Carcinogenicity

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 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 (Adler et al., 2011).

Regulatory context

The approaches for evaluating the carcinogenic potential of substances, including whether carcinogenicity studies should be conducted, differ substantially across sectors.

Despite variations in testing schemes, the two-year bioassay study in rodents represents the gold standard across all sectors (see OECD Series on Testing and Assessment). Its adequacy to predict cancer risk in humans, however, is the subject of considerable debate, with notable challenges and uncertainty associated with extrapolating from rodents to humans, with quantitative risk estimation and limited accuracy.

Furthermore, these studies are extremely time and resource-consuming and the high animal burden has raised ethical concerns (Paules et al., 2011). While in vitro and in vivo genotoxicity tests contribute to the assessment of genotoxic carcinogens (see Genotoxicity section), there is a lack of tests available for the assessment of non-genotoxic carcinogens. For all these reasons, there is a strong demand for alternative strategies and methods in this area.

EURL ECVAM has carried out an analysis of carcinogenicity testing requirements and assessment approaches across different sectors. This consisted of:

• a systematic review of the different regulatory testing schemes (Table 1);
• an analysis of the number of animals used per sector;
• an estimation of the number of carcinogenicity and genotoxicity studies conducted or waived in respect of the number of substances authorized per sector per year;
• a review of the type of justifications for waiving the two-year bioassay (Madia et al., 2016).

Results from this analysis provide context for initiatives aimed at:

1. reducing the need for animal use where animal testing is still a requirement;
2. ensuring an adequate hazard identification and characterization in sectors where animal use is banned or limited; and
3. where existing methods are not suitable.

EURL ECVAM validated test methods

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.

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.

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.

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 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 (JaCVAM) 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.

More information on the validated cell transformation assay methods for carcinogenicity testing are available here:

• Bhas Cell Transformation Assay
• Three Cell Transformation Assays

Further studies aiming at of the development of an automated scoring systems of transformed foci and further exploitation of the assay are ongoing (Urani et al., 2013; Callegaro et al., 2017a; Callegaro et al., 2017b)

Development and optimisation of alternative methods and approaches

EURL ECVAM-ESTIV workshop on the way forward

As resulted from Madia et al., 2016, there is an increased need to develop novel alternative approaches to the two-year rodent bioassay for the carcinogenicity assessment of substances where the rodent bioassay is still a basic requirement, as well as for those substances where animal use is banned or limited or where information gaps are identified within legislation. The current progress in this area was addressed in a EURL ECVAM- ESTIV workshop held in October 2016, in Juan les Pins (Corvi et al., 2017).

A number of initiatives were presented and discussed, including data-driven, technology-driven and pathway-driven approaches. Despite a seemingly diverse range of strategic developments, commonalities are emerging. For example, providing insight into carcinogenicity mechanisms is becoming an increasingly appreciated aspect of hazard assessment and is suggested to be the best strategy to drive new developments.

Thus, now more than ever, there is a need to combine and focus efforts towards the integration of available information between sectors. Such cross-sectorial harmonisation will aid in building confidence in new approach methods leading to increased implementation and thus a decreased necessity for the two-year rodent bioassay.

Non-genotoxic carcinogenicity

Non-genotoxic carcinogens contribute to an increased cancer risk by a variety of mechanisms that are not yet included in international regulatory approaches. To address this need, an integrated approach to testing and assessment (IATA) of non-genotoxic carcinogens is beginning to be developed internationally under the auspices of the OECD.

An expert working group has in fact been set up to examine the current international regulatory requirements and their limitations in respect to non-genotoxic carcinogenicity, and how an IATA could be developed to assist regulators in their assessment of non-genotoxic carcinogenicity.

Moreover, the working group is tasked to review, describe and assess relevant in vitro assays with the aim of tentatively organising them into levels of testing, following the adverse outcome pathway format, such as that possible structure(s) of the future IATA(s) can be created.

Some preliminary work to this activity has already been described in a publication of Jacobs and colleagues (Jacobs et al., 2016). EURL ECVAM is an active player of the group.

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.

The results of this workpackage are published in Doktorova et al., 2014 and Herwig et al., 2016.

See also Vinken et al., 2008 for chemical selection.

Scientific tools and databases

The EURL ECVAM Genotoxicity and Carcinogenicity Consolidated Database is a structured master database that compiles available genotoxicity and carcinogenicity data for Ames positive chemicals originating from different sources.

By using a harmonised format to capture the information, this database represents a powerful resource for data analysis that can be used to guide a thorough evaluation of genotoxicity and carcinogenicity.