ngin - Norfolk Genetic Information Network
2 August 2002

PUSZTAI VS AVENTIS

"...in science we need the evidence and not comforting and unsupported statements." - Dr Arpad Pusztai

1. Statement by Dr Arpad Pusztai for the Chardon LL Hearings
2. Aventis's response to Pusztai
3. Pusztai's response to Aventis

See also:
*Royal Society-Pusztai statements discussed in ACS publication (The Vortex) http://ngin.tripod.com/250702a.htm
*CAN SCIENCE GIVE US THE TOOLS FOR RECOGNIZING POSSIBLE HEALTH RISKS OF GM FOOD? Nutrition and Health, 2002, Vol 16, pp.73-84
http://ngin.tripod.com/230702c.htm

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1.Statement by Dr Arpad Pusztai for the Chardon LL Hearings

Aberdeen, Scotland, 23 October 2000

I am a "Summa cum Laude" graduate in Chemistry (Eotvos Lorand University, Budapest) 1953, BSc in Physiology (University of London), PhD in Biochemistry (University of London) 1960 and a Fellow of the Royal Society of Edinburgh in 1988.  I have worked at the Rowett Research Institute from 1963 till my retirement in 1990 as a Principal Scientific Officer and from 1990 to 1999 as a Senior Scientific Fellow of the Rowett after which my contract was not renewed.

The main area of my expertise has been in establishing the salient biochemical, physiological, nutritional, metabolic/hormonal, immunological and microbiological features of the interactions between biologically active food/feed components and the mammalian gastrointestinal tract.  I have published close to 300 papers in peer-reviewed top international science journals and written/edited 8 scientific books.  Since 1999 I have been lecturing at Universities and at Conferences all over the world, writing scientific papers and other articles and acting as a consultant to scientists and others, helping to write research proposals and/or participating in programmes of their work.

The focus in my present statement will be a scientific critique of the relevant parts of some of the documents submitted by Aventis to the UK Government in support of their application.  However, before my detailed criticisms are given one needs to understand that the studies and results described in this submission have never been properly published in peer-reviewed scientific journals.  These have, therefore, never been subjected to scientific scrutiny in keeping with the age-old tradition of science.  In fact, in this respect Aventis just follows the tradition of the GM biotechnology companies' general practice.  Indeed, as in a recent article in Science (1) the author commented that during the nearly eighteen years of the existence of GM crops he could only find seven peer-reviewed published papers on their health effects by computer aided literature search of four different scientific data bases.  Of these, two are from our laboratory.  This was therefore an independent confirmation of my statement to the OECD Conference in Edinburgh earlier this year that this is a very poor record for an industry which claims to save mankind from starvation in the 21st century.

As appropriate for my expertise and competence, and because the emphasis placed by Aventis in their submission on a 10/14 day rat feeding study in which the effect on growth, organ weights and haematological and other parameters of a preparation of the PAT protein, included in the diet at two different concentrations was recorded, I shall concentrate on the details of their rat study.  It is my intention to show that the design and execution of these experiments was considerably flawed and consequently their results do not stand up to a critical scientific evaluation and cannot be regarded as proper supporting evidence for their application.  In my opinion, therefore, had these results been written up as a scientific paper and submitted for peer-review, this paper would have most likely been deemed not to have reached a sufficiently high standard to be acceptable for publication by any major nutrtitional journal.

1. Substantial equivalence.
 
i.   In the absence of specification of the origin, the conditions of cultivation and generation of the different GM and non-GM samples in the documents strict scientific comparisons cannot be made.  However, even under these conditions the analytical values given in the document "Bundle 10/4; GSM/PS2" by Aventis for the composition of T14 and T25 maize showed many statistically significant differences in fat and carbohydrate contents between GM and non-GM grain samples and fat, protein and fibre between silage made of GM and non-GM maize samples.  As these values can only be used for rough comparisons it is highly pertinent that the Company's values still indicated significant compositional differences.  Thus, the conclusion of Aventis that T25 maize is not "materially different" from current commercial varieties is not valid.

ii.  For the record, differences in growth conditions can have serious impact on composition. Scientifically valid comparisons can only be made when the parent, non-GM line is grown together, side-by-side, with the GM line as this is the only way to establish whether the composition of the parent and GM lines is the same or not.  As the origin of the lines analysed in the Aventis document is not recorded, no valid comparisons can be made.  Comparisons with historical or literary values, particularly as their range can be very wide, has no scientific validity.

iii. It has been pointed out by many that substantial equivalence is a flawed, non-scientific and crude concept.  It provides an excuse for the GM biotechnology companies not to carry out expensive and time-consuming animal nutritional and toxicological tests to establish whether biological effect on the consumers of the GM crop-based foodstuff is substantially equivalent to its non-GM counterpart.  It therefore allows them on one hand to claim that because the GM crops are substantially identical there is no need for complex biological testings while one the other hand it makes it possible for them to claim that since the GM crops are novel containing genes from other than the parent line they are patentable.  However, reasonable scientific opinion is turning against this concept of substantial equivalence because due to the occurrence of unintentional and unpredictable genetic changes in the plants as a result of the incorporation of the vector in the plant genome and its positioning effects, we cannot precisely know which of the hundreds of components of the crop may carry deleterious, toxic or allergenic properties.  Therefore comparisons of crude protein, carbohydrates, fats and other nutrients can only be a starting point for the establishment of equivalence.  Minor and unexpected constituents in the GM crop with potentially high biological activity, when eaten, may have considerable and disproportionally harmful effects in the digestive system of humans/animals.  As for most these are unknown they are usually not included in the analytical comparisons.  Their presence, therefore, can only be revealed from nutritional, toxicological, immunological and metabolic studies which makes it imperative that such animal studies are performed with a flawless design and experimentation.

2.  Repeated dose oral toxicity (14 day feeding) study in rats:  Bundle 10/3.  RCC project 616307
 
i.   It appears from the description of the project that the rationale of this study was to assess the cumulative toxicity of the PAT-PROTEIN when given orally to rats in their diet for 14 days.  The study is also meant to provide a rational basis for toxicologic risk assessment in man.  As Chardon LL maize, both the meal and silage, are apparently to be mainly used in ruminant nutrition it is not clear how this study will help to determine whether this crop can be safely used in ruminants.  Additionally, although the testing by Aventis of this gene product, PAT-PROTEIN, can be commended, this study is no substitute for the nutritional testing of the entire GM crop, the actual GM maize, seed, plant and silage in all target animal species.  Without such studies the potentially harmful, unintended and unpredictable effects of the gene transfer, the effects of other components of the vector and the gene insertion (positioning effect) cannot be established or excluded.  In other words this study has the same relevance to the health effects of GM maize as testing the wheels of a car and drawing conclusions about the whole car's roadworthiness.  In the followings I intend to show that the claim based on this study that there is no evidence of toxicity for PAT-PROTEIN in rats will not stand up to rigorous critical examination and in the absence of similar studies with ruminant animals such a claim  may have even less validity with other animals.  This is particularly serious because no histopathology studies of any of the organs have so far been carried out (p. 14).

ii.  Specific major criticisms:

Materials and Methods - Experimental Design

a.  No valid conclusions can be drawn from a feeding study carried out with 5 rats per group in which the starting weight of the rats varies by more than ± 20% (53 to 82 g for males and 50 to 74 for females; p. 15) because for any differences to reach significance they must exceed ± 20%.  To achieve this in a 14 day study should require catastrophic conditions in the experiments which would not be allowed by Home Office rules. For information in most of our experiments the starting weight of the rats was usually 80 ± 1 g (which is less than 2%).
b.  As 5 rats per group were not housed individually the feed intakes of the individual rats could not be monitored (no statistics).  Moreover, feed intakes were not measured daily but only on 1 (pre), 3, 7, 10 and 14 days.  Also it is not clear that, as the rats were fasted on the 14th day, how it was possible to measure their feed intakes on this last day.  These are particularly serious flaws in the design because of the huge differences in the starting weights of the rats that almost certainly led to major differences in feed intakes (pp 18 & 19).
c.  The source of the PAT-PROTEIN is not given (p. 17).  Without this it is impossible to know whether the feeding study has any relevance to Chardon LL or not.
d.  As rats in group 1 were fed a different diet (full rat chow) from the other 3 groups (p. 18), for statistical (or other) comparisons group 1 was not appropriate.  In the statistical studies groups 2 and 3 ought to have been compared to rats in group 4.
e.  Rather curiously, although the main target organ of the PAT-PROTEIN fed to rats was the digestive tract (and pancreas), the weights of these were not measured (p. 30).  This is a major experimental design fault particularly as there are now 3 major independent studies carried out with 3 different GM foodstuffs which indicated that serious lesions occurred in the gastrointestinal tract of the test animals (2,3)!
f.  Urine and blood samples from the rats were obtained after the experiment and from animals after 18 h of fasting (p. 19).  Their relevance to the experiment is at best questionable, at worst the results might be misleading.
g.  Pathology (p. 29).  The results are still pending after 5 years?

Results

a.  The starting weight and the feed intake of group 3 rats (high PAT-PROTEIN) was the highest but they ended up with the lowest final body weight (pp 37 & 39).  This indicates an elevated metabolic activity possibly induced by the PAT-PROTEIN.  Our statistical analysis (ANOVA) of the data of individual body weights after the first day and at the end of the experiment on pp. 91 & 92 shows that the weight gain for both male and female rats on the high PAT-PROTEIN diet (group 3) was significantly less than that of either group 4 (control) and group 2 (low PAT-PROTEIN diet).  Thus, when it is properly calculated according to the basic principles of nutritional science, PAT-PROTEIN reduces feed conversion efficiency and therefore it is potentially harmful.  Thus, the conclusion on p. 34 that "there were no differences which could be attributed to treatment with the test article" is not valid.
b.  The statement on p. 35 that "there were no changes on ...., clinical biochemistry and urine analysis after 14 days" is not valid.  According to their own results there were differences between the groups in glucose, cholesterol, triglyceride and phospholipid levels indicating an increased metabolic functional load in the rats.  It is unusual, to say the least, that these differences are dismissed by the authors of the study as incidental and unrelated to the treatment.  Our ANOVA statistical analysis in fact revealed that the urine output in rats on the high PAT-PROTEIN diet was significantly reduced that again indicated treatment-related effects.
c.  It is true that there were no significant differences in the organ weight data but as it has already been alluded to (Materials and Methods; a.) the huge differences in the starting weight of the rats of this study would have required disastrous changes in organ weights before they could have reached statistical significance.  However, it is clear that rats fed the high level PAT-PROTEIN diet (group 3) had the lowest liver, thymus and spleen weights of all other groups (even though the differences were not significant).  This is of particular importance because the macroscopical findings (p. 79) indicated thymus focus/foci in 20 - 40% of the animals fed diets containing the PAT-PROTEIN (even at the lower level of dietary inclusion).

In conclusion: The design and execution of this 14 day rat feeding study was seriously flawed and, contrary to the conclusions reached by the authors, indicated serious treatment-related effects due to the inclusion of a sample of PAT-PROTEIN of unspecified origin.  Thus, the results of the study could not be taken as evidence that the PAT-PROTEIN introduced by genetic engineering into Chardon LL maize showed no toxicity for the rat.  The results are even less convincing that feeding to Chardon LL maize could not have had harmful effects for the growth and health of rats (which according to Aventis' statement is a model for humans).  Moreover, as apparently Aventis have not carried out direct feeding trials with cattle (the intended main target animal) in which they are fed with rations containing unprocessed whole maize plant or silage prepared from it, these studies are irrelevant.  Finally, a recent publication (4) showed that DNA survives in intact form or slightly fragmented in both unprocessed and ensiled maize products and therefore cattle fed on these would be exposed to DNA used in the genetic engineering process.  Thus, without extensive feeding trials with ruminants to show that it has no harmful effects to cattle and/or to humans eating meat products derived from them, the introduction of Chardon LL maize into national seed listing would be highly irresponsible and Aventis' application for such must be rejected.  In instances when no data are provided by a GM biotechnology company the precautionary principle must be applied.  Moreover, even when these are done by the company in the future, for the suspension of this principle there is an overriding need for independent confirmation of the data in compliance with the Edinburgh OECD Conference's conclusions that all work concerning GM food should be done "openly, transparently and inclusively".
 

References

1. JL Domingo (2000).  Health risks of genetically modified foods: Many opinions but few data.  Science, 288, 1748-1749.
2. NH Fares, AK El-Sayed (1998).  Fine structural changes in the ileum of mice fed on delta-endotoxin treated potatoes and transgenic potatoes.  Natural Toxins, 6, 219-233.
3. SWB Ewen, Pusztai A (1999)  Effects of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine.  The Lancet, 354, 1353-1354.
4. A Chiter, JM Forbes, GE Blair (2000).  DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food.  FEBS Lett. 24098, 1-5.

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2. Aventis's response to Pusztai

Subject: Re: Chardon LL
Date: Thu, 13 Jun 2002 16:48:34 +0100
From: "Ray Foulk" <gmo.campaign@virgin.net>
To: "A.Pusztai" <A.Pusztai@freenet.co.uk>

Dear Arpad,

Thank you for your email. After some legal deliberations Mr Alesbury has ruled that we at the GMO Campaign have the unusual right to submit further responses to the Chardon LL Hearing at this late stage. The public hearing as such is now closed. Mr Alesbury has allowed 14 days from today's date for us to submit our written response to what Avantis said about you and us. We would be extremely grateful if you would consider what Mr Smith (lawyer for Aventis) had to say and what response you could provide. If you would like to discuss this over the telephone please let me know the number and I will call you at our expense.

I'm not sure if you are up to speed on the transcripts, but as I understand it, the relevant passage appears on pages 105-13, 29 May. The site address for the May 29 transcript is:

http://www.defra.gov.uk/planth/pvs/chardon/transc.htm

Best wishes, Ray Foulk

ps. In case you cannot download the transcript, there follows a copy of what appears to be the offending section (pp 103-13).
 

MR SMITH: Turning now, then, to the rat toxicity study. A summary of the study is actually found in the submissions to the T25 open hearing. Again, a copy of which is on website. It should be noted that in June 1999 the Royal Society concluded that Dr Pusztai had produced no convincing evidence of adverse effects from GM potatoes on the growth rate of rats or their immune functions. I refer to the review of data on possible toxicity on GM potatoes, June 1999; there is a reference there. In any event, Dr Pusztai's analysis concerned potatoes, containing an experimental line which was still in laboratory development, with potential insect-controlling properties, and not a commercially available herbicide-tolerant maize. So any comments would have limited reference to Chardon LL.

Turning to the study conducted by Aventis, this has been criticised for its relevance and design. On the first point, there was a criticism that PAT protein from canola should have been used but was not used in the study. This criticism is misplaced and results from a possible misreading of the paper by Dr Pusztai. The protein was actually generated in a microbial system in E.coli in order to produce enough evidence for the study. This is an accepted practice, and a separate study confirmed that there were no distinguishable differences in PAT protein, whether it came from bacteria, canola or T25 maize. Thus, the study is relevant in the evaluation of the potential toxicity of PAT protein expressed in T25 maize. On the second point of design, the study was performed by one of the most well known and established Swiss contract laboratories, which conducts toxicological studies and should be familiar to anyone with a wide experience in toxicology. The study used was a standard OECD protocol. The protocol is widely used and was generally accepted within the scientific community at the time, and as such the use of this protocolshould not be something for which Aventis is now criticised, six years later. The study objective was to assess the cumulative toxicity of Pat protein when fed to laboratory rats over a period of 14 days. The results would determine, if there were an indication of mammalian toxicity, that might justify further study.

As DEFRA's own GM Policy and Regulation Unit conclude in their report: "The fact that these studies were conducted for 14 days has been criticised by some observers as being too short, but this is a recognised process whereby test animals are fed large amounts of a specific substance over a relatively short period and monitored for any toxic effects. After two weeks of this regime, there was no apparent effect on the animals, and so confidence can be gained that PAT is not acutely toxic." There was no evidence of toxicity despite comments from Dr Pusztai in this hearing, which seemed to be misplaced and based upon a rather confused analysis of the study. Indeed, from the critique, it is evident that the interpretation of the data is not very thorough, and this suggests a lack of experience on his part with the interpretation of toxicity studies. In particular, several comments seemed to indicate that it was being evaluated as a nutritional study and was considered to study nutritional parameters, such as the daily measurement of feed intake or determination of feed conversion efficiency. The objective of a nutritional study is obviously very different from a toxicity study and, as such, this is why the design is different.

Such a view of the study is completely misplaced, as it was an oral toxicity study, not a nutritional feeding study. The parameters evaluated were those consistent with an evaluation of potential oral toxicity to the PAT protein. In this respect, the study was extreme, in that it used dosages that were equivalent to 100 and 1,000 and 1,000 times the concentration of protein to be found in GM herbicide-tolerant oilseed rape meal, which as such contains the highest concentration of the protein.The conclusion was that, even at the 1,000-fold concentration, there was no evidence of toxicity. In addition, Dr Pusztai commented that no histopatholgy studies were conducted, yet later in his submissions references are made to microscopical findings. In fact, the study clearly included histopatholgy examinations, and the pathology report was included as an appendix on pages 192 to 244 of the study. It would appear from this that Dr Pusztai had a copy of the wrong report, perhaps the preliminary report, not the final report that was submitted for the part C application. Aventis would have of course provided the GMO campaign group with a copy of the final report, which included the pathology report, if they had been asked or, more sensibly, if these questions had been put to Aventis before the hearing.

There was also some testimony giving the impression that there were only five animals per test per group and that more should have been used. In fact, there were ten per group, caged in groups of five males and five females, and there were no premature deaths. The numbers used were, therefore, perfectly adequate. There was also submitted that it was not appropriate to include comparisons of rats fed a standard diet with those fed the low protein diet, two of which contained the PAT protein. This suggests that Dr Pusztai appears to have missed the point about the control group. In fact, the standard diet group was included as a control group to check for any possible effects of the low protein diet. By including this group in the statistical analyses, treatment responses that may have been attributable solely to a low-protein diet, irrespective of the presence of PAT protein, would have been discerned. It was thus a control for a different type of low-protein diet, rather than a control for the PAT protein per se. The data should be evaluated in comparison with the group 4, which is the low-protein diet supplemented by soyamin, and this is how it has been viewed both by Aventis and the regulators. Feed intake measurements were criticised as only having only been taken on days 1, 3, 7, 10 and 14, rather than daily.Again, Dr Pusztai links daily measurements with nutritional studies. However, this is standard practice in routine toxicology studies. Again this is not a study for nutrition. The suggestion made is that weighing/measuring the pancreas and the digestive tract within the organs weighed is important because of studies on other GM crops in which lesions were found in the gastrointestinal tract of the test animals. However, it is again worth noting that Dr Pusztai's analysis concerned a particular GM line, potato crop, and not T25 maize, so any comments have limited relevance to Chardon LL. Further, from a technical perspective, weighting the pancreas and digestive tract is not easy. There is a great risk of errors due to dissection procedures for the pancreas. Variably digested food in the gut is not easily removed without disturbing the histopatholgy. This latter aspect, histopathology, is considered to be more relevant than organ weight. In the Research and Consulting Company study, histopathological examinations did not show any evidence of histopathological changes in the pancreas and/or gut. Dr Pusztai claims that with other GMOs examinations have revealed lesions, major proliferative changes in mice fed Bt toxin in potato or by itself. Yet, this is not seen by taking the weight, but by microscopic examinations. In the RCC study, histopathology did not show any negative effects. Comment is also made on lower liver, thymus and spleen weights in group 3 animals, ie the high PAT dose. The interpretation is based on the absolute organ weights. However, the body weights of the animals in group 3 are also slightly lower, though not statistically significant. This is the reason why organ weights are also presented as ratios to body weight and ratios to brain weight. When looking at this relative mode of expression, it is evident that there is no effect on organ weights. The conclusion in the report is perfectly valid. The challenge again suggests a lack of relevant experience in terms of toxicology data. Dr Pusztai also claims, on the basisof a statistical comparison, ANOVA, of the difference between starting and ending weights of the rats, that PAT protein reduces feed conversion efficiency and therefore is potentially harmful. Again he brings up feed conversion efficiency as a parameter, as if it were a nutritional study; that is wrong. There were no statistically or toxicologically significant differences in body weight gains or food consumption over the 14-day period. Similarly, no dose response was seen in body weight gains. Feed conversion efficiency is not a parameter measured in acute toxicity studies.

Feed efficiency values, which are expressed as a percentage of grammes of feed consumed per grammes of body weight gained, are such that even if an effect is to be claimed, which Aventis does not support, as this study was not designed to measure feed efficiency, there is in fact a higher feed efficiency at 50,000 parts per million PAT does level, not a lower one, as suggested. Dr Pusztai claims that an ANOVA statistical analysis of urine output showed a significant reduction of output in group 3, the high PAT, implying some adverse toxicological effect. This is wrong. In the absence of significant changes in the urine -- osmolality -- and blood -- creatinine, urea and uric acid levels -- and kidney weight or histopathology, this change is certainly not an adverse toxicological effect. It can be related to lower water consumption or urine retention during the collection of urinary samples.

Notwithstanding any comments from ACAF, DEFRA has confirmed in its report on GM maize that taking these factors into account, the data did imply that the dose of PAT given to rats was some 5,000-fold greater than normal dietary exposure. If this is the case, and it is still to be confirmed, then the study is a test of acute toxicity and would allow a no-observed adverse effect level to be set above any reasonable level for concern. It is important to note that Aventis concluded, based upon the results of the study, that for a daily consumption of 7,600 milligrammes per kilogramme body weight of pure PAT proteinthere was no observable toxic effect. To deliver this quantity of PAT protein for a human subject, some 15,674 tonnes of grain maize would need to be consumed every day. Aventis submits that this demonstrates that T25 was found to be non-toxic within acceptable scientific parameters.

Moving next then to the poultry feeding study, a summary of which is reproduced in Aventis's submissions to the T25 open hearing; again, which is available on the web and on ACRE's website. The chicken feeding study has been criticised for its relevance in assessing the feed safety, and there was a suggestion that mortality was increased with use of the GM maize. In fact, the study was not intended to assess feed safety, as this was not considered necessary in view of the assessment of substantial equivalence and the previous toxicity study. Nevertheless, a chicken study was conducted to assess the nutritive value of the maize. It was chosen because it is a target species for grain maize and because it was regarded by the authors as a very sensitive test species, due to the fifteen-fold increase in body weight which occurs during the first 18 days of the life of a broiler chicken. With such rapid growth, any inadequacies in the diet will be observed quickly.

Specific criticisms have been made by reviewers from the University of Bristol, suggesting that far too few replicates had been used to be able to pick significant differences in either body weight or mortality. The authors of that study have commented that discussions of trends referred to by the reviewers are not relevant to the assessment of the treatments, and only sound statistical analysis is able to determine if any differences have occurred. The results showed that the GM maize had no effect upon body weight, feed intake, feed intake to body weight grain, or percentage mortality rates. The reviewers, however, suggest that the differences in body weights between treatments at the end of the experiment would have to be very large to be detectable, because of the number of pens and replicates used.However, this seems to ignore the fact that such an assessment is only one of several live weight assessments analysed in the study. The broiler has a rapid growth rate and is consequently very sensitive to nutrient effects, and the study design used is normally able to detect the differences quoted as desirable by the reviewers, ie an 80-gramme difference at 42 days. This is exemplified in the 0-18 day growth period, when the broiler's weight increases fifteen-fold and therefore a far more sensitive phase in which to look for differences. During this period, body weights differed by only 6 grammes, which is less than 1 per cent, whilst the standard error of the mean body weights was only 2 to 2.5 per cent. Such small variances are thus well within that required by the reviewers. It is easy to become distracted by discussion of variant and precision from the most important point of the results, which is the equivalence of the means for the treatment groups. These in fact showed less than an 18-gramme body weight difference at 42 days, ie 0.7 per cent, much lower than the difference of 78 grammes quoted by the reviewers to have been found to be significant in similar studies of their own.

On the question of mortality, the difference between the two groups was 3.5 per cent with the non-GM and 7 per cent with the GM maize. However, the range of mortality observed was well within the norm for the fast-growing strain of bird used in the study. With these birds, their high growth rate associated with a healthy diet normally results in a small number that suffer sudden death, know in the industry as "flip-over". This was confirmed by the postmortems carried out at the time, which demonstrated that most deaths were attributed to heart failure, and not to feed-related reasons, to do either with the GM or non-GM maize. The study reflected best practice at the time in merely replicating that of the industry standard. It is accepted that at the time a mortality rate of up to 8 per cent was standard in the industry for raising broilers under commercial production techniques. The 5 to 8 per cent mortality rate quoted in the study is from male birds at the research facility at the time the study was conducted, back in1996. It is widely recognised that males have a higher mortality rate than female broilers. In addition, mortality rates have dropped over the past five years due to changes in management practices, including the use of darkness periods each 24-hour cycle. The 4 per cent industry average stated by the reviewers is the current industry average and probably incorporates both males and females in the figure. In fact, the lack of use of darkness periods was another criticism, but it is simply a fact that at the time the protocol was developed continuous lighting was standard practice in the industry in North America. Thus, the mortality that occurred during the study was not surprising or suspicious and was not indicative of any adverse effect. It is accepted scientific practice that wholesomeness studies are best observed using animals with a fast development cycle. Any adverse treatment-related effects will therefore be exhibited fairly swiftly and data can be generated much faster than it would take to conduct studies upon larger, as well as more costly, animals such as cattle.

As DEFRA observed, whilst some people have expressed concern over the mortality rates in the birds fed GM maize, compared that of birds fed non-GM maize, both are within the expected range for chickens reared in these conditions, ie up to 8 per cent. DEFRA then go on to consider the proper question for scientific scrutiny, as to whether or not this difference in mortality is biologically significant. DEFRA observed that the numbers of chickens dying was very small, irrespective of their diets, and the mortality varied a great deal between the different rearing-rooms used in the experiment. This sort of variation among mortality data is typical of such experiments; it does not mean that it is not possible to prove that small differences between birds fed on different diets have not arisen simply by chance. Again, as DEFRA observe, in most cases, where a dietary factor affects mortality rates in chickens, the surviving chickens in a test group show evidence of other impacts, such as decreased weight gain. In the reported experiment, there were no significant differences noted in weight gain and feed intake among the survivingchickens. The lack of difference in weight gain between the two groups indicates that the difference in mortality rates was more likely to be due to chance than to any dietary effect. DEFRA concluded that the weight gain and feed intake results from this test support the hypothesis that the two maize diets are nutritionally equivalent.

Further, ACAF advised on 8th November 2001 in relation to the chicken study that the results of the chicken study did not raise any obvious safety concerns. Again, this study is to be seen as just one piece in a much larger jigsaw, that of the overall regulatory assessment. Aventis stands by the results which are consistent with other studies and analysis in demonstrating the equivalence of T25 maize to its non-GM counterpart.

However, even if specific criticism is now made of the way the study was constructed, the data was submitted in good faith to DEFRA and ACRE and was accepted six years ago. There is no reason why Aventis as a company should have anticipated that ACRE, or any other body, should later take issue with the findings of the study, after a positive review back in 1996/1997. At worst, if the doubts now expressed about the robustness of the study's protocol were to be attributed some weight, then the proper conclusion to be drawn would be that this particular study has nothing to add to the regulatory assessment, as from a statistical perspective its findings are unable to be validated without further work. In such circumstances, Aventis would submit that, in contrast to the statements made by Friends of the Earth and its scientific witnesses from the University of Bristol, the study tells us nothing in scientific terms about whether the sample group of birds fed T25 maize may have had a higher mortality rate than that of the control group fed a conventional corn diet. It must be emphasised that, even if the study is removed from equation, which Aventis would contest, the evidence before ACRE, ACAF and ACNFP was conclusive in not raising any doubts over the safety the GM maize for use as an animal feed.

MR ALESBURY: I wonder, thinking of some of the things that have been said to me, what do you say to those whom I understand to say: "while it may be true to observe that the difference in the mortality rate was not statistically significant, the very fact that there was in fact twice as high a mortality rate in the GM-fed chickens as in the others at least called for the matter to be investigated further"? Then the question is asked, "Has that been done?", or the comment made, "But that was not done". I am not wanting to push the line further from my own standpoint, but I am aware that such comment is made, and I am not one hundred per cent sure that you have answered that specific point.

MR SMITH: I think it has been seen in the context of what was the standard industry practice and the standard level of mortality, both for the particular study and the laboratory and also generally amongst the broiler chicken industry, as I understand it. The question therefore is whether that would have put you on notice of anything in particular.

MR ALESBURY: One has heard that sort of point being made, you see.

MR SMITH: Also, I will take you slightly forward, to where we were looking under 23.46, in terms of dietary factors which may also affect mortality rates, another factor brought into the equation. Also, just looking in the roundness in terms of the evaluation of the study, at the time, it was submitted, as I understand it, it was conducted to standard protocols which were seen as best practice. The fact that they may or may not have changed since then - people are making comments now the plain fact is that with it was submitted according to standard protocols, the results submitted, assessed by the regulatory bodies at that stage.

MR ALESBURY: Yes, I can see the points about these comments being made with hindsight, but quite a lot of people have been saying -- not only here, but one has heard comments on the radio and so forth -- you will be aware of what I am talkingabout -- They have been saying that, at the least, it suggested that more work needed to be done. Anyway, that is what you say in answer to that point. Okay. Right, Further Scientific Study, The Cattle Feeding Study.

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3. Pusztai responds to Aventis

This is in response to relevant parts (23.20 - 23.37) of the oral submission of Aventis Cropscience UK, containing new evidence, delivered by Herbert Smith to the Chardon LL Hearing concerning the controversy of the Aventis rat toxicity study.

Before considering the relevant technical parts of the new submission two general issues need to be dealt with in some detail.

First of these (in 23.21), before any scientific points could be dealt with, the submission had a reference to the Royal Society's dismissal of our GM potato work. However, in view of their own argument this seems rather pointless and certainly irrelevant.  According to them, "Dr Pusztai's analysis concerned potatoes, containing an experimental line still in laboratory development with potential insect-controlling properties and not a commercially available herbicide tolerant maize, so any comments would have limited relevance to CHARDON LL".  In view of this what could have been the reason for starting their rebuttal of my original submission with this reference to my potato work?  I expect, one can reasonably assume that the only reason could have been their hope of discrediting my criticism of their rat toxicity work by implying that because the Royal Society criticised my work, my criticism of Chardon LL must also be worthless.  I am afraid, this line of argument is not only illogical but it also lacks scientific merit.  Maybe to legalistic minds this argument appears to carry weight but in science we deal with facts and results and therefore we must concentrate on the scientific worth of the design of the Aventis experiments, the methods used and the conclusions reached.  In a similar vein, the fact, that in contrast to the Royal Society working group's meagre nutritional publication output, I published near to 300 peer-reviewed papers in top nutritional and biochemical journals is neither here or there, what counts is how well the Aventis rat toxicity study was done.

Second, following the argument from the first point leads us neatly to the Aventis' chief "scientific" argument, that is their study was a rat toxicity and not a nutritional study and therefore Dr Pusztai's nutritional experience is irrelevant.  The submission is full of what for the lack of a better word can be regarded as name-calling and unsupported statements, such as his "criticism is misplaced and results from Dr Pusztai's misreading the paper", or  "based upon a confused evaluation of the study" or "his interpretation of the data is not very thorough and suggests a lack of experience..." or "Dr Pusztai had a copy of the wrong report..." or Dr Pusztai appears to have missed the point..." or "The challenge again suggests a lack of relevant experience in terms of toxicology data".  I can only suggest that this submission was put together by someone who is used to personalizing scientific debates rather than concentrating on facts.  In contrast, my original review was factual and so will this reply be.

In the followings I shall deal with the points raised in the same order as in the Aventis' submission:

23.22. I am afraid, in the Aventis original submission the source of the PAT protein was not clearly specified.  I am rather glad that this has now been clarified so that what I suspected before has now become confirmed.  So I can now use my full and valid scientific criticism because as it is now stated: "The protein was actually generated in a microbial system (E. coli)...".  This may be an accepted practice for Aventis but as the PAT protein was not in fact isolated from the actual GM crop, all studies in which this recombinant was used cannot be accepted as valid, at least not without further validation experiments.  In evolutionary terms E. coli and crop plants are eons apart and therefore they deal differently with the emerging proteins synthesized on the ribosomes.  This is called post-translational modification (proteolytic processing, amidation, glycosylation, etc) and the scientific literature is full of data showing how different the two systems are.  If needed, I can provide some relevant references.   Moreover, in a review I commented on this business by saying: "Although testing of the PAT protein can be commended, this study is no substitute for the nutritional testing of the entire GM plant, seeds, vegetative parts and silage in all target animal species.  Without these the potentially harmful, unintended and unpredictable effects of the gene transfer, other components of the vector and gene insertion (positioning effects) cannot be established or excluded".

23.23. On the second point of design, the study was performed by one of the most well-  known and established Swiss contract laboratories..."   This is irrelevant!  What is important whether it was well-done or not and not who says so.  Nobody questions the credentials of this laboratory but in science we have something called peer-review in which other scientists subject the work to independent review and pass it or not for to be of sufficiently high standard for publication.  Clearly, the work of this laboratory has not gone through such a process and therefore Aventis has to resort to boost its credentials by making references and comments to standard OECD protocols and such.

23.24. The 14 days' feeding study for assessing the toxicity should have been satisfactory if all the protocols, designs and monitoring of the feeding experiment had been properly designed and executed.  In that case there would not be any need to refer to the opinion of other bodies, such as DEFRA.    This also raises the point to which the Aventis' submission keep coming back how the toxic effects were monitored by the Swiss laboratory?  See next para!

23.25. There are many comments in the submission that this is a toxicity and not a nutritional study.  Curiously, despite assertions to the contrary, most of the parameters measured in the study were nutritional.  Thus, measurements of feed intake (23.29), organ weights (23.30/31), body weight gains, food consumption again (23.33), urine output (23.35), etc were done by the Swiss laboratory.  And they were right as indeed, it is commonly agreed that changes in these parameters could be indicators of toxic effects.   Perhaps, the confusion was in the mind of the Aventis lawyers because scientists would know the difference between these two.  However, it would have been helpful if rather than reiterating that their study was toxicity and not nutritional had they defined in the submission what it was that  Aventis  called toxicity and what parameters they considered as truly reflecting this toxicity.  One is left with the impression that their toxicity meant rat death.    True, in LD50 tests by giving increasing doses of the PAT protein (or anything to be tested) to groups of rats (usually mice) to reach a concentration at which half of the experimental animals died is standard toxicological practice (not readily permitted by the UK Home Office).  However, nobody of right mind would even consider to use in food/feed a transgene coding for an acute poison and, I am sure, Aventis would not consider it either.   In any case, as the Swiss laboratory used recombinant PAT protein and not that isolated from the actual transgenic crop invalidates their conclusions of no toxic effects because the stability of the recombinant is different from the true transgenic PAT protein.  Indeed on p.53 (23.3) it was shown that the recombinant PAT protein is readily broken down in the gut and therefore it does not matter whether you increase the concentration of any food component by tenfold if it breaks down and cannot exert its potential effect.  The only valid test would have been carried out with the PAT protein isolated from Chardon LL.

23.26. I am afraid, I could only commented on what was presented to me and others in the Chardon LL enquiry.  Thus, any additional evidence by Aventis could not have been anticipated by myself or anybody else.

23.27.  They are a bit disingenious:  The comment was that it was difficult to draw valid conclusions from a feeding study carried out with 5 rats per group.  However, they left out the next part of the sentence: in which the starting weight of the rats varied by more than 20% each way (53 to 82 g for males and 50 to 74 g for females) rather than the usual 2%.  For any differences to reach significance they needed to exceed the 20% each way and to achieve this in a 14 day study should have required catastrophic experimental conditions.    Incidentally, the physiology of male and female rats is so different that they could not be regarded as a single group.  For all these reasons the number of rats used per group was not adequate!

23.28. This is an interesting admission by Aventis that I was indeed right that the rats in group 2 and 3 should have been compared to group 4 and not group 1 as it was given in the original Aventis submission.  At least it is satisfactory that now even Aventis realized that their original comparison was wrong as I suggested in my criticque.

23.29. This comment about that feed intake measurements in toxicology trials should be done only on 1,3,7,10 and 14 days is irrelevant.

23.30. This is a smear on the professionalism of the Swiss laboratory scientists who carried out the dissections that they could not reliably and reproducibly dissect the pancreas and the intestines from their experimental rats.  Of course histology is important but, I am afraid, a simple comment that the RCC study did not show any negative histopathology effects is not enough in itself.  One needs to be given all the experimental conditions, methods used and some results illustrated before on can be satisfied that everything was alright with the study.  Again, in science we need the evidence and not comforting and unsupported statements.

23.31. The large differences in the starting weight of the rats prevented finding of significant differences in organ weights which should have differed by more than 20% to show up such differences.

23.32. The simple fact is that the weight gain for both male and female rats on the high PAT protein diet (group 3;  65.2 for males and 43.6 g for females, respectively) was significantly less than that of either the control (group 4; 72.8 and 48.8 g for males and females, respectively) or group two (the lower PAT protein diet; 73.4 and 44.4 g for males and females respectively).  Anyone with simple arithmetic skills can calculate it for themselves.

23.33. The same as before no matter how authorative the statement may sound that there were no statistically or toxicologically significant differences in body weight gains of food consumption.

23.34. Again, their statement does not agree with the figures given in the original submission by Aventis.

23.35. Thus, Aventis does not deny that there is a significant difference in urine output between the groups but tries to give an explanation for a de facto difference

23.36. I am afraid, on the evidence presented by Aventis the DEFRA statement cannot be accepted that no adverse effects were observed in the rat toxicity study.

23.37. My original conclusion that the design and execution of this rat toxicity study by Aventis was poor and, contrary to the authors' conclusions, the results indicated treatment-related effects induced by the PAT protein.  These results therefore could not be taken as final evidence that the transfer its gene into maize represented no risk for the rat and, by inference, for humans.
 


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