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Glyphosate & Glyphosate Formulations

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Research Overview

Status: Ongoing
Substances: Glyphosate
Nominated: December 1984

Background Information

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.

Test Results

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.

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

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.

Methods

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-FacilitatedText-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 vitro 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 battery of in vitro assays, will be tested using the following in vitro 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

United States
International

Informational Resources

The informational resources below provide additional details on NTP's research on glyphosate and glyphosate formulations.

Presentations
Supporting Documents

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