rasH2 mouse(R)

Formal name: CBYB6F1-Tg(HRAS)2Jc
Coat color: Wild color (Agouti)
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The rasH2 mouse is a genetically engineered mouse with the HRAS (c-Ha-ras) gene, developed by CIEA, inserted. The inserted gene is a prototype prepared by removal of the point mutation sites from two HRAS (c-Ha-ras) genes obtained from human malignant sarcoma and human urinary bladder, followed by their recombination. Animals obtained by injection of the prototype gene into the pronucleus oocyte and by backcrossing these animas more than 20 times with the C57BL/6J strain formed the original breed. Stable conditions are maintained by tandem array integration of three copies of the gene, which is located in chromosome number 15E3. Since mice used in carcinogenicity studies must detect the carcinogenicity of many unspecified compounds in each organ, an F1 hybrid (F1 hybrid with the BALB/cByJ strain) with a wider range of susceptibility than the inbred line was prepared.

1. At ICH4 (S1B), it was decided to use this mouse in tests to replace long-term carcinogenicity studies (1997).
2. The high reproducibility and stability of data obtained with the rasH2 mouse were verified in ILSI/HESI international confirmatory studies (1997-2000).
3. At an ILSI/HESI workshop in 2003, regulatory authorities in Japan, the United States and EU submitted their policies for acceptance of data from carcinogenicity studies on both genotoxic and non-genotoxic substances using the rasH2 mouse as application data.
4. At the Congress of the American Society of Toxicologic Pathology in 2009, the results of a questionnaire survey on the alternate mouse carcinogenicity test method were published and the usefulness of the rasH2 mouse was confirmed.

1. High uniformity of genetic background
Backcrossing of the original breeding colony has reached more than 20 generations and very high genetic uniformity is maintained. To prevent the appearance of substrains in the international production bases of the rasH2 mouse at CLEA Japan (Shizuoka, Japan) and Taconic-Artemis in the United States, the breeding colonies are renewed every 5 years (or 10 generations) by recovery of breeding stock from cryopreserved embryos at CIEA. All of the candidate breeding stock animals are tested for the integrated gene by Southern blotting to confirm that there are no mutations. All animals supplied are identified as Tg/non-Tg by double PCR.
2. High reproducibility and stability of carcinogenicity
Carcinogenicity monitoring is performed using a standard positive control substance (MNU: N-methyl-N-nitrosourea) in carcinogenicity studies using this mouse and periodic tests are performed to ensure that no changes in sensitivity have occurred. In the most recent monitoring (2010), no differences in carcinogenic sensitivity were found between animals produced by CLEA Japan and Taconic-Artemis, and the reproducibility of previous data was confirmed.

In the same way as with long-term carcinogenicity studies, short-term studies consist of a preliminary study and the 26-week actual study. The protocol is based on that prepared immediately after the ILSI/HESI international confirmatory studies, but some revisions were gradually made in the United States.
1. Preliminary study
The study period is 4 weeks (28 days) with administration on consecutive days. With non-Tg mice, it is necessary to use groups of 10 animals of each gender and also animals for a toxicokinetics (TK) study. The objective of the study is to determine the maximum tolerated dose (MTD) and maximum feasible dose (MFD) values.
2. 26-week actual study
The study period is 26 weeks with administration on consecutive days. It is necessary to establish five groups: a negative control group, three dose groups and a positive control group. The animals used are 25 Tg animals of each gender in each group. In addition, it is also necessary to have animals for TK (non-Tg animals) in the negative control and three dose groups. The standard positive control substance is N-methyl-N-nitrosourea (MNU) at 75 mg/kg (vehicle is citrate buffer pH 4.5, prepared at time of use) administered once intraperitoneally.