An Interlaboratory Collaborative Study on a Cell Transformation Assay Using Bhas 42 Cells

Noriho Tanaka1, Kiyoshi Sasaki1, Kumiko Hayashi1, Ayako Sakai1, Shin Asada1, Dai Muramatsu1, Sachiko Kuroda1, Fukutaro Mizuhashi2, Miho Nagai2, Hiroshi Suzuki3, Tadashi Imamura3, Masumi Asakura4, Hidetaka Satoh5, Akiko Sakamoto5, Ryosuke Nakao5, Hideki Hirose6, Nana Ishii6 and Makoto Umeda1

1Hatano Research Institute, Food and Drug Safety Center, 2Biosafety Research Center, Foods, Drugs and Pesticides, 3Ina Research Inc, 4Japan Bioassay Research Center, 5Japan Food Research Laboratories, 6Mitsubishi Chemical Safety Institute Ltd.

AATEX 14(1):831-848, 2009

A sensitive cell transformation assay for tumor initiators as well as promoters has been developed using a v-Ha-ras-transfected BALB/c 3T3 cell line, Bhas 42. In order to establish the method as a routine procedure, we have conducted a NEDO Project for the confirmation of its usefulness. Six laboratories joined in this project and examined nine chemicals, so as to be tested for a chemical by three laboratories. Judgments on carcinogenic compounds, N-methyl-N'-nitro-N-nitrosoguanidine and benzo[a]pyrene, were positive in the initiation assay and negative in the promotion assay. Although benz[a]anthracene is a carcinogen and pyrene is reported to be non-carcinogenic to animals, they gave positive results in both initiation and promotion assays. Anthracene, a non-carcinogen, was negative in both initiation and promotion assays. Mezerein, lithocholic acid and methapyrilene hydrochloride, known to act as tumor promoters, were judged positive in the promotion assay and negative in the initiation assay. Phorbol, a negative control for tumor-promoting phorbol esters, was shown negative in both initiation and promotion assays. In conclusion, this inter-laboratory collaborative study demonstrated that the Bhas 42 cell transformation assay is reproducible and applicable to the detection of both initiators and promoters, leaving the possibility of more improvement by the modification of the protocol and guidance for the procedure.

Key words: transformation, Bhas 42 cells, v-Ha-ras, tumor-initiator, tumor-promoter


(AATEX: Altern. Animal Test. EXperiment.: Alternatives to Animal Testing and EXperimentation)