"The term carcinogen denotes a chemical substance or a mixture of chemical substances which induce cancer or increase its incidence" (UNECE, 2004, p. 167). An alternate definition is that carcinogenic substances are ones that "induce tumors (benign or malignant), increase their incidence or malignancy, or shorten the time of tumor occurrence when they are inhaled, injected, dermally applied, or ingested" (Maurici, et al., 2005, p. 177).
Carcinogens are classified according to their mode of action as genotoxic or nongenotoxic carcinogens. Genotoxic carcinogens initiate carcinogenesis by direct interaction with DNA, resulting in DNA damage or chromosomal aberrations that can be detected by genotoxicity tests (OECD, 2006). Nongenotoxic carcinogens are agents that, at least initially, directly interact with DNA. These indirect modifications to DNA structure, amount, or function may result in altered gene expression or signal transduction (OECD, 2006). In animal studies, most potent mutagens are also found to be carcinogenic (Maurici, et al., 2005, p. 177). Substances that induce tumors in animals are considered as presumed or suspected human carcinogens until convincing evidence to the contrary is presented (UNECE, 2004, p. 167).
The Animal Test(s)
The conventional test for carcinogenicity is the long-term rodent carcinogenicity bioassay described in Organisation for Economic Cooperation and Development (OECD) Test Guideline (TG) 451. The objective of this test is "to observe test animals for a major portion of their life span for the development of neoplastic lesions during or after exposure to various doses of a test substance by an appropriate route of administration." The study is usually conducted using two species - rats and mice of both sexes. The animals are dosed by oral, dermal, or inhalation exposures, based upon the expected type of human exposure. Dosing typically lasts around two years. Certain animal health features are monitored throughout the study, but the key assessment resides in the full pathological analysis of the animal tissues and organs when the study is terminated.
Two endpoints in animal bioassays, carcinogenicity and chronic toxicity, can be combined to reduce animal use, as described in OECD TG 453.
The International Life Sciences Institute (ILSI) Health and Environmental Science Institute's (HESI) Alternatives to Carcinogenicity Testing Technical Committee coordinated a large-scale research program to characterize a number of transgenic rodent models proposed for use in human cancer risk assessment (Robinson & MacDonald, 2001). None of these models were considered sufficient as standalone assays. Most could detect genotoxic compounds that a genotoxicity test battery would already detect, but better detection of nongenotoxic carcinogens is still needed (Goodman, 2001).
Regulators at a 2003 ILSI-HESI workshop on the use of transgenic animals for carcinogenicity testing concluded that these assays should be integrated with traditional test methods (ILSI-HESI, 2003). The regulators considered the p53+/- and Tg.RasH2 models useful in providing data for regulatory purposes and the Tg.AC model useful in evaluating dermal products.
Regulatory Requirements & Test Guidelines
The UN Globally Harmonized System (GHS) classifies carcinogens under two categories based on the strength of the evidence: Category 1 chemicals are known or presumed human carcinogens (Category 1A if based on human data and 2A if based on animal data); Category 2 chemicals are suspected human carcinogens (UNECE, 2004, p. 167). According to GHS guidance, chemical-induced tumorigenesis involves genetic changes; thus, chemicals that are mutagenic in mammals may warrant being classified as carcinogens.
The GHS describes other "important factors" to be taken into consideration in carcinogen hazard classification, such as the location and number of tumors, tumor type and characteristics, responses in both sexes and/or multiple species, relevance of the mode of action to humans, and more. The OECD's guidance on these factors is provided in the 2001 Harmonized Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures (ENV/JM/MONO(2001)6), and in the 2005 Proposal for Guidance on How to Consider Important Factors in Classification of Carcinogenicity (ENV/JM/HCL(2005)2/REV). The 2005 OECD guidance discusses various frameworks for assessing the "important factors" and states that "the weight of evidence analysis called for in GHS is an integrative approach which considers important factors in determining carcinogenic potential along with the strength of evidence analysis."
OECD TGs 451, 452, and 453 provide information for conducting carcinogenicity and chronic toxicity studies. The OECD Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies (ENV/JM/MONO(2002)19) "provides broad guidance on approaches to hazard assessment and on some of the problems and pitfalls that may arise during an assessment...."
The US Environmental Protection Agency's (EPA) revised its Guidelines for Carcinogen Risk Assessment (EPA/630/P-03/001B) in 2005. The revised guidelines use five descriptors (Carcinogenic to Humans, Likely to be Carcinogenic To Humans, Suggestive Evidence of Carcinogenic Potential, Inadequate Information to Assess Carcinogenic Potential, Not Likely to Be Carcinogenic to Humans) that are followed by a weight of evidence narrative to describe the carcinogenic potential of a substance. The EPA provides additional information on its Web page Evaluating Pesticides for Carcinogenic Potential.
The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and drug regulatory authorities provide guidance on testing for the carcinogenic potential of new drugs. Long-term toxicity studies such as carcinogenicity testing are usually conducted concurrently with clinical trials. Jena, et al. (2005) provide a good overview of carcinogenicity testing for drug development.
The International Agency for Research on Cancer (IARC), part of the World Health Organization (WHO), provides Monographs on the Evaluation of Carcinogenic Risks to Humans and has evaluated the carcinogenic risk of more than 900 substances. "The IARC Monographs are recognized as an authoritative source of information" and may be used by national and international authorities in making risk assessments.
Non-animal Alternative Methods
Non-animal methods include cell-based assays and computational prediction models. Mutagenicity and genotoxicity assays can be used to indicate possible carcinogenic substances, and the two in vitro methods described below (cell transformation and gap junction intercellular communication) can be used to identify possible carcinogens, including nongenotoxic carcinogens. Compared to the in vivo carcinogenicity assays, in vitro methods are significantly faster and less expensive, but current methods are not considered sufficient to serve as full animal replacements at this time.
Cell transformation assays (CTA) are based on detecting phenotypic changes induced by chemicals in mammalian cell cultures. The most widely used of these assays are the Syrian hamster embryo (SHE) assay, the low-pH SHE assay, the Balb/c 3T3 assay, and the C3H10T1/2 assay (Maurici, et al., 2005). The SHE assay is believed to detect early steps of carcinogenesis, and the Balb/c and C3H10 assays later carcinogenic changes (OECD, 2006). These assays determine the cytotoxicity of test substances by measuring effects on morphology, colony-forming ability, and/or growth rate (Combes, et al., 1999). "Accumulated evidence strongly supports the assumption that cellular and molecular processes involved in cell transformation in vitro are similar to those of in vivo carcinogenesis" (Combes, et al., 1999; OECD, 2006). An ECVAM prevalidation project on SHE and Balb/3T3 assays is under way. The gap junction intercellular communication (GJIC) method is based on the disruption of the intercellular exchange of low-molecular-weight molecules through the gap junctions of adjacent cells; this disruption can result in abnormal cell growth and behavior (Maurici, et al., 2005a). The assay appears to be a good candidate for screening for nongenotoxic carcinogens and tumor promotors, but it still needs to be standardized and validated.
(Quantitative) (and qualitative) structure-activity relationship models ((Q)SARs and SARs) and expert systems have been developed to predict carcinogenicity. Several recent publications have reviewed models, such as TOPKAT, CASE, and DEREK, used by regulatory authorities (Cronin, et al., 2003; OECD, 2007). In general, the computation of carcinogenicity is complex, and predictive capability has been limited. The US FDA funded the development of MultiCASE based on data from regulatory submissions, and it was reported to have improved predictivity (Cronin, et al., 2003).
Mutagenicity/genotoxicity assays are the most commonly used in vitro test systems to predict carcinogenicity. Mutagenicity refers to the induction of transmissible changes in the structure of the genetic material of cells or organisms (Maurici, et al., 2005b). Mutations may involve a single gene or a group of genes. Genotoxicity is a broader term that refers to changes to the structure or number of genes via chemical interaction with DNA and/or nonDNA targets such as the spindle apparatus and topoisomerase enzymes (Maurici, et al., 2005b). The term genotoxicity is generally used unless a specific assay is being discussed. In use for over 30 years, genotoxicity assays are employed in a tier-testing approach that starts with Tier I in vitro tests, followed by Tier II in vivo genotoxicity tests to determine the biological relevance of chemicals that are positive in the in vitro tests. Common genotoxicity testing batteries include assays that measure mutations as well as structural and numerical chromosome aberrations (as reviewed in Maurici, et al., 2005b).
Eight in vitro genotoxicity test methods, four of which are commonly used, have been adopted at the EU level with OECD guidelines (see table below). These four in vitro assays include two mutagenicity test methods based on bacterial cells (the bacterial reverse mutation test [Ames test], OECD TG 471; and the Escehrichia coli reverse mutation assay, OECD TG 472), as well as two methods based on mammalian cells (the in vitro mammalian chromosome aberration test, OECD TG 473; and the in vitro mammalian cell gene mutation test, OECD TG 476). Additionally, the European Centre for the Validation of Alternative Methods (ECVAM) validated the in vitro micronucleus test for genotoxicity testing in 2006 as an alternative to the in vitro chromosome aberration assay (ESAC statement: 17 November 2006; revised OECD TG 487, in preparation).
Numerous other in vitro genotoxicity tests, including the in vitro Comet assay, are being developed but are not yet validated.
Table 1. OECD TGs for in vitro genotoxicity and mutagenicity testing
||Bacterial Reverse Mutation Test (Ames Test)
||Genetic Toxicology: Escherichia coli, Reverse Assay
||In Vitro Mammalian Chromosome Aberration Test
||In Vitro Mammalian Cell Gene Mutation Test
||Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells
||Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
||Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
||Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells in vitro
||In vitro Mammalian Cell Micronucleus Test
A recent analysis of the performance of the most common in vitro genotoxicity tests for prediction of carcinogenicity has been published (Kirkland, et al., 2005, p. 200). In this assessment, a battery of three in vitro genotoxicity assays--the Ames test, the mouse lymphoma assay (MLA), and the in vitro micronucleus (MN) or chromosomal alterations (CA) test--discriminated between rodent carcinogens and noncarcinogens when all three tests were positive or all three were negative (Kirkland, et al., 2005). The sensitivity of the data was high, but the specificity of the mammalian assays was poor. In fact, 75-90% of rodent noncarcinogens were positive in one or more of the assays, resulting in a high number of false positive results. Because of this, it isn't possible at this time to rely on current in vitro genotoxicity tests alone, without the Tier II in vivo genotoxicity tests.
Validation and Acceptance of Non-animal Alternative Methods
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and ECVAM have not formally validated any alternative methods for carcinogenicity testing at this time.
ECVAM is conducting a prevalidation/validation study on three cell transformation assays: SHE cells at pH 6.7; SHE cells at pH 7.0; and Balb/c 3T3 cell line. An OECD TG is being drafted simultaneously. The prevalidation study phase was completed in 2010, and the Validation Management Team concluded that "standardised protocols are now available that should be the basis for future use. The SHE pH 6.7, and the SHE pH 7.0 protocols and the assays system themselves are transferable between laboratories, and are reproducible within- and between-laboratories. For the Balb/c 3T3 method, some clarifications and modifications to the protocol were needed to obtain reproducible results. Overall, three methods have shown to be valuable to detect rodent carcinogens." The results from the prevalidation studies are currently undergoing peer review with the ESAC.
Due to the multiple stages of carcinogenesis, the long in vivo time period required, the multiple mechanisms, and the need for metabolic conversion of some substances, existing cell-based assays can be used only in a tiered testing scheme or test battery as a partial replacement for the animal bioassays (Maurici, et al., 2005a). An ECVAM panel could not provide an estimated date for the total replacement of animal testing for carcinogenicity at the EU level (Maurici, et al., 2005a). An ECVAM panel concluded that total replacement of animal testing for genotoxicity is not feasible within the next 12 years (Maurici et al., 2005b). The following diagram represents the timeline for validation of non-animal alternatives for carcinogenicity testing proposed by the panel in 2005.