How did the US EPA and IARC reach diametrically opposed conclusions on the genotoxicity of glyphosate-based herbicides?

Abstract

Background
The US EPA considers glyphosate as “not likely to be carcinogenic to humans.” The International Agency for Research on Cancer (IARC) has classified glyphosate as “probably carcinogenic to humans (Group 2A).” EPA asserts that there is no convincing evidence that “glyphosate induces mutations in vivo via the oral route.” IARC concludes there is “strong evidence” that exposure to glyphosate is genotoxic through at least two mechanisms known to be associated with human carcinogens (DNA damage, oxidative stress). Why and how did EPA and IARC reach such different conclusions?

Results
A total of 52 genotoxicity assays done by registrants were cited by the EPA in its 2016 evaluation of technical glyphosate, and another 52 assays appeared in the public literature. Of these, one regulatory assay (2%) and 35 published assays (67%) reported positive evidence of a genotoxic response. In the case of formulated, glyphosate-based herbicides (GBHs), 43 regulatory assays were cited by EPA, plus 65 assays published in peer-reviewed journals. Of these, none of the regulatory, and 49 published assays (75%) reported evidence of a genotoxic response following exposure to a GBH. IARC considered a total of 118 genotoxicity assays in six core tables on glyphosate technical, GBHs, and aminomethylphosphonic acid (AMPA), glyphosate’s primary metabolite. EPA’s analysis encompassed 51 of these 118 assays (43%). In addition, IARC analyzed another 81 assays exploring other possible genotoxic mechanisms (mostly related to sex hormones and oxidative stress), of which 62 (77%) reported positive results. IARC placed considerable weight on three positive GBH studies in exposed human populations, whereas EPA placed little or no weight on them.

Conclusions
EPA and IARC reached diametrically opposed conclusions on glyphosate genotoxicity for three primary reasons: (1) in the core tables compiled by EPA and IARC, the EPA relied mostly on registrant-commissioned, unpublished regulatory studies, 99% of which were negative, while IARC relied mostly on peer-reviewed studies of which 70% were positive (83 of 118); (2) EPA’s evaluation was largely based on data from studies on technical glyphosate, whereas IARC’s review placed heavy weight on the results of formulated GBH and AMPA assays; (3) EPA’s evaluation was focused on typical, general population dietary exposures assuming legal, food-crop uses, and did not take into account, nor address generally higher occupational exposures and risks. IARC’s assessment encompassed data from typical dietary, occupational, and elevated exposure scenarios. More research is needed on real-world exposures to the chemicals within formulated GBHs and the biological fate and consequences of such exposures.

Full text

Benbrook Environ Sci Eur (2019) 31:2
https://doi.org/10.1186/s12302-018-0184-7
RESEARCH
How did theUS EPA andIARC
reach diametrically opposed conclusions
onthegenotoxicity ofglyphosate-based
herbicides?
Charles M. Benbrook*
Abstract
Background: The US EPA considers glyphosate as “not likely to be carcinogenic to humans.” The International Agency
for Research on Cancer (IARC) has classified glyphosate as “probably carcinogenic to humans (Group 2A).” EPA asserts
that there is no convincing evidence that “glyphosate induces mutations in vivo via the oral route.” IARC concludes
there is “strong evidence” that exposure to glyphosate is genotoxic through at least two mechanisms known to be
associated with human carcinogens (DNA damage, oxidative stress). Why and how did EPA and IARC reach such dif-
ferent conclusions?
Results: A total of 52 genotoxicity assays done by registrants were cited by the EPA in its 2016 evaluation of techni-
cal glyphosate, and another 52 assays appeared in the public literature. Of these, one regulatory assay (2%) and 35
published assays (67%) reported positive evidence of a genotoxic response. In the case of formulated, glyphosate-
based herbicides (GBHs), 43 regulatory assays were cited by EPA, plus 65 assays published in peer-reviewed journals.
Of these, none of the regulatory, and 49 published assays (75%) reported evidence of a genotoxic response follow-
ing exposure to a GBH. IARC considered a total of 118 genotoxicity assays in six core tables on glyphosate technical,
GBHs, and aminomethylphosphonic acid (AMPA), glyphosate’s primary metabolite. EPA’s analysis encompassed 51 of
these 118 assays (43%). In addition, IARC analyzed another 81 assays exploring other possible genotoxic mechanisms
(mostly related to sex hormones and oxidative stress), of which 62 (77%) reported positive results. IARC placed consid-
erable weight on three positive GBH studies in exposed human populations, whereas EPA placed little or no weight
on them.
Conclusions: EPA and IARC reached diametrically opposed conclusions on glyphosate genotoxicity for three primary
reasons: (1) in the core tables compiled by EPA and IARC, the EPA relied mostly on registrant-commissioned, unpub-
lished regulatory studies, 99% of which were negative, while IARC relied mostly on peer-reviewed studies of which
70% were positive (83 of 118); (2) EPA’s evaluation was largely based on data from studies on technical glyphosate,
whereas IARC’s review placed heavy weight on the results of formulated GBH and AMPA assays; (3) EPA’s evaluation
was focused on typical, general population dietary exposures assuming legal, food-crop uses, and did not take into
account, nor address generally higher occupational exposures and risks. IARC’s assessment encompassed data from
typical dietary, occupational, and elevated exposure scenarios. More research is needed on real-world exposures to
the chemicals within formulated GBHs and the biological fate and consequences of such exposures.
Keywords: Glyphosate, Roundup, Genotoxicity, Cancer, IARC , US EPA, Co-formulants, Occupational exposure,
Regulation
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Open Access
*Correspondence: charlesbenbrook@gmail.com
Benbrook Consulting Services, Enterprise, OR, USA
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Page 2 of 16
Benbrook Environ Sci Eur (2019) 31:2
Background
Markedly different judgements have been reached by the
US Environmental Protection Agency (EPA), European
regulators, and the International Agency for Research
on Cancer (IARC) regarding the potential of glyphosate-
based herbicides (GBHs) to cause or contribute to human
cancer. e EPA and European regulators have concluded
that glyphosate technical poses no significant cancer risks
to the general public, based on currently approved, food-
crop uses and the levels of dietary exposure expected in
the general population (including residues in drinking
water and beverages) [1–3].
In the IARC summary of the rationale for its Group
2A “probably carcinogenic to humans” classification of
glyphosate and GBHs, the Working Group wrote “there
is strong evidence that glyphosate can operate through
two key characteristics of known human carcinogens,
and that these can be operative in humans”: genotoxicity
(DNA damage) and oxidative stress [4].
In reference to the “strong evidence” of genotoxic-
ity in its summary statement, the IARC Working Group
highlighted a study in an exposed, human population
(presumably Bolognesi et al. [5]) in which “markers of
chromosomal damage (micronucleus formation) were
significantly greater after exposure than before exposure
in the same individuals” [4].
Also according to the IARC Working Group, there is
“strong evidence” that glyphosate, GBHs, and glypho-
sate’s major metabolite AMPA can induce oxidative
stress in animal studies and in invitro human cell assays.
Moreover, IARC stressed that observed oxidative stress
in several assays was ameliorated by administration of
an antioxidant, lending further support to this second
mechanism of action (e.g. [6, 7]).
In March 2015, IARC classified glyphosate and GBHs
as “probably carcinogenic to humans” [8]. is unex-
pected classification set off intense debate across and
among key scientific and regulatory institutions, the orig-
inal registrant of GBHs (Monsanto), and scientists pub-
lishing research on, or relevant to the assessment of GBH
carcinogenicity [9–14].
IARC initially released its full Monograph Volume 112
report on glyphosate and GBH carcinogenicity on July
29, 2015, and subsequently issued a slightly revised, final
version in January 2017 [4]. A summary of the IARC’s
classification decision was first published online March
25, 2015 in Lancet Oncology [8]. As the case with all
IARC reviews, the Working Group assessing glyphosate
and GBH carcinogenicity relied predominantly on pub-
licly available, peer-reviewed studies. e basis of IARC’s
Group 2A “probably carcinogenic to humans” classifica-
tion is summarized in Sect.6.4 “Rationale” in the Mono-
graph Volume 112 report, and reads in part:
“In making this overall evaluation, the Working
Group noted that the mechanistic and other rel-
evant data support the classification of glyphosate
in Group 2A. In addition to limited evidence for the
carcinogenicity of glyphosate in humans and suffi-
cient evidence for the carcinogenicity of glyphosate
in experimental animals, there is strong evidence
that glyphosate can operate through two key char-
acteristics of known human carcinogens, and that
these can be operative in humans” [4].
Focus ofEPA andEFSA glyphosate risk assessments
Recent regulatory judgements on glyphosate cancer risk
in the US and Europe are based upon an assessment of
general population, dietary exposures under typical
conditions, and do not take into account, nor reflect a
detailed evaluation of the sometimes much-higher levels
of exposure that occur in a variety of occupational mixer/
loader and applicator scenarios [15], e.g., hand-held,
backpack, ATV, and truck-mounted sprayers that require
a person to hold and direct an application wand. Such
applications lead to much higher dermal exposures com-
pared to applicators working inside tractor or sprayer
cabs. In addition, applying a GBH many days per year for
several hours per day inevitably leads to greater, routine
dermal exposures, as well as more numerous incidents
during which significantly greater than normal exposures
occur because of a leaky hose or value, wind conditions, a
spill, or other unusual or unforeseen circumstance.
EPA’s comprehensive report on the carcinogenicity
of glyphosate was released in September 2016 [3]. After
presenting a detailed assessment of registrant-conducted
animal bioassays, published epidemiological studies,
and the genotoxicity database on glyphosate technical
(but not GBHs, see below for why), Sect.6.6 of the EPA
report contrasts the information and evidence the agency
reviewed relative to the five different cancer classifica-
tions set forth in its 2005 cancer guidelines: carcinogenic
to humans; likely to be carcinogenic to humans; sugges-
tive evidence of carcinogenic potential; inadequate infor-
mation to assess carcinogenic potential; and, not likely to
be carcinogenic to humans [16]. ese classifications are
roughly equivalent to IARC’s categories: Group 1 (carci-
nogenic to humans); Group 2A (probably carcinogenic);
Group 2B (possibly carcinogenic); Group 3 (not classifi-
able); and, Group 4 (probably not carcinogenic) [17].
Based on EPA’s weight-of-the-evidence review of data
mostly from studies on glyphosate technical, the agency
concluded that “e strongest support is for ‘not likely to
be carcinogenic to humans’ at doses relevant for human
health risk assessment” (emphasis added) [3]. Such “rel-
evant doses,” as discussed in the EPA assessment, arise
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Benbrook Environ Sci Eur (2019) 31:2
from residues in food and beverages following legal,
labelled food-crop applications of a GBH.
Other regulatory authorities have mostly concurred
with the EPA’s judgement, for essentially the same rea-
sons [1, 2, 18]. Regulatory agencies other than the US
EPA cite mostly the same set of registrant-conducted
studies as EPA, supplemented to one degree or another,
by a portion of the studies in the peer-reviewed literature
that formed the primary basis of IARC’s review.
Since March of 2015, papers have been published gen-
erally supporting and/or defending the IARC determina-
tion [9, 10, 19–21], while others have criticized IARC’s
conclusion and/or addressed why the EPA and other reg-
ulatory-agency judgements should be accepted as more
surely based on “sound science” [2, 14, 22, 23]. A few
papers have attempted to explain why IARC and the US
EPA reached such different conclusions [11, 24]. IARC
distributed a January 2018 “Briefing Note for IARC Sci-
entific and Governing Council Members” responding to a
number of criticisms and erroneous claims regarding the
deliberations of the Working Group [25].
GBHs should be thefocus ofrisk assessments
It is important to emphasize that in the case of glypho-
sate, the vast majority of registrant-conducted animal
studies, and all the chronic studies, have dosed animals
with technical glyphosate. Hence, the key toxicologi-
cal parameters embedded in EPA and other regulatory,
human-health risk assessments reflect risks stemming
from exposure to glyphosate technical in the absence of
co-formulants.
is is inappropriate for three reasons: (1) formulated
GBHs account for all commercial uses and human expo-
sures (no herbicide products contain just glyphosate),
(2) regulators are aware that the co-formulants in GBHs
markedly alter the absorption, distribution, metabolism,
excretion, and possibly the toxicity, of the glyphosate in
formulated GBHs [1–3]; and (3) multiple studies report
that formulated GBHs are more toxic than glyphosate
technical (see Table3 for references and accompanying
discussion).
e three major pillars of the EPA and IARC evalua-
tions of GBH oncogenic risk to humans are animal bioas-
says, epidemiological studies in exposed populations, and
genotoxicity studies useful in determining whether there
are plausible mechanism(s) through which exposure to
GBHs might trigger or accelerate cancerous cell growth.
Differences exist in the EPA’s and IARC’s assessments of
animal bioassay and epidemiological data, but by far the
most pronounced, and consequential differences arise in
the area of GBH genotoxicity.
Taking full account of differences in the absorption, dis-
tribution, metabolism, excretion, and toxicity of technical
or “pure” glyphosate, in contrast to formulated, glypho-
sate-based herbicides (GBHs) poses many challenges
for regulators, scientists, and glyphosate manufactur-
ers. According to the European Food Standards Agency
(EFSA), the purity of glyphosate technical in its evalua-
tion and risk assessment ranges between 95% and 98.3%
glyphosate [2]. Impurities including N-nitroso-glypho-
sate and formaldehyde make up no more than 1.1% of
technical glyphosate [2]. Formulated GBHs, on the other
hand, typically contain between 2 and 60% glyphosate
and a fraction of 1% to 25% adjuvants, surfactants, and
other co-formulants.
is analysis strives to shed light on why EPA and
IARC reached diametrically opposed judgements regard-
ing GBH genotoxicity. e science base cited and relied
upon by the EPA and IARC in their full evaluation
reports are contrasted, and also compared to the set of
studies addressed in several Monsanto-commissioned
review articles [14, 22, 26, 27]. Differences in the evi-
dentiary foundation and technical focus of the EPA and
IARC, as they undertook their genotoxicity assessments,
are described, as are the ways these differences altered
the weight accorded to different studies and lines of
evidence.
Methods
Section 5.3 of the September 2016 EPA evaluation of
glyphosate carcinogenicity [3] includes seven tables set-
ting forth the assays the agency considered in the follow-
ing areas:
• Table 5.1. Invitro test for gene mutation in bacte-
ria: glyphosate technical (hereafter Bacterial Reverse
Mutation Studies);
• Table5.2. Invitro mammalian gene mutation assays:
glyphosate technical;
• Table5.3. Invitro tests for chromosomal aberrations
in mammalian cells—glyphosate technical;
• Table5.4. Invitro tests for micronuclei induction in
mammalia cells—glyphosate technical;
• Table5.5. Invivo tests for chromosomal aberrations
in mammals—glyphosate technical;
• Table5.6. Invivo tests for micronuclei induction in
mammals—glyphosate technical; and
• Table5.7. Assays for detecting primary DNA dam-
age—glyphosate technical (hereafter DNA Damage).
Each of these seven tables reports the Test/Endpoint;
Test [assay] System; Route of Administration; Doses/
Concentration; Test Material Purity (when known);
Results; References; and, Comments. “Results” typically
are “positive” or “negative,” and sometimes specify the
conditions under which a positive/negative response
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Benbrook Environ Sci Eur (2019) 31:2
was reported (e.g., “Negative ± S9”; “Positive, Statis-
tically significant [p < 0.05] increase in MN at 15 and
20mg/L”).
e assay-by-assay information in these core tables
was moved into an Excel workbook, in which the follow-
ing data were recorded: Year, Author, Result, whether the
assay was conducted by a registrant (Regulatory) or was
published in the Public Literature, and Comments. All of
the above information is recorded as reported in the EPA
tables noted above. e assay results described in the
EPA tables were chosen by the agency, and usually were
also noted in each study’s abstract.
Each of this paper’s tables is organized according to
whether an assay was conducted with glyphosate techni-
cal (Glyphosate), or a formulated GBH. Also included is
whether a study or assay was cited by EPA 2016, IARC
2017, and/or a Monsanto-commissioned review. Addi-
tional file 1: Tables S1–S7 include the above informa-
tion for the studies and assays cited by EPA in seven core
tables (Tables5.1–5.7).
In addition, the EPA listed studies on glyphosate-based
formulations separately in Appendix F, “Genotoxicity
Studies with Glyphosate Based Formulations” [3]. Assays
in Tables F.1, F.2, F.3, F.4 and F.5 were added to Additional
file 1: Tables S1–S7, and incorporated in the present
analysis.
Summary statistics by type of genotoxicity test and
assay system were calculated for studies on glyphosate
technical and formulated GBHs. For regulatory studies,
public literature studies, and all studies, the number of
studies, number of positives, and percent positive were
calculated.
A similar Excel worksheet was constructed from
all glyphosate-related genotoxicity studies and assays
cited in Volume 112 of the IARC Monograph series in
“Sect.4.2 Mechanisms of carcinogenesis” [4]. e IARC
Working Group organized its assessment of genotoxicity
data in six core tables:
• Table4.1 Genetic and related effects of glyphosate in
exposed humans;
• Table4.2 Genetic and related effects of glyphosate,
AMPA, and glyphosate-based formulations in human
cells invitro;
• Table4.3 Genetic and related effects of glyphosate,
AMPA, and glyphosate-based formulations in non-
human mammals invivo;
• Table4.4 Genetic and related effects of glyphosate,
AMPA, and glyphosate-based formulations in non-
human mammalian cells invitro;
• Table4.5 Genetic and related effects of glyphosate,
AMPA, and glyphosate-based formulations in non-
mammalian systems invivo; and
• Table 4.6 Genetic and related effects of glyphosate
and glyphosate-based formulations on non-mamma-
lian systems invitro.
Each of the above six, IARC tables covers studies and
assays done on glyphosate technical, as well as any stud-
ies/assays conducted using a formulated GBH. A few
studies testing the primary glyphosate metabolite ami-
nomethylphosphonic acid (AMPA) are also included in
the IARC tables. As in the case of the core EPA tables,
the IARC Working Group selected the assays to describe
from each study. With a few exceptions entailing indeter-
minate results, each assay was designated as “positive” or
“negative” for genotoxicity. Hence, the information in this
paper’s tables on assays considered by the IARC Working
Group reflect that Working Group’s judgements regard-
ing which of the assays were scientifically valid, relevant,
and which indicated genotoxic potential versus those that
did not.
For these six IARC tables, the following information
was recorded in the Excel workbook: Category of Study,
Citation (lead author(s) and year), End-point Studied,
Test/Assay, Response/Results, and Comments. In addi-
tion, the tables note whether a given study/assay was
cited in a Monsanto-commissioned Review (yes/no) [14,
22, 26, 27] and/or in the EPA’s September 2016 report
[3]. Summary statistics are calculated by IARC assay
category. Additional file1: Table S8 lists the genotoxic-
ity studies and assays considered by the IARC Working
Group and records the information described above.
Assays cited by IARC and recorded in Additional file1:
TableS8 were then added to Additional file1: TablesS1–
S7, allowing comparison of the number of studies and
assays in each of the categories that were cited by EPA,
IARC, or both.
In addition to the assays cited by IARC in Tables4.1–
4.6, additional studies are discussed in the narrative of
the IARC Monograph 112 in Sects. 4.2.2–4.2.4. ese
studies are related to mechanisms of genotoxicity other
than those listed in the core tables. e majority of these
studies explore sex hormone disruption and oxidative
stress. For studies cited in the narrative sections of IARC
2017, the following information was recorded in Addi-
tional file1: TableS9: Citation (lead author(s) and date),
Category, Study type, End-point studied, and Result.
e data in Additional file 1: Tables S1–S9 are inte-
grated and summarized in Additional file 1: TableS10.
is table lists all assays considered by the EPA and
IARC, and breaks them down by source (Regulatory or
Public Literature) and result (Total Number, Number
Positive, and Percent Positive). Finally, Additional file1:
TableS11 reports, for the core IARC and EPA tables, the
number and results of assays considered by IARC but not
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Benbrook Environ Sci Eur (2019) 31:2
by EPA, and the number considered by EPA but not by
IARC.
Note that many of the studies cited by IARC and EPA
report results in more than one test system or assay.
Other studies cover assays testing glyphosate technical,
AMPA, and formulated GBHs. As a result, the total num-
ber of reported assay results for glyphosate technical,
AMPA, and GBHs exceeds the number of studies cited
by EPA, IARC, or in any, or all of the four Monsanto-
commissioned reviews [14, 22, 26, 27]. Also, EPA and
IARC utilize somewhat different classification systems
and methods for recording assay results, altering the
number of assay results reported from a few studies (~ 9
assays), and account for minor differences in the assay
counts across Additional file1: Tables S1–S11.
roughout this paper, a “negative” assay is one
reported as negative across all dose levels, as well as
across all alternative ways a given assay was conducted;
a “positive” assay is one where the authors reported, and
IARC and/or EPA concurred, that there was at least one
statistically significant genotoxic response at one or more
dose levels.
Results
Table1 provides an overview of the database available as
of early 2015 for evaluation of the genotoxicity of glypho-
sate and glyphosate-based herbicides (GBHs). e seven
categories of studies in Table1 cover assays cited by EPA
and/or IARC in their analyses [3, 4]. Across all catego-
ries, a total of 52 regulatory assays were cited by EPA on
glyphosate technical. Another 52 assays appeared in the
public literature and were cited by EPA, IARC, or both,
for a total of 104 assays on glyphosate technical.
Of the total 104 assays on glyphosate technical, one
regulatory assay and 35 published assays reported posi-
tive evidence of a genotoxic response, for a total of 36
positive assays in the case of glyphosate technical. us,
for glyphosate technical just 2% of the regulatory assays
and 67% of the assays published in peer-reviewed jour-
nals reported positive results.
While EPA did not consider any assays on AMPA,
IARC’s analysis did include five assays on the genotoxic-
ity of this major metabolite of glyphosate (all positive).
In the case of formulated GBHs, 43 regulatory assays
and 65 assays from published literature were cited by
EPA and/or IARC, for a total of 108. Of these 108, none
of the regulatory assays reported evidence of a positive
genotoxic response, while 49 published assays did (75%).
Bacterial reverse mutation assays accounted for 51 out
of the 95 (54%) regulatory assays submitted to EPA on
glyphosate technical and formulated GBHs. ere were a
total of 29 assays on glyphosate technical exploring direct
DNA damage cited by EPA, mostly COMET and sister
chromatid exchange assays. Of these 29 assays, 27 were
reported in public literature studies.
Table 2 reports the number of assays on glyphosate
technical, AMPA, and formulated GBHs cited by IARC
in the following six categories of studies:
• Exposed humans category (GBHs);
• Human cells invitro categories (glyphosate technical,
AMPA, GBHs);
• Non-human mammals invivo categories (glyphosate
technical, AMPA,GBHs);
• Non-human mammalian cells in vitro categories
(glyphosate technical, AMPA, GBHs);
• Non-mammalian systems invivo categories (glypho-
sate technical, AMPA, GBHs); and
• Non-mammalian systems invitro categories (glypho-
sate technical, GBHs).
In addition, for each category of study, Table2 reports
the number of assays that were also cited in one or more
of the four Monsanto-commissioned reviews [14, 22, 26,
27] and by EPA in its September 2016 report [3].
Across all categories of studies in its six core tables,
IARC considered 118 assays. Of these, Monsanto-com-
missioned reviews cited 84 (71%) and the EPA cited
51 (43%). In general, both scientists and regulators [3]
place greater emphasis on mammalian assay systems, in
contrast to non-mammalian systems, in evaluating the
mechanisms of toxicity in humans following exposure
to pesticides. As shown in Table2, the IARC Working
Group cited 54 mammalian assays, and 49 (91%) and 40
(74%) of these were cited in the Monsanto-commissioned
reviews and by the EPA, respectively.
Of the 51 assays cited by both the EPA and IARC, 24
report the impact of exposures to GBHs, but these were
given little weight in the EPA’s assessment of the geno-
toxicity of glyphosate (see “Discussion”). erefore, EPA’s
conclusion regarding the genotoxicity of glyphosate was
based predominantly on the agency’s review of 27 assays
out of the 118 assays assessed by IARC (51 total IARC
and EPA assessed assays, minus 24 on GBHs not focused
on by EPA).
Hence, in its evaluation of glyphosate genotoxicity,
the EPA took fully into account the results of only 23%
of the assays considered by IARC (27/118). In addition,
Additional file1: TableS11 shows that the EPA took into
account 61 registrant-commissioned assays on glypho-
sate technical and 48 registrant assays on GBHs that
were not considered by the IARC Working Group. Of
these 109 assays, seven (6.4%) were positive. In terms of
focus, 54 of the 109 assays (49.5%) were from bacterial
reverse mutation studies (all negative), and 31 (28.4%)
were invivo micronuclei induction assays (30 negative).
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Benbrook Environ Sci Eur (2019) 31:2
Additional file1: TableS11 also shows that IARC ana-
lyzed 21, 41, and 5 published assays (total 67), respec-
tively, on glyphosate technical, GBHs, and AMPA that
were not reviewed or given weight by the EPA, 55 (82%)
of which were positive.
In order to accurately describe the full genotoxic-
ity database evaluated by IARC, several additional
studies and assays cited in the Volume 112 narrative
report have been added to Additional file1: TableS9
and are summarized in Additional file 1: Table S10.
There were 82 assays involving other potential mecha-
nisms of genotoxicity (mainly sex hormone disruption
and oxidative stress) that were evaluated by the IARC
Working Group and referenced in the narrative sec-
tion of IARC 2017. These include an additional 53
assays on glyphosate technical, 4 on AMPA, and 25
on formulated GBHs, 77% of which reported positive
results.
When included in the overall analysis (see Additional
file1: TableS10), these additional assays bring the total
number considered by IARC or EPA to 306, 51% of
which report positive results. 211 of these 306 assays
are from the public literature, 74% of which reported
one or more positive result.
Table 1 Genotoxicity assays onglyphosate andformulated GBHs byregistrants (“Reg.”) andinpublic literature (“Pub.”)
1. Table includes assays on glyphosate technical cited in EPA’s 2016 “Glyphosate Issue Paper: Evaluation of Carcinogenic Potential,” Sect.5: Data Evaluation of Genetic
Toxicity, Table5.1, 5.2, 5.3, 5.4, 5.5, 5.6, and 5.7. Assays on formulated GBHs considered by EPA come from Tables F.1, F.2, F.3, F.4 and F.5 in Appendix F: “Genotoxicity
Studies with Glyphosate Based Formulations” [3]
2. Also included are additional assays on glyphosate technical, AMPA, and formulated GBHs from IARC Monograph 112 on the carcinogenicity of glyphosate [4] from
Tables4.1, 4.2, 4.3, 4.4, 4.5, or 4.6
3. Additional le1: TablesS1–S7 list all assays in the core tables from EPA 2016 [3] and IARC 2017 [4] based on genotoxicity assay type
Assay type andcompound
tested Number ofassays Number ofpositives Percent positive
Reg. Pub. Total Reg. Pub. Total Reg. (%) Pub. (%) Total (%)
Bacterial reverse mutation
Glyphosate technical 23 4 27 0 0 0 0 0 0
Formulated GBHs 28 3 31 0 1 1 0 33 3
In vitro and in vivo mammalian gene mutation
Glyphosate technical 4 2 6 0 1 1 0 50 17
Formulated GBHs 0 1 1 0 1 1 0 100 100
In vitro chromosomal aberration
Glyphosate technical 4 5 9 0 3 3 0 60 33
AM PA 0 1 1 0 1 1 0 100 100
Formulated GBHs 0 2 2 0 1 1 0 50 50
In vitro micronuclei induction in mammals
Glyphosate technical 0 6 6 0 4 4 0 67 67
In vivo chromosomal aberration
Glyphosate technical 5 2 7 0 2 2 0 100 29
Formulated GBHs 0 8 8 0 6 6 0 75 75
In vivo micronuclei induction in mammals
Glyphosate technical 14 6 20 1 2 3 7 33 15
AM PA 0 1 1 0 1 1 0 100 100
Formulated GBHs 15 13 28 0 7 7 0 54 25
DNA damage
Glyphosate technical 2 27 29 0 23 23 0 85 79
AM PA 0 3 3 0 3 3 0 100 100
Formulated GBHs 0 38 38 0 33 33 0 87 87
All assays
Glyphosate technical 52 52 104 1 35 36 2 67 35
AM PA 0 5 5 0 5 5 0 100 100
Formulated GBHs 43 65 108 0 49 49 0 75 45
All tested compounds 95 122 217 1 89 90 1 73 41
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e tendency of scientists to not disseminate or seek
publication of “negative” studies is likely not a factor
in the case of registrant-commissioned lab studies of
glyphosate and GBHs, the majority of which are submit-
ted to regulators and report mostly negative results. A
substantial share (~ 27%) of published assays on glypho-
sate or GBHs done by scientists not working for pesti-
cide manufacturers reported negative results. ere is no
way of knowing how many additional assays have been
done by registrants with negative or positive results that
were never submitted to regulators, nor published. It is
also not possible to project the number of assays done by
scientists not working for industry that showed positive
or negative signs of genotoxicity, but were not published.
Discussion
In the IARC Working Group’s “Exposed Humans” table,
three studies are cited assessing chromosomal aberra-
tions, 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]. e intro-
duction 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 glypho-
sate-based formulations that contain various con-
Table 2 Number of genotoxicity assays cited in core tables by IARC 2017, EPA 2016, or in Monsanto-commissioned
reviews
1. IARC totals are from the detailed accounting of all studies considered by the IARC Work ing Group in the glyphosate section of M onograph 112 [4]. I nformation on
the studies are taken from Tables4.1–4.6 and the discussion of studies on oxidative stress in humans in Sect.4.2.3 (a) (i) of the monograph
2. “MON Reviews” include four published studies: Brusick etal. [22]; Kier and Kirkland [27]; Heydens etal. [26]; and Williams etal. [14]. The references in these four,
Monsanto-commissioned reviews were cross-checked against the list of IARC studies
3. “EPA Sept 2016” refers to the September 12, 2016 “Glyphosate Issue Paper: Evaluation of Carcinogenic Potential” [3]. All studies cited in this EPA document, including
in its Appendix F which contains studies on formulated glyphosate-based herbicides, were cross-checked against the studies cited by IARC and in the Monsanto-
commissioned reviews
Assay type andcompound tested Number ofstudies considered
byIARC Cited by
MON reviews EPA Sept 2016
Exposed humans
Formulated GBHs 5 5 0
Human cells in vitro
Glyphosate 10 8 7
AM PA 2 2 0
Formulated GBHs 4 2 3
Non-human mammals in vivo
Glyphosate 11 10 10
AM PA 1 1 0
Formulated GBHs 13 13 13
Non-human mammalian cells in vitro
Glyphosate 5 5 5
AM PA 1 1 0
Formulated GBHs 2 2 2
Non-mammalian systems in vivo
Glyphosate 12 7 1
AM PA 2 2 0
Formulated GBHs 40 21 4
Non-mammalian systems in vitro
Glyphosate 8 3 4
Formulated GBHs 2 2 2
Total all categories 118 84 51
As percent of total IARC 71% 43%
Total of all mammalian categories 54 49 40
As percent of total IARC 91% 74%
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Page 8 of 16
Benbrook Environ Sci Eur (2019) 31:2
centrations of the glyphosate as well as other com-
ponents 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 gen-
otoxicity evaluation are summarized in table form
in Appendix F. As described in Sect.7.0 of this docu-
ment, glyphosate formulations are hypothesized to
be more toxic than glyphosate alone. e agency is
collaborating with NTP [National Toxicology Pro-
gram] to systematically investigate the mechanism(s)
of toxicity for glyphosate and glyphosate formula-
tions. 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 glypho-
sate 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.
Dierential toxicity
EPA regards such differences as “hypothesized,” despite
many studies reporting that GBHs are, in general, more
toxic than glyphosate technical [30–32], and sometimes
by large margins [33, 34], as shown in Table3. e exam-
ples of differential toxicity in Table 3 are based on the
levels of glyphosate technical triggering a defined, posi-
tive 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 lim-
ited 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.
e 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 mam-
malian cells [31, 35]. Many co-formulants are more toxic
than technical glyphosate [31, 36] and synergistic activ-
ity 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 glypho-
sate, the impact of the surfactant(s) on the movement of
glyphosate through human skin and into cells, and pos-
sible synergistic impacts [30,37–39].
e 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 rela-
tively high doses that are not relevant for human health
risk assessment” [3].
Need toaddress andmitigate unusual andhigh
occupational exposures
e EPA’s September 2016 evaluation of glyphosate carci-
nogenicity is largely focused on typical, expected dietary
exposures facing the general public. e 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 glypho-
sate 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 inadvert-
ently leads to significant exposure to spray solution; and,
Table 3 Examples ofthedierential toxicity oftechnical glyphosate andformulated GBHs inhuman cell assays
Assay/marker Glyphosate technical Formulated GBHs Dierential Source
Viability of human peripheral blood mononuclear cells 1640 μg/mL 56.4 μg/mL 29 Martinez et al. [40]
LC 50 in HepG2 cells (ppm) 19,323 62 312 Mesnage et al. [33]
LC 50 in JEG3 cells (ppm) 1192 32 37 Mesnage et al. [33]
1/LC 50 JEG3 cells (ppm) [glyphosate IPA-salt; Roundup Classic] 0.000082 0.0081 99 Defarge et al. [32]
DNA damage to peripheral blood mononuclear cells 250 μM 5 μM [Roundup 360 PLUS] 50 Wozniak et al. [34]
LC 50 in human HepaRG cells 2 mg/mL 0.04–0.1 mg/mL 20–50 Rice et al. [41]
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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.
ere 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:
• A child playing with a dog that has recently spent
time in an area sprayed with a GBH;
• sugar cane harvesters in Central America work-
ing 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;
• 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
• 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
ai women during their seventh month of pregnancy.
Kongtip etal. [42] reported the number of women fall-
ing 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.
e IARC Working Group placed considerable weight
on the genotoxicity studies in human populations
exposed to formulated GBHs (80% of which were posi-
tive), while the EPA did not. ese 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 toxic-
ity of formulated GBHs in contrast to technical glypho-
sate, 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.
e data generated by such research and biomonitor-
ing 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 Equip-
ment (PPE) are warranted.
Why somany bacterial reverse mutation studies?
Over one-half (51 out of 95) of all registrant-commis-
sioned genotoxicity studies on glyphosate and GBHs
report the results of bacterial reverse mutation assays
(aka Ames tests). e 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 bac-
terial 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 test-
ing 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 sup-
port their proprietary GBH brands and hence had to
fulfil EPA data requirements (e.g., Syngenta, Chemi-
nova). Still, dozens of bacterial reverse mutation studies
were conducted after data requirements were fulfilled
and after there was widespread recognition among reg-
ulators and companies that glyphosate, and GBHs pose
virtually no risk of genotoxicity in bacterial reverse
mutation assays. e scientific and regulatory “added
value” of so many bacterial reverse mutation stud-
ies 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.
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Dierent 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 evi-
dence of a genotoxic response, while 67% of the studies
in peer-reviewed journals reported one or more posi-
tive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:
• e reported result occurred at an excessive dose
level;
• the dose was toxic to cells via a non-genotoxic mech-
anism; and/or
• the route of administration is regarded as not rel-
evant in human-health risk assessment.
Dozens of examples of the above judgements are
described in the Monsanto-commissioned, comprehen-
sive reviews of glyphosate and GBH genotoxicity assays
[14, 22, 26, 27]. In general when compared to stud-
ies 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. e criteria and decision process regulators apply
Fig. 1 a, b Range of glyphosate concentrations in maternal and umbilical cord serum. This figure was created using data from Kongtip et al. [42,
see Figure 1 for publication.eps]
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Benbrook Environ Sci Eur (2019) 31:2
in determining whether the authors of regulatory stud-
ies are justified in dismissing a given positive result are
generally unknown. is is an area in need of further
research.
Genotox studies published post‑EPA andIARC reviews
e most recent genotoxicity study evaluated by the
IARC Working Group [4]and EPA [3]was published in
January 2015 [43]. From February 2015 through Decem-
ber 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.
ese studies lend further support to the IARC Work-
ing 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 oxida-
tive stress), thereby possibly triggering or accelerating the
progression of cancerous cell growths.
e database supporting assessments of the genotox-
icity of glyphosate and GBHs continues to evolve. Ghisi
Nde et al. [44] conducted a meta-analysis of studies
reporting the formation of micronuclei following expo-
sure to glyphosate and/or GBHs. e 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 syn-
ergistic effect on the induction of primary DNA breaks.
is 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 conduct-
ing invitro assays comparing the genotoxicity of glypho-
sate technical and several glyphosate formulations, as
well as conducting a comprehensive literature review of
the current database on glyphosate genotoxicity. eir
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 for-
mulations. In addition, GBHs were substantially more
toxic than glyphosate alone. e glyphosate formulations
studied decreased cell viability by more than 90% at con-
centrations “approximately 20- to 50-fold lower than
glyphosate” [41].
Debate likely topersist
e 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 for-
mulated 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 penetra-
tion 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 reg-
istrants in accord with testing guidance from regulators,
and by scientists not affiliated with, or funded by pesti-
cide registrants or their allied organizations.
Conclusions
According to EPA, glyphosate technical does not pose
oncogenic risk at “relevant” levels of exposure, i.e. those
levels likely to occur among members of the general
public from “typical” dietary exposures. In reaching
its “not likely to be oncogenic” conclusion, the EPA (1)
largely ignored epidemiological studies, some of which
reported elevated, statistically significant odds ratios
among cohorts that were relatively more highly exposed
to GBHs and, (2) placed little or no weight on multiple
invivo GBH genotoxicity assays that reported DNA dam-
age and/or oxidative stress in laboratory animals and
exposed human cohorts.
IARC, on the other hand, placed considerable weight
on studies linking use of, and exposure to GBHs to can-
cer, as well as evidence of DNA damage and oxidative
stress in human populations exposed to GBHs. e sci-
ence base reviewed by IARC included adverse impacts
among some people much more heavily exposed than
a typical person in the US or Europe. Hence, the IARC
Working Group’s weight-of-evidence judgement that
GBHs are “probably carcinogenic to humans” is most
appropriately applied to those humans who are relatively
more heavily exposed to GBHs. In fact, had the IARC
Working Group restricted its assessment of GBH onco-
genicity in ways comparable to the limits embedded in
the assessments conducted by the EPA and EFSA, it is
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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
Citation Compound tested Dose/concentration Test target Assay Result
Genotox assay type
Mammalian in vivo
Kasuba et al. [48] Glyphosate technical 0.5, 2.91 and 3.5 μg/mL Human CBMN assay, oxidative stress Positive, oxidative stress (all
conc.)
Kasuba et al. [48] Glyphosate technical 0.5, 2.91 and 3.5 μg/mL Human HepG2 cells, oxidative stress Positive, oxidative stress (all
conc.)
Milic et al. [49] Glyphosate technical 0.1, 0.5, 1.75 and 10 mg/kg/day Wistar rat COMET, DNA damage, leuko-
cytes, liver cells Positive, DNA damage in leuko-
cytes and liver cells (all conc.)
Mammalian in vitro
Luaces et al. [50] Formulated GBH (Roundup
Full II®)280, 420, 560, 1120 μmol/L Armadillo Chromosomal aberrations Positive (all conc.)
Luo et al. [51] Formulated GBH (Roundup®) Not provided in abstract Human L-02 hepatocytes, oxidative
stress Positive, oxidative stress
Kwiatkowska et al. [52] Glyphosate technical 0.1–10 mM Human blood cells Comet assay, DNA damage Positive, DNA damage (at
0.5–10 mM doses)
Townsend et al. [53] Glyphosate technical 0.1 μM–15 mM Human Raji cells Comet assay, DNA damage Positive, DNA damage (at doses
above 1 mM)
De Almeida et al. [54] Formulated GBHs (Roundup®
and Wipeout®)10–500 μg/mL Human cancer cell lines Comet assay, DNA damage Positive, DNA damage (all conc.)
Rice et al. [41] Glyphosate technical and
formulated GBHs 0.0007–33 mM Human HepaRG and HaCaT
cell lines Cell Titer-Glo, ROS-Glo and
JC10, oxidative stress Positive, oxidative stress (pure
glyphosate ≥ 10 mM, formula-
tions all conc.)
Rossi et al. [55] Formulated GBH (Roundup
Full II®)0.026–0.379 mL/day Armadillo Chromosomal aberrations Positive (all conc.)
Santovito et al. [56] Glyphosate technical 0.5, 0.1, 0.05, 0.025 and
0.0125 μg/mL Human lymphocytes Chromosomal aberration and
micronuclei Positive (all conc.)
Szepanowski et al. [57] Glyphosate technical and
formulated GBH 0.0005–0.005% of culture
medium Human ganglia cultures Myelination Positive (all conc. of GBH)
Wozniak et al. [34] Glyphosate technical, formu-
lated GBH (Roundup 360
PLUS®) and AMPA
1 to 1000 μM Human cells Breaks, peripheral mononu-
clear cells, DNA damage Positive, DNA dam-
age (GBH ≥ 5 μM,
glyphosate ≥ 250 μM,
AMPA ≥ 500 μM)
Non-mammalian in vivo
Schaumburg et al. [58] Formulated GBH (Roundup®) 50, 100, 200, 400, 800, 1600 μg/
egg Salvator merianae (reptile) Comet assay, DNA damage Positive, DNA damage
(≥ 100 μg/egg)
Soloneski et al. [45] Formulated GBH (Credit®) and
formulated dicamba herbi-
cide (Banvel®)
5–10% formulation of GBHs Rhinella arenarum larvae
(amphibian) SCGE, DNA breaks Positive, DNA breaks (above 5%)
Vieira et al. [59] Formulated GBHs (in situ
exposure) Not provided in abstract Prochilodus lineatus (fish) DNA damage, erythrocyte
abnormalities Positive
Burella et al. [60] Formulated GBH (Roundup®) 750, 1250, 1750 μg/egg Caiman latirostris (reptile) Comet, embryos, DNA damage Positive, DNA damage (all conc.)
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Table 4 (continued)
Citation Compound tested Dose/concentration Test target Assay Result
de Moura et al. [61] Formulated GBH (Roundup®) Not provided in abstract Leiarius marmoratusx/Pseudo-
platystoma reticulatum (fish) Micronucleus Positive (≥ 1.357 mg/L)
Hong et al. [62] Glyphosate technical 0, 4.4, 9.8, 44 and 98 mg/L Eriocheir sinensis (crab) Haemocyte DNA damage Positive (> 4.4 mg/L)
Lopez Gonzalez et al. [63] Formulated GBHs (Roundup
Full II® and PanzerGold®)500, 750, 1000 μg/egg Caiman latirostris (reptile) Micronucleus, embryos Positive, embryos (≥ 1000 μg/
egg)
Hong et al. [64] Formulated GBH (Roundup®) 0.35, 0.70, 1.40, 2.80 and
5.60 mg/L Macrobrachium nipponensis
(shrimp) Micronucleus Positive (≥ 1.40 mg/L)
Non-mammalian in vitro
Baurand et al. [65] Formulated GBH (Roundup
Flash®)Range around EC50 values Cantareus aspersus (snail) DNA damage Positive (≥ 30 mg)
Perez-Iglesias et al. [66] Glyphosate technical 100, 1000 and 10,000 μg/g Leptodactylus latinasus (frog) Hepatic melanomacrophages–
erythrocyte nuclear abnor-
malities
Positive (all conc.)
Bailey et al. [67] Formulated GBH
(TouchDown®)2.7%, 5.5%, or 9.8% glyphosate Caenorhabditis elegans (nema-
tode) Mitochondrial chain com-
plexes, oxidative stress Positive, oxidative stress (≥ 5.5%)
de Brito Rodrigues et al. [68] Formulated GBHs (Roundup®
and glyphosate AKB 480) Low doses not specified, high
doses up to 37.53 mg/L Danio rerio (fish) Genotoxicity Negative (lethal at high doses
but no genotoxicity observed)
Bollani et al. [69] Formulated GBHs (in situ
exposure) Glyphosate ≤ 13.6 μg/L,
AMPA ≤ 9.75 μg/L Allium cepa (plant) Micronucleus Positive
Santo et al. [70] Formulated GBH (Roundup®) 5.0 mg/L Danio rerio (fish) Micronucleus, oxidative stress Positive, oxidative stress
Glyphosate technicalaNumber of studies 11
Number positive 11
Percent positive 100%
Formulated GBHsbNumber of studies 19
Number positive 18
Percent positive 95%
All new studies Number of studies 27
Number positive 26
Percent positive 96%
a Includes studies published after 02/01/2015, the publication date of Marques etal. [43], the most recent study cited in IARC [4] or EPA [3]
b Note that since some studies conducted assays on BOTH glyphosate technical and formulated GBHs, the number of studies on glyphosate technical plus formulated GBHs exceeds the total number of new studies
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 14 of 16
Benbrook Environ Sci Eur (2019) 31:2
likely that the IARC Working Group would not have clas-
sified glyphosate as Group 2A, “probably carcinogenic to
humans.”
Ongoing research, regulatory risk assessments, and
debate over glyphosate and GBH carcinogenicity and
genotoxicity should be focused on studies relevant to the
biological impacts triggered by exposures to widely used,
formulated GBHs.
e laws and policies governing EPA regulatory deci-
sion-making direct the agency to carefully assess expo-
sures and typical, expected risks to the general public and
environment from pesticide applications made in accord
with label directions and required safety precautions.
Much less effort has been invested by EPA in assuring
that occupational and worker-risk assessments are based
on accurate exposure and toxicological data. Such scenar-
ios should include when, where, how, and how frequently
and heavily a formulated GBH is applied by a given per-
son. Risk assessments, pesticide label directions, pesti-
cide applicator training and certification curricula, and
health-related warnings to applicators should address
scenarios when hoses leak, spills occur, the wind blows
in unexpected ways, clothes are drenched and need to be
handled safely, and other unusual circumstances leading
to higher-than-normal exposures.
While unusually high-exposure scenarios generally
arise from some combination of equipment malfunction,
operator error or carelessness, or working in or near a
recently treated field or area, the frequency of such epi-
sodes is a function of the diversity and number of appli-
cations made. In the case of glyphosate-based herbicides,
the world’s most widely used pesticide ever, such rela-
tively high-exposure episodes occur tens of thousands of
times on a daily basis in the US and hundreds of thou-
sands, if not millions of times globally.
IARC’s evaluation relied heavily on studies capable of
shedding light on the distribution of real-world expo-
sures and genotoxicity risk in exposed human popula-
tions, while EPA’s evaluation placed little or no weight on
such evidence.
Additional le
Additional le1. Additional tables.
Abbreviations
AMPA: aminomethylphosphonic acid; EFSA: European Food Safety Authority;
EPA: United States Environmental Protection Agency; GBH: glyphosate-based
herbicide; IARC : International Agency for Research on Cancer; NTP: National
Toxicology Program.
Authors’ contributions
CB designed the study, compiled the data, carried out the analysis, and wrote
the paper. The author read and approved the final manuscript.
Acknowledgements
The author wishes to thank the 10 reviewers for their many constructive com-
ments on ways to strengthen and clarify the paper, as well as their attention
to detail, and also appreciates the assistance of Rachel Benbrook in the design
of the tables and figures in this paper, and in securing and properly citing
references.
Competing interests
CB has served since September 2016 as an expert witness for plaintiffs in US
litigation involving the impact of glyphosate-based herbicides on non-Hodg-
kin lymphoma. His work as an expert witness has included assessment of the
genotoxicity databases relied upon by the EPA and IARC in their evaluations
of GBH carcinogenicity. Lawyers involved in the litigation had no role in the
decision to write this paper, nor in reviewing or commenting on its content. In
the past, CB has also served as an expert witness in litigation focused on
whether foods containing ingredients from genetically-engineered crops can
be labeled “natural” without misleading consumers. His work since 1981 on
pesticide use, risks, and regulation has been supported by state and federal
agencies, the US Congress, the US National Academy of Sciences, foundations,
NGOs, pesticide manufacturers, and the food industry, including organic food
and farming companies and organizations.
Availability of data and materials
The 11 additional tables used to conduct the analysis reported herein are
available in Additional file 1.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Funding
CB thanks the Ceres Trust for the grant to the Children’s Environmental Health
Network that supported the preparation and publication of this manuscript.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 23 November 2018 Accepted: 28 December 2018
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