Biological Relevance and Health Concerns of Genotoxicity
October 24-25, 2012
John M. Clayton Hall Conference Center
University of Delaware
Speakers Bios, Abstracts & Presentations
Dr. Richard Albertini
University of Vermont
Dr. Albertini received the M.D. and Ph.D. degrees from the University of Wisconsin, Madison in 1963 and 1972, respectively. Between these dates he also received clinical training in Internal Medicine. Dr. Albertini's thesis research involved development of a method for quantifying HPRT mutations in diploid fibroblasts – a pursuit he then enlarged to studies of in vivo mutations of this same gene in human lymphocytes. A by-product of these studies begun as a graduate student is a blood test for the detection of Lesch-Nyhan heterozygosity in women.
Dr. Albertini has authored or co-authored approximately 190 papers and co-edited four books/journal issues. Among his awards are the Alexander Hollaender Award of the Environmental Mutagen Society in 1990, the St. George Medal of the American Cancer Society in 1990, was voted a University of Vermont Scholar for the year 2004-2005 and inducted into the Vermont Academy of Arts and Sciences in 2005. He was President of the Environmental Mutagen Society during 1983-1984 and the editor-in-chief of Environmental and Molecular Mutagenesis from 1988 to 1993. Dr. Albertini reviews papers in his area of expertise for many journals and has served on review and oversight boards for academia, industry and government. He currently consults for several industrial organizations.
Genetic toxicology: A tapestry of basic and clinical sciences
Genetic Toxicology is a true tapestry in the literary sense of the word – "an intricate combination of things or sequences of events". The applications of genetic toxicology have evolved over the past decades being primarily engaged in hazard identification to latter uses in devising and conducting molecular epidemiological studies. Data from both applications, when integrated with related information, have become essential elements for making human risk assessments for both cancer and inherited disorders. The juxtaposition of genetic toxicology with epidemiology has also lead to the use of the former to judge the biological plausibility of the latter. Genetic toxicology is also maturing sufficiently to impact the practice of medicine. Diagnostically, it may provide data for effective medical decision making while, therapeutically, it allows monitoring of interventions for disease prevention. Most of the applications and tools of genetic toxicology are too well known to be repeated. However, newer tools or better understandings of older tools are now allowing more realistic interpretations of studies aimed at hazard identification. Potential pitfalls, or perhaps unrealized opportunities, may lie ahead in some approaches to molecular epidemiology. The long-standing argument concerning threshold effects for mutagenic carcinogens, so important for making risk assessments, may be moving from semantics to experimentation, at least in the area of endogenous mutagens. The biological plausibility of the relationships of chemicals as diverse as 1,3-butadiene and formaldehyde to human leukemia can now be tested by using newer tools of genetic toxicology. Examples of the use of these tools for medical decision making and for monitoring anti-mutagenic interventions are also available. Although the enormous potential of "omics" of various sorts to enlarge the scope of genetic toxicology is recognized, their consideration will not be part of this discussion as the focus is on protection of the genome and the health consequences of its damage.
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Maik Schuler, Ph.D.
Pfizer Global Research and Development
Dr. Schuler received his Ph.D. from the University of Kaiserslautern in Germany where he worked on the detection of chromosomal damage induced by environmental and synthetic estrogens. During a postdoctoral fellowship with Dr. David Eastmond at the University of California, he developed fluorescence in situ hybridization methods for the detection of aneugenic and clastogenic damage in vitro and in rodent target tissues in vivo. Dr. Schuler joined Pfizer Global Research and Development in 2000 and is responsible for the risk management of early drug development candidates and development of novel risk management tools. He is involved in the International Working Groups on Genetic Toxicology (IWGT), the OECD in vitro micronucleus testing guideline working group, and the ILSI/HESI efforts developing follow-up strategies for in vitro genetic toxicology positive findings.
Mechanistic follow-up to in vitro micronucleus positives
The in vitro micronucleus assay has been extensively used for the genotoxicity screening of chemical compounds. Following the finalization of an OECD guideline (TG487) and with the implementation of the revised testing guideline ICH S2(R1), the in vitro micronucleus assay has now become an integral part of the regulatory in vitro genetic toxicology testing battery of pharmaceuticals. Using examples from the pharmaceutical industry, the presentation will show the impact of scoring criteria on weak increases in micronuclei and show approaches to distinguishing aneugenic from clastogenic modes of action. In addition, the presentation will provide examples on the impact of apoptosis induction on the interpretation of micronucleus results in the human lymphoblastoid cell line TK6 and identify follow-up approaches. Lastly, using kinase inhibitors as examples, approaches will be shown that can identify molecular targets for micronucleus induction in human U2OS cells.
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Professor Dr. Werner K. Lutz, born in Zürich, Switzerland, studied Chemistry at the Swiss Federal Institute of Technology (ETH Zürich) and received his doctoral degree in 1972. He was then trained in Experimental Medicine at the University of Zürich and was a Postdoc in Immune Biology at Rockefeller University in New York.
In 1975, he returned to Switzerland to join the new Institute of Toxicology of the ETH and the University of Zürich as a Research Group Leader for Chemical Carcinogenesis. He became Lecturer for Toxicology in 1980 and Professor in 1988. In 1994, he was appointed to the Chair of Toxicology at the Institute of Pharmacology and Toxicology of the University of Würzburg, Germany. He spent sabbatical leaves at Rutgers University, New Brunswick NJ (1988) and at the CIIT (now: The Hamner Institutes; 2003) in the Research Triangle Park NC. He retired in spring 2009.
Werner Lutz is well known for his investigations on DNA adduct formation of genotoxic carcinogens, defining in 1979 of the "Covalent Binding Index", CBI, as a quantitative indicator of carcinogenic potency. Mechanism-based cancer risk assessment formed the focus of his subsequent work. In 1990 he published a highly cited Commentary in the Journal "Carcinogenesis" on dose-response relationships in chemical carcinogenesis, which was seminal for the understanding of shapes of dose-response curves in this field. This also formed the basis to address other aspects of dose-response relationships in toxicology in general, including analysis of nonlinear and nonmonotonic shapes, as well as consideration of individual susceptibility as a basis for dose-incidence relationships. Among the 157 publications listed in PubMed, 53 included the medical subject heading "dose-response".
Dose response modeling and identifying thresholds: Mechanistic and statistical considerations
Debate on low-dose extrapolation is often complicated by misleading data presentation (logarithmic dose scaling), different definitions of “threshold” regarding the putative slope above control (>0 [monotonic increase], 0 [mathematical threshold], or <0 [nonmonotonic]), lack of distinction between the two types of response variable (continuous [biomarkers] vs. binary [incidence]), and misinterpretation of statistical test results. Exemplified by genotoxicity data and in considerations of the multi-stage process of chemical carcinogenesis in individuals, the following statements will be presented and discussed:
Continuous response variables: Proportionality ("linearity") between dose and response at lowest dose is a reasonable approximation for the dose-response relationship of reaction rates or biomarker concentrations. Deviation from linearity can be observed if the response is modulated, e.g., by saturation, inhibition, or induction of the processes involved. Superposition of the dose-response relationships of all contributing factors can result in an apparent threshold or even a nonmonotonic shape of the dose-response curve, but never in a mathematical threshold with slope 0 up to a break point and slope >0 thereafter.
Binary response variable: Risk assessment in humans is based on incidence data, i.e., on the question whether or not a defined effect is observed in an individual. The dose response relationship is given by a sequence of individual threshold doses required to produce the disease. The shape of the flight of stairs is the result of multiplicative combination of factors that determine individual susceptibility. For a complex disease such as carcinogenesis, the individual time-to-tumor is determined by multiplicative combination of susceptibility factors, the dose response can be approximated by a lognormal curve. To what extent an apparent threshold observed for a critical biomarker in animals can be applied for a risk assessment in humans, depends on the variability of the protecting factor(s) among individuals.
Statistics can indicate differences in data fit of different models but cannot prove any one model to be true. The Lutz & Lutz threshold test merely indicates whether a hockey stick model with initial slope zero provides a better fit to the data than a straight line (linearity). Statistical significance to reject linearity indicates a clear upward convex shape of the dose-response curve but is no proof that the – mechanistically unrealistic – alternate hypothesis is true.
Kerry Dearfield, Ph.D.
Overview of food safety regulations
The food supply in the United States is among the safest in the world; the U.S. government regulates the nation’s food producers and manufacturers to assure safe food and reduce foodborne diseases. The Centers for Disease Control (CDC) estimate that 1 in 6 Americans (or 48 million people) become sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases annually. In the U.S., several Federal agencies are responsible for the safety of the food supply, including USDA’s Food Safety and Inspection Service (FSIS), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). Each of these agencies has specific statutes that determine their food safety mandate. For example, FSIS regulates the safety and wholesomeness of the meat, poultry and egg products in the U.S. food supply. FDA regulates the safety of all other food commodities as well as feed and drugs for food producing animals. EPA regulates pesticide residues that can end up in different foods. These agencies are involved in many activities such as monitoring the food supply, safety testing, and performing risk assessments to assess the safety of food. Specific areas examined include bacterial and chemical contamination of food, food additives, veterinary drug residues, pesticide residues, and dietary supplements. Emerging issues, such as nanomaterial additions to food, also are being assessed. Because the U.S. food supply is tied closely to the global economy, international efforts to ensure food safety and effective trade are also part of the Federal government’s attention.
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Ms. Beyer received her BS from the University of California at Berkeley, Berkeley, CA (1977) and her MS in Environmental Health Science with a specialty in Occupational Health and Safety from the Harvard School of Public Health, Boston, MA (1982). She is a Diplomate of the American Board of Toxicology.
Ms. Beyer has made numerous presentations at meetings of the Society of Toxicology, in addition to presenting "Structure-Function Claim Development & Substantiation in a Competitive World" at the American Herbal Products Association Tele-Seminar: Structure/Function Claims: How to Craft Smart and Lawful Marketing Info and Labels, June 28. 2007. She has coauthored multiple publications including "Effects of MTBE on the reported incidence of Leydig cell tumors in Sprague-Dawley rats: Range of possible Poly-3 results." Regul. Toxicol. Pharmacol (2008) and “Historical perspective on the use of animal bioassays to predict carcinogenicity: Evolution in design and recognition of utility.” Crit. Rev. Toxicol. (2008). She is presently secretary of the Toxicology Committee of the American Industrial Hygiene Association.
Safety testing for dietary supplements
Use of dietary supplements is common among the U.S. adult population. Over 40% used supplements in 1988–1994, and over one-half in 2003–2006. This presentation will provide a brief overview of the requirements for testing of dietary supplements for safety, with an emphasis on the genetox testing. Dietary supplements are regulated primarily by FDA under the Dietary Supplement Health and Education Act (DSHEA) (October 1994). DSHEA regulates the safety of dietary supplements (vitamins, minerals, herbs or other botanicals, amino acids, or substances in the diet), primarily through Pre-marketing Notifications (PMNs) for new dietary ingredients (NDIs). PMNs must be filed at least 75 days before the product goes to market and must demonstrate that the NDI is safe through providing information on the history of use or other evidence of safety such as testing results. Relatively few PMNs have been filed and most have been rejected by FDA, many for a lack of information showing that the product is safe. Although not specifically codified by FDA, tests conducted typically include in vitro genotoxicity studies, animal studies, and clinical studies. Some examples of FDA's assessments will be provided to give some sense of what is required, which can be gleaned from reviewing submissions that are either "accepted" or rejected, as provided in the database created by the American Herbal Products Association (AHPA). In addition, the National Toxicology Program (NTP) has tested or is in the process of testing a number of dietary supplements identified based on potential toxicity associated with high dose or prolonged use. Some of these results will be presented and discussed in the context of relevant PMNs and FDA's reaction to them. For example, in response to a filing for a supplement consisting of tarragon extract, FDA noted that tarragon contains two compounds, estragole and methyleugenol, which, as determined by NTP, caused cancer in rats and mice but do not induce mutations in the types of bacterial mutagenesis assays used in the test results provided to FDA. Finally some compounds, which are generally recognized as safe (GRAS) in food, have been identified as being of potential health concern when present in dietary supplements (e.g., estragole) based on a concern for cancer; we will look at this apparent conundrum.
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Thomas C. Zebovitz, Ph.D.
Dr. Zebovitz earned his BS in Chemistry from University of Delaware (1977), and then did Master’s work at University of Minnesota, working on Prof. Eddie Leete. He earned his PhD at the University of Vermont, contributing to the synthetic chemical knowledge of dimeric indole alkaloids under Prof. Martin Kuehne (1984). He then moved to Columbia University for postdoctoral study with Prof. Gilbert Stork. There, he worked on the chemical synthesis of vitamin D analogues using a new radical coupling technology developed in Prof. Stork’s labs.
Dr. Zebovitz has authored or co-authored 20 peer-reviewed publications and edited a book on flavor and fragrance chemicals. He has 9 US and international patents.
Food contact substances: Safety at the consumer, technology, and regulatory interfaces
Since the days of the general store, where it was practically nonexistent, food packaging has come to play an important role in the transport, presentation, and safety of almost all food sold in the US. In the Food and Drug Administration Modernization Act of 1997, Congress established the Food Contact Notification Program for authorizing the safe use of food contact substances. Today, all packaging contacting food must have FDA clearance before it is released into the market. This presentation will cover the types of food additives and ingredients, how to determine the regulatory status of a food contact substance, and the options for premarket review of the safety of food contact substances.
Presentation not available
Dr. Goodman received an S.B. in Environmental Engineering in 1996 from the Massachusetts Institute of Technology (Cambridge, MA); she earned an Sc.M. in Epidemiology and a Ph.D. in Environmental Health Sciences/Toxicology in 2000 and 2002, respectively, from the Johns Hopkins Bloomberg School of Public Health (Baltimore, MD). From 2002 to 2004, she was a Cancer Prevention Fellow at the National Cancer Institute in the Laboratory of Human Carcinogenesis. Here she conducted several molecular epidemiology studies on colon, breast, and prostate cancer and was instrumental in the development of "Polymorphism Interaction Analysis," a statistical tool for cancer risk assessment.
Dr. Goodman has authored and co-authored more than 60 original research articles, review articles, and book chapters on a wide variety of topics related to epidemiology, toxicology, and risk assessment, including weight-of-evidence analyses of several chemicals. She also has presented scientific findings to community groups and state and federal regulatory and legislative bodies, such as US EPA's Clean Air Scientific Advisory Committee, the National Toxicology Program's Board of Scientific Councilors, and US Congress. She is an active member in the Society of Risk Analysis and has held elected positions in the Society of Toxicology and the American Board of Epidemiology. She is an editorial board member of Carcinogenesis and The Open Biomarkers Journal. In 2012, Dr. Goodman received the International Dose-Response Society Outstanding New Investigator Award.
Overview of the controversy surrounding bisphenol toxicity
Many of the controversies surrounding low-dose endocrine disruption (ED) are similar to those that are emerging in the field of genotoxicology. Therefore, an understanding of ED issues may be informative for chemicals that test positive in some, but not all, genotoxicity assays. Bisphenol A (BPA), the most studied and debated endocrine disruptor, is a good example of current controversies. Two major issues are how to evaluate effects at low doses and how to synthesize conflicting test results. Proponents of ED at low doses assert that standard toxicity tests miss effects not immediately evident as apical endpoints; look only for traditional toxicities, missing endpoints uniquely relevant to ED; and don’t allow for the exploration of non-linear dose-response curves. They suggest that studies using novel methods are able to address these issues and, although none have been validated, EPA is considering whether these new methods should be required. Low-dose proponents also generally assume that statistically significant biological perturbations indicate causation of an adverse effect. Further, they evaluate studies differently depending on whether the results are positive or negative. For BPA, some toxicity studies have reported low-dose endocrine activity, but they are countered by null findings in more numerous studies; have not been replicated; do not exhibit coherence and plausibility; do not show consistency across species, doses, and time points; and/or are from studies using non-oral exposure routes. Statistical associations in epidemiology studies are not coherent with the toxicity, mechanistic, and pharmacokinetic BPA data. Several government bodies and expert panels around the world conclude that the evidence does not support low oral doses of BPA adversely affecting human health. Nonetheless, citing a precautionary approach, some government agencies have banned certain products made with BPA. Rather than being precautionary, these bans may lead to increased health risks from the widespread use of replacement chemicals that have not been tested as thoroughly as BPA.
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Mr. Seipelt received his BS in Forensic Chemistry from Ohio University in Athens, OH (1983). Since joining Abbott in 1985, he has held various positions in the Analytical R&D laboratories focusing primarily on analytical method development and the use of mass spectrometry in nutritional research. Projects included trace organic analysis for food safety, high throughput quantitative analysis of micronutrients, and proteomics applications for nutritional ingredients.
An active member of the American Society for Mass Spectrometry and AOAC International, Mr. Seipelt is a regular presenter at conference meetings and the co-author of several publications. Within AOAC International, he serves on the Stakeholder Panel on Infant Nutrition and Adult Nutritionals (SPIFAN), the Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM), and is member of the Method Centric Committee on Melamine.
Food contact materials: Considerations for infant formula packaging
Food packaging is regulated in many countries based on the assessment of the identity and amount of chemical components that migrate from the food contact materials into the food. The regulations define safe levels of exposure to these chemicals as a result of their use in food packaging. Although there is typically no differentiation drawn between infant formulas and general foods in the regulations, infant formulas can present unique technical challenges in the assessment of packaging materials due to the unique population served by these products. Increasingly, lower exposure limits are requested for infant formula packaging, challenging the technology used in typical packaging qualification. Current challenges and considerations for infant formula packaging will be discussed.
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Dr. Kristi Muldoon Jacobs is currently a toxicology reviewer at the Office of Food Additive Safety (OFAS) at CFSAN, and the lead of the Division of food contact notifications (DFCN) Genetox Evaluation Team, and OFAS Structure Activity Relationship (SAR) team. In her current position she is responsible for reviewing safety information and toxicology data in premarket notifications for food-contact substances, providing recommendations for remedying deficiencies, and offering advisory opinions to industry. As team leader for the Genetox and SAR teams she has had extensive experience using non-standard techniques for identifying data needs and filling data gaps, as well as using weight of evidence approaches to overall safety assessments for DFCN.
Dr. Muldoon Jacobs received her BS in biochemistry from Richard Stockton College of NJ (1999) and a Ph.D in Biomedical Sciences from the University of Medicine and Dentistry of NJ (2006). As a graduate student she studied ribosome structure-function in yeast using forward and directed mutation studies. Her postdoctoral training was done at the NIH National Cancer Institute in the Radiation Oncology Branch where she studied reactive oxygen signaling effects on epigenetics and carcinogenesis in cell culture and animal models.
Dr. Muldoon Jacobs consistently publishes peer-reviewed publications in her field, has authored over 100 review memorandum for DFCN, and has represented the FDA at several conferences as an invited speaker. She was awarded the CFSAN outstanding new reviewer award in 2010, an exceptional achievement award in 2009, and several NIH service awards. She is an active member in the OECD genetox and cell transformation working groups as well as the OECD endocrine disruptor group. She is also an expert member for ILSI TTC working groups. She is a member of SOT and ACR.
Genotoxicity and safety assessment: Purposes and approaches
Genetic toxicity information is critical for the safety assessment of all substances. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about a substance’s potential for carcinogenicity. Genetic toxicity assessments are conducted using a weight of the evidence approach considering all information including: the quality of the genetic toxicity studies, the array of positive and negative genetic toxicity test results, other available safety data, and the chemical structure of the substance.
OFAS has published guidance for stakeholders which outlines tests that are generally recommended for a genetic toxicity evaluation. The testing recommendations are exposure based, and the amount of testing increases with exposure. For substances and constituents with exposures less than 0.025 µg/kg bw/day, OFAS recommends an updated literature search and an evaluation of the compound structure; at 0.025 µg/kg bw/day two in vitro Genetox tests are recommended; in the case of a positive result, or at 2.5 µg/kg bw/day a follow up in vivo test is recommended. A discussion of the specific tests and examples of how these tests are evaluated and interpreted will be presented.
To the extent feasible, discussions or explanations that predict toxicity based on structure/activity relationships may be incorporated into the safety assessment of substances. When appropriate, expert analysis, or computer-assisted quantitative structure/activity techniques may be used to relate the chemical structure of a substance with potential genotoxicity or carcinogenicity. In some cases of low exposures, where data suggests the compound is carcinogenic, read-across approaches can be used to identify analogs useful for risk assessment. Such information should not be considered as a substitute for actual data, but may be useful in developing an overall strategy for assessing the safety of a substance or performing risk assessment for compounds with low exposures.
Presentation not available
Dr. Maier’s career is focused on health risk assessment and risk policy. He is experienced in pharmaceutical, food, environmental and occupational toxicology and risk assessment. As a commercial pilot, Dr. Maier’s risk assessment practice includes assessment of human risk factors.
Dr. Maier earned a BS in Chemistry and a BS in Biology from Metropolitan State College, Denver, CO, a MS in Environmental Health and a PhD in Toxicology from Colorado State University, Fort Collins, CO. He also holds certificates in economics and project management.
Dr. Maier has authored or co-authored publications primarily in the areas of molecular toxicology and chemical risk assessment. He is certified in general toxicology by the American Board of Toxicology.
Transcriptional activation as a feferee for in vitro genotoxicity
In the safety assessment of food-contact substances, when cumulative daily intake exceeds 150 µg per person per day, FDA guidance (July 2007) recommends “a test for gene mutations in bacteria” and “an in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells” OR “an in vitro mouse lymphoma thymidine kinase+/- gene mutation assay” and “an in vivo test for chromosomal damage using mammalian hematopoietic cells.” In practice, this recommendation is often fulfilled with the GLP Ames and the chromosomal aberrations (CA) assays, with and without S9 metabolic activation.
Of particular interest to coating developers are diglycidyl ethers of diphenols (DGEs) used to make epoxy-based food-contact substances. DGEs are especially problematic because they have oxirane moieties that are alerting for mutagenicity and thus must be shown to be non-genotoxic. Although known to be non-carcinogenic, DGEs such as bisphenol-A diglycidyl ether (BADGE), consistently produce base-pair substitutions in tester strains TA100 and TA1535 of the Ames assay. In addition, DGEs are often cytotoxic enough to cause non-genotoxic, cytotoxicity-induced chromosomal aberrations in the CA assay.
When first-tier in vitro genotoxicity screening tests are positive, food contact material innovators need a way to decide if these tests may be false positive and might be overcome with more expensive and time-consuming second-tier in vivo assays such as the single cell gel electrophoresis assay (comet assay) and/or the in vivo micronucleus assay.
We evaluated the Ames II microformat, the mouse lymphoma microtitre screen and the GreenScreen HC®* GADD45 transcriptional activation assays for their ability to correctly predict the outcome of second-tier in vivo assays. The Ames II and the GreenScreen assays correctly foretold negative mutagenicity results in vivo. The Ames II microformat however, was not consistent with results from traditional Ames assay that it is supposed to emulate. The mouse lymphoma microtitre screen consistently produced results opposite the second-tier comet assay. Although our sample size was small, the GreenScreen assay correctly refereed positive Ames/CA or mouse lymphoma microtitre/CA first-tier screening results as ultimately being either positive or negative for mutagenicity and/or clastogenicity in second-tier in vivo assays.
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Research and Development - Environmental, Health and Safety
Dr. Gould received her BS from University of Minnesota, Twin Cities, MN in Biology and Chemistry (1990); and her Ph.D. in Toxicology from Rutgers University and University of Medicine and Dentistry of New Jersey, New Brunswick, NJ. The focus of her research was on the effect of polychlorinated biphenyls on chicken embryo growth (1995). Her post-doctoral training was done at the Chemical Industry Institute of Toxicology in Research Triangle Park, NC where she examined the estrogenic potential of bisphenol A in vitro and in multigenerational rat studies.
Dr. Gould has presented a number of posters and oral presentations related to occupational toxicology at SOT, Society for Risk Assessment and the Occupational Toxicology Roundtable. She has authored a journal article on dermal sensitization, and several Worker Environmental Exposure Limits (WEEL) as a member of the American Industrial Hygiene Association WEEL committee. Dr Gould is active in the Mid Atlantic Society of Toxicology, currently serving as president. She is a Diplomat of the American Board of Toxicology.
Evaluation and assignment of occupational exposure control bands to chemicals with
limited toxicity data
In the pharmaceutical industry, the synthesis of the final drug molecule includes multiple chemical reactions, which result in the isolation of chemical intermediates. These are distinct molecular entities that have limited hazard information which are generally not chemically, pharmacologically or toxicologically similar to the well-studied drug. Companies are required by the various chemical regulations (e.g., US OSHA) to provide hazard information and safe handling guidance for employees. Each isolated intermediate is evaluated for hazards by computer structure activity relationship analysis using DEREK software. Additionally, limited toxicity testing including the Ames, acute oral toxicity, dermal irritation, skin irritation and skin sensitization tests are performed. Based on these results, an Exposure Control Band (ECB) is assigned. This is an air concentration range of the compound that dictates what engineering controls and personal protective equipment are needed in order to ensure safe handling. For employees that work with Ames positive compounds, an ECB is derived using the concept of the threshold of toxicological concern and considerations of the appropriate risk level. To provide a scientific justification for the end points for the other four tests, a comparison of the ECB assignments with the literature and government publications (e.g., UK COSSH) is described. Based on the approaches described in this presentation, the health of Bristol-Myers Squibb workers is protected when handling pharmaceutical intermediates controlled to the appropriate hazard-based ECB.
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Impact of new mutagenicity data on marketed drugs: A case study
Recently, new mutagenicity data for reagents commonly used in the synthesis of pharmaceuticals triggered mutagenicity testing and analytical work to determine the presence or absence of these potential impurities. In all cases, the new mutagenicity data was discovered on an MSDS from our vendor, presumably in preparation to register EDAC in the REACH process. A case study for one of these compounds, EDAC [1-(3-dimethylaminopropyl)-3-ethylcabodiimide hydrochloride, CAS # 25952-53-8] will be discussed including risk evaluation. EDAC is a widely used reagent for forming amide bonds during the synthesis of pharmaceutical compounds. In the presence of water EDAC rapidly degrades to a urea derivative. Mutagenicity data for EDAC and the urea derivative (EDAU) will be presented as well as data from a 1-month repeat dose genotoxicity assay to further investigate genotoxic potential. Data from Pig-a erythrocyte mutation, micronucleus induction in blood, and Comet DNA damage assessment in liver will be presented. Internal processes put in place to mitigate risk from future instances of new mutagenicity data will be highlighted.
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Acceptable daily exposure (ADE) value as a harmonized approach in
toxicology risk assessment
An effort to develop a clear guidance on conducting toxicological assessments to support quality risk management programs was recently initiated by European Medicines Agency (EMA). Any technical guidance needs to be consistent, providing a rationale, scientific approach, using concepts of risk assessment congruous with those articulated in ICH Q9, to ultimately identify exposure limits for cross-contamination that protect patients. The concern arises that in the absence of any guidance and with a plethora of toxicological tools being available a lack of harmonized interpretation could occur both in pharmaceutical industry and National Competent Authorities. In this presentation, the concept of Risk-MaPP (Risk-based Manufacture of Pharmaceutical Products), an ISPE (International Society of Pharmaceutical Engineering) good practices guidance document will be elaborated with particular emphasis on deriving an Acceptable Daily Exposure (ADE) for pharmaceuticals. The application of the Threshold of Toxicological Concern (TTC) concept for compounds with limited or no toxicity information to support pharmaceutical manufacturing operations will also be highlighted.
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Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, NCI, NIH, DHHS, Bethesda, MD
B.S. (Honors) and M.S. in Biochemistry from University of Stirling, UK in 1976 and 1978, respectively. Her M.S. thesis was on Nucleic acid synthesis during the germination of Aspergillus nidulans. In 1979 she completed a Certificate in Education, University of Manchester, UK. Dr. Sinha went on to complete a Ph.D. in Nutritional Science from University of Maryland, College Park, Maryland in 1987. There thesis was on Age, nutrition, and bone metabolism: Analyses of effects using a short-term in vivo bone model.
Dr. Sinha was awarded a fellowship from the Japanese Foundation for Cancer Research in 2001 and 1997, Sigma Xi Distinguished Lectureship 2005; and invited to present at the prestigious 38th International Princess Takamatsu Symposium, Tokyo, Japan. Dr. Sinha has received the NIH Award of Merit, Technology Transfer Award, DCEG Special Appreciation Award, and several performance awards.
Dr. Sinha has co-authored over 180 peer-reviewed publications. She was one of the founding members of the steering committee of the Molecular Epidemiology Group of the American Association for Cancer Research from 1997-1998. She was a member of the working group for the IARC Monograph on the Evaluation of Carcinogenic Risks to Humans, (Vol. 92), and was a reviewer for the American Institute for Cancer Research/World Cancer Research Fund report Food, Nutrition, Physical Activity, and Prevention of Cancer (2007 and 1997 editions). She was the president of the 8th International Conference on Carcinogens and Mutagenic–substituted Aryl Compounds and edited a special issue of Mutation Research – Fundamental and Molecular Mechanisms in 2002. She serves on the Editorial Board of Cancer Epidemiology, Biomarkers and Prevention, and the journal of the Japanese Cancer Association, Cancer Science .
Epidemiology of food carcinogens in red and processed meat intake
Epidemiologic evidence suggests that high intake of red meat and processed meat, especially cooked well-done at high temperatures, increases risk for certain types of cancers. Meat may contain a variety of potentially carcinogenic/mutagenic compounds, such as heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) in meat cooked at high temperatures, heme iron, and nitrate/nitrite in processed meat. With a meat-cooking questionnaire and quantitative databases, we estimated intake of three HCAs: 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a marker of PAHs: benzo[a]pyrene (B[a]P), total mutagenic activity of meat, heme iron, and nitrate/nitrite in multiple prospective studies and evaluated odds ratios (OR) or hazards ratios (HR) and 95% confidence intervals (CI) for various cancers. We found in case-control studies that heterocyclic amines (HCAs) were significantly associated with tumors of the colorectum, breast, stomach, pancreas, and lung and that benzo[a]pyrene (B[a]P) was associated with an increased risk of colorectal adenoma. In contrast, we did not find associations with either bladder or Non-Hodgkin's lymphoma. In a prospective study, the NCI Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, we found a modest increased risk for colorectal adenomas with high intake of red meat, well-done red meat, two HCAs (MeIQx and PhIP), and B[a]. We also observed an association between higher intake of processed meats such as bacon and sausage with increased risk of colorectal adenomas. There was no association with intake of total meat, red meat, or white meat. However, the highest category of very well-done meat consumption (>10 g/day) was associated with a significant increased incidence of prostate cancer when compared to those who did not consume well-done meat. Furthermore, intake of PhIP was associated with a significant increased incidence of prostate cancer in men in the highest quintile of intake. The carcinogenic potential of these compounds depends on phase I and II xenobiotic metabolizing enzymes (XMEs); therefore, we evaluated the role of 29 single nucleotide polymorphisms in phase I and II XME genes in relation to advanced colorectal adenoma from the PLCO study. Although there were no main effects of the SNPs on adenoma risk, there were suggestive interactions with HCAs. In summary, even though the results from case-control studies lend support to an association between meat cooking-mutagens and cancer larger prospective studies are not consistent. Other components of red meat, such as heme iron, fat, nitrite/nitrosamine need to be explored further.
Presentation not available
Interference with the UVC-induced DNA damage response by tumor promoters
Abstract coming soon.
Presentation in PDF
Comparison of closely related human cell lines and their p53 functionality in the in vitro micronucleus assay
Abstract coming soon.
Presentation in PDF
David Jacobson-Kram, Ph.D., D.A.B.T.
US-Food and Drug Administration
Center for Drug Evaluation and Research
Office of New Drugs
S2(R1), finally final; M7, impurity thoughts
ICH S2(R1) was finalized in November of 2011 and published in the Federal Register in June of 2012. The revised guideline included surprisingly few changes since its original publication in 1995 and 1997. Major changes include the option of performing an in vitro micronucleus assay as a mammalian clastogenicity test, a choice between performing the genetic toxicology battery as described in the original guideline or choosing instead to perform an in vivo study with two endpoints and lowering the top concentration in in vitro mammalian cell assays to 1 mM or 0.5 mg/ml, whichever is lower. In an effort to reduce the use of animals, positive control animals do not have to be included in every iteration of an in vivo assay.
The initiation of the ICH M7 expert working group was 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, a 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 to 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) how should specifications be set higher for drugs that consumed for less than a lifetime.
Presentation not available
Current Employment and responsibilities:
Dr. Yoshifumi Uno is currently the General Manager of Safety Department I of Safety Research Laboratories at Mitsubishi Tanabe Pharma Corporation, in Japan. His current research covers all fields of toxicology including safety pharmacology, and he has responsibility for preclinical toxicity evaluation in the stage of pharmaceutical candidate selection at the company. Prior to joining Mitsubishi Tanabe Pharma, he was the Manager of genetic toxicology for Mitsubishi Chemical Corporation (now Mitsubishi Chemical Holdings Corporation). He was also a lecturer of genetic toxicology at Tokyo University of Agriculture and Technology (1994-2006).
Education and post-doc training:
Dr. Uno received his BS (1984) and MS (1986) from Tokyo University of Agriculture and Technology, Tokyo, Japan, and Ph.D. (1994) from Gifu University, Gifu, Japan. He has a license of D.V.M. (Doctor of Veterinary Medicine).
Publications, professional affiliations, awards, etc:
Dr. Uno has authored or co-authored about 30 peer-reviewed publications. He is the Executive Board member of the Japanese Environmental Mutagen Society (JEMS), the Steering Committee member of the International Workshops on Genotoxicity Testing (IWGT), the Validation Management Team member of the Japanese Center for the Validation of Alternative Methods (JaCVAM) International Comet Assay Validation Studies, and the Editorial Board member of Genes and Environment, an official journal of JEMS. He is also a member of the JEMS/Mammalian Mutagenicity Study group (MMS) and the Japanese Cancer Association. He has been the recipient of JEMS Encouragement Award (1999) from JEMS.
Update of the status of the JaCVAM organized international in vivo comet assay validation study
The in vivo rodent alkaline comet assay is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese JaCVAM, with cooperation of the U.S. NICEATM and ICCVAM, the European ECVAM, and the Japanese JEMS/MMS, organized an international validation study to evaluate the reliability of the assay using liver and stomach as target organs following multiple treatments and to assess its ability to identify genotoxic carcinogens, thereby serving as a potential predictor of rodent carcinogenicity. The ultimate goal of this exercise is to establish an OECD guideline. The validation effort involved 4 phases. The study protocol was optimized in the 1st to 3rd phases. In the 4th phase-1st step, assay reproducibility was confirmed among laboratories using four coded test chemicals and the positive control ethyl methanesulfonate. In the 4th phase-2nd step, the predictive capability for detecting genotoxic carcinogens was investigated using 40 selected coded chemicals with known genotoxic and carcinogenic activity. The validation study involved 14 laboratories in Japan, North America, and Europe. Classification of results as positive or negative was based on a pre-established statistical approach that considered dose response and increased migration at individual dose levels. For genotoxic carcinogens, 13 of 19 tested were positive in at least one tissue. The six negatives included two aneugens and a cross-linker, which represent modes of activity not expected to be detected by the comet assay. After excluding those three, assay sensitivity was 81% (13/16). For non-genotoxic non-carcinogens, 7 of 8 tested were judged to be negative giving an assay specificity of 88%. Five of the 6 (83%) non-carcinogens with genotoxic activity and 7 of 7 (100%) of the non-genotoxic carcinogens were judged to be negative. Therefore, the concordance for expected assay results is calculated to be 80% (32/40) or 86% (32/37 excluding two aneugens and a cross-linker), indicating that the in vivo comet assay is highly capable of identifying genotoxic chemicals and therefore serve as a potentially reliable predictor of rodent carcinogenicity. A combination comet and micronucleus assay would therefore be recommended for screening the in vivo genotoxic potential of test chemicals.
Presentation in PDF
Genetic toxicology testing under REACH
REACH (Registration, Evaluation, Authorization, and Restriction of Chemical substances) is a single regulatory system in the European Union for new and existing chemicals. All substances manufactured or imported into the EU are affected by this legislation with a few exceptions. Registration requirements for substances are dependent upon production volume while authorization is required for substances of high concern (e.g., carcinogenic, mutagenic, or reprotoxic, category 1 and 2). Tests for genotoxicity are an integral part of REACH data requirements. A bacterial mutagenicity assay (Ames test) is required for all substances with production volume of 1 ton/year or more. Additional tests for cytogenetic damage and gene mutations using mammalian cell cultures are required if the Ames test is positive or when the production volume is ≥10 tons/year. If all the in vitro assays are negative, no further in vivo testing is required with few exceptions (e.g., metabolism considerations). Positive results in one or more of the in vitro assays trigger the consideration for in vivo studies for clastogenicity (micronucleus, chromosomal aberrations, or Comet) and/or mutagenicity (transgenic animals, UDS, or Comet). No further testing is generally required if the in vivo test results are negative (the substance declared as non-genotoxic) or positive (the substance declared as somatic as well as germ cell mutagen). Tests for genotoxicity in germ cells are required only in rare exceptions based on expert judgment and to resolve any uncertainties. For substances that are classified as category 1 or 2 mutagens, a test for carcinogenicity is not normally required based on the default assumption that a genotoxic mechanism for carcinogenicity is likely for these substances.
Presentation not available
Dr. Kim 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).
Dr. Kim 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). Jim became a DABT in 2007.
Updates for the HESI IVGT project committee
The Health and Environmental Sciences Institute (HESI) is a non-profit industry-sponsored research institute, with the mission of engaging scientists from academic, government and industry to identify and resolve global health and environmental issues. HESI 's scientific programs bring together scientists from around the world from academia, industry, and regulatory agencies and other governmental institutions, to address and reach consensus on scientific questions that have the potential to be resolved through creative application of intellectual and financial resources. This “tripartite” approach forms the core of every HESI scientific endeavor.
The Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing Project Committee (IVGT) was established in 2006 with the mission of: 1) improving the scientific basis of the interpretation of results from in vitro genetic toxicology tests for purposes of more accurate human risk assessment, 2) developing follow-up strategies for determining the relevance of in vitro test results to human health, and 3) providing a framework for integration of in vitro testing results into a risk-based assessment of the effects of chemical exposures on human health. The IVGT is now broadening its active project areas to address other important issues in genetic toxicology. In November of 2012, the IVGT will petition the HESI Program Strategy and Stewardship Committee to transition to a Technical Committee status with a broader mission and additional objectives. The Technical Committee will be called the Genetic Toxicology Technical Committee (GTTC). GTTC will continue to work on 1) the Improvement of Existing Assays (e.g. cell repository network and cell characteristics for genotoxicity evaluation) and 2) the Quantitative Evaluation of Genotoxicity (e.g. Workshop on In Vitro to In Vivo Extrapolation). In addition, the Committee is currently forming new Workgroups in the following areas: 1) Interpretation of Recently Implemented Assays (in vitro micronucleus, Comet assay, transgenic animals), 2) Evaluation of New Models in Germ Cells, 3) Evaluation of New Compound Genotoxicity (nanomaterials, biologics), 4) Framework for Adoption of New Test Methods, and 5) Clean Sheet Testing Strategy. The GTTC will provide genetic toxicologists with an ongoing forum for discussions of critical issues, and the ability to collaborate on projects important for these discussions.
OECD test guidelines for genetic toxicology – Overview of ongoing
revisions and updates
This presentation will provide a condensed but comprehensive oversight of the ongoing effort to revise and update the entire set of the test guidelines (TGs) on genotoxicity of the Organisation for Economic Co-operation and Development (OECD). The presentation will give an update of the ongoing work of the Expert Group as per October 2012. Included is information on the projected future fate of the individual TGs; highlights of the most relevant expected study design and conceptual changes including but not limited to selection of cell lines; selection of top dose level (particularly for the in vitro mammalian cell based assays); considerations of statistical power and sample sizes; number of dose levels to be included in the studies; use of automated systems of analysis; and verification of laboratory proficiency.
As many in the audience of this meeting know, the OECD TGs for the testing of chemicals are almost universally applied to in vitro and in vivo studies used for safety assessment. These data are generally accepted by regulatory agencies, with stronger adherence by the OECD member countries falling under the OECD Mutual Acceptance of Data (MAD) for studies conducted under OECD TGs and Good Laboratory Practice (GLP). The guidelines also have taken on a central role for the everyday professional life of many of us. The first set of TGs on genotoxicity was adopted 30 years ago (1983-1986), and revisited in 1997, when TG 486 was added and TG 472 was combined with TG 471. For the past 15 years there have been no revisions or updates, with the exception of the adoption of two new guidelines; TG 487 (in 2010) and TG 488 (in 2011). Meanwhile the experiences from using the TGs have increased in parallel to the advances in scientific knowledge, and there has been an opportunity for retrospective evaluation of the actual predictability and reliability of the guidelined studies. Enough evidence and conceptual change has gathered over time to prompt a review of the current set of OECD TGs on genotoxicity, which are presently under study by an expert group to the OECD. This group will ultimately work its way to a recommendation of an updated set of TGs for member country approval.