CONSUMER, FARMER & PET OWNER ALERT

 Crops genetically engineered to resist the herbicide Roundup ( glyphosate) put all at risk---livestock, pets, human consumers and the plants themselves

Don M. Huber, Professor Emeritus of Purdue University writes a cover letter about his letter to Vilsack plus the text and images from his original letter regarding his concerns about glyphosate:

 

This cover letter is provided to explain the reasoning and concerns that were conveyed in a letter which I sent to Secretary of Agriculture, Thomas Vilsack on January 17, 2011 (Attachment 1). The letter was not intended for public distribution; however, the letter was ‘leaked’ and subsequently posted on the internet from which it soon became public knowledge world-wide. Once it was widely distributed, I gave permission for subsequent postings in order to keep it consistent. My busy meeting and travel schedule has delayed getting further information on this matter out publicly to the many individuals who have requested it. The scientific data on this newly recognized organism is being prepared for formal publication.

I wrote the letter to Secretary Vilsack for a very simple reason: we are experiencing a large number of problems in production agriculture in the U.S. that appear to be intensified and sometimes directly related to genetically engineered (GMO) crops, and/or the products they were engineered to tolerate – especially those related to glyphosate (the active chemical in Roundup® herbicide and generic versions of this herbicide). We have witnessed a deterioration in the plant health of corn, soybean, wheat and other crops recently with unexplained epidemics of sudden death syndrome of soybean (SDS), Goss’ wilt of corn, and take-all of small grain crops the last two years. At the same time, there has been an increasing frequency of previously unexplained animal (cattle, pig, horse, poultry) infertility and spontaneous abortions. These situations are threatening the economic viability of both crop and animal producers.

Incidence of high infertility and spontaneous abortions in the various animal species is becoming more common. Often, all previously known causes of these conditions can be ruled out as factors for these particular farm operations (Attachment 2). Detailed examination for the newly recognized organism has shown its presence in all of the cases examined to date. Koch’s postulates have been completed for animals to verify the cause/effect relationship with this newly culturable organism. A search for the source of animal infections revealed a high population of this newly discovered electron microscopic sized organism in soybean meal and corn products. The organism appears compatible, and probably synergistic, with other microorganisms such as Fusarium solani fsp. glycines, the cause of SDS of soybeans and also with gram positive bacteria. The organism also is in a very high population in Goss’ wilt infected corn caused by the gram positive bacterium Clavibacter michiganensis subsp. nebraskensis.

Although most corn hybrids have been genetically resistant to Goss’ wilt, preliminary research in 2010 demonstrated that the application of glyphosate herbicide, or the surfactant from glyphosate formulations, nullified this resistance and rendered them fully susceptible to this pathogen (Fig. 1). This disease was commonly observed in many Midwestern U.S. fields planted to RR corn in 2009 and 2010, while adjacent non-GMO corn had very light to no infections in spite of the high inoculum present in no-till crop residues (Figure 2). The increased Goss’ wilt in 2010 was a major contributor to the estimated almost one billion bushels of corn ‘lost’ last year (based on USDA August estimated yields and actually harvested crop reported by USDA in January) in spite of generally good harvest conditions.

Increased severity of plant diseases after glyphosate is applied (Fig. 3) is well documented and, although rarely cited, the increased disease susceptibility is the herbicidal mode of action of glyphosate (Johal andRahe,1988, 1990; Johal and Huber, 2009; Schafer et al, 2009, 2010). The loss of disease resistance in Roundup Ready® sugar beets when glyphosate was applied prompted researchers at the USDA sugar beet laboratory to include a precautionary statement in their paper, e.g. “Precautions need to be taken when certain soil-borne diseases are present if weed management for sugar beet is to include post-emergence glyphosate treatments” (Larson et al, 2006).

The loss of genetic resistance in Roundup Ready® corn hybrids to Goss’ wilt (Clavibacter michiganensis subsp. nebraskensis) (Figs. 2, 3), synergistic relationship of the newly recognized electron microscopic organism causing infertility and abortions in animals with gram+ bacteria, and high populations of the new EM organism in RR corn leaves and silage creates a concern for the deregulation of Roundup Ready® alfalfa which is productive in many areas only because of its genetic resistance to bacterial wilt caused by Clavibacter michiganensis subsp. insidiosum. This disease could make alfalfa unprofitable for production and, if the EM organism is associated with it in alfalfa as it is in corn, also unsafe for animal feed and their products such as milk for human consumption. The loss of alfalfa, the United State’s most valuable forage crop and fourth most economically important crop, could strike a mortal blow to struggling dairy and beef operations.

Extensive research has shown that this potent tool for weed management, glyphosate, is also a strong immobilizer (chelator) of essential plant nutrients to impair nutrient uptake, translocation, and physiological efficiency at only a fraction of the labeled herbicidal rate (Ekers, Ozturk, Cakmak, Zobiole, Jolly et al., 2004). Glyphosate is a powerful biocide to harm beneficial soil organisms important for nutrient recycling, N-fixation, nutrient availability, and natural disease control (Kremer & Means, Zobiole et al, Dick et al) with a resultant increase in diseases of corn, soybeans (Fig. 3), wheat and other crops. The close relationship between mineral nutrition and disease severity is well documented (Datnoff et al, 2007). These activities can have deleterious effects on plant nutrition, disease susceptibility, and nutritional quality of the crop produced.

Deleterious effects of GM crops also are vividly demonstrated in reports from livestock producers in the U.S. Although some of these reports are anecdotal because of limited analytical techniques to verify the cause, some producers have been able to resume economical operations by changing feed sources to non-GMO crops. Replicated independent research is needed in this area, especially in light of the serious toxicological concerns raised recently that show potential human and animal toxicity from very low levels of residual glyphosate in food/feed that are many times lower than permitted in U.S. food and feed products (Seralini et al., 2011). The recent Indian Supreme Court’s independent analysis and Ruling that GMO egg plant posed a significant health risk to humans needs further evaluation in the U.S. (AgroNews, 2011).

I feel I would be totally irresponsible to ignore my own research and the vast amount of published research now available that support the concerns we are seeing in production agriculture, without bringing it to the attention of the Secretary of Agriculture with a request for him to initiate the much needed independent research. Many producers can’t wait an additional 3-10 years for someone to find the funds and neutral environment to conduct such critical research (Attachment 2. Entomologists letter to EPA).

Based on the scientific evidence currently accumulating, I do not believe it is in the best interests of the agricultural producer or consuming public for regulatory agencies to approve more GMO crops, particularly Roundup Ready® alfalfa and sugar beets, until independent research can establish their productivity when predisposed to potentially severe diseases, the irrelevance of the new EM organism, and their nutritional equivalency. In my letter, I asked the Secretary to allocate the necessary resources to do this, and requested that he exercise the utmost caution in deregulating these crops until such findings resolve the concerns expressed in the letter, if they do.

Don M. Huber

Professor Emeritus, Purdue University

 

References cited

AgroNews. 2011. India: Signs of food toxicity in GE eggplant. Scoop.co.nz 2011-1-18. [http://news.agropages.com/News/NewsDetail---3369.htm] Nib, 24 Jnuary 111.

Bellaloui, N., reddy, K.N., Zablotowicz, R.M., Abbas, H.K., and Abel, C.A. 2009. Effects of glyphosate application on seed iron and root ferric (III) reductase in soybean cultivars. J. Agric. Food Chem. 57:9569-9574.

Bott, S., Tesfamariam, T., Kania, A., Eman, B., Aslan, N., Roemheld, V., and Neumann, G. 2011, Phytotoxicity of glyphosate soil residues re-mobilise4d by phosphate fertilization. Plant Soil 315:2-11. DOI 10, 1007/s11104-010-06989-3.

Cakmak, I., Yazici, A., Tutus, Y., Ozturk, L. 2009. Glyphosate reduced seed and leaf concentrations of calcium, magnesium, manganese, and iron in non-glyphosate resistant soybean. European J. Agron. 31:114-119.

Datnoff, L.E., elmer, W.H., and Huber, D.M. 2007. Mineral Nutrition and Plant Disease. APS Press, St. Paul, Mn. 278. 278 pages.

Eker, S., Ozturk, L., Yazici, A., Erenoglu, B., Roemheld, V., and Cakmak, I. 2006. Foliar-applied glyphosate substantially reduced uptake and transport of iron and manganese in sunflower (Helianthus annuus L.) plants. J. Agric. Food Chem. 54:100019-10025.

Fernandez, M.R., Zentner, R.P., Basnyat, P., Gehl, D., Selles, F., and Huber, D.M. 2009. Glyphosate associations with cereal diseases caused by Fusarium spp. in the Canadian Prairies. European J. Agon. 31:133-143.

Johal, G.R. and Rahe, J.E. 1984. Effect of soilborne paltn-pathogenic fungi on the herbicidal action of glyphosate on bean seedlings. Phytopathology 74:950-955.

Johal, G.R. and Rahe, J.E. 1990. Role of phytoalexins in the suppression of resistance of Phaseolus vulgaris to Colletotrichum lindemuthianum by glyphosate. Canad. J. Plant Pathol. 12:225-235.

Johal, G.R. and Huber, D.M. 2009. Glyphosate effects on diseases of plants. European J. Agron. 31:144-152.

Kremer, R.J. and Means, N.E. 2009. Glyphosate and glyphosate-resistant crop interactions with rhizosphere microorganisms. European J. Agron. 31:153-161.

Larsen, R.L., Hill, A.L., Fenwick, A., Kniss, A.R., Hanson, L.E., and Miller, S.D. 2006. Influence of glyphosate on Rhizoctonia and Fusarium root rot in sugar beet. Pest Manag. Sci. 62:1182-1192.

Ozturk, L., Yazici, A., Eker, S., gokmen, O., roemheld, V., and Cakmak, I. 2008. Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots. New Phytol. 177:899-906.

Schafer, J.R., Westhoven, A.M., Kruger, G.R., Davis, V.M., Hallett, S.G., and Johnson, W.G. 2009. Effect of growth media on common lambsquarter and giant ragweed biotypes response to glyphosate. Proc. Northcentral Weed Sci. Soc. 64:102.

Schafer, J.R., Hallett, S.G., and jophnson, W.G. 2010. Role of soil-borne fungi in the response of giant ragweed (Ambrosia trifida) biotypes to glyphosate. Proc. Northcentral Weed Sci. Soc. 65:.

Seralini, G-E., Mesnage, R., Clair, E., Gress, S., de Vendomois, J.S., Cellier, D. 2011. Genetically modified crops safety assessments: present limits and possible improvements. Environ. Sci. Europe 23:10-20. http://www.enveurope.com/content/23/1/10

Tesfamariam, T., Bott, S., Cakmak, I., Roemheld, V., and Neumann, G. 2009. Glyphosate in the rhizosphere – role of waiting times and different glyphosate binding forms in soils for phytoxicity to non-target plants. European J. Agron. 31:126-132.

Yamada, T., Kremer, R.J., Camargo e Castro, P.R., and Wood, B.W. 2009. Glyphosate interactions with physiology, nutrition, and diseases of plants: Threat to agricultural sustainability? European J. Agron. 31:111-113.

Zobiole, L.H.S., Oliveira, R.S.Jr., Huber, D.M., Constantin, J., Castro, C., Oliveira, F.A., Oliveira, A. Jr. 2010. Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans. Plant Soil 328:57-69.

Zobiole, L.H.S., Oliveira, R.S. Jr., Kremer, R.J., Constantin, J., Yamada, T., Castro, C., Oliveiro, F.A., and Oliveira, A. Jr. 2010. Effect of glyposate on symbiotic N2 fixation and nickel concentration in glyphosate-resistant soybeans. Applied Soil Ecol. 44:176-180.

Attachment 1: Letter to Secretary of Agriculture Thomas Vilsak

CONFIDENTIAL and URGENT

1/17/11

The Honorable Thomas Vilsack
United States Secretary of Agriculture

Dear Secretary Vilsack:

A team of senior plant and animal scientists have recently brought to my attention the discovery of an electron microscopic pathogen that appears to significantly impact the health of plants, animals, and probably human beings. Based on a review of the data, it is widespread, very serious, and is in much higher concentrations in Roundup Ready (RR) soybeans and corn—suggesting a link with the RR gene or more likely the presence of Roundup. This organism appears NEW to science!

This is highly sensitive information that could result in a collapse of US soy and corn export markets and significant disruption of domestic food and feed supplies. On the other hand, this new organism may already be responsible for significant harm (see below). My colleagues and I are therefore moving our investigation forward with speed and discretion, and seek assistance from the USDA and other entities to identify the pathogen’s source, prevalence, implications, and remedies.

We are informing the USDA of our findings at this early stage, specifically due to your pending decision regarding approval of RR alfalfa. Naturally, if either the RR gene or Roundup itself is a promoter or co-factor of this pathogen, then such approval could be a calamity. Based on the current evidence, the only reasonable action at this time would be to delay deregulation at least until sufficient data has exonerated the RR system, if it does.

For the past 40 years, I have been a scientist in the professional and military agencies that evaluate and prepare for natural and manmade biological threats, including germ warfare and disease outbreaks. Based on this experience, I believe the threat we are facing from this pathogen is unique and of a high risk status. In layman’s terms, it should be treated as an emergency.

A diverse set of researchers working on this problem have contributed various pieces of the puzzle, which together presents the following disturbing scenario:

Unique Physical Properties

This previously unknown organism is only visible under an electron microscope (36,000X), with an approximate size range equal to a medium size virus. It is able to reproduce and appears to be a micro-fungal-like organism. If so, it would be the first such micro-fungus ever identified. There is strong evidence that this infectious agent promotes diseases of both plants and mammals, which is very rare.

Pathogen Location and Concentration

It is found in high concentrations in Roundup Ready soybean meal and corn, distillers meal, fermentation feed products, pig stomach contents, and pig and cattle placentas.

Linked with Outbreaks of Plant Disease

The organism is prolific in plants infected with two pervasive diseases that are driving down yields and farmer income—sudden death syndrome (SDS) in soy, and Goss’ wilt in corn. The pathogen is also found in the fungal causative agent of SDS (Fusarium solani fsp glycines).

Implicated in Animal Reproductive Failure

Laboratory tests have confirmed the presence of this organism in a wide variety of livestock that have experienced spontaneous abortions and infertility. Preliminary results from ongoing research have also been able to reproduce abortions in a clinical setting.

The pathogen may explain the escalating frequency of infertility and spontaneous abortions over the past few years in US cattle, dairy, swine, and horse operations. These include recent reports of infertility rates in dairy heifers of over 20%, and spontaneous abortions in cattle as high as 45%.

For example, 450 of 1,000 pregnant heifers fed wheatlege experienced spontaneous abortions. Over the same period, another 1,000 heifers from the same herd that were raised on hay had no abortions. High concentrations of the pathogen were confirmed on the wheatlage, which likely had been under weed management using glyphosate.

Recommendations

In summary, because of the high titer of this new animal pathogen in Roundup Ready crops, and its association with plant and animal diseases that are reaching epidemic proportions, we request USDA’s participation in a multi-agency investigation, and an immediate moratorium on the deregulation of RR crops until the causal/predisposing relationship with glyphosate and/or RR plants can be ruled out as a threat to crop and animal production and human health.

It is urgent to examine whether the side-effects of glyphosate use may have facilitated the growth of this pathogen, or allowed it to cause greater harm to weakened plant and animal hosts. It is well-documented that glyphosate promotes soil pathogens and is already implicated with the increase of more than 40 plant diseases; it dismantles plant defenses by chelating vital nutrients; and it reduces the bioavailability of nutrients in feed, which in turn can cause animal disorders. To properly evaluate these factors, we request access to the relevant USDA data.

I have studied plant pathogens for more than 50 years. We are now seeing an unprecedented trend of increasing plant and animal diseases and disorders. This pathogen may be instrumental to understanding and solving this problem. It deserves immediate attention with significant resources to avoid a general collapse of our critical agricultural infrastructure.

Sincerely,

COL (Ret.) Don M. Huber
Emeritus Professor, Purdue University
APS Coordinator, USDA National Plant Disease Recovery System (NPDRS)

 

Attachment 2. Letter from a Veterinarian

Hello, my name is ___________. I am a veterinarian in Michigan.

I am working with a sow herd that has had elevated death loss for over two years and very poor reproductive performance for the last 6-8 months. I have done extensive diagnostics (primarily at Iowa State) and can find nothing infectious that is routinely found to explain the problem.

I suspect there is a toxin involved; I have done extensive testing on liver, feed, and water but can find no evidence of those compounds either. We have had a few individuals mention that the use of GMO crops could be contributing to these problems.

The producer recently saw your article to the secretary of agriculture and forwarded it to me. We are very intrigued by the organism you mention. Could you tell me if any laboratory is looking for this agent? How do we go about finding it? We are at the end of our rope and cannot figure this out. Any help you can give us would be greatly appreciated.

Attachment 3. Letter from 26 University Entomologists to EPA

Public Submission: EPA-HQ-OPP-2008-0836-0043. Docket EPA-HQ-OPP-2008-0836

Docket Title Evaluation of the Resistance Risks from Using a Seed Mix Refuge with Pioneer's Optimum AcreMax 1 Corn Rootworm-Protected Corn

Document EPA-HQ-OPP-2008-0836-0001; Public Submission EPA-HQ-OPP-2008-0836-0043

Public Submission Title Anonymous public comment Receipt Date 02/09/2009

Doc. Legacy ID EPA-HQ-OPP-2008-0836-0032(0900006480849377) Track No. 8084de39

General Comment

Comment The following statement has been submitted by 26 leading corn insect scientists working at public research institutions located in 16 corn producing states. All of the scientists have been active participants of the Regional Research Project NCCC-46 "Development, Optimization, and Delivery of Management Strategies for Rootworms and Other Below-ground Insect Pests of Maize" and/or related projects with corn insect pests. The statement may be applicable to all EPA decisions on PIPs, not just for the current SAP. It should not be interpreted that the actions and opinions of these 26 scientists represent those of the entire group

of scientists participating in NCCC-46. The names of the scientists have been withheld from the public docket because virtually all of us require cooperation from industry at some level to conduct our research.

Statement:

"Technology/stewardship agreements required for the purchase of genetically modified seed explicitly prohibit research. These agreements inhibit public scientists from pursuing their mandated role on behalf of the public good unless the research is approved by industry. As a result of restricted access, no truly independent research can be legally conducted on many critical questions regarding the technology, its performance, its management implications, IRM, and its interactions with insect biology. Consequently, data flowing to an EPA Scientific Advisory Panel from the public sector is unduly limited."

GMO RISKS

Title : Submission from Norway on the Risks of GMOs to Biodiversity and Human
Health
Date : 19 April 2010

THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE

Dear Friends and colleagues,

RE: Submission from Norway on the Risks of GMOs to Biodiversity and Human Health

At its fourth meeting in 2008, Parties to the Cartagena Protocol on Biosafety
established an Ad Hoc Technical Expert Group (AHTEG) on Risk Assessment and Risk
Management. The AHTEG is considering, among other things, the framework to
identify GMOs or specific traits that may have adverse effects on the
conservation and sustainable use of biological diversity, including risks to
human health.

Governments and relevant organizations were invited to submit scientifically
sound information on the types of GMOs or traits that may have adverse effects
on biological diversity and human health that would be compiled and included in
a synthesis report for consideration by the AHTEG and Parties.

In its submission, Norway highlighted information from scientific studies which
raise "early warning" signs on the effects of GMOs on biological environments
and on human health.

It noted that GMOs harbouring Bacillus thuringiensis (Bt) Cry endotoxins may
cause unintended direct adverse effects on biological diversity including but
not limited to insects, aquatic life, soil microbes, and their food web
dynamics, as well as on the sustainable use of biological diversity related to
crop plants and their progenitors important for sustainable agricultural
production and food security. Similar caution was expressed towards GMOs with
genes that confer herbicide tolerance as well as GM plants with tolerance to
abiotic stresses such as tolerance to drought and cold and GMOs with stacked
events.

In addition, Norway recommended caution with regard to GM fish with traits such
as cold tolerance, increased growth rate or high tolerance to environmental
pollutants. It also noted that GM trees with long life-spans would be a
challenge for risk assessment. Norway also expressed caution with regard to GM
viruses with altered traits and host specificity and was concerned about GM
pharmplants entering the food chain.

Given the broad uncertainties surrounding the current scientific knowledge on
the impacts of novel organisms into complex environments, Norway called for the
adoption of the precautionary approach as well as for further studies,
especially long term studies, to be conducted.

The AHTEG is holding its second meeting from 19-23 April in Slovenia, where it
will among other items on the agenda, have the synthesis report of the
submissions available for its consideration.

With best wishes,
 
Third World Network

Review

Genetically modified crops safety assessments: present limits and possible improvements

Gilles-Eric Séralini1*, Robin Mesnage1, Emilie Clair1, Steeve Gress1, Joël S de Vendômois2 and Dominique Cellier3

• * Corresponding author: Gilles-Eric Séralini criigen@unicaen.fr

Author Affiliations

1 Laboratory of Biochemistry - IBFA, University of Caen, Esplanade de la Paix, 14032 Caen, Cedex, France

2 CRIIGEN, Paris, France

3 University of Rouen LITIS EA 4108, 76821 Mont-Saint-Aignan, France

 

For all author emails, please log on.

Environmental Sciences Europe 2011, 23:10 doi:10.1186/2190-4715-23-10

 

The electronic version of this article is the complete one and can be found online at: http://www.enveurope.com/content/23/1/10

 

- Received: 17 January 2011
- Accepted: 1 March 2011
- Published: 1 March 2011

 

© 2011 Séralini et al; licensee Springer.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

We reviewed 19 studies of mammals fed with commercialized genetically modified soybean and maize which represent, per trait and plant, more than 80% of all environmental genetically modified organisms (GMOs) cultivated on a large scale, after they were modified to tolerate or produce a pesticide. We have also obtained the raw data of 90-day-long rat tests following court actions or official requests. The data obtained include biochemical blood and urine parameters of mammals eating GMOs with numerous organ weights and histopathology findings.

Methods

We have thoroughly reviewed these tests from a statistical and a biological point of view. Some of these tests used controversial protocols which are discussed and statistically significant results that were considered as not being biologically meaningful by regulatory authorities, thus raising the question of their interpretations.

Results

Several convergent data appear to indicate liver and kidney problems as end points of GMO diet effects in the above-mentioned experiments. This was confirmed by our meta-analysis of all the in vivo studies published, which revealed that the kidneys were particularly affected, concentrating 43.5% of all disrupted parameters in males, whereas the liver was more specifically disrupted in females (30.8% of all disrupted parameters).

Conclusions

The 90-day-long tests are insufficient to evaluate chronic toxicity, and the signs highlighted in the kidneys and livers could be the onset of chronic diseases. However, no minimal length for the tests is yet obligatory for any of the GMOs cultivated on a large scale, and this is socially unacceptable in terms of consumer health protection. We are suggesting that the studies should be improved and prolonged, as well as being made compulsory, and that the sexual hormones should be assessed too, and moreover, reproductive and multigenerational studies ought to be conducted too.

Background, aim, and scope

Recently, an ongoing debate on international regulation has been taking place on the capacity to predict and avoid adverse effects on health and the environment for new products and novel food/feed (GMOs, chemicals, pesticides, nanoparticles, etc.). The health risk assessments are often, but not always, based on the study of blood analyses of mammals eating these products in subchronic tests, and more rarely in chronic tests. In particular, in the case of GMOs, the number and nature of parameters assessed, the length of the necessary tests, the statistics used and their interpretations are the subject of controversies, especially in the application of Organization of Economic Cooperation and Development (OECD) norms. Confusion is perceived even in regulatory agencies, as in the European Food Safety Authority (EFSA) GMO panel working group and its guidelines. Doubt has arisen on the role and necessity of animal feeding trials in safety and nutritional assessments of GM plants and derived food and feed [1]. Based on the literature data, EFSA first admitted (p. S33) that for other tests than GMOs: "For 70% (57 of 81) of the studies evaluated, all toxicological findings in the 2-year tests were seen in or predicted by the 3-month subchronic tests". Moreover, they also indicated (p. S60) that "to detect effects on reproduction or development [...] testing of the whole food and feed beyond a 90-day rodent feeding study may be needed." We fully agree with these assumptions. This is why we think that in order to protect large populations from unintended effects of novel food or feed, imported or cultivated crops on a large scale, chronic 2-year and reproductive and developmental tests are crucial. However, they have never been requested by EFSA for commercial edible crops. We therefore wish to underline that in contrast with the statements of EFSA, all commercialized GMOs have indeed been released without such tests being carried out, and as it was the case recently with maize stacked events without 90-day in vivo mammalian tests being conducted. GM stacked events have the cumulated characteristics of first generation of GMOs (herbicide tolerance and insecticide production), which are mostly obtained by hybridization. For instance, Smarstax maize contains two genes for herbicide tolerance and six genes for insecticide production. In fact, this contradictory possibility was already highlighted in the same review by EFSA (p. S60), when substantial equivalence studies and other analyses were performed: "animal feeding trials with rodents [...] adds little if anything [...], and is not recommended." This is why, in this work we will analyze and review deficiencies in GMO safety assessments, not only performed by biotech companies, but also by regulatory agencies.

We will focus on the results of available 90-day feeding trials (or more) with commercialized GMOs, in the light of modern scientific knowledge. We also suggest here an alternative to conventional feeding trials, to understand the biological significance of statistical differences. This approach will make it possible to avoid both false negative and false positive results in order to improve safety assessments of agricultural GMOs before their commercialization for cultivation and food/feed use and imports.

Overview of the safety studies of GMOs performed on mammals

Our experience in scientific committees for the assessment of environmental and health risks of GMOs and in biological, biostatistical research, and medicine, as well as in the research relative to side effects [2-6] allowed us to review and criticize mammalian feeding trials with GMOs and make new proposals. Mammalian feeding trials have been usually but not always performed for regulatory purposes in order to obtain authorizations or commercialization for GM plant-derived foods or feed. They may have been published in the scientific literature afterwards; however, without public access to the raw data.

We have obtained, following court actions or official requests, the raw data of several 28- or 90-day-long safety tests carried out on rats. The thing we did was to thoroughly review the longest tests from both a biostatistical and a biological point of view. Such studies often analyze the biochemical blood and urine parameters of mammals eating GMOs, together with numerous organ weights and histopathology. We have focused our review on commercialized GMOs which have been cultivated in significant amounts throughout the world since 1994 (Table 1). We observe and emphasize that all the events in Table 1 correspond to soybean and maize which constitute 83% of the commercialized GMOs, whilst other GMOs not displayed in the table, but still commercialized, are canola or cotton. However, they are not usually directly consumed [7]. Only Sakamoto's and Malatesta's studies have been more than 90 days long (104 weeks and 240 days with blood analyses in Japanese for the first one). Moreover, such tests are not obligatory yet for all GMOs. No detailed blood analysis is available for Malatesta's study, as it mostly includes histochemistry at the ultrastructural level; moreover, the latter tests have not been used to obtain the commercial release by the firm. However, this work has been performed by researchers independent from the GMO industry; it is an important element to take into account for an objective interpretation of the facts, as pointed out in the case of the risk assessments conducted by regulatory agencies with Bisphenol A. For instance in the latter case, it was observed that none of the industry-funded studies showed adverse effects of Bisphenol A, whereas 90% of government-funded studies showed hazards at various levels and various doses [8]. However, regulatory agencies still continue to refer only to industry-funded studies because they are supposed to follow OECD norms, even if such standards are not always appropriate for the detection of environmental hazards [9]. In this paper, Myers et al. showed that hundreds of laboratory animals and cell culture studies were rejected by regulatory authorities because they did not follow the Good Laboratory Practices (GLP). The Food and Drug Administration and EFSA have based their final decision on two industry-funded studies, claiming that they were superior to the others because they followed GLP. Yet, GLP are based on ancient paradigms. They have serious conceptual and methodological flaws, and do not take into account the latest knowledge in environmental sciences. For example, in the case of Bisphenol A assessment, the animal models used are known to be insensitive to estrogen (CD-1 mouse). Also, assays and protocols in some OECD guidelines are out of date and insensitive. It is obvious that new product assessments should be based on adapted studies using state-of-the-art experiments. The significant gap between scientific knowledge and regulations should be filled also in the case of GMOs [9]. Therefore, some tests presented here show controversial results or statistically significant results that were not considered as biologically significant by EFSA, raising the question of their interpretation.

Table 1. Review of the longest chronic or subchronic toxicity studies in mammals fed with commercialized GM soybean and maize representing more than 80% of edible GMOs (2010).

First of all, the data indicating no biological significance of statistical effects in comparison to controls have been published mostly by companies from 2004 onwards, and at least 10 years after these GMOs were first commercialized round the world. This is a matter of grave concern. Moreover, only three events were tested for more than 90-days in feeding experiments or on more than one generation. This method was not performed by industries which conducted 90-day tests (with blood and organ analyses), but it was in some cases only. However, a 90-day period is considered as insufficient to evaluate chronic toxicity [1,5]. All these commercialized cultivated GMOs have been modified to contain pesticides, either through herbicide tolerance or by producing insecticides, or both, and could therefore be considered as "pesticide plants." Almost all GMOs only encode these two traits despite claims of numerous other traits. For instance, Roundup ready crops have been modified in order to become insensitive to glyphosate. This chemical together with adjuvants in formulations constitutes a potent herbicide. It has been used for many years as a weed killer by blocking aromatic amino acid synthesis by inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Most Roundup ready plants have been modified thanks to the insertion of a mutated EPSPS gene coding for a mutated enzyme, which is not inhibited by glyphosate. Therefore, GM plants exposed to glyphosate-based herbicides such as Roundup do not specifically degrade glyphosate. They can even accumulate Roundup residues throughout their life, even if they excrete most of such residues. Glyphosate and its main metabolite AMPA (with its own toxicity) are found in GMOs on a regular and regulatory basis [10,11]. Therefore, such residues are absorbed by people eating most GM plants (as around 80% of these plants are Roundup tolerant). On the other hand, about 20% of the other GMOs do synthesize new insecticide proteins through the insertion of mutated genes derived from Bacillus thuringiensis (Bt).

Usually, pesticides are tested over a period of 2 years on a mammal, and this quite often highlights side effects. Additionally, unintended effects of the genetic modification itself cannot be excluded, as direct or indirect consequences of insertional mutagenesis, creating possible unintended metabolic effects. For instance, in the MON810 maize, the insertion of the transgene in the ubiquitine ligase gene caused a complex recombination event, leading to the synthesis of new RNA products encoding unknown proteins [12]. Thus, genetic modifications can induce global changes in the genomic, transcriptomic, proteomic, or metabolomic profiles of the host. The frequency of such events in comparison to classical hybridization is by nature unpredictable. In addition, in a plant producing a Cry1Ab-modified toxin, a metabolomic study [13] revealed that the transgene introduced indirectly 50% changes in osmolytes and branched amino acids.

Review of statistical effects after GMO consumption

Some GMOs (Roundup tolerant and MON863) affect the body weight increase at least in one sex [2,14]. It is a parameter considered as a very good predictor of side effects in various organs. Several convergent factors appear to indicate liver and kidney problems as end points of GMO diet effects in these experiments [2,5,15,16]. This was confirmed by our meta-analysis of all in vivo studies published on this particular topic (Table 2). The kidneys are particularly affected, concentrating 42% of all parameters disrupted in males. However, other organs may be affected too, such as the heart and spleen, or blood cells [5].

Table 2. Meta-analysis of statistical differences with appropriate controls in feeding trials

Liver parameters

For one of the longest independent tests performed, a GM herbicide-tolerant soybean available on the market was used to feed mice. It caused the development of irregular hepatocyte nuclei, more nuclear pores, numerous small fibrillar centers, and abundant dense fibrillar components, indicating increased metabolic rates [17]. It was hypothesized that the herbicide residues could be responsible for that because this particular GM plant can absorb the chemicals to which it was rendered tolerant. Such chemicals may be involved in the above-mentioned pathological features. This became even clearer when Roundup residues provoked similar features in rat hepatic cells directly in vitro [18]. The reversibility observed in some instances for these parameters in vivo [19] might be explained by the heterogeneity of the herbicide residues in the feed [20]. Anyway, these are specific parameters of ultrastructural dysfunction, and the relevance is clear. The liver is reacting. The Roundup residues have been also shown to be toxic for human placental, embryonic, and umbilical cord cells [21-23]. This was also the case for hepatic human cell lines in a comparable manner, inducing nuclei and membrane changes, apoptosis and necrosis [24].

The other major GMO trait has to do with the mutated (mBt) insecticidal peptidic toxins produced by transgenes in plants. In this case, some studies with maize confirmed histopathological changes in the liver and the kidneys of rats after GM feed consumption. Such changes consist in congestion, cell nucleus border changes, and severe granular degeneration in the liver [16]. Similarly, in the MON810 studies, a significantly lower albumin/globulin ratio indicated a change in hepatic metabolism of 33% of GM-fed male rats (according to EFSA opinion on MON810 and [5]). Taken together, the results indicate potential adverse effects in hepatic metabolism. The insecticide produced by MON810 could also induce liver reactions, like many other pesticides. Of course, the mCry1Ab and other mBt (mutated Bt toxins derived from native Bacillus thuringiensis toxins) in GMOs are proteic toxins; however, these are modified at the level of their amino acid sequence by biotechnologies and introduced by artificial vectors, thus these could be considered as xenobiotics (i.e., a molecule foreign to life). The liver together with the kidneys are the major reactive organs in case of food chronic intoxication.

Kidney parameters

In the NK603 study, statistically significant strong urine ionic disturbances and kidney markers could be explained by renal leakage [5], which is well correlated with the effects of glyphosate-based herbicides (like Roundup) observed on embryonic kidney cells [23]. This does not exclude metabolic effects indirectly due to insertional mutagenesis linked to the plant transformation. Roundup adjuvants even stabilize glyphosate and allow its penetration into cells, which in turn inhibit estrogen synthesis as a side effect, cytochrome P450 aromatase inhibition [21]. This phenomenon changes the androgen/estrogen ratio and may at least, in part, explain differential impacts in both sexes.

Kidney dysfunctions are observed with mBt maize producing mutated insecticides such as in MON863. For instance, we quote the initial EFSA report: "Individual kidney weights of male rats fed with the 33% MON863 diet were statistically significantly lower compared to those of animals on control diets", "small increases in the incidences of focal inflammation and tubular regenerative changes in the kidneys of 33% MON863 males." This was confirmed by the company tests [25] and another counter analysis revealed disrupted biochemical markers typical of kidney filtration or function problems [2]. The first effects were not always but sometimes greater than the ones with non-isogenic maize (called reference lines), which contain different salts, lipids, or sugars. Moreover, both results described are different between males and females; this is quite usual in liver or kidney pesticide reactions. These facts do not exclude that such effects can be considered as treatment-related. Other studies also confirmed effects on kidneys. Tubular degeneration and not statistically significant enlargement in parietal layer of Bowman's capsules were also observed with GM maize fed rats [16].

Last but not least, a total of around 9% of parameters were disrupted in a meta-analysis (Table 2). This is twice as much as what could be obtained by chance only (generally considered as 5%). Surprisingly, 43.5% of significant different parameters were concentrated in male kidneys for all commercialized GMOs, even if only around 25% of the total parameters measured were kidney-related. If the differences had been distributed by chance in the organs, not significantly more than 25% differences would have been found in the kidney. Even if our own counter analysis is removed from the calculation, showing numerous kidney dysfunctions [2], around 32% of disturbances are still noticed in kidneys.

Discussion

Need for chronic tests and other tests

Chronic toxicity tests (both with males and females) and reproductive tests with pregnant females and then with the developing progeny over several generations (none of these steps exist at present) are called as a whole the Toxotest approach (or Risk management test, see "Details on the new suggested Toxotest approach"). This could address the long-term physiological or pathological relevance of the previous observations. The physiological interpretations of 90-day-based effects are otherwise somewhat limited. These studies should be complementary to the present regulations or the Safotest and the sentinel test suggested by EFSA [1]. The Toxotest could provide evidence of carcinogenic, developmental, hormonal, neural, and reproductive potential dysfunctions, as it does for pesticides or drugs. Additionally, it is obvious that the 90-day-long trials on mature animals performed today cannot scientifically replace the sensitivity of developmental tests on neonates. A good example is the gene imprinting by drugs that will be revealed only at maturity; this is an important subject of current research, and many findings have been reported for some chemicals such as bisphenol A [26,27]. Even transgenerational effects occur after epigenetic imprinting by a pesticide [28]. These effects cannot be detected by classical 90-day feeding trials and will be visible after many decades by epidemiology in humans if any, as illustrated in the case of diethylstilbestrol, which induced female genital cancers among other problems in the second generation [29]. The F3 multigenerational study for a GMO (Table 1) was too rarely performed. This is why, because of the number of parameters disrupted in adult mammals within 90 days, the new experiments should be systematically performed to protect the health of billions of people that could consume directly or indirectly these transformed products.

The acute toxicity approach (less than a month of investigations on rodents with high doses) may give effects which are more proportional to the dose, as it might correspond to a rapid poisoning of the animals, generally with force-fed experiments. However, for many pesticide studies in the scientific literature, some long-term side effects of pesticides at environmental doses are described, which are not apparent in short-term experiments [30]. Classical toxicology is quite often based on the concept of revealing linear dose-responses as defined by Paracelsus, which generally fails to evidence U or J curves observed after hormonal sex-specific disruptions. Moreover, the effects of mixtures are also neglected in long-term studies, when supposed active principles of pesticides are not assessed with their adjuvants, which also are present as residues in GMOs. Such pesticides may have the capacity to disrupt the "cell web", i.e., to interfere with a signaling pathway, and this could be unspecific. For instance Roundup is known to disrupt the EPSPS in plants, but is also known to interact with the mammalian ubiquist reductase [21] common and essential to cytochromes P450, a wide class of detoxification enzymes. The so-called Roundup active principle, glyphosate, acts in combination with adjuvants to increase glyphosate-mediated toxicity[21,31], and this may apply to other environmental pollutants [22]. Moreover, all new metabolites in edible Roundup ready GMOs, as acetyl-glyphosate for the new GAT GMOs, have not been assessed for their chronic toxicity [11], and we consider this as a major oversight in the present regulations.

Therefore, as xenobiotic effects are complex, the determination of their toxic effects cannot be determined using a single method, but rather converging pieces of evidence. In GMO risk assessment, the protocols must be optimized to detect side effects, in particular for herbicide-treated GM plants. These cannot be reduced to GM assessment on one side and herbicide residues with any diet on the other side, but unfortunately this has been the case, and this approach has been promoted up to now by regulatory authorities.

In fact, it is impossible, within only 13 weeks, to conclude about the kind of pathology that could be induced by pesticide GMOs and whether it is a major pathology or a minor one. It is therefore necessary to prolong the tests, as suggested by EFSA, since at least one third of chronic effects visible with chemicals are usually new in comparison to the ones highlighted in subchronic studies [1]. The so-called Toxotests, which are supposed to include the studies of chronic pathologies in particular, should be performed on three mammalian species, with at least one non-rodent, similar to the type of rodents used for pesticides and drugs. However, the chronic feeding tests for GMOs cannot be based on the no observed adverse effect level, nor on the lowest observed adverse effect level approach, as in classical toxicology. There are several reasons for that. There is not only one chemical, but also several unknown metabolites and components, in Roundup tolerant varieties for instance, and therefore toxicity is enhanced thanks to the fact that they are mixed together. There is also no possibility of increasing the doses of GMOs in an equilibrated diet over an acceptable level. The diets should be rather representative of an equilibrated diet with GMOs like it could be the case in a real population in America. To prolong 90-day subchronic tests with three normal doses of GM in the diet (11%, 22%, 33% for instance) is the solution.

Sex- or dose-specific pathological effects are common

When there is a low or environmental dose impregnation of the feed (with a pesticide GM plant for instance), the chronic effects could be more differentiated according to the sex, the physiological status, the age, or the number of intakes over such and such a period of time in the case of a drug. These parameters (chronic intake, age of exposure, etc.) are more decisive for pathologies like cancers, than the actual quantity of toxin ingested in one intake. This is in part because the liver, kidney, and other cytochrome P450-rich organs are concerned for long-term metabolism and detoxification, and this phenomenon is hormone dependent. It is also due to the process of carcinogenesis or hormone-sensitive programming of cells [32]. The liver for instance is a sex differentiated organ as far as its enzymatic equipment is concerned [4]. An effect in subchronic or chronic tests cannot be disregarded on the rationale that it is not linear to the dose (or dose-related) or not comparable in genders. This would not be scientifically acceptable. However, this reasoning was adopted both by companies and EFSA for several GMOs, as underlined by Doull et al. [33]. Indeed, most xenobiotics or pollutants may have non-linear effects, and/or may have sex- and age-specific impacts.

One of the pivotal requirements for regulators nowadays, in order to interpret a significant difference as biologically relevant, is to observe a linear dose-response. This allows them to deduce a causality. However, this dose-response cannot be studied with only two points, which is nonetheless the case for all major commercial GMOs today, which are given in the diet in 11% and 33% concentrations only, in subchronic tests. This is true overall if no preliminary data has been obtained to choose the given doses, which is the case in regulatory files. As we have already emphasized, most of pathological and endocrine effects in environmental health are not directly proportional to the dose, and they have a differential threshold of sensitivity in both sexes [34]. This is, for instance, the case with carcinogenesis and endocrine disruption.

Improving the knowledge on impacts of modified Bt toxins

One of the interpretations of the side effects observed (Tables 1 and 2) would be that the insecticide toxins in maize lines may have more pleiotropic or specific actions than originally supposed. The toxins could generate particular metabolites, either in the GM plant or in the animals fed with it. The Bt toxins in GMOs are new and modified, truncated, or chimerical in order to change their activities/solubility in comparison to wild Bt. For instance, there is at least a 40% difference between the toxin in Bt176 and its wild counterpart [10]. None of the modified Bt toxins have been authorized separately for food or feed, neither has the wild Bt, and neither have they been tested by themselves on animal or human health to date. Even if some studies were performed, the receptors have not been cloned and the signaling pathways have not been identified as yet, nor required for authorizations, and the metabolism of these proteins in mammals are unknown [35]. Thus, the argument about "safe use history" of the wild Bt protein (not designed for direct consumption, in contrast to several GMOs) cannot, on a sound scientific basis, be used for direct authorizations of the above-cited GM corns, overall without in vivo chronic toxicity tests (or Toxotest approach), as it is requested for a pesticide. Some improvements may even be included with regard to pesticide legislation, since these human modified toxins considered as xenobiotics are continuously produced by the plants devoted to consumption.

The proteins usually compared (modified Bt toxins and wild ones) are not identical, and the tests on human cells of Bt proteins are not performed nor are they requested by authorities. Their stability has been assessed in vitro, and GM insecticide toxins are never fully digested in vivo [36]. If some consumers suffer from stomach problems or ulcers, the new toxins will possibly act differently; the digestion in children could be affected too; however, these GMOs could be eaten anywhere and all proteins are never fully decomposed in amino acids by the digestive tract.

Details on the new suggested Toxotest approach

The suggested Toxotest would basically include an extension of the existing 90-day tests, but with at least three doses plus controls (0%, 11%, 22%, 33% GMOs for instance; today the equilibrated diets tested contain 0%, 11%, and 33% GMOs in the best regulatory tests). The purpose would be to characterize scientifically the dose-response approach. The latter cannot be taken seriously with only two GM doses. The final goal is the best health protection for the population without really possible clinical trials, in our case for practical and ethical reasons. There is also no epidemiological follow-up for lack of traceability and labeling in GM-producing American countries. In addition, the fact that the Toxotest includes the best possible toxicological approach will also be in favor of the biotechnology economy and the European Community because it is more expensive to address an issue concerning a whole population afterwards, rather than to work with laboratory animals beforehand; it is also more ethical to work on rats and other mammalian experiments, in order to get the relevant information, rather than to give pesticide plants directly to humans on a long-term basis.

As previously underlined, the health effects such as those suggested in Table 2 (if any, are revealed by adapted studies, such as Safotests or Toxotests), could only be due to two possibilities:

Firstly, the side effects may be directly or indirectly due to a pesticide residue and/or its metabolites. The direct effect is about the pesticide effect on the consumer, and the indirect one is about a metabolism disruption that it has provoked within the plant first. This could not be visible by a detailed compositional analysis, such as the one performed to be assessed by a substantial equivalence study. This concept is not a well-defined one (how many cultivations of crops, over how many years, under which climate, and to measure what precise parameters).

Secondly, the pathological signs may be due to the genetic transformation itself, its method provoking either insertional mutagenesis or a new metabolism by genetic interference. This is the reason why separating intended effects (the direct genetic trait consequence itself) from unintended effects (linked to biotechnology, e.g., insertional mutagenesis), such as spiking the control diet with the purified toxin in the Toxotest approach, is clearly inadequate. It could work in the case of a direct action of the toxin in mammals, but conversely one could not conclude, between an insertional mutagenesis and a specific metabolic action in the plant due to the toxin. However, this is more a research question about the mode of genesis of an effect on health, and new research avenues could be, for instance, to compare the GM diet with or without herbicide treatment in long-term tests with the isogenic control diet including herbicide residues added. This is only necessary for the understanding of the potential signs of toxicity and not for a conclusion of the Safotest or the Toxotest, which would rather suggest, if positive, excluding immediately the corresponding GMO from food and feed.

Improvement of statistical analysis

A serious experimental design is based on a proper choice of the groups, with only one question studied per experiment if possible, and balanced sample sizes. In several authorized GMOs, the sample sizes appear inadequate in 90 days: ten animals per group for the measurement of biochemical parameters out of 20, as performed by the major stakeholders, and accepted by EFSA for MON863, MON810, or NK603 for instance. This is too limited a size to ensure that parametric statistical methods used by the company are reliable. Moreover, an important discrepancy between GMO-treated rats (40 measured out of 80) and the total number of animals (400) renders more difficult the evidencing of relevant effects, and confusion factors are brought in at the same time with six different reference diets in addition to the two normal control groups as performed in three commercialized GMOs at least [5,6]. This introduces new uncontrolled sources of variability about the effects of the diets and new unnecessary questions not relevant to the GMO safety. The representation of a standard diet with multiple sources could have been studied with only one control group of the same size than the GMO group, eating a mix of six different regular non-GM diets.

Several questions have been raised by companies and authorities as well as comments on statistically significant effects that would supposedly not be biologically meaningful. A subjective part is introduced at this level because it is necessary to take into account the context and the general and detailed knowledge of toxicology and endocrine disruption, as EFSA underlines. This might be highly expert dependent. This is why, to avoid or prevent any misunderstanding, we suggest, in addition to a new statistical approach based on classical methods, to analyze the 90-day tests, even with control and reference diets called the "SSC method" (according to the initials of the authors in [2]).

Briefly, following the necessity to model and analyze the growth curves, multivariate data analysis and data mining of all parameters can be used to correlate, cluster, and select meaningful variables. This kind of approach is not performed at all today. Thereafter, the detailed comparison between GM-treated and control groups, fed with the near isogenic line (because the real isogenic line does not often exists anymore), will necessarily be followed by the study of specific diet effects, when there are non-substantially equivalent diets for reference groups. For that purpose, the controls will be first compared using multivariate inference with reference groups, and thereafter, similarly GMO-treated groups with reference groups. The significant differences linked to the GMO and/or the composition of the diet will be classified according to organ and function. The results will appear more clearly than with the simple statistics accepted today by the authorities (that is, comparison of the highest GM dose group with the mean value of all six control groups), and will reveal in addition new information, as it can be demonstrated.

As recommended by EFSA, an appropriate and relevant statistical analysis is crucial. It should follow the following series of steps, allowing the use of several methods depending on the questions raised:

• Obtaining and modeling the growth curves and feed consumption, assessed by non-linear regression, validation, and statistical comparisons in order to test if the curves are significantly different, thus taking into account individual variability. This necessitates the use of time series analysis, selection models, and non-parametric tests, Akaike Information Criteria and related methods. Water consumption should also be an important factor to follow-up and therefore better understand kidney and urine data.

• The study of dose-response predictions using non-linear regression should be the goal, but the only two doses generally used in these tests do not make it possible to evidence linearity as we indicated. Moreover, in the cases where there are not dose-related trends or relationships using the two doses mentioned, the absence of linear dose-response curves cannot be a reason to neglect the effects. For instance, as previously cited, U or J curves may be characteristic of endocrine effects [37], and spiky irregular curves may be detected in carcinogenesis.

• Simultaneous analysis of all observed variables: multivariate data analysis, principal component analysis, correlations analysis, factorial analysis and clustering

• Multivariate comparisons of the different variables: hypothesis testing, multiple ways ANOVA, MANOVA, and others to determinate if the groups differ relative to the different questions: specific GMO effect or diet effect per se. To evidence a detail, when comparing two mean values, SEM should be calculated to determine confidence intervals; however, SD have been used up to now by the company for MON863 and NK603 files for instance.

Apart from empirical curves in some instances, ANOVA and univariate hypothesis testing only the GMO effect, none of the other statistical approaches is currently used nor requested by the authorities.

Human tests and post-market monitoring

For the record, it must be said that very few tests on humans have been carried out up to now. Moreover, epidemiological studies are not feasible in America, since there is no organized traceability of GMOs anywhere on the continent, where, by far, most of edible GMOs are cultivated (97%). As a consequence, a post-market monitoring (PMM) is offered to the population. The Cartagena Biosafety Protocol identifying GMOs at the borders of a country has now been signed by over 150 countries, including the member states of the European Union. PMM may have some value in detecting unexpected adverse effects. It could therefore be considered as a routine need. This approach makes it possible to collect information related to risk management. It can be relied upon as a technique for monitoring adverse events or other health outcomes related to the consumption of GM plant-derived foods, provided that the Toxotest approach, together with the SSC method, should have already been applied. The PMM should be linked with the possibility of detecting allergenicity reactions to GMOs in routine medicine, thanks to the very same routine cutaneous tests that should be developed prior to large-scale commercialization. A screening of serum banks of patients with allergies could be also put forward in order to search for antibodies against the main GMOs and not only their transgenic proteins, since they may induce secondary allergenic metabolites in the plant not visible in the substantial equivalence study.

The traceability of products from animals fed on GMOs is also crucial. The reason for this is because they can develop chronic diseases which are not utterly known today. Such possible diseases could be linked to the hepatorenal toxicity observed in some GMO-related cases (Table 1).

Moreover, labeling animals fed on GMOs is therefore necessary because some pesticide residues linked to GMOs could pass into the food chain and also because nobody would want to eat disabled or physiologically modified animals after long-term GMOs ingestion, even if pesticides residues or DNA fragments are not toxic nor transmitted by themselves.

Conclusion

Transcriptomics, proteomics and other related methods are not ready yet for routine use in the laboratories, and moreover they may be inappropriate for studying toxicity in animals, and could not in any way replace in vivo studies with all the physiological and biochemical parameters that are measured with organs weight, appearance, and histology. By contrast, afterwards, new approaches could well help to explain pathological results or action mechanisms of pesticides present in the GM plants or GM-fed animals, if found.

To obtain the transparency of raw data (including rat blood analyses) for toxicological tests, maintained illegally confidential, is crucial. It has also become crucial to apply objective criteria of interpretation like the criteria described here: sex-specific side effects or non-linear ones. Such data can be put online on the EFSA website with a view to provide a fuller review to the wider scientific community, and in order to better inform the citizen to make biotechnologies more socially acceptable. Since fundamental research is published on a regular basis, it should be the same for this kind of applied research on long-term health effects, as suggested by the CE/2001/18 and the corresponding 1829/2003 regulations.

We can conclude, from the regulatory tests performed today, that it is unacceptable to submit 500 million Europeans and several billions of consumers worldwide to the new pesticide GM-derived foods or feed, this being done without more controls (if any) than the only 3-month-long toxicological tests and using only one mammalian species, especially since there is growing evidence of concern (Tables 1 and 2). This is why we propose to improve the protocol of the 90-day studies to 2-year studies with mature rats, using the Toxotest approach, which should be rendered obligatory, and including sexual hormones assessment too. The reproductive, developmental, and transgenerational studies should also be performed. The new SSC statistical method of analysis is proposed in addition. This should not be optional if the plant is designed to contain a pesticide (as it is the case for more than 99% of cultivated commercialized GMOs), whilst for others, depending on the inserted trait, a case-by-case approach in the method to study toxicity will be necessary.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

GES designed and coordinated the review. RM participated in the drafting of the manuscript and final version. EC, SG, JSV and DC helped the writing, compiling the literature, revising in details and proofreading the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank the CRIIGEN scientific committee for helpful discussions and structural support, as well as the Risk Pole (MRSH-CNRS, University of Caen, France). We acknowledge the French Ministry of Research for financial support and the Regional Council of Basse-Normandie. We are grateful to Herrade Hemmerdinger for the English revision of this manuscript.

References

1. 1.         EFSA: Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials.

Food Chem Toxicol 2008, 46:S2-70.

1. 2.         Séralini GE, Cellier D, Spiroux J: New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity.

Arch Environ Contam Toxicol 2007, 52:596-602. PubMed Abstract | Publisher Full Text

1. 3.         Séralini G-E: Comment on Transgenic aubergines put on ice.

Naturenews 2009.

1. 4.         Séralini GE, Spiroux J, Cellier D, Sultan C, Buiatti M, Gallagher L, Antoniou M, Dronamraju KR: How subchronic and chronic health effects can be neglected for GMOs, pesticides or chemicals.

Int J Biol Sci 2009, 5:438-443. PubMed Abstract | PubMed Central Full Text

1. 5.         Spiroux J, Roullier F, Cellier D, Séralini GE: A comparison of the effects of three GM corn varieties on mammalian health.

Int J Biol Sci 2009, 5:706-726. PubMed Abstract | PubMed Central Full Text

1. 6.         Spiroux J, Cellier D, Vélot C, Clair E, Mesnage R, Séralini GE: Debate on GMOs health risks after statistical findings in regulatory tests.

Int J Biol Sci 2010, 6:590-598. PubMed Abstract | PubMed Central Full Text

1. 7.         James C: Global Status of Commercialized Biotech/GM Crops.

ISAAA Brief 41 2009.

1. 8.         Vom Saal FS, Hughes C: An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment.

Environ Health Perspect 2005, 113:926-933. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 9.         Myers JP, vom Saal FS, Akingbemi BT, Arizono K, Belcher S, Colborn T, Chahoud I, Crain DA, Farabollini F, Guillette LJ Jr, Hassold T, Ho SM, Hunt PA, Iguchi T, Jobling S, Kanno J, Laufer H, Marcus M, McLachlan JA, Nadal A, Oehlmann J, Olea N, Palanza P, Parmigiani S, Rubin BS, Schoenfelder G, Sonnenschein C, Soto AM, Talsness CE, Taylor JA, Vandenberg LN, Vandenbergh JG, Vogel S, Watson CS, Welshons WV, Zoeller RT: Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

Environ Health Perspect 2009, 117:309-315. PubMed Abstract | PubMed Central Full Text

1. 10.      Séralini G-E: Ces OGM qui changent le monde. France: Flammarion; 2004.

1. 11.      EFSA: Modification of the residue definition of glyphosate in genetically modified maize grain and soybeans, and in products of animal origin on request from the European Commission.

EFSA Journal 2009, 7:42.

1. 12.      Rosati A, Bogani P, Santarlasci A, Buiatti M: Characterisation of 3' transgene insertion site and derived mRNAs in MON810 YieldGard maize.

Plant Mol Biol 2008, 67:271-81. PubMed Abstract | Publisher Full Text

1. 13.      Manetti C, Bianchetti C, Casciani L, Castro C, Di Cocco ME, Miccheli A, Motto M, Conti F: A metabonomic study of transgenic maize (Zea mays) seeds revealed variations in osmolytes and branched amino acids.

J Exp Bot 2006, 57:2613-2625. PubMed Abstract | Publisher Full Text

1. 14.      Zhu Y, Li D, Wang F, Yin J, Jin H: Nutritional assessment and fate of DNA of soybean meal from roundup ready or conventional soybeans using rats.

Arch Anim Nutr 2004, 58:295-310. PubMed Abstract | Publisher Full Text

1. 15.      Vecchio L, Cisterna B, Malatesta M, Martin TE, Biggiogera M: Ultrastructural analysis of testes from mice fed on genetically modified soybean.

Eur J Histochem 2004, 48:448-454. PubMed Abstract

1. 16.      Kilic A, Akay MT: A three generation study with genetically modified Bt corn in rats: biochemical and histopathological investigation.

Food Chem Toxicol 2008, 46:1164-1170. PubMed Abstract | Publisher Full Text

1. 17.      Malatesta M, Caporaloni C, Gavaudan S, Rocchi MB, Serafini S, Tiberi C, Gazzanelli G: Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean.

Cell Struct Funct 2002, 27:173-180. PubMed Abstract | Publisher Full Text

1. 18.      Malatesta M, Perdoni F, Santin G, Battistelli S, Muller S, Biggiogera M: Hepatoma tissue culture (HTC) cells as a model for investigating the effects of low concentrations of herbicide on cell structure and function.

Toxicol In Vitro 2008, 22:1853-1860. PubMed Abstract | Publisher Full Text

1. 19.      Malatesta M, Tiberi C, Baldelli B, Battistelli S, Manuali E, Biggiogera M: Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean.

Eur J Histochem 2005, 49:237-242. PubMed Abstract

1. 20.      Arregui MC, Lenardon A, Sanchez D, Maitre MI, Scotta R, Enrique S: Monitoring glyphosate residues in transgenic glyphosate-resistant soybean.

Pest Manag Sci 2004, 60:163-166. PubMed Abstract | Publisher Full Text

1. 21.      Richard S, Moslemi S, Sipahutar H, Benachour N, Séralini GE: Differential effects of glyphosate and roundup on human placental cells and aromatase.

Environ Health Perspect 2005, 113:716-720. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 22.      Benachour N, Sipahutar H, Moslemi S, Gasnier C, Travert C, Séralini GE: Time- and dose-dependent effects of roundup on human embryonic and placental cells.

Arch Environ Contam Toxicol 2007, 53:126-133. PubMed Abstract | Publisher Full Text

1. 23.      Benachour N, Séralini GE: Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells.

Chem Res Toxicol 2009, 22:97-105. PubMed Abstract | Publisher Full Text

1. 24.      Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC, Séralini GE: Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines.

Toxicology 2009, 262:184-191. PubMed Abstract | Publisher Full Text

1. 25.      Hammond B, Lemen J, Dudek R, Ward D, Jiang C, Nemeth M, Burns J: Results of a 90-day safety assurance study with rats fed grain from corn rootworm-protected corn.

Food Chem Toxicol 2006, 44:147-160. PubMed Abstract | Publisher Full Text

1. 26.      Braniste V, Jouault A, Gaultier E, Polizzi A, Buisson-Brenac C, Leveque M, Martin PG, Theodorou V, Fioramonti J, Houdeau E: Impact of oral bisphenol A at reference doses on intestinal barrier function and sex differences after perinatal exposure in rats.

Proc Natl Acad Sci USA 2009, 107:448-453. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 27.      Braun JM, Yolton K, Dietrich KN, Hornung R, Ye X, Calafat AM, Lanphear BP: Prenatal bisphenol A exposure and early childhood behavior.

Environ Health Perspect 2009, 117:1945-1952. PubMed Abstract | PubMed Central Full Text

1. 28.      Anway MD, Cupp AS, Uzumcu M, Skinner MK: Epigenetic transgenerational actions of endocrine disruptors and male fertility.

Science 2005, 308:1466-1469. PubMed Abstract | Publisher Full Text

1. 29.      Wise LA, Palmer JR, Rowlings K, Kaufman RH, Herbst AL, Noller KL, Titus-Ernstoff L, Troisi R, Hatch EE, Robboy SJ: Risk of benign gynecologic tumors in relation to prenatal diethylstilbestrol exposure.

Obstet Gynecol 2005, 105:167-173. PubMed Abstract | Publisher Full Text

1. 30.      Hernandez AF, Casado I, Pena G, Gil F, Villanueva E, Pla A: Low level of exposure to pesticides leads to lung dysfunction in occupationally exposed subjects.

Inhal Toxicol 2008, 20:839-849. PubMed Abstract | Publisher Full Text

1. 31.      Benachour N, Moslemi S, Sipahutar H, Séralini GE: Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination.

Toxicol Appl Pharmacol 2007, 222:129-140. PubMed Abstract | Publisher Full Text

1. 32.      Melnick R, Lucier G, Wolfe M, Hall R, Stancel G, Prins G, Gallo M, Reuhl K, Ho SM, Brown T, Moore J, Leakey J, Haseman J, Kohn M: Summary of the National Toxicology Program's report of the endocrine disruptors low-dose peer review.

Environ Health Perspect 2002, 110:427-431. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 33.      Doull J, Gaylor D, Greim HA, Lovell DP, Lynch B, Munro IC: Report of an Expert Panel on the reanalysis by of a 90-day study conducted by Monsanto in support of the safety of a genetically modified corn variety (MON 863).

Food Chem Toxicol 2007, 45(11):2073-85. PubMed Abstract | Publisher Full Text

1. 34.      Goldsmith JR, Kordysh E: Why dose-response relationships are often non-linear and some consequences.

J Expo Anal Environ Epidemiol 1993, 3:259-276. PubMed Abstract

1. 35.      Then C: Risk assessment of toxins derived from Bacillus thuringiensis-synergism, efficacy, and selectivity.

Environ Sci Pollut Res Int 2010, 17:791-797. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 36.      Paul V, Guertler P, Wiedemann S, Meyer HH: Degradation of Cry1Ab protein from genetically modified maize (MON810) in relation to total dietary feed proteins in dairy cow digestion.

Transgenic Res 2010, 19(4):683-689. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 37.      Andrade AJ, Grande SW, Talsness CE, Grote K, Chahoud I: A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): non-monotonic dose-response and low dose effects on rat brain aromatase activity.

Toxicology 2006, 227:185-192. PubMed Abstract | Publisher Full Text

1. 38.      Malatesta M, Caporaloni C, Rossi L, Battistelli S, Rocchi MB, Tonucci F, Gazzanelli G: Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean.

J Anat 2002, 201:409-415. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

1. 39.      Malatesta M, Biggiogera M, Manuali E, Rocchi MB, Baldelli B, Gazzanelli G: Fine structural analyses of pancreatic acinar cell nuclei from mice fed on genetically modified soybean.

Eur J Histochem 2003, 47:385-388. PubMed Abstract

1. 40.      Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA, Delaney B: Subchronic feeding study of herbicide-tolerant soybean DP-356O43-5 in Sprague-Dawley rats.

Food Chem Toxicol 2008, 46:2201-2213. PubMed Abstract | Publisher Full Text

1. 41.      Sakamoto Y, Tada Y, Fukumori N, Tayama K, Ando H, Takahashi H, Kubo Y, Nagasawa A, Yano N, Yuzawa K, Ogata A: A 104-week feeding study of genetically modified soybeans in f344 rats.

Shokuhin Eiseigaku Zasshi 2008, 49:272-282. PubMed Abstract | Publisher Full Text

1. 42.      Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA, Delaney B: Subchronic feeding study of grain from herbicide-tolerant maize DP-O9814O-6 in Sprague-Dawley rats.

Food Chem Toxicol 2009, 47:2269-2280. PubMed Abstract | Publisher Full Text

1. 43.      Hammond B, Dudek R, Lemen J, Nemeth M: Results of a 13 week safety assurance study with rats fed grain from glyphosate tolerant corn.

Food Chem Toxicol 2004, 42:1003-1014. PubMed Abstract | Publisher Full Text

1. 44.      Hammond BG, Dudek R, Lemen JK, Nemeth MA: Results of a 90-day safety assurance study with rats fed grain from corn borer-protected corn.

Food Chem Toxicol 2006, 44:1092-1099. PubMed Abstract | Publisher Full Text

1. 45.      MacKenzie SA, Lamb I, Schmidt J, Deege L, Morrisey MJ, Harper M, Layton RJ, Prochaska LM, Sanders C, Locke M, Mattsson JL, Fuentes A, Delaney B: Thirteen week feeding study with transgenic maize grain containing event DAS-O15O7-1 in Sprague-Dawley rats.

Food Chem Toxicol 2007, 45:551-562. PubMed Abstract | Publisher Full Text

1. 46.      He XY, Huang KL, Li X, Qin W, Delaney B, Luo YB: Comparison of grain from corn rootworm resistant transgenic DAS-59122-7 maize with non-transgenic maize grain in a 90-day feeding study in Sprague-Dawley rats.

Food Chem Toxicol 2008, 46:1994-2002. PubMed Abstract | Publisher Full Text

1. 47.      Malley LA, Everds NE, Reynolds J, Mann PC, Lamb I, Rood T, Schmidt J, Layton RJ, Prochaska LM, Hinds M, Locke M, Chui CF, Claussen F, Mattsson JL, Delaney B: Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats.

Food Chem Toxicol 2007, 45:1277-1292. PubMed Abstract | Publisher Full Text

1. 48.      Appenzeller LM, Malley L, Mackenzie SA, Hoban D, Delaney B: Subchronic feeding study with genetically modified stacked trait lepidopteran and coleopteran resistant (DAS-O15O7-1xDAS-59122-7) maize grain in Sprague-Dawley rats.

Food Chem Toxicol 2009, 47:1512-1520. PubMed Abstract | Publisher Full Text