In the IARC Working Group’s “Exposed Humans” table, three studies are cited assessing chromosomal aberrations, DNA strand breaks, and micronuclei formation in five populations of exposed people [5, 28, 29]. Positive evidence of genotoxicity was reported in four of the five populations. Only Bolognesi et al. [5] was cited in the EPA’s Appendix F Table F.5. “Other assays for detecting DNA damage—glyphosate formulations” [3]. The introduction to Appendix F states the following:
“While the focus of this analysis is to determine the genotoxic potential of glyphosate, the agency has identified numerous studies conducted with glyphosate-based formulations that contain various concentrations of the glyphosate as well as other components of the end use products and are presented in Tables F.1–F.5” [3].
In the introduction to Sect. 5 on genotoxicity in its 2016 report, the EPA writes:
“Studies conducted with glyphosate formulations that were identified and considered relevant for genotoxicity evaluation are summarized in table form in Appendix F. As described in Sect. 7.0 of this document, glyphosate formulations are hypothesized to be more toxic than glyphosate alone. The agency is collaborating with NTP [National Toxicology Program] to systematically investigate the mechanism(s) of toxicity for glyphosate and glyphosate formulations. However, the focus of this section [Sect. 5 on genotoxicity] is the genotoxic potential of glyphosate technical” [3].
In the above passages, the EPA makes clear that it based its judgment regarding the genotoxicity of glyphosate and GBHs predominantly on studies conducted with glyphosate technical. EPA’s choice of words in discussing differences in the toxicity of formulated GBHs in contrast to glyphosate technical is hard to square with the results of multiple, published studies.
Differential toxicity
EPA regards such differences as “hypothesized,” despite many studies reporting that GBHs are, in general, more toxic than glyphosate technical [30,31,32], and sometimes by large margins [33, 34], as shown in Table 3. The examples of differential toxicity in Table 3 are based on the levels of glyphosate technical triggering a defined, positive genotoxicity response in a given assay, in contrast to the amount of glyphosate in a GBH that triggers the same response. Accordingly, such comparisons are limited to a specific assay and marker of biological response and should be interpreted as only one of many indicators of the relative toxicity of a dose of glyphosate in a GBH compared to the same dose of glyphosate in the absence of co-formulants.
Table 3 Examples of the differential toxicity of technical glyphosate and formulated GBHs in human cell assays
The reasons why the glyphosate in GBHs is more toxic than the same amount of glyphosate technical are generally agreed upon. Most of the surfactants used in the formulation of GBHs are designed to accelerate the movement of glyphosate across plant surface membranes and also foster the movement of glyphosate into mammalian cells [31, 35]. Many co-formulants are more toxic than technical glyphosate [31, 36] and synergistic activity may occur in some exposure scenarios with certain formulations. Accordingly, differential toxicity arises from variable combinations of the innate toxicity of the surfactant(s) in a GBH compared to technical glyphosate, the impact of the surfactant(s) on the movement of glyphosate through human skin and into cells, and possible synergistic impacts [30, 37,38,39].
The EPA goes on to state that it placed greater weight on in vivo genotoxicity assays than on those testing in vitro exposures, especially for the same endpoint and that the only positive in vivo results were seen “at relatively high doses that are not relevant for human health risk assessment” [3].
Need to address and mitigate unusual and high occupational exposures
The EPA’s September 2016 evaluation of glyphosate carcinogenicity is largely focused on typical, expected dietary exposures facing the general public. The EPA’s analysis does not encompass occupational and unusual exposure scenarios, nor circumstances where some problem, error, mistake, land use factor, or quirk of nature leads to an unusually high GBH-exposure episode.
Periodically, the EPA issues a report covering glyphosate exposure and health-impact incident reports. For example, between 2002 and 2008, a total of 271 incident reports were compiled by EPA, 36% of which involved neurological symptoms, 29.5% dermal irritation, rash, or hives, and 14% respiratory duress [15]. Common causes of such incidents include a slow leak in a hose or fitting on a backpack sprayer, leading to the drenching over several hours of the applicator’s neck, back, and/or legs; repair of an equipment breakdown that inadvertently leads to significant exposure to spray solution; and, routine maintenance and service of spraying equipment and tank cleanup procedures. In the case of large-scale spray equipment used to apply a GBH in farm fields or large areas, a person repairing a leaky fitting or valve, or dealing with clogged nozzles or a blown hose, can be heavily exposed in a matter of seconds.
There is a vast array of unusual circumstances leading to elevated- to very-high exposure episodes, compared to typical, “general population” exposures, that do not involve equipment malfunction. Some examples include the following:
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A child playing with a dog that has recently spent time in an area sprayed with a GBH;
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sugar cane harvesters in Central America working in a recently burnt field that had been sprayed 7–10 days earlier with a GBH, creating a possibly toxic mix of smoke and GBH residues;
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an applicator on an ATV or driving a truck-mounted sprayer that covers an area via a concentric-circle spray pattern on a windy day, and
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workers in a rice field adjacent to an irrigation ditch recently treated with a GBH for weed control.
More data needed on the distribution of exposure levels
Across all occupationally exposed populations, there is a distribution of glyphosate exposure levels ranging from modestly above typical, background levels, to manyfold higher. Only a few studies have reported sufficient data to gain some sense of the distribution of exposure levels in an exposed population. One such study focused on 82 Thai women during their seventh month of pregnancy. Kongtip et al. [42] reported the number of women falling within progressively higher levels of glyphosate in maternal serum and umbilical cord blood. Levels varied by some 100- to 200-fold, as evident in Fig. 1a, b.
The IARC Working Group placed considerable weight on the genotoxicity studies in human populations exposed to formulated GBHs (80% of which were positive), while the EPA did not. These studies reflect high-end, real-world human-exposure scenarios more closely than any other category of study. It is true that the populations in these studies lived in or near, or worked around areas heavily treated with formulated GBHs, but it is also highly likely that millions of people around the world applying a GBH on any given day, or living near areas where substantial volumes of GBHs are applied, are also exposed to elevated levels because of application equipment problems, wind conditions, human error, or negligence.
Further research is urgently needed to quantify urine and serum levels of glyphosate following known, high-exposure scenarios. In light of the heightened toxicity of formulated GBHs in contrast to technical glyphosate, research is also needed to determinate the levels of major GBH surfactants and adjuvants in urine and blood, as well as their rate of skin penetration, metabolism and excretion. Such data are essential to sort out whether, and to what degree, GBH adjuvants and surfactants account for the genotoxicity and/or other adverse health effects of GBHs, in contrast to exposure to glyphosate technical.
The data generated by such research and biomonitoring will be valuable for regulators and GBH registrants in two ways. First, it will help guide future changes in co-formulants to limit use of those known to increase risks through one or more mechanisms. Second, these data will help sharpen worker-risk assessments and identify under what conditions, and for what uses, additional worker-safety precautions and Personal Protective Equipment (PPE) are warranted.
Why so many bacterial reverse mutation studies?
Over one-half (51 out of 95) of all registrant-commissioned genotoxicity studies on glyphosate and GBHs report the results of bacterial reverse mutation assays (aka Ames tests). The EPA requires just one bacterial reverse mutation assay on a pesticide active ingredient like glyphosate.
It is not clear why registrants focused so heavily on bacterial reverse mutation assays (54% of total assays), nearly all of which report the same result (negative). Scientists not affiliated with the industry and publishing in peer-reviewed journals pursued different genotoxicity testing priorities and published only seven bacterial reverse mutation assay results (one positive), or 5.7% of the 122 assays reported in public literature.
In addition to Monsanto, other pesticide companies developed their own set of toxicology studies to support their proprietary GBH brands and hence had to fulfil EPA data requirements (e.g., Syngenta, Cheminova). Still, dozens of bacterial reverse mutation studies were conducted after data requirements were fulfilled and after there was widespread recognition among regulators and companies that glyphosate, and GBHs pose virtually no risk of genotoxicity in bacterial reverse mutation assays. The scientific and regulatory “added value” of so many bacterial reverse mutation studies is questionable, other than increasing the number of negative studies supporting the safety of glyphosate and GBHs. In vitro bacterial reverse mutation assays cost much less to run than nearly all other genotoxicity assays and hence would be among the least expensive options to increase the number of negative assays.
Different outcomes in regulatory and public literature studies
Table 1 reports that across all genotoxicity assays on glyphosate technical, just 2% of studies sponsored by glyphosate registrants reported some positive evidence of a genotoxic response, while 67% of the studies in peer-reviewed journals reported one or more positive result. Given that the same basic genotoxicity assay systems were used in carrying out most regulatory and public literature studies, this big difference in outcomes begs for an explanation.
In some cases, the authors of regulatory studies report some evidence of a genotoxic response in a given assay, but then classify the study as “negative” because of the following:
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The reported result occurred at an excessive dose level;
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the dose was toxic to cells via a non-genotoxic mechanism; and/or
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the route of administration is regarded as not relevant in human-health risk assessment.
Dozens of examples of the above judgements are described in the Monsanto-commissioned, comprehensive reviews of glyphosate and GBH genotoxicity assays [14, 22, 26, 27]. In general when compared to studies in the peer-reviewed literature, regulatory studies tend to place more weight on factors that can arguably turn a positive assay result into a negative, or equivocal one. The criteria and decision process regulators apply in determining whether the authors of regulatory studies are justified in dismissing a given positive result are generally unknown. This is an area in need of further research.
Genotox studies published post-EPA and IARC reviews
The most recent genotoxicity study evaluated by the IARC Working Group [4] and EPA [3] was published in January 2015 [43]. From February 2015 through December 24, 2018, at least 27 additional studies have been published addressing possible mechanisms of genotoxic action for glyphosate and/or formulated GBHs (see Table 4). All but one of the 27 studies in Table 4 reported one or more positive result: 18 positives arising from DNA damage, 6 associated with oxidative stress, and two with other genotoxicity mechanisms.
Table 4 Studies published since February 1, 2015 and the completion of the EPA [3] and IARC [4] reviews of glyphosate and formulated GBH genotoxicity These studies lend further support to the IARC Working Group’s conclusion that there is “strong evidence” that formulated GBHs can trigger cell damage through at least two mechanisms of action (DNA damage and oxidative stress), thereby possibly triggering or accelerating the progression of cancerous cell growths.
The database supporting assessments of the genotoxicity of glyphosate and GBHs continues to evolve. Ghisi Nde et al. [44] conducted a meta-analysis of studies reporting the formation of micronuclei following exposure to glyphosate and/or GBHs. The team reports that both glyphosate and GBHs increase the frequency of micronuclei formation. Soloneski et al. [45] conducted a study in toads comparing the genotoxic impacts of a GBH, a dicamba-based herbicide, and a combination of these two, formulated herbicides, and concluded that the combination of GBH + dicamba herbicide led to a synergistic effect on the induction of primary DNA breaks. This result is worrisome given that well over one-half of the soybeans planted in the US in 2018 were genetically engineered to resist both GBHs and dicamba, and around two-thirds of national acreage will likely be sprayed in 2019 with this same mixture of herbicides [46, 47].
Currently the National Toxicology Program is conducting in vitro assays comparing the genotoxicity of glyphosate technical and several glyphosate formulations, as well as conducting a comprehensive literature review of the current database on glyphosate genotoxicity. Their full report has not been published, but a poster presented at the 2018 Society of Toxicology Conference reported the results of several assays on human HepaRG and HeCaT cell lines [41]. CellTiter-Glo, ROS-Glo, and JC10 assays on both cell lines revealed significant impacts on cell viability and alteration of mitochondrial membrane potential for both glyphosate and glyphosate-based formulations. In addition, GBHs were substantially more toxic than glyphosate alone. The glyphosate formulations studied decreased cell viability by more than 90% at concentrations “approximately 20- to 50-fold lower than glyphosate” [41].
Debate likely to persist
The scientific debate over the genotoxicity and oncogenic potential of GBHs is ongoing. While both the EPA and EFSA consider the glyphosate database to be essentially complete relative to current testing requirements, critical knowledge and data gaps persist in three areas: (1) well-designed 2-year feeding studies in mice and rats fed formulated GBHs; (2) data on occupational exposures and risk under a diversity of scenarios, including atypical but recurrent handling and application scenarios that lead to markedly elevated exposures; and (3) modern, rigorous data on the rate of skin penetration of the glyphosate and co-formulants in GBHs, in contrast to rates of penetration from studies conducted using technical glyphosate. Ideally, to build confidence in study results, each of the above sets of studies should be undertaken both by registrants in accord with testing guidance from regulators, and by scientists not affiliated with, or funded by pesticide registrants or their allied organizations.