Emilie Clair, Robin Mesnage, Steeve Gress, Nicolas Defarge, Manuela Malatesta, Didier Hennequin and Joël Spiroux de Vendômois
The health effects of a Roundup-tolerant NK603 genetically modified (GM) maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone (from 0.1 ppb of the full pesticide containing glyphosate and adjuvants) in drinking water, were evaluated for 2 years in rats. This study constitutes a follow-up investigation of a 90-day feeding study conducted by Monsanto in order to obtain commercial release of this GMO, employing the same rat strain and analyzing biochemical parameters on the same number of animals per group as our investigation. Our research represents the first chronic study on these substances, in which all observations including tumors are reported chronologically. Thus, it was not designed as a carcinogenicity study. We report the major findings with 34 organs observed and 56 parameters analyzed at 11 time points for most organs.
Biochemical analyses confirmed very significant chronic kidney deficiencies, for all treatments and both sexes; 76% of the altered parameters were kidney-related. In treated males, liver congestions and necrosis were 2.5 to 5.5 times higher. Marked and severe nephropathies were also generally 1.3 to 2.3 times greater. In females, all treatment groups showed a two- to threefold increase in mortality, and deaths were earlier. This difference was also evident in three male groups fed with GM maize. All results were hormone- and sex-dependent, and the pathological profiles were comparable. Females developed large mammary tumors more frequently and before controls; the pituitary was the second most disabled organ; the sex hormonal balance was modified by consumption of GM maize and Roundup treatments. Males presented up to four times more large palpable tumors starting 600 days earlier than in the control group, in which only one tumor was noted. These results may be explained by not only the non-linear endocrine-disrupting effects of Roundup but also by the overexpression of the EPSPS transgene or other mutational effects in the GM maize and their metabolic consequences.
Our findings imply that long-term (2 year) feeding trials need to be conducted to thoroughly evaluate the safety of GM foods and pesticides in their full commercial formulations.
Genetically modifiedGMORoundupNK603RatGlyphosate-based herbicidesEndocrine disruption
Empirical natural and social sciences produce knowledge (in German: Wissenschaften schaffen Wissen) which should describe and explain past and present phenomena and estimate their future development. To this end quantitative methods are used. Progress in science needs controversial debates aiming at the best methods as basis for objective, reliable and valid results approximating what could be the truth. Such methodological competition is the energy needed for scientific progress. In this sense, ESEU aims to enable rational discussions dealing with the article from G.-E. Séralini et al. (Food Chem. Toxicol. 2012, 50:4221–4231) by re-publishing it. By doing so, any kind of appraisal of the paper’s content should not be connoted. The only aim is to enable scientific transparency and, based on this, a discussion which does not hide but aims to focus methodological controversies. -Winfried Schröder, Editor of the Thematic Series “Implications for GMO-cultivation and monitoring” in Environmental Sciences Europe.
There is an ongoing international debate as to the necessary length of mammalian toxicity studies, including metabolic analyses, in relation to the consumption of genetically modified (GM) plants . Currently, no regulatory authority requires mandatory chronic animal feeding studies to be performed for edible genetically modified organisms (GMOs), or even short-term studies with blood analyses for the full commercial formulations of pesticides as sold and used, but only for the declared active principle alone. However, several 90-day rat feeding trials have been conducted by the agricultural biotechnology industry. These investigations mostly concern GM soy and maize that are engineered either to be herbicide-tolerant (to Roundup (R) in 80% of cases), or to produce a modified Bt toxin insecticide, or both. As a result, these GM crops contain new pesticide residues for which new maximum residue levels (MRL) have been established in some countries.
Though the petitioners conclude in general that no major physiological changes is attributable to the consumption of the GMO in subchronic toxicity studies [2, 3, 4, 5], significant disturbances have been found and may be interpreted differently [6,7]. A detailed analysis of the data in the subchronic toxicity studies [2, 3, 4, 5] has revealed statistically significant alterations in kidney and liver function that may constitute signs of the early onset of chronic toxicity. This may be explained at least in part by pesticide residues in the GM feed [6,7]. Indeed, it has been demonstrated that R concentrations in the range of 103 times below the MRL can induce endocrine disturbances in human cells  and toxic effects thereafter . This may explain toxic effects seen in experiments in rats in vivo  as well as in farm animals . 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 [12, 13, 14]. Furthermore, this toxic effect was reproduced by the application of R herbicide directly to hepatocytes in culture .
More recently, 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 investigation because of metabolic modifications . However, in contrast with the study we report here, none of these previous investigations have included a detailed follow-up of the animals, including multiple (up to 11) blood and urine sampling over 2 years, and none has investigated either the GM NK603 R-tolerant maize or Roundup.
Furthermore, evaluation of long-term toxicity of herbicides is generally performed on mammalian physiology employing only their active principle, rather than the complete formulations as used in agriculture. This was the case for glyphosate (G) , the declared active chemical constituent of R. It is important to note that G is only able to efficiently penetrate target plant organisms with the help of adjuvants present in the various commercially used R formulations . Even if G has shown to interact directly with the active site of aromatase at high levels , at low contaminating levels, adjuvants may be better candidates than G to explain the toxicity or endocrine disruptive side effects of R on human cells [8,20] and also in vivo for acute toxicity . In this regard, it is noteworthy that the far greater toxicity of full agricultural formulations compared to declared supposed active principles alone has recently been demonstrated also for six other major pesticides tested in vitro . When G residues are found in tap water, food, or feed, they arise from the total herbicide formulation although little data is available as to the levels of the R adjuvants in either the environment or food chain. Indeed, adjuvants are rarely monitored in the environment, but some widely used adjuvants (surfactants) such as nonylphenol ethoxylates, another ethoxylated surfactant like POEA present in R, are widely found in rivers in England and are linked with disruption of wildlife sexual reproduction . Adjuvants are found in groundwater . The half-life of POEA (21 to 42 days) is even longer than for G (7 to 14 days) in aquatic environments . As a result, the necessity of studying the potential toxic effects of total chemical mixtures rather than single components has been strongly emphasized [26, 27, 28]. On this basis, the regular measurement of only G or other supposed active ingredients of pesticides in the environment constitute at best markers of full formulation residues. Thus, in the study of health effects, exposure to the diluted whole formulation may be more representative of environmental pollution than exposure to G alone.
With a view to address this lack of information, we performed a 2-year detailed rat feeding study. Our study was designed as a chronic toxicity study and as a direct follow-up to a previous investigation on the same NK603 GM maize conducted by the developer company, Monsanto . A detailed critical analysis of the raw data of this subchronic 90-day rat feeding study revealed statistically significant differences in multiple organ function parameters, especially pertaining to the liver and kidneys, between the GM and non-GM maize-fed group [3,7]. However, Monsanto’s authors dismissed the findings as not ‘biologically meaningful’ , as was also the case with another GM corn . The European Food Safety Authority (EFSA) accepted Monsanto’s interpretation on NK603 maize , like in all other cases.
Our study is the first and to date the only attempt to follow up Monsanto’s investigation and to determine whether the differences found in the NK603 GM maize-fed rats, especially with respect to liver and kidney function, were not biologically meaningful, as claimed, or whether they developed into serious diseases over an extended period of time.
The Monsanto authors adapted Guideline 408 of the Organization for Economic Co-operation and Development (OECD) for their experimental design . Our study design was based on that of the Monsanto investigation in order to make the two experiments comparable, but we extended the period of observation from Monsanto’s 90 days to 2 years. We also used three doses of GMOs (instead of Monsanto’s two) and Roundup to determine treatment dose response, including any possible non-linear as well as linear effects. This allowed us to follow in detail the potential health effects and their possible origins due to the direct or indirect consequences of the genetic modification itself in the NK603 GM maize, or due to the R herbicide formulation used on the GM maize (and not G alone), or both. Because of recent reviews on GM foods indicating no specific risk of cancer [2,16], but indicating signs of hepatorenal dysfunction within 3 months [1,7], we had no reason to adopt a carcinogenesis protocol using 50 rats per group. However, we prolonged to 2 years the biochemical and hematological measurements and measurements of disease status, as allowed, for example, in OECD protocols 453 (combined chronic toxicity and carcinogenicity) and 452 (chronic toxicity). Both OECD 452 and 453 specify 20 rats per sex per group but require only 50% (ten per sex per group, the same number that we used in total) to be analyzed for biochemical and hematological parameters. Thus, these protocols yield data from the same number of rats as our experiment. This remains the highest number of rats regularly measured in a standard GM diet study, as well as for a full formulated pesticide at very low environmentally relevant levels.
We used the Sprague-Dawley strain of rat, as recommended for chronic toxicology tests by the National Toxicology Program in the USA , and as used by Monsanto in its 90-day study . This choice is also consistent with the recommendation of the OECD that for a chronic toxicity test, rats of the same strain should be used as in studies on the same substance but of shorter duration . We then also tested for the first time three doses (rather than the two usually employed in 90-day protocols) of the R-tolerant NK603 GM maize alone, the GM maize treated with R, and R alone at very low environmentally relevant doses, starting below the range of levels permitted by regulatory authorities in drinking water and in GM feed.
Overall, our study is the first in-depth life-long toxicology study on the full commercial Roundup formulation and NK603 GM maize, with observations on 34 organs and measurement of 56 parameters analyzed at 11 time points for most organs, and utilizing 3 doses. We report here the major toxicological findings on multiple organ systems. As there was no evidence in the literature on GM food safety evaluation to indicate anything to the contrary, this initial investigation was designed as a full chronic toxicity and not a carcinogenicity study. Thus, we monitored in details chronologically all behavioral and anatomical abnormalities including tumors. A full carcinogenicity study, which usually focuses only on observing incidence and type of cancers (not always all tumors), would be a rational follow-up investigation to a chronic toxicity study in which there is a serious suspicion of carcinogenicity. Such indications had not been previously reported for GM foods.
Our findings show that the differences in multiple organ functional parameters seen from the consumption of NK603 GM maize for 90 days [3,7] escalated over 2 years into severe organ damage in all types of test diets. This included the lowest dose of R administered (0.1 ppb, 50 ng/L G equivalent) of R formulation administered, which is well below permitted MRLs in both the USA (0.7 mg/L)  and European Union (100 ng/L) . Surprisingly, there was also a clear trend in increased tumor incidence, especially mammary tumors in female animals, in a number of the treatment groups. Our data highlight the inadequacy of 90-day feeding studies and the need to conduct long-term (2 years) investigations to evaluate the life-long impact of GM food consumption and exposure to complete pesticide formulations.
2.1 Biochemical analyses of the maize feed
Standard biochemical compositional analysis revealed no particular differences between the different maize types and diets, the GM and non-GM maize being classified as substantially equivalent, except for transgene DNA quantification. For example, there was no difference in total isoflavones. In addition, we also assayed for other specific compounds, which are not always requested for establishing substantial equivalence. This analysis revealed a consistent and statistically significant (p < 0.01) decrease in certain phenolic acids in treatment diets, namely ferulic and caffeic acids. Ferulic acid was decreased in both GM maize and GM maize + R diets by 16% to 30% in comparison to the control diet (889 ± 107, 735 ± 89, respectively, vs. control 1,057 ± 127 mg/kg) and caffeic acid in the same groups by 21% to 53% (17.5 ± 2.1, 10.3 ± 1.3 vs. control 22.1 ± 2.6 mg/kg).
2.2 Anatomopathological observations and liver parameters
All rats were carefully monitored during the experiment for behavior, appearance, palpable tumors, and infections. At least ten organs per animal were weighed and up to 34 analyzed postmortem, at the macroscopic and/or microscopic levels (Table 1). Due to the large quantity of data collected, it cannot all be shown in one report, but we present here the most important findings. There was no rejection by the animals of the diet with or without GM maize, nor any major difference in body weight (data not shown).
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|
|Animals measured/group/sex||10/10 SD rats (200 rats measured)||10/20 SD rats (200 rats measured/total 400)||At least 10 rodents|
|Duration in months||24 (chronic)||3 (subchronic, 13 weeks)||3|
|Doses by treatment||3||2||At least 3|
|Treatments + controls||GMO NK603, GMO NK603 + Roundup, Roundup, and closest isogenic maize||GMO NK603 + Roundup, closest isogenic maize, and 6 other maize lines non substantially equivalent||GMOs or Chemicals (in standard diet or water)|
|Animals by cage (same sex)||1 to 2||1||1 or more|
|Monitoring/week||2||1||1 or more|
|Organs and tissues studied||For high dose and controls|
|Organs weighted||10||7||At least 8|
|Histology/animal||34||17/36||At least 30|
|Feed and water consumptions||Measured||For feed only||At least feed|
|Behavioral studies (times)||2||1 (no protocol given)||1|
|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|
|Number of blood samples/animal||11, each month (0 to 3) then every 3 months||2, weeks 4 and 13||1, at the end|
|Urine parameters studied||16||18||7 if performed|
|Number of urine samples||11||2||Optional, last week|
|Liver tissue parameters||6||0||0|
|Roundup residues in tissues||Studied||Not studied||Not mandatory|
|Microbiology in feces or urine||Yes||Yes||No|
|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.
The most affected organs in males were the liver, hepatodigestive tract, and kidneys (Table 2; Figure 1A,B,C,D,E,F,G,H,I). Liver abnormalities such as hepatic congestions and macroscopic and microscopic necrotic foci were 2.5 to 5.5 times more frequent in all treatments than in control groups, where only two rats out of ten were affected with one abnormality each. For instance, there were 5 abnormalities in total in the GMO 11% group (2.5 times higher than controls) and 11 in the GMO 22% group (5.5 times greater). In addition, by the end of the experiment, Gamma GT hepatic activity was increased, particularly in the GMO + R groups (up to 5.4 times higher), this probably being reflective of liver dysfunction. Furthermore, cytochrome P450 activity generally increased in the presence of R (either in drinking water or in the GM maize-containing diet) according to the dose and up to 5.7 times greater at the highest dose.
Summary of the most frequent anatomical pathologies observed
|Organs and associated pathologies||Controls||GMO 11%||GMO 22%||GMO 33%||R (A)||R (B)||R (C)||GMO 11% + R||GMO 22% + R||GMO 33% + R|
|Kidneys, CPN||3 (3)||4 (4)||5 (5)||7 (7)||6 (6)||5 (5)||3 (3)||5 (5)||4 (4)||4 (4)|
|Liver||2 (2)||5 (4)||11 (7)||8 (6)||11 (5)||9 (7)||6 (5)||5 (4)||7 (4)||6 (5)|
|Hepatodigestive tract||6 (5)||10 (6)||13 (7)||9 (6)||23 (9)||16 (8)||9 (5)||9 (6)||13 (6)||11 (7)|
|Pituitary||9 (6)||23 (9)||20 (8)||8 (5)||22 (8)||16 (7)||13 (7)||19 (9)||9 (4)||19 (7)|
|Mammary glands||10 (5)||22 (8)||10 (7)||16 (8)||26(10)||20(10)||18 (9)||17 (8)||16 (8)||15 (9)|
|Mammary tumors||8 (5)||15 (7)||10 (7)||15 (8)||20 (9)||16(10)||12 (9)||10 (6)||11 (7)||13 (9)|
After the number of pathological abnormalities, the number of rats affected out of the initial ten is indicated in parentheses. Only marked or severe chronic progressive nephropathies (CPN) are listed in male animals, excluding two nephroblastomas in groups consuming GMO 11% and GMO 22% + Roundup. Hepatodigestive pathological signs in males concern the liver, stomach, and small intestine (duodenum, ileum, or jejunum). Pathological signs in liver are mostly congestions, macroscopic spots, and microscopic necrotic foci. In females, pituitary dysfunctions include adenomas, hyperplasias, and hypertrophies. Mammary fibroadenomas and adenocarcinomas are the major tumors detected; galactoceles and hyperplasias with atypia were also found and added to the pathological signs in mammary glands.
Anatomopathological observations in rats fed GMO treated or not by Roundup and effects of Roundup alone. Macroscopic (A to D) and microscopic (A’ and C’) photographs show male left kidneys and livers (E to I) and female pituitaries (J to M), in accordance to Table 2. The number of each animal and its treatment is specified. Macroscopic pale spots (I) and microscopic necrotic foci in liver (G clear-cell focus, H basophilic focus with atypia), and marked or severe chronic progressive nephropathies, are illustrated. In females, pituitary adenomas (K to M) are shown and compared to control (J, rat number and C for control). Apostrophes after letters indicate organs from the same rat.
Transmission electron microscopic observations of liver samples confirmed changes for all treated groups in relation to glycogen dispersion or appearance in lakes, increase of residual bodies and enlargement of cristae in mitochondria (Figure 2, panels 2 to 4). The GM maize-fed groups either with or without R application showed a higher heterochromatin content and decreased nucleolar dense fibrillar components, implying a reduced level of mRNA and rRNA transcription. In the GMO + R group (at the highest dose), the smooth endoplasmic reticulum was drastically increased and nucleoli decreased in size, becoming more compact. In the R alone treatment groups, similar trends were observed, with a partial resumption of nucleolar activity at the highest dose.
Ultrastructure of hepatocytes in male rats from groups presenting the greatest degree of liver pathology. (1) Typical control rat hepatocyte (bar 2 μm 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) Details of treatment effects with 22% dietary GMO (bar 1 μm). a, cluster of residual bodies (asterisks); b, mitochondria show many enlarged cristae (arrows). (4) Hepatocytes of animal fed GM maize (GMO) at 22% of total diet. Large lakes of glycogen occur in the cytoplasm. M, mitochondria.
Degenerating kidneys with turgid inflammatory areas demonstrated the increased incidence of marked and severe chronic progressive nephropathies, which were up to two fold higher in the 33% GM maize or lowest dose R treatment groups (Table 2; Figure 1, first line).
2.3 Biochemical analyses of blood and urine samples
Biochemical measurements of blood and urine were focused on 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% females, and 100% controls). Statistical analysis of results employed OPLS-DA 2-class models built between each treated group per sex and controls. Only models with an explained variance R2(Y) ≥ 80%, and a cross-validated predictive ability Q2(Y) ≥ 60%, were used for selection of the discriminant variables (Figure 3), when their regression coefficients were significant at a 99% confidence level. Thus, in treated females, kidney failures appeared at the biochemical level (82% of the total disrupted parameters). Levels of Na and Cl or urea increased in urine with a concomitant decrease of the same ions in serum, as did the levels of P, K, and Ca. Creatinine and creatinine clearance 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 discriminant variables were kidney-related, but the disrupted profiles 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.
Orthogonal partial least squares-discriminant analysis (OPLS-DA) for biochemical data (females fed 33% GMO versus controls). (A) First, detailed examples of significant 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. (B) Wider view of OPLS-DA regression coefficients for predictive component, with jack-knifed confidence intervals at 99% confidence level, indicating discriminant parameters versus controls at month 15. 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. Profiles evidence kidney ion leakages and sex hormonal imbalance versus controls.
Percentage variation of parameters indicating kidney failures of female animals
|Discriminant variables||GMO 11%||GMO 22%||GMO 33%||GMO 11% + R||GMO 22% + R||GMO 33% + R||R (A)||R (B)||R (C)|
|Serum decrease or increase||Na||−1||−4a||−6a||2||1||1||−7||0||−3|
OPLS-DA was performed on 48 variables at month 15. Here, we show mean differences (%) of variables (adiscriminant at 99% confidence level) indicating kidney parameters of female animals, together with sex hormones. Male kidney pathologies are already illustrated in Figure 1.
Furthermore, in females (Table 3), the androgen/estrogen balance in serum was modified by GM maize and R treatments (at least 95% confidence level, Figure 3). For male animals at the highest R treatment dose, levels of estrogens were more than doubled.
2.4 Tumor incidence
Tumors are reported in line with the requirements of OECD chronic toxicity protocols 452 and 453, which require all ‘lesions’ (which by definition include tumors) to be reported. These findings are summarized in Figure 4. The results are presented in the form of real-time cumulative curves (each step corresponds to an additional tumor in the group). Only the growing largest palpable growths (above a diameter of 17.5 mm in females and 20 mm in males) are presented (for example, see Figure 5A,B,C). These were found to be in 95% of cases non-regressive tumors (Figure 5D,E,F,G,H,I,J) and were not infectious nodules. These arose from time to time; then, most often disappeared and were not different from controls after bacterial analyses. The real tumors were recorded independently of their grade, but dependent on their morbidity, since non-cancerous tumors can be more lethal than those of cancerous nature, due to internal hemorrhaging or compression and obstruction of function of vital organs, or toxins or hormone secretions. These tumors progressively increased in size and number, but not proportionally to the treatment dose, over the course of the experiment (Figure 4). As in the case of rates of mortality (Figure 6), this suggests that a threshold in effect was reached at the lower doses. Tumor numbers were rarely equal but almost always more than in controls for all treated groups, often with a two- to threefold increase for both sexes. Tumors began to reach a large size on average 94 days before controls in treated females and up to 600 days earlier in two male groups fed with GM maize (11 and 22% with or without R).
Largest non-regressive tumors in rats fed GMO treated or not by 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%, and 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 administered in drinking water at three increasing doses, same symbols, environmental (A), MRL in some agricultural GMOs (B), and half of minimal agricultural levels (C), see ‘Methods’). 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 large tumors; white, small internal tumors; grey, metastases.