Using Short-Term Tests to Predict Carcinogenic Activity in the Long-Term Bioassay

A method for classifying chemicals with respect to carcinogenic potential based on short-term test results is presented. The method utilizes the logistic regression model to translate results from short-term toxicity assays into predictions of the likelihood that a chemical will be carcinogenic if tested in a long-term bioassay. The proposed method differs from previous approaches in two ways. First, statistical confidence limits on probabilities of cancer rather than central estimates of those probabilities are used for classification. Second, the method does not classify all chemicals in a data base with respect to carcinogenic potential. Instead, it identifies chemicals with highest and lowest likelihood of testing positive for carcinogenicity in the bioassay. A subset of chemicals with intermediate likelihood of being positive remains unclassified, and will require further testing, perhaps in a long-term bioassay. Two data bases of binary short-term and long-term test results from the literature are used to illustrate and evaluate the proposed procedure. A cross-validation analysis of one of the data sets suggests that, for a sufficiently rich data base of chemicals, the development of a robust predictive system to replace the bioassay for some unknown chemicals is a realistic goal.     

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.     

Measurement of DNA-excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: its possible use in carcinogenicity screening.

When suspensions of freshly isolated rat hepatocytes were exposed to a number of carcinogenic compounds, it was possible to measure an increased UDS by a rapid procedure via liquid-scintillation counting. For a number of carcinogenic compounds and some of their non-carcinogenic structural analogues a good correlation between the carcinogenic property and the ability to induce UDS was demonstrable. Out of 12 carcinogenic compounds, belonging to several different chemical classes, 10 gave rise to an increased UDS, whereas only 2 compounds, the polycyclic aromatic hydrocarbons benzo[alpha]pyrene and benz[alpha]anthracene, did not. All 4 noncarcinogenic compounds tested were negative. Possibly this method can be of value as a routine screening test, in combination with other short-term test systems, thus improving the predictive value of screening in vitro with respect to carcinogenicity.     

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.     

Animal carcinogenicity studies: 2. Obstacles to extrapolation of data to humans

Due to limited human exposure data, risk classification and the consequent regulation of exposure to potential carcinogens has conventionally relied mainly upon animal tests. However, several investigations have revealed animal carcinogenicity data to be lacking in human predictivity. To investigate the reasons for this, we surveyed 160 chemicals possessing animal but not human exposure data within the US Environmental Protection Agency chemicals database, but which had received human carcinogenicity assessments by 1 January 2004. We discovered the use of a wide variety of species, with rodents predominating, and of a wide variety of routes of administration, and that there were effects on a particularly wide variety of organ systems. The likely causes of the poor human predictivity of rodent carcinogenicity bioassays include: 1) the profound discordance of bioassay results between rodent species, strains and genders, and further, between rodents and human beings; 2) the variable, yet substantial, stresses caused by handling and restraint, and the stressful routes of administration common to carcinogenicity bioassays, and their effects on hormonal regulation, immune status and predisposition to carcinogenesis; 3) differences in rates of absorption and transport mechanisms between test routes of administration and other important human routes of exposure; 4) the considerable variability of organ systems in response to carcinogenic insults, both between and within species; and 5) the predisposition of chronic high dose bioassays toward false positive results, due to the overwhelming of physiological defences, and the unnatural elevation of cell division rates during ad libitum feeding studies. Such factors render profoundly difficult any attempts to accurately extrapolate human carcinogenic hazards from animal data.     

Animal carcinogenicity studies: implications for the REACH system

The 2001 European Commission proposal for the Registration, Evaluation and Authorisation of Chemicals (REACH) aims to improve public and environmental health by assessing the toxicity of, and restricting exposure to, potentially toxic chemicals. The greatest benefits are expected to accrue from decreased cancer incidences. Hence the accurate identification of chemical carcinogens must be a top priority for the REACH system. Due to a paucity of human clinical data, the identification of potential human carcinogens has conventionally relied on animal tests. However, our survey of the US Environmental Protection Agency's (EPAs) toxic chemicals database revealed that, for a majority of the chemicals of greatest public health concern (93/160, i.e. 58.1%), the EPA found animal carcinogenicity data to be inadequate to support classifications of probable human carcinogen or non-carcinogen. A wide variety of species were used, with rodents predominating; a wide variety of routes of administration were used; and a particularly wide variety of organ systems were affected. These factors raise serious biological obstacles that render accurate extrapolation to humans profoundly difficult. Furthermore, significantly different International Agency for Research on Cancer assessments of identical chemicals, indicate that the true human predictivity of animal carcinogenicity data is even poorer than is indicated by the EPA figures alone. Consequently, we propose the replacement of animal carcinogenicity bioassays with a tiered combination of non-animal assays, which can be expected to yield a weight-of-evidence characterisation of carcinogenic risk with superior human predictivity. Additional advantages include substantial savings of financial, human and animal resources, and potentially greater insights into mechanisms of carcinogenicity.     

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.     

Genetic toxicology data in the evaluation of potential human environmental carcinogens.

In 1969, the International Agency for Research on Cancer (IARC) initiated the Monographs Programme to evaluate the carcinogenic risk of chemicals to humans. Results from short-term mutagenicity tests were first included in the IARC Monographs in the mid-1970s based on the observation that most carcinogens are also mutagens, although not all mutagens are carcinogens. Experimental evidence at that time showed a strong correlation between mutagenicity and carcinogenicity and indicated that short-term mutagenicity tests are useful for predicting carcinogenicity. Although the strength of these correlations has diminished over the past 20 years with the identification of putative nongenotoxic carcinogens, such tests provide vital information for identifying potential human carcinogens and understanding mechanisms of carcinogenesis. The short-term test results for agents compiled in the EPA/IARC Genetic Activity Profile (GAP) database over nearly 15 years are summarized and reviewed here with regard to their IARC carcinogenicity classifications. The evidence of mutagenicity or nonmutagenicity based on a 'defining set' of test results from three genetic endpoints (gene mutation, chromosomal aberrations, and aneuploidy) is examined. Recommendations are made for assessing chemicals based on the strength of evidence from short-term tests, and the implications of this approach in identifying mutational mechanisms of carcinogenesis are discussed. The role of short-term test data in influencing the overall classification of specific compounds in recent Monograph volumes is discussed, particularly with reference to studies in human populations. Ethylene oxide is cited as an example.     

Quantification of the predictivity of some short-term assays for carcinogenicity in rodents.

A statistical procedure is described for assessing the predictive performance of short-term tests for carcinogenicity in which the actual number of chemicals tested is taken into consideration. The method is then applied to several widely used short-term assays.     

The second National Toxicology Program comparative exercise on the prediction of rodent carcinogenicity: definitive results

Chemical carcinogenicity has been the target of a large array of attempts to create alternative predictive models, ranging from short-term biological assays (e.g. mutagenicity tests) to theoretical models. Among the theoretical models, the application of the science of structure-activity relationships (SAR) has earned special prominence. A crucial element is the independent evaluation of the predictive ability. In the past decade, there have been two fundamental comparative exercises on the prediction of chemical carcinogenicity, held under the aegis to the US National Toxicology Program (NTP). In both exercises, the predictions were published before the animal data were known, thus using a most stringent criterion of predictivity. We analyzed the results of the first comparative exercise in a previous paper [Mutat. Res. 387 (1997) 35]; here, we present the complete results of the second exercise, and we analyze and compare the prediction sets. The range of accuracy values was quite large: the systems that performed best in this prediction exercise were in the range 60-65% accuracy. They included various human experts approaches (e.g. Oncologic) and biologically based approaches (e.g. the experimental transformation assay in Syrian hamster embryo (SHE) cells). The main difficulty for the structure-activity relationship-based approaches was the discrimination between real carcinogens, and non-carcinogens containing structural alerts (SA) for genotoxic carcinogenicity. It is shown that the use of quantitative structure-activity relationship models, when possible, can contribute to overcome the above problem. Overall, given the uncertainty linked to the predictions, the predictions for the individual chemicals cannot be taken at face value; however, the general level of knowledge available today (especially for genotoxic carcinogens) allows qualified human experts to operate a very efficient priority setting of large sets of chemicals.     

Naturalness and predictivity of classifications

It is shown for binary (1, 0) data that there is a direct relationship between a new measure of predictivity in classification and the Simple Matching Coefficient. This predictivity measure is related to the variance of a cluster in phenetic space and to the mean variance of the proportion of 1 states in the members of the cluster. It can be considered a form of 'squared predictivity'. Maximizing the measure leads to Variance Clustering, and if weighted by the number of cluster members, to Sums of Squares Clustering. The relations to the Unweighted Average Pair Group and Unweighted Centroid Pair Group methods are also elucidated.     

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

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.     

Short-term testing--are we looking at wrong endpoints?

Short-term testing has been performed and interpreted on the basis of correlation between these tests and animal carcinogenicity. This empirical approach has been the only feasible one, due to a lack of knowledge of the actual genetic endpoints of relevance in carcinogenicity. However, the rapidly growing information on genetic alterations actually involved in carcinogenicity and in particular activation of oncogenes, provides facts of basic importance for the strategy of short-term testing. The presently used sets of short-term tests focus on standard genetic endpoints, mainly point mutations and chromosomal aberrations. Little attention has been paid in that connection to other endpoints, which have been shown or suspected to play an important role in carcinogenicity. These endpoints include gene amplification, transpositions, hypomethylation, polygene mutations and recombinogenic effects. Furthermore, indirect effects, for instance via radical generation and an imbalance of the nucleotide pool, may be of great significance for the carcinogenic and cocarcinogenic effects of many chemicals. Modern genetic and molecular technology has opened entirely new prospects for identifying genetic alterations in tumours and in its turn these prospects should be taken advantage of in order to build up more sophisticated batteries of assays, adapted to the genetic endpoints actually demonstrated to be involved in cancer induction. Development of new assay systems in accordance with the elucidation of genetic alterations in carcinogenicity will probably constitute one of the most important areas in genetic toxicology in the future. From a regulatory point of view the prerequisite for a development in this direction will be a flexibility of the handling of questions concerning short-term testing also at a bureaucratic level.     

Efficiency of the prediction of carcinogenic activities of chemical substances based on scoring somatic mutations in the soybean Glycine max (L.) Merrill

The efficiency of scoring somatic mutations in soybean (Glycine max (L.) Merrill) leaves as a test for carcinogenic activity of chemical substances in rodents has been evaluated. The efficiency of the test used alone or as part of a battery of tests has been estimated. The mutagenic activities of some chemical substances estimated using the soybean test are presented. Selective information on the carcinogenic activities of substances obtained in special carcinogenicity tests has been used as a quantitative measure of the efficiency of the tests with soybean leaves. To estimate the weight of evidence for the presence of this activity in the tested substances, a special function has been used whose values are uniquely related to the complete information, which is the sum of a priori information and the information obtained after testing. In general, the results have shown that the somatic mutation score test using soybean leaves is at least as efficient as the well-known tests that are generally used now, such as the Ames test and the chromosome aberration score test using mammalian cells in vitro. This test may be promising for the formation of efficient short-term test batteries.     

The carcinoGENOMICS project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.     

A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds

Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73–82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy‐three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost‐effective stand‐alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively     

Predictive assay for rodent carcinogenicity using in vivo biochemical parameters: operational characteristics and complementarity.

111 chemicals of known rodent carcinogenicity (49 carcinogens, 62 noncarcinogens), including many promoters of carcinogenesis, nongenotoxic carcinogens, hepatocarcinogens, and halogenated hydrocarbons, were selected for study. The chemicals were administered by gavage in two dose levels to female Sprague-Dawley rats. The effects of these 111 chemicals on 4 biochemical assays (hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450)) were determined. Composite parameters are defined as follows: CP = [ODC and P450), CT = [ALT and ODC), and TS = [DD or CP or CT]. The operational characteristics of TS for predicting rodent cancer were sensitivity 55%, specificity 87%, positive predictivity 77%, negative predictivity 71%, and concordance 73%. For these chemicals, the 73% concordance of this study was superior to the concordance obtained from published data from other laboratories on the Ames test (53%), structural alerts (SA) (46%), chromosome aberrations in Chinese hamster ovary cells (ABS) (48%), cell mutation in mouse lymphoma 15178Y cells (MOLY) (52%), and sister-chromatid exchange in Chinese hamster ovary cells (SCE) (60%). The 4 in vivo biochemical assays were complementary to each other. The composite parameter TS also shows complementarity to all 5 other predictors of rodent cancer examined in this paper. For example, the Ames test alone has a concordance of only 53%. In combination with TS, the concordance is increased to 62% (Ames or TS) or to 63% (Ames and TS). For the 67 chemicals with data available for SA, the concordance for predicting rodent carcinogenicity was 47% (for SA alone), 54% (for SA or TS), and 66% (for SA and TS). These biochemical assays will be useful: (1) to predict rodent carcinogenicity per se, (2) to 'confirm' the results of short-term mutagenicity tests by the high specificity mode of the biochemical assays (the specificity and positive predictivity are both 100%), and (3) to be a component of future complementary batteries of tests for predicting rodent carcinogenicity.     

Efficiency of the prediction of carcinogenic activities of chemical substances based on scoring somatic mutations in the soybean <Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill

The efficiency of scoring somatic mutations in soybean (Glycine max (L.) Merrill) leaves as a test for carcinogenic activity of chemical substances in rodents has been evaluated. The efficiency of the test used alone or as part of a battery of tests has been estimated. The mutagenic activities of some chemical substances estimated using the soybean test are presented. Selective information on the carcinogenic activities of substances obtained in special carcinogenicity tests has been used as a quantitative measure of the efficiency of the tests with soybean leaves. To estimate the weight of evidence for the presence of this activity in the tested substances, a special function has been used whose values are uniquely related to the complete information, which is the sum of a priori information and the information obtained after testing. In general, the results have shown that the somatic mutation score test using soybean leaves is at least as efficient as the well-known tests that are generally used now, such as the Ames test and the chromosome aberration score test using mammalian cells in vitro. This test may be promising for the formation of efficient short-term test batteries.