Carcinogenicity

 

    1. ECVAM validated test methods
    2. Development and optimisation of alternative methods
    3. Publications

 

Background

 

Substances are defined as carcinogenic if after inhalation, ingestion, dermal application or injection they induce (malignant) tumours, increase their incidence or malignancy, or shorten the time of tumour occurrence. It is generally accepted that carcinogenesis is a multihit/ multi-step process from the transition of normal cells into cancer cells via a sequence of stages and complex biological interactions, strongly influenced by factors such as genetics, age, diet, environment, hormonal balance, etc.
Since the induction of cancer involves genetic alterations which can be induced directly or indirectly, carcinogens have conventionally been divided into two categories according to their presumed mode of action: genotoxic carcinogens and non-genotoxic carcinogens.
Genotoxic carcinogens have the ability to interact with DNA and/or the cellular apparatus (such as e.g. the spindle apparatus and topoisomerase enzymes) and thereby affect the integrity of the genome, whereas non-genotoxic carcinogens exert their carcinogenic effects through other mechanisms that do not involve direct alterations in DNA.
The two-year cancer bioassay in rodents is widely regarded as the gold-standard to evaluate cancer hazard and potency, although it is generally known that this test has its limitations to predict human cancer risk.

 

Test Method

COUNCIL REGULATION (EC) No 440/2008

Test Method

OECD Test Guideline

endpoint

Vitro/vivo

Carcinogenicity studies

B.32

TG 451

Carcinogenicity

vivo

Combining chronic toxicity/carcinogenicity studies

B.33

TG 453

Carcinogenicity

vivo


The complexity of the carcinogenicity process makes it difficult to develop in vitro alternative test models that mimic the full process, especially for non-genotoxic chemicals. The challenge in developing in vitro alternatives is also heightened because of the complexity of the number of target organs. It is expected that an integrated approach involving multiple in vitro models will be needed, but a better understanding of the entire process is needed before this will be possible (Adler et al., Alternative (non-animal) methods for cosmetics testing: current status and future prospects,  Arch. Toxicol. 85:367–485, 2011). While in vitro and in vivo genotoxicity tests contribute to the assessement of genotoxic carcinogens (see section on genotoxicity), there is a lack of tests available for the assessment of non-genotoxic carcinogens.


Alternative test methods and approaches

 

1. ECVAM validated test methods

 

 

1.1 Cell transformation assays

  

The in vitro cell transformation assays (CTAs) have been shown to closely model some key stages of the in vivo carcinogenesis process and have been in use for more than four decades to screen for potential carcinogenicity as well as investigate mechanisms of carcinogenicity. Moreover, they are faster and more cost efficient than the in vivo rodent carcinogenicity assay, providing a useful approach for screening of chemicals with respect to their carcinogenic potential. 

CTAs are considered to provide additional useful information to more routinely employed tests for assessing carcinogenic potential and are therefore listed in various recent  guidelines and testing strategies for such purposes (SCCP 2006; Jacobson-Kram and Jacobs, 2005; ECHA, 2008; Pfuhler et al., 2010). Since regulatory agencies may receive and review CTA data and these assays are used for internal risk assessment of various chemicals, there was a need within the scientific community for standardisation of these test methods and technical guidance on their conduct and use. 

This need was already addressed in 1998 by a workshop organised by ECVAM on CTAs (Combes et al., 1999) and by the OECD that produced a detailed review paper (DRP) on the CTAs for the detection of chemical carcinogens (OECD, 2007). As with some other assays with a long history of use, CTAs had not undergone formal validation in accordance with current standards (OECD GD 34, 2005 pdf icon).

Therefore, ECVAM coordinated an international study that was designed to address issues of CTA protocols standardisation, transferability and reproducibility. The study assessed to protocol variants for the SHE CTA (at pH 6.7 and pH 7.0) and the BALB/c 3T3 assay.

This study was peer reviewed by the EURL ECVAM Scientific Advisory Committee (ESAC) that issued an ESAC opinion, leading to the publication of an EURL ECVAM Recommendation on three CTAs for assessment of the carcinogenic potential of chemical substances. An OECD draft Test Guideline on the CTA in SHE cells is in progress.

The complete study results as well as the recommended CTA protocols and photo catalogues developed during the ECVAM study are published in a special issue of Mutation Research on CTA (Corvi and Vanparys; 2012). See also the pdf icon EURL ECVAM DB-ALM SHE CTA protocol and EURL ECVAM DB-ALM BALB/c 3T3 protocol and the corresponding photo catalogues.

 

The Japanese Centre for the Validation of Alternative Methods coordinated the validation of the Bhas 42 CTA, a system derived from the BALB/c 3T3 CTA. The study addressed two protocols: a 6-well method and the 96-well method and has been peer reviewed by ESAC. Based on the validation report and the ESAC Opinion EURL ECVAM issued a Recommendation on the Bhas 42 CTA


2. Development and optimisation of alternative methods

The potential of new approaches such as omics and in silico methods are also being explored.

3.1 CarcinoGENOMICS FP6 project 

EURL ECVAM has been involved in the CARCINOGENOMICS FP6 project, which aimed at developing toxicogenomics- and metabolomics-based in vitro tests to detect potential genotoxicants and carcinogens. 

Two tests have been selected for further optimization: a toxicogenomics-based test in HepaRG cells for the liver and a toxicogenomics-based test in RPTEC/TERT1 cells for the kidney. The optimisation/prevalidation work package was coordinated by EURL ECVAM and aimed at 1) further developing the two test models by testing 15 additional chemicals; 2) assessing test models transferability and reproducibility using the same agreed SOPs and 3) develop dedicated bioinformatics tools to serve as basis for future validations of omics-based tests.