Major Pesticides Are More Toxic to Human Cells Than Their Declared Active Principles
Robin Mesnage, 1 Nicolas Defarge, 1 Joël Spiroux de Vendômois, 2 and Gilles-Eric Séralini
Pesticides are used throughout the world as mixtures called formulations. They contain adjuvants, which are often kept confidential and are called inerts by the manufacturing companies, plus a declared active principle, which is usually tested alone.The chronic tests of pesticides may not reflect relevant environmental exposures if only one ingredient is tested alone.We tested the toxicity of 9 pesticides, comparing active principles and their formulations, on three human cell lines (HepG2, HEK293, and JEG3). Glyphosate, isoproturon, fluroxypyr, pirimicarb, imidacloprid, acetamiprid, tebuconazole, epoxiconazole, and prochloraz constitute, respectively, the active principles of 3 major herbicides, 3 insecticides, and 3 fungicides. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. Fungicides were the most toxic from concentrations 300–600 times lower than agricultural dilutions, followed by herbicides and then insecticides, with very similar profiles in all cell types. Despite its relatively benign reputation, Roundup was among the most toxic herbicides and insecticides tested. Most importantly, 8 formulations out of 9 were up to one thousand times more toxic than their active principles.Our results challenge the relevance of the acceptable daily intake for pesticides because this norm is calculated from the toxicity of the active principle alone. Chronic tests on pesticides may not reflect relevant environmental exposures if only one ingredient of these mixtures is tested alone.
This is the first time that all these formulated pesticides have been tested on human cells well below agricultural dilutions. The three different cell types reacted very similarly and the toxicities were observed on several biomarkers; this confirmed our results. Moreover, these are very consistent with several studies on cell lines [1, 25], where placental JEG3 cells were found to be the most sensitive. In this study , adjuvants were also more cytotoxic through the disruption of membrane and mitochondrial respiration than from an activation of apoptotic pathways. Primary cells are in some case up to 100 times more sensitive, for instance, neonate umbilical cord vein cells . We also study here short exposures (24 h), but we have previously demonstrated a time-amplifying effect: the differential toxicity between the AP glyphosate and Roundup is increased by 5 times in 72 h . It appears that, with cell lines and short exposures, we underestimate by far the direct toxicity of the products in the long term. In this case in vivo, the metabolism may reduce the toxic effect, but this can be compensated or amplified by bioaccumulation and/or the combined effect of the AP with adjuvants. For instance, in this experiment, after 24 h, 63 ppm of Roundup was found to be toxic to cells, but in our previous experiment, after two years in rats, only 0.1 ppb of Roundup was found to be sufficient to provoke pathologies .
Adjuvants in pesticides are generally declared as inerts, and for this reason they are not tested in long-term regulatory experiments. It is thus very surprising that they amplify up to 1000 times the toxicity of their APs in 100% of the cases where they are indicated to be present by the manufacturer (Table 1). In fact, the differential toxicity between formulations of pesticides and their APs now appears to be a general feature of pesticides toxicology. As we have seen, the role of adjuvants is to increase AP solubility and to protect it from degradation, increasing its half-life, helping cell penetration, and thus enhancing its pesticidal activity  and consequently side effects. They can even add their own toxicity . The definition of adjuvants as “inerts” is thus nonsense; even if the US Environmental Protection Agency has recently changed the appellation for “other ingredients,” pesticide adjuvants should be considered as toxic “active” compounds.
In the scientific literature, in contrast with regulatory beliefs, some harmful effects of the adjuvants present in this study are reported. In the formulations (Table 1) Starane 200, Opus, and Eyetak, the adjuvants include solvent naphtha (a petroleum distillate), which is known to have developmental effects in rodents . Xylene (in Eyetak) has long been associated with cardiac and central nervous system diseases in humans . 1-Methyl-2-pyrrolidinone (in Confidor) is a developmental toxicant and caused malformations, incomplete ossification of skull, and decreased fetal body weights in rats . N,N-Dimethyldecanamide (Maronee adjuvant) has been characterized as a developmental toxicant in rodents  but is insufficiently studied for reproductive toxicity. The distinction between AP and “declared inert” compounds appears to be a regulatory assumption with no toxicological basis, from this experiment and others. Even industry and regulators contradict themselves in the classification of APs and inert compounds. For example, 1,2-benzisothiazoline-3-one is classed as an inert ingredient in the pesticide Polysect in particular and as an active ingredient in cleaning products .
All this does not exclude the toxicity of APs alone. Glyphosate inserted in the aromatase active site of mammalian cells disrupts steroidogenesis . Imidacloprid alters the developing immunity in rats . Fluroxypyr (ester 1-methylheptyl) has never been tested in human cells before this study but appears to be toxic from 22 ppm in formulation; its ADI is only 0.8 ppm/day (DG SANCO, 2013). It also appears here that prochloraz is the main toxicant of the tested formulation.
It is commonly believed that Roundup is among the safest pesticides. This idea is spread by manufacturers, mostly in the reviews they promote [39, 40], which are often cited in toxicological evaluations of glyphosate-based herbicides. However, Roundup was found in this experiment to be 125 times more toxic than glyphosate. Moreover, despite its reputation, Roundup was by far the most toxic among the herbicides and insecticides tested. This inconsistency between scientific fact and industrial claim may be attributed to huge economic interests, which have been found to falsify health risk assessments and delay health policy decisions .
In conclusion, our results challenge the relevance of the ADI, because it is calculated today from the toxicity of the AP alone in vivo. An “adjuvant factor” of at least a reduction by 100 can be applied to the present calculation of the ADI if this is confirmed by other studies in vivo. As an example, the present ADI for glyphosate is 0.3 ppm; for glyphosate-based herbicides it would be 3 ppb or less. However, this will never replace the direct study of the commercial formulation with its adjuvants in regulatory tests. Anyway, an exposure to a single formulated pesticide must be considered as coexposure to an active principle and the adjuvants. In addition, the study of combinatorial effects of several APs together may be very secondary if the toxicity of the combinations of each AP with its adjuvants is neglected or unknown. Even if all these factors were known and taken into account in the regulatory process, this would not exclude an endocrine-disrupting effect below the toxicity threshold. The chronic tests of pesticides may not reflect relevant environmental exposures if only one ingredient is tested alone.