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Development and evaluation of a multiple endpoint in vigo genotoxicity assayin the rat


The objectives are:

i) to develop test methodologies for an increased coverage of genetic endpoints in organs of rats exposed in vivo to chemical carcinogens;

ii) to determine the degree of correlation between short-term (genetic) and long-term (neoplastic) endpoints in a target tissue for chemical-induced tumourigenesis in the rat.

The animal model used for the mutagenesis and tumourigenesis studies is the Granuloma Pouch Assay (GPA). In this system, a number of short-term genetic endpoints can be determined in a subcutaneous, fibroblastic target tissue for tumourigenesis (fibrosarcoma). Long before the appearance of tumours, i.e. up to 5 days after carcinogen exposure, the target tissue, which consists of transiently growing fibroblasts can be isolated, brought into single cell suspension, and subcultured in vitro for the analysis of a variety of genetic changes at the cellular and/or molecular level.

The genetic endpoints investigated in the present study include (i) Restriction Site Mutations (RSM) in the a-haemoglobin gene and in proto-oncogenes or tumour suppressor genes, such as, H-ras, N-ras, c-myc, p53, (ii) HPRT gene mutations, and (iii) structural chromosome aberrations. In other related investigations, the occurrence of numerical chromosome aberrations (aneuploidy) in GPA tissue cells is being analysed as well. It is intended to further develop and calibrate these short-term methodologies using reference carcinogenic compounds, and to assess their practical incorporation in a multiple endpoint in vivo assay.

The second part of the studies involves an analysis of the degree of correlation between the occurrence of short-term genetic events in granuloma pouch cells and the induction of fibrosarcomas in granuloma pouch tissue. The approach is, first, to compare the relative frequencies of gene and/or chromosome mutations in the short-term range with the incidence of fibrosarcoma after carcinogen exposure. Second, to compare the nature and location of DNA changes occurring at RSM sites in the original fibroblastic target population with RSM sites and oncogenic changes occurring in fibrosarcoma DNA.

On the basis of results obtained so far in the participating laboratories, and in combination with comparative studies performed elsewhere, it is expected that procedures for assessing various genetic endpoints in vivo in the rat will become available at the end of the study. Likewise, the present investigations are expected to contribute to an increase in our knowledge about the involvement of genetic events in the process of chemical-induced tumourigenesis.

Funding Scheme

CSC - Cost-sharing contracts


9,Antonie Van Leeuwenhoeklaan 9
3720 BA Bilthoven

Participants (1)

United Kingdom
Singleton Park
SA2 8PP Swansea