Bacillus tlturingiensis subsp. israelensis September 2007dissemination.echa.europa.eu/Biocides/ActiveSubstances/0005-18/... · equipment between every production nm, the control of

Sumitomo Chemical Agro Eurnpe (fo1· Valent BioSciences Corporation)

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

September 2007

SECTION IIIA 4 ANALYTICAL METHODS Official use ouly

IIIA 4.1 Methods for analysis of the micro-organism as manufactured

IIIA 4.1.1 The methods of analysis for Bti (Strain AM65-52) are confidential to Method to preserve and Valent Bios ciences and is presented in the confidential attachment maintain the master seed under Point IIIA 4.1.1. stock

Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted

Evaluation by Rapporteur Membe1· State Da te September 2007

Materials a nd methods Not applicable Conc.lusion Not applicable

Reliability Not applicable Acceptability Not applicable

Remar ks See evaluation of Confidential Infonnation

Comments from ...

Date Results and disc.ussion

Conc.lusion

Reliability Acceptability Remarks



September 2007

SECTION IIIA 4 ANALYTICAL METHODS Official use only

IIIA 4.1 Methods for analysis of the micrn-organism as manufactured

IIIA 4.1.2 Characterisation of strain or serotype within Bacillus thuringiensis Method to detect, isolate species is commonly perfonned using classicaI techniques such as; and enumerate the micro- c1ystal morphology, biochemical reactions and bioassays. Rec.ent organism advances in mole.cular biology have allowed the development of

specific DNA based methods capable of distinguishing individual strains and isolates, including Valent Bioscience strain AM65-52.

The methods used are described in the reports summarised under Point IIIA 1.3.4-01 and IIIA 1.3.4-04 which are included with all other confidential information in the confidential attachment.

Bti (Strain AM65-52) is a Gram positive, spore forming rod-shaped bacterium that produces a crystalline protein inclusion (parasporal crystal toxin) which is responsible for toxicity to the larvae of some Dipteran insects upon ingestion. The following study describes methodology to detect and quantify the toxins present in the parasporal crystal.

IIIA 4.1.2-01 Method to detect, isolate and enumerate the micro-organism

Reference Coddens, M. (1990) 'VectoBac' Technical Powder (EPA Registration Number 275-54) Product che1nistty Based on Bacillus thuringiensis, subspecies israelensis , Strain AM65-52 (ATCC-SD-12796) as the Active Ingredient. Abbott Laboratories, unpublished repo1t no. VTP-03.

The infonnation in this report is confidential to Valent Biosciences and is presented in the confidential attaclunent m1der Point IIIA 4.1.2-01 .

Evaluation by Competent Authorities Use separate "evaluation boxes" to provide tt<1nsparency as to the comments and views submitted

Evaluation by Rapporteur Member State

Date September 2007 Materials and methods Not applicable Conclusion Not applicable Reliability Not applicable Ac.ceptability Not applicable Remarks See evaluation of Confidential Infonnation

Comments from ...

Date Results and discussion Conclusion Reliability Ac.ceptability

Remarks



September 2007


IIIA 4.1 Methods for analysis of the micro-organism as manufactured

IIIA 4.1.3 JUSTIFICATION FOR NON-SUBMISSION OF DATA Method to differ entiate mutant or GM str ains

Other existing data [ I Technically not feasible [ I Scientific.ally unjustified [X)

Limited exposure [ I Other justification [ I Detailed justification: Bti (Strain AM65-52) originates from a natural wild strain of the

bacteria and has not been genetically modified nor is it the result of a spontaneous or an induced mutation. Methods to differentiate mutant or GM strains are therefore not required.

Under taking of intended Not applicable. data submission [ I

EVALUATION BY COMPETENT AUIBORITIES

EVALUATION BY RAPPORTEUR MEMBER STATE

Date September 2007

Evaluation of applicant 's Pattially acceptable justification

Conclusion The applicant has presented a method to identify to strain level. The absence of mutations is regulated through the manufactming process in which substantial detail has been provided as complimentary infonnation.

However the Applicant has no proof of mutations which might have occwTed in the past untill this methodology has been developed in 2008

Rema1·ks Analytical methods to be completed

COMMENTS FROM OTHER MEMBER STATE

Date

Evaluation of applicant's justification

Conclusion

Remarks

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


September 2007



IIIA 4.1.4 A strain alteration can occur within a strain through genetic mutation or Method to detect plasmid loss. In addition, plasmid and gene transfer may theoretically spontaneous c.hanges to occur between a strain and a contaminating organism, if the the micrn-organism contaminant is of a type amenable to these genetic transfers. To ensure

that no modifications occur to the production culture during growth and fe1mentation, all production runs are inoculated with sub-samples of the original strain. Serial transfers from the original strain are minimized. Mass transfers of the culture are used to avoid selection of a rare mutant. To ensure no contaminants are present, stringent sterility controls and precisely controlled grov.rth conditions are used throughout the production process. Strain purity checks are routinely done at eve1y transfer stage. The culture maintenance process previously described was developed to minimise the number of transfers and the possibility of genetic drift and loss of plasmids dlll'ing growth.

Because of the mass-transfer inoculations, the possibility that a gene mutation( s) might occw· during the limited time of a fermentation, and that the mutant population would reach detectable levels, is very low. Spontaneous mutations have been found to occw· in Bacillus thuringiensis bacteria at the levels of one mutation per 107 to 1011

cells 1, and many of these mutations are deleterious or lethal to the cell. Furthennore, the chance for a mutation to be manifested as an altered crystal protein is even lower because the c1ystal protein is just one of thousands of gene products of the Bacillus thuringiensis microorganism. At such low frequency, non-mutated cells would mask any surviving mutant that occurs during the fermentation process.

Bacillus thuringiensis bacteria are capable of plasmid and gene transfer. It is theoretically possible that these events could occur during fe1mentation, if a contaminant organism of a type amenable to these transfers was present with the Bacillus thuringiensis culture. However, this is unlikely due to the rigorous sterilization of fermentation equipment between every production nm, the control of source inoculum, and the constant monitoring of mns to ensw·e a pure culture during fem1entation.

Plasmid loss could also occur during fermentation, and could negatively affect the activity of the final product, if the toxin-bearing plasmids are lost. Therefore, all production mus are monitored for acceptable biological activity as described wider Point IIIA 4.1.5.

I Prescott, L.M., Harley, J .P. and Klein, D.A. 1990. Microbiology. W.C. Brown. IA. pp 345.

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


September 2007

Evaluation by Competent Authorities

Use separate "evaluation boxes" to provide transparency as to the comments and views submitted


Date September 2007

Materials and methods Not applicable

Conclusion Absence of mutations

Reliability Not applicable

Acceptability Pa1tially acceptable. It is rather difficult to evaluate whether mutations are occmTing if a suitable method for unambiguous identification of the microbe at strain level is not available

Remar ks See remarks to Section IIlA 4 .1.3. Such a method is now available.

Comments from ...

Date

Results and discussion

Conclusion

Reliability

Acceptability

Remar ks



September 2007



IIIA 4.1.5 The content of Bti (Strain AM65-52) is described in terms of its Method to defme conten t biopotency (ITU/g) towards larvae of the insect species Aedes aegyp ti. of the mic.ro-organism The methodology used to determine biopotency is contained in the

following Abbott Laboratories Standard Procedure:

Bioassay for Bacillus thuringiensis Serotype H-14, Number. B-6-8.

This bioassay is confidential to Valent BioSciences and is presented in the confidential attachment under Point IIIA 4.1.5.

Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted


Da te September 2007 Materials and methods Not applicable Conc.lusion Not applicable Reliability Not applicable Acceptability Not applicable

Remarks See evaluation of Confidential Infonnation

Comments from ... Date

Results and discu ssion Conc.lusion

Reliability Acceptability

Remarks

Sumitomo Chemical Agro Europe (fo1· Valent BioSciences Corporation)


September 2007


IIIA 4.1 Methods fo1· analysis of the micro-organism as manufactured

IIIA 4.1.6 Human or mammalian pathogens may occur as beta-exotoxins (Type I Method to show control and Type II), bacterial contaminants or emetic/diatThoeal enterotoxins. of tnicrobial and othe1· Type I and Type II Beta-exotoxins are adenosine triphosphate (ATP) toxic impurities analogues that are water soluble and heat stable metabolites formed

during the vegetative grov.rth phase of some Bacillus thuringiensis strains. They are inhibitors of RNA polymerase and act competitively with natural ATP in various biological processes and as such can be toxic. The presence of bacterial pathogens may arise due to microbial contamination, whilst enterotoxins are considered important because they are characteristic of the Bacillus cereus species which is closely related to Bacillus thuringiensis.

Each lot of fermentation solids and soluble concentrate is tested for mammalian safety using the mouse safety test prior to addition of other fo1mulation ingredients using Standard Test Method B-0612. Sterility testing procedures used for microbial purity and sterility monitoring of the se.ed stock and fennentors are perfonned using. Basic Operating Procedure BOP.N.0834.006.03.

Details of these methods are confidential to Valent Bios ciences and are presented in the confidential attachment under Point IIIA 4.1.6.

Enterotoxins can be detected using the TECRA Bacillus Dia1Thoeal Enterotoxin Visual Immm1oassay kit produced by Tecra Intemational Pty Ltd. The TECRA BDE VIA system detects the 40-45kDa protein refel1'ed to as the BDE nonhemolytic enterotoxin or NHE. The sensitivity of the TECRA BDE system is 2 ng/ml. Details of the method used are provided in the smnmaries presented under Point IIIA 2.8-04.

The absence of Type I and Type II beta-exotoxin is dete1mined by High Performance Liquid Chromatography (HPLC) as described in the following rep01ts. Periodic monitoring of production batches is perfonned to provide assurance that beta-exotoxins are not produced.

IIIA 4.1.6-01 Method to show control of microbial and other toxic impmities

Reference Nair, A. (2005) High Perfonnance Liquid Chromatography Assay for Type I and Type II P-Exotoxin and their Dephosphorylated Variants in 'VectoBac' Sluny. Valent Biosciences, tmpublished report no. VBC-LG-C-01-02-0005.

Data p rotection Yes

Data owner Valent Biosciences

Companies with letter of None access

C1i te1ia for data Submitted on an existing a.s. for the pmpose of entiy into Annex I. protection

Guideline study US EPA Guideline 885-1200.

GLP No

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' sluny (R-4486, R-4487 and 4488) for potential beta-exotoxin content. Identification was made with reference standards for both Type I (sodimn thurin~iensin, Lot 12-313-BD, ouritv 79.8%) and Tvoe II (HD-12,



SECTION IIIA 4 ANALYTICAL METHODS

September 2007

Official use only

serotype 8ab, variety mon isoni) exotoxin, using UV detection at 260 run. The change in HPLC retention time of the reference materials following dephosphorylation was also measured.

Results The 'VectoBac' shlll'ies all tested negative for the presence of both Type I and Type II fomlS ofbeta-exotoxin. Positive controls showed the presence of the respective beta-exotoxins. The results are Sllllllllarised in Table IIIA 4.1.6-01 .

Applicant's Summary The HPLC method described was suitable for the detection of Type I and and conclusion Type II beta-exotoxins and confirmed that these substances were not

present in representative batches of 'VectoBac' slurry.

Reliability 1.

Deficiencies No.

Table lllA 4.1.6-01 HPLC analysis of beta-exotoxins in 'VectoBac' slurries

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

Soditun thtu'ingiensin Type I standard 6.3 minutes Not detected

Positive strain ofBt (k) for Type I exotoxin (NRD-12) 6.1 minutes Not detected

Negative strain ofBt for Type I exotoxin (HD-1) Not detected Not detected

HD-12 Type II beta-exotoxin standard Not detected 1. 8 minutes

'V ectoBac' shmy batch R-4486 Not detected Not detected

'VectoBac' shmybatchR-4487 Not detected Not detected

'V ectoBac' shmy batch R-4488 Not detected Not detected

Evaluation by Competent. Authorities

Use separate "evaluation boxes" to provide transparency as to the comments and views submitted

Evaluation by Rapportem· Membe1· State

Date September 2007 Materials a nd methods Not applicable Conclusion Not applicable Reliability Not applicable Acceptability Not applicable Remar ks See evaluation of Confidential Infonnation

Comments from ...

Date Results and discu ssion Conclusion Reliability Ac.ceptability

Remarks



September 2007




Reference Chang, W. (1994). Detennination of 13-Exotoxin in 'VectoBac' TGAI, Abbott Laboratories, unpublished report No. 82-2435-62.

Data p rotection Yes

Data owner Valent Biosciences

Companies with letter of None access

C1·ite1ia for data Submitted on an existing a.s. for the pwpose of ently into Annex I. protec.tion

Guideline study Not stated

GLP No

Deviations None

Materials and Methods A high perfonnance liquid chromatographic method with ultraviolet detection at 260 nm (HPLC/UV) was developed and validated for the analysis ofbeta-exotoxin as a limit test in 'VectoBac' TGAI.

Results Recove1y was tested at a concentration of 10 mg/kg. Mean recovery for beta-exotoxin was 95% and so was within guideline requirements (70 -110%; RSD ~ IIIA 4.1.6-02.

Good linearity was observed in the range of 1.25 to 12. 5 ~t.g/mL for beta-exotoxin standards in 2.5M K2HP04 (r

2= 0.9999) . Analysis of conti·ol samples showed no significant interference at the retention time of beta -exotoxin therefore the method was considered to be specific. The limit of quantification, defined as the lowest concentration at which an acceptable recove1y is obtained, is 10 mg/kg. The relative standard deviation measUl'ed with respect to recoveries following foit ification at 10 mg/kg was 5.3%. The values obtained show the method has satisfacto1y precision.

Applicant's Summar y A method for detennination of beta-exotoxin in Bti was validated and is and conclusion considered suitable to measlll'e the level of these substances in

'VectoBac' TGAI.

Reliability 1.

Deficiencies No.

Table lllA 4.1.6-02 Recovery data from method validation of beta-exotoxin in 'VectoBac' TGAI

Analyte Matdx Fortification Rec.over' rate(%) RSD

Reference level (mg/kg)• (%)

n mean ran2e

Beta- 'VectoBac' 10 95 86 - 101 5.3 5 'VectoBac' TGAI

exotoxin TGAI

a Limit of quantification is 10 mg/kg (defined as the lowest validated fortification level).



Evaluation by Competent Autho1·ities

September 2007

Use separate "evaluation boxes" to provide transparency as to the conunents and views submitted

Evaluation by Rapportem· Member State

Date September 2007

Materials and methods Not applicable

Condusion Not applicable

Reliability Not applicable

Acceptability Not applicable

Remarks See evaluation of Confidential Infonnation

Comments from ...

Date Results and discu ssion Condusion Reliability Acceptability Remar ks



September 2007




Reference Campbell, D.P., Dieball, D.E., Brackett, J.M. (1987). Rapid HPLC Assay for the beta-exotoxin of Bacillus thuringiensis, J. Agric. Food Chem. 1987, 35, 156-158.

Data p rotection No, published research

Data o·wner Not applicable

Companies with letter of Not applicable access

C1·ite1·ia for data Not applicable protection

Guideline study Not applicable

GLP No

Deviations Not applicable

Materials and Methods Information on the suitability ofHPLC as a means of detecting and quantifying beta-exotoxin content in Bac;//us thuringiensis fennentation beers is given in this published repo1t.

Analysis was perfom1ed using high perfonnance liquid chromatographic with ultraviolet detection at 260 nm (HPLC/UV).

Results Recovery was tested by spiking reference beta-exotoxin (lot 635-19) into formulated samples at levels up to I 0% of the nominal concentration. Recoveries were 97. 0 to 100 .4 % based on peak height and peak area. Detector response was linear over the range of0.00 to 0.56 mg/mL (r2= 1.000). Specificity was confirmed by heating a mixture ofbeta-exotoxin at pH 3 and monitoring the decrease in beta-exotoxin peak and cotTesponding increase in dephosphorylated-exotoxin peak follov.iing cleavage of the phosphate ester linkage. Fwt her confumation was obtained by coITelation with results from house fly bioassay. Information on sensitivity was not repo1ted. Precision was dete1mined by analysing eight replicate samples which gave a relative standard deviation of0.4% based on peak height and peak area.

Applican t's Summary HPLC was shown to be a specific method for dete1mination of beta-and conclusion exotoxin in Bti formulations and is capable of detecting both

phospho1ylated and dephospho1ylated fonns of the toxin.

Reliability 2 .

Deficiencies No.



Evaluation by Competent Autho1·ities

September 2007

Use separate "evaluation boxes" to provide transparency as to the conunents and views submitted

Evaluation by Rapportem· Member State

Date September 2007

Materials and methods HPLCmethod

Condusion The method is specific for detemlining beta-exotoxin

Reliability 2

Acceptability Acceptable

Remarks None

Comments from ...

Date Results and discu ssion Condusion Reliability Acceptability Remar ks



September 2007



IIIA 4.1.7 Testing for microbial pathogen contaminants is contained in the Method to show presence following standard procedures: of human and

STM.0309600: Standard Procedure - Colifo1m Enumeration & mammalian pathogens

Identification From Product

STM0323700: Standard Procedure - Salmonella Tests for Isolation & ID From Product

STM.0154800: SP - Clostridium perfringens Enumeration

STM.0329100: SP - Total Aerobic Microbial Count, Pour Plate, Spread Plate, Rodac Plate

STM.0323800: Standard Procedure - Pseudomonas aernginosa Enumeration and ID from Product

STM.0154600: S. Procedure - Staphylococcus aureus Tests for Enumeration and ID from Product

STM.0042100: Total Combined Yeast and Mould Com1t

STM.0154700: Standard Procedure - Enterococci Screening

The info1mation concerned is confidential to Valent Bios ciences and is presented in the confidential attachment under Point IIIA 4.1. 7.

Evaluation by Competent Authorities Use separate "evaluation boxes" to provide transparency as to the comments and views submitted Evaluation by Rapportem· Member State

Date September 2007 Materials and methods Not applicable Conclusion Not applicable Reliability Not applicable Acceptability Not applicable Remar ks All the tests are Internal Standard Operating Procedures

Comments from ...

Da te Results and discussion Conclusion Reliability Acceptability Remarks



September 2007


IIIA 4.2 Methods to determine and quantify r esidues (viable and non-viable)

IIIA 4.2.1 JUSTIFICATION FOR NON-SUBMISSION OF DATA Food

Other existing data [ l Technically not feasible [ l Scientifically unjustified [X]

Limited exposure [X] Other justification [ l Detailed justification: 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. Contamination of food items is not anticipated following use and furthermore Bti (Strain AM65-52) is hannless to non-target species and humans.

MRL's in food are not established for Bti (Strain AM65-52).

Monitoring methods in food are therefore not relevant.

Undertaking of intended Not applicable. data submission [ l

EVALUATION BY COMPETENT AUTHORITIES


Date September 2007

Evaluation of applicant's Contamination of food items can occur when aerial spray is used and via justification horizontal drainage from ponds

Conclusion Methods would be required if food items have been in contact.

Remar ks None


Date

Evaluation of applicant's j ustific.a tio n

Conclusion

Remarks



September 2007



IIIA 4.2.2 JUSTIFICATION FOR NON-SUBMISSION OF DATA Feed


Limited exposure [X ] Other justification [ l Detailed justification: 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. Contamination of feed items is not anticipated following use and furthermore Bti (Strain AM65-52) is hannless to non-target species and humans.

MRL's in feed are not established for Bti (Strain AM65-52) .

Monitoring methods in feed are therefore not relevant.




Date September 2007

Evaluation of applicant's See IIIA 4 .2. l justification

Conclusion See IIIA 4.2.1

Remar ks None


Date


Conclusion

Remarks



September 2007



IIIA 4.2.3 JUSTIFICATION FOR NON-SUBMISSION OF DATA Animal tissue


Limited exposure [X ] Other justification [ l Detailed justification: 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. Contamination of animal tissues is not anticipated following use and furthermore Bti (Strain AM65-52) is hannless to non-target species and humans.

MRL's in animal tissues are not established for Bti (Strain AM65-52).

Monitoring methods in animal tissues are therefore not relevant.




Date September 2007

Evaluation of applicant's See IIIA 4.2. l justification

Conclusion See IIIA 4.2.1

Remarks None


Date


Conclusion

Remarks



September 2007


IIIA 4.2 Methods to determine and quantify 1·esidues (viable and non-viable)

IIIA 4.2.4 JUSTIFICATION FOR NON-SUBMISSION OF DATA Soil

Other existing data [ l Technically not feasible [ l Scientific.ally unjustified [X]

Limited exposure [X] Other justification [ l Detailed justification: The use pattem of the product means there is negligible potential for Bti

(Strain AM65-52) vegetative cells, spores or parasporal crystals to enter soil at concentrations significantly greater than those present naturally. Furthermore Bti (Strain AM65-52) is readily inactivated in soil and is harmless to non-target ten-estrial species .

Methods in soil are therefore not considered relevant.




Date September 2007

Evaluation of applicant's Partially acceptable. If the concentration of the Product is not significantly higher justification than those present naturally, how can the product be effective?

Conclusion Methods would be needed if contamination of foodstuff occurs

Remarks None


Date


Conclusion

Remarks



September 2007



IIIA 4.2.5 JUSTIFICATION FOR NON-SUBMISSION OF DATA Wate1·

Other existing data [ l Technic.ally not feasible [ l Scientific.ally unjustified [X]

Limited exposure [X] Other justification [ l Detailed justification: Bti (Strain AM65-52) is rapidly inactivated in water by sorption onto

paiticulate matter and is harmless to non-target species and humans. Furthermore, Bti (Strnin AM65-52) is not used on water bodies that are intended for drinking water.

Monitoring methods for Bti (Strain AM65-52) in water ai·e therefore not considered necessary.




Date September 2007

Evaluation of applicant's Partially acceptable. If the Product is rapidly inactivated, how can the treatment be justification effective? Mosquito larvae feed on microorganisms and orgai1ic pa1i icles using

mouthparts modified into "bmshes" which draw food into the mouth. Thus the mosquito will still ingest the microbe, although it is not accessible to other non-targets.

Conclusion Methods would be needed if contamination of foodstuff occurs.

Remar ks None


Date


Conclusion

Remarks



September 2007



IIIA 4.2.6 JUSTIFICATION FOR NON-SUBMISSION OF DATA Air

Other existing data [ l Technically not feasible [ l Scientific.ally unjustified [X]

Limited exposure [X] Other justification [ l Detailed justification: The use pattem of the product means there is negligible potential for Bti

(Strain AM65-52) vegetative cells, spores or parasporal crystals to enter the air at concentrations significantly greater than those present naturally. Furthermore, Bti (Strnin AM65-52) is hamtless to non-target species and humans.

Methods in air are therefore not considered relevant.




Date September 2007

Evaluation of applicant's Partially acceptable. In the case of aerial spray this would be the case when justification inappropriate use is made

Conclusion Methods would be needed if inappropriate use is made via aerial spray

Remarks None


Date


Conclusion

Remarks

Bacillus tlturingiensis subsp. israelensis September 2007dissemination.echa.europa.eu/Biocides/ActiveSubstances/0005-18/... · equipment between every production nm, the control of

Documents