Lab rat and animal testing

In vitro tests on three GMO-associated pesticides show alarming toxicity of 2,4-D but miss toxic effects of glyphosate that show up in other types of study

Testing of toxic chemicals like pesticides in cell lines can provide useful information but may not accurately predict their ability to damage DNA and cause cancer in living organisms, research published in the journal Food and Chemical Toxicology shows.

Tests on three GMO-associated pesticides in cell lines showed that 2,4-D had high carcinogenic potential (an outcome reflected in other types of test) but missed the cancer-triggering effects of glyphosate that show up in many studies on living humans or animals. Results on dicamba are suspected of underestimating the full extent of the pesticide’s effects on carcinogenicity.

The findings come as international and national pesticide assessment bodies such as the US Environmental Protection Agency (EPA) and the Organisation for Economic Cooperation and Development (OECD) are moving towards computer modelling and in vitro studies in an attempt to reduce dependency on animal testing.

GM crop-associated pesticides tested

For the research, scientists led by Dr Michael Antoniou and Dr Robin Mesnage at King's College London tested three GM crop-associated pesticides alone and in combination using a testing system called ToxTracker. The ToxTracker assay system consists of mouse cells known as embryonic stem cells, with genes inserted whose expression can provide a readout of potential carcinogenic outcomes. If a chemical tested in this system stimulates expression of these “reporter” genes, then it is scored as a potential carcer-causing agent.     

The three pesticides tested by Dr Antoniou’s team were 2,4-D, dicamba, and glyphosate, all of which are used on GM herbicide-tolerant crops. As a result of the widespread planting of these crops in certain countries, exposure to these pesticides is increasing.

ToxTracker is designed to measure several carcinogenic properties of substances, from a list of 10 identified by the International Agency for Research on Cancer (IARC). The properties targeted by ToxTracker are DNA damage, oxidative stress (a destructive imbalance in the body), and the unfolded protein response – all of which can trigger or indicate the onset of cancer.

The manufacturers of ToxTracker aim to take advantage of the regulatory shift away from animal testing, stating that their method “can contribute to a mechanism-based, animal-free, cancer hazard assessment of chemicals”.

In the research, the pesticides were tested in their pure forms, without the additives (co-formulants or adjuvants) that they are mixed with in commercial formulations. These additives are known to be toxic in their own right and to enhance the toxicity of the isolated active ingredient.

The scientists’ aim was to mirror regulatory practices, which only consider the isolated declared “active” pesticide ingredient and not the complete formulations as sold and used. They also wanted to see if ToxTracker could identify the enhanced toxicity that can result from pesticide mixtures, as compared with the individual pesticides tested alone.


The ToxTracker test showed that 2,4-D was a strong inducer of oxidative stress and an unfolded protein response, but damaged DNA only to a limited extent. The link between 2,4-D and oxidative stress is confirmed by human epidemiological studies, as well as studies in animals and cell lines. IARC too stated that there is strong evidence from mechanistic studies that 2,4-D causes oxidative stress.

However, IARC considered that there was inadequate evidence for 2,4-D carcinogenicity in humans, as epidemiological studies showed mixed results. Thus IARC concluded that 2,4-D is only “possibly carcinogenic to humans”.

ToxTracker accurately predicted 2,4-D’s ability to cause oxidative stress.


In the ToxTracker test, dicamba induced moderate oxidative stress but, in common with the other two pesticides tested, did not cause much damage to DNA in the cell lines. However, in an epidemiological study in humans, dicamba was found to be associated with the development of cancer in the liver and liver bile duct.

Dr Antoniou’s team concluded that the pathways by which dicamba exerts potential carcinogenic effects are likely to be indirect, without involving DNA damage through oxidative stress.

It appears that ToxTracker may not have revealed the full extent of dicamba’s potential to cause cancer, which would need to be further investigated in animal tests.


The most surprising result involved glyphosate, which ToxTracker did not predict to be carcinogenic or genotoxic in any of the tests. This rang alarm bells because, as Dr Antoniou and colleagues state in their study, it was at odds with a large body of evidence from other model systems suggesting that glyphosate has genotoxic and carcinogenic effects. There is also plenty of evidence showing that glyphosate or its commercial formulations can cause oxidative stress, as demonstrated in a large range of organisms, including human cells, rats, mice, plants, worms, frogs, and aquatic organisms.

Indeed, a separate study in living rats led by Drs Mesnage and Antoniou show that both glyphosate and its commercial formulations (various Roundup herbicides) cause oxidative stress and DNA damage – both indications of cancer-causing potential. GMWatch reported on this study in April this year.

Why, then, didn't the ToxTracker tests in cell lines find that glyphosate was carcinogenic? The study in rats shows that the mechanism by which glyphosate causes oxidative stress and DNA damage in living animals is indirect, via inflammation and organ damage. Isolated cell lines cannot reveal these effects because the full range of complex interactions that are needed to produce them simply do not happen. So cell line tests cannot mimic what actually occurs in a living body.

The scientists conclude that studies in cell lines can provide valuable predictive information on carcinogenicity potential, but cannot as yet replace studies in laboratory animals with real-life exposure scenarios.

Mixture effects

ToxTracker did not identify strongly increased toxicity from the mixture of the three herbicides, over and above what could be seen by looking at the most toxic of the three (2,4-D) alone.

The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D, with dicamba and glyphosate only playing a minor role.

Study lead Dr Michael Antoniou comments, “Given the strong evidence from studies in live animals that mixture effects can be more toxic at lower levels than effects of isolated substance tested alone, it is questionable whether testing in cell lines can give a fully accurate picture of mixture toxicity.”

Dr Antoniou’s team is following up this line of inquiry with studies on the effects of pesticide mixtures in living animals.  

Commenting on the full range of results from the research, on the pesticides tested alone and in combination, Dr Antoniou said, “We would all welcome a situation in which animal tests were unnecessary. But as long as humankind continues to use known and suspected toxins on our farms and food, we need to use animal tests because the currently available alternatives are not able to predict the full range of effects on living organisms such as humans and other animals.”

It’s a conclusion that is shared by experts interviewed by science journalist Sharon Lerner in an article for The Intercept about the EPA’s intention to phase out the use of animal experiments in assessing toxic chemicals. Lerner writes, “Without the tests on rats, mice, and rabbits currently used to gauge the toxicity of chemicals and set safe levels, public health and environmental advocates worry that the policy shift will leave EPA unable to limit chemicals at all.” She quotes Jennifer Sass, a senior scientist at the Natural Resources Defense Council, as saying, “It effectively will mean you can’t regulate.”

GM crops

The principle also applies to GM crops. Currently short 90-day animal feeding studies are required in the EU for assessing the safety of single-trait GM crops, though stacked trait GM crops (with multiple GM traits engineered into them) do not have to be subjected to animal testing, as EFSA believes that feeding trials with just the single-trait GM crops that go into making the stacked trait crop are sufficient. This, however, ignores the issue of combinatorial effects of multiple GM traits.

Now the GMO industry is attempting to remove the requirement for even the 90-day animal feeding trial by deregulating a new generation of GM crops produced using gene editing. This will leave us at the mercy of whatever assumptions and arguments GMO developers choose to make about their products, without the hard biological evidence provided by the experimental animals that consume them.

Whether the product is a GM crop or a pesticide, abandoning animal studies would create serious risks for public health.