Specific feedback to the company’s claims on its findings through Bt Brinjal tests and trials:

It is utterly meaningless to comment on the company’s claims apparently based on their studies and trials with Bt Brinjal. This is because no protocols are described for the tests nor any numbers or tables presented. However, from whatever’s put up on the MoEF’s website,:

1         Is there data on how many different ways is brinjal consumed by different communities in India and how it is fed to animals? How has the impact assessment taken this on board? What happens in all those cases where the brinjal is consumed directly, both by human beings and by animals?

2         The tests related to allergenecity and toxicity prescribed as part of biosafety testing are obviously inadequate as the experience with Bt Cotton in Indiashows. Despite being cleared as safe, Bt Cotton is reported to be causing widespread allergies in cotton growing belts of the country. Therefore, the protocols for such tests need to be re-looked at to capture the real adverse potential and such revised and better protocols applied for Bt Brinjal testing, especially given that it is a food crop with the toxin consumed in large quantities with no or very little processing.

3         Feeding tests done on goats do not capture the potential hazards as goats are known to be hardy animals, compared to sheep for instance. The protocol used in the case of Bt Cotton was to feed goats with cotton seed and the results apparently showed that there is no difference between feeding the goats with Bt Cotton seed and non-Bt Cotton seed. There were no multi-generational feeding tests done. What was not clear however was what the exact research protocol was - how old was the cotton seed, for instance? It is now clear that the tests did not capture the reality of farmers grazing their animals on Bt Cotton plants and not seeds. They also do not in any way predict what could happen with sheep. In the case of Bt Brinjal, there was no change in the testing regime from the Bt Cotton testing regime, despite such valuable lessons emerging from the field and despite this being a vegetable!

4         It is not enough to understand the effect of the Bt gene alone while understanding the impacts on human health and environment. It is important to capture the effects of the other genes transferred too. For this, a set of tests have to be evolved and undertaken.

5         It is surprising that the company says that the Bt toxin rapidly degrades in the soil. Published literature shows that this is not the case. There are many studies that show that Bt toxin can persist in the soil and retain its insecticidal activity. It is in any case known that the half life period of Cry1Ac toxin in plant tissue in the case of Bt Cotton is around 41 days. In such a case, why are the studies done by the company showing that the protein presence was non-detectable? At what stage of the crop was the test done?

6         What is the implication of growing Bt Brinjal in terms of the next crop, given the potential impacts on soil?

7         It is also surprising that pollen flow studies were done for just one year in two locations. Other information from India on pollen flow in Brinjal has results that should make any regulator sit up and take a cautious approach. The protocols used for devising Minimum Standards for Seed Production and Certification should be used here, since they have the worst case scenario built into the framework.

8         Such pollen flow studies should begin by listing out the wild species and related [compatible] species available in India in various regions of brinjal cultivation and check the effect of Bt Brinjal growth on such species, in a controlled environment [and not in farmers’ fields]. Where is the data on associated biodiversity [like insects, birds, animals, microbes etc.] which depend on brinjal and its related crops [both wild, related and cultivated] and where are the impact studies on such associated biodiversity?

9         No detailed molecular characterization has been provided by the company. This is important, since we now know that developers cannot control where the transgene insert lands and that DNA rearrangements occur, with the potential to affect the spatial and temporal expression patterns of nearby genes.

10     Bt protoxins differ immunologically from the truncated proteins used for testing purposes. There is evidence that the toxic portion of Cry1A proteins can have a different 3-D conformation depending on whether it is part of the protoxin or in its free state. DNA structurally associated with the protoxin is released during the proteolysis process that generates the toxic fragment from the protoxin. If safety testing was performed on truncated versions of bacterial surrogate proteins rather than the full-length plant-produced Bt proteins that people are actually exposed to, such testing is absolutely inadequte. It has been found often that biosafety testing does not take into account such a difference and it is not clear how the tests were conducted here.

11     It is obvious that investigations have not been carried out to check whether the bacteria in the GM agro-ecosystems have 'picked up' DNA sequence fractions of kanamycin resistance reporter genes or streptomycin-resistance reporter genes.

12     What do the "isolated instances of necropsy" findings in all treatments indicate and what is the company's explanation, in the case of Sub-Chronic Oral Toxicity studies in rats? How many such instances in Bt-treated rats and how many in non-Bt treated?

13     Where is the data on how the Bt Brinjal affects children?

14     Where is the data on the cultural diversity that exists with regard to the cooking of brinjal in this country? Brinjal is also used for medicinal purposes in India. What impact would Bt Brinjal have on such use? Where is data related to socio-cultural importance of Brinjal in different communities in India and the possible impact of Bt Brinjal on the same?

15     Where is data on quantified protein expression related to pest incidence in the complete growing season of the crop? Given that the expression of the toxin is highest in the fruit, the consumed part, what implications does this have for human health for particular hybrids?

16     Deeper investigations into what the farmers have observed during field trials of Bt Brinjal of color change in the fruits as the day passes have to be taken up.

17     There is no data that shows that pesticide use does come down with Bt Brinjal by how much? How does it compare with NPM and organic practices?

FINALLY, WHERE ARE INDEPENDENT RESEARCH PROJECTS BY THE REGULATORS THEMSELVES TO OBJECTIVELY TEST FOR RESULTS ON EACH OF THE ABOVE ISSUES?
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Annexure 2:

USE OF BRINJAL IN AYURVEDA AND OTHER TRADITIONAL SYSTEMS OF MEDICINE

Brinjal is one of the important medicinal plant in the traditional ayurveda system of medicine in our country.  Different species of brinjal ( apart from Solanum melongena which is also used widely as vegetable all over the country) are used in the ayurveda preparations, especially the roots and stem of the plant.

The most required species are given below:

• Solonam xanthocarpum Schrad& wendal. -  Dasamoolam (bhaishajyaratnavali) Indukantham ghritham ( Sahasrayogam)
• Solanam indicum Linn - Dasamoolam ( bhaishajyaratnavali), Dhanwantharam kwadham (ashtangahridayam)
• Solanam nigram Linn - (kantakari) - asthama, rheumatism, heart diseases, hydrophobia, etc It is the major ingredient in Kantakaryavalehyam (Sarngadharasamhitha).
• Withania somnifera - Aswagandha  is a major rejuvenative , cardiotonic, effective against neurological complaints, sexual debility, emaciation, etc. It is the major ingredient in Aswagandharishta ( ref.Bhishajyaratnavali),  Balarishtam (Bhaishajyaratnavali), Aswagandhadi lehyam (Bhaishajyaratnavali)

Other species which comes only in a few formulae and are required in lesser quantity, but a very significant ingredient, are the following:  

• Solanam anguivi  Lim - (mullan kathiri)- Cardiotonic, skin diseases, diuretic, gynecological disorders
• Solanam melongena Linn - (vazhuthina) - analgesic, aphrodesiac, liver complaints
• Solanam torvum Swartz -
• Solanum incanum Willd -
• Solanam surrattanse Burm.f. ( Thudavalam)Rheumatism, Hypertension, rhinopathy, heart diseases
• Solanam trilobatum Linn - cough, bronchitis
• Solanam viarum linn - birth control, hormone supports

Apart from the ayurveda system, in sidha or unani system, brinjal is also widely used in folklore practices and some species are considered as home remedies.

Hence maintaining the purity of the naturally available varieties of Brinjal is important for the Ayurvedic and other Indian systems of medicine. 

Dr.Anand P.K.V
Lecturer
Vaidyaratnam Ayurveda Collage
Ollur - Thaikkattussery
Thrissur 680322, Kerala.
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Annexure 4: Transgene products and Bt toxins 

- a note by Dr Arpad Pusztai, in the context of the Bt Brinjal development in India

The physiological or immunological interactions with vertebrate organisms of bacterial versions of cry proteins, or that of the recombinant protein produced in bacteria have been studied. These revealed that the toxin form of Cry1Ac is a potent antigen in mice, following gastric administration. Specific serum IgG and IgM antibodies and locally produced IgA and IgG antibodies to the toxin were detected. The Cry1Ac protein could also be taken up from the intestinal mucosa to be processed in peripheral lymphoid organs. Several human cell cultures, including colonic epithelial and liver cells, demonstrated a number of cytotoxic reactions when exposed to Bt toxins, and immunologic sensitization of farm workers has been well documented. Accordingly, it would be unwise to use Bt toxin-containing foodstuffs in human or animal diets.

Bt toxins remain in existence in ecosystems for long times that helps to build up resistance in target insects, and cause potentially harmful effects on non-target invertebrate species.GM-crops designed for increased pest resistance, such as the Bt toxin crops are not sufficiently selective and specific for their major pests and, by inflicting damage to beneficial insects, they destroy the natural balance between pests and useful organisms.  It also must be emphasized that Bt toxins expressed  in transgenic plants have never  been systematically tested in mammalian or other vertebrate organisms, neither have the effects of the integration of cry genes in vertebrate cells/organisms been studied. Cry proteins bind to the intestinal mucosa, and other cell types and available cell surface receptors in the consumers. Furthermore, the Bt toxins may conceivably enter into complexes with macromolecules in the producer plant or in the gastro-intestinal tract and these may have biological activities and receptor specificities that differ from that of cry toxins or the unattached plant or intestinal macromolecules. 

It is also possible that when the transgenic DNA from GM plant food/feed, is taken up, integrated and expressed in the vertebrate organisms, like the alpha-amylase inhibitor gene from beans when expressed in peas may appear in a number of post-translationally modified forms with altered functions and immune/allergenic properties. Intracellular complex formation or other forms of interactions with endogenous gene products may confer unanticipated activities and effects to the cry gene products.

For all the above reasons and because GM crops developed by transgenic splicing techniques present unique and irreversible risks, no new GM crops should be allowed to be cultivated, commercially traded or incorporated into human food or animal feeds unless, as a minimum, it was first shown by the following testing methods that the crop presents no unacceptable harm:

a.       the comparison of the GM- and isogenic lines should include investigations with novel and up-to-date analytical techniques, such as proteomic analysis (2D electrophoresis and mass spectrometric analysis of relevant components)

b.       a full biochemical, nutritional and toxicological comparison of the in planta produced Bt toxin, and not that of a bacterially produced recombinant surrogate, with that of the original whose gene was used for the transformation, must be done

c.       microarray analysis of all novel RNA species in the genetically modified plant must be performed

d.       full molecular biological examination should be carried out with particular attention to the possiblity of secondary DNA insertions into the plant genome

e.       a full metabolomic NMR, etc analysis of the transformed plant is obligatory

f.        the stability to degradation by acid or pepsin or other proteases/hydrolases of GM products, foreign DNA, including the gene construct, promoter, antibiotic resistance marker gene, etc, must be established in the gut of animals in vivo, and not in vitro as done presently.

g.       with GM lectins, including the Bt-toxins the presence/absence of epithelial binding in the gut should also be demonstrated by immunohistology.

h.       an investigation of the nutritional, immunological, hormonal properties and allergenicity of GM-crop itself must be carried out on laboratory animals in short- and long-term experiments

Animal testing is but a first step. If the animals did not suffer any health harm, and only then, the results will have to be validated with human volunteers in clinical double-blind, placebo-controlled drug-type tests but keeping in mind that the harm can be most acute in the young, elderly and sick, particularly those suffering from HIV, hepatitis or other viral diseases.
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Annexure 5:

GM Brinjal Contains Bt Toxin Linked to Hundreds of Allergy Cases and Thousands of Sheep Deaths

It would be unthinkable and irresponsible to approve the genetically modified brinjal

Dr. Mae-Wan Ho and Prof. Joe Cummins find no published studies nor experimental details on safety tests in the application for field releases of the Bt brinjal and raise serious questions

Bt brinjal a test case for other GM food crops

The Indian subsidiary of US seeds corporation Monsanto, Maharashtra Hybrid Seed, has developed genetically modified (GM) brinjal resistant to fruit and shoot borer and is applying for large-scale test releases [1]. Brinjal, an eggplant, is widely consumed in India and recognized for its health promoting properties such as reducing serum levels of cholesterol. Field trials of other GM crops, including mustard and potatoes, will follow the brinjal test releases.

            The GM brinjal contains the same Cry1Ac toxin from the soil bacterium Bacillus thuringiensis as the widely cultivated GM cotton that has been implicated recently in major health controversies in India. Hundreds of farm workers and cotton handlers developed allergic reactions [2] (More illnesses linked to Bt crops, SiS30) and thousands of sheep died from toxic reactions after grazing on the post-harvest GM cotton fields [3] (Mass deaths in sheep grazing on Bt cotton, SiS30).

            These controversies on the health hazards of Bt crops corroborate findings dating back to the 1980s, which linked Bt bacteria and spores producing a mixture of Cry proteins to allergic reactions [4]. Cry1Ac itself has been identified as a potent systemic and mucosal immunogen [5] and adjuvant comparable to cholera toxin [6]. Thus, not only can the Bt toxin provoke immune reactions to itself, it can also sensitize a person to develop allergies to other components in the diet. At least 12 dairy cows died in Germany after feeding on GM maize containing a gene coding for a protein similar to Cry1Ac [7] (Cows ate GM maize and died SiS21).

            Cry1Ac is not the only Bt transgenic protein linked to serious health problems. Dozens of villagers fell ill in the south of the Philippines when a Bt maize with Cry1Ab came into flower in 2003, and five have died since [8] (GM ban long overdue, dozens ill & five deaths in the Philippines, SiS 29). Illnesses and death associated with numerous other GM crops with different transgenes have been reported in many species. The most dramatic recent example is the severe stunting and premature deaths in the litter of female rats fed GM soya throughout their pregnancy [8], and the debilitating inflammation of the lungs in mice tested with a transgenic pea containing a normally harmless bean protein [9] (Transgenic pea that made mice ill, SiS 29).

A comprehensive public enquiry into the health hazards of GM crops is long overdue, as is a global ban while the enquiry is in place. It is unthinkable and irresponsible to release yet another GM crop with a transgenic protein that has already been implicated in so many illnesses and fatalities.

            The Report accompanying the application for field release [1] provides such a superficial description of theGM brinjal and unpublished experiments on environmental and health impacts that it would never have passed muster in Europe; which is not to say that Europe’s regulatory system is adequate. We concentrate on health impact studies that, according to the company, show Bt brinjal is as safe as non Bt brinjal.

Toxicological studies raise worrying questions

Toxicological studies were all unpublished experiments conducted (except for one) at Intox Pvt Ltd., and amounted to bland assurances that none of the tests caused any toxicity.

However, some statements in the Report should be examined carefully. On p. 7, it states (emphasis added): “Acute oral administration of transgenic Bt brinjal expressing CrylAc protein to Sprague Dawley rats at the limiting dose of 5000mg/kg did not cause any toxicity.” What exactly is the limiting dose? Does it mean that beyond 5 000 mg/kg the Bt brinjal was in fact acutely toxic? After all, that is equivalent to a person weighing 50 kg eating a medium-size brinjal, which is not unusual.

The next paragraph reports the results of subchronic oral toxicity study, where it states that “the no-observed-adverse-effect (NOAEL) of transgenic Bt brinjal expressing Cry1Ac protein in Sprague Dawley rat, following oral administration for 90 days was found to be more than 1000 mg/kg body weight. This study demonstrates that Bt brinjal expressing Cry1Ac protein is non-toxic to the study animal by oral route.” 

The designation of “NOAEL” (no-observed-adverse-effect-level) is worrying as it has no scientific precedent. Does that mean doses higher than 1 000mg/kg body weight could be toxic? So, a person weighing 50 kg eating a quarter of a brinjal a day might be putting herself in danger?

The “allergenicity” studies, unpublished and conducted by another company, Rallis India Limited, contained even less details to support the statement of “no differences between the allergenicity or inflammatory characteristics of the 5 brinjal extracts tested including transgenic Bt brinjal and non transgenic brinjal.”

The same goes for the “primary skin irritation test”, and the “mucous membrane irritation test”, both conducted by Intox Pvt. Ltd.

Nutritional studies highly questionable

Another series of  “nutritional studies”, involved “compositional analysis”, which, the company claims, shows that Bt brinjal is “substantially equivalent” to “control brinjal” and thus “the food and feed derived from Bt brinjal will also be substantially equivalent to the food and feed derived from non-Bt counterpart.” Again, there are no experimental details given whatsoever.

Compositional studies have long been rejected by the European public as a demonstration of “substantial equivalence”, and “substantial equivalence” itself is widely seen as unscientific and unacceptable as a principle of risk assessment [10] (The Case for a GM-free Sustainable World).

Another series of unpublished feeding studies with Bt brinjal on fish, chickens cows, goats and rabbits are reportedly, carried out in a variety of companies and institutions, all demonstrating “no significant differences” between Bt and non-Bt brinjal.

In the only case (chickens) where the amount of Bt brinjal eaten is stated, it constituted 5 or 10 percent of the diets. That is equivalent to little more than a mouthful of Bt brinjal at each meal for a human being.

No molecular data

There are no molecular data in the Report to indicate where and in what form the transgenes have inserted into the brinjal genome, and whether the insert has remain stable, which would have been required under the European directive for deliberate release. It is now generally accepted that genetic modification is “event-specific”, the transformation causing a lot of collateral mutational damage to the genome including [10-12] as well as the tendency of the integrated insert to be unstable [10-11, 13-15] (The Case for A GM-Free Sustainable World; Living with the Fluid Genome;Trangenic lines proven unstable;Unstable transgenic lines illegal).

The only molecular information provided is that the Cry1Ac gene is driven by an “enhanced CaMV 35S promoter” (no further details), and two antibiotic resistance marker genes are present: the nptII gene coding for neomycin phosphotransferase II (NPTII) (kanamycin resistance) derived from the prokaryotic transposon Tn5; and the aad gene coding for aminoglycoside adenyl transferase (AAD) (spectinomycin and streptomycin resistance) isolated from bacterial transposon Tn7. The aad gene is under the control of a bacterial promoter and hence not expressed in Bt brinjal, though it would be fully active in bacteria.

Horizontal gene transfer not considered

There is strong likelihood that the two antibiotic resistance marker genes will spread to pathogenic bacteria in all environments by horizontal gene transfer [16-18] (FAQs on genetic engineering;Recent evidence confirms risks of horizontal gene transfer) and hence exacerbate resistance to antibiotics that are currently used in human and veterinary medicine. Horizontal gene transfer is not considered at all in the Report.

            There is evidence that such resistance markers may spread to bacteria in the gut of animals including human beings [19]  (DNA in GM food and feed, SiS 23), as well as to bacteria in the soil and water [16] simply because DNA does not break down fast enough in all environments.

Conclusion

In conclusion, it would be courting disaster to release yet another GM crop with a transgenic protein that has already been implicated in so many illnesses and fatalities. The company’s dossier is highly unsatisfactory and incomplete, and raises some serious safety questions. It can give no comfort to farmers and cotton handlers who have suffered allergic reactions to Bt cotton, nor to farmers who have lost their sheep to Bt cotton.

            Instead of approving more GM crops, regulatory authorities in India should start a comprehensive enquiry into the health impacts of Bt cotton and impose a ban on further releases of all GM crops.

References

1. Maharashtra Hybrid Seed, Company DEVELOPMENT OF FRUIT AND SHOOT BORER TOLERANT BRINJAL 2006 http://www.envfor.nic.in/divisions/csurv/geac/macho.pdf
2. Ho MW. More illnesses linked to Bt crops. Science in Society 2006, 30, 8-10, http://www.i-sis.org.uk/isisnews.php
3. Ho MW. Mass death in sheep grazing on Bt cotton. Science in Society 2006, 30, 12-13, http://www.i-sis.org.uk/isisnews.php
4. Bernstein IL, Bernstein JA, Miller M, Tierzieva S, Bernstein DI, Lummus Z, Selgrad MJK, Doerfler DL, Seligy VL. Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides. Environmental Health Perspectives 1999, 107 (7), http://www.ehponline.org/members/1999/107p575-582bernstein/bernstein-full.html
5. Vázquez-Padrón R, Moreno-Fierros L, Neri-Bazan L, de la Riva G and López-Revilla R. Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice.  Life Sci. 1999, 64, 1897-912.
6. Vazquez RI, Moreno-Fierros L, Neri-Bazan L, De La Riva GA and López-Revilla R. Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant. Scand J Immunol 1999, 578-84.
7. Ho MW and Burcher S. Cows ate GM maize and died. Science in Society 2004, 21, 4-6, http://www.i-sis.org.uk/isisnews.php
8. Ho MW. GM ban long overdue, dozens ill and five deaths in the Philippines. Science in Society 2006, 29, 28-29, http://www.i-sis.org.uk/isisnews.php
9. Ho MW. Transgenic pea that made mice ill. Science in Society 2006, 29, 26-27, http://www.i-sis.org.uk/isisnews.php
10. Ho MW, Lim LC et al. The Case for A GM-Free Sustainable World, Independent Science Panel Report, TWN and ISIS, Penang and London, 2003, republished as GM-Free, Vital Health Publishing, Ridgefield, CT, 2004, translated into Spanish, French, German, Portuguese, Chinese and Indonesian, http://www.i-sis.org.uk/onlinestore/books.php#232
11. Ho MW. Living with the Fluid Genome, TWN, ISIS, Penang, London, 2003, http://www.i-sis.org.uk/onlinestore/books.php#232
12. Latham JR, Wilson AK, Steinbrecher RA. The mutational consequences of plant transformtion. J Biomed Biotech 2006, Article ID 25476, pp. 1-7.
13. 13.  Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani N, Kobilinsky A, Romanuk M, Bertheau Y. Characterization of commercial GMO inserts: a source of useful material to study genome fluidity. Poster presented at ICPMB: International Congress for Plant Molecular Biology (n°VII), Barcelona, 23-28th June 2003. Poster courtesy of Pr. Gilles-Eric Seralini, Président du Conseil Scientifique du CRII-GEN, www.crii-gen.org
14. 14.  Ho MW. Transgenic lines proven unstable. Science in Society 2003, 20, 35, http://www.i-sis.org.uk/isisnews.php
15. 15.  Ho MW. Unstable transgenic lines illegal. Science in Society 2004, 21, 23, http://www.i-sis.org.uk/isisnews.php
16. Ho MW. Recent evidence confirms risks of horizontal gene transfer. ISIS contribution to ACNFP/Food Standards Agency Open Meeting 13 November 2002, http://www.i-sis.org.uk/FSAopenmeeting.php
17. de Vries J, Herzfeld T and Wackernagel W. Transfer of plastid DNA from tobacco to the soil bacterium Acinetobacter sp. by natural transformation. Molecular Microbiology 2004, 53, 323-34.
18. Nielsen K, van Elsas J and Smalla K. Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants.  Appl Environ Microbiol. 2000, 66,1237-42.
19. Ho MW. DNA in GM food and feed. Science in Society 2004, 23, 34-36, http://www.i-sis.org.uk/isisnews.php
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READ ON

Bt brinjal 1: Feedback on Bt Brinjal biosafety & beyond
Letter to the Chairperson of India's GM regulatory body

Bt brinjal 2: Signatories to the letter [1-120]

Bt brinjal 3: Signatories to the letter [121-270]

Bt brinjal 4: Annex 1
Letter to India's Minister for the Environment

Bt brinjal 5: More annexes
includes: a note by Dr Arpad Pusztai
a note from an Ayurveda expert (vaidya) 
comments on Monsanto Mahyco's claims
comments by Dr. Mae-Wan Ho and Prof. Joe Cummins