The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay’s endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen’s mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA −derived variant MT clones induced by benzo(a)pyrene.