Obtaining approval to use microbes as feed or food additive or as a production strain can be challenging. The European (EU / EFSA) and US (US-FDA) regulatory agencies now have strict guidelines for submitting dossiers. Applying the regulatory guidelines and rules to generated genomic and other data often presents both technical and interpretative challenges. In this article, we provide an overview of these regulatory requirements and the challenges that you can encounter in the process, and summarize the differences between the EFSA and FDA approaches to this subject.

  • Written by Adalberto Costessi (Product Manager Genomics & Regulatory Affairs) and Derek Butler (Commercial Director)

Common issues with microbial genome sequence data and its interpretation

Both the EFSA and FDA nowadays require that the complete genome of the organisms you work with are sequenced and analysed. However, a number of technical challenges is encountered. Especially when analysing larger genomes such as those of fungi or yeast. In addition, it is often extremely difficult to extract and purify genomic and plasmid DNA of sufficient quality to allow long read sequencing. This is particularly important when using the PacBio or Oxford Nanopore sequencing platforms. The presence of plasmids in a strain and determining the correct sequence of these plasmids can be very challenging, because in many cases multiple plasmids are present that carry homologous genes and genetic elements.

In addition, in cases where a potential trait of concern is found, such an episomal plasmid or a partial antimicrobial resistance gene, the interpretation of this information is crucial to the final outcome. Are these traits a safety issue for the use of the microbe as a feed or food additive or as a production strain? Or are they normal biological characteristics of the genus or species which don’t cause safety concerns? And how will this information be evaluated by the regulatory authorities? To address these issues effectively, it is important to have a good understanding of the current rules and guidelines from the regulatory authorities.

Safety assessment for feed additive applications: EU vs. USA

Not surprisingly, different countries apply different approaches when it comes to the safety assessment of food and feed products and ingredients. Consequently, their regulatory procedures and requirements are also different, despite some overlap. Here, we discuss the systems in place in the European Union and the United States specifically for microorganisms used as feed additives or as production organisms for additives like enzymes or vitamins.

The EU and USA have different starting points, resulting in quite different procedures. First of all, in the EU, unlike the USA, the risk assessment (science) and risk management (policy) are strictly separated. EFSA (European Food Safety Authority) is an independent agency responsible for the science-based risk assessment of any new feed additive. Following this assessment, the decision if an additive is approved for the EU market is taken by the risk managers, namely the European Commission, Member States and European Parliament, who are also responsible for defining the overall food and feed policy. In the USA, however, both tasks of risk assessment and risk management are performed by the FDA (U.S. Food and Drug Administration).

In the EU, a feed additive must always undergo formal review and authorisation before it is placed on the market and is used, and there is one clear procedure for this purpose. To pursue authorisation, the notifier (i.e. the producer of the substance) submits a dossier to the EU Commission including data to support safety (for animals, humans and the environment), as well as efficacy (i.e. improvement of the characteristics of the feed, improvement of animal production, performance or welfare, or meeting of a nutritional need). EFSA reviews the data and publishes the conclusions of their risk assessment in a public “Scientific opinion”, based on which the risk managers decide on a final approval, which is published in the form of an “Implementing regulation”.

In the USA, all feed additives are in principle also subject to premarket review and approval by the FDA. Importantly, only safety is considered in the USA, because any claimed positive effect on animal production or welfare would classify the substance as a veterinary drug according to the US regulations. In which case, a very different and stricter regulatory system applies.

There is one important exception to the general rule of premarket approval in the USA, which is more and more often used for microorganisms with no safety concerns: the self-determined and notified GRAS, also known as “GRAS notice” or “GRAS notification”. GRAS stands for “Generally Regarded As Safe”. If sufficient data about the safety of the microorganism is available – for at least 6 months – in the public domain, the notifier can prepare a dossier and self-determine that the microorganism is safe for the specific intended use. The FDA will review the data and provide a response by means of a letter to the notifier. Importantly, in the case of a GRAS notice, the notifier holds the final responsibility for the safety of the product.

EFSA requirements for feed applications

EU’s regulatory framework includes a number of very detailed guidance documents developed by EFSA. An important tool worth mentioning is EFSA’s QPS (Qualified Presumption of Safety) system. Often, the QPS system is misinterpreted as the counterpart of the American GRAS notice. However, this is incorrect. The QPS system is a tool for safety assessment that EFSA established in order to increase the efficiency of their safety assessment for microorganisms. Based on solid scientific data, EFSA established a QPS list which includes microbial taxonomic units for which sufficient evidence is available to conclude that they pose no safety risks. If a species of interest is on the QPS list, the notifier does not need to provide detailed safety studies, like tolerance studies in target animals or toxicological studies, which are not only expensive, but require many test animals. The QPS list is regularly updated by EFSA, based on research in the scientific literature, as well as information provided by notifiers in their applications.

For the characterisation and approval of microorganisms for feed applications, EFSA makes a distinction between additives containing viable microorganisms (i.e. probiotics, feed sileage agents) and additives produced by microorganisms and further purified (i.e. enzymes, vitamins), as shown in the table below [1].

A critical requirement for the European legislation is that any new product must not contribute to the spread of antimicrobial resistance in the food chain and the environment. Therefore, phenotypic and genotypic assessment of antimicrobial resistance is always required.

Food additives containing viable microorganisms Food additives produced by production organisms
Bacteria Fungi-yeast Bacteria Fungi-Yeast
Identification x x x x
Antimicrobial Susceptibility x x
Antimicrobial production x x x x
Toxigenicity and pathogenicity x x x x
Genetic modification For GMMs only For GMMs only
Absence of the production strain x x
Presence of DNA from the production strain Where relevant Where relevant
Compatibility with other authorised additives Where relevant Where relevant

Taken from [1]. GMM, genetically modified microorganisms.

Whole genome sequencing

Whole genome sequencing (WGS) plays an important role in many of the requirements mentioned above. First of all, whole genome sequencing (WGS) provides the information required to unequivocally identify the strain that is used. EFSA requires WGS for bacteria and yeasts; for filamentous fungi this is also recommended. Only when WGS is not available for filamentous fungi can the notifier perform identification by comparing 18S rRNA genes, ITS regions and other characteristic genes.

Moreover, WGS has the advantage that it also provides information which enables further characterisation of the strains, specifically regarding potential functional traits, such as:

  • Antimicrobial susceptibility. Microbial food additives should not add to the pool of antimicrobial resistance (AMR) genes that are already present in the gut bacterial population or otherwise increase the spread of antimicrobial resistance. This can be checked by searching the genome for the presence of known AMR genes.
  • Plasmids and mobile elements. The presence of AMR genes is not an issue if they are present on the chromosomal genome, and therefore cannot be easily transferred horizontally. However, AMR genes on episomal plasmids are a clear hazard according to European legislation. Specific data analysis and literature research can make a difference in these cases.
  • The production of antimicrobials. The microorganisms should not produce antimicrobials that are clinically relevant. This must be assessed with laboratory assays on culture supernatants.
  • Toxigenicity and pathogenicity. The microorganisms should not cause diseases or produce toxic components. The genome can be searched for example for the presence of genes involved in the production of known toxic metabolites and the presence of virulence factors.
  • Genetic modifications. For genetically modified production strains, the genome sequence must be analysed to assess if the modifications introduced are correct and stable over time.

Dossier filing for EFSA and FDA applications

For final approval of a product, a safety assessment dossier with all the necessary evidence needs to be filed on the microorganisms used as production strains, probiotics, live biotherapeutics, novel foods, feed additive production and fermentation among other food & feed applications. For this purpose, it is essential that the appropriate whole genome sequencing and other data is generated that is compliant with regulatory requirements. Based on this data, a solid dossier is built with rigorous scientific justification to support the safety of strains introduced intentionally in the food chain.

Want to know more? BaseClear regularly organises experts meetings to discuss challenges and opportunities in strain safety assessment. Our  next experts meeting on Strain Safety Assessment is scheduled for Tuesday April 21, 2020 and will be hosted online. And, of course, BaseClear can assist you in all steps in product registration. Contact our specialists to discuss you specific request and needs.

References

  1. EFSA: Guidance on the characterisation of microorganisms used as feed additives or as production organisms, http://www.efsa.europa.eu/en/efsajournal/pub/5206

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