Sumitomo Chemical Agro Eurnpe Bacillus …dissemination.echa.europa.eu/Biocides/ActiveSubstances/...Sumitomo Chemical Agro Eurnpe (fo1· Valent BioSciences Corporation) Bacillus tlturingiensis

Sumitomo Chemical Agr o Eu rnpe (fo1· Valent BioSciences Corporation)

Bacillus tlturingiensis subsp. israelensis Ser otype H-14 Strain AM65-52

September 2007

SECTION IHA 2 BIOLOGICAL PROPERTIES O F THE MICRO-ORGANISM Official use ouly

IHA 2.1 History of the micr o-or ganism and its uses, natural occ.unence and geographical distt·ibution

IHA 2.1.1 During the years 197 5 and 197 6 a survey was conducted in Israel for Histo1ical backgr ound biocontrol agents against mosquitoes. In this survey, a small pond was

encountered in a dried river-bed in the n01th central Negev Desert, near Kibbutz Zeelim. This mosquito breeding site contained a ve1y dense population of dead and dying exclusively Cu/ex pipiens larvae appearing as a "thick carpet" on the water surface in an epizootic situation. A sample collected from the edge of the pool, containing dead and de.composing larvae, water and silty mud was taken to the laboratory and refrigerate.cl. Bacteria were isolated from this sample and purified to a single colony designated ONR 60A, from which Strain AM65-52 is derived. Sub-samples were taken from the homogenate of the parent sample and cultured on standard media and then processed for larvicidal activity. The larvicidal activity of this strain was tested in 197 6 and found to be effective against five species of mosquitoes belonging to the generaAedes. Cu/ex Anopheles and Uranotaenia. Clones of this strain were delivered through the World Health Organisation (WHO) to Dr. de Baijac, at the reference laborato1y of the Pasteur Institute in Paris where it was identified as Bacillus thuringiensis subsp . israelensis. 1

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Materials a nd methods Not applicable

Conclusion Historical data leading to description of strnin AM65-52 since 1965.

Reliability Not applica ble

Acceptability Acceptable

Remar ks No molecular approaches were available in the seventies to identify colony ONR60A from which the strain AM65-52 has been derived.

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de Barjac, H., Sutherland, D.J . eds. 1990. Bacterial Control of Mosquitoes and Black Flies, Rutgers University Press, pp 3-9.

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September 2007

SECTION IHA 2 BIOLOGICAL PROPERTIES OF THE MICRO-ORGANISM Official use only

IHA 2.1 History of the micro-organism and its uses, natural occ.utTence and geographical distt·ibution

IHA 2.1.2 Bacillus thwingiensis subsp israelensis is a conunon naturally occurring Origin and natural micro-organism with worldwide distribution. The species has been occurence detected both in soil and on insects and plants and will be indigenous to

intended areas of application.

The origin of the strnin used for the production of 'VectoBac' products is confidential to Valent Biosciences and infonnation relating to this is contained in the confidential attaclunent under Point IIIA 2.1.2.

Infom1ation on the natural occw1·ence of Bacillus thuringiensis species in soil and on plants is given in the following reports and also is discussed in the book by Glare, T.R. and O'Callaghan, M. 2

IHA 2.1.2-01 Origin and natural occurence Official use only

Reference Martin, P.A.W., Travers, R.S. Worldwide Abundance and Distribution of Bacillus thuringiensis Isolates. Applied and Enviromnental Microbiology, Oct. 1989. p 2437-2442.

Data p rntection No, published research

Data owner Not applicable

Companies with letter of Not applicable access

Criteria for data Not applicable protection

Guideline study Not applicable

GLP No

Deviations Not applicable

Materials and Methods Over 1000 soils samples from a variety of locations worldwide were analysed for the presence of Bacillus thwingiensis species using an acetate selection technique followed by crystal morphology and biochemical profile identification techniques. Experimental details ru·e described in the report.

Results Between 55.6 ru1d 94.3% of the soil samples taken from locations within New Zealand, Asia, Emope, South America, Central America and the United Sates contained at least one Bacillus thuringiensis isolate, with samples from Asia showing the most abundance. Characterisation according to biochemical type showed that the subspe.cies israelensis was the most conunon type overall accotmting for 20.4% of isolates out of a total of 8916 isolates tested.

Applicant 's Summa1-y and Bacillus thuringiensis was shown to be a ubiquitous soil micro-organism conclusion with worldwide distJ·ibution. Within the samples tested a high proportion

(20.4%) was shown to be characteristic of the subspecies israelensis.

Reliability 2.

Deficiencies No.

2 Glare, T.R. and O'Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety, Jolm Wiley and Sons Ltd, pp 17-19.

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September 2007


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Materials and methods For the origin of the strain reference is made to textbook. For the natural occwTence over 1000 soils samples from a variety of locations worldwide were analysed for the presence of Bacillus thuringiensis species using an acetate selection technique followed by crystal morphology and biochemical profile identification techniques

Cond usion Bacillus thuringiensis subsp israelensis is a common naturally occwTing micro-organism with worldwide distribution. The species has been detected both in soil and on insects and plants. The species (not the strain AM65-52) will be indigenous to intended areas of application.

Reliability 1


Remar ks For the origin of the strain historica l data repo1t ed. Unfortw1ately there are no experimental details showing the identity of the actual isolate with previous one.

For the natural occwTence, the data show that the subspecies israelensis (not the strain AM65-52) accounts for 20.4% of the isolates out of8916 tested from various patts of the world. We do not share the opinion that 20.4% is a "high propo1t ion" of the tested population. In our view 20.4% represents only a higher proportion than the other ones.

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September 2007


IHA 2.1 History of the micr o-or ganism and its uses, natur al occ.utTence and geographical distt·ibution

IHA 2.1.2-02 Origin and nat.ural occurence

Refer ence Smith, R.A., Couche, G.A. The Phylloplane as a Source of Bacillus thuringiensis. Applied and Environmental Microbiology, Jan. 1991. p 311-315.

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GLP No


Materials and Methods Leaf samples were taken from the canopy of a variety of trees within the US. Potential bacteria were removed from the leaf surfaces and examined microscopically after plating to determine those of the Bacillus thuringiensis species. Standard strnins for the Bacillus thuringiensis subspecies kurstaki, israelensis and teneb1·ionis were used for comparison. Crystal protein analysis was pe1formed using SDS-P AGE and strains were also analysed for biological activity against 3rd instar Trichoplusia ni. Experimental details are described in the repo11.

Results The recove1y of Bacillus thuringiens;s from leaf surfaces was high with 50 to 70% of trees analysed showing positive for the organism. The density of Bacillus thuringiensis recovered ranged from 3 cfu/cm2 to 100 cfu/cm2 and it was considered that the presence of the bacteria was natural growth and not the result of a casual isolate or the result of residues from agricultural sprays.

Crystal protein analysis showed that strains of the sub-species kurstaki, israelensis and tenebJionis were present in the extracts in addition to strains that showed a unique protein pattem. Eleven out of sixteen isolates tested for biological activity showed a positive reaction against the Lepidopteran species tested. The presence of Bacillus thuringiensis on samples of twf and prairie grass was also reported.

Applicant's Summary and Bacillus thuringiensis species were shown to be naturally present on the conclusion surface of plants.

Reliability 2.

Deficiencies No.



September 2007

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Materials and methods Microbiological (microscopy and cultural) and biochemical tests.

Condusion Bt is shown to be natural occuni.ng in the phylloplane

Reliability 2


Remarks The work is limited to the canopy of trees within US. Therefore to avoid generalization, the conclusion should be limited to the ecosystem tested.

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September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES O F THE MICRO-ORGANISM Official use only

IIIA 2.1 History of the micr o-or ganism and its uses, natur al occ.utTence and geographical distt·ibution

IIIA 2.1.2-03 Origin and nat.ural occurence

Refer ence Meadows, M.P. et al. Distribution, Frequency, and Diversity of Bacillus thwingiensis in an Animal Feed Mill. Applied and Enviromnental Microbiology, Apr. 1992. p 1334-1350.

Data p rotection No, published research



C1i te1ia for data Not applicable protection


GLP No


Materials a nd Methods Samples of residue were collected from an animal feed mill located in East Sussex, UK. The mill processed wheat, barley, maize gluten and wheat-feed commodities originating from the UK and other areas. Samples were taken from numerous locations throughout the mill and included a variety of residues, for example, grain dust, whole grains, insect webbings, inse.ct cadavers, mammal faeces and bird fae.ces. Bacillus thuringiensis bacteria were extracted from the samples with water and cultured on nutrient agar. Classification of the bacteria was perfonned on the basis of parasporal c1ystal morphology and the spectra of toxicity to lepidopteran (Heliothis virescens, Pieris brassicae and Spodoptera littoralis) and dipteran (Aedes aegypti) insect larvae.

Results Bacillus thuringiensis was widely distributed within the range of samples analysed, as shown in Table IIIA 2 .1.2-01 . The samples shown to contain Bacillus thwingiensis also contained Bacillus cereus although overall Bacillus thwingiensis was isolated more frequently. A total of 4 77 colonies of Bacillus thuringiensis were isolated based on the production of parasporal c1ystals.

A diverse population of Bacillus thuringiensis was observed based on insect larvae toxicity and c1ystal morphology. A total of 55% of isolates were not toxic to the insect orders tested, 40% were toxic to Lepidoptera and 20%, 1 % and 1 % were toxic only to Pieris brassicae, Heliothis virescens, and Spodoptera littoralis, respectively. Isolates having bipyramidal parasporal c1ystal mo1phology typical of Bacillus thuringiensis subsp. kurstaki. were most common. Itregular spherical and rectangular m01phology typical of Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. tenebrionis, respectively were also present but were less prevalent. The distribution of larval toxicity and c1ystal morphology is summarised in Table IIIA 2.1.2-02.

Applicant's Summary Bacillus thuringiensis was widely distributed in a range of natmally and conclusion occmTing matrices sampled from an animal feed store. Analysis of

isolates showed a wide diversity of Bacillus thuringiensis species based on insect larvae toxicity and parasporal c1ystal morphology.

Reliability 2 .

Deficiencies No.

Sumitomo Chemical Agro Europe (for Valent BioSciences Corporation)

Bacillus thuringiensis subsp. israelensis Serotype H-14 Strain AM65-52

September 2007

Table IIIA 2.1.2-01 Distribution of Bacillus thuringiensis in samples collected from an animal feed

mill

Sample material Number of samples Number of samples yielding Bacillus thuringiensis

Settled dust 18 14 Debris 9 6 Insects 7 4 Grain 6 3 Insect webbing 3 3 Guano 3 3 Mammalian faeces 2 2 Bird nests 2 1

Table IIIA 2.1.2-02 Distribution of larval toxicity and crystal morphology within Bacillus

thuringiensis isolates

Group Toxicity to: No.

isolates No. isolates with morphology

P. brassicae A. aegypti H. virescens S. littoralis BP IP IS R S 1 - - - - 263 106 74 27 20 36 2 + - - - 96 72 16 1 3 4 3 - + - - 22 7 9 1 3 2 4 - - + - 7 4 2 0 1 0 5 - - - + 7 5 0 0 0 2 6 + + - - 33 22 8 2 1 0 7 + - + - 7 7 0 0 0 0 8 + - - + 7 6 1 0 0 0 9 - + + - 6 4 1 1 0 0 10 - + - + 1 1 0 0 0 0 11 - - + + 0 0 0 0 0 0 12 + + + - 23 15 0 8 0 0 13 + + - + 1 0 1 0 0 0 14 + - + + 2 2 0 0 0 0 15 - + + + 0 0 0 0 0 0 16 + + + + 2 2 0 0 0 0

BP = Bipyramidal, IP = Irregular pointed, IS = Irregular spherical, R = Rectangular, S = Spherical.



September 2007

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Materials and methods Cultural methods by plating on nutrient agar. Classification based on c1ystal motphology and spectrum of toxicity.

Conclusion Bacillus thuri.ngiensis was widely distributed in a range of naturally occwTing matric.es sampled from an animal feed store.

Reliability 2


Remar ks None

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September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES OF THE MICRO-ORGANISM Official use only

IIIA 2.1 History of the micro-organism and its uses, natural occ.utTence and geographical distt·ibution

IIIA 2.1.2-04 Origin and nat.ural occurence

Reference Hansen, B.M. et al. Molecular and Phenotypic Characterisation of Bacillus thuringiensis Isolated from Leaves and Insects. Journal of Inve1tebrate Pathology 71, 106 - 114 (1998).

Data p rotection No, published research



C1i te1ia for data Not applicable protection


GLP No


Materials a nd Methods Bacillus thuringiensis bacteria were isolated from cabbage inhabiting insects (8 isolates) and cabbage leaves (50 isolates) collected from field grown cabbage in Denmark. Crystal mo1phology of the isolates was examined by phase-contrast microscopy and serotyping was perfonned against recognized flagella antigens. Insect bioassay was performed using the larvae of Pieris brassicae and Trichoplusia ni. These characterisation methods were supplemented by colony hybridization, PCR and random amplified polymo1phic DNA (RAPD) molecular analysis methods however data from these analyses are not relevant to this annex point and are not smrunarised.

Results A diverse population of Bacillus thuringiensis was observed in the insect isolates, with the subspe.cies aizawai, kurstaki and colmerilindiana identified, in addition to unknown or non-motile species. A summary of the serotype, crystal m01phology and bioassay results are given in Table IIIA 2. 1.2-03.

A wider diversity in Bacillus thuringiensis populations was observed in leaf isolates, with the subspecies kurstaki found most often. A summary of the serotype, crystal m01phology and bioassay results are given in Table IIIA 2 .1.2-04.

Applicant 's Summary A wide diversity of Bacillus thuringiensis species were isolated from and conclusion insects and plant smfaces and were identified based on flagella

serotyping, insect larvae toxicity and parasporal crystal mo1phology methods.

Reliability 2.

Deficiencies No.


Bacillus tlturingiensis subsp. israelensis Serotype H-14 Strain AM65-52

Table lllA 2.1.2-03 Characterisation of insect Bacillus thuringiensis isolates

Isolate Isolated from Sero type Crystal morphology

360 Delia radicum (adult) aizawai round 293 Delia radicum (adult) kurstaki, bipyramidal 295 Delia radicum (adult) unknown round 294 Delia radicum (adult) unknown round 316/317 Delia flora/is (larvae) kurstaki, bipyramidal 287 Aleochara bilineata (adult) colimeri/indiana round 285 Pie1is brassicae (la1vae) non-motile bipyramidal 1 Positive result attributed when at least 80% laivae killed compared to control results.

September 2007

Bioassay1

-+ -+ + -+

Table lllA 2.1.2-04 Distribution of larval toxicity and crystal morphology within Bacillus thuringiensis isolates

Isolate Sero type Crystal morphology 1 Bio assay Numerous (28) kurstaki, biovramidal + 319 canadensis/colmeri round -330 oswaldocmzi round -324 fulmokaensis round -323 canadensis round -349 israelensis round -303/305 non-motile round -300 non-motile round Nmnerous (14) unknovm round 1 Positive result attributed when at least 80% laivae killed compared to control results.

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Date September 2007 Materials and methods Methods crystal mo1phology, serotyping, bioassay, RAPD molecular analysis.

Conc.lusion A wide diversity of Bacillus thuringiensis species were isolated from insects and plant surfaces.

Reliability 2


Remarks None

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Reliability Acceptability

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September 2007


IIIA 2.2 Information on the target organism(s)

IIIA 2.2.1 Bti (Strain AM65-52) is a Gram positive, spore forming rod-shaped Description of the target bacterium that produces a crystalline protein inclusion which is toxic to organism larvae of some Dipteran insects upon ingestion.

The biocidal use of Bti (Strain AM 65-52) is for control of mosquito (for example Aedes spp and Cu/ex spp) and black fly (Simuliidae) larvae in contaminated water habitats and filter fly midges (for example Sylvicola spp, Metriocnemus hygropetricus, Orthocladiusfuscimanus, Psychoda altemata and P. severini) in sewage treatment plants.

IIIA 2.2.2 The mode of action of Bti (Strain AM65-52) results from toxic proteins Mode of action contained in parasporal crystals. The crystals are taken up via ingestion

and under the alkali conditions present in the larvae gut the crystal dissolves releasing the active protein delta endotoxins (C1y4Aal, C1y4Bal, CrylOAal, Cryl lAal and CytlAal; revised version as per Crickmore et al 3) that induce disintegration of the larvae gut epithelium and consequent death of the larvae.

A detailed description of the mode of action of Bacillus thuringiensis subsp israelensis is given in the book by de Brujac, H. and Sutherland, D.J.4

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Date September 2007 Materials and methods Not applicable Conclusion Bti (Strain AM65-52) is a Gram positive, spore fom1ing rod-shaped bacterium that

produces a c1ystalline protein inclusion which is toxic to larvae of some Dipteran insects upon ingestion. The mode of action of Bti (Strain AM65-52) results from toxic proteins contained in parasporal c1ystals.

Reliability 1 Ac.ceptability Acceptable Remar ks None

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Remarks

3 http://www.lifesci.sussex.ac. uk/home/neil _ crickmore/B ti

4 de Barjac, H. ru1d Sutherland, D.J. eds. 1990. Bacterial Control of Mosquitoes and Black Flies, Rutgers University Press, pp 45-62.



September 2007


IIIA 2.3 Bti (Strain AM65-52) exhibits specific toxicity to Dipteran insects upon Host specificity r ange ingestion as demonstrated in the following report, and has a limited and effects on species impact on non-target organisms and is non-toxic to mammalian species. other than the ta1·get Further infonnation relating to the effects on non-target species and on organism toxic effects on manunalian species are contained within Section IIIA 7

and Section IIIA 8, respectively.

Smith, R.A., Cooper, R.D. (1990). 'VectoBac' Technica l Powder (EPA Registration Number 275-54) Product Chemistry Based on Bacillus thuringiensis, subspecies israelensis Strain AM65-52 (ATCC-SD-1276) as the Active Ingredient. Abbott Laboratories, unpublished report no. VTP-02 .

The infonnation in this repo1t is confidential to Valent Bios ciences and is presented in the confidential attachment under Point IIIA 2. 3.

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Mate1·ials and methods lllfotmation considered confidential by the Applicant

Condusion Considered confidential by the Applicant Reliability 1 Acceptability Acceptable Remarks None

Comments from ... Date Results and discu ssion Condusion Reliability Acceptability Remar ks



September 2007

SECTION IIIA 2 BIOLOGI CAL PROPERTIES OF THE MICRO-ORGANISM Official use only

IIIA 2.4 Bacillus thuringiensis cultures are fow1d in nature in one of two states, Development stages/life either as vegetative cells that are actively growing and dividing, or as cycle of the micro- spores . The spores are a resistant, metabolically inactive, resting f01m organism with a completely different fine structure, chemical composition and

enzymatic constitution from the vegetative cells. The life-cycle of Bacillus thuringiensis follows the characteristic process of spore fonnation (spornlation) typical of Bacillus cultures, with the exception that insect toxin containing parasporal bodies are fonned dw·ing spomlation. Spore fonnation normally commences when vegetative growth ceases due to a lack of nutrients or a shift in the environment to conditions less favotll'able for vegetative growth. Bacillus spores are resistant to desiccation, heat, ultraviolet itrndiation and other envit-onmental factors such as chemical disinfectants and can survive in envit-onments protected from sunlight (e.g. soil) for many months. Degradation of the insect toxins, however, is more rapid and is generally measUl'ed it1 days in most situations. In water, Bacillus thuringiensis is rarely detected for more than a few days after application and on foliage Bacillus thuringiensis and its associated insect toxins do not persist.



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Da te September 2007 Materials and methods Not applicable Conclusion Descriptive paragraph

Reliability Not applicable Acceptability Not applicable Remarks No reference to specific studies

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September 2007


IIIA 2.5 Bacillus thuringiensis species are not infective within populations of the Infectiveness, disper sal target organism and do not multiply substantially in the cadaver. Re-and colonisation ability infection in the field after application is not expected to occur. Bacillus

thuringiensis species can be considered a nati.u·al pa1t of the micro flora in the environment as detailed under Point IHA 2 .1.

Toxins of human health and/or environmental concern consist of beta-exotoxins, bacterial contarninants or emetic/dian-hoeal enterotoxins. The production of Bti (Strnin AM65-52) using submerged pure cultllre fermentation is considered to be free of these hazardous human and/or animal pathogens and there is no expectation that these human and/or animal toxins will be formed as a consequence of the nati.u·al life-cycle of the organism following release into the environment.

Infectivity Despite many years of broad scale application of Bacillus thuringiensis based products, very few associated human health problems have been reported and proven cases of Bacillus thuringiensis causing clinical infections in hmnans remain extremely rare. These have been two occupational health incidents where accidental exposure to Bacillus thuringiensis resulted in conditions requiring treatment with antibiotics. Bacillus thuringiensis isolates have be.en recovered from patients with severe btun wounds but these isolates were different from those used in commercial preparations.

Monitoring of public health and bacterial cult1.Ires collected for routine pmposes in communities exposed to Bacillus thuringiensis spraying has raised few concerns. Where Bacillus thuringiensis was recovered from patients, it could not be determined as a cause of illness. Considering the extensive use of Bacillus thuringiensis ah'eady made for pest control and its ubiquitous nat1.Iral occurrence in the environment, reports of human infections are rare indicating a low risk. 56

Dispersal Dispersal via transrnission between insects will be poor or non-existent since the mode of action of Bti (Strain AM65-52) is not dependent upon infection. Recycling in the target population is not expected since Bacillus thuringiensis has a toxic rather than a pathogenic mode of action. For these reasons dispersal ·will lead to dilution of any toxic effects.

Colonisation ability The srudies summarised in Section IHA 7. I show that vegetative cells of Bacillus thwingiensis have a lirnited survival time in the environment and spores do not genninate readily. It is therefore considered highly unlikely that Bacillus thuringiensis will colonise areas of intended use above levels that may occur nati.u·ally.

5 Glare, T .R. and O 'Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety, John Wiley and Sons Ltd, pp 59.

6 Siegel, J.P. The Mammalian Safety of Bacillus thwingiensis Based Insecticides. Jownal oflnve1tebrate Pathology. 77, 13-21 (2001).

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Materials and methods Partly considered as confidential pa1t ly refen·ed in textbooks.

Condusion Bacillus thuringiensis species are not infective within populations of the target organism and do not multiply substantially in the cadaver. Despite many years of broad scale application of Bacillus thuringiensis based products, very few associated human health problems have been reported and proven cases of Bacillus thUl'ingiensis causing clinical infections in humans remain extremely rare. These have been two occupational health incidents where accidental exposure to Bacillus thwingiensis resulted in conditions requiring treatment with antibiotics Dispersal via transmission between insects will be poor or non-existent since the mode of action of Bti (Strain AM65-52) is not dependent upon infection. Recycling in the target population is not expected since Bacillus thuringiensis has a toxic rather than a pathogenic mode of action. Vegetative cells of Bacillus tlnaingiensis have a limited survival time in the environment and spores do not genninate readily.

Reliability I


Remarks None

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September 2007


IIIA 2.6 Bacillus anthracis and Bacillus cereus are bacterial species related to Relationships to known Bacillus thuringiensis. Bacillus anthracis is known to cause antlu·ax in plant 0 1· animal or human humans and animals, whilst Bacillus cereus is knovm to ca.use gastro-pathogens intestinal disorders in humans. Methods to distinguish Bti (Strain

AM65-52) from these other Bacillus species and strains are described under Point IIIA l.3.4/04.

There are no other active metabolites and degradation products that are known to contribute to the toxicity of Bti (Strain AM65-52). The presence ofbeta-exotoxins and enterotoxins which may be produced by other Bacillus thuringiensis subspecies is monitored and controlled during production and do not occur in Bti (Strain AM65-52). Studies to show the absence ofbeta.-exotoxin and enterotoxins are presented wider Point IIIA 2.8.

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Conclusion There is sufficient experimental evidence to consider that Bti AM65-52 has no relationship to human pathogens, and that. there is absence of beta toxin and enterotoxin

Reliability 2 Acceptability Acceptable

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September 2007


IIIA 2.7 Gene transfer to and frorn Bacillus thuringiensis bacteria are a natural Genetic. stability and events in the environment, however gene transfer will only take place in factors affecting it the presence of metabolically active bacteria. Bacillus thuringiensis is

present primarily in its spore form in the environment and therefore opportunity for gene transfer rnay be regarded as negligible.

Furthermore, plasmid transfer has been shown to be difficult in non-sterile soils and therefore natural gene transfer rnay not be conunon. 7


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Da te September 2007 Materials and methods References to textbook Conc.lusion Gene transfer negligible, plasmid transfer difficult Reliability 112 Acceptability Acceptable

Remarks None

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Results and discussion Conc.lusion

Reliability Acceptability

Remarks

7 Glare, T.R. and O'Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety, John Wiley and Sons Ltd, pp 99.



September 2007

SECTION IHA 2 BIOLOGICAL PROPERTIES OF THE MI CRO-ORGANISM Official use only

IHA 2.8 Human or mammalian toxins may occur as beta -exotoxins or Information on the emetic/diall'hoeal enterotoxins. Beta-exotoxins are adenosine production of triphosphate (ATP) analogues that are water soluble and heat stable metabolites (especially metabolites fo1med dw-ing the vegetative grov.rth phase of some Bacillus toxins) thuringiensis strains. They are inhibitors of RNA polymerase and act

competitively with natural ATP in various biological processes and as such can be toxic. Enterotoxins are considered important because they are characteristic of the Bacillus cereus species which is closely related to Bacillus thuringiensis.

Bti (Strain Al\165-52) is in a phylogenetic grouping containing Bacillus thuringiensis strains which are not toxigenic to vertebrates. These strnins are distinctly separate from the clusters of other Bacillus isolates (e.g. Bacillus anthracis and Bacillus cereus) tliat f01m toxic metabolites.

The fonnation of toxic metabolites is discussed in the following review document.

IHA 2.8-01 Info1·mation on the production of metabolites (especially toxins)

Reference Goodyear, A. (2005) Bacillus thuringiensis subsp. israelensis, Strain AM65-52: Lack of Metabolites of Concern Expert Review for EU Dossier. Valent Bios ciences, unpublished rep01t no. 22-1-5.TOX.

Data p rntection Yes

Data o·wner Valent Biosciences

Companies with letter of None access

C1·ite1·ia for data Submitted on an existing a.s. for the pmpose of entry into Annex I. protection

Guideline study Not applicable (review of published literature)

GLP No (review of published literature)

Deviations None

Materials and Methods Not applicable, review of published literature.

Results Metabolites that are of concern for human health and/or the environment There are three types of metabolites that might be considered of concem for human health.

Some strains of Bacillus thuringiensis (Bt) have been found to produce ~-exotoxins. These are heat stable nucleotides, or their hydrolysis products, which inhibit RNA polymerase by competitive inhibition of ATP and are toxic to almost all life fonns. Strain AM65-52 has been tested for the ability to synthesize ~-exotoxins using analysis for tlie toxins by HPLC and with a bioassay using the Musca domestica fly. Both methods showed that the exotoxins are not produced by Bt subsp. israelensis, strain AM65-52.

Bt isolates have been found to produce Bacillus cereus type enterotoxius. This is reviewed below and it is concluded that, under n01mal use conditions, Bt subsp. israelensis, strain Al\165-52 would not produce amounts of the enterotoxins that cause either vomiting or diall'hoea. Testing specific to Valent Biosciences Corp. manufactw-ing with Bf subsp. israelensis, strain Al\165-52 also shows the lack of enterotoxin production in the product.

Consideration also has to be given to whether the o endotoxin is toxic to



September 2007


humans. Alkaline conditions are required to solubilise the large inert protoxin of o endotoxin. In addition, suitable proteases are needed to convert the protoxin to the smaller o endotoxin. Whilst these conditions may prevail in the gut of insects, they are not found in the mammalian gut. Data indicate that o endotoxin proteins are readily degraded within two minutes by simulated gastric fluid and within ten minutes in the presence of the gastric enzyme, pepsin. As a result, extensive man:unalian safety tests against a range of small mammals have demonstrated a very low safety risk from direct exposW'e to Bt spores and o endotoxins. Studies are presented on the acute toxicity of Bt subsp. israelensis st.rain AM65-52, containing the o endotoxin. These show the low acute toxicity to mammals. When proteins are toxic, they ai·e known to act via acute mechanisms and at very low dose levels. Therefore, since no significant acute effects were observed, even at relatively high dose levels, the delta-endotoxin is not considered toxic to man:unals. The o endotoxins have been evaluated by a1nino acid sequencing and show no similarities to any known allergens.

As a general test for any other toxic metabolites or toxic residues from the fomentation process ea.ch batch of technical slW'ry is tested by the EPA mammalian mouse safety test. The ma.n:unalian mouse safety test was specifically instigated by the US EPA to ensW'e that Bacillus anthracis is not accidentally present. In this test a mouse is given a subcutaneous injection of the production sluny containing a concentration of> 106

spores and observed for 7 days. If the mouse shows any sign of infection or injwy the batch will be rejected.

Information regarding closely related SQecies Bt is closely related to Bacillus cereus, the only known difference between the species being the production of the o endotoxin. The pathogenic effects of Bacillus cereus are caused by metabolites. Bf is also related to Bacillus anthracis which produces the anthrax toxin.

The literature available to assess potential effects of Bt on humans is extensive, and has been extensively reviewed, which is consistent with the history of use, extent of production ai1d global adoption of Bf for pest control. The body of studies available suggests the continued use of Bt, tmder the present conditions of use, poses negligible risk to human health. Equally as compelling as the available studies demonstrating product safety, are the relatively few reports in the literature of clinical infections potentially caused by Bf. Available data support the contention that Bt is highly w1likely to infect humans exposed. There are ve1y few reported incidences of adverse effects associated with Bt exposure to humans and only one where the observed effect was associated with occupational exposure to Bf, in which it could not be conclusively detennined it was ca.used by Bt.

Large scale epide1niological studies have been conducted on populations exposed to aerially applied con:unercial fo1mulations of Bt subsp. kursfaki.. In both studies exposme was confumed and no adverse effects attributable to Bf exposW'e were observed in the populations examined. If Bt were a vimlent human pathogen or did commonly produce toxins at levels which might be expected to produce ill effects in exposed individuals rn1der field conditions the frequency of occtUTence of reported effects associated with use would be expected to be much greater than that observed. At a minimum, it would be expected that some patterns would be observed in over 30 years of use. The fa.ct that it is not, suggests the potential for ill effect from exposure is possibly overstated.

Formation of toxic metabolites among closely related s11ecies Bacillus cereus is known to be responsible for two types of foodbome poisoning



September 2007


• Vomiting which is caused by the ingestion of a heat stable toxin which consists of a cyclic peptide.

• DiatThoea which is ca used by heat labile enterotoxins of which 3 have been identified. All three components are understood to be necessary for enterotoxic activity to be fully expressed, however a binary combination of components can have some biological activity (Granum and Lund 1997).

Both symptoms are rarely reported together. It is known that the peptide responsible for the emetic effect is denatured by digestive enzymes and that onset of vomiting is normally observed soon (0.5 to 5 how-s) after ingestion of contaminated food. This suggests the Bacillus cereus emetic toxin must be present at an elevated concentration (105

- 108 cells g-1) at

the time of ingestion to produce a gastroenteritic event. The U.S. Food & Dmg Administration also indicates that these levels of Bacillus cereus would be required to possibly cause food poisoning events.

Analysis of isolates of Bacillus species have demonstrated that toxin production is liinited to a few closely related strains. Bacillus cereus includes strains that are considered innocuous (some strnins are used as animal probiotics). Bacillus cereus var. toyoi NCIMIS 40112/CNCM I-1012 at a rninimum of 1 x 101° CFU/g is registered in the EU as a feed additive.

It is likely that enterotoxins produced during fermentation will be degraded by proteases released in the lysed cultures after fermentation is completed. This has been confumed in a study which tested for one of the enterotoxins. Even if any enterotoxins were present in the active ingredient or formulation, they are also unlikely to survive either the low pH or digestion by proteolytic enzymes in the stomach. Enterotoxin is produced following ingestion of an infective dose of spores. The germination of spores, growth and toxin production have to occur within a certain period of time as the onset of symptoms is usually 6-24 hours after consumption. Enterotoxin may also be produced by ingestion of vegetative cells but, unlike spores, most cells will not survive the acid pH in the stomach.

It is important to recognise that all Bacillus cereus strains do not have an equal ability to cause diaiThoea. Bacillus cereus is frequently present in the gastrointestinal tract and it has been proposed that there is a symbiotic relationship between Bacillus cereus (and related species) and the invertebrate host that only occasionally results in a pathogenic effect.

The amotmts of enterotoxin produced and cytotoxic activity against cells have been shown to vary amongst strains in an in vitro assay using Caco-2 cells. The enterotoxin producing genes are not expressed tmder all conditions. A number of studies indicate that enterotoxin production in culture is promoted through availability of starch and under conditions of optimal pH and temperature. Viable spores of Bt subsp. kurstaki. have been shovm to produce enterotoxin detectable in commercial assays and characterized as cytotoxic based on results of assays with Vero cells. However, the same strain gave negative results when evaluated with a rabbit ileal loop test. Vegetative cells of Bt, Bt spores and Bt enterotoxin extracted from culture medium failed to elicit a positive response in more than 2 of 7 animals tested. The Bt spores and cells from this culture were also fed to monkeys and no effect was observed.

The discrepancy between observed enterotoxin producing ability in in vitro and in vivo test systems shows that there may be a fundamental difference in the expression of enterotoxins produced by Bt and Bacillus cereus strains. This conclusion is suooorted by the results of DNA



September 2007


analysis. DNA from over 300 strains of Bt, Bacillus cereus and Bacillus anthracis have been analysed by amplified fragment length polymorphism (AFLP). This technique produces a visual finge1print of the DNA and the data obtained have been used to construct a phyllogenetic tree which shows that the strains could be placed into 3 clusters, each of which contained 3 or 4 branches. The DNA of Bt subsp. israelensis strain AM65-52 has been analysed by the same technique and the results placed them into Cluster I Branch A. This is a phyllogenetic grouping consisting of many Bacillus thuringiensis strains which are not toxigenic to vertebrates . It is distinctly separate from pathogenic and toxigenic Bacillus isolates which are included in a different cluster. TI1e analysis also showed that Bt is only distantly related to Bacillus anthracis and there is consequently no likelihood that Bt will produce toxins similar to those which cause anthrax.

The conclusion that pathogenic strains present their genotypes in one cluster has been reached by various other researchers. Pathogenic strains, including B. anthracis, have been shown to be more closely related to one another than to environmental isolates . All emetic strains, as well as B. anthracis, seem to represent a single clone. The levels of DNA relatedness between B. cereus and Bf strains range between 65 and 70%. This result suggests that many of the Bt serovars are genetically distinct but closely related.

Applicant's Summary It is concluded that Bt subsp. israelensis strain AM65-52 is very unlikely and conclusion to present a hazard to humans through the production of toxic metabolites

and this conclusion is consistent with many years experience of the use of this strain as an insecticide.

Reliability I.

Deficiencies No.



Evaluation by Rapporteur Member State

Date September 2007 Materials a nd methods Not applicable Conclusion Bt subsp. israelensis strain AM65-52 is unlikely to present a hazard to humans

through the production of toxic metabolites Reliability Not applicable (it is a review document)

Acceptability Not applicable (it is not an experimental paper) Remarks none

Comments from ... Date Results and disc.ussion

Condusion Reliability Acceptability Remarks



September 2007


IIIA 2.8-02 Info1·mation on the production of metabolites (especially toxins)

Reference Nair, A. (2005) High Performance Liquid Chromatography Assay for Type I and Type II ~-Exotoxin and their dephosphorylated variants in 'VectoBac' Sluny. Valent BioSciences, tmpublished report no. VBC-LG-C-01 -02-0005.

Data protection Yes

Data owner Valent Biosciences


C1·iteria for data Submitted on an existing a.s. for the purpose of entry into Annex I. protection

Guideline study US EPA Guideline 885-1200.

GLP Yes

Deviations None

Materials a nd Methods A reversed phase HPLC method (C18 column with potassium phosphate (pH 3) mobile phase) was used to analyse three batches of 'VectoBac' shury (R-4486, R-4487 and 4488) for potential beta-exotoxin content. Identification was made with reference standards for both Type I (sodiwn thuringiensin, Lot 12-313-BD, purity79.8%) and Type II (HD-12, serotype 8ah, variety monisoni) exotoxin, using UV detection at 260 nm. The change in HPLC retention time of the reference materials following dephosphorylation was also measured.

Results The 'VectoBac' shmies all tested negative for the presence of both Type I and Type II forms ofbeta-exotoxin. Positive contrnls showed the presence of the respective beta-exotoxi.ns. The results are summarised in Table IIIA 2.8-01.

Applicant's Summary The HPLC method described was shown to be suitable for the detection of and conclusion Type I and Type II beta-exotoxins and confirmed that these substances

were not present in representative batches of 'VectoBac' sluny.

Reliability I.

Deficiencies No.

Table lllA 2.8-01 HPLC analysis of beta-exotoxins in 'VectoBac' slurries

Sample HPLC retention time HPLC retention time beta-exotoxin type I beta-exotoxin type II

Sodiwn thuringiensin Type I standard 6.3 minutes Not detected

Positive strain ofBt (k) for Type I exotox.in {NRD-12) 6.1 minutes Not detected

Negative strain ofBt for Type I exotoxi.t1 (HD-I) Not detected Not detected

HD-12 Type II beta-exotoxin standard Not detected 1.8 minutes

'VectoBac' sluny batch R-4486 Not detected Not detected

'VectoBac' slunybatchR-4487 Not detected Not detected

'VectoBac' sluny batch R-4488 Not detected Not detected



September 2007

Evaluation by Competent Authoti ties


Evaluation by Rapporteur Member State Date September 2007

Materials and methods HPLC analysis

Condusion The HPLC method described seems to be suitable for the detection of Type I and Type II beta-exotoxins and suggets that these substances are not present in some batches of 'VectoBac' slwry.

Reliability 2

Acceptability acceptable

Remarks none

Comments from ...

Date Results and discu ssion Condusion Reliability Acceptability Remar ks



September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES OF THE M ICRO-ORGANISM Official use. only


Reference Chang, W. (1994) Determination of~-Exotoxin in ' VectoBac' TGAI. Valent Biosciences, unpublished report no. 82-2435-62.

Data p rotection Yes



C1i te1·ia for data Submitted on an existing a.s. for the pmpose of entry into Annex I. protection

Guideline study Not stated

GLP No

De via tio ns None

Materials and Methods A reversed phase HPLC method (C18 column with gradient elution using 0.5% potassium dihydrogen phosphate (pH 2.5) and 0.5% potassium dihydrogenphosphate/acetinitrile (1: 1 v/v) mobile phase) was used to analyse three representative batches of 'VectoBac' Technical Grade active ingredient (TGAI) (lot 71-182-W5, lot 72-21 l -W5 and lot 72-212-W5) for potential beta-exotoxin content following isolation and concentration using aminopropyl solid phase extraction (SPE). Identification was made with a reference standard for Type I exotoxin (sodimn thmingiensin, Lot 12-313-BD, purity 80.7%) using UV detection at 260 run. The limit of detection was 0.5 ppm.

Results The 'VectoBac' TGAI batches 71-182-W5, 72-21 l -W5 and 72-212-W5 tested negative for the presence of Type I beta-exotoxin at a limit of detection of 0.5 ppm. The results are show11 in Table IIIA 2.8-02.

Applicant's Summary Type I beta-exotoxin was not detected in representative batches of and conclusion 'VectoBac' TGAI using analysis by HPLC.

Reliability 1.

Deficiencies No.

Table lllA 2.8-02 HPLC analysis of beta-exotoxins in 'VectoBac' TGAI

'V ectoBac' TGAI batch number Beta-exotoxin content (ppm)

71-182-W5 Not detected, less than 0.5 ppm





September 2007




Materials and methods Reverse phase HPLC

Condusion Type I beta-exotoxi.t1 was not detected in representative batches of 'VectoBac' TGAI usi.t1g analysis by HPLC

Reliability 3 (unpublished, no GLP, no guidelines) Ac.ceptability acceptable

Remarks none

Comments from ...

Date Results and discussion Condusion Reliability Acceptability Remarks



September 2007



Reference Bowman, L. (2004) Summary Report: Detection ofEnterotoxin in Valent Biosciences Bt Fermentation Beers and Bt Products. Valent Biosciences, unpublished repo1t, munber not stated.

Data protection Yes



C1i te1·ia for data Submitted on an existing a.s. for the pmpose of entry into Annex I. protection

Guideline study US EPA Guideline 885-1200.

GLP No

De via tio ns None

Materials and Methods It has been repo1t ed in the literatme that certain Bacillus thuringiensis strains contain the genes necessaiy for producing enterotoxins and, under certain growth conditions, the production of enterotoxin can be detected8

.

However, the following report shows that production of these enterotoxins does not occur in Bti (Strain AM65-52) final fennentation sluny, or in the final 'VectoBac' products.

The purpose of the studies was to detennine if enterotoxin proteins ai·e detectable in the final fermentation beers used for 'VectoBac' products, or in the final 'VectoBac' products themselves.

The test method used was the TECRA Bacillus Diall'hoeal Enterotoxin Visual Immunoassay kit produced by Tecra International Pty Ltd. The TECRA BDE VIA system detects the 40-45kDa protein refell'ed to as the BDE nonhemolytic enterotoxin or NHE. The sensitivity of the TECRA BDE system is 2 ng/ml. For the TECRA system, all dilutions were made using 25 mM Tris buffer at pH 8.0 as the diluent.

Enterotoxin Testing of Production Beers Enterotoxins are produced in log or early stationaiy phase cultures. It is unlikely that enterotoxins will be produced during the harvesting and recove1y process, when the nutrients are depleted and growth has ceased. Any enterotoxins will be either removed or degraded by proteases in the lysed cultmes after fermentation is completed. Therefore if enterotoxins are not present in the final beers they would not be expected to be present in the final products.

To detennine whether enterotoxins were produced in commercial production processes, final fennentation beers from completed Abbott production runs of the various product strains were collected and tested for the presence of soluble enterotoxin. These samples were collected by manufacturing personnel and immediately frozen prior to transfer to VBC for analysis. The samples were thawed (if necessary), centrifuged for 20 minutes at 15000 rpm (26,900 x g) in an SS-34 rotor ai1d filtered through

8 Damgaard, P.H. Diall'hoeal Enterotoxin Production by Strains of Bacillus thuringiensis Isolated from Collllllercial Bacillus thwingiensis-Based Insecticides. FEMS Immunology and Medical Microbiology 12, (1995), 245-250.



September 2007

SECTION IHA 2 BIOLOGICAL PROPERTIES OF THE MICRO-ORGANISM

a 0.2 µm filter. If the samples did not clarify well in the first centrifugation step, they were centrifuged a second time for 40 minutes prior to the filtration step. The filtered materials were then diluted then analysed using the TECRA VIA test kit.

The 'VectoBac' samples evaluated were the final be.ers of the following production nms:

'VectoBac' (B.t. subsp. israelensis, Strain AM65-52) nm 4413 5-25-04 'VectoBac' (B.t. subsp. israelensis, Strain AM65-52) VS-138 Tank 1 (soy medium) 5-24-04

In addition, Bacillus cereus strain UW-85, known to be enterotoxin positive, was grown in tryptic soy broth (TSB) in flasks for use as a positive control in the test. Both fresh and frozen broth (-20°C) was included to test if freezing reduced the ability to detect the presence of enterotoxin. As additional controls, fresh and frozen flask broth of B. t. israelensis, Strain AM65-52, grown in TSB, was also included. Since Bacillus sphael'icus does not produce enterotoxin9

, the following two Bacillus sphael'icus fe1mentation final beers were included in the test as negative controls: 'VectoLex' (B. sp haericus strain 2362) nms 6040 T-911 5-22-04 and 6041 T-911 5-24-04.

Bt kurstaki HD-1 DiPel 2675 and Bt aizawai XenTari 2672 fem1entation beers were also analysed as part of the study, however the results are not relevant to this dossier.

Enterotoxin Testing of Finished Products Finished products were tested for enterotoxin to confirm results seen with the fe1mentation beers and to verify that enterotoxin would not be concentrated in the downstream processing of the product.

The 'VectoBac' sample evaluated was the product: 'VectoBac' TP (B.t. subsp. israelensis, Strain AM65-52) Lot# 70-150-W5 .

B. cereus UW-85 flask broth, grown in brain/heait infusion broth (BHI) was included as a positive control. Other products were tested but the results are not relevant to th.is dossier. For the 'VectoBac' TP (technical powder) a 10% (w/v) solution in 25 mM Tris buffer was prepared, centrifuged, and filtered through a 0.2 ~un filter. Dilutions of 1/10 and 11100 of this sample were analysed.

Results The results are presented as they appeai·ed on the test plate in relation to the colom coded test key. Values of 1 or 2 are considered negative for NHE enterotoxin. Values of3, 4, or 5 are considered positive for NHE enterotoxin. The results for the fe1mentation beers ai·e shown in Table IIIA 2.8-03 and the visual image from the respective test plate is presented in Figure IIIA 2.8-01. The results for the finished products are shown in Table IIIA 2.8-04 and the visual image from the respective test plate is shown in Figme IIIA 2.8-02. The results from this study showed no enterotoxin was detected in fermentation beers from productions rnns or in the finished products. The comparison of the fresh and frozen TSB broth samples indicates that there is little if any loss of detectible enterotoxin that occw·s as a result of freezing the beer samples prior to testing. Therefore, it was deemed acceptable to have sainples collected and frozen prior to use if necessaiy.

Applicant's Summary The final fe1mentation beers, used for Valent Bios ciences Bacillus and conclusion thuringiensis products, test negative for enterotoxin according to the Tecra

9 Yuan, Z. et al. Detection ofEnterotoxin Genes in Mosquito-Larvicidal Bacillus Species. current Microbiology Vol. 45 (2002), pp. 221-225.

Official use only

Sumitom o Chemical Agr o Eu rnpe (fo1· Valent BioSciences Corporation)

Bacillus tlturingiensis subsp. israelensis Ser otype H -14 Strain AM65-52

September 2007

SECTION IHA 2 BIOLOGICAL PROPE RTIES OF THE M ICRO-ORGANISM Official use only

VIA test system. The corresponding final products also test negative for the presence of enterotoxins according to the TECRA VIA test system.

Reliability 1.

Deficiencies No.

Table lllA 2.8-03 Enterotoxin test results for final fermentation beers

Dilution Sample

1/100 1/10 115 Full str ength

Internal Negative Control 1 1 1

Internal Positive Control 4 4 4

B . cereus broth TSB 4 5 5

Bt israelensis broth TSB 1 1 1

B . cereus broth TSB frozen 3 4 4

Bt israelensis broth TSB frozen 1 1 1

B . sphaericus 'Vectolex' 6040 1 1 1

B . sphaericus 'Vectolex' 6041 1 1 1

Bt israelensis 'VectoBac' 4413 1 1 1

Bt israelensis 'VectoBac' VS-138 1 1 1

Figure llM 2.7.2-01 Visualisation of enterotoxin test results for final fermentation beers

1:100

1: 10

1: 5

FS

2 3 4 5 6 7 8 9 10 11 12

Batch No. ~ .... 1.rc 14203057 TECRA®

8.0.E VISUAL IMMUNOASSAY

Use By: 7 JAN2005 0 \ ~) 0

ANTIBODY COA 1 ': '.' REPl'">VA\. ._ Rtsea, ~:>L>th . !It ~

h-

Negative Control 7. B. sphaericus Vectolex Run 6040 B. sphaericus Vectolex Run 6041 B.t. kurstaki DiPel Rtm 2675

Positive Control 8. B. cereus broth, grown on TSB 9.

1

4

5

1

4

2

1

1

1

1

1. 2. 3. 4. 5. 6.

B.t. israelensis broth, grown on TSB I 0. B. cereus, grown on TSB and frozen 11 .

B.t. israelensis 'VectoBac' Run 4413 B.t. aizawai Rw1 Xentari Rw1 2672

B.t. israelensis broth , grown on TSB and frozen 12. B.t. israelensis Run VS-138 (Soy medium)

Sumitomo Chemical Agro Europe (for Valent BioSciences Corporation)

Bacillus thuringiensis subsp. israelensis Serotype H-14 Strain AM65-52

September 2007

Table IIIA 2.8-04 Enterotoxin test results for finished products

Sample Dilution

1/100 1/10 Internal Negative Control 1 1 Internal Positive Control 4 4 B. cereus BHI 5 5 Bt israelensis ‘Teknar’ 1 1 Bt israelensis ‘VectoBac’ TP 1 1

Figure IIIA 2.8-02 Visualisation of enterotoxin test results for finished products



September 2007




Materials and methods Visual Immunoassay (TECRA test)

Conclusion Test negative for enterotoxin according to the T ecra VIA test system. The coITesponding final products also test negative f the presence of enterotoxins according to the TECRA VIA test system

Reliability 2

Acceptability acceptable

Remar ks none

Comments from ...

Date Results and discussion Conclusion Reliability Acceptability Remar ks



September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES OF THE M ICRO-ORGANISM Official use. only

IIIA 2.9 Infonnation on resistance/sensitivity to antibiotics/anti-microbial agents Antibiotics and other used in human or veterinary medicine is given in the repo1t summarised anti-microbial agents under Point IIIA 1.3.4-01.

The results showed that Bti (Strain AM65-52) is susceptible to some antibiotics but not others. Susceptibility or resistance was based on comparing zones of inhibition with a control strain of staphylococcus aureus.


Use separate "evaluation boxes" to provide trnnsparency as to the conunent.s and views submitted


Da te Sept.ember 2007

Materials and methods Considered confidential by the Applicant Conc.lusion Bti (Strain AM65-52) is susceptible to some antibiotics but not to others.

Susceptibility or resistance was based on comparing zones of inhibition with a control strain of staphylococcus aureus.

Reliability 1


Remar ks The study quoted (point IIIA 1.3.4-01) does not repo1t the experimentaI design

Comments from ...

Date Results and discu ssion Conc.lusion Reliability Acceptability Remar ks



September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES OF THE MI CRO-ORGANISM Official use only

IIIA 2.10 JUSTIFICATION FOR NON-SUBMISSION OF DATA Robustness to environmental factor s

Other existing data [X) Technically not feasible [ I Scientifically unjustified [ I Limited exposure [ I Other justification [ I Detailed justification: Infonnation concerning the behaviour of Bti (Strain AM65-52) in the

environment is presented in Section 7.

Undertaking of intended Not applicable. data submission [ I

EVALUATION BY COM PETENT AUIBORITIES

EVALUATION BY RAPPORTEUR MEMBER STATE

Date September 2007

Evaluation of applicant's Acceptable justification

Conclusion Not applicable

Remar ks None

COMMENTS FROM OTHER MEMBER STATE

Date

Evaluation of applicant's j ustific.a tio n

Conclusion

Remarks



September 2007

SECTION IIIA 2 BIOLOGICAL PROPERTIES OF THE MI CRO-ORGANISM Official use only

IIIA 2.11 JUSTIFICATION FOR NON-SUBMISSION OF DATA Effects on mate1i als, substances and products

Other existing data [ I Technically not feasible [ I Scientifically unjustified [X)

Limited exposure [ I Other justification [ I Detailed justification: Bti (Strain AM65-52) has a specific mode of action against larvae of

certain species of Dipteran insects. The mode of action results from toxic proteins contained in parasporal c1ystals. The c1y stals are taken up via ingestion and under the alkali conditions present in the larvae gut the crystal dissolves releasing the active protein delta endotoxins. There are no other active metabolites and degradation products that are knovm to contribute to the toxicity of Bti (Strnin AM65-52).

Bti (Strain AM65-52) is used for the control of mosquito and black fly larvae in water habitats and filter fly midges in sewage treatment plants. Due to its specific mode of action, Bti (Strnin AM65-52) will have no other effects on the intended area of use.

Undertaking of intended Not applicable. data submission [ I

EVALUATION BY COM PETENT AUIBORITIES

EVALUATION BY RAPPORTEUR MEMBER STATE

Date September 2007

Evaluation of applicant's acceptable justification

Conclusion Bti (Strain AM65-52) has a specific mode of action against larvae of certain species of Dipteran insects.

Remar ks none

COMMENTS FROM OTHER MEMBER STATE

Date

Evaluation of applicant's justification

Conclusion

Remarks

Sumitomo Chemical Agro Eurnpe Bacillus …dissemination.echa.europa.eu/Biocides/ActiveSubstances/...Sumitomo Chemical Agro Eurnpe (fo1· Valent BioSciences Corporation) Bacillus tlturingiensis

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