Incidence of spontaneous non-neoplastic lesions in transgenic CBYB6F1-Tg(HRAS)2Jic mice.

Since 2003, the Tg.rasH2 model has been accepted by regulatory agencies worldwide for 26-week short-term carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice. However, over the decade, the number of actual studies conducted with alternative mouse models has remained low. The primary cause for low acceptance of this model has been lack of a historical database for the incidence of spontaneous lesions. Recently, we published the historical control database on spontaneous tumors in the Tg.rasH2 mice. The purpose of this publication is to present a large database pertaining to the non-neoplastic spontaneous lesions noted in Tg.rasH2 mice from studies conducted at our facility. Lesions that are considered unique in Tg.rasH2 mice are skeletal muscle myopathy, vascular anomalies involving various organs, and mesenteric arterial thrombosis. Other notable lesions are extramedullary hematopoiesis of spleen, subacute inflammatory foci in the liver, and infiltration of histiocytes in the lungs.

Historical control data of spontaneous tumors in transgenic CByB6F1-Tg(HRAS)2Jic (Tg.rasH2) mice.

The Tg.rasH2 mouse is a hemizygous transgenic mouse, approved by regulatory agencies for carcinogenicity assessment. However, the absence of a historical database for the incidence of spontaneous neoplasms has subsequently led to reluctance by some pharmaceutical companies to adopt the use of this short-term carcinogenicity assay. Our laboratory has generated a database summarizing the mortality, body weights, and the incidence of spontaneous tumors in 1420 male and female mice assigned to 26 studies conducted at our facility. In addition, we present the incidence of tumors in positive control mice treated with urethane from these studies. Mortality in the vehicle-treated Tg.rasH2 mouse was low (average of 1% in each study). The most common spontaneous tumors in the Tg.rasH2 mice were alveolar bronchiolar adenoma of the lungs (10.14% in males and 5.77% in females) and hemangiosarcoma of the spleen (3.66% in both males and females). The incidence of all other tumors was generally very low. In the positive control, urethane-treated animals, the incidence of alveolar bronchiolar adenomas and alveolar bronchiolar carcinomas in the lungs was 93.69% and 42.88% in males and 92.43% and 72.79% in females, respectively. In addition, the incidence of splenic hemangiosarcomas in urethane-treated males was 89.18% and 92.25% in females. The 6-month Tg.rasH2 assay is more precise, faster, and more economical than the conventional 2-year mouse assays because of the low incidence of background tumors, very high survival, shorter duration, and the lower number of animals used.

Reduction in the number of animals and the evaluation period for the positive control group in Tg.rasH2 short-term carcinogenicity studies.

The lack of a clear guidance on the adequate number of animals used for positive controls in the short-term (26-weeks) transgenic mouse carcinogenicity studies has resulted in the use of high number of animals. In our earlier Tg.rasH2 studies, 25 mice/sex were used in the urethane-positive control dose groups that were sacrificed by 18 weeks. Based on a robust response, several of our protocols for Tg.rasH2 studies with 15 mice/sex and terminal sacrifice at 17 ± 1 weeks were submitted and accepted by the Carcinogenicity Assessment Committee of the US Food and Drug Administration since we demonstrated close to 100% response for the development of lung and splenic tumors (target organs) in 500 mice/sex. These 500 mice/sex included 17 groups of 25 mice/sex and 5 groups of 15 mice/sex.  The objective of this investigation was to determine whether the number of animals can be further reduced along with the shortened duration of exposure to urethane. Accordingly, 10 Tg.rasH2 mice/sex/group were administered a total of 3 intraperitoneal (IP) injections of urethane (1000 mg/kg per day) on study days 1, 3, and 5, and the presence of tumors in the lungs and spleen was evaluated after 8, 10, 12, 14, or 16 weeks. Our results demonstrate that 100% of the mice at 8 weeks had developed lung tumors, whereas close to 100% of the mice at 14 weeks had developed splenic tumors. Based on the development of lung tumors alone in 100% of the mice, we recommend that 10 mice/sex are sufficient and that these mice can also be sacrificed as early as 10 ± 1 weeks following the administration of urethane.

Genotoxic assessment and toxicity evaluation of peginesatide in CByB6F1 hybrid mice.

Peginesatide is a PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA) that was designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Clinical use of peginesatide is anticipated to result in chronic dosing in chronic kidney disease (CKD) patients, and the nonclinical data to support development should include an evaluation of carcinogenic potential evaluation. Peginesatide was not mutagenic or clastogenic in a standard genotoxicity battery of tests. Doses for a rasH2 transgenic mouse carcinogenicity assay were defined in a 28-day study in the wild-type littermates of the rasH2 transgenic mouse strain, using intravenous doses of 1-25 mg/kg on days 1 and 22. The findings were consistent with exaggerated pharmacology, including polycythemia, with associated increases in hemoglobin level and extramedullary hematopoiesis and bone marrow hypercellularity.