Emulsifiers, gut microbiota & biological responses: Unjustified conclusions based on flawed research
In March, 2015 a study was published by Chassaing et al. in Nature entitled “Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome” (1). The investigators gave wild-type (C57Bl/6) mice, germ-free mice or mice predisposed to developing colitis (strains Il10-/- and Tlr5-/-) 1.0% sodium carboxymethylcellulose (CMC) , polysorbate 80 (P80) or sodium sulfite in the only source of drinking water for 12 weeks and examined a number of different biological responses. The investigators also tested the effect of lower concentrations of CMC and P80 (0.1% and 0.5%) on selected parameters. Controls received regular drinking water, and all animals had free access to feed (Purina Rodent Chow 5001®). Based on their results, the authors concluded that the emulsifiers altered the microbial flora of the gastrointestinal (GI) tract, and induced epithelial cell damage, increased tight-junction permeability, low grade inflammation, shortening of the colon and obesity/metabolic syndrome in wild-type mice and colitis in mice predisposed to the disorder. The changes did not occur in wild-type mice provided water containing sodium sulfite or in germ-free mice provided water containing the emulsifiers. Transfer of microbiota from emulsifier–treated mice to germ–free control mice induced many of the pathological changes observed in emulsifier –treated mice, suggesting that “emulsifier-induced changes in the microbiota have a role in driving the inflammation and metabolic changes promoted by these food additives.” Based on the results of their research, the authors hypothesized that dietary emulsifiers may have contributed to the post- and mid-twentieth century increase in the incidence of inflammatory bowel disease, metabolic disease and other chronic inflammatory diseases and that the testing of substances that have been given GRAS status may be inadequate. These comments are unjustified based on the design and results of this study.
The design flaws in this study are serious enough to question its validity. Important design flaws include: a) no measurement of water consumption, b) no removal of feces to prevent coprophagia (and re-cycling of test substance), and c) inappropriate controls. At the concentrations tested, the emulsifiers may have affected the taste and viscosity of the drinking water, stimulating or depressing water intake. The investigators should have ensured from the outset that water consumption of exposed animals was similar between and within groups, taking steps to equalize water intake if necessary. Further, mice should have been housed in cages with wire bottoms to allow fecal matter to drop through and remain inaccessible to prevent ingestion of the fecal material, which would be expected to contain high amounts of test material and bacteria. Because overall consumption of the test material from water and feces may not have been the same among treated animals, the doses of material that individual mice may have received were likely to have been extremely variable. The fact that the results were highly variable among animals in each treatment group and skewed by results from a few animals supports this hypothesis. Further, water with or without sodium sulfite is not the best control for the molecules that were tested in the study, which are large, hygroscopic molecules that are difficult to digest and increase fecal bulk. In order to prove that the results were due to ingestion of an emulsifier in water, the investigators should have used a control material that has similar physical properties as the emulsifier (other than the property of emulsification), housed the mice in wire wages to prevent accumulation of feces in cages, and ensured that water intake was similar within and between groups (or administered the materials by gavage rather than drinking water).
While the authors did find some evidence of some changes in the gastrointestinal tract (e.g. altered microflora, shortened colon, and increased FITC-labelled dextran permeability and inflammatory mediators) they did not demonstrate that such changes were adverse. It is not surprising that the gut microflora were altered in CMC or P80 exposed animals compared to animals provided water or water containing sodium sulfite, as the gut microflora varies with diet (2). It is logical that the gut microbiota would change in response to ingestion of a large amount of material that is difficult to digest. Microbes that have lower energy or nutritive requirements or could possibly degrade some of the material would tend to thrive at the expense of other organisms. The fact that colon length was shortened is likely an adaptive response to help the animals eliminate the large amount of bulk. The change in colon length, as well as several other findings, were reversed after a six week recovery period. An increase in intestinal permeability in response to ingestion of certain food ingredients (even some that are perceived as “healthy”) is common and insufficient to cause intestinal disease (3,4). The finding of increased fecal lipocalin 2 ( LNC2 ) and myeloperoxidase in colonic tissue is unclear, given that there was no difference in neutrophil recruitment between emulsifier-treated and control animals. The only indication that an adverse effect occurred in the colon was a subjective, total score for epithelial damage and inflammatory infiltrate in stained sections that was not scored in a blinded manner. The reversibility of the histopathological findings was not assessed. The conclusion that the findings were toxicological in nature would be more convincing if the changes were still observed in animals allowed to recover from exposure and the histological slides were scored in a blinded fashion.
The conclusion that the data are consistent with the emulsifiers causing metabolic syndrome in the animals is not supported by the data. Metabolic syndrome is a multifaceted syndrome, the diagnosis of which is dependent on the presence of at least three of the following five criteria: central or abdominal obesity, increased triglycerides, increased HDL cholesterol, fasting glucose ≥ 100 mg/dL and blood pressure ≥ 130/85 mm Hg. The fact that feed intake, body weight, fat pad mass and fasting blood glucose is increased in wild-type or Tlr5-/- mice ingesting drinking water containing 1.0 % P80 or CMC compared to normal drinking water with or without sodium sulfite is interesting, but is not sufficient for a diagnosis of metabolic syndrome. The reason for the increases in food consumption, body weight and fasting blood glucose is unclear, but may be related to the design flaws mentioned above, strains of rats used in the study, or diet. In studies conducted by the National Toxicology Program (NTP), increased food consumption or body weight or histological changes in the GI tract did not occur in in B6C3F1 mice exposed to up to 5% P80 in the diet (approximately 7500 mg/kg bw/day) for 13 weeks or two years (5). Furthermore, rats administered 2.5-10% CMC in feed (approximately 2500 – 10,000 mg/kg bw/day) for three months or 5% CMC in feed (approximately 6000 mg/kg bw/day) for 201-250 days do not exhibit increases in body weight or histological changes in the GI tract (6). This suggests that the doses administered in the Chassaing et al. study were considerably higher than theoretical (perhaps due to feces ingestion or increased water consumption) or the design of the study predisposed the mice to developing the changes that were observed.
The assertion that the results of this study challenge the GRAS status of CMC and P80 is without merit due to the high doses administered, issues with study design and lack of confirmatory findings in longer term toxicity studies with equal or higher doses. The doses of CMC or P80 in mice provided 0.1 -1.0% of these substances in drinking water were approximately 250 -2500 mg/kg bw/day, assuming that the mice consumed a normal amount of drinking water. As mentioned above, it is also possible that the mice received even higher doses by ingesting feces or drinking more water than usual. Doses of 250 -2500 mg/kg bw/day in mice are 10-100 times greater than the acceptable daily intakes (ADIs) for P80 or CMC of 25 mg/kg bw/day (8), set by the Joint FAO/WHO Expert Committee on Food (JECFA) after applying appropriate safety factors to no observable adverse effect levels (NOAELs) from safety studies in rodents, dogs and/or guinea pigs. The results of the study do not challenge the conclusion that P80 and CMC are safe at the ADI of 25 mg /kg bw/day and this study should be viewed with skepticism, due to the limitations discussed above.
References
Chassaing, B., Koren, O., Goorrich, J.K., Poole, A. C., Srinivasan, A., Ley, R. E. and Gewirtz, A. T. 2015. Nature. Doi:10.1038/nature14232.
Graf, D., DiCagno, R., Fak, F., Flint, F.J., Nyman, M., Saarela, M. and Watzl, B. 2015. Composition of diet to the composition of the human gut microbiota. Microbial Ecology in Health & Disease. 26: 26164.
Kosińska, A. and Andlauer, W. 2013. Modulation of tight junction integrity by food components. Food Research International 54: 951-960.
Turner, J.R. 2009. Intestinal mucosal barrier function in health and disease. Nature Reviews/Immunology 9:799-809.
S. Department of Health and Human Services (USDHHS). 1992. NTP technical report on the toxicology and carcinogenesis studies of polysorbate 80 (CAS No. 9005-65-6) in F344/N rats and B6C3F1 mice (feed studies). NIH publication 92-3146.
Bär, A., Til, H.P. and Timonen, M. 1995. Subchronic oral toxicity study with regular and enzymatically depolymerized sodium carboxymethylcellulose in rats. Food Chemical Toxicology 11:909-917.
Rowe, V.K., Spencer, H.C., Adams, E.M., and Irish, D.D. 1944. Response of laboratory animals to cellulose glycolic acid and its sodium and aluminum salts. Food Res. 9, 175-182.
World Health Organization. 1974.; 17.Toxicological evaluation of certain food additives with a review of general principles and of specifications (Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 539.