Annexes: Analysis of detailed answers of MAHYCO to my report, p 66-84 of GEAC report

1/ My name is Prof Seralini and I am from the University of Caen, Mahyco has made two mistakes in this first sentence. This tells long about the precisions of the answers.

First of all numerous criticisms that I have raised in my report of January 2009 have not been answered in GEAC report (for instance the signatures of the research files to be valid, but also a lot of scientific points). The major point is that it could be criminal and a scientific nonsense to wait for final proofs of chronic toxicity within three month tests in mammals. Accordingly, there is absolutely no scientific reference for proven safe chronic consumption for any GMO, nor for Bt brinjal. GMOs are not labeled nor traced in America, no epidemiological serious study can thus be available; and the numerous chronic dietary pathologies observed there may be attributable to any or several food and feed contaminants.

Details answering to annotated remarks in the Table:

Seralini: Bt brinjal has been modified to produce an unknown chimeric insecticide toxin containing Cry1Ab and Cry1Ac modified sequences. In the toxicity tests on target and non-target insects, this chimeric toxin has not been used but instead, an improper Cry1Ac toxin was used because this control was easier. This could also make these tests not valid.

GEAC point in favour of Mahyco: The crylAc gene inserted in Bt brinjal event EE-1 has been constructed by combining the first 1398 nucleotides of the crylAb gene (corresponding to amino acids 1 to 466) (Fischhoff et. al., 1987) with nucleotides number 1399 to 3534 of the crylAc gene (corresponding to amino acids 467 to 1178). The resultant protein encoded by this gene is 99.4% identical to native Cry1Ac from Bacillus thuringiensis sub sp. kurstaki. This difference of 0.6% is attributed to the difference in presence of one amino acid at position 766 i.e. serine in place of leucine.

S: This has not been proven by resequencing three different brinjal samples after growth in the plant. The transgene described is well chimeric, it was maybe the genetic construction used but several research articles have shown (Bertheau Y.'s group; Buiatti's group... ) that commercialized GMOs have mutated sequences during or after insertion in the plant. Moreover, only one amino-acid change in a protein may have a folding and a pathological incidence, like in pathogenic prion or several genetic diseases. The experiences made with the recombinant protein not extracted from the Bt brinjal are thus invalid, because they were made with an improper protein out of a chimeric Cry1Ab-Cry1Ac gene which only resembles to a mutated Cry1AC, this is recognized by Mahyco.

G: The molecular characterization studies (Western blot) have confirmed that Cry1Ac protein expressed in EE-1 brinjal is equivalent to native B.t.k Cry1Ac as also Bollgard cotton event 531 already approved in India.

S: Every scientist in molecular and cellular biology knows that a Western blot is not sufficient at all to characterize a protein, anyway this figure is not shown in the file precisely enough, and the protein from Bt cotton has not been assessed for long term consumption.

G: The EC-II concluded that the argument that this protein is unknown is incorrect as detailed characterization has been undertaken. The issue has been raised on the presumptions that the inserted construct is going to produce unknown chimeric protein. Based on the scientific facts, it is evident that large chunk of gene is cry 14c and the expressed protein is also the same as per the experiments shown. Hence the control is appropriate.

S: "Large chunk of gene" is not an entire gene! You are potentially feeding billions of meals with that improperly characterized protein! According to my previous arguments, this is still completely wrong. There are no scientific data of sequencing the transgene appropriately inside the plant, and Mahyco already admits a difference anyway.

2. S: Two unnecessary antibiotic marker genes, called NPTII (neomycin phosphotransferase II) and aad (coding resistance to streptomycin or spectinomycin) have been used in Bt brinjal. Antibiotic resistance is recognized to be a major health problem and the commercialization of such a food should be forbidden.

G: Antibiotic resistance markers have been extensively used in the production of GM plants. The health issues related to antibiotic marker genes have already been addressed by numerous peer reviewed publications and studies as well as reviews by regulatory authorities worldwide such as US FDA, Health Canada, EFSA, FSANZ etc. Though, the antibiotic resistance genes produce enzymes that can degrade antibiotics, it has been well researched and proven that the enzymes from these genes are produced at such low levels that is absolutely ineffective on the antibiotic. Numerous studies have also been carried out on the fate of antibiotic resistance marker from GE plants in digestives tracts. It has been well established that the probability of transfer of transgenic from GM plant material to bacteria (including that normally inhabit stomach and intestine) is unlikely because of a series of well established barriers. All the above is supported by experimental evidence.

The EC-II concluded that the two genes used in Bt brinjal Event EE1 i.e npt II and aad genes have already been accepted for use by regulatory authorities around the world, such as USA, EU, Australia, Philippines etc and that the crops containing the same have a history of safe use for more than a decade.

S: The facts are 1/ these genes are useless to be added in all cells of a genetically engineered food or feedstuffs, 2/ millions of people are deceased or sick or had amputations because of capture of antibiotic resistance genes in the environment; they did develop antibiotic resistant infections, 3./ all the experimental evidence non referenced above is not in the real life. These genes should be removed before commercialization; this is also the recommendation from European Union.

3. S: Cooked forms of Bt brinjal are supposed not to contain Cry1Ac although the specificity and sensitivity of the assay does not form a part of the dossier. Thus this cannot be accepted as proof that the Bt toxin is not present in cooked Bt brinjal.

G: All cooked forms of Bt brinjal have been tested using ELISA for the presence of the Bt protein, which is an established and accepted method for testing the presence of the protein. The ELISA kit is based on a monoclonal antibody which specifically recognizes Cry1Ac/Cry1Ab antigen only. It will not detect any other Bt protein. The Cry1Ac ELISA plate is highly specific for Cry1Ac residues and Cry1Ab residues in plant extracts. It has a sensitivity of below 1%, i.e. the plate can detect presence of Cry1Ac. The limit of quantification (LOQ) is 0.625 ng/ml and limit of detection (LOD) is 0.046 ng/ml. Further it has been demonstrated that Cry1Ac protein is heat labile and thus is not expected to be present in any cooked form of the brinjal. Thus ELISA results have confirmed the same.

S: This is a convincing answer. However, undetectable secondary metabolites of Cry1Ac or disruptions of metabolism due to Cry1Ac justify completely toxicity experiments with cooked Bt brinjal or food/feed from it.

G: The EC-II was of the view that no additional information regarding toxicity and allergenicity needs to be generated as the Bt proteins have a history of safe use since last more than one century, starting from whole bacteria to purified proteins.

S: Not at all, there is no authorization for adding Bt in food as an ingredient in the world, nor serious chronic toxicity study with a Bt protein in food. And if that was for a Bt protein, that does not exclude effects with this new mutated toxin in Bt brinjal.

3bis S: It is also expected that cooking degrades at least in part the Bt toxin. However there is no information on toxicity and allergenicity of the resulting products.

G: Bt protein behaves like any other protein during the cooking process. Further it breaks down into common amino acids in the digestive system, which are part of the normal diet and are neither toxic nor allergic. The Cry1Ac protein has been extensively tested internationally in various digestive assays and found to be safe.

The EC-II was of the view that no additional information regarding toxicity and allergenicity needs to be generated.

S: To think that every protein is degraded in individual amino acids in the digestive tract is scientifically wrong. There is always a part non digested and smaller undegraded peptides, every physician and person looking at his feces knows that. Once again, the experiments with the improper Cry1Ac protein, as it was admitted by Mahyco's answer above, are not valid for this purpose, unless laxity is admitted. We are not in an approximate proof of a research but in common life potentially feeding billions of humans and animals!!

4. S: Studies for toxicity assessment and nutritional effects in mammals have been limited to a maximum of 90 days period or less, which is not adequate and there is a need for long term studies for assessment of chronic effects.

G: Safety assessment of a GM food crop requires an integrated stepwise and case by case approach. The evaluation of possible toxicity and allergenicity of gene products as well as consideration of the nutritional aspects is undertaken in a comprehensive manner through a multitude of tests. As per FAO/ WHO expert consultations and Codex principles, toxicity studies up to a 90 day repeat exposure study are sufficient to indicate the safety of the GM crops. Similarly, the guidelines have been provided for assessment of allergenicity and comparison of nutritional aspects. The studies undertaken in Bt brinjal comply with the international guidelines as well as ICMR Guidelines accepted by GEAC.

S: The scientific history has been written first believing that the planet earth was flat. Who can believe that a new genetically engineered fruit producing an uncharacterized insecticide may be given to millions of beings without being tested more than three months on rats?

G: The EC-II concluded that no long term studies are required because of the following reasons:

• The Cry1Ac protein inserted into Bt brinjal event EE-1 has been extensively studied for its safety. It has been well established that the Cry1Ac protein cannot cause any toxic effect in mammals because of lack of highly specific receptors and alkaline environment in the gut of mammals.

S: Arguments already have proven wrong above.

• G: Cry1Ac protein has a history of safe use for human and animal consumption as GM crops such as Bt maize and Bt potato containing Cry proteins including Cry1Ac protein have been consumed by millions of people without any adverse effects.
• S: Arguments already proven wrong above.
• G: It has been reported that 90 -110 days of age (mating age) of rats is considered equivalent to 21-25 years age of humans (Laboratory Animals in the Study of Nutrition' in LAIS Centre
• News- No - 30, 1993).

S: We hope that people will not be living only 25 years anyway, thus it is crucial to prolong the tests; moreover this is a too much approximate calculation. Developing small animals (from gestation to 2 months) should be used also, babies being more sensitive than adults.

• G: Cry1Ac protein has shown to be rapidly degraded (in 30 seconds) in simulated digestive fluids and thus is not detectable even in the short term studies.

S: This experiment has not been done with the good protein (see above) which is not the exact Cry1Ac in Bt brinjal, moreover it could be processed differently in the plant than the recombinant protein used in these experiments.

4 Sub-chronic feeding study in Goats

a-S: There was significantly lower hay consumption in Bt group in week 11 in comparison to non
Bt group. The authors of the experiment do not conclude anything problematic from this.

a-G: Inter-animal variability and intra-animal variability at different time intervals in the feed/hay consumption is commonly observed in the feeding experiments. Such differences are expected in dynamic biological systems but such differences should be checked only if they are statistically significantly different. In the present study, there is variability in the feeding pattern of animals at different intervals of time e.g. hay consumption of Bt brinjal treated group males during week 11 was reflected due to decreased consumption in only one male goat. This particular animal has shown similar variability in the hay consumption as seen in the goats with normal diet without brinjal as well as with non Bt brinjal. As the differences were not of any statistical significance, the decreased hay consumption of Bt brinjal treated males during week 11, was considered as incidental and not related to Bt brinjal treatment.

S: The difference pointed out IS statistically significant according to the original Mahyco's file. The authors of the experiment could explain that 1/ It is only due to one animal. 2/ It is not biologically significant. If 1/ is true, that means that there is a crucial lack in the protocol of the experiment taking a dramatically insufficient number of goats, and thus no conclusion can be driven out of this invalid experiment. 2/ There is no proof at all in the experiment that it is not of biological relevance. Any sign of toxicity should be considered since these are dramatically short experiments insufficient to study chronic toxicity. If all statistical signs are neglected one after the other on an arbitrary subjective basis, there is no point at all to undertake such experiments. More investigations are needed.

G: The EC-II opined that it is inappropriate to quote insignificant observations out of context as many such observations seen at one time point do not persist at the next time point or not observed in the other sex.

S: Sorry, but to wait for a persistent sign of toxicity in both sexes is wrong (Seralini et al., 2009), since carcinogenesis or endocrine disruption for instance is settling by waves and sex-differentiated.

b-S: The feeding trial consisting of six male and six female but on page 323 it has been stated that the trial consisted of 6 male and 3 females.

b-G: The phrase reads as 12 animals (6 males and 3 females).
The EC-II noted that this is clearly a typo graphical error. The entire dossier provides data on all six males and six females.

S: Admitted, but not very serious for a commercial file. Proofread please next time.

c-S: The prothrombin time as well as total bilirubin was significantly higher in the GM-fed males at termination, and alkaline phosphatase was significantly lower.

c-G: As regards the prothrombin formation time, the values in the pre treated male goats as well as Bt brinjal treated animals were within the physiological ranges. These marginal changes in the Bt are a normal variation and of no statistical significance. Although some differences were noted in the total bilirubin, AST and ALT values in control Non -Bt brinjal and Bt brinjal treated groups in comparison with the Normal diet without brinjal but even the higher total bilirubin values were within the physiological control range. Additionally, there were no alterations in the hepatic parameters or histopathology of the liver. Therefore these changes were considered as normal variation and not related to Bt brinjal treatment.

S: We are speaking about the statistical differences that I cited, that are not by definition within the physiological variations a priori, otherwise there is no point to do this experiment. The authors of the experiment are confused between significant statistical differences, they always say that these are within physiological variations whatever these are, without demonstration, and they affirm that there is no biological significance. This point clearly cannot be assessed during 90 days only, since any sign of toxicity should be taken into account in this context. Correlations with other parameters may exist or not at the first beginning of a pathology, and if it is the case it may be an indication, but the contrary cannot be BY ANY MEANS a proof of safety. Theoretical reasoning and parameters taken into account are not clear. Histopathological changes cannot be assessed by the referee and the counter-expertise, and thus are not valid to cite.

G: The EC-II noted that no statistically significant changes have been observed in the parameters mentioned and the values are within the normal physiological ranges. Further significant changes given only at a single time point and not given at the end of the study are considered to be transient changes. These changes are also not associated with any histopathological changes and therefore do not warrant any attention.

S: Again, sorry, but transient significant changes within the first 90 days (this is the case) may be signs for settling of a chronic pathology. Who is a medical doctor contradicting that within GEAC or in the world? Who could sign in GEAC that he has examined organs and histopathological slides of this experiment? For any histopathologist a sign may warrant attention. The last sentence reveals, I am sorry, a crucial laxity.

d-S: Growth curve in Bt fed -males are below the others from week 7. They gain a lot less weight. The feed consumption is lot less, even 25% less (week 5) only for this transgenic fed group. This is important although not clearly reported in the summary and obviously significant after curves observation. This appears to be a sex dependent effect like for endocrine diseases. Bt brinjal as an animal feed, or human food that it will be mostly, cannot be considered as safe with such results.

d-G: Barring the intra-animal variability, there is no statistical significance difference obtained in the feed consumption, body weight and body weight gains and the hay consumption (except for the incidence of decrease hay consumption in males during week 11) of Bt brinjal treated group and the Non - Bt brinjal treated group and Normal diet group. The scale used in the growth curves very small, even a subtle change is seen with magnification.

S: Some statistical changes are effectively revealed by Mahyco in the original file.

G: The EC-II noted that the test product findings need to be compared with the concurrent control and/or normal control findings, baseline control data/historical control data and published references wherever applicable and in this study, the generally accepted procedures have been duly complied with.

S: Sorry, but the historical control data do not mean anything in this experiment. In science to raise a conclusion the treated animals should be first compared with the appropriate controls in the experiment, these having similar living conditions and closely related diets, except for the GMO. Once again, it is wrong within 90 days to search for a final proof of toxicity to stop Bt brinjal, we are preoccupied by any sign of toxicity that warrant attention, and then we need to ask for further investigations since billions of lifes will be concerned.

5 Sub-chronic feeding study in rabbits

a-S: There was a reduction of consumption at week 6 in the male Bt group in comparison to non Bt, the GM fed males consumed less in general, in the female group at week 11 (due to one animal, but the groups are too limited in numbers of animals unfortunately to calculate a real statistical significance) as if the Bt brinjals were less palatable. The females consumed less Bt brinjal.

a-G: The data related to food consumption demonstrates that the average food consumption in the Bt brinjal and Non -Bt brinjal treatment group- males were 95 and 104 grams/rabbit/day, respectively. The decrease is very marginal (9 grams) and such variations are expected in dynamic biological systems.

S: A new light is shed with parallel signs occurring in goats, this is preoccupying.

G: In respect of variations reported in the sub-chronic feed study in rabbits (5 a to c), the EC-II opined that in general toxicology and in safety testing, the results are compared between test and control groups. If differences do occur which is but expected in dynamic biological systems such differences should be checked for:
i. Are they statistically significantly different.

S: Yes, we noticed only these ones admitted by Mahyco with the proper closest isogenic controls.

G: ii. If yes, are the values or data in the normal physiological range.

S: This is too subjective to be taken into account since it was not defined a priori. The other controls eating other kind of diets not substantially equivalent but richer or poorer in vitamins, ions, minerals, proteins, amino acids, lipids, sugars... do not represent the physiological range but other treatments.
Thus we cannot answer to that in the protocol or from the back of a head.

G: iii. If there are more than one dose group then one should look for dose dependent changes in the parameters.

S: Absolutely not, sorry, it is completely and scientifically wrong, since endocrine disruption, carcinogenesis, or immune answers for instance are not linear within two doses chosen a priori, which is the case in this protocol.

G: iv. Even if there are significant differences and they are in physiological range then one should correlate biochemical and haematological data with histopathological changes.

S: If this is possible OK, otherwise one should compare the patterns occurring in all mammalian models, which are preoccupying here. Moreover, if there is no correlation at all, never forget that in science and medicine the first signs of any pathology are not correlated, but there can be wild sporadic disruptions of variable parameters. Once again, it is wrong to search for a final proof of chronic toxicity within 90 days; we are just looking for the first signs of a possible chronic pathology, because billions of people are potentially concerned with such a diet during their entire life.

G: v. If there are significant differences and they are outside physiological ranges and they are associated with parallel histological changes e.g. elevation of AST, ALT with liver cell necrosis or changes in haematological parameters with corroborative changes in bone marrow. Then it is assumed that the test material could result in cellular changes. Significant changes given only at a single time point and not given at the end of the study are considered to be transient changes.

S: Of course transient changes should be taken into account and not a priori neglected just because they are transient, it could be an heavy medical mistake to consider that there is nothing to see, without following the experiment after 90 days and during 2 years, like for any chronic search of a side effect for a drug in a laboratory mammal.

G: In view of the above, the EC-II concluded that the findings of the study are in order and the variations observed are not attributable to Bt treatment.

S: There is no reason to conclude that sorry, as explained above. We conclude that there is no proof of toxicity, but possible signs of the beginning of a chronic pathology, and that the experiments should be prolonged in mammals before giving this very new insecticide-producing plant in billions of meals.

b-S: There was at interim blood sampling an increase in albumin and total bilirubin in GM fed males versus adequate controls, and of total bilirubin and lactose dehydrogenase in GM fed females; at terminal blood sampling again a significant increase of total bilirubin in males and females GM fed, increases in hepatic markers alanine and aspartate aminotransferases and sodium levels in GM fed males, a decrease of glucose levels in GM fed females. The authors of the study claim all the above differences as incidental and not treatment related, with no scientifically acceptable reasons.

b-G: The changes in the total bilirubin were normal variations in biological systems. All the values were within the normal ranges and do not signify any organ pathology and hence were considered as incidental and not related to Bt brinjal treatment.

S: All these are very preoccupying changes which are statistically significant changes according to Mahyco (between GM-fed animals and appropriate controls). The reasoning exposed in the previous paragraph is not understandable a priori for all the reasons already explained in a detailed manner by the referee.

c-S: The platelet count was significantly reduced during the experiment as well as mean corpuscular haemoglobin concentration in the blood of Bt fed males in comparison to their controls, an increase
in haematocrit value; prothrombin time was increased in females.

c-G: The minor variations in various blood parameters are physiological changes which are observed even in the Normal diet treatment and the Non-Bt brinjal treatment and hence considered as not related to the Bt brinjal treatment. Further the testing lab as well as experts have re-examined the data and informed that all the figures are within the normal ranges (e.g. the normal range of
platelet count is between 1,00,000 to 5,00,000).

S: The statistical changes first count between GM-fed animals and their closest controls. This should be considered in relation to disruption of similar parameters in the other mammalian model. The normal range has not to be decided a posteriori independently of experimental conditions, strand, etc... Otherwise there is no need to put controls in the protocol!

6. Feeding study in (lactating, crossbred) cows:

a-S: It has been claimed that Bt toxin is not detected in blood but there was only a short description of the method of detection and its limits and efficiency as well as repeatability were not indicated.

a.-G: The method of detection that has been used is ELISA based on a monoclonal antibody which specifically recognizes Cry1Ac/Cry1Ab antigen only. It will not detect any other Bt protein. It has a sensitivity of below 1%. The limit of Quantification (LOQ) is 0.0625 ng/ml and Limit of Detection (LOD) is 0.046ng/ml.

S: OK, good, but other direct an indirect metabolites are not proven to be detected by this method.

G: The EC-II noted that the test has been standardized by M/s Mahyco and the required information has been provided.

S: OK, but not for the question just mentioned above.

b-S: More milk production (14.3%) by GM fed cows, almost as if they were treated by a light hormone, in 42 days.

b-G: The data clearly indicates that there was no significant difference in weekly yield and feed intake between the cows fed transgenic and non -transgenic brinjal fruits.

S: The power of the statistical method is too bad to detect such changes with such limited number of animals, thus the conclusion is maintained.

G: The EC-II reiterated that insignificant changes have been unnecessarily highlighted.

S: Sorry for GEAC, the necessity is to reveal any sign of problem within a so short period, overall when a committee is legally responsible for giving advices to the government giving authorizations for this product in billions of meals. To my mind, the composition of the GEAC committee is too scientifically light, as well as from a medical point of view, to be maintained as such.

c-S: The ash content of the milk varied significantly for Bt brinjal - fed cows between the second and fourth week, by the end of the experiment they had significantly more roughage dry matter intake (10.5%).

c-G: The variations in ash content in milk in different weeks were also there in cows fed with non-transgenic brinjal fruits.

S: No, we speak about statistical differences with appropriate controls. Look again the results.

G: The EC-II noted that all the values of ash content were within the normal range in both the groups.

S: No, there were statistically different with appropriate controls. Look please!!

d-S: It cannot be concluded from this experiment that there are no metabolic changes after Bt brinjal consumption in lactating cows and thus this feed cannot be considered as safe.

d-G: There was no significant difference between the cows fed transgenic and non-transgenic brinjal fruits and differences in weekly yield and feed intake.

S: Yes there were statistical changes with appropriate controls admitted by Mahyco. Anybody can take the time to check in the raw data.

G: The results of the study clearly demonstrated that consumption of Bt brinjal by cows did not result in any metabolic changes and showed no adverse effects.

S: This is wrong, sorry. Discussed above.

7. Sub-chronic oral toxicity study in (Sprague Dawley) Rats:

a-S: The first experiment of 14 days with rats allowed to the company to test two doses of Bt brinjal is a badly designed experiment from a scientific point of view, increasing control animals by 2 in regard to treated rats. This was unexplained.

a-G: All protocols were designed as per the guidelines issued by DBT and approved by RCGM. Two controls (non-Bt counterpart and commercial Brinjal) were used in the study to demonstrate that there were no differences between non-Bt hybrid developed by the applicant and commercially available Brinjal.

S: In any serious scientific experiment, if GMO potential toxicity is really studied, only this parameter should be changed first. A mixture of substantially equivalent commercial brinjals could have been given in controls, but the number of studied animals should be the same in GM and non GM-fed rats by the end to avoid statistical problems and bad power of discrimination of any effect!

G: (a-b-c) The EC-II noted that increasing control animals in no way affect the scientific credibility of the experiments. The reviewer has unnecessarily tried to correlate common and incidental observations to the effect of Bt brinjal which is very unfortunate and indicates lack of familiarity with the subject.

S: The reviewer has more than 20 years familiarity with carcinogenesis experiments, or toxin effects in vivo in rats, more than 10 years familiarity in assessing commercial files of GMOs as an expert for governments. More than 10 research papers assessing GMO residues toxicity and several books published in major editors on this subject are also in his CV. How many GEAC members have similar experience?

G: For example circling observed due to internal ear infection is a common observation in all rodents in cages and has taken place in both Bt and non Bt group. However, the same has been highlighted only in case of Bt group to mislead the readers about safety of Bt brinjal. Regarding the minor difference in clinical parameters, the EC-II reiterated its observations cited earlier (refer Point 5).

S: The referee didn't speak about that, sorry, read again.

b-S: Circling disorder and diarrhea were noticed only in the Bt brinjal group, males and females.

b-G: Circling was observed in one rat from non-Bt brinjal control group and one rat from Bt brinjal group. This is due to internal ear infection which is commonly noticed rodents housed in cages. This does not affect normal living of rats. Hence it is not related to Bt treatment Diarrhoea seen in only one female and two males out of ten animals in each group of rats, for only four days out of 90 days of study is an incidental observation and not related to treatment.

S: This conclusion is somewhat arbitrary, no further analysis has been performed. An impact on the immune system of a treatment may always render the animals more sensitive to infections. No cytology analysis was available, which is a common analysis when animals are sick in an OCDE standard test.

c-S: Moreover liver weight and relative liver to body weight ratio decreased in the dose range study in females, by 13% apparently significantly. Bt brinjal cannot be considered safe for rats considering these results. For the rats fed with Bt brinjal water consumption was 8-21% more than the non Bt brinjal group for some periods. The significance of this claimed to be null. However, all the scientific committees consulted agree with companies that statistical significant differences have been reported during 90 day studies between control and treated rats with different GMOs on numerous parameters, including blood composition and detoxification organs such as kidneys.

c-G: Minor differences in clinical parameters of Bt brinjal fed animals have been quoted out of context as such variations are fairly common in biological systems. Many observations seen at one time point do not persist at the next time point or not observed in the other sex or not significant against the non-Bt brinjal.

S: 13% of liver weight significant difference, for instance, in comparison to appropriate controls, is not minor at all, but the sign of a mammalian physiology that could have heavy nutritional problems within a so short period. Biological systems should be assessed in comparing in this case GM-fed rats to their closest appropriate controls. Otherwise think about this question: what could be considered as preoccupying for GEAC? Significant differences only at one point out of two in an experiment should be considered as a potential problem to be explored, otherwise a laxist interpretation may lead to a criminal attitude towards billions of people (these tests last 90 days only!). Sorry, but to avoid to consider an effect because it is only in one sex is stupid, non scientific, and criminal also. Numerous hormonal imbalances begin in one sex first, numerous cancers are sex-dependant, and not only for breast or gonads, but liver and kidney are biochemically sexually differentiated (Seralini et al., 2009).

8.  Primary skin irritation tests on (New Zealand white) rabbits:

S: Three rabbits only were treated with Bt brinjal on a total of 12; this is not serious at all.

G: In the study, the Bt treatment was compared with three sets of controls, which does not in any way affect the results obtained with Bt treatment. The study was conducted in 2004 as per the "Guidelines for toxicity and allergenicity evaluation of Transgenic Plants", 1998.

S: Sorry, three rabbits only were treated with Bt brinjal on a total of 12; this is not serious at all. To test immune problems seriously in a whole human situation? Revise your physiology.

G: The EC-II concluded that since the non allergenicity of Bt protein has been demonstrated through a series of studies using pure protein...

S: It was proven above that the "purified protein" was not the toxin in the Bt brinjal. This argument is invalid.

G: ...this study is not much relevant and not required as per the new Guidelines for safety assessment of foods derived from GE plants, 2008 adopted by GEAC.

S: This is not correct, as indicated.

9.  S: Mucous membrane irritation test and measurement of allergenicity in young adult Brown
Norway rats are very limited tests to assess allergenicity for a whole population, a serum bank to
assess antibodies potentially reacting with Bt brinjal residues could have been an alternative.

G: Allergenicity assessment is undertaken using a weight of evidence approach based on FAO/WHO Consultation on Biotechnology and Food Safety. Internationally accepted methods such as the source of gene, heat stability, pepsin digestion, amino acid homology testing using bioinformatics have been used in allergenicity assessment of Cry1Ac protein in Bt brinjal.

S: Cry1Ac as such is not present in Bt brinjal, the modified Cry1Ac toxin in Bt brinjal may have reactivity. This was not tested, it is a severe lack in this Mahyco's and GEAC assessment.

G: In addition, allergenicity tests in young adult Brown Norway rats were undertaken as per the regulatory requirements stipulated as per the "Guidelines for toxicity and allergenicity evaluation of Transgenic Plants", 1998.
S: Three animals only are ridiculous in comparison to the goal of immune safety testing that should be reached.

G: The EC-II concluded that the allergenicity assessment of Bt brinjal event EE-1 has been done as per the internationally accepted Codex guidelines. Further, the requirement for testing in young adult brown Norway rats is not mandatory in India as per the newly adopted Guidelines for safety assessment of foods derived from genetically engineered plants, 2008. This requirement has been dispensed with as there are no validated animal models for allergenicity assessment of GM foods.

S: A screening of a patient's serum bank as been proposed by numerous authors in this case; the development of specific allergy tests in humans is also possible for medical doctors.

10. GM brinjal consumption by birds (Feeding study in broiler chickens):

a-S: 40 unsexed chickens received 5% Bt brinjal in their diet, 40 others, 10%, and 200 received different non GM diets. This was not a good design to detect any unintended GM effect in these conditions. In particular 10% is too low a percentage to clearly see unintended effects.

a-G: The experimental design consisted seven dietary treatments with two levels (5 and 10%) each of Bt, non Bt parental and a commercial brinjal along with a corn soya control treatment. Each diet was fed ad libitum to five replicated groups of eight unsexed chicks in both the grown phases i.e. starting 0 to 3 weeks and finishing 4 to 6 weeks phases following completely randomized design. The experimental design is a widely accepted design and many research papers have been published in international journals with the similar design.

S: But still what I have said is exact and several moratoriums have been put in the world; moreover no country has grown nor tested Bt brinjal; moreover other GMOs proofs of safety are widely discussed and generally found insufficient all over the world. These papers have been invalidated by numerous scientists in the world.

G: The test diets contained 5 or 10% brinjal meal in dried powder form as part of total daily diet that for 42 days (starting from 1 day -old to 42nd day) ad libitum without any interruption in between. The 10 g dried brinjal meal in 100g of feed is equivalent to 67 grams of fresh brinjal for a broiler of about 1000g body weight. Hence, these levels of inclusions are much higher than generally consumed by human being.

S: Not within 42 days at all for chronic safety testing, this is the main point of this written debate.

G: (a-b) The EC-II opined that broiler chicken is the representative model and the experimental design is in conformity with the accepted guidelines. The EC-II was of the view that the reviewer has unnecessarily' chosen to highlight insignificant issues. All the six groups have lower and insignificant differences compared to the control group.

S: GEAC always opines Mahyco's assessments in this file, thank you, we all know that. The answer is not relevant, Mahyco's one was better.
b-S: Moreover there is only one species of bird studied for a limited period of time.

b-G: Poultry birds especially broiler chickens are used as model animals for bio-safety studies because of faster growth rate. Also since the broiler chickens are more vulnerable to any toxic / anti-nutritional factor (s) present in feed/feedstuffs they serve as good model animals. Six weeks (0-42 days) experimental period, when rate of growth is maximum, is sufficient to exhibit the toxicity of any substance, if any. Being rapid growth, 37 times of initial body weight, in the present context, in 42 day old broiler chickens were the most suited birds for conducting this experiment.

S: For environmental assessment concerning non-target species of birds, if bird biodiversity is a concern in India, this is insufficient, sorry.

c-S: The feed intake for GM-fed broilers (10% Bt -brinjal) was 10% lower than in the corresponding control (10% non Bt brinjal in the diet) at different weeks (21-35 days of age) and then higher, the implication of this is a differential metabolism between both groups but the experimental report did not calculate the statistical significance of this difference. The blood glucose was also significantly different in the Bt groups.

c-G: The feed intake for GM-fed broilers was compared statistically week -wise and phase-wise. The mean values did not differ statistically/significantly either when analysed on weekly basis or on phase basis. The blood glucose was also significantly different in the Bt group in comparison to control diet, which was also evident in all the treatments fed brinjal from any source. Therefore, it is the characteristic of brinjal as a whole, not for Bt brinjal alone.

S: The other brinjals are not demonstrated chemically equivalent at all to the Bt brinjal and its control, they may contain less water or more sugars, thus this scientific reasoning is invalid in this case.

G: (c-d) As regards the intra-animal variability in the results, the EC-II reiterated that such differences are expected in dynamic biological system. The EC-II further reiterated its observations in Point 5.

S: If such statistical differences are expected from this design, that means that GEAC opines that potential toxicity signs may be evidenced, since there is no point to consider that all significant results are obtained just by chance only, in a well powerful design of a statistical study. And if this was possible, the contrary also. In order to decide, drastically wrong scientific assessments should be avoided such as: a statistically significant effect has to be present in both sexes to be a clinical sign of a potential chronic pathology, or should be linear to the dose between two doses chosen a priori to be assessed, or the effect has to be outside of the range of any other diet, not chemically equivalent, but containing other brinjals. All these reasonings have been used by error by Mahyco. Sorry to repeat again, but Mahyco finally always uses these arguments at the final points of each experiment of this debate, that GEAC repeats, and that are fully wrong for the all scientific serious community.

d-S: The authors of the experiment write that there is no significant difference due to Bt brinjal consumption by chickens, but these differences lead instead to the conclusion that the Bt brinjal cannot be considered as safe according to this experiment.

d-G: The reviewers have interpreted the results based on the observations and statistical analyses. All the response criteria, related to growth and welfare evaluated in this study were not affected by feeding the brinjal of any source. Neither the heterophile to lymphocyte ratio nor the yield of visceral and immune organs, humoral and cell mediated immune response were affected with Bt brinjal. Hence, the conclusions drawn were based on scientific observations, rather than mere assumptions.

S: There is no other thing for counter-expertise than the raw data of this experiment and Mahyco's statistical analysis. The counter-expertise has been base on pathological / endocrinological interpretation of these data. Going to the raw data, the confidence has been lost with the criteria of interpretation used by Mahyco. All the statistical data between Bt brinjal fed groups and closest appropriate control are confirmed by Mahyco in the raw data, whatever the comments are.

11. Feeding studies on common carps:

a-S: There were numerous unnecessary non-transgenic control groups masking the significant effects between the two closest groups, Bt and non Bt. There were only 6 pools of 60 fishes (360) receiving Bt brinjal in the feed on a total of 24 pools, i.e. 1440 fishes, instead of having two main groups. This disproportion can mask a lot of significant effects if only a small group is compared with all the others.

a-G: The four groups of brinjal used in feeding trial for common carp were taken for better results to actually compare with each other. Secondly, the numbers of fishes per pool were enough (60/pool) for statistical analysis with two replicates (60 + 60 =120) for each test concentration (15%, 30% and 45%) of four groups of brinjal used totalizing 360 fishes for each brinjal feeding trial. Thus, there was no harm to undertake the comparative study of Bt and Non-Bt with other groups of same type of brinjal available in the market. This was taken up for the comparative studies without adversely affecting the objectives.

S: OK, but the point above is not answered. There were two many different non Bt brinjal groups that can mask the results. Instead, Mahyco should have made two equivalent groups in numbers (720), one fed with Bt brinjal, the other fed with a mixture of commercial brinjals equilibrated to be substantially equivalent to the first group (except for the GM character and toxin), in order to assess the effects of several brinjals in comparison.

G: The EC-II opined that the objection of disproportionate sample sizes between treatment group and control group is very unfortunate and unscientific.

S: Thank you, then this experiment is invalid and the effects of Bt brinjal release in the environment is at best unknown because there are no valid studies on aquatic life, and any brinjal cultivation will put Bt brinjal residues in contact with surface waters and finally with aquatic life. A moratorium should be pronounced immediately for Bt brinjal release in the environment. At worse, statistical signs are real signs of chronic pathology and this Bt brinjal release should be forbidden.

G: Comparison was made with Bt-brinjal group with non-Bt brinjal group, local variety and with a group that does not contain any brinjal. This means the Bt group is compared with three times its size. This is very robust and logical, since data in each group is provided separately with its own statistics reported. No pooling of the control group was done to mask the observations as claimed. Further growth was similar in all the groups, which nullify the possibility of any apprehension regarding the comparison between Bt and non-Bt brinjal.

S: The pooling was not done in the calculations, but in the statistical comparisons and the interpretations, when any significant effect was in the range of any other non chemically equivalent diet, it was considered as null. This is wrong, since a diet with more or less lipids, salts or sugars may induce pathologies as worse as some GM effects.

b-S: Average feed conversion and efficiency ratios were significantly higher in the Bt group versus closest control, at 45% brinjal in the diet. No safety can be concluded.

b-G: The Feed Conversion Ratio (FCR) and Feed Efficiency Ratio (FER) ranged between 2.8 + 0.02 to 3.3 + 0.10 and 0.35 + 0.02 to 0.30 + 0.02 respectively in all the feeding trials of four groups of brinjal. Furthermore, FCR and FER in the Bt group versus the closest control, at 45% brinjal in the diet were recorded as 3.1+0.2 & 3.3+0.10 and 0.32+0.03, 0.30+0.02 respectively which clearly demonstrate that there is no significant difference in feed conversion and efficiency among them.

S: Crude values never demonstrate a significant difference or not, sorry. Again this is a crucial mathematical and interpretation error. The "nose opinion" cannot replace the statistical calculations done by Mahyco itself.

G: The only significant difference with respect FCR found at 45% level of feeding is unusually higher for fish. This difference might have been caused due to erroneous recording of feed intake data for the Bt brinjal fed group at 45% level. This can be explained as:

FCR = Feed intake/Weight gain

The weight gain was similar in all the groups as explained earlier (table 9). Hence FCR value will only increase if feed intake value increases. Feed intake value is calculated after collecting the refusal. If something is leached out into water then it is not estimated. There is maximum possibility of overlooking this factor, which otherwise gives a wrong impression of higher feed intake. Inclusion of 45% brinjal in a pelleted feed may definitely change the texture of the feed leading to leaching of the dry matter. The water stability of that diet was beyond the scope of the study.

S: Thus Mahyco recognizes at best an important mistake in the data recording. This experiment should be considered as invalid and the underlined conclusion above is confirmed.

G: The EC-II opined that these minor differences are in no way linked with the safety issues.

S: There is no scientific reason to decide that a priori. Science does not function with such a priori authority decisions.

12. LIMITED TESTS OF BT BRINJAL ON SOME SOIL MICROFLORA:

a-S: Limited environmental studies of Bt brinjal risks have been performed on an extremely little part of soil microflora, collembolan, nematodes and earthworms. It is almost impossible through a few species measurements to get a whole view of a complicated ecosystem, moreover varying a lot from place to place in India. In addition, statistical tests that have been chosen appear to be limited, grossly inadequate as we have demonstrated in other studies (Seralini et al., Arch. Env. Contam. Tox. 52, 596 -602, 2007). There are some severe limitations to the studies performed or that can be performed:

• Culture media used do not allow for sure all bacteria and fungi to be measured.
• Not all groups of invertebrates or insects have been taken into account.
• Bt brinjal was cultivated only during 5 months before testing soil fertility, but most effects can appear after long term cultivations with pesticides treatments.
• New Bt insecticide present in the soil due to GM brinjal and produced by it may be partially linked to particles and be released after rain or environmental changes, this has not been assessed either.
• Significant differences have been observed in colembolla and earthworms populations between Bt and non Bt real control fields. Two additional controls mask the effects, by the end of the experiment (120 or 150 days), these don't have to be persistent to be of biological relevance since evolutions and reactions may exist in these complicated ecosystems that could alter in a long-term soil life and fertility.
• Mortality of beings is often an insufficient parameter measured, reproduction capacity or physiological parameters are more pertinent for non-acute but chronic effects.

a-G: The soil microflora studies and effects on Collembola, nematodes and earthworms in Bt brinjal field plots have been carried out in almost every growing season since 2003 over at least 50 locations in India and not a single instance of reduced soil fertility has been reported. The studies have been repeated in large scale trials conducted by IIVR as per the directive of GEAC. These studies demonstrated that Bt brinjal does not have any significance effects on soil microflora both fungi and bacteria and soil invertebrates such as earthworm, collembola and nematodes. No Cry1Ac protein was detected in any of the soil samples of Bt brinjal field plots.

S: It is not Cry1Ac which is in Bt brinjal but a modified form of Cry1Ac, this was admitted by Mahyco above. Moreover, the scientific hypothesis that all the toxicity is due to the toxin is a limited one, the genetic modification may have induced unknown metabolic changes either.

G: The EC-II opined that it is not possible to culture many soil fungi and bacteria and indicator species measured provide a framework for evaluation of soil effects. It was concluded that sufficient soil impact analysis has been undertaken by M/s Mahyco and IIVR. Therefore, hypothetical "evolutions and reactions" are not a justification for invalidating the studies conducted. Further the Bt cotton expressing the same gene is being grown extensively in the country since 2002. Bt cotton and Bt corn containing the same gene are being grown in more than 20 countries worldwide and no adverse reports have been reported.
S: Same answer, the toxin may not be the only reason for toxicity. Moreover a crucial debate is open on the environmental effects of Bt cotton in India that should be scientifically investigated for a Court with external experts to decide.

13. Bt TOXICITY TESTS FOR NON TARGET INSECTS

a-S: Effects on honey bees (7 days) or larvae survivals were considered non significant at 20 ppm of Bt (NOEL: chosen as the No Observable Effect Level). Ladybird beetles, or green lacewing larvae, also beneficial insects, gave similar results for the company after 30 days. Unfortunately these tests are not relevant since they have been conducted with Cry1Ac which is not the insecticide produced by the Bt brinjal at all. As anyone can see, they are also very limited in time and doses.

a-G: The results reported by the applicant are comparable and relevant as they are tested with the protein that has been demonstrated to be biochemically and functionally similar to the one produced in Bt brinjal event EE-1 through a series of tests.

S: No, it is a mutated modified Cry1Ac, this was admitted by Mahyco at the beginning of this written debate.

G: The effects on honeybees, ladybird beetles or green lacewing are tested by adding the Cry1Ac protein at doses lethal to target insect pests and represent the direct effects, if any. The dose of 20 ppm is around 338 fold higher than the dose of 0.059 ppm required to kill the target insect or stop its growth.

S: This is why the chronic real effects have not been assessed at all in these very limited tests.

G: The EC-II was of the view that the ecotoxicology tests for non-target insects have been always carried out using bacterially produced proteins as it is extremely difficult to extract the required protein from the plant material.

S: GEAC should not carry on his shoulders the technical problems of Mahyco to help them. If Mahyco is a potent serious biotechnological company, it is possible to extract a modified toxin produce in such important levels in the brinjal (16-17 mg/kg).

G: The study reports submitted by the applicant have been reviewed and accepted by regulatory authorities in many countries viz. United States Environment Protection Agency, Canadian Food Inspection Agency, Office of Gene Technology Regulator, Australia, European Food Safety Agency, etc.

S: This is not a proof. These organisms have not published their analyses in peer-reviewed scientific journals. Bt brinjal has not been cultivated at large scales in any of these countries (www.isaaa.org) and no scientific observation has been made.

b-S: Field trials are an inadequate basis to assess impacts on the agrosystem:

• Studies of long term effects are lacking,
• Studies on beneficial insects (e.g. natural enemies of target pests), as well as studies of abundance of secondary pests (which would have to be sprayed with insecticides) are lacking.

• Indirect effects (e.g. does the Bt toxin affect organisms that eat the target insect) are important in this regard.
• No laboratory studies have been performed to evaluate other lepidoptera insects.

b-G: The field trials are well accepted methods for evaluating the impact on agro system. Extensive field trials have been conducted on Bt brinjal event EE1 over a period of five years at multiple locations representing different agro-climatic conditions. During these field trials the non-target insects (includes moths and butterflies) and beneficial insects have been recorded throughout the crop growth period. A total of 17 non-target and beneficial insect species are being recorded in these field trials comprising of insects from orders Lepidoptera, Coleoptera, Thysanoptera, Homoptera and Diptera, besides spiders (Arachnida).

S: Congratulations. All the crude data are not reported in the file.

G: As regards long term effects, the reduction in insecticide use in a Bt crop will enhance the survival and reproduction of predators and parasitoids in an agroecosystem, which was already reported in several studies from Australia, China and the USA (Head et al . 2005, Chen et al., 2007). Laboratory studies have been performed to evaluate the impact on other lepidopteran insect as well and reported in the literature (Mendelsohn et al., 2003).

S: Once again, the scientific hypothesis that says that all possible side effects should be due to the modified insecticide toxin or to the reduction (unmeasured either in the raw data, by the way) in chemical insecticides is wrong.

G: The EC-II concluded that data generated during field trials coupled with laboratory and greenhouse evaluation and information on the biology of brinjal is adequate to assess the impact of Bt brinjal event EE1 on agro systems.

S: GEAC always finds that Mahyco assessment is sufficient by authoritarian decision, we all know that.

c-S: The harmful increase in secondary pests takes place after many years. Brinjal has also many insect pests (for example, sucking pests like whiteflies) that will not be controlled by this Bt toxin, and may increase over time. Thus will in turn increase chemical insecticide use compared to initial years of Bt brinjal use. This situation is difficult to predict, and would require monitoring after commercialization.

c-G: In the Bt resistance management program, the sustainability of efficacy of currently available insecticides will be considered, as Bt crops are part of holistic IPM program in a crop.

S: Thank you to agree with my point. Could these raw data (that should also have been measured during the important field trials of course) be given to the Court for counter-expertise, before any decision is taken.

G: The EC-II opined that appropriate post release monitoring mechanisms and IRM strategies would address this issue.

S: So everybody agrees with that, and that the pre-monitoring raw data should be given (Full nature of chemical insecticides - and fungicides, herbicides? - removed or added from controls, quantities released each year on controls, and on Bt brinjal fields).

14.  Bt toxicity tests for target insects:

a-S: The toxicity of Bt toxin Cry1Ac to the larvae of a target fruit and shoot borer lepidopteran insect, Leucinodes orbonalis Gwen has been evaluated by the company. The Cry1Ac was from a commercial formulation and not purified from the Bt brinjal (surrogate protein), thus modifications of the protein in amino acids, structure and post-transcriptional modifications such as potential glycosylations have not been taken into account, limiting the significance of the results.

a-G: The objective of the test is to study bio efficacy of the Cry1Ac protein on target fruit and shoot borer. Both commercial Cry1Ac protein and lypholized Bt brinjal powder have been found to be effective against fruit and shoot borer. The use of commercial Cry1Ac protein does not in any way limit the significance of the results as the protein produced is the same as present in Bt brinjal event EE-1.

S: Not at all, we have already admitted that a lot of times, this is a too much gross scientific approximation. If this could be tentatively admitted in a research paper, it cannot be in a real life for billions of individuals eating that.

G: The EC-II concluded that the insect bioassays for bio efficacy against fruit and shoot borer have been undertaken as per the protocols approved by regulatory agencies. Further, the results of insect bioassays have been confirmed in field trials conducted in more than 50 locations across various agroclimatic zones in the country.

S: This does not answer to the scientific problems previously raised.

b-S: Some lyophilized transgenic fruit powder was also used in bioassays but these lasted only 7 days. There were 12-14 fold variations in the results. The Bt protein was significantly toxic in this regard; this was the goal of the GM brinjal on this insect.

b-G: Bioassays have been done for seven days to demonstrate a dose response with Bt brinjal powder which is comparable to artificial diet bioassays. In an artificial diet bioassay, one can hold the diet with no bacterial and viral infections to a maximum of 8-10 days. The 12-14 fold variation presented in the results is a natural response observed in insect populations.

S: Thus the samples are insufficient to have good statistical results.

15. Pollen flow studies:

a-S: Cross pollination or pollen flow is a very small part of contamination possible by GM plants.
Thus, pollen flow study alone has little impact on environmental risk assessment of dissemination per se because:

• First, the seeds can be contaminated during the production when bought or taken by agricultural workers,
• The transportation and spreading of seeds for cultures is not full closed and cannot be restricted temporally to a particular designed field,
• The cultivation can imply the sharing of workers or tools or even machines that bring contamination of pollen or seeds from one field to another,
• The insects, birds, other animals such as rodents or mammals will bring fruits or parts of flowers or fruits from one place to another,
• The harvest is made by tools that are shared and may mix the productions at low levels,
• The storage is made in places that cannot be always fully dedicated to GM or non GM plants,
• The markets or transformation factories or cookers may mix the fruits or seeds.

a-G: Pollen flow is a natural phenomenon in plants, which cannot be controlled and thus its impact needs to be evaluated. Issues related to dissemination mentioned by the reviewer are external factors, several of which can be controlled and the extent to which this aspect needs to be monitored is a trade related issue and not a part of environmental risk assessment.

S: All these points become finally a huge part of environmental risks, thank you to agree with that. Thus, the monitoring plans to address these issues should be given, and reviewed, and tested also, in order to see if there are credible ways to prevent these risks.

G: The EC-II concluded that the pollen flow studies for four years as well as other environmental safety studies provide enough evidence of the safety of Bt brinjal to the environment. Other issues raised by the reviewer are hypothetical and out of the scope of the environmental risk assessment.

S: All the other issues raised by the reviewer are very pragmatic and real, happened for other files in the States of America or in Europe, and are of concern for 150 countries having signed the Carthagena protocol, these are considered as part of environmental risks. The fact that GEAC does not consider these risks means that the Indian government should claim a moratorium on commercial Bt brinjal release immediately and built another Committee with agronomic experience to assess that. This was already carefully studied by European authorities.

b-S: The sampling procedures are crude and limited and do not take into account the form and size of the field and the environment.

b-G: The sampling procedures are as per the standard practices and approved by RCGM and IIVR.

S: Numerous scientific studies since 2000 have proven that these pollen flows vary a lot with the size, the environment, and the form of the fields. These raw data should be included in the experiments.

c-S: A maximum of 50 meters from the source has been studied for dissemination, this is not significant in comparison to the well known wave's effects of pollen disseminations depending on the wind blowing and insects, and this has been demonstrated for several pollens (maize, oilseed rape...). Thus the assessment was incomplete and not extensive. Pollen flow rates depend on a number of factors not addressed by the applicants. For example, in addition to proximity of fields, the relative size of brinjal fields can influence the rate and level of pollen contamination. Small conventional (non-GMO) brinjal fields planted near large Bt brinjal fields will have higher rates of contamination than large conventional brinjal fields in otherwise similar situations. Therefore, smaller conventional brinjal farmers may be at greater risk of higher levels of contamination than larger farmers.
Further, the applicant did not consider that levels of contamination may be additive over time if a farmer saves non-GMO brinjal seed, and if neighbouring Bt brinjal farmers continue to plant Bt brinjal. If more than one brinjal crop is planted in a year, this would accelerate this trend.

c-G: In the pollen flow studies conducted initially, the distance of 50 metres was used, but the studies were repeated as per the recommendations EC-I and conditions stipulated by GEAC up to 300 metres the pollen flow was observed only up to 30 metres. Dissemination of pollen is dependent on wind blowing and insects, but most importantly it is crop specific and depends on the reproductive biology of that particular crop. Keeping in mind the size, pollen production, morphology and viability as well as environmental factors, pollen flow studies are designed. In no way, the results obtained in maize, oilseed etc. can be compared with brinjal. Size of field or repeated cultivation has no effect on the pollen flow.

S: Of course it has, if the pollen flow is taken in the wide sense, i.e. contamination with pollen over time, and not only release of pollen during the culture. And even that is dependent on the field size, environment (wind, rivers, specific animals transporting...), etc... As explained.

d-S: The analysis of pollen flow also neglects other very important routes of contamination (e.g. by mixing seeds). Based on data from other countries on other genetically engineered crops, it seems likely that routes of contamination such as seed mixing are important. For example, in the U.S., levels (concentration) and rates (percent of the total crop) of contamination of soybean, a crop with low out-crossing rates similar to brinjal, were as high as for crops like corn that outcross at much higher rates. Since out-crossing occurs by pollen flow, these data suggest that other means of contamination are likely to be important ("A Growing Concern", Union of Concerned Scientists, 2004, http://www.ucsusa.org/assets/documents /food_and_agriculture/seedreport_fullreport.pdf).

d-G: The concerns regarding seed mixing are trade related issues and not a part of environmental risk assessment.

S: These should be assessed anyway because these create concerns in environmental risks assessments anyway, and monitoring plans should be given before any commercial releases.

e-S: Gene flow to wild weedy relatives may result in environmental harm. This important route of possible environmental harm is widely recognized, but apparently not considered by the applicant. Gene flow from Bt brinjal in India may occur with the sexually compatible wild weedy relative Solanum insanum. Another sexually compatible relative, and the progenitor species of brinjal, S. incanum, probably also occurs in India. Gene flow from GMO crops has occurred from a large scale field trial of creeping bentgrass (Agrostis stolonifera) in the U.S., and from commercialized canola in Canada - in both cases involving a gene for glyphosate herbicide tolerance. Transfer of the modified Cry1 gene to these wild relatives may lead to harm to Lepidoptera or other non-target organisms that feed on these wild plants or the wild plants may become weedier due to suppression of herbivorous insects that may help keep their growth in check. Whether these possibilities occur depends on a number of factors that have not been tested by the applicant. For example, it must be determined whether these wild species grow in areas where brinjal is cultivated, which would allow gene flow to occur. Harm from such gene flow can only be determined through appropriate tests such as determining which organisms feed on these wild species, and whether they are sensitive to the Bt toxin. It should be noted that GM crops containing a Bt gene have not been commercialized in proximity to wild relatives anywhere in the world. Finally, gene flow to wild relatives may in some cases lead to reduce genetic diversity of the wild species. This is especially true for wild relative that grow near the crop, and occurs through the phenomenon of gene swamping when the crop is more numerous than the wild relative.

e-G: The crossability of different species of brinjal in India has been studied and reviewed by Rao, 1979. It has been reported that there is no natural crossing among cultivated and wild species of brinjal including S. incanum and S. insanum.

S: Please use more recent references with modern and more precise technologies of assessment.

G: Under forced crossing situations, even if crossing was possible, the viability and subsequent development of stable crosses have not been successful. Particularly in case of S. incanum, the crossability studies have been repeated by Indian Institute of Vegetable Research. It has been indicated that there was very limited crossing when S. incanum was used as female parent, whereas in the earlier study (2007-08), no crosses could be obtained. It can be concluded that gene flow from S. melongena to wild relatives of brinjal is not possible under natural conditions.

S: The science under this very important affirmation is really too light, these crossing have a lot of chances to occur in real life with several relatives.

f-S: It is recognized that brinjal wild relatives may provide important pest resistance genes for brinjal diseases and insects, as well as other desirable traits. The possible reduction of such diversity could have negative implications for further improvement of the brinjal crop, and should therefore be carefully considered.

f-G: Genetic improvement by conventional plant breeding has not been successful due to the lack of resistance to FSB in brinjal germplasm. Some wild Solanum species showed high levels of resistance, but it has proved to be impossible to incorporate the genes for resistance from wild species into commercial cultivars due to breeding incompatibilities (Dhankhar et al., 1982).

S: This does not answer the question at all that remains an entire real problem, sorry. The above answer from Mahyco and GEAC even shows that the conservation of brinjal biodiversity is crucial as indicated, and thus should be preserved by an interdiction of Bt brinjal release.