A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

A comparison of the lowest effective concentration in culture media for detection of chromosomal damage in vitro and in blood or plasma for detection of micronuclei in vivo.

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro.

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential.

In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.

A weight of evidence assessment of the genotoxicity of 2,6-xylidine based on existing and new data, with relevance to safety of lidocaine exposure.

Lidocaine has not been associated with cancer in humans despite 8 decades of therapeutic use. Its metabolite, 2,6-xylidine, is a rat carcinogen, believed to induce genotoxicity via N-hydroxylation and DNA adduct formation, a non-threshold mechanism of action. To better understand this dichotomy, we review literature pertaining to metabolic activation and genotoxicity of 2,6-xylidine, identifying that it appears resistant to N-hydroxylation and instead metabolises almost exclusively to DMAP (an aminophenol). At high exposures (sufficient to saturate phase 2 metabolism), this may undergo metabolic threshold-dependent activation to a quinone-imine with potential to redox cycle producing ROS, inducing cytotoxicity and genotoxicity. A new rat study found no evidence of genotoxicity in vivo based on micronuclei in bone marrow, comets in nasal tissue or female liver, despite high level exposure to 2,6-xylidine (including metabolites). In male liver, weak dose-related comet increases, within the historical control range, were associated with metabolic overload and acute systemic toxicity. Benchmark dose analysis confirmed a non-linear dose response. The weight of evidence indicates 2,6-xylidine is a non-direct acting (metabolic threshold-dependent) genotoxin, and is not genotoxic in vivo in rats in the absence of acute systemic toxic effects, which occur at levels 35 × beyond lidocaine-related exposure in humans.

A weight of evidence assessment of the genotoxic potential of 4-methylimidazole as a possible mode of action for the formation of lung tumors in exposed mice.

4-Methylimidazole (4-MeI) is a byproduct formed during the cooking of foods containing carbohydrates and amino acids, including the production of flavors and coloring substances, e.g., class III and IV caramel colors, used in many food products with extensive human exposure. Two-year rodent bioassays via oral exposure conducted by the National Toxicology Program reported evidence of carcinogenicity only in B6C3F1 mice (increased alveolar/bronchial neoplasms). In 2011, the International Agency for Research on Cancer classified 4-MeI as Group 2B, "possibly carcinogenic to humans". An expert panel was commissioned to assess the genotoxic potential of 4-MeI and the plausibility of a genotoxic mode of action in the formation of lung tumors in mice when exposed to high doses of 4-MeI. The panel defined and used a weight-of-evidence (WOE) approach that included thorough evaluation of studies assessing the genotoxic potential of 4-MeI. The panelists categorized each study, consisting of study weight, degree of technical performance, study reliability, and contribution to the overall WOE. Based on the reviewed studies' weighted contribution, the panel unanimously concluded that the WOE supports no clear evidence of in vivo genotoxicity of 4-MeI and no association for a genotoxic mode of action in the formation of mouse lung tumors.

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.

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.

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.

An updated review of the genotoxicity of respirable crystalline silica.

Human exposure to (certain forms of) crystalline silica (CS) potentially results in adverse effects on human health. Since 1997 IARC has classified CS as a Group 1 carcinogen [1], which was confirmed in a later review in 2012 [2]. The genotoxic potential and mode of genotoxic action of CS was not conclusive in either of the IARC reviews, although a proposal for mode of actions was made in an extensive review of the genotoxicity of CS by Borm, Tran and Donaldson in 2011 [3]. The present study identified 141 new papers from search strings related to genotoxicity of respirable CS (RCS) since 2011 and, of these, 17 relevant publications with genotoxicity data were included in this detailed review.Studies on in vitro genotoxic endpoints primarily included micronucleus (MN) frequency and % fragmented DNA as measured in the comet assay, and were mostly negative, apart from two studies using primary or cultured macrophages. In vivo studies confirmed the role of persistent inflammation due to quartz surface toxicity leading to anti-oxidant responses in mice and rats, but DNA damage was only seen in rats. The role of surface characteristics was strengthened by in vitro and in vivo studies using aluminium or hydrophobic treatment to quench the silanol groups on the CS surface.In conclusion, the different modes of action of RCS-induced genotoxicity have been evaluated in a series of independent, adequate studies since 2011. Earlier conclusions on the role of inflammation driven by quartz surface in genotoxic and carcinogenic effects after inhalation are confirmed and findings support a practical threshold. Whereas classic in vitro genotoxicity studies confirm an earlier no-observed effect level (NOEL) in cell cultures of 60-70 µg/cm2, transformation frequency in SHE cells suggests a lower threshold around 5 µg/cm2. Both levels are only achieved in vivo at doses (2-4 mg) beyond in vivo doses (> 200 µg) that cause persistent inflammation and tissue remodelling in the rat lung.

A review of the genotoxicity of trimethylolpropane triacrylate (TMPTA).

Trimethylolpropane triacrylate (TMPTA) is a trifunctional acrylate monomer which polymerizes rapidly when exposed to sources of free radicals. It is widely used as a reactive diluent and polymer building block in the formulation of overprint varnishes, inks and a variety of wood, plastic and metal coatings. TMPTA has been tested in a range of in vitro and in vivo genotoxicity tests. There is no clear evidence of induction of gene mutations by TMPTA in bacteria or mammalian cells in vitro, but there is evidence of clastogenicity from induction of small colony tk mutants in the mouse lymphoma assay, and also induction of micronuclei and chromosomal aberrations. However, TMPTA was negative in bone marrow or blood micronucleus tests in vivo following oral or repeated dermal application, and did not induce comets in bone marrow or liver of mice following intravenous administration, which would have achieved plasma (and therefore tissue) concentrations estimated to exceed those inducing clastogenic effects in vitro. It is concluded that TMPTA is not genotoxic in vivo.

Origin of the TTC values for compounds that are genotoxic and/or carcinogenic and an approach for their re-evaluation.

The threshold of toxicological concern (TTC) approach is a resource-effective de minimis method for the safety assessment of chemicals, based on distributional analysis of the results of a large number of toxicological studies. It is being increasingly used to screen and prioritize substances with low exposure for which there is little or no toxicological information. The first step in the approach is the identification of substances that may be DNA-reactive mutagens, to which the lowest TTC value is applied. This TTC value was based on the analysis of the cancer potency database and involved a number of assumptions that no longer reflect the state-of-the-science and some of which were not as transparent as they could have been. Hence, review and updating of the database is proposed, using inclusion and exclusion criteria reflecting current knowledge. A strategy for the selection of appropriate substances for TTC determination, based on consideration of weight of evidence for genotoxicity and carcinogenicity is outlined. Identification of substances that are carcinogenic by a DNA-reactive mutagenic mode of action and those that clearly act by a non-genotoxic mode of action will enable the protectiveness to be determined of both the TTC for DNA-reactive mutagenicity and that applied by default to substances that may be carcinogenic but are unlikely to be DNA-reactive mutagens (i.e. for Cramer class I-III compounds). Critical to the application of the TTC approach to substances that are likely to be DNA-reactive mutagens is the reliability of the software tools used to identify such compounds. Current methods for this task are reviewed and recommendations made for their application.

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay.

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow® DNA Damage Kit-p53, γH2AX, Phospho-Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals' predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146-161, 2017. © 2017 Wiley Periodicals, Inc.

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.

A review of the carcinogenic potential of glyphosate by four independent expert panels and comparison to the IARC assessment.

The International Agency for Research on Cancer (IARC) published a monograph in 2015 concluding that glyphosate is "probably carcinogenic to humans" (Group 2A) based on limited evidence in humans and sufficient evidence in experimental animals. It was also concluded that there was strong evidence of genotoxicity and oxidative stress. Four Expert Panels have been convened for the purpose of conducting a detailed critique of the evidence in light of IARC's assessment and to review all relevant information pertaining to glyphosate exposure, animal carcinogenicity, genotoxicity, and epidemiologic studies. Two of the Panels (animal bioassay and genetic toxicology) also provided a critique of the IARC position with respect to conclusions made in these areas. The incidences of neoplasms in the animal bioassays were found not to be associated with glyphosate exposure on the basis that they lacked statistical strength, were inconsistent across studies, lacked dose-response relationships, were not associated with preneoplasia, and/or were not plausible from a mechanistic perspective. The overall weight of evidence from the genetic toxicology data supports a conclusion that glyphosate (including GBFs and AMPA) does not pose a genotoxic hazard and therefore, should not be considered support for the classification of glyphosate as a genotoxic carcinogen. The assessment of the epidemiological data found that the data do not support a causal relationship between glyphosate exposure and non-Hodgkin's lymphoma while the data were judged to be too sparse to assess a potential relationship between glyphosate exposure and multiple myeloma. As a result, following the review of the totality of the evidence, the Panels concluded that the data do not support IARC's conclusion that glyphosate is a "probable human carcinogen" and, consistent with previous regulatory assessments, further concluded that glyphosate is unlikely to pose a carcinogenic risk to humans.

Genotoxicity Expert Panel review: weight of evidence evaluation of the genotoxicity of glyphosate, glyphosate-based formulations, and aminomethylphosphonic acid.

In 2015, the International Agency for Research on Cancer (IARC) published a monograph concluding there was strong evidence for genotoxicity of glyphosate and glyphosate formulations and moderate evidence for genotoxicity of the metabolite aminomethylphosphonic acid (AMPA). These conclusions contradicted earlier extensive reviews supporting the lack of genotoxicity of glyphosate and glyphosate formulations. The IARC Monograph concluded there was strong evidence of induction of oxidative stress by glyphosate, glyphosate formulations, and AMPA. The Expert Panel reviewed the genotoxicity and oxidative stress data considered in the IARC Monograph, together with other available data not considered by IARC. The Expert Panel defined and used a weight of evidence (WoE) approach that included ranking of studies and endpoints by the strength of their linkage to events associated with carcinogenic mechanisms. Importantly, the Expert Panel concluded that there was sufficient information available from a very large number of regulatory genotoxicity studies that should have been considered by IARC. The WoE approach, the inclusion of all relevant regulatory studies, and some differences in interpretation of individual studies led to significantly different conclusions by the Expert Panel compared with the IARC Monograph. The Expert Panel concluded that glyphosate, glyphosate formulations, and AMPA do not pose a genotoxic hazard and the data do not support the IARC Monograph genotoxicity evaluation. With respect to carcinogenicity classification and mechanism, the Expert Panel concluded that evidence relating to an oxidative stress mechanism of carcinogenicity was largely unconvincing and that the data profiles were not consistent with the characteristics of genotoxic carcinogens.

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9.

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546-558, 2016. © 2016 Wiley Periodicals, Inc.

Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests.

In 2008 we published recommendations on chemicals that would be appropriate to evaluate the sensitivity and specificity of new/modified mammalian cell genotoxicity tests, in particular to avoid misleading positive results. In light of new data it is appropriate to update these lists of chemicals. An expert panel was convened and has revised the recommended chemicals to fit the following different sets of characteristics: • Group 1: chemicals that should be detected as positive in in vitro mammalian cell genotoxicity tests. Chemicals in this group are all in vivo genotoxins at one or more endpoints, either due to DNA-reactive or non DNA-reactive mechanisms. Many are known carcinogens with a mutagenic mode of action, but a sub-class of probable aneugens has been introduced. • Group 2: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests. Chemicals in this group are usually negative in vivo and non-DNA-reactive. They are either non-carcinogenic or rodent carcinogens with a non-mutagenic mode of action. • Group 3: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce gene mutations in mouse lymphoma cells, chromosomal aberrations or micronuclei, often at high concentrations or at high levels of cytotoxicity. Chemicals in this group are generally negative in vivo and negative in the Ames test. They are either non-carcinogenic or rodent carcinogens with an accepted non-mutagenic mode of action. This group contains comments as to any conditions that can be identified under which misleading positive results are likely to occur. This paper, therefore, updates these three recommended lists of chemicals and describes how these should be used for any test evaluation program.

"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.

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays.

Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of "misleading" positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of "misleading" positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by "misleading positive" genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.