Long term toxicity of a Roundup herbicide and a Roundup-tolerant

genetically modied maize

Gilles-Eric Sralini
a,
, Emilie Clair
a
, Robin Mesnage
a
, Steeve Gress
a
, Nicolas Defarge
a
,
Manuela Malatesta
b
, Didier Hennequin
c
, Jol Spiroux de Vendmois
a
a
University of Caen, Institute of Biology, CRIIGEN and Risk Pole, MRSH-CNRS, EA 2608, Esplanade de la Paix, Caen Cedex 14032, France
b
University of Verona, Department of Neurological, Neuropsychological, Morphological and Motor Sciences, Verona 37134, Italy
c
University of Caen, UR ABTE, EA 4651, Bd Marchal Juin, Caen Cedex 14032, France
a r t i c l e i n f o
Article history:
Received 11 April 2012
Accepted 2 August 2012
Available online xxxx
Keywords:
GMO
Roundup
NK603
Rat
Glyphosate-based herbicides
Endocrine disrupting effects
a b s t r a c t
The health effects of a Roundup-tolerant genetically modied maize (from 11% in the diet), cultivated
with or without Roundup, and Roundup alone (from 0.1 ppb in water), were studied 2 years in rats. In
females, all treated groups died 23 times more than controls, and more rapidly. This difference was vis-
ible in 3 male groups fed GMOs. All results were hormone and sex dependent, and the pathological pro-
les were comparable. Females developed large mammary tumors almost always more often than and
before controls, the pituitary was the second most disabled organ; the sex hormonal balance was mod-
ied by GMO and Roundup treatments. In treated males, liver congestions and necrosis were 2.55.5
times higher. This pathology was conrmed by optic and transmission electron microscopy. Marked
and severe kidney nephropathies were also generally 1.32.3 greater. Males presented 4 times more large
palpable tumors than controls which occurred up to 600 days earlier. Biochemistry data conrmed very
signicant kidney chronic deciencies; for all treatments and both sexes, 76% of the altered parameters
were kidney related. These results can be explained by the non linear endocrine-disrupting effects of
Roundup, but also by the overexpression of the transgene in the GMO and its metabolic consequences.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
There is an ongoing international debate as to the necessary
length of mammalian toxicity studies in relation to the consump-
tion of genetically modied (GM) plants including regular meta-
bolic analyses (Sralini et al., 2011). Currently, no regulatory
authority requests mandatory chronic animal feeding studies to
be performed for edible GMOs and formulated pesticides. How-
ever, several studies consisting of 90 day rat feeding trials have
been conducted by the biotech industry. These investigations
mostly concern GM soy and maize that are rendered either herbi-
cide tolerant (to Roundup (R) in 80% of cases), or engineered to
produce a modied Bt toxin insecticide, or both. As a result these
GM crops contain new pesticide residues for which new maximal
residual levels (MRL) have been established in some countries.
If the petitioners conclude in general that there is no major
change in genetically modied organism (GMO) subchronic toxic-
ity studies (Domingo and Gin Bordonaba, 2011; Hammond et al.,
2004, 2006a,b), signicant disturbances have been found and
may be interpreted differently (Sralini et al., 2009; Spiroux de
Vendmois et al., 2010). Detailed analyses have revealed altera-
tions in kidney and liver functions that may be the signs of early
chronic diet intoxication, possibly explained at least in part by
pesticide residues in the GM feed (Sralini et al., 2007; Spiroux
de Vendmois et al., 2009). Indeed, it has been demonstrated that
R concentrations in the range of 10
3
times below the MRL induced
endocrine disturbances in human cells (Gasnier et al., 2009) and
toxic effects thereafter (Benachour and Seralini, 2009), including
in vivo (Romano et al., 2012). After several months of consumption
of an R-tolerant soy, the liver and pancreas of mice were affected,
as highlighted by disturbances in sub-nuclear structure (Malatesta
et al., 2008a, 2002a,b). Furthermore, this toxic effect was repro-
duced by the application of R herbicide directly to hepatocytes in
culture (Malatesta et al., 2008b).
0278-6915/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.08.005
Abbreviations: GM, genetically modied; R, Roundup; MRL, maximal residual
levels; GMO, genetically modied organism; OECD, Organization for Economic Co-
operation and Development; GT, glutamyl-transferase; PCA, principal component
analysis; PLS, partial least-squares; OPLS, orthogonal partial least-squares; NIPALS,
Nonlinear Iterative Partial Least Squares; OPLS-DA, Orthogonal Partial Least Squares
Discriminant Analysis; G, glycogen; L, lipid droplet; N, nucleus; R, rough endoplas-
mic reticulum (on microscopy pictures only); U, urinary; UEx, excreted in urine
during 24 h; APPT, Activated Partial Thromboplastin Time; MCV, Mean Corpuscular
Volume; PT, Prothrombine Time; RBC, Red Blood Cells; ALT, alanine aminotrans-
ferase; MCHC, Mean Corpuscular Hemoglobin Concentration; A/G, Albumin/Glob-
ulin ratio; WBC, White Blood Cells; AST, aspartate aminotransferase.

Corresponding author. Tel.: +33 (0)231565684; fax: +33 (0)231565320.

E-mail address: [email protected] (G.-E. Sralini).
Food and Chemical Toxicology xxx (2012) xxxxxx
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Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005
Since then, long-term and multi-generational animal feeding
trials have been performed with some possibly providing evidence
of safety, while others conclude on the necessity of further investi-
gations because of metabolic modications (Snell et al., 2011).
However, none of these studies have included a detailed follow-
up of the animals with up to 11 blood and urine samples over
2 years, and none has investigated the NK603 R-tolerant maize.
Furthermore, toxicity evaluation of herbicides is generally per-
formed on mammalian physiology through the long-term study
of only their active principle, rather than the formulation used in
agriculture, as was the case for glyphosate (Williams et al., 2000),
the active herbicide constituent of R. It is important to note that
glyphosate is only able to efciently penetrate target plant organ-
isms with the help of adjuvants present in the various commer-
cially used R formulations (Cox, 2004). When R residues are
found in tap water, food or feed, they arise from the total herbicide
formulation, which is the most commonly used mixture in agricul-
ture; indeed many authors in the eld have strongly emphasized
the necessity of studying the potential toxic effects of total chem-
ical mixtures rather than single components (Cox and Surgan,
2006; Mesnage et al., 2010; Monosson, 2005). Even adjuvants
and not only glyphosate or other active ingredients are found in
ground water (Krogh et al., 2002), and thus an exposure to the di-
luted whole formulation is more representative of an environmen-
tal pollution than the exposure to glyphosate alone in order to
study health effects.
With a view to address this lack of information, we have per-
formed a 2 year detailed rat feeding study. The actual guideline
408 of the Organization for Economic Co-operation and Develop-
ment (OECD) was followed by some manufacturers for GMOs even
if it was not designed for that purpose. We have explored more
parameters and more frequently than recommended in this stan-
dard (Table 1) in a long-term experiment. This allowed us to follow
in details potential health effects and their possible origins due to
the direct or indirect consequences of the genetic modication it-
self in GMOs, or due to the formulated herbicide mixture used on
GMOs (and not glyphosate alone), or both. Because of recent re-
views on GMOs (Domingo and Gin Bordonaba, 2011; Snell et al.,
2011) we had no reason to settle at rst for a carcinogenesis pro-
tocol using 50 rats per group. However we have prolonged the bio-
chemical and hematological measurements or disease status
recommended in combined chronic studies using 10 rats per group
(up to 12 months in OECD 453). This remains the highest number
of rats regularly measured in a standard GMO diet study. We have
tested also for the rst time 3 doses (rather than two in the usual
90 day long protocols) of the R-tolerant NK603 GM maize alone,
the GM maize treated with R, and R alone at very low environmen-
tally relevant doses starting below the range of levels permitted by
regulatory authorities in drinking water and in GM feed.
2. Materials and methods
2.1. Ethics
The experimental protocol was conducted in accordance with the regulations of
our ethics in an animal care unit authorized by the French Ministries of Agriculture
and Research (Agreement Number A35-288-1). Animal experiments were per-
formed according to ethical guidelines of animal experimentations (CEE 86/609 reg-
ulation). Concerning eld studies of plant species, no specic permits were
required, nor for the locations/activities. The maize grown (MON-00603-6 com-
monly named NK603) was authorized for unconned release into the environment
and use as a livestock feed by the Canadian Food Inspection Agency (Decision Doc-
ument 2002-35). We conrm that the location is not privately-owned or protected
in any way and that the eld studies did not involve endangered or protected spe-
cies. The GM maize was authorized for import into the European Union (CE 258/97
regulation).
2.2. Plants, diets and chemicals
The varieties of maize used in this study were the R-tolerant NK603 (Monsanto
Corp., USA), and its nearest isogenic non-transgenic control. These two types of
maize were grown under similar normal conditions, in the same location, spaced
at a sufcient distance to avoid cross-contamination. The genetic nature, as well
as the purity of the GM seeds and harvested material, was conrmed by qPCR anal-
ysis of DNA samples. One eld of NK603 was treated with R at 3 L ha
1
(Weather-
MAX, 540 g/L of glyphosate, EPA Reg. 524-537), and another eld of NK603 was not
treated with R. Corns were harvested when the moisture content was less than 30%
and were dried at a temperature below 30 C. From these three cultivations of
Table 1
Protocol used and comparison to existing assessment, and to non-mandatory regulatory tests.
Treatments and analyses In this work Hammond et al., 2004 Regulatory tests
Treatments + controls GMO NK603, GMO NK603 +
Roundup, Roundup, and
closest isogenic maize
GMO NK603 + Roundup, closest isogenic
maize, and six other maize lines non
substantially equivalent
GMOs or chemicals
(in standard diet or water)
Doses by treatment 3 2 At least 3
Duration in months 24 (chronic) 3 (subchronic: 13 weeks) 3
Animals measured/group/sex 10/10 SD rats (200 rats measured) 10/20 SD rats (200 rats measured/total
400)
At least 10 rodents
Animals by cage (same sex) 12 1 1 or more
Monitoring/week 2 1 1 or more
Feed and water consumptions Measured For feed only At least feed
Organs and tissues studied For high dose and controls
Histology/animal 34 17/36 At least 30
Organs weighted 10 7 At least 8
Electronic microscopy Yes No No
Behavioral studies (times) 2 1 (no protocol given) 1
Ophtalmology (times) 2 0 2
Number of blood samples/
animal
11, each month (03) then every 3 months 2, weeks 4 and 13 1, at the end
Blood parameters 31 (11 times for most) 31 (2 times) At least 25 (at least 2 times)
Plasma sex steroids Testosterone, estradiol No No, except if endocrine effects suspected
Liver tissue parameters 6 0 0
Number of urine samples 11 2 Optional, last week
Urine parameters studied 16 18 7 if performed
Microbiology in feces or urine Yes Yes No
Roundup residues in tissues Studied Not studied Not mandatory
Transgene in tissues Studied Not studied Not studied
The protocol used in this work was compared to the regulatory assessment of NK603 maize by the company (Hammond et al., 2004), and to non mandatory regulatory in vivo
tests for GMOs, or mandatory for chemicals (OECD 408). Most relevant results are shown in this paper.
2 G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx
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maize, laboratory rat chow was made based on the standard diet A04 (Safe, France).
The dry rat feed was made to contain 11, 22 or 33% of GM maize, cultivated either
with or without R, or 33% of the non-transgenic control line. The concentrations of
the transgene were conrmed in the three doses of each diet by qPCR. All feed for-
mulations consisted in balanced diets, chemically measured as substantially equiv-
alent except for the transgene, with no contaminating pesticides over standard
limits. All secondary metabolites cannot be known and measured in the composi-
tion. However we have measured isoavones and phenolic acids including ferulic
acid by standard HPLC-UV. All reagents used were of analytical grade. The herbicide
diluted in the drinking water was the commercial formulation of R (GT Plus, 450 g/L
of glyphosate, approval 2020448, Monsanto, Belgium). Herbicides levels were as-
sessed by glyphosate measurements in the different dilutions by mass
spectrometry.
2.3. Animals and treatments
Virgin albino Sprague-Dawley rats at 5 weeks of age were obtained from Harlan
(Gannat, France). All animals were kept in polycarbonate cages (820 cm
2
, Genestil,
France) with two animals of the same sex per cage. The litter (Toplit classic, Safe,
France) was replaced twice weekly. The animals were maintained at 22 3 C under
controlled humidity (4565%) and air purity with a 12 h-light/dark cycle, with free
access to food and water. The location of each cage within the experimental room
was regularly moved. This 2 year life-long experiment was conducted in a GPL envi-
ronment according to OECD guidelines. After 20 days of acclimatization, 100 male
and 100 female animals were randomly assigned on a weight basis into 10 equiv-
alent groups. For each sex, one control group had access to plain water and standard
diet from the closest isogenic non-transgenic maize control; six groups were fed
with 11, 22 and 33% of GM NK603 maize either treated or not with R. The nal three
groups were fed with the control diet and had access to water supplemented with
respectively 1.1 10
8
% of R (0.1 ppb of R or 50 ng/L of glyphosate, the contaminat-
ing level of some regular tap waters), 0.09% of R (400 mg/kg, US MRL of glyphosate
in some GM feed) and 0.5% of R (2.25 g/L, half of the minimal agricultural working
dilution). This was changed weekly. Twice weekly monitoring allowed careful
observation and palpation of animals, recording of clinical signs, measurement of
any tumors that may arise, food and water consumption, and individual body
weights.
2.4. Biochemical analyses
Blood samples were collected from the tail vein of each rat under short isou-
rane anesthesia before treatment and after 1, 2, 3, 6, 9, 12, 15, 18, 21 and
24 months: 11 measurements were obtained for each animal alive at 2-years. It
was rst demonstrated that anesthesia did not impact animal health. Two aliquots
of plasma and serum were prepared and stored at 80 C. Then 31 parameters were
assessed (Table 1) according to standard methods including hematology and coag-
ulation parameters, albumin, globulin, total protein concentration, creatinine, urea,
calcium, sodium, potassium, chloride, inorganic phosphorus, triglycerides, glucose,
total cholesterol, alanine aminotransferase, aspartate aminotransferase, gamma
glutamyl-transferase (GT), estradiol, testosterone. In addition, at months 12 and
24 the C-reactive protein was assayed. Urine samples were collected similarly 11
times, over 24 h in individual metabolic cages, and 16 parameters were quantied
including creatinine, phosphorus, potassium, chloride, sodium, calcium, pH and
clairance. Liver samples at the end made it possible to perform assays of CYP1A1,
1A2, 3A4, 2C9 activities in S9 fractions, with glutathione S- transferase and gam-
ma-GT.
2.5. Anatomopathology
Animals were sacriced during the course of the study only if necessary because
of suffering according to ethical rules (such as 25% body weight loss, tumors over
25% body weight, hemorrhagic bleeding, or prostration), and at the end of the study
by exsanguination under isourane anesthesia. In each case, the following organs
were collected: brain, colon, heart, kidneys, liver, lungs, ovaries, spleen, testes, adre-
nals, epididymis, prostate, thymus, uterus, aorta, bladder, bone, duodenum, esoph-
agus, eyes, ileum, jejunum, lymph nodes, lymphoreticular system, mammary
glands, pancreas, parathyroid glands, Peyers patches, pituitary, salivary glands, sci-
atic nerve, skin, spinal cord, stomach, thyroid and trachea. The rst 14 organs (at
least 10 per animal depending on the sex, Table 1) were weighted, plus any tumor
that arose. The rst nine organs were divided into two parts and one half was
immediately frozen in liquid nitrogen/carbonic ice. The remaining parts including
other organs were rinsed in PBS and stored in 4% formalin before anatomopatholog-
ical study. These samples were used for further parafn-embedding, slides and HES
histological staining. For transmission electron microscopy, kidneys, livers and tu-
mors were cut into 1 mm
3
fragments. Samples were xed in pre-chilled 2% parafor-
maldehyde/2.5% glutaraldehyde in 0.1 M PBS pH 7.4 at 4 C for 3 h and processed as
previously described (Malatesta et al., 2002a).
2.6. Statistical analysis
Biochemical data were treated by multivariate analysis with the SIMCA-P (V12)
software (UMETRICS AB Umea, Sweden). The use of chemometrics tools, for exam-
ple, principal component analysis (PCA), partial least-squares to latent structures
(PLS), and orthogonal PLS (OPLS), are robust methods for modeling, analyzing and
interpreting complex chemical and biological data. OPLS is a recent modication
of the PLS method. PLS is a regression method used in order to nd the relationship
between two data tables referred to as X and Y. PLS regression (Eriksson et al.,
2006b) analysis consists in calculating by means of successive iterations, linear
combinations of the measured X-variables (predictor variables). These linear com-
binations of X-variables give PLS components (score vectors t). A PLS component
can be thought of as a new variable a latent variable reecting the information
in the original X-variables that is of relevance for modeling and predicting the re-
sponse Y-variable by means of the maximization of the square of covariance
(Max cov
2
(X,Y)). The number of components is determined by cross validation. SIM-
CA software uses the Nonlinear Iterative Partial Least Squares algorithm (NIPALS)
for the PLS regression. Orthogonal Partial Least Squares Discriminant Analysis
(OPLS-DA) was used in this study (Weljie et al., 2011; Wiklund et al., 2008). The
purpose of Discriminant Analysis is to nd a model that separates groups of obser-
vations on the basis of their X variables. The X matrix consists of the biochemical
data. The Y matrix contains dummy variables which describe the group member-
ship of each observation. Binary variables are used in order to encode a group iden-
tity. Discriminant analysis nds a discriminant plan in which the projected
observations are well separated according to each group. The objective of OPLS is
to divide the systematic variation in the X-block into two model parts, one linearly
related to Y (in the case of a discriminant analysis, the group membership), and the
other one unrelated (orthogonal) to Y. Components related to Y are called predic-
tive, and those unrelated to Y are called orthogonal. This partitioning of the X data
results in improved model transparency and interpretability (Eriksson et al., 2006a).
Prior to analysis, variables were mean-centered and unit variance scaled.
3. Results
3.1. Mortality
Control male animals survived on average 624 21 days, whilst
females lived for 701 20, during the experiment, plus in each case
5 weeks of age at the beginning and 3 weeks of stabilization period.
After mean survival time had elapsed, any deaths that occurred
were considered to be largely due to aging. Before this period,
30% control males (three in total) and 20% females (only two) died
spontaneously, while up to 50% males and 70% females died in
some groups on diets containing the GM maize (Fig. 1). However,
the rate of mortality was not proportional to the treatment dose,
reaching a threshold at the lowest (11%) or intermediate (22%)
amounts of GM maize in the equilibrated diet, with or without
the R application on the plant. It is noteworthy that the rst two
male rats that died in both GM treated groups had to be euthanized
due to kidney Wilms tumors that were over 25% of body weight.
This was at approximately a year before the rst control animal
died. The rst female death occurred in the 22% GM maize feeding
group and resulted from a mammary broadenoma 246 days be-
fore the rst control. The maximum difference in males was 5
times more deaths occurring during the 17th month in the group
consuming 11% GM maize, and in females 6 times greater mortal-
ity during the 21st month on the 22% GM maize diet with and
without R. In the female cohorts, there were 23 times more
deaths in all treated groups compared to controls by the end of
the experiment and earlier in general. Females were more sensitive
to the presence of R in drinking water than males, as evidenced by
a shorter lifespan. The general causes of death represented in his-
togram format (Fig. 1) are linked mostly to large mammary tumors
in females, and other organic problems in males.
3.2. Anatomopathological observations
All rats were carefully monitored for behavior, appearance, pal-
pable tumors, infections, during the experiment, and at least 10 or-
gans per animal were weighted and up to 34 analyzed post
mortem, at the macroscopic and/or microscopic levels (Table 1).
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All data cannot be shown in one report, and the most relevant are
described here. There was no rejection by the animals of the diet
with or without GMOs, nor any major difference in the body
weight.
The largest palpable growths (above a diameter of 17.5 mm in
females and 20 mm in males) were found to be in 95% of cases
non-regressive tumors, and were not infectious nodules. These
growths progressively increased in size and number, but not pro-
portionally to the treatment dose over the course of the experi-
ment (Fig. 2). As in the case of rates of mortality, this suggests
that a threshold in effect was reached at the lowest doses. They
were rarely equal but almost always more frequent than in con-
trols for all treated groups, often 23 times more in both sexes. Tu-
mors began to reach a large size on average 94 days before in
treated females, and up to 600 days earlier in 2 male groups eating
the GM maize (11 and 22% with or without R).
In female animals, the largest tumors were in total 5 times more
frequent than in males after 2 years, with 93% being mammary tu-
mors. Adenomas, broadenomas and carcinomas were deleterious
to health due to a very large size, rather than the grade of the
tumor itself. Large tumor size caused impediments to either
breathing or nutrition and digestion because of their thoracic or
abdominal location and also resulted in hemorrhaging. In addition,
one metastatic ovarian cystadenocarcinoma and two skin tumors
were identied. Metastases were observed in only 2 cases; one in
a group fed with 11% GM maize, and another in the highest dose
of R treatment group.
Up to 14 months, no animals in the control groups showed any
signs of tumors whilst 1030% of treated females per group devel-
oped tumors, with the exception of one group (33% GMO + R). By
the beginning of the 24th month, 5080% of female animals had
developed tumors in all treated groups, with up to 3 tumors per
animal, whereas only 30% of controls were affected. The R treat-
ment groups showed the greatest rates of tumor incidence with
80% of animals affected with up to 3 tumors for one female, in each
group. A summary of all mammary tumors at the end of the exper-
iment, independent of the size, is presented in Table 2. The same
trend was observed in the groups receiving R in their drinking
water; all females except one (with metastatic ovarian carcinoma)
presented, in addition mammary hypertrophies and in some cases
hyperplasia with atypia (Table 2).
The second most affected organ in females was the pituitary
gland, in general around 2 times more than in controls for most
treatments (Table 2). At this level again, adenomas and/or hyper-
Fig. 1. Mortality of rats fed GMO treated or not with Roundup, and effects of Roundup alone. Rats were fed with NK603 GM maize (with or without application of Roundup) at
three different doses (11, 22, 33% in their diet: thin, medium and bold lines, respectively) compared to the substantially equivalent closest isogenic non-GM maize (control,
dotted line). Roundup was administrated in drinking water at 3 increasing doses, same symbols (environmental (A), MRL in agricultural GMOs (B) and half of minimal
agricultural levels (C), see Section 2). Lifespan during the experiment for the control group is represented by the vertical bar SEM (grey area). In bar histograms, the causes of
mortality before the grey area are detailed in comparison to the controls (0). In black are represented the necessary euthanasia because of suffering in accordance with ethical
rules (tumors over 25% body weight, more than 25% weight loss, hemorrhagic bleeding, etc.); and in hatched areas, spontaneous mortality.
4 G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx
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plasias and hypertrophies were noticed. For all R treatment groups,
7080% of animals presented 1.42.4 times more abnormalities
than controls in this gland.
The big palpable tumors in males (in kidney, and mostly skin)
were by the end of the experimental period on average twice as
frequent as in controls, in which one skin broma appeared during
the 23rd month. At the end of the experiment, internal non-palpa-
ble tumors were added, and their sums were lower in males than
in females. They were not really different from controls, although
slightly above in females (Histograms Fig. 2).
The most affected organs in males were the liver, together with
the hepatodigestive tract and kidneys (Table 2 and Fig. 3). Hepatic
congestions, macroscopic and microscopic necrotic foci were 2.5
5.5 times more frequent in all treatments than in control groups.
Gamma GT hepatic activity was increased in particular for GMO + R
groups (up to 5.4 times), this being probably due to a liver disorder.
Fig. 2. Largest non-regressive tumors in rats fed GMO treated or not by Roundup, and effects of Roundup alone. The symbols of curves and treatments are explained in the
caption of Fig. 1. The largest tumors were palpable during the experiment and numbered from 20 mm in diameter for males and 17.5 mm for females. Above this size, 95% of
growths were non-regressive tumors. Summary of all tumors are shown in the bar histograms: black, non regressive largest tumors; white, small internal tumors; grey,
metastases.
Table 2
Summary of the most frequent anatomical pathologies observed.
Organs and associated pathologies Controls GMO 11% GMO 22% GMO 33% GMO 11% + R GMO 22% + R GMO 33% + R R (A) R (B) R (C)
Males, in liver 2 (2) 5 (4) 11 (7) 8 (6) 5 (4) 7 (4) 6 (5) 11 (5) 9 (7) 6 (5)
In hepatodigestive tract 6 (5) 10 (6) 13 (7) 9 (6) 9 (6) 13 (6) 11 (7) 23 (9) 16 (8) 9 (5)
Kidneys, CPN 3 (3) 4 (4) 5 (5) 7 (7) 5 (5) 4 (4) 4 (4) 6 (6) 5 (5) 3 (3)
Females, mammary tumors 8 (5) 15 (7) 10 (7) 15 (8) 10 (6) 11 (7) 13 (9) 20 (9) 16 (10) 12 (9)
In mammary glands 10 (5) 22 (8) 10 (7) 16 (8) 17 (8) 16 (8) 15 (9) 26 (10) 20 (10) 18 (9)
Pituitary 9 (6) 23 (9) 20 (8) 8 (5) 19 (9) 9 (4) 19 (7) 22 (8) 16 (7) 13 (7)
After the number of pathological abnormalities, the number of rats reached is indicated in parentheses. In male animals pathological signs are liver congestions, macroscopic
spots and microscopic necrotic foci. Hepatodigestive pathological signs concern the liver, stomach and small intestine (duodenum, ileum or jejunum). Only marked or severe
chronic progressive nephropathies (CPN) are listed, excluding two nephroblastomas in groups consuming GMO 11% and GMO 22% + Roundup. In females, mammary
broadenomas and adenocarcinomas are the major tumors detected; galactoceles and hyperplasias with atypia are also found and added in mammary glands pathological
signs. Pituitary dysfunctions include adenomas, hyperplasias and hypertrophies. For details of the various treatment groups see Fig. 1.
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In addition, cytochrome activities also generally increased in the
presence of R (in drinking water or GM diet) according to the dose
up to 5.7 times at the highest dose. Transmission electron micro-
scopic observations of liver samples conrmed changes for all trea-
ted groups in relation to glycogen dispersion or appearance in
lakes, increase of residual bodies and enlargement of cristae in
mitochondria (Fig. 4). The GM maize fed groups either with or
without R application (in plants) showed a reduced transcription
in mRNA and rRNA because of higher heterochromatin content,
and decreased nucleolar dense brillar components. In the
GMO + R group (at the highest dose) the smooth endoplasmic
reticulum was drastically increased and nucleoli decreased in size,
Fig. 3. Anatomopathological observations in rats fed GMO treated or not by Roundup, and effects of Roundup alone. Macroscopic and microscopic photographs show male
livers (AE) and left kidneys (FI
0
), female mammary glands (JP) and pituitaries (QT), according to Table 2. The number of each animal and its treatment is specied.
Macroscopic pale spots (D) and microscopic necrotic foci in liver (C clear-cell focus, E basophilic focus with atypia), and marked or severe chronic progressive nephropathies,
are illustrated. In females, mammary tumors (J,J
0
,N adenocarcinoma and K,K
0
,L,L
0
,O,P broadenomas) and pituitary adenomas (RT) are shown and compared to controls (C
after the rat number).
6 G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx
Please cite this article in press as: Sralini, G.-E., et al. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modied maize. Food
Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005
becoming more compact. For R treatment alone similar trends
were observed, with a partial resumption of nucleolar activity at
the highest dose.
Degenerating kidneys with turgid inammatory areas demon-
strate the increased incidence of marked and severe chronic pro-
gressive nephropathies, which were up to 2-fold higher in the
33% GM maize or lowest dose R treatment groups (Table 2 and
Fig. 3).
3.3. Biochemical analyses
For the different corns and diets, the study of the standard chem-
ical composition revealed no particular difference; this is why they
were classied as substantially equivalent, except for transgene
DNA quantication. For instance, there was no difference between
total isoavones. In addition, other specic compounds not always
requested for substantial equivalence establishment were assayed.
Among phenolic acids, the only consistent and signicant (p < 0.01)
results concerned ferulic acid that was decreased in both GM and
GM + R diets by 1630% in comparison to the control diet
(889 107, 735 89 respectively vs control 1057 127 mg/kg)
and caffeic acid by 2153% (17.5 2.1, 10.3 1.3 vs control
22.1 2.6 mg/kg).
For biochemical measurements in rats, statistical analysis was
performed on the results obtained from samples taken at the
15th month time point, as this was the last sampling time when
most animals were still alive (in treated groups 90% males, 94% fe-
males, and 100% controls). OPLS-DA 2-class models were built be-
tween each treated group per sex and controls. Only models with
an explained variance R
2
(Y) P80%, and a cross-validated predic-
tive ability Q
2
(Y) P60%, were used for selection of the discrimi-
nant variables (Fig. 5A), when their regression coefcients were
signicant at 99% condence level. Thus, in treated females, kidney
failures appeared at the biochemical level (82% of the total dis-
rupted parameters). Ions (Na, Cl) or urea increased in urine.
Accordingly, the same ions decreased in serum (Fig. 5B) as did
the levels of P, K and Ca. Creatinine or clairance decreased in urine
for all treatment groups in comparison to female controls (Table 3).
In GM maize treated males (with or without R), 87% of discrimi-
nant variables were kidney related, but the disrupted proles were
less obvious because of advanced chronic nephropathies and
deaths. In summary, for all treatments and both sexes, 76% of the
discriminant variables versus controls were kidney related.
Moreover, in females (Table 3) the androgen/estrogen balance
in serum was modied by GM maize and R treatments (at least
95% condence level, Fig. 5B), and for male animals at the highest
R-treatment dose, levels of estrogens were more than doubled.
4. Discussion
This report describes the rst life-long rodent (rat) feeding
study investigating possible toxic effects rising from an R-tolerant
Fig. 4. Ultrastructure of hepatocytes in male rats from groups presenting the greatest degree of liver pathology. (1) Typical control rat hepatocyte (Bar 2 lm except in 4). (2)
Effects with Roundup at the lowest dose. Glycogen (G) is dispersed in the cytoplasm. L, lipid droplet; N, nucleus; R rough endoplasmic reticulum. (3) Hepatocytes of animal
fed GM maize (GMO) at 22% of total diet. Large lakes of glycogen occur in the cytoplasm. M, mitochondria. (4) Details of treatment effects with 22% dietary GMO (Bar 1 lm).
(a) Cluster of residual bodies (asterisks). (b) Mitochondria show many enlarged cristae (arrows).
G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx 7
Please cite this article in press as: Sralini, G.-E., et al. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modied maize. Food
Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005
GM maize (NK603) and a complete commercial formulation of R-
herbicide.
Our data show that, as is often the case for hormonal diseases,
most observed effects in this study were not proportional to the
dose of the treatment (GM maize with and without R application;
R alone), non-monotonic and with a threshold effect (Vandenberg
et al., 2012). Similar degrees of pathological symptoms were no-
ticed in this study to occur from the lowest to the highest doses
suggesting a threshold effect. This corresponds to levels likely to
arise from consumption or environmental exposure, such as either
11% GM maize in food, or 50 ng/L of glyphosate in R-formulation as
can be found in some contaminated drinking tap waters, and
which fall within authorized limits.
The lifespan of the control group of animals corresponded to the
mean rat lifespan, but as is frequently the case with most mam-
mals including humans (WHO, 2012), males on average died before
females, except for some female treatment groups. All treatments
in both sexes enhanced large tumor incidence by 23-fold in com-
Fig. 5. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) for biochemical data (females fed 33% GMO versus controls). (A) OPLS-DA regression coefcients for
predictive component, with jack-knifed condence intervals at 99% condence level, indicate discriminant parameters versus controls at month 15 (Abbreviations: U Urinary,
UEx Excreted in urine during 24 h, APPT Activated Partial Thromboplastin Time, MCV Mean Corpuscular Volume, PT Prothrombine Time, RBC Red Blood Cells, ALT ALanine
aminoTransferase, MCHC Mean Corpuscular Hemoglobin Concentration, A/G Albumin/Globulin ratio, WBC White Blood Cells, AST aspartate aminotransferase). (B) In this
case, detailed examples of signicant discriminant variables distribution between females fed 33% GMO (bold line) and controls (dotted line). On x axis: animals; on y axis:
serum or urine biochemical values for Na, Cl, estradiol, testosterone. Proles evidence kidney ion leakages and sex hormonal imbalance versus controls.
8 G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx
Please cite this article in press as: Sralini, G.-E., et al. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modied maize. Food
Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005
parison to our controls but also for the number of mammary tu-
mors in comparison to the same Harlan Sprague Dawley strain
(Brix et al., 2005), and overall around 3-fold in comparison to the
largest study with 1329 Sprague Dawley female rats (Chandra
et al., 1992). In our study the tumors also developed considerably
faster than the controls, even though the majority of tumors were
observed after 18 months. The rst large detectable tumors oc-
curred at 4 and 7 months into the study in males and females
respectively, underlining the inadequacy of the standard 90 day
feeding trials for evaluating GM crop and food toxicity (Sralini
et al., 2011).
Suffering inducing euthanasia and deaths corresponded mostly
in females to the development of large mammary tumors. These
appeared to be clearly related to the various treatments when
compared to the control groups. These tumors are generally known
to be mostly estrogen-dependent (Harvell et al., 2000). We ob-
served a strikingly marked induction of mammary tumors by R
alone, a major formulated pesticide, even at the very lowest dose
administered. R has been shown to disrupt aromatase which syn-
thesizes estrogens (Richard et al., 2005), but to also interfere with
estrogen and androgen receptors in cells (Gasnier et al., 2009). In
addition, R appears to be a sex endocrine disruptor in vivo, also
in males (Romano et al., 2010). Sex steroids are also modied in
treated rats. These hormone-dependent phenomena are conrmed
by enhanced pituitary dysfunction in treated females. An estrogen
modied feedback mechanismmay act at this level (Popovics et al.,
2011; Walf and Frye, 2010). The similar pathological proles pro-
voked by the GM maize containing R residues may thus be ex-
plained at least by R residues themselves, knowing that the
medium dose of the R treatment corresponds to acceptable levels
of this pesticide residues in GMOs.
Interestingly, in the groups of animals fed with the NK603 with-
out R application, similar effects with respect to enhanced tumor
incidence and mortality rates were observed. A possible explana-
tion for this nding is the production of specic compound(s) in
the GM feed that are either directly toxic and/or cause the inhibi-
tion of pathways that in turn generate chronic toxic effects. This is
despite the fact that the variety of GM maize used is this study was
judged by industry and regulators as being substantially equivalent
to the corresponding non-GM closest isogenic line. As the total
chemical composition of the GM maize cannot be measured in de-
tails, the use of substantial equivalence is insufcient to highlight
potential unknown toxins and therefore cannot replace long-term
animal feeding trials for GMOs. A cause of the effects of the effects
could be that the NK603 GM maize used in this study is engineered
to overexpress a modied version of the Agrobacterium tumefaciens
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Ham-
mond et al., 2004) allowing the R tolerance. The modied EPSPS
is not inhibited by glyphosate by contrast to the wild enzyme. This
enzyme is known to drive the rst step of aromatic amino acid bio-
synthesis in the plant shikimate pathway; in addition estrogenic
isoavones and their glycosides are also products of this pathway
(Duke et al., 2003). They were not disturbed in our study. By con-
trast, the levels of caffeic and ferulic acids in the GM diets, which
are also secondary metabolites from this pathway, but not always
measured in regulatory tests, are signicantly reduced. This may
lower their protective effects against carcinogenesis and even
mammalian tumors (Kuenzig et al., 1984; Baskaran et al., 2010).
Moreover, these phenolic acids and in particular ferulic acid may
modulate estrogen receptors or the estrogenic pathway in mam-
malian cells (Chang et al., 2006). This does not exclude the action
of other unknown metabolites. This explanation also corresponds
to the fact that the observed effects of NK603 and R are not addi-
tive and reached a threshold. This implies that both the NK603
maize and R may cause hormonal disturbances in the same
biochemical and physiological pathway.
As expected, mammary tumors in males occurred far less fre-
quently than in females. Death in male rats was mostly due to
the development of severe hepatorenal insufciencies, conrming
the rst signs of toxicity observed in 90 day feeding trials with
NK603 maize (Spiroux de Vendmois et al., 2009). In females, kid-
ney ion leakages were evidenced at the biochemical levels at
month 15, when severe nephropathies were evidenced in dead
male animals afterwards, at the anatomopathological level. Early
signs of toxicity at month 3 in kidney and liver were also observed
for 19 edible GM crops containing pesticide residues (Sralini et al.,
2011). As a matter of fact, only elderly male rats are sensitive to
chronic progressive nephropathies (Hard and Khan, 2004). The dis-
turbed kidney parameters may have been induced by the reduction
of phenolic acids in our study, since caffeic and ferulic acids are
benecial in the kidney as they prevent oxidative stress (Srinivasan
et al., 2005; U Rehman and Sultana, 2011). Accordingly, we previ-
ously demonstrated that plant extracts containing ferulic and caf-
feic acids were able to promote detoxication of embryonic kidney
cells after R contamination (Gasnier et al., 2011). It is thus possible
that NK603 consumption by reducing these compounds may well
provoke an early aging of kidney physiology in this study, like R
by oxidative stress.
Disturbances that we found to occur in the male liver are
characteristic of a chronic intoxication, conrmed by alterations
Table 3
Percentage variation of parameters indicating kidney failures of female animals.
Discriminant variables GMO 11% + R GMO 22% + R GMO 33% + R GMO 11% GMO 22% GMO 33% R (A) R (B) R (C)
Urinary decrease Clairance 4 11 20 20 20 19 20 24 40
Creatinine 5 32 37 19 37 36 43 23 1
Creatinine ex 5 11 19 18 17 21 21 22 39
Urinary increase Urea 12 18 15 15 12 1 0 13 32
Na 25 33 30 52 2 95 62 65 91
Na ex 24 50 68 50 24 125 108 51 7
Cl 14 35 28 46 5 101 67 56 94
Cl ex 20 63 70 51 31 138 121 48 13
Serum decrease Na 2 1 1 1 4 6 7 0 3
Cl 1 2 2 5 7 6 8 1 4
P 6 11 13 17 18 20 32 9 13
K 4 5 10 2 4 0 4 8 5
Ca 4 3 3 2 2 5 6 3 6
Gonads Estradiol 8 1 2 5 2 25 26 73 39
Testosterone 5 9 27 56 17 81 97 72 10
OPLS-DA was performed on 48 variables at month 15. Here we showed mean differences (%) of variables (discriminant at 99% condence level, in bold character) indicating
kidney parameters of female animals, together with sex hormones. Male kidney pathologies are already illustrated in Table 2.
G.-E. Sralini et al. / Food and Chemical Toxicology xxx (2012) xxxxxx 9
Please cite this article in press as: Sralini, G.-E., et al. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modied maize. Food
Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005
in biochemical liver and kidney function parameters. The observa-
tion that liver function in female animals is less affected may be
due to their physiology being better adapted to estrogen metabo-
lism. Furthermore, liver enzymes have been clearly demonstrated
as sex-specic in their expression patterns, including in a 90-day
rat feeding trial of NK603 maize (Spiroux de Vendmois et al.,
2009). However, in a long-term study, evidence of early liver aging
was observed in female mice fed with R-tolerant GM soy (Malates-
ta et al., 2008a). In the present investigation, deeper analysis at an
ultrastructural level revealed evidence of impediments in tran-
scription and other defects in cell nuclear structure that were com-
parable in both sexes, and dose-dependent in hepatocytes in all
treatments. This is consistent with the well-documented toxic ef-
fect of very low dilutions of R on apoptosis, mitochondrial function,
and cell membrane degradation inducing necrosis of hepatocytes,
and other cell lines (Benachour and Seralini, 2009; Benachour
et al., 2007; Gasnier et al., 2010; Peixoto, 2005).
The disruptions of at least the estrogen-related pathways and/
or enhancement of oxidative stress by all treatments need further
investigations. This can be addressed through the application of
transcriptomic, proteomic and metabolomic methods to analyze
the molecular proles of kidneys and livers, as well as the GM
NK603 maize (Jiao et al., 2010; Zhou et al., 2009; Zolla et al.,
2008). Other possible causes of observed pathogenic effects may
be due to disturbed gene expression resulting from the transgene
insertional, general mutagenic or metabolic effects (Latham et al.,
2006; Wilson et al., 2006) as has been shown for MON810 GM
maize (Rosati et al., 2008). A consequent disruption of general
metabolism in the GMO cannot be excluded, which could lead,
for example, to the production of other potentially active com-
pounds such as miRNAs (Zhang et al., 2012) or leukotoxin diols
(Markaverich et al., 2005).
In conclusion, it was previously known that glyphosate con-
sumption in water above authorized limits may provoke hepatic
and kidney failures (EPA). The results of the study presented here
clearly demonstrate that lower levels of complete agricultural gly-
phosate herbicide formulations, at concentrations well below of-
cially set safety limits, induce severe hormone-dependent
mammary, hepatic and kidney disturbances. Similarly, disruption
of biosynthetic pathways that may result from overexpression of
the EPSPS transgene in the GM NK603 maize can give rise to com-
parable pathologies that may be linked to abnormal or unbalanced
phenolic acids metabolites, or related compounds. Other muta-
genic and metabolic effects of the edible GMO cannot be excluded.
This will be the subject of future studies, including transgene and
glyphosate presence in rat tissues. Reproductive and multigenera-
tional studies will also provide novel insights into these problems.
This study represents the rst detailed documentation of long-
term deleterious effects arising from the consumption of a GM R-
tolerant maize and of R, the most used herbicide worldwide.
Altogether, the signicant biochemical disturbances and physi-
ological failures documented in this work conrm the pathological
effects of these GMO and R treatments in both sexes, with different
amplitudes. We propose that agricultural edible GMOs and formu-
lated pesticides must be evaluated very carefully by long term
studies to measure their potential toxic effects.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgments
We thank Michael Antoniou for English assistance and con-
structive comments on the manuscript, as well as Herrade Hem-
merdinger for proofreading. We gratefully acknowledge the
Association CERES, the Foundation Charles Leopold Mayer pour
le Progrs de lHomme, the French Ministry of Research, and CRI-
IGEN for their major support.
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Please cite this article in press as: Sralini, G.-E., et al. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modied maize. Food
Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005