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Gollapudi B.B.,Dow Chemical Company | Johnson G.E.,University of Swansea | Hernandez L.G.,National Health Research Institute | Pottenger L.H.,Dow Chemical Company | And 11 more authors.
Environmental and Molecular Mutagenesis | Year: 2013

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL10) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data. © 2012 Wiley Periodicals, Inc.

PubMed | ILSI Health and Environmental science Institute HESI, Environmental Health Science and Research Bureau, National Institute for Public Health and the Environment RIVM, University of Swansea and 10 more.
Type: | Journal: Environmental and molecular mutagenesis | Year: 2016

For several decades, regulatory testing schemes for genetic damage have been standardized where the tests being utilized examined mutations and structural and numerical chromosomal damage. This has served the genetic toxicity community well when most of the substances being tested were amenable to such assays. The outcome from this testing is usually a dichotomous (yes/no) evaluation of test results, and in many instances, the information is only used to determine whether a substance has carcinogenic potential or not. Over the same time period, mechanisms and modes of action (MOAs) that elucidate a wider range of genomic damage involved in many adverse health outcomes have been recognized. In addition, a paradigm shift in applied genetic toxicology is moving the field toward a more quantitative dose-response analysis and point-of-departure (PoD) determination with a focus on risks to exposed humans. This is directing emphasis on genomic damage that is likely to induce changes associated with a variety of adverse health outcomes. This paradigm shift is moving the testing emphasis for genetic damage from a hazard identification only evaluation to a more comprehensive risk assessment approach that provides more insightful information for decision makers regarding the potential risk of genetic damage to exposed humans. To enable this broader context for examining genetic damage, a next generation testing strategy needs to take into account a broader, more flexible approach to testing, and ultimately modeling, of genomic damage as it relates to human exposure. This is consistent with the larger risk assessment context being used in regulatory decision making. As presented here, this flexible approach for examining genomic damage focuses on testing for relevant genomic effects that can be, as best as possible, associated with an adverse health effect. The most desired linkage for risk to humans would be changes in loci associated with human diseases, whether in somatic or germ cells. The outline of a flexible approach and associated considerations are presented in a series of nine steps, some of which can occur in parallel, which was developed through a collaborative effort by leading genetic toxicologists from academia, government, and industry through the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC). The ultimate goal is to provide quantitative data to model the potential risk levels of substances, which induce genomic damage contributing to human adverse health outcomes. Any good risk assessment begins with asking the appropriate risk management questions in a planning and scoping effort. This step sets up the problem to be addressed (e.g., broadly, does genomic damage need to be addressed, and if so, how to proceed). The next two steps assemble what is known about the problem by building a knowledge base about the substance of concern and developing a rational biological argument for why testing for genomic damage is needed or not. By focusing on the risk management problem and potential genomic damage of concern, the next step of assay(s) selection takes place. The work-up of the problem during the earlier steps provides the insight to which assays would most likely produce the most meaningful data. This discussion does not detail the wide range of genomic damage tests available, but points to types of testing systems that can be very useful. Once the assays are performed and analyzed, the relevant data sets are selected for modeling potential risk. From this point on, the data are evaluated and modeled as they are for any other toxicology endpoint. Any observed genomic damage/effects (or genetic event(s)) can be modeled via a dose-response analysis and determination of an estimated PoD. When a quantitative risk analysis is needed for decision making, a parallel exposure assessment effort is performed (exposure assessment is not detailed here as this is not the focus of this discussion; guidelines for this assessment exist elsewhere). Then the PoD for genomic damage is used with the exposure information to develop risk estimations (e.g., using reference dose (RfD), margin of exposure (MOE) approaches) in a risk characterization and presented to risk managers for informing decision making. This approach is applicable now for incorporating genomic damage results into the decision-making process for assessing potential adverse outcomes in chemically exposed humans and is consistent with the ILSI HESI Risk Assessment in the 21st Century (RISK21) roadmap. This applies to any substance to which humans are exposed, including pharmaceuticals, agricultural products, food additives, and other chemicals. It is time for regulatory bodies to incorporate the broader knowledge and insights provided by genomic damage results into the assessments of risk to more fully understand the potential of adverse outcomes in chemically exposed humans, thus improving the assessment of risk due to genomic damage. The historical use of genomic damage data as a yes/no gateway for possible cancer risk has been too narrowly focused in risk assessment. The recent advances in assaying for and understanding genomic damage, including eventually epigenetic alterations, obviously add a greater wealth of information for determining potential risk to humans. Regulatory bodies need to embrace this paradigm shift from hazard identification to quantitative analysis and to incorporate the wider range of genomic damage in their assessments of risk to humans. The quantitative analyses and methodologies discussed here can be readily applied to genomic damage testing results now. Indeed, with the passage of the recent update to the Toxic Substances Control Act (TSCA) in the US, the new generation testing strategy for genomic damage described here provides a regulatory agency (here the US Environmental Protection Agency (EPA), but suitable for others) a golden opportunity to reexamine the way it addresses risk-based genomic damage testing (including hazard identification and exposure). Environ. Mol. Mutagen., 2016. 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Fenech M.,CSIRO | Holland N.,University of California at Berkeley | Zeiger E.,Errol Zeiger Consulting | Chang W.P.,Taipei Medical University Hospital | And 6 more authors.
Mutagenesis | Year: 2011

The International Human Micronucleus (HUMN) Project (www.humn.org) was founded in 1997 to coordinate worldwide research efforts aimed at using micronucleus (MN) assays to study DNA damage in human populations. The central aims were to (i) collect databases on baseline MN frequencies and associated methodological, demographic, genetic and exposure variables, (ii) determine those variables that affect MN frequency, (iii) establish standardised protocols for performing assays so that data comparisons can be made more reliably across laboratories and countries and (iv) evaluate the association of MN frequency with disease outcomes both cross-sectionally and prospectively. In the first 10 years of the HUMN project, all of these objectives were achieved successfully for the MN assay using the cytokinesis-block micronucleus (CBMN) assay in human peripheral blood lymphocytes and the findings were published in a series of papers that are among the most highly cited in the field. The CBMN protocol and scoring criteria are now standardised; the effect of age, gender and smoking status have been defined, and it was shown prospectively using a database of almost 7000 subjects that an increased MN frequency in lymphocytes predicts cancer risk. More recently in 2007, the HUMN coordinating group decided to launch an equivalent project focussed on the human MN assay in buccal epithelial cells because it provides a complementary method for measuring MN in a tissue that is easily accessible and does not require tissue culture. This new international project is now known as the human MN assay in exfoliated cells (HUMNxL). At present, a database for >5000 subjects worldwide has been established for the HUMNxL project. The inter-laboratory slide-scoring exercise for the HUMNxL project is at an advanced stage of planning and the analyses of data for methodological, demographic, genetic, lifestyle and exposure variables are at a final stage of completion. Future activities will be aimed at (i) defining the genetic variables that affect MN frequencies, (ii) validation of the various automated scoring systems based on image analysis, flow cytometry and laser scanning cytometry, (iii) standardisation of protocols for scoring micronuclei (MNi) in cells from other tissues, e.g. erythrocyte and nasal cells and (iv) prospective association studies with pregnancy complications, developmental defects, childhood cancers, cardiovascular disease and neurodegenerative diseases. © The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.

Zeiger E.,Errol Zeiger Consulting | Hoffmann G.R.,College of the Holy Cross, Worcester
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2012

A recent report (Calabrese et al., Mutat. Res. 726 (2011) 91-97) concluded that an analysis of Ames test mutagenicity data provides evidence of hormesis in mutagenicity dose-response relationships. An examination of the data used in this study and the conclusions regarding hormesis reveal a number of concerns regarding the analyses and possible misinterpretations of the Salmonella data. The claim of hormesis is based on test data from the National Toxicology Program using Salmonella strain TA100. Approximately half of the chemicals regarded as hormetic, and the majority of the specific dose-responses identified as hormetic, were actually nonmutagenic. We conclude that the data provide no evidence of hormetic effects. The Ames test is an excellent measure of bacterial mutagenicity, but the numbers of revertant (mutant) colonies on the plate are the result of a complex interaction between mutagenicity and toxicity, which renders the test inappropriate for demonstrating hormesis in bacterial mutagenicity experiments. © 2012 Elsevier B.V..

Haseman J.K.,J.K. Haseman Consulting | Growe R.G.,International Federation for Family Health | Zeiger E.,Errol Zeiger Consulting | McConnell E.E.,Toxpath Inc. | And 2 more authors.
Regulatory Toxicology and Pharmacology | Year: 2015

A rat carcinogenicity bioassay (CaBio) of quinacrine was reanalyzed to investigate its mode of tumor induction. Quinacrine's effects in the rat uterus when administered as a slurry in methylcellulose were contrasted with the human clinical experience which uses a solid form of the drug, to determine the relevance of the tumors produced in the rat to safe clinical use of quinacrine for permanent contraception (QS). A review was performed of the study report, dose feasibility studies, and clinical evaluations of women who had undergone the QS procedure. The top three doses of quinacrine in the CaBio exceeded the maximum tolerated dose, and produced chronic damage, including inflammation, resulting in reproductive tract tumors. Chronic inflammation was significantly correlated with the tumors; there was no evidence of treatment-related tumors in animals without chronic inflammation or other reproductive system toxicity. Because such permanent uterine damage and chronic toxicity have not been observed in humans under therapeutic conditions, we conclude that this mode of action for tumor production will not occur at clinically relevant doses in women who choose quinacrine for permanent contraception. © 2015 The Authors.

PubMed | National Institute of Public Health and the Environment RIVM, University of Campinas and Errol Zeiger Consulting
Type: | Journal: Advances in biochemical engineering/biotechnology | Year: 2016

There is ongoing concern about the consequences of mutations in humans and biota arising from environmental exposures to industrial and other chemicals. Genetic toxicity tests have been used to analyze chemicals, foods, drugs, and environmental matrices such as air, water, soil, and wastewaters. This is because the mutagenicity of a substance is highly correlated with its carcinogenicity. However, no less important are the germ cell mutations, because the adverse outcome is related not only to an individual but also to population levels. For environmental analysis the most common choices are in vitro assays, and among them the most widely used is the Ames test (Salmonella/microsome assay). There are several protocols and methodological approaches to be applied when environmental samples are tested and these are discussed in this chapter, along with the meaning and relevance of the obtained responses. Two case studies illustrate the utility of in vitro mutagenicity tests such as the Ames test. It is clear that, although it is not possible to use the outcome of the test directly in risk assessment, the application of the assays provides a great opportunity to monitor the exposure of humans and biota to mutagenic substances for the purpose of reducing or quantifying that exposure.

Zeiger E.,Errol Zeiger Consulting
Issues in Toxicology | Year: 2014

Cancer is a disease that is the result of a progression of cell and tissue changes leading to a tumor. In the animal, the progression of the tumor and the tissue it appears in will differ according to the specific carcinogen and the animal in which it is being tested. Not all tissues that are affected by a carcinogen develop tumors; tissue-specific and immunological factors will affect the progression and survival of the altered cells. As a consequence, no single in vitro test system will mimic the entire cancer process. When commercial organizations screen chemicals under development for potential carcinogenicity, a positive response in any one of the screening tests (usually the Ames test or a chromosome damage test) may be sufficient to remove a substance from further development. The alternative would be to perform a definitive cancer assay in the hopes that it would not be carcinogenic, and this is done only if the perceived commercial return from the chemical is substantial enough to justify the risk. Although numbers are not publically available, many chemicals are declared to be presumptive carcinogens, and not tested further in animals, based on the in silico and in vitro test results. As noted above, none of the in vitro tests measure cancer induction, but positive results in any of the tests are predictive for cancer, albeit with varying degrees of accuracy. Unfortunately, negative results in these tests are not predictive of non-carcinogenicity. Many testing schemes favored by regulatory authorities require two or three in vitro tests that measure different genetic endpoints (e.g., gene mutation; chromosome aberrations). In addition, tests that measure DNA damage (e.g., comet assay) and cell transformation are increasingly being used. One challenge associated with these multiple genotoxicity tests is that each test has its unique false positives. Therefore, as more tests are added to the battery, the proportion of both true positives and false positives will increase. If a positive response in any of the test systems is sufficient to label a substance as potential carcinogen, the use of multiple test systems could have the effect of discarding a high proportion of noncarcinogenic substances, or, alternatively lead to additional in vivo tests being performed in an attempt to distinguish the true positives from false positives. Current approaches are moving towards integrating the results of QSAR analysis, genetic toxicity tests, and tests for other precarcinogenic mechanisms (i.e., genetic recombination; cell transformation, in addition to toxicogenomics) to refine the estimation of the probability of carcinogenicity or noncarcinogenicity.

Zeiger E.,Errol Zeiger Consulting
Environmental and Molecular Mutagenesis | Year: 2010

The currently used genetic toxicity testing battery (the Ames Salmonella test, the in vitro mammalian cell mouse lymphoma assay and/or the in vitro mammalian cell chromosome assay, and the rodent bone marrow chromosome aberration or micronucleus assay) had its origins in the early-to-mid 1970s. By the late 1970s, a large number of genetic tests had been proposed or recommended by the US-EPA for identifying germ cell mutagens and carcinogens. After a number of modifications that were primarily directed toward minimizing the number of tests used, the test battery reached its current state in the mid-1980s. This test battery, with some minor modifications in the timing or ordering of the tests is mandated by regulatory authorities worldwide. Although it would be intellectually satisfying to presume that this compendium of tests was developed and selected for regulatory screening based solely on scientific grounds, it was actually based on a combination of scientific data, theoretical considerations, chance, and advocacy, and not always in equal proportions. The evolution of the current genetic toxicity test battery, and some of the activities and considerations that directed this evolution are described. © 2010 Wiley-Liss, Inc.

Zeiger E.,Errol Zeiger Consulting
Methods in Molecular Biology | Year: 2013

Bacterial mutagenicity tests, specifically the Salmonella and E. coli reverse mutation (Ames) test, are widely used and are usually required before a chemical, drug, pesticide, or food additive can be registered for use. The tests are also widely used for environmental monitoring to detect mutagens in air or water. Their use is based on the showing that a positive result in the test was highly predictive for carcinogenesis. This chapter describes the Salmonella and E. coli tests, presents protocols for their use, and addresses data interpretation and reporting. © Springer Science+Business Media, New York 2013.

Kirkland D.,Kirkland Consulting | Zeiger E.,Errol Zeiger Consulting
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2014

A Workshop sponsored by EURL ECVAM was held in Ispra, Italy in 2013 to consider whether the in vitro mammalian cell genotoxicity test results could complement and mitigate the implications of a positive Ames test response for the prediction of in vivo genotoxicity and carcinogenicity, and if patterns of results could be identified. Databases of Ames-positive chemicals that were tested for in vivo genotoxicity and/or carcinogenicity were collected from different sources and analysed individually (Kirkland et al., in this issue). Because there were overlaps and inconsistent test results among chemicals in the different databases, a combined database which eliminated the overlaps and evaluated the inconsistencies was considered preferable for addressing the above question. A database of >700 Ames-positive chemicals also tested in vivo was compiled, and the results in in vitro mammalian cell tests were analysed. Because the database was limited to Ames-positive chemicals, the majority (>85%) of carcinogens (103/119) and in vivo genotoxins (83/88) were positive when tested in both in vitro gene mutation and aneugenicity/clastogenicity tests. However, about half (>45%) of chemicals that were not carcinogenic (19/28) or genotoxic in vivo (33/73) also gave the same patterns of positive mammalian cell results. Although the different frequencies were statistically significant, positive results in 2 in vitro mammalian cell tests did not, per se, add to the predictivity of the positive Ames test. By contrast, negative results for both in vitro mammalian cell endpoints were rare for Ames-positive carcinogens (3/119) and in vivo genotoxins (2/88) but, were significantly more frequent for Ames-positive chemicals that are not carcinogenic (4/28) or genotoxic in vivo (14/73). Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential. © 2014 The Authors.

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