Multi-generational exposure to glyphosate and glyphosate-based herbicides induces neurobehavioral alterations in Sprague-Dawley rats

Technical Summary: Multi-generational Exposure to Glyphosate and Glyphosate-Based Herbicides Induces Neurobehavioral Alterations in Sprague-Dawley Rats

Problem Statement
Glyphosate is the most widely used herbicide globally, with ubiquitous environmental and occupational exposure detected in human biomonitoring studies. While regulatory bodies have established "safe" thresholds such as the Acceptable Daily Intake (ADI) and No Observed Adverse Effect Level (NOAEL), evidence linking glyphosate and glyphosate-based herbicides (GBHs) to neurobehavioral disturbances—specifically anxiety, depression, and cognitive impairment—remains a subject of intense investigation. A critical gap exists in current toxicological literature: most pre-clinical studies focus on short-duration, single-generation exposures to the pure compound glyphosate, rather than the commercial formulations (containing adjuvants and surfactants) to which humans are actually exposed. Furthermore, the potential for multi-generational neurotoxic effects, consistent with the Developmental Origins of Health and Disease (DOHaD) framework, has not been comprehensively characterized at regulatory-relevant doses. This study addresses these gaps by evaluating the neurobehavioral impacts of transgenerational exposure to glyphosate and two distinct commercial GBH formulations (Roundup Bioflow and Ranger Pro) in Sprague-Dawley rats.

Methodology
The study was conducted as the neurobehavioral arm of the Global Glyphosate Study (GGS) at the Cesare Maltoni Cancer Research Center. The experimental design involved a multi-generational exposure paradigm spanning three generations (F0, F1, and F2) of Sprague-Dawley rats.

• Test Substances: Three substances were administered via drinking water: pure glyphosate, the EU commercial formulation Roundup Bioflow (MON 52276), and the US commercial formulation Ranger Pro (EPA 524–517).
• Dosing: Targeted daily oral doses of 0, 0.5 (EU ADI), 5, and 50 (EU NOAEL) mg/kg body weight/day were administered. F0 pregnant dams were treated from gestational day 6 through lactation; F1 offspring were exposed postnatally and during mating; F2 offspring were exposed from weaning until sacrifice at 1 year of age.
• Subjects: The study utilized 240 F2 offspring (120 males, 120 females) across 10 experimental groups (Control, 3 glyphosate doses, 3 Roundup Bioflow doses, 3 Ranger Pro doses).
• Behavioral Assessment: At 51–53 weeks of age, animals underwent a comprehensive battery of tests:
  1. Open Field Test: Assessed general activity, exploration, anxiety-like behavior (thigmotaxis, freezing), and grooming microstructure.
  2. Balance Beam Test: Evaluated gross motor coordination and sensorimotor function.
  3. Grip Strength Test: Measured forelimb and hindlimb neuromuscular tone.
  4. Passive Avoidance Test: Assessed short-term memory and emotional learning regarding aversive stimuli.
  5. Irwin Test: A functional observational battery for detecting behavioral and physiological changes.
• Statistical Analysis: Data were analyzed using mixed-model ANOVA with treatment and sex as fixed effects and rat ID as a random effect.

Key Results
The study found that multi-generational exposure to glyphosate and GBHs produced significant, dose-dependent neurobehavioral alterations, particularly regarding anxiety and neuromuscular tone, with commercial formulations often exhibiting greater toxicity than glyphosate alone.

• Anxiety-Like Behavior: All three compounds altered open field exploration consistent with increased anxiety. This was evidenced by a significant increase in the ratio of distance traveled in the wall zone versus the center, increased time spent in the wall zone, longer duration of wall-zone visits, and increased freezing/immobility in the wall zone.
  • Formulation Differences: Roundup Bioflow and Ranger Pro generally caused broader and more severe anxiety-like behaviors than glyphosate alone. For instance, all doses of the commercial formulations increased wall-zone time in males, whereas glyphosate effects were more selective.
  • Grooming: All formulations reduced the proportion of "syntactic" (ordered, cephalocaudal) grooming episodes in males, a marker of stress-evoked chaotic grooming.
  • Sex Differences: Males were generally more affected than females across multiple anxiety metrics.
• Motor Coordination and Neuromuscular Tone:
  • Balance Beam: No significant effects were observed on gross motor coordination or the number of falls for any treatment group.
  • Grip Strength: While general motor activity was unaffected, modest deficits in neuromuscular tone were observed. Roundup Bioflow and Ranger Pro (specifically at the 5 mg/kg/d dose) significantly reduced hindlimb grip strength in males compared to controls. Glyphosate alone showed a main effect on forelimb strength but no significant dose-specific contrasts against controls.
• Memory and Physiological Screening:
  • Passive Avoidance: No significant effects were found on short-term memory or the ability to form associations with aversive stimuli. Animals in all groups retained normal fear memory.
  • Irwin Test: No overt physiological or neurological abnormalities (e.g., tremors, convulsions, altered reflexes) were detected in either the undisturbed or handling phases.
• Body Weight: Exposure did not significantly affect body weight, except for a minor, isolated difference in the Ranger Pro male group which did not correlate with behavioral outcomes.

Significance and Claims
The authors claim that this study provides a uniquely translational assessment of neurodevelopmental risks by simulating real-world, multi-generational human exposure scenarios. The primary significance of the findings lies in three areas:

1. Safety Thresholds: The study demonstrates that significant neurobehavioral impairments (increased anxiety and reduced neuromuscular tone) occur at doses currently considered "safe" by regulatory agencies (spanning the EU ADI to the NOAEL). This suggests that current regulatory thresholds may not be sufficiently protective against the chronic, transgenerational neurotoxicity of these herbicides.
2. Formulation Toxicity: The results substantiate the hypothesis that commercial GBH formulations are more toxic than the active ingredient (glyphosate) alone. The adjuvants and surfactants in Roundup Bioflow and Ranger Pro appear to enhance or modify the toxicological profile, leading to broader behavioral deficits than glyphosate alone. This implies that risk assessments based solely on the pure compound may underestimate the actual risk of human exposure to formulated products.
3. Sex-Specific Susceptibility: The study highlights a clear sex-dependent vulnerability, with male offspring exhibiting greater susceptibility to anxiety-like behaviors and neuromuscular deficits than females.

The authors conclude that these findings support growing concerns regarding the safety of glyphosate-based herbicides, particularly in relation to anxiety disorders and neurodevelopmental conditions, and advocate for the inclusion of multi-generational neurobehavioral assessments in future regulatory evaluations.