Cluster analysis in predicting the carcinogenicity of chemicals using short-term assays

Cluster analysis can be a useful tool for exploratory data analysis to uncover natural groupings in data, and initiate new ideas and hypotheses about such groupings. When applied to short-term assay results, it provides and improves estimates for the sensitivity and specificity of assays, provides indications of association between assays and, in turn, which assays can be substituted for one another in a battery, and allows a data base containing test results on chemicals of unknown carcinogenicity to be linked to a data base for which animal carcinogenicity data are available. Cluster analysis was applied to the Gene-Tox data base (which contains short-term test results on chemicals of both known and unknown carcinogenicity). The results on chemicals of known carcinogenicity were different from those obtained when the entire data base was analyzed. This suggests that the associations (and possibly the sensitivities and specificities) which are based on chemicals of known carcinogenicity may not be representative of the true measures. Cluster analysis applied to the total data base should be useful in improving these estimates. Many of the associations between the assays which were found through the use of cluster analysis could be 'validated' based on previous knowledge of the mechanistic basis of the various tests, but some of the associations were unsuspected. These associations may be a reflection of a non-ideal data base. As additional data becomes available and new clustering techniques for handling non-ideal data bases are developed, results from such analyses could play an increasing role in strengthening prediction schemes which utilize short-term tests results to screen chemicals for carcinogenicity, such as the carcinogenicity and battery selection (CPBS) method (Chankong et al., 1985).     

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.     

Knowledge-based battery design of short-term tests based on dose information

A construction of batteries of short-term tests (STTs) is described which is based on a classification of 73 chemicals in regard to their carcinogenicity. The 73 chemicals were studied within the U.S. National Toxicology Program (Ashby and Tennant, 1988). The batteries are validated using the classification of 35 additional chemicals. They are defined by logically structured combinations of rules. The single rules are defined by the z-scores of the logarithmic values of the limiting doses obtained from the 4 in vitro STTs used in the study by Ashby and Tennant. The limiting dose is defined as the lowest effective dose or the highest ineffective dose (Waters et al., 1987). The batteries are constructed by minimizing the number of disagreements with the classification by Ashby and Tennant. Compared with the results obtained from single STTs, 2 batteries of 3 STTs have higher concordances with the carcinogenicity data, namely 70% for the NTP data and 74-77% for the independent test data. In addition, a theoretical result shows that the proposed battery design, for a large enough learning set of chemicals, leads to results which are replicated with high probability on a large enough validation set. Based on the first results obtained with a limited number of chemicals it is concluded that the knowledge-based battery design is worth further development.     

The carcinogenicity prediction and battery selection (CPBS) method: a Bayesian approach

Recently, a large number of relatively inexpensive in vitro short-term tests have been developed to help predict the carcinogenicity of chemicals. The carcinogenicity prediction and battery selection (CPBS) method utilizes the results of such short-term tests to screen for chemicals that are most likely to cause cancer. The method is an integrated approach for analyzing large, often sparsely filled, data bases containing short-term test results, which often have only marginal representation of known non-carcinogens. The CPBS method is developed for the purpose of (i) determining the reliability and predictive capability of individual and batteries of short-term tests, and (ii) developing a strategy for formulating and selecting optimally preferred batteries of short-term tests for screening chemicals for further testing. The term 'optimally preferred' connotes the best acceptable combination of tests in terms of trade-offs among the multiple attributes of each test and resulting battery (e.g., cost, sensitivity, specificity, etc). The CPBS method consists of 5 major tasks: (1) data consolidation, (2) parameter estimation, (3) predictivity calculation, (4) battery selection and (5) risk assessment. Although there is a great need for more research and improvement, the CPBS method at its present stage should add an important method to the maze of the thousands of new chemicals that are introduced into drugs, foods, consumer goods and to the environment every year. This method should also provide an enhanced identification procedure for classifying chemicals more accurately as suspected carcinogens or non-carcinogens.     

The uses of carcinogen-DNA adduct measurement in establishing mechanisms of mutagenesis and in chemoprevention

DNA adducts generated by carcinogenic chemicals reflects human exposure and DNA adducts are related to tumor formation. Most chemical carcinogens require activation to reactive intermediates that bind to nucleophilic centers in proteins and nucleic acids thereby forming covalent adducts. Also, many of the chemicals considered carcinogenic for humans form covalent DNA adducts. Therefore, such DNA damage is generally considered to be causative and linked to tumor formation. In this article we have summarized the work done for many years on the role of DNA adduct formation as an indicator of their carcinogenicity. We have also addressed the important role for measurement of DNA adducts in studies with potential chemopreventive agents for which it is central to have a marker that can be measured more rapidly than changes in cancer incidence.     

Screening for carcinogens by the DNA synthesis inhibition test using human fibroblasts

The Ames test has probably been the most common prescreening test for potential carcinogens. This system, however, occasionally presents false-positive or false-negative results for certain kinds of chemicals. We chose 24 agents, most of which showed a discrepancy in their results with the animal carcinogenicity test and the Ames test, and screened them by the DNA synthesis inhibition test using human fibroblasts. Among 22 agents, 13 (59%) showed consistent results, 6 (27%) showed discrepant results with those obtained by animal tests and 3 (14%) showed ambiguous results. The majority of the DNA synthesis inhibition test results for these chemicals were consistent with the results obtained by whole animal tests, suggesting that the DNA synthesis inhibition test with mammalian cells in culture is a reasonably reliable prescreening test for potential carcinogens.     

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.     

Methods of identifying carcinogenic factors in medication, food and cosmetics]

The removal of carconogenic factors would be a most efficient measure to prevent cancer. As far as known chemicals are concerned, every effort is made to avert them, or at least to reduce the exposure to such compounds, but is necessary to detect unknown chemicals, especially those, drugs and foodstuffs for example, to which large populations are exposed. Giving suspected chemicals to laboratory animals is a standard carcinogenicity test. Studies of the carcinogenicity of unknown chemicals in animals are time consuming, expensive and cumbersome. This is why other means of establishing carcinogenicity are sought for. Several rapid tests are available to-day to select suspected carcinogens. These methods aim primarily at determining with chemicals--at the cell or tissue level--certain changes that would appear essential to trigger the carcinogenic process, such as somatic mutations. Studies are used on the mutagenicity of chemicals for bacteria of the Salmonella type, for yeast and cultured mammalian cells, together with the induction of recessive lethal mutations in Drosophila and of the unscheduled repair synthesis of DNA and the transformation of mammalian cells in vitro. Although there is an unequivocal correlation between the activity of chemicals in such tests and their carcinogenicity, discrepancies are found. Thus, the in vivo tests on laboratory animals remain the most reliable method to determine carcinogenicity. Whereas direct extrapolation of experimental data to human pathology is impossible, the experimental evidence of the carcinogenicity of any chemical should allow us to draw constructive conclusions. We shall never be able to reject drugs which produce the expected results and cannot be replaced by other drugs. But we can must the drugs whose beneficial effects are not exceptional and which can be replaced by other chemicals. As for the chemicals used in food additives and cosmetics, and recognized as carcinogenic in animals, they should be totally given up. Any decision made should be based on animal studies.     

Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP.

An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04).     

Artificial intelligence and Bayesian decision theory in the prediction of chemical carcinogens

Two procedures for predicting the carcinogenicity of chemicals are described. One of these (CASE) is a self-learning artificial intelligence system that automatically recognizes activating and/or deactivating structural subunits of candidate chemicals and uses this to determine the probability that the test chemical is or is not a carcinogen. If the chemical is predicted to be carcinogen, CASE also projects its probable potency.

The second procedure (CPBS) uses Bayesian decision theory to predict the potential carcinogenicity of chemicals based upon the results of batteries of short-term assays. CPBS is useful even if the test results are mixed (i.e. both positive and negative responses are obtained in different genotoxic assays). CPBS can also be used to identify highly predictive as well as cost-effective batteries of assays.

For illustrative purposes the ability of CASE and CPBS to predict the carcinogenicity of a carcinogenic and a non-carcinogenic polycyclic aromatic hydrocarbon is shown. The potential for using the two methods in tandem to increase reliability and decrease cost is presented.     

Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test

The genotoxicity of 9 chemicals used as epoxy resin hardeners was examined in the DNA repair test with rat hepatocytes. DNA repair synthesis was elicited by 7 chemicals, i.e., 4-aminodiphenyl ether, 4,4-diaminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether, 1,3-phenylenedi-4-aminophenyl ether, 4,4′-diaminodiphenyl methane and 4,4′-methylene-bis(2-chloroaniline).The positive results obtained with 4 epoxy resin hardeners of unknown carcinogenicity, i.e., 4-aminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether and 1,3-phenylene-di-4-aminophenyl ether suggest that they may be carcinogens. The genotoxicity of 1,4-phenylene-di-4-aminophenyl ether, of unknown carcinogenicity, and 4,4′-diaminodiphenyl sulfone, for which there is no sound proof of carcinogenicity, was not confirmed in the DNA repair test. The result with 4,4′-diaminodiphenyl sulfone was in agreement with its lack of mutagenicity in Salmonella typhimurium.     

Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics

Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals.     

Animal carcinogenicity studies: 1. Poor human predictivity

The regulation of human exposure to potentially carcinogenic chemicals constitutes society's most important use of animal carcinogenicity data. Environmental contaminants of greatest concern within the USA are listed in the Environmental Protection Agency's (EPA's) Integrated Risk Information System (IRIS) chemicals database. However, of the 160 IRIS chemicals lacking even limited human exposure data but possessing animal data that had received a human carcinogenicity assessment by 1 January 2004, we found that in most cases (58.1%; 93/160), the EPA considered animal carcinogenicity data inadequate to support a classification of probable human carcinogen or non-carcinogen. For the 128 chemicals with human or animal data also assessed by the World Health Organisation's International Agency for Research on Cancer (IARC), human carcinogenicity classifications were compatible with EPA classifications only for those 17 having at least limited human data (p = 0.5896). For those 111 primarily reliant on animal data, the EPA was much more likely than the IARC to assign carcinogenicity classifications indicative of greater human risk (p < 0.0001). The IARC is a leading international authority on carcinogenicity assessments, and its significantly different human carcinogenicity classifications of identical chemicals indicate that: 1) in the absence of significant human data, the EPA is over-reliant on animal carcinogenicity data; 2) as a result, the EPA tends to over-predict carcinogenic risk; and 3) the true predictivity for human carcinogenicity of animal data is even poorer than is indicated by EPA figures alone. The EPA policy of erroneously assuming that tumours in animals are indicative of human carcinogenicity is implicated as a primary cause of these errors.     

Invited contribution: An objective approach to the development of short-term tests predictive of carcinogenicity

The Carcinogenicity Prediction and Battery Selection procedure was developed to address two problems: (1) the identification of highly predictive, yet cost-effective, batteries of short-term tests and (2) the objective prediction of the potential carcinogenicity of chemicals based upon the results of short-term tests even when a mixture of positive and negative results is obtained. In the present report the usefulness of the Carcinogenicity Prediction and Battery Selection procedure is demonstrated using benzo[a]pyrene, benzoin and diethylstilbestrol as examples. In addition, its applicability in the analysis of all the possible outcomes of a battery is illustrated together with an analysis of the worth of additional testing.Abbreviations B[a]P benzo[a]pyrene - CASE Computer-Automated Structure Evaluation - CPBS Carcinogenicity Prediction and Battery Selection - DEHP diethylhexylphthalate - DES diethylstilbestrol - NTA nitrilotriacetate - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

The unique role of rodents in the detection of possible human carcinogens and mutagens

Some of the probable reasons underlying the observation that not all chemicals shown to be genotoxic in vitro are capable of eliciting tumours in rodents or humans are discussed using appropriate examples. It is suggested that a substantial proportion of the resources currently available for conducting rodent carcinogenicity bioassays should be employed in the short-term evaluation in vivo of some of the many hundreds of chemicals recently defined as genotoxic in vitro, rather than in the protracted evaluation of a few chemicals, often of unknown activity in vitro, for carcinogenicity. A decision tree approach to the evaluation of chemicals for human mutagenic/carcinogenic potential is presented which is at variance with the construction and philosophy of many of the current legislative guidelines. The immediate need for the adoption of one of the available short-term in vivo liver assays, and/or the development of a short-term in vivo rodent assay capable of concomitantly monitoring different genetic end-points in a range of organs or tissues is emphasized.     

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

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.     

Carcinogen prediction in the laboratory: a personal view.

Although carcinogens can be divided into various categories, i.e. viruses, physical agents and synthetic and naturally occurring chemicals, it is the latter that give rise to the greatest concern because of their number, quantity and distribution. Present methods of testing chemicals for potential carcinogenicity rely in the main on administration of these at maximally tolerated dose levels to animals, usually rodents, for the animals' lifetime. Such tests would be economically impractical for all chemicals to which man is exposed. New methods have recently been introduced to screen large numbers of chemicals quickly and cheaply which rely on the unifying hypothesis that all carcinogenic chemicals are electrophiles or must be converted to such by metabolism. These methods will be reviewed and compared with traditional methods of carcinogenicity testing, particularly as to their role in attempting to predict hazard to man.     

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.     

Dose-response and threshold-mediated mechanisms in mutagenesis: statistical models and study design

The objective of this paper is to review the use, in mutagenesis, of various mathematical models to describe the dose-response relationship and to try to identify thresholds. It is often taken as axiomatic that genotoxic carcinogens could damage DNA at any level of exposure, leading to a mutation, and that this could ultimately result in tumour development. This has led to the assumption that for genotoxic chemicals, there is no discernible threshold. This assumption is increasingly being challenged in the case of aneugens. The distinction between 'absolute' and 'pragmatic' thresholds is made and the difficulties in determining 'absolute' thresholds using hypothesis testing approaches are described. The potential of approaches, based upon estimation rather than statistical significance for the characterization of dose-response relationships, is stressed. The achievement of a good fit of a mathematical model to experimental data is not proof that the mechanism supposedly underlying this model is operating. It has been argued, in the case of genotoxic chemicals, that any effects produced by a genotoxic chemical which augments that producing a background incidence in unexposed individuals will lead to a dose-response relationship that is non-thresholded and is linear at low doses. The assumptions underlying this presumption are explored in the context of the increasing knowledge of the mechanistic basis of mutagenicity and carcinogenicity. The possibility that exposure to low levels of genotoxic chemicals may induce and enhance defence and repair mechanisms is not easily incorporated into many of the existing mathematical models and should be an objective in the development of the next generation of biologically based dose-response (BB-DR) models. Studies aimed at detecting or characterizing non-linearities in the dose-response relationship need appropriate experimental designs with careful attention to the choice of biomarker, number and selection of dose levels, optimum allocation of experimental units and appropriate levels of replication within and repetition of experiments. The characterization of dose-response relationships with appropriate measures of uncertainty can help to identify 'pragmatic' thresholds based upon biologically relevant criteria which can help in the regulatory process.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.