Trans-disciplinary diagnosis for an in-depth reform of regulatory expertise in the field of environmental toxicology and security.

Repeated health and environmental scandals, the loss of biodiversity and the recent burst of chronic diseases constantly remind us the inability of public authorities and risk assessment agencies to protect health and the environment. After reviewing the main shortcomings of our evaluation system of chemicals and new technologies, supported by some concrete examples, we develop a number of proposals to reform both the risk assessment agencies and the evaluation processes. We especially propose the establishment of an independent structure, a High Authority of Expertise, supervising, either at European level or at national level, all the evaluation agencies, and ensuring the transparency, the methodology and the deontology of the expertise. In addition to modifying the evaluation protocols, both in their nature and in their content, especially in order to adapt them to current pollutants such as endocrine disruptors, we propose a reform of the expertise processes based on transparency, contradiction, and greater democracy, including close collaboration between the institutional and scientific parties on the one hand and the whole civil society on the other. All the proposals we make are inspired by the desire to prevent, through appropriate mechanisms, the human, health, ecological, but also economic consequences of contemporary technological choices.

Impact of Antibiotics on Efficacy of Cry Toxins Produced in Two Different Genetically Modified Bt Maize Varieties in Two Lepidopteran Herbivore Species, Ostrinia nubilalis and Spodoptera littoralis.

The insecticidal crystal proteins from Bacillus thuringiensis (Bt) are widely-used biopesticides that are used both as Bt spore-crystal preparations in sprayable formulations and as activated toxins in genetically modified (GM) plants. Models for their modes of action have been proposed but many issues remain unresolved. Among those is the role of commensal gut bacteria in target insect death: previous studies showed that antibiotics attenuate the toxicity of Bt sprays. We tested whether antibiotics interfere with the effects of GM plant-produced Bt toxins in larvae of two Lepidopteran species, the European corn borer Ostrinia nubilalis and the cotton leafworm Spodoptera littoralis. The larvae were reared on artificial diet with or without antibiotics and, thereafter, fed two varieties of Bt GM maize in comparison to conventional non-Bt maize leaves sprayed with antibiotic solution and/or with a Bt formulation. Antibiotics significantly reduced or delayed the toxicity of Cry toxins, although to a lesser extent than previously reported for Bt-sprays. This supports the hypothesis that Cry toxins induce mortality by themselves in the absence of Bt bacteria and spores, and of commensal gut bacteria. However, larvae that were not treated with antibiotics died faster and at a higher rate which was further compounded by plant variety and species sensitivity. These findings support a hypothesis that toxicemia alone can inflict significant mortality. However, in the absence of antibiotics, the gut bacteria likely enhance the Cry toxin effect by inflicting, additionally, bacterial septicemia. This has important implications in field situations where antibiotic substances are present-e.g., from manure of animals from conventional production systems-and for ecotoxicological testing schemes of Bt toxins and nontarget organisms that are often using artificial diets enriched with high concentrations of antibiotics.

Sex-dependent impact of Roundup on the rat gut microbiome.

A growing body of research suggests that dysbiosis of the gut microbiota induced by environmental pollutants, such as pesticides, could have a role in the development of metabolic disorders. We have examined the long-term effects of 3 doses of the Roundup(R) herbicide (made of glyphosate and formulants) on the gut microbiota in male and female Sprague-Dawley rats. A total of 141 bacteria families were identified by a 16S sequencing analysis approach. An OPLS-DA analysis revealed an increased Bacteroidetes family S24-7 and a decreased Lactobacillaceae in 8 out of the 9 females treated with 3 different doses of R (n = 3, for each dose). These effects were confirmed by repetitive sequence-based PCR fingerprinting showing a clustering of treated females. A culture-based method showed that R had a direct effect on rat gut microbiota. Cultivable species showed different sensitivities to R, including the presence of a high tolerant or resistant strain identified as Escherichia coli by 16S rRNA sequencing. The high tolerance of this E. Coli strain was explained by the absence of the EPSPS gene (coding glyphosate target enzyme) as shown by DNA amplification. Overall, these gut microbiome disturbances showed a substantial overlap with those associated with liver dysfunction in other studies. In conclusion, we revealed that an environmental concentration of R (0.1 ppb) and other two concentrations (400 ppm and 5,000 ppm) have a sex-dependent impact on rat gut microbiome composition and thus warrants further investigation.

Dig1 protects against locomotor and biochemical dysfunctions provoked by Roundup.

BACKGROUND: Plant medicinal extracts may be claimed to prevent or cure chemical intoxications. Few of these are tested for their mechanisms of actions in vivo and for their cellular impacts. In 2011, we demonstrated that hepatic cell mortality induced by environmentally realistic levels of the widely used herbicide Roundup (R) in vitro can be almost entirely prevented by plant extracts called Dig1 (D, Digeodren). METHODS: We tested the in vivo effects of D alone (1.2 ml/kg bw/d), but also prior to and during 8 days of R intoxication (at 135 mg/kg bw/d) in a total of 4 groups of 40 adult Sprague-Dawley male rats each. After treatments, horizontal and vertical locomotor activities of the animals were measured by use of actimeters. Brain, liver, kidneys, heart and testes were collected and weighted. Body weights as well as feed and water consumption were recorded. Proteins, creatinine, urea, phosphate, potassium, sodium, calcium, chloride ions, testosterone, estradiol, AST and ALT were measured in serum. In liver S9 fractions, GST, GGT, and CYP450 (1A2, 2C9, 2C19, 2D6, 3A4) were assessed. RESULTS: D did not have any physiological or biochemical observable impact alone at 2 %. Out of a total of 29 measured parameters, 8 were significantly affected by R absorption within only 8 days. On these 8 parameters, only 2 were not restored by D (GGT activity and plasmatic phosphate), 5 were totally restored (horizontal and vertical locomotor activities, CYP2D6 activity, plasmatic Na + and estradiol), and the 6th was almost restored (plasmatic K+). The specificities of the toxic effects of R and of the therapeutic effects of D treatment were thus demonstrated, both at the behavioural and biochemical levels. CONCLUSIONS: D, without any side effect observable in these conditions, presented strong preventive and therapeutic properties in vivo after a short-term intoxication by the widely used pesticide Roundup.

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels.

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.

Laboratory Rodent Diets Contain Toxic Levels of Environmental Contaminants: Implications for Regulatory Tests.

The quality of diets in rodent feeding trials is crucial. We describe the contamination with environmental pollutants of 13 laboratory rodent diets from 5 continents. Measurements were performed using accredited methodologies. All diets were contaminated with pesticides (1-6 out of 262 measured), heavy metals (2-3 out of 4, mostly lead and cadmium), PCDD/Fs (1-13 out of 17) and PCBs (5-15 out of 18). Out of 22 GMOs tested for, Roundup-tolerant GMOs were the most frequently detected, constituting up to 48% of the diet. The main pesticide detected was Roundup, with residues of glyphosate and AMPA in 9 of the 13 diets, up to 370 ppb. The levels correlated with the amount of Roundup-tolerant GMOs. Toxic effects of these pollutants on liver, neurodevelopment, and reproduction are documented. The sum of the hazard quotients of the pollutants in the diets (an estimator of risk with a threshold of 1) varied from 15.8 to 40.5. Thus the chronic consumption of these diets can be considered at risk. Efforts toward safer diets will improve the reliability of toxicity tests in biomedical research and regulatory toxicology.

Major pesticides are more toxic to human cells than their declared active principles.

Pesticides are used throughout the world as mixtures called formulations. They contain adjuvants, which are often kept confidential and are called inerts by the manufacturing companies, plus a declared active principle, which is usually tested alone. We tested the toxicity of 9 pesticides, comparing active principles and their formulations, on three human cell lines (HepG2, HEK293, and JEG3). Glyphosate, isoproturon, fluroxypyr, pirimicarb, imidacloprid, acetamiprid, tebuconazole, epoxiconazole, and prochloraz constitute, respectively, the active principles of 3 major herbicides, 3 insecticides, and 3 fungicides. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. Fungicides were the most toxic from concentrations 300-600 times lower than agricultural dilutions, followed by herbicides and then insecticides, with very similar profiles in all cell types. Despite its relatively benign reputation, Roundup was among the most toxic herbicides and insecticides tested. Most importantly, 8 formulations out of 9 were up to one thousand times more toxic than their active principles. Our results challenge the relevance of the acceptable daily intake for pesticides because this norm is calculated from the toxicity of the active principle alone. Chronic tests on pesticides may not reflect relevant environmental exposures if only one ingredient of these mixtures is tested alone.

Conflicts of interests, confidentiality and censorship in health risk assessment: the example of an herbicide and a GMO.

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.

Republished study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

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.

Answers to critics: Why there is a long term toxicity due to a Roundup-tolerant genetically modified maize and to a Roundup herbicide.

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.

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

The health effects of a Roundup-tolerant genetically modified 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 2-3 times more than controls, and more rapidly. This difference was visible in 3 male groups fed GMOs. All results were hormone and sex dependent, and the pathological profiles 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 modified 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.