ABSTRACT
Arylboronic acids and esters (referred to collectively as arylboronic compounds) are commonly used intermediates in the synthesis of pharmaceuticals but pose a challenge for chemical syntheses because they are often positive for bacterial mutagenicity in vitro. As such, arylboronic compounds are then typically controlled to levels that are acceptable for mutagenic impurities, that is, the threshold of toxicological concern (TTC). This study used ICH M7 guidance to design and conduct a testing strategy to investigate the in vivo relevance of the in vitro positive findings of arylboronic compounds. Eight arylboronic compounds representing a variety of chemical scaffolds were tested in Sprague Dawley and/or Wistar rats in the in vivo Pig-a (peripheral blood reticulocytes and mature red blood cells) and/or comet assays (duodenum and/or liver). Five of the eight compounds were also tested in the micronucleus (peripheral blood) assay. The arylboronic compounds tested orally demonstrated high systemic exposure; thus the blood and bone marrow were adequately exposed to test article. One compound was administered intravenously due to formulation stability issues. This investigation showed that arylboronic compounds that were mutagenic in vitro were not found to be mutagenic in the corresponding in vivo assays. Therefore, arylboronic compounds similar to the scaffolds tested in this article may be considered non-mutagenic and managed in accordance with the ICH Q3A/Q3B guidelines. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.
Subject(s)
Boronic Acids/toxicity , Esters/toxicity , Mutagens/toxicity , Animals , Bone Marrow/drug effects , Comet Assay/methods , Duodenum/drug effects , Erythrocytes/drug effects , Liver/diagnostic imaging , Male , Micronucleus Tests/methods , Mutagenesis/drug effects , Mutagenicity Tests/methods , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reticulocytes/drug effectsABSTRACT
Splenic tumors have been reported in rat cancer bioassays with para-chloroaniline (PCA) and aniline. Development of these tumors is hypothesized to be due to hematotoxicity via the formation of methemoglobin (MetHb) and not direct DNA reactivity. To evaluate the mode of action (MOA) for tumor formation a transgenic rodent (TGR) in vivo gene mutation assay in Big Blue® TgF344 rats was performed with parallel micronuclei analysis in peripheral blood. Male rats were gavaged daily for 28 d to 0.5, 15, and 60 mg/kg PCA and 100 mg/kg aniline, the base molecular structure of PCA. On test day 10, the 60 mg/kg PCA dose was reduced to 30 mg/kg due to toxicity. On test day 4 and 29 peripheral blood micronucleus analysis was performed and on test day 29 clinical chemistry, hematology, and MetHb measurements were taken. At study termination, on test day 31, spleen, bone marrow, and liver (control tissue) were analyzed for cII transgene mutant frequency (MF). Repeat gavage exposure to PCA and aniline for 28 d did not produce an increase in cII transgene MF in analyzed tissues. An increase in micronuclei was seen at both time points at ≥15 mg/kg PCA and 100 mg/kg aniline. At the same dose levels, significant reductions in red blood cells, increases in absolute reticulocytes (ABRET), and increased levels of MetHb were observed. Together these results support that generation of micronuclei and tumorigenicity following exposure to PCA and aniline is due to compensatory mechanisms (e.g. increased cellular turnover) and not direct DNA reactivity. Environ. Mol. Mutagen. 59:785-797, 2018. © 2018 Wiley Periodicals, Inc.
Subject(s)
Aniline Compounds/toxicity , Micronuclei, Chromosome-Defective/chemically induced , Micronucleus Tests , Mutagens/toxicity , Animals , Biomarkers , Blood Cells/drug effects , Blood Cells/metabolism , Bone Marrow/drug effects , Case-Control Studies , Dose-Response Relationship, Drug , Hematologic Tests , Liver/drug effects , Mutagenicity Tests , Mutation Rate , Rats , Spleen/drug effectsABSTRACT
Allura Red AC is an approved food color additive internationally with INS number 129, in the United States as food color subject to batch certification "Food, Drug, and Cosmetic" (FD&C) Red No. 40, and in Europe as food color additive with E number 129. In their evaluation of the color (2013), the European Food Safety Authority (EFSA) expressed concerns of potential genotoxicity, based primarily on one genotoxicity study that was not conducted according to Guidelines. The present in vivo genotoxicity study was conducted according to OECD Guidelines in response to EFSA's request for additional data. The animal species and strain, and the tissues examined were selected specifically to address the previously reported findings. The results show clear absence of genotoxic activity for Allura Red AC, in the bone marrow micronucleus assay and the Comet assay in the liver, stomach, and colon. These data addressed EFSA's concerns for genotoxicity. The Joint WHO/FAO Committee on Food Additives (JECFA) (2016) also reviewed the study and concluded that there is no genotoxicity concern for Allura Red AC. Negative findings in parallel genotoxicity studies on Tartrazine and Ponceau 4R (published separately) are consistent with lack of genotoxicity for azo dyes used as food colors.
Subject(s)
Azo Compounds/pharmacology , Food Coloring Agents/pharmacology , Animals , Comet Assay , DNA Damage/drug effects , Food Safety , Male , Mice , Mice, Inbred ICR , Micronucleus TestsABSTRACT
Tartrazine is approved as a food color additive internationally with INS number 102, in the United States as food color subject to batch certification "Food, Drug, and Cosmetic" (FD&C) Yellow No. 5, and in Europe as food color additive with E number 102. In their evaluation of the color (2013), the European Food Safety Authority (EFSA) expressed concerns of potential genotoxicity, based primarily on one genotoxicity study that was not conducted according to Guidelines. The present in vivo genotoxicity study was conducted according to OECD Guidelines in response to EFSA's request for additional data. The animal species and strain, and the tissues examined were selected specifically to address the previously reported findings. The results of this study show clear absence of genotoxic activity for Tartrazine, in the bone marrow micronucleus assay and the Comet assay in the liver, stomach, and colon. These data addressed EFSA's concerns for genotoxicity. The Joint WHO/FAO Committee on Food Additives (JECFA) (2016) also reviewed these data and concluded that there is no genotoxicity concern for Tartrazine. Negative findings in parallel genotoxicity studies on Allura Red AC and Ponceau 4R (published separately) are consistent with lack of genotoxicity for azo dyes used as food colors.
Subject(s)
Food Coloring Agents/pharmacology , Tartrazine/pharmacology , Animals , Comet Assay , DNA Damage/drug effects , Europe , Food Safety , Male , Mice , Mice, Inbred ICR , Micronucleus Tests , Mutagenicity TestsABSTRACT
In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e-cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e-cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e-cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190-198, 2017. © 2017 Wiley Periodicals, Inc.
Subject(s)
Electronic Nicotine Delivery Systems/adverse effects , Neoplasms/pathology , Animals , Carcinogens/toxicity , Cell Transformation, Neoplastic , In Vitro Techniques , MiceABSTRACT
Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Escherichia coli/drug effects , Mutagenicity Tests/methods , Mutagens/toxicity , Research Design , Salmonella typhimurium/drug effects , Control Groups , Dose-Response Relationship, Drug , Escherichia coli/genetics , Mutagenicity Tests/standards , Mutagens/chemistry , Research Design/standards , Salmonella typhimurium/geneticsABSTRACT
The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay's endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen's mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA -derived variant MT clones induced by benzo(a)pyrene.
Subject(s)
Carcinogens/toxicity , Cell Transformation, Neoplastic/genetics , Animals , Cell Line , Cricetinae , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA Methylation/drug effects , DNA Methylation/genetics , MesocricetusABSTRACT
Cigarette smoking remains a major health risk worldwide. Development of newer tobacco products requires the use of quantitative toxicological assays. Recently, v-Ha-ras transfected BALB/c3T3 (Bhas 42) cell transformation assay was established that simulates the two-stage animal tumorigenesis model and measures tumor initiating and promoting activities of chemicals. The present study was performed to assess the feasibility of using this Bhas 42 cell transformation assay to determine the initiation and promotion activities of cigarette smoke condensate (CSC) and its water soluble fraction. Further, the modulating effects of selenium and arsenic on cigarette smoke-induced cell transformation were investigated. Dimethyl sulfoxide (DMSO) and water extracts of CSC (CSC-D and CSC-W, respectively) were tested at concentrations of 2.5-40 µg mL(-1) in the initiation or promotion assay formats. Initiation protocol of the Bhas 42 assay showed a 3.5-fold increase in transformed foci at 40 µg mL(-1) of CSC-D but not CSC-W. The promotion phase of the assay yielded a robust dose response with CSC-D (2.5-40 µg mL(-1)) and CSC-W (20-40 µg mL(-1)). Preincubation of cells with selenium (100 nM) significantly reduced CSC-induced increase in cell transformation in initiation assay. Co-treatment of cells with a sub-toxic dose of arsenic significantly enhanced cell transformation activity of CSC-D in promotion assay. The results suggest a presence of both water soluble and insoluble tumor promoters in CSC, a role of oxidative stress in CSC-induced cell transformation, and usefulness of Bhas 42 cell transformation assay in comparing tobacco product toxicities and in studying the mechanisms of tobacco carcinogenesis.
Subject(s)
Arsenic/toxicity , Cell Line, Transformed/drug effects , Selenium/toxicity , Smoking/adverse effects , Toxicity Tests/methods , Animals , Carcinogenicity Tests/methods , Cell Transformation, Neoplastic/drug effects , Dimethyl Sulfoxide/chemistry , Dose-Response Relationship, Drug , MiceABSTRACT
The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.
Subject(s)
Carcinogens/analysis , Comet Assay/methods , Comet Assay/standards , DNA Damage , Animals , Europe , Guidelines as Topic , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Societies, Scientific , Stomach/drug effects , United StatesABSTRACT
The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.
Subject(s)
Carcinogens/analysis , Comet Assay/methods , Comet Assay/standards , Animals , DNA Damage , Ethyl Methanesulfonate , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Stomach/drug effectsABSTRACT
In the international validation study of the in vivo rat alkaline comet assay (comet assay), the Japanese Center for the Validation of Alternative Methods (JaCVAM) provided three coded chemicals to BioReliance, 1,3-dichloropropene, ethionamide and busulfan, to be tested in a combined in vivo comet/micronucleus assay. Induction of DNA damage (comet) in liver, stomach and jejunum (1,3-dichloropropene only) cells, and induction of MNPCEs in bone marrow, were examined in male Sprague-Dawley (Hsd:SD) rats following oral administration of the test chemical for three consecutive days. A dose range finding (DRF) test was performed with each chemical to determine the maximum tolerated dose (MTD). Based on the results of the DRF test; 1,3-dichloropropene was tested at 50, 100 and 200 mg/kg/day; ethionamide was tested at 125, 250 and 500 mg/kg/day, and busulfan was tested at 10, 20 and 40 mg/kg/day. The results indicated that 1,3-dichloropropene induced DNA damage only in liver cells at all three test article doses, while no effects were observed in the stomach and jejunum cells. Additionally, it did not increase MNPCEs in the bone marrow. 1,3-Dichloropropene was concluded to be negative in the MN assay but positive in the comet assay. Ethionamide did not induce DNA damage in liver. However, in stomach, statistically significant decreases (although still within historical range) in % tail DNA at all test article doses compared to the vehicle control were observed. There was no increase in MNPCEs in the bone marrow. Thus, ethionamide was concluded to be negative in the comet/MN combined assay. Busulfan did not induce DNA damage in any of the organs tested (liver and stomach) but it did induce a significant increase in MNPCEs in the bone marrow. Busulfan was concluded to be negative in the comet assay but positive in the MN assay.
Subject(s)
Comet Assay/methods , Micronucleus Tests/methods , Administration, Oral , Allyl Compounds/toxicity , Animals , Bone Marrow/drug effects , Busulfan/toxicity , DNA Damage/drug effects , Dose-Response Relationship, Drug , Ethionamide/toxicity , Hepatocytes/drug effects , Hydrocarbons, Chlorinated , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Stomach/drug effectsABSTRACT
The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.
Subject(s)
Carcinogenicity Tests/methods , Carcinogens/toxicity , Cell Transformation, Neoplastic , Spectroscopy, Fourier Transform Infrared , Animals , Carcinogens/classification , Embryo, Mammalian/drug effects , Embryo, Mammalian/pathology , Mesocricetus , Multivariate Analysis , Principal Component AnalysisABSTRACT
As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.
Subject(s)
Comet Assay/methods , Comet Assay/standards , DNA Damage , DNA , Animals , DNA/analysis , DNA/chemistry , DNA/isolation & purification , Education , HumansABSTRACT
As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET(CD59-) and RBC(CD59-), respectively) in peripheral blood of male Sprague Dawley(®) rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET(CD59-) and RBC(CD59-) (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.
Subject(s)
Membrane Proteins/genetics , Mutagens/toxicity , Urethane/toxicity , Animals , Cells, Cultured , Comet Assay , Male , Micronuclei, Chromosome-Defective , Micronucleus Tests , Mutagenesis , Mutation , Rats, Sprague-Dawley , Reticulocytes/drug effects , Reticulocytes/metabolism , Reticulocytes/pathologyABSTRACT
14-Hydroxycodeinone (14-HC) is an α,ß-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay.
Subject(s)
Comet Assay/methods , Oxycodone/analogs & derivatives , Administration, Oral , Animals , Chlorambucil/toxicity , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/toxicity , DNA Damage/drug effects , DNA Damage/radiation effects , Dose-Response Relationship, Drug , Hempa/toxicity , Liver/drug effects , Liver/radiation effects , Male , Mice, Inbred ICR , Mitomycin/chemistry , Mitomycin/toxicity , Oxycodone/administration & dosage , Oxycodone/chemistry , Oxycodone/toxicity , Stomach/drug effects , X-RaysABSTRACT
ToxCast is a multiyear effort to develop a cost-effective approach for the US EPA to prioritize chemicals for toxicity testing. Initial evaluation of more than 500 high-throughput (HT) microwell-based assays without metabolic activation showed that most lacked high specificity and sensitivity for detecting genotoxicants. Thus, EPA initiated a pilot project to investigate the use of standard genotoxicity endpoints using medium-throughput genotoxicity (MTG) assays in the context of a large testing program. Twenty-five chemicals were selected from the ToxCast program based in part on their known genotoxicity. The two MTG assays used were the Ames II(™) assay and 96-well In Vitro MicroFlow(®) Micronucleus (MN) assay. The Ames II assay showed a reasonable correlation with published Ames test data and industry submissions, though specificity was much better than sensitivity due to restraints on top concentrations as prescribed by ToxCast. Overall concordance was 73% both with and without metabolic activation. The flow MN assay had concordances of 71% and 58% with and without metabolic activation, respectively, when compared to published data and submissions. Importantly, a comparison of results without S9 from the MTG assays to an HT ToxCast p53 activation assay showed a fairly good degree of concordance (67%). The results reported here indicate that assays for genotoxicity endpoints can be conducted in a MT format and have the potential to add to the interpretation of results from large-scale testing programs such as EPA's ToxCast program. Inherent limitations such as the top concentrations used in large scale testing programs are discussed. Environ. Mol. Mutagen. 56:468-476, 2015. © 2014 Wiley Periodicals, Inc.
Subject(s)
Micronuclei, Chromosome-Defective/chemically induced , Mutagenicity Tests/methods , Mutagens , Salmonella typhimurium/drug effects , Animals , CHO Cells , Cricetulus , Flow Cytometry , Hep G2 Cells , Humans , Liver/drug effects , Liver/metabolism , Mutagens/chemistry , Mutagens/classification , Mutagens/toxicity , Rats , Reproducibility of Results , Salmonella typhimurium/genetics , Sensitivity and Specificity , United States , United States Environmental Protection AgencyABSTRACT
There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays.
Subject(s)
Comet Assay/methods , Lymphocytes/drug effects , Animals , Ethyl Methanesulfonate/toxicity , Female , Freezing , Humans , Male , Methyl Methanesulfonate/toxicity , Mice, Inbred ICR , Organ Specificity , Rats, Sprague-Dawley , Reproducibility of Results , Rodentia , Tail/physiologyABSTRACT
Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure-activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53-competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)-compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non-GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL-positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay.
Subject(s)
Chromosome Aberrations , Mutagenicity Tests/methods , Animals , CHO Cells , Cricetinae , Cricetulus , DNA Damage/genetics , Humans , Lymphocytes/metabolismABSTRACT
The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 µm. Resultant data were analysed using Fisher's linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.
Subject(s)
Carcinogens/classification , Cell Transformation, Neoplastic , Mutagens/classification , Animals , Biomarkers/metabolism , Carcinogens/toxicity , Cells, Cultured , Cricetinae , Data Interpretation, Statistical , Embryo, Mammalian/cytology , Hydrogen-Ion Concentration , Linear Models , Mesocricetus , Mutagenicity Tests/methods , Mutagens/toxicity , Spectroscopy, Fourier Transform InfraredABSTRACT
The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.