The most comprehensive multigenerational study on glyphosate and Roundup herbicides has found dose-dependent anxiety-like behavior in rats exposed to doses regulators call “safe”. The findings, which will soon be peer-review published, add fuel to multiple smaller studies that have shown glyphosate to have a possible role in autism spectrum disorder.
Glyphosate-based herbicides are sprayed on everything from soybean fields to suburban sidewalks. Regulators in the United States and Europe have long maintained that the doses most people encounter are safe. However, this new comprehensive study, posted this month as a preprint by researchers from the Ramazzini Institute, University of California Santa Cruz, George Mason University and Boston College suggests that assumption deserves a harder look — at least when it comes to the brain.
The study exposed rats to glyphosate or one of two commercial formulations, Roundup Bioflow and Ranger Pro, continuously across three generations — starting in the womb and continuing for a full year of life. The doses spanned the European Union’s Acceptable Daily Intake, the level regulators consider safe for lifelong human exposure, up to the EU’s No Observed Adverse Effect Level, the highest dose at which no harm was supposed to occur.
By the time the third generation of rats reached one year old, roughly analogous to middle age in a human, the exposed animals were behaving differently. In a standard test of exploratory behavior called the open field test, treated rats — especially males, and especially those exposed to the branded herbicide formulations rather than glyphosate alone — spent more time hugging the walls of the test arena, moved less freely into open space, froze more often, and groomed themselves in a disorganized, fragmented way rather than the smooth head-to-tail sequence typical of an undisturbed rat. Taken together, the researchers say, these are classic markers of heightened anxiety in rodents. Male rats exposed to the commercial formulations also showed modest reductions in hind-limb grip strength, suggesting subtle effects on neuromuscular function. Memory, general motor coordination and overt neurological signs were unaffected.
“Our toxicological findings raise important concerns on the neurobehavioral effects of glyphosate-based herbicides, including autism spectrum disorders, even at doses currently considered “safe””, Dr. Daniele Mandrioli, Principal Investigator of the Global Glyphosate Study and senior author of the manuscript, stated on Tuesday.
Why autism enters the conversation
The paper does not claim to have found direct evidence of autism in rats — it did not run the tests, such as assessments of social behavior, that researchers typically use to model autism-like traits in animals. What it does is add a data point to a growing body of human and animal research that researchers say raises the question of whether glyphosate belongs on the list of environmental exposures worth scrutinizing for a possible role in neurodevelopmental conditions, autism spectrum disorder among them.
That broader link rests mostly on other studies, which the new paper cites as context. Epidemiological work has found associations between glyphosate exposure, measured in urine, and cognitive impairment, depressive symptoms and lower performance on social-perception tests in adolescents. Separate animal studies — not part of this new paper — have reported that mice exposed to glyphosate-based herbicides during gestation display autism-like social deficits in adulthood. A 2025 review scoped out the existing evidence connecting glyphosate herbicide exposure to autism spectrum disorder specifically, describing it as an emerging but still incomplete area of research.
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The authors of the new comprehensive rat study argue that their findings are relevant to that conversation because anxiety, disrupted grooming patterns and altered sensory reactivity to novel environments are behavioral domains that overlap with traits seen in autism spectrum disorder, and because the neurobiological pathways implicated elsewhere in glyphosate research — disrupted serotonin signaling, neuroinflammation, gut microbiome disturbances that affect brain chemistry — are pathways also implicated in autism research more broadly.
What made this study different
The new paper is one arm of something larger: the Global Glyphosate Study, a multi-institutional research program that is the most comprehensive toxicological evaluation of glyphosate and its commercial formulations ever undertaken, examining not just neurobehavior but cancer risk, organ toxicity, endocrine disruption and developmental effects across the same multigenerational cohort of animals. Other arms of that program, including a carcinogenicity study, have already been published and showed that glyphosate-based herbicides cause multiple types of cancer; this neurobehavioral analysis is the latest installment. The European Food Safety Authority (EFSA) is currently performing a full review on the results of the carcinogenicity arm, which may lead to changes in regulation surrounding the chemical in the EU.
Most prior animal research on glyphosate’s neurological effects has looked at a single generation, exposed for a matter of weeks, and tested the chemical in its pure form. This study did the opposite on every count: three generations, continuous exposure from before birth through one year of age, and — critically — testing of the commercial products people are actually exposed to, not just the active ingredient.
That distinction turned out to matter. Across nearly every measure, the branded formulations Roundup Bioflow and Ranger Pro produced broader and often larger effects than glyphosate by itself, even when the dose of glyphosate was identical. The researchers point to this as evidence that the inactive ingredients in commercial herbicides — the surfactants and adjuvants that help the products stick to and penetrate plant leaves — are not biologically inert in mammals, a concern other laboratories have raised for years but that regulatory risk assessments, which typically evaluate glyphosate alone, do not fully capture.
The male-skewed pattern is also notable. In outcome after outcome, when a sex difference emerged, it was male rats that were more affected — a pattern that echoes the roughly four-to-one male skew in autism spectrum disorder diagnoses in humans, though the study’s authors are careful not to draw a direct line between the two.
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