State-of-the-art tests show damage from mixtures of pesticides, when each is present at a level regulators claim is safe

Consumption of a mixture of pesticides commonly found in foods in the EU, when tested at levels deemed safe by regulators, has been revealed to cause ill health effects that are completely missed by the standard safety tests required for regulatory assessments, a new study shows.

The study implies that the pesticide regulatory system, which relies on toxicity tests done by manufacturers to decades-old designs, is unsafe by design.

In the study, led by Drs Michael Antoniou and Robin Mesnage at King's College London, rats were fed a mixture of the six pesticides (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) that are most frequently found in foods in the EU.

Each pesticide was administered at its regulatory permitted EU acceptable daily intake (ADI), the level that regulators assume can safely be ingested over a lifetime without ill effects. So the expectation was that no effects or signs of toxicity would be seen.

Insofar as the standard safety tests were concerned, that was the case. Histological analysis – microscopic examination of tissue – showed there was a non-significant increase in the incidence of liver and kidney lesions (the increase might have reached statistical significance if larger numbers of animals were used and/or the study lasted for longer). And a blood serum analysis only detected a small reduction in levels of a substance called creatinine – a larger reduction can be a sign of liver or kidney damage, or muscle disease.

Omics analysis shows different picture

Not much harm was visible from these tests – certainly not enough to make any regulator ban a pesticide. But the researchers didn't stop there. They applied in-depth molecular analytical techniques called "omics" to search for more subtle changes that could predict the development of disease – with very different results.

The results showed that the low-dose mixture of pesticides tested caused changes in the metabolites of the gut and blood, with consequences to liver function, with the whole picture being suggestive of an oxidative stress response. Oxidative stress is an imbalance in the body that can lead to conditions such as non-alcoholic fatty liver disease, DNA damage, and cancer.

There were no changes in the populations of bacteria present in the gut. However, the researchers also tested the pesticides individually and as a mixture in vitro (in the laboratory, not in living animals) to see if they affected gut bacterial growth. When the six pesticide active ingredients were tested individually, they did not affect the growth of the four bacterial strains tested. However, when all six pesticides were tested as a mixture, the resulting chemical cocktail was found to inhibit growth of certain strains.

The changes in blood metabolite levels did not reflect a disease state, but probably an adaptive response to oxidative stress, which can lead to the development of a disease state if the damage produced exceeds the capacity for repair.

Moreover, omics analysis of gene expression (transcriptomics) in the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways.

In summary, the omics analyses revealed a very consistent pattern of biochemical and gene expression in the gut, blood and liver that clearly indicated the pesticide mixture subjected the experimental animals to potentially harmful oxidative stress.

Crucially, these changes would not be detected using conventional biochemical and histopathological investigations, which regulators currently rely on for pesticide risk assessment. Thus the results highlight the advantages of incorporating omics methods into the OECD test guidelines that industry follows to test the toxicity of its products.

Another study shows omics succeeds where standard toxicity tests fail

These latest results build on the findings of a previous study in rats, in which Drs Antoniou and Mesnage found that glyphosate and the glyphosate-based herbicide Roundup disrupt the gut microbiome by the same mechanism by which the chemical acts as a weedkiller. These effects too were found to happen at low doses that regulators claim to be safe.

As with the new research on the pesticide mixture, this previous study on Roundup and glyphosate was able to reveal adverse effects even over the relatively short exposure period of 90 days because the researchers used omics to measure the composition of the blood and contents of the gut, which highlighted a potentially damaging oxidative stress response.

Dr Antoniou commented on both studies: “Our findings demonstrate the need for regulators to stop dragging their feet and urgently adopt omics methods as part of the pesticide risk assessment, to gain a more accurate picture of the toxicity of chemical pollutants and thus better protect public health.”
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The new study:

Mesnage, R., Teixeira, M., Mandrioli, D. et al. Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats. Commun Biol 4, 471 (2021). https://doi.org/10.1038/s42003-021-01990-w

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