NK603

NK603 is a variety of Roundup Ready corn made by Monsanto that was deregulated in the U.S. in 2000 and commercialized in 2001 under the brand name "Roundup Ready 2." It has been genetically engineered to survive being sprayed with the herbicide glyphosate, the active ingredient in Monsanto's herbicide Roundup. Roundup Ready crops, and genetically modified organisms are controversial around the world. A study published in 2012 linked this variety of GM maize to tumors in rats.

Study Links NK603 to Tumors in Rats
On September 19, 2012, a team led by Gilles-Eric Séralini of the University of Caen published a study called "Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize" in the journal Food and Chemical Toxicology. The study acknowledged "major support" from the Association CERES, the Foundation "Charles Leopold Mayer pour le Progrès de l’Homme," the French Ministry of Research, and CRIIGEN. According to the study, there have been previous studies that led researchers to believe that consuming Roundup Ready crops, including residual amounts of the glyphosate (Roundup) sprayed on them, could lead to health problems.


 * "Detailed analyses have revealed alterations in kidney and liver functions that may be the signs of early chronic diet intoxication, possibly explained at least in part by pesticide residues in the GM feed (Séralini et al., 2007; Spiroux de Vendômois et al., 2009). Indeed, it has been demonstrated that R [Roundup] concentrations in the range of 103 times below the MRL [maximum residual level] induced endocrine disturbances in human cells (Gasnier et al., 2009) and toxic effects thereafter (Benachour and Seralini, 2009), including in vivo (Romano et al., 2012). After several months of consumption of an R-tolerant soy, the liver and pancreas of mice were affected, as highlighted by disturbances in sub-nuclear structure (Malatesta et al., 2008a, 2002a,b). Furthermore, this toxic effect was reproduced by the application of R herbicide directly to hepatocytes in culture (Malatesta et al., 2008b).

The study acknowledges that "long-term and multi-generational animal feeding trials have been performed with some possibly providing evidence of safety, while others conclude on the necessity of further investigations because of metabolic modifications (Snell et al., 2011)" but adds that "none of these studies have included a detailed followup of the animals with up to 11 blood and urine samples over 2 years, and none has investigated the NK603 R-tolerant maize."

Study Design
The study began by growing Monsanto's NK603 Roundup Ready corn and "its nearest isogenic non-transgenic control" in the same location, similar conditions, spaced "at a sufficient distance to avoid cross-contamination." One field of NK603 was treated with 3 liters/hectare of Roundup WeatherMAX®, which contains 540 grams/liter of glyphosate as well as inactive adjuvants like surfactants that improve the ability of the glyphosate to penetrate cells. Another field of NK603 was not treated with Roundup. Following harvest, the researchers had three groups of corn: non-GMO untreated corn, GMO untreated corn, and GMO treated corn. From that, they formulated three different sets of rat chow (one with each group of corn). The rat chow was made "based on the standard diet A04 (Safe, France)." "The dry rat feed was made to contain 11, 22 or 33% GM maize, cultivated either with or without R [Roundup], or 33% of the non-transgenic control line."

The study used 200 rats, 100 male and 100 female. The rats used were Virgin albino Sprague-Dawley rats, obtained at 5 weeks of age and kept in polycarbonate cages with two rats of the same sex per cage. After 20 days, the 100 male and 100 female rats were "randomly assigned on a weight basis into 10 equivalent groups." A control group of each sex had access to plain water and standard non-GMO feed. Six groups received feed formulated with 11, 22 and 33% GM NK603 maize either treated or not with Roundup. The remaining three groups ate the control diet but drank water supplemented with Roundup in formulation. One drank water contaminated at 0.1 ppb of Roundup, a level found in some tap waters. The second drank water at 0.09% Roundup (400mg/kg, the U.S. maximum residue level of glyphosate in some GM feed). The third group drank water contaminated with Roundup at a rate of 0.5%, which is half the minimal amount used in agriculture. The animals' health was monitored twice weekly, blood and urine tests were performed 11 times at intervals of one to three months, and the study lasted two years. During the study, any rats who were severely suffering were put down as required by research ethics laws and their organs were collected for analysis.

Results
"Control male animals survived on average 624 ± 21 days, whilst females lived for 701 ± 20, during the experiment, plus in each case 5 weeks of age at the beginning and 3 weeks of stabilization period. After mean survival time had elapsed, any deaths that occurred were considered to be largely due to aging." Of the 20 control rats (10 male, 10 female), "30% control males (three in total) and 20% females (only two) died spontaneously prior to the time at which their deaths would be attributed to old age."

"Up to 14 months, no animals in the control groups showed any signs of tumors whilst 10–30% of treated females per group developed tumors, with the exception of one group (33% GMO + R). By the beginning of the 24th month, 50–80% of female animals had developed tumors in all treated groups, with up to 3 tumors per animal, whereas only 30% of controls were affected. The R [Roundup] treatment groups showed the greatest rates of tumor incidence with 80% of animals affected with up to 3 tumors for one female, in each group."

The most affected organ in females was the mammary, followed by the pituitary gland. In males, the most affected organ was the liver, together with the hepatodigestive tract and kidneys. The study includes several grotesque photos of rats with large tumors.

The researchers performed a statistical analysis on lab results obtained in the study's 15th month, as that was the last time when most of the rats were alive (in treated groups 90% males, 94% females, and 100% controls). They found:


 * "OPLS-DA 2-class models were built between each treated group per sex and controls. Only models with an explained variance R2(Y)P80%, and a cross-validated predictive ability Q2(Y)P60%, were used for selection of the discriminant variables (Fig. 5A), when their regression coefficients were significant at 99% confidence level. Thus, in treated females, kidney failures appeared at the biochemical level (82% of the total disrupted parameters). Ions (Na, Cl) or urea increased in urine. Accordingly, the same ions decreased in serum (Fig. 5B) as did the levels of P, K and Ca. Creatinine or clairance decreased in urine for all treatment groups in comparison to female controls (Table 3). In GM maize treated males (with or without R), 87% of discriminant variables were kidney related, but the disrupted profiles were less obvious because of advanced chronic nephropathies and deaths. In summary, for all treatments and both sexes, 76% of the discriminant variables versus controls were kidney related.


 * "Moreover, in females (Table 3) the androgen/estrogen balance in serum was modified by GM maize and R treatments (at least 95% confidence level, Fig. 5B), and for male animals at the highest R-treatment dose, levels of estrogens were more than doubled."

Although the treatment groups showed increased mortality and morbidity compared to the controls, the results were not in proportion to the amount of GM maize and Roundup each group consumed. The study authors wrote:


 * "Our data show that, as is often the case for hormonal diseases, most observed effects in this study were not proportional to the dose of the treatment (GM maize with and without R application; R alone), non-monotonic and with a threshold effect (Vandenberg et al., 2012). Similar degrees of pathological symptoms were noticed in this study to occur from the lowest to the highest doses suggesting a threshold effect. This corresponds to levels likely to arise from consumption or environmental exposure, such as either 11% GM maize in food, or 50 ng/L of glyphosate in R-formulation as can be found in some contaminated drinking tap waters, and which fall within authorized limits."

Additionally, they found that "All treatments in both sexes enhanced large tumor incidence by 2–3-fold in comparison to our controls but also for the number of mammary tumors in comparison to" other studies of the same type of rats (Brix et al., 2005 and Chandra et al., 1992). In the Seralini study, the tumors in the treatment groups developed "considerably faster" than in the controls. Significantly, the first large detectable tumors occurred "at 4 and 7 months into the study in males and females respectively, underlining the inadequacy of the standard 90 day feeding trials for evaluating GM crop and food toxicity (Séralini et al., 2011)." (emphasis added)

The authors link the large number of mammary tumors to estrogen, writing that "even at the very lowest dose administered. R has been shown to disrupt aromatase which synthesizes estrogens (Richard et al., 2005), but to also interfere with estrogen and androgen receptors in cells (Gasnier et al., 2009). In addition, R appears to be a sex endocrine disruptor in vivo, also in males (Romano et al., 2010). Sex steroids are also modified in treated rats. These hormone-dependent phenomena are confirmed by enhanced pituitary dysfunction in treated females."

For rats fed Roundup Ready corn (NK603) that was grown without any use of Roundup, "similar effects with respect to enhanced tumor incidence and mortality rates were observed." The authors provide a hypothesis for why this occurred and conclude that "both the NK603 maize and R may cause hormonal disturbances in the same biochemical and physiological pathway."

Criticism and Evaluation of the Study
As with any study critical of GMOs, the study was immediately met with criticism when it was published.

Feed Quantities, Growth Rates, Rat Breed, and Statistical Methods
Dr. Tom A. Sanders, head of the nutritional sciences research division at King's College London, questioned why the study did not include how much the rats were given to eat, or what their growth rates were, saying: "This strain of rat is very prone to mammary tumors particularly when food intake is not restricted. The statistical methods are unconventional ... and it would appear the authors have gone on a statistical fishing trip."

Dr. Michael Antoniou of CRIIGEN (the Committee of Research & Independent Information on Genetic Engineering), an organization that supported the study, is a colleague of Dr. Sanders' at Kings College London. He vigorously refuted Dr. Sanders' criticism, commenting that the same breed of rat, the Sprague-Dawley or SD, was used in the original glyphosate toxicity studies. He continued, "In addition, many studies -- including many from industry -- on GM foods use SD rats. Based on this history of use, it was appropriate to use this strain too. If it was the wrong strain to use here then it was wrong in many previous GM food safety feeding studies conducted by industry and upon which marketing approval was granted."

Sustainable Food Trust (SFT), an organization whose mission is to serve the public interest by ensuring that peer-reviewed results on the outcome of different farming systems are subjected to public scrutiny, assimilated other responses from scientists to Dr. Sanders' claim of the authors having gone on a "statistical fishing trip": "The statistical analysis was one of a number of valid methods that could have been used to evaluate a diverse set of data sets. An expert statistician was part of the research team and this was certainly not a "fishing trip". Significance in many liver and kidney parameters are shown and highlighted in the Tables 1 and 2."

Then "Why Aren't North Americans Dropping Like Flies?"
Mark Tester, a research professor at the Australian Centre for Plant Functional Genomics at the University of Adelaide, asked why no previous studies had similar findings and said: "If the effects are as big as purported, and if the work really is relevant to humans, why aren't the North Americans dropping like flies? GM has been in the food chain for over a decade over there - and longevity continues to increase inexorably."

SFT responded, "Americans have been eating GM food (soya, maize) for only a relatively short time in significant quantities in processed foods. So it may be too short a period for long-term effects such as tumour formation to be noticeable. However, we should also note that there is no labelling of GM foods in the USA and no monitoring of the population for ill-effects, so if GM food were causing ill health this would be going undetected."

Statistical Significance
Michael Hansen, senior scientist at Consumers Union, found the sample size of 10 rats of each sex per group too small but also noted that for each of six morbidities (the three most common in each rat sex), in every case except two, the control rats were healthier than each of the various treatment groups. In a few other cases, the control group and a treatment group were equal. But in every other case, the control rats were healthier than the rats exposed to NK603 and/or Roundup. While this proves nothing as it lacks statistical significance, it "suggests that there might be something there" because if it were random, one would expect that the number of control group rats afflicted with each morbidity would sometimes be more and sometimes be less than the number of sick rats in the treatment groups. Therefore, he feels this study shows there is a need for further study with a statistically significant sample size of rats.

While this study lacks statistical significance and should have had a larger sample size, Hansen called it a better designed study than previous studies because the corn used in the control and the treatment groups were "near isolines" (similar varieties of corn) and they were grown in the same place at the same time. Additionally, Hansen pointed out that previous studies, which were all less well-designed than this one, all used the same sample size. Thus, anyone who accepts the previous results but not the results of this study due to sample size is being inconsistent. And SFT notes, "This study used more rats in test groups, for a far longer duration, than any previous investigation employed by industry to obtain approval for NK603 GM maize and other GM crop products."

Journal Retracts Study
Elsevier retracted the study from the journal on November 28, 2013. A statement announcing the retraction noted that "no evidence of fraud or intentional misrepresentation of the data" was found. However, there was concern with the small sample size and the strain of rats used. "Ultimately," the statement continued, "the results presented (while not incorrect) are inconclusive, and therefore do not reach the threshold of publication for Food and Chemical Toxicology."

Michael Hansen of Consumers Union responded to the criticism of the Seralini study as follows:


 * "The two main criticisms of the Séralini et al. study were that they used too few rat per group and that they used a strain of rat (Sprague Dawley, aka SD) that is prone to mammary tumors as they age. Both criticisms are off base. This study took blood and other biochemical measurements on 10 rats per group, the same number of rats that Monsanto took measurements on in their 90 day feeding study, which was published in the same journal eight years before the Séralini study. If ten rats is too small a sample size to demonstrate health problems, how come ten rats is a sufficient sample size to demonstrate no safety concerns? As for the strain of rat use,Séralini used the same strain (Sprague Dawley) that was used in the Monsanto feeding study. In addition, the same strain of rat was used in a Monsanto-sponsored two-year feeding study of rats fed glyphosate as part of a reregistration process in Europe. Why is use of SD rats bad when Séralini uses them, but ok when Monsanto and other biotech companies use them?


 * "However, both the French Food Safety Agency (ANSES) and the European Food Safety Authority (EFSA) have agreed with Dr. Séralini that such long-term safety assessment should be done on GE foods. Indeed, the ANSES report on the Séralini study notes, “ANSES recommends initiating studies and research on the long-term effects of GMOs in combination with plant protection products ... [and] calls for public funding on the national and European level to enable large-scale studies and research for consolidating knowledge of insufficiently documented health risks.” At a meeting in December, the “EFSA board meeting on Thursday last week there was agreement that long-term studies were needed and it was now just a question of how to fund them.” If the Séralini study is so flawed, why have ANSES and EFSA functionally agreed with its call for independently-funded long-term feeding studies on GE crops? On June 28, 2013 the European Commission announced they were spending 3 million Euros to fund a two-year carcinogenicity study on the same GE corn variety (NK603) that Dr. Séralini and colleagues used."

Monsanto's Studies
Monsanto performed studies feeding NK603 corn to both pigs and rats. The pig study focused on "performance and carcass characteristics" whereas the rat study focused on safety.

Safety Study on Rats
In 2004, Monsanto published the results of a 90 day led by Bruce G. Hammond. Male and female Sprague–Dawley derived rats were obtained at six weeks of age. Three varieties of corn were used: "test," "control," and "reference control." Test corn was Monsanto's NK603 Roundup Ready corn. Control corn was the same corn without the NK603 trait. These two varieties of corn were grown in Ohio during the 1999 season. Roundup was applied to the NK603 Roundup Ready corn at the commercial application rate. Reference control corn was commercial non-GMO corn grown in various parts of the US during the 1999 season. The 400 rats in the experiment were divided into ten groups. Each group had 20 rats of each sex. Six groups received various reference control corns. The remaining four were as follows: One group received feed containing 11% control corn and 22% corn used in standard feed formulations. A second group received feed containing 33% control corn. A third group received feed with 11% NK603 corn and 22% corn used in standard feed formulations. The last group received feed with 33% NK603 corn. The scientists found very few statistically significant differences between the test and the control rodents at the end of the 90-day study and concluded that NK603 is "as safe and nutritious as existing commercial corn hybrids."

Although the study used 20 rats per sex per group, blood and urine were collected for analysis from only 10 rats per sex per group. All animals were sacrificed and dissected at the end of the study. According to Michael Hansen of Consumers Union, the only relevant data in the study is from the groups consuming Roundup Ready corn and the control groups consuming nearly identical corn without the Roundup Ready trait (i.e. the "reference controls" are irrelevant). Therefore, the relevant figures are the high dose test group as compared to the high dose control group, and the low dose test group compared to the low dose control group.

Reference control groups were used to dismiss differences in data between the test and control groups. For example, the study reads:


 * "Urinalysis: There were no differences between the control and treated groups that were considered to be test article related (data not shown). Urine phosphorous and potassium were increased slightly in males from the high dose Roundup Ready corn group when compared to the control group, but were within the mean +/- 2 standard deviations of the population of reference controls."

Deregulation
On January 11, 2000, Monsanto submitted a petition to deregulate NK603 to the USDA Animal and Plant Health Inspection Service (APHIS). Specifically, they requested "an extension of the determination of nonregulated status issued previously for Roundup Ready corn line GA21." On June 21, 2000, the USDA published a notice in the Federal Register that an environmental assessment was available for review, soliciting public comments. The USDA conducted an environmental assessment (EA) under the National Environmental Policy Act (NEPA) and issued a "finding of no significant impact" (FONSI). NK603 was deregulated on September 29, 2000.

At the time of deregulation, the USDA wrote in the Federal Register:
 * "Like the antecedent organism [GA21], corn line NK603 has been genetically engineered to express an enzyme, 5-enolpyruvyishikimate3-phosphate synthase (EPSPS), that imparts tolerance to the herbicide glyphosate. Corn was the source of the EPSPS enzyme in the anticident organism, while a functionally equivalent EPSPS enzyme in NK603 was derived from Agrobacterium sp. strain CP4. The subject corn line and the antecdent organism were developed through use of the particle acceleration method, and expression of the added genes in NK603 and the antecedent organism is controlled in part by gene sequences derived from the plant pathogen Agrobacterium tumefaciens.


 * "Corn line NK603 and the antecedent organisms were genetically engineered using the same transformation method and contain a functionally equivalent enzyme which makes the plants tolerant to the herbicide glyphosate."

Commercialization
Monsanto commercialized the NK603 trait as "Roundup Ready 2" in 2001. NK603 is included in the following products:

Monsanto:
 * Roundup Ready® Corn 2
 * YieldGard® Corn Borer with Roundup Ready® Corn 2 (MON 810 and NK603)
 * YieldGard® Rootworm with Roundup Ready® Corn 2 (MON 863 and NK603)
 * YieldGard® Plus with Roundup Ready® Corn 2 (MON 810, MON 863, and NK603)
 * Genuity™ VT Double PRO™ (Mon89034 and NK603)

Dow Agrosciences and Pioneer Hi-Bred:
 * Herculex® I with Roundup Ready® Corn 2 (TC1507 + NK603)
 * Herculex® RW Roundup Ready® Corn 2 (DAS 59122-7 and NK603)
 * Herculex® Xtra Roundup® Corn 2 (TC1507, DAS 59122-7, and NK603)

Related SourceWatch articles

 * Roundup Ready Corn
 * Roundup Ready Crops
 * Glyphosate
 * Glyphosate Resistant Weeds
 * GE Corn
 * Biotechnology
 * GMO Policies by Country
 * Genetically Modified Organisms
 * Monsanto

External articles

 * "GM maize linked to tumours," The Connexion, September 19, 2012.
 * Ben Hirschler and Kate Kelland, "Study on Monsanto GM corn concerns draws skepticism," Chicago Tribune, September 19, 2012.
 * Elinor Zuke, "Monsanto Roundup weedkiller and GM maize implicated in ‘shocking’ new cancer study," The Grocer, September 19, 2012.
 * Carolyn Lochhead, "French study rocks GMO labeling debate," San Francisco Chronicle, September 19, 2012.
 * Hubert Nguyen, "Genetically Modified food linked to tumors [scientific study]," Ubergizmo, September 19, 2012.
 * Hank Campbell, "GM Maize Causes Tumors In Rats? Here Is How Experts Responded," Science 2.0, September 19, 2012.
 * Gilles-Eric Séralini, Emilie Clair, Robin Mesnage, Steeve Gress, Nicolas Defarge, Manuela Malatesta, Didier Hennequin, Joël Spiroux de Vendômois, "Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize," Food and Chemical Toxicology, Available online September 19, 2012.