Development and Regulatory Challenges for Peptide Therapeutics.

There has been an increased interest in and activity for the use of peptide therapeutics to treat a variety of human diseases. The number of peptide drugs entering clinical development and the market has increased significantly over the past decade despite inherent challenges of peptide therapeutic discovery, development, and patient-friendly delivery. Disparities in interpretation and application of existing regulatory guidances to innovative synthetic and conjugated peptide assets have resulted in challenges for both regulators and sponsors. The Symposium on Development and Regulatory Challenges for Peptide Therapeutics at the 40th Annual Meeting of the American College of Toxicology held in November of 2019 focused on the following specific topics: (1) peptide therapeutic progress and future directions, and approaches to discover, optimize, assess, and deliver combination peptide therapeutics for treatment of diseases; (2) toxicological considerations to advance peptide drug-device combination products for efficient development and optimal patient benefit and adherence; (3) industry and regulatory perspectives on the regulation of synthetic and conjugated peptide products, including exploration of regulatory classifications, interpretations, and application of the existing guidances International Council for Harmonisation (ICH) M3(R2) and ICH S6(R1) in determining nonclinical study recommendations; and (4) presentation of the 2016 Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee working group assessment of genotoxicity testing requirements. Perspectives were shared from industry and regulatory scientists working in the peptide therapeutics field followed by an open forum panel discussion to discuss questions drafted for the peptide therapeutics scientific community, which will be discussed in more detail.

EURL ECVAM Genotoxicity and Carcinogenicity Database of Substances Eliciting Negative Results in the Ames Test: Construction of the Database.

The bacterial reverse mutation test (Ames test) is the most commonly used genotoxicity test; it is a primary component of the chemical safety assessment data required by regulatory agencies worldwide. Within the current accepted in vitro genotoxicity test battery, it is considered capable of revealing DNA reactivity, and identifying substances that can produce gene mutations via different mechanisms. The previously published consolidated EURL ECVAM Genotoxicity and Carcinogenicity Database, which includes substances that elicited a positive response in the Ames test, constitutes a collection of data that serves as a reference for a number of regulatory activities in the area of genotoxicity testing. Consequently, we considered it important to expand the database to include substances that fail to elicit a positive response in the Ames test, i.e., Ames negative substances. Here, we describe a curated collection of 211 Ames negative substances, with a summary of complementary data available for other genotoxicity endpoints in vitro and in vivo, plus available carcinogenicity data. A descriptive analysis of the data is presented. This includes a representation of the chemical space formed by the Ames-negative database with respect to other substances (e.g. REACH registered substances, approved drugs, pesticides, etc.) and a description of the organic functional groups found in the database. We also provide some suggestions on further analyses that could be made.

Strategies in genotoxicology: Acceptance of innovative scientific methods in a regulatory context and from an industrial perspective.

The tests used and the general principles behind test strategies are now often over 30 years old. It may be time by now, given that our knowledge of genetic toxicology has improved and that we also technically are better able to investigate DNA damage making use of modern molecular biological techniques, to start thinking on a new test strategy. In the present paper, it is discussed that the time is there to consider a new approach for genotoxicity assessment of substances. A fit for all test strategy was discussed making use of the most recent technological methods and techniques. It was also indicated that in silico tools should be more accepted by regulatory institutes/bodies as supporting information to better conclude which tests should be required for each separate substance to demonstrate its genotoxic potency. Next to that there should be a good rationale for performing in vivo studies. Finally, the need for germ cell genotoxicity testing, essential when classification and labeling of substances is mandatory, was discussed. It was suggested to change the GHS for genotoxicity classification and labelling from in vivo tests in germ cells into in vivo tests in somatic cells. Quantitative genotoxicology was also discussed. It appeared that we are currently at a transition, where the science developing to justify carrying out human health risk assessments based on genetic toxicology data sets supported by mechanistic data and exposure data. However, implementation will take time, and acceptance will be supported through the development of numerous case studies. Major remaining questions are: is genetic damage a relevant endpoint in itself, or should the risk assessment be carried out on the apical endpoint of cancer and which genotoxic endpoint should be used to derive the point of departure (PoD) for the human exposure limit?

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro.

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT).

The working group reached complete or majority agreement on many issues. Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens. TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity. A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing. Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required. In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017). Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route. The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline. The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.

Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co-Operation and Development (OECD) criteria.

The forward gene mutation mouse lymphoma assay (MLA) is widely used, as part of a regulatory test battery, to identify the genotoxic potential of chemicals. It identifies mutagens capable of inducing a variety of genetic events. During the 1980s and early 1990s, the U.S. National Toxicology Program (NTP) developed a publicly available database (https://tools.niehs.nih.gov/cebs3/ui/) of MLA results. This database is used to define the mutagenic potential of chemicals, to develop structure-activity relationships (SAR), and to draw correlations to animal carcinogenicity findings. New criteria for MLA conduct and data interpretation were subsequently developed by the International Workshop for Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development (OECD). These recommendations are included in a new OECD Test Guideline (TG490). It is essential that early experimental data be re-examined and classified according to the current criteria to build a curated database to better inform chemical-specific evaluations and SAR models. We re-evaluated more than 1900 experiments representing 342 chemicals against the newly defined acceptance criteria for background mutant frequency (MF), cloning efficiency (CE), positive control values (modified for this evaluation due to lack of colony sizing), appropriate dose selection, and data consistency. Only 17% of the evaluated experiments met all acceptance criteria used in this re-evaluation. Results from 211 chemicals were determined to be uninterpretable, 92 were positive, and 39 equivocal. The authors could not classify any responses as negative because colony sizing was not performed for any of these experiments and it is clear, based on many experiment with unacceptably low background and positive control MFs, that mutant colony recovery was often suboptimal. This re-evaluation provides a curated database for the MLA. A similar curation should be done for other widely used genetic toxicology assays, but will be more difficult for certain assays (e.g., in vitro chromosomal aberrations) because important parameters such as level of cytotoxicity were often not evaluated/reported. Environ. Mol. Mutagen. 59:829-841, 2018. © 2018 Wiley Periodicals, Inc.

Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria.

Skin tumors have been observed in C3H/HeJ mice following treatment with high and strongly irritating concentrations of 2-ethylhexyl acrylate (2-EHA). Dermal carcinogenicity studies performed with 2-EHA are reviewed, contrasting the results in two mouse strains (C3H/HeJ and NMRI) under different dosing regimens. Application of contemporary evaluation criteria to the existing dermal carcinogenicity dataset demonstrates that 2-EHA induces skin tumors only at concentrations exceeding an maximum tolerated dose (MTD) and in the immune-dysregulated C3H/HeJ mouse model. Overall, the available chronic toxicity and genotoxicity data on 2-EHA support a non-genotoxic chemical irritant mechanism, whereby chronic irritation leads to inflammation, tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice and leads to tumor formation. Tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model, and, even in the immune compromised C3H/HeJ model, did not induce skin tumors at doses which did not exceed the MTD.

2-Hydroxypyridine-N-oxide (HOPO): Equivocal in the ames assay.

2-Hydroxypyridine-N-oxide (HOPO) is a useful coupling reagent for synthesis of active pharmaceutical ingredients. It has been reported to be weakly mutagenic in the Ames assay (Ding W et al. []: J Chromatogr A 1386:47-52). According to the ICH M7 guidance (2014) regarding control of mutagenic impurities to limit potential carcinogenic risk, mutagens require control in drug substances such that exposure not exceeds the threshold of toxicological concern. Given the weak response observed in the Ames assay and the lack of any obvious structural features that could confer DNA reactivity we were interested to determine if the results were reproducible and investigate the role of potentially confounding experimental parameters. Specifically, Ames tests were conducted to assess the influence of compound purity, solvent choice, dose spacing, toxicity, type of S9 (aroclor vs phenobarbital/ß-napthoflavone), and lot variability on the frequency of HOPO induced revertant colonies. Initial extensive testing using one lot of HOPO produced no evidence of mutagenic potential in the Ames assays. Subsequent studies with four additional lots produced conflicting results, with an ∼2.0-fold increase in revertant colonies observed. Given the rigor of the current investigation, lack of reproducibility between lots, and the weak increase in revertants, it is concluded that HOPO is equivocal in the bacterial reverse mutation assay. It is highly unlikely that HOPO poses a mutagenic risk in vivo; therefore, when it is used as a reagent in pharmaceutical synthesis, it should not be regarded as a mutagenic impurity, but rather a normal process related impurity. Environ. Mol. Mutagen. 59:312-321, 2018. © 2018 Wiley Periodicals, Inc.

Recommendations for the evaluation of complex genetic toxicity data sets when assessing carcinogenic risks to humans.

Genotoxicity testing plays an important role in the assessment of carcinogenic and heritable risks. In many cases, experts charged with assessing genotoxicity test results need to evaluate widely varying numbers and types of bioassays of differing quality, conducted in a variety of cells and species using a wide range of protocols. The recommendations in this article were initially prepared as general guidelines to assist experts involved in the 2016 Joint Food and Agricultural Organization and World Health Organization Meeting on Pesticide Residues (JMPR) in their evaluation of the human health risks associated with exposure to pesticide residues in the diet. A weight of evidence approach is recommended in which studies are evaluated based on quality, reproducibility and consistency, significance of the genetic alteration, phylogenetic relevance to humans, type (in vivo vs. in vitro), and relevance of the route of administration. Using the recommended approach, the most weight will generally be given to high quality in vivo studies of gene and chromosome mutations (including aberrations) in humans or mammals exposed to the chemical through a physiologically relevant route such as oral or dermal administration or by inhalation. The guidelines are intended to give reviewers flexibility in evaluating all relevant scientific information, and allow them to use their best scientific judgment in reaching conclusions about the significance of the genotoxicity results. The use of these guidelines and the associated weighting considerations should facilitate the evaluation of complex and sometimes contradictory data sets, and provide more consistency in evaluations across risk assessments. Environ. Mol. Mutagen. 58:380-385, 2017. © 2017 Wiley Periodicals, Inc.

International regulatory requirements for genotoxicity testing for pharmaceuticals used in human medicine, and their impurities and metabolites.

The process of developing international (ICH) guidelines is described, and the main guidelines reviewed are the ICH S2(R1) guideline that includes the genotoxicity test battery for human pharmaceuticals, and the ICH M7 guideline for assessing and limiting potentially mutagenic impurities and degradation products in drugs. Key aspects of the guidelines are reviewed in the context of drug development, for example the incorporation of genotoxicity assessment into non-clinical toxicity studies, and ways to develop and assess weight of evidence. In both guidelines, the existence of "thresholds" or non-linear dose responses for genotoxicity plays a part in the strategies. Differences in ICH S2(R1) protocol recommendations from OECD guidelines are highlighted and rationales explained. The use of genotoxicity data during clinical development and in assessment of carcinogenic potential is also described. There are no international guidelines on assessment of potentially genotoxic metabolites, but some approaches to safety assessment are discussed for these. Environ. Mol. Mutagen. 58:296-324, 2017. © 2017 Wiley Periodicals, Inc.

Current methods in risk assessment of genotoxic chemicals.

Chemical contaminants and residues are undesired chemicals occurring in consumer products such as food and drugs, at the workplace and in the environment, i.e. in air, soil and water. These compounds can be detected even at very low concentrations and lead frequently to considerable concerns among consumers and in the media. Thus it is a major challenge for modern toxicology to provide transparent and versatile tools for the risk assessment of such compounds in particular with respect to human health. Well-known examples of toxic contaminants are dioxins or mercury (in the environment), mycotoxins (from infections by molds) or acrylamide (from thermal treatment of food). The process of toxicological risk assessment of such chemicals is based on i) the knowledge of their contents in food, air, water etc., ii) the routes and extent of exposure of humans, iii) the toxicological properties of the compound, and, iv) its mode(s) of action. In this process quantitative dose-response relationships, usually in experimental animals, are of outstanding importance. For a successful risk assessment, in particular of genotoxic chemicals, several conditions and models such as the Margin of Exposure (MoE) approach or the Threshold of Toxicological Concern (TTC) concept exist, which will be discussed.

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.

Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

An integrative test strategy for cancer hazard identification.

Assessment of genotoxic and carcinogenic potential is considered one of the basic requirements when evaluating possible human health risks associated with exposure to chemicals. Test strategies currently in place focus primarily on identifying genotoxic potential due to the strong association between the accumulation of genetic damage and cancer. Using genotoxicity assays to predict carcinogenic potential has the significant drawback that risks from non-genotoxic carcinogens remain largely undetected unless carcinogenicity studies are performed. Furthermore, test systems already developed to reduce animal use are not easily accepted and implemented by either industries or regulators. This manuscript reviews the test methods for cancer hazard identification that have been adopted by the regulatory authorities, and discusses the most promising alternative methods that have been developed to date. Based on these findings, a generally applicable tiered test strategy is proposed that can be considered capable of detecting both genotoxic as well as non-genotoxic carcinogens and will improve understanding of the underlying mode of action. Finally, strengths and weaknesses of this new integrative test strategy for cancer hazard identification are presented.

Principles and procedures for implementation of ICH M7 recommended (Q)SAR analyses.

The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.

"Aspartame: A review of genotoxicity data".

Aspartame is a methyl ester of a dipeptide of aspartic acid and phenylalanine. It is 200× sweeter than sucrose and is approved for use in food products in more than 90 countries around the world. Aspartame has been evaluated for genotoxic effects in microbial, cell culture and animal models, and has been subjected to a number of carcinogenicity studies. The in vitro and in vivo genotoxicity data available on aspartame are considered sufficient for a thorough evaluation. There is no evidence of induction of gene mutations in a series of bacterial mutation tests. There is some evidence of induction of chromosomal damage in vitro, but this may be an indirect consequence of cytotoxicity. The weight of evidence from in vivo bone marrow micronucleus, chromosomal aberration and Comet assays is that aspartame is not genotoxic in somatic cells in vivo. The results of germ cell assays are difficult to evaluate considering limited data available and deviations from standard protocols. The available data therefore support the conclusions of the European Food Safety Authority (EFSA) that aspartame is non-genotoxic.