Granulocytosis, a Paraneoplastic Syndrome Associated With Non-Glandular Squamous Cell Carcinomas (NGSCC) in the Tg.rasH2 Studies.

Non-glandular squamous cell carcinoma (NGSCC) is an extremely rare tumor in Tg.raH2 mice. There have been 5 NGSCC in 1615 control male mice (0.31%) and 2 NGSCC in 1560 control female mice (0.13%) on 26-week carcinogenicity studies, with a range of 0 to 1 of per group per sex in each study without statistical significance in 52 male and 51 female studies conducted in Tg.rasH2 mice. Every case of NGSCC was accompanied by profound granulocytosis.

Assessment of the Carcinogenic Potential of Pretomanid in Transgenic Tg.rasH2 Mice.

Pretomanid is a nitroimidazooxazine antimycobacterial drug that was approved as part of a three-drug oral regimen, consisting of bedaquiline, pretomanid, and linezolid, for 6-months treatment of adults with pulmonary extensively drug-resistant tuberculosis or with complicated forms of multidrug-resistant tuberculosis by the food and drug administration in the United States and regulatory bodies in over 10 other countries. Nitroaromatic compounds as a class carry a risk of genotoxicity and potential carcinogenicity based on reactive metabolite formation. A battery of good laboratory practice genotoxicity studies on pretomanid indicated that the compound was not genotoxic, however its hydroxy imidazole metabolite (M50) was genotoxic in the Ames assay. To assess the in vivo carcinogenic potential of pretomanid, hemizygous Tg.rasH2 mice were administered pretomanid once daily by oral gavage for 26 weeks. Male mice were given pretomanid in vehicle at doses of 0, 5, 15 and 40 mg/kg/day and female mice were given pretomanid in vehicle at doses of 0, 10, 30 and 80 mg/kg/day. Positive control mice of both sexes received intraperitoneal injections of urethane at 1000 mg/kg on Days 1, 3 and 5. There were no pretomanid-related early deaths, tumors, non-neoplastic microscopic findings, or gross necropsy findings at any dose level. The positive control gave the anticipated response of lung tumors. Oral administration of pretomanid to mice produced plasma exposure to the parent compound (high dose AUC of pretomanid 3 times the clinical AUC at the maximum recommended human dose) and exposure to the M50 metabolite (less than 10% of pretomanid) at all dose levels in both sexes. These data show that pretomanid was not carcinogenic in a transgenic mouse model at systemic exposures greater than human therapeutic exposures.

Chemical-induced lung tumor in Tg-rasH2 mice: a novel mouse tumor model to assess immune checkpoint inhibitors combined with a chemotherapy drug.

In subcutaneous tumor models, changes in the tumor microenvironment can lead to differences in therapeutic treatment responses between the subcutaneous and parent tumors. Accordingly, we generated a lung carcinogenesis model that combines genetically modified mice (Tg-rasH2 mice) with two-stage chemical carcinogenesis as an alternative to the subcutaneous tumor model. In this model, Tg-rasH2 mice were treated with 1-ethyl-1-nitrosourea, followed by butylhydroxytoluene. Mice developed lung adenomas five weeks after treatment initiation. Subsequently, anti-mouse PD-1 antibody (α-mPD-1) or isotype control was administered intraperitoneally twice a week for 4 weeks. Tumor growth was examined by measuring the relative tumor area in serially sliced lung histopathological specimens. No statistically significant differences were observed in the relative lung tumor areas between treated and control groups. A second experiment then examined the antitumor efficacy of α-mPD-1 combined with gemcitabine in a mouse model. Mice were treated identically as in Experiment 1, except that the treated group received once-weekly intraperitoneal injections of 10 mg/kg gemcitabine. In contrast to Experiment 1, the combined treatment significantly reduced the relative tumor areas in the lungs. This result also resembles that of a phase III clinical trial (ORIENT-12), showing that patients with non-small-cell lung carcinoma benefited from combination treatment with gemcitabine and the anti-human PD-1 antibody sintilimab. Thus, this mouse model could be a feasible means to preclinically evaluate the antitumor efficacy of different immunotherapy and chemotherapy drug combinations.

Carcinogenicity assessment of baricitinib in Tg.rasH2 mice and Sprague-Dawley (Crl:CD) rats.

Baricitinib is a potent and selective Janus kinase (JAK)1 and JAK2 inhibitor, and is approved for the treatment of moderately to severely active RA in adults in Europe, Japan, and other countries. This study evaluated the carcinogenic potential of baricitinib in Tg. rasH2 mice and Sprague-Dawley (Crl:CD) rats. Baricitinib was administered daily by oral gavage to Crl:CD rats for up to 94 weeks (dose levels of 0, 1, 3, or 8 mg/kg for males and 0, 3, 8, or 25 mg/kg for females) and to Tg. rasH2 mice for 26 weeks (dose levels of 0, 15, 40, or 300 mg/kg for males and 0, 10, 30, or 150 mg/kg for females). Baricitinib was well tolerated with no incidence of compound-related neoplasms at any dose levels in rats and mice. In mice, non-neoplastic events observed were bone marrow hypocellularity and increased adipocytes. In rats, baricitinib administration was associated with a dose-dependent increase in survival, with a decreased incidence of neoplasm (hematopoietic and mammary), potentially secondary to drug-related decreased weight gain. The incidence of proliferative changes such as neoplastic and hyperplastic lesions in the mammary glands of females and in the livers of males and females also decreased. In conclusion, baricitinib is not considered to be carcinogenic.

Twenty-six-week oral carcinogenicity study of 3-monochloropropane-1,2-diol in CB6F1-rasH2 transgenic mice.

The carcinogenic potential of 3-monochloro-1,2-propanediol (3-MCPD) was evaluated in a short-term carcinogenicity testing study using CB6F1 rasH2-Tg (rasH2-Tg) mice. 3-MCPD is found in many foods and food ingredients as a result of storage or processing and is regarded as a carcinogen since it is known to induce Leydig cell and kidney tumors in rats. Male and female rasH2-Tg mice were administered 3-MCPD once daily by oral gavage at doses of 0, 10, 20, and 40 mg/kg body weight (bw) per day for 26 weeks. As a positive control, N-methyl-N-nitrosourea (MNU) was administered as a single intraperitoneal injection (75 mg/kg). In 3-MCPD-treated mice, there was no increase in the incidence of neoplastic lesions compared to the incidence in vehicle control mice. However, 3-MCPD treatment resulted in an increased incidence of tubular basophilia in the kidneys and germ cell degeneration in the testes, with degenerative germ cell debris in the epididymides of males at 20 and 40 mg/kg bw per day. In 3-MCPD-treated females, vacuolation of the brain and spinal cord was observed at 40 mg/kg bw per day; however, only one incidence of vacuolation was observed in males. Forestomach and cutaneous papilloma and/or carcinoma and lymphoma were observed in most rasH2 mice receiving MNU treatment. We concluded that 3-MCPD did not show carcinogenic potential in the present study using rasH2-Tg mice. The findings of this study suggest that the carcinogenic potential of 3-MCPD is species specific.

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Immunohistochemical characterization of urethane-induced lung tumors in CB6F1-Tg rasH2 mice.

The purpose of this study was to define the histopathological characteristics of pulmonary neoplastic lesions, especially focusing on the origin of tumor cells, in urethane-treated Tg rasH2 mice. Bronchiolar-alveolar adenomas/adenocarcinomas were observed in the lungs from all of the urethane-treated animals. Immunohistochemically, these tumors showed an alveolar epithelial type II (AE2) cell phenotype demonstrating positive staining of surfactant protein C (SP-C). Cells expressing Clara cell 10 (CC10), a Clara cell marker, were also observed in a scattered manner in some tumors. Several SP-C and CC10 double-positive cells were observed in these tumors. Most of the urethane-induced pulmonary tumors were considered to have an AE2 cell phenotype, but the presence of SP-C and CC10 double positive cells in the tumors of Tg rasH2 mice suggests that some tumors arose from bronchioalveolar stem cells, which are known to express both SP-C and CC10.

Regulatory Forum Opinion Piece*: Retrospective Evaluation of Doses in the 26-week Tg.rasH2 Mice Carcinogenicity Studies: Recommendation to Eliminate High Doses at Maximum Tolerated Dose in Future Studies. A Response to the Counterpoints.

We recently conducted a retrospective analysis of data collected from 29 Tg.rasH2 carcinogenicity studies conducted at our facility to determine how successful was the strategy of choosing the high dose of the 26-week studies based on an estimated maximum tolerated dose (MTD). As a result of our publication, 2 counterviews were expressed. Both counterviews illustrate very valid points in their interpretation of our data. In this article, we would like to highlight clarifications based on several points and issues they have raised in their papers, namely, the dose-level selection, determining if MTD was exceeded in 26-week studies, and a discussion on the number of dose groups to be used in the studies.

Accumulation of Mott cells in the spleen in a CB6F1-Tg rasH2 mouse.

Mott cells are a variant form of plasma cells in humans and laboratory animals. This report describes the morphological characteristics of Mott cells observed in a 33-week-old female CB6F1-Tg rasH2 mouse. Microscopically, a large number of round cells with abundant eosinophilic globules, which were variable in size, were observed in the spleen and were densely distributed in the red pulp adjacent to the marginal zone. A few similar cells were present in the submandibular lymph node and bone marrow. Neither systemic nor local chronic inflammatory changes were seen in this animal. These cells were positive for mouse immunoglobulins. Ultrastructurally, the dilated rough endoplasmic reticulum had a homogenous substances with an intermediate electron density. On the basis of the above findings, these cells were identified as Mott cells. The present lesion is thought to be a spontaneous lesion, an unusual appearance of Mott cells without any associated pathological conditions.

Trend analysis of body weight parameters, mortality, and incidence of spontaneous tumors in Tg.rasH2 mice.

Carcinogenicity studies have been performed in conventional 2-year rodent studies for at least 3 decades, whereas the short-term carcinogenicity studies in transgenic mice, such as Tg.rasH2, have only been performed over the last decade. In the 2-year conventional rodent studies, interlinked problems, such as increasing trends in the initial body weights, increased body weight gains, high incidence of spontaneous tumors, and low survival, that complicate the interpretation of findings have been well established. However, these end points have not been evaluated in the short-term carcinogenicity studies involving the Tg.rasH2 mice. In this article, we present retrospective analysis of data obtained from control groups in 26-week carcinogenicity studies conducted in Tg.rasH2 mice since 2004. Our analysis showed statistically significant decreasing trends in initial body weights of both sexes. Although the terminal body weights did not show any significant trends, there was a statistically significant increasing trend toward body weight gains, more so in males than in females, which correlated with increasing trends in the food consumption. There were no statistically significant alterations in mortality trends. In addition, the incidence of all common spontaneous tumors remained fairly constant with no statistically significant differences in trends.

alpha-Glycosyl Isoquercitrin (AGIQ) and its lack of carcinogenicity in rasH2 mice.

alpha-Glycosyl Isoquercitrin (AGIQ), is used in Japan as a food additive and was granted generally recognized as safe (GRAS) status in 2005 (FEMA) and 2007 (FDA). The safety and toxicity information for AGIQ is sparse and therefore, the carcinogenicity potential of AGIQ was examined in the CByB6F1-Tg(HRAS)2Jic (rasH2) model. One hundred female and male rasH2 mice, each, were allocated to one of four designated dose groups; 0 (control)%, 1.5%, 3.0% or 5.0% AGIQ. Animals were administered the diets for six months and an additional 10 females and 10 males, each, were administered a positive control, N-methyl-N-nitrosourea (MNU). Body weights and clinical observations were collected. A full screen necropsy, organ weights, clinical chemistry, urinalysis and histopathology were performed. The positive control animals elicited appropriate responses specific to this strain (rasH2) of mice. There were statistically significant sporadic non-dose-dependent changes in clinical chemistries without corresponding pathological correlation. No microscopic AGIQ-related findings were noted; the range of pathology observations were all considered background findings, either specific to rasH2 mice or common to inbred strains of mice. Therefore, under the study conditions, the no-observed-adverse-effect level (NOAEL) was determined to be more than 5.0% (7215.4 mg/kg BW/day in male mice and 14685.5 mg/kg/day in female mice).

Assessment of Pig-a, Micronucleus, and Comet Assay Endpoints in Tg.RasH2 Mice Carcinogenicity Study of Aristolochic Acid I.

A newly developed in vivo Pig-a gene mutation assay displays great potential for integration into genotoxicity tests. To obtain more evidence for application of the Pig-a assay, we integrated this assay, micronucleus test in peripheral blood (MN-pb test) and bone marrow (MN-bm test), as well as a Comet assay into a transgenic RasH2 mice carcinogenicity study. Fourteen male RasH2 mice and five wild-type (WT) mice were treated with a strong mutagen aristolochic acid I at a dose of 5 mg/kg/day for 4 consecutive weeks. Mice recovered in 5 weeks. Peripheral bloods were collected for Pig-a assay, MN-pb test, and Comet assay at several time points, while bone marrow and target organs were harvested for the MN-bm test and pathological diagnosis after mice were euthanized. Finally, 13 of the 14 RasH2 mice developed squamous cell carcinomas in the forestomach, while there were no carcinomas in the WT mice. Pig-a mutant frequencies (MFs) consecutively increased throughout the study to a maximum value of approximately 63-fold more than background. These frequencies were relative to the incidence, size, and malignant degree of tumors. Micronucleated reticulocytes increased from Day 1 to Day 49, before returning to background levels. No positive responses were observed in either the MN-bm test or the Comet assay. Results suggested that, when compared with the other two tests, the Pig-a assay persistently contributed to sustaining MFs, enhanced detection sensitivity due to the accumulation of Pig-a mutations, and demonstrated better predictability for tumorigenicity. Environ. Mol. Mutagen. 61:266-275, 2020. © 2019 Wiley Periodicals, Inc.

A 28-day oral gavage toxicity study of 3-monochloropropane-1,2-diol (3-MCPD) in CB6F1-non-Tg rasH2 mice.

3-Monochloro-1,2-propanediol (3-MCPD) is a well-known contaminant of foods containing hydrolyzed vegetable protein. However, limited toxicity data are available for the risk assessment of 3-MCPD and its carcinogenic potential is controversial. To evaluate the potential toxicity and determine the dose levels for a 26-week carcinogenicity test using Tg rasH2 mice, 3-MCPD was administered once daily by oral gavage at doses of 0, 25, 50, and 100 mg/kg body weight (b.w.)/day for 28 days to male and female CB6F1-non-Tg rasH2 mice (N = 5 males and females per dose). The standard toxicological evaluations were conducted during the in-life and post-mortem phase. In the 100 mg/kg b.w./day group, 3 males and 1 female died during the study and showed clinical signs such as thin appearance and subdued behavior accompanied by significant decreases in mean b.w. Microscopy revealed tubular basophilia in the kidneys, exfoliated degenerative germ cells in the lumen of the seminiferous tubule of the testes, vacuolation in the brain, axonal degeneration of the sciatic nerve, and cardiomyopathy in the 100, ≥25, ≥50, 100, and 100 mg/kg b.w./day groups, respectively. In conclusion, 3-MCPD's target organs were the kidneys, testes, brain, sciatic nerve, and heart. The "no-observed-adverse-effect level" (NOAEL) of 3-MCPD was ≤25 and 25 mg/kg b.w./day in males and females, respectively.