DOCSLIB.ORG

SCIENTIFIC OPINION Draft Guidance on Aneugenicity Assessment

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SCIENTIFIC OPINION Draft Guidance on Aneugenicity Assessment SCIENTIFIC OPINION ENDORSED: 20 February 2020 doi:10.2903/j.efsa.2020.eNNNNN Draft guidance on aneugenicity assessment EFSA Scientific Committee (SC) Simon John More, Vasileios Bampidis, Claude Bragard, Thorhallur Ingi Halldorsson, Antonio F. Hernández-Jerez, Susanne Hougaard Bennekou, Kostas Koutsoumanis, Kyriaki Machera, Hanspeter Naegeli, Søren Saxmose Nielsen, Josef Schlatter, Dieter Schrenk, Vittorio Silano, Dominique Turck, Maged Younes, Gabriele Aquilina, Margherita Bignami, Claudia Bolognesi, Riccardo Crebelli, Rainer Gürtler, Francesca Marcon, Elsa Nielsen, Christiane Vleminckx, Maria Carfì, Carla Martino, Daniela Maurici, Juan Parra Morte, Annamaria Rossi and Diane Benford Abstract The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that exhibit aneugenicity but do not induce gene mutation or clastogenicity. The Scientific Committee confirmed that the preferred approach is to perform an in vivo bone marrow micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, needs to be validated to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be drafted for substances that are aneugenic but do not induce gene mutations or clastogenicity, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to set up health-based guidance values, some approaches to risk assessment are proposed such as calculating the margin of exposure between a relevant reference point and the estimated human dietary exposure. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment. © 20YY European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords aneugenicity, micronucleus test, genotoxicity in vivo and in vitro www.efsa.europa.eu/efsajournal 1 EFSA Journal 20YY;volume(issue):eNNNNN SCIENTIFIC OPINION ENDORSED: 20 February 2020 doi:10.2903/j.efsa.2020.eNNNNN Requestor: European Food Safety Authority Question number: EFSA-Q-2019-00263 Correspondence:[email protected] www.efsa.europa.eu/efsajournal 2 EFSA Journal 20YY;volume(issue):eNNNNN 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Panel members: Simon John More, Diane Benford, Vasileios Bampidis, Claude Bragard, Thorhallur Ingi 18 Halldorsson, Antonio F. Hernández-Jerez, Susanne Hougaard Bennekou, Kostas Koutsoumanis, Kyriaki 19 Machera, Hanspeter Naegeli, Søren Saxmose Nielsen, Josef Schlatter, Dieter Schrenk, Vittorio Silano, 20 Dominique Turck, and Maged Younes. 21 Acknowledgements: The EFSA Scientific Committee wishes to acknowledge the following hearing experts 22 for the views provided to this scientific output: Roland Froetschl, Uta Herbst, David Kirkland, Wim Mennes, 23 Christina Tlustos; the observers Raffaella Corvi (Directorate General - Joint Research Centre) and Frank Le 24 Curieux (European Chemicals Agency - ECHA); the EFSA staff Esraa Elewa, Paola Manini, and Fabiola Pizzo. 25 Suggested citation: EFSA Scientific Committee, More SJ, Bampidis V, Bragard C, Halldorsson TI, 26 Hernández-Jerez AF, Hougaard Bennekou S, Koutsoumanis K, Machera K, Naegeli H, Nielsen SS, Schlatter 27 J, Schrenk D, Silano V, Turck D, Younes M, Aquilina G, Bignami M, Bolognesi C, Crebelli R, Gürtler R, Marcon 28 F, Nielsen E, Vleminckx C, Carfì M, Martino C, Maurici D, Parra Morte J, Rossi A, and Benford D, 2020. Draft 29 Guidance on aneugenicity assessment, scientific guidance. EFSA Journal 2020;volume(issue):NNNN, XXX 30 pp. doi:10.2903/j.efsa.2020.NNNN 31 ISSN: XXXX-XXXX 32 © 20YY European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of 33 European Food Safety Authority. 34 Reproduction of the images listed below is prohibited and permission must be sought directly from the 35 copyright holder: Figure 2: © Elsevier 36 www.efsa.europa.eu/efsajournal 3 EFSA Journal 20YY;volume(issue):eNNNNN 37 TABLE OF CONTENTS 38 Abstract ................................................................................................................................... 1 39 Keywords ................................................................................................................................. 1 40 1. Background and Terms of Reference as provided by EFSA......................................................... 6 41 1.1. Audience and degree of obligation ................................................................................. 7 42 2. Introduction....................................................................................................................... 7 43 3. In vivo follow-up of in vitro aneugens..................................................................................... 8 44 4. Evidence for thresholds in aneugenicity ................................................................................11 45 4.1. Theoretical considerations ...........................................................................................11 46 4.2. Threshold in aneuploidy – empirical evidence .................................................................11 47 5. Risk assessment of substances that are aneugenic but not clastogenic ......................................12 48 5.1. Setting up reference points for aneugenicity ...................................................................12 49 5.2. Hazard characterisation for substances that are aneugenic but not clastogenic....................13 50 5.2.1. Data-rich substances, in vivo dose–response data are available for aneugenicity ...........14 51 5.2.2. Data-rich substances, in vivo data are not sufficient to set up a dose–response 52 relationship for aneugenicity ...............................................................................................14 53 5.2.3. Data-poor substances, i.e. gaps in the toxicological database .....................................14 54 5.3. Risk characterisation for substances that are aneugenic but not clastogenic ........................14 55 5.4. Risk assessment for combined exposure to multiple substances that are aneugenic but not 56 clastogenic ...........................................................................................................................15 57 6. Conclusions.......................................................................................................................16 58 6.1. Question 1: What is the most appropriate in vivo follow-up for substances that are aneugenic 59 in vitro? ...............................................................................................................................16 60 6.2. Question 2: How should risk to human health be assessed for a substance exhibiting 61 aneugenicity? .......................................................................................................................16 62 7. Recommendations .............................................................................................................17 63 8. References........................................................................................................................19 64 Annex A. Methods to detect aneuploidy.......................................................................................22 65 A.1. Detection of aneuploidy in mitotic cells ..............................................................................22 66 A.2. Detection of aneuploidy in interphase cells .........................................................................22 67 A.3. Micronucleus assay techniques in tissues other than bone marrow and peripheral blood ..........24 68 Annex A. References ..............................................................................................................26 69 Annex B. Examples of active substances (possibly) causing aneugenic effects ....................................30 www.efsa.europa.eu/efsajournal 4 EFSA Journal 20YY;volume(issue):eNNNNN 70 Annex C. List of Abbreviations.....................................................................................................34 71 72 www.efsa.europa.eu/efsajournal 5 EFSA Journal 20YY;volume(issue):eNNNNN 73 1. Background and Terms of Reference as provided by EFSA 74 The genotoxicity testing strategy 1 indicated in the EFSA Scientific Committee opinion is designed to 75 investigate the genotoxic potential of substances through the detection of three genotoxic endpoints: gene 76 mutations, structural chromosomal aberrations (i.e. clastogenicity) and numerical
Load more

Recommended publications
  • Comet Assay Modifications for Its Application in Food Safety
    Facultad de Farmacia y Nutrición Comet assay modifications for its application in food safety Modificaciones del ensayo del cometa para su aplicación en seguridad alimentaria José Manuel Enciso Gadea Facultad de Farmacia y Nutrición El presente trabajo de investigación titulado: Comet assay modifications for its application in food safety Modificaciones del ensayo del cometa para su aplicación en seguridad alimentaria que presenta D. José Manuel Enciso Gadea para aspirar al grado de Doctor por la Universidad de Navarra en el Programa de Doctorado en Alimentación, Fisiología y Salud, ha sido realizado en el Departamento de Farmacología y Toxicología de la Facultad de Farmacia y Nutrición, bajo la dirección de la Dra. Adela López de Cerain Salsamendi y la co-dirección de la Dra. Amaya Azqueta Oscoz y la Dra. Ariane Vettorazzi Armental. Pamplona, 8 de febrero de 2018 Dra. Amaya Azqueta Oscoz Dra. Ariane Vettorazzi Armental Dra. Adela López de Cerain Salsamendi This work received financial support from: The “Asociación de Amigos” of the University of Navarra (Predoctoral grant). Ministry of Economy, Industry and Competitiveness (Spain). BIOGENSA Project: “Aplicación de una nueva estrategia de evaluación de genotoxicidad en ingredientes funcionales y en frituras de restauración colectiva” (AGL2015-70640-R) University of Navarra (PIUNA). Project: “Efecto cancerígeno de la ocratoxina A: influencia del sexo en el mecanismo de acción” [PIUNA 2012]. Acknowledgements / Agradecimientos Con estas breves palabras deseo expresar mi más sincero agradecimiento a todas aquellas personas que, de una manera u otra, me han ayudado a que esta tesis doctoral sea una realidad. En primer lugar, a la Universidad de Navarra, por haberme formado tanto en el plano intelectual como en el humano, y la Asociación de Amigos de la misma, por haberme dotado con los medios económicos para la consecución de este proyecto.
    [Show full text]
  • Principles and Methods for the Risk Assessment of Chemicals in Food
    WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE EHC240: Principles and Methods for the Risk Assessment of Chemicals in Food SUBCHAPTER 4.5. Genotoxicity Draft 12/12/2019 Deadline for comments 31/01/2020 The contents of this restricted document may not be divulged to persons other than those to whom it has been originally addressed. It may not be further distributed nor reproduced in any manner and should not be referenced in bibliographical matter or cited. Le contenu du présent document à distribution restreinte ne doit pas être divulgué à des personnes autres que celles à qui il était initialement destiné. Il ne saurait faire l’objet d’une redistribution ou d’une reproduction quelconque et ne doit pas figurer dans une bibliographie ou être cité. Hazard Identification and Characterization 4.5 Genotoxicity ................................................................................. 3 4.5.1 Introduction ........................................................................ 3 4.5.1.1 Risk Analysis Context and Problem Formulation .. 5 4.5.2 Tests for genetic toxicity ............................................... 14 4.5.2.2 Bacterial mutagenicity ............................................. 18 4.5.2.2 In vitro mammalian cell mutagenicity .................... 18 4.5.2.3 In vivo mammalian cell mutagenicity ..................... 20 4.5.2.4 In vitro chromosomal damage assays .................. 22 4.5.2.5 In vivo chromosomal damage assays ................... 23 4.5.2.6 In vitro DNA damage/repair assays ....................... 24 4.5.2.7 In vivo DNA damage/repair assays ....................... 25 4.5.3 Interpretation of test results ......................................... 26 4.5.3.1 Identification of relevant studies............................. 27 4.5.3.2 Presentation and categorization of results ........... 30 4.5.3.3 Weighting and integration of results .....................
    [Show full text]
  • Flow Micronucleus, Ames II, Greenscreen and Comet June 28, 2012 EPA Computational Toxicology Communities of Practice
    State of the Art High-throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet June 28, 2012 EPA Computational Toxicology Communities of Practice Dr. Marilyn J. Aardema Chief Scientific Advisor, Toxicology Dr. Leon Stankowski Principal Scientist/Program Consultant Ms. Kamala Pant Principal Scientist Helping to bring your products from discovery to market Agenda 11am-12 pm 1. Introduction Marilyn Aardema 5 min 2. In Vitro Flow Micronucleus Assay - 96 well Leon Stankowski, 10 min 3. Ames II Assay Kamala Pant 10 min 4. GreenScreen Assay Kamala Pant 10 min 5. In Vitro Comet Assay - 96 well TK6 assay Kamala Pant 10 min 6. Questions/Discussion 15 min 2 Genetic Toxicology Testing in Product Development Discovery/Prioritization Structure activity relationship analyses useful in very early lead identification High throughput early screening assays Lead Optimization Screening versions of standard assays to predict results of GLP assays GLP Gate Perform assays for regulatory GLP Gene Tox Battery submission according to regulatory guidelines Follow-up assays Additional supplemental tests to to solve problems investigate mechanism and to help characterize human risk 3 High Throughput Genotoxicity Assays • Faster • Cheaper • Uses Less Chemical/Drug • Non-GLP • Predictive of GLP assay/endpoint • Mechanistic Studies (large number of conc./replicates) • Automation 4 Example of Use of Genotoxicity Screening Assays: EPA ToxCast™ Problem: Tens of thousands of poorly characterized environmental chemicals Solution: ToxCast™– US EPA program intended to use: – High throughput screening – Genomics – Computational chemistry and computational toxicology To permit: – Prediction of potential human toxicity – Prioritization of limited testing resources www.epa.gov/ncct/toxcast 5 5 BioReliance EPA ToxCast Award July 15, 2011 • Assays – In vitro flow MN – In vitro Comet – Ames II – GreenScreen • 25 chemicals of known genotoxicity to evaluate the process/assays (April-June 2012) – In vitro flow MN – In vitro Comet – Ames II 6 6 Agenda 11am-12 pm 1.
    [Show full text]
  • In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin
    CORE Metadata, citation and similar papers at core.ac.uk Provided by idUS. Depósito de Investigación Universidad de Sevilla toxins Article In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin Leticia Díez-Quijada, Ana I. Prieto, María Puerto, Ángeles Jos * and Ana M. Cameán Area of Toxicology, Faculty of Pharmacy, University of Sevilla, C/Profesor García González 2, 41012 Sevilla, Spain; [email protected] (L.D.-Q.); [email protected] (A.I.P.); [email protected] (M.P.); [email protected] (A.M.C.) * Correspondence: [email protected]; Tel.: +34-954-556-762 Received: 10 April 2019; Accepted: 31 May 2019; Published: 4 June 2019 Abstract: The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04–2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays.
    [Show full text]
  • Monograph No. 27 Aneuploidy in Chinese Hamster Ocytes Following in Vivo Treatments with Trimethoxybenzoic Compounds and Their Analogues
    ECETOC T025 17 November 1999 Monograph No. 27 Aneuploidy August 1997 European Centre for Ecotoxicology Avenue E Nieuwenhuyse 4 (Bte 6) and Toxicology of Chemicals B - 1160 Brussels, Belgium Monograph No. 27 Aneuploidy August 1997 ISSN-0773-6347-27 Brussels, August 1997 © ECETOC copyright 1997 ECETOC Monograph No. 27 © Copyright - ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals), 4 Avenue E. Van Nieuwenhuyse (Bte 6), 1160 - Brussels, Belgium. All rights reserved. No part of this publication may be reproduced, copied, stored in a retrieval system or transmitted in any form of by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the copyright holder. Applications to reproduce, store, copy or translate should be made to the Secretary General. ECETOC welcomes such applications. References to the document, its title and summary contained in data retrieval systems may be copied or abstracted. The content of this document has been prepared and reviewed by experts on behalf of ECETOC with all possible care and from the available scientific information. It is provided for information only. ECETOC cannot accept any responsibility or liability and does not provide a warranty for any use or interpretation of the material contained in the publication. Aneuploidy CONTENTS SUMMARY .................................................................................................................................................... 1 1. BACKGROUND ......................................................................................................................................
    [Show full text]
  • Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus (MN) and Comet Assay Protocol
    The Journal of Toxicological Sciences (J. Toxicol. Sci.) 149 Vol.35, No.2, 149-162, 2010 Original Article Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol Leslie Recio1, Cheryl Hobbs1, William Caspary2 and Kristine L. Witt2 1Genetic Toxicology Division, ILS, Inc., PO Box 13501, RTP, NC 27709, USA 2National Toxicology Program, NIEHS, RTP, NC 27709, USA (Received October 15, 2009; Accepted November 25, 2009) ABSTRACT — The in vivo micronucleus (MN) assay has proven to be an effective measure of geno- toxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic dam- DJHXVLQJWKH01DVVD\SURYLGHVDOLPLWHGJHQRWR[LFLW\SUR¿OH7KHin vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues fol- lowing administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophospha- mide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24- hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last admin- LVWUDWLRQ$OOFRPSRXQGVLQGXFHGVLJQL¿FDQWLQFUHDVHVLQPLFURQXFOHDWHGUHWLFXORF\WHV 015(7 LQWKH SHULSKHUDOEORRGRIPLFHDQGDOOEXW$&0LQGXFHG015(7LQUDWV(06DQG$&0LQGXFHGVLJQL¿FDQW increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced '1$GDPDJHZDVGHWHFWHGLQOHXNRF\WHVDQGGXRGHQXPFHOOV96DVSLQGOH¿EHUGLVUXSWLQJDJHQWZDV negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
    [Show full text]
  • Structural Chromosome Aberrations Cause Swelling of the Nucleus Kenji Takeshita1,2*, Hiroaki I
    Takeshita et al. Genes and Environment (2016) 38:22 DOI 10.1186/s41021-016-0047-7 RESEARCH Open Access Structural chromosome aberrations cause swelling of the nucleus Kenji Takeshita1,2*, Hiroaki I. Ogawa1 and Toshinari Maeda1 Abstract Background: Carcinogens are known to cause swelling of the mammalian cell nucleus. However, the mechanism of the swelling and its toxicological significance have not been fully elucidated. Since nuclear swelling (NS hereafter) has been frequently observed in chromosomal aberration (CA hereafter) tests (in vitro), the relationship between NS and CAs was investigated in this study. Results: In a short-term CA test using the fibroblast CHL cell line, the appearance of NS increased in a dose-dependent manner after exposure to six types of clastogens (mitomycin C, methyl methane sulfonate, 1-methyl-3-nitro-1-nitrosoguanidine, benzo[a]pyrene, cyclophosphamide monohydrate, and 9,10-dimethyl-2-benzanthracene), and a strong correlation was found between NS (%) and CAs (%) at each dosage. Therefore, we hypothesized that clastogens cause NS in cultured mammalian cells, since the mouse lymphoma L5178Y cell line is known to have a similar sensitivity to clastogens. Thus, we measured NS for 14 compounds (clastogens) that are known to induce structural CAs, 4 aneugens, and 12 non-mutagenes. Almost all clastogens caused NS of more than 5 %, which increased in a dose-dependent manner. Among the aneugens, colchicine, and diethylstilbestrol caused the same level of NS % as the clastogens, while carbendazim and trichlorfon caused a similar level of NS % as the clastogens only at higher levels of cytotoxicity. Almost all the non-mutagens caused less than 5 % NS.
    [Show full text]
  • Thresholds for in Vivo Mutagens
    COMMITTEE ON MUTAGENICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT GUIDANCE STATEMENT : THRESHOLDS FOR IN VIVO MUTAGENS. Introduction 1. The general advice of the COM when considering the risk assessment of chemicals which are mutagenic in vivo has been that it is prudent to assume a non threshold dose response. Thus it is assumed that any exposure to an in vivo mutagen is associated with some damage to DNA and consequently an increased risk of mutation leading to an increased risk of adverse health effects albeit that this may be small. In such instances the Committee has recommended that exposures be reduced to a low as is reasonably practicable. The Committee has previously considered specific chemicals, on a case-by-case basis, with regard to deviations from its general approach to in vivo mutagens.(COM., 2001) 2. The Committee considered draft guidance statements at its June and October meetings in 2009 and its March 2010 meeting. The Committee agreed that advice should be given on terminology used to define threshold in vivo mutagens, some examples of in vivo mutagens considered to exhibit a threshold should be provided, as well as information on an approach to the identification of threshold doses for in vivo mutagens. Thresholds for in-vivo mutagens Definitions 3. A number of definitions are used in this document and in the published literature regarding thresholds for in vivo mutagens.(Kirsch-Volders et al., 2009) . True Threshold: A point in the dose response relationship for mutagenicity where a slope of zero changes into a slope of greater than zero.
    [Show full text]
  • Comet Assay Revised Validation Report.Pdf
    Title: Report of the JaCVAM initiative international validation study of the in vivo rodent alkaline Comet assay for the detection of genotoxic carcinogens: the 4th (definitive) phase-1st step Issued: Yoshifumi Uno, D.V.M., Ph.D., and a Validation Management Team (VMT) member Date: November 19 , 2012 Status: Draft version-3 Notes: this document is prepared to summarize the in vivo Comet assay validation process and results in the 4th (definitive) phase-1st step. The methods are briefly mentioned in this document, because the details are described in the study protocol and the study plan. An article for submission to a scientific journal will be provided separately based on this document, the study protocols and the study plans (and the other documents if necessary and available). 99 A Table of Contents Item Page 1. Introduction …………………………………………………………………… .101 2. Background and Purpose …………... ………………………………………101 3. Experimental Period ……………………………………… ……………………..102 4. Participant Laboratories………………………………………………………… .102 5. Success Criteria in the Study Plan……………………………………………… ..102 6. Materials and Methods ………………………………………………………… ..102 7. Results …………………………………………………………………………… 105 8. Discussion …………………………………………………….............................116 9. References ……………………………………… ..……………………………….119 Appendix Appendix 1: Study protocol (version 14) …………………………..…………………..……….120 Appendix 2: Study plan………………………………………………………………................130 Appendix 3: Study reports in testing facilities (Not included) Appendix 4: Statistical analysis results (Not included) 100 1. Introduction 1. The in vivo rodent alkaline Comet assay is used worldwide for detecting DNA damage as evidenced by strand breaks. The assay can be applied to the investigation of genotoxic potential of test chemicals, and is currently identified as a second in vivo genotoxicity assay in the ICH-S2(R1) guidance (2012) along with the more usual in vivo micronucleus test in bone marrow or peripheral blood.
    [Show full text]
  • 213535Orig1s000 NON-CLINICAL REVIEW(S)
    CENTER FOR DRUG EVALUATION AND RESEARCH APPLICATION NUMBER: 213535Orig1s000 NON-CLINICAL REVIEW(S) DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION CENTER FOR DRUG EVALUATION AND RESEARCH PHARMACOLOGY/TOXICOLOGY NDA REVIEW AND EVALUATION Application number: 213535 Supporting document: 001 Applicant’s letter date: 7/15/2019 CDER stamp date: 7/15/2019 Product: Risdiplam (RO7034067) Indication: Spinal muscular atrophy (b) (4) Applicant: Genentech Review Division: Neurology 2 Reviewer: Ed Fisher Supervisor: Lois Freed Acting Division Director: Nicholas Kozauer Project Manager: Stephanie Parncutt Disclaimer Except as specifically identified, all data and information discussed below and necessary for approval of N213535 are owned by Genentech or are data for which Genentech has obtained a written right of reference. Any data or information described or referenced below from reviews or publicly available summaries of a previously approved application are for descriptive purposes only and are not relied upon for approval of N213535. Note: All figures and tables in this review were excerpted from the sponsor’s submission 1 Reference ID: 4646220 NDA # 213535 Ed Fisher TABLE OF CONTENTS 1 EXECUTIVE SUMMARY...........................................................................................3 2 DRUG INFORMATION............................................................................................10 3 STUDIES REVIEWED.............................................................................................10
    [Show full text]
  • Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and P-N,N-Bis (2-Chloroethyl) Aminophenylacetic Acid in Cell Cultures in Vitro
    Aneugenic Potential of the Nitrogen Mustard Analogues Melphalan, Chlorambucil and p-N,N-bis (2-Chloroethyl) Aminophenylacetic Acid in Cell Cultures In Vitro aM. Efthimiou, aC. Andrianopoulos, aG. Stephanou, aN. A. Demopoulos, bS. S. Nikolaropoulos aDivision of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Greece bLaboratory of Medicinal Chemistry, Department of Pharmacy, University of Patras, Greece ABSTRACT Melphalan (MEL), chlorambucil (CAB) and p-N,N-bis(2- chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above drugs and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with Fluorescence In Situ Hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (α-satellite) was applied to identify chromosomes in micronuclei and an X-chromosome specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with aneugenic potential, was investigated in C2C12 mouse cell line by double immunofluorescence of α- and γ-tubulin. We found that the studied drugs increased MN frequency in a linear dose- dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X chromosome were also induced. Abnormal Key words: nitrogen mustard analogues, chromosome metaphase cells were linearly increased with concentration and malsegregation, abnormal centrosome number characterized by abnormal centrosome number.
    [Show full text]
  • Non-Ionizing Radiation, Part 2: Radiofrequency Electromagnetic Fields Volume 102
    NON-IONIZING RADIATION, PART 2: RADIOFREQUENCY ELECTROMAGNETIC FIELDS VOLUME 102 This publication represents the views and expert opinions of an IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, which met in Lyon, 24-31 May 2011 LYON, FRANCE - 2013 IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS 4. OTHER RELEVANT DATA Data on specific absorption rate (SAR) and distribution of radiofrequency (RF) radiation inside tissues and organs and at the subcellular level are presented elsewhere in this Volume (Section 1.3, Dosimetry). 4.1 Genetic and related effects in these studies DNA damage was measured in peripheral blood lymphocytes and buccal cells 4.1.1 Humans by means of the alkaline or neutral single-cell gel electrophoresis assay (comet assay), which reveals During the past decades, extensive research alkali-labile lesions and single- and double- efforts have focused on determining the extent strand breaks in DNA, or by use of cytogenetic of DNA damage in eukaryotic and prokaryotic tests for chromosomal aberrations, micronucleus cells exposed to RF radiation. Several published formation and sister-chromatid exchange (SCE). reviews concluded that: (i) the existing data are The studies reviewed below are summarized in not sufficiently strong to suggest that RF radiation Table 4.1 and Table 4.2 (with details of the expo­ is directly genotoxic; (ii) exposure to RF radiation sure conditions). probably does not enhance the damage induced by known genotoxic agents; and (iii) some of the (a) Peripheral blood lymphocytes reported “adverse effects” may be attributed to (i) Occupational exposure hyperthermia induced by RF radiation (Brusick et al., 1998; Verschaeve & Maes, 1998; Moulder Garaj-Vrhovac et al.
    [Show full text]