Conclusion Facts Found by Inspection of the
Safety Assessment of GM Roundup Tolerant Soybean
Monsanto's Dangerous Logic as seen in the Application Documents submitted to Health Ministry of Japan
Technology and Man magazine1nov00
Masaharu Kawata Assistant Professor School of Science Nagoya University, Japan
Katsumi Yamada, translator of the first version and this version. <firstname.lastname@example.org>
What is herbicide resistant soybean by Monsanto?
In growing soybean, well-planned weed and pest control is important to get desired harvest. If soybean itself had herbicide resistance, low input cultivation would be possible and dust cropping could be much simpler. Monsanto had endeavored, in vain, till I 990s to achieve this goal by creating soybean mutant that is resistant to their best selling
organo-phosphoric herbicide Roundup in which the glyphosate is the active ingredient. The resistant strains created, however, had seriously hampered enzymatic activity of EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase: one of the enzymes work to synthesize aromatic amino acid, Tyrosine, Phenylalanine and Tryptphan) and the soybean failed to thrive.
The genetic engineering technology was becoming popular at the time, and was naturally employed in introducing gene from different organism into soybean. Herbicide resistant bacterium was found in the glyphosate factory sewage of Monsanto USA. This Agrobacterium tumefaciens CP4 strain is a kind of soil bacterium, which could synthesize aromatic amino acid in the presence of glyphosate. The amino acids sequence of the enzyme is largely different from that of any plants and is called class II EPSPS (refered to as CP4EPSPS hereafter).
Inserting the bacterial genes into plant genome generally does not work by itself because genetic switch called promoter for prokaryotes and eukaryote are different. A powerful promoter from "Cauliflower Mosaic virus" called 35S promoter was connected to the gene insert . Then connected is a small protein called "signal peptide" which carries the CP4EPSPS protein to where the enzyme is supposed to function, in this case chloroplast. This signal peptide gene was taken from petunia. A part of plant cancer virus gene called NOS that signals to stop reading the gene is also required. Thus created "Roundup tolerant soybean gene cassette" is a completely artificial one that never existed in natural life kingdom nor could have evolved naturally.
In addition to these modifications of the genetic construct, Monsanto had to change genetic codons for efficient expression of the CP4ESPS gene in soybean plant. The 239(17.51%) nucleotides out of 1,365 total were manually converted into different bases (though mostly in the third letter) in order for the protein synthetic machinery of soybean cell to decipher the bacterial gene across species barrier. Thus, the Roundup Tolerant soybean came to possess a gene unlike either the prokaryotic or the eukaryotic gene. It is with reason that genetically modified plants are called "the Frankenstein plants" in Europe. Focal point of safety assessment is whether such soybean with artificially modified genes is the same as the conventional non modified soybean.
The soybeans used for analyses and animal feed tests were grown without herbicide application".
The Roundup Ready soybean marketed is usually sprayed with the herbicide Roundup. It was a surprise to find that both the genetically modified soybean 40-3-2 strain and the parent strain A5403 used for feed tests were NOT sprayed with Roundup herbicide in their cultivation. What Monsanto had produced with Roundup application was minimal amount enough to test glyphosate residues in the harvested forage, hay and seed. Several tons of soybean used in safety assessments was not produced with the Roundup. The reason is not stated in the documents.
The data obtained with such samples may not be valid to guarantee the safety of soybean that human and animals consume in the real life, not just because the residual glyphosate is a toxin that kills plants by inhibiting plant enzyme EPSPS but the effects on other metabolic pathways must also be taken into account when such artificial genes are inserted. For consumers, the test results obtained by using different sample other than what is marketed is meaningless.
The protein CP4EPSPS analysed is from E.coli. not from RR soybean!
It is expected that CP4EPSPS protein expressed in the bio-engineered soybean has the same amino acid sequence as the soil bacterium from which the gene was extracted. This can only be verified when soybean produced protein is isolated and the amino acid sequence is determined, because exchanging genes between bacteria and higher organism can sometimes result in partial change of amino acid and/or post-translational modification after expression. It was our presumption before the inspection of the documents that the amino acid sequence of the soybean CP4EPSPS was determined but, to our surprise, it was not.
What Monsanto has sequenced was only 15 amino acids from N-terminal of the protein that was expressed in E.coli. The rest of the sequence was an assumption from the nucleotide sequence of the bacterial DNA. They determined only 3.3% of expected total of 455 amino acids and that the protein is not of soybean! ELISA test described in the documents is the only method to verify antigenic equivalence of proteins. But antigenic similarity itself does not prove that the amino acid sequences are the same. The real sequence of CP4EPSPS protein in the soybean that we are eating is still unknown.
Acute toxicity test on rats is also carried out by using the protein expressed in E.coli.
CP4EPSPS protein used for acute toxicity test on rats also come from that produced by E.coli harbouring CP4EPSPS plasmid. What Monsanto says in the application document is that extracting large amount of CP4EPSPS protein from soybean is difficult. This is an unacceptable excuse because there is a possibility that the inserted gene work differently in soybean than in the original bacterium, therefore the expressed product may be different from that of soybean. Moreover, according to the application document, 0.238mg of CP4ESPS protein is detected in one gram of genetically modified 40-3-2 soybean which is good enough concentration to extract with no difficulty. This again is the typical "for the roundup" approach by Monsanto. This kind of problem could be resolved if all CP4ESPS amino acid sequence in soybean had been sequenced and confirmed equal as the bacterium. The experiment appears to have been conducted on the presumption that the other soybean proteins are the same as the non-GM soybean as long as the CP4EPSPS is not toxic. If so, this is too easy and one-sided approach. The core of this problem is whether or not the soybean gene gets affected by insertion of foreign gene. The series of experiments described are fundamentally invalid .
Insufficient feeding experiments and intentional neglect of "inappropriate" data.
Animal feeding test is important for safety assessment. Monsanto conducted these experiments on such animals as rat, cow, chicken, catfish and quail. However, the scale of experiment is much less than adequate. For example, in rat experiments, raw and toasted soybean both genetically modified and non-modified were fed to only 10 rats each group and feeding period is only 28 days. Toxicity across generation or chronic toxicity will not be measured by these limited experiments.
Even with these far from satisfactory experiments, the data for body and organ weight of lever, kidney and testicles show obvious difference in the male rats between both groups, wild strain A5403 and bio-engineered strain 40-3-2 soybean.
Raw soybean fed group showed no difference. But toasted soybean 40-3-2 fed male group weighed 6.7% less body weight than A5403 fed group and 13% less than commercial feed mix fed group at the end of test period of 28 days. Though this difference is described as statistically significant in the data sheet, the conclusion ignores these results and states that "no statistical significance is observed."
The experiments are far from satisfactory in its samples and the statistic method used. Our group transcribed all raw data and redid statistical analysis using Turkey multiple method. The result again showed the apparent growth obstacle for the body and kidney weight in male rats group fed with toasted 40-3-2 soybean. I wondered why there is no such difference in female rats group. The answer to this question seemed to be the amount of the feed intake where male took 25-30g/day, female rats took only 18-20g (approx. 70% of male)/day. It is highly possible that female rats also showed significant growth difference if experiment is conducted in much larger scale and with longer feeding period.
Misinterpretation, false conclusion and disregard of data.
Chemical analysis of the components from both normal and genetically modified is important to certify so called substantial equivalence.
We found a highly intentional misinterpretation by ignoring obvious difference between A5403 and 40-3-2 hybrid in the documents. Raw soybeans showed no difference in the analysis between genes modified 30-4-2 and non-modified A5403 soybean. Difference is observed in toasted soybeans. Besides such main components like water, protein, fat, fibre and ash, trypsin-inhibitor, lectin and urease which are called harmful physiologically active substance as feed are detected in the analysis. Urease is used as an indicator of protein denaturation by heat treatment.
Obvious difference appeared after toasting at actual feed processing condition (108 degrees Celsius, 30min). While the concentration of total protein and potassium were not changed, the concentration of trypsin-inhibitor, urease, and lectin are significantly higher in the toasted the glyphosate-tolerant bean 30-4-2 compared to that of A5403 normal bean. These physiologically active substances remained active even after heat treatment in the genetically modified soybean, though those of herbicide sensitive normal bean were easily denatured and inactivated. The high activity of these elements does not usually satisfy as feed.
Monsanto took this result as "the modified soybeans are not toasted sufficiently in the experiment" and returned and asked for re-treatment of the sample to Texas A & M laboratory who processed the beans. Monsanto ordered the condition of re-toast at 220 degrees Celsius for 25min, which is considerably higher than normal processing of 100 degrees Celsius, 10 minutes. However re-toasting further widened the difference in the activity between the two strains. The hybrid 61-67-1, which is another genetically modified soybean inserted with bacterial CP4EPSPS, showed high heat resistant property.
Scientist would usually conclude in such case that there is substantial difference between the two. But Monsanto dared to challenge this common practice and concluded again the second toasting was still not enough. In the end, they toasted twice further and got the result they wanted, i.e. all proteins were denatured and inactivated. With this result, they concluded that genetically modified and non-modified soybeans have equivalent properties.
No protein can withstand repeated heat treatment and stay active. This is a common knowledge of protein chemistry. The argument at normal feed processing condition is required and no more, no less. Monsanto based their argument on their presumption that "they can't be different" and their need that "they shouldn't be different". Their translation of the experiment is based on "the conclusion is safe" attitude and not at all scientific. The English data volume did not show analysis data of third and fourth heat treatment,
but the summary volume in Japanese, as if there were data, has a graph showing after loss of activity and stated that "the data from insufficient heat treatment is not adopted" and "No substantial difference observed." If you review only summary volume in Japanese and not look into English data volume, you would be ushered to the conclusion of "Safe."
However we could found in the first and the second analyses data of toasted soybean a fact indicative of regular heat treatment. Granulated soybean, when heated, loses weight as water and other volatile components evaporate, and as the result, relative concentration of non-volatile substance such as total protein and ash increases. The data shows clearly that the gene modified 40-3-6 and 61-67-1 and non-modified A5403 gone through same level of heat treatment. The decrease of water content also certifies this fact.
Monsanto requested slackened herbicide residue level
Monsanto's concluded that the residual herbicide in crop increases, therefore the safety standard should be slackened. Adopting the Roundup tolerant soybean would increase the herbicide concentration in the soybean plants and seeds, because the herbicide is directly sprayed on the plant by postemergence application before harvest. The Monsanto studied in detail what would be the results by changing factors like spraying times, concentration of the active ingredient glyphosate, duration of harvest after spraying and growing locations. The data show clearly that the concentration of glyphosate and AMPA (a degraded substance of glyphosate) in forage and hay increase greatly by postemergence application of the herbicide compared to that of conventional preemergence application, although the residual concentration in the plant differed from place to place. The largest value of the combined glyphosate and AMPA was 40.187 ppm in forage which is higher than the US safety standard of 15 ppm in forage and hay in 1994 when FDA and USDA accepted the application documents. The maximum combined concentration of glyphosate and AMPA in soybean seed was 13.178 ppm, which is less than 20 ppm of the US standard at that time. The concentration residual glyphosate increased in accordance with the application increased from twice to three times. Then cultivating Roundup ready soybean may sometimes violates the US safety standard. We found a surprising description in the document to dissolve the problem.
In final conclusion, Monsanto say that "the maximum combined glyphosate and AMPA residue level of approximately 40 ppm in soybean forage resulting from these new uses exceeds the currently established tolerance of 15 ppm. Therefore, an increase in the combined glyphosate and AMPA tolerance for residues in soybean forage will be requested." They know very well that adoption of herbicide tolerance crop needs slackened safety standards. In effect, the US tolerance standard of combined glyphosate and AMPA in soybean forage was changed to 100 ppm after they approved the genetically engineered soybean.
As to Japanese government, they revised the safety standard of combined glyphosate and AMPA in soybean seed to 20ppm in April 2000 from what used to be 6 ppm according to the request of US government. Japan could import soybean from USA without violation of the law by this decision.
Thus, Monsanto, in their rush to verify safety, patch worked the results of experiments and analyses that are full of voids like a puzzle and asserted safe with manipulation of the results. They requested even the revision of safety standard. We have managed to find facts showing inadequate and incomplete safety assessment described above in the application document by Monsanto even in our limited work under difficult situation. The process of genetic recombination and the results of other animal experiments remained uninspected by us.
Monsanto informed US soybean importing countries in May 2000 that they found Roundup resistant soybean has two extra fragments of the CP4EPSPS gene in the genome.
They were there since the first FDA approval in 1992, and all the GM soybeans supplied worldwide contain this gene fragments. Monsanto asserts that these fragmented genes do not create unknown protein since they have any open reading frame or termination signal around them. But such basic facts comes to light 8 years after the approval is a sure indication of how incomplete the genetic recombination of crop is, ana now dangerous safety assessment can be to rely only on company s information and data. We doubt it very much if at all government experts in charge at the Japanese Ministry of Health and Welfare for safety assessment had a good to have concluded as safe on the bases of such incomplete application.