Protocol development for mutagenicity testing of fungal culture extracts

Project Code BPI09-100

Project Lead

Deena Errampalli - Agriculture and Agri-Food Canada

Objective

To develop and validate protocols for mutagenicity testing of fungal culture extracts for regulatory purposes and product development

Summary of Results

Background

Many fungi are developed as biological control agents for the management of agricultural insect pests, diseases, and weeds. As part of the regulatory requirements for the registration of microbial pesticides with fungal active ingredients, there is a need to demonstrate that fungal active ingredients proposed for registration of pest control products do not produce genotoxic metabolites that pose a threat to consumers and the environment.

However, there is a substantial cost associated with the detailed investigation of specific fungal metabolites, and published data to meet this requirement is available for only very few fungal species. This poses a potential barrier to commercialize promising new biocontrol agents.

A solution to this constraint may lie in the testing of crude extracts or filtrates of fungal cultures. The direct testing of culture extracts or filtrates in established test systems would allow for an efficient process to determine whether a particular fungal strain produces genotoxic substances that pose a risk to human health and environment.

The purpose of this project, which was developed in consultation with regulators from the Microbial and Biochemical Evaluation Section of Health Canada’s Pest Management Regulatory Agency (PMRA), was to develop methods for the establishment of a validated standard protocol for mutagenicity testing of fungal cultures.

Approaches

The approach taken in this project was based on the Ames test, a bioassay developed by Ames et al. in the 1970s to evaluate the mutagenicity potential of chemicals. This assay uses certain deficient bacterial strains which, due to a genetic mutation are unable to grow without supplemental nutrients. When these bacteria are exposed to a mutagenic substance, mutations reversing the genetic defect occur in some bacterial cells, allowing these bacteria to grow in non-supplemented media. The number of bacteria growing in non-supplemented media can therefore be used as a measure of mutagenicity of the test substance.

Advanced commercial test kits of this Ames bioassay are now available, which allows for relatively low cost high throughput sample testing. Although typically used to test purified compounds, the kits can also be used on more complex test substances. As such, it has been proposed by the European Union-funded REBECA project as a screening tool to determine whether fungal biological control agents produce genotoxic/mutagenic metabolites which require further attention in the regulatory risk assessment.

Culture filtrates and extracts from six test fungi, as well as two positive control and two negative control fungi (ie known to either produce or not produce mutagenic metabolites) were tested with the new microplate fluctuation protocol (MPF) developed by Xenometrix for evaluation of the mutagenic activity of the samples. The secondary metabolites produced in liquid culture media were tested directly as filtrates or as extracts, obtained with solvents such as dichloromethane or ethyl acetate.

Positive controls were known mutagen-producing fungi: (Aspergillus flavus strain 63-67, which produces aflatoxins, and Metarhizium robertsii ASREF 2575, which has been previously shown to produce mutagenic compounds). Pure aflatoxin AFB1 served as an additional positive control. Negative controls were Penicillum camemberti (which has a long history of safe use on various cheeses such as Brie and Camembert) and a strain of Aspergillus flavus strain 63-66 which does not produce aflatoxins. These test items were included to validate that the test can differentiate between know mutagen producers and fungi which do not produce any metabolites of concern.

The test fungi were five biopesticide strains (Metarhizium anisopliae BIPESCO5, Beauvaria brogniartii BIPESCO2, Beauvaria bassiana ARSEF 5808, Trichoderma harzianum JAT1977, Clonostachys rosea ACM941), and a strain of Fusarium graminearum known to produce a toxic metoabolite, dexonivalenol.

The assays were performed with two different, deficient Salmonella typhimurium strains which are used to detect frame-shift and point mutation activity associated with metabolic transformation.

Results

The study demonstrated that a commercial microtitre plate-based Ames bioassay can differentiate between fungal strains which produce mutagenic metabolites and those which do not.

As expected all samples of aflatoxin AFB1, as well as culture filtrates and extracts of the aflatoxin producing A. flavus strain 63-67 tested positive for mutagenic activity.

In the case of M. robertsii strain ASREF 2575, a more differentiate pattern was observed, as results were influenced by the choice or growing medium, the tester strain used, and the presence or absence of metabolic transformation.

The tested Fusarium graminearum strain, known to produce deoxynivalenol (DON, vomitoxin), tested negative for mutagenic activity in this test. Deoxynivalenol is not thought to be mutagenic.

None of the tested negative controls and biocontrol fungi showed any mutagenic activity in this assay.

Summarizing, the project developed and demonstrated a protocol which can detect potential hazards posed by mutagenic substances in fungal culture filtrates and extracts, confirming that this method can be used to identify fungal strains that may pose a risk to human and environmental health if used as biocontrol agents.

These findings have potential applications both in the development of new biocontrol strains as well as in the regulatory context. Researchers and prospective registrants can use this method to detect potential future regulatory challenges at an early stage of product discovery and development. In the regulatory context, this protocol may be used to confirm or rule out any concerns about mutagenic metabolites being produced by an organism proposed for registration as a biological control agent.

The Pest Management Centre has shared the results from this project with Health Canada scientists at PMRA and will provide recommendations on how the new validated protocol might be used in the regulatory evaluation of microbial biopesticides with fungal active ingredients proposed for registration.

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