28. Page 22: “No cry1Ac was detected in the soil samples” this contradicts what is known and what the available literature says about this protein and it is interesting to note that the EC2 is ready to accept this finding as is without wondering if something was wrong with the methodology adopted or with the finding itself.
29. Page 22: Point f: Flavour analysis NOT done by CFTRI the response that the recently adopted guidelines do not require such a study is a faulty argument and nullifies the very process taken up earlier.
30. Page 23, Point i: Foliage studies with goats: GEAC deciding to dispense with this study is not based on any new investigations and is in fact based on falsified information (Refer to Annexure 9 for more information on this). Therefore, this justification is not tenable and the EC2’s readiness to rationalize this on these grounds arouses suspicions.
31. Page 23: Point j: Dispensing with the skin sensitization test is not on scientific grounds but only on an argument that recent guidelines do not need it! This kind of selective arguments with regard to suggestions of the earlier Committee are not acceptable picking up some points and negating some has no scientific basis.
32. Page 23: Point k: The NIN director looked at only 3 studies, as per data obtained under Right To Information. Even here, he raised questions that remain unanswered. This is certainly non-compliance on the part of regulators themselves, leave alone the crop developer!
33. Page 24: Socio-economic impact assessment of Bt Brinjal - the NCAP study is yet to come in. In fact, the GEAC deciding to clear Bt Brinjal before this study has been finalized is indeed very hasty and unjustifiable.
34. Page 25, Point 2.4.: Compliance with the 2008 guidelines. The recasting of these guidelines has been supported by USAID in India and the only apparent reason why this would have been done is to benefit American business interests and not to protect Indians, their health and environment from risks associated with GMOs.
35. Checklist Point 8 of the new 2008 guidelines: Assessment of possible allergenicity the EC2 says “Not applicable as the Bt protein is neither known to be allergenic nor has sequence homology with any known allergen” this is a very faulty premise for not testing since there could be novel proteins (not necessarily the Bt protein) through the GE process and that is what Prescott et al (2005) show with GM peas shows; there is also scientific literature that shows that the Bt protein could be as potent as the Cholera toxin.
36. Page 29 Conclusions at the end of the section on Compliance to Guidelines says, “Since everything is as per guidelines, no additional studies need to be prescribed for safety assessment””¦This is absolutely unscientific and does not form part of a scientific evaluation process.
37. SECTION III review of bt brinjal biosafety assessment dossier: Given that the EC2 ignored the points raised by the Director NIN from his earlier analysis of three studies on Bt Brinjal and given that there is no evidence of even a single point raised now on the biosafety data of Mahyco, there can only be one conclusion that this Expert Committee did not look at the raw data from the biosafety dossiers of Bt Brinjal. Otherwise, how can there be a scientific evaluation of 1000 PLUS pages by 16 scientists, without a single point being raised, unless there has been no review of such data or unless there is something very fishy!
38. Page 30: Point 3.1.2.: not clear what the last line means: “transformation method was a modified method developed at Mahyco”. If that’s the case, are there any implications for any of the studies being cited being irrelevant for this new transformation method?
39. Page 30-31: 3.1.3. description of Cry1Ac gene and protein: (a) This section ignores the fact that Bt Brinjal does not have the Cry1Ac gene but a chimeric gene. (b) It further ignores scientific evidence that the Bt protoxin is known to bind to (mucosal surface) surface protein in mammalian intestine too (Vazquez-Padron et al, 2000). (c) Finally, this section ignores the fact that it is not just individual genes in a transgene that are a cause for concern in r-DNA technology but the process of genetic engineering itself and transgenic expression of non-native proteins that are a potential cause for concern (“Diversity in translational and post-translational modification pathways between species could potentially lead to discrete changes in the molecular architecture of the expressed protein and subsequent cellular function and antigenicity”¦These investigations, however, demonstrate that transgenic expression of non-native proteins in plants may lead to the synthesis of structural variants with altered immunogenicity.” Prescott et al, 2005).
40. Page 31: “For Cry proteins to be active, it requires alkaline conditions”, says the EC2 report: Vazquez-Padron et al (2000) have shown that binding of protoxin to mucosal surface in mammalian gut happened too.
41. Page 31: on CaMV 35S promoter and alpha subunit of the beta-conglycinin gene of soybean: The EC2 says that “both the regulatory sequences introduced into EE1 event are not capable of causing any disease” There is at least one published paper in the Lancet that questions this in addition to a published paper where recombination between viral genes in GM plants and infecting viruses has been demonstrated (Wintermantel and Schloez, 1996).
42. Page 32: nptII gene and aad gene: the concern is not just with the expression of these genes in the plant but with the possibility of Horizontal Gene Transfer and therefore, the response of the EC2 is inadequate.
43. Page 33: Point 3.1.5 Expression of Cry1Ac protein and its quantification: “The levels of Cry1Ac protein were found to vary between 5 to 47 ppm in shoots and fruits”, notes the EC2 report. “Mean molt inhibitory concentration (MIC95 for Leucinodes orbonalis has been calculated to be 0.059 ppm for Cry1Ac”. In May 2007, the Director, Department of Animal Husbandry (AHD), Andhra Pradesh, sent a letter to the GEAC (ref: No 3531/Epid/2006.dated 9/5/2007), where he reported : “the Bt protein levels detected in the samples of Bt cotton bolls and leaves sent for analysis to different laboratories was recorded as 5 microgram/gm. This level is within the tolerable range which is said to be “5-10 microgram/gm”. On this basis, it justified that this level of protein expression in Bt Cotton is tolerable for sheep/goats. In such a case, this clearly shows that the Bt protein far exceeds the “tolerable range” in Bt Brinjal. (Annexure 7 is a letter based on the letter of the Director-AHD, GoAP)
44. Page 33: Conclusions: “The EC-II opined that the insect resistance trait is stably integrated in the brinjal genome and there is no evidence or likelihood of genetic instability”. This is a faulty argument since the genetic instability in Bt Brinjal is not about integration of the Bt gene. The crop developer might be interested in that aspect alone but regulators should obviously go beyond this as several studies point to this aspect.
45. Page 34: Point 3.2.1: Crossability studies: There are many studies from India which are centred around inter-specific hybrids which also provide an indication of crossability. Nishio et al. (1984) observed that crossing S. melongena with S. incanum, S. macrocarpon, S. integrifolium, S. gilo and S. nodiflorum was compatible. The S. viarum has been utilized in breeding experiments involving brinjal (S. melongena) and viable interspecific hybrids have been realized (Nandakumar, 1983). Nasrallah et al. (1963) found that S. melongena was crossed with S. gilo and S.indicum, the F1’s of these crosses were highly sterile and S. melongena was not able to be crossed with S. mammosum and S. ciliatum. S. melongena cultivar could cross easily with S. incanum and S. integrifolium. It gave hybrids with S. gilo and S. indicum when used as a female parent (Rao, 1968). Anis (1994) reported that the cross S. melongena x S. incanum yielded seedless fruits. The per cent survival of the hybrid S. incanum x S. melongena was better than the parents. Preneetha (2002) evaluated the interspecific F1 hybrids (EP 45 x S. viarum, EP 65 x S. viarum, CO 2 x S. viarum and MDU 1 x S. viarum) and found that the F1 hybrid plants resembled their corresponding female parents morphologically.
The IIVR crossability studies however report findings that are in variance with this existing knowledge. The IIVR crossability study did not use S. viarum species or insanum at all and there are issues of serious concern with the design of the crossability study itself, as prescribed by the EC1 with a focus on S indicum.
The Crossability Study b/w S melongena and S indicum that was taken up by IIVR further cites the following in its report: “Rao (1979) carried out a comprehensive survey of inter-specific hybrids of Solanum. Ten species were chosen: S. melongena, S. melongena var. insanum, S. incanum, S integrifolium, S. gilo, S. zuccagnium, S. xanthocarpum, S. indicum, S. sisymbrifolium and S khasianum. Crosses were attempted in all possible combinations. The results are summarized in Table 1 (No Table 1 is in the report however!). Out of 90 pos sible combinations, there was no fruit set in 47, and only parthenocarpic fruits in four. In the remaining 39 crosses which resulted in fruit set, only 24 gave rise to plants which reached the flowering stage”.
46. Page 35: Outcrossing: Wide range of outcrossing is acknowledged based on available literature (2 to 48% outcrossing) by the EC2. However, the results of the pollen flow studies are at great variance with this knowledge and the EC2 did not deem it fit for further investigation. The crop developer’s pollen flow studies were taken up even as back-crossing programme was underway! In Brinjal, literature shows that 60-70% fruit setting happens through pollination by insects while 30-40% is by selfing. Insect activity is therefore a predominant variable and it is not clear whether this has been factored in into the pollen flow studies taken up on Bt Brinjal.
47. Further, it is pointed out that Bt brinjal pollen traveled to a maximum distance of 30 meters and that there has been 0.14% to 2.7% outcrossing as per Mahyco’s pollen flow studies. EC2 classifies this as “limited outcrossing”! What is limited outcrossing in a country of smallholdings? Isolation distance and changed planting time is being suggested to minimize outcrossing who is liable for this? How can small farmers maintain isolation, that too in vegetable plots which themselves are very small, usually leased?
48. Page 36: “Further, the EC-II opined that even if there is a very small influx of pollen originating from Bt brinjal varieties, it is not of any consequence, as the Bt protein has been extensively tested for its safety to the environment and food/feed and thus pollen transfer to other cultivated brinjal would not pose any safety risk”. This is not an acceptable stand the EC2 cannot decide on the rights of people who want to remain GM-Free like this! The Bt protein testing that they are referring to is of Cry1Ac and not the chimeric gene used in Bt Brinjal.
49. Page 36: Horizontal Gene Transfer: “Horizontal gene transfer from plants to animals (including humans) or microorganisms is extremely unlikely; Similarly, gene transfer from brinjal, or any other plant, to microorganisms is extremely unlikely”, says the EC2 report. This is simply not true (Annexure 3) and this kind of a superficial response without any scientific basis and testing especially in the current instance of Bt Brinjal is not acceptable.
50. Page 37: Aggressiveness studies: Did they happen during MLRTs or Large Scale Trials? There might be a difference depending on this given how many plants were actually planted per plot in the first instance.
51. 3.2.3.: Impact on non-target organisms and Specificity of Cry proteins: No in vitro studies have been taken up with Bt Brinjal, especially at the highest levels of Bt protein expression from this GMO. Further, this analysis once again is a reductionist analysis centred around only Cry1Ac and refuses to acknowledge that other changes in the plant could also result in unpredictable impacts.
52. Page 38-Table 3.1: Laboratory based eco-toxicology experiments: It appears that a surrogate protein has been used in all the studies cited in the table and that itself can make these studies invalid. Further, the EC2 says that “No adverse effects were found at the above levels, which are significantly higher than that would be present in the fields” this is not true since the protein levels in Bt Brinjal are supposed to reach up to 47 ppm!
53. Page 40: Soil impact studies Despite the EC1’s recommendation, no study has been taken up on subsequent crop impact to this day. (Page 3 of EC1 report: The changes in fertility and impact on next crop may also be recorded. In other words carry over effects of residues of Bt brinjal should be investigated). Further, the soil impacts studies in 2007 and 2008 were taken up by Mahyco and not IIVR!
54. On absence of detection of Bt protein and such findings from these Bt Brinjal-related studies, the EC2 observes that findings are in agreement with numerous studies that have shown only target pest impacts and no other impacts. These findings are in fact at great variance with findings from numerous other studies including a recent IARI study with Bt Cotton in India. It is not possible to agree with a finding that says “No Cry1Ac protein was detected in any soil samples”. There must be something seriously wrong with the methodology if that were the case! This contradicts numerous other findings!
55. Page 40 last para The EC2 says: “It was further noted that cry1Ac gene has been derived from a common soil bacterium and therefore it is expected that soil microorganisms are already exposed to these proteins within the environment”. This is a very unscientific statement since Bt as an organism is certainly present in the soil, but the protein is not in constant expression; and our farm soils certainly do not have Bt as a protein that is being expressed by GM plants on a large scale. To even equate all these in a superficial, unscientific way is unacceptable on the part of the EC2.
56. Page 41: “Large scale cultivation of Bt Cotton since 2002 without any toxic effects reconfirms that Cry1Ac protein has no deleterious effect on soil microflora”, argues the EC2 report! On what basis is this being concluded? What studies have been done to show that there are no toxic effects from such large scale cultivation? What about the IARI and UAS-Dharwad experimental studies which do show impacts of Bt Cotton on soil?
57. Box 3.3 on Possible accumulation and persistence of Bt protein in soil: Half life in different studies cited here is reported to run into several days - 9.3 days to 40 days depending on the soils and incorporation of plant material etc. If this is the case from available literature, how come there has been no detection of Bt protein in the Bt Brinjal studies conducted by the company and IIVR?
58. Point 3.3. on Page 43: Food and Feed Safety Assessment: An Independent Expert Committee (October 2006) noted the following on the genes and vector used in Bt Brinjal. “Though Cry1Ac gene was earlier considered generally innocuous, recent published evidence indicates that Cry1Ac protein from Bacillus thurengiensis is a potent systemic and mucosal adjuvant as potent as the cholera toxin which enhances mostly serum and intestinal IgG antibody responses specifically at the large intestine (Vazquez et al, 1999). Also another study (Vazquez-Padron et al, 2000) demonstrates the possible interaction invivo of Cry proteins with animal bowel. According to Moreno-Fierros et al (2000), caution needs to be exercised while using Cry-containing plants and plant products for human use. Recent reports on CaMV 35S (Myhre et al, 2006) note that promoter gene expression in human enterocyte-like cells might have GE food implications. Regarding the aad gene used in developing Bt Brinjal [streptomycin resistant gene], this Committee notes that according to the EFSA, this is a potentially dangerous marker to animals and human beings and should not be used in the case of GM plants used as food. The Agrobacterium tumefaciens medium was used for the transformation process of development of Bt Brinjal. Strains of agrobacterium were earlier implicated in incidence of bronze wilt in cotton in the US (McGraw, 2000). It is not clear whether its potential impacts have been studied carefully in this case”.
59. Page 43, Point 3.3.1. Toxicity and allergenicity of purified Cry1Ac protein: All such evidence is irrelevant in the current instance as Bt Brinjal has a chimeric protein. The references are from 1993, 1996 and 1999 while the allergen database could have increased subsequently. Further, while a positive finding of homology may indicate allergenicity, a negative finding may not be a useful indicator of safety! This approach of individual genes and citing safety studies around them is completely inadequate since several studies with GM foods with these genes incorporated into them have shown numerous adverse impacts.
60. nptII protein: The half life in simulated intestinal fluids from a 1993 study cited by EC2 is two to five minutes. Can we conclude that there is no safety implication from this, with this information? Further, the implications from HGT of the gene cannot be ruled out.
61. Page 45: Point 3.3.2.: Toxicity and allergenicity of Bt Brinjal: Acute oral toxicity test: “proteins that are non-toxic by the oral route are not expected to be toxic by the dermal or pulmonary route”. This is something that is routinely used in biosafety dossiers by crop developers, their sponsors (ABSPII, for example) and even “Expert Committees”. However, the example of Ricin, a phytotoxin in castor, is worth mentioning here as an illustration to the contrary. Toxicity of Ricin differed with the route of challenge in experimental studies and the most lethal was the inhalation route, compared to other methods of exposure routes. The toxicity of ricin by the oral route is reported to be several orders lower than by pulmonary or injected routes1.
62. Point 3.3.2.: the sub-chronic oral toxicity study in Sprague Dawley rats is described as “This study provided information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time”. This is an interesting description and admission about this study since the EC2 is also arguing that sub-chronic studies are long enough! Prof Seralini however pointed out to: “Circling disorder and diarrhea were noticed only in the Bt brinjal group, males and females. Moreover liver weight as well as relative liver to body weight ratio decreased in the dose range study in females, by 13% apparently significantly. For the rats fed Bt brinjal water consumption was 8-21% more than the non Bt brinjal group for some periods”.
63. Page 46: Point 3.3.3.: Alkaloid content: “Alkaloid profile of Bt and non-Bt is the same with not much appreciable variation in their relative abundances”, states the EC2 report. Prof Seralini has calculated that the difference is upto 237% and no statistical significance tests have been conducted. The EC2 is however not hesistant to classify this as “not much appreciable variation” and this is unscientific.
64. Page 47: Detailed compositional analysis: The EC2 report says that the control substance was collected from ‘near-isogenic line’: what does that mean, near-isogenic line?
65. Page 47: Feeding studies on Rabbits: “It was concluded based on the health, growth and physio-pathological parameters analysed during the experiment that there were no significant differences between the groups fed with Bt brinjal containing cry1Ac gene and control non-Bt brinjal fruit”. However, this is not the conclusion in the study. As per Report of Study No. 4418/05, dated 14/7/2006, as contained in Volume 3 of Bt Brinjal biosafety dossiers on the GEAC website:
“6. Haemotology: There were no changes observed in between Control Non Bt Brinjal (G2) and transgenic Bt Brinjal containing Cry1Ac gene (G3) groups except for an incidental but not biologically significant reduction in platelet count in G3 males at interim blood sampling and significant increase in Hct, reduced MCHC in G3 males and increased prothrombin time in G3 females at terminal blood sampling”.
“7. Clinical Chemistry: There were no changes observed in between Control Non Bt Brinjal (G2) and transgenic Bt Brinjal containing Cry1Ac gene (G3) groups except for an incidental but not biologically significant increase in albumin, and total bilirubin in G3 males and increased total bilirubin, lactose dehydrogenase in G3 females at interim blood sampling and significant increase in the AST, ALT, Total Billirubin and Sodium levels in G3 males and increased total bilirubin and decreased glucose levels in G3 females at terminal blood sampling”. G3 group in this study is Bt Brinjal-fed animals’ group and the results by the admission of the crop developer itself are the above whereas the EC2 chooses to falsify the findings by saying that “there were no significant differences between the groups”.
These differences are discounted by the study scientists as thus: “these changes are considered incidental and not related to transgenic Bt Brinjal feeding since the changes were marginal and of no biological significance”. Beyond this, no rationale is available or provided and the explanation provided by EC2 on Page 59 under Issue 9 is simply not applicable here. Bt Brinjal cannot be considered safe just because the EC2 concludes so without any scientific basis, falsifying even the findings of the crop developer!