【部分译文】：
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 .
--对孟山都的转基因抗除草剂大豆安全性评估的检查结果：在作为提交给日本申请的文件中，孟山都的危险逻辑显而易见
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，孟山都对转基因食品标明的做法生效了。日本的消费者可以按标签在任何店铺和商店选择非转基因食品。然而，还是会存在这样的消费者，他们因对厚生省的批准的安全证书的信心方面的原因而依旧不敢选择非转基因食品，或者还存在这样的情况：采购了含转基因配方的加工过的食品却不知道。人工转基因有未知的危险，对种植的转基因作物的安全评估再谨慎也不过分。本文是对有由JAPAN- MONSANT递交的转基因抗除草剂大豆安全性评估的申请文件而得出的检查报告，在1996年，这些转基因耐除草剂大豆被日本厚生省批准作物食品和被日本农业省批准作为动物饲料。
(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）信息披露有名无实
递交给厚生省的申请文件存放在食品安全协会，食品安全协会是属于政府部门以外的一个组织。检查活动要到该组织设在东京和大阪的办事处进行。然而，每星期只有三天允许进行检查，上午10点到12点，下午13点到16点，不许拷贝和照相。为了检查精准，我们只能采用手工抄写的方法了，就好像回到了日本江户武士时期的旧时代。这部孟山都为他的耐除草剂大豆而递交的申请由十卷组成，堆起来高一米。而且，第三章以后都是用英语写的。在规定的时间里我们根本不可能抄得全。为了到达最高效率，我们总共40个人10天聚焦重点设法完成了500多页。信息披露法律在今年四月颁布，我们密切注意信息披露是变得容易还是以公司秘密为借口，依旧决定安全评估只面向几个官僚和他们的学者，从而使得信息披露愈加困难。除非安全评估面向公众开放，否则对遗传工程会更加的担心。
(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）孟山都的抗除草剂大豆是什么？
在大豆生长过程中，妥善安排控制杂草和害虫事务对获得理想的收成是很重要的。要是大豆本身抗除草剂，给作物喷洒除草剂做起来就不复杂，那么低耕作投入就成为了可能。孟山都一直为此徒劳地努力着，直到20世纪90年代大豆的突变异种的问世，才达到了这个目标。这种大豆的突变异种对孟山都自己卖得最好的有机碳酸盐除草剂有耐药性。这种除草剂的有效成分是草甘膦。这个被创造出来的抗性品系的酶EPSPS的活性受损严重（5-enol-pyruvylshikimate-3-phosphate 合酶：一种合成芳香族氨基酸，酪氨酸，苯基丙氨酸，色氨酸的酶）从而导致大豆本身的生长缺陷。那时很流行的遗传工程就很自然的被用来把其他不同的有机体的基因引入大豆。据说在美国的孟山都草甘膦工厂的废水里发现了耐除草剂的细菌。 这个被命名为CP4品种的根癌土壤杆菌是一种在有草甘膦的场合能合成芳香族氨基酸土壤细菌。这种酶的氨基酸排列的序列不同于任何植物，因此被命名为class II EPSPS (也叫做CP4EPSPS 来世)。
仅仅把细菌的基因插入到基因组，细菌的基因还不能起作用，因为叫作原核生物的发起者以及真核细胞的发起者的基因开关是不同的。然后，一个来自“花椰菜花叶病毒”的强大的发起者，叫作35S发起者，被连接到了目标基因。接着基因工程就把携带CP4EPSPS 蛋白质的叫作“信号肽”的小蛋白质，连接到被期望能起作用的酶上，本例中是叶绿素。信号肽的基因来自牵牛花。被称为NOS的植物肿瘤病毒基因的一部分，用来产生基因读出时的停止信号的东西也是必须的。因此，被创造出来的“抗除草剂大豆的基因”是完全彻底的人造基因，这种基因在自然界根本不存在（图一），在自然界的进化过程中也不会出现。
除变动基因结构外，孟山都还必须人为地改动基因的密码子，为的是让大豆植物中的CP4ESPS基因有效转化。1365种核苷酸基中的239种核苷酸基（占17.51%）被手动地改为其他基（尽管绝大多数在第三个字母上的）为了让大豆细胞的蛋白质的合成机制能够跨过物种障碍解释病毒基因。因此，抗除草剂大豆就有了一种既不像原核细胞的基因也不像真核细胞基因的基因。正由于这样，在欧洲，转基因植物被叫做“弗兰肯斯坦植物”（ 弗兰肯斯坦--毁灭创造者自己之物）。安全评估的焦点集中在人工转基因大豆是不是等同于传统意义上的大豆。
【原文和链接如下】
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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