WHO SPECIFICATIONS AND EVALUATIONS FOR PUBLIC …

1 Page 1 of 41 WHO SPECIFICATIONS AND EVALUATIONS FOR PUBLIC health pesticides bacillus thuringiensis subspecies israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 Page 2 of 41 TABLE OF CONTENTS Page DISCLAIMER 3 INTRODUCTION 4 PART ONE SPECIFICATIONS FOR bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 bacillus thuringiensis subsp. israelensis strain AM65-52 INFORMATION 6 bacillus sphaericus strain ABTS-1743 INFORMATION 8 bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 GRANULES (APRIL 2016) 10 PART TWO EVALUATIONS OF bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 2015 FAO/WHO evaluation REPORT ON bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 25 SUPPORTING INFORMATION 28 ANNEX 1: HAZARD SUMMARY PROVIDED BY THE PROPOSER 33 ANNEX 2: REFERENCES 41 Page 3 of 41 Disclaimer1 WHO SPECIFICATIONS are developed with the basic objective of promoting, as far as practicable, the manufacture, distribution and use of pesticides that meet basic quality requirements.

2 Compliance with the SPECIFICATIONS does not constitute an endorsement or warranty of the fitness of a particular pesticide for a particular purpose, including its suitability for the control of any given pest, or its suitability for use in a particular area. Owing to the complexity of the problems involved, the suitability of pesticides for a particular purpose and the content of the labelling instructions must be decided at the national or provincial level. Furthermore, pesticides which are manufactured to comply with these SPECIFICATIONS are not exempted from any safety regulation or other legal or administrative provision applicable to their manufacture, sale, transportation, storage, handling, preparation and/or use. WHO disclaims any and all liability for any injury, death, loss, damage or other prejudice of any kind that may be arise as a result of, or in connection with, the manufacture, sale, transportation, storage, handling, preparation and/or use of pesticides which are found, or are claimed, to have been manufactured to comply with these SPECIFICATIONS .

3 Additionally, WHO wishes to alert users to the fact that improper storage, handling, preparation and/or use of pesticides can result in either a lowering or complete loss of safety and/or efficacy. WHO is not responsible, and does not accept any liability, for the testing of pesticides for compliance with the SPECIFICATIONS , nor for any methods recommended and/or used for testing compliance. As a result, WHO does not in any way warrant or represent that any pesticide claimed to comply with a WHO specification actually does so. _____ 1 This disclaimer applies to all SPECIFICATIONS published by WHO. Page 4 of 41 INTRODUCTION WHO establishes and publishes SPECIFICATIONS * for technical material and related formulations of PUBLIC health pesticides with the objective that these SPECIFICATIONS may be used to provide an international point of reference against which products can be judged either for regulatory purposes or in commercial dealings.

4 From 2002, the development of WHO SPECIFICATIONS follows the New Procedure, described in the Manual for Development and Use of FAO and WHO SPECIFICATIONS for pesticides . This New Procedure follows a formal and transparent evaluation process. It describes the minimum data package, the procedure and evaluation applied by WHO and the experts of the FAO/WHO Joint Meeting on Pesticide SPECIFICATIONS (JMPS). WHO SPECIFICATIONS now only apply to products for which the technical materials have been evaluated. Consequently, from the year 2002 onwards the publication of WHO SPECIFICATIONS under the New Procedure has changed. Every specification consists now of two parts, namely the SPECIFICATIONS and the evaluation report(s): Part One: The specification of the technical material and the related formulations of the pesticide in accordance with chapters 4 to 9 of the above-mentioned manual. Part Two: The evaluation Report(s) of the pesticide, reflecting the evaluation of the data package carried out by WHO and the JMPS.

5 The data are provided by the manufacturer(s) according to the requirements of chapter 3 of the above-mentioned manual and supported by other information sources. The evaluation Report includes the name(s) of the manufacturer(s) whose technical material has been evaluated. evaluation reports on SPECIFICATIONS developed subsequently to the original set of SPECIFICATIONS are added in a chronological order to this report. WHO SPECIFICATIONS under the New Procedure do not necessarily apply to nominally similar products of other manufacturer(s), nor to those where the active ingredient is produced by other routes of manufacture. WHO has the possibility to extend the scope of the SPECIFICATIONS to similar products but only when the JMPS has been satisfied that the additional products are equivalent to that which formed the basis of the reference specification . SPECIFICATIONS bear the date (month and year) of publication of the current version.

6 EVALUATIONS bear the date (year) of the meeting at which the recommendations were made by the JMPS. * Footnote: The publications are available on the Internet under ( ). Page 5 of 41 PART ONE SPECIFICATIONS bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 Page bacillus thuringiensis subsp. israelensis strain AM65-52 INFORMATION 6 bacillus sphaericus strain ABTS-1743 INFORMATION 8 bacillus thuringiensis subsp. israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 GRANULES (APRIL 2016) 10 Page 6 of 41 WHO SPECIFICATIONS FOR PUBLIC health pesticides bacillus thuringiensis subspecies israelensis strain AM65-52 INFORMATION Scientific name bacillus thuringiensis subsp. israelensis strain AM65-52. Shorthand terms Bt: all subspecies of bacillus thuringiensis Bti: all strains of bacillus thuringiensis subspecies israelensis (flagella serotype H-14) Bti AM65-52: the strain to which the CIPAC code 770 applies CIPAC number 770 Identity tests Identification is based upon the following tests.

7 (i) Microscopic examination: gram-positive rods; presence of spores and parasporal crystalline inclusions. (ii) SDS-PAGE analysis of molecular weight profile of the endotoxin protein crystals. (iii) Agarose-GE analysis of the plasmid profile (Note 1) Definition of active ingredient A mixture of free endotoxin protein crystals produced by Bti strain AM65-52 and the spores and cells bearing them. Page 7 of 41 Measurement of active ingredient activity Bioassay with fourth instar larvae of Aedes aegypti (strain Bora Bora), results expressed as international toxic units (ITU)/mg product, relative to a reference Bti material. Note: the only reference standard currently available is Valent BioSciences Corp. strain AM65-52, lot # 093-177-W502, which has a biopotency of 6388 ITU/mg. Note 1 : To identify to strain level, genomotyping methodology may be used. Bacterial strains can be characterized by comparing their genomic DNA to an array of genomic DNA fragments originating from a mixture of different strains of the same species.

8 Background information on the potential of the technology and its resolution is given in the following references (Salama et al. 2000, Leavis et al. 2007, Vlaminckx et al. 2007). This current hybridization technology has permitted the assaying of thousands of nucleic acid sequences in a single reaction on a solid substrate. Such a massively parallel system offers the opportunity of diagnostic applications for strain identification through a comparative process. Page 8 of 41 WHO SPECIFICATIONS FOR PUBLIC health pesticides bacillus sphaericus strain ABTS-1743 INFORMATION Scientific name bacillus sphaericus strain ABTS-1743. Shorthand terms Bs: All subspecies of bacillus sphaericus (serotype H5a5b) Bs, strain ABTS-1743: The strain to which CIPAC code 978 applies and the subject of the current evaluation CIPAC number 978 Identity tests Identification is based upon the following tests. (i) Microscopic examination: gram-positive rods, presence of a spherical spore within a visible swelling at one end of the cell, prior to lysis.

9 (ii) SDS-PAGE analysis provides a profile of the two Bs crystalline endotoxin proteins of sizes 51kDa and 42kDa. (iii) Genomotyping analysis determines the overall genetic relatedness to highly similar strains within the same species (Note 1). Definition of active ingredient The active ingredient of Bs strain ABTS-1743 is defined as a mixture of the cells, spores, and the crystalline inclusion associated with the spores. Page 9 of 41 Measurement of active ingredient activity The content of active ingredient activity (biopotency) is measured and expressed as Bs International Toxic Units (BsITU) per mg of product. Bioassay with early L3 third instar larvae of Culex quinquefasciatus. Results expressed as international toxic units (ITU)/mg product, relative to a reference ABTS-1743 material. Note: The only reference standard currently available is Valent BioSciences Corporation Bs strain ABTS-1743, Lot # 089-273-W501, which has a biopotency of 1639 Bs ITU/mg.

10 Note 1 : To identify to strain level, genomotyping methodology may be used. Bacterial strains can be characterized by comparing their genomic DNA to an array of genomic DNA fragments originating from a mixture of different strains of the same species. Background information on the potential of the technology and its resolution is given in the following references (Salama et al. 2000, Leavis et al. 2007, Vlaminckx et al. 2007). This current hybridization technology has permitted the assaying of thousands of nucleic acid sequences in a single reaction on a solid substrate. Such a massively parallel system offers the opportunity of diagnostic applications for strain identification through a comparative process. Page 10 of 41 WHO SPECIFICATIONS FOR PUBLIC health pesticides bacillus thuringiensis subspecies israelensis strain AM65-52 + bacillus sphaericus strain ABTS-1743 GRANULES WHO specification 770+978/GR (April 2016) This specification , which is PART ONE of this publication, is based on an evaluation of data submitted by the manufacturer whose name is listed in the evaluation report (770+978/2015).