Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize

Long term toxicity of a Roundup herbicide a, S x Espla and Received 11 April 2012 Accepted 2 August 2012 Available online xxxx with or without Roundup, and Roundup alone (from 0.1 ppb in water), were studied 2 years in rats. In de Vendômois et al., 2009). Indeed, it has been demonstrated that R concentrations in the range of 103 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). Abbreviations: GM, genetically modified; R, Roundup; MRL, maximal residual levels; GMO, genetically modified 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. Food and Chemical Toxicology xxx (2012) xxx–xxx Contents lists available at Food and Chemi journal homepage: www.elsev E-mail address: criigen@unicaen.fr (G.-E. Séralini). 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 modified (GM) plants including regular meta- bolic analyses (Séralini 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 modified 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 modified organism (GMO) subchronic toxic- ity studies (Domingo and Giné Bordonaba, 2011; Hammond et al., 2004, 2006a,b), significant disturbances have been found and may be interpreted differently (Séralini et al., 2009; Spiroux de Vendômois 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 (Séralini et al., 2007; Spiroux Keywords: GMO Roundup NK603 Rat Glyphosate-based herbicides Endocrine disrupting effects 0278-6915/$ - see front matter � 2012 Elsevier Ltd. A http://dx.doi.org/10.1016/j.fct.2012.08.005 Please cite this article in press as: Séralini, G.-E., Chem. Toxicol. (2012), http://dx.doi.org/10.1016 females, all treated groups died 2–3 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- files 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- ified by GMO and Roundup treatments. In treated males, liver congestions and necrosis were 2.5–5.5 times higher. This pathology was confirmed by optic and transmission electron microscopy. Marked and severe kidney nephropathies were also generally 1.3–2.3 greater. Males presented 4 times more large palpable tumors than controls which occurred up to 600 days earlier. Biochemistry data confirmed very significant kidney chronic deficiencies; 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. a r t i c l e i n f o Article history: a b s t r a c t The health effects of a Roundup-tolerant genetically modified maize (from 11% in the diet), cultivated genetically modified maize Gilles-Eric Séralini a,⇑, Emilie Clair a, Robin Mesnage Manuela Malatesta b, Didier Hennequin c, Joël Spirou aUniversity of Caen, Institute of Biology, CRIIGEN and Risk Pole, MRSH-CNRS, EA 2608, bUniversity of Verona, Department of Neurological, Neuropsychological, Morphological cUniversity of Caen, UR ABTE, EA 4651, Bd Maréchal Juin, Caen Cedex 14032, France ll rights reserved. et al. Long term toxicity of a Ro /j.fct.2012.08.005 and a Roundup-tolerant teeve Gress a, Nicolas Defarge a, de Vendômois a nade de la Paix, Caen Cedex 14032, France Motor Sciences, Verona 37134, Italy SciVerse ScienceDirect cal Toxicology ier .com/locate / foodchemtox undup herbicide and a Roundup-tolerant genetically modified maize. Food 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 modifications (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 efficiently 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 field 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. views on GMOs (Domingo and Giné Bordonaba, 2011; Snell et al., 2011) we had no reason to settle at first 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 first time 3 doses (rather than two in the usual 90 day long protocols) of the R-tolerant NK603 GM maize alone, the GMmaize 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 field studies of plant species, no specific permits were required, nor for the locations/activities. The maize grown (MON-00603-6 com- monly named NK603) was authorized for unconfined release into the environment and use as a livestock feed by the Canadian Food Inspection Agency (Decision Doc- ument 2002-35). We confirm that the location is not privately-owned or protected in any way and that the field studies did not involve endangered or protected spe- cies. The GM maize was authorized for import into the European Union (CE 258/97 treated with R. Corns were harvested when the moisture content was less than 30% ry t am MO aiz ubs (su 0/2 00) or feed only At least feed 7/3 o (no , w 1 (2 o 8 es ot ot 2 G.-E. Séralini et al. / Food and Chemical Toxicology xxx (2012) xxx–xxx 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 modification it- self in GMOs, or due to the formulated herbicide mixture used on GMOs (and not glyphosate alone), or both. Because of recent re- Table 1 Protocol used and comparison to existing assessment, and to non-mandatory regulato Treatments and analyses In this work H Treatments + controls GMO NK603, GMO NK603 + Roundup, Roundup, and closest isogenic maize G m s Doses by treatment 3 2 Duration in months 24 (chronic) 3 Animals measured/group/sex 10/10 SD rats (200 rats measured) 1 4 Animals by cage (same sex) 1–2 1 Monitoring/week 2 1 Feed and water consumptions Measured F Organs and tissues studied Histology/animal 34 1 Organs weighted 10 7 Electronic microscopy Yes N Behavioral studies (times) 2 1 Ophtalmology (times) 2 0 Number of blood samples/ animal 11, each month (0–3) then every 3 months 2 Blood parameters 31 (11 times for most) 3 Plasma sex steroids Testosterone, estradiol N Liver tissue parameters 6 0 Number of urine samples 11 2 Urine parameters studied 16 1 Microbiology in feces or urine Yes Y Roundup residues in tissues Studied N Transgene in tissues Studied N The protocol used in this work was compared to the regulatory assessment of NK603 mai tests for GMOs, or mandatory for chemicals (OECD 408). Most relevant results are show Please cite this article in press as: Séralini, G.-E., et al. Long term toxicity of a Ro Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005 For high dose and controls 6 At least 30 At least 8 No protocol given) 1 2 eeks 4 and 13 1, at the end times) At least 25 (at least 2 times) No, except if endocrine effects suspected 0 Optional, last week 7 if performed No studied Not mandatory studied Not studied and were dried at a temperature below 30 �C. From these three cultivations of ests. mond et al., 2004 Regulatory tests NK603 + Roundup, closest isogenic e, and six other maize lines non tantially equivalent GMOs or chemicals (in standard diet or water) At least 3 bchronic: 13 weeks) 3 0 SD rats (200 rats measured/total At least 10 rodents 1 or more 1 or more 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 sufficient distance to avoid cross-contamination. The genetic nature, as well as the purity of the GM seeds and harvested material, was confirmed by qPCR anal- ysis of DNA samples. One field of NK603 was treated with R at 3 L ha�1 (Weather- MAX, 540 g/L of glyphosate, EPA Reg. 524-537), and another field of NK603 was not ze by the company (Hammond et al., 2004), and to non mandatory regulatory in vivo n in this paper. undup herbicide and a Roundup-tolerant genetically modified maize. Food Virgin albino Sprague-Dawley rats at 5 weeks of age were obtained from Harlan Animals were sacrificed during the course of the study only if necessary because spontaneously, while up to 50% males and 70% females died in mica 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 isoflurane 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, Peyer’s patches, pituitary, salivary glands, sci- atic nerve, skin, spinal cord, stomach, thyroid and trachea. The first 14 organs (at least 10 per animal depending on the sex, Table 1) were weighted, plus any tumor that arose. The first 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 paraffin-embedding, slides and HES histological staining. For transmission electron microscopy, kidneys, livers and tu- 2.4. Biochemical analyses Blood samples were collected from the tail vein of each rat under short isoflu- 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 first 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 quantified 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 (Gannat, France). All animals were kept in polycarbonate cages (820 cm2, 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 (45–65%) 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 final 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. 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 confirmed 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 isoflavones 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 G.-E. Séralini et al. / Food and Che mors were cut into 1 mm3 fragments. Samples were fixed 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). Please cite this article in press as: Séralini, G.-E., et al. Long term toxicity of a Ro Chem. Toxicol. (2012), http://dx.doi.org/10.1016/j.fct.2012.08.005 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 first two male rats that died in both GM treated groups had to be euthanized due to kidney Wilm’s tumors that were over 25% of body weight. This was at approximately a year before the first control animal died. The first female death occurred in the 22% GM maize feeding group and resulted from a mammary fibroadenoma 246 days be- fore the first 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 2–3 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- 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-