If a “sign of toxicity” may only provoke a reaction, pathology or a poisoning, a so-called “toxic effect” is without doubt deleterious on a short or a long term. Clearly, the statistically significant effects observed here for all three GM maize varieties investigated are signs of toxicity rather than proofs of toxicity, and this is essentially for three reasons. Firstly, the feeding trials in each case have been conducted only once, and with only one mammalian species. The experiments clearly need to be repeated preferably with more than one species of animal. Secondly, the length of feeding was at most only three months, and thus only relatively acute and medium-term effects can be observed if any similar to what can be derived in a process such as carcinogenesis 19, 20
or after endocrine disruption in adults 21
. Proof of toxicity is hard to decide on the basis of these conditions. Longer-term (up to 2 years) feeding experiments are clearly justified and indeed necessary. This requirement is supported by the fact that cancer, nervous and immune system diseases, and even reproductive disorders for examples can become apparent only after one or two years of a given intervention treatment under investigation, but they will not be evident in all cases after three months of administration when first signs of toxicity may be observed 22, 23
. In addition, large effects (e.g. 40% increase in triglycerides) in all likelihood will be missed with the protocol of the current studies, since they are limited by the number of animals used in each feeding group and by the nature of the parameters studied. Thirdly, the statistical power of the tests conducted is low (30%) because the experimental design of Monsanto (see Materials and Methods). However, it is important to note that these short-term (3-month) rat feeding trials are the only tests conducted on the basis of which regulators determine whether these GM crop/food varieties are as safe to eat as conventional types. Given that these GM crops are potentially eaten by billions of people and animals world-wide, it is important to discuss whether the experimental design, the statistical analyses and interpretations originally undertaken are appropriate and sufficient.
Any differences observed in comparison with the isogenic variety, has to be taken into account as a potential physiological disruption. This is particularly valid since any statistically differences that are observed are highly unlikely to be arising from population variation as in the case of humans due to the genetic homogeneity of the rat strain used in these studies. Moreover, the standardized conditions of rat maintenance employed, which are stated to be in accordance with OECD standards 24, 25
, make the diet the only factor of variation in the protocol. Thus, the GM maize component of the test diet is the major factor of difference if one directly compares treated rats and controls. This is indicated by stars in the Tables expressing the total characteristics of GM-linked physio-pathological profiles. The other results that are encompassed by frames in the Tables highlight that effects from the GM maize are over and above those observed for any of the six different diets; for instance, over that observed with a diet richer in salt or sugar over the 3-month feeding period. These additional “control” diets could have been avoided with an experimental design that truly focused on the general question of GM toxicity.
The first observation that we were able to make was that there is a good general concordance between our data and the results of Monsanto as presented in their original confidential reports, in particular on the proportion of statistically significant observations. However, the methodology we employed revealed different effects, which completely changed the interpretation of the experimental results. For instance, the sex differences are fully taken into account in our study, which contrasts with the first published comments of these data 18, 26, 27
. We evaluated and took note of differences in the reaction of male and female rats to the GM maize test diets based on accepted and now classical knowledge of endocrinology 28
, embryology 29, 30
, physiology 31, 32
, enzymology or hepatology 33
demonstrating sex-specific physio-pathological effects. Indeed, our present results fully confirmed the sex-specific distribution of effects on kidney and liver parameters for all rats in all three studies analyzed here. An identical effect in both sexes would have been exceptional, like with strong or acute toxicity. This is obviously not the case here. In addition, we considered equally important effects that were neither time nor dose related, even if we detailed these when observed in the results. The proof for a linear dose dependency, as requested by Doull and coll. 4
to determine the significance of effects, is impossible with only two feeding points with no prior standardization. Furthermore, a metabolic reaction either physiological or pathological is not necessarily linear in its response 34, 35
. Again, this does not invalidate a description of effects appearing at the higher GM feed doses.
Even if the significant differences are around 5% of all comparisons for each GM corn, we believe that they either constitute a very good possibility to represent signs of toxicity, or at the very least should be considered as sufficiently strong evidence to justify a repeat of the experiments incorporating longer feeding times, for several reasons. Firstly, the arguments of Hammond and coll. 18, 26, 27
from Monsanto and Doull and co-workers 4
cannot demonstrate that the statistically significant GM-feed linked differences are not physiologically relevant 2
. Secondly, very few GM-feed effects appear only at the low dose or after the shortest (5 week) feeding period; 8.6% for NK 603, 6.6% for MON 810, 14.7% for MON 863 (Tables , , and ref. 5
). Thirdly, the marked sex difference effects observed for the GM maize feeding groups, in several instances, are found for physiological markers in all rats. Therefore, there is little probability that these effects were a random, chance occurrence. Fourthly, our stringent statistical tools allowed differentiation of GM-feed impacts from differences arising from variation in the composition of other reference diet. This is the first time that such an analysis has been conducted. Fifthly, there is a lack of cancer, hormonal or hepatic functional marker measurements (for example, oncogene expression, sex steroid hormone levels, cytochrome P450 levels), that could have provided explanatory insight into the results. The lack of availability of this type of data may be of benefit to those that doubt the current observations provide evidence of potential signs of toxicity. Sixthly, the physiological and biochemical parameters found to be disrupted in these feeding studies frequently provide a coherent, GM-specific picture of events, which corresponds and is in support of the generally admitted concept held by industry and regulators that GM crops and food should be considered on a case by case basis. Seventhly, several double-framed outcomes encompass all dietary effects only after the 3 month period of feeding. Last but not least, the most marked and most numerous effects are on organs involved in detoxification like the kidney and liver, usually reached after a diet-linked toxicity.
For instance in the NK 603 study statistically significant strong urine ionic disturbances and kidney markers imply renal leakage. This includes creatinine (increased urinary clearance), together with its diminution in the blood, and the decrease in urea nitrogen. Blood creatinine reduction has in some cases been found to be associated with muscle problems. It is therefore perhaps of note that the heart, as a very representative muscle organ was affected in the GM feeding groups. The possibility of renal porosity as evidenced by these data may be due to the presence of residues of Roundup herbicide, that are present in GM crop varieties such as the NK 603 maize investigated here. We have previously demonstrated that glyphosate-based herbicides such as Roundup are highly toxic at very low concentrations to human embryonic kidney cells 36
, inducing a decrease in viability, noticeably via inhibition of mitochondrial succinate dehydrogenase.
The deficiency in kidney function we highlight to be present in male rats is different between animals fed NK 603 and MON 863. The latter is characterized by an increase in plasma creatinine levels and retention of ions, which were associated with a chronic interstitial nephropathy, as originally admitted in the Monsanto MON 863 report and by Hammond and coll. 18
. However, this disturbance in kidney function was dismissed in their conclusions because the strain of rat used in the feeding studies is apparently sensitive to this type of pathology, especially during aging, which was not the case here. However, this reasoning was admitted by various regulatory authorities (EFSA, CGB in France). These arguments again appear flawed as the rats were still relatively young, 5 months by the end of the experimental period and therefore below the age when they might be expected to spontaneously develop kidney diseases. More importantly, these kidney effects are clearly MON 863-specific since they are not observed with all three GM maize varieties and the control groups, and therefore could not have arisen from an inherent genetic predisposition of the strain of rat used, which in addition was the same in all cases. Overall, no kidney parameters in male animals are disrupted in the MON 810 feeding group, even though sensitivity to toxics appears in general to be greater in this sex 37, 38
. An additional contributory factor to this disturbance in kidney function could arise from either novel unintended toxic effect caused by the inherent mutagenic effect of the GM technology, or possibly due to the new mutant forms of Bt toxin produced by MON 863, which is completely different from that engineered into MON 810. However, MON 810-fed females have a slight kidney weight enhancement, which may correspond with a mild hyperplasia usually seen in association with immune inflammatory processes. A re-evaluation of the histological slides from these animals would be of interest to test this hypothesis. Furthermore, analysis of some pertinent markers of kidney function such as arterial tension or angiotensin levels are lacking from these studies. This type of investigation including controls where animals are fed a normal diet spiked with the corresponding purified Bt toxin, would allow a more rational and precise interpretation of the results.
In the case of the MON 863 feeding trials, which have previously been discussed 5
and are at the center of a debate 2, 4
, new results have been obtained by the re-evaluation of the data with more powerful statistical methods as we present here. In female rats, there is a risk of becoming pre-occupied with the reactions already ascribed to the GM feeding group since several parameters indicate increases in circulating glucose and triglyceride levels, with liver function parameters disrupted together with a slight increase in total body weight 5
. This physiological state is indicative of a pre-diabetic profile. We demonstrate here that in female animals triglycerides profile, creatinine or urine chloride excretion are differentially and specifically altered over time in comparison to control groups, depending on the GMO dose. All these disruptions and differences taken together could be interpreted as clear signs of toxicity.
The effects found after only 5 weeks of feeding or at lower 11% feed dose, cannot be neglected simply on the basis that they are less frequently observed. Compensation or recuperation could occur after tissues are harmed, as possibly observed in the case of mice fed a diet containing Roundup Ready GM soy 39
. Peak inflammatory processes may occur in damaged tissues, followed by a regeneration phase as observed after bacteria/viral infection or a chemical toxic insult 40, 41
. For instance, urine potassium decreases in male rats over time in the GM MON 863 group at the 11% feed dose, which was not observed in all but one of the controls. This effect is specifically time-dependent and thus does not appear to be artefactual. This type of punctual regeneration may be part of a carcinogenic process, and clearly even if total recovery occurs, this should not be taken as a sign that the GM feed is safe.