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ItalianMicrobiology
Microbiological Unit works on a wide variety of product categories based on Microorganisms: Biopestides, Biocides, Biostimulants.
The staff of BioTecnologie BT gained significant experience, over the years, in performing GLP studies on Biopesticides:
– Physical-chemical characterisation
– Active substance content determination
Measuring the Colony Forming Unit (CFU) or by direct microscopic count and determination of the viability.
– Methods validation
Shelf life studies
Cold Stability Testing (0°C) according to CIPAC MT 39.3
Accelerated Storage Stability according to CIPAC MT 46.3
Shelf Life at different Temperatures
Test on treated seeds
Uniformity of distribution CIPAC MT175
Adehsion to treated seed CIPAC MT194
Seed load (CFU determination)
5 Batches analysis
The analyses of at least 5 representative batches of production, or pilote-scale batches, is necessary to determine the content of the pure active substance, microbial contaminants and pathogens.The manufacturing processes have the potential of producing unwanted microorganisms in addition to the desired Microbial Pest Control Agents which include pathogens, their associated toxins and other metabolic by-products of health concern.
Search and quantification of contaminants and phatogens are performed according to OECD No. 65 “Issue paper on microbial contaminant limits for microbial pest”.
Residues
– In plants, plant products, foodstuffs, feedingstuffs and environmental samples
The laboratory can develop methods to determine residues coming from Plant Protection Products based on microorganims.
– Analytical method validation for residues determination
The laboratory carries out routinely methods validation in matrices resulting from ecotoxicological studies where the tested formulations are based on microorganisms.
MIC determination of antifungal and antimicrobial substances for yeasts, moulds and bacterial strains
- Agar diffusion method using strips test
- Broth dilution method following EUCAST or CLSI guidelines
MIC is defined as the lowest concentration of an antimicrobial or antifungal that can inhibit the visible growth of a microorganism (fungi, bacteria and yeast) representing a quantitative estimate of the susceptibility of an organism to a certain antimicrobial agent.
Quantification of Bacillus thuringiensis δ-endotoxins content by SDS PAGE and densitometric analysis
The aim of test is the quantification of Bacillus thuringiensis δ-endotoxins in technical powders, broths and final concentrates resulting from fermentation processes. The assay makes use of electrophoretic separation of the soluble proteins based on their molecular weights. After separation, the relevant protein is quantified by densitometric analysis carried out through specific equipment BIO-RAD Gel Doc 2000TM and its associated software Quantity One.
Biological Compatibility with other products for the combined use
The aim of this test is to evaluate the effects of different products with a microbial formulated product after one or more contact times by the determination of Colony Forming Units (CFU) of the different contact solutions compared with the CFU of the control.
5 Batches analysis
Analytical profile of batches of technical materials and formulated products
Analyses of at least 5 representative batches of production or pilote-scale batches is necessary to determine the content of the pure active substance, microbial contaminants and pathogens.The manufacturing processes have the potential of producing unwanted micro-organisms in addition to the desired microbial pest control agent (MPCA) which include pathogens, their associated toxins and other metabolic by-products of health concern.
Contaminants and pathogens for Bacteria based technical materials/formulated products:
– Viable yeasts and moulds
– Anaerobic spore formers
– Pseudomonas aeruginosa
– Coliforms or Escherichia coli
– Staphylococcus aureus
– Listeria monocytogenes
– Vibrio cholerae
– Shigella spp.
– Salmonella spp.
Contaminants and pathogens for Fungi based technical materials/formulated products:
– Aerobic plate count
– Anaerobic spore formers
– Pseudomonas aeruginosa
– Coliforms or Escherichia coli
– Staphylococcus aureus
– Listeria monocytogenes
– Shigella spp.
– Salmonella spp.
STUDY DESIGN
Analysis of the 5 batches, in terms of determination of the active ingredient content, are carried out by an analytical method validated according to SANCO/3030/99 rev. 4 (11/07/2000) on 1 of the 5 batches
Determination of the active substance*
3 samples (as 3 independent weighings) of each batch
Progressive dilutions, for each of the 3 samples, until reaching the appropriate dilution factors
Quantification/detection of microbial contaminants and pathogens*
1 sample of each batch
Progressive dilutions of the sample until reaching the appropriate dilution factors or sample enrichment
* Additional determinations, upon Sponsor’s request, can be carry out on one batch within a shelf life study
LIMITS
Viable yeasts and moulds: < 103 CFU/g or mL
Anaerobic spore formers: < 105 CFU/g or mL
Aerobic plate counts: < 105 CFU/g or mL
Listeria monocytogenes: Absence in 25 g/25 mL
Shigella spp.: Absence in 25 g/25 mL
Pseudomonas aeruginosa: Absence in 1 g/1 mL
Salmonella spp.: Absence in 25 g/25 mL
Vibrio cholerae: < 1 CFU/ 25 g
Coliforms: < 10 CFU/g or mL or E. coli: Absence in 1 g/1 mL
Staphylococcus aureus: Absence in 1 g/1 mL
Study includes GLP managment and reporting (if required)
REFERENCES AND GUIDELINES
SANCO/12116/2012 rev.0 (September 2012) and ISO methods
Methods validation
Validation of the analytical method in the formulated product
STUDY DESIGN
Validation of the analytical method in the formulated product
Linearity
5 solutions (in the appropriate range of concentration) from 5 different weighings of technical material reference item
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Precision
5 solutions (as 5 independent weighings of formulated product)
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Accuracy or Interference check
2 Fortification levels on the blank formulation: High and Low
Low level: 1 solution of blank sample added with known amount of technical material reference item
High level: 1 solution of blank sample added with known amount of technical material reference item Progressive dilutions, for each solution, until reaching the appropriate dilution factors
or
Interferences check
Blank formulation: 2 samples
Validation of the analytical method in the aqueous solutions
Linearity
5 solutions in Standard water (in the appropriate range of concentration) from 5 different weighings of technical material reference item
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Precision
5 solutions Standard water (as 5 independent weighings of formulated product)
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Accuracy or Interference check
2 Fortification levels on the blank formulation: High and Low
Low level: 1 solution in Standard water of blank sample added with known amount of technical material reference item
High level: 1 solution in Standard water of blank sample added with known amount of technical material reference item Progressive dilutions, for each solution, until reaching the appropriate dilution factors
or
Interferences check
Blank formulation: 2 samples
Study includes GLP management and reporting
REFERENCES AND GUIDELINES
SANCO/3030/99 rev. 4 (11/07/2000) – Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414.
Analytical method validation for determination of the active ingredient in technical materials
STUDY DESIGN
Linearity
5 solutions (in the appropriate range of concentration) from 5 different weighings of technical material reference item
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Precision
5 solutions (as 5 independent weighings of formulated product)
Progressive dilutions, for each of the 5 solutions, until reaching the appropriate dilution factors
Interferences check
Blank formulation: 2 samples
Study includes GLP management and reporting
REFERENCES AND GUIDELINES
SANCO/3030/99 rev. 4 (11/07/2000) – Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414.
Other tests
Determination of the Minimal Inhibitory Concentration (MIC) using E-test
MIC is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism (fungi and yeast) representing a quantitative estimate of the susceptibility of an organism to a certain antimicrobial agent. E-test consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along plastic test strips which, placed on the surface of agar plate, can release the agent gradually. After the incubation and the microorganism’s growth, the point at which the drop-shaped inhibition zone intersects the graded test strip is defined as the MIC of the antibiotic. A reference standard is used for the quality check of reagents, media and inoculum.
STUDY DESIGN
The MIC value is determined by agar diffusion procedure using E-TEST in which specify the name of substance, the number and the name of strains, the number and the name of the reference strain. 3 replicates are carried out and MIC value is expressed as µg/mL.
Study includes GLP management and reporting (if required)
REFERENCES AND GUIDELINES
Antimicrobial Susceptibility Testing 9302553C – en – 2012/01 and Etest® for MIC determination of antifungal agents (AB BIODISK)
Quantification of δ-endotoxins content
The aim of test is the quantification of Bacillus thuringiensis δ-endotoxins in technical powders, broths and final concentrates resulting from fermentation processes. The assay makes use of electrophoretic separation of the soluble proteins based on their moleculare weights. After separation, the relevant protein is quantified by densitometric analysis carried out through specific equipment BIO-RAD Gel Doc 2000TM and its associated software Quantity One.
STUDY DESiGN
The Internal method forsees 5 standard concentrations and 1 test item concentration with 3 replicates for each concentration.
RESULTS
% of CRY 1 and % of CRY 2
Study finder
Scientific Contact
Simona Coranelli
E-mail: scoranelli@biotecnologiebt.it
Phone: +39 075 895 0045 – Ext. 236
Business Contact
Katy Lazzari
E-mail: klazzari@biotecnologiebt.it
Phone: +39 075 895 0045 – Ext. 246
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