Genotoxicity

 

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

    4. Relevant publications

 

Background 

 

Genetic alterations in somatic and germ cells are associated with serious health effects, which in principle may occur even at low exposure levels. Mutations in somatic cells may cause cancer if mutations occur in proto-oncogenes, tumour suppressor genes and/or DNA damage response genes, and are responsible for a variety of genetic diseases. Accumulation of DNA damage in somatic cells has also been proposed to play a role in degenerative conditions such as accelerated aging, immune dysfunction, cardiovascular and neurodegenerative diseases. Mutations in germ cells can lead to spontaneous abortions, infertility or heritable damage to the offspring and possibly to the subsequent generations. 

The assessment of the genotoxicity represents an important component of the safety assessment of substances, relevant in the context of EU and international regulations aiming at the protection of human health and the environment (cfr.reg.). The results of the genotoxicity tests form the scientific basis for risk assessment and are used for classification and labelling (C&L) of chemical substances in the EU (the Dangerous Substances Directive 67/548/EEC and Regulation (EC) No. 1272/2008 on the classification, labelling and packaging (CLP) of substances and mixtures), and across the world (UN Globally Harmonised System (GHS)). 

For an adequate evaluation of the genotoxic potential 3 endpoints need to be assessed: gene mutation, structural chromosome aberrations, numerical chromosome aberrations. Therefore, a battery of tests is needed for an adequate coverage of all endpoints (Table 1). 

  • The assessment of genotoxic hazard to humans usually requires that the substance is tested first in the in vitro testing battery. According to some legislations and guidance documents (e.g. REACH, CLP, Cosmetics Directive), if a substance is negative in the in vitro battery it is classified as non genotoxic, while a positive outcome in one or more of the in vitro tests requires confirmation by appropriate follow up in vivo testing (Table 1). Other regulatory requirements (e.g. ICH for pharmaceuticals, VICH for veterinary drugs) foresee that the in vitro testing battery is always followed up by in vivo testing. This clearly implies that, although several in vitro tests for genotoxicity assessment are available at different stages of development and acceptance, they cannot be considered to fully replace animal tests needed to evaluate the safety of substances (See: Adler et al., Alternative (non-animal) methods for cosmetics testing: current status and future prospects,  Arch. Toxicol. 85:367–485, 2011.). Based on an analysis of regulatory requirements for this endpoint within different pieces of EU legislation, EURL ECVAM proposes a pragmatic approach to improve the traditional genotoxicity testing paradigm that offers solutions in both the short- and medium-term and that draws on the considerable experience of 40 years of regulatory toxicology testing in this area (EURL ECVAM strategy to avoid and reduce animal use in genotoxicity testing, released in December 2013). In its strategy to avoid and reduce animal use in genotoxicity testing, EURL ECVAM considers that efforts should be directed towards the overall improvement of the current testing strategy for better hazard and risk assessment approaches, which either avoids or minimises the use of animals, whilst satisfying regulatory information requirements, irrespective of regulatory context. Several opportunities for the improvement of genotoxicity testing have been identified which aim to 
    • enhance the performance of the in vitro testing battery so that fewer in vivo follow-up tests are necessary and
    • guide more intelligent in vivo follow-up testing to reduce unnecessary use of animals. 

The implementation of this strategic plan will rely on the cooperation of EURL ECVAM with other existing initiatives and the coordinated contribution from various stakeholders.


Table 1 – Test methods most commonly used for genotoxicity/mutagenicity testing

 

Test Method

COUNCIL REGULATION (EC) No 440/2008

Test Method

OECD Test Guideline*

endpoint

In vitro/ in vivo

Bacterial reverse mutation test

(Ames test)

B.13-14

TG 471

Gene mutations

vitro

In vitro mammalian chromosome aberration test

B.10

Updated TG 473

Structural aberrations

vitro

Mammalian cell gene mutation test

B.17

TG 476 (under revision)

Gene mutations

vitro

In vitro mammalian cell micronucleus test


Updated TG 487

Structural and numerical aberrations

vitro

Mammalian erythrocyte micronucleus test

B.12

Updated TG 474

Structural and numerical aberrations

vivo

Mammalian bone marrow chromosome aberration test

B.11

Updated TG 475

Structural aberrations

vivo

Transgenic rodent somatic and germ cell gene mutation assays


TG 488

Gene mutations

vivo

In vivo mammalian alkaline comet assay


TG

DNA damage

vivo

 

Alternative test methods and approaches


1. ECVAM validated test methods

 

1.1 Micronucleus Test (MNT) in vitro

ECVAM has successfully validated the Micronucleus Test (MNT) in vitro, based on a retrospective evaluation of existing data. A Validation Management Team was established to pool together the existing data, which were used to assess the validity of the MNT in vitro on the basis of ECVAM’s modular validation approach. The primary focus was to evaluate the potential of the MNT in vitro as an alternative to the standard Chromosome Aberration Test (CAT) in vitro (Corvi et al., 2008).

 

On the basis of a peer review of the retrospective validation, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the MNT in vitro is a scientifically valid alternative to the CAT in vitro for genotoxicity testing. The in vitro MNT has gained widespread international regulatory acceptance, as it offers significant advantages over the in vitro CAT. It is less expensive and time-consuming, requires less investment in training, allows a greater statistical power, and has the potential to enhance the basic package of in vitro tests to detect aneugens. The successful validation of the MNT in vitro led to EU regulatory acceptance and to the quick integration of the test into the REACH legislation for use as part of the tier 1 genotoxicity test battery. Furthermore, the validation study has supported the finalisation of the Test Guideline 487 and its regulatory acceptance by the Organisation for Economic Co-operation and Development (OECD).

 

2. Test methods under validation by ECVAM

 

2.1 Genotoxicity Assays in 3D

ECVAM has been a partner in a project led by Cosmetics Europe, which aimed to establish and validate new methods for genotoxicity testing in reconstructed human 3D skin models (micronucleus test and comet assay) (Aardema et al., 2010, Reus et al., 2013). The comet assay is now being validated in a joint effort between Cosmetic Europe and BFR.

 

2.2 Comet Assay

ECVAM has been involved in the Validation Management Team for the validation of the comet assay in vitro and in vivo, which is coordinated by the Japanese Centre for the Validation of Alternative Methods (JaCVAM). The validation of the in vivo assay has now been finalised and the respective OECD TG has been approved by the Working Group of National Coordinators (WNT) in April 2014. 

3. Development and optimisation of alternative methods

False positives

The high false positive rate of the established in vitro genotoxicity tests leads to an increased number of follow up in vivo tests needed for the confirmation of these results. To address this issue, ECVAM organised a workshop in April 2006. The workshop aimed at discussing how to improve current in vitro tests and at reviewing the development of new tests. The recommendations of this workshop (Kirkland et al., 2007) have paved the way for several international initiatives (e.g. Cosmetics Europe, ILSI/HESI, ECVAM, NC3Rs, JaCVAM). This report is among the 10 most cited Mutation Research publications since 4 years. 

Following one of the workshop recommendations, ECVAM coordinated an expert group that established recommended lists of gentoxic and non-genotoxic chemicals for assessment of the performance of new and improved genotoxicity tests (Kirkland et al., 2008). This chemical list is used world wide by test developers and other organisations.

During the ECVAM workshop, testing at high concentrations was identified as one possible source of false positives. To address this issue an analysis of published data for top concentration considerations in mammalian cell genotoxicity testing was carried out which suggested that the top concentration could be reduced without any loss of sensitivity in detecting rodent carcinogens (Parry et al., 2010).

These topics were highly debated during the ICH and OECD guideline revisions.

Some OECD Test Guidelines (TGs) for genotoxicity have recently been revised and other are still under revision (Table 1) taking also into account the knowledge asquired during the last decades of testing and the recent activities related to false positives. The recommendations made in the revised TGs will most probably enhance the quality of the data that will be produced.

The Ames test conducted in bacteria is the most commonly used genotoxicity test within the in vitro battery as it is considered able to reveal DNA reactivity and DNA reactive compounds. Despite the many activities on false positive results in in vitro mammalian cell tests, positive results in the Ames test have not been analysed in the same way as for mammalian cell tests. In this context, EURL ECVAM held a workshop in January 2013 and initiated a project with the aim of 

  • evaluating the predictivity of the Ames for in vivo genotoxicity and carcinogenicity when considered alone or in association with a mammalian cell assay for the detection of chromosome damage and/or gene mutations, and 
  • better characterising the cases where the Ames test seems to lead to irrelevant (false positive) results (e.g. chemical classes, type of bacterial strains, magnitude of effects).   

 

 

Reduction

Since substantial in vivo testing is still required by chemical authorities for confirmation of the genotoxic prediction in vitro, it became clear that it is crucial to address issues related to the reduction and refinement of genotoxicity tests. The collection of relevant data might be considered as a basis for possible amendments of guidelines to reduce animal consumption. Recommendations on opportunities to reduce the number of animals in genotoxicity tests are published by Pfuhler and colleagues in an ECVAM workshop report (Pfuhler et al., 2009).

 

 

4. Relevant publications

available here