How GMO-Free is "GMO-Free"? Decoding Global Regulations

4 Crucial Questions about GMO Testing New Labeling Laws in the USA

Spot On is a publication of Romer Labs Division Holding GmbH, distributed free-of-charge.

ISSN: 2414-2042

Editors:

Joshua Davis, Cristian Ilea

Contributors:

Pavlo Futernyk, Donna Houchins, Christina Huber

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Research: Kurt Brunner

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©Copyright 2019, Romer Labs®

How GMO-Free is "GMO-Free"? Decoding Global Regulations, Labeling Conventions, and Testing Requirements for Genetically Modified Crops

As technologies to produce GM plants have developed, regulations that govern their cultivation, import and export have multiplied and adapted. R&D specialist Donna Houchins of Romer Labs breaks down some trends in important areas, such as labeling, and discusses how producers and traders can stay compliant amid this shifting regulatory landscape.

By Donna Houchins, Research & Development Specialist, Romer Labs

Labeling Laws for GMOs in the United States

By Christina Huber, Product Manager, Romer Labs

The Science of GMOs: Some Important Terms

By Christina Huber, Product Manager, Romer Labs

GMO Testing: 4 Important Questions

Are you testing protein or DNA? How do methods for testing raw materials differ from those of processed materials? These are just two considerations that anyone looking to test for GMOs needs to keep in mind. Product manager Christina Huber offers some practical answers.

Romer

By Christina Huber, Product Manager, Romer Labs®

Photo: hauged

GMOs: staying on top of the latest regulations and testing methods

Genetically modified organisms became a part of how the world feeds itself more than 20 years ago. Since then, the human population has increased from 5.8 to 7.7 billion people. The increased production of food became possible because of effective and precise agronomy, developments in biotechnologies and, in part, because of the creation and use of GMOs.

We have made remarkable scientific progress since the first commercial GM soybean was developed. Recent research moves GMO development from herbicide or insect resistance to modified nutritional content and the production of drugs, from addition of single traits to complex modifications and genome editing.

As the technology enabling these modifications has advanced, those of us in the analytical community have faced considerable challenges to meet ever more complex testing demands, in part necessitated by new regulations. The USA serves as an instructive example: GMOs were largely unregulated there, yet 2020 will see the implementation of their first nationwide labelling laws. The shifting regulatory landscape is, however, only one element of this complexity; technical challenges abound in GMO testing. From determining the appropriate method for testing raw and processed materials to understanding the advantages and limitations of different technologies, producers (as well as importers and exporters) are faced with an often-intimidating array of considerations.

In this issue of Spot On, our R&D specialist Donna Houchins provides an overview of regulations on plant transformation techniques, including cuttingedge technologies of genome editing such as CRISPR-Cas9. We’ll also dissect the new labelling law in the USA and discuss the crucial differences between process and product testing. Our product manager Christina Huber offers up a few important questions to ask yourself when choosing a GMO testing approach: Are you testing protein or DNA? How do methods for testing raw materials differ from those of processed materials? Are you looking for specific traits or do you just need to confirm that the sample is GMO-free?

For us at Romer Labs, it’s more than just a pleasure to keep you informed about the latest in diagnostic solutions; it’s also our job. With this in mind, we hope that you enjoy this issue of Spot On

Pavlo Futernyk Managing Director, Romer Labs Ukraine

How GMO-Free is "GMO-Free"?

Decoding Global Regulations, Labeling Conventions, and Testing Requirements for Genetically Modified Crops

As technologies to produce GM plants have developed, regulations that govern their cultivation, import and export have multiplied and adapted. R&D specialist Donna Houchins of Romer Labs breaks down some trends in important areas, such as labeling, and discusses how producers and traders can stay compliant amid this shifting regulatory landscape.

By Donna Houchins, Research & Development Specialist, Romer Labs®

Genetically modified crops have achieved broad popularity around the world. Corn, soybeans, cotton and canola are the most commonly commercialized GMO crops. Currently, 229 different corn traits or trait stacks, 62 cotton traits or trait stacks, and 41 different soy and canola traits or trait stacks have an approval for food, feed, or cultivation in at least one country worldwide. These approvals vary with each approving country. Many crops are planted with the intention of being exported; they may reach countries in which the approvals or labeling laws differ from that of their country of origin.

With the greatest amount of acreage devoted to GMO crop production, countries such as the USA, Brazil, Argentina, India and Canada have implemented extensive approvals for food, feed, and cultivation of many events and stacks. In contrast, much exported material may reach countries with differing approvals or labeling requirements. For example, most European Union member countries have banned the cultivation of GM crops. Only a few countries such as Spain and Portugal permit the planting of genetically modified crops. However, the European Union is the world’s leading importer of GM-crops. It receives more than 30 million tons of biotech corn and soy imports intended for livestock feed each year 1. For this robust trade in GMO crops to conform to legal requirements, producers who plant and export GMOs must have a thorough knowledge of the regulations of different countries. Each country independently decides whether cultivation, import and the use in food or feed is allowed, and sets restrictions on permitted events and stacks. In addition, each country sets its own labeling laws governing when these crops are sold. Some countries allow the cultivation and import of GM crops (e.g. US), some only ban the cultivation but allow import (e.g. Austria) and some ban both cultivation and import (e.g. Russia).

Authorized vs non-authorized crops

When a crop arrives at its destination country, it is often tested before it can be transported to its final destination. These tests may reveal GM crops that are not authorized in the receiving country but may be authorized in the country of origin. Another common scenario is that GM crops arrive in a country unknown or unapproved.

Laws forbidding the import of a GM crop are the primary barrier to entry. In general, only approved events are allowed for import. Unauthorized events will not be allowed to enter the receiving country and will be shipped back to the country of origin, resulting in high costs for the exporter. Unauthorized events can also cause high costs due to recall within their country of origin if they reach unintended markets (for example, a crop found in food that only has approvals for feed). A regulation in force in the EU serves as an example: at the “EU Register of Authorized GMOs2,” a list of all approved, withdrawn and pending crops can be found. Exporters must consider this list when shipping to the European Union. If a crop is being shipped to Europe and it is found to contain an unapproved event, it must be shipped back to its country of origin. Other countries have similar lists and rules for import.

A brief case study: how GMO labeling requirements differ in the EU and the USA

Different labeling laws in each country represent the second barrier to GMO imports. These vary widely from country to country. The European Union has one of the strictest laws regarding GM crops. In the EU, every shipment of food and feed containing GMOs must be labeled, irrespective of the ease of detection: GMO traits are known to be difficult to detect in highly refined oils. The threshold for unintentional or technically unavoidable contamination with GMO can be up to 0.9% per ingredient. This is an extremely important

1 https://gmo.geneticliteracyproject.org/FAQ/where-are-gmos-grown-and-banned/

2 View the EU Register of Authorised GMOs at http://ec.europa.eu/food/dyna/gm_register/index_en_new.cfm.

For this robust trade in GMO crops to conform to legal requirements, producers who plant and export GMOs must have a thorough knowledge of the regulations of different countries.

The new law in the US states that every ingredient may contain up to 5% GMO if it was technically unavoidable and unintentional. Any intentional use of GMO ingredients must be labeled regardless of the level.

qualification; it means that a food shipment could feasibly contain 0.6% of GM corn and 0.7% of GM soy and would nevertheless not fall under the regulation of labeling, as 0.9% are allowed per ingredient. However, if two different traits of GM corn are used and one contains 0.5% and the other 0.6%, labeling would be necessary as the sum of GM corn would amount to 1.1%.

In contrast, the USA has recently released a new labeling law, which is scheduled to take effect in January 2020 for large manufacturers and January 2021 for small ones. Prior to this, the United States had not had a nationwide labeling law. This new law states that every ingredient may contain up to 5% GMO if it was technically unavoidable and unintentional (For more about the new labeling law, see inset below). However, any intentional use of GMO ingredients must be labeled regardless of the level. Therefore, producers in the US who intend to ship to the EU will need to make sure that none of their shipments include more than 0.9% per ingredient in order not to require labeling of the products in the EU, although their own regulation would permit up to 5% of unavoidably or unintentionally added GMOs. In Brazil, the threshold is set at 1%. Japan also uses a 5% threshold. Argentina and Canada do not have any labeling laws regarding GMOs. It is incumbent upon exporters to take care

that their shipments meet the labeling requirements of their destination countries and not just their home countries, as the receiving country may have more stringent labeling requirements. Recalls or shipment rejections in these situations can lead to burdensome, extra costs.

Product-based vs. process-based GMOs

Products produced by new gene editing tools such as CRISPR-Cas9 may be regulated differently in different countries. CRISPR stands for “clustered regularly interspaced short palindromic repeats”. Cas9 refers to an enzyme that uses the CRISPR sequences as a guide to recognize and cleave specific strands of DNA. This new technique has given researchers the ability to edit the genome of any organism with a high degree of precision. When used in plant breeding, the difference between the standard GMO development tools and CRISPR-Cas9 is that in the standard technologies, biolistics (the “gene gun”) or foreign bacteria (typically an Agrobacterium) are used to incorporate the gene, whereas in CRISPR-Cas9, precise genome edits are made without the use of foreign organisms.

The results of CRISPR-Cas9 can be indistinguishable from the results of traditional plant breeding techniques, mutagenesis, or naturally occurring random

Labeling Laws for GMOs in the United States

By Christina Huber, Product Manager, Romer Labs

The United States Department of Agriculture (USDA) has filed a new labeling standard regarding GMOs. Up to now, no standard was in place for bioengineered (BE) food. This new standard will be implemented beginning in January 2020, except for small food manufacturers whose implementation date is delayed by a year. Bioengineered food is defined in the US as anything that contains detectable genetic material modified through laboratory techniques and whose modification can neither be found in nature nor have happened through conventional breeding. Food producers will have to label any food that is bioengineered or contains BE ingredients. Exceptions for labeling are made (A) if the food is modified but it cannot be detected, such as oils and sugars, (B) for several foods such as meat, catfish, domesticated birds or egg products, (C) for products derived from animals such as eggs and milk. Every ingredient can consist of GMOs up to 5% if the contamination was not intentional; a common example is that of a truck transporting non-BE crops after unloading BE crops.

However, every ingredient that intentionally contains

GMOs must be labeled, regardless of the amount of GMO. For labelling, food producers have four options:

• Using the “bioengineered” symbol;

• Using a printed text stating “bioengineered food” or “contains bioengineered food ingredients”;

• Using an electronic or digital link accompanied by a statement such as “scan here for more food information” accompanied by a telephone number;

• Sending an immediate text message to the consumer’s mobile device with the bioengineered food disclosure.

mutations. This has, in turn, occasioned a debate about whether crops that are altered using CRISPR-Cas9 are considered GMOs. The definition of a “GMO” that triggers the implementation of labeling law requirements differs from country to country, and therefore, some jurisdictions consider CRISPR-Cas9 traits to be GMOs, while others do not. For example, the legal definition of “bioengineered” in the United States is “product-based”, and defines bioengineered food as anything that contains detectable genetic material modified through laboratory techniques and whose modification can neither be found in nature nor have happened through conventional breeding. This definition is included in the new labeling law that will go into effect in 2020.

The European Union’s definition is “process based”, and defines a genetically modified organism as “an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination”. Under these definitions, the United States does not consider traits emerging from CRISPR-Cas9 to be GMO, whereas the European Union does.

Traceability

In the European Union, every batch of GMO food or feed requires tracking along every step of the supply chain. Should there be reasonable suspicion that the food or feed has been contaminated with a GM crop, traceability measures would ensure that authorities are able to track down the crop and, if necessary, withdraw it from the market. The operators must inform customers by writing if a product is genetically modified or if it contains GM-ingredients. It must include information on the unique identifier or identifiers for these GMOs and the operators must ensure that this information is passed on to those who would be next in the supply chain. Every operator must keep a record of all supply chain transactions for a period of five years. This information must identify who was previous and next in each supply chain. The EU is the only jurisdiction with these tracking requirements.

Ensuring legal conformity through GMO testing

Due to the extreme degree of variance in labeling laws and approvals around the world, testing for GMOs is key in facilitating the movement of grain from country to country. It may also be used to meet specific quality control criteria set in place by grain producers and exporters, or to meet contract requirements from purchasers. Typical tests performed by exporters and grain

handlers include lateral flow devices and ELISA test kits. Both of these methods test for specific novel proteins that are produced by the GMO plant of interest, and may be quantitative or qualitative. These are rapid tests that are typically performed at grain handling facilities. PCR testing for the specific DNA modification of interest may also be undertaken. This is a laboratory test that may detect the promoter and terminator in the DNA inserted into the GMO plant, or may be specific to a particular event.

GMO definitions and labeling: a complex landscape

Not only do legal requirements differ from country to country, but also the definition of what a GMO is varies drastically. The different laws show the need to test for GMOs, as they could otherwise lead to unexpectedly high costs if, for example, an exporter attempts to transfer a crop to a country in which it is not approved; usually, in such a case, the exporter would have to bear the costs of the return.. New labeling laws are being implemented in the USA and other countries are currently discussing how new plant editing techniques will be regulated. To help those in the global grain trade to adapt to the complexities in GMO regulation, a broad palette of testing options can provide clarity and reduce risk. The following article goes into greater detail about common GMO testing solutions.

The Science of GMOs: Some Important Terms

By Christina Huber, Product Manager, Romer Labs

Events? Traits? Stacked events? Here’s a quick look at some key terms related to GMOs.

Trait: A trait is the novel characteristic feature a plant expresses after the DNA has been inserted into the plant's genome. For example, a trait may increase a plant’s resistance to a specific herbicide.

Event: An event refers to the insertion of DNA into the plant’s genome. The newly recombined DNA then generates a new strain of transgenic plant.

Stacked event: A stacked event is a combination of two or more GMO events that introduce several traits into a crop. Stacked events result from the simultaneous insertion of several events into its genome, for example, herbicide resistance and drought resistance to increase efficiency.

Trade name: The trade name is the name under which the GM crop is sold on the market commercially.

Due to the extreme degree of variance in labeling laws and approvals around the world, testing for GMOs is key in facilitating the movement of grain from country to country.

GMO Testing:

4 Important Questions

Are you testing protein or DNA? How do methods for testing raw materials differ from those of processed materials? These are just two considerations that anyone looking to test for GMOs needs to keep in mind. Product manager Christina Huber offers some practical answers.

By Christina Huber, Product Manager, Romer Labs®

As discussed in the previous article, different countries have different regulations regarding approvals for trading with genetically modified plants or cultivating them within their jurisdiction. Hence, the criteria for testing vary significantly among countries.

This article lays out some basic considerations when choosing the right test format to determine whether a sample is derived from a genetically modified plant. Genetic modification refers to any artificial alteration of the DNA in a plant, either by editing the genome or by inserting foreign fragments of DNA. The modified gene will convey a new trait, such as insect resistance, herbicide resistance or other agriculturally relevant features. One of the first questions that you will encounter is

whether you want to test DNA or protein. Complementary to this decision, you have to decide whether the raw commodity or processed material will be tested. A third matter is your target: are you looking for a specific GMO trait, or do you simply want to know whether your crop is GMO-free? Finally, factors such as sensitivity, speed and ease-of-use play a large role as well. All these decisions will help you in choosing the right format from the most commonly applied testing options: polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) or lateral flow devices (LFD).

1) DNA or protein?

As the plant is being modified, its DNA sequence is changed. The alteration can be determined by iden-

tifying the artificial alteration of the DNA itself or by detecting the expressed protein, which does not occur naturally in the crop in question.

Nucleic acid-based testing approaches like PCR amplify a region of interest of the plant genome to obtain billions of copies of small DNA fragments using so called primers and DNA polymerase as enzyme. Previously, those DNA fragments could be separated and visualized on an agarose gel but nowadays more sophisticated instruments allow for the direct measurement of the amplification reaction based on fluorescence techniques. Not only is a PCR approach qualitative, it also allows for the quantification of GM material in a sample.

For the detection of proteins, immunoassays are used. These immunological tests use specific antibodies that can bind the protein. Two different testing formats are available – lateral flow device (LFD) and enzyme-linked immunosorbent assay (ELISA). Both systems work in a sandwich-format, meaning that one antibody is immobilized on a surface that specifically binds the protein of interest. Once the protein has bound to the immobilized antibody, a labeled secondary antibody can bind, thereby forming a sandwich and giving a color signal. If this protein of interest is not available, no sandwich can be formed and therefore no color will be visible, indicating that the tested plant is GMO-free. In most cases, ELISA uses a 96-well plate coated with the specific antibody that allows for the simultaneous analysis of multiple samples. LFDs are strips that have a test and a control line coated with the specific antibody to capture the protein of interest. If the test line appears, the result is positive, indicating the presence of the target protein. These tests exist in a quantitative or qualitative format; an LFD reader is required to quantify the result.

In sum, DNA testing will read the altered DNA sequence, whereas protein testing identifies the new protein derived from this specific DNA sequence.

The specific qualities of the sample to be analyzed also plays a role in determining whether a DNA-based or protein-based method best fits your needs. Moving on, we will look at the different requirements for testing raw commodities or processed materials.

2) How does testing for raw materials differ from that of processed materials?

When it comes to determining the appropriate testing technique, a further question beyond the target (DNA or protein) arises: does the matrix consist of raw or processed material? This is particularly relevant for protein testing. Raw materials express the intact protein, meaning that both PCR and immunological tests

can be used. However, once a raw material has been processed, its proteins denature with the result that they change their natural structure and, in most cases, cannot be detected by immunological tests. Heat treatment, for example, is a process that can lead to the denaturation of proteins. However, this process does not change the DNA sequence; therefore, PCR can be used to detect the inserted gene even in some processed materials.

More testing options are open to you if your sample is a raw commodity: both DNA- and protein-based approaches will detect the genetic modification. If, however, your sample is a heavily processed material in which the protein may have been denatured, your options are restricted to DNA testing.

3) Is there a specific trait in my sample? Is it GMO-free? What exactly do you need to know?

Sometimes, it is necessary to test a sample for specific traits in order to determine whether it contains plants not permitted in certain regions. It may also be necessary to determine whether the sample is completely free of GMOs, regardless of any particular trait.

To test for the presence of a specific GMO, it is important to know which proteins are expressed in the GMO of interest. This information can be acquired from the producer or from sources such as the ISAAA webpage (http://www.isaaa.org/), where all approved GMOs are listed.

The next step is to determine the desired test format (PCR, ELISA or LFD) based on the desired specificity, time-to-result, type of material, and laboratory equipment available. Analysts need to be careful here, as there are many GMO products that contain not just one inserted gene but several (called stacked events); therefore, if you want to test for a specific GMO, you need to test for all inserted genes (or expressed proteins) associated with that particular plant.

Another strategy for GMO testing is to determine whether the plant contains any GMO materials at all. This type of testing is not specific to any particular event. To detect whether a sample is completely free of GMOs, it is sufficient to test only for one event of

To detect whether a sample is completely free of GMOs, it is sufficient to test only for one event of each crop.

each crop; this is effective even considering GMO crops with stacked events, as proving the presence of only one event is sufficient to prove the presence of a GMO. All three techniques are appropriate for this kind of test. PCR would provide the widest range, as, for example, the NOS-terminator or 35S promoter is very often inserted. When testing with LFD strips, a combinational strip can detect several proteins at once.

These two approaches depend solely on the needs of the customer, as for some it might be more important whether the crop is GMO-free (as in a country were cultivation is banned but illegal planting might occur). Others might want to know specifically which plant they have been planting.

4) How can you choose the right testing method when taking other factors such as the testing environment into account?

Another factor that plays a role in choosing the right testing format is the testing environment. Will you have a laboratory and trained personnel available or would you rather have a quick test that does not require any laboratory equipment? Table 1 summarizes the available testing formats and their advantages and disadvantages.

Let’s go through the table step-by-step. As the reference method for GMO testing, PCR is the most suitable method if you want to test any processed material. This technique also provides the highest degree of sensitivity and detects virtually any GMO. However, one significant drawback is that laboratory equipment and trained personnel are needed. To perform a PCR, especially when quantification is desired, exact pipetting skills are essential, because imprecise pipetting could lead to wrong or inconclusive results.

The major advantage of an ELISA is the ability to test

multiple samples simultaneously. This is important when you want to carry out single-seed testing. However, as this is a protein-based test, it can only be used for raw materials.

If you compare all three techniques in terms of timeto-result, LFD is the fastest method. LFD strips can be used on-site and do not require sophisticated instrumentation. So, if you want to test an incoming shipment and need fast results, it would be very reasonable to choose an LFD test, as results are available within 10 minutes; no laboratory personnel and little equipment are needed. This technique also does not require any laboratory skills, as the procedure is designed to be easy to use.

So which method works best? Your decision is ultimately a function of your desired time-to-result and the facilities, equipment and personnel at your disposal.

Conclusion: the value of customized solutions for GMO testing

All in all, you must take into account several factors to find the right testing strategy. It depends on the material that you want to test (raw or processed), whether you need fast results, whether you have the capacity to set up a laboratory with equipment and personnel, and whether you want to know specifically which GMO trait you have or if you want to know whether your crop is GMOfree. There is no right or best technique, as your decision depends on your answer to all these questions. Some may choose LFDs in order to meet tight deadlines, whereas others may want more specific and detailed results using PCR. Still others, such as research institutions, may want to test their research-trial field and do single-seed testing of multiple samples using ELISA. Taking all these elements into consideration will help you make the right choice when it’s time to test your crop.

Your choice of test method is ultimately a function of your desired time-toresult and the facilities, equipment and personnel at your disposal.

Table 1. Characteristics of GMO testing techniques

Source: Romer Labs