I n t e r n a t i o n a l J o u r n a l o f B i o l o g i c a l S c i e n c e s
2009; 5(7):706-726
© Ivyspring International Publisher. All rights reserved Research Paper
A Comparison of the Effects of Three GM Corn Varieties on Mammalian Health
Joël Spiroux de Vendômois1, François Roullier1, Dominique Cellier1,2 and Gilles-Eric Séralini1,3
1.CRIIGEN, 40 rue Monceau, 75008 Paris, France
2.University of Rouen LITIS EA 4108, 76821 Mont-Saint-Aignan, France
3.University of Caen, Institute of Biology, Risk Pole CNRS, EA 2608, 14032 Caen, France
Correspondence to: Prof. Gilles-Eric Séralini, Institute of Biology, EA 2608, University of Caen, Esplanade de la Paix, 14032 Caen Cedex, France. Phone +33 2 31 56 56 84; Fax +33 2 56 53 20; Email: criigen@unicaen.fr.
Received: 2009.07.23; Accepted: 2009.11.17; Published: 2009.12.10
Abstract
We present for the first time a comparative analysis of blood and organ system data from
trials with rats fed three main commercialized genetically modified (GM) maize (NK 603,
MON 810, MON 863), which are present in food and feed in the world. NK 603 has been
modified to be tolerant to the broad spectrum herbicide Roundup and thus contains resi-
dues of this formulation. MON 810 and MON 863 are engineered to synthesize two differ-
ent Bt toxins used as insecticides. Approximately 60 different biochemical parameters were
classified per organ and measured in serum and urine after 5 and 14 weeks of feeding. GM
maize-fed rats were compared first to their respective isogenic or parental non-GM
equivalent control groups. This was followed by comparison to six reference groups, which
had consumed various other non-GM maize varieties. We applied nonparametric methods,
including multiple pairwise comparisons with a False Discovery Rate approach. Principal
Component Analysis allowed the investigation of scattering of different factors (sex, weeks
of feeding, diet, dose and group). Our analysis clearly reveals for the 3 GMOs new side ef-
fects linked with GM maize consumption, which were sex- and often dose-dependent. Ef-
fects were mostly associated with the kidney and liver, the dietary detoxifying organs, al-
though different between the 3 GMOs. Other effects were also noticed in the heart, adrenal
glands, spleen and haematopoietic system. We conclude that these data highlight signs of
hepatorenal toxicity, possibly due to the new pesticides specific to each GM corn. In addi-
tion, unintended direct or indirect metabolic consequences of the genetic modification
cannot be excluded.
Key words: GMO, toxicity, GM corn, rat, NK 603, MON 810, MON 863
1. Introduction
There is a world-wide debate concerning the safety and regulatory approval process of genetically modified (GM) crops and foods [1, 2]. In order to sci-entifically address this issue, it is necessary to have access to toxicological tests, preferably on mammals, performed over the longest time-scales involving de-tailed blood and organ system analyses. Furthermore, these tests should, if possible, be in accordance with OECD guidelines. Unfortunately, this has been a challenge since usually these are regulatory tests performed confidentially by industry prior to com-mercialization of their GM crops, pesticides, drugs or chemicals. As a result, it is more instructive to inves-tigate the available data that allows comparisons of several GMOs consumptions on health effects. This will allow the most appropriate statistical analyses to
comparisonsbe performed in order to avoid possible false positive as well as false negative results. The physiological criteria used to either accept or reject any GM signifi-cant effect as relevant should be made clear. Here we discuss sex-related, temporal, linear and non-linear dose effects which are often involved in the estab-lishment of chronic and endocrine diseases.
We investigated three different GM corn namely NK 603, MON 810 and MON 863, which were fed to
rats for 90 days. The raw data have been obtained by European governments and made publically avail-able for scrutiny and counter-evaluation. These stud-ies constitute a model to investigate possible sub-chronic toxicological effects of these GM cereals in mammals and humans. These are the longest in vivo tests performed with mammals consuming these GMOs. The animals were monitored for numerous blood and organ parameters. One corn (NK 603) has been genetically engineered to tolerate the broad spectrum herbicide Roundup and thus contains resi-dues of this formulation. The two other types of GM maize studied produce two different new insecticides namely modified versions of Cry1Ab (MON 810) and Cry3Bb1 (MON 863) Bacillus thuringiensis-derived proteins. Therefore, all these three GM maize contain novel pesticide residues that will be present in food and feed. As a result, the potential effects on physio-logical parameters, due either to the recognized mutagenic effects of the GM transformation process or to the presence of the above mentioned novel pes-ticides within these plants can be evaluated in animal feeding studies.
2. Materials and Methods
2.1. Experimental design
The three animal feeding studies were con-ducted in two different laboratories and at two dif-ferent d
ates; at Monsanto (Missouri, USA) for NK 603 and MON 810 (June 7, 2000) and at Covance Labora-tories Inc. (Virginia, USA) for MON 863 (March 14, 2001) on behalf of Monsanto. The young adult male and female rats, approximately 4-6 week-old, were of the Sprague-Dawley albino strain Crl:CD(SD)IGS BR®, (obtained from Charles River Laboratories Inc., NY, USA). The animals (400 per GMO; 200 for each sex) were randomized for similar body weight dis-tribution. In fact, there were only two treated groups for each sex (20 animals each consuming specific GM maize feed). Only 10 rats were measured per group for blood and urine parameters and served as the ba-sis for the major statistical analyses conducted. In ad-dition, the investigators claimed that OECD guide-lines and standards were followed. For each type of GM maize, only two feeding doses were tested per sex. This consisted of either 11 or 33% GM maize in an otherwise equivalent equilibrated diet; that is when the diet contained only 11% GM maize, the dif-ference was made up by adding 22% non-GM maize (varieties not indicated). There were also two com-parative control groups fed diets containing similar quantities of the closest isogenic or parental maize variety. Furthermore, groups of animals were also fed with diets containing one of six other normal (non-GM) reference maize lines; the same lines for the NK 603 and MON 810 tests, but different types for the MON 863 trials. We note that these unrelated, different non-GM maize types were not shown to be substantially equivalent to the GMOs. The quantity of some sugars, ions, salts, and pesticide residues, do in fact differ from line
to line, for example in the non-GM reference groups. This not only introduced unnecessary sources of variability but also increased considerably the number of rats fed a normal non-GM diet (320) compared to the GM-fed groups (80) per transformation event, which considerably unbalances the experimental design. A group con-sisting of the same number of animals fed a mixture of these test diets would have been a better and more appropriate control. In addition, no data is shown to demonstrate that the diets fed to the control and ref-erence groups were indeed free of GM feed.
2.2. Data collection
The raw biochemical data, necessary to allow a statistical re-evaluation, should be made publically available according to European Union Directive CE/2001/18 but unfortunately this is not always the case in practice. On this occasion, the data we re-quired for this analysis were obtained either through court actions (lost by Monsanto) to obtain the MON 863 feeding study material (June 2005), or by courtesy of governments or Greenpeace lawyers. We thank the Swedish Board of Agriculture, May 30, 2006 for making public the NK 603 data upon request from Greenpeace Denmark and lawyers from Greenpeace Germany, November 8, 2006 for MON 810 material. This allowed us to conduct for the first time a precise and direct side-by-side comparison of these data from the three feeding trials with these GMOs.
Approximately 80 different biochemical and weight parameters, including crude and relative measures (Table A, Annexes), were evaluated in se-rum and urine after 5 and 14 weeks of feeding. We classified these per organ (markers by site of synthe-sis or regulation). These organs weighed at the end of the experimental period, along with the whole body were: adrenal glands, brain, gonads, heart, kidneys,
liver, and spleen. In addition, some parameters measured were related to bone marrow (blood cells) and pancreas (glucose) function. Unfortunately, some important measurements serving as markers for liver function were not conducted for technical or un-known reasons. This included gamma glutamyl transferase after 90 days feeding, cholesterol and triglyceride levels in the NK 603 and MON 810 trials, and cytochrome P450 family members in all cases. In addition, important sex difference markers were also ignored such as blood sex or pituitary hormone lev-els. Furthermore, it is well known and present in OECD guidelines that measurements should be con-ducted for at least 3 different experimental points to study dose- or time-related effects. Contrastingly and for reasons that are not stated, in all three studies for all three GMOs, only 2 doses and periods of feeding were measured, which makes it difficult to evaluate dose and cumulative effects. We have in a first in-stance indicated lacking values for different parame-ters (Annexes, Tables B, C, D).
2.3. Statistical power related to the experimen-tal design
The most fundamental point to bear in mind from the outset is that a sample size of 10 for bio-chemical parameters measured two times in 90 days is largely insufficient to ensure an acceptable degree of power to the statistical analysis performed and presented by Monsanto. For example, concerning the statistical power in a t test at 5%, with the comparison of 2 samples of 10 rats, there is 44% chance to miss a significant effect of 1 standard deviation (SD; power 56%). In this case to have a power of 80% would ne-cessitate a sample size of 17 rats. Therefore, the statis-tical power is insufficient in these studies to allow an a priori dismissal of all significant effects. Indeed, this is true overall with the amplitude of the effects that can usually be observed within three months, in the case of usual chronic toxicity appearing after one year of treatment. Hence, the lack of rejection of the null hypothesis at 5% does not mean that this hypothesis is true. Thus, the assessment of statistical power is absolutely necessary to understand the undetectable size effect; the statistical power depends on the sam-ple and effect size, and the level of the test. This is exemplified when Monsanto performed one-way analysis of variance (ANOVA) calculations at 5% with a sample size of 10 animals for 10 groups. In this case the probability of not detecting a medium size effect [3] (0.5 SD for a t test for instance) is about 70% (power of the test 30%). However, the fact is that within 90 day
s, a chronic toxicity has a maximum chance of giving rise to a medium rather than large size effects. The disturbance of parameters at the be-ginning of a disease is generally less important than at its end or as time progresses. Therefore, the proto-col has to be drastically improved at this level, and as a result we consider that based on the analysis as presented by Monsanto that it fails to demonstrate that the consumption of these GM maize feeds was indeed safe as claimed. Any sign of toxicity should be taken into consideration to justify the prolongation of the experiment, or, if this is not possible, to reassess the statistical analysis, and to propose a scientifically valid physiological interpretation of any findings re-lating to disturbed functional parameters on a per organ basis. This was the ultimate objective of this investigation.
In reality, in their report containing the raw data and statistical analysis, Monsanto did not apply in any case their chosen and described statistical meth-ods. Only parametric tests (one-way ANOVA under homoscedasticity hypothesis and Student t tests on contrasts) were employed. Moreover, to select sig-nificant results, they only contrasted the data sets from the 33% GM maize feeding groups (for NK 603 and MON 810) with all reference groups. Moreover, their biological interpretation of statistically signifi-cant results differs from case to case. In particular, sex differences were frequently used to reject pathologi-cal significance, despite the fact that this was without measuring effects on s
ex hormone levels. They also used the lack of linear dose-related effects, which is almost inevitable given that only two feeding doses were measured, to declare the diet as safe, as pro-posed for MON 863 GM maize [4]. In the MON 863 experiments, the authors still failed to apply their de-clared methodology, which was slightly different. The ANOVA and contrast analysis (33% GM feeding dose versus controls) were in this case the determin-ing criteria for evaluation of statistical significance, but only if the mean of the 33% GM feeding group was outside the range of the mean of the reference cohorts. All this increases noticeably the risks of false negative results.
Consequently, based on the clear inadequacy of the statistical power used to refute toxic effects (for instance the unquestionable large size effects in this study), knowing also that billions of people and ani-mals can consume these products prior to the per-formance of appropriate in vivo safety evaluation, we applied an appropriate, experimentally validated sta-tistical analytical methodology [5], elements of which are described below.
2.4. Statistical methods employed
We first repeated the same statistical analysis as
conducted by Monsanto to verify descriptive statistics (sample size, means, and standard deviation)
and ANOVA per sex, per variable and for each of the three GMO. For all that, the normality of the residues was tested using the Shapiro test and the homosce-dasticity (homogeneity of the variances) using the Bartlett test. In the case where the Shapiro and Bart-lett tests were non significant (*p > 0.05 and **p > 0.01, respectively) we performed an ANOVA [6, 7], and in the case of heteroscedasticity the approximate Welch method was used. In the case where the Shapiro test was significant, we performed the Kruskal-Wallis rank sum test [7, 8].
We then analyzed the effects of the GM maize varieties on each sex and each diet by pairwise com-parisons of the parameters of GM-fed rats versus control groups, and subsequently to the unrelated non-GM maize reference groups. The statistical dif-ferences between reference and control groups were calculated in order to study the effects of the different normal diets per se (due to differences in salts, sugars, minerals, vitamins, pesticides, etc composition), and indicated by contrast to Monsanto's work (see legend Table 1). In order to select the appropriate two-tailed comparison test [7], we again studied first normality (Shapiro test) and variance equality (F test). Accord-ing to the results, we performed the adapted test; that is, an unpaired t test, a Welch corrected t test or a Mann-Whitney test (which is generally more appro-priate with a sample size of 10). To perform multiple pairwise comparisons, we used the False Discovery Rate approach (FDR, [9]) to calculate a
djusted p-values, in order to limit the rate of false positives to 5%. We preferred Benjamini and Yekutieli's method [10] rather than that of Benjamini and Hochberg [11] as the parameters under investigation are not inde-pendent. In addition, after centering and scaling the data, Principal Components Analysis (PCA, [12]) was performed in order to study the scattering of the dif-ferent factors (sex, period, diet, dose and group). Fi-nally, we established per group for each rat and by parameter the representations and paired tests cor-responding to the temporal changes between the two feeding periods.
We used the R language [7] version 2.5 for all statistical computations [13] with the appropriate package: pwr package for power studies, the bio-conductor's multtest package for FDR [14-15] and the ADE4 package [16, 17] for multivariate analysis.
3. Results
We have previously reported indications of tox-icity in rats fed with MON 863 GM maize for 90 days [5]. However, these signs of toxicity alone do not constitute proof of adverse health effects. We have therefore extended our initial analysis on the MON 863 feeding data by collectively compiling the sig-nificant differences observed in the physiological and biochemical parameters measured in feeding tr
ials of rats with each of the three GM maize varieties MON 863, MON 810 and NK 603 (Tables 1, 2; Annex Table E). When we then initially compare all p-values in our calculations with those of Monsanto (significant and non significant differences, Annex Table E), we obtain ratios of 432/452 (NK 603), 435/450 (MON 810) and 442/470 (MON 863). By employing our sta-tistical methods even if we reached a concordance with Monsanto’s results (Annex Table E), the level of precision of the main effects and their interpretation are highly different. Therefore, we then progressed to consider only relative differences over 5% (Tables 1 and 2).
3.1. NK 603
We first evaluated the results for the NK 603 feeding trials. The observations shown in Table 1 with relative differences versus controls reveal that of 23 significantly different effects that are supposed to be due to this GM maize, 18 are in males (raw means with SEM; Annex Table F). The repartition of effects is thus sex-dependent. In addition, in general liver (Fig. 1) and kidney (Fig. 2) parameters in all rats are sex differentially expressed. This is evident not only in the experiments involving NK 603, independently of the treatment at week 14, but also at week 5 (data not shown), but similarly observed in the MON 810 and MON 863 feeding tests (Annex Fig. A- Fig. D).
Males are clearly more sensitive than female animals to show physiological disturbances when fed NK 603. This is not observed for all three GM maize varieties. Moreover, most effects appear to be dose-dependent since 83% of male effects emerge only at the 33% feeding level (15/18), the highest GM maize concentration in the diet (Table 1). The maxi-mal mean differences are observed in male kidney parameters.
Urine phosphorus, for instance, is importantly disturbed in a dose-dependent manner and at both 5 and 14 week periods of feeding and hence reproduci-ble over time. The significant effect at this level does not appear to be a false positive result (week 5, 33%, adjusted p<0.003 for FDR calculated according to Benjamini and Yekutieli), considering that all pa-rameters were not independent. Comparable results were also obtained for relative lymphocyte and neu-trophil differences (all for males, week 14, 33%, ad-justed p<0.005).
Table 1. Differences between NK 603-fed rats and controls. Study of the GMO effects, which are indicated by mean differences (%) for each parameter with the corresponding control group per sex and per dose. The significant differences versus controls (*p < 0.05, **p < 0.01), for all the parameters measured in the subchronic feeding tests, are presented. The parameters were grouped by organs according to the sites of synthesis or classical indicators of dysfunction. They were indicat
ed for all groups only if they showed at least for one sex or one diet a significant and relatively ± 5% difference to the mean. The animals were male (m) or female (f) young adult rats fed during 5 or 14 weeks with the GM maize NK 603 (11 or 33% in the diet) and compared with controls fed with a ‘‘substantially equivalent’’ isogenic maize line. The parameters were measured for 10 rats, except for the organ weights (20 rats), obtained only at the end of the experiment. In single-boxed numbers, we indicate the statistical differences between GMO-fed rats and controls, which are not found between the mean of the six reference groups and controls. A difference between reference and control groups could indicate an effect of the diet per se. In double-boxed numbers, among the effects due to the GMO, are indicated the statistical differences between the GMO groups and the mean of the six reference groups (which have not even eaten a genetically linked variety of maize as the control and the GMO treated groups). (p): Differences for the indicated parameters are not significant by a non-parametric test but by a parametric one; all other differences by both. “Lar Uni Cell” means percent of large unnucleated cell count.

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