Four industry-sponsored GMO feeding studies are probably fraudulent
Date Added to website 11th February 2014
Following the FCT retraction of the Seralini long-term rat feeding and toxicology study, and the myriad of consequential complaints from the science community, a number of scientists have directed scrutiny towards some other studies published in FCT which -- presumably -- are accepted as soundly-based and as showing conclusively that GMOs are safe. Veterinarian Ena Valikov has looked at a study published in FCT by Hammond et al in 2004, purporting to show that Roundup-tolerant has no significant effect on animal health when it is incorporated into a test diet. What she discovered was shocking, and included a failure to rule out the allergenicity of the Roundup Ready transgene; a failure to consider pleiotropic effects resulting from the GM insertion process; experimental design flaws including the use of invalid reference or control groups; the introduction of selection bias into the choice of animals to be tested; the use of invalid statistics; a failure to address signs of kidney and liver toxicity; and a failure to consider the pleiotropic effects of Roundup herbicide in the sampled groups of rats.
From all points of view the study by Hammond et al is at best an inconclusive demonstration of the safety of the GMO variety being studied, and at worst it is actually fraudulent. From all points of view the study is vastly inferior to the study by Seralini et al which was retracted by Wallace Hayes, the Editor of FCT, on the grounds that it was "inconclusive."
This is the clearest possible demonstration of the hypocrisy and double standards employed by this Elsevier journal, which has failed to respond to calls for the study by Hammond et al (2004) to be retracted.
And it gets worse. There are three other studies (2006, 2006 and 2008) by the same research team, all using similar techniques and all claiming to demonstrate the safety of another GMO crop being put into the approvals process. These too should be retracted, since they have clearly not been assessed properly in the peer review process.
And it gets worse. The four studies cited (including those involving MON 810, MON 863 and MON 88017) used similar experimental designs and analytical methods, and yet all of them were accepted at face value by European regulators, including EFSA, as demonstrating beyond reasonable doubt that these varieties are as safe as their conventional counterparts. All three of these named varieties have been given consent for use in food and feed in Europe, and MON810 has been given consent for cultivation. This demonstrates -- not for the first time -- that EFSA is both biased and incompetent, in that the body has failed to address the obvious shortcomings of these Monsanto-funded studies in spite of these defects being pointed out to them repeatedly by NGOs and independent scientists.
This provides clear evidence of the corruption of the scientific process both in the publishing world and in the world of GMO regulation.
Dear Food and Chemical Toxicology -- Shouldn't you retract Hammond's GMO study?
by Ena Valikov
http://beachvethospital.blogspot.com/2014/01/dear-food-and-chemical-toxicology.html
Jan 16th 2014
"Results of a 13 week safety assurance study with rats fed grain from glyphosate tolerant (Round-Up-Ready) corn."
Veterinary medical perspectives
Hammond is not conclusive
Food and Chemical Toxicology, the journal which recently retracted the infamous Seralini study, also published four safety assurance studies by Hammond et al.
The feeding trial I will discuss on Sprague-Dawley rats (same rat strain as Seralini's) by Hammond et. al. was published in 2004 and cited by Seralini, but curiously has not drawn any attention. It is the "mirror" piece of science that triggered Seralini's investigation. The full text of the study is linked in the title.
The study is a safety assessment to ensure that the Round Up Ready corn is as safe as conventional corn involving two components. Firstly, it is an evaluation of safety of the newly introduced trait, Round Up Resistance conferred by the Round Up Ready transgene (CP4 EPSPS). Secondly, it is an assessment for possible toxicity due to unintended pleiotropic effects resulting from the insertion of the transgene. [*]
It is my opinion, the study does not provide robust evidence to support either claim and is just as inconclusive as Seralini, and should likewise be retracted. The following dissection should clarify the reasons why.
Safety of introduced CP4 EPSPS trait
Allergenicity of Round Up Ready(CP4 EPSPS) transgene protein has not been ruled out.
Committees of global experts have created decision trees largely based on assessment of IgE-mediated food allergenicity for risk assessment.[8] The WHO guidelines state that a transgene with greater than 6 amino acid homology to a known allergenic epitope or > 35% of the sequence, should be further screened on 25 individual serum samples with high IgE titers to that air borne or food borne allergen.[2] CP4 EPSPS protein has homology of 7 contiguous amino acids ( 7 amino acid chain is clearly longer than 6) to a known and prevalent allergen- dust mite allergen (der p 7).[1] Hammond cites Harrison et al, a study on digestibility of CP4 EPSPS protein as evidence for lack of allergenicity of the introduced transgene. While digestibility is a preliminary step in screening novel proteins, it is not a substitute for immunologic studies. Hammond also cites studies on Round Up Ready soybeans as evidence of lack of allergenicity. However, studies on Round Up Ready soybeans are not a substitute for studies on Round Up Ready corn. Moreover, a targeted analysis on serum from patients with dust mites allergies was not performed on 25 serum samples in the studies. The largest inhibition ELISA on CP4 EPSPS was reported for Round Up Ready soybeans on a trivial sample of 4 [9] and thus the studies would not uncover cross reactivity between Cp4 EPSPS and der p 7 in soybeans.
Research to verify lack of cross-reactivity is not cited by Hammond et al, and a literature search failed to uncover such studies on 25 serum samples with high levels of IgE to dust mites.[9] Inhibition ELISA studies can and should be conducted to assess for the presence of allergenic cross-reactivity between the EPSPS CP4 protein in corn and der p 7 to definitively rule out allergenicity [10] This is a minimal standard of risk assessment for known allergens, while everyone accepts that even this standard leaves large gaps since not all allergies are IgE mediated.
Toxicity due to pleotropic effects resulting from insertion into the corn genome [*]
Random integration of a transgene may cause gene disruptions leading to sequence changes, production of new proteins or formation of new metabolites or altered levels of existing metabolites that could compromise safety. This includes the potential production of new allergens or toxins.[5]
A proteomics study on maize demonstrated that 43 proteins were up- or down-regulated in transgenic seeds with respect to their controls specifically related to the insertion of a single gene into a maize genome by particle bombardment.[4] Non-targeted "omics" profiling studies might be able to detect unintended and unexpected changes that targeted compositional analysis underpinning the current risk assessment by Hammond et al would not, making this safety assurance study more conclusive. [11]
Experimental design flaws
Invalid reference groups.
The experiment was conducted on 400 rats fed two doses of corn (11% & 33%). A balanced experimental design using 400 rats would consist of 50 rats/sex/dose on transgenic corn compared to 50 rats/sex/dose of its isogenic parent line grown side by side.
See Table 1. Hammond's reference groups A-F are grown in random geographic locales such as Indiana, Iowa and Colorado, while the genetically engineered corn was grown in Ohio!
Agronomic factors (soil, fertilizers), and environmental influences (location, weather, stress) are factors that should be considered during "GE versus non-GE" evaluations. [3] The environment has been shown to play an important effect in the protein, gene expression and metabolite levels of maize samples tested, where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed.[5] Approximately 100 total proteins were differentially expressed as a consequence of environmental influence in a proteomics study. [4]
Thus reference control A-F in Table 1 are inappropriate reference groups used to establish invalid reference ranges. Shrinking the experimental group to 80 (20/sex/dose) while increasing the control group to 320 animals serves to increase type II error (false negatives), reducing sensitivity of detecting adverse health effects.
I could stop right here. If the reference ranges for non-GE corn are invalid, it isn't possible to compare GE and non-GE for statistical differences within 2 standard deviations as the study does. But I shall go on.
Data is reported for HALF the animals.
Elementary school arithmetic for non-scientists. Number of experimental animals (N) in Tables should be 20 because there were 20 rats/sex/group. Don't feel too bad if you aren't finding it easy, because the "esteemed" scientists and editors at Food and Chemical Toxicology evidently flunked elementary school arithmetic as well.
There is a very good reason a system of blinding has been used for hundreds of years in research to prevent bias. Scientists often want a particular outcome from an experiment, and it is possible to choose the animals to test in such a way as to achieve desired results. In a non-blinded experiment, where the researchers know which animals are exposed to which food, and test results are published for half the animals ( N 4-10, instead of 20 in Tables 2,3,4), the researchers are free to cherry pick the rats based on overt clinical signs. If the transgenic corn was engineered with a metabolite toxic to the kidneys, the most affected rats would be identifiable, as they would be drinking excessively and urinating excessively. Rats with liver disease could likewise be avoided for sampling because their white skin would glow yellow due to jaundice. Bias does not have to be due to deliberate deception- human nature makes scientists vulnerable to confirmation bias.
Data and conclusions of a non-blinded experiment reporting data for half the animals should be declared invalid by design. I could stop right here, but I shall go on.
Statistics are invalid
Tables 2-4 do not report precise number of rats. Means and standard deviations cannot be calculated for ranges of rats.
A study with four times as many control/reference animals (N=320) as experimental (N=80) is intuitively grossly imbalanced which raises questions about the accuracy of the statistics cited. Unfortunately, statistics are not my strong suit.
Kidney toxicity
Sprague Dawley rats suffer from spontaneous kidney disease, chronic progressive nephropathy (CPN) which confounds toxicology studies. CPN primarily affects males and is felt to be exacerbated by ad libidum feeding and high protein diets.
Blood tests associated with kidney function (BUN, creatinine) are not significantly elevated until advanced stages of disease, which makes the "normal" tests reported in Tables 4 & 5 meaningless. A urinalysis does indicate progression and loss of renal function by declining urine specific gravity, elevated levels of urine protein/ albumin, and casts- prior to elevation of blood tests. These inexpensive and crucial functional tests are not published in this study nor any Hammond et al feeding trial. I could stop right here, but I shall go on.
The findings reported in Table 7 are vague and not descriptive of the specific histopathological changes. The earliest lesions of CPN in young rats are convolutions of a single proximal tubule showing basophilia and crowded nuclei, representing simple tubule hyperplasia, with thickened basement membrane (especially of the Bowman's capsule), which is not reported in this study. The study instead reports mononuclear cell infiltrate, which is generally a feature of inflammatory/ immune mediated processes. However, CPN is not an inflammatory or vascular disease, and it has no immunological or autoimmune basis. [6] Regeneration is reported in 17/20 rats, which is disproportionately high to the number of animals in whom degeneration is reported, while these processes occur simultaneously. Thus without a review of histopathology slides by independent pathologists assurances of absence of reno-toxicity can not be made.
Furthermore, pathologists recommend histopathological grading on a scale of 0-4 (kidneys graded by the study pathologist as minimal = 1 (less than 25% of renal tubules affected); mild = 2 (T 25–50%); moderate =3 (T 50–75%); or marked = 4 (T 75%); [7] [6] which likewise was not done in this study, making a valid comparison of severity between the groups impossible.
In any case, sample sizes of 9-10 for whom results are published are too low to parse toxic effects fron confounding spontaneous kidney disease - unless the potential toxin has a very strong effect in a very short period of time.
So, if you recall the main, and I believe, valid criticism of Seralini as having sample sizes too small to draw conclusions, the same is the case with this study. The sample size is too small to rule out kidney toxicity, separate and apart from the other flaws I listed above. The difference is that Hammond et al reaches the questionable conclusion that this GMO corn does not cause harm, even as half the laboratory animals disappeared, while Seralini accounts for all of his rats but concludes that the corn is harmful.
I could stop right here, but I shall go on.
Liver toxicity
Tables 2,3,4 report normal liver enzymes which would suggest that the corn is not hepatotoxic. Unfortunately they do not.
Liver enzyme tests are not liver function tests-they are tests of inflammation. In fact, liver enzymes can appear relatively normal in the face of liver failure. Bilirubin is a marker of jaundice most often associated with hepato-biliary disease. In cats the most common form of liver disease is known as hepatic lipidosis or fatty liver disease. The same disease is recognized in rats and in people, and it is called non-alcoholic fatty liver disease (NAFLD). Its prevalence rate is rising- especially among kids.
I present an illustrative case report with blood work and a urinalysis in a cat with imminent liver failure, in whom liver enzymes were nearly normal. The test most crucial for diagnosis was serum and urine bilirubin.
Total bilirubin (t.bili) in Table 4 is reported for 4-6 out of 20 male rats and 7-8 out of 20 female rats. Bilirubin values in the urine are not published for the rats at all. These are red flags!
The study reports mulifocal chronic inflammation in most of the rats in Table 7 which could be histopathological indicators of chronic inflammatory hepatitis. Histopathological inflammation should cause elevation of liver enzymes, interestingly reported to be within normal ranges, which doesn't really make sense.
The liver represents a suitable model for monitoring effects of a diet, due to its key role in controlling the whole metabolism. A study examining effects of Round Up Ready soy diet were studied on liver of female mice in order to elucidate possible interference with ageing. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. The study demonstrated that GM soybean intake can influence some liver features during ageing while liver enzymes (the tests reported by Hammond et al) were not afffected at all. The mechanisms remain unknown, underlining the importance of investigating long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions. [12] [13]
I hope I've clarified why this study by Hammond et al does not meet its stated goal of reassuring me of safety of this crop. The other three studies linked above relied on the same flawed experimental design.
In spite of lack of " conclusiveness" factor I outlined above, this study has not been retracted, which suggests that the agricultural biotechnology science establishment is promoting scientific double standards .
I could go on -- but I won't. Hopefully, I've given you enough food for thought.
References
1.BMC Structural Biology Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens Gijs A Kleter1 and Ad ACM Peijnenburg1 http://link.springer.com/article/10.1186%2F1472-6807-2-8/fulltext.html
2.Evaluation of Allergenicity of Genetically Modified Foods Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology January 2001 Food and Agriculture Organization of the United Nations (FAO) Rome, Italy http://www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf
3. ISB NEWS REPORT • MAY 2010 Molecular Profiling Techniques as Tools to Detect Potential Unintended Effects in Genetically Engineered Maize. Eugenia Barros. http://researchspace.csir.co.za/dspace/bitstream/10204/4465/1/Barros1_2010.pdf
4. J Proteome Res. 2008 May;7(5):1850-61. doi: 10.1021/pr0705082. Epub 2008 Apr
5. Proteomics as a complementary tool for identifying unintended side effects occurring in transgenic maize seeds as a result of genetic modifications. Zolla L, Rinalducci S, Antonioli P, Righetti PG. http://www.ncbi.nlm.nih.gov/pubmed/18393457 http://www.ask-force.org/web/Genomics/Zolla-Proteoimics-Complementary-Tool-GMO-2008.pdf 5. Comparison of two GM maize varieties with a near isogenic non-GM variety using transcriptomics, proteomics and metabolomics. Eugenia Barros,Sabine Lezar, Mikko J. Anttonen, , Jeroen P. van Dijk, Richard M. Röhlig, Esther J. Kok3, and Karl-Heinz Engel http://researchspace.csir.co.za/dspace/bitstream/10204/4436/1/Barros_2010.pdf
6.Toxicologic Pathology, 32:171–180, 2004A Contemporary Overview of Chronic Progressive Nephropathy in the Laboratory Rat, and Its Significance for Human Risk Assessment GORDON C. HARD1 AND KANWAR NASIR KHAN http://tpx.sagepub.com/content/32/2/171.full.pdf+html
7. Toxicol. Sci. (2012) 128 (2): 346-356. Chemically Exacerbated Chronic Progressive Nephropathy Not Associated with Renal Tubular Tumor Induction in Rats: An Evaluation Based on 60 Carcinogenicity Studies by the National Toxicology Pgram Ronald L. Melnick, Kathleen M Burns, Jerrold M. Ward and James Huff http://toxsci.oxfordjournals.org/content/128/2/346.long
8. Environ Health Perspect. 2003 Jun;111(8):1114-21. Clinical and laboratory investigation of allergy to genetically modified foods. Bernstein JA, Bernstein IL, Bucchini L, Goldman LR, Hamilton RG, Lehrer S, Rubin C, Sampson HA. http://www.ncbi.nlm.nih.gov/pubmed/12826483
9.http://www.allergome.org/script/dettaglio.php?id_molecule=2952&year=1
10. Clinical and Molecular Allergy 2007; 5:2 Assessment of allergen cross-reactivity Rob C Aalberse http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797810/
11.Molecular profiling — a tool for addressing emerging gaps in the comparative risk assessment of GMOs Jack A. Heinemann, Brigitta Kurenbach,, David Quist http://www.sciencedirect.com/science/article/pii/S0160412011001322
12. Histochem Cell Biol. 2008 Nov;130(5):967-77. Epub 2008 Jul 22. A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing. Malatesta M, Boraldi F, Annovi G, Baldelli B, Battistelli S, Biggiogera M, Quaglino D. http://www.ncbi.nlm.nih.gov/pubmed/18648843
13. Cell Struct Funct. 2002 Aug;27(4):173-80.Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean. Malatesta M, Caporaloni C, Gavaudan S, Rocchi MB, Serafini S, Tiberi C, Gazzanelli G. http://www.ncbi.nlm.nih.gov/pubmed/12441651 Sourcehttp://www.ncbi.nlm.nih.gov/pubmed/12441651
Footnotes:
[*] The study as designed by Hammond et al does not permit evaluation for pleotropic effects since there is a confounding factor that has not been segregated out. The corn was treated with Round Up- a mixture of glyphosate, AMPA and proprietary adjuvants. These additional chemicals make it impossible to tease away any potential metabolic toxicity caused by transgene insertion on the genome from chemical toxicity of Round Up and its adjuvants. It is also the reason that Seralini in his retracted paper came up with the "complicated design" separating rats into groups exposed to transgenic corn alone ( to evaluate effects of transgene insertion on the genome in isolation of Round Up) and those exposed to Round Up in the drinking water. Posted 3 weeks ago by Ena Valikov
http://beachvethospital.blogspot.com/2014/01/dear-food-and-chemical-toxicology.html
Studies by Hammond et al in FCT -- all should be retracted on grounds of experimental bias / flawed experimental design and inconclusive results.
Results of a 13-week safety assurance study with rats fed grain from corn rootworm-protected, glyphosate-tolerant MON 88017 corn. Healy C, Hammond B, Kirkpatrick J. Food Chem Toxicol. 2008 Jul;46(7):2517-24. doi: 10.1016/j.fct.2008.04.005. Epub 2008 Apr 13. PMID: 18492601
Results of a 90-day safety assurance study with rats fed grain from corn borer-protected corn. (MON810) Hammond BG, Dudek R, Lemen JK, Nemeth MA. Food Chem Toxicol. 2006 Jul;44(7):1092-9. Epub 2006 Feb 17. PMID: 16487643
Results of a 90-day safety assurance study with rats fed grain from corn rootworm-protected corn. (MON863) Hammond B, Lemen J, Dudek R, Ward D, Jiang C, Nemeth M, Burns J. Food Chem Toxicol. 2006 Feb;44(2):147-60. Epub 2005 Aug 9. PMID: 16084637
Results of a 13 week safety assurance study with rats fed grain from glyphosate tolerant corn. Hammond B, Dudek R, Lemen J, Nemeth M. Food Chem Toxicol. 2004 Jun;42(6):1003-14. PMID: 15110110