Status: Preprint — not yet peer-reviewed. The findings described below come from a manuscript posted to Research Square, a preprint server, in July 2026. It has not completed peer review and has not been published in a scientific journal. Preprints have not undergone the scrutiny of independent expert reviewers, and their conclusions can change — or fail to hold up — during that process. Readers should treat these results as preliminary.
Overview
A multigenerational rat study from the Global Glyphosate Study (GGS), a multi-institutional research program with researchers from the Cesare Maltoni Cancer Research Center at the Ramazzini Institute, University of California Santa Cruz, George Mason University and Boston College, reports dose-dependent anxiety-like behavior in rats exposed continuously across three generations to glyphosate or one of two commercial glyphosate-based herbicide (GBH) formulations: Roundup Bioflow and Ranger Pro. The GGS is the most comprehensive toxicological evaluation of glyphosate and its commercial formulations conducted to date, and this neurobehavioral analysis is one arm of that larger program.
Study design
- Species/strain: Sprague-Dawley rats
- Exposure route: Continuous oral administration in drinking water
- Generational span: F0 dams exposed starting gestational day 6; exposure continued through F1 and into F2 offspring, which were tested at approximately one year of age
- Test substances: Glyphosate alone (99% purity); Roundup Bioflow (MON 52276, the EU formulation); Ranger Pro (EPA 524–517, the US formulation)
- Doses: Targeted at 0, 0.5, 5, and 50 mg/kg body weight/day of glyphosate — spanning the EU’s Acceptable Daily Intake (0.5 mg/kg/day) up to the EU’s No Observed Adverse Effect Level (50 mg/kg/day)
- Sample size: 240 F2 animals total (120 males, 120 females) across 10 exposure groups, with 12 animals per sex per group
- Behavioral battery: Open field test, forelimb/hindlimb grip strength, balance beam, passive avoidance (short-term memory), and the Irwin functional observation battery
- Statistical approach: Mixed-model ANOVA (4 dose levels × 2 sexes) per formulation, with rat ID as a random effect; significant effects followed by pairwise Student’s t-tests against controls; significance threshold set at p ≤ 0.05
Key findings
Open field test (anxiety-like behavior). All three substances produced dose-dependent increases in thigmotaxis — the tendency of rodents to stay near the walls of an open arena rather than explore the center, a well-established proxy for anxiety in rodent behavioral pharmacology. Specific outcomes affected included the ratio of wall-zone to total distance traveled, time spent in the wall zone, average duration of wall-zone visits, freezing time, and total time immobile in the wall zone. Total distance traveled and average locomotor speed were unaffected by any treatment, indicating the effect was specific to exploratory/anxiety-related behavior rather than general motor suppression.
Grooming microstructure. All three substances reduced the proportion of “syntactic” (orderly, cephalocaudal) grooming relative to total grooming events, primarily in males (p < 0.0001 for all three formulations in males). Disorganized grooming is considered a stress-sensitive behavioral marker in rodent studies.
Sex differences. Where sex differences emerged, males were consistently more affected than females across the open field and grip strength measures.
Formulation differences. Roundup Bioflow and Ranger Pro produced broader and generally larger effects than glyphosate alone at equivalent glyphosate doses, implicating the surfactants and other co-formulants in the commercial products.
Grip strength. Roundup Bioflow at 5 mg/kg/day significantly reduced hindlimb grip strength in males (p = 0.0498) versus controls; Ranger Pro showed a similar non-significant trend (p = 0.109) at the same dose. Glyphosate alone showed a significant main effect on forelimb grip strength (p = 0.0338) but no individual dose contrast reached significance.
Null findings. No treatment affected balance beam performance (motor coordination), passive avoidance latency (short-term associative memory), or any of the 24 Irwin test parameters assessing overt neurological/physiological function. Body weight was not affected by glyphosate or Roundup Bioflow exposure.
The autism question
The autism connection in the paper is drawn from surrounding literature and concerns are raised from the study’s own data:
- Epidemiological studies (e.g., the Ecuador-based ESPINA cohort) have linked urinary glyphosate levels in adolescents to lower performance on social-perception tests.
- Separate rodent studies not part of this paper — including work by Buchenauer et al. and by Pu et al. — have reported autism-relevant social deficits or autism-like behaviors in offspring following gestational or maternal glyphosate/GBH exposure.
- A 2025 scoping review cataloged the existing (and still limited) evidence connecting GBH exposure to autism spectrum disorder specifically.
Dr. Daniele Mandrioli, the Global Glyphosate Study’s principal investigator, said the toxicological findings raise important concerns on the neurobehavioral effects of glyphosate-based herbicides, including for autism spectrum disorder, even at doses currently classified as safe.
The authors argue the anxiety and grooming-disruption phenotypes they observed are relevant to autism research because they overlap behaviorally with traits studied in autism models, and because mechanisms implicated elsewhere in glyphosate research — serotonergic disruption, neuroinflammation, and gut microbiome perturbation — are also active areas of autism etiology research.
One pattern worth noting on its own terms: the male-skewed effect sizes observed across multiple outcomes mirror the roughly 4-to-1 male-to-female skew in human autism diagnoses, though the authors do not draw a causal line between the two and no mechanism for that parallel was tested here.
Context: the broader Global Glyphosate Study program
This neurobehavioral paper is one of several arms of the GGS examining the same multigenerational exposure cohort for different endpoints. A separate arm of the program, evaluating carcinogenicity in the same rat colony under a similar multigenerational exposure design, has already been published and reported that glyphosate and glyphosate-based herbicides caused multiple types of tumors in exposed animals. Other published GGS arms have examined the gut microbiome and metabolome, comparative genotoxicity, and bacterial/fungal community composition in the cecal microbiome. Taken together, the program’s organizing premise is that glyphosate’s real-world toxicological profile can only be captured by testing commercial formulations — not glyphosate in isolation — across continuous, multigenerational exposure, rather than the single-generation, short-duration protocols more commonly used in regulatory toxicology.
Bottom line
At doses spanning current EU regulatory thresholds, continuous multigenerational exposure to glyphosate and two commercial glyphosate-based herbicide formulations produced measurable, dose-dependent increases in anxiety-like behavior and modest, sex-specific reductions in neuromuscular grip strength in rats — with the branded formulations generally producing larger effects than glyphosate alone. The manuscript is a preprint and has not yet been peer-reviewed or published in a journal.