RESUMEN
Recently, the formation of genotoxic and carcinogenic N-nitrosamines impurities during drug manufacturing of tetrazole-containing angiotensin-II blockers has been described. However, drug-related (complex) nitrosamines may also be generated under certain conditions, i.e., through nitrosation of vulnerable amines in drug substances in the presence of nitrite. An investigation of valsartan drug substance showed that a complex API-related N-nitrosamine chemically designated as (S)-2-(((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)(nitroso)amino)-3-methylbutanoic acid (named 181-14) may be generated. 181-14 was shown to be devoid of a mutagenic potential in the Non-GLP Ames test. According to ICH M7 (R1) (2018), impurities that are not mutagenic in the Ames test would be considered Class 5 impurities and limited according to ICH Q3A (R2) and B (R2) (2006) guidelines. However, certain regulatory authorities raised the concern that the Ames test may not be sufficiently sensitive to detect a mutagenic potential of nitrosamines and requested a confirmatory in vivo study using a transgenic animal genotoxicity model. Our data show that 181-14 was not mutagenic in the transgenic gene mutation assay in MutaTMMice. The data support the conclusion that the Ames test is an adequate and sensitive test system to assess a mutagenic potential of nitrosamines.
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Mutágenos , Nitrosaminas , Animales , Daño del ADN , Ratones , Mutagénesis , Mutágenos/toxicidad , Valsartán/químicaRESUMEN
The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.
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Proteínas de la Membrana/genética , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Mutación , Reticulocitos/efectos de los fármacos , Animales , Ácidos Aristolóquicos/toxicidad , Clorambucilo/toxicidad , Eritrocitos/efectos de los fármacos , Masculino , Melfalán/toxicidad , Ratas , Ratas Wistar , Tiofenos/toxicidad , Tiotepa/toxicidad , Factores de TiempoRESUMEN
5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.
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Desoxiuridina/análogos & derivados , Proteínas de la Membrana/genética , Micronúcleo Germinal/efectos de los fármacos , Mutagénesis/genética , Animales , Antivirales/efectos adversos , Antivirales/química , Antivirales/uso terapéutico , Daño del ADN/efectos de los fármacos , Desoxiuridina/química , Desoxiuridina/farmacología , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Ratones , Mutagénesis/efectos de los fármacos , Mutágenos/efectos adversos , Mutágenos/química , Mutación/efectos de los fármacos , Nucleósidos de Pirimidina/química , Ratas , Reticulocitos/efectos de los fármacosRESUMEN
The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional 'small molecule' drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo, as this would ensure that the relevant 'free' linker forms stemming from in vivo catabolism are tested.
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Guías como Asunto , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Péptidos/toxicidad , Animales , Humanos , Mutágenos/efectos adversos , Péptidos/efectos adversos , Péptidos/uso terapéuticoRESUMEN
The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.
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Inhibidores mTOR , Sirolimus , Ratones , Animales , Síndrome , Sistema Nervioso Central/metabolismo , Encéfalo/metabolismo , Serina-Treonina Quinasas TOR , Adenosina TrifosfatoRESUMEN
The concept of thresholds in genotoxicity has been open for debate in the last decades. The micronucleus (MN) test contributed to a large extent in understanding the dose-response relationship for aneugens and clastogens. The threshold concept for aneuploidy is well accepted by the scientific community based on the data and for mechanistic reasons. The concept of threshold for clastogens is still challenging. Acceptance is based on a case-by-case basis together with thorough mechanistic understanding of the different steps from the mutagen-target interactions to MN formation for this class of genotoxicants. This review summarises the significant achievements in the assessment of threshold for genotoxins using the MN test and concludes with an overview of knowledge gaps and recommendations.
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Aneugénicos/toxicidad , Daño del ADN , Resistencia a Medicamentos , Mutágenos/toxicidad , Aneuploidia , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/ultraestructura , Pruebas de Micronúcleos , Sensibilidad y EspecificidadRESUMEN
In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug development.
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Apoptosis , Citometría de Flujo/métodos , Linfocitos/citología , Micronúcleos con Defecto Cromosómico , Adulto , Animales , Apoptosis/efectos de los fármacos , Tamaño del Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Dexametasona/farmacología , Fluoresceínas/metabolismo , Fluorescencia , Humanos , Indoles/metabolismo , Linfocitos/efectos de los fármacos , Pruebas de Micronúcleos , Persona de Mediana Edad , Ratas , Succinimidas/metabolismo , Adulto JovenRESUMEN
The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials.
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Daño del ADN , Linfocitos/efectos de los fármacos , Mutágenos/toxicidad , Piel/efectos de los fármacos , Línea Celular , Humanos , Linfocitos/ultraestructura , Pruebas de Micronúcleos , Técnicas de Cultivo de Órganos , Piel/ultraestructuraRESUMEN
The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring.
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Recuento de Células/métodos , Recuento de Células/tendencias , Citometría de Flujo/métodos , Citometría de Flujo/tendencias , Animales , Células Cultivadas , Humanos , Pruebas de Micronúcleos/tendenciasRESUMEN
Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.
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Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/tendencias , Animales , Línea Celular , Aberraciones Cromosómicas , Guías como Asunto , Humanos , Pruebas de Micronúcleos/métodos , Valor Predictivo de las PruebasRESUMEN
Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids lagging behind in anaphase and are not included in the daughter nuclei at telophase. The mechanisms of MN formation are well understood; their possible postmitotic fate is less evident. The MN assay allows detection of both aneugens and clastogens, shows simplicity of scoring, is widely applicable in different cell types, is internationally validated, has potential for automation and is predictive for cancer. The cytokinesis-block micronucleus assay (CBMN) allows assessment of nucleoplasmic bridges, nuclear buds, cell division inhibition, necrosis and apoptosis and in combination with FISH using centromeric probes, the mechanistic origin of the MN. Therefore, the CBMN test can be considered as a "cytome" assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. The toxicological relevance of the MN test is strong: it covers several endpoints, its sensitivity is high, its predictivity for in vivo genotoxicity requires adequate selection of cell lines, its statistical power is increased by the recently available high throughput methodologies, it might become a possible candidate for replacing in vivo testing, it allows good extrapolation for potential limits of exposure or thresholds and it is traceable in experimental in vitro and in vivo systems. Implementation of in vitro MN assays in the test battery for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified.
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Carcinógenos/toxicidad , Pruebas de Micronúcleos/métodos , Mutágenos/toxicidad , Animales , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Medición de Riesgo/métodosRESUMEN
Diclofenac is a non-steroidal anti-inflammatory drug discovered several decades ago, which has since been used by an estimated one billion patients and has demonstrated an acceptable safety profile. In support of its marketing approval, a comprehensive set of genotoxicity studies were conducted in vitro and in vivo. Despite the fact that these studies preceded both Good Laboratory Practice (GLP) requirements and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines on genotoxicity testing, they were conducted using the best scientific principles and are considered appropriate by contemporary standards. In addition to bacterial mutagenicity and mammalian in vitro assays, repeat-dose somatic, germ cell and dominant lethal assays were conducted. These data are made available for the first time to offer researchers an opportunity to review the existing data set that unequivocally demonstrates that diclofenac sodium is not genotoxic. This is further substantiated by long-term bioassay data demonstrating that diclofenac sodium has no carcinogenic potential in rodents. However, more recently, new studies have been published showing a genotoxic potential for diclofenac in novel or modified in vitro test systems. These new publications are discussed in the context of the existing comprehensive data package.
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Diclofenaco/toxicidad , Animales , Carcinógenos/toxicidad , Línea Celular Tumoral , Cricetulus , Femenino , Células Germinativas/efectos de los fármacos , Masculino , Mamíferos , Ratones , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , RatasRESUMEN
The following reference genotoxic agents were tested in the in vitro micronucleus test, at Novartis, Basel, Switzerland. Mitomycin C, 5-fluoruracil, colchicine and etoposide were tested in the human lymphoblastoid cell line TK6, with and without cytokinesis block (in the presence of cytochalasin B). This was done in support of the toxicity measures recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) and was part of an international collaborative work. As toxicity measures, detecting cytostasis and cell death, relative cell counts (RCC), relative increase in cell counts (RICC), and relative population doubling (RPD) were used for treatments in the absence of cytokinesis block, and replication index (RI) or cytokinesis-blocked proliferation in the presence of cytokinesis block. All four reference agents were positive in the assay with and without cytokinesis block at concentrations giving approximately 50% toxicity or less as assessed by all of the toxicity measures used. Accordingly, the results of this work support the use of relative population doubling and relative increase in cell counts, as well as relative cell counts, as appropriate measures of toxicity for the non-cytokinesis-blocked in vitro micronucleus assay.
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Pruebas de Micronúcleos/métodos , Mutágenos/toxicidad , Recuento de Células , Línea Celular , Proliferación Celular , Colchicina/toxicidad , Citocalasina B/farmacología , Citocinesis , Etopósido/toxicidad , Fluorouracilo/toxicidad , Guías como Asunto , Humanos , Linfocitos/efectos de los fármacos , Pruebas de Micronúcleos/normas , Mitomicina/toxicidad , SuizaRESUMEN
The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the requirements for OECD approval are demonstrations of assay reliability, including reproducibility within and among laboratories. Experiments reported herein address the reproducibility of the rat blood Pig-a assay using the reference mutagens chlorambucil and melphalan. These agents were evaluated for their ability to induce Pig-a mutant erythrocytes in three separate studies conducted across two laboratories. Each of the studies utilized a common treatment schedule: 28 consecutive days of exposure via oral gavage. Whereas one laboratory studied Crl:CD(SD) rats, the other laboratory used Wistar Han rats. One or two days after cessation of treatment blood samples were collected for mutant reticulocyte and mutant erythrocyte measurements that were accomplished with the same analytical technique whereby samples were depleted of wildtype erythrocytes via immunomagnetic separation followed by flow cytometric enumeration of mutant phenotype cells (MutaFlow®). Dunnett's test results showed similar qualitative outcomes within and between laboratories, that is, each chemical and each study demonstrated statistically significant, dose-related increases in mutant reticulocyte and erythrocyte frequencies. Benchmark dose analysis (PROAST software) provided a means to quantitatively analyze the results, and the relatively tight, overlapping benchmark dose confidence intervals observed for each of the two chemicals indicate that within and between laboratory reproducibility of the Pig-a assay are high, adding further support for the development of an OECD test guideline.
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Bioensayo/métodos , Laboratorios , Mutación/genética , Animales , Clorambucilo/farmacología , Eritrocitos/efectos de los fármacos , Masculino , Melfalán/farmacología , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Reticulocitos/efectos de los fármacosRESUMEN
In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.
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Rutas de Resultados Adversos , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Aneuploidia , Animales , Aurora Quinasa A/antagonistas & inhibidores , Rotura Cromosómica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Humanos , Pruebas de Mutagenicidad/métodos , Mutación/efectos de los fármacosRESUMEN
Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retro-orbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50mg/kg methyl methanesulfonate (MMS) once by gavage 4h before blood sampling. Another group (six animals) was treated twice orally with 10mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 microg/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats.
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Daño del ADN , Formaldehído/toxicidad , Mutágenos/toxicidad , Animales , Sangre , Ensayo Cometa , Exposición por Inhalación/efectos adversos , Masculino , Pruebas de Micronúcleos , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Intercambio de Cromátides HermanasRESUMEN
In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict the mutagenic and carcinogenic potential of compounds for regulatory purposes and/or to follow-up positive results from in vitro testing. These tests are widely used and consume large numbers of animals, with a foreseeable marked increase as a result of the EU chemicals legislation (REACH), which may require follow-up of any positive outcome in the in vitro standard battery with appropriate in vivo tests, regardless of the tonnage level of the chemical. A 2-day workshop with genotoxicity experts from academia, regulatory agencies and industry was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) in Ranco, Italy from 24 to 25 June 2008. The objectives of the workshop were to discuss how to reduce the number of animals in standard genotoxicity tests, whether the application of smarter test strategies can lead to lower animal numbers, and how the possibilities for reduction can be promoted and implemented. The workshop agreed that there are many reduction options available that are scientifically credible and therefore ready for use. Most of these are compliant with regulatory guidelines, i.e. the use of one sex only, one administration and two sampling times versus two or three administrations and one sampling time for micronucleus (MN), chromosomal aberration (CA) and Comet assays; and the integration of the MN endpoint into repeat-dose toxicity studies. The omission of a concurrent positive control in routine CA and MN tests has been proven to be scientifically acceptable, although the OECD guidelines still require this; also the combination of acute MN and Comet assay studies are compliant with guidelines, except for sampling times. Based on the data presented at the workshop, the participants concluded that these options have not been sufficiently utilized to date. Key factors for this seem to be the uncertainty regarding regulatory compliance/acceptance, lack of awareness, and an in many cases unjustified uncertainty regarding the scientific acceptance of reduction options. The workshop therefore encourages the use and promotion of these options as well as the dissemination of data related to reduction opportunities by the scientific community in order to boost the acceptance level of these approaches. Furthermore, experimental proof is needed and under way to demonstrate the credibility of additional options for reduction of the number of animals, such as the integration of the Comet assay into repeat-dose toxicity studies.
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Alternativas a las Pruebas en Animales/legislación & jurisprudencia , Bienestar del Animal/legislación & jurisprudencia , Mutágenos/toxicidad , Proyectos de Investigación/legislación & jurisprudencia , Pruebas de Toxicidad , Animales , Daño del ADN , Unión Europea , Femenino , Agencias Gubernamentales , Masculino , Pruebas de Mutagenicidad/normas , Mutágenos/clasificación , Proyectos de Investigación/normas , Pruebas de Toxicidad/ética , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
During the last two decades the micronucleus (MN) test has been extensively used as a genotoxicity screening tool of chemicals and in a variety of exploratory and mechanistic investigations. The MN is a biomarker for chromosomal damage or mitotic abnormalities since it can originate from chromosome fragments or whole chromosomes that fail to be incorporated into daughter nuclei during mitosis (Fenech et al., Mutagenesis 26: 125-132, 2011; Kirsch-Volders et al., Arch Toxicol 85: 873-899, 2011). The simplicity of scoring, accuracy, amenability to automation by image analysis or flow cytometry and the readiness to be applied to a variety of cell types either in vitro or in vivo made it a versatile tool that contributed to a large extent in our understanding of key toxicological issues related to genotoxins and their effects at the cellular and organism levels. Recently, the final acceptance of the in vitro MN test Organization for Economic Cooperation and Development (OECD) guideline 487 (OECD, Guideline for testing of chemicals: in vitro mammalian cell micronucleus test 487: in vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris, 2010) together with the standard in vivo MN test OECD guideline 474 (OECD, Guideline for the testing of chemicals no. 474 mammalian erythrocyte micronucleus test. Organization for Economic Cooperation and Development, Paris, 1997) further positioned the assay as a key driver in the determination of the genotoxicity potential in exploratory research as well as in the regulatory environment. This book chapter covers to some extent the protocol designs and experimental steps necessary for a successful performance of the MN test and an accurate analysis of the MN by the flow cytometry technique.
Asunto(s)
Citometría de Flujo/métodos , Pruebas de Micronúcleos/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Cultivadas , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Mutágenos/toxicidadRESUMEN
A database of the micronuclei counts was built up for historical negative control data from human lymphocyte in vitro micronuclei tests (MnVit) carried out in 8 laboratories with experience of the method. The mean incidence of micronucleated cells (mnt)/1000 cells ranged from 2.2/1000 to 15.9/1000. There were no large differences in incidence between the presence or absence of S9 mix or between different treatment lengths. There was also little evidence that different solvents affected the numbers of micronuclei appreciably. A number of laboratories did show significant inter-experiment variability, indicating that there remained unidentified factors affecting frequencies. Donor variance may be one such factor. Inter-individual variability may explain some of these differences. The approximate 7.5-fold difference in mnt/1000 scores in a relatively small group of experienced laboratories illustrates the potential complications that can arise if a metric like a fold increase was considered the only biologically important finding. Although there is inherent variability between experiments, it was evident that within a laboratory the overall laboratory mean remains constant over time. It is believed that these findings will provide help to laboratories conducting studies using human lymphocytes in the MnVit and to those involved in the assessment of MnVit results.