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.

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.

Sucralose Non-Carcinogenicity: A Review of the Scientific and Regulatory Rationale.

Regulatory authorities worldwide have found the nonnutritive sweetener, sucralose, to be noncarcinogenic, based on a range of studies. A review of these and other studies found through a comprehensive search of electronic databases, using appropriate key terms, was conducted and results of that review are reported here. An overview of the types of studies relied upon by regulatory agencies to assess carcinogenicity potential is also provided as context. Physiochemical and pharmacokinetic/toxicokinetic studies confirm stability under conditions of use and reveal no metabolites of carcinogenic potential. In vitro and in vivo assays reveal no confirmed genotoxic activity. Long-term carcinogenicity studies in animal models provide no evidence of carcinogenic potential for sucralose. In studies in healthy adults, sucralose was well-tolerated and without evidence of toxicity or other changes that might suggest a potential for carcinogenic effects. In summary, sucralose does not demonstrate carcinogenic activity even when exposure levels are several orders of magnitude greater than the range of anticipated daily ingestion levels.

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.

A perspective on testing of existing pharmaceutical excipients for genotoxic impurities.

Guidance recommendations by the Committee for Medicinal Products for Human Use (CHMP) and Pharmaceutical Research and Manufacturers of America (PhRMA) acknowledge the presence of potential toxic impurities in some pharmaceutical ingredients and have proposed setting limits on impurities with genotoxic activity as a means to protect patients in clinical trials and for marketing of the approved products. Recently, there have been suggestions that drug excipients, including existing products, should also be subjected to the same testing procedures and intake limits as proposed for active ingredients. This report is an attempt to put such recommendations into the proper perspective regarding the likelihood of protecting or improving public health.

Expert panel report on a study of Splenda in male rats.

A recent study in rats investigated the retail sweetener product, Granulated SPLENDA No Calorie Sweetener (Splenda) (Abou-Donia et al., 2008. Splenda alters gut microflora and increases intestinal P-glycoprotein and cytochrome P-450 in male rats. J. Toxicol. Environ. Health A, 71, 1415-1429), which is composed of (by dry weight) maltodextrin ( approximately 99%) and sucralose ( approximately 1%). The investigators reported that Splenda increased body weight, decreased beneficial intestinal bacteria, and increased the expression of certain cytochrome P450 (CYP450) enzymes and the transporter protein, P-glycoprotein (P-gp), the latter of which was considered evidence that Splenda or sucralose might interfere with the absorption of nutrients and drugs. The investigators indicated that the reported changes were attributable to the sucralose present in the product tested. An Expert Panel conducted a rigorous evaluation of this study. In arriving at its conclusions, the Expert Panel considered the design and conduct of the study, its outcomes and the outcomes reported in other data available publicly. The Expert Panel found that the study was deficient in several critical areas and that its results cannot be interpreted as evidence that either Splenda, or sucralose, produced adverse effects in male rats, including effects on gastrointestinal microflora, body weight, CYP450 and P-gp activity, and nutrient and drug absorption. The study conclusions are not consistent with published literature and not supported by the data presented.

Critical assessment of the genetic toxicity of naphthalene.

Studies demonstrating that naphthalene produces respiratory tract tumors in mice and rats raised the question of whether humans are at risk for cancer, at environmental or workplace concentrations of naphthalene. Arguments in favor of a threshold-dependent mode of action for tumor induction have been based on the facts that naphthalene does not appear to bind to DNA in vivo and that the rodent tumors occurred at high dose levels associated with substantial target site toxicity. A summary of more than 45 publications describing results for naphthalene in genetic toxicology test methods shows that 80% of the studies reported found no evidence of genotoxicity for naphthalene and that some of the studies which reported positive finding were technically unsuited to study this class of chemicals and, therefore, generated unreliable data. The remaining positive findings for naphthalene were all consistent with secondary DNA effects produced by toxicity from naphthalene alone or one of its metabolites. Based on the data reviewed in this report, it is not apparent that genetic lesions produced by naphthalene or any of its metabolites drive the tumorigenic activity.

Possible genotoxic modes of action for naphthalene.

This report provides a summary of deliberations conducted under the charge for members of Module D participating in the Naphthalene State-of-the-Science Symposium (NS(3)), Monterey, CA, October 9-12, 2006. The charge directed the panel to ascertain to the best of its ability a consensus judgment of the state-of-the-science concerning the potential for a genotoxic mode of action for naphthalene and its metabolites, with implications for low-dose extrapolations of cancer risk estimates for exposed populations. Where scientific uncertainties remained, the panel was asked to identify which scientific uncertainties (if any) could be resolved through targeted, timely, cost-effective research. The report provides a brief summary of naphthalene genotoxicity; identifies those areas where there is a general scientific consensus regarding the effects of naphthalene; identifies areas of uncertainty regarding the effects of naphthalene; and key questions that currently limit our ability to assess the genotoxic risks of naphthalene. The report also outlines a set of six studies that could resolve some of these key uncertainties.

A review of the genotoxicity of ethylbenzene.

Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (IARC class 2B). It induces tumours in rats and mice, but neither the relevance of these tumours to humans nor their mechanism of induction is clear. Considering the carcinogenic potential of ethylbenzene, it is of interest to determine whether there is sufficient data to characterize its mode of action as either genotoxic or non-genotoxic. A review of the currently available genotoxicity data is assessed. Ethylbenzene is not a bacterial mutagen, does not induce gene conversion or mutations in yeast and does not induce sister chromatid exchanges in CHO cells. Ethylbenzene is not clastogenic in CHO or rat liver cell lines but was reported to induce micronuclei in SHE cells in vitro. No evidence for genotoxicity has been seen in humans exposed to relatively high levels of ethylbenzene. Mouse lymphoma gene mutation studies produced a mixed series of responses that have proved difficult to interpret. An increase in morphological transformation of SHE cells was also found. Results from a more relevant series of in vivo genotoxicity studies, including acute and sub-chronic micronucleus tests and the mouse liver UDS assay, indicate a lack of in vivo genotoxic activity. The composite set of results from both in vitro and in vivo tests known to assess direct damage to DNA have been predominantly negative in the absence of excessive toxicity. The available data from the standard battery of genotoxicity assays do not support a genotoxic mechanism for ethylbenzene-induced kidney, liver or lung tumors in rats and mice.

Analysis of genotoxicity and the carcinogenic mode of action for ortho-phenylphenol.

Ortho-phenylphenol (OPP) and its sodium salt (SOPP) are commercial products that have wide human exposure and have been shown in several studies to be rodent carcinogens. Genetic toxicology data were assessed in an attempt to understand the carcinogenic mode of action of OPP and SOPP. More than 130 studies were evaluated to determine if OPP, SOPP, or any of their enzymatic or nonenzymatic breakdown products react directly with DNA to induce mutation, changes in chromosome structure or number, DNA repair, or nonspecific DNA damage including strand breakage or covalent binding. The genotoxicity databases for OPP and SOPP are not only large but heterogeneous, requiring weight-of-evidence methods to arrive at a conclusion regarding their genotoxic properties and potential. Evidence derived from the available studies leads to the conclusion that study results showing OPP/SOPP directly interacting with DNA are equivocal. Clastogenicity was the most consistent type of genetic toxicity produced by OPP/SOPP (and their break-down products) and was consistently associated with other intracellular preneoplastic toxicity produced at super-threshold concentrations. The weight of evidence from the combined database supports the hypothesis that OPP/SOPP-induced DNA damage is a threshold-dependent response associated with target tissue toxicity, most likely induced by their breakdown products phenylhydroquinone and phenylbenzoquinone. It is possible that this threshold-dependent clastogenicity could contribute to the carcinogenic mode of action for OPP or SOPP.

Atrazine and breast cancer: a framework assessment of the toxicological and epidemiological evidence.

The causal relationship between atrazine exposure and the occurrence of breast cancer in women was evaluated using the framework developed by Adami et al. (2011) wherein biological plausibility and epidemiological evidence were combined to conclude that a causal relationship between atrazine exposure and breast cancer is "unlikely". Carcinogenicity studies in female Sprague-Dawley (SD) but not Fischer-344 rats indicate that high doses of atrazine caused a decreased latency and an increased incidence of combined adenocarcinoma and fibroadenoma mammary tumors. There were no effects of atrazine on any other tumor type in male or female SD or Fischer-344 rats or in three strains of mice. Seven key events that precede tumor expression in female SD rats were identified. Atrazine induces mammary tumors in aging female SD rats by suppressing the luteinizing hormone surge, thereby supporting a state of persistent estrus and prolonged exposure to endogenous estrogen and prolactin. This endocrine mode of action has low biological plausibility for women because women who undergo reproductive senescence have low rather than elevated levels of estrogen and prolactin. Four alternative modes of action (genotoxicity, estrogenicity, upregulation of aromatase gene expression or delayed mammary gland development) were considered and none could account for the tumor response in SD rats. Epidemiological studies provide no support for a causal relationship between atrazine exposure and breast cancer. This conclusion is consistent with International Agency for Research on Cancer's classification of atrazine as "unclassifiable as to carcinogenicity" and the United States Environmental Protection Agency's classification of atrazine as "not likely to be carcinogenic."