Use of Genetic Toxicology Testing for Predicting Carcinogenicity: A Multi-Stakeholder Approach

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Carcinogenicity

Use of Genetic Toxicology Testing for Predicting Carcinogenicity: A Multi-Stakeholder Approach

By Marilyn J. Aardema, Procter & Gamble
Published: December 6, 2007
About the Author(s)
Dr. Marilyn J. Aardema. 2010-present, Marilyn Aardema Consulting, LLC; 1985-2010, Procter & Gamble Co, Central Product Safety Department, Genetic Toxicology Research Group; 1994 Principle Scientist. BS Hope College Holland, MI; Ph.D. Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, TN. Served on numerous professional committees, societies, task forces including: ILSI-HESI subcommittees, International Working Group on Genotoxicity Testing Procedures (IWGT), European Cosmetic Association (COLIPA) genotoxicity task force, European Center for Validation of Alternative Methods (ECVAM) committees, Environmental Mutagen Society committees, Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) committees, International Congress of Harmonization (ICH) Genetox Task Force, NIEHS review committees.

Marilyn J. Aardema
Email: mjaardema@me.com

Genetic toxicology tests are routinely used for predicting the carcinogenicity of chemicals and there are numerous in vitro genotoxicity assays that have been in use for over 30 years. So one might ask why a completely non-animal approach isn’t possible today? While the current in vitro genotoxicity assays have a high sensitivity (80-90% of rodent carcinogens are detected), the problem is that the rate of “false positive” or “irrelevant positive” results (not relevant to humans) in current in vitro genotoxicity tests is extremely high with 75-90% of rodent non-carcinogens shown to be positive in one or more of the standard in vitro genotoxicity tests. This was highlighted in a series of papers where data for over 700 chemicals that had genotoxicity and rodent carcinogenicity data were examined (Kirkland et al., 2005; 2006). The problem of “false positive results” in in vitro genotoxicity assays was first identified in the 1990s in smaller analyses by the US National Toxicology Program but didn’t lead to significant changes in testing practices. Our more recent analyses with a larger database covering a wider variety of chemical classes demonstrates the wider scope of the problem and has prompted a renewed interest within the scientific community to address this problem. In vitro genotoxicity tests were designed to be sensitive, Tier I screens for hazard as part of a battery which consists of Tier II in vivo genotoxicity assays and Tier III tests for carcinogenicity and in vivo germ cell mutagenicity. Positive results in in vitro genotoxicity tests demonstrate the intrinsic genotoxic activity of a chemical or drug, but often under conditions that are extreme and not relevant or attainable in in vivo genotoxicity assays or for human exposures. To interpret biological relevance of this in vitro activity, other data, such as negative results from Tier II and Tier III in vivo tests, is needed. Thus the high frequencies of false positive results from of in vitro genotoxicity tests constitute a major factor leading to unnecessary animal testing and delayed or halted development of new products. Initiatives such as the European Union (EU) Registration, Evaluation, Authorisation and Restriction of Chemical (REACH) substances legislation and the EU 7th Amendment Cosmetics Directive (which bans in vivo genotoxicity tests for cosmetics as of 2009), as well as the general goal of the scientific community to reduce/eliminate animal tests, have led to a variety of efforts over the last few years to develop better non-animal approaches to address the genotoxicity of chemicals. Importantly, the interest within the chemical and cosmetic industries to address this problem and eliminate animal testing has coincided with the interest within the pharmaceutical industry to reduce the time/effort to resolve in vitro positive results even though there has not been as high an emphasis to eliminate animal testing. These combined goals and the urgent timing have led to an extensive multi-stakeholder approach that is leading the way forward in the field of genetic toxicology to reducing/eliminating animal tests. These initiatives, and the key scientific learnings and policy approaches that are evolving to move this area forward, are reviewed below.

European Centre for the Validation of Alternative Methods (ECVAM).

A workshop was held April 26-28, 2006 that has resulted in the publication: “How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM workshop” (Kirkland et al., 2007a). A group of genotoxicity experts from academia, government and industry reviewed data from the currently available assays, as well as discussed: (i) existing methods that might have a reduced tendency to false positive results, (ii) potential modifications to further improve these assays, and (iii) new test systems that show promise of improved specificity without sacrificing sensitivity. The workgroup concluded that a collaborative research program is needed to identify, further develop and evaluate new cell systems or modification of existing systems to obtain appropriate sensitivity but improved specificity. The workgroup also concluded there is a need to refocus on the detection of human carcinogens and in vivo genotoxins that are DNA reactive, rather than trying to expand genotoxicity assays to try and detect every type of rodent carcinogen as was done in the past, which likely contributes to the high false positive problem we are dealing with today. This has led to a project to develop a database of genotoxic carcinogens and definitive in vivo genotoxins that will be used for evaluating new and modified tests in collaboration with COLIPA (see below). Also toward the goal of developing non-animal tests for carcinogenicity prediction, ECVAM has recently completed a retrospective validation of the in vitro micronucleus assay, which sets an important precedence for expediting the validation of new methods. ECVAM also is conducting a prevalidation study of the Syrian hamster embryo (SHE) and Balb/3T3 cell transformation assays and sponsoring studies, in collaboration with COLIPA, to develop the Comet assay in 3D human skin models (see below).

European Cosmetic Toiletry and Perfumery Association (COLIPA).

COLIPA represents the interests of the cosmetic, toiletry and perfumery industry in Europe. To address the upcoming EU 7th amendment ban on the use of in vivo genotoxicity tests for cosmetics, COLIPA is sponsoring parallel projects to improve current tests and develop new ones. To design the appropriate approach for improving current in vitro genotoxicity tests, COLIPA sponsored a review of the factors known or thought to be involved in the high false positive rate of these assays (Kirkland et al., 2007b). This review highlighted the fact that many types of DNA damage arise in vitro through events that can be categorized as ‘overload of normal physiology’ (e.g., generation of ROS) which may not be expected to occur in normal human exposures. The challenge is obtaining evidence in support of such mechanisms especially without the use of in vivo assays. Since many of the approaches to providing this evidence are based on only a few published examples, validated approaches do not exist and it was concluded that there is an urgent need for developing consensus approaches that do not rely on animal studies. Finally, it was noted that the future of in vitro testing may lie with completely new assays rather than investigations to interpret the relevance of results from existing assays. With this as background, COLIPA is starting a 3 year project (“Reduction in the “false positive” rate of in vitro mammalian cell genotoxicity assays”) in collaboration with ECVAM and the UK’s The National Centre for the Replacement, Refinement and Reduction of Animals in Research, at Covance, UK, to compare different cell lines, cytotoxicity parameters, etc. In addition, COLIPA started a 2.5 year project in April 2004 to develop genotoxicity assays in 3D human skin tissue models. We published a method for the assessment of micronuclei in EpiDermTM 3D human skin tissue models (Curren et al., 2006) and L’Oreal scientists published a method for assessing Comet in 3D EpiSkin® (Flamand et al., 2006) which will be further evaluated in different laboratories and 3D skin models in this COLIPA project. ECVAM is funding a portion of the Comet project.

International Life Sciences Institute-Health and Environmental Sciences Institute (ILSI-HESI).

An ILSI-HESI emerging issues subcommittee on Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing was formed March 2005. Two international workshops were held, June 2006 and June 2007 to define the issue and possible approaches. The goals of these meetings were to start to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The proceedings of the June 2006 workshop were recently published (Thybaud et al., 2007a). Based on the number of other ongoing initiatives in this area (reviewed in this commentary), the IVGT project has focused on initiating two projects. One is the development of a decision tree for follow-up strategies and weight of evidence approaches to assist in risk-based decision making including how to evaluate DNA reactivity and develop quantitative information to extrapolate laboratory data to estimate the degree of human risk. The other project is an examination of new and emerging technologies that may improve prediction of effects in humans and to support the evaluation and development of new technologies and approaches. Participation by ILSI-HESI member companies in these activities is currently being defined.

Other ILSI initiatives that impact the use of genetic toxicology testing/data for prediction of carcinogenicity are:

  • Biological Significance of DNA Adducts
  • Cancer Hazard Identification Strategies
  • Application of Genomics to Mechanism-based Risk Assessment
  • New ILSI Europe initiative: Application of the margin of exposure (MOE) approach to genotoxic carcinogenesis in food
International Working Group on Genotoxicity Testing Procedures (IWGT).

This is a regular discussion forum that meets to discuss issues related to the conduct and interpretation of specific assays as well as testing approaches. At the 2005 International Congress on Environmental Mutagens (ICEM) meeting, a workgroup of recognized international authorities from industry, government, and academia met to develop recommendations for interpretation of results from genotoxicity tests and a strategy for follow-up testing when positive in vitro results are obtained (Thybaud et al., 2007b).  A decision tree was developed that includes criteria for (i) assessing when the pattern and magnitude of positive results were such that there was very low or no concern (e.g., nonreproducible or marginal responses) and no further testing would be needed, (ii) selecting appropriate follow-up testing, and (iii) determining a sufficient weight of evidence to attribute a level of risk. There was discussion of genotoxicity that arises from events other than direct DNA reactivity that may have a non-linear, or threshold, dose-response where it may be possible to determine an exposure level below which there is negligible risk to humans. The next official IWGT meeting is scheduled for 2009.

International Congress on Harmonization (ICH).

The S2 ICH “Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use” is currently being revised. Topics included in these discussions are (i) approaches to reduce irrelevant positive results in the in vitro tests by reducing the top concentration from 10 mM to 1 mM or even lower, (ii) limitation of the maximum cytotoxicity in the in vitro mammalian cell assays, and (iii) the potential to avoid in vitro chromosome damage tests if two end-points/organs are evaluated in vivo. Also being discussed are ways to reduce animal usage by integrating genotoxicity end-points in general toxicity studies and to eliminating the need for positive control groups in standard in vivo genotoxicity tests.

European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC).

A workshop sponsored by European Chemical Industry Council (CEFIC) Long-range Research Initiative (LRI) entitled: “Refinement of Mutagenicity/Genotoxicity Testing” was held April 23-24, 2007, in Malta. Participants included experts from industry, government and academia. The objective of the workshop was to review the challenges of current in vitro testing, and the development of new and refined methods including those involving genomics, systems biology, 3D tissue models, and genetically modified cells. Issues such as limitation of animal use, and high rate of irrelevant positive results in vitro, were key to the discussions. A publication of the proceedings of this workshop is in preparation, which may lead to work funded by CEFIC LRI.

Conclusion

This is an exciting time in the field of genetic toxicology due to momentum within the scientific community to address the problems leading to false positive results in in vitro genotoxicity assays and thereby reduce, and where possible, eliminate animal use. There is willingness among scientists from multiple sectors globally to change the fundamental tests in the current paradigm and even consider new approaches. The multi-stakeholder projects described above constitute an important way forward.

©2007 Marilyn Aardema

References
Curren, R.D., Mun, G.C., Gibson, D.P. & Aardema, M.J. (2006). Development of a Method for Assessing Micronuclei Induction in a 3-D Human Skin Model EpiDermTM. Mutat. Res. 607, 61-87.

Flamand, N., Marrot, L., Belaidi, J.P., Bourouf, L., Dourille, E., Feltes, M. & Meunier, J.R. (2006). Development of genotoxicity test procedures with Episkin®, a human reconstructed skin model: towards new tools for in vitro risk assessment of dermally applied compound? Mutat. Res. 606, 39-51.

Kirkland, D., Aardema, M., Henderson, L. & Müller, L. (2005). Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat. Res. 584, 1-256.

Kirkland, D., Aardema, M., Müller, L. & Hayashi, M. (2006). Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: II. Further analysis of mammalian cell results, relative predictivity and tumour profiles. Mutat. Res. 608, 29-42.

Kirkland, D., Pfuhler, S., Tweats, D., Aardema, M., Corvi, R., Darroudi, F., Elhajouji, A., Glatt, H., Hastwell, P., Hayashi, M., Kasper, P., Kirchner, S., Lynch, A., Marzin, D., Maurici, D., Meunier, J.R., Müller, L., Nohynek, G., Parry, J., Parry, E., Thybaud, V., Tice, R., van Benthem, J., Vanparys, P. & White, P. (2007a). How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop. Mutat. Res. 628, 31-55.

Kirkland, D.J., Aardema, M., Banduhn, N., Carmichael, P., Fautz, R., Meunier, J.R. & Pfuhler, S. (2007b). In vitro approaches to develop weight of evidence (WoE) and mode of action (MoA) discussions with positive in vitro genotoxicity results. Mutagenesis. 22, 161-175.

Thybaud, V., Aardema, M., Casciano, D., Dellarco, V., Embrye, M., Gollapudi, B., Hayashi, M., Holsapple, M.P., Jacobson-Kram, D., Kasper, P., MacGregor, J.T. & Rees, R. (2007a). Relevance and follow-up of positive results in in vitro genetictoxicity assays: An ILSI-HESI initiative. Mutat. Res. 633, 67–79.

Thybaud, V., Aardema, M.J., Clements, J., Dearfield, K., Galloway, S., Hayashi, M., Jacobson-Kram, D., Kirkland, D., MacGregor, J., Marzin, D., Ohyama, W., Schuler, M., Suzuki, H. & Zeiger, E. (2007b). Strategy for genotoxicity testing: follow-up testing and evaluation of results for positive or equivocal results. Report of IWGT workshop. Mutat. Res. 627, 41–58.