& Regulatory Issues in Genetic Toxicology
September 14-15, 2011
John M. Clayton Hall Conference Center
University of Delaware
Speakers Bios, Abstracts & Presentations
Thomas Hartung, M.D., Ph.D.
Johns Hopkins Bloomberg School of Public Health, CAAT, Baltimore
In 1991 Dr. Hartung received a PhD in Biochemical Pharmacology from the University of Konstanz, Germany, and an MD in Toxicology in 1992 from the University of Tubingen. He completed his medical internship at the University of Freiburg in surgery at the hospital of Singen, Germany.
Dr. Hartung joined the faculty at University of Konstanz in 1994, where he served as an Assistant Professor of Biochemical Pharmacologyuntil 1999, and then as an Associate Professor until 2002. He is an honorary full professor of Pharmacology and Toxicology at Konstanz since 2003, which he continues as a joint appointment. From 1996 to 2002, Dr. Hartung also served as the CEO of the Steinbeis Technology Transfer Center for In Vitro Pharmacology and Toxicology (InPuT).
In 2002, Dr. Hartung became the Head of the European Centre for Alternative Methods (ECVAM) at the European Commission Joint Research Centre in Italy. As Head of ECVAM, he was integral in accelerating the alternative methods validation process, and in establishing a network of more than 400 experts from all stakeholder groups to facilitate global regulatory harmonization in toxicity testing. He has authored more than 350 scientific articles.
Toxicity testing for the 21st Century: Impact on genetic toxicology, carcinogenicity and the 3R’s
For a general public, possible carcinogenic effects of substances are the most prominent environmental health concern. The rodent bioassay, standardized 40 years ago, has produced as many problems as it solved: It costs more than $1 million per substance, lasts several years, 53% of all substances tested are positive and mice and rat predict each other with only 57%. Genetic toxicity testing as a type of first tier has addressed some problems, but especially in combination, the in vitro battery of tests as used today further amplifies the number of false-positive results and leaves open the problem of non-genotoxic carcinogens. While fine-tuning of test strategies has some promise, they do not fundamentally change the situation.
Some pragmatic solutions such as exposure-tailored testing, thresholds of toxicological concern, grouping and read-across help, but cannot satisfy fully from a scientific and precautionary point of view.
Two novel concepts are emerging:
In Europe in the context of REACH, integrated testing strategies (ITS) are favored, which can be mapped on modes of action of substances. Their promise lies in the integration of results but they are too often falsely understood as only larger batteries of simple tests or combination of tests, which each alone did not succeed to replace the animal test. A major challenge is the systematic composition and validation of ITS based on targeted test development.
In the US strongly prompted by the Toxicity Testing in the 21st Century vision document by the NRC 2007 and a possible TSCA reauthorization, a concept of pathways of toxicity (PoT) is emerging. It is suggesting an even finer resolution of analysis than mode of action type cell approaches: the perturbed physiological pathways or the molecular targets of toxic action shall form the basis for PoT-based high-throughput assays. This requires in the end mapping the Human Toxome, the entirety of PoT. Pilot initiatives are starting to address this challenge. The concepts of PoT identification, annotation and validation as well as the governance of a Human Toxome database are under discussion. This new approach needs to be complemented by quality assurance; traditional validation does only in part accommodate the needs of the new approach. The idea to exploit concepts of evidence-based medicine led to the creation of the Evidence-based Toxicology (EBT) Collaboration in the US in March 2011, which is shaping this process. A similar initiative in Europe is in preparation for 2012. EBT offers a concept to analyze traditional and novel approaches in toxicology and help the transition into toxicology for the 21st century.
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Marilyn J. Aardema, Ph.D.
Dr. Marilyn Aardema is currently the Chief Scientific Officer for Toxicology at the BioReliance Corporation. In addition, Marilyn is President of Marilyn Aardema Consulting, LLC. In her CSO role, Marilyn is responsible for providing scientific leadership within BioReliance, for setting business strategy, providing regulatory and scientific advice to colleagues and clients. Marilyn was previously at The Procter & Gamble Company in the Central Product Safety organization where she managed overall genetic toxicology battery design and risk assessment/external defense of key ingredients for diverse products worldwide. In addition, Marilyn pursued various research projects including studying mechanisms of genotoxicity, aneuploidy, and development of improved genotoxicity testing approaches including most recently the development of a novel micronucleus assay in 3D human skin models.
Dr. Marilyn Aardema received her Ph.D. in Genetics from The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and B.S. from Hope College.
Marilyn has more than 75 publications on topics including thresholds, aneuploidy, mechanisms of genotoxicity, new assay development/validation, toxicogenomics, cell transformation, performance of genetic toxicology assays, animal alternatives, design of new testing approaches, germ cell aneuploidy, micronucleus assay, regulatory guidelines. Marilyn is active in the Genetic Toxicology Association (GTA), has served on numerous Environmental Mutagen Society committees, member of The Society of Toxicology; past member of European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) Task Forces on aneuploidy and threshold-mediated mechanisms, Chair of the Mutagenicity Subcommittee of the American Industrial Health Council (AIHC); member of the International Congress on Harmonization (ICH 2) 1990’s US Pharmaceutical Manufacturer's Association Genetic Toxicology Task Force; is an OECD US Expert; member of various International Life Science Institute (ILSI)-Health and Environmental Sciences Institute (HESI) Subcommittees including serving as rapportuer of the Application of Genomics to Mechanism Based Risk Assessment Genotoxicity Working Group and the current ILSI-HESI steering team on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing; member of various European Center for Validation of Alternative Methods (ECVAM) Committees including the Establishment of Timetables for the Phasing out of Animal Experiments for Cosmetics and the current Cell Transformation Steering Committee; past member of the European Cosmetics Association (COLIPA) Animal Alternatives Genotoxicity Subgroup chairing the 3D skin genotoxicity assay project; various International Workshop on Genotoxicity Testing (IWGT) committees including the recent Invitro Cytogenetics Assay Working Group and Integration Working Group, past member of the Institute for Invitro Sciences (IIVS) Scientific Advisory Panel and AltTox Editorial Board.
Overview of ECVAM Validation of Cell Transformation Assays and 3D Skin Models for Genotoxicity Assessment
Assessment of carcinogenicity is a key consideration in the evaluation of safety of chemicals and pharmaceuticals. The standard approach is the conduct of the 2-year rodent bioassay which is expensive, time-consuming and uses a large number of animals. There is a need for validated alternative tests for carcinogenicity screening and mechanistic studies. In vitro cell transformation assays (CTAs) are a fast, cost efficient tool for carcinogenicity screening. Because of the utility of CTAs, an Organisation for Economic Co-operation and Development (OECD) detailed review paper (DRP) of CTAs was recently prepared (OECD, 2007). OECD concluded that the performances of the Syrian hamster embryo (SHE) and Balb/c 3T3 CTAs were sufficient and should be developed into official OECD test guidelines. To help with this, the European Centre for the Validation of Alternative Methods (ECVAM) initiated a formal prevalidation study of the Syrian Hamster Embryo (SHE) and Balb/c3T3 CTAs. In this exercise, within-laboratory reproducibility, test method transferability, between-laboratory reproducibility and development of a standardized protocol were addressed. This presentation will review the OECD and ECVAM cell transformation projects.
3D Skin Models for Genotoxicity Assessment
There is increasing interest in the use of 3D tissue models for genotoxicity testing. This is being driven by a number of factors including the emphasis by various stakeholders on the 3Rs. 3D tissue models prepared from normal human cells provide potential improvements over standard in vitro assays including more realistic exposures, normal metabolism and DNA repair. One example is the recently developed reconstructed human skin micronucleus assay (RSMN) in the EpiDermTM model (MatTek Corp) for use as an alternative to Tier II in vivo genotoxicity assays. The RSMN assay has been shown to be reproducible with known genotoxins/carcinogens and non-genotoxins/carcinogens in laboratories in the United States as well as Europe. This work has prompted others to investigate the use of 3D tissue models for a variety of testing purposes that will be described.
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Dr. Paul Carmichael has over 20 years experience in Toxicology and Cancer Research; largely in the academic arena, before joining Unilever, from Imperial College London, in 2003. He has published over seventy research papers in peer-reviewed scientific journals and has served on numerous committees such as the UK Environmental Mutagen Society, British Toxicology Society, COLIPA (currently Co-Chair of Genotoxicity Task Force) and was a past recipient of the Upjohn Cancer Investigator Award from the American Association for Cancer Research. He is a Fellow of the British Toxicology Society and the Society of Toxicology in the US. His interests cover the safety evaluation and risk assessment of genotoxic agents and the development of new approaches for 'Toxicity Testing in the 21st Century'. He has a B.Sc. degree from the University of Surrey in Toxicology and a Ph.D. in Biochemistry from King's College.
Toxicity testing in the 21st Century (TT21C): Genetic Toxicology as a prototype pathway - a perspective from the personal care product industry
IThe NAS/NRC report on TT21C (Krewski et al., 2010) offers an attractive future paradigm for toxicologists based on toxicity pathway perturbations and human exposure, rather than apical end points measured in experimental animals. In the context of a risk-based approach to the safety assessment of ingredients in consumer products we are exploring the practical application of this paradigm through a collaborative research programme with the Hamner Institutes. Work investigating the individual elements of: (a) exposure and consumer use assessment, (b) fast, high-content-information in vitro assays in human cells, (c) dose-response assessments, (d) computational models of the circuitry of relevant toxicity pathways and (e) pharmacokinetic models supporting in vitro to in vivo extrapolations, are being brought together to craft novel risk assessments for putative ‘genotoxic’ case-study chemicals; maintaining exposure below the levels that significant pathway perturbations occur. A combination of techniques are being employed and combined from Cellomics high content imaging for dose-response assessments of DNA damage and underlying threshold characteristics, to bioavailability measures derived from bloodspot and/or micro-dosing determinations in human subjects. We anticipate that this prototype toxicity pathway research will provide scientific evidence to support the future application of the TT21C principles, and will foster greater development of these much-needed methodologies.
Dr. Andrew Kligerman is currently a research biologist in the Integrated Systems Toxicology Division at the U.S. EPA in Research Triangle Park, NC. He has been a cytogeneticists and genetic toxicologist at the EPA for over 24 years. He is currently doing a rotation with the National Center for computational Toxicology at the EPA, where he is investigating the sensitivity and specificity of high-throughput tests for determining the genetic toxicology of chemicals. For the vast majority of his research career at EPA, he has studied the genotoxicity of important environmental and commodity chemicals. For the previous 10 years his research has concentrated on investigating the mode of action of arsenicals in inducing genetic damage and cancer. Prior to joining the U. S. EPA, Dr. Kligerman was a program leader at EHRT, Inc. and staff cytogeneticists at C.I.I.T.
Dr. Kligerman received his BS from Duke University in Zoology (1971). He attended Cornell University where he obtained an MS (1974) and Ph.D. (1977) in Animal Cytogenetics in the Laboratory of Dr. Stephen Bloom studying SCEs and chromosome breakage in the mud minnow. Dr. Kligerman did a Post-doctoral fellowship at Duke University in the Department of Pathology under Dr. George Michalopolous developing co-culture methods using primary rat hepatocytes and human fibroblasts to study genetic damage.
Dr. Kligermanhasauthored or co-authored over 100 peer-reviewed publications. He is an editorial board member of Mutation Research and Environmental and Molecular Mutagenesis. He is active in EMS, twice previously as a counselor and now as Co-chair of the EMS Finance Committee. He has previously been President and Vice President of GEMs. Dr. Kligerman has received the US EPA’s Bronze Medal and Levels I and II Scientific Achievement Awards and the EMS Special Service Award.
Evaluating ToxCast Assays for their Ability to Detect Genotoxicity
It has become evident over the past several decades, that though the standard battery of genotoxicity tests including bacterial and in vitro mammalian mutagenesis and in vitro and in vivo clastogenicity assays have been quite useful in screening out potent genotoxicants during the development and manufacturing processes, the number of industrial and environmental chemicals that need to be tested overwhelms our ability to test them in a careful and efficient manner using the present battery of assays. This was clearly pointed out by the National Academy of Sciences’ report entitled Toxicity Testing in the Twenty-first Century: a Vision and a Strategy. With this in mind, the U.S. EPAset out on an ambitious project to develop chemical screening tools. One such tool is the Toxicity Forecaster (ToxCast™). ToxCast™ is a multi-year effort to develop a cost-effective approach for prioritizing the thousands of chemicals that need toxicity testing. With more than 600 assays, Phase I of ToxCast™ was designed to screen a large number of toxicity endpoints using approximately 300 well-studied chemicals, mostly pesticides. Phase II, which is currently underway, will look at an additional 700 chemicals. However, only a few of the assays were designed to measure endpoints that were directly related to genetic toxicology, and most if not all systems lacked efficient metabolic activation capabilities. The goals of the present analysis were 1) to determine which Phase I chemicals were genotoxic without metabolic activation, and 2) to evaluate how some of the high-throughput assays performed in identifying direct-acting genotoxicants. A variety of information was used including various data bases, industry submissions, and the open literature to classify the chemicals according to their reported ability to induce point mutations, chromosome breakage, and/or chromosomal numerical changes. The chemicals were then assigned a numerical value from +5 (highly active) to -5 (no activity) for each endpoint. Results from several assays will be presented to illustrate their ability to detect genotoxicants, but to date, none of the assays investigated for genotoxicity shows high specificity or sensitivity for any of the endpoints. [This abstract does not necessarily reflect US EPA policy.]Back to Top ↑
Thomas A. Rosenquist
State University of New York/ Department of Pharmacological Sciences.
Dr. Rosenquist received his BS from Northern Illinois University, Dekalb, IL (1982), and Ph.D. in Biochemistry/Developmental Biology from University of Wisconsin, Madison, WI (1989) studying C. elegans with Dr. Judith Kimble (molecular genetics of sex determination). His Post-doctoral training was at the University of California, San Francisco under the supervision of Dr. Gail Martin (mouse gene-targeting techniques).
Recent publications relevant to aristolochic acid nephropathy
1. Rosenquist TA. (2011) Genetic loci that affect aristolochic acid nephrotoxicity in the mouse. Am J Physiol Renal Physiol. 300(6):F1360-7.
2. Rosenquist TA, et al. (2010) Cytochrome P450 1A2 detoxicates aristolochic acid in the mouse. Drug Metab Dispos. 38(5):761-8.
3. Shibutani S, et al. (2010). Detoxification of aristolochic acid I by O-demethylation; less nephrotoxicity and genotoxicity of aristolochic acid I in rodents. Int J Cancer. 127(5):1021-7.
4. Grollman AP, et al. (2007) Aristolochic acid and the etiology of endemic (Balkan) nephropathy. PNAS (USA) 104(29):12129-34.
Aristolochic Acid Nephropathy and Associated Upper Urothelial Cancer: An Environmental and Iatrogenic Disease
Aristolochic acid (AA), a principal component of Aristolochia species, was shown recently to be the toxin responsible for the clinical syndromes known as Chinese herb nephropathy and endemic (Balkan) nephropathy (EN). Both disorders are associated with a high incidence of upper urinary tract urothelial carcinomas (UUC) and appear to constitute a single disease entity, designated aristolochic acid nephropathy (AAN). Based on the traditional use of Aristolochia in herbal remedies, we posited that AAN represents a long-overlooked iatrogenic disease and an international public health problem of considerable magnitude (Adv Mol Tox 3, 211, 2010). Pursuing this hypothesis, we conducted studies of 100 patients with UUC residing in Taiwan, or in regions of Croatia, Bosnia and Serbia where EN is prevalent. DNA was obtained, with informed consent, from renal cortical and tumor tissues following nephroureterectomy. Aristolactam (Al)-DNA adducts in the renal cortex were quantified by 32P- post-labeling techniques. Chip-sequencing technology was utilized to establish the pattern of mutations in the TP-53 gene. Al- DNA adducts were detected in the majority of patients with UUC. The TP53 mutation spectrum was dominated by A:TàT:A transversions located almost exclusively on the non-transcribed DNA strand, reflecting a failure to excise AL-DNA adducts by global genomic nucleotide excision repair. This factor may account for the remarkable persistence of these adducts in human tissues. Thus, aristolochic acid joins aflatoxin and vinyl chloride as one the few human carcinogens with a definitive TP53 mutational signature. Coupled with the use of AL-DNA adducts as biomarkers, the presence of a mutational signature establishes an etiological role for AA in UUC. Public health authorities in countries where Aristolochia has been used are encouraged to initiate screening programs to detect UUC and to implement measures that will reduce human exposure to this nephrotoxic and carcinogenic herb. (Supported by NIEHS).
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Dr. Tarasova received her BS and MS in Chemistry from Lomonosov University (Moscow, Russia). She obtained her Ph.D. in Bioorganic Chemistry and Chemistry of Natural Compounds from Lomonosov University in 1980. Her Post-doctoral training was done at Institute of Biochemical Genetics of Copenhagen University (Denmark) under the supervision of Prof. Bent Foltman.
Dr. Tarasova has authored or co-authored over 100 peer-reviewed publications and holds 13 patents in the area of drug discovery. She is a member of Chemistry and Structural Biology Steering Committee of NCI, recipient of several Federal Technology Transfer Awards, NCI Director’s Innovation Awards and serves as Executive Editor, Current Pharmaceutical Design: Peptides and Peptidomimetics as Anti-Cancer Therapeutics.
Yes, We Can: Developing Drugs for the “Undruggable”
In spite of increasing investment in drug discovery from the industry, academia and government, the number of novel drugs approved by FDA has been declining during the past 12 years. Possible reasons include inability of most commonly used approaches to produce inhibitors for “non-druggable” targets and a wide spread commitment to small molecules that frequently fail to provide sufficient selectivity towards intended molecular target. Peptide therapeutics are promising alternatives to small molecules. The advantages of peptides are in chemical and biological diversity that provide for high activity, high specificity, little unspecific binding to molecular structures other than intended target, minimal drug-drug interactions, less accumulation in the tissue and lower toxicity. Peptides are well suited for targeting protein-protein interactions that represent a particularly attractive way of developing new drugs for many disorders. The task proved to be challenging for small molecules because of large interacting surfaces that need to be blocked by a synthetic compound. In spite of the fact that there are more than 60 marketed peptides worldwide, around 270 peptides in clinical phase testing, and about 400 in advanced preclinical phases, the innate shortcomings continue hampering wider use and development of peptides and peptidomimetics as therapeutics. The difficulties are attributed to peptide’s low stability in circulation, low oral bioavailability, poor membrane penetration, rapid clearance from the body and risks of immunological effects. We and others have succeeded recently in developing metabolically stable cell permeable peptide analogs with rigid and predictable structures amenable to rational design. General applicability of these rational approaches was confirmed by generation of selective antagonists of insulin-like growth factor 1 receptor, STAT transcription factors, Wnt signaling pathway, RAS oncogene and other non-druggable targets.
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Kurt A. Black, Ph.D., DABT
Amgen Inc., Comparative Biology and Safety Sciences
Genotoxic Impurity Limit for an ICH S9 Oncology Indication
Current regulatory guidances on genotoxic impurities in pharmaceuticals recommend limits based on negligible increases in the risk for development of cancer over a lifetime of exposure. Higher levels of genotoxic impurities may be justified in certain circumstances, such as for treatment of life-threatening conditions. Recently, The International Conference on Harmonisation issued Guidance on Nonclinical Evaluation for Anticancer Pharmaceuticals (ICH S9) that provides recommendations for nonclinical safety programs to support development of pharmaceuticals for patients with advanced cancer. Flexibility in the approach to the nonclinical safety assessment is recommended as compared to indications for diseases with long life expectancy. Specifically, ICH S9 states that limits for genotoxic impurities that are based on lifetime exposure are not appropriate for pharmaceuticals used to treat patients with advanced cancer, and that higher limits can be justified. These principles were used to set a limit for a genotoxic impurity in Compound A, which is intended for treatment of malignancies with limited life expectancy. Compound A was negative in an Ames test. Impurity X is an intermediate in the synthesis of Compound A and also occurs as a drug substance impurity and degradant. In silico assessment of Impurity X predicted that it would be mutagenic, and subsequent testing in an Ames test confirmed the prediction. Higher limits than the defaults stated in the genotoxic impurity guidances were specified for Impurity X in the Compound A drug substance and justified based on the nature of the patient population. Although such an approach is justified for advanced cancer indications, as described in ICH S9, such higher limits would not necessarily be appropriate in other circumstances, for example, if the oncologic agent was administered to healthy volunteers in clinical pharmacology studies. Also, the appropriateness of limits set for a genotoxic impurity in a pharmaceutical initially developed for treatment of advanced cancer would need to be reevaluated if the compound was subsequently developed for chronic administration as maintenance therapy to minimize the risk of recurrence of cancer.
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Control of a Low Level Mutagenic Impurity: An Industry Case Study
Targeted industry approaches to determine the risk of the presence of low level mutagenic impurities in pharmaceutical products has evolved significantly over the past decade. Quality by design approaches to process and product development coupled with in-silico tools to assess structural features associated with potential mutagenicty has enabled the process chemist in partnership with the genetic toxicologist to better assess mutagenic impurity risk. This presentation will provide a brief overview of these approaches and present an industry case study.
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Dr. Jacobson-Kram served as the VP of the Toxicology and Laboratory Animal Health Division at BioReliance Corporation, a contract testing laboratory from 1988 until 2003. Currently, he serves as the Associate Director of Pharmacology and Toxicology in FDA’s Office of New Drugs. Over the past twenty years he has served as principal and co-principal investigator on several N.I.H. grants and government contracts and published widely in the areas of genetic and molecular toxicology.
Dr. Jacobson-Kram has served as council member, treasurer and chairman of the Genetic Toxicology Association, executive council member to the Environmental Mutagen Society, Editor of Cell Biology and Toxicology, and as a member of N.I.H. special study sections. In 1996 he became a Diplomat of the American Board of Toxicology (DABT).
The current state of regulation of genotoxic impurities: ICH M7, (Q) SAR predictions and complete CDER consistency
The initiation of the ICH M7 expert working group is designed to bring global consistency in the way in which genotoxic impurities (GTIs) in human drugs are regulated. Guidance has been published by the EMA, draft guidance was published by CDER and PhRMA published a “white paper” on their perspective. All publications agree that limits on GTIs should be set during development (clinical trials) as well as for marketed products. There is general agreement that 1) a negative bacterial mutation assay is sufficient to qualify a impurity, 2) (Q) SAR predictions should be used raise or obviate concern over an alerting structure, 3) reducing the concentration of an alerting structure to a level such that the quantity of the impurity consumed is below the appropriate threshold of toxicological concern (TTC) eliminates the need for qualification. Outstanding issues include 1) what types and how many (Q) SAR programs should be used routinely, 2) should alerting structures expected to act by common mechanisms of action be summed or regulated individually and 3) should specifications be set higher for drugs that consumed for less than a lifetime.
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Warren received his B.S. in Biology from Purdue University and M.S. in Forensic Analytical Chemistry from the University of Illinois. He received his Ph.D. in Cellular Biochemistry from the University of Michigan, studying keratinocyte biochemistry and chemical irritancy mechanisms in the laboratory of Dr. Isadore Bernstein. He then served as a post-doctoral fellow in Dr. Walter Piper's laboratory at Michigan, focusing on the biochemical mechanisms of lead toxicity to erythroid precursor cells. He then trained in Dr. Robert Chapin's laboratory in reproductive toxicology as a Senior Staff Fellow at the National Institute of Environmental Health Sciences (NIEHS).
Dr. Ku has published forty papers, five book chapters and is co-inventor on two patents. He has been primary or co-author on fifty presentations at professional scientific meetings, and invited speaker or program chair for twenty scientific programs. He has been a member of the Society of Toxicology, Environmental Mutagen Society, Genetic Toxicology Association and the Society for Biomolecular Sciences. Dr. Ku has chaired or served on several scientific review and advisory panels or boards, including the NIH Toxicology Study Section, Center for the Evaluation of Risks to Human Reproduction, PhRMA DruSafe Genetic Toxicology Working Group, Genetic Toxicology Association and the International Workshop on Genotoxicity Testing.
ICH M7: Opportunities for Improved Safety and Quality Integration to Manage Risk of Low Level Mutagenic Impurities
The positioning of genotoxic impurities as an ICH guideline topic (M7) was formalized through a concept paper in 2009, and an Expert Working Group (EWG) is currently working on developing a harmonized guideline for their evaluation and control. The M7 EWG is composed of safety and quality topic leads and experts to develop a Step 1 technical consensus document. The ICH M7 effort presents an opportunity to review and consider existing guidance content, evaluate new information and experiences since their introduction, and improve the integration of safety and quality aspects for detection, risk management and control. This presentation will provide an overview of current status and progress including some brief historical context, introduce the EWG, review the objectives of ICH M7, and define the current key issues for safety and quality topic areas. These include refining considerations and elements for conducting assessments, using in silico tools to identify concerning structures, applying the TTC and alternative risk approaches to limit carcinogenic risk (clinical development, marketed products) and defining process/product control strategy elements.
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FDA Perspective on Control Strategies for Genotoxic Impurities
This presentation will outline the types of strategies that may be used to control genotoxic impurities in active ingredients and drug products. Once the acceptable level of a specific genotoxic impurity in a given drug has been determined, the approach to controlling/monitoring the manufacturing process will be selected, to assure that each batch will conform. This talk will define terms and describe the variety of control strategies that are used in practice. These include monitoring in finished products, monitoring in earlier synthetic intermediates taking into account the ability of the manufacturing process to consistently reduce the impurity level, and reliance on the control provided by the manufacturing process itself. Some of the challenges caused by the need to control genotoxic impurities at levels that are frequently far below the ICH Q3A/B thresholds will be identified. Risk-driven approaches that are recommended in existing guidances (e.g., ICH Q8 and Q9) will be discussed.
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Rosalie Elespuru, Ph.D.
FDA/CDRH/OSEL/Division of Biology Genomics Lab
Genotoxicity Assessment of Nanomaterials: Update on In Vitro Approaches
Nanomaterials have unique properties that make them challenging to assess. Importantly, whether the standard genotoxicity assays can correctly identify genotoxic potential of nanomaterials has not been established. EMS held a workshop in October 2010 with a goal of Refining Strategies and Tests for Hazard Identification of nanomaterials.
The workshop began with a plenary session providing summaries of approaches to assessment of nanoparticles for genotoxicity in Europe, the US, Canada and Japan. This was followed by two break-out sessions evaluating the usefulness of standard in vitro and in vivo assays. A third break-out group addressed whether new technologies or new research would be useful in meeting the goal of development of a genotoxicity testing scheme for nanomaterials. A final plenary session brought groups back together for discussion and development of consensus statements. Summaries from the workshop related to in vitro genotoxicity tests will be presented. [Summaries from the workshop related to in vivo tests will be presented by Stefan Pfuhler.] One of the most important issues related to an appropriate genotoxicity test battery for nanomaterials involves the role of bacterial mutagenicity testing. Some reports in the literature indicate that bacteria generally exclude nanomaterials, and hence bacterial tests may be non-informative concerning potential genotoxicity outcomes. Other studies show positive mutagenicity effects in bacteria. These discrepancies will be summarized and addressed. In addition, results from experiments in my lab addressing the response of Salmonella typhimurium TA100 to some nanomaterials, including several sizes of nano-Ag, will be presented. Further discussion of the issues surrounding the genotoxicity assessment of nanomaterials will be encouraged by an interactive session with GTA attendees.
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Genotoxicity Assessment of Nanomaterials: In Vivo Aspects
Nanomaterials have proven to be a challenging group of materials for genetic toxicologists. In order to discuss the progress made for nanomaterials in this field, a workshop was held on October 23, 2010 in Forth Worth Texas, USA, which aimed at developing recommendations for a genotoxicity testing scheme for nanomaterials, as well as the identification of potential research needs. One of the three break-out groups addressed the usefulness of standard in vivo genetox tests for these materials, and the summary of the outcome will be presented [Summaries from the workshop related to in vitro genotoxicity tests will be presented by Rosalie Elespuru] and put into context with findings in our own lab. It has been speculated that nanomolecular size, itself, leads to cellular alterations that can induce DNA damage. In an effort to understand potential modes of action (MoA) and whether those would be picked up by the in vivo assays used, we tested nano/microsize gold (2, 20 and 200nm) and latex particles (20 and 1000nm), nanosized amorphous silica (15nm and 55nm), as well as quartz (DQ12) in a comet assay/micronucleus combination study. We measured DNA damage in liver, lung and blood lymphocytes and micronuclei in circulating reticulocytes after 3 consecutive iv injections to male Wistar rats (n=4-8) at 48, 24 and 4h before sacrifice. The results demonstrated a lack of genotoxic potential for all tested sizes of gold and latex particles in all parameters/tissues investigated. The 15nm and 55nm size silica particles, however, did increase the number of micronucleated reticulocytes. These results were corroborated by findings in the comet assay, as a small but reproducible increase in DNA damage was observed in liver cells. The results obtained in this study support the conclusion that the nanosize of the particles, alone, does not trigger genotoxic activity and that these effects which were obtained at extreme doses after i.v. injection were picked up in the comet/NM combination study. Supporting data will be shown that suggest that the genotoxic effects were secondary to inflammation.
Further discussion of the issues surrounding the genotoxicity assessment of nanomaterials will be encouraged by an interactive session with GTA attendees.
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Kirsten Achilles-Poon, B.S.
Genentech, Inc., Development Sciences/Safety Assessment
Genotoxicity Testing of an Antibody Drug Conjugate: A Case Study of T-DM1
Antibody drug conjugates (ADCs), or immunoconjugates, are hybrid molecules usually comprised of monoclonal antibodies conjugated with potent cytotoxic agents. Due to the promise of a targeted therapy and an improved therapeutic index compared to traditional chemotherapies, a number of ADCs are in clinical development for the treatment of cancer. In these cases, tumors expressing specific antigens can be selectively targeted by the antibody portion of the ADC and, upon antigen binding, the cytotoxin can be internalized and released resulting in cell death. Ideally, to minimize off target toxicity, the target antigen would be over-expressed in tumor compared to normal tissue. Since ADCs contain both biologic and small molecule components, standard approaches for pre-clinical safety evaluation for either component may not be appropriate or adequate, and regulatory expectations are continuing to be defined. These attributes have presented challenges in developing a strategy for genotoxicity testing of ADCs. The genotoxicity testing strategy of T-DM1, an ADC currently in ongoing clinical trials for treatment of metastatic breast cancer, will be presented to illustrate the complexities of genetic toxicity evaluations for ADCs in oncology indications.
Presentation not available
Genetic Toxicology of Oligonucleotide-based Therapeutics
Oligonucleotide-based therapeutic programs are currently distributed throughout drug developmental pipelines from early research to late stage clinical trials. Oligo-based drug classes include miRNA, siRNA, antisense and aptamers that are designed to affect proteins involved in disease process without direct genomic DNA interaction. The oligonucleotide safety working group (OSWG) is comprised of members from industry, academia and regulatory agencies and provides a forum for discussion of safety related issues unique to this therapeutic class. This presentation will summarize the discussions and recommendations of the OSWG genotoxicity subcommittee. Discussions focused on identifying potential genotoxic liabilities of established and novel oligonucleotide chemistries. Additionally, the group discussed what circumstances, if any, call for use of currently available genetic toxicology tests.
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Stephen Dertinger, Ph.D.
Interlaboratory Pig-a Mutation Assay Collaborative Trial Update
An international collaborative trial was established in order to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (approximately 20 minutes per sample) limited the number of cells per sample that could be interrogated for the mutant phenotype. It was therefore desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or non-genotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed. In order to evaluate this method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS) or melphalan (MEL). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte counts. 1,3-PS and MEL were found to increase Pig-a mutation and micronucleated reticulocyte frequencies in both study designs. While the greatest induction of micronuclei was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents interrogated per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the international trial will benefit from the inclusion of studies that are based on both acute and protracted dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.
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Managing Risk of Human Genotoxic Metabolites During Drug Development
Drug candidates are designed to avoid the potential for biotransformation to reactive metabolites which confer genotoxicity. Despite this effort, on occasion a human metabolite of genotoxic concern is identified during drug development. These cases, although infrequent, present significant challenges. Regulatory guidance is limited, with no recommendations related to the risk assessment of human genotoxic metabolites. In addition, quantitation of human risk may not be possible early in development as the necessary data have not been generated. Ultimately, managing the risk of genotoxic metabolites requires multidisciplinary input and case by case consideration. However, the complexity and uniqueness of each individual case can be put into a common framework to aid in decision making. This common framework will be presented along with case studies to illustrate the challenges, practical considerations and strategies that can be used to assess human risk and support critical drug development decisions.
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Jim received his B.A. in Biology from the Johns Hopkins University (1993), M.H.S in Molecular Microbiology and Immunology (1994), and Ph.D. in Toxicology from the Johns Hopkins School of Public Health (2001).
Jim has authored or co-authored publications on cytochrome P4501B1, and his work with the committee activities noted above. He is an active member of the Society of Toxicology, Environmental Mutagen Society, and the Teratology Society. After serving as the Secretary/Treasurer for the Korean Toxicologists Association in America SOT SIG, he is now the current Vice-President (President-Elect).
Update for the HESI IVGT Project Committee
In vitro assays are key elements of the battery of genotoxicity assays. During the past 15 years, accumulated evidence has shown that a large number of the mammalian in vitro positive findings were not confirmed in in vivo genotoxicity and/or carcinogenicity studies, raising the question of their relevance to humans. The mission of the In Vitro Genetic Toxicity (IVGT) Testing Project Committee of the ILSI Health and Environmental Sciences Institute (HESI) is to improve the scientific basis for interpretation of in vitro genetic toxicology data for purposes of more accurate human risk assessment. It is increasingly accepted that positive results should not be considered in isolation, and that weight of evidence and mode of action approaches should be preferred. To this aim, the IVTG Committee 1) elaborates recommendations for follow-up testing strategies, 2) considers how quantitative data can be used to extrapolate from in vitro to in vivo exposures and from rodent to human risk assessment, 3) supports the improvement of existing assays, and 4) facilitates the evaluation and development of new technologies and approaches that would improve the prediction of effects in humans. Scientists from 15-20 industrial companies, as well as academic institutes and regulatory authorities are involved in this effort that could lead to innovations in risk assessment.
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In 2009 he was awarded the new investigator travel award from the Genetic Toxicology Association for evaluating interlaboratory consistency of mitotic index data generated via flow cytometry. He is an active member of EMS, ISAC, and has participated in the Pig-a Assay Validation Breakout sessions hosted by ILSI-HESI. Since 2010 he has volunteered as the assistant treasurer for the GTA, and has just recently been accepted into the Joint Graduate Program of Toxicology, which is a cooperative effort of Rutgers University, the Robert Wood Johnson Medical School, and the Environmental and Occupational Health Sciences Institute.
Integration of Pig-A Gene Mutation Into Subchronic Toxicity Studies: the Dose Makes the Poison
During the “Stage 3” Pig-a interlaboratory validation trials, we assessed the genotoxicity of 4-nitroquinilone-1-oxide (4NQO) across 4 endpoints in multiple tissues: Pig-a mutant (i.e., CD59 negative) red blood cells (RBCs) and reticulocytes (RETs) by flow cytometry (FCM), micronucleated RETs (mnRETs) by FCM, DNA damage in blood, liver, and stomach via the alkaline Comet assay, and chromosome aberrations in peripheral blood lymphocytes. Integration to this degree conforms to the 3Rs initiative by reducing animal use during drug development. This 28-day oral gavage study in male Sprague-Dawley rats utilized doses up to and exceeding the MTD, with standard clinical chemistry and hematology analyses performed at termination. Biologically meaningful increases were observed only for Pig-a mutant RBCs/RETs and mnRETs, with the latter results potentially confounded by erythropoesis. A follow-up study was performed at the same cumulative doses administered in a single bolus, or split over 3 equal daily doses. The 3 daily dose regimen, which mimics the dosing protocol for a micronucleus/Comet combination assay, proved to be the most sensitive in detecting dose-dependent increases across most endpoints (i.e., Comet tail intensity in all three tissues, mnRETs, Pig-a mutant RBCs and RETs, and micronucleated bone marrow PCEs, which was added to this acute study). The Pig-a mutational endpoint in the 28-day Stage 3 study was the most robust in terms of hazard identification, and was the only persistent response during the recovery period after the acute treatments. Overall, these findings support the development of the flow cytometric Pig-a assay for use in regulatory genotoxicity studies, as compounds with a predominantly mutagenic mode of action can be identified with minimal impact to ongoing toxicological studies. However, these findings also demonstrate the caution that must be employed when integrating genotoxicity endpoints into subchronic studies, as none of the other in vivo endpoints used in the 28-day study (all recommended by ICH S2(R)), provided clear indications of genotoxicity for this known mutagen and carcinogen.
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