Integration of cytogenetic assays with toxicology studies (original) (raw)
Related papers
Mutagenesis
Genotoxicity testing is an important part of standard safety testing strategies. Animal studies have always been a key component, either as a mandatory part of the regulatory test battery, or to follow-up questionable in vitro findings. The strengths and weaknesses of in vivo assays is a continuous matter of debate, including their capacity to predict (human) carcinogenicity. We have therefore analysed the sensitivity of five routinely used in vivo tests to determine, in addition to other aspects, which tests or combination of tests best identify 73 chemicals classified as IARC Group 1 and 2A carcinogens. The in vivo tests included the micronucleus (MN), unscheduled DNA synthesis (UDS), comet, Pig-a and transgenic rodent assays (TGR). The individual assays detect 74.2% (49/66, MN), 64.3% (9/14, UDS), 92.1% (35/38, comet), 82.4% (14/17, Pig-a) and 90.3% (28/31, TGR) of the probable and confirmed human carcinogens that were tested in these assays. Combining assays that cover different genotoxicity endpoints and multiple tissues, e.g. the bone marrow MN and the liver comet assays, increases the sensitivity further (to 94%). Correlations in terms of organ-specificity for these assays with human cancer target organs revealed only a limited correlation for the hematopoietic system but not for other organs. The data supports the use of the comet and TGR assays for detection of 'site-of-first-contact' genotoxicants, but these chemicals were generally also detected in assays that measure genotoxicity in tissues not directly exposed, e.g. liver and the hematopoietic system. In conclusion, our evaluation confirmed a high sensitivity of the five in vivo genotoxicity assays for prediction of human carcinogens, which can be further increased by combining assays. Moreover, the addition of the comet to the in vivo MN test would identify all DNA reactive human carcinogens. Importantly, integration of some of the study readouts into one experiment is an animal-saving alternative to performing separate experiments.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2005
The performance of a battery of three of the most commonly used in vitro genotoxicity tests--Ames+mouse lymphoma assay (MLA)+in vitro micronucleus (MN) or chromosomal aberrations (CA) test--has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75-95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results.
IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans
Mutation Research/Reviews in Mutation Research, 2000
The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.
Mutation Research/Reviews in Mutation Research, 1999
Ž . 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 Ž . short-term test results for agents compiled in the EPArIARC 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 Ž 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. q Volume 428, Nos. 1 and 2. 0165-1110r99r$ -see front matter q 1999 Elsevier Science B.V. All rights reserved.
Data selection and treatment of chemicals tested for genotoxicity and carcinogenicity
Environmental Health Perspectives, 1991
A datbase contaning q live and qu e Of Arlmenl in tbe fikofgenniy and carcinogenicity has boee deped. By a zg resultofthesde performed by the U.& Natonullsckgy Program, or by a simia program develped inapn, orrepretdinthesentc l e, amsull perfmonedbg priat oag dons, Infomation has bo e d reling o3389 chmcls, Ideried by their CAS number. Th studies consdered for the database Indude three gnotkicty/mug dty short-term test (STl), namely, twoin Wtv (Samonella, gene muation amy, andmu mmaliacelsh/man cy roosome abe r amy) and onein Wvo, the rodent bone murew moceuns asy. lb ipte the pome value ofthoe SIT a forr n the rut ofannalllong-term ayse so beencolected. V* hae re-evauatedan the genety sesand the majorityofthose casestudied In diferent labotoris con resultshasbeen resolved; a pro orn questionable cases is, however, stil present in the da e. In total, 289 (85S%) of the che ahave been tested in the Salmonella amy; 399(413%) have beept In the in vit caberdon amy; 319 (9A%) hae been tested in the in Wwo rodent bon nawel mi rmkclensI ay; 71 (2L2%) ofthechenulcaw b haeenested In theix Wso Ianial long-term biosy. For 1lll ch as tested in the Salmn a , 30,650 qustudies have been Included in the d thus alowing a possble la n ofmut i ding tother mutagenic potency. One thousn nnebnddchb (5.1%) vesbown positive rnts hi at leone ofthe four d ass, thus leaving 1$89 chemical (43.9%) with neptive result By ng the oat between gsy,siown by the thrme STIs condered, and n w have demonstrated that the podtive peitivity nreas to a value of 95.6% if the three ST1b are conidered together (two Ui itrv and oneix wvoSTIT); sibmiry, the negAive p reichtivity risestoavalueof89.6% with the sam three assys. The accuracy, or the dance, of the SIT sand the cacin_!-t resuts was92.5% for the three SM Abough the results colected are of high interest for sdentific and practil actons, the aim of the present study is to prepare a genotoodcity/cardnogenicity database for a further quantitat scture-activity relationship (QSAR) study based on a computer cbemistry analysis.
Mutagenesis, 1989
A series of multivariate statistical methods have been used to explore the results of a set of four in vitro short-term tests (STT) on 73 chemicals reported by the US National Toxicology Program (NTP). Cluster analysis showed that the mouse lymphoma mutation (MLY) and sister-chromatid exchange (SCE) were similar in performance, as were the Salmonella (STY) and chromosomal aberration test (CHA). The lack of association between tests using the same genetic end-point or at the same phylogenetic level found in previous analyses was confirmed in this study. Factor analysis was used to derive a scale of genetic damage. This measure was contrasted with rodent carcinogenicity; only a limited association was found (rank correlation coefficient, rs = 0.32). Linear discriminant analysis was used to study whether the STTs could be used to complement one another. The combination of STY with the other STTs did not improve significantly the prediction of rodent carcinogenicity of STY alone. In the entire set of chemicals, 33% were negative in STY and positive in at least two other STT, and 11% was negative in STY and positive in the three other tests. SCE and MLY were complementary to STY for identifying the most genotoxic chemicals, but CHA was not a useful complement. The presence of potential electrophilic sites in the chemicals was highly correlated with the STY results, but did not improve the ability of STY to identify genotoxic chemicals or predict rodent carcinogens. In conclusion, the other in vitro STTs did not complement STY for predicting carcinogenicity, but were an important complement for describing the potential genotoxicity of chemicals.
How accurate is in vitro prediction of carcinogenicity?
British Journal of Pharmacology, 2011
Positive genetic toxicity data suggest carcinogenic hazard, and this can stop a candidate pharmaceutical reaching the clinic. However, during the last decade, it has become clear that many non-carcinogens produce misleading positive results in one or other of the regulatory genotoxicity assays. These doubtful conclusions cost a lot of time and money, as they trigger additional testing of apparently genotoxic candidates, both in vitro and in animals, to discover whether the suggested hazard is genuine. This in turn means that clinical trials can be put on hold. This review describes the current approaches to the 'misleading positive' problem as well as efforts to reduce the use of animals in genotoxicity assessment. The following issues are then addressed: the application of genotoxicity testing screens earlier in development; the search for new or improved in vitro genotoxicity tests; proposed changes to the International Committee on Harmonisation guidance on genotoxicity testing [S2(R1)]. Together, developments in all these areas offer good prospects of a more rapid and cost-effective way to understand genetic toxicity concerns.
Quantitative Approaches to Assess Key Carcinogenic Events of Genotoxic Carcinogens
Toxicological research, 2018
Chemical carcinogenesis is a multistep process. Genotoxic carcinogens, which are DNA-reactive, induce DNA adduct formation and genetic alterations in target cells, thereby generating mutated cells (initiation). Subsequently, preneoplastic lesions appear through clonal proliferation of the mutated cells and transform into tumors (promotion and progression). Many factors may influence these processes in a dose-dependent manner. Therefore, quantitative analysis plays an important role in studies on the carcinogenic threshold of genotoxic carcinogens. Herein, we present data on the relationship between key carcinogenic events and their deriving point of departure (PoD). Their PoDs were also compared to those of the carcinogenesis pathway. In an experiment, the liver of rats exposed to 2-amino-3,8-dimethylimidazo-(4,5-)quinoxaline (MeIQx) was examined to determine the formation of MeIQx-DNA adducts, generation of mutations at transgene, and induction of preneoplastic glutathione -transfe...
Toxicology Mechanisms and Methods, 2005
The advent of the industrial revolution has seen a significant increase in the number of new chemical entities (NCEs) released in the environment. It becomes imperative to check the toxic potential of NCEs to nontarget species before they are released for commercial purposes because some of these may exert genotoxicity, mutagenicity, or carcinogenicity. Exposure to such compounds produces chemical changes in DNA, which are generally repaired by the DNA repair enzymes. However, DNA damage and its fixation may occur in the form of gene mutations, chromosomal damage, and numerical chromosomal changes and recombination. This may affect the incidence of heritable mutations in man and may be transferred to the progeny or lead to the development of cancer. Hence, adequate tests on NCEs have to be undertaken for the risk assessment and hazard prediction. Compounds that are positive in tests that detect such damages have the potential to be human mutagens/carcinogens. Only long-term animal bioassays, involving lifetime studies on animals, were used earlier to classify substances as mutagens/carcinogens. These tests were cumbersome and time consuming and required a lot of facilities and personnel. Short-term tests, therefore, were brought into practice. A "battery" of three to four of these short-term tests has been proposed now by a number of regulatory authorities for the classification of compounds as mutagenic or carcinogenic. This review deals with the current status of these short-term tests.
Qualitative and quantitative approaches in the dose–response assessment of genotoxic carcinogens
Mutagenesis, 2015
Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on lowdose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2′-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose-response assessment must be approved on the basis of scientific judgment.