible cancer model (Lindsay 1979). The F344 was favored for early tumor transplantation studies (Dunning & Curtis 1957) because of its size and low spontaneous tumor rate (with the exception of the high rate of Leydig cell tumors). Dr. Dunning provided F344 breeding stock to Walter Heston at the NCI who, in turn, provided breeding stock to the NIH Division of Research Services in 1951(Cameron et al., 1985). After that the NIH colony was used as the source of F344 rats for cancer research in the 1960s. What followed were some comparisons of the F344 rat response with the Sprague Dawley, the ACI rat and the Osborne Mendel rat with the ultimate conclusion that the F344 provided a more consistent response to a spectrum of chemical carcinogen classes. This led to the selection of the inbred F344 rat as the rat choice for the NCI bioassay program, largely based on the sensitivity of the F344 to chemically induced liver tumors (Cameron et al., 1985).
The NCI cancer bioassay program transferred to the NTP in 1970. The initial emphasis of the bioassay hazard assessment program was on carcinogenicity but a substantial amount of refinement occurred from the origin of the early NCI bioassays to the contemporary study design. The majority of the studies from the early 1970’s have been relatively well standardized, thereby allowing meaningful retrospective study of tumor responses. The default group size has been 50 males and 50 females per dose along with increased group sizes to accommodate interim evaluations and special studies. The earliest studies involved a high dose that was the estimated maximum tolerated dose (MTD) and a lower dose that was one-half the MTD plus control groups. This approach evolved to include an additional lower dose but the conceptual reliance on an MTD has remained a part of the testing paradigm. More recent modifications of the NTP rodent bioassay testing approach include more emphasis on non-cancer endpoints, incorporation of mechanistic endpoints into studies, use of molecular biology to better understand the relevance of the observed responses, and a default study design incorporating in utero exposure to assess the effects of chemical exposure through the entire life cycle.
The basic NTP testing scenario is to have the toxicity and carcinogenicity studies conducted at contract research laboratories using F344 rats from the NTP colony and with study data submitted to NTP for quality assurance and pathology peer review. A draft technical report is next prepared by NTP scientists and made publicly available for comment. The draft report is peer reviewed by an external panel of scientific experts who either endorse the conclusions of the NTP or recommend modification of those conclusions.
In their cancer bioassay technical report conclusions regarding carcinogenic responses, the NTP uses five categories of evidence of carcinogenic activity to summarize the strength of evidence observed in each species and sex. There are two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major flaws (inadequate study). For the judgment of clear evidence there is a dose-related increase of malignant neoplasms, a dose-related increase in a combination of benign and malignant neoplasms, or a dose-related increase in benign neoplasms where there is evidence of progression to malignancy. In a some evidence determination the data show a treatment-relat