ORIGINAL ABSTRACT

Human exposure to low-level mixtures of herbicides is widespread, yet regulatory assessments still rely on single-compound, high-dose studies. This study aimed to determine whether prenatal-to-adolescent exposure to regulatory doses of glyphosate (GLY) alone or combined with 2,4-D and dicamba (COMB) alters liver function in rats. Pregnant Wistar dams (n=5/group) received drinking-water containing (i) no herbicide (BLANK), (ii) GLY at the EU acceptable daily intake (ADI, 0.5mg/kg bw/day; GLY 1), (iii) GLY at the EU NOAEL (50mg/kg bw/day; GLY 2) or (iv) the COMB mixture (each herbicide at its EU ADI). Offspring (≈10/sex/group) continued the same exposure until 90 days post-weaning. Serum lipids and liver enzymes were quantified; livers were weighed and examined histologically. In COMB-exposed males, ALAT, ASAT and ALP increased significantly; females showed elevated ALAT, ASAT, cholesterol and triglycerides. GLY 2 reproduced this pattern in males, while GLY 1 heightened ALAT and ALP in males and triglycerides in females. Liver weight was unchanged, but histology revealed dose-related centrilobular vein dilation, granular degeneration, micro-vacuolar change and focal necrosis, most severe in the COMB group. Continuous exposure from gestation to young adulthood to herbicide doses at or below current "safe" limits produced clear biochemical and structural liver injury, amplified by the glyphosate plus 2,4-D and dicamba mixture and with sex-specific sensitivity. These findings challenge the adequacy of single-chemical risk assessments and underscore the need to re-evaluate mixture toxicity, particularly for vulnerable populations.