Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Establishing a quantitative framework for regulatory interpretation of genetic toxicity dose-response data: Margin of exposure case study of 48 compounds with both in vivo mutagenicity and carcinogenicity dose-response data.

Quantitative relationships between carcinogenic potency and mutagenic potency have been previously examined using a benchmark dose (BMD)-based approach. We extended those analyses by using human exposure data for 48 compounds to calculate carcinogenicity-derived and genotoxicity-derived margin of exposure values (MOEs) that can be used to prioritize substances for risk management. MOEs for 16 of the 48 compounds were below 10,000, and consequently highlighted for regulatory concern. Of these, 15 were highlighted using genotoxicity-derived (micronucleus [MN] dose-response data) MOEs. A total of 13 compounds were highlighted using carcinogenicity-derived MOEs; 12 compounds were overlapping. MOEs were also calculated using transgenic rodent (TGR) mutagenicity data. For 10 of the 12 compounds examined using TGR data, the results similarly revealed that mutagenicity-derived MOEs yield regulatory decisions that correspond with those based on carcinogenicity-derived MOEs. The effect of benchmark response (BMR) on MOE determination was also examined. Reinterpretation of the analyses using a BMR of 50% indicated that four out of 15 compounds prioritized using MN-derived MOEs based on a default BMR of 5% would have been missed. The results indicate that regulatory decisions based on in vivo genotoxicity dose-response data would be consistent with those based on carcinogenicity dose-response data; in some cases, genotoxicity-based decisions would be more conservative. Going forward, and in the absence of carcinogenicity data, in vivo genotoxicity assays (MN and TGR) can be used to effectively prioritize substances for regulatory action. Routine use of the MOE approach necessitates the availability of reliable human exposure estimates, and consensus regarding appropriate BMRs for genotoxicity endpoints.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Compilation and use of genetic toxicity historical control data.

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include:

In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop.

Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.

Strategies in case of positive in vivo results in genotoxicity testing.

At the 2009 International Workshop on Genotoxicity Testing in Basel, an expert group gathered to provide guidance on suitable follow-up tests to describe risk when basic in vivo genotoxicity tests have yielded positive results. The working group agreed that non-linear dose-response curves occur in vivo with at least some DNA-reactive agents. Quantitative risk assessment in such cases requires the use of (1) adequate data, i.e., the use of all available data for the selection of reliable in vivo models to be used for quantitative risk assessment, (2) appropriate mathematical models and statistical analysis for characterizing the dose-response relationships and allowing the use of quantitative and dose-response information in the interpretation of results, (3) mode of action (MOA) information for the evaluation and analysis of risk, and (4) reliable assessments of the internal dose across species for deriving acceptable margins of exposure and risk levels. Hence, the elucidation of MOA and understanding of the mechanism underlying the dose-response curve are important components of risk assessment. The group agreed on the need for (i) the development of in vivo assays, especially multi-endpoint, multi-species assays, with emphasis on those applicable to humans, and (ii) consensus about the most appropriate mathematical models and statistical analyses for defining non-linear dose-responses and exposure levels associated with acceptable risk.

Utility of a next-generation framework for assessment of genomic damage: A case study using the pharmaceutical drug candidate etoposide.

We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals.

Heritable hazards of smoking: Applying the "clean sheet" framework to further science and policy.

All the cells in our bodies are derived from the germ cells of our parents, just as our own germ cells become the bodies of our children. The integrity of the genetic information inherited from these germ cells is of paramount importance in establishing the health of each generation and perpetuating our species into the future. There is a large and growing body of evidence strongly suggesting the existence of substances that may threaten this integrity by acting as human germ cell mutagens. However, there generally are no absolute regulatory requirements to test agents for germ cell effects. In addition, the current regulatory testing paradigms do not evaluate the impacts of epigenetically mediated intergenerational effects, and there is no regulatory framework to apply new and emerging tests in regulatory decision making. At the 50th annual meeting of the Environmental Mutagenesis and Genomics Society held in Washington, DC, in September 2019, a workshop took place that examined the heritable effects of hazardous exposures to germ cells, using tobacco smoke as the example hazard. This synopsis provides a summary of areas of concern regarding heritable hazards from tobacco smoke exposures identified at the workshop and the value of the Clean Sheet framework in organizing information to address knowledge and testing gaps.

Utility of a next generation framework for assessment of genomic damage: A case study using the industrial chemical benzene.

We recently published a next generation framework for assessing the risk of genomic damage via exposure to chemical substances. The framework entails a systematic approach with the aim to quantify risk levels for substances that induce genomic damage contributing to human adverse health outcomes. Here, we evaluated the utility of the framework for assessing the risk for industrial chemicals, using the case of benzene. Benzene is a well-studied substance that is generally considered a genotoxic carcinogen and is known to cause leukemia. The case study limits its focus on occupational and general population health as it relates to benzene exposure. Using the framework as guidance, available data on benzene considered relevant for assessment of genetic damage were collected. Based on these data, we were able to conduct quantitative analyses for relevant data sets to estimate acceptable exposure levels and to characterize the risk of genetic damage. Key observations include the need for robust exposure assessments, the importance of information on toxicokinetic properties, and the benefits of cheminformatics. The framework points to the need for further improvement on understanding of the mechanism(s) of action involved, which would also provide support for the use of targeted tests rather than a prescribed set of assays. Overall, this case study demonstrates the utility of the next generation framework to quantitatively model human risk on the basis of genetic damage, thereby enabling a new, innovative risk assessment concept. Environ. Mol. Mutagen. 61:94-113, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

Application of quantitative microbial risk assessments for estimation of risk management metrics: Clostridium perfringens in ready-to-eat and partially cooked meat and poultry products as an example.

The U.S. Department of Agriculture, Food Safety and Inspection Service is exploring quantitative risk assessment methodologies to incorporate the use of the Codex Alimentarius' newly adopted risk management metrics (e.g., food safety objectives and performance objectives). It is suggested that use of these metrics would more closely tie the results of quantitative microbial risk assessments (QMRAs) to public health outcomes. By estimating the food safety objective (the maximum frequency and/or concentration of a hazard in a food at the time of consumption) and the performance objective (the maximum frequency and/or concentration of a hazard in a food at a specified step in the food chain before the time of consumption), risk managers will have a better understanding of the appropriate level of protection (ALOP) from microbial hazards for public health protection. We here demonstrate a general methodology that allows identification of an ALOP and evaluation of corresponding metrics at appropriate points in the food chain. It requires a two-dimensional probabilistic risk assessment, the example used being the Monte Carlo QMRA for Clostridium perfringens in ready-to eat and partially cooked meat and poultry products, with minor modifications to evaluate and abstract required measures. For demonstration purposes, the QMRA model was applied specifically to hot dogs produced and consumed in the United States. Evaluation of the cumulative uncertainty distribution for illness rate allows a specification of an ALOP that, with defined confidence, corresponds to current industry practices.

An evaluation of the mode of action framework for mutagenic carcinogens case study: Cyclophosphamide.

In response to the 2005 revised US Environmental Protection Agency (EPA) Cancer Guidelines, a Risk Assessment Forum's Technical Panel has devised a strategy in which genetic toxicology data combined with other information are assessed to determine whether a carcinogen operates through a mutagenic mode of action (MOA). This information is necessary for EPA to decide whether age-dependent adjustment factors (ADAFs) should be applied to the cancer risk assessment. A decision tree has been developed as a part of this approach and outlines the critical steps for analyzing a compound for carcinogenicity through a mutagenic MOA (e.g., data analysis, determination of mutagenicity in animals and in humans). Agents, showing mutagenicity in animals and humans, proceed through the Agency's framework analysis for MOAs. Cyclophosphamide (CP), an antineoplastic agent, which is carcinogenic in animals and humans and mutagenic in vitro and in vivo, was selected as a case study to illustrate how the framework analysis would be applied to prove that a carcinogen operates through a mutagenic MOA. Consistent positive results have been seen for mutagenic activity in numerous in vitro assays, in animals (mice, rats, and hamsters) and in humans. Accordingly, CP was processed through the framework analysis and key steps leading to tumor formation were identified as follows: metabolism of the parent compound to alkylating metabolites, DNA damage followed by induction of multiple adverse genetic events, cell proliferation, and bladder tumors. Genetic changes in rats (sister chromatid exchanges at 0.62 mg/kg) can commence within 30 min and in cancer patients, chromosome aberrations at 35 mg/kg are seen by 1 hr, well within the timeframe and tumorigenic dose range for early events. Supporting evidence is also found for cell proliferation, indicating that mutagenicity, associated with cytotoxicity, leads to a proliferative response, which occurs early (48 hr) in the process of tumor induction. Overall, the weight of evidence evaluation supports CP acting through a mutagenic MOA. In addition, no data were found that an alternative MOA might be operative. Therefore, the cancer guidelines recommend a linear extrapolation for the risk assessment. Additionally, data exist showing that CP induces mutagenicity in fetal blood and in the peripheral blood of pediatric patients; thus, the ADAFs would be applied.

Genomics-based food-borne pathogen testing and diagnostics: possibilities for the U.S. Department of Agriculture's Food Safety and Inspection Service.

The use of genomic technologies at the U.S. Department of Agriculture could enhance inspection, monitoring, and risk assessment capabilities within its Food Safety and Inspection Service (FSIS). Molecular assays capable of detecting hundreds of microbial DNA sequences within a single food sample that identify food-borne pathogens of concern and characterize their traits most relevant to human health risk are of great interest for FSIS. For example, a high-density assay, or combination of assays, could screen FSIS inspected food for pathogens relevant to public health (e.g., Salmonella, Listeria, and toxic E. coli) as well as their associated virulence factors and antibiotic resistance genes. Because most genotype assays can be completed in one working day with a minimum of reagents, use of such assays could potentially save FSIS a significant amount of cost/time for analyses. Further, a genotype assay can detect specific microbial traits relevant to human health risk based on the DNA sequence of toxin producing genes, antibiotic resistance alleles, and more. By combining rapid analysis with specific data on human health risks, information from such high-density genotype assays could provide expanded support for test and hold situations, recalls, outbreak management, and microbial risk assessments (e.g., provide data needed for food-borne illness source attribution). Environ. Mol. Mutagen.

Next generation testing strategy for assessment of genomic damage: A conceptual framework and considerations.

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. 58:264-283, 2017. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Use of genetic toxicology information for risk assessment.

Genetic toxicology data are used worldwide in regulatory decision-making. On the 25th anniversary of Environmental and Molecular Mutagenesis, we think it is important to provide a brief overview of the currently available genetic toxicity tests and to outline a framework for conducting weight-of-the-evidence (WOE) evaluations that optimize the utility of genetic toxicology information for risk assessment. There are two major types of regulatory decisions made by agencies such as the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA): (1) the approval and registration of pesticides, pharmaceuticals, medical devices, and medical-use products, and (2) the setting of standards for acceptable exposure levels in air, water, and food. Genetic toxicology data are utilized for both of these regulatory decisions. The current default assumption for regulatory decisions is that chemicals that are shown to be genotoxic in standard tests are, in fact, capable of causing mutations in humans (in somatic and/or germ cells) and that they contribute to adverse health outcomes via a "genotoxic/mutagenic" mode of action (MOA). The new EPA Guidelines for Carcinogen Risk Assessment [Guidelines for Carcinogen Risk Assessment, USEPA, 2005, EPA Publication No. EPA/630/P-03/001F] emphasize the use of MOA information in risk assessment and provide a framework to help identify a possible mutagenic and/or nonmutagenic MOA for potential adverse effects. An analysis of the available genetic toxicity data is now, more than ever, a key component to consider in the derivation of an MOA for characterizing observed adverse health outcomes such as cancer. We provide our perspective and a two-step strategy for evaluating genotoxicity data for optimal use in regulatory decision-making. The strategy includes integration of all available information and provides, first, for a WOE analysis as to whether a chemical is a mutagen, and second, whether an adverse health outcome is mediated via a mutagenic MOA.

Perfluorooctane Sulfonate Plasma Half-Life Determination and Long-Term Tissue Distribution in Beef Cattle (Bos taurus).

Perfluorooctane sulfonate (PFOS) is used in consumer products as a surfactant and is found in industrial and consumer waste, which ends up in wastewater treatment plants (WWTPs). PFOS does not breakdown during WWTP processes and accumulates in the biosolids. Common practices include application of biosolids to pastures and croplands used for feed, and as a result, animals such as beef cattle are exposed to PFOS. To determine plasma and tissue depletion kinetics in cattle, 2 steers and 4 heifers were dosed with PFOS at 0.098 mg/kg body weight and 9.1 mg/kg, respectively. Plasma depletion half-lives for steers and heifers were 120 ± 4.1 and 106 ± 23.1 days, respectively. Specific tissue depletion half-lives ranged from 36 to 385 days for intraperitoneal fat, back fat, muscle, liver, bone, and kidney. These data indicate that PFOS in beef cattle has a sufficiently long depletion half-life to permit accumulation in edible tissues.

Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays(☆).

This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity.

Genotoxicity risk assessment: a proposed classification strategy.

Recent advances in genetic toxicity (mutagenicity) testing methods and in approaches to performing risk assessment are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. The US Environmental Protection Agency (EPA) first published Guidelines for Mutagenicity Risk Assessment in 1986 that focused mainly on transmissible germ cell genetic risk. Somatic cell genetic risk has also been a risk consideration, usually in support of carcinogenicity assessments. EPA and other international regulatory bodies have published mutagenicity testing requirements for agents (pesticides, pharmaceuticals, etc.) to generate data for use in genotoxicity risk assessments. The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process. A classification strategy for agents based on inherent genotoxicity, dose-responses observed in the data, and an exposure analysis is proposed. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim.

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data. Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.