Glyphosate & Glyphosate Formulations
Nominated: December 1984
Glyphosate is the most widely used herbicide in the United States and worldwide. It is applied as a formulation (or mixture) with other substances that help plants to absorb glyphosate. Glyphosate acts as an herbicide by preventing susceptible plants from making proteins that are needed for growth. Over the past 25 years, use of glyphosate has risen dramatically due to development of glyphosate-resistant genetically modified crops. Most people are exposed to residual amounts of glyphosate by ingestion of food or water. Individuals who regularly handle glyphosate products as part of their occupation may experience higher exposures. Glyphosate is also used in residential settings.
In 1992, NTP reported that rodents exposed to glyphosate in feed showed little evidence of toxicity, and there was no evidence of glyphosate causing damage to DNA. Since then, several public health agencies have reviewed the scientific literature to learn whether exposure to glyphosate is a cancer hazard for humans.
- In March 2015, the International Agency for Research on Cancer (IARC) concluded that glyphosate is a likely human carcinogen based on studies in humans and animals. They also reported that glyphosate-based formulations are often more toxic than glyphosate alone.
- In November 2015, the European Food Safety Authority (EFSA) concluded that glyphosate is unlikely to pose a carcinogenic hazard to humans.
- In May 2016, the Joint Food and Agricultural Organization of the United Nations/World Health Organization Meeting on Pesticide Residues concluded that glyphosate is unlikely to pose a carcinogenic risk to humans from exposure in the diet.
- Currently, the United States Environmental Protection Agency (EPA) is completing a new human health risk assessment on glyphosate including an evaluation of its cancer-causing potential.
Due to the multiple interpretations of evidence on the potential health risks of glyphosate exposure, major public concern about exposure risks, and reported differences in the toxicity of different glyphosate products, NTP is conducting more research on glyphosate and its formulations. We are testing the potential genetic and mechanistic toxicity, and will also examine the published scientific literature for data on the effects of glyphosate on non-cancer outcomes.
NTP Research Plan
NTP is currently pursuing glyphosate and glyphosate formulations research. Human exposure to glyphosate usually occurs in the form of glyphosate-based formulations. Few studies have made side-by-side comparisons of the toxicity of glyphosate and glyphosate-based formulations using the same experimental protocols and endpoints. Also, there have been few direct comparisons of the toxicity of different glyphosate products.
Many existing studies of glyphosate and glyphosate-based formulations have focused on whether they induce DNA damage (genetic toxicity) and/or oxidative stress, as both are mechanisms that contribute to carcinogenesis (Smith et al., 2016).
As part of the research plan, NTP will use in vitro and in vitro approaches to further investigate whether glyphosate and glyphosate-based formulations can induce genetic toxicity and/or oxidative stress. Furthermore, NTP will use a transcriptomics (changes in gene expression) approach in vitro to examine whether any other biological perturbations are caused by the test articles. This research effort to interrogate key outcomes will aid interpretation of the existing literature on glyphosate and glyphosate-based formulations.
Specific aims of NTP include the following:
- Evaluate whether glyphosate is genotoxic (causes DNA damage)
- Evaluate whether glyphosate induces oxidative damage
- Compare the effects of glyphosate on measures of genotoxicity, oxidative stress, and cell viability to the effects of glyphosate-based formulations
- Identify data gaps on the effects of glyphosate and glyphosate-based formulations on human health outcomes other than cancer.
NTP will use in vitro approaches to assess the genotoxicity and oxidative stress-inducing properties of glyphosate and glyphosate-based formulations. NTP will test glyphosate and several glyphosate-based formulations used for either agricultural or residential purposes. Agricultural formulations will be procured as available. Residential products available at home improvement stores will be selected to span a range of percentage of glyphosate in the formulations.NTP will also test (aminomethyl) phosphonic acid (AMPA), a metabolite of glyphosate that is produced by the mammalian microbiome. A screening strategy using cellular assays will identify test articles for potential follow-up experiments that may include short-term animal studies.
NTP will use the Sciome Workbench for Interactive, Computer-Facilitated Text-mining (SWIFT) software to describe the existing published literature evidence base for health outcomes investigated in connection to glyphosate exposure. This approach will help identify data gaps in the assessment of the effects of glyphosate and glyphosate-based formulations on human health.
Planned NTP Studies
In Vitro Screening Assays
Glyphosate, glyphosate-based formulations, AMPA, and positive and negative controls will be tested in in vitro screening assays using 384-well plates by the NTP Laboratory Branch. The battery of assays includes assays for oxidative stress, DNA damage, and cell viability. Furthermore, the effects of the test articles on multiple cellular pathways will be assessed using a transcriptomics assay. Several human cell lines will be used for testing, including metabolically active liver derived cells (human HepaRG cells), a lymphoblastoid cell line derived from B cell lymphocytes (TK6 cells), and skin cells (a keratinocyte cell line called HaCaT). Robust dose-response data will be generated. A benchmark dose analysis will be used to identify the concentration of test article at which biological effects first become evident, as well as the AC50.
Genetic Toxicity Testing
Glyphosate, AMPA, and glyphosate-based formulations that are active in an in vitro screening assay for DNA damage will be tested in the following in vivo genotoxicity assays, in the presence or absence of an exogenous rat liver metabolic activation system:
- Bacterial mutagenicity assays with S. typhimurium tester strains TA100, TA98, TA97, TA1535, and E. coli tester strain WP2.
- Micronucleus assay (TK6 cells)
- Comet assay (TK6 cells)
- Modified comet assay (TK6 cells): If a test article is active in the comet assay, it will be tested in a modified comet assay in which a DNA glycosylase is used to detect the presence of DNA bases that have undergone oxidative alteration
Glyphosate and at least one glyphosate-based formulation, selected based on data from the battery of in vitro assays, will be tested using the following in vivo genotoxicity assays:
- Combined micronucleus and comet assay via oral gavage using male and female Harlan Sprague Dawley rats and B6C3F1 mice.
- For the comet assay, several tissues will be evaluated in both species.
- If a test article is active in the comet assay, the assay will also be conducted as a modified comet assay in which a DNA glycosylase is used to detect the presence of DNA bases that have undergone oxidative alteration.
Significance and Expected Outcomes
The short-term studies described here will provide data on whether glyphosate and glyphosate-based formulations induce genotoxicity and oxidative stress, inform whether the test systems employed are able to discriminate among different test articles and perhaps form hypotheses related to determinants of toxicity that could be further investigated. The robust dose-response data generated in these experiments will provide effect level estimates that will hopefully aid ongoing risk evaluations.
The in vitro screening assays are expected to be completed in mid-2018 and results will be made available to the public on the NTP web site. The results of genetic toxicity testing are expected to be available by mid-2019.
Research at Other Agencies
The informational resources below provide additional details on NTP's research on glyphosate and glyphosate formulations.
- NTP Board of Scientific Counselors Meeting, Research Triangle Park, NC, June 15, 2016
- NTP Board of Scientific Counselors Meeting, Research Triangle Park, NC, December 7, 2017
- Environmental Mutagenesis & Genomics Society Annual Meeting, Washington, DC, September 19-23, 2019
- National Toxicology Program (US). NTP technical report on toxicity studies of glyphosate (CAS no. 1071-83-6) administered in dosed feed to F344/N rats and B6C3F1 mice. (NTP Toxicity Reports Series; No. 16). 1992. http://
ntp.niehs.nih.gov/ results/ pubs/ shortterm/ reports/ abstracts/ tox016/index.html
- International Agency for Research on Cancer (FR). Some organophosphate insecticides and herbicides. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; Vol. 112). 2015. http://
monographs.iarc.fr/ ENG/ Monographs/ vol112/ index.php
- Mason RP. Imaging free radicals in organelles, cells, tissue, and in vitro with immuno-spin trapping. Redox Biol. 2016 Aug;8:422-9. PubMed PMID: 27203617; PubMed Central PMCID: PMC4878322. https://
www. ncbi. nlm. nih. gov/ pubmed/ 27203617
- Food and Agriculture Organization of the United Nations. Pesticide residues in food 2016: special session of the joint FAO/WHO meeting on pesticide residues. (FAO Plant Production and Protection Paper; No. 227:1-123). 2016. http://
www. fao. org/ publications/ card/ en/ c/ 22b948ec-af63-45c9-8de1-34153d2482c5/
- Howard BE, Phillips J, Miller K, Tandon A, Mav D, Shah MR, et al. SWIFT-Review: a text-mining workbench for systematic review. Syst Rev. 2016; 5(1): 87. PubMed PMID: 27216467; PubMed Central PMCID: PMC4877757. https://
www. ncbi. nlm. nih. gov/ pubmed/ 27216467
- EPA's evaluation of the carcinogenic potential of glyphosate. Environmental Protection Agency (US); 2018. https://
cfpub. epa. gov/ si/ si_ public_ record_ Report. cfm? dirEntryId =337935
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