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Research Article Open Access
Open Access
Review Article
Environmental & Analytical
Toxicology
Antoniou et al., J Environ Anal Toxicol 2012, S:4
http://dx.doi.org/10.4172/2161-0525.S4-006
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
*Corresponding authors: Claire Robinson, Research director, Earth Open Source,
London, UK, E-mail: claire.robinson@earthopensource.org
John Fagan, Director, Earth Open Source, E-mail: jfagan64@gmail.com
Received June 01, 2012; Accepted June 21, 2012; Published June 23, 2012
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al.
(2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence of
Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006.
doi:10.4172/2161-0525.S4-006
Copyright: © 2012 Antoniou M, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Teratogenic Effects of Glyphosate-Based Herbicides: Divergence of
Regulatory Decisions from Scientific Evidence
M Antoniou1, MEM Habib2, CV Howard3, RC Jennings4, C Leifert5, RO Nodari6, CJ Robinson7* and J Fagan8*
1Head, Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King’s College London School of Medicine, UK
2Professor of entomology, former director, Institute of Biology, UNICAMP, and former provost of extension and community affairs, UNICAMP, São Paulo, Brazil
3Professor, Centre for Molecular Biosciences, University of Ulster, Northern Ireland
4Afliated research scholar, Department of History and Philosophy of Science, University of Cambridge, UK
5Research development professor for ecological agriculture at the University of Newcastle, UK. Interests: director and trustee of the Stockbridge Technology Centre Ltd
(STC), UK
6Professor, Center for Agricultural Sciences (department of plant science), Federal University of Santa Catarina, Brazil
7Research director, Earth Open Source, London, UK. Interests: editor, GM Watch, UK
8Director, Earth Open Source. Interests: employed at a GMO testing and certication company
Keywords: Glyphosate; Roundup; Teratogenicity; Teratogenic
eects; Malformations; Risk assessment; Reproductive toxicity
Introduction
An investigation (Paganelli et al.) of the toxicity of a commercial
Roundup® herbicide formulation and its active ingredient glyphosate
found that these substances caused severe malformations in embryos
of the South African clawed frog Xenopus laevis and chickens.
In frogs, dilutions of 1/5000 of the formulation (equivalent to
430 μM of glyphosate) were sucient to induce malformations,
including shortening of the anterior−posterior axis, microcephaly,
microphthalmia, cyclopia, and craniofacial malformations at tadpole
stages. Embryos injected with pure glyphosate showed similar
phenotypes, suggesting that glyphosate itself, rather than a surfactant
or other adjuvant present in the formulation, was responsible for these
developmental abnormalities. Roundup® produced similar eects
in chicken embryos, which showed a loss of rhombomere domains,
reduction of the optic vesicles, and microcephaly.
rough the use of reporter gene assays and phenotypic rescue
via administration of an antagonist, the authors conrmed that the
mechanism by which glyphosate and Roundup caused the observed
teratogenic eects in Xenopus embryos was via disruption of the
retinoic acid signalling pathway. is resulted in dysregulation of
the shh, slug and otx2 regulatory genes, which are crucial to the
development of the central nervous system [1]. e study, while not
a classical toxicological study, is relevant to human risk assessment
because the retinoic acid signalling pathway is a central signalling
pathway in embryonic development that operates in virtually all
vertebrates, whether amphibians, birds, or mammals.
Other Studies Showing Malformations from Glyphosate
and Roundup Exposure
Paganelli et al.’s study was one among several to nd malformations
from glyphosate and Roundup exposure. Jayawardena et al. (2010)
found nearly 60% malformations in tadpoles of the tree frog Polypedates
cruciger treated with an environmentally relevant concentration of 1
ppm Roundup. Eects included kyphosis, scoliosis, and edema [2].
Relyea (2012) found that environmentally relevant concentrations of
Roundup induced relatively deeper tails similar to the adaptive changes
caused by the presence of a predator in the tadpoles of the wood frog
(Rana sylvatica or Lithobates sylvaticus) and leopard frog (R. pipiens
or L. pipiens) [3]. A study on tadpoles of Scinax nasicus (Lajmanovich
et al., 2005) found that exposure to glyphosate herbicide caused
craniofacial and mouth deformities, eye abnormalities and bent, curved
tails, as well as mortality. Malformations and mortality increased with
dose and time of exposure. A 2-day exposure to 3.07 mg/l glyphosate
Abstract
The publication of a study in 2010, showing that a glyphosate herbicide formulation and glyphosate alone caused malformations in
the embryos of Xenopus laevis and chickens through disruption of the retinoic acid signalling pathway, caused scientic and regulatory
controversy. Debate centred on the effects of the production and consumption of genetically modied Roundup Ready® soy, which is
engineered to tolerate applications of glyphosate herbicide. The study, along with others indicating teratogenic and reproductive effects
from glyphosate herbicide exposure, was rebutted by the German Federal Ofce for Consumer Protection and Food Safety, BVL, as well
as in industry-sponsored papers. These rebuttals relied partly on unpublished industry-sponsored studies commissioned for regulatory
purposes, which, it was claimed, showed that glyphosate is not a teratogen or reproductive toxin.
However, examination of the German authorities’ draft assessment report on the industry studies, which underlies glyphosate’s EU
authorisation, revealed further evidence of glyphosate’s teratogenicity. Many of the malformations found were of the type dened in the
scientic literature as associated with retinoic acid teratogenesis. Nevertheless, the German and EU authorities minimized these ndings
in their assessment and set a potentially unsafe acceptable daily intake (ADI) level for glyphosate. This paper reviews the evidence
on the teratogenicity and reproductive toxicity of glyphosate herbicides and concludes that a new and transparent risk assessment
needs to be conducted. The new risk assessment must take into account all the data on the toxicity of glyphosate and its commercial
formulations, including data generated by independent scientists and published in the peer-reviewed scientic literature, as well as the
industry-sponsored studies.
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 2 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
herbicide caused only 10% mortality but caused malformations in 55%
of the test animals [4].
Malformations have also been found in mammals treated with
glyphosate herbicides. A toxicological study by Dallegrave et al. (2003)
found that the ospring of pregnant rats dosed with 500, 750 and
1000mg/kg Roundup on days 6–15 aer fertilisation had increased
skeletal abnormalities, including at doses that were not maternally
toxic. Malformations consisted of the absence of bones or parts of
bones, shortened and bent bones, asymmetry, fusions, and cles.
e percentage of altered foetuses increased with dose. e authors
concluded that the formulated product was more toxic than the
technical glyphosate evaluated by the World Health Organisation [5]
and tested in the industry-sponsored teratogenicity studies described
in Germany’s 1998 dra assessment report on glyphosate.
Scientic and Political Controversy in Europe
In Europe, the publication in 2010 of the study by Paganelli et al.
[1] coincided with rising concern over the 40 million tons of soy that
are imported each year, mostly to feed livestock. Much of this soy is
the genetically modied (GM) Roundup Ready® variety [6], which is
engineered to tolerate applications of glyphosate herbicide. Scientic
and political debate had continued for many years over the public
health, environmental, and socioeconomic consequences of GM
soy cultivation in producer countries [7]. More recently, concerns
expanded to include potential risks to animal and human health posed
by glyphosate residues in the animal feed and human food chain [8,9].
Residues of up to 17 mg/kg of glyphosate have been found in harvested
soybean crops [10].
As the existing EU approval of glyphosate dated from 2002 [11]
and was valid for ten years, a new review was due in 2012. In response
to a Parliamentary question, John Dalli, EU Commissioner for health
and consumer policy, stated that the date might be brought forward if
the new evidence justied it [12].
German Authorities and Industry Respond
Commissioner Dalli gave the task of assessing Paganelli et al.’s
ndings to the German regulatory authorities. As the “rapporteur”
member state for glyphosate, Germany was responsible for liaising
between industry, the European Commission, and the member states
in the authorisation process. In October 2010 Germany’s Federal Oce
for Consumer Protection and Food Safety, BVL, issued an anonymous
rebuttal of the study, which stated:
ere is a huge and reliable database for developmental toxicity
of glyphosate and no evidence of teratogenicity has been obtained.
In particular, studies in rats and rabbits failed to reveal craniofacial
malformations as … would be expected if a substance aects mainly
the neural crest [13].
While the identity of the “huge and reliable database” is not dened
by BVL, the studies on rats and rabbits to which BVL referred were
those commissioned by manufacturers of glyphosate herbicides and
summarised in Germany’s 1998 dra assessment report on glyphosate.
Germany’s summary of, and commentary on, these largely unpublished,
non-peer-reviewed industry-sponsored studies in the dra assessment
report form the basis of the EU’s current authorisation of glyphosate,
which dates from 2002 [11].
BVL concluded that Paganelli et al.’s ndings “do not put the
current risk assessment for glyphosate and glyphosate-based PPP
[plant protection products – pesticides] into question with regard to
human health” [13].
Based on BVL’s assessment, Commissioner Dalli said there was no
need to restrict or ban glyphosate [14]. e Commission did not bring
the glyphosate review forward, or even keep to the expected date of 2012.
Instead, in November 2010 it issued a Directive delaying the review
of glyphosate until 2015 [15]. In response to a query from Friends of
the Earth Germany as to the reason for the delay, BVL replied that the
Commission and the European Food Safety Authority (EFSA) had too
heavy a workload to review glyphosate and had not nalized the rules
for renewing the approval of certain pesticides, including glyphosate
(personal communication from BVL to Friends of the Earth Germany).
BVL’s response to Paganelli et al. was followed in 2011 by a similar
response from industry. Employees of Monsanto, Dow, and Syngenta,
all manufacturers of glyphosate herbicides, published a letter in the
same journal that published Paganelli et al.’s original paper [16].
e Monsanto/Dow/Syngenta letter, published alongside a reply
from Andres Carrasco, lead author of the Paganelli et al. Paper [17],
stated: “Glyphosate does not cause adverse reproductive eects in
adult animals or birth defects in ospring of these adults exposed to
glyphosate, even at very high doses” [16].
Claims of No Teratogenicity Assessed
In order to test BVL’s claim of the absence of teratogenicity in
the industry studies on glyphosate, we obtained from the German
authorities the dra assessment report on glyphosate that they
compiled in 1998. e industry toxicological data summarised in the
dra assessment report are not publicly available and are claimed by
Monsanto to be commercially condential, though Pesticide Action
Network Europe is pursuing disclosure through the courts (personal
communication).
Examination of the dra assessment report revealed that the
industry toxicological studies on rabbits and rats that BVL said showed
“no evidence of teratogenicity” did, in fact, report malformations from
glyphosate exposure [12].
In the dra assessment report, the German authorities
concluded, based on the industry studies, “Glyphosate does not cause
teratogenicity”, but added that higher doses of glyphosate caused
“reduced ossication and a higher incidence of skeletal and/or visceral
anomalies” in rats and rabbit foetuses, as well as a reduced number of
viable fetuses [18]. e latter is consistent with increased incidence of
malformations. e German authorities do not dene what they mean
by “higher” doses, but the industry-sponsored teratogenicity studies
typically use three doses: low, mid, and high dose. For details of the
dose levels used, see Table 1.
However, in the industry-sponsored studies, malformations were
found not only at high, maternally toxic doses, as the Commission’s
health and consumer aairs division, DG SANCO, stated in its 2002
review report on glyphosate [11], but also at lower doses. In some cases,
eects at lower doses were statistically signicant [12], though statistical
signicance at lower doses is dicult to obtain in standardised industry
studies performed for regulatory purposes, which use small numbers of
animals per group.
Table 1, below, shows the range of malformations found in the
industry teratogenicity studies on glyphosate, as summarised by
the German authorities in the dra assessment report. e studies
were conducted as long ago as the 1980s and 1990s. Many of the
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 3 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
malformations found are consistent with descriptions of retinoic
acid-induced teratogenesis in the literature. For example, the
increased heart malformations and abnormalities noted in the dra
assessment report are cited as characteristic of retinoic acid-induced
teratogenicity by Lammer et al. [19], Kessel [20], and Huang et al.
[21]. e supernumerary, distorted, and rudimentary ribs noted in
the dra assessment report are consistent with Kessel’s (1992) citation
of the generation of supernumerary ribs and rib malformations as
characteristic of retinoic acid-induced teratogenicity [20]. Absent
postcaval lobe of the lungs, as mentioned in the dra assessment
report, is consistent with the lung dysmorphogenesis caused by
retinoic acid administration or deprivation as cited in Malpel [22],
Wilson et al. [23], Shenefelt [24] and Dickman et al. [25]. e reduced
ossication of cranial centres and sacro-caudal vertebral arches, as well
as the undened skeletal malformations, cited in the dra assessment
report are consistent with the cranial and skeletal malformations cited
by Lammer et al. [19], Kessel [20] and Huang et al. [21] as characteristic
of retinoic acid-induced teratogenicity.
General Observations on the Dra Assessment Report
Maternal toxicity
roughout the dra assessment report, German regulators
dismissed ndings of malformations in industry studies by claiming
that the eects were due to maternal toxicity. In its 2002 report
that forms the basis of the EU authorisation of glyphosate, the EU
Commission’s health and consumer division, DG SANCO, followed the
German regulators’ lead, discounting the developmental abnormalities
on the grounds that they were conned to “maternally toxic doses”
[11], though how this conclusion was reached is unclear.
e general reasoning behind this conclusion is that poisoning
of the mother with any substance (including commonly ingested
substances like salt and caeine) could aect the development of the
foetus and therefore such malformations are not a direct and specic
eect of the substance on the foetus. Germany and DG SANCO argued
that the studies report maternal toxicity and therefore the foetal
abnormalities were due to maternal toxicity. However, the studies
failed to dierentiate between maternal toxicity and compound-specic
teratogenicity. At the high doses used, both could be taking place.
It is unfortunate that the standardised industry studies performed
for regulatory purposes use only a small number of animals per group.
Given this restriction, relatively high doses of the test compound are
used and maternal toxicity eects are common. ere is a high risk that
this type of study design can miss compound-specic eects that occur
at low- and medium- frequency.
An equally valid conclusion that could be drawn from the industry
studies is that maternal toxicity could be obscuring a compound-
specic teratogenic eect and may not be the only cause of the
observed malformations. is argues that another study should have
been conducted employing larger groups of animals and lower, more
realistic doses administered over a longer, preferably lifelong, period
before the possibility of compound-specic teratogenic eects could
be eliminated and before glyphosate could be deemed free from
teratogenic eects.
Paumgartten (2010) supports this conclusion, stating that in cases
of maternal toxicity, it is not possible to know whether an eect on the
embryo is due to non-specic maternal poisoning or to a direct action
of the chemical at doses that also adversely aect the mother. In the
latter case, the chemical would be a developmental toxin [26].
Dallegrave et al. (2007) demonstrated that this issue is relevant to
glyphosate formulations. e authors examined the eects of Roundup
on reproduction in male and female ospring of rats treated during
pregnancy and lactation with doses of Roundup that were too low
to induce maternal toxicity. ey found that Roundup at these doses
induced adverse reproductive eects in male ospring, showing that
this herbicide formulation is a reproductive toxin at non-maternally
toxic doses [27].
is argues for the need to redesign regulatory tests to use larger
groups of animals and more realistic doses over longer exposure
periods, preferably beginning exposures prenatally.
Nonlinear Dose-Response
roughout the dra assessment report, German regulators
assumed that only eects that follow a linear dose-response relationship
are valid. But this assumption is not supported by current knowledge.
Dose-response relationships can be complex and nonlinear, especially
where the endocrine system is involved. A large body of evidence
indicates that for some compounds, toxic eects are found at low doses
but not at higher doses, and that dierent toxic eects can be found at
dierent doses [28-31].
Roundup and glyphosate have been found to be endocrine
disruptors [27,32], and therefore, nonlinear dose-responses may apply
for some endpoints. Indeed, a neurotoxicity study found not only that
Roundup was more toxic than glyphosate and produced eects at
concentrations as low as 10 ppb or 0.01 micrograms/L (equivalent to
a glyphosate concentration of 0.5 nM), but also found “unusual” dose-
response relationships with both substances, suggesting that low-dose
eects may not be predictable from eects at high doses [33].
We conclude that it is not in accord with current scientic
understanding to dismiss ndings of increased developmental
malformations on the grounds of a nonlinear dose-response
relationship. However, given that that current practice by regulatory
agencies assumes that the dose-response relationship should be linear,
we indicate, where relevant in Table 1, where such a relationship is
observed.
Historical control data
e German regulators repeatedly discounted ndings of
malformations in industry studies by referring to historical control data
instead of the concurrent controls measured in the studies themselves.
However, the use of this historical control data set is questionable
from two perspectives. First, the dra assessment report fails to disclose
the historical control data used. It does not provide the individual
data points or statistical measures of the variability within the dataset.
Second, the dra assessment report fails to present evidence that
demonstrates the validity of this historical control dataset.
e use of historical, instead of concurrent, controls is contrary to
rigorous scientic methodology, which is designed to tightly control
for variables. It articially introduces variables into the dataset,
potentially masking any eect caused by the substance being tested.
Potential variables include:
• strain of animal, involving a dierent genetic background and
sensitivity.
• substance tested, introduced by dierent manufacturing
processes and storage conditions.
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 4 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
Study
author and
date
Submitter company Experimental animal/
exposure route
Doses
used
mg/kg
bw/d
Effects found Dose-related effects/Statistical
signicance
Suresh,
1993 Feinchemie Rabbits/gavage 0, 20, 100,
500 Dilated heart
Linear dose-response relationship.
Signicantly elevated at all doses, including
low dose
Unspecied “major visceral malformations”
Linear dose-response relationship.
Increased in all treatment groups,
signicantly increased at highest dose
Extra 13th rib
Linear dose-response relationship.
Statistically signicant increase at highest
dose
Brooker et
al., 1991 Monsanto/Cheminova Rabbits/gavage 0, 50, 150,
450
Heart malformations (only type specied is
interventricular septal defect)
Effect found at highest dose. No information
provided by Germany on statistical
signicance
Embryonic deaths Signicant at all doses, though no clear
dose/response relationship
Bhide and
Patil, 1989 Barclay/Luxan Rabbits/route
unstated
0, 125,
250, 500 Heart malformation (ventricular septal defect)
Linear dose-response relationship. No
statistical analysis provided by authors.
Increased heart malformations found in all
treatment groups
Lungs: postcaval lobe absent
Linear dose-response relationship. No
statistical analysis provided by authors.
Dose-dependent increases found in all
treatment groups
Kidneys absent
Linear dose-response relationship. No
statistical analysis provided by authors.
Dose-dependent increases found in all
treatment groups
Rudimentary 14th rib, unilateral
No statistical analysis provided by authors.
Dose-dependent increases found in mid-
and high-dose groups
Number of viable foetuses per litter decreased
and number of non-viable implants increased
Linear dose-response relationship in case of
non-viable implants. No statistical analysis
provided by authors. Effects found at high
dose level.
Tasker et al.,
1980 Monsanto/Cheminova Rabbits/gavage 0, 75, 175,
350 Increased number of deaths in dams
Linear dose-response relationship. 1, 2,
and 10 deaths in low, mid- and high-dose
treatment groups respectively (no. of rabbits
per group: 16 or 17). 75 mg/kg stated by
Germany to be NOAEL
Anon., 1981 Alkaloida Rats and rabbits/oral
feeding
0, 10.5,
50.7,
255.3
Increased number of foetal deaths Effect seen at two upper dose levels
Zhu et al.,
1984 Barclay Mice/gavage 80, 420,
1050
Germany comments that there is “No evidence
of dose-related toxic effects” and “no …
structural malformations” but that description of
experiment was “poor”
Data not provided by Germany
Brooker et
al., 1991 Monsanto/Cheminova Rats/gavage
0, 300,
1000,
3500
Distortions affecting thoracic ribs
Dose-dependent increases found in
mid- and high-dose groups. Statistically
signicant at high dose
Reduced ossication of one or more cranial
centres
Dose-dependent increases found in mid-
and high-dose groups.
Reduced ossication of sacro-caudal vertebral
arches
Dose-dependent increases found in mid-
and high-dose groups
Unossied sternebrae Increases found in all treated groups,
statistically signicant at high dose
Skeletal variations
Dose-dependent increases found in all
treated groups, statistically signicant in
mid-dose and high-dose groups.
Tasker and
Rodwell,
1980
Monsanto/Cheminova Rats/gavage
0, 300,
1000,
3500
Unossied sternebrae Increase found at highest dose level
Unspecied malformations Increase at highest dose level
No. of viable foetuses per litter and mean foetal
weight decreased Effects found at highest dose level
Early resorption of embryos Data not provided by Germany
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 5 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
total number of foetuses with major visceral anomalies was
high in all treated groups, including the low dose level of 20
mg/kg glyphosate, and was signicantly increased at the
highest dose level of 500 mg/kg. e percentage of foetuses
with dilated heart was signicantly elevated at all dose levels.
Skeletal variations, anomalies and malformations were found
but there was no clear dose-response pattern. ere was a
dose-related increase in the occurrence of an extra 13th rib in
all glyphosate-treated groups; in the high dose group this was
statistically signicant.
e German regulators dismissed the ndings on the grounds that
the actual number of foetuses with dilated heart was small, that there was
no increase in foetuses with heart dilation in the mid-dose over the low-
dose group, that almost no other so organ malformations occurred,
and that the supposed consequences of this heart malformation were
“equivocal”. ey concluded that the low dose of 20 mg/kg bw/d and
even the mid dose of 100 mg/kg bw/d were NOAELs (No Observable
Adverse Eect Levels) [37].
b. e UK’s Pesticides Safety Directorate (PSD) commented,
“e increased incidences of abnormalities … are of concern,
particularly the heart eects which are also reported in other
rabbit studies with glyphosate… e interpretation of this
nding must rely on comparison with historical control data”
[38].
c. In fact, no NOAEL was found in this study, as a statistically
signicant increase in the dilated heart malformation was
found even at the lowest dose of 20 mg/kg. erefore, the
German regulators should have asked for further tests at
lower doses to establish a true NOAEL. eir comment that
the number of foetuses with abnormalities was small merely
identies a shortcoming of the standardised industry studies
performed for regulatory purposes. Larger numbers of animals
are preferable. If the number of animals used is small, any eect
will only be seen in a few animals and statistical signicance
will be dicult to obtain. is is especially true at lower doses,
where observable eects will be smaller and/or less frequent.
e German regulators’ dismissal of the heart malformations on
the grounds that no other so organ malformations were found is
invalid, as toxic agents can have organ-specic eects. eir argument
that the heart dilation malformation had “equivocal” consequences and
could therefore be dismissed is scientically and clinically indefensible.
• diet for the experimental animals, which can vary in
composition and contaminants.
• pathogens in the environment.
• year and laboratory in which the experiments were performed,
for unknown reasons [34].
In order to demonstrate that the use of these historical control data
for glyphosate is valid, German regulators must disclose the datasets
used and demonstrate the relevance of each data point included in
the dataset. In the absence of such documentation, we consider the
conclusions of the dra assessment report to be questionable.
Several reviews state that concurrent control groups are the
most valid controls and warn against the biasing eect of conducting
comparisons with historical control data [34-36]. Cue (2011) stated
that using such data can lead to Type II errors [36]. In the case of
glyphosate, this would be a false negative, in which a nding of toxicity
was overlooked.
In rare instances, the use of historical control data is acceptable,
such as where eects observed are borderline, showing only a marginal
increase over concurrent controls, or in the case of rare tumours, where
data other than historical data is unavailable. Nevertheless, extreme
care must be taken in selecting the data points included in the historical
control dataset. Specically, all sources of variability in the historical
control data must be identied and controlled for [34]. ere is no
evidence in the dra assessment report that the German regulators did
this in the case of glyphosate.
Analysis of industry-sponsored studies from the dra
assessment report
Below we analyse selected industry-sponsored teratogenicity
studies from the German regulators’ dra assessment report (see Table
1 for a summary of all the industry teratogenicity studies cited in the
dra assessment report). For each study analysed, we present: (a) a
condensed version of the German regulators’ summary of the ndings;
(b) the comments of the UK’s Pesticides Safety Directorate (PSD),
where relevant; and (c) our comments.
Increased skeletal, visceral, and heart malformations
With regard to a study in rabbits by Suresh:
a. e German regulators stated that this study found that the
Suresh,
1991 Feinchemie Rats/gavage 0, 1000
Increase in delayed ossication (caudal
vertebral arch, forelimb proximal and hindlimb
distal phalanges) found in treatment group, but
increase in delayed ossication of skull found in
control group
Conicting data led Germany to conclude
that the NOAEL for developmental toxicity
was 1000 mg/kg
Bhide, 1986 Barclay/Luxan Rats/gavage 0, 100,
500
No effects found but Germany commented
that “serious reporting deciencies” and lack of
statistical analysis led it to consider the study as
supplementary information only
In spite of lack of reliable data, Germany
derived a NOAEL for developmental toxicity
of 500 mg/kg
Anon., 1981 Alkaloida Rats and rabbits/oral
feeding
22, 103,
544
Germany commented that description of study
is so “poor” that it only considered the study
as supplementary information. There were
“no malformations recorded” and effects on
foetuses were “not observed” but it is unclear
from Germany’s summary whether this was due
to poor reporting by the study’s authors or if
there was an actual absence of effects
In spite of lack of reliable data, Germany
derived a NOAEL for developmental toxicity
of 544 mg/kg
Source for all studies: Rapporteur member state Germany (1998) Monograph on glyphosate, German Federal Agency for Consumer Protection and Food Safety (BVL).
Vol 3-1 Glyphosat 05: pp. 9–20
Table 1: Malformations, embryonic deaths, and maternal deaths in industry-sponsored teratogenicity studies on glyphosate.
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 6 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
Increased heart malformations and embryonic deaths
With regard to a study in rabbits by Brooker et al.:
a. e German regulators stated that this study on the eects
of glyphosate on pregnancy in rabbits found a signicant
increase in embryonic deaths in all treatment groups.
However, they argued that comparison with historical control
data showed that the incidence in the concurrent control
group was untypically low and that therefore the increase
was not signicant. In addition, they questioned its biological
signicance, arguing that a clear dose-response relationship
was not shown. e German regulators did, however, state that
an increase in late embryonic deaths at the highest dose level
had been reported in another study. ey noted the increased
incidence of heart malformations in the high dose group, but
stated that this was within the range of historical control data.
ey added that anomalies of the heart were found in other
teratogenicity studies with glyphosate in rabbits, but concluded
that a possible eect on the occurrence of visceral anomalies
was “equivocal” [39].
b. e UK PSD commented: “e increased levels of embryonic
death/post-implantational loss at all dose levels are of concern,
as are the reports of heart defects… a more robust argument
should be presented before these ndings can be dismissed”
[38].
c. Again, the German regulators used historical control data and
an inappropriate model for toxicity dose-response to dismiss
heart malformations. We believe that this conclusion was
not justied and that the increase in late embryonic deaths
required investigation because malformed foetuses are oen
spontaneously aborted or are born dead. e relevance of this
observation to humans is suggested by a study of farm families
in Ontario, Canada, which found a higher than normal rate of
miscarriages and pre-term deliveries associated with glyphosate
herbicide exposure [40].
Decrease in viable foetuses, increase in malformations
With regard to a study in rabbits by Bhide and Patil:
a. e German regulators stated that this study found a decreased
number of viable foetuses per litter and increased embryonic
deaths. e number of visceral and skeletal malformations was
increased in the high-dose group [41].
b. e UK PSD commented: “Another study with equivocal
evidence of heart defects” [42].
c. Dose-dependent increases in lung and kidney malformations
were found across all treatment groups. Increased frequency
of heart malformations was found in all treatment groups.
Increased skeletal (rudimentary 14th rib) malformations,
typical of retinoic acid embryopathy, were found in the mid-
dose and high-dose groups.
German regulators incorrectly stated that the teratogenic NOAEL
was the mid dose of 250 mg/kg bw/d. In fact, the data showed increases
in most defects even at the low dose of 125 mg/kg bw/d. e authors
of this study did not provide an analysis of statistical signicance and
groups of only 15 animals were used, making it dicult to achieve
statistical power at lower doses. e data presented in this study suggest
that it would be more appropriate to declare the mid dose, possibly even
the low dose of 125 mg/kg, as the LOAEL (lowest observed adverse
eect level) and to state that no NOAEL was found.
Increased foetal deaths
With regard to a study in rats and rabbits by Anon:
a. e German regulators stated that this oral feeding study was
poorly recorded and was only considered as supplementary
information. No malformations were recorded, but there were
more foetal deaths at the two upper dose levels (50.7 and 255.3
mg/kg bw/d). ey stated that it was dicult to understand
why an increase in foetal deaths would occur at doses far below
those at which foetal eects were found in gavage studies
and concluded that it was “doubtful” whether this eect was
treatment-related [43].
b. e UK PSD commented, “ough this study is questioned
[by German regulators] for showing evidence of foetotoxicity
at lower doses than other studies, the study by Brooker (see
above) may also indicate foetotoxicity at 50 mg/kg bw/d” [42].
c. e German regulators’ assumption that low-dose ndings
were non-treatment-related because oral feeding resulted in
dierent eects than gavage is not defensible. As was pointed
out by the UK’s PSD, another study supported this study’s
ndings [42]. ere is no explanation in the dra assessment
report as to whether, or how, this disagreement was resolved,
and thus the issue remains open for discussion.
Increased unossied sternebrae
With regard to a study in rats by Tasker and Rodwell:
a. e German regulators stated that this study found a higher
number of foetuses with malformations at the highest dose
level (3500 mg/kg bw/d), but considered that this was within
the range of historical control data and not treatment-
related. Specically, there were more foetuses with unossied
sternebrae in the high-dose group. While they accepted that
this eect was treatment-related, they concluded that it was
“rather a developmental variation than a malformation” [44].
b. e German regulators again used historical control data
to dismiss evidence of teratogenicity. Given the ndings of
malformations in other studies, this is not justied. To dene
unossied sternebrae as a “developmental variation” rather
than a malformation is scientically unjustiable. Unossied
sternebrae in the rat are clearly dened as a skeletal deformity
in e Handbook of Pesticide Toxicology [45].
Increased skeletal malformations
With regard to a study in rats by Brooker et al.:
a. e German regulators stated that this gavage study in rats
found increased incidence of reduced ossication and increased
skeletal malformations at the mid and high doses but added
that the results were within the range of historical control
data. ey stated that maternal toxicity was a confounding
factor and described the signicance of the malformations as
“equivocal” [46].
b. Again, the German regulators used historical control data and
maternal toxicity to minimize the signicance of malformations.
However, these malformations are consistent with the ndings
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 7 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
of Paganelli et al. and are associated with disturbances in the
retinoic acid signalling pathway [1].
PSD’s conclusion
e UK PSD’s overall conclusion on the industry-sponsored rabbit
teratogenicity studies was: “Taken in isolation, none of the ndings …
would be clearly of concern. However, overall there is an indication
of a pattern” (our emphasis). e PSD asked the German regulatory
authorities to make available the historical control data against which
they compared the ndings of these studies [42], but we have been
unable to locate any published statement indicating whether the PSD
saw this data or, if it did, how it responded.
Following the deliberations of the German regulators and the UK’s
PSD, the issue of glyphosate-mediated teratogenicity was considered by
the EU Commission’s ECCO scientic review panel. e ECCO panel
noted “the incidence of heart malformations”, but dismissed them on
the grounds that they were “within the range of the historical control
data” [47]. Again, it is unclear from the panel’s statement whether it saw
the historical control data and, if so, whether it systematically assessed
the validity of that data set. Subsequently, in 2002, the EU Commission
authorised glyphosate.
Misleading “Safe” Level Set For Glyphosate?
e central purpose of a pesticide risk assessment is to establish
an Acceptable Daily Intake (ADI), a level of exposure deemed safe for
humans over a long period. In the case of glyphosate, the ADI was
calculated from the dataset provided by industry-sponsored studies,
some of which are discussed above. e level that should be used to
set the ADI is the highest dose at which no adverse eect is observed
(NOAEL), which is also lower than the lowest dose at which adverse
eects are observed (LOAEL). is level should be selected from “the
most appropriate study in the most sensitive species”, as the German
regulators note [48].
e German regulators set the ADI for glyphosate at 0.3 mg/kg
bw/d [49]. is ADI was accepted by the European Commission in its
nal report [11].
But this ADI is incorrect. e German authorities arrived at this
ADI by excluding certain studies from the ADI process. First, they
excluded the mid-term teratogenicity studies on rabbits discussed
above, on the grounds that only long-term studies should be used to
set safe chronic exposure levels. Second, they claimed that the most
sensitive species for chronic exposure was the rat [50], providing
another reason to exclude the rabbit teratogenicity studies.
However, while mid-term studies are generally discounted in ADI
calculations because they are considered less sensitive than long-term
studies, in this case, the mid-term rabbit studies found toxic eects at
lower doses than the long-term studies in rats. erefore, the mid-term
rabbit studies were found to be more sensitive, and the rabbit was a
more appropriate species. ese data clearly indicate that the rabbit
studies should have been used to set the ADI.
e exclusion of the toxicity studies in rabbits has introduced
signicant bias into the data used by the German regulators to calculate
the ADI. e German authorities cited as their starting point for
establishing the ADI a LOAEL of 60 mg/kg bw/d from a two-year study
in rats, which found signicant toxicity at that level (Suresh et al., 1996).
is was stated to be the lowest dose at which toxicity was observed.
ey then identied the highest NOAEL below that level – 31 mg/kg
bw/d – as the one from which the ADI should be calculated. Applying
the usual 100-fold safety factor, the German regulators proposed an
ADI for glyphosate of 0.3 mg/kg bw/d [49].
e German authorities ignored the LOAEL of 20 mg/kg identied
by Suresh et al. in rabbits, a value three times lower than their chosen
LOAEL of 60 mg/kg bw/d [51].
e reason given by the German regulators for not adopting the 20
mg/kg LOAEL (Suresh et al., 1993) for setting the acceptable operator
(applicators’) exposure level (AOEL), is that it is a mid-term rather
than long-term experiment and therefore more suitable for setting this
type of level [52].
However, we propose that given the greater sensitivity of the rabbit
model system to glyphosate exposure, the LOAEL of 20 mg/kg bw/d
(Suresh et al., 1993) should have been the starting point for the ADI
and for the applicators’ AOEL. Indeed, this study found no NOAEL,
as even the lowest dose produced toxic eects [51]. If the LOAEL of
20 mg/kg were used, employing the same procedure as the German
regulators, the highest NOAEL below this dose from their approved
list of studies is 10 mg/kg [49]. Applying the customary 100-fold safety
factor to this value results in a more objectively accurate ADI of 0.1 mg/
kg bw/d, one-third of the ADI suggested by the German authorities
and subsequently adopted by the EU Commission.
e ADI According To Peer-Reviewed Studies
Two mammalian toxicological studies suggest that the LOAEL for
glyphosate should be even lower than the ADI of 0.1 mg/kg bw/d that
we derive from the industry-sponsored studies.
Romano et al. (2010) found that Roundup is a potent endocrine
disruptor and disturbed the reproductive development of rats with
exposure during puberty. Adverse eects, including delayed puberty
and reduced testosterone production, were found at all dose levels,
including the LOAEL of 5 mg/kg. ere was a clear dose-response
relationship [53].
Benedetti et al. (2004) found that Glyphosate-Biocarb caused
“irreversible” damage to rat liver cells, including at the LOAEL of 4.87
mg/kg, with a clear dose-response relationship [54].
No dose below these LOAELs was tested in these two studies
[53,54], so the NOAEL will be lower. Hypothetically, if the NOAEL
were conservatively assumed to be 2.5 mg/kg bw/d, applying the 100-
fold safety margin would result in an ADI of 0.025 mg/kg bw/d. is is
twelve times lower than the ADI proposed by the German regulators,
which is currently in force in the EU and used as a basis for the
maximum residue limit for food and feed.
ese studies used a species (rats) and exposure route (oral) that
are accepted by industry, EU regulators and the Organisation for
Economic Cooperation and Development (OECD). ey tested specic
glyphosate formulations, so it is not known whether their ndings can
be extrapolated to other formulations. However, this raises the crucial
question of why formulations are approved on the basis of industry
tests on, and a regulatory assessment of, only the isolated ingredient,
glyphosate.
Papers Defending Glyphosate’s Safety
In their rebuttal of Paganelli et al.’s study, the authors from
Monsanto/Dow/Syngenta state:
e GLP [Good Laboratory Practice] studies that Paganelli
et al. infer as untrustworthy “industry-funded studies” have been
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 8 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
exhaustively reviewed by multiple government scientic regulators,
oen comprised of academic expert scientists and all of which
have strongly supported the conclusions put forth in those studies.
Glyphosate does not cause adverse reproductive eects in adult animals
or birth defects in ospring of these adults exposed to glyphosate, even
at very high doses [16].
Given the evidence we present here from both academic and
industry-sponsored studies, this argument is unconvincing. e data
clearly show that glyphosate does cause adverse reproductive eects
and malformations in laboratory animals.
Even if one accepts the position proposed by Monsanto/Dow/
Syngenta, that only studies conducted according to GLP should be
considered, this argument does not stand up to scrutiny, as some of
the studies in the industry dossier on glyphosate are too old to utilize
GLP [55].
Williams et al. (2000), in a paper that is frequently cited as evidence
of the safety of Roundup and glyphosate, also cite the GLP status of
industry studies to back their claim that glyphosate is not a reproductive
toxin. However, some of the studies that they cite are, in fact, non-
GLP: for example, Schroeder (1981) and Tasker (1980) [55]. Moreover,
they fail to cite other studies from the same industry dossier – Suresh
(1993), Brooker (1991), and Bhide and Patil (1989) [56], which found
teratogenic eects from glyphosate, as detailed above.
It is important to note that GLP is not a measure of scientic
reliability or validity, but a set of laboratory management rules
instituted by regulators in the 1970s and 1980s to combat fraud
in industry testing. Interestingly, the move to GLP standards was
prompted by a high-prole case of fraud involving toxicological tests
on glyphosate for regulatory purposes conducted by a laboratory under
contract to Monsanto in late 1970s. However, the implementation of
GLP failed to prevent a second major case of fraud, which came to
light in the 1990s. is case also involved glyphosate at a dierent
laboratory under contract to Monsanto, but this time involved residue
tests [57,58]. While Monsanto said it later replaced the fraudulent tests
[59], this history shows that industry-sponsored testing can be prone to
fraud and that GLP cannot be assumed to prevent it.
Both Williams et al. (2000) and the authors from Monsanto/Dow/
Syngenta cite World Health Organisation (WHO) reports in support of
glyphosate’s safety [60,56,16]. However, the WHO relies on data from
industry studies [60], which, as shown above, in fact provide evidence
of teratogenicity.
In addition, the study by Williams et al. (2000) was co-authored
by Ian C. Munro, whose aliation was listed as the chemical industry
consulting rm Cantox [56]. Cantox states that its mission is to
“protect client interests while helping our clients … bring products
to market” [61]. Williams et al. published their paper in the journal
Regulatory Toxicology and Pharmacology, which was investigated by
a US Congressional Committee in 2008 over its industry sponsorship
in relation to its role in the FDA’s decision allowing the endocrine-
disrupting chemical bisphenol A in infant formula and other foods
[62,63].
A Monsanto-funded review by Williams et al. (2012), co-authored
by two representatives of the chemical industry consulting rm
Exponent, argued for the unreliability of Dallegrave’s study (2003)
partly on the claimed basis that the malformations found were artefacts
of histopathological xation and processing [64]. But these hypothetical
arguments could be countered by the clear dose-response relationship
(as required by regulators) found by Dallegrave [5]. Williams et al. also
argued that the malformations were only “signs of a developmental
delay that correct themselves within a brief period” [64]. e authors
failed to provide citations of any experimental evidence upon which
this claim was based and whether the malformations would indeed
“correct themselves” without resulting in lasting damage to the
developing central nervous system and other organs and systems. us
this assertion remains unsubstantiated.
e argument used by Williams et al. (2012), is similar to the
German authorities’ redenition of a malformation as a “developmental
variation” [44]. Indeed, in a discussion of an unpublished mammalian
toxicology study on glyphosate (IRDC, 1980a), Williams et al. followed
the German authorities in dening the observed unossied sternebrae
in treated rats as not a malformation but “a variation, possibly related
to the reduced foetal weights and a developmental delay” [64].
With regard to Paganelli et al.’s study [1], Williams et al. (2012)
stated that the glyphosate solution tested was not pH-adjusted and
thus the malformations “may have been due to the acidic nature of the
test compound” [64]. However, this hypothetical argument is spurious
since at the dilutions used, the pH of the buered test solution was not
changed by the addition of herbicide.
BVL’s Response to Dallegrave et al.
In its response to Paganelli et al. [1], BVL dismissed Dallegrave
et al.’s study, which found malformations in the ospring of rats
treated with Roundup, on the basis that “there were no craniofacial
malformations” [13]. But this is a misrepresentation of Dallegrave’s
study, which stated, “e most frequent skeletal alterations observed
were incomplete skull ossication and enlarged fontanel[le]” [5]. Both
are craniofacial malformations. erefore, contrary to BVL’s assertion,
Dallegrave et al.’s study provides clear evidence that a glyphosate
herbicide can cause craniofacial malformations.
Moreover, by focusing on craniofacial malformations, BVL ignored
the broad range of malformations associated with disturbances in the
retinoic acid signalling pathway during development, which were found
from glyphosate exposure by Dallegrave et al. [5], Paganelli et al. [1]
and in the industry studies (see above). For example, a malformation
found by Dallegrave et al. in a dose-dependent relationship was “caudal
vertebrae: absent” [5]. is malformation is associated with the retinoic
acid signalling pathway. Exposure of mouse embryos to retinoic acid
at a similar period of development has been found to produce agenesis
of caudal vertebrae, caused by the down-regulation of posterior Hox
genes [20].
Reports and Studies from South America
Paganelli et al. stated that they were prompted to conduct their
study by reports and studies indicating high rates of human birth
defects in regions of South America dedicated to growing GM Roundup
Ready soy [1].
For example, an epidemiological study carried out in Itapua,
Paraguay, found a higher rate of malformations in the ospring of
women exposed in pregnancy to pesticides, compared with controls.
e malformations observed included craniofacial defects, anencephaly,
microcephaly, hydrocephalus, myelomeningocele, cle palate, anotia,
polydactyly, syndactyly, and congenital heart defects [65].
Many of these malformations are of the same type as those
observed by Paganelli et al. in frogs and chickens, and are associated
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 9 of 13
J Environ Anal Toxicol Toxicology of Pesticides ISSN:2161-0525 JEAT an open access journal
with the retinoic acid pathway. e authors do not mention glyphosate,
and most agrochemical applications use mixtures of pesticides, so a
sole causative agent cannot be identied. However, Itapua is an area of
intensive Roundup Ready soy cultivation [66].
A study commissioned by the provincial government of Chaco,
Argentina, a region of intensive GM soy production, showed a threefold
increase in birth defects in the province and a fourfold increase in
cancer in the locality of the agricultural town of La Leonesa in the last
decade, coinciding with the expansion of GM soy and the associated
application of pesticides. e authors named glyphosate as a pesticide
of concern and noted that complaints from residents were highest in
regions where GM crops are planted [67].
A study of birth defects in seven regions of Argentina found that
Cordoba, an area of intensive planting of GM soy where pesticides are
heavily used, had a higher incidence of spina bida, microtia, cle lip
with cle palate, polycystic kidney, postaxial polydactyly and Down’s
syndrome than other regions [68]. Many of these defects are of the type
associated with disturbances in the retinoic acid signalling pathway,
though it is not possible to identify a sole causative agent.
Epidemiological Studies in North America
Epidemiological studies carried out in North America show an
association between exposure to glyphosate herbicides and adverse
reproductive and developmental outcomes. In Canada, the Ontario
Farm Family Health Study found a higher than normal rate of
miscarriages and pre-term deliveries associated with glyphosate
exposure [40,69]. An epidemiological study carried out in the USA
found that the children of pesticide applicators exposed to glyphosate
herbicides had an increased incidence of ADHD (attention decit
hyperactivity disorder) [70]. e nding suggested that glyphosate
herbicide impacts neurological development.
Rull et al. provided evidence of an association between maternal
exposure to glyphosate herbicides and anencephaly, a type of neural
tube defect, as well as with neural tube defects (NTDs) in general
[71,72]–consistent with retinoic acid-linked teratogenicity. e study
found that maternal glyphosate herbicide exposure was associated
with anencephaly using one type of analytical model (polytomous
conventional multiple pesticide model), but not with another
(hierarchical polytomous or single pesticide model).
e data showed modest associations between glyphosate and
NTDs for both single and multiple pesticide models, with an odds ratio
(OR) of 1.5 for both. For the hierarchical model the OR was 1.4. e
authors’ criteria for signicant eects were that the OR should be greater
than or equal to 1.4 and the lower limit of the condence interval (CI)
should be greater than or equal to 0.9. e OR requirement was met for
glyphosate and NTDs using both models, but both models delivered
CIs of 0.8, just below the cut-o value [71,72].
ese results indicate a modest association between glyphosate
herbicide exposure and NTDs and are in disagreement with the
interpretation put forward by Williams et al. that the data shows “no
eect” on NTDs. Williams et al. disagree with Rull et al.’s classication
of glyphosate as an organophosphate [64], although chemically, it falls
into that category of compounds.
Some studies that Williams et al. (2012) cite in their review in
defence of the safety of glyphosate herbicides are unpublished industry-
sponsored studies [64]. It should be noted, however, that the industry
teratogenicity studies examined glyphosate and not the commercial
herbicide formulations, which are the substances under examination
in epidemiological and most laboratory studies from the peer-reviewed
literature. Crucially, these are also the substances to which humans are
exposed. Studies have found that, although glyphosate itself is toxic,
the formulations are more toxic than glyphosate alone [32,33,73,74].
Even the industry-sponsored studies on glyphosate alone show cause
for concern, as shown above.
Genotoxicity of Glyphosate
While the EU Commission’s 2002 review report on glyphosate
concludes that it is “not genotoxic” [11], it is dicult to understand
how this position can be maintained. Studies indicate that glyphosate
herbicides are genotoxic and thus have the potential to increase the risk
of birth defects and cancer. Cytogenetic monitoring of crop sprayers
in Cordoba, Argentina revealed that the number of chromosomal
aberrations in peripheral blood cells was signicantly higher in the
exposed group in comparison to the unexposed group. e pesticides
most commonly used by the exposed group were glyphosate,
cipermetrine, and atrazine [75].
An epidemiological study on Ecuadorian populations showed that
people exposed to aerial glyphosate spraying showed a higher degree of
DNA damage than a control population living 80 km away [76]. Mañas
et al. found that glyphosate was genotoxic in the comet assay in Hep-2
cells and in the micronucleus test at 400 mg/kg in mice [77].
Glyphosate herbicides and glyphosate’s main metabolite, AMPA,
altered cell cycle checkpoints in sea urchin embryos by interfering with
the DNA repair machinery [78-80]. e failure of cell cycle checkpoints
is known to lead to genomic instability and cancer in humans.
Glyphosate and AMPA have also been found to cause irreversible
damage to DNA that may increase the risk of cancer [77,81]. AMPA
damaged DNA in human cells at doses of 2.5-7.5mM and caused
chromosomal breaks at 1.8mM [81].
e surfactants and other adjuvants in glyphosate formulations
enhance the toxic eects of glyphosate, as they enable it to penetrate
more easily through the cell membrane [79,82]. e adjuvants alone
are also toxic [73].
Farm Family Exposure Study
e Monsanto/Dow/Syngenta authors cited the Farm Family
Exposure Study (FFES) [83], as evidence that the doses used by
Paganelli et al. and the suggestion of a link between glyphosate herbicide
exposure and birth defects in Argentina are unrealistic [16]. e FFES
measured urinary glyphosate concentrations for farmers, their spouses,
and their children. e study concluded that the maximum systemic
dose to spouses in the FFES was only 0.04 μg/kg body weight, with
more than 95% of the spouse exposures below the limit of detection
[83]. e Monsanto/Dow/Syngenta authors stated that this exposure
scenario was “similar” to that of the populations in Argentina and
other soy-producing regions of South America that were the focus of
concern in Paganelli et al.’s study [16].
However, it is dicult to envisage how these two scenarios are similar.
e US-based FFES measured urinary glyphosate concentrations the
day before, the day of, and for three days following a single glyphosate
application, which was carried out by tractor and boom sprayer. In
the US, it is usual for farmers to conduct spraying from the relatively
protected environment of an enclosed air-conditioned cab. People
living in South American GM soy-producing regions are exposed not
once but frequently during the growing season and application is oen
carried out from the air, leading to problems of dri.
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 10 of 13
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Moreover, any evaluation of the eects of pesticide exposures
must take into consideration the eects of repeated and continuous
exposures. Bolognesi (2003) found that chromosomal damage caused
by pesticides was temporary in short or time-limited exposures but
cumulative in continuous exposures to agrochemicals [84].
e FFES authors acknowledged that the nature of their study
may have led participating farmers to take extra care in their work.
erefore it may not have reected real conditions, even in the US
(a representative of the study was present with the farmer at the time
of application). Also, the FFES was sponsored by members of the
pesticide industry: Bayer, Dow, DuPont, FMC, Monsanto, Syngenta,
and the American Chemistry Council. One author, Acquavella, was
an employee of Monsanto; another was an employee of the industry
consulting rm, Exponent. ese links with the pesticide industry
create a risk of bias.
For these reasons, the FFES may not reect realistic conditions.
Mage (2006) stated in a critique of the FFES that a study that randomly
and frequently assesses glyphosate burdens in farm families over a long
period of time would provide a more realistic assessment of exposure
[85].
Our concerns are supported by another study, which is not
mentioned by the authors of the Monsanto/Dow/Syngenta rebuttal. In
a study investigating pesticide exposure in farm and non-farm families
in Iowa, USA, Curwin et al. (2007) found that 75% of farmers, 67% of
wives, and 81% of farmers’ children were carrying urinary burdens of
more than 900 ppb of glyphosate (0.9 mg/kg bw) [86]. In contrast, the
FFES reported average urinary burdens of glyphosate ranging from 1
to 6.4 ppb on dierent days of the study for farmers, and with averages
close to 0 ppb for wives and children (less than 25% of subjects were
reported to have any detectable urinary glyphosate burden) [83].
Court Cases on Glyphosate Herbicide Exposure
e safety of glyphosate herbicides has been successfully
challenged in several court cases. In New York in 1996, a court ruled
that Monsanto was no longer allowed to market Roundup as safe,
non-toxic, biodegradable or environmentally friendly [87]. In France
in 2007, Monsanto was forced to withdraw advertising claims that
Roundup was biodegradable and leaves the soil clean aer use [88]. In
March 2010, in a case brought by residents, a court in Santa Fe province,
Argentina instituted a regional ban on the spraying of glyphosate and
other agrochemicals in populated areas on grounds of “severe damage
to the environment and to the health and quality of life of the residents”
[89].
In June 2012 criminal charges were brought by aected residents
against two soy producers and a crop-spraying airplane pilot, in
a case heard by a court in Cordoba, Argentina. Plaintis charged
the defendants with malicious contamination over the spraying of
glyphosate and other agrochemicals in Ituzaingó, an area on the
outskirts of Cordoba reportedly characterized by a high incidence of
cancer and birth defects [90].
Relevance of Dierent Exposure Routes
BVL and Monsanto/Dow/Syngenta dismissed Paganelli et al.’s
study on the grounds that it used inappropriate exposure routes. ey
object to injection and culture on the grounds that they are “highly
articial”, “do not reect human exposure” [13], and are “irrelevant”
[16], to human risk assessment. is argument is also used by Williams
et al. (2012) in defence of the safety of glyphosate [64].
e standard that is being invoked is not named but is likely to be
the OECD standardised protocols for industry studies performed for
regulatory purposes, which prefer oral, dermal or inhalation exposure
routes [91].
OECD guidelines exist to guide industry on how to conduct
standardised tests performed for regulatory purposes, but it is not
credible to suggest that they represent the only valid or the most
scientically rigorous route to acquiring information about a chemical’s
toxicity.
In the case of Paganelli et al.’s study [1], injection of the treated
group with glyphosate and of the control group with water claried that
only one substance-glyphosate-could have caused the malformations.
e absence of malformations in the water-injected controls showed
that the trauma of injection did not cause the malformations.
Two studies comparing oral dosing with injection presented
ndings that challenge assumptions about dierent exposure routes:
• A study comparing the eects of bisphenol A (BPA)
administered to rats by oral dosing and injection found that
aer two hours, the level of active BPA in the blood was the
same between orally dosed and injected groups. Both exposure
routes resulted in the same pre-cancerous toxic eects on the
prostate seven months aer exposure. e study concluded
that the internal received dose, not route of exposure, is the
critical factor, and that therefore, the injection exposure route
should be acceptable for human risk assessment [92,93].
• A study comparing the toxicokinetics of glyphosate
administered to rats by oral dosing and injection found that
when given orally, glyphosate was more slowly absorbed but
took longer to clear from blood, leading to the possibility
that it could be distributed to the tissues, causing systemic
toxic eects [94]. e oral route is favoured by industry and
regulators on the claimed grounds that it better reects real
human exposures. us, based on this study, experiments
using injection could be assumed to result in less toxic eects
than those using oral methods.
While doses received by dierent tissues may vary according to
exposure route, this should be tested and not assumed. It seems critically
important to conduct biomonitoring studies on exposed populations
to discover how much glyphosate and its main metabolite, AMPA, is
present in tissues and to investigate the potential for bioaccumulation.
An in vitro study on human buccal cells attempted to mirror
human exposures to glyphosate herbicide through inhalation. e
study found that glyphosate and Roundup caused DNA damage in the
cells aer a single 20-minute exposure at a dose corresponding to a
450-fold dilution of the concentration used in agriculture. Roundup
was more toxic than glyphosate alone. e authors concluded that
inhalation may cause DNA damage in exposed individuals and that the
DNA damage was caused directly by the substances instead of being an
indirect result of cell toxicity [95].
While the Monsanto/Dow/Syngenta authors condemn in vitro
methods as “unvalidated”, this value judgement only raises the
question: validated by whom? We interpret this statement to mean that
these in vitro tests do not conform to OECD standardised protocols
for industry toxicological studies. However, outside the narrow context
of industry testing for regulatory purposes, such in vitro tests are an
important tool. For example, in the pre-clinical phase of pharmaceutical
drug development, if a potential drug gives a positive micronucleus test
Citation: Antoniou M, Habib MEM, Howard CV, Jennings RC, Leifert C, et al. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence
of Regulatory Decisions from Scientic Evidence. J Environ Anal Toxicol S4:006. doi:10.4172/2161-0525.S4-006
Page 11 of 13
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in vitro, then development is discontinued. Also, such in vitro tests
add valuable evidence to ndings from laboratory in vivo and human
epidemiological studies. Regarding glyphosate and Roundup, studies
of all these types suggest that both substances are genotoxic and have
toxic eects on development and reproduction.
Unrealistically High Doses?
In their response to Paganelli et al., the Monsanto/Dow/Syngenta
authors argue that the researchers used “inappropriately high” and
“unrealistic” doses, far higher than the already high doses that have
been shown in other studies not to cause malformations [16].
Considering rst Paganelli et al.’s frog embryo injection experiments,
calculations based on Monsanto/Dow/Syngenta’s own paper show that
the doses were not inappropriately high. e Monsanto/Dow/Syngenta
authors stated that a 400 mg/kg dose of glyphosate, delivered through
feeding, results in a blood concentration of 4.6 μg/ml. Animal studies
typically use between 50 and 500 mg/kg bw/d doses. Making a linear
extrapolation (as the Monsanto/Dow authors do for other purposes),
a 50 mg/kg dose should result in a blood concentration of 0.575 μg/
ml, or 575 μg/L. erefore, the range of blood concentrations achieved
in animal studies would be in the range of 575–5750 μg/L. Clearly,
the concentrations achieved in the frog embryos (690–950 μg/L) are
comparable to the blood concentrations typically achieved in animal
feeding studies.
Regarding Paganelli et al.’s frog embryo culture experiments, the
Monsanto/Dow/Syngenta authors stated that the concentrations used
were 9-15 times greater than the acute LC50 value for frog embryos of
the same species. Monsanto/Dow cites as its authority for this argument
a study by Edginton et al. [96], However, Edginton used a dierent
glyphosate formulation, with a potentially dierent LC50 value.
Moreover, the low mortality rate found by Paganelli et al. counters the
Monsanto/Dow/Syngenta authors’ claim that the doses used were even
close to the LC50 value.
Regarding Paganelli et al.’s experiments with chicken eggs, using
the Monsanto/Dow/Syngenta authors’ own estimate that 20μL of
a 1/4500 dilution of glyphosate-formulated product translates to 2
μg glyphosate injected into the egg, and assuming that the volume
of a chicken egg is approximately 35 ml, the actual concentration of
glyphosate within the egg would be 57 μg/L. is is much lower than the
blood concentrations of glyphosate that would be expected in animal
toxicity studies (575–5750 μg/L, see above), according to Monsanto/
Dow’s own calculation methods.
Further countering the claim that Paganelli et al. used unrealistically
high doses or doses higher than the LC50 value is new, as yet unpublished
data obtained by the same researchers. In these culture experiments
with embryos of Xenopus laevis, the same methodology was followed
as in the original culture experiments detailed in Paganelli et al. [1]
A dierent commercial formulation of Roundup was used (Gleba
from Gleba S.A., instead of the Roundup Classic used in the original
experiments) and batches of embryos were cultured in progressively
lower dilutions. e same malformations as were observed in the
original experiments were reproduced in a dose-dependent manner,
even at dilutions of 1/500,000 (4.30 μM). is dilution produced
developmental abnormalities in 17% of the embryos, with no lethality
[97].
Conclusion
Studies published in the peer-reviewed scientic literature have
raised major concerns regarding the potential for glyphosate and its
commercial formulations to cause birth defects and other reproductive
problems. In addition, a debate has emerged over the reported eects
on human health of herbicide application in regions that produce
GM glyphosate-tolerant crops and about the safety of food and feed
produced from these crops.
Regulatory authorities and industry aliates have defended the
use of glyphosate largely by citing the industry-sponsored toxicological
tests conducted for regulatory purposes, which they claimed showed
no evidence of teratogenicity. However, the German authorities’ dra
assessment report revealed that even these industry tests contained
clear evidence of glyphosate-mediated teratogenicity and reproductive
toxicity. Many of the malformations observed in these studies are of the
type associated with the retinoic acid signalling pathway. Paganelli et
al. [1] showed that this was the mechanism through which glyphosate
and Roundup exercise their teratogenic eects.
It is noteworthy that these industry tests were commissioned by
the same companies that stand to prot from regulatory authorization.
Regrettably, this system possesses an inherent risk of bias and makes
it especially important that the regulatory assessment is rigorous.
Yet in the EU, the evidence suggests that this was not the case. e
signicance of clear teratogenic eects of glyphosate in rabbits and rats
found in tests commissioned by industry were minimized by German
regulators. A scientically rigorous assessment was further impeded
by the outdated design of the standard tests, which are not suciently
sensitive to detect eects from realistic exposures. As a result, the
German authorities suggested, and the EU adopted, an acceptable daily
intake (ADI) for glyphosate that is unreliable and could potentially
result in exposures that cause harm to humans.
Another relevant factor is that the industry teratogenicity tests were
on glyphosate, the presumed active ingredient of the herbicide, and not
on the herbicide formulations as sold and used, even though studies
indicate that the formulations are more toxic for certain endpoints
than glyphosate alone.
A substantial body of evidence demonstrates that glyphosate
and Roundup cause teratogenic eects and other toxic eects on
reproduction, as well as genotoxic eects. From an objective scientic
standpoint, attempts by industry and government regulatory bodies to
dismiss this research are unconvincing and work against the principle
that it is the responsibility of industry to prove that its products are
safe and not the responsibility of the public to prove that they are
unsafe. e precautionary principle would suggest that glyphosate and
its commercial formulations should undergo a new risk assessment,
taking full account of the entirety of the peer-reviewed scientic
literature as well as the industry-sponsored studies. Experience to date
suggests that the new risk assessment should be conducted with full
public transparency by scientists who are independent of industry.
Disclaimer
The opinions expressed are those of the individual authors and do not reect
the policies of organizations with which they are associated.
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