Identification of the structural requirements for mutagencitiy, by incorporating molecular flexibility and metabolic activation of chemicals. II. General Ames mutagenicity model

The tissue metabolic simulator (TIMES) modeling approach is a hybrid expert system that couples a metabolic simulator together with structure toxicity rules, underpinned by structural alerts, to predict interaction of chemicals or their metabolites with target macromolecules. Some of the structural alerts representing the reactivity pattern-causing effect could interact directly with the target whereas others necessitated a combination with two- or three-dimensional quantitative structure-activity relationship models describing the firing of the alerts from the rest of the molecules. Recently, TIMES has been used to model bacterial mutagenicity [Mekenyan, O., Dimitrov, S., Serafimova, R., Thompson, E., Kotov, S., Dimitrova, N., and Walker, J. (2004) Identification of the structural requirements for mutagenicity by incorporating molecular flexibility and metabolic activation of chemicals I: TA100 model. Chem. Res. Toxicol. 17 (6), 753-766]. The original model was derived for a single tester strain, Salmonella typhimurium (TA100), using the Ames test by the National Toxicology Program (NTP). The model correctly identified 82% of the primary acting mutagens, 94% of the nonmutagens, and 77% of the metabolically activated chemicals in a training set. The identified high correlation between activities across different strains changed the initial strategic direction to look at the other strains in the next modeling developments. In this respect, the focus of the present work was to build a general mutagenicity model predicting mutagenicity with respect to any of the Ames tester strains. The use of all reactivity alerts in the model was justified by their interaction mechanisms with DNA, found in the literature. The alerts identified for the current model were analyzed by comparison with other established alerts derived from human experts. In the new model, the original NTP training set with 1341 structures was expanded by 1626 proprietary chemicals provided by BASF AG. Eventually, the training set consisted of 435 chemicals, which are mutagenic as parents, 397 chemicals that are mutagenic after S9 metabolic activation, and 2012 nonmutagenic chemicals. The general mutagenicity model was found to have 82% sensitivity, 89% specificity, and 88% concordance for training set chemicals. The model applicability domain was introduced accounting for similarity (structural, mechanistic, etc.) between predicted chemicals and training set chemicals for which the model performs correctly.     

Carbamates and ICH M7 classification: Making use of expert knowledge

Carbamates are widely used in the chemical industry so understanding their toxicity is important to safety assessment. Carbamates have been associated with certain toxicities resulting in publication of structural alerts, including alerts for mutagenicity. Structural alerts for bacterial mutagenicity can be used in combination with statistical systems to enable ICH M7 classification, which allows assessment of the genotoxic risk posed by pharmaceutical impurities. This study tested a hypothetical bacterial mutagenicity alert for carbamates and examined the impact it would have on ICH M7 classifications using (Q)SAR predictions from the expert rule-based system Derek Nexus and the statistical-based system Sarah Nexus. Public datasets have a low prevalence of mutagenic carbamates, which highlighted that systems containing an alert for carbamates perform poorly for achieving correct ICH M7 classifications. Carbamates are commonly used as protecting groups and proprietary datasets containing such compounds were also found to have a low prevalence of mutagenic compounds. Expert review of the mutagenic compounds established that mutagenicity was often only observed under certain (non-standard) conditions and more generally that the Ames test may be a poor predictor for the risk of carcinogenicity posed by chemicals in this class. Overall a structural alert for the in vitro bacterial mutagenesis of carbamates does not benefit workflows for assigning ICH M7 classification to impurities.     

Validation of the (Q)SAR combination approach for mutagenicity prediction of flavor chemicals

Most exposure levels of flavor in food are considered to be extremely low. If at all, genotoxic properties should be taken into account in safety evaluations. We have recently established a (quantitative) structure–activity relationship, (Q)SAR, combination system, which is composed of three individual models of mutagenicity prediction for industrial chemicals. A decision on mutagenicity is defined as the combination of predictive results from the three models. To validate the utility of our (Q)SAR system for flavor evaluation, we assessed 367 flavor chemicals that had been evaluated mainly by JECFA and for which Ames test results were available. When two or more models gave a positive evaluation, the sensitivity was low (19.4%). In contrast, when one or more models gave a positive evaluation, the sensitivity increased to 47.2%. The contribution of this increased sensitivity was mainly due to the result of the prediction by Derek for Windows, which is a knowledge-based model. Structural analysis of false negatives indicated some common sub-structures. The approach of improving sub-structural alerts could effectively contribute to increasing the predictability of the mutagenicity of flavors, because many flavors possess categorically similar functional sub-structures or are composed of a series of derivatives.     

Genotoxicity studies OF p-dimethylaminoazobenzene (DAB)

The potential genotoxicity of the rodent liver carcinogen p-dimethylaminoazobenzene (DAB) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for testing chemicals. DAB was clearly positive in both the bacterial reverse mutation test (Ames test) and in vitro chromosomal aberration test in the presence of metabolic activation, whereas it was weakly positive at toxic doses in the rat bone marrow micronucleus test. It has been reported that DAB was clearly positive in in vivo genotoxicity tests, i.e., a mouse alkaline single cell gel electrophoresis (comet) assay and a young rat liver micronucleus test. These results suggest that the test system using the liver is effective for in vivo genotoxicity assessment of chemicals that show mutagenicity in in vitro genotoxicity tests in the presence of metabolic activation.     

Genotoxicity assessment of some cosmetic and food additives

α-Hexylcinnamaldehyde (HCA) and p-tert-butyl-alpha-methylhydrocinnamic aldehyde (BMHCA) are synthetic aldehydes, characterized by a typical floral scent, which makes them suitable to be used as fragrances in personal care (perfumes, creams, shampoos, etc.) and household products, and as flavouring additives in food and pharmaceutical industry. The aldehydic structure suggests the need for a safety assessment for these compounds. Here, HCA and BMHCA were evaluated for their potential genotoxic risk, both at gene level (frameshift or base-substitution mutations) by the bacterial reverse mutation assay (Ames test), and at chromosomal level (clastogenicity and aneuploidy) by the micronucleus test. In order to evaluate a primary and repairable DNA damage, the comet assay has been also included. In spite of their potential hazardous chemical structure, a lack of mutagenicity was observed for both compounds in all bacterial strains tested, also in presence of the exogenous metabolic activator, showing that no genotoxic derivatives were produced by CYP450-mediated biotransformations. Neither genotoxicity at chromosomal level (i.e. clastogenicity or aneuploidy) nor single-strand breaks were observed. These findings will be useful in further assessing the safety of HCA and BMHCA as either flavour or fragrance chemicals.     

Evaluation of the genotoxicity of (-)-hydroxycitric acid (HCA-SX) isolated from Garcinia cambogia

(-)-Hydroxycitric acid (HCA) is widely used as an ingredient for nutritional supplements aimed at reducing food intake, appetite, and body weight. In this study, the genotoxicity of HCA was evaluated using three tests: a bacterial reverse mutation assay (Ames test), an in vitro chromosomal aberration (CA) test, and an in vivo micronucleus (MN) test. HCA was negative by the Ames test in the presence or absence of a microsomal metabolizing system. HCA did not induce mutagenic activity in the Ames test, and no significant mutagenic potency was indicated by CA tests. However, HCA significantly and dose-dependently increased the number of MNPCEs (micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes) and PCE/(PCE + NCE) ratios according to the MN test. These results suggest that HCA preferentially induce micronuclei.     

Induction of the P-450 I family of proteins by polycyclic aromatic hydrocarbons: possible relationship to their carcinogenicity

The hypothesis has been put forward that mutagenic polycyclic aromatic hydrocarbons which induce the P-450 I family of cytochromes, the major enzyme system responsible for their activation, are likely to be carcinogenic. In order to test this hypothesis, rats have been pretreated with a number of polycyclic aromatic hydrocarbons of different mutagenic and carcinogenic potency and hepatic P-450 I activity was monitored using chemical probes such as the O-deethylation of ethoxyresorufin and metabolic activation of Glu-P-1 to mutagens, and immunologically employing polyclonal antibodies against purified rat P-450 I A1. All compounds studied enhanced P-450 I activity and induced P-450 I apoproteins but the extent of induction was very markedly different. The results are discussed with reference to the mutagenicity of these chemicals in the Ames test and their carcinogenicity in the classical mouse skin model. A relationship appears to exist between carcinogenicity of polycyclic aromatic hydrocarbons and their ability to induce hepatic P-450 I activity.     

Genotoxicity studies of 2,6-dinitrotoluene (2,6-DNT)

The potential genotoxicity of the rodent liver carcinogen 2,6-dinitrotoluene (2,6-DNT) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for the testing of chemicals by performing the bacterial reverse mutation (Ames) assay, the in vitro chromosomal aberration assay, and the in vivo comet assay (alkaline single cell gel electrophoresis) in rat liver. In the Ames assay, 2,6-DNT was moderately positive irrespective of metabolic activation. In the in vitro chromosomal aberration assay, under conditions where the test substance would precipitate out, weak structural aberrations were observed with or without S9 mix at each dose at which the cell growth rate was about 40 to 50%. The in vivo comet assay yielded positive results in rat liver; that is to say, the increases in % tail DNA in liver in the 25 and 50 mg/kg groups were observed statistically significantly and dose-dependent. Our findings are in accordance with previous findings in the in vivo/in vitro unscheduled DNA synthesis (UDS) assay in rat liver and in a young rat liver micronucleus assay, although the rat bone marrow micronucleus assay gave negative results. These results suggest that test systems using liver are a useful method for the in vivo genotoxicity assessment of chemicals that require metabolic activation.     

猫眼草水煎液体外致突变性的研究

目的检验猫眼草水煎液的致突变性;改进经典的A-mes试验体系使之适应于中药体外致突变性检验。方法通过经典的Ames试验检测猫眼草的体外致突变性;通过哺乳动物骨髓细胞染色体畸变试验检测猫眼草的致畸作用;改进的Ames试验通过增设含补充组氨酸(含量对应于猫眼草水煎液中组氨酸浓度)的阴性对照,排除样品中组氨酸成分对试验结果的影响。结果猫眼草水煎液在经典的Ames试验中为强阳性;对哺乳动物骨髓细胞染色体致畸作用为阴性;在改进的Ames试验中猫眼草水煎液的致突变性为阴性。结论经典的Ames试验不适合猫眼草水煎液致突变性检测,改进后的Ames实验体系适合。猫眼草水煎液在体外和体内均没有致突变性。     

Mutagenicity in V79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines

The aim of this investigation was to study the correlation between carcinogenicity in small rodents and mutagenic potency of aromatic amines, as measured by the induction of 6-thioguanine resistance in V79 Chinese hamster cells. It has been previously shown that the carcinogenic potency of these compounds is not correlated to their ability to induce DNA breakage, SCEs, or point mutations in bacteria, but a correlation exists with autoradiographic DNA repair test (in primary hepatocyte cultures). Twelve aromatic amines were tested and the rat liver S9 fraction was routinely incorporated in the mutation assay; mouse liver and hamster liver S9 fractions were also used as metabolizing systems. The comparison of the ranks of mutagenic and oncogenic potencies by means of the Spearman test shows no correlation between carcinogenicity and V79 cell mutagenicity of the tested aromatic amines. There was a generally low mutagenicity seen for aromatic amines in V79 cells. In some cases this could be attributed to an insufficient metabolic activation by rat S9. For example, benzidine, which was inactive when assayed in the presence of rat S9, became mutagenic when in the presence of mouse S9. On the other hand, hamster S9, which has been shown to be the best activating system for 2-acetylaminofluorene in the Ames test, did not activate this compound in V79 cells. Inadequate metabolic activation of the standard system (rat S9) used in this work could explain the low mutagenicity and the lack of correlation observed between mutagenicity and carcinogenicity. A second possibility is that point mutation is not the essential end point for the initiating activity of aromatic amines during the carcinogenic process. A third possibility is that the activity of some aromatic amines is not restricted to the initiation step in carcinogenesis. Chronic treatments with the sublethal doses often result in significant promoting activities, which could mask efficiently the initiating potential of the same chemicals.     

Characterization and validation of an in silico toxicology model to predict the mutagenic potential of drug impurities

Control and minimization of human exposure to potential genotoxic impurities found in drug substances and products is an important part of preclinical safety assessments of new drug products. The FDA's 2008 draft guidance on genotoxic and carcinogenic impurities in drug substances and products allows use of computational quantitative structure-activity relationships (QSAR) to identify structural alerts for known and expected impurities present at levels below qualified thresholds. This study provides the information necessary to establish the practical use of a new in silico toxicology model for predicting Salmonella t. mutagenicity (Ames assay outcome) of drug impurities and other chemicals. We describe the model's chemical content and toxicity fingerprint in terms of compound space, molecular and structural toxicophores, and have rigorously tested its predictive power using both cross-validation and external validation experiments, as well as case studies. Consistent with desired regulatory use, the model performs with high sensitivity (81%) and high negative predictivity (81%) based on external validation with 2368 compounds foreign to the model and having known mutagenicity. A database of drug impurities was created from proprietary FDA submissions and the public literature which found significant overlap between the structural features of drug impurities and training set chemicals in the QSAR model. Overall, the model's predictive performance was found to be acceptable for screening drug impurities for Salmonella mutagenicity.     

Skin sensitization: modeling based on skin metabolism simulation and formation of protein conjugates

A quantitative structure-activity relationship (QSAR) system for estimating skin sensitization potency has been developed that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins. A training set of diverse chemicals was compiled and their skin sensitization potency assigned to one of three classes. These three classes were, significant, weak, or nonsensitizing. Because skin sensitization potential depends upon the ability of chemicals to react with skin proteins either directly or after appropriate metabolism, a metabolic simulator was constructed to mimic the enzyme activation of chemicals in the skin. This simulator contains 203 hierarchically ordered spontaneous and enzyme controlled reactions. Phase I and phase II metabolism were simulated by using 102 and 9 principal transformations, respectively. The covalent interactions of chemicals and their metabolites with skin proteins were described by 83 reactions that fall within 39 alerting groups. The SAR/QSAR system developed was able to correctly classify about 80% of the chemicals with significant sensitizing effect and 72% of nonsensitizing chemicals. For some alerting groups, three-dimensional (3D)-QSARs were developed to describe the multiplicity of physicochemical, steric, and electronic parameters. These 3D-QSARs, so-called pattern recognition-type models, were applied each time a latent alerting group was identified in a parent chemical or its generated metabolite(s). The concept of the mutual influence amongst atoms in a molecule was used to define the structural domain of the skin sensitization model. The utility of the structural model domain and the predictability of the model were evaluated using sensitization potency data for 96 chemicals not used in the model building. The TIssue MEtabolism Simulator (TIMES) software was used to integrate a skin metabolism simulator and 3D-QSARs to evaluate the reactivity of chemicals thus predicting their likely skin sensitization potency.     

Mutagenicity of aromatic amines and amides with chemical models for cytochrome P450 in Ames assay

Chemical models for cytochrome P450, consisting of water-insoluble or water-soluble iron porphyrin plus an oxidant, have been used to detect the mutagenicity of promutagens in genotoxicity assays. The procedure for using chemical models for cytochrome P450 as substitutes for the S9 mix in the Ames assay have been already established. Aromatic amines and amides require metabolic activation by cytochrome P450 when they exert their mutagenicity in Salmonella typhimurium strains. In this study, we optimized the conditions of the assay using a water-soluble chemical model, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron(III) pentachloride (4-MPy), plus tert-butyl hydroperoxide (t-BuOOH), magnesium monoperoxyphthalate, or iodosylbenzene, by comparing the mutagenicity of 2-aminofluorene (AF) in the Ames test. The model with 4-MPy/t-BuOOH showed the highest AF mutagenic potency. The chemical model activated 2-naphthylamine, 4-aminobiphenyl, and benzidine in S. typhimurium TA98. In aromatic amides, the model with 4-MPy/t-BuOOH weakly activated 2-acetylaminofluorene (AAF). To detect higher mutagenicity of aromatic amides, we used a higher concentration of 4-MPy/t-BuOOH by a factor of 5 over that used for aromatic amines, and then detected the mutagenicity of AAF, 2-acetylaminoanthracene, and 2-acetylamino-9-fluorenone. Furthermore, we concluded that the AAF mutagenicity in the presence of 4-MPy/t-BuOOH is derived from N-hydroxylacetylamino compounds.     

Comparative evaluation of 11 in silico models for the prediction of small molecule mutagenicity: role of steric hindrance and electron-withdrawing groups

The goal of this investigation was to perform a comparative analysis on how accurately 11 routinely-used in silico programs correctly predicted the mutagenicity of test compounds that contained either bulky or electron-withdrawing substituents. To our knowledge this is the first study of its kind in the literature. Such substituents are common in many pharmaceutical agents so there is a significant need for reliable in silico programs to predict precisely whether they truly pose a risk for mutagenicity. The predictions from each program were compared to experimental data derived from the Ames II test, a rapid reverse mutagenicity assay with a high degree of agreement with the traditional Ames assay. Eleven in silico programs were evaluated and compared: Derek for Windows, Derek Nexus, Leadscope Model Applier (LSMA), LSMA featuring the in vitro microbial Escherichia coli–Salmonella typhimurium TA102 A-T Suite (LSMA+), TOPKAT, CAESAR, TEST, ChemSilico (±S9 suites), MC4PC and a novel DNA docking model. The presence of bulky or electron-withdrawing functional groups in the vicinity of a mutagenic toxicophore in the test compounds clearly affected the ability of each in silico model to predict non-mutagenicity correctly. This was because of an over reliance on the part of the programs to provide mutagenicity alerts when a particular toxicophore is present irrespective of the structural environment surrounding the toxicophore. From this investigation it can be concluded that these models provide a high degree of specificity (ranging from 71% to 100%) and are generally conservative in their predictions in terms of sensitivity (ranging from 5% t o 78%). These values are in general agreement with most other comparative studies in the literature. Interestingly, the DNA docking model was the most sensitive model evaluated, suggesting a potentially useful new mode of screening for mutagens. Another important finding was that the combination of a quantitative structure–activity relationship and an expert rules system appeared to offer little advantage in terms of sensitivity, despite of the requirement for such a screening paradigm under the ICH M7 regulatory guideline.     

Mutagenicity evaluation of pesticide analogs using standard and 6-well miniaturized bacterial reverse mutation tests

The Ames test is widely used in the mutagenicity evaluation of new and existing chemicals as a part of a compound selection strategy, regulatory control, the equivalence assessment, carcinogenic potential measurement etc. Intensification of the chemical industry and synthesis of plenty of new molecules has led to the necessity of tests with a higher throughput capacity. The 6-well miniaturized bacterial reverse mutation test and the standard Ames test were compared using 14 technical grade active ingredients (TGAIs) of pesticides. With some exceptions, the responses obtained in the miniscreen Ames are similar to those seen in the standard method: 4 overall test outcomes were negative and 9 were positive in both test versions, but 1 discordant result between the miniscreen and standard version. Comparison of the standard and the miniscreen Ames test resulted in 98% of concordance across five strains and conditions (±S9). The overall judgment is that the miniscreen Ames test can be used to assess the mutagenicity of pesticide analogs. It has the advantage of decreasing the number of materials and animals (for S9) and keeping a high-test performance.     

N-Nitrosodiethylamine mutagenicity at low concentrations

N-Nitrosodiethylamine (NDEA) requires metabolic activation by cytochrome P450 enzymes, leading to electrophile species that react in DNA. Although, carcinogenicity is not an end point in genotoxicity assays, NDEA has been considered a weak carcinogen. In this study, we carried out an analysis of the mutagenicity at low concentrations of NDEA. Using SOS chromotest in the presence of metabolic activation, we detected positive mutagenicity response for NDEA doses between 0.75 and 36.46 microg/ml. In Ames test, using more sensitive strains in the presence of S9 metabolic activation mixture (S9 mix), positive results were also detected for NDEA doses between 1.01 x 10(-3) and 50.64 x 10(-3 microg per plate. Our results indicate that NDEA mutagenicity can be detected at low concentrations when more sensitive conditions are used.     

遗传毒性评价Ames试验方法的比较分析

Ames试验是一项体外致突变性试验,被广泛用于药物、食品、化学品和农药的遗传毒性检测,以评价受试物致突变的可能性。药物、食品、化学品和农药的指导原则和国家标准中Ames试验方法不尽相同,就这几者Ames试验方法的主要异同点进行比较分析,以期能熟练掌握Ames试验实施要点,更加规范实施检测工作,不断提高试验质量。     

An evaluation of the genotoxicity of the antitussive drug Dextromethorphan

Dextromethorphan (DMP) is an effective and widely used antitussive drug. While DMP has over a 50 year safe-marketing history, the only available genotoxicity data was an unpublished, negative Ames assay (Roche). Lack of a complete genotoxicity profile on DMP, specifically covering the chromosomal damage endpoint, prompted a regulatory request for an in vitro chromosome aberration assay. In accordance with EC and CPMP Guidance, we evaluated data for a number of chemicals with a structural relationship to DMP. DMP contains no structural alerts for genotoxicity or carcinogenicity using the Deductive Estimation of Risk from Existing Knowledge (DEREK) software tool, confirming the negative results obtained in the existing Ames assay. This is also consistent with the mostly negative genotoxicity and carcinogenicity data available on structurally related chemicals including morphine, codeine, nalbuphine, buprenorphine, naloxone, hydromorphone, levorphanol, and oxycodone. A state-of-the-science, in vitro chromosome aberration assay was also conducted, which demonstrated a lack of genotoxicity for DMP. The overall weight of evidence for DMP and its structural analogues, supports the conclusion that this class of phenanthrene-based chemicals, and DMP, in particular, are not genotoxic in vitro or in vivo, and do not represent a carcinogenic risk to patients.