【部分譯文】：
Inspection of the Safety Assessment of Genetically Modified, the Roundup Tolerant Soybean:
Monsanto’s Dangerous Logic as seen in the Application Document submitted to Japan .
--對孟山都的轉基因抗除草劑大豆安全性評估的檢查結果：在作為提交給日本申請的文件中，孟山都的危險邏輯顯而易見
by: Masaharu Kawata
(Assistant Professor, School of Science , Nagoya University , Japan )
Mandatory labeling of genetically modified food became effective this April in Japan . Japanese consumers can then be able to choose non-genetically modified food by the label in any shop and store. But,there would be consumers who dare not to choose it for their confidence in Health and Welfare Ministry's safety approval or who purchase processed food containing genetically modified ingredient without knowing it. Safety assessment of genetically modified crop whose unknown risk with artificially modified gene is raised cannot be too cautious. This is the report of inspection carried out on Safety Assessment Application Documents submitted by JAPAN- MONSANT for their herbicide torelant soybean that was approved as food by the Japanese Ministry of Health and Welfare and as animal feed by the Ministry of Agriculture in 1996.
--在日本，今年四月，孟山都對轉基因食品標明的做法生效了。日本的消費者可以按標簽在任何店鋪和商店選擇非轉基因食品。然而，還是會存在這樣的消費者，他們因對厚生省的批準的安全證書的信心方面的原因而依舊不敢選擇非轉基因食品，或者還存在這樣的情況：采購了含轉基因配方的加工過的食品卻不知道。人工轉基因有未知的危險，對種植的轉基因作物的安全評估再謹慎也不過分。本文是對有由JAPAN- MONSANT遞交的轉基因抗除草劑大豆安全性評估的申請文件而得出的檢查報告，在1996年，這些轉基因耐除草劑大豆被日本厚生省批準作物食品和被日本農業省批準作為動物飼料?！ ?/p>
(1) Information disclosure is but nominal
The application submitted by Monsanto for "Roundup Ready soybean" consists of 10 volumes, which pile up to 1 meter high. Moreover, the third section on are all in English. It was impossible for us to transcribe all of them in given time. We were all told 40 people in 10 days and managed more than 500 pages focusing on important points for best possible effectiveness. When information disclosure law enacted this April, we must watch closely whether it would make things easier or even harder with excuse of company secrets and keep the decision making of safety assessment to a few bureaucrats and their academics. Unless safety assessment is made open to public, concerns over genetic engineering will be even worse.
--（1）信息披露有名無實
遞交給厚生省的申請文件存放在食品安全協會，食品安全協會是屬于政府部門以外的一個組織。檢查活動要到該組織設在東京和大阪的辦事處進行。然而，每星期只有三天允許進行檢查，上午10點到12點，下午13點到16點，不許拷貝和照相。為了檢查精準，我們只能采用手工抄寫的方法了，就好像回到了日本江戶武士時期的舊時代。這部孟山都為他的耐除草劑大豆而遞交的申請由十卷組成，堆起來高一米。而且，第三章以后都是用英語寫的。在規定的時間里我們根本不可能抄得全。為了到達最高效率，我們總共40個人10天聚焦重點設法完成了500多頁。信息披露法律在今年四月頒布，我們密切注意信息披露是變得容易還是以公司秘密為借口，依舊決定安全評估只面向幾個官僚和他們的學者，從而使得信息披露愈加困難。除非安全評估面向公眾開放，否則對遺傳工程會更加的擔心?！ ?/p>
(2) What is herbicide resistant soybean by Monsanto?
In growing soybean, well-planned weed and pest control is important to get desired harvest. Low input cultivation becomes possible if soybean itself has herbicide resistance and dust cropping is done without complexity. Monsanto has endeavored, in vain, till 1990s to achieve this goal by creating soybean mutant, which is resistant to their best selling organic-phosphate herbicide Roundup in which the glyphosate is its active ingredient. The resistant strains created had seriously damaged enzymatic activity of EPSPS(5-enol-pyruvylshikimate-3-phosphate synthase : one of the enzymes work to synthesize aromatic amino acid, Tyrosine, Phenylalanine, Tryptphan) and caused growth defect of soybean itself. The genetic engineering technology was becoming popular then, and was naturally selected tried to introduce gene from different organism to soybean. Purported herbicide resistant bacterium was found in the sewage water of the glyphosate factory of Monsanto in USA . This Agrobacterium tumefaciens named as 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 (refer to as CP4EPSPS hereafter).
The bacterial gene generally can not work in plant cell by just inserting it to the genome, because the genetic switch called promoter of prokaryotes and that of eukaryote is different. Then a powerful promoter from "Cauliflower Mosaic virus" called 35S promoter was connected to the target gene. Next gene engineering was to connect a small protein called "signal peptide" which carries the CP4EPSPS protein to where the enzyme is supposed to function, in this case chloroplast. This gene of signal peptide was taken from flower petunia. A part of plant cancer virus gene called NOS, which make a signal to stop gene read through is also required. Thus created "Roundup tolerant soybean gene" is a completely artificial gene that never exists in natural kingdom (figure 1) which would be never existed in natural evolution.
In addition to these modifications of the genetic construct, Monsanto artificially had to change genetic codons for efficient translation of the CP4ESPS gene in soybean plant. The 239 nucleotides out of total of 1,365 (17.51%) were manually converted to other bases (though mostly in the third letter) in order for the protein synthetic machinery of soybean cell to decipher the bacterial gene across the species barrier. Thus, the Roundup Tolerant soybean came to possess a gene unlike either the prokaryotic gene or the eukaryotic gene. It is with reason that gene modified plants are called "the Frankenstein plants" in Europe . Focal point of safety assessment is whether such soybean with artificially modified gene is the same as the conventional one.
--（2）孟山都的抗除草劑大豆是什么？
在大豆生長過程中，妥善安排控制雜草和害蟲事務對獲得理想的收成是很重要的。要是大豆本身抗除草劑，給作物噴灑除草劑做起來就不復雜，那么低耕作投入就成為了可能。孟山都一直為此徒勞地努力著，直到20世紀90年代大豆的突變異種的問世，才達到了這個目標。這種大豆的突變異種對孟山都自己賣得最好的有機碳酸鹽除草劑有耐藥性。這種除草劑的有效成分是草甘膦。這個被創造出來的抗性品系的酶EPSPS的活性受損嚴重（5-enol-pyruvylshikimate-3-phosphate 合酶：一種合成芳香族氨基酸，酪氨酸，苯基丙氨酸，色氨酸的酶）從而導致大豆本身的生長缺陷。那時很流行的遺傳工程就很自然的被用來把其他不同的有機體的基因引入大豆。據說在美國的孟山都草甘膦工廠的廢水里發現了耐除草劑的細菌。 這個被命名為CP4品種的根癌土壤桿菌是一種在有草甘膦的場合能合成芳香族氨基酸土壤細菌。這種酶的氨基酸排列的序列不同于任何植物，因此被命名為class II EPSPS (也叫做CP4EPSPS 來世)?！ ?/p>
僅僅把細菌的基因插入到基因組，細菌的基因還不能起作用，因為叫作原核生物的發起者以及真核細胞的發起者的基因開關是不同的。然后，一個來自“花椰菜花葉病毒”的強大的發起者，叫作35S發起者，被連接到了目標基因。接著基因工程就把攜帶CP4EPSPS 蛋白質的叫作“信號肽”的小蛋白質，連接到被期望能起作用的酶上，本例中是葉綠素。信號肽的基因來自牽?；ā１环Q為NOS的植物腫瘤病毒基因的一部分，用來產生基因讀出時的停止信號的東西也是必須的。因此，被創造出來的“抗除草劑大豆的基因”是完全徹底的人造基因，這種基因在自然界根本不存在（圖一），在自然界的進化過程中也不會出現?！ ?/p>
除變動基因結構外，孟山都還必須人為地改動基因的密碼子，為的是讓大豆植物中的CP4ESPS基因有效轉化。1365種核苷酸基中的239種核苷酸基（占17.51%）被手動地改為其他基（盡管絕大多數在第三個字母上的）為了讓大豆細胞的蛋白質的合成機制能夠跨過物種障礙解釋病毒基因。因此，抗除草劑大豆就有了一種既不像原核細胞的基因也不像真核細胞基因的基因。正由于這樣，在歐洲，轉基因植物被叫做“弗蘭肯斯坦植物”（ 弗蘭肯斯坦--毀滅創造者自己之物）。安全評估的焦點集中在人工轉基因大豆是不是等同于傳統意義上的大豆。
【原文和鏈接如下】
http://www2.odn.ne.jp/~cdu37690/ProblemsinGMFpermit.htm
Inspection of the Safety Assessment of Genetically Modified, the Roundup Tolerant Soybean:
Monsanto’s Dangerous Logic as seen in the Application Document submitted to Japan .
by: Masaharu Kawata
(Assistant Professor, School of Science , Nagoya University , Japan )
Mandatory labeling of genetically modified food became effective this April in Japan . Japanese consumers can then be able to choose non-genetically modified food by the label in any shop and store. But,there would be consumers who dare not to choose it for their confidence in Health and Welfare Ministry's safety approval or who purchase processed food containing genetically modified ingredient without knowing it. Safety assessment of genetically modified crop whose unknown risk with artificially modified gene is raised cannot be too cautious. This is the report of inspection carried out on Safety Assessment Application Documents submitted by JAPAN- MONSANT for their herbicide torelant soybean that was approved as food by the Japanese Ministry of Health and Welfare and as animal feed by the Ministry of Agriculture in 1996.
(1) Information disclosure is but nominal
The application submitted by Monsanto for "Roundup Ready soybean" consists of 10 volumes, which pile up to 1 meter high. Moreover, the third section on are all in English. It was impossible for us to transcribe all of them in given time. We were all told 40 people in 10 days and managed more than 500 pages focusing on important points for best possible effectiveness. When information disclosure law enacted this April, we must watch closely whether it would make things easier or even harder with excuse of company secrets and keep the decision making of safety assessment to a few bureaucrats and their academics. Unless safety assessment is made open to public, concerns over genetic engineering will be even worse.
(2) What is herbicide resistant soybean by Monsanto?
In growing soybean, well-planned weed and pest control is important to get desired harvest. Low input cultivation becomes possible if soybean itself has herbicide resistance and dust cropping is done without complexity. Monsanto has endeavored, in vain, till 1990s to achieve this goal by creating soybean mutant, which is resistant to their best selling organic-phosphate herbicide Roundup in which the glyphosate is its active ingredient. The resistant strains created had seriously damaged enzymatic activity of EPSPS(5-enol-pyruvylshikimate-3-phosphate synthase : one of the enzymes work to synthesize aromatic amino acid, Tyrosine, Phenylalanine, Tryptphan) and caused growth defect of soybean itself. The genetic engineering technology was becoming popular then, and was naturally selected tried to introduce gene from different organism to soybean. Purported herbicide resistant bacterium was found in the sewage water of the glyphosate factory of Monsanto in USA . This Agrobacterium tumefaciens named as 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 (refer to as CP4EPSPS hereafter).
The bacterial gene generally can not work in plant cell by just inserting it to the genome, because the genetic switch called promoter of prokaryotes and that of eukaryote is different. Then a powerful promoter from "Cauliflower Mosaic virus" called 35S promoter was connected to the target gene. Next gene engineering was to connect a small protein called "signal peptide" which carries the CP4EPSPS protein to where the enzyme is supposed to function, in this case chloroplast. This gene of signal peptide was taken from flower petunia. A part of plant cancer virus gene called NOS, which make a signal to stop gene read through is also required. Thus created "Roundup tolerant soybean gene" is a completely artificial gene that never exists in natural kingdom (figure 1) which would be never existed in natural evolution.
In addition to these modifications of the genetic construct, Monsanto artificially had to change genetic codons for efficient translation of the CP4ESPS gene in soybean plant. The 239 nucleotides out of total of 1,365 (17.51%) were manually converted to other bases (though mostly in the third letter) in order for the protein synthetic machinery of soybean cell to decipher the bacterial gene across the species barrier. Thus, the Roundup Tolerant soybean came to possess a gene unlike either the prokaryotic gene or the eukaryotic gene. It is with reason that gene modified plants are called "the Frankenstein plants" in Europe . Focal point of safety assessment is whether such soybean with artificially modified gene is the same as the conventional one.
(3) A mystery of "The samples used for analysis and animal dietary test were cultivated without herbicide application".
The Roundup Ready soybean marketed is usually applied with the herbicide Roundup. But surprisingly enough, our inspection revealed that both the gene modified soybean 40-3-2 strain and conventional strain A5403 were NOT sprayed with Roundup herbicide in their cultivation. Monsanto produced only small amount sample with Roundup on the side to test residual glyphosate in the harvested forage, hay and seed. All the soybean of a few thousand kilograms used in safety experiments was harvested not sprayed. The reason is not stated in the documents.
The data obtained with such samples may be therefore not valid to guarantee safety of soybean that human and animals take in the real life, not just because of the residue glyphosate is a toxin to kill plants by inhibiting plant enzyme EPSPS. Effects on other metabolic pathway must be taken into account particularly when such artificial genes are inserted. For consumers, the test results using different sample than marketed soybean may be meaningless.
(4) Incomplete analysis of introduced protein CP4EPSPS.
It is expected that CP4EPSPS protein expressed in the bio-engineered soybean have the same amino acid sequence as the soil bacterium from which the gene was originated. 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 amino acid change and/or post-translational modification after expression. Before inspection we presumed that amino acid sequence of soybean CP4EPSPS was determined. However, to my surprise, it was not.
What Monsanto has determined was only 15 amino acids from N-terminal of the protein, which was expressed in E.coli. The rest of the sequence was presumed one from the nucleotide sequence of the bacterial DNA. Then only 3.3% of expected total of 455 amino acids was decided, and 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 the amino acid sequences are the same. The true face of CP4EPSPS protein in the soybean that we are taking is still unknown.
(5) The E.coli expressed protein is used for acute toxicity test too on rat.
CP4EPSPS protein used for acute toxicity test on rat is also came from that produced by E.coli harboring CP4EPSPS plasmid. Monsanto excuses in the application document that to obtain large amount of CP4EPSPS protein from soybean is difficult. This is unacceptable because there is a possibility that the inserted gene work differently in soybean than was in the original bacterium, and therefore the expression 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 enough concentration to extract without problem. This again is the typical "All for the conclusion" approach by Monsanto. This kind of problem could be resolved if all CP4ESPS amino acid sequence in soybean had been analyzed and confirmed equal as the bacterium. The experiment looks like 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 the soybean gene gets affected from insertion of foreign gene or not. The series of experiments described is incoherent on the fundamentals.
(6) Insufficient feeding experiment and intentional neglect of “wrong “ data.
Animal feeding test is important for safety assessment. Then Monsanto conducted the experiments for animals as rat, cow, chicken, catfish and quail. However, the scale of experiment is less than adequate.
For example, in rat experiments, raw and toasted soybean both genetically modified and non-modified were fed to mere 10 rats each group and feeding period is only limited to 28 days. Toxicity across generation or chronic toxicity may not be detectable by these limited experiment size and duration.
Under these insufficient experiments however, the data for body and organ weight of lever, kidney and testicles show obvious difference in the male rats between both groups, wild A5403 and bio-engineered 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 periods, 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 sample size 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.
(7) Misguided interpretation and disregard of data in chemical analysis.
Chemical analysis of the components from both normal and genetically modified is important to certify so-called substantial equivalence.
We found a highly intended misinterpretation ignoring obvious data difference between A5403 and 40-3-2 hybrid in the documents. Analysis of raw soybeans showed no difference between gene modified 30-4-2 and non-modified A5403 soybean. Difference is observed in toasted soybeans. Besides such main components like water content, protein, fat, fiber and ash, the analysis detected trypsin-inhibitor, lectin and urease which are called harmful physiologically active substance as feed. Urease is used an indicator of protein denaturation by heat treatment.
Obvious difference appeared when after toasted with actual feed processing condition (108℃, 30min). The concentration of total protein and potassium were not changed, but concentration of trypsin-inhibitor, urease, and lectin have significantly higher in the toasted 30-4-2, the glyphosate-tolerant bean 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 the feed standard.
Monsanto took this result as "only the modified soybeans are toasted insufficiently in the experiment", and returned and asked re-treatment of the sample to Texas A & M who processed the beans. Monsanto ordered the condition of re-toast as 220 ℃ for 25min, which is considerably higher than normal processing of 100℃,10 minutes. However re-toasting further widened the difference in the activity between the two strains. Another hybrid 61-67-1,which is another genetically modified soybean inserted with bacterial CP4EPSPS, showed highly resistant property to the heat.
Scientist would usually conclude in such case that there is substantial difference between the two. But Monsanto dared to challenge this common sense, and concluded again the second toasting is still not enough. In the end, they toasted twice further and got the result they wanted, 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 " they can't be different" and their need "there shouldn't be difference". Their translation of the experiment is "Safe is the conclusion" attitude and not at all scientific. The English data volume did not show analysis data of third and fourth heat treatment, but the Japanese summary volume, as if there were data, has a graph showing after loss of activity and described that "data from insufficient heat treatment is not adopted" and "No substantial difference observed." If you review only Japanese summary volume 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, lose 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 certify this facts.
(8) Residual herbicide in crop increase, then the safety standard should be changed high level, Monsanto’s conclusion.
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 exeeds 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 higher 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 old standard of 6 ppm according to the request of US government. Of course Japan become could import soybean from USA without worrying about of 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, if necessary, even the revision of safety standard. We found the 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 experiment remained not inspected yet.
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, and how 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 sense to have concluded as safe on the bases of such incomplete application.
The safety assessment of the Monsanto Roundup ready soybean needs to be reassessed, is our conclusion.
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