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expert reaction to study investigating content of and contaminants in laboratory rodent diets

The diets of rodents in food safety and toxicology studies is the topic of a paper published in the journal PLOS ONE, in which the authors report the presence of contaminants including heavy metals, pesticides, and genetically modified organisms in the feed or lab rodents. The authors go on to suggest that such contaminants could affect the reliability of toxicity testing in general.

 

Commenting on the GMO aspect of the study, Prof Cathie Martin, Group Leader, John Innes Centre, said:

“The conclusions of Seralini et al. in this current manuscript are highly speculative. For their interpretations to be compelling, Seralini et al. would need to do feeding experiments with ‘non-contaminated chow’ and show that there is a significantly lower level of cancer incidence in those animals on the non-contaminated chow (using an appropriate number of animals, over an appropriate amount of time) – this study has not done that.

“Seralini et al conclude that one cannot trust historical control data, but the tumour incidence of Sprague Dawley rats was published several decades ago (CANCER RESEARCH 33, 2768-2773, November 1973), when the contamination values for rodent chow were likely to be very different. Reports of the high cancer incidence of Sprague-Dawley rats were made long before the advent of GMO crops, so it cannot be concluded that GMOs are the cause of the high tumour incidence. The high tumour incidence in Sprague Dawley rats, which underpins the recommendation that they should not be used for studies beyond 18 months old, is most likely their high level of inbreeding.

“It is strange that the conclusion of Seralini et al. is that one cannot trust any experiments done with ‘control’ rats because of dietary contaminants. A more reasonable conclusion would be that no one can trust data purporting to show that glyphosate and glyphosate resistant corn cause tumors in Sprague-Dawley rats, when they have a high inherent incidence of tumors in animals older than the recommended 18 months (whatever the cause).”

 

Prof. Andy Smith, Senior Scientist at the MRC Toxicology Unit, University of Leicester, said:

“The possible low level contamination of rodent diets and bedding has been recognised for decades in toxicological investigations but the data in the study of Mesnage and colleagues are useful additions to our background knowledge. With all the controls used, including historical controls, any such ’contaminants’ are unlikely to affect conclusions drawn from most toxicity tests in rats. In addition, it is very well established over many years that different strains of rats and mice develop particular diseases over their life, just as some people are more likely than others to develop some types of cancer. In contrast to the suggestions of the authors, the use of this reproducible genetic variation, seen in historical controls and independent of diet, rather than being detrimental, has been exploited to greatly help our understanding of the risks to humans from the use of chemicals and consumption of novel foods.”

 

Prof. Tamara Galloway, Professor of Ecotoxicology, University of Exeter, said:

“This is an interesting study that sets out to determine whether food chow intended for laboratory rodents is contaminated with residues of pesticides, metals and/or industrial chemicals. The chemical analysis has been performed by accredited laboratories and appears robust and of good quality. The authors found that a number of the test diets contained residues of pesticides, metals and industrial chemicals, and this is perhaps unsurprising given their widespread use, but it is reassuring to see that the concentrations of these compounds are below regulatory limits.

“In the paper, the authors have calculated a hazard quotient, which they then compare with acceptable daily intakes (ADI) calculated for human consumption. Since the ADI is calculated using a safety factor of 100, a direct comparison of the two estimates could give the wrong impression, since the safety factor is not taken into account.

“The authors do not measure the biological or health effects of the food and hence it is not possible to say anything from the paper on the likelihood that food contamination might have a role to play in the high incidence of health problems in laboratory rodents, as speculated by the authors. The discussion speculates beyond the evidence presented in the paper.”

 

Prof. Alan Boobis, Professor of Biochemical Pharmacology, Imperial College London, said:

“The paper of Mesnage et al provides a useful survey of the levels of a number of contaminants present in the feed of laboratory rodents.  However, I believe the title is potentially misleading in the use of the term “toxic levels”. Firstly, in general the levels of individual contaminants are extremely low. Note that a hazard quotient (HQ) of 1 would represent exposure at 1% of the critical no observed adverse effect level (NOAL).  Secondly, it is assumed that these low levels would all exhibit dose additivity (HQs were summed), regardless of mode of action or potential site of effect.  This is contrary to the approach that EFSA, and others, recommend for risk assessment of combined exposures.  However, even in this situation, the summed HQs are in all cases less than 100, indicating that total exposure is below the NOAEL. No information is provided on whether there is any relationship between the level of contamination found and background rates of pathology in the rodent populations.  However, such information would only be indicative, and to verify the conclusions of the authors, a specific intervention study would be necessary, using diets with a range of verified levels of contaminants. The use of laboratory animals for this purpose would be questionable.”

 

Dr Nick Plant, Reader in Molecular Toxicology, University of Surrey, said:

“A comprehensive assessment of potential contaminants in animal chow is welcome, and this study has been undertaken in a robust manner by nationally accredited laboratories. It is not unsurprising that some level of contamination exists given what is known about agricultural practices worldwide, and indeed it is pleasing to see that in the main the levels are only just above the limit of detection and below the regulatory levels. As such, this work is reassuring.

“The authors’ comment that such contamination may contribute to spontaneous pathologies in chronic studies may be justified, although this would need further study to prove. However, their assertion that this invalidates the use of historical data is questionable. Historical data provides a wealth of information on the background rate of pathology, even if this is driven (at least in part) by variable food contaminants. Not only that, animal studies always include a control group who will be fed the same diet as the treatment groups, proving an internal control for diet-induced effects.

“In the paper the authors refer to a hazard quotient (HQ). This is a way of putting a single value on the level of several different hazards, in this case the hazards represented by the various components of the animal feed.  In feeding experiments, the hazard quotient should be compared to the level of feed at which no observed adverse effect is expected (the NOAEL). However in this paper the authors compare the hazard quotient instead to a different measure, the acceptable daily intake (ADI), which is usually 100-fold lower than the level at which no adverse effect is expected. This means that the hazard quotient calculated in this paper will substantially overestimate the cumulative risk of the feed.

“In summary, while this is a useful piece of work and adds to the body of evidence that allows us to interpret animal experiments correctly, it does not support the concerns raised by the authors on the use of historical data to interpret such studies.”

 

Prof. Tony Dayan, Emeritus Toxicologist, said:

“Professor Mesnage and his colleagues have reported analyses of single samples of 13 different rodent diets. The nature of the data and interpretation of the results is not straightforward in this very complex area of experimental toxicology.

“It appears to have been assumed that the samples of diets were representative samples which had not been contaminated prior to analysis. It may be more important that the quality of the feed provided for animals in regulatory tests has to comply with legally binding Good Laboratory Practice regulations (GLP), which include regular analyses to demonstrate amongst other things that contamination with heavy metals, pesticides and other substances is kept below strict limits. No information is provided to show that the batches reported in the new work were of a quality compliant with GLP and so might have been used in carcinogenicity or other formal tests.

“Professor Mesnage and colleagues make many comparisons between their findings and ADI (‘Acceptable Daily Intake’) and HQ (‘Hazard Quotient’) values, suggesting that the results may indicate risks to rodents. Those concepts and the calculated values are empirical factors derived and successfully employed over many years specifically to assess potential risks to humans. There is no experimental justification or experience to apply them to rodents as done here.

“In attributing many of the diseases that may affect aging experimental rodents to the widespread chemical contamination of their diets claimed here Professor Mesnage and colleagues have not taken into consideration the considerable amount of evidence linking those disorders to the considerable over-nutrition that follows the ad lib feeding of the rodents. That is widely considered to be a major factor in the rising incidence of background lesions in elderly rats and mice.

“We rely to a large extent on the results of various experiments in rodents to predict and evaluate potential chemical hazards in humans and other species but many other aspects of the substances tested and the circumstances and extent of exposure are also evaluated in suggesting real risks in practice. The breadth and complexity of risk assessment has been overlooked in the conclusions suggested in this paper.”

 

Prof. Maurice Moloney, CEO of the Global Institute for Food Security, Canada (An Institute of the University of Saskatchewan), said:

“The underlying hypothesis of this paper is the contention that the development of tumorous growths and carcinomas on the widely used Sprague-Dawley rat is the result of the toxicological effects of contaminants carried over into the feed pellets of lab animals from agricultural cultivation. Special emphasis is placed on the GM nature of some of the source materials and to one particular herbicide, glyphosate, or when formulated ‘RoundUp’. If this hypothesis were upheld, the authors believe that it means that most of the toxicological studies using this strain of rats are invalid, because of the intrinsic pernicious nature of the feed itself rather than the chemical (or other treatment) being tested. The previous study from this group was retracted from the Journal of Food and Chemical Toxicology, based on serious flaws identified after publication that should have been corrected in peer review, but which rendered the work inconclusive. The current work is no less inconclusive than the previous study, but nevertheless does offer some new data not heretofore published.

“The actual measured data are probably all accurate and expected given the source materials. The group has monitored for a large number of common pesticides. It is unsurprising what was found based on the formulation of commercial rat chow. Perhaps the biggest surprise is that relatively few were found at toxicologically meaningful levels. The lumping together of GMOs or glyphosate with organophosphates or heavy metals is “guilt by association”. These results show no correlation, let alone causality with either GMOs or glyphosate. Linking these to pyrimiphos (an acetylcholine esterase inhibitor) or a heavy metal like lead, both of which do seem to have residues that could be toxicologically meaningful, is unwarranted and misleading in my opinion.

“The best evidence for interference of GM sources and glyphosate would be toxicological data with these rats in the absence of these agents. Although not presented here, those data do exist, because the GM trait and use of glyphosate is a post-1990 phenomenon. Yet, the literature is replete with reports of Sprague-Dawley rats decades earlier showing exactly the same pathologies when the rats are used in long-term studies for 18 months to 2 years. Clearly, this is not related to GMOs or glyphosate.  Whether the levels of organophosphates or certain heavy metals are pertinent is simply not shown here as no controls are provided. These controls could have been performed if the authors had availed themselves of pesticide-free ingredients and screened for low heavy-metal residues. Without this, the paper lacks even correlation and certainly does not establish causation. In short, it cannot displace the current well-supported hypothesis that these pathologies are essentially genetically controlled and which appear in each new generation of Sprague-Dawley rats.

“In summary, if this paper is an attempt to support the previous (now retracted) study by this group, it falls far short of doing so. The new paper suggests we ought to doubt an entire testing system but does not provide the evidence to back up such doubt.

“By analogy, this is like a football team that is consistently disallowed goals, because they fall prey to an offside trap, calling into question all the laws of football. They do this rather than simply imposing discipline on their strikers not to move until the ball is in play. This paper lacks discipline in its discussion of the data, even though in the case of organophosphates and heavy metals, it might have discovered something that warrants further investigation. Food with minimal known toxin residues would help to ensure that test animals are treated ethically. For Sprague-Dawley rats, an ethical prerequisite is to use them only as intended for 90 day trials. Beyond that, as we saw in the revoked paper in the Journal Food and Chemical Toxicology, the test animals are subjected to significant distress, most likely due to a well-known genetic predisposition to form tumours.”

 

Dr Graham Tobin, Technical Director, Harlan Laboratories, Europe/ROW, said:

“The authors bring needed attention to the often overlooked role of diet in laboratory animal safety and toxicology studies.

“Contaminant levels in diets are a function of their ingredient composition, and the geographical source of those ingredients. A primary source of variation in contaminant levels between the different diets in this study was likely related to the diet formulations. It is known that certain ingredients are more likely to contain specific contaminants.

“Diets fed in safety and toxicology studies must be monitored for contaminant residues in order to meet Good Laboratory Practices (GLP). In regards to the 13 diets analyzed for this study, no diet exceeded current established contaminant criteria in any of these standards used in the US and Europe.

“The argument is flawed on the following basis:

“1. No data or compelling literature support was presented to demonstrate the contaminant levels found in the diets will contribute to these specific pathologies within rats.

“2. The safety assessment was predicated on human ADI levels that are not properly scaled to assess safety risks in rodents.

“3. The argument is too simplistic, ignoring other factors such as genetic predisposition, and practical experience. It overlooks the well-established effect of dietary restriction to improve survival and reduce pathology; this beneficial effect occurs in the absence of a reduction in contaminant intake per unit body weight.

“This paper addresses an important subject, but does a disservice to laboratory animal science by inflating the potential risk of typical dietary contaminant levels through use of an inappropriate metric.  On the basis of the evidence presented it certainly seems inadvisable and unwarranted to apply the adjective ‘toxic’ to the levels of contaminants reported here.”

 

 

‘Laboratory rodent diets contain toxic levels of environmental contaminants: implications for regulatory tests’ by Robin Mesnage et al. published in PLOS ONE on Thursday 2 July 2015.

 

Declared interests

Prof Cathie Martin: “I co-founded a spin-out company, Norfolk Plant Sciences, with the objective of commercialising health-promoting tomato products. These tomato products will, in some cases, have been produced by genetic modification. I am a holder of a patent (granted in the USA) giving me freedom to operate in producing tomatoes biofortified in flavonoids.”

Prof. Andy Smith: Member of the Expert Committee on Pesticides, Member of Panel and Working Groups of EFSA.

Prof. Tamara Galloway: “I’m a Professor of Ecotoxicology at the University of Exeter. I’m a member of the Hazardous Substance Advisory Committee which provides expert advice on how to protect the environment, and human health via the environment, from potentially hazardous substances.”

Prof. Alan Boobis: “I have no direct or indirect financial interests related to the subject of the paper. I have a number of indirect interests, involving collaborative research and participation in scientific advisory committees, addressing the interpretation of results from toxicity studies in laboratory rodents.”

Dr Nick Plant: “I am a member of the UK Committee on Toxicity, which is an independent body that advises Government on the risks associated with chemicals in food, consumer products and the environment. As such, we have assessed the risk of contaminants in various products, although never animal chow.”

Prof. Tony Dayan: “In the past 40 years I have been a toxicological adviser to many official agencies in Britain, Europe and WHO and to a large number of national and international chemical, food, pesticide and pharmaceutical companies. In my partial retirement over the past 10 years I have not been involved with practical carcinogenicity testing in animals for any specific company or industry.”

Prof. Maurice Moloney: “The Institute’s core benefaction is from Potash Corp (a fertilizer, not pesticide, company) and the taxpayers (Govt) of Saskatchewan.  Our research is at arms- length from all donors, by its founding charter. Previously, I have directed Rothamsted Research and CSIRO’s life science portfolio. Both organisations have a history of collaboration with conventional agribusiness, biotechnology companies and agroecology proponents.”

Dr Graham Tobin has been involved in laboratory animal nutrition for many years in his work at Harlan Laboratories.

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