EUROPEAN COMMISSION
HEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL
Directorate E – Safety of the food chain
Unit E.3 - Chemicals, contaminants, pesticides
Bacillus subtilis
SANCO/10184/2003 - rev. 3
19 June 2006
EU-RESTRICTED
COMMISSION WORKING DOCUMENT - DOES NOT NECESSARILY REPRESENT
THE VIEWS OF THE COMMISSION SERVICES
DRAFT
Review report for the active substance Bacillus subtilis QST 713
Finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 14
July 2006 in view of the inclusion of Bacillus subtilis in Annex I of Directive 91/414/EEC.
1.
Procedure followed for the evaluation process
This review report has been established as a result of the evaluation of the new active substance
Bacillus subtilis strain QST 713 (hereafter "Bacillus subtilis"), made in the context of the work
provided for in Articles 5 and 6 of Directive 91/414/EEC concerning the placing of plant protection
products on the market, with a view to the possible inclusion of this substance in Annex I to the
Directive.
In accordance with the provisions of Article 6(2) of Directive 91/414/EEC, the German authorities
received on 19 April 2000 an application from AgraQuest, hereafter referred to as the applicant, for
the inclusion of the active substance Bacillus subtilis in Annex I to the Directive. The German
authorities indicated to the Commission on 28 May 2000 the results of a first examination of the
completeness of the dossier, with regard to the data and information requirements provided for in
Annex II and, for at least one plant protection product containing the active substance concerned, in
Annex III to the Directive. Subsequently, and in accordance with the requirements of Article 6(2), a
dossier on Bacillus subtilis was distributed to the Member States and the Commission.
The Commission referred the dossier to the Standing Committee on the Food Chain and Animal
Health in the meeting of the working group ‘legislation’ thereof on 01 September 2000, during
which the Member States confirmed the receipt of the dossier.
-2-
In accordance with the provisions of Article 6(3), which requires the confirmation at Community
level that the dossier is to be considered as satisfying, in principle, the data and information
requirements provided for in Annex II and, for at least one plant protection product containing the
active substance concerned, in Annex III to the Directive and in accordance with the procedure laid
down in Article 20 of the Directive, the Commission confirmed in its Decision 2001/6/EC 1 of 12
December 2000 that these requirements were satisfied.
Within the framework of that decision and with a view to the further organisation of the work
related to the detailed examination of the dossier provided for in Article 6(2) and (4) of Directive
91/414/EEC, it was agreed between the Member States and the Commission that Germany, as
rapporteur Member State and Sweden, as co-rapporteur Member State, would carry out the detailed
examination of the dossier and report the conclusions of the examination accompanied by any
recommendations on the inclusion or non-inclusion and any conditions relating thereto, to the
Commission as soon as possible and at the latest within a period of one year.
Germany submitted to the Commission on 15 May 2001 the report of its detailed scientific
examination, hereafter referred to as the draft assessment report, including, as required, a
recommendation concerning the possible inclusion of Bacillus subtilis in Annex I to the Directive.
On receipt of the draft assessment report, the Commission forwarded it for consultation to all the
Member States as well as to AgraQuest being the sole applicant on 23 July 2001.
Further discussion between the Rapporteur Member State and the Co-rapporteur Member State
were organised, to review the draft assessment report and the comments received thereon in
particular on each of the following disciplines :
-
identity and biological properties ;
-
fate and behaviour in the environment ;
-
ecotoxicology ;
-
mammalian toxicology ;
-
residues and analytical methods ;
-
regulatory questions.
The report of the peer review (i.e. full report) was circulated, for further consultation, to Member
States and the sole applicant on 14 October 2003.
The dossier, draft assessment report and the peer review report (i.e. full report) including in
particular an outline resumé of the remaining technical questions, were referred to the Standing
Committee on the Food Chain and Animal Health, and specialised working groups of this
Committee, for final examination, with participation of experts from all Member States. This final
1
OJ No L2, 05.01.2001, p. 25
-3-
examination took place from March 2003 to July 2006, and was finalised in the meeting of the
Standing Committee on 14 July 2006.
The present review report contains the conclusions of this final examination; given the importance
of the (revised) draft assessment report, the peer review report (i.e. full report) and the comments
and clarifications submitted after the peer review as basic information for the final examination
process, these documents are considered respectively as background documents A, B and C to this
review report and are part of it.
The review of Bacillus subtilis did not reveal any open questions or concerns, which would have
required a consultation of the Scientific Committee on Plants or of the European Food Safety
Authority which has taken over the role of that Committee.
2.
Purposes of this review report
This review report, including the background documents and appendices thereto, have been
developed and finalised in support of the Directive 2007/6/EC concerning the inclusion of Bacillus
subtilis in Annex I to Directive 91/414/EEC, and to assist the Member States in decisions on
individual plant protection products containing Bacillus subtilis they have to take in accordance
with the provisions of that Directive, and in particular the provisions of article 4(1) and the uniform
principles laid down in Annex VI.
This review report provides also for the evaluation required under Section A.2.(b) of the above
mentioned uniform principles, as well as under several specific sections of part B of these
principles. In these sections it is provided that Member States, in evaluating applications and
granting authorisations, shall take into account the information concerning the active substance in
Annex II of the directive, submitted for the purpose of inclusion of the active substance in Annex I,
as well as the result of the evaluation of those data.
In parallel with the provisions of Article 7(6) of Regulation 3600/92 for existing active substances,
the Commission and the Member States will keep available or make available this review report for
consultation by any interested parties or will make it available to them on their specific request.
Moreover the Commission will send a copy of this review report (not including the background
documents) to the applicant.
The information in this review report is, at least partly, based on information which is confidential
and/or protected under the provisions of Directive 91/414/EEC. It is therefore recommended that
this review report would not be accepted to support any registration outside the context of Directive
91/414/EEC, e.g. in third countries, for which the applicant has not demonstrated possession of
regulatory access to the information on which this review report is based.
-4-
3.
Overall conclusion in the context of Directive 91/414/EEC
The overall conclusion from the evaluation is that it may be expected that plant protection products
containing Bacillus subtilis will fulfil the safety requirements laid down in Article 5(1)(a) and (b) of
Directive 91/414/EEC. This conclusion is however subject to compliance with the particular
requirements in sections 4, 5, 6 and 7 of this report, as well as to the implementation of the
provisions of Article 4(1) and the uniform principles laid down in Annex VIB of Directive
91/414/EEC, for each Bacillus subtilis containing plant protection product for which Member
States will grant or review the authorisation.
Furthermore, these conclusions were reached within the framework of the uses which were
proposed and supported by the sole data submitter and mentioned in the list of uses supported by
available data (attached as Appendix IV to this Review Report).
Extension of the use pattern beyond those described above will require an evaluation at Member
State level in order to establish whether the proposed extensions of use can satisfy the requirements
of Article 4(1) and of the uniform principles laid down in Annex VIB of Directive 91/414/EEC.
Given the results of the evaluation of the information submitted for ecotoxicology, particular
conditions have been provided for as explained in section 6 of this report, which need short term
attention from the Member States when granting new authorisations or varying existing provisional
authorisations.
4.
Identity and biological properties
The main properties of Bacillus subtilis are given in Appendix I.
The review has established that for the active substance notified by the applicant (AgraQuest), none
of the manufacturing impurities considered are, on the basis of information currently available, of
toxicological or environmental concern.
5.
Endpoints and related information
In order to facilitate Member States, in granting or reviewing authorisations, to apply adequately the
provisions of Article 4(1) of Directive 91/414/EEC and the uniform principles laid down in Annex
VIB of that Directive, the most important end points as identified during the evaluation process are
listed in Appendix II.
-5-
6.
Particular conditions to be taken into account on short term basis by Member States
in relation to the granting of authorisations of plant protection products containing
Bacillus subtilis
On the basis of the proposed and supported uses, no particular issues have been identified as
requiring short term attention from the Member States.
7.
List of studies to be generated
No further studies were identified which were considered at this stage, and under the current
inclusion conditions necessary in relation to the inclusion of Bacillus subtilis in Annex I.
8.
Information on studies with claimed data protection
For information of any interested parties, Appendix III gives information about the studies for
which the applicant has claimed data protection and which are not present in the original dossier
neither mentioned in the draft report. This information is only given to facilitate the operation of the
provisions of Article 13 of Directive 91/414/EEC in the Member States. It is based on the best
information available to the Commission services at the time this review report was prepared; but it
does not prejudice any rights or obligations of Member States or operators with regard to its uses in
the implementation of the provisions of Article 13 of the Directive 91/414/EEC neither does it
commit the Commission.
9.
Updating of this review report
The technical information in this report may require periodic updating to take account of technical
and scientific developments as well as of the results of the examination of any information referred
to the Commission in the framework of Articles 7, 10 or 11 of Directive 91/414/EEC. Such
adaptations will be examined and finalised in the Standing Committee on the Food Chain and
Animal Health, in connection with any amendment of the inclusion conditions for Bacillus subtilis
in Annex I of the Directive.
-6Bacillus subtilis
APPENDIX I
Identity and biological properties
23 May 2006
APPENDIX I
IDENTITY AND BIOLOGICAL PROPERTIES
BACILLUS SUBTILIS
Intended Uses:
Biocontrol of plant pathogenic fungi in viticulture and
horticulture
Known or new organism:
Bacterium Bacillus subtilis (Cohn 1872)
GMO
No
Taxonomy:
The genus Bacillus belongs to the family Bacillaceae among
the group gram-positive eubacteria.
Species, subspecies, strain:
Strain QST 713, identical with strain AQ 713
Culture collection N° NRRL B -21661
Identification / detection:
Using the available morphological, physiological and
biochemical data, the strain QST 713 was clearly identified as
Bacillus subtilis. Besides the basically relevant positive
Catalase reaction inherent to all Bacillus species, further
biochemical key parameters identifying strain QST 713 as B.
subtilis are e.g.: positive Voges-Proskauer reaction and
growth in 7 % NaCl.
Strain discrimination of Bacillus subtilis QST 713 is possible
by ribotyping.
Methods of analysis:
The species is identified by microscopy using classical
morphological (cellular and colonial morphology) criteria and
by using physiological and biochemical criteria.
Mode of action:
The mode of action of B. subtilis is fungistatic and fungitoxic
by disruption of hyphae following contact with the fungal
pathogen at the leaf surface. Besides antagonism nutrient
competition is involved in the mode of action and more
importantly B. subtilis induces systemic resistance response
of the plant, indicated by enhanced peroxidase production.
-7Bacillus subtilis
APPENDIX I
Identity and biological properties
23 May 2006
Life cycle:
All spore-formers, including members of the Genus Bacillus,
undergo a cycle consisting of several discernible phases:
germination, outgrowth, multiplication, and sporulation. The
primary cell formed at the end of outgrowth can, under some
conditions, such as insufficient nutrients, divide
asymmetrically and proceed directly to sporulation or, in time
of favourable conditions, such as sufficient nutrients, can
divide symmetrically and proceed through many divisions
before sporulating.
The endospore plays a dominant role in the biology and the
life-cycle of B. subtilis and relatives. It is a dormant structure
which enables the micro-organism to survive when
environmental conditions turn unfavourable for vegetative
growth and is a vehicle for dispersal by dust and air streams,
as it is easily blown up. The global distribution of Bacillus spp.
may largely be derived from the endospore-forming
capability. Basically the endospore is the most heat tolerant
bacterial life-form, enduring temperatures >80°C or even
>100°C. The endospore does not present an obligate stage
in the life-cycle, vegetative growth by cell-division may be
predominant - or even the norm, unless e.g. lack of nutrients
occurs. In a dry state endospores can remain viable for
several years.
Host specificity:
B. subtilis is not characterised by a distinct host specificity
since growth is not dependant upon a host but upon supply
with decomposable organic matter. Moreover the endospore
is prevalent in all environmental compartments and B. subtilis
is not geographically restricted.
Known opportunist:
B. subtilis is considered an opportunist with no pathogenic
potential. In some cases B. subtilis was isolated from surgical
wounds or tumour drainages; only highly immunosuppressed
patients were reported to have suffered from dissipating
infections.
Toxin production:
B. subtilis produces different exo-enzymes contributing to the
decay of organic matter. The extracellular enzyme subtilisin is
known to elicit allergic or hypersensitive reactions in
individuals repeatedly exposed to it however its toxigenic
properties are assessed to be very low. B. subtilis does not
produce significant quantities of extracellular enzymes or
toxins and is generally considered to have a low degree of
virulence to humans.
Resistance:
Up to now there is no indication of decreasing efficacy of the
Bacillus subtilis strain in SerenadeTM WP against fungal
pathogens to be controlled. The mode of action of strain QST
713 of Bacillus subtilis has been demonstrated to rely on a
broader base than single site action, since it includes
diverging mechanisms not easily to overcome by pathogens.
The risk on the occurrence of development of resistance is to
be classified as low.
-8Bacillus subtilis
APPENDIX I
Identity and biological properties
23 May 2006
Resting stages:
The endospore of B. subtilis is a dormant structure which
enables the micro-organism to survive when environmental
conditions turn unfavourable for vegetative growth and is a
vehicle for dispersal by dust and air streams, as it is easily
blown up.
Production control:
Each ”seed” (liquid media with suspended cells) transfer is
checked for purity both microscopically and by streak plating.
The completed fermentation material (broth) of each
fermentation run (batch) is tested by counts of colony forming
units (cfu) of B. subtilis, microscopic examination, optical
density and is tested for contaminants by plating analysis,
esp. with regard to human pathogens. Content of cfu and
contaminants may additionally be determined for the
Technical Powder. The test results showed no detectable
levels of human pathogens or other contaminants.
-1Bacillus subtilis
APPENDIX II
END POINTS AND RELATED INFORMATION
23 May 2006
APPENDIX II
END POINTS AND RELATED INFORMATION
BACILLUS SUBTILIS
1 Hazard evaluation
Hazard to humans
Pathogenicity:
No evidence of adverse effects from acute studies - except
minor and transient effects after intratracheal challenge.
Infectivity:
No evidence of adverse effects from acute studies. B. subtilis
infections are only reported from immuno-deficient patients.
Toxicity:
Rat LD50 oral:
> 1.13 x 108 cfu/animal
Rat LD50 intratracheal:
> 1.2 x 108 cfu/animal
Rabbit LD50 dermal
> 2.3-2.7 x 1011 cfu/animal
Rat LD50 intravenous:
> 9.4 x 106 cfu/animal
Rat 28 d inhalation (nose-only): no clinical effects during
exposure of 5 x 108 cfu/animal and during recovery; no viable
spores in lungs and drained lymph nodes 8 w after final
exposure
Irritation, Sensitisation:
Rabbit: Very slight irritating effects (skin, eye)
Based on the sensitising property of the formulation: R43
Genotoxicity:
No evidence of mutagenicity for secondary metabolites
Medical reports:
Limited database: No adverse health effects observed among
personnel involved in laboratory investigations. B. subtilis is
capable of producing subtilisin which may cause allergic
reactions after repeated exposure. B. subtilis has been
reported to be associated with food poisoning and infections
in immuno-deficient patients.
Formulation:
Rat LD50 oral:
> 5000 mg/kg bw
(~ 2.5 x 1010 cfu/kg bw)
Rat LD50 inhalation:
> 0.63 mg/l air; 4 h
(~ 5 x 108 cfu/kg bw)
Rabbit LD50 dermal
> 2000 mg/kg bw
(~ 1 x 1010 cfu/ kg bw)
Skin sensitisation (Buehler test): positive (R43)
-2Bacillus subtilis
APPENDIX II
END POINTS AND RELATED INFORMATION
23 May 2006
Hazard to the environment
Impact on non-targets:
Different studies have shown, that populations of B. subtilis
are influenced by biotic environmental factors. Introduced B.
subtilis populations are subject to competition by the
indigenous microflora (bacteria and fungi) and may also be
affected by infectious agents like phages. As a result, high
initial population numbers resulting from application of
Bacillus subtilis will decline and reach a natural equilibrium.
A 5-day acute oral toxicity and pathogenicity test with
bobwhite quail showed that QST 713 technical has a low
toxicity to birds. The LD50 is >5000 mg/kg/d equivalent to
>1011 cfu/kg/d. There is no evidence of pathogenicity or
replication of the QST 713 strain of Bacillus subtilis for birds;
the same is true for mammals. Thus the intended use of
Bacillus subtilis should not pose a risk to terrestrial
vertebrates.
Bacillus subtilis QST 713 TP is of low toxicity to aquatic
organisms. In addition to that no signs of infectivity were
observed in the histological investigations.
O. mykiss
LC50; 4d:
1.4 x 107 cfu/mL
D.magna
NOEC21 d:
7.9 x 105 cfu/mL
P. pugio
NOEC30d
3.7 x 106 cfu/mL
No evidence of toxicity of Bacillus subtilis QST 713 to
honeybees at doses applied at practical use ;
5-day-Dietary LC50: ~ 8900 ppm
equivalent to ~ 1.8 x 108 cfu/mL diet.
Bacillus subtilis has no unacceptable influence on non-target
arthropods, represented by species of two ecological groups.
The risk to earthworms can be considered acceptable.
Eisenia fetida:
LC50 > 1000 mg formulation/kg dry wt soil
NOEC 316 mg formulation/kg dry wt soil
Formulation:
SerenadeTM WP (5 x 109 cfu/g)
-3Bacillus subtilis
APPENDIX II
END POINTS AND RELATED INFORMATION
23 May 2006
2 Exposure assessment and risk evaluation
Operator exposure
Application method:
FCTM, HCTM, HCHH
Operator exposure models:
In relation to the results of the available acute toxicity studies,
sufficient margins of safety exist (calculation on the basis of
cfu / German model)
Exposure of the environment
Natural occurrence, background
level:
B. subtilius is a ubiquitous -not geographically restrictedinhabitant of the soil, from which it is spread to associated
environments, including plants and plant materials (straw,
composts), foods (cereals, esp. dried spices), animals and
their faeces (by ingestion of spores) and is also naturally
found in aquatic environments (fresh water, estuarine and
coastal waters). Although B. subtilis is commonly found in soil
it occurs in almost any environment, including niches in
kitchen and bathrooms.
The magnitude of occurrence of B. subtilis in the soil is not
definitely stated in the supplied literature. Indications for their
general prevalence can be derived from high levels of
presumably soil-born Bacillus spp. spores in straw
approaching 105 cfu/g, and from the high numbers of Bacillus
spp. found in coastal waters (where they constitute up to 20
% of total bacterial population) and from the major
contribution of their endospores in estuarine and coastal
sediments (achieving up to 80 % of the heterotrophic flora).
Application method:
Spraying
Post release control:
Not required
-4Bacillus subtilis
APPENDIX II
END POINTS AND RELATED INFORMATION
23 May 2006
Consumer exposure
Residues:
Residues of B. subtilis strain QST 713 on crops, feedingstuffs
or foodstuffs are not expected at relevant concentrations:
- With regard to its natural global distribution and nonpathogenic character B. subtilis cells left on the surface of
treated areas or plant products do not imply health or
environmental impacts.
- B. subtilis has been used for enzyme production on a large
industrial scale, and is even used for food production without
having caused health or environmental hazards or damages.
- B. subtilis does not produce toxins.
- B. subtilis has no special attachment ability to plants or plant
products, i.e. there is no compatibility comparable to hostpathogen interactions.
- The unfavourable environmental conditions prevailing on
the leaf surface and the dependence of B. subtilis on organic
matter supply are restricting its growth. In addition, in
processing of raw products no growth or sporulation of B.
subtilis is expected to occur.
3 Formulation
Technical specification:
Packaging:
The product Serenade WP is a water dispersible powder
(WP) containing 100 g/kg (5x109 cfu/g) bacillus subtilis QST
175.
At room temperature the product is stable for 3 years.
Storage at 40 °C for 8 weeks has been determined not to
alter physico-chemical and technical properties of this
product.
The product will be packed in 10 kg gussetted paper bag with
high density polyethylene inlet, three different quality kraft
paper layers comprising the outer envelope with a 30 cm
diameter open mouth. Glue is applied to mouth opening and
heat sealed.
-5Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Appendix III
BACILLUS SUBTILIS
List of studies which were submitted during the evaluation process and were
not cited in the draft assessment report:
Annex
point/
reference
number
Author(s)
Year
AIIB-1
Anonymous
2004
AIIB-1
Heins, S.
2004
AIIB-1
Lehman, L.
2002
AIIB-1.4.2
AIIB-2.2.2
AIIB-2.8
AIIB-4.1
AIIB-2.2.2
Manker, D.C.
Abo-El-Dahab and
El-Goorani, M.A.
1964
AIIB-2.2.2
AIIB-2.8
AIIB-2.9
Berdy et al.
1985
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Documentation related to sample group
NG-0914853 prepared for AgrarQuest.
Report-no. not stated
not GLP, unpublished
BWS2005-13
Comparison of Bacillus subtilis QST 713 to
additional Bacillus subtilis strains by
Ribotyping using the enzyme PvuII.
Report-no. not stated
not GLP, unpublished
BWS2005-12
Strain discrimination of Bacillus subtilis
QST 713 by Ribotyping.
Statement
Report-no. not stated
not GLP, unpublished
BWS2002-2
Chemical Characterisation of QST 713.
Not GLP, unpublished
BWS2005-32
CHE2005-1532
Antagonistic effect of a Bacillus subtilis
strain upon Erwinia amylovora, Vol. 54,
1285-1286.
Report-no. not stated
not GLP, published
BWS2005-16
CRC Handbook of Antibiotic Compounds.
CRC Press, Boca Raton, FL, U.S.A.
not GLP, published
BWS2005-27
Date of
submission
Year/Month/Day
2004-12-8
2004-12-08
2002-07-26
2001-04-19
2004-12-08
2002-09-16
-6Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
AIIB-2.2.2
Farabee, G.J. and
Lockwood, J.L.
1958
AIIB-2.2.2
Földes, T.,
Banhegyi, I.,
Herpai, Z., Varga,
L. and Szigeti, J.
2000
AIIB-2.2.2
Krebs, B., Höding, 1998
B., Kübart, S.,
Alemayehu
Workie, M., Junge,
H., SchmiedeKnecht, G.,
Grosch, R.,
Bochow, H. and
Hevesi, M.
Kunst, F. et al.
1997
AIIB-2.2.2
Year
AIIB-2.2.2
Lin, D., Qu, L.-J.,
Gu, H. and Chen,
Z.
2001
AIIB-2.2.2
AIIB-2.8
Manker, D.C.
2001
AIIB-2.2.2
Rumbolz, J.
2004
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Date of
submission
Year/Month/Day
Inhibition of Erwinia amylovora by
bacterium sp. isolated from fire blight
cankers.
Phytopathology, Vol. 48, 209-211
not GLP, published
BWS2005-15
Isolation of Bacillus subtilis strains from the
rhizosphere of cereals and in vitro
screening for antagonism against
phytopathogenic, food-borne pathogenic
and spoilage micro-organisms.
Journal of Applied Microbiology, 89, 840846
not GLP, published
BWS2005-19
Use of Bacillus subtilis as biocontrol agent.
I. Activities and characterisation of Bacillus
subtilis strains.
Journal of Plant Diseases and Protection,
105 (2), 181-197
not GLP, published
BWS2005-17
2004-12-08
The complete genome sequence of the
Gram-positive bacterium Bacillus subtilis.
Nature, Vol. 390, 249-256
not GLP, published
BWS2005-24
A 3.1-kb genomic fragment of Bacillus
subtilis encodes the protein inhibiting
growth of Xanthomonas oryzae pv. Oryzae.
Journal of Applied Microbiology 91, 10441050
not GLP, published
BWS2005-21
Chemical Characterisation of QST 713.
not GLP, unpublished
BWS2005-32
Serenade WP (Bacillus subtilis, strain QST
713) - Expert Statement not GLP, unpublished
BWS2005-14
2002-09-16
2004-12-08
2004-12-08
2004-12-08
2001-04-19
2004-12-18
-7Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
AIIB-2.2.2
Sharga, B.M. and
Lyon, G.D.
AIIB-2.2.2
Thimon, L., Maget- 1992
Dana, R. and
Michel, G.
AIIB-2.2.2
Vollenbroich, D.,
Pauli, G., Özel, M.
and Vater, J.
AIIB-2.6
O`Donnell A.G.,
1980
Norris J.R.,
Berkely R.C.W.,
Kaneko N., Nozaki
R.
AIIB-2.6
Heins, S.
2001
AIIB-2.6
Sneath, P.H.A.
1986
AIIB-2.8
AIIA-4.1
Manker, D.C.
2002
1998
1997
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Bacillus subtilis BS 107 as an antagonist of
potato blackleg and soft rot bacteria.
Canadian Journal of Microbiology (44),
777-783
not GLP, published
BWS2005-18
Surface active properties of antifungal
lipopeptides produced by Bacillus subtilis.
JAOCS, Vol. 69 (1), 92-93
not GLP, published
BMF2000-144
Antimycoplasma properties and application
in cell culture of surfactin, a lipopeptide
antibiotic from Bacillus subtilis.
Applied and Environmental Microbiology,
63(1), 44-49
not GLP, published
BWS2005-20
Characterization of Bacillus subtilis,
Bacillus pumilus, Bacillus licheniformis and
Bacillus amyloliquefaciens by Pyrolysis
Gas-Liquid Chromatography,
Deoxyribonucleic Acid-Deoxyribonucleic
Acid Hybridization, Biochemical and API
Systems
not GLP, published
International Journal of Systematic
Bacteriology BWS2002-1
Report: Morphological and Physiological
Differentiation of Bacillus subtilis From
Pathogenic Bacillus species
not GLP, published
Stellungnahme
BMF2001-55
Bergey`s manual of Systematic
Bacteriology, Vol. 2, page 1133
not GLP, published
BWS 2002-8
Analysis of Serenade for Presence of
Subtilisin.
Not GLP, unpublished
BWS2002-6
CHE2005-1583
Date of
submission
Year/Month/Day
2004-12-08
2000-04-19
2004-12-08
2002-06-19
2001-04-23
2002-10-02
2002-06-19
-8Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
AIIB-2.8
Anonymous
2001
AIIB-2.8
Anonymous
2002
AIIB-2.8
Bloy, C.
2002
AIIB-2.8
Grivot, S.
2000
AIIB-2.8
Manker, D.C.
2002
AIIB- 2.9.
Lehman, L.
2001
AIIB-5.2.3
Pedersen, P.B.,
Bjornvad, M.E.,
Rasmussen, M.D.
and Petersen, J.E.
2002
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Side effects of Serenade (as B. subtilis
QST 713) on Fermentation and Sensorial
Properties of Wine - Translation and
summary of studies performed in 1999 2001.
GLP, unpublished
BWS2005-33
Side effects of Serenade (as B. subtilis
QST 713) on Fermentation and Sensorial
Properties of Wine - Translation and
summary of studies performed in 2000 2002.
GLP, unpublished
BWS2005-34
Study of unintentional effects of
experimental fungicide compounds
"Serenade WP" and "Serenade AS" on
production and quality of must and wines.
Report-no. VCE 0001 GBI
not GLP, unpublished
BWS2005-35
Etude des effets non intentionnels de
produits phytopharmaceutiques
(fongicides) sur lè laboration et la qualite
des vins.
not GLP, unpublished
BWS2005-36
Analysis of Serenade for Presence of
Subtilisin.
not GLP, unpublished
BWS2005-6
Antibiotic Susceptibility Testing of Bacillus
subtilis QST 713.
AgraQuest, Inc., 1530 Drew Avenue, Davis,
CA 95616, U.S.A
Report-no. not stated
not GLP, unpublished
Cytotoxic potential of industrial strains of
Bacillus SP.
not applicable
Regul. Toxicol. Pharmacol., Vol. 36, pp.
155-161
Report-no. not applicable
not GLP, published
Date of
submission
Year/Month/Day
2002-06-18
2002-06-18
2002-06-18
2002-06-18
2002-06-19
2001-04-23
2002-12-01
-9Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
AIIB-5.2.3
Boer de, A.S. and
Diderichsen, B.
Year
1991
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
On the safety of Bacillus subtilis and B.
amyloliquefaciens: A review.
Date of
submission
Year/Month/Day
2000-04-19
not applicable
Appl. Microbiol. Biotechnol., Vol. 36, pp. 1-4
Report-no. not applicable
not GLP, published
AIIB-5.2.3
Frommer, W.,
Archer, L. and
Brunius, G.
1989
Safe Biotechnology III. Safety precautions
for handling Microorganisms of different
classes.
2000-04-19
not applicable
Appl. Microbiol. Biotechnol., Vol. 30, pp.
541-552
Report-no. not applicable
not GLP, published
AIIB-5.2.3
U.S.EPA
1997
Final decision document, TSCA section 5
(H) (4) exemption for Bacillus subtilis+
Attachment 1.
2004-12-08
not applicable
EPA-TOX
Report-no. not applicable
not GLP, published
TOX2006-1274
AIIB5.2.5.1
Arts, J.H.E. and
Tap, S.H.M.
2004
Sub-acute (4-week) inhalation toxicity study,
including an 8-week recovery study, with
Serenade Biofungicide in rats.
2005-01-13
TNO, AJ Zeist, Netherlands
AgraQuest, Inc., 1530 Drew Avenue, Davis,
CA 95616, U.S.A
Report-no. 5435
GLP, unpublished
AIIB-6.2.2
Ryder Fox, J.
2001
Serenade WP Residues on Wine Grapes.
not GLP, unpublished
RIP2002-985
2002-06-17
AIIB 7.1.1
Bochow, H. and
Gantcheva, K.
1995
Soil introductions of Bacillus subtilis as
biocontrol agent and its population and
acivity dynamic
not GLP, published
Acta Horticulturae 382
BOD2002-551
2002-06-17
- 10 Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
AIIB 7.1.1
Casida, L. E., Jr.
1988
AIIB 7.1.1
Milus, E. A. and
Rothrock, C. S
1993
AIIB 7.1.1
Pandey, A., Palni,
L. M. S. and Bisht,
D.
2001
AIIB 7.1.1
Pantastica-Caldas, 1992
M. and Duncan, K.
E.
AIIB 7.1.1
Tokuda, Y., Ano,
T. and Shoda, M.
1993
IIB, 8.2
Drottar, K.R.,
Flaggs, R.S.,
Krueger, H.O.
2001
IIB, 8.2
Machado, M.W.
2001
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Response in soil of Cupriavidus necator
and other copper-resistant bacterial
predators of bacteria to addition of water,
soluble nutrients, various bacterial species,
or Bacillus thuringiensis spores and
crystals
not GLP, published
Appl. Environ. Microbiol. 54
BOD2002-554
Rhizosphere colonization of wheat by
selected soil bacteria over diverse
environments
Canadian Journal of Microbiology 39
not GLP, published
BOD2002-555
Dominant fungi in the rhizosphere of
established tea bushes and their
interaction with the dominant bacteria
under in situ conditions
Microbiol. Research 156
not GLP, published
BOD2002-556
Population dynamics of bacteriophage and
Bacillus subtilis in soil
not GLP, published
Ecology 73 (5)
BOD2002-557
Survival of Bacillus subtilis NB22, an
Antifungal-Antibiotic Iturin Producer, and its
transformant in Soil-Systems
J. of Fermentation and Bioengineering 75
(2)
BOD2002-558
QST 713 Technical: A Five-Concentration
Toxicity and Pathogenicity Test with the
Rainbow Trout (Oncorhynchus mykiss)
489A-108
GLP
unpubl.
WAT2002-442
QST 713 Technical Powder – Infectivity
and Pathogenicity to Grass Shrimp
(Palaemonetes pugio) during a 30-Day
Static Renewal Test
13759.6101
GLP
unpubl.
WAT2002-446
Date of
submission
Year/Month/Day
2002-06-17
2002-06-17
2002-06-17
2002-06-17
2002-06-17
2002-06-17
2002-06-17
- 11 Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
IIB, 8.2
Drottar, K.R.,
Flaggs, R.S.,
Krueger, H.O.
2001
IIB 8.3
Mayer, D.F.
2000
IIB, 8.3
IIIB, 10.3
Jimenez, D.R. and
Richards, K.B.
2003
IIB, 8.3
IIIB, 10.3
Jimenez, D.R.
2004
IIB, 8.3
IIIB, 10.3
Jimenez, D.R.
2004
IIB, 8.3
Shimanuki, H. and
Cantwell, G.
1978
IIB, 8.3
Shabanov, M. and
Balabanov, V.A.
1983
IIB, 8.3
Krieg, A.
1973
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
QST 713 Technical: A 21-Day Life-Cycle
Toxicity and Pathogenicity Test with the
Cladoceran (Daphnia magna)
489A-107A
GLP
unpubl.
WAT2002-449
Honey Bee Field Study of Serenade
Biofungicide Wettable Power in Alfalfa
GLP
unpubl.
BIE2002-14
Evaluation of the dietary effect(s) of QST
713 technical powder on larval honeybee
development (Apis mellifera L.).
CAR 158-03
GLP, unpublished
BIE2005-76
Safety of the Bacillus subtilis - based
biofungicide, Serenade, to the honeybee
(Apis mellifera L.).
not GLP, unpublished
BIE2005-77
Discussion of the results of honeybee
studies conducted with QST 713 technical
and Serenade products.
not GLP, unpublished
BIE2005-91
Diagnosis of honeybee diseases, parasites
and pests.
ARS-NE 87, pp 1-3
not GLP, published
BIE2005-78
Aspergilosis in bees in Bulgaria and it’s
causes.
Acta Microbiologica, Vol. 12, pp 77-82
not GLP, published
BIE2005-79
Über die toxische Wirkung von Bacillus
cereus- und Bacillus thuringiensis-Kulturen
auf die Honigbiene (Apis mellifera).
Zeitschrift f. Pfl.krankh. und Pfl.sch., Vol.
80, pp 483-486
not GLP, published
BIE2005-80
Date of
submission
Year/Month/Day
2002-06-17
2000-07-27
2005-10-05
2002-10-05
2005-10-05
2005-10-05
2005-10-05
2005-10-05
- 12 Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
IIB, 8.3
Cano, R.J. et al.
1994
IIB, 8.3
Gilliam, M. and
Valentine, D.K.
1976
IIB, 8.3
Gilliam, M.
1978
IIB, 8.3
Gilliam, M.,
Buchmann, SL.,
Lorenz, B.J. and
Schmalzel, R.J.
1990
IIB, 8.3
Gilliam, M.
1985
IIB, 8.3
Reynaldi, F.J., De
Giusti, M.R. and
Alippi, A.M.
2004
IIB, 8.3
Gokte, N. and
Swarup, G.
2004
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Bacillus DNA in fossil bees: An ancient
symbiosis?.
Applied and Enviromental Entomology, Vol.
60, pp 2164-2167
not GLP, published
BIE2005-81
Bacteria isolated from the intestinal
contents of foraging worker honey bees
Apis mellifera: The genus Bacillus.
Journal of Invertebrate Pathology, Vol. 28,
No. 2, pp 275-276
not GLP, published
BIE2005-82
Bacteria belonging to the genus Bacillus
isolated from selected organs of queen
honey bees, Apis mellifera.
Journal of Invertebrate Pathology, Vol. 31,
pp 389-391
not GLP, published
BIE2005-83
Bacteria belonging to the genus Bacillus
associated with three species of solitary
bees.
Apidologie (1990) 21, 99-105
not GLP, published
BIE2005-193
Microbes from Apiarian Sources: Bacillus
spp. in frass of the greater wax moth.
Journal of Invertebrate Pathology, Vol. 45,
pp 218-224
not GLP, published
BIE2005-84
Inhibition of growth of Ascospaera apis by
Bacillus and Paenibacillus strains isolated
from honey.
Revista Argentina de Microbiologia, Vol. 36
no. 1, pp 52-55
not GLP, published
BIE2005-85
On the potential of some bacterial biocides
against root-knot and cyst.
Indian Journal of Nematology, Vol. 18, pp.
152-153
not GLP, published
BIE2005-86
Date of
submission
Year/Month/Day
2005-10-05
2005-10-05
2005-10-05
2005-10-05
2005-10-05
2005-10-05
2005-10-05
- 13 Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
IIB, 8.3
Heins, S.D.,
2004
Manker, D.C.,
Jimenez, D.R.,
McCoy, R.J.,
Marrone, P.G. and
Orjala, J.E.
Assie, L.K., Deleu, 2002
M., Arnaud, L.,
Paquot, M.,
Thonart, P.,
Gaspar, C. and
Haubruge, E.
Peng, C.Y.-S.,
1992
Mussen, E., Fong,
A., Montague, M.A.
and Tyler, T.
IIB, 8.3
IIB, 8.3
IIB, 8.3
Pfleeger, T.G.,
Fong, A., Hayes,
R., Ratsch, H. and
Wickliff, C.
IIB 8.4,
IIIB, 10.4
Bryan et al.
IIB 8.4,
IIIB, 10.4
Ipach, R.
IIB 8.4,
IIIB, 10.4
Ipach, R.
Year
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Date of
submission
Year/Month/Day
Strain of Bacillus for controlling plant
diseases and corn rootworm.
US Patent 6,291425 B1
not GLP, published
BIE2005-87
2005-10-05
Insecticide activity of surfactins and iturins
from from a biopesticide Bacillus subtilis
Cohn (S499 Strain).
Med. Fac. Landbouww Univ. Gent 67/3
not GLP, published
BIE2005-88
Effects of chlortetraycline of honey bee
worker larvae reared in vitro.
Journal of Invertebrate Pathology Vol. 6 pp,
127-133
not GLP, published
BIE2005-89
1996 Field evaluation of the EPA (Kenaga)
nomogram, a method for estimating wildlife
exposure to pesticides residues on plants.
Environmental Toxicology and Chemistry
Vol. 15, pp. 535-543
not GLP, published
BIE2005-90
2000 Bacillus subtilis strain QST 713: A Dietary
Pathogenicity and Toxicity Study With the
Parasitic Hymenopterian (Nasonia
vitripennis)
GLP
unpubl.
ANA2002-288
2000a Effects of Serenade WP on predatory
mites (Typhlodromus pyri) under typical
vine culture conditions on grape vines,
Germany
GLP
unpubl.
ANA2002-286
2000b Effects of Serenade WP on predatory
mites (Typhlodromus pyri) under typical
vine culture conditions on grape vines,
Germany
GLP
unpubl.
ANA2002-287
2005-10-05
2005-10-05
2005-10-05
2002-06-17
2002-06-17
2002-06-17
- 14 Bacillus subtilis
APPENDIX III
List of studies
23 May 2006
Annex
point/
reference
number
Author(s)
Year
AIIB-8.5
Kristufek, V.,
Ravasz, K. & Pizl,
V.
1993
AIIB-8.5
AIIIB-10.5
Stäbler, D.
2002
AIIIB-2.2
Gingras
2001
Title
Source (where different from company)
Company, Report No.
GLP or GEP status (where relevant)
Published or not
BVL Registration Number
Date of
submission
Actinomycete communities in earthworm
guts and surrounding soil
Pedobiologia 37, 379-384
ARW 2002-167
Acute Toxicity of QST 713 WP (Serenade
WP) on Earthworms, Eisenia Fetida Using
an Artificial Soil Test
20011062/01
ARW2002-117
2002-06-17
Storage stability of Qst 713 strain of dried
Bacillus subtilis with residual fermentation
media identified as Qst 713 WP.
Final Report
Project No. L08726 SN9
GLP, unpublished
PHY2002-297
Year/Month/Day
2002-05-03
2000-04-19
- 15 Bacillus subtilis
APPENDIX IV
List of uses supported by available data
23 May 2006
APPENDIX IV
List of uses supported by available data
BACILLUS SUBTILIS strain QST 713
Crop and/
Member
Product
F
Pests or
PHI
or situation
State
name
G
Group of pests
(days)
or
controlled
I
(c)
or
Country
Formulation
Product application rate per
treatment (g)
Type
Northand
SouthEurope
SerenadeTM
WP
F
Venturia
inaequalis
(scab)
Conc.
method
growth
number
of as
kind
stage &
season
min max
(d)
(e)
WP
5 x 109
cfu/g
spraying
Northand
SouthEurope
SerenadeTM
WP
F
Monilia
BBCH
10 to 72
during
blossom
WP
5 x 109
cfu/g
spraying
1 - 16
(h)
(i)
kg /hl
water l/ha
kg /ha
min max
min max
min
max
max. 5
days
1
(i.e. 1012
cfu)
500 1.500
5 - 15
---
*
max. 5
days, on
fruit
(BBCH
70 – 89)
5 to 7
days
1
(i.e. 1012
cfu)
500 1.500
5 - 15
---
*
interval
between
applications
(f)
Erwinia
amylovora
(fire blight)
Orchards,
stone fruits
Application rate per treatment
/
(b)
(a)
Orchards,
Apple,
Pear
Application
Remarks:
4
BBCH
55 to 69
4
BBCH
70 to 84
4
BBCH
85 to 89
2
**
- 16 Bacillus subtilis
APPENDIX IV
List of uses supported by available data
23 May 2006
Crop and/
Member
Product
or situation
State
name
F
Pests or
G
Group of pests
or
or
controlled
Country
I
(c)
PHI
Formulation
Product application rate per
treatment (g)
Type
Middleand
SouthEurope
SerenadeTM
WP
F
Uncinula
necator
(Oidium)
Conc.
method
growth
number
of as
kind
stage &
season
min max
(d)
(e)
WP
5 x 109
cfu/g
spraying
(a)
(b)
(d)
(e)
(f)
Northand
SouthEurope
SerenadeTM
WP
F,
G
Bremia
lactucae
WP
5 x 109
cfu/g
spraying
BBCH
55 to 75
1-8
BBCH
68 to 81
1-4
after
planting
--
(days)
(h)
(i)
kg /hl
water l/ha
kg /ha
min max
min max
min
max
max. 5
days
1
(i.e. 1012
cfu)
400 –
1.200
4 - 12
---
*
5 to 7
days
1
(i.e. 1012
cfu)
400 –
1.200
4 - 12
---
**
interval
between
applications
(f)
Botrytis
cinerea
Lettuce
Application rate per treatment
/
(b)
(a)
Grapevines
Application
Remarks:
EU and Codex classification
Outdoor or field use (F), glasshouse application (G) or indoor application (I)
internationally (GIFAP) agreed codes
cfu = colony forming units
according to BBCH scale; grapevines:
55 inflorescences swelling, flowers closely pressed together;
68: 80% of flowerhoods fallen;
75: berries pea-sized, bunches hang;
81: beginning of ripening: berries begin to develop variety- specific colour;
Application timing: exact timing depends on local conditions: spray when infestation will occur or according to local extension service
(g) minimum pre-harvest interval not relevant, no residues
(h) product stated as active substance expressed in colony forming units (cfu)
* spray interval max. 5 days; use product in spraying sequence with other fungicides
** spray interval 5 to 7 days, up to the day of harvest