*Chaufan, G, Coalova, I & Rios de Molina Mdel, C 2014 oxidative stress glyphosate, AMPA and glyphosate formulation human (HepG2 cell line) Glyphosate Commercial Formulation Causes Cytotoxicity, Oxidative Effects, and Apoptosis on Human Cells Differences With its Active Ingredient. International journal of toxicology, 33(1), 29–38 Shows formulation increases ROS and has toxic effects not seen in glyphosate alone *Elie-Caille, C, Heu, C, Guyon, C & Nicod, L 2010 oxidative stress glyphosate human keratinocyte (HaCaT cell line) Morphological damages of a glyphosate-treated human keratinocyte cell line revealed by a micro-to nanoscale microscopic investigation. Cell biology and toxicology, 26(4), 331–39 Shows the timeline of membrane damage and ROS production in human keratinocytes *Gasnier, C, Dumont, C, Benachour, N, Clair, E, Chagnon, MC & Seralini, GE 2009 genotoxicity glyphosate and glyphosate formulations human (HepG2 cell line) Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology, 262(3), 184–91 Shows effects are on formulation not glyphosate concentration *Gehin, A, Guillaume, YC, Millet, J, Guyon, C & Nicod, L 2005 oxidative stress glyphosate and round-up human keratinocyte (HaCaT cell line) Vitamins C and E reverse effect of herbicide-induced toxicity on human epidermal cells HaCaT: a biochemometric approach. International journal of pharmaceutics,288(2), Shows effects are due to formulation; uses human keratinocyte cell line 26
*Mladinic, M, Berend, S, Vrdoljak, AL, Kopjar, N, Radic, B & Zeljezic, D 2009a genotoxicity glyphosate human (lymphocyte cell line) Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environmental and molecular mutagenesis, 50(9), 800–7 Shows no clear dose dependent effect *Mladinic, M, Perkovic, P & Zeljezic, D 2009b genotoxicity glyphosate human (lymphocyte cell line) Characterization of chromatin instabilities induced by glyphosate, terbuthylazine and carbofuran using cytome FISH assay. Toxicology letters, 189(2), 130–7 Cytome FISH assay; shows no hazardous effect on DNA at low concentrations *Monroy, CM, Cortes, AC, Sicard, DM & de Restrepo, HG 2005 genotoxicity glyphosate human (GM38 and fibrosarcoma HT1080 cell lines) Cytotoxicity and genotoxicity of human cells exposed in vitro to glyphosate.Biomedica, 25(3), 335–45 Suggests MOA not limited to plants Prasad, S, Srivastava, S, Singh, M & Shukla, Y 2009 genotoxicity glyphosate swiss albino mice Clastogenic effects of glyphosate in bone marrow cells of Swiss albino mice. Journal of toxicology, 2009 Shows positive clastogenic and cytotoxic effects in mouse bone marrow
*Rank, J, Jensen, AG, Skov, B, Pedersen, LH & Jensen, K 1993 genotoxicity glyphosate isopropylamine salt and Roundup in vitro and in vivo Genotoxicity testing of the herbicide Roundup and its active ingredient glyphosate isopropylamine using the mouse bone marrow micronucleus test, Salmonella mutagenicity test, and Allium anaphase-telophase test. Mutation Research/Genetic Toxicology, 300(1), 29–36 Shows negative effects for glyphosate in three genotoxicity tests *Considered by EFSA ()
B – IARC 112 The studies that were referenced in the IARC monograph that the OCS Tier 1 assessment concluded required further assessment to determine their relevance to the carcinogenicity classification of glyphosate are presented below in Table 7. The APVMA will rely on international assessments of these studies to determine whether glyphosate should be placed under formal reconsideration. Table 7: List of studies recommended by the OCS for further assessment to determine if relevant to carcinogenicity classification of glyphosate Author(s) Year Endpoint Formulation type (if stated) Species Title/publication details Comments Website *Alavanja, MC, Samanic, C, Dosemeci, M, Lubin, J, Tarone, R, Lynch, CF, Knott, C, Thomas, K, Hoppin, JA, Barker, J, Coble, J, Sandler, DP & Blair, A. 2003 Carcinogenicity/ epidemiology unknown formulation human Use of agricultural pesticides and prostate cancer risk in the Agricultural Health Study cohort. American Journal of Epidemiology,157(9), 800–14 No direct reference to glyphosate in abstract, increased risk to ‘other pesticides’ only seen in subjects with a FHx of prostate cancer *Astiz, M, de Alaniz, MJ, & Marra, CA. 2009b oxidative stress glyphosate rat Antioxidant defense system in rats simultaneously intoxicated with agrochemicals. Environmental toxicology and pharmacology, 28(3), 465–73 Glyphosate administered alone and in combo with other a.i.’s; unclear if results are for combo; in vivo rat model Astiz, M, Hurtado de Catalfo, GE., García, MN, Galletti, SM, Errecalde, AL, de Alaniz, MJ, & Marra, CA. 2013 oxidative stress glyphosate wistar rat Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol. Ecotoxicology and environmental safety, 91, 129–38 Oxidative stress seen in testicular cells; investigates antioxidant treatment after administration; unclear if administered in combo
Benachour, N, & Séralini, GE. 2009 MOA Roundup human (umbilical, embryonic, placental cell lines) Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells.Chemical research in toxicology, 22(1), 97–105 Uses glyphosate formulations, investigates metabolites Benachour, N, Sipahutar, H, Moslemi, S, Gasnier, C, Travert, C, & Séralini, GE. 2007 MOA Roundup (bioforce) human (embryonic and placental cell lines) Time-and effects of roundup on human embryonic and placental cells. Archives of Environmental Contamination and Toxicology,53(1), 126–33 Uses glyphosate formulations, investigates toxicity and *Bolognesi, C, Carrasquilla, G, Volpi, S, Solomon, KR, & Marshall, EJP. 2009 glyphosate + cosmo-flux human Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate.Journal of Toxicology and Environmental Health, Part A, 72(15-16), 986–97 Columbian aerial spray program; uses formulation as exposure to glyphosate; measurement of binucleated lymphocytes with micronuclei as DNA damage Brewster, DW, Warren, J, & Hopkjns, WE. 1991 metabolism glyphosate SD rat Metabolism of glyphosate in Sprague-Dawley rats: tissue distribution, identification, and quantitation of materials following a single oral dose.Toxicological Sciences, 17(1), 43–51 Tissue distribution study, shows no persistence in body after single oral dose
Brown, LM, Burmeister, LF, Everett, GD, & Blair, A. 1993 unknown formulation human Pesticide exposures and multiple myeloma in Iowa men. Cancer Causes & Control, 4(2), 153–6 No direct reference to glyphosate or roundup; shows little evidence of association between pesticides and multiple myeloma Cattani, D, Cavalli, VLDLO, Rieg, CEH, Domingues, JT, T, Tasca, CI, & Zamoner, A. 2014 oxidative stress Roundup rat Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity. Toxicology, 320, 34–45 Uses formulation; neurotoxic effects on rat hippocampus Çavuşoğlu, K, Yapar, K, Oruç, E, & Yalçın, E. 2011 oxidative stress Roundup SA mouse Protective effect of Ginkgo biloba L. leaf extract against glyphosate toxicity in Swiss albino mice.Journal of medicinal food, 14(10), Uses formulation; i.p. to mice; studies the effect of Ginkgo against effects seen Chruscielska, K, Brzezinski, J, Kita, K, Kalhorn, D, Kita, I, Graffstein, B, & Korzeniowski, P. 2000 toxicity Glyphosate. Evaluation of chronic activity and possible far-reaching effects. Part 1. Studies on chronic toxicity. Pestycydy, 3 Chronic toxicity study review Coalova, I, de Molina, MDCR, & Chaufan, G. 2014 oxidative stress atanor + impacto (adjuvant) human (Hep-2 cell line) Influence of the spray adjuvant on the toxicity effects of a glyphosate formulation. Toxicology in Vitro,28(7), 1306–11 Uses formulation and adjuvant on Hep-2 cell line; shows toxicity and ROS
*Dimitrov, BD, Gadeva, PG, Benova, DK, & Bineva, MV. 2006 genotoxicity Roundup mouse (bone marrow) Comparative genotoxicity of the herbicides Roundup, Stomp and Reglone in plant and mammalian test systems. Mutagenesis, 21(6), 375–82 Comparative study using glyphosate formulation; negative results *Engel, LS, Hill, DA, Hoppin, JA, Lubin, JH, Lynch, CF, Pierce, J, Samanic, C, Sandler, DP, Blair, A & Alavanja, MC. 2005 unknown formulation human Pesticide use and breast cancer risk among farmers’ wives in the agricultural health study. American Journal of Epidemiology,161(2), 121–35 Uses formulation; glyphosate not directly referenced in the abstract; no clear association with breast cancer *Eriksson, M, Hardell, L, Carlberg, M, & Åkerman, M. 2008 unknown formulation human Pesticide exposure as risk factor for non‐Hodgkin lymphoma including histopathological subgroup analysis. International Journal of Cancer, 123(7), 1657–63 Uses formulation; results were not adjusted for multiple exposures; shows increased risk of NHL for glyphosate *Considered by EFSA ()
SD: Sprague Dawley; MTD: maximum tolerated dose; HD: high dose; MD: median dose; LD: low dose; M: males; F: females; GLP: good laboratory practice; OECD (TG): the organisation for economic co-operation and development (testing guideline) Discussion and OCS conclusion All of the studies evaluated by were assessed and cited by the EFSA RAR (). Based on a review of the supplementary raw data provided within the documents and in parallel with the conclusions of the EFSA RAR (), the OCS conclusions are consistent with and the EFSA RAR () that the evidence indicates that glyphosate is not carcinogenic in animals. As part of the review of , the cited review paper by was considered. All of the studies reviewed by were referenced by the IARC monograph. Five of the studies cited by were reviewed previously by the OCS in 2005 (; ; ; ; ). The OCS weight-of-evidence conclusion in 2005 was that glyphosate is not mutagenic or carcinogenic and it is unlikely that exposure to glyphosate is associated with an increased risk of NHL. Of the remaining studies cited by , four (; ; ; ) were considered during the Tier 1 assessment as not appropriate for review due to a lack of reference to glyphosate in the abstract and the remaining 12 were indicated in the proposed Tier 2, Part 2 review of the monograph. However, as these papers were critically evaluated by the international JMPR assessment of glyphosate, the proposed Tier 2, Part 2 assessment will not be undertaken by the OCS.