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Background The insertion of a transgene into a plant organism can, in addition to the intended effects, lead to unintended effects in the plants. To uncover such effects, we compared maize grains of two genetically modified varieties containing NK603 (AG8025RR2, AG9045RR2) to their non-transgenic counterparts (AG8025conv, AG9045conv) using high-throughput RNA sequencing. Moreover, in-depth analysis of these data was performed to reveal the biological meaning of detected differences. Results Uniquely mapped reads corresponded to 29,146 and 33,420 counts in the AG8025 and AG9045 varieties, respectively. An analysis using the R-Bioconductor package EdgeR revealed 3534 and 694 DEGs (significant differentially expressed genes) between the varieties AG8025RR2 and AG9045RR2, respectively, and their non-transgenic counterparts. Furthermore, a Deseq2 package revealed 2477 and 440 DEGs between AG8025RR2 and AG9045RR2, respectively, and their counterparts. We were able to confirm the RNA-seq results by the analysis of two randomly selected genes using RT-qPCR (reverse transcription quantitative PCR). PCA and heatmap analysis confirmed a robust data set that differentiates the genotypes even by transgenic event. A detailed analysis of the DEGs was performed by the functional annotation of GO (Gene Ontology), annotation/enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) ontologies and functional classification of resulting key genes using the DAVID Bioinformatics Package. Several biological processes and metabolic pathways were found to be significantly different in both variety pairs. Conclusion Overall, our data clearly demonstrate substantial differences between the analyzed transgenic varieties and their non-transgenic counterparts. These differences indicate that several unintended effects have occurred as a result of NK603 integration. Heatmap data imply that most of the transgenic insert effects are variety-dependent. However, identified key genes involved in affected pathways of both variety pairs show that transgenic independent effects cannot be excluded. Further research of different NK603 varieties is necessary to clarify the role of internal and external influences on gene expression. Nevertheless, our study suggests that RNA-seq analysis can be utilized as a tool to characterize unintended genetic effects in transgenic plants and may also be useful in the safety assessment and authorization of genetically modified (GM) plants.

We have studied the long-term toxicity of a Roundup-tolerant GM maize (NK603) and a whole Roundup pesticide formulation at environmentally relevant levels from 0.1 ppb. Our study was first published in Food and Chemical Toxicology (FCT) on 19 September, 2012. The first wave of criticisms arrived within a week, mostly from plant biologists without experience in toxicology. We answered all these criticisms. The debate then encompassed scientific arguments and a wave of ad hominem and potentially libellous comments appeared in different journals by authors having serious yet undisclosed conflicts of interests. At the same time, FCT acquired as its new assistant editor for biotechnology a former employee of Monsanto after he sent a letter to FCT to complain about our study. This is in particular why FCT asked for a post-hoc analysis of our raw data. On 19 November, 2013, the editor-in-chief requested the retraction of our study while recognizing that the data were not incorrect and that there was no misconduct and no fraud or intentional misinterpretation in our complete raw data - an unusual or even unprecedented action in scientific publishing. The editor argued that no conclusions could be drawn because we studied 10 rats per group over 2 years, because they were Sprague Dawley rats, and because the data were inconclusive on cancer. Yet this was known at the time of submission of our study. Our study was however never attended to be a carcinogenicity study. We never used the word ‘cancer’ in our paper. The present opinion is a summary of the debate resulting in this retraction, as it is a historic example of conflicts of interest in the scientific assessments of products commercialized worldwide. We also show that the decision to retract cannot be rationalized on any discernible scientific or ethical grounds. Censorship of research into health risks undermines the value and the credibility of science; thus, we republish our paper.

Background 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. Results 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. Conclusion 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.

Our recent work (Séralini et al., 2012) remains to date the most detailed study involving the life-long consumption of an agricultural genetically modified organism (GMO). This is true especially for NK603 maize for which only a 90-day test for commercial release was previously conducted using the same rat strain (Hammond et al., 2004). It is also the first long term detailed research on mammals exposed to a highly diluted pesticide in its total formulation with adjuvants. This may explain why 75% of our first criticisms arising within a week, among publishing authors, come from plant biologists, some developing patents on GMOs, even if it was a toxicological paper on mammals, and from Monsanto Company who owns both the NK603 GM maize and Roundup herbicide (R). Our study has limits like any one, and here we carefully answer to all criticisms from agencies, consultants and scientists, that were sent to the Editor or to ourselves. At this level, a full debate is biased if the toxicity tests on mammals of NK603 and R obtained by Monsanto Company remain confidential and thus unavailable in an electronic format for the whole scientific community to conduct independent scrutiny of the raw data. In our article, the conclusions of long-term NK603 and Roundup toxicities came from the statistically highly discriminant findings at the biochemical level in treated groups in comparison to controls, because these findings do correspond in an blinded analysis to the pathologies observed in organs, that were in turn linked to the deaths by anatomopathologists. GM NK603 and R cannot be regarded as safe to date.

In mid-September 2012, a scientific team headed by Gilles-Eric Séralini at the University of Caen in France published the results of a long-term study with rats which had been fed ge-netically modified glyphosate-tolerant NK603 maize…The Federal Institute for Risk Assessment (BfR) has evaluated the study in terms of its relevance for the evaluation of the health risk of genetically modified glyphosate-tolerant maize NK603 and also with regard to the evaluation of the health risk of the glyphosate-containing formulation. On the basis of the publication, the BfR has come to the conclusion that the authors’ main statements are not sufficiently corroborated by experimental evidence, due to deficiencies in the study design and in the presentation and interpretation of the study results. Therefore, the main conclu-sions of the authors are not supported by the presented incomplete data. The study does not comply with internationally recognised standards for long-term carcinogenicity studies. The rat strain used shows a relatively high spontaneous tumour rate, especially for mammary and pituitary tumours, and the number of animals used was too small and insufficient for assess-ing the claimed differences between the test groups and the control group. Also the authors’ hypothesis that the observed effects could result from adverse effects on the endocrine sys-tem is not sufficiently supported by the data presented. Furthermore, the BfR criticises that the glyphosate dose administered was not determined in the studies with the glyphosate-containing plant protection product Roundup. Due to these deficiencies the BfR has asked the authors to provide the complete study report including the individual animal data. More-over, it has asked specific questions in order to allow for a further evaluation of the reported effects.

Den 19. september 2012 ble det publisert en artikkel i det tyske tidsskriftet «Food and Chemical Toxicology» som vakte oppmerksomhet i store deler av verden. Artikkelen bygger på en toårig fôringsstudie med rotter og konkluderer med en antatt økt hyppighet for ulike svulstdannelser og nyrelidelser blant dyrene knyttet til den genmodifiserte maisen NK603 og plantevernmidlet Roundup som inneholder glyfosat. Grunnet den store oppmerksomheten ga Mattilsynet i et brev datert 19.10.2012 (ref. 2012/225329) VKM i oppdrag å vurdere den aktuelle publikasjonen fra Séralini et al. «Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize», og å gi svar på følgende spørsmål: Gir den aktuelle publikasjonen av Séralini et al. grunnlag for å revurdere konklusjonen i VKMs tidligere helserisikovurdering av NK603? VKM opprettet i november en tverrfaglig ad hoc-gruppe for å utarbeide dette dokumentet som svar til Mattilsynet. Rapporten fra ad hoc-gruppen ble vurdert og godkjent av Faggruppen for genmodifiserte organismer på deres faggruppemøte 7.12.2012. VKM konkluderer med at studien til den franske forskeren Gilles-Eric Séralini og medarbeidere har alvorlige statistiske mangler, mangler i design, metodikk og analyse. Den tilfredsstiller derfor ikke godkjente vitenskapelige standarder. VKM mener det ikke er behov for å revurdere sin tidligere risikovurdering av den genmodifiserte maisen NK603 på bakgrunn av Séralini og medarbeidernes studier.