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  • From: "GlobalCirclenet" <webmaster@globalcircle.net>
  • To: livingontheland@lists.ibiblio.org
  • Subject: [Livingontheland] Flaws in Monsanto's Safety Assessment of Roundup Ready Soybeans
  • Date: Thu, 07 Aug 2003 09:59:28 -0600


Flaws in Monsanto's Safety Assessment of Roundup Ready Soybeans
THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE
28 July 2003

Dear Friends and colleagues,

RE: Flaws in RR soybean safety assessment

We wish to bring to you an important document which found that the safety
assessment application made by Monsanto to the Japanese Health Ministry for
approval of Roundup Ready soybeans were "inadequate and incomplete".

Monsanto has maintained that there is no difference between GM soybeans and
its conventional strains. But according to a Japanese scientist, whose
report is attached below, safety tests conducted by Monsanto are riddled
with flaws that include: testing of proteins not derived from the GM plant;
soybeans used for tests were not produced with Roundup, therefore the data
obtained with such samples may not be valid to guarantee the safety of
soybean that human and animals consume in real life; insufficient feeding
experiments; and intentional neglect of "inappropriate" data which have a
bearing on the final conclusions.

We hope special attention will be given to the report as it is important to
be aware that the safety of RR soybean is in question and governments in
the process of approving RR soybeans should take heed of this important
information. We also urge governments that have approved the RR soybean to
review their decision.

With best wishes,

Lim Li Lin and Chee Yoke Heong
Third World Network
121-S Jalan Utama
10450 Penang
Malaysia
Email: twnet@po.jaring.my
Website: www.twnside.org.sg
---
REF: Doc.TWN/Biosafety/2002/C

Fraudulent 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

Masaharu Kawata
Assistant Professor
School of Science
Nagoya University, Japan

What is herbicide resistant soybean by Monsanto?

In growing soybean, well-planned weed and pest control is important to get
the desired harvest. If soybean itself had herbicide resistance, low input
cultivation would be possible and dust cropping could be much simpler.
Monsanto had endeavoured, in vain, till 1990s 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
execus 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 celcius, 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 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 celcius for 25min, which
is considerably higher than normal processing of 100 degrees celcius, 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, 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 basis of such incomplete
application.






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