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1.
Transfusion ; 64(6): 1097-1108, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38716879

RESUMEN

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.


Asunto(s)
Pruebas de Mutagenicidad , Ratas Sprague-Dawley , Animales , Masculino , Ratas , Pruebas de Mutagenicidad/métodos , Antígenos CD59/genética , Reticulocitos/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Proteínas de la Membrana/genética , Mutagénesis/efectos de los fármacos , Mutágenos/toxicidad
2.
Altern Lab Anim ; 51(1): 55-79, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36821083

RESUMEN

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Productos de Tabaco , Nicotiana/toxicidad , Productos de Tabaco/toxicidad , Nicotina/toxicidad , Aerosoles/toxicidad , Técnicas In Vitro
3.
Regul Toxicol Pharmacol ; 136: 105263, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36228836

RESUMEN

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).


Asunto(s)
Nanopartículas del Metal , Reproducibilidad de los Resultados , Nanopartículas del Metal/química , Titanio/toxicidad , Titanio/química , Ensayo Cometa , Daño del ADN , Mutágenos/toxicidad , ADN
4.
Mutagenesis ; 30(3): 335-42, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25934985

RESUMEN

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.


Asunto(s)
Proteínas de la Membrana/genética , Mutágenos/toxicidad , Uretano/toxicidad , Animales , Células Cultivadas , Ensayo Cometa , Masculino , Micronúcleos con Defecto Cromosómico , Pruebas de Micronúcleos , Mutagénesis , Mutación , Ratas Sprague-Dawley , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Reticulocitos/patología
5.
Drug Test Anal ; 15(10): 1175-1188, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35830202

RESUMEN

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Productos de Tabaco , Nicotiana , Aerosoles , Técnicas In Vitro
6.
Environ Mol Mutagen ; 62(3): 227-237, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33608913

RESUMEN

The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig-a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described.


Asunto(s)
Pruebas de Mutagenicidad , Mutágenos/farmacología , Mutación/genética , Reticulocitos/efectos de los fármacos , Animales , Bioensayo , Citometría de Flujo , Masculino , Mutágenos/toxicidad , Ratas , Reticulocitos/patología , Roedores/genética
7.
Artículo en Inglés | MEDLINE | ID: mdl-32087853

RESUMEN

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.


Asunto(s)
Escherichia coli/efectos de los fármacos , Mutagénesis , Pruebas de Mutagenicidad/normas , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Animales , Bancos de Muestras Biológicas/organización & administración , Bases de Datos de Compuestos Químicos/provisión & distribución , Escherichia coli/genética , Guías como Asunto , Humanos , Cooperación Internacional , Mutágenos/clasificación , Salmonella typhimurium/genética , Tokio
8.
Artículo en Inglés | MEDLINE | ID: mdl-31708075

RESUMEN

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to seek consensus on difficult or conflicting approaches to genotoxicity testing based upon experience, available data, and analysis techniques. At the 2017 IWGT meeting in Tokyo, one working group addressed the sensitivity and selectivity of the bacterial strains specified in the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471 to recommend possible modification of the test guideline. Three questions were posed: (1) Although TA100 is derived from TA1535, does TA1535 detect any mutagens that are not detected by TA100? (2) Among the options of Salmonella TA1537, TA97 or TA97a, are these strains truly equivalent? (3) Because there is a choice to use one of either E. coli WP2 uvrA, E. coli WP2 uvrA pKM101, or Salmonella TA102, are these strains truly equivalent? To answer these questions, we analyzed published bacterial mutation data in multiple strains from large (>10,000 compound) databases from Leadscope and Lhasa Limited and anonymized data for 53 compounds tested in TA1535 and TA100 provided by a pharmaceutical company. Our analysis involved (1) defining criteria for determining selective responses when using different strains; (2) identifying compounds producing selective responses based upon author calls; (3) confirming selective responses by visually examining dose-response data and considering experimental conditions; (4) using statistical methods to quantify the responses; (5) performing limited additional direct-comparison testing; and (6) determining the chemical classes producing selective responses. We found that few mutagens would fail to be detected if the test battery did not include Salmonella strains TA1535 (8/1167), TA1537 (2/247), TA102 (4/46), and E. coli WP2 uvrA (2/21). Of the mutagens detected by the full TG471 strain battery, 93% were detected using only strains TA98 and TA100; consideration of results from in vitro genotoxicity assays that detect clastogenicity increased this to 99%.


Asunto(s)
Guías como Asunto , Pruebas de Mutagenicidad/normas , Escherichia coli/genética , Salmonella/genética
9.
Artículo en Inglés | MEDLINE | ID: mdl-30744809

RESUMEN

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.


Asunto(s)
Médula Ósea/efectos de los fármacos , Colon/efectos de los fármacos , Ensayo Cometa/métodos , Hígado/efectos de los fármacos , Mutágenos/toxicidad , Mutación , Estómago/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Daño del ADN , Femenino , Masculino , Ratones , Pruebas de Micronúcleos , Ratas
10.
Toxicol Sci ; 164(2): 391-416, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29701824

RESUMEN

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.


Asunto(s)
Pruebas de Mutagenicidad/métodos , Nanoestructuras/toxicidad , Animales , Aberraciones Cromosómicas , Ensayo Cometa , Humanos , Técnicas In Vitro/métodos , Pruebas de Mutagenicidad/normas , Mutación
11.
Artículo en Inglés | MEDLINE | ID: mdl-29307374

RESUMEN

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.


Asunto(s)
Núcleo Celular/genética , Proyectos de Investigación/normas , Línea Celular , Humanos , Micronúcleos con Defecto Cromosómico , Pruebas de Micronúcleos , Control de Calidad
12.
Mutat Res ; 627(1): 36-40, 2007 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-17157054

RESUMEN

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe and the United States, met on September 9, 2005, in San Francisco, CA, USA. This meeting of the MLA Workgroup was devoted to reaching a consensus on issues involved with 24-h treatment. Recommendations were made concerning the acceptable values for the negative/solvent control (mutant frequency, cloning efficiency and suspension growth) and the criteria to define an acceptable positive control response. Consensus was also reached concerning the use of the global evaluation factor (GEF) and appropriate statistical trend analysis to define positive and negative responses for the 24-h treatment. The Workgroup agreed to continue their support of the International Committee on Harmonization (ICH) recommendation that the MLA assay should include a 24-h treatment (without S-9) in those situations where the short treatment (3-4 h) gives negative results.


Asunto(s)
Linfoma/genética , Pruebas de Mutagenicidad/métodos , Mutación , Timidina Quinasa/genética , Animales , Ratones , Mutágenos/toxicidad , Factores de Tiempo
13.
Environ Mol Mutagen ; 47(1): 1-5, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15991242

RESUMEN

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).


Asunto(s)
Bioensayo/normas , Pruebas de Mutagenicidad/normas , Timidina Quinasa/genética , Animales , Linfoma/enzimología , Linfoma/genética , Ratones , Mutación
14.
Environ Mol Mutagen ; 57(1): 17-27, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26407646

RESUMEN

During interlaboratory validation trials for the Pig-a gene mutation assay we assessed the genotoxicity of 4-nitroquinoline-1-oxide (4NQO) across endpoints in multiple tissues: induction of Pig-a mutant red blood cells (RBCs) and reticulocytes (RETs); micronucleated RETs (MN RETs); and DNA damage in blood and liver via the alkaline Comet assay (%tail intensity [TI]). In a previous subchronic toxicity study with 28 daily doses, biologically meaningful increases were observed only for Pig-a mutant RBCs/RETs while marginal increases in the frequency of MN RET were observed, and other clastogenic endpoints were negative. Follow up acute studies were performed using the same cumulative doses (0, 35, 70, 105, and 140 mg/kg) administered in a bolus, or split over three equal daily doses, with samples collected up to 1 month after the last dose. Both of the acute dosing regimens produced similar results, in that endpoints were either positive or negative, regardless of 1 or 3 daily doses, but the three consecutive daily dose regimen yielded more potent responses in TI (in liver and blood) and Pig-a mutant frequencies. In these acute studies the same cumulative doses of 4NQO induced positive responses in clastogenic endpoints that were negative or inconclusive using a subchronic study design. Additionally, a positive control group using combination doses of cyclophosphamide and ethyl methanesulfonate was employed to assess assay validity and potentially identify a future positive control treatment for integrated genetic toxicity studies.


Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Pruebas de Toxicidad Aguda , Pruebas de Toxicidad Subcrónica , 4-Nitroquinolina-1-Óxido/administración & dosificación , Administración Oral , Animales , Ensayo Cometa/métodos , Análisis Mutacional de ADN/métodos , Masculino , Pruebas de Micronúcleos/métodos , Pruebas de Mutagenicidad/métodos , Mutágenos/administración & dosificación , Mutación , Ratas , Pruebas de Toxicidad Aguda/métodos , Pruebas de Toxicidad Subcrónica/métodos
15.
Environ Mol Mutagen ; 56(5): 468-76, 2015 06.
Artículo en Inglés | MEDLINE | ID: mdl-25537651

RESUMEN

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.


Asunto(s)
Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de Mutagenicidad/métodos , Mutágenos , Salmonella typhimurium/efectos de los fármacos , Animales , Células CHO , Cricetulus , Citometría de Flujo , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Mutágenos/química , Mutágenos/clasificación , Mutágenos/toxicidad , Ratas , Reproducibilidad de los Resultados , Salmonella typhimurium/genética , Sensibilidad y Especificidad , Estados Unidos , United States Environmental Protection Agency
16.
Artículo en Inglés | MEDLINE | ID: mdl-25953398

RESUMEN

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Although the assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans, currently it is optimized only for measuring CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. An expert workgroup formed by the International Workshop on Genotoxicity Testing considered the state of assay development and the potential of the assay for regulatory use. Consensus was reached on what is known about the Pig-a assay and how it should be conducted, and recommendations were made on additional data and refinements that would help to further enhance the assay for use in hazard identification and risk assessment.


Asunto(s)
Anemia Hemolítica , Eritrocitos , Citometría de Flujo , Hemoglobinuria , Proteínas de la Membrana , Mutación , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patología , Animales , Anticuerpos/química , Educación , Eritrocitos/metabolismo , Eritrocitos/patología , Citometría de Flujo/métodos , Citometría de Flujo/normas , Hemoglobinuria/metabolismo , Hemoglobinuria/patología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratas
17.
Environ Mol Mutagen ; 56(3): 277-85, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25482136

RESUMEN

Genetic toxicity tests currently used to identify and characterize potential human mutagens and carcinogens rely on measurements of primary DNA damage, gene mutation, and chromosome damage in vitro and in rodents. The International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI) Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing held an April 2012 Workshop in Washington, DC, to consider the impact of new understanding of biology and new technologies on the identification and characterization of genotoxic substances, and to identify new approaches to inform more accurate human risk assessment for genetic and carcinogenic effects. Workshop organizers and speakers were from industry, academe, and government. The Workshop focused on biological effects and technologies that would potentially yield the most useful information for evaluating human risk of genetic damage. Also addressed was the impact that improved understanding of biology and availability of new techniques might have on genetic toxicology practices. Workshop topics included (1) alternative experimental models to improve genetic toxicity testing, (2) Biomarkers of epigenetic changes and their applicability to genetic toxicology, and (3) new technologies and approaches. The ability of these new tests and technologies to be developed into tests to identify and characterize genotoxic agents; to serve as a bridge between in vitro and in vivo rodent, or preferably human, data; or to be used to provide dose response information for quantitative risk assessment was also addressed. A summary of the workshop and links to the scientific presentations are provided.


Asunto(s)
Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Animales , District of Columbia , Epigénesis Genética/efectos de los fármacos , Genómica/métodos , Humanos , Medición de Riesgo
18.
Environ Mol Mutagen ; 40(4): 292-9, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12489120

RESUMEN

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytotoxicity measures and their determination, 2) use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4) concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry. Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and soft agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft agar or microwell version of the assay was used. In the absence of sufficient data to make a definitive determination, the Workgroup continued to endorse the International Committee on Harmonization recommendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection.


Asunto(s)
Linfoma/enzimología , Pruebas de Mutagenicidad/métodos , Mutación , Timidina Quinasa/genética , Animales , Educación , Guías como Asunto , Ratones , Factores de Tiempo
19.
Mutat Res ; 540(2): 127-40, 2003 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-14550497

RESUMEN

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.


Asunto(s)
Bioensayo/normas , Linfoma/metabolismo , Timidina Quinasa/análisis , Animales , Ratones , Pruebas de Mutagenicidad/normas
20.
Environ Mol Mutagen ; 52(9): 738-47, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22020836

RESUMEN

As part of the Stage III Pig-a multilaboratory validation trial, we examined the induction of CD59-negative reticulocytes and total red blood cells (RET(CD59-) and RBC(CD59-) , respectively) in male Sprague Dawley(®) rats treated with 4-nitroquinoline-1-oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg(-1) day(-1) (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg(-1) day(-1) . The largest increases observed for the genetic toxicology endpoints (fold-increase compared to control, where significant; all at 5.00 mg kg(-1) day(-1) on Day 29) were: RET(CD59-) (21X), RBC(CD59-) (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies.


Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Aberraciones Cromosómicas/inducido químicamente , Proteínas de la Membrana/genética , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/ultraestructura , Antígenos CD59/genética , Calibración , Ensayo Cometa/métodos , Ensayo Cometa/normas , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Laboratorios/normas , Hígado/efectos de los fármacos , Hígado/ultraestructura , Masculino , Pruebas de Micronúcleos/métodos , Pruebas de Micronúcleos/normas , Pruebas de Mutagenicidad/métodos , Pruebas de Mutagenicidad/normas , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los Resultados , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Reticulocitos/ultraestructura , Medición de Riesgo , Estómago/efectos de los fármacos , Estómago/ultraestructura , Factores de Tiempo
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