Animal study: Genetically modified crops safety assessments: present limits and possible improvements

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Genetically modified crops safety assessments: present limits and

possible improvements

Gilles- Séralini1*, Robin Mesnage1, Emilie Clair1, Steeve Gress1,

Joël S de Vendômois2 and Dominique Cellier3

* Corresponding author: Gilles- Séralini criigen@...

Author Affiliations

1 Laboratory of Biochemistry - IBFA, University of Caen, Esplanade de la

Paix, 14032 Caen, Cedex, France

2 CRIIGEN, Paris, France

3 University of Rouen LITIS EA 4108, 76821 Mont-Saint-Aignan, France

For all author emails, please log on.

Environmental Sciences Europe 2011, 23:10 doi:10.1186/2190-4715-23-10

The electronic version of this article is the complete one and can be

found online at: http://www.enveurope.com/content/23/1/10

Received: 17 January 2011

Accepted: 1 March 2011

Published: 1 March 2011

This is an Open Access article distributed under the terms of the

Creative Commons Attribution License

(http://creativecommons.org/licenses/by/2.0), which permits unrestricted

use, distribution, and reproduction in any medium, provided the original

work is properly cited.

Abstract

Purpose

We reviewed 19 studies of mammals fed with commercialized genetically

modified soybean and maize which represent, per trait and plant, more

than 80% of all environmental genetically modified organisms (GMOs)

cultivated on a large scale, after they were modified to tolerate or

produce a pesticide. We have also obtained the raw data of 90-day-long

rat tests following court actions or official requests. The data

obtained include biochemical blood and urine parameters of mammals

eating GMOs with numerous organ weights and histopathology findings.

Methods

We have thoroughly reviewed these tests from a statistical and a

biological point of view. Some of these tests used controversial

protocols which are discussed and statistically significant results that

were considered as not being biologically meaningful by regulatory

authorities, thus raising the question of their interpretations.

Results

Several convergent data appear to indicate liver and kidney problems as

end points of GMO diet effects in the above-mentioned experiments. This

was confirmed by our meta-analysis of all the in vivo studies published,

which revealed that the kidneys were particularly affected,

concentrating 43.5% of all disrupted parameters in males, whereas the

liver was more specifically disrupted in females (30.8% of all disrupted

parameters).

Conclusions

The 90-day-long tests are insufficient to evaluate chronic toxicity, and

the signs highlighted in the kidneys and livers could be the onset of

chronic diseases. However, no minimal length for the tests is yet

obligatory for any of the GMOs cultivated on a large scale, and this is

socially unacceptable in terms of consumer health protection. We are

suggesting that the studies should be improved and prolonged, as well as

being made compulsory, and that the sexual hormones should be assessed

too, and moreover, reproductive and multigenerational studies ought to

be conducted too.

Background, aim, and scope

Recently, an ongoing debate on international regulation has been taking

place on the capacity to predict and avoid adverse effects on health and

the environment for new products and novel food/feed (GMOs, chemicals,

pesticides, nanoparticles, etc.). The health risk assessments are often,

but not always, based on the study of blood analyses of mammals eating

these products in subchronic tests, and more rarely in chronic tests. In

particular, in the case of GMOs, the number and nature of parameters

assessed, the length of the necessary tests, the statistics used and

their interpretations are the subject of controversies, especially in

the application of Organization of Economic ation and Development

(OECD) norms. Confusion is perceived even in regulatory agencies, as in

the European Food Safety Authority (EFSA) GMO panel working group and

its guidelines. Doubt has arisen on the role and necessity of animal

feeding trials in safety and nutritional assessments of GM plants and

derived food and feed [1]. Based on the literature data, EFSA first

admitted (p. S33) that for other tests than GMOs: " For 70% (57 of 81) of

the studies evaluated, all toxicological findings in the 2-year tests

were seen in or predicted by the 3-month subchronic tests " . Moreover,

they also indicated (p. S60) that " to detect effects on reproduction or

development [...] testing of the whole food and feed beyond a 90-day

rodent feeding study may be needed. " We fully agree with these

assumptions. This is why we think that in order to protect large

populations from unintended effects of novel food or feed, imported or

cultivated crops on a large scale, chronic 2-year and reproductive and

developmental tests are crucial. However, they have never been requested

by EFSA for commercial edible crops. We therefore wish to underline that

in contrast with the statements of EFSA, all commercialized GMOs have

indeed been released without such tests being carried out, and as it was

the case recently with maize stacked events without 90-day in vivo

mammalian tests being conducted. GM stacked events have the cumulated

characteristics of first generation of GMOs (herbicide tolerance and

insecticide production), which are mostly obtained by hybridization. For

instance, Smarstax maize contains two genes for herbicide tolerance and

six genes for insecticide production. In fact, this contradictory

possibility was already highlighted in the same review by EFSA (p. S60),

when substantial equivalence studies and other analyses were performed:

" animal feeding trials with rodents [...] adds little if anything [...],

and is not recommended. " This is why, in this work we will analyze and

review deficiencies in GMO safety assessments, not only performed by

biotech companies, but also by regulatory agencies.

We will focus on the results of available 90-day feeding trials (or

more) with commercialized GMOs, in the light of modern scientific

knowledge. We also suggest here an alternative to conventional feeding

trials, to understand the biological significance of statistical

differences. This approach will make it possible to avoid both false

negative and false positive results in order to improve safety

assessments of agricultural GMOs before their commercialization for

cultivation and food/feed use and imports.

Overview of the safety studies of GMOs performed on mammals

Our experience in scientific committees for the assessment of

environmental and health risks of GMOs and in biological, biostatistical

research, and medicine, as well as in the research relative to side

effects [2-6] allowed us to review and criticize mammalian feeding

trials with GMOs and make new proposals. Mammalian feeding trials have

been usually but not always performed for regulatory purposes in order

to obtain authorizations or commercialization for GM plant-derived foods

or feed. They may have been published in the scientific literature

afterwards; however, without public access to the raw data.

We have obtained, following court actions or official requests, the raw

data of several 28- or 90-day-long safety tests carried out on rats. The

thing we did was to thoroughly review the longest tests from both a

biostatistical and a biological point of view. Such studies often

analyze the biochemical blood and urine parameters of mammals eating

GMOs, together with numerous organ weights and histopathology. We have

focused our review on commercialized GMOs which have been cultivated in

significant amounts throughout the world since 1994 (Table 1). We

observe and emphasize that all the events in Table 1 correspond to

soybean and maize which constitute 83% of the commercialized GMOs,

whilst other GMOs not displayed in the table, but still commercialized,

are canola or cotton. However, they are not usually directly consumed

[7]. Only Sakamoto's and Malatesta's studies have been more than 90 days

long (104 weeks and 240 days with blood analyses in Japanese for the

first one). Moreover, such tests are not obligatory yet for all GMOs. No

detailed blood analysis is available for Malatesta's study, as it mostly

includes histochemistry at the ultrastructural level; moreover, the

latter tests have not been used to obtain the commercial release by the

firm. However, this work has been performed by researchers independent

from the GMO industry; it is an important element to take into account

for an objective interpretation of the facts, as pointed out in the case

of the risk assessments conducted by regulatory agencies with Bisphenol

A. For instance in the latter case, it was observed that none of the

industry-funded studies showed adverse effects of Bisphenol A, whereas

90% of government-funded studies showed hazards at various levels and

various doses [8]. However, regulatory agencies still continue to refer

only to industry-funded studies because they are supposed to follow OECD

norms, even if such standards are not always appropriate for the

detection of environmental hazards [9]. In this paper, Myers et al.

showed that hundreds of laboratory animals and cell culture studies were

rejected by regulatory authorities because they did not follow the Good

Laboratory Practices (GLP). The Food and Drug Administration and EFSA

have based their final decision on two industry-funded studies, claiming

that they were superior to the others because they followed GLP. Yet,

GLP are based on ancient paradigms. They have serious conceptual and

methodological flaws, and do not take into account the latest knowledge

in environmental sciences. For example, in the case of Bisphenol A

assessment, the animal models used are known to be insensitive to

estrogen (CD-1 mouse). Also, assays and protocols in some OECD

guidelines are out of date and insensitive. It is obvious that new

product assessments should be based on adapted studies using

state-of-the-art experiments. The significant gap between scientific

knowledge and regulations should be filled also in the case of GMOs [9].

Therefore, some tests presented here show controversial results or

statistically significant results that were not considered as

biologically significant by EFSA, raising the question of their

interpretation.

Table 1. Review of the longest chronic or subchronic toxicity studies in

mammals fed with commercialized GM soybean and maize representing more

than 80% of edible GMOs (2010).

First of all, the data indicating no biological significance of

statistical effects in comparison to controls have been published mostly

by companies from 2004 onwards, and at least 10 years after these GMOs

were first commercialized round the world. This is a matter of grave

concern. Moreover, only three events were tested for more than 90-days

in feeding experiments or on more than one generation. This method was

not performed by industries which conducted 90-day tests (with blood and

organ analyses), but it was in some cases only. However, a 90-day period

is considered as insufficient to evaluate chronic toxicity [1,5]. All

these commercialized cultivated GMOs have been modified to contain

pesticides, either through herbicide tolerance or by producing

insecticides, or both, and could therefore be considered as " pesticide

plants. " Almost all GMOs only encode these two traits despite claims of

numerous other traits. For instance, Roundup ready crops have been

modified in order to become insensitive to glyphosate. This chemical

together with adjuvants in formulations constitutes a potent herbicide.

It has been used for many years as a weed killer by blocking aromatic

amino acid synthesis by inhibition of 5-enolpyruvylshikimate-3-phosphate

synthase (EPSPS). Most Roundup ready plants have been modified thanks to

the insertion of a mutated EPSPS gene coding for a mutated enzyme, which

is not inhibited by glyphosate. Therefore, GM plants exposed to

glyphosate-based herbicides such as Roundup do not specifically degrade

glyphosate. They can even accumulate Roundup residues throughout their

life, even if they excrete most of such residues. Glyphosate and its

main metabolite AMPA (with its own toxicity) are found in GMOs on a

regular and regulatory basis [10,11]. Therefore, such residues are

absorbed by people eating most GM plants (as around 80% of these plants

are Roundup tolerant). On the other hand, about 20% of the other GMOs do

synthesize new insecticide proteins through the insertion of mutated

genes derived from Bacillus thuringiensis (Bt).

Usually, pesticides are tested over a period of 2 years on a mammal, and

this quite often highlights side effects. Additionally, unintended

effects of the genetic modification itself cannot be excluded, as direct

or indirect consequences of insertional mutagenesis, creating possible

unintended metabolic effects. For instance, in the MON810 maize, the

insertion of the transgene in the ubiquitine ligase gene caused a

complex recombination event, leading to the synthesis of new RNA

products encoding unknown proteins [12]. Thus, genetic modifications can

induce global changes in the genomic, transcriptomic, proteomic, or

metabolomic profiles of the host. The frequency of such events in

comparison to classical hybridization is by nature unpredictable. In

addition, in a plant producing a Cry1Ab-modified toxin, a metabolomic

study [13] revealed that the transgene introduced indirectly 50% changes

in osmolytes and branched amino acids.

Review of statistical effects after GMO consumption

Some GMOs (Roundup tolerant and MON863) affect the body weight increase

at least in one sex [2,14]. It is a parameter considered as a very good

predictor of side effects in various organs. Several convergent factors

appear to indicate liver and kidney problems as end points of GMO diet

effects in these experiments [2,5,15,16]. This was confirmed by our

meta-analysis of all in vivo studies published on this particular topic

(Table 2). The kidneys are particularly affected, concentrating 42% of

all parameters disrupted in males. However, other organs may be affected

too, such as the heart and spleen, or blood cells [5].

Table 2. Meta-analysis of statistical differences with appropriate

controls in feeding trials

Liver parameters

For one of the longest independent tests performed, a GM

herbicide-tolerant soybean available on the market was used to feed

mice. It caused the development of irregular hepatocyte nuclei, more

nuclear pores, numerous small fibrillar centers, and abundant dense

fibrillar components, indicating increased metabolic rates [17]. It was

hypothesized that the herbicide residues could be responsible for that

because this particular GM plant can absorb the chemicals to which it

was rendered tolerant. Such chemicals may be involved in the

above-mentioned pathological features. This became even clearer when

Roundup residues provoked similar features in rat hepatic cells directly

in vitro [18]. The reversibility observed in some instances for these

parameters in vivo [19] might be explained by the heterogeneity of the

herbicide residues in the feed [20]. Anyway, these are specific

parameters of ultrastructural dysfunction, and the relevance is clear.

The liver is reacting. The Roundup residues have been also shown to be

toxic for human placental, embryonic, and umbilical cord cells [21-23].

This was also the case for hepatic human cell lines in a comparable

manner, inducing nuclei and membrane changes, apoptosis and necrosis [24].

The other major GMO trait has to do with the mutated (mBt) insecticidal

peptidic toxins produced by transgenes in plants. In this case, some

studies with maize confirmed histopathological changes in the liver and

the kidneys of rats after GM feed consumption. Such changes consist in

congestion, cell nucleus border changes, and severe granular

degeneration in the liver [16]. Similarly, in the MON810 studies, a

significantly lower albumin/globulin ratio indicated a change in hepatic

metabolism of 33% of GM-fed male rats (according to EFSA opinion on

MON810 and [5]). Taken together, the results indicate potential adverse

effects in hepatic metabolism. The insecticide produced by MON810 could

also induce liver reactions, like many other pesticides. Of course, the

mCry1Ab and other mBt (mutated Bt toxins derived from native Bacillus

thuringiensis toxins) in GMOs are proteic toxins; however, these are

modified at the level of their amino acid sequence by biotechnologies

and introduced by artificial vectors, thus these could be considered as

xenobiotics (i.e., a molecule foreign to life). The liver together with

the kidneys are the major reactive organs in case of food chronic

intoxication.

Kidney parameters

In the NK603 study, statistically significant strong urine ionic

disturbances and kidney markers could be explained by renal leakage [5],

which is well correlated with the effects of glyphosate-based herbicides

(like Roundup) observed on embryonic kidney cells [23]. This does not

exclude metabolic effects indirectly due to insertional mutagenesis

linked to the plant transformation. Roundup adjuvants even stabilize

glyphosate and allow its penetration into cells, which in turn inhibit

estrogen synthesis as a side effect, cytochrome P450 aromatase

inhibition [21]. This phenomenon changes the androgen/estrogen ratio and

may at least, in part, explain differential impacts in both sexes.

Kidney dysfunctions are observed with mBt maize producing mutated

insecticides such as in MON863. For instance, we quote the initial EFSA

report: " Individual kidney weights of male rats fed with the 33% MON863

diet were statistically significantly lower compared to those of animals

on control diets " , " small increases in the incidences of focal

inflammation and tubular regenerative changes in the kidneys of 33%

MON863 males. " This was confirmed by the company tests [25] and another

counter analysis revealed disrupted biochemical markers typical of

kidney filtration or function problems [2]. The first effects were not

always but sometimes greater than the ones with non-isogenic maize

(called reference lines), which contain different salts, lipids, or

sugars. Moreover, both results described are different between males and

females; this is quite usual in liver or kidney pesticide reactions.

These facts do not exclude that such effects can be considered as

treatment-related. Other studies also confirmed effects on kidneys.

Tubular degeneration and not statistically significant enlargement in

parietal layer of Bowman's capsules were also observed with GM maize fed

rats [16].

Last but not least, a total of around 9% of parameters were disrupted in

a meta-analysis (Table 2). This is twice as much as what could be

obtained by chance only (generally considered as 5%). Surprisingly,

43.5% of significant different parameters were concentrated in male

kidneys for all commercialized GMOs, even if only around 25% of the

total parameters measured were kidney-related. If the differences had

been distributed by chance in the organs, not significantly more than

25% differences would have been found in the kidney. Even if our own

counter analysis is removed from the calculation, showing numerous

kidney dysfunctions [2], around 32% of disturbances are still noticed in

kidneys.

Discussion

Need for chronic tests and other tests

Chronic toxicity tests (both with males and females) and reproductive

tests with pregnant females and then with the developing progeny over

several generations (none of these steps exist at present) are called as

a whole the Toxotest approach (or Risk management test, see " Details on

the new suggested Toxotest approach " ). This could address the long-term

physiological or pathological relevance of the previous observations.

The physiological interpretations of 90-day-based effects are otherwise

somewhat limited. These studies should be complementary to the present

regulations or the Safotest and the sentinel test suggested by EFSA [1].

The Toxotest could provide evidence of carcinogenic, developmental,

hormonal, neural, and reproductive potential dysfunctions, as it does

for pesticides or drugs. Additionally, it is obvious that the

90-day-long trials on mature animals performed today cannot

scientifically replace the sensitivity of developmental tests on

neonates. A good example is the gene imprinting by drugs that will be

revealed only at maturity; this is an important subject of current

research, and many findings have been reported for some chemicals such

as bisphenol A [26,27]. Even transgenerational effects occur after

epigenetic imprinting by a pesticide [28]. These effects cannot be

detected by classical 90-day feeding trials and will be visible after

many decades by epidemiology in humans if any, as illustrated in the

case of diethylstilbestrol, which induced female genital cancers among

other problems in the second generation [29]. The F3 multigenerational

study for a GMO (Table 1) was too rarely performed. This is why, because

of the number of parameters disrupted in adult mammals within 90 days,

the new experiments should be systematically performed to protect the

health of billions of people that could consume directly or indirectly

these transformed products.

The acute toxicity approach (less than a month of investigations on

rodents with high doses) may give effects which are more proportional to

the dose, as it might correspond to a rapid poisoning of the animals,

generally with force-fed experiments. However, for many pesticide

studies in the scientific literature, some long-term side effects of

pesticides at environmental doses are described, which are not apparent

in short-term experiments [30]. Classical toxicology is quite often

based on the concept of revealing linear dose-responses as defined by

Paracelsus, which generally fails to evidence U or J curves observed

after hormonal sex-specific disruptions. Moreover, the effects of

mixtures are also neglected in long-term studies, when supposed active

principles of pesticides are not assessed with their adjuvants, which

also are present as residues in GMOs. Such pesticides may have the

capacity to disrupt the " cell web " , i.e., to interfere with a signaling

pathway, and this could be unspecific. For instance Roundup is known to

disrupt the EPSPS in plants, but is also known to interact with the

mammalian ubiquist reductase [21] common and essential to cytochromes

P450, a wide class of detoxification enzymes. The so-called Roundup

active principle, glyphosate, acts in combination with adjuvants to

increase glyphosate-mediated toxicity[21,31], and this may apply to

other environmental pollutants [22]. Moreover, all new metabolites in

edible Roundup ready GMOs, as acetyl-glyphosate for the new GAT GMOs,

have not been assessed for their chronic toxicity [11], and we consider

this as a major oversight in the present regulations.

Therefore, as xenobiotic effects are complex, the determination of their

toxic effects cannot be determined using a single method, but rather

converging pieces of evidence. In GMO risk assessment, the protocols

must be optimized to detect side effects, in particular for

herbicide-treated GM plants. These cannot be reduced to GM assessment on

one side and herbicide residues with any diet on the other side, but

unfortunately this has been the case, and this approach has been

promoted up to now by regulatory authorities.

In fact, it is impossible, within only 13 weeks, to conclude about the

kind of pathology that could be induced by pesticide GMOs and whether it

is a major pathology or a minor one. It is therefore necessary to

prolong the tests, as suggested by EFSA, since at least one third of

chronic effects visible with chemicals are usually new in comparison to

the ones highlighted in subchronic studies [1]. The so-called Toxotests,

which are supposed to include the studies of chronic pathologies in

particular, should be performed on three mammalian species, with at

least one non-rodent, similar to the type of rodents used for pesticides

and drugs. However, the chronic feeding tests for GMOs cannot be based

on the no observed adverse effect level, nor on the lowest observed

adverse effect level approach, as in classical toxicology. There are

several reasons for that. There is not only one chemical, but also

several unknown metabolites and components, in Roundup tolerant

varieties for instance, and therefore toxicity is enhanced thanks to the

fact that they are mixed together. There is also no possibility of

increasing the doses of GMOs in an equilibrated diet over an acceptable

level. The diets should be rather representative of an equilibrated diet

with GMOs like it could be the case in a real population in America. To

prolong 90-day subchronic tests with three normal doses of GM in the

diet (11%, 22%, 33% for instance) is the solution.

Sex- or dose-specific pathological effects are common

When there is a low or environmental dose impregnation of the feed (with

a pesticide GM plant for instance), the chronic effects could be more

differentiated according to the sex, the physiological status, the age,

or the number of intakes over such and such a period of time in the case

of a drug. These parameters (chronic intake, age of exposure, etc.) are

more decisive for pathologies like cancers, than the actual quantity of

toxin ingested in one intake. This is in part because the liver, kidney,

and other cytochrome P450-rich organs are concerned for long-term

metabolism and detoxification, and this phenomenon is hormone dependent.

It is also due to the process of carcinogenesis or hormone-sensitive

programming of cells [32]. The liver for instance is a sex

differentiated organ as far as its enzymatic equipment is concerned [4].

An effect in subchronic or chronic tests cannot be disregarded on the

rationale that it is not linear to the dose (or dose-related) or not

comparable in genders. This would not be scientifically acceptable.

However, this reasoning was adopted both by companies and EFSA for

several GMOs, as underlined by Doull et al. [33]. Indeed, most

xenobiotics or pollutants may have non-linear effects, and/or may have

sex- and age-specific impacts.

One of the pivotal requirements for regulators nowadays, in order to

interpret a significant difference as biologically relevant, is to

observe a linear dose-response. This allows them to deduce a causality.

However, this dose-response cannot be studied with only two points,

which is nonetheless the case for all major commercial GMOs today, which

are given in the diet in 11% and 33% concentrations only, in subchronic

tests. This is true overall if no preliminary data has been obtained to

choose the given doses, which is the case in regulatory files. As we

have already emphasized, most of pathological and endocrine effects in

environmental health are not directly proportional to the dose, and they

have a differential threshold of sensitivity in both sexes [34]. This

is, for instance, the case with carcinogenesis and endocrine disruption.

Improving the knowledge on impacts of modified Bt toxins

One of the interpretations of the side effects observed (Tables 1 and 2)

would be that the insecticide toxins in maize lines may have more

pleiotropic or specific actions than originally supposed. The toxins

could generate particular metabolites, either in the GM plant or in the

animals fed with it. The Bt toxins in GMOs are new and modified,

truncated, or chimerical in order to change their activities/solubility

in comparison to wild Bt. For instance, there is at least a 40%

difference between the toxin in Bt176 and its wild counterpart [10].

None of the modified Bt toxins have been authorized separately for food

or feed, neither has the wild Bt, and neither have they been tested by

themselves on animal or human health to date. Even if some studies were

performed, the receptors have not been cloned and the signaling pathways

have not been identified as yet, nor required for authorizations, and

the metabolism of these proteins in mammals are unknown [35]. Thus, the

argument about " safe use history " of the wild Bt protein (not designed

for direct consumption, in contrast to several GMOs) cannot, on a sound

scientific basis, be used for direct authorizations of the above-cited

GM corns, overall without in vivo chronic toxicity tests (or Toxotest

approach), as it is requested for a pesticide. Some improvements may

even be included with regard to pesticide legislation, since these human

modified toxins considered as xenobiotics are continuously produced by

the plants devoted to consumption.

The proteins usually compared (modified Bt toxins and wild ones) are not

identical, and the tests on human cells of Bt proteins are not performed

nor are they requested by authorities. Their stability has been assessed

in vitro, and GM insecticide toxins are never fully digested in vivo

[36]. If some consumers suffer from stomach problems or ulcers, the new

toxins will possibly act differently; the digestion in children could be

affected too; however, these GMOs could be eaten anywhere and all

proteins are never fully decomposed in amino acids by the digestive tract.

Details on the new suggested Toxotest approach

The suggested Toxotest would basically include an extension of the

existing 90-day tests, but with at least three doses plus controls (0%,

11%, 22%, 33% GMOs for instance; today the equilibrated diets tested

contain 0%, 11%, and 33% GMOs in the best regulatory tests). The purpose

would be to characterize scientifically the dose-response approach. The

latter cannot be taken seriously with only two GM doses. The final goal

is the best health protection for the population without really possible

clinical trials, in our case for practical and ethical reasons. There is

also no epidemiological follow-up for lack of traceability and labeling

in GM-producing American countries. In addition, the fact that the

Toxotest includes the best possible toxicological approach will also be

in favor of the biotechnology economy and the European Community because

it is more expensive to address an issue concerning a whole population

afterwards, rather than to work with laboratory animals beforehand; it

is also more ethical to work on rats and other mammalian experiments, in

order to get the relevant information, rather than to give pesticide

plants directly to humans on a long-term basis.

As previously underlined, the health effects such as those suggested in

Table 2 (if any, are revealed by adapted studies, such as Safotests or

Toxotests), could only be due to two possibilities:

Firstly, the side effects may be directly or indirectly due to a

pesticide residue and/or its metabolites. The direct effect is about the

pesticide effect on the consumer, and the indirect one is about a

metabolism disruption that it has provoked within the plant first. This

could not be visible by a detailed compositional analysis, such as the

one performed to be assessed by a substantial equivalence study. This

concept is not a well-defined one (how many cultivations of crops, over

how many years, under which climate, and to measure what precise

parameters).

Secondly, the pathological signs may be due to the genetic

transformation itself, its method provoking either insertional

mutagenesis or a new metabolism by genetic interference. This is the

reason why separating intended effects (the direct genetic trait

consequence itself) from unintended effects (linked to biotechnology,

e.g., insertional mutagenesis), such as spiking the control diet with

the purified toxin in the Toxotest approach, is clearly inadequate. It

could work in the case of a direct action of the toxin in mammals, but

conversely one could not conclude, between an insertional mutagenesis

and a specific metabolic action in the plant due to the toxin. However,

this is more a research question about the mode of genesis of an effect

on health, and new research avenues could be, for instance, to compare

the GM diet with or without herbicide treatment in long-term tests with

the isogenic control diet including herbicide residues added. This is

only necessary for the understanding of the potential signs of toxicity

and not for a conclusion of the Safotest or the Toxotest, which would

rather suggest, if positive, excluding immediately the corresponding GMO

from food and feed.

Improvement of statistical analysis

A serious experimental design is based on a proper choice of the groups,

with only one question studied per experiment if possible, and balanced

sample sizes. In several authorized GMOs, the sample sizes appear

inadequate in 90 days: ten animals per group for the measurement of

biochemical parameters out of 20, as performed by the major

stakeholders, and accepted by EFSA for MON863, MON810, or NK603 for

instance. This is too limited a size to ensure that parametric

statistical methods used by the company are reliable. Moreover, an

important discrepancy between GMO-treated rats (40 measured out of 80)

and the total number of animals (400) renders more difficult the

evidencing of relevant effects, and confusion factors are brought in at

the same time with six different reference diets in addition to the two

normal control groups as performed in three commercialized GMOs at least

[5,6]. This introduces new uncontrolled sources of variability about the

effects of the diets and new unnecessary questions not relevant to the

GMO safety. The representation of a standard diet with multiple sources

could have been studied with only one control group of the same size

than the GMO group, eating a mix of six different regular non-GM diets.

Several questions have been raised by companies and authorities as well

as comments on statistically significant effects that would supposedly

not be biologically meaningful. A subjective part is introduced at this

level because it is necessary to take into account the context and the

general and detailed knowledge of toxicology and endocrine disruption,

as EFSA underlines. This might be highly expert dependent. This is why,

to avoid or prevent any misunderstanding, we suggest, in addition to a

new statistical approach based on classical methods, to analyze the

90-day tests, even with control and reference diets called the " SSC

method " (according to the initials of the authors in [2]).

Briefly, following the necessity to model and analyze the growth curves,

multivariate data analysis and data mining of all parameters can be used

to correlate, cluster, and select meaningful variables. This kind of

approach is not performed at all today. Thereafter, the detailed

comparison between GM-treated and control groups, fed with the near

isogenic line (because the real isogenic line does not often exists

anymore), will necessarily be followed by the study of specific diet

effects, when there are non-substantially equivalent diets for reference

groups. For that purpose, the controls will be first compared using

multivariate inference with reference groups, and thereafter, similarly

GMO-treated groups with reference groups. The significant differences

linked to the GMO and/or the composition of the diet will be classified

according to organ and function. The results will appear more clearly

than with the simple statistics accepted today by the authorities (that

is, comparison of the highest GM dose group with the mean value of all

six control groups), and will reveal in addition new information, as it

can be demonstrated.

As recommended by EFSA, an appropriate and relevant statistical analysis

is crucial. It should follow the following series of steps, allowing the

use of several methods depending on the questions raised:

• Obtaining and modeling the growth curves and feed consumption,

assessed by non-linear regression, validation, and statistical

comparisons in order to test if the curves are significantly different,

thus taking into account individual variability. This necessitates the

use of time series analysis, selection models, and non-parametric tests,

Akaike Information Criteria and related methods. Water consumption

should also be an important factor to follow-up and therefore better

understand kidney and urine data.

• The study of dose-response predictions using non-linear regression

should be the goal, but the only two doses generally used in these tests

do not make it possible to evidence linearity as we indicated. Moreover,

in the cases where there are not dose-related trends or relationships

using the two doses mentioned, the absence of linear dose-response

curves cannot be a reason to neglect the effects. For instance, as

previously cited, U or J curves may be characteristic of endocrine

effects [37], and spiky irregular curves may be detected in carcinogenesis.

• Simultaneous analysis of all observed variables: multivariate data

analysis, principal component analysis, correlations analysis, factorial

analysis and clustering

• Multivariate comparisons of the different variables: hypothesis

testing, multiple ways ANOVA, MANOVA, and others to determinate if the

groups differ relative to the different questions: specific GMO effect

or diet effect per se. To evidence a detail, when comparing two mean

values, SEM should be calculated to determine confidence intervals;

however, SD have been used up to now by the company for MON863 and NK603

files for instance.

Apart from empirical curves in some instances, ANOVA and univariate

hypothesis testing only the GMO effect, none of the other statistical

approaches is currently used nor requested by the authorities.

Human tests and post-market monitoring

For the record, it must be said that very few tests on humans have been

carried out up to now. Moreover, epidemiological studies are not

feasible in America, since there is no organized traceability of GMOs

anywhere on the continent, where, by far, most of edible GMOs are

cultivated (97%). As a consequence, a post-market monitoring (PMM) is

offered to the population. The Cartagena Biosafety Protocol identifying

GMOs at the borders of a country has now been signed by over 150

countries, including the member states of the European Union. PMM may

have some value in detecting unexpected adverse effects. It could

therefore be considered as a routine need. This approach makes it

possible to collect information related to risk management. It can be

relied upon as a technique for monitoring adverse events or other health

outcomes related to the consumption of GM plant-derived foods, provided

that the Toxotest approach, together with the SSC method, should have

already been applied. The PMM should be linked with the possibility of

detecting allergenicity reactions to GMOs in routine medicine, thanks to

the very same routine cutaneous tests that should be developed prior to

large-scale commercialization. A screening of serum banks of patients

with allergies could be also put forward in order to search for

antibodies against the main GMOs and not only their transgenic proteins,

since they may induce secondary allergenic metabolites in the plant not

visible in the substantial equivalence study.

The traceability of products from animals fed on GMOs is also crucial.

The reason for this is because they can develop chronic diseases which

are not utterly known today. Such possible diseases could be linked to

the hepatorenal toxicity observed in some GMO-related cases (Table 1).

Moreover, labeling animals fed on GMOs is therefore necessary because

some pesticide residues linked to GMOs could pass into the food chain

and also because nobody would want to eat disabled or physiologically

modified animals after long-term GMOs ingestion, even if pesticides

residues or DNA fragments are not toxic nor transmitted by themselves.

Conclusion

Transcriptomics, proteomics and other related methods are not ready yet

for routine use in the laboratories, and moreover they may be

inappropriate for studying toxicity in animals, and could not in any way

replace in vivo studies with all the physiological and biochemical

parameters that are measured with organs weight, appearance, and

histology. By contrast, afterwards, new approaches could well help to

explain pathological results or action mechanisms of pesticides present

in the GM plants or GM-fed animals, if found.

To obtain the transparency of raw data (including rat blood analyses)

for toxicological tests, maintained illegally confidential, is crucial.

It has also become crucial to apply objective criteria of interpretation

like the criteria described here: sex-specific side effects or

non-linear ones. Such data can be put online on the EFSA website with a

view to provide a fuller review to the wider scientific community, and

in order to better inform the citizen to make biotechnologies more

socially acceptable. Since fundamental research is published on a

regular basis, it should be the same for this kind of applied research

on long-term health effects, as suggested by the CE/2001/18 and the

corresponding 1829/2003 regulations.

We can conclude, from the regulatory tests performed today, that it is

unacceptable to submit 500 million Europeans and several billions of

consumers worldwide to the new pesticide GM-derived foods or feed, this

being done without more controls (if any) than the only 3-month-long

toxicological tests and using only one mammalian species, especially

since there is growing evidence of concern (Tables 1 and 2). This is why

we propose to improve the protocol of the 90-day studies to 2-year

studies with mature rats, using the Toxotest approach, which should be

rendered obligatory, and including sexual hormones assessment too. The

reproductive, developmental, and transgenerational studies should also

be performed. The new SSC statistical method of analysis is proposed in

addition. This should not be optional if the plant is designed to

contain a pesticide (as it is the case for more than 99% of cultivated

commercialized GMOs), whilst for others, depending on the inserted

trait, a case-by-case approach in the method to study toxicity will be

necessary.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

GES designed and coordinated the review. RM participated in the drafting

of the manuscript and final version. EC, SG, JSV and DC helped the

writing, compiling the literature, revising in details and proofreading

the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank the CRIIGEN scientific committee for helpful discussions and

structural support, as well as the Risk Pole (MRSH-CNRS, University of

Caen, France). We acknowledge the French Ministry of Research for

financial support and the Regional Council of Basse-Normandie. We are

grateful to Herrade Hemmerdinger for the English revision of this

manuscript.

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