Applicant:

Prof. Pablo Steinberg

Department of Food Toxicology and Replacement / Complementary Methods to Animal Testing,

Institute for Food Toxicology and Analytical Chemistry

University of Veterinary Medicine Hannover

Bischofsholer Damm 15, 30173 Hannover, Germany

The aim of the European Community Regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals is to improve the protection of human health and the environment through the better and earlier identification of the toxic properties of chemical substances, including their carcinogenic potential. In this context thousands of chemicals have to be investigated in the next few years. In this context it is generally agreed that the number of animal experiments will drastically increase. In order to avoid the use of a very high number of animals for long-term carcinogenicity studies it is imperative that an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is developed.

The BALB/c-3T3 cell transformation assay and the soft agar colony formation assay are two well established in vitro test systems to determine whether a compound is carcinogenic or not, whereby they have two different biological endpoints. Briefly, in the case of the classic BALB/c-3T3 cell transformation assay BALB/c-3T3 cells (clone A31-1-1) are incubated with the test compound and after six weeks the number of transformed cell colonies is detected by making use of a stereo microscope. The soft agar colony forming assay is used to determine whether a cell line treated with a test 5 compound shows anchorage-independent growth. Nowadays this assay takes one week to be performed and colony growth is quantified by a fluorimetric assay (see our own previous work under 2.2).

The aims of the present research project are:

1) to combine the BALB/c-3T3 cell transformation assay with the soft agar assay;

2) to simplify the soft agar assay;

3) to couple the assay to a metabolizing system. In second 2-year research period we want to automate the developed test system. With the help of this combined in vitro assay one could determine for the first time the carcinogenic potential of a high number of chemicals within a short period of time without animal testing.