Response to the Final Conclusions of the Expert Committee (EC-II) of the Genetic Engineering Approval Committee on Bt Brinjal

Dr. Johannes Manjrekar
Microbiology Department and Biotechnology Centre
The Maharaja Sayajirao University of Baroda

Below are my reactions to some of the important points presented in the final conclusions of the report on Bt brinjal produced by the Expert Committeee-II (EC-II) appointed by the Genetic Engineering Approval Committee (GEAC). While by no means exhaustive, these comments will hopefully be sufficient to convey the inadequate and unsatisfactory nature of not only the studies carried out on Bt brinjal, but the unacceptable procedure by which the studies and their assessment have been done.

Conclusion 1. “The current practices of extensive use of chemical pesticides besides being expensive and unsustainable are also harmful to health and the environment.”

While this statement is no doubt true, the EC-II does not at any point even consider the efficacy or potential of other approaches, such as integrated Pest Management (IPM) Strategies or the experiences of organic farmers. The projections on potential outcomes and benefits of Bt brinjal are based entirely on comparisons with farming practices involving heavy pesticide use, with the implicit assumption that no other strategies and alternatives are available. The assumptions and projections of EC-II are questionable in the context of the mixed experiences with Bt cotton. It may be pointed out, for instance, that a study by Navdanya reports a 13-fold (!!) increase in expenditure on pesticides in areas of Vidharba under Bt cotton cultivation over the period 2004-2007. It is also worth remembering that distress of Bt cotton farmers in Vidharba has been claimed as an important contributing factor to the unprecedentedly high incidence of farmer suicides in this region. Surely a thoughtful assessment of the experiences so far with Bt cotton should have provided part of the background to evaluations of the potential costs and benefits of Bt brinjal introduction.

Conclusion 2. “The RCGM had considered and examined the biosafety data generated by M/s Mahyco and concluded that Bt brinjal is effective in controlling target pests, safe to the environment, non-toxic as determined by toxicity and animal feeding tests, non- allergenic”¦”

A preliminary but absolutely crucial point to be made regards the reliance of EC-II on data provided by Mahyco, the very company whose product is being evaluated. Good regulatory practice must surely involve mechanisms by which crucial safety evaluations are based on studies by completely independent agencies, rather than by the party seeking to have its product approved. This essential general principle apart, Monsanto, Mahyco’s collaborator from whom the transgene construct introduced into Bt brinjal was obtained, has an unsavory history of dishonest dealings with serious implications for consumers and general populations. This history includes fudging and concealing of unfavourable data in its own studies, and bribing of government officials to clear its products. To uncritically accept data from studies carried out by Mahyco itself is therefore astonishingly naïve on the part of EC-II, to state it mildly.
Related to the reliance on data provided by Mahyco is the matter of the composition of the EC-II itself. Incredibly, several members of the EC-II have strong ties to Mahyco through funding, participation in evaluations for Mahyco, and development of the Bt brinjal. Such blatant conflict of interest casts a strong shadow over the impartiality and objectivity of the evaluation itself. It is a completely unacceptable situation which severely undermines the credibility of the entire process.

Regarding the Mahyco study itself, a number of points need to be made:

i)    The study used bacterially produced Cry1Ac protein, while the transgene inserted in Bt brinjal is a chimaeric construct which is a Cry1Ab-Cry1Ac fusion. The EC-II claims that the Cry1Ac protein can be used as a surrogate for the chimaeric Cry1Ab-Cry1Ac protein because they are 99.4% identical in sequence. Contrary to EC-II’s assertion that this difference is due to a single amino acid change, it is easy to see that for a protein of the size of Cry1Ab-Cry1Ac, this would involve 6 or 7 amino acid differences. Any biologist worth their salt knows that multiple amino acid differences are capable of causing significant alterations in protein structure, which could be global, affecting overall protein folding and tertiary structure, or local, affecting for instance the epitope properties (i.e. ability to elicit immune responses) of small peptide segments of the protein. EC-II says that it was necessary to use surrogate protein because of the low levels at which the transgenic protein is produced in brinjal. Even if this is the case, it is difficult to understand why the study did not use bacterially produced chimaeric protein identical in primary sequence to the transgenic protein. A further problem with use of the surrogate Cry1Ac protein is that, being bacterially produced, it is not self-evident that the mature protein is identical to that produced in the transgenic plant, since proteins may be subject to numerous modifications inside the cells of plants after they have been synthesised. EC-II says that this problem was adequately addressed in the study by western blotting, which showed a transgenic Cry1Ab-Cry1Ac band of identical mobility to the Cry1Ac band. While this indicates that the transgenic protein may not have large oligosaccharide chains attached to it (glycosylation), it leaves unanswered the possibility of other post-translational modifications. There are a large number of such modifications which occur in plants, and many of these might escape detection in a western blot. Moreover, small modifications can affect immune properties of proteins; for instance, antibodies have been raised which can distinguish small modifications such as acetylation on single amino acid residues. The claim that the surrogate bacterially synthesised Cry1Ac protein is for all practical purposes identical to the plant-produced chimaeric protein is thus not settled by the experimental data, and hence unwarranted. Even if one were to accept the need to use a surrogate protein, in a well designed study this protein would be tested in a mixture with plant extract to at least attempt simulation of conditions under which the protein is produced in the plant, which could involve not only post-translation modifications such as the ones referred to above, but even unpredictable interactions with other cellular proteins or other biomolecules. Unfortunately, the Mahyco study has not troubled to take such concerns into account, casting doubt on the value of the data. Equally unfortunately, the EC-II does not appear to be alive to such concerns any more than the experimenters who carried out the study.

ii)    By far the most serious concerns about the Mahyco study relate to the basic design, particularly in terms of scale and numbers. The small numbers of rats used in the toxicity studies and the small numbers of brinjals used in the “compositional analysis” severely confound any attempts at statistical analysis of the results to the extent that only large, gross effects would appear as statistically significant in the data. In other words, the study has little sensitivity to uncover effects which might have manifested as statistically significant in larger samples. The EC-II simply sidesteps these objections and proceeds to defend the validity of the study. It is not clear why the Mahyco researchers chose to be so parsimonious about the number of rats and plants used in their study. The study is shoddy and superficial in a number of other aspects that have been criticised by a number of people, to the extent that no reviewer for a respectable journal would have considered it for publication. The EC-II fails to make a convincing defence against the methodological criticisms of the study beyond bland assertions about the reliability of the results.

iii)    One major concern with the study was its limited time period. In light of some research reports that GM foods can cause tissue damage and reproductive problems (manifested in subsequent generations), one might have expected the EC-II to concede the possibility that additional, longer-term studies are required to be certain about the absence of toxic effects of the transgenic protein. However, the EC-II is adamant in maintaining that the acute oral toxicity testing regime is adequate. Its argument is little more than repeated assertions that a limited study of this nature is adequate according to internationally accepted standards and that it satisfies Indian guidelines. While the EC-II repeatedly asserts that the study “conforms to accepted international standards”, it is difficult to resist the impression that the EC-II is either unaware of, or completely indifferent to, the considerable controversy and debate regarding what constitutes acceptable standards, and whether existing mechanisms and criteria for safety evaluation are adequate. It is precisely because of such controversies that only a few countries presently permit the planting of transgenic crops. From the tone of the EC-II report anyone would be excused for believing that a wide international consensus exists on GM foods, and that the cultivation of GM food crops has been permitted in many countries. The EC-II assertions on safety and “accepted standards” notwithstanding, in light of the recent reports about possible harmful effects of genetically modified crops, and specifically of crops containing the Bt transgene, it is essential that longer term, far more thorough and well designed animal studies be carried out than what was presented in the Mahyco study. The Precautionary Principle is something the EC-II does not appear to have in its vocabulary. It must be mentioned that in India there have been reports of a large number of livestock dying in Andhra Pradesh after feeding on Bt cotton plants. The evidence strongly suggests that Bt cotton was indeed the cause of the deaths, and a follow-up study in which animals were fed with Bt and non-Bt cotton found that animals died in the Bt-fed groups but not in the non-Bt-fed groups. Even if the EC-II showed greater skepticism towards these results than it has towards the data from the Mahyco study, it is astonishing that it has not seen fit to even consider these alarming indications as worthy of further investigation. (The Monsanto Bollgard cotton which is the suspect in these cases contains the same transgene as that used in Bt brinjal.) One would have expected any responsible committee with serious concern about safety issues to have initiated further investigations of these cases and awaited their results before rushing to declare its satisfaction with the safety of the Cry1Ab-Cry1Ac transgenic protein.

Conclusion 5.  “The three genes introgressed by M/s Mahyco”¦approved by regulatory agencies worldwide”¦All the inserted genes and their regulatory sequences”¦have a history of safe use”¦”

The assertion about “the history of safe use” is untenable because of the absence of adequate studies. Apart from the possibility of unintended and unforeseen effects of the Bt toxin itself on humans or animals, many safety concerns revolve not merely around the Bt protein, but also about potential undesirable effects of the accompanying sequences in the transgenic constructs.

i)    One such major concern is the possibility of the horizontal transfer of transgenic DNA to other organisms. In the case of antibiotic genes (two of which are included in the Bt brinjal transgenic construct), the worry is obvious, namely that the DNA coding for antibiotic resistance could be transferred to pathogenic microorganisms, thereby accelerating the spread of antibiotic resistance. This is particularly a concern with transgenic food plants since they would be raised in large numbers, thereby increasing the chances that the antibiotic resistance genes could find their way into other organisms, such as gut microflora (after being eaten by humans or animals), or soil organisms which could take up DNA from waste plant material. The EC-II dismisses this concern with some astonishing statements (p.70). First they make the point that “it has been well researched” that the level of antibiotic degrading enzymes produced by the transgenic plant are too low to have any effect on the antibiotics, an odd point to make since this is quite irrelevant to the problem of transfer of antibiotic resistance to microorganisms. They then proceed to assert that “it has been well established that the probability of transfer of transgenic from GM plant material to bacteria”¦is unlikely because of a series of well established barriers [sic]. All the above is supported by experimental evidence.” This is a patently false assertion, given that there are a variety of evidences that horizontal DNA transfer must have occurred repeatedly in the course of evolution, and that a number of studies have directly described instances of such DNA uptake, including by gut microorganisms. One is left wondering whether the EC-II members can be genuinely ignorant of the discussions and evidence for horizontal gene transfer, or whether they have chosen to simply ignore it. Whatever the case, it is disturbing that they can come so blithely to such a confident conclusion in a matter that is generally considered a serious concern, and does little to inspire confidence in their scientific judgement.

ii)    A second concern with transgenics is about their genetic stability. On this matter too the EC-II inveighs with great confidence. “This [the statement that the ‘expression of the cry1Ac gene is consistent during the entire life of the crop’] demonstrates that the insect resistance trait is stably integrated in the brinjal genome and there is no evidence or likelihood of genetic instability.” [Emphasis mine.] Again the arguments and assertions of the proven safety of the elements of the transgenic construct do not give the impression that EC-II members are aware of recent discussions of the likelihood of genetic instability being triggered by the insertion of transgenic constructs in plants. Genetic rearrangements have been described at the sites of integration of transgenes in several Bt transgenics, and the CaMV promoter which is also used in the Bt brinjal transgenic has been suspected of acting as a recombination hotspot that could trigger genomic rearrangements.

Conclusion 9. The statement that the transgenic insertion in Bt brinjal “confers no advantage to recipients in terms of aggressiveness or growth characteristics” is hardly warranted by the data in the Mahyco study. It would require much better designed and careful ecological studies to arrive at such a conclusion. The subsequent arguments to claim that even if gene flow occurred to wild species it would confer no fitness advantage, can hardly be described as better than silly.

Conclusion 11. “Cry1Ac protein rapidly degrades (in 30 seconds) in simulated gastric and intestinal fluids and is not detectable even in short term digestibility studies.”

Such an in vitro test for CryA protein stability can at best be used as in initial indicator of the stability in vivo. Any experienced researcher is only too well aware of the limitations of in vitro studies and the difficulty of simulating realistic in vivo conditions in a test tube. Without additional in vivo studies, such experiments with “simulated gastric and intestinal fluids” have little meaning. In this context, it is worth mentioning that CryAc protein has been observed experimentally to survive and indeed bind to mammalian gut. Moreover, in any study on the fate of a protein after ingestion, it would be necessary not only to look at the stability of the entire protein, but also to examine the degree of degradation that occurs, what kind of peptide fragments are generated on what time scale, and whether these breakdown products might have effects distinct from the intact protein (such as, for instance, their ability to evoke immune responses). None of these issues have been addressed in the study beyond the cursory in vitro examination of Cry1Ac “digestibility”.

Conclusion 12. “The detailed compositional analysis”¦demonstrates that Bt brinjal event EE-1 is substantially equivalent to its non transgenic counterpart.”  

Clearly, more sophisticated compositional analysis is required to claim “substantial equivalence” (a controversial concept that has been frequently criticised). The EC-II offhandedly dismisses suggestions for additional compositional studies using techniques such as proteomic analysis, claiming that various ‘omics’ technologies are not sufficiently developed for useful application to evaluation of GM crops, and need further development before it is worth applying them to “routine safety assessments of food and feed derived from GM crops”. While a number of techniques have certain limitations, ‘omics’ techniques are already very widely used in research, and few scientists would dare to claim that they are incapable of yielding useful information on various kinds of molecular profiles. That the EC-II should think of evaluation of a transgenic food crop before its approval as “routine safety assessment” is not only revealing, but consistent with the overall tone and scientific rigour of its own report!

Conclusion 16. Though there are numerous other points in the EC-II report that deserve comment, I shall restrict myself to one more, namely the statement in this conclusion that Cry1Ac has a “history of safe use for human and animal consumption as GM crops containing Cry1A proteins including Cry1Ac have been consumed by millions of people for over two decades without any adverse effects reported in the published literature.”

This is a logically false statement, since adverse effects are going to be reported only when studies are carried out to look for them. In case of humans, this would require epidemiological studies, which have not been carried out for GM foods. Perhaps the EC-II needs to be reminded in this context that peanuts and wheat were eaten by many more millions of people for many centuries before their allergenic properties for some people were identified in the scientific literature.

In summary, the report of the EC-II is deeply flawed and quite unacceptable on numerous counts, only some of which have been discussed here. It is absolutely essential that better designed and executed studies be carried out by neutral researchers or agencies, and that this research be assessed by an evaluating agency also consisting of individuals who are not vulnerable to suspicions of bias and where the question of conflict of interest of any of its members never arises. Until much more careful, scientifically rigorous evaluations are made through credible mechanisms, the present report must be rejected in toto and approval to Bt brinjal MUST await fresh testing. It would be nothing short of reckless and deeply irresponsible to approve the cultivation of Bt brinjal in India on the basis of the unsatisfactory process of evaluation that has occurred so far.