^fcDsrx ' O ' UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION MEMORANDUM Date: January 13, 2019 SUBJECT: Glyphosate: Response to Comments on the Proposed Interim Decision Regarding the Human Health Risk Assessment PC Code: 417300; 103601; 103603; 103604; 103605; 103607;103608;103613 Decision No.: 558081 Petition No.: NA Risk Assessment Type: NA TXR No.: NA MRU) No.: NA DP Barcode: D455459 Registration No.: NA Regulatory Action: Registration Review Case No.: *178 CAS No.: 1071-83-6; 38641-94-0; 70393-85-0; 114370-14-8; 40465-76-7; 69254-40-6; 34494-04-7; 70901-12-1 40 CFR: §180.364 FROM: Monique M. Perron, Sc.D., Toxicologist Risk Assessment Branch 1 (RAB1) Health Effects Division (HED; 7509P) THROUGH: Christine L. Olinger, Branch Chief RAB1/HED (7509P) TO: Steven Peterson, Chemical Review Manager Dana Friedman, Branch Chief Pesticide Re-evaluation Division (PRD; 7508P) The Office of Pesticide Programs received hundreds of thousands of public comments on the proposed interim decision (PID) for glyphosate as part of registration review. Comments regarding the human health risk assessment came from a wide array of stakeholders. Topics relating to human health included concerns with the agency's cancer assessment, toxicological studies, protection of children, and detections of glyphosate. These comments regarding the human health risk assessment for glyphosate have been previously addressed in the Glyphosate: Response to Comments on the Raman Health Draft Risk Assessment (D448021; M. Perron; 23 - APR-2018) and did not result in changes to the agency's risk assessment. During the public comment period, 65 open literature studies were also identified for the agency's consideration (Appendix A). Of these, 23 were previously identified and considered by Page 1 of 13 ------- the agency as part of two open literature searches conducted to support the draft human health risk assessment for registration review. The agency reviewed the open literature as part of the Glyphosate Systematic Review of Open Literature (D417703; TXR 0056885; M. Perron; 12- DEC-2017) for hazard identification and characterization purposes in order to identify studies that could potentially impact the human health risk assessment. A fit-for-purpose systematic review was also executed to obtain relevant and appropriate open literature studies with the potential to inform the human carcinogenic potential of glyphosate and detailed in the Revised Glyphosate Issue Paper: Evaluation of Carcinogenic Potential (D444689; TXR 0057688; G. Akerman; 12-DEC-2017). The remaining 42 studies identified during the public comment period were primarily journal articles published since these searches were conducted. The majority of the studies did not warrant detailed evaluation for a variety of reasons, such as no effects seen from glyphosate exposure, administration via a non-relevant route, effects seen at doses higher than the current points of departure, only one dose was tested eliminating the potential for dose-response evaluation, lack of glyphosate measurements, and exposure or biomonitoring studies that would not impact risk estimates. Furthermore, several studies were conducted in vitro or evaluated biochemical or molecular effects that are difficult to translate into in vivo effects. These studies are typically considered as part of mode of action/adverse outcome pathway (MOA/AOP) analyses. As a result, in vivo studies are given more weight at this time. For the in vivo studies identified, the most common limitations/deficiencies were related to the nature of the test substance(s) used for exposure. Many of the studies used commercial formulations or dilutions; however, direct measurements of the active ingredient were not conducted in order to determine actual dose concentrations and/or identification information was not provided for the formulation used. There are numerous glyphosate formulations and providing a general product name, such as Roundup, does not provide the agency with information to ascertain the exact formulation used and determine all of its chemical components. Additionally, several studies were conducted in other countries and utilized formulations that are not registered in the United States. As a result, the active ingredients and other components of the formulation are unknown, and any potential effects cannot be attributed to glyphosate and/or defined glyphosate concentrations. Several in vitro genotoxicity studies were identified for consideration. Although positive results were observed in some of these studies, there would be no impact on the agency's weight of evidence evaluation of the genotoxic potential of glyphosate since there is sufficient evidence in the existing database (described in the Revised Glyphosate Issue Paper: Evaluation of Carcinogenic Potential; D444689; TXR 0057688; G. Akerman; 12-DEC-2017) to conclude that the in vitro effects claimed by the study authors do not lead to genotoxicity in vivo. Two additional journal articles on the association between glyphosate exposure and non- Hodgkin lymphoma (NHL) were identified for detailed review (D455531; D. Miller; 6-JAN- 2020;). The following are brief summaries of these journal articles and the agency's reviews: • Zhang et al. (2019) is a review article summarizing epidemiological studies between 2001 and 2018 on the association between glyphosate exposure and NHL. All of the data/information included in the article was previously considered by the agency as part Page 2 of 13 ------- of the Revised Glyphosate Issue Paper: Evaluation of Carcinogenic Potential (D444689; TXR 0057688; G. Akerman; 12-DEC-2017); however, the authors conducted their own meta-analysis to incorporate recently published risk estimates for the Agricultural Health Study (AHS) cohort (Andreotti et al., 2018) based on their a priori hypothesis that the highest exposures to glyphosate will lead to increased risk of NHL in humans1. In its detailed review of this study, the agency identified concerns with the meta-analysis as performed by the authors. A supplemental analysis by the agency indicated that a lower non-statistically significant meta-risk ratio of 1.14 (95% CI: 0.87-1.50) would be obtained if the Andreotti et al. (2018) study is properly incorporated into the meta- analysis2. Therefore, the meta-analyses performed by Zhang et al. (2019) would not impact the conclusions presented in the agency's revised issue paper. • Leon et al. (2019) is a pooled analysis of NHL in agricultural cohorts from France, Norway, and the United States (the AHS cohort). For overall NHL malignancies and NHL subtypes, except diffuse large B-cell lymphoma (DLBCL), glyphosate risk estimates were less than 1. For DLBCL a somewhat elevated, but non-statistically significant risk estimate of 1.36 (95% CI: 1.00-1.85) was observed. While the analysis benefited from a combined cohort of more than 300,000 farmers and farmworkers from different countries, only the cohort from the United States used actual measurement instruments (self-administered questionnaire) for glyphosate exposure where a smaller risk estimate for the DLBCL subtype of 1.2 (95% CI: 0.72-1.98) was obtained. Furthermore, the nature and characteristics of the three cohorts differed in substantive ways and it is not clear that the statistical adjustments made were adequate to account for these differences. Therefore, none of the open literature studies identified for the agency's consideration were found to have an impact on the glyphosate hazard characterization, cancer assessment, or human health risk assessment. The agency will continue to monitor the open literature for studies that use scientifically sound and appropriate methodology and relevant routes of exposure that have the potential to impact the risk evaluation of glyphosate. 1 Here, higher exposures correspond to higher levels, longer durations, and/or greater lags/longer latencies. 2We note, however, that the meta-estimate in our Revised Glyphosate Issue paper was 1.27 (95% CI: 1.01. 1.59) while our updated estimate after incorporating the Andreotti et al. (2018) paper is 1.14 (95% CI: 0.87, 1.50). See D. Miller; D455531; 6-JAN-2020 for details and information. Page 3 of 13 ------- Appendix A. Studies identified for the agency's consideration during the public comment period for the Proposed Interim Decision (PID). Author Year Title Part of previous agency open literature reviews? Comments Aiassa, D 2019 Evaluation of genetic damage in pesticide applicators from the province of Cordoba, Argentina No ecological epidemiological study; no glyphosate-specific estimates; would be assigned low quality ranking according to process detailed in revised issue paper on evaluation of carcinogenic potential Alvarez-Moya, C 2013 Comparison of the in vivo and in vitro genotoxicity of glyphosate isopropylamine salt in three different organisms Yes -- Astiz, M 2009 Antioxidant defense system in rats simultaneously intoxicated with agrochemicals Yes -- Baier, Carlos J 2017 Behavioral impairments following repeated intranasal glyphosate-based herbicide administration in mice No Argentina formulation; micropipette administration into nostrils (not a relevant route); one dose tested; no glyphosate measurements Benbrook, CM 2019 How did US EPA and IARC reach diametrically opposed conclusions on the genotoxicity of glyphosate-based herbicides? No review paper; examined list of studies published since agency's evaluation; supplemental tables comparing EPA and IARC yielded one study to reexamine (Gasnier et al. 2009 included in table below) Bolognesi, C 2009 Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate Yes -- Bolognesi, C 1997 Genotoxic Activity of Glyphosate and Its Technical Formulation Roundup Yes -- Page 4 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Bolognesi, C 2011 Micronuclei and pesticide exposure No Review article for biomonitoring studies for several pesticides; already considered the one study on glyphosate (Bolognesi et al. 2009) in previous systematic review Brendler-Schwaab, S 2005 The in vivo comet assay: use and status in genotoxicity testing No No glyphosate information Caballero, M 2018 Estimated Residential exposure of agricultural chemicals and premature mortality by Parkinson's disease in Washington state No Glyphosate exposure assessed indirectly using geospatial information on residential address and crop data from WA State from 2011-2015; residential address was based only on address listed in the death registry for 2011-2015 so the analysis was cross-sectional in nature and was unable to assess potential lifetime exposure based on either changes in WA State agriculture or changes in residential address; statistical analysis only considered the demographic variables sex, race, marital status, and education indicating limited ability to assess confounding or control for co-exposure to pesticides and other environmental factors; given these limitations, the modest risk estimate of 1.3 (95% CI: 1.0- 1.62) may reflect residual confounding and is based on a low quality exposure assessment that did not directly assess individual exposure or allow the investigators to assess lifetime exposure Page 5 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Cavalli, VL 2013 Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells Yes -- Dimitroy, BD 2006 Comparative genotoxicity of the herbicides Roundup, Stomp and Reglone in plant and mammalian test systems Yes -- Gasnier, C 2009 Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Yes In previous open literature review considered non-relevant; reexamination indicated it is relevant; in vitro study evaluating glyphosate alone and formulations for cytotoxicity, estrogenic activity, anti-androgenic activity and aromatase disruption; only effect reported for glyphosate alone was a statistically significant, non-concentration dependent increase in anti-androgenic activity Gehin, A 2005 Vitamins C and E reverse effect of herbicide- induced toxicity on human epidermal cells HaCat: a biochemometric approach Yes -- Ghisi, N 2016 Does exposure to glyphosate lead to an increase in the micronuclei frequency? A systematic and meta- analytic review Yes -- Gillezeau, C 2019 The evidence of human exposure to glyphosate: a review No Exposure study; review article; no impact on risk estimates Grisolia, C 2002 A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides Yes -- Heu, C 2012 Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells Yes -- Page 6 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Jiang, Xiao 2018 A commercial Roundup formulation induced male germ cell apoptosis by promoting the expression of XAF1 in adult mice. No Formulation by gavage at 60, 180 or 540 mg/kg/day and in vitro exposures; no glyphosate measurements; potential effects only seen at dose higher than current POD Kasuba, V 2017 Effects of low doses of glyphosate on DNA damage, cell proliferation and oxidative stress in the HepG2 cell line No In vitro study evaluating DNA damage, cell proliferation and oxidative stress in HepG2 cells tested at 3 concentrations of glyphosate; non-statistically significant increase in proliferative response; primary DNA damage (comet assay) decreased relative to negative control; increase in MN and effects on lipid peroxidation were not concentration dependent; no convincing glyphosate-related effects Kojima, H 2010 Endocrine-disrupting Potential of Pesticides via Receptors and Aryl Hydrocarbon Receptor No Mini-review; only mention of glyphosate is in one table and it was negative for all receptors Kongtip, P 2017 Glyphosate, and paraquat in maternal and fetal serum in Thai Women No Exposure study; no impact on risk estimates Kubsad, D 2019 Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology No Intraperitoneal injection of glyphosate at 25 mg/kg/day in pregnant females; not a relevant route Kwiatkowska, M 2016 DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study) No Report primary DNA damage at high concentrations of glyphosate (0.5 mM and higher) and increase in DNA methylation of p53 and pl6 promoters; methylation was statistically significantly increased at both concentrations for the p53 promoter, but not pi6; not enough concentrations tested to make a conclusion on impact of glyphosate on p53 methylation. Page 7 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Leon, M 2019 Pesticide use and risk of non-Hodgkin lymphoid malignancies in agricultural cohorts from France, Norway and the USA: a pooled analysis from the AGRICOH consortium No Detailed review performed (D. Miller; 6- JAN-2020; D455531); summarized in text above Lioi, M 1998 Genotoxicity and oxidative stress induced by pesticide exposure in bovine lymphocyte cultures in vitro Yes -- Lueken, A 2004 Synergistic DNA damage by oxidate stress (induced by H202) and nongenotoxic environmental chemicals in human fibroblasts Yes -- Manas, F 2009 Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetics test Yes -- Manas, F 2009 Genotoxicity of glyphosate by the comet assay and cytogenetic test Yes -- Manservisi, F 2019 The Ramazzini Institute 13- week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on development and endocrine system No Glyphosate and formulation; no blinding to dose; small sample size for developmental evaluation (n=8/dose); generally no adverse effects observed; only one dose tested; no glyphosate measurement Mao, Q 2018 The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on the microbiome No Glyphosate and formulation; no glyphosate measurement; evaluated changes in microbiome; no link to adverse apical outcomes Martinez, A 2019 Effects of glyphosate and aminomethylphosphonic acid on an isogeneic model of the human blood- brain barrier No In vitro study; no glyphosate measurements Mensah, PK 2015 Ecotoxicology of glyphosate and glyphosate-based herbicide - toxicity to wildlife and humans No Book chapter; all references on human health effects from glyphosate already considered Page 8 of 13 ------- Part of previous Author Year Title agency open literature reviews? Comments Mesnage, R 2016 Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide No Belgium formulation; proteome and metabolome data; changes may reflect adaptive liver effects; no link to adverse apical outcomes Milesi, MM 2018 Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats No Argentina formulation by diet at 2 or 200 mg/kg/day; potential effects at dose higher than current POD Milic, M 2018 Oxidative stress, cholinesterase activity, and DNA damage in the liver, whole blood, and plasma of Wistar rats following a 28-day exposure to glyphosate No Glyphosate administered via gavage at 0.1, 0.5, 1.75, and 10 mg/kg/day; glyphosate was not measured in dose preparations and small sample size (n=5) used; no change in body and liver weights; plasma and liver ROS and plasma GSH levels similar to controls, no change in GSH activity in blood, inconsistent choline sterase data and no neurotoxicity MOA/AOP for glyphosate Study measured glyphosate/AMPA metabolite excretion levels in NAFLD Mills, PJ 2019 Glyphosate excretion is associated with steatohepatitis and advanced liver fibrosis in patients with fatty liver disease No patients who were either NASH (Non- Alcoholic Steatohepatitis) or not NASH patients and compared glyphosate and AMPA measurement in urine; no subjects without NAFLD; problems with temporality; single urine measurement only; no information collected on dietary intake or occupation Mills, PJ 2017 Excretion of the herbicide Glyphosate in older adults between 1993-2016 No Exposure/biomonitoring study; no impact on risk estimates Niemann, L 2015 A critical review of glyphosate findings in human urine sample and comparison with the exposure of operators and consumers No Review article for exposure; no impact on risk estimates; calculations show glyphosate exposure well below Page 9 of 13 ------- Part of previous Author Year Title agency open literature reviews? Comments acceptable daily intake (ADI) and acceptable operator exposure levels (AOEL) Parvez, S 2018 Glyphosate exposure in pregnancy and shortened gestational length: a prospective Indiana birth cohort study No Glyphosate levels in urine and drinking water; cross-sectional study evaluating correlation; no AMPA metabolite measured; small sample size; limited diversity; no evaluation of other chemical exposures (except smoking, caffeine, and alcohol) Panzacchi, S 2018 The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: study design and first in-life endpoints evaluation No Glyphosate and formulation; no glyphosate measurement; no adverse effects observed Perry, M 2019 Historical evidence of glyphosate exposure from a US agricultural cohort No Exposure/biomonitoring; no impact on risk estimates Pham, T 2019 Perinatal exposure to glyphosate and a glyphosate- based herbicide affect spermatogenesis in mice No Glyphosate and Belgium formulation; no glyphosate measurement; small sample size for some parameters; no adverse effect in several parameters; small magnitude of change and/or lack of dose response in others; no incidence or severity scores reported for histopathological evaluations Portier, C 2016 Difference in the carcinogenic evaluation of glyphosate between the IARC and EFSA Yes -- Prasad, S 2009 Clastogenic Effects of Glyphosate in Bone Marrow Cells of Swiss Albino Mice Yes -- Richard, S 2005 Differential Effects of Glyphosate and Roundup on Human Placenta Cells and Aromatase Yes -- Page 10 of 13 ------- Part of previous Author Year Title agency open literature reviews? Comments Roustan, A 2014 Genotoxicity of mixtures of glyphosate and atrazine and their environmental transformation products before and after photoactivation Yes -- Saleh, SM 2018 Hepato-morphology and biochemical studies on the liver of albino rats after exposure to glyphosate- Roundup No Egypt formulation; no glyphosate measurements; histopathological findings cannot be interpreted without incidence or severity scoring Authors report chromosomal aberrations and micronuclei formation in human Santovito, A 2018 In vitro evaluation of genomic damage induced by glyphosate on human lymphocytes No lymphocytes treated in vitro with 0.025 (ig/mL glyphosate and above; effects reported are inconsistent with other findings in the literature; sample size too small to make reliable conclusions for low test concentration Schimpf, MG 2015 Neonatal exposure to a glyphosate based herbicide alters the development of the rat uterus No sc injection (not a relevant route); formulation not registered in US; no glyphosate measurements; only one dose tested Sena de Souza, J 2019 Maternal glyphosate-based herbicide exposure alters antioxidant-related genes in the brain and serum metabolites of male rat offspring No Brazil formulation; gene expression profiles; no link to adverse apical outcomes Brazil formulation; no glyphosate Sena de Souza, J 2017 Perinatal exposure to glyphosate-based herbicide alters the thyrotrophic axis and causes thyroid hormone homeostasis imbalance in male rats No measurements; no changes in T3 or T4; TSH decreased rather than increased and presented a flat dose response; hormone measurement calibration information not reported; gene and metabolomic data Seneff, S 2018 Is Glyphosate a key factor in mesoamerican nephropathy? No Review article; hypothesis generating paper Sivikova, K 2005 Cytogenetic effect of technical glyphosate on cultivated bovine peripheral lymphocytes Yes -- Page 11 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Stur, E 2019 Glyphosate-based herbicides at low doses affect canonical pathways in estrogen positive and negative breast cancer cell lines No Brazil formulation testing with breast cancer cell lines; no glyphosate measurements Szepanowksi, F 2018 Differential impact of pure glyphosate and glyphosate-based herbicide in a model of peripheral nervous system myelination No No differences in myelination in cultures treated with glyphosate compared to vehicle control Teleken, JL 2019 Glyphosate-based herbicide exposure during pregnancy and lactation malprograms the male reproductive morphofunction in F1 offspring No Brazil formulation; no glyphosate measurements/only one dose tested that is above current POD Thongprakaisang, S 2013 Glyphosate induces human breast cancer cells growth via estrogen receptors Yes -- Townsend, M 2017 Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity No DNA damage in vitro only at concentrations that the authors report as several orders of magnitude larger than those attainable in vivo Vigfusson, N 1980 The effect of the pesticides, Dexon, Captan and Roundup, on sister-chromatid exchanges in human lymphocytes in vitro Yes -- Von Ehrenstein, 0 2019 Prenatal and infant exposure to ambient pesticides and autism spectrum disorder in children: population based case-control study No Assumed exposure from agricultural application records (no actual measurements) other exposure pathways not considered; addresses at time of enrollment may not reflect an individual's exposure over pregnancy Walsh, L 2000 Roundup Inhibits Steroidogenesis by Disrupting Steroidogenic Acute Regulatory (StAR) Protein Expression Yes -- Wang, L 2019 Glyphosate induces benign monoclonal gammopathy and promotes multiple myeloma progression in mice No incorrect EPA chronic reference dose cited; only one dose tested that is above current POD Page 12 of 13 ------- Author Year Title Part of previous agency open literature reviews? Comments Wozniak, E 2018 The mechanism of DNA damage induced by Roundup 360 PLUS, glyphosate and AMP A in human peripheral blood mononuclear cells - genotoxic risk assessment (Accepted Manuscript) No In vitro study using human lymphocytes; DNA damage at glyphosate concentrations of 250 (.iM and higher Zhang, L 2019 Exposure to glyphosphate-based herbicides and risk for non-Hodgkin lymphoma: A meta-analysis and supporting evidence No Detailed review performed (D. Miller; 6- JAN-2020; D455531); summarized in text above Page 13 of 13 ------- |