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The in vitro Bhas 42 Cell Transformation Assay (CTA) is used to assess the carcinogenic potential of chemicals. The method uses a type of cell line that is able to recapitulate to some extent the multi-stage cell transformation process that happens when normal cells transform into cancer cells.

In 2012, the Hadano Research Institute Food and Drug Safety Center finalised a validation study of the method. The study was supervised by a validation management team at the Japanese Centre for the Validation of Alternative Methods (JaCVAM).

Upon completion JaCVAM requested EURL ECVAM to coordinate the ESAC peer review of the validation study. This process was completed in December 2012 and we fully endorsed their opinion.

The results of the validation study, the peer review of ESAC and our recommendation can be found on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.

Read more about the Bhas 42 Cell Transformation Assay on TSAR


[collapsed]Carcinogenesis, is a complex multistage process in which normal cells transform into cancer cells.

The changes happen at various different stages and generally involve four different steps. These include a block in cellular differentiation, acquisition of immortality and then tumourigenicity and finally full malignancy and metastasis.

A chemical can induce carcinogenesis through initiation and promotion. If a chemical can be shown to affect either it is considered positive and therefore a potential carcinogen.[/collapse]

Bhas 42 Cell Transformation Assay

[collapsed]The Bhas 42 Cell Transformation Assay has the ability to model to some extent the multistage cell transformation process of carcinogenicity.

The assay consists of two parts, one that assesses whether a chemical can cause initiation, the other promotion.

If a chemical is shown to be positive in either (or both) assay it is considered to be a potential carcinogen. A negative result in both assays suggests absence of transforming activity and therefore carcinogenicity.

Two variants of the assay were assessed in the validation study with the only major difference being that one used a 6-well approach and the other a 96 well approach.[/collapse]

Animal study replacement

[collapsed]The complexity of carcinogenicity means that in vitro models such as the Bhas Cell Transformation Assay are unlikely to fully model real world biological processes as a stand-alone test. However, in combination with other information sources, the assay can provide indications regarding hazards and risk.

As the method is reliable and relevant, is based on a cell line, and does not involve any animals, it is likely to have benefits from a 3Rs perspective.

Indeed, it is recommended that data from such assays should be considered in combination with other information sources before any animal experimentation is carried out.[/collapse]

Validation study

[collapsed]The validation study considered the transferability and the within- and between-laboratory reproducibility of the method.

It also considered whether the assay could identify potential carcinogenicity of substances and if it could discriminate between tumour initiators or promoters.[/collapse]

Validation study outcomes

[collapsed]On the basis of data from the validation study and other published sources, both versions of the method (the 6- and 96-well versions) are considered standardised, transferable and reproducible between laboratories.

In addition, the method can identify potential carcinogens and may provide some information on whether substances are tumour initiators or promoters.[/collapse]

EURL ECVAM recommendations

[collapsed]On the basis of the data from the validation study and the additional published information that was supplied, we recommended the following:

  • The method is reliable and relevant for the identification of potential carcinogens and may provide information on whether a substance is a tumour initiator or promoter.
  • It is unlikely that the method will fully replace any particular animal-based test method. Rather it should be used as part of a wider integrated testing strategy or as a component in a Weight of Evidence approach.
  • Depending on the context and the extent of other information available, data from the Bhas 42 cell transformation assay may be sufficient for decision-making and in specific circumstances this might mean no animal based testing will be needed. However, in many sectors it is likely that data alone from this assay will be insufficient and will therefore contribute to partial replacement and reduction of animal tests.
  • The assay is based on a cell line meaning that no experimental animals are needed to run the method. This means it has additional benefits in terms of 3Rs over some other cell transformation assays (e.g. SHE CTA, which uses hamster embryo cells).
  • The 96-well format of the assay should be amenable to automation.
  • An OECD Test Guideline should be developed for this method.

Before embarking on animal experiments for carcinogenicity detection, data from the Bhas 42 cell transformation assay (in combination with complementary information) should be considered if available. This is to avoid unnecessary animal testing.[/collapse]