Carcinogenicity is a complex, multi-stage biological process by which normal cells are transformed into cancer cells through an accumulation of changes that have occurred at the cellular, genetic and epigenetic level.
A minimum of four phenotypic stages appears to be involved in cell transformation. These include (from primary to fully malignant cells): 1) a block in cellular differentiation; 2) acquisition of immortality; 3) acquisition of tumorigenicity; 4) full malignancy.
The assay is based on the change of the phenotypic features of transformed foci undergoing the first steps of the conversion from normal cells to neoplastic-like cells with oncogenic properties.
Regulatory bodies across the globe each require candidate molecules selected for further development or use (in multiple industries) to be evaluated for potential toxicities. One of the multiple endpoints to be considered is carcinogenicity and the Organisation for Economic Co-operation and Development (OECD) have internationally approved guidelines for performing biological assays to evaluate the carcinogenic potential of a molecule.
In vitro methods for evaluating this complex endpoint are limited with the Cell Transformation Assay (CTA) being one of the few methods available. An in vitro Cell Transformation Assay (CTA) protocol consists of an initiator assay and a promoter assay to detect tumour-promoting activity of chemical carcinogens.
The three CTAs evaluated by ECVAM in the early 2000’s were amalgamated into two. This was because two versions of the Syrian Hamster Embryo (SHE) cell assay were evaluated but found to perform to such a similar level, only one was required going forward and a Guidance Document was published (OECD Guidance Document 214). The other CTA was the in vitro Bhas 42 cell assay which, after the initial ECVAM evaluation, was further developed to improve its performance and a Guidance Document was published (OECD Guidance Document 231).
SHE cell CTA
The SHE cell cell transformation assay (CTA) can be performed at either pH 6.7 or pH 7.0 as both were shown to be equivalent. As embryo cells are utilized, the assay has two stages: the initiation stage and the promotion stage and because they are embryonic, the cells are metabolically competent (OECD, 2015).
Although the assay gives a prediction of transformation potential, it cannot be used as a stand-alone test. This means test results derived from the SHE cell CTA would be expected to be used as part of a testing strategy and/or in a weight of evidence approach using other information such as genotoxicity data, structural-activity analysis and pharmaco-/toxicokinetic information to the assessment of carcinogenic potential (OECD, 2015).
Bhas 42 cell CTA
The Bhas 42 cell promoter transformation assay is a sensitive short-term system for predicting chemical carcinogenicity related to transformation promotion activity. Bhas42 cells were established from BALB/c 3T3 A31-1-1 cells by the transfection of v‑Ha‑ras gene.
As the Bhas 42 cell line is already considered to be initiated, this method only requires the promotor assay (OECD, 2016). Similarly to many Gentoxicity assays, the Bhas 42 CTA also requires a metabolic activation system (such as RegenSys / Mutazyme / S9). Although the assay gives a prediction of transformation potential, the assay cannot be used as a stand-alone test. This means test results derived from the Bhas 42 CTA would be expected to be used as part of a testing strategy and/or in a weight of evidence approach using other information such as genotoxicity data, structural-activity analysis and pharmaco-/toxicokinetic information to the assessment of carcinogenic potential (OECD, 2016).
To be completed.