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. 2025 Jun 3:14:102063.
doi: 10.1016/j.toxrep.2025.102063. eCollection 2025 Jun.

Genotoxic activity of glyphosate and co-formulants in glyphosate-based herbicides assessed by the micronucleus test in human mononuclear white blood cells

Khadija Ramadhan Makame  1   2 , Yazen Aljaber  1 , Moustafa Sherif  3 , Balázs Ádám  4 , Károly Nagy  1
Affiliations

Genotoxic activity of glyphosate and co-formulants in glyphosate-based herbicides assessed by the micronucleus test in human mononuclear white blood cells

Khadija Ramadhan Makame et al. Toxicol Rep. .

Abstract

Glyphosate-based herbicides (GBHs) are widely used and contribute to soil, water, and air contamination. Despite differing assessments of its carcinogenic potential, glyphosate toxicity may be enhanced by the co-formulants (adjuvants) used to improve its effectiveness. In this study, we investigated the genotoxic effects of glyphosate, alkyl dimethyl betaine (adjuvant A), and polyethoxylated tallow amine (adjuvant B) on human peripheral white blood cells using a cytokinesis block micronucleus (CBMN) assay. The experiments tested Glyphosate (0.1, 1, 10, and 100 μM) and adjuvants (at concentrations matching their levels in respective GBHs) in whole blood samples. The samples were exposed for 4 and 20 h with and without S9 metabolic treatment. The results showed that glyphosate and adjuvant A caused a statistically significant increase in the frequency of binucleated cells with micronuclei (BNMN%) only at 100 μM after 4-hour exposure without S9 treatment. Adjuvant B, however, induced a statistically significant increase in BNMN% starting at 1 μM after 4-hour exposure without S9 treatment. No significant effects were observed after 4 h of exposure with S9 or 20 h of exposure, with or without S9. The proliferation index (PI) showed no significant changes. This study concluded that the co-formulants in GBHs can induce genotoxic effects at low concentrations and short exposure times. This indicated that some surfactants in GBHs may be more toxic than glyphosate.

Keywords: Adjuvant; Co-formulant; Genotoxicity; Glyphosate; Glyphosate-based herbicide; Micronucleus.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Karoly Nagy reports financial support was provided by Hungarian Academy of Sciences. Khadija Ramadhan Makame reports financial support was provided by Stipendium Hungaricum Scholarship Programme PhD grant. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Examples of a mononucleated cell (A), binucleated cell (B), multinucleated cell (C), and binucleated cell with micronucleus (D), as seen under high-resolution optical microscope (ZEISS®, Germany) at 1000x magnification.
Fig. 2
Fig. 2
Schematic representation of the cytokinesis-block micronucleus (CBMN) assay protocol used for evaluating the genotoxicity of the test agents in human mononuclear white blood (HMWB) cells.
Fig. 3
Fig. 3
Percentage of binucleated cells with micronuclei (BNMN%) observed after a 4 and 20-hour exposures to varying concentrations of glyphosate and two co-formulants, adjuvant A and adjuvant B, without S9 treatment (S9-). Data points represent the mean ± standard error of the mean (SEM) from three independent experiments. A statistically significant (* p < 0.05, ** p < 0.01) increase was analyzed by comparing the frequency of binucleated cells with micronuclei induced by various doses of test chemicals to the background level of untreated cells by ANOVA with Dunnett’s post hoc test.
Fig. 4
Fig. 4
Percentage of binucleated cells with micronuclei (BNMN%) observed after a 4 and 20-hour exposures to varying concentrations of glyphosate and two co-formulants, adjuvant A and adjuvant B, with S9 treatment (S9+). Data points represent the mean ± standard error of the mean (SEM) from three independent experiments. A statistically significant increase was analyzed by comparing the frequency of binucleated cells with micronuclei induced by various doses of test chemicals to the background level of untreated cells by ANOVA with Dunnett’s post hoc test.
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