Regulation (EU) No 528/2012 concerning the making available on the market and
use of biocidal products
Evaluation of active substances
Assessment Report
Bacillus sphaericus 2362 - Serotype
H5a5b - Strain ABTS1743
PT18
(Insecticide)
July 2014
ITALY
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CONTENTS
1. STATEMENT OF SUBJECT MATTER AND PURPOSE ................................................................... 2
1.1 PROCEDURE FOLLOWED .......................................................................................................................... 2 1.2 PURPOSE OF THE ASSESSMENT REPORT ............................................................................................... 2
2. OVERALL SUMMARY AND CONCLUSIONS .................................................................................... 4
2.1 PRESENTATION OF THE ACTIVE SUBSTANCE ........................................................................................... 4 2.1.1 Identity, Biological, Physico-Chemical Properties & Methods of Analysis ...................... 4 2.1.2 Intended Uses and Efficacy ................................................................................................................... 5 2.1.3 Classification and Labelling ................................................................................................................... 6
2.2 SUMMARY OF THE RISK ASSESSMENT .................................................................................................. 6 2.2.1 Human Health Risk Assessment .......................................................................................................... 6 2.2.1.1 Hazard identification ........................................................................................................................ 6 2.2.1.2 Exposure assessment and Risk characterization .............................................................. 11
2.2.2 Environmental Risk Assessment ....................................................................................................... 14 2.2.2.1 Fate and distribution in the environment ............................................................................. 14 2.2.2.2 Effects assessment .......................................................................................................................... 15 2.2.2.3 PBT and POP assessment ............................................................................................................. 20 2.2.2.4 Exposure assessment ..................................................................................................................... 20 2.2.2.5 Risk characterisation ...................................................................................................................... 23
2.2.3 Assessment of endocrine disruptor properties .......................................................................... 25 2.3 OVERALL CONCLUSIONS ....................................................................................................................... 26 2.4 LIST OF ENDPOINTS .............................................................................................................................. 26
Appendix I: List of endpoints............................................................................................................................... 27 Chapter 1: Identity, Physical and Chemical Properties, Classification and Labelling ................. 27 Chapter 2: Methods of Analysis ....................................................................................................................... 29
Chapter 3: Impact on Human Health ............................................................................................................ 30 Chapter 4: Fate and Behaviour in the Environment ................................................................................ 32
Chapter 5: Effects on Non-target Species ................................................................................................... 32 Chapter 6: Other End Points............................................................................................................................. 34
Appendix II: List of Intended Uses .................................................................................................................... 35
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STATEMENT OF SUBJECT MATTER AND PURPOSE 1.
1.1 Procedure followed
This assessment report has been established as a result of the evaluation of the active
substance Bacillus sphaericus 2362, Serotype H5a5b, Strain ABTS1743 as product-type PT
18 (insecticide), carried out in the context of the work programme for the review of existing
active substances provided for in Article 89 of Regulation (EU) No 528/2012, with a view to
the possible approval of this substance.
The micro-organism, Bacillus sphaericus 2362- Serotype H5a5b Strain ABTS 1743 was
notified as an existing active substance, by Sumitomo Chemical Agro Europe SAS (for Valent
BioSciences Corporation), hereafter referred to as the applicant, in product-type PT18.
Commission Regulation (EC) No 1451/2007 of 4 December 20071 lays down the detailed
rules for the evaluation of dossiers and for the decision-making process.
In accordance with the provisions of Article 7(1) of that Regulation, Italy was designated as
Rapporteur Member State to carry out the assessment on the basis of the dossier submitted
by the applicant. The deadline for submission of a complete dossier for Bacillus sphaericus
2362- Serotype H5a5b Strain ABTS 1743 as an active substance in Product Type PT18 was
30 April 2006, in accordance with Annex V of Regulation (EC) No 1451/2007.
On 30 April 2006, Italy’s competent authorities received a dossier from the applicant. The
Rapporteur Member State accepted the dossier as complete for the purpose of the
evaluation on 9 November 2006.
On 19 January 2009, the Rapporteur Member State submitted to the Commission and the
applicant a copy of the evaluation report, hereafter referred to as the competent authority
report.
In order to review the competent authority report and the comments received on it,
consultations of technical experts from all Member States (peer review) were organised by
the Agency. Revisions agreed upon were presented at the Biocidal Products Committee and
its Working Groups meetings and the competent authority report was amended accordingly.
1.2 Purpose of the assessment report
The aim of the assessment report is to support the opinion of the Biocidal Products
Committee and a decision on the approval of Bacillus sphaericus 2362- Serotype H5a5b
Strain ABTS 1743 for product-type PT18, and, should it be approved, to facilitate the
authorisation of individual biocidal products. In the evaluation of applications for product-
authorisation, the provisions of Regulation (EU) No 528/2012 shall be applied, in particular
the provisions of Chapter IV, as well as the common principles laid down in Annex VI.
1 Commission Regulation (EC) No 1451/2007 of 4 December 2007 on the second phase of the 10-year work programme referred to in Article 16(2) of Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market. OJ L 325, 11.12.2007, p. 3
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For the implementation of the common principles of Annex VI, the content and conclusions
of this assessment report, which is available from the Agency web-site shall be taken into
account.
However, where conclusions of this assessment report are based on data protected under
the provisions of Regulation (EU) No 528/2012, such conclusions may not be used to the
benefit of another applicant, unless access to these data for that purpose has been granted
to that applicant.
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OVERALL SUMMARY AND CONCLUSIONS 2.
2.1 Presentation of the Active Substance
2.1.1 Identity, Biological, Physico-Chemical Properties & Methods of Analysis
Bacillus sphaericus 2362 Serotype H5a5b Strain ABTS 1743 (hereafter abbreviated to Bs
2362) is the biological insecticide active substance of the biocide product ‘VectoLex’ WG and
is manufactured by submerged pure culture fermentation. The technical grade powder
contains nominally 99% Bs 2362 with low and high limits of 97 and 100%, respectively. The
formulated product ‘VectoLex’ WG contains 51.2 % of the technical grade active substance,
with lower and higher limits of 46% and 64% by weight, respectively. The technical powder
contains 1.5 x 1010 CFU/g MPCA and the MPCP contains 650 IU/mg product. The minimum
biopotency is 600 ITU/mg. The identification of Bs 2362 at strain level has been achieved by
genomotyping and is available at product authorization. A summary description of the
method applied to Bs 2362 is attached to the CAR as an Addendum. Genetic stability is
ensured through manufacturing directions for the fermentation process. During Culture
Maintenance and Preparation of Stock Culture a minimal number of transfers between the
parental stock culture and the working culture lines is carried out. Each transfer series and
working culture line is subjected to a battery of tests to ensure the purity and genetically
unchanged culture.
Bs 2362 is a spore forming rod-shaped bacterium that produces during sporulation a
crystalline protein Bin inclusion which is toxic to larvae of some Dipteran insects upon
ingestion. Bs 2362 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. Bacillus
sphaericus is a common naturally occurring micro-organism with worldwide distribution.
The species has been detected both in soil and water and will be indigenous to intended
areas of application.
According to the general knowledge of the B. sphaericus, the toxicity of highly toxic strains,
including strain Bs 2362, is primarily due to the production of the crystalline binary toxin Bin
during sporulation. The binary toxin consists of two components of Bin: BinA (42 kDa) and
BinB (51 kDa). Both toxins are required in equimolar amounts to exert maximal biological
activity. After ingestion by targets, BinA and BinB the crystalline inclusions are solubilised
under the alkaline conditions in the larval gut. This is followed by activation by gut
proteases, producing the 39 and 43-kDa active forms of BinA and BinB, respectively. Target
specificity is determined by the specific binding of the binary toxin to the midgut epithelial
cells. BinB component has been found to act as the primary binding component and directs
the regional binding of BinA. The receptor of the binary toxin has been identified as a
glycosyl-phosphatidylinositol-anchored alfa-glucosidase in Culex pipiens. Binding to the
receptor is followed by various sorts of ultrastructural changes in the epithelial cells,
suggesting membrane pore formation. In vitro assays with artificial membranes have shown
that mainly BinA, and to a lesser extent BinB, cause membrane channel formation. The
precise role of BinA is still unclear, however there is some evidence showing that BinA alone
is toxic to Culex mosquito cells when administered at high doses, suggesting that this
component may act as a toxic subunit. So far, the detailed molecular mechanism underlying
the mosquito-larvicidal activity has not been defined.
The binary toxins are not homologous with delta-endotoxins of B. thuringiensis, except for
the Cry34/cry35 binary toxins isolated from B. thuringiensis strains active against the
western corn rootworm (a beetle larva).
Bacillus sphaericus is not related to Bacillus cereus the bacterial species known to cause gastro-
intestinal disorders in humans. Bacillus cereus like enterotoxin genes and enterotoxin products
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are not detected in Bacillus sphaericus. There are no other known metabolites and degradation
products that may contribute to the toxicity of Bs 2362. Bacillus sphaericus species are not
known to produce beta-exotoxins (adenosine triphosphate (ATP) analogues) that are formed
during the vegetative growth phase of some Bacillus thuringiensis strains.
Bs 2362 exhibits specific toxicity to some dipteran insect larvae upon ingestion and due to
the specific alkaline conditions of the insect gut and specific binding sites of the mosquito
larvae, has a very limited impact on non-target organisms and is non-toxic to mammalian
species. Adequate methodology exists for the identification of Bs 2362 both as active
substance and in the technical grade p r o d u c t . The methods presented for the analysis
of Bs 2362 are considered to be appropriate for determination of Bs 2362 in the formulated
product. The methods contain information confidential to Valent BioSciences and are shown
in the confidential attachment.
Methods of analysis in food and feed are not considered relevant since the biocidal use of
Bs 2362 is for the control of mosquitoes in water habitats. Bs 2362 is not used on water
bodies used for the abstraction of treated drinking water, i.e. clean drinking waters.
Vegetative cells of Bs have a limited survival time in the environment and spores do not
germinate readily, making it highly unlikely that Bs will multiply and colonise areas of
intended use above levels that may occur naturally. Since Bs is a naturally occurring
organism, methods for determining residues of Bs 2362 in environmental compartments
are not considered necessary.
2.1.2 Intended Uses and Efficacy
Bs 2362 is a biological larvicide. The intended field of use is Pest Control (Main Group 3)
under Product Type 18 (insecticide).
The ‘VectoLex’ WG biocidal use is for the control of mosquitoes (principally Culex and
Anopheles species) in a range of aquatic breeding habitats, such as stagnant and standing
ponds, flood and irrigation water, ditches, storm water retention areas, tidal water and salt
marshes, sewerage settling ponds and water with moderate to high organic content. The
following uses have not been assessed: application to clean purified drinking water, or water
intended for direct human consumption; intentional spray of food crops, processed foods or
surfaces likely to be used to store, process or present food; application for air spray by
planes, helicopters or other flying vehicles; application of irrigation systems were overheads
sprinklers are used; application to soil.
The product can also be used in rice paddies only up to one month before harvest (this is the
period of time, at the end of the cropping cycle, during which the crop is growing in dry
conditions, therefore excluded from intended uses).
Information is available from a series of field and laboratory experiments to show that
‘VectoLex’ WG is effective under a range of conditions against a variety of mosquito species
with the exception of Stegomyia subg.(like Aedes albopictus). The tests were performed at a
range of rates, with the target species present between the 1st and early 4th instar larval
growth stage. In the tests conducted at rates of 600 g/ha or less an acceptable efficacy of
the MPCP was demonstrated. In conclusion, ‘VectoLex’ WG is effective against mosquito
larvae of many species and the results of the studies support the label recommendations.
‘VectoLex’ WG is not an adulticide and application when larvae are present up to the early
4th instar growth stage is necessary for effective control.
Resistance can develop. The resistance to Bs in target species can be managed using mixtures of
Bti and Bs or rotating MPCPs containing either Bti or Bs.
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In addition, in order to facilitate the work of Member States in granting or reviewing
authorisations, the intended uses of the substance, as identified during the evaluation
process, are listed in Appendix II.
2.1.3 Classification and Labelling
Proposal for the classification and labelling of the active substance The classification and labelling of Bs 2362 according to Regulation (EC) No 1272/2008 (CLP
Regulation) is not required as the active substance is a living micro-organism not covered
under the Regulation. It is not biohazardous according to Directive 2000/54/EC on the
protection of workers from risks related to exposure to biological agents at work. However,
based on the precautionary principle all micro-organisms should be considered to have the
potential to provoke sensitising reactions.
2.2 Summary of the Risk Assessment
2.2.1 Human Health Risk Assessment
2.2.1.1 Hazard identification
The potential for ‘VectoLex’ WG (Bs 2362) to cause adverse effects in humans is considered
below.
Concerns in relation to bacteria and human health arise from two sources:
(1) A potential to cause a direct toxic effect.
(2) A potential to cause infection in humans.
The safety of Bacillus sphaericus 2362, Serotype H-5a5b, Strain ABTS 1743 to mammals has
been extensively evaluated with high levels of activated and inactivated spores administered
by various parenteral or oral routes of exposure, and injection of the spore-free entomocidal
toxin. There is no evidence to lead to a conclusion that the limited exposures following use of
the biocidal product could result in a direct toxic effect in humans.
No adverse reactions in individuals as a result of contact with Bacillus sphaericus during its
development, manufacture, preparation or field application have been documented or
reported. The manufacturer performs regular health monitoring of operators at the
fermentation plants. The Medical Director responsible for the plant confirmed no
abnormalities and no human health related or other adverse reactions to Bacillus sphaericus
2362, Serotype H-5a5b, Strain ABTS 1743 .
The ability of Bs 2362 to remain viable in mammalian tissue may lead to detection in
humans, particularly in environments where the microbial agent is used for insect vector
control. No confirmation that Bacillus sphaericus has been causative in the infection process
has been established.
The term “infection” can be defined variously to indicate either the harmless presence of
micro- organisms within living tissue or true infectious disease. The former may be referred
to as “persistence” which can result from distribution throughout the body following
inadvertent oral or other exposure, and is no different to the colonization of the mammalian
body by many micro- organisms after birth. True infectious disease – where the microbes
penetrate host defences, multiply within the host organism and disrupt functional or
structural homeostasis (“infective”) is a more relevant definition in risk assessment.
Persistence can be measured by rates of clearance for individuals or from specific tissues
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and is typically a period of days to weeks for Bacillus sphaericus in mammalian tissue.
There have been no reports of infective activity in cases where humans have been exposed
directly (i.e. spraying preparations) to Bacillus sphaericus. In terms of mammalian infection,
the specific toxicity of the parasporal body is important because it is not activated in
mammals.
Discussion of human infection in relation to Bacillus species is presented in IIIA 5.1.4 with
details of a number of patients examined for the presence of Bacillus species in a general
hospital. It concludes that occurrence of Bacillus species in cultures and smears from a wide
variety of conditions should not be considered a contamination effect associated with
transient bacteraemia but rather evaluated within the hospital environment with
compromised patients as potentially pathogenic. However, the strains from human sources
require close identification and the pathogenicity of various strains needs to be determined.
There are no indications that Bacillus sphaericus is involved in human infectivity, toxicity, or
pathogenicity.
Therefore, the risk of Bs 2362 causing true infectious disease in mammals, including
humans, is considered to be negligible. Animal testing using a variety of conventional
toxicity tests and a range of infectivity protocols has been completed to confirm that Bacillus
sphaericus, Serotype H-5a5b, Strain 2362 has no adverse effects. The overall assessment of
Bacillus sphaericus indicates no evidence of toxicity/infectivity or pathogenicity in relation to
human health and safety considerations.
Infectivity and pathogenicity
A number of acute administration studies were completed to investigate possible infectivity
or pathogenicity via oral, ocular, intravenous, intraperitoneal or intranasal routes of
administration. The studies confirmed that Bs 2362 did not induce or cause infectious
disease following oral, ocular, intravenous, intraperitoneal or intranasal administration to
rats, but that the test organisms were eliminated from the body over time. A further series
of studies were performed in mice with active and inactivated Bs 2362 spores and filtered
(i.e. spore-free) entomocidal toxin.
In an oral infectivity study, a single administration of Bs 2362 to rats at approximately 108
CFU resulted in no deaths or adverse clinical signs. The test compound was neither toxic,
pathogenic nor infectious to rats following a 21-day test period. One treated rat had the test
microbe present in lungs on day 1 and a second animal on day 7. Spores were present in
faeces on Day 1, clearing by Day 14 of the study.
A single intranasal instillation of Bs 2362 to young rats at a dose level of approximately 1.4 x
1010 CFU of the technical material was not associated with clinical signs of reaction to
treatment or adverse effects on bodyweight. All rats survived to their scheduled interim
termination points. The presence of high numbers of bacterial spores was expected in the
lungs due to the route of exposure. Infectivity was generally absent in most other tissues,
with bacteria present in lymph nodes and spleen on day 1 and in the liver of one animal on
Day 7. Total clearance was achieved from all tissues other than lungs by Day 49.
A single intravenous injection of Bs 2362 to young rats at a dose level of ca 107 CFU of the
technical material was not associated with clinical signs of reaction to treatment or adverse
effects on bodyweight. All rats survived to their scheduled interim termination points. There
was no evidence of pathogenicity. The test microbe was effectively cleared from most tissues
by Day 49.
A series of studies were based on intraperitoneal injection of activated and inactivated Bs
2362 spores in mice. Mice were also treated with the filtered (i.e. spore-free) entomocidal
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toxin. Injection of spores at high levels (≥108 CFU) was associated with rapid mortality.
Clearance of spores from the spleen was exponential and was almost complete after 67
days. The spore-free toxin was not lethal. There was evidence that the spores were activated
in organ homogenates but no conclusive evidence that multiplication took place in mouse
tissues. There was no evidence that the presence of Bacillus sphaericus in mouse tissues
was associated with any ill-effects. Injection to immuno-deficient mice (i.e athymic) was also
not associated with any discernible ill-effects, and no change in clearance rates. The authors
concluded that while there have been reports in the literature of Bacillus sphaericus in
wounds in humans, and in patients presenting with other disease, the animal data show no
indication that Bs 2362 was in any way causative in the infection process.
Twelve female New Zealand White rabbits received 4.48 x 108 CFU of Bs 2362 into the
conjunctival cul-de-sac of one eye and the eyes were swabbed for the presence of test
organisms for up to eight weeks. Swabs were streaked onto brain-heart infusion plates,
incubated and counted. There was no histological evidence of infection. Moderate
heterophilic conjunctivitis was observed in both treated and control eyes and was considered
within normal limits. There was no evidence of other ocular lesions. Bacillus sphaericus was
recovered up to 8 weeks after exposure.
Toxicity, irritancy and sensitization
The basic acute studies confirmed Bacillus sphaericus 2362, Serotype H-5a5b, Strain ABTS
1743 to be of low oral, dermal and inhalation toxicity.
A single oral administration of Bs 2362 to rats at 5000 mg/kg bw resulted in no deaths or
adverse clinical signs. The test compound was neither toxic nor pathogenic to rats.
An acute dermal toxicity study showed that the 14 day median lethal dermal dose level
(LD50) of Bs 2362 was greater than 2000 mg/kg bw for male and female rabbits.
A single, four-hour inhalation exposure (nose-only) study in rats to the maximum practical
concentration of the test material of 0.09 mg/L (equivalent to 3.6 x 106 spores/L) resulted
in no deaths during exposure or during the 14 day post exposure observation period. The
nature of the test material is such that traditional measures to generate an inhalable
atmosphere (grinding/micronising, dissolving in aqueous or organic solvents, heating to
generate a vapour) are impossible without destroying the test material. There were no
clinical signs of reaction to treatment. The nares of all ten test animals were positive for the
presence of the test organism at necropsy, but it was not viable.
Bs 2362 was tested in a four-hour dermal irritation test in the rabbit. Slight signs of
transient irritation were recorded in some animals. Bs 2362 is mild irritating to the skin in a
standard dermal irritation test.
In a 4-hour dermal irritation study, the test material produced transient very slight erythema
in rabbits, which persisted for up to 24 hours after dosing. Very slight oedema was also
briefly observed in one animal. ‘VectoLex’ WG was not considered to be irritant according to
EU classification guidance.
Bs 2362 was tested in a guideline eye irritation study in the rabbit. There was slight
transient irritation, but all signs had reverted to zero by day 10. Bs 2362 is mild irritating to
the eye. In an eye irritation study, transient slight conjunctival irritation was apparent in all
rabbits within an hour of dosing. All reactions had resolved within 72 hours of dosing.
Overall , ‘VectoLex’ WG is not considered an ocular irritant. A maximization test in the guinea
pig using either active or inactivated Bacillus sphaericus suspension did not induce signs of
skin sensitization, and a maximization study on the formulation also did not induce signs of
skin sensitization.
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The medical records for production plant operators indicate no adverse reactions in
individuals as a result of contact with Bs 2362 during its development, manufacture,
preparation or filed application have been documented or reported. There have been no
medical surveillance abnormalities or reports to the Occupational Health Services to date
regarding health related or other adverse reactions.
Sensitization
There was no evidence that ‘VectoLex’ WG induced delayed contact hypersensitivity by
dermal contact in a maximization test.
Both respiratory and skin sensitization, the potential to cause sensitizing reactions can be
attributed to Bs 2362 on the basis of the precautionary principle, because it is a micro-organism.
However, it should be stressed that information on workers, including agricultural workers and
others exposed to this micro-organism do not indicate that Bs 2362 is a respiratory and/or skin
sensitizer. The lack of suitable method to define with certainty the sensitization potential of
microorganisms in general needs to be overcome to allow more reliable assessments.
Genotoxicity
The need for genotoxicity studies has been assessed, and it has been concluded that studies
are not required. The requirement for genotoxicity testing of microbials should be based on
the characteristics of the micro-organism in question, their infectivity potential of
mammalian cells, the known natural occurrence and previous human exposure to the micro-
organism, and the genotoxicity potential of toxins and metabolic by- products. The
guidelines currently in place for genotoxicity testing have been developed to test chemicals.
The use of these guidelines poses certain problems when testing microbials. It is recognized
that the physicochemical properties of a substance (e.g., volatility, pH, solubility, stability,
its purity, etc.) can sometimes make standard test conditions inappropriate. This becomes
even more apparent as one considers microbial organisms. Standard mutagenicity and
genotoxicity assays are not considered appropriate for many living micro-organisms nor
does the risk they pose often warrant such testing. Further discussion and a waiver request
for genotoxicity testing based on testing impracticalities is presented at Point IIIA 5.4. Cell
culture studies are required for viruses and viroids or specific bacteria and protozoa with
intracellular replication. This is not applicable to Bs 2362 which does not replicate in warm-
blooded organisms and consequently no cell culture studies are presented for Bs 2362. An
Ames test is presented on the related organism Bacillus thuringiensis subspecies aizawai,
which was negative in S. typhimurium strains TA98, TA100, TA1535, TA1537, and E.coli
strain WP2uvrA.
Short-term toxicity
Acute studies with Bs 2362 have demonstrated that it does not induce infectious disease
following single dose inhalation or oral administration in rats. The organism is detected in
various tissues following exposure and the organisms do not appear to proliferate but are
eliminated from the body over time. It is proposed that repeat exposure to the organism
would simply result in a cumulative dose, which would be the same as one larger dose. The
acute inhalation (LC50) was greater than the maximum practical atmosphere of 0.09 mg/L
(equivalent to 3.6 x 106 spores/L) and the acute oral (LD50) is greater than 5000 mg/kg
(equivalent to 108 CFU). Two repeat-dose studies (one inhalation, one oral) were performed
on a formulation of the related Bacillus thuringiensis (‘VectoBac’ 12 AS, a formulation
containing 106 Bti AM65-52 spores/mL). The results from the studies are entirely consistent
with what would be anticipated for Bacillus sphaericus, based on acute oral and inhalation
studies, and on oral and inhalation infectivity studies. These studies have been used to
bridge from Bacillus thuringiensis to Bacillus sphaericus.
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In the rat study, groups of four male and four female rats were repeatedly exposed to a test
atmosphere within a concentration range of 1.2 x 106 spores/L air (recorded on Day 7) to
1.8 x 106 spores/L air (recorded on Day 12) over a period of 14 days; daily exposure was for
four hours. ‘VectoBac’ 12 AS (containing Bacillus thuringiensis subsp israelensis) was not
found to be toxic to rats by the inhalation route when repeatedly administered at up to 1.8 x
106 spores/L air. There were no mortalities during the study, no treatment-related adverse
clinical signs of reaction and no changes in various in-life parameters (rectal temperatures,
bodyweights, food consumption, organ weights). Post-mortem examinations (macroscopic or
microscopic pathology) revealed no treatment-related changes.
In a 90-day dog study, groups of dogs were dosed for 90 consecutive days resulting in no
mortality or treatment-related adverse clinical signs. There were no changes in the various
in-life parameters investigated (behaviour, body temperatures, bodyweights or weight gain,
food consumption and physiological function). No effects of treatment were apparent for any
of the haematology, clinical chemistry or urinalysis parameters. Pathological examination
and terminal necropsy revealed no effects of ‘VectoBac’ 12 AS administration. No evidence
for sub-acute toxicity of Bacillus thuringiensis subsp israelensis was found in the dog dosed
at circa 106 Bti spores/mL.
The acute toxicity studies conducted with ‘VectoLex’ WG indicate the product is of low toxicity by
oral, dermal and inhalation routes, and shows limited irritation effects below the threshold
for classification. There were no indications the product has the potential to elicit delayed
contact hypersensitivity. The oral LD50 of ‘VectoLex’ WG, containing the active ingredient Bacillus
sphaericus H-5a5b, Strain 2362, was determined to be greater than 5050 mg/kg bw in rats. The
dermal LD50 of ‘VectoLex’ WG, containing the active ingredient Bacillus sphaericus H-5a5b,
Strain 2362, was determined to be greater than 5050 mg/kg bw in rats. Rats exposed to a
respirable atmosphere containing the maximum achievable concentration of 0.435 mg
‘VectoLex’ WG /L for 4 hours (nominal 22.9 mg/L) tolerated the exposure without adverse effect.
It should be noted that the nature of the test material precluded milling, use of solvents or other
techniques traditionally used in inhalation studies to achieve higher concentrations. The
acute inhalation LC50 of the test material is greater than the maximum achievable dose level of
0.435 mg/L (4h) when administered undiluted as an aerosol to albino rats.
Percutaneous absorption
No study on percutaneous absorption were submitted.
Summary of mammalian toxicity
The basic acute studies confirmed Bacillus sphaericus 2362 to be of low oral, dermal and
inhalation toxicity. Bs 2362 was not irritating to the skin or eye, and was not a skin
sensitizer.
A number of acute administration studies were completed to investigate possible infectivity
or pathogenicity via oral, ocular, intravenous, intraperitoneal or intranasal routes of
administration with dose levels of between 107 and 1010 CFU. The studies confirmed that Bs
did not induce or cause infectious disease following oral, ocular, intravenous, intraperitoneal
or intranasal administration to rats, but that the test organisms were eliminated from the
body over time. Direct injection of spores at high levels (≥108 CFU) was associated with
rapid mortality. The spore-free toxin was not lethal by injection. There was evidence that the
spores were activated in organ homogenates but no conclusive evidence that multiplication
took place in living tissues. There was no evidence that the presence of Bs 2362 in tissues
was associated with any ill-effects. Injection to immuno-deficient (i.e athymic) mice was also
not associated with any discernible ill-effects, and no change in clearance rates. It was
concluded that Bs 2362 was well tolerated by the test species used showed no propensity to
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multiply within the host and was rapidly eliminated without causing adverse effects.
Data show that no adverse effects occur following direct human exposure during spraying. The
formulations have been in use for several decades, according to the manufacturer, with no
adverse findings reported. Given that there is no scientifically defined effect of exposure, other
than incidental presence in tissues, followed by clearance (which occurs with many other
common, commensal and symbiotic bacteria following exposure), it is not possible to define a
level of exposure that is associated with an effect. It is therefore not possible to define a no-
effect level either. The regulations do not require short-term or chronic testing for bacteria,
consequently there are no limit-tests available that could be used to set an AEL. Based on the
lack of pathogenicity and infectivity of Bs 2362 and adverse effect in the available data derivation
of AEL is not necessary.
The summary of findings from laboratory studies and published literature is that Bacillus
sphaericus 2362, Serotype H5a5b, Strain ABTS 1743 poses no quantifiable risk to human
health in respect of its use as a microbial insecticide. Bacillus sphaericus 2362, Serotype
H5a5b, Strain ABTS 1743 is therefore unlikely to cause human disease and can be classified
as a Group 1 biological agent according to the German and Swiss technical guidelines on
biological substances (TRBA 466; available in English at the link:
http://www.baua.de/en/Topics-from-A-to-Z/Biological-Agents/TRBA/pdf/TRBA-
466.pdf? blob=publicationFile&v=3) .
2.2.1.2 Exposure assessment and Risk characterization
‘VectoLex’ WG is used for the control of mosquito larvae (principally Culex and Anopheles
species while it is not enough effective against Aedes subgenus Stegomyia species) in a
range of aquatic breeding habitats, such as stagnant and standing ponds, flood and
irrigation water, ditches, storm water retention areas, tidal water and salt marshes,
sewerage settling ponds and water with moderate to high organic content. It is not applied
to drinking water reservoirs or water intended for human consumption or directly to crops.
‘VectoLex’ WG (Bs 2362) is applied as a spray treatment for the control of mosquito larvae in
water habitats.
Data show that no adverse effects occur following direct human exposure during spraying.
The formulations have been in used for several decades, according to the manufacturer, with
no adverse findings reported. Given that there is no scientifically defined effect of exposure,
other than incidental presence in tissues, followed by clearance (which occurs with many
other common, commensal and symbiotic bacteria following exposure), it is not possible to
define a level of exposure that is associated with an effect. It is therefore not possible to
define a no-effect level either. The data show that there are no adverse effects following
actual exposure of humans to related Bacillus species, and therefore an AEL is unnecessary.
Though the derivation of AEL for systemic effect is not necessary, as micro-organisms may
have the potential to provoke sensitising reactions a qualitative assessment of local risks
was performed.
RISK CHARACTERIZATION FOR PROFESSIONAL USERS
(a) Active substance
The setting of critical endpoints for Bs 2362 is not considered relevant as described above.
Bacillus sphaericus products are manufactured by a third party for Valent BioSciences
Corporation. Potential inhalation, oral or dermal exposures during manufacture, packing,
cleaning or maintenance are subject to engineering controls, administrative procedures
designed to prevent exposure and the wearing of protective equipment in accordance with
industrial health and safety legislation. The potential for exposure to Bs 2362 is therefore
Bs2362 Product-type 18 July 2014
12
negligible.
Risk characterization for production / formulation of a.s.
The risk to professional workers is negligible. No adverse reactions in individuals as a
result of contact with Bs 2362 during its development, manufacture, preparation or field
application have been documented or reported. There have been no medical surveillance
abnormalities or reports to the Occupational Health Services at the manufacturing plant
from employees to date regarding health related or other adverse reactions.
(b) Biocidal product
Critical end point(s)
Findings from laboratory studies, published literature, regulatory reviews and medical
surveillance reports from production areas conclude that Bs 2362 poses no quantifiable risk
to human health in respect of its use as a microbial insecticide. The setting of critical
endpoints for the biocidal product ‘VectoLex’ WG is therefore not considered relevant.
Relevant exposure paths
‘VectoLex’ WG is used for the control of mosquito larvae in water habitats. The potential for
professional workers to be exposed to ‘VectoLex’ WG during use is summarised below.
Inhalation exposure
‘VectoLex’ WG is a water dispersible granule (WG) formulation. Professional users could
be exposed by inhalation during mixing/loading of the spray solution and during application.
However, the formulation is non-dusty which will reduce the potential for inhalation
exposure during mixing/loading. Users are required to wear a dust mask to reduce
inhalation exposure during mixing/loading and during application if not in enclosed tractor
cabs or aircraft. Only users wearing protective equipment are permitted in areas being treated.
Dermal exposure
‘VectoLex’ WG is a WG formulation. Professional users could be exposed dermally
during mixing/loading of the spray solution and during application, including when acting as
ground markers for aerial spraying. However, the formulation is a granule which will reduce
the potential for dermal exposure of the hands during mixing/loading as the particles will not
adhere to gloved hands. Users are required to wear long-sleeved shirt, long trousers,
shoes and socks, and water-proof gloves to reduce dermal exposure during
mixing/loading and application. Only users wearing protective equipment are permitted in
areas being treated.
Oral exposure
‘VectoLex’ WG is not likely to reach the mouth of professional users. Therefore, the risk
during use is considered to negligible.
Risk characterisation for the biocidal product
The potential for professional workers to be exposed to Bs 2362 is small due to the physical
nature of the product and the use of personal protective equipment during application. Bs
2362 poses no quantifiable risk to human health and therefore the likelihood of adverse
health effects occurring in humans through inadvertent inhalation, dermal or oral exposure
will be negligible.
Overall assessment of the risk to professionals for the use of the active
substance in the biocidal product
Bs 2362 poses no quantifiable risk to human health and the risks to professional workers
through either manufacture or use of the active substance or formulated product are
negligible.
Bs2362 Product-type 18 July 2014
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RISK CHARACTERIZATION FOR NON-PROFESSIONAL USERS
Non-professional users may be exposed during mixing/loading and during application because
the use of personal protective equipment cannot be assumed. However, the ‘VectoLex’ WG
formulation is a granule which will reduce the potential for inhalation exposure and dermal
exposure of the hands during mixing/loading and ‘VectoLex’ WG will only be used
infrequently by non-professional users thereby reducing the overall potential for exposure.
Consequently, the risk to non-professional users is considered to be negligible.
The potential for the active substance in ‘VectoLex’ WG (Bs2362 ) to cause adverse effects
in humans is considered below. Data show that there are no adverse effects following actual
exposure of humans to different Bacillus species (oher than B.anthrax and B. cereus) and
therefore an AEL for the active substance is unnecessary. However calculations on AEL using
the UK model have shown that with/without PPE the use is safe (see Addendum to CAR).
Consequently, the risk to non-professional users is considered to be negligible and estimates
of the actual level of exposure of non-professional users are not relevant. AEL was not derived due
to lack of adverse effects.
INDIRECT EXPOSURE AS A RESULT OF USE
(a) Active substance
Critical endpoint(s)
The setting of critical endpoints for Bs 2362 is not considered relevant as described above.
Relevant exposure paths
Bs 2362 is manufactured under strict engineering and procedural control and the possibility of
indirect exposure to the active substance during manufacture is negligible.
Risk characterization for production / formulation of a.s.
Bs 2362 poses no quantifiable risk to human health and the possibility of indirect exposure
during manufacture is negligible due to the controls in place in the manufacturing plant.
(b) Biocidal product
Critical end point(s)
Findings from laboratory studies, published literature, regulatory reviews and medical
surveillance reports from production areas conclude that Bs 2362 poses no quantifiable risk
to human health in respect of its use as a microbial insecticide. The setting of critical
endpoints for the product ‘VectoLex’ WG is therefore not considered relevant.
(c) Relevant exposure paths
Inhalation exposure
Non-users are not expected to be close to the spray during application. The risk of
inhalation exposure of non-users to spray drift during application or to residues after
application via the environment is considered to be negligible.
Dermal exposure
Non-users are not expected to be close to the spray during application. The risk of dermal
exposure of non-users to spray drift during application or to residues after application via
the environment is considered to be negligible.
Oral exposure
Bs 2362 is not to be applied directly to food or feed commodities and is not to be used on
water bodies which are treated drinking water. The risk of oral exposure to residues
during or after application is therefore considered to be negligible.
Bs2362 Product-type 18 July 2014
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Risk characterization for non-users
Non-users are not expected to be close to the spray during application of ‘VectoLex’ WG and
will not be exposed to food and feed commodities or drinking water containing residues of
the active substance, Bs 2362. The possibility of indirect exposure to Bs 2362 is therefore
extremely small. Bs 2362 poses no quantifiable risk to human health and therefore the
likelihood of adverse effects occurring in humans through any indirect exposure will be
negligible.
Overall assessment of the risk to non-users for the use of the active substance in
biocidal product
Bs 2362 poses no quantifiable risk to human health and the risks to non-users through
indirect exposure are negligible.
The use patterns of ‘VectoLex’ WG does not result in the direct application of the product to
food crops, processed foods or surfaces likely to be used to store, process or present food.
There is no application to clear drinking water. In rice crops and/or if a direct application
contaminates surrounding food crops, an interval of 30 days before harvest should be
considered. Application to irrigation water at point of introduction of rice is allowed.
COMBINED EXPOSURE
Bs 2362 poses no quantifiable risk to human health and the risks to professional workers
through either manufacture or use of the active substance or formulated product are
negligible.
Bs 2362 poses no quantifiable risk to human health and the risks to non-professional users
through use of the formulated product are negligible.
Bs 2362 poses no quantifiable risk to human health and the risks to non-users through
indirect exposure are negligible.
2.2.2 Environmental Risk Assessment
2.2.2.1 Fate and distribution in the environment
Bs 2362 is used for the control of some mosquitoes in a range of aquatic breeding habitats,
such as stagnant and standing ponds, flood and irrigation water, ditches, storm water
retention areas, tidal water and salt marshes, sewerage settling ponds and water with
moderate to high organic content. Bs 2362 is not intended for application to soil, although it
is a naturally occurring soil bacteria. Inadvertent application of Bs 2362 to soil via spray drift
will be minimal and is unlikely to increase levels of the bacteria above those that may
naturally occur in the soil environment.
No data has been provided about the world distribution of Bs in the soil environment. The
only data presented take into account the background levels of mixed populations of Bacillus
thuringiensis and Bs. A ratio of about 1:1 is reported by Park et al.2 on the amount of Bt and
Bs, showing mosquitocidal activity, in mosquito breeding sites of Florida. Therefore it could
be assumed, even if only tentatively, that the population of Bs in soil is 2x102 – 4.9x104
CFU/g soil as evaluated for Bt3. Bs 2362 spore and toxin activity is affected by exposure to
UV light.
2 H.-W Park, Hayes,S.R., Mangum C.M., 2008 - Distribution of mosquitocidal Bacillus thuringiensis and Bacillus sphaericus from sediment samples in Florida. Journal of Asia-Pacific Entomology 11: 217–220 3 P.A.W Martin, 1991 – Dynamics of Bacillus thuringiensis turnover in soil, p.315. Abs.: The General Meeting of the American Society for Microbiology, 1991. Am. Soc. Microbiol.
Bs2362 Product-type 18 July 2014
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2.2.2.2 Effects assessment
Spread, mobility, multiplication and persistence in air, soil and water of the active substance
Following application to water bodies, levels of Bacillus sphaericus spores and insecticidal activity decline in the water phase primarily as a result of sedimentation4 and although levels in the sediment phase increase temporarily as a consequence, the bacteria is not persistent and does not multiply in sediment. Re-cycling of the bacteria in the larval cadaver has been reported to be a reason for extended insecticidal activity due to use of the larval cadaver as a food source by healthy larvae. However, this effect is unlikely to be a significant factor under field conditions, since larval cadavers are quickly removed from the water column (they drown within 48 hours) and Bacillus sphaericus being an aerobic organism is not able to compete effectively with naturally occurring anaerobic bacilli. Any residual deposits of the originally ingested protein toxins in the larval cadaver gut would not support re-cycling of the toxin under field conditions5.
The insecticidal action of Bacillus sphaericus can persist for periods up to 9 months under
ideal conditions in clear water, but where levels of organic matter are high the insecticidal
activity is more limited due to microbial degradation of the protein toxin. Settling out of
spores and toxin crystals also results in a gradual decline in insecticidal activity in the
treated water. Once the spores and toxin protein have settled onto the sediment the
insecticidal activity is rapidly lost in the organic rich environment. Sunlight can denature the
toxin protein and inactivate the spores of Bacillus sphaericus. The degree to which sunlight
will effect insecticidal activity will depend on the depth of the water body and the clarity of
the water.
Spore viability is reduced under acidic conditions and the insecticidal toxin protein is
denatured under alkali conditions6. Data on UV light effects are confirmed by Cokmus et al.
(2000 Doc. IIIA 7.1.2-04). According to this study, the spores and the insecticidal toxin (42
kDa protein) of Bacillus sphaericus are inactivated by expsoure to UV light, with this effect
more pronounced at low spore concentrations. UV absorbing materials have a protective
effect and prolong spore viability. The effect of sunlight on Bs 2362 in the environment is
therefore expected to cause a gradual decline in the concentration of spores and toxins and
reduce the potential for the organism to multiply in the environment.
Airborne concentrations of Bs 2362 are expected to be negligible following application
to water bodies.
Data on toxins
The life-cycle of Bs 2362 follows the characteristic process of spore formation (sporulation) typical
of Bacillus cultures, with the exception that insect toxin containing parasporal bodies are formed
during sporulation. Spore formation normally commences when vegetative growth ceases due
to a lack of nutrients or a shift in the environment to conditions less favourable for
vegetative growth. The insecticidal action of Bs 2362 can persist for periods up to 9 months
under ideal conditions in clear water, but where levels of organic matter are high the insecticidal
activity is more limited due to microbial degradation of the protein toxin. Settling out of the toxin
crystals also results in a gradual decline in insecticidal activity in treated water. Once the toxin
protein has settled onto the sediment the insecticidal activity is rapidly lost. Sunlight can
4 E. W. Davidson, Urbina M., Payne J., Mulla M.S., Darwazeh H., Dulmage H.T. Correa J.A., 1984 - Fate of Bacillus sphaericus 1593 and 2362 Spores Used as Larvicides in the Aquatic Environment. Applied And Environmental Microbiology, 47: 125-129 5 D.Klein, Uspensky, I., Braun, S. (2002): Tightly bound binary toxin in the cell wall of Bacillus sphaericus. Applied and Environmental Microbiology, July 2002, p. 3300-3307. 6 de Barjac, H., Sutherland, D.J. eds. 1990. Bacterial Control of Mosquitoes and Black Flies, Rutgers University Press, pp 284-291
Bs2362 Product-type 18 July 2014
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inactivate the toxin protein but the degree to which sunlight will affect insecticidal activity will
depend on the depth of the water body and the clarity of the water.
Effect of the active substance on the Aquatic compartment
Toxicity to fish
Bs 2362 is not considered to be acutely toxic to fish.
Toxicity to invertebrates
Bs 2362 is not considered to be acutely toxic to aquatic invertebrates.
A literature review summarised Bs 2362 studies from a wide range of non–target aquatic
organisms, these included species from the Odonata (Tarnetrum corruptum and Enallagma
civile), Ephemeroptera (Callibatis pacificus), Heteroptera (Notonecta undulata, N. unifasciata and
Buenoa sp.), Coleoptera (Dytiscid beetles), Diptera (Chironomus crassicaudatu, Glyptotendipes
paripes and Toxorhynchites sp) and Crustacea (Daphnia similis and Streptocephalus
dichotomus). The results indicated that Bs 2362 primary powder and formulations can be used
to control mosquito larvae with minimal adverse impact on the environment.
In a series of studies, predatory stonefly larvae (P. media) were shown to acquire Bs 2362 by
eating midges that had been fed with spores of the Bacillus. Spores were also shown to be
consumed by both leaf shredding stoneflies (P. proteus) and cranefly (T. abdominalis). The three
aquatic insects used in these studies were unaffected by the consumption of Bs 2362
spores at levels lethal to mosquitoes. Of the three consumer organisms, cranefly larvae
uniquely possessed an alkaline foregut (pH 11.5) that presented conditions conducive to the
solubilisation of the Bs active protein delta endotoxins. Failure of these conditions to elicit an
adverse response in the cranefly larvae was considered likely to have been due to the
absence of specific binding sites. A potency assay performed with Bacillus sphaericus spores
recovered from cranefly faeces demonstrated that the toxicity of voided spores to target mosquitoes was substantially reduced, compared to the toxicity of the stock preparation initially
dosed.
Effects on algal growth
Based on nominal exposure concentrations the 120-h EC50 of Bs 2362 to the green alga
Selenastrum capricornutum was observed to be >2.2 mg/L (> 1.73 x 108 CFU/L; >6.3 x 103 ITU/L
(the highest concentration tested).
Bs2362 Product-type 18 July 2014
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Toxicity to aquatic plants
No studies have been performed with aquatic plants.
Terrestrial compartment
AQUATIC COMPARTMENT
Test organism Test
substance Duration Effective concentration
Effects on fish
Onchorhynchus
mykiss
ABG-6184
Technical
(4x1010
CFU/g)
96-h
EC50> 15.5 mg/L (>6.2x108 CFU/L)
NOEC: 15.5 mg/L (6.2x108 CFU/L)
(Surprenant, 1986a)
Lepomis
macrochirus
ABG-6184
Technical
(4x1010
CFU/g)
96-h
LC50> 15.5 mg/L
NOEC: 15.5 mg/L (6.2x108 CFU/L)
(Surprenant, 1986b)
Cyprinodon
variegatus
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
LC50> 100 mg/L (7.9x109 CFU/L;
2.8x105 ITU/L)
NOEC: 22 mg/L (1.7x109 CFU/L;
6.3x104 ITU/L) (Bowman, 1989)
Effects on freshwater invertebrates
Daphnia magna ABG-6184
Technical
(4x1010
CFU/g)
48-h
EC50>15.5 mg/L (>6.2x108 CFU/L)
(Suprenant, 1986c)
NOEC: 15.5 mg/L (6.2x108 CFU/L)
Crassostrea
virginica
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
EC50>42 mg/L (> 2.5x109
CFU/L;>1.2x105 ITU/L)
NOEC: 15 mg/L (8.9x108 CFU/L; 4.3x104
ITU/L)
(Dionne, 1990)
Mysidopsis
bahia
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
LC50: 71 mg/L (5.6x109 CFU/L; 2.02x105
ITU/L)
NOEC: 50 mg/L (3.9x109 CFU/L;
1.4x105 ITU/L) (Forbis, 1990)
Effects on algal growth
Selenastrum
capricornutum
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
120-h
EC50> 2.2 mg/L (>1.73x108 CFU/L;
>6.3x103 ITU/L)
NOEC: 2.2 mg/L (1.73x108 CFU/L;
6.3x103 ITU/L) (Forbis, 1989)
Effects on aquatic plants
No data
presented
Bs2362 Product-type 18 July 2014
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Toxicity to birds
An acute oral toxicity study was performed using Mallard duck (Anas platyrhynchos) aged 16
days, previously acclimated to laboratory conditions for 14 days. Birds were dosed with Bs 2362
in a saline solution via a rubber catheter. Following dosing birds were observed for 29
days. Bacillus sphaericus technical showed no apparent pathogenicity, toxicity or effect upon
survival of young mallards when administered by oral gavage at 9000 mg/kg (>3.6 x 1011
CFU/kg bw) followed by a further 29 day observation period. The LD50 was therefore >9000
mg/kg (>3.6 x 1011 CFU/kg bw).
A short-term avian dietary toxicity study was conducted in which diets containing Bs 2362 were
fed to mallard ducks aged 15 days, previously acclimated to laboratory conditions for 14 days.
Following five days dietary exposure at Bs 2362 dietary concentrations of 1, 7.5 and 20%,
the birds were observed for a further 25 days. Following a total of 30 days of exposure, at
the maximum dosage of 3.7 g MPCA/bird/day (2.9 x 10 11 CFU/bird/day; 1.1
x 107 ITU/bird/day), there was no apparent pathogenicity or effect upon survival of young
mallards.
An avian injection pathogenicity study was performed with mallard duck (Anas platyrhynchos)
aged 18 days. The study was considered unacceptable due to the absence of
recognized international guidelines. Bs 2362 was given to the birds as a single
intraperitoneal injection according to individual body weights. Following injection the birds
were observed for 30 days. During the observation period four of the Bs 2362 birds died, one
of the birds that died had lesions consistent with trauma due to injection, another died after
falling from its cage. Thus it is not likely that these birds died as a result of the Bs 2362
treatment. The results of the study indicate that intraperitoneal injection of 1.1 times the
adjusted host equivalent of Bs 2362 to mallard ducks resulted in an LD50 >1500 mg/kg bw
(> 1.25 x 1010 CFU/kg bw. Little toxicity occurred which was directly related to treatment
with the active substance. The Bs 2362 did not appear to grow to any great extent or to
replicate in the tissues of mallard ducks.
Bees Two bee studies were performed with caged adult honey bees. In experiment one, bees in three replicate cages per treatment were given water and sucrose ad libitum. Controls were given plain sucrose and three other sets were given Bs 2362 treated sucrose with concentrations
of 104
to 108
spores per mL. The study duration was 15 days. In experiment two, bees were treated identically except that in addition each cage was given a supply of commercial pollen substitute ad libitum. The study duration was 28 days. Isolation of viable Bs 2362 organisms in the intestinal tract of most bees examined confirmed the ingestion of the test material. The number of colonies was not quantified; however, it was observed that there was no evidence for multiplication of Bs2362 in the bee. Under the experimental conditions,
feeding Bacillus sphaericus technical material in sucrose concentrations of 104
to 108
spores per mL had no effect on adult honey bee longevity. Even though the strain is not specified and the study does not match any EU Guideline, the honey bee gut pH is said to be weakly acidic (Chauvin, 19627). Therefore the Bs binary toxin is likely to be not effective.
Toxicity to earthworms
Bs 2362 is not considered to be acutely toxic to earthworms. A 30-day earthworm study gave an
LC50 >1000 ppm dry weight soil (1.7 x 106 ITU/kg dw soil). Exposure was via soil and treated
food. Under the conditions of the study Bs 2362 was neither toxic nor pathogenic to the
earthworm Eisenia fetida.
7 R. Chauvin (1962) Nutrition de l’abeille. Ann.Nutr. Aliment. 16:41-63.
Bs2362 Product-type 18 July 2014
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Effects on soil non-target micro-organisms
No study presented.
Other flora and fauna
No specific studies were carried out to determine whether Bs 2362 has an impact on other flora and
fauna.
TERRESTRIAL COMPARTMENT
Test
organism
Test
substance Duration Effective Concentration
Effects on birds
Mallard duck ABG-6184
Technical
(4x1010
CFU/g)
1
treatment;
mortality
checked up
to 30 days
NOEC: 9000 mg/kg bw (3.6x1011 CFU/kg bw)*
(Henck, 1986a)
Mallard duck ABG-6184
Technical
(7.9x1010
CFU/g;
2844
ITU/mg)
29 day-
dietary
exposure
No pathogenicity nor mortality observed after 30
days of a diet containing a maximum dosage of 3.7
g MPCA/bird/day (2.9x1011 CFU/bird/day; 1.1x107
ITU/bird/day) (Grimes & Jaber, 1989)
Effects on earthworms
Eisenia
fetida
VectoLex
Technical
Powder
(850
ITU/mg)
30-d NOEC: 1000 ppm (1.7 x 106 ITU/kg dry weight soil)
(Rodgers, 2006)
Effects on honey bee
Honeybee
adults
Bs
unknown
strain
1.4x1012
CFU/g
15 and 28
days
NOEC: 108 CFU/mL ** (Vandenberg, 1986)
Effects on soil non-target micro-organisms
No data
presented
Effects on arthropods other than bees
No test
carried out
Effects on terrestrial plants
No test
carried out
* The reliability of the paper is questionable since only one treatment was done by oral gavage; the study is not
conducted under any shared international guideline
** The study does not match any guideline and was not conducted under GLP conditions. The strain is not specified.
Bs2362 Product-type 18 July 2014
20
Non compartment specific effects relevant to the food chain (secondary
poisoning)
No specific studies were carried out to determine whether Bs 2362 residues have an
impact on secondary poisoning. However, secondary poisoning in the aquatic and
terrestrial compartments is assessed in Document IIC of the CAR.
2.2.2.3 PBT and POP assessment
Not applicable
2.2.2.4 Exposure assessment
There are no formulation components or properties of the formulation that are considered to
affect the fate and distribution of the active substance, Bs 2362, in the environment. The
following data on Bs 2362 are appropriate to describe the environmental fate of ‘VectoLex’WG.
Expected environmental population density and concentrations in soil and water
In order to perform a risk assessment for non-target organisms, the predicted
environmental population densities (EEDs) of Bs 2362 and the predicted environmental
concentrations (PECs) of toxins are calculated for soil and water, based on application
rates both on soil and in water of 1.5 kg/ha ‘VectoLex’ WG and a maximum of 5 repetitions
at a minimum interval of 7 days. As a worst case, no adsorption, interception and
degradation are assumed for spores and toxins between applications. For risk assessment
the load of a single application of ‘VectoLex’WG will be related to the top 5 cm of soil to
achieve the theoretical soil concentration of 1.5x104 CFU/g soil, and to 30 cm water depth
to achieve the theoretical water concentration of 3.8x106 CFU/L.
Soil
No data are available on degradation of spores and toxins in soil. Therefore, the data have
been calculated assuming a worst case of no degradation between applications.
Assumptions:
MPCP addition to soil
incorporation into the top 5 cm layer
soil density of 1.5 g/ cm³
no adsorption
no degradation
plant interception: 0 %.
One application
EEDS=1.5x104 CFU/g
PECS= 1.3 ITU/g.
Five applications
EEDS=7.7x104 CFU/g
PECS= 6.5 ITU/g.
Water
Only a set of data useful to derive a half-life value in water has been found in scientific
open literature (Yousten et al., 1992, Doc. IIIA, 7.1.2/01). A DT50 value has been
calculated by the RMS, by using the data plotted in the Yousten et al. (1992) paper for the
degradation of Bs spores in pond water at 30°C.
No studies on degradation of toxins in water has been found, and therefore the RMS
Bs2362 Product-type 18 July 2014
21
assumed no degradation between applications.
Assumptions:
MPCP addition to water
no adsorption on sediments
water depth 30 cm
interception 0%
DT50 spores = 25 d
One application
EEDSW=3.8x106 CFU/L
PECSW= 3.3x102 ITU/L.
Five applications
EEDSW=1.4x107 CFU/L
PECSW= 1.6 x103 ITU/L.
The values of the EEDs and PECs were calculated without taking into account the
adsorption of the spores and the toxins on the sediments (Step-1 model). However, the
adsorption cannot be excluded and therefore a specific STEP-2 model for the
water/sediments system was developed and is reported in the following paragraph.
EEDs and PECs in the water – sediment system
Prior to any risk assessment in the water-sediment systems, PECs (Predicted
Environmental Concentration, for toxin) and EEDs (Expected Environmental Density, for
CFU of micro-organisms) have to be calculated. Different models for calculation of the
PECs in the water-sediment systems have been developed (FOCUS, 2006), and are
currently used for inclusion of chemical active substances in Annex I under the Council
Directive 91/414/EEC.
For micro-organisms, no agreed models have been developed. A straightforward first
approach could be the calculation at time zero of the Colony Forming Units (CFU) following
the addition of the microbial pest control product (MPCP), under the conditions of neither
growth nor death of the microbes. In this case, which can be thought of as a step-1
approach, the microbial population density will be both over-estimated in water and under-
estimated in sediments, giving rise to a non-realistic worst case for water and a non-
realistic best case for sediments. Furthermore, it would be impossible to evaluate any
effect due to repeated applications of MPCP at time intervals.
A tentative step-2 approach could be carried out if the kinetics and the adsorption
behaviour of the system are known, i.e. if the first-order rate constants k and the
adsorption constant Kads of the two processes are known or can be inferred from reliable
data. In this case a simple two-compartment system can be assumed, with degradation
occurring exclusively in the water phase and the adsorption in the sediment phase.
In this last case the equations used to calculate both EED and PEC were the following:
KD = KOCx%OC/100
CL(t) CL,0 e
k
deg rad
1 KD
LS
LL
t
CL,0 e K dissip t
Kdissip kdegrad
1 KD LS
LL
Cs(t) KD CL(t)
Bs2362 Product-type 18 July 2014
22
where:
CL(t) is the total amount of CFU or ITU in water CFU/ha or ITU/ha
CS(t) is the total amount of CFU or ITU in sediments CFU/ha or ITU/ha
Cl,0 is the total amount of CFU or ITU in water at time zero following CFU/ha or
ITU/ha
instantaneous adsorption
LS is the thickness of the sediments m
LL is the depth of the water pounding on sediments m
kdegr is thefirst-order degradation constant day-1
KOC is the linear adsorption constant normalized to organic carbon content ml g-1 OC
Kdissip is the dissipation constant day-1
VL is the volume of wate ponding on sediments L ha-1
WS is the weight of sediments g ha-1
% OC is the percentage of organic carbon in sediments % δ is the bulk density of sediments kg L-1
The final results of the step-2 model calculations are now summarized in the following
table
WATER SEDIMENTS
µg MPCA L-1 ITU L-1 CFU L-1 µg MPCA g-1 ITU g-1 CFU g-1
One application
1.9x10 2.4x10 2.8x105 9.5x10-1 1.2 1.4x104
Five applications
6.7x10 1.2x102 1.1x106 3.3 6.1 5.0x104
EEDs in Sewage Treatment Plant
No specific studies on micro-organisms were carried out to assess the biological effects of
Bs 2362 on the STP microbial community.
Calculations of CFU amount in water following a STP treatment has been performed in a
similar manner to the disposal of general industrial chemicals as laid down in the Technical
Guidance Documents (TGD) for the Risk Assessment of Existing and New Notified
Industrial Chemicals (1996), with some necessary modifications. Therefore, the local spore
density (EED) of the biocide in surface water has been calculated ignoring elimination
processes like volatilisation, degradation or sedimentation in a sewage treatment plant
(STP)
Following the step-1 approach in case of one application, the concentration in STP-
untreated waste water, EEDlocal, influent, EEDlocal, sw, EEDlocal, sed are:
EEDlocal, influent = 3.8x106 [CFU/L]
EEDlocal, sw =3.8x105 [CFU/L]
EEDlocal, sed = 3.3x107 [CFU/kg sed]
Analogously, following the same steps of calculations, in case of 5 applications the
different EEDs will be:
EEDlocal, influent, = 1.6x107 [CFU/L]
EEDlocal, sw = 1.6x106 [CFU/L]
Bs2362 Product-type 18 July 2014
23
EEDlocal, sed = 1.2x108 [CFU/kg sed]
Following the step-2 approach, in case of one application:
EEDlocal, influent = 2.8x105 [CFU/L]
EEDlocal, sw = 2.8x104 [CFU/L]
EEDlocal, sed = 2.5x106 [CFU/kg sed]
and in case of 5 applications:
EEDlocal, influent =1.2x106 [CFU/L]
EEDlocal, sw =1.0x105 [CFU/L]
EEDlocal, sed =8.7x106 [CFU/kg sed]
Non compartment specific exposure relevant to the food chain (primary and
secondary poisoning)
2.2.2.5 Risk characterisation
‘VectoLex’ WG is a bacterial product based on spores and crystals of Bs 2362. The specific
biocidal use is for the control of mosquito larvae (principally Culex and Anopheles species
while it is not efficient enough against Aedes subgenus Stegomyia species) in a range of
aquatic breeding habitats, such as stagnant and standing ponds, flood and irrigation
water, ditches, storm water retention areas, tidal water and salt marshes, sewage settling
ponds and water with moderate to high organic content.
Bacillus sphaericus 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
crystals. The crystals are ingested and, under the alkali conditions present in the larval
gut and for the activity of gut proteases, the crystals dissolve, releasing the active toxin
(principally a 42 kDa protein). There are no other active metabolites and degradation
products that are known to contribute to the toxicity of Bacillus sphaericus. Due to its
specific mode of action, Bacillus sphaericus will have no other effects on the intended area
of use.
To evaluate risk assessment for aquatic compartment, PEC/PNEC ratio has to be
calculated: if PEC/PNEC ratio < 1, no refinement is required, if PEC/PNEC >1, a STEP-2
calculation (Document II B) has to be made. Expected Environmental Density (EED) is
used as a metric owing to the fact that the calculations are made with CFU of a micro-
organism instead of weight or concentration of a chemical. Similarly, PNED is used instead
of PNEC when dealing with microbial densities.
For Bs 2362, the value of PNEDsw is extrapolated from the NOEC obtained for Daphnia in
48-h survival and reproduction test, where NOEC= 15.5 mg/L (6.2x108 CFU/L). Applying
an AF of 100, PNEDsw is 0.15 mg/L (6.2x106 CFU/L). EEDsw/PNEDsw ratios for 1 and 5
treatments are indicated in following table. The AF applied takes into consideration that
the different aquatic organisms tested give no effect , and that the chronic studies done in
the field show similarly no effect (see Lacey and Merrit, 2003)8,
Aquatic compartment. EEDsw/PNED sw ratio based on the most sensitive species
(STEP-1)
8 L.A.Lacey, R.W.Merrit (2003) The safety of bacterial microbial agents used for blackfly and mosquito control in aquatic environments. In “Environmental impacts of microbial insecticides”, H.M.T.Hokkanen and A.E. Hagek Eds., Kluwer Academic Publ., pp 151-168.
Bs2362 Product-type 18 July 2014
24
AQUATIC COMPARTMENT
Test organism NOEC (48-h acute)
mg/L[CFU/L]
AF PNEDsw
(CFU/L)
STEP EEDsw
(CFU/L)
EEDsw/
PNEDs
w
Daphnia
magna 15.5 mg/L[6.2x108]
10
0 6.2x106 1
1 appl.
3.8x106
5 appl.
1.4x107
6.1x10-
1
2.2 >1
As shown in the previous table, a value lower than 1 after one treatment (EEDSW
=3.8x106, EEDSW/PNEDSW <1) was calculated and a value > 1 resulted after 5 applications
(EEDSW = 1.4x107 ; EEDsW/PNEDSW = 2.2). The value of the ratio EEDsw/PNEDsw following
5 applications (assuming no dissipation) is > 1. This result suggests to provide tentatively
a refinement of the risk assessment, taking into account a more realistic dissipation
assumption. Assuming a DT50,diss =25 days (Yousten et al., 1992 in Doc. IIIA 7.1.2-01), a
STEP-2 calculation according to the model developed for AM 65-52 can be done.
Aquatic compartment. EEDsw/PNED sw ratio based on the most sensitive species
(STEP-2)
AQUATIC COMPARTMENT
Test organism NOEC (48-h acute)
mg/L[CFU/L]
AF PNEDsw
(CFU/L)
STEP EEDsw
(CFU/L)
EEDsw/
PNEDsw
Daphnia
magna 15.5 mg/L[6.2x108]
10
0 6.2x106 2
1 appl.
2.8x105
5 appl.
1.0x106
4.5x10-2
1.6x10-1
According to this STEP-2 calculation, 5 treatments can be done.
Sediment EED/PNED calculation
A quantitative risk assessment for sediments, using the STEP-2 approach used for surface
water, has been made and is shown in the next table.
EED values for sediments (STEP-2)
EEDsed [CFU/g] PNEDsed
STEP-2
1 application 5 applications
1.42 x 104 5.0 x 104 n.s
n.s.: not stated
EEDsed in STEP-2 has been calculated assuming the MPCA dissipation. However, no PNEDsed
value is available, so that a EEDsed/PNEDsed cannot be calculated.
In conclusion, it can be assumed that VectoLex used at field maximum rate does not have any
adverse effect on the aquatic phase of the water-sediment system. No reliable conclusion can
Bs2362 Product-type 18 July 2014
25
be drawn on the sediment phase.
Sewage treatment plants (STP)
No specific study on micro-organisms was carried out to assess biological effects of Bs 2362 on
STP microbial community.
According to the calculations of EED local,sw and EED local,sed (following the STEP-2 approach), the
amount of Bs 2362 is 2.8x104 CFU/L and 2.5x106 CFU/kg sed for 1 application and 1.0x105
CFU/L and 8.7x106 CFU/kg sed following 5 applications, respectively.
Due to the lack of data on the effects on microbial community in sewage treatment plants,
no conclusion can be drawn about the use of VectoLex WG in STP.
EED values for STP (STEP-2)
1 application 5 applications
EEDloc,sw [CFU/L] 2.8 x 104 1.0 x 105
EEDloc,sed [CFU/kg] 2.5 x 106 8.7 x 106
EED/PNED calculation for terrestrial compartment
EED/PNED (or PEC/PNEC if the NOEC is expressed as ITU/kg dw soil ) ratio at local level below
1 indicates negligible risk for the environment.
The PNED (PNEC) for terrestrial organisms can take into account the value of acute toxicity
obtained for earthworms, corrected by an AF equal to 1000. Due to the lack of DT50,soil value, it
is assumed the worst case of no degradation between replications.
Test
organism
NOEC (mg
MPCA/kg dw
soil)
AF PECsoil
[ITU/kg
dry
weight
soil]
PECsoil
[ITU/kg dw
soil)]
PECsoil/
PNECsoil
Eisenia
fetida
1000
(1.7x106
ITU/kg dry
weight soil)
1000
1.7 x103
1 application:
1.3x103
5 applications
6.5 x103
1 application:
= 0.76
5 applications
= 3.8
Therefore only one application can be done, posing no unacceptable risks to earthworms.
However, VectoLex is not intended for use on soil.
The overall conclusion on evaluation of risk assessment for terrestrial compartment is that the
use of VectoLex WG poses no unacceptable risk to terrestrial organisms.
2.2.3 Assessment of endocrine disruptor properties
Not applicable.
Bs2362 Product-type 18 July 2014
26
2.3 Overall conclusions
The outcome of the assessment for Bs 2362 in product-type 18 is specified in the BPC
opinion following discussions at the sixth meeting of the Biocidal Products Committee
(BPC-6).
2.4 List of endpoints
The most important endpoints, as identified during the evaluation process, are listed in
Appendix I.
Bs2362 Product-type 18 July 2014
27
Appendix I: List of endpoints
Chapter 1: Identity, Physical and Chemical Properties, Classification and Labelling
Active substance (ISO Common Name) Bacillus sphaericus 2362, Serotype H5a5b,
Strain ABTS-1743 (abbreviated to Bs 2362 in
this dossier)
Product-type PT 18 (Insecticide)
IDENTITY OF THE MICRO-ORGANISM
Name and species description, strain characterization
Common name of
the micro-
organism
Bacillus sphaericus 2362, Serotype H5a5b,
Strain ABTS-1743 (abbreviated to Bs 2362
in this dossier).
Taxonomic name
and strain and
indication whether
it is a stock
varient, a mutant
strain or a GMO.
Species: Bacillus sphaericus
Subspecies: Bacillus sphaericus is a heterogeneous
species of bacteria that contains strains
belonging to at least five different DNA
homology groups. The bacteria in these
homology groups are phenotypically
similar to an extent that it has not been
practical to establish each as a new
Serotype: H5a5b
Strain: ABTS-1743
Genus: Bacillus
Family: Bacillaceace
Bs (Strain 2362) 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.
Collection and
culture reference
number
ATCC safe
deposit No.
American Type Culture Collection.
SD-1170
Strain
designation
Bacillus sphaericus, Serotype H5a5b,
Strain ABTS-1743, ATCC No.1170
Production
strain/product
Production strain for ‘VectoLex’ WG
Identity
Chemical name (IUPAC) Not applicable, it is a micro-organism
Chemical name (CA) Not applicable, it is a micro-organism
CAS No Not applicable
EC No Not applicable
Other substance No. Not applicable
Minimum purity of the active substance ‘VectoLex’ WG contains approximately 512 g/kg
Bs2362 Product-type 18 July 2014
28
as manufactured (g/kg or g/l) Bs 2362 as the active ingredient. Other
components in ‘VectoLex’ WG are confidential to
Valent BioSciences are detailed in the
confidential attachment under Point IIIB 1.4.
The technical grade fermentation slurry
contains nominally 99% Bs 2362. The
formulated product ‘VectoLex’ WG contains
51.2% of the technical grade active substance,
with lower and higher limits of 46 and 64% by
weight, respectively.
The minimum potency is 600 Bsp units/mg.
Identity of relevant impurities and
additives (substances of concern) in the
active substance as manufactured (g/kg)
No relevant impurities. For microbial
contaminants, their presence is monitored
through manufacturing directions for the
fermentation process.
Molecular formula Not applicable
Molecular mass Not applicable
Structural formula Not applicable
Biological, Physical and chemical properties
Resistance to antibiotics Bs2362 is naturally resistant to streptomycin
(>20 mg/ml) and chloramphenicol (16
ug/ml). It is sensitive to gentamycin,
penicillin, erythromycin, sulfamethoxozole
Genetic stability Genetic stability is ensured through
manufacturing directions for the
fermentation process.
Melting point (state purity) Not applicable
Boiling point (state purity) Not applicable
Temperature of decomposition Not applicable
Appearance (state purity) Not applicable
Relative density (state purity) Not applicable
Surface tension Not applicable
Vapour pressure (in Pa, state
temperature)
Not applicable
Henry’s law constant (Pa m3 mol -1) Not applicable
Solubility in water (g/l or mg/l, state
temperature)
Not applicable
Solubility in organic solvents (in g/l or
mg/l, state temperature)
Not applicable
Stability in organic solvents used in
biocidal products including relevant
breakdown products
Not applicable
Partition coefficient (log POW) (state
temperature)
Not applicable
Bs2362 Product-type 18 July 2014
29
Hydrolytic stability (DT50) (state pH and
temperature)
Not applicable
Dissociation constant Not applicable
UV/VIS absorption (max.) (if absorption
> 290 nm state at wavelength)
Not applicable
Photostability (DT50) (aqueous, sunlight,
state pH)
Not applicable
Quantum yield of direct
phototransformation in water at > 290
nm
Not applicable
Flammability Not flammable
Explosive properties Not explosive
Proposal for the classification and labelling of the active substance
Hazard symbol: Not classified
Risk phrases Not classified
Safety phrases 24/25
Proposal for the classification and labelling of the biocidal product
Hazard symbol: Not classified
Risk phrases Not classified
Safety phrases Not classified
Chapter 2: Methods of Analysis
Analytical methods for the active substance
Technical active substance (principle of
method)
Characterization is achieved by (a) visual
morphology, (b) flagella antigen serotyping,
(c) biochemical profile, (d) Genomotyping for
identification of a.s. at strain level
Impurities in technical active substance
(principle of method)
For microbial contaminants the enumeration
of colony forming units on selective or
appropriate media is used
Analytical methods for residues
Soil (principle of method and LOQ) For viable residues enumeration of colony
forming units on selective or appropriate
media is used, coupled to characterization
(see above) and genomotyping for
identification of a.s. at strain level
Air (principle of method and LOQ) Not applicable
Water (principle of method and LOQ) For viable residues enumeration of colony
forming units on selective or appropriate
Bs2362 Product-type 18 July 2014
30
media is used, coupled to characterization
(see above) and genomotyping for
identification of a.s. at strain level
Body fluids and tissues (principle of
method and LOQ)
For viable residues enumeration of colony
forming units on selective or appropriate
media is used, coupled to characterization
(see above) and genomotyping for
identification of a.s. at strain level
Food/feed of plant origin (principle of
method and LOQ for methods for
monitoring purposes)
Not applicable
Food/feed of animal origin (principle of
method and LOQ for methods for
monitoring purposes)
Not applicable
Chapter 3: Impact on Human Health
Basic information
(Annex IIIA,
Section 5.1)
No adverse reactions in individuals as a result of
contact with Bs 2362 during its development,
manufacture, preparation or field application have
been documented or reported. Quality control of the
Sensitisation: A maximization test in the guinea pig using either
(Annex IIIA, Section 5.2.1) Bs 2362 suspension did not induce signs of skin
sensitization.
Acute oral toxicity, pathogenicity
and infectivity:
(Annex IIIA, Section 5.2.2.1,
Annex IIIB, Section 7.1.1)
A single oral administration of Bs 2362 to rats at 5000
mg/kg bw resulted in no deaths or adverse clinical
signs. The test compound was found to be neither
toxic nor pathogenic to rats.
The oral LD50 of ‘VectoLex’ WG ABG-6491, containing the active substance Bs 2362, was determined to be
Acute inhalation toxicity,
pathogenicity and infectivity:
(Annex IIIA, Section 5.2.2.2,
Annex IIIB, Section 7.1.2)
A single, four-hour inhalation exposure (nose-only) study in rats to the maximum practical concentration of the test material of 0.09 mg/L (equivalent to 3.6 x
106 spores/L) resulted in no deaths during exposure or during the 14 day post exposure period. There were no clinical signs of reaction to treatment.
A single intranasal instillation of Bs 2362 to young rats
at a dose level of approximately 1.4 x 1010 CFU of the technical material was not associated with clinical signs of reaction to treatment or adverse effects on bodyweight.
The acute inhalation (LC50) of ‘VectoLex’ WDG (ABG-6491) was greater than the maximum achievable
f
Bs2362 Product-type 18 July 2014
31
Intraperitoneal/subcutaneous
single dose: (Annex IIIA,
Section 5.2.2.3, Annex IIIB,
Section 7.1.3)
A single intravenous injection of Bs 2362s to young
rats at a dose level of approximately 107 CFU of the technical material was not associated with clinical signs of reaction to treatment or adverse effects on bodyweight.
Injection of spores i n m i c e at high levels (≥108
CFU) was associated with rapid mortality.
Clearance of spores from the spleen was exponential
and was almost complete after 67 days. The spore-free
toxin was not lethal. Injection to immuno-deficient
mice (i.e athymic) was also not associated with any
discernible ill-effects, and no change in clearance rates.
l ll
In vitro genotoxicity:
(Annex IIIA, Section
5.2.3)
The need for genotoxicity studies has been assessed,
and it has been concluded that studies are not
required. Standard mutagenicity and genotoxicity
assays are not considered appropriate for many living
micro-organisms nor does the risk they pose often
Cell culture study:
(Annex IIIA, Section 5.2.4)
Cell culture studies are required for viruses and viroids
or specific bacteria and protozoa with intracellular
replication. This is not applicable to Bacillus sphaericus
which does not replicate in warm- blooded organisms.
Information on short-term
toxicity and pathogenicity: (Annex IIIA, Section 5.2.5)
The acute inhalation (LC50) was greater than the
maximum practical atmosphere of 0.09 mg/L
(equivalent to 3.6 x 106 spores/L) and the acute oral
(LD50) is greater than 5000 mg/kg (equivalent to 108
Dermal toxicity: An acute dermal toxicity study showed that the 14 day
median lethal dermal dose level (LD50 ) of Bacillus
sphaericus was greater than 2000 mg/kg bw for male and female rabbits. Bacillus sphaericus was also tested in a four-hour dermal irritation test in the rabbit. Slight signs of transient irritation were recorded in some animals. Bacillus sphaericus is not irritating to the skin in a standard dermal irritation test.
The dermal LD50 of ‘VectoLex’ WG containing the active
substance Bacillus sphaericus was determined to be /
Specific-toxicity, pathogenicity
and infectivity: (Annex IIIA, Section 5.3)
Bs 2362 was tested in a guideline eye irritation study
in the rabbit. There was slight transient irritation, but
all signs had reverted to zero by Day 10. Bs 2362 is
mild irritating to the eye.
‘VectoLex’ WDG is considered a mild ocular irritant.
Transient slight conjunctival irritation was apparent in
Genotoxicity – in vivo studies
in germ cells: (Annex IIIA,
Section 5.4)
In vivo testing of Bs 2362 is not indicated.
Bs2362 Product-type 18 July 2014
32
Chapter 4: Fate and Behaviour in the Environment
Spread, mobility, multiplication and
persistence in air, soil and water (Annex IIIA, Section 7.1 and 7.2)
No data has been provided about the world
distribution of Bs in the soil environment.
The only data presented take into account
the background levels of mixed populations
of Bacillus thuringiensis and Bs. A ratio of
about 1:1 is reported on the amount of Bt
and Bs, showing mosquitocidal activity, in
mosquito breeding sites of Florida. Therefore
it could be assumed, even if only tentatively,
that the population of Bs in soil is 2x102 –
4.9x104 CFU/g soil as evaluated for Bt.
Bs 2362 spore and toxin activity is affected
by exposure to UV light.
Following 1 application:
EEDSW,time=0= 2.8x105 CFU/L
PECSW,time=0= 2.4x10 ITU/L
EEDSed,time=0= 1.4x104 CFU/g
PECSed,time=0= 1.2 ITU/g
Following 5 applications with intervals of 7
days:
EEDSW,time=0= 1.0x106 CFU/L
PECSW,time=0= 1.2x102ITU/L
EEDSed,time=0= 5.0x104CFU/g
PECSed,time=0= 6.0 ITU/g
Airborne concentrations of Bs 2362 are
expected to be negligible following application to water bodies and sewage.
Chapter 5: Effects on Non-target Species
Bs2362 Product-type 18 July 2014
33
TERRESTRIAL COMPARTMENT
Test
organism
Test
substance Duration Effective Concentration
Effects on birds
AQUATIC COMPARTMENT
Test organism Test
substance Duration Effective concentration
Effects on fish
Onchorhynchus
mykiss
ABG-6184
Technical
(4x1010
CFU/g)
96-h
EC50> 15.5 mg/L (>6.2x108 CFU/L)
NOEC: 15.5 mg/L (6.2x108 CFU/L)
(Surprenant, 1986a)
Lepomis
macrochirus
ABG-6184
Technical
(4x1010
CFU/g)
96-h
LC50> 15.5 mg/L
NOEC: 15.5 mg/L (6.2x108 CFU/L)
(Surprenant, 1986b)
Cyprinodon
variegatus
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
LC50> 100 mg/L (7.9x109 CFU/L; 2.8x105
ITU/L)
NOEC: 22 mg/L (1.7x109 CFU/L; 6.3x104
ITU/L) (Bowman, 1989)
Effects on freshwater invertebrates
Daphnia magna ABG-6184
Technical
(4x1010
CFU/g)
48-h
EC50>15.5 mg/L (>6.2x108 CFU/L)
(Suprenant, 1986c)
NOEC: 15.5 mg/L (6.2x108 CFU/L)
Crassostrea
virginica
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
EC50>42 mg/L (> 2.5x109 CFU/L;>1.2x105
ITU/L)
NOEC: 15 mg/L (8.9x108 CFU/L; 4.3x104
ITU/L)
(Dionne, 1990)
Mysidopsis
bahia
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
96-h
LC50: 71 mg/L (5.6x109 CFU/L; 2.02x105
ITU/L)
NOEC: 50 mg/L (3.9x109 CFU/L; 1.4x105
ITU/L) (Forbis, 1990)
Effects on algal growth
Selenastrum
capricornutum
ABG-6184
Technical
(7.9x1010
CFU/g; 2844
ITU/mg)
120-h
EC50> 2.2 mg/L (>1.73x108 CFU/L;
>6.3x103 ITU/L)
NOEC: 2.2 mg/L (1.73x108 CFU/L; 6.3x103
ITU/L) (Forbis, 1989)
Effects on aquatic plants
No data
presented
Bs2362 Product-type 18 July 2014
34
Mallard
duck
ABG-
6184
Technical
(4x1010
CFU/g)
1
treatment;
mortality
checked
up to 30
days
NOEC: 9000 mg/kg bw (3.6x1011 CFU/kg bw)*
(Henck, 1986a)
Mallard
duck
ABG-
6184
Technical
(7.9x1010
CFU/g;
2844
ITU/mg)
29 day-
dietary
exposure
No pathogenicity nor mortality observed after 30
days of a diet containing a maximum dosage of 3.7
g MPCA/bird/day (2.9x1011 CFU/bird/day; 1.1x107
ITU/bird/day) (Grimes & Jaber, 1989)
Effects on earthworms
Eisenia
fetida
VectoLex
Technical
Powder
(50
ITU/mg)
30-d NOEC: 1000 ppm (5x104 ITU/kg dry weight soil)
(Rodgers, 2006)
Effects on honey bee
Honeybee
adults
Bs
unknown
strain
1.4x1012
CFU/g
15 and 28
days
NOEC: 108 CFU/mL ** (Vandenberg, 1986)
Effects on soil non-target micro-organisms
No data
presented
Effects on arthropods other than bees
No test
carried out
Effects on terrestrial plants
No test
carried out
* The reliability of the paper is questionable since only one treatment was done by oral gavage; the study is not conducted under any shared international guideline ** The study does not match any guideline and was not conducted under GLP conditions. The strain is not
specified.
Chapter 6: Other End Points
None
Bs2362 Product-type 18 July 2014
35
Appendix II: List of Intended Uses
1. Use and product type
‘VectoLex’ WG is a biological larvicide used as an insecticide (Product Type 18) for Pest
Control (Main Group 3).
2. Details of intended use
‘VectoLex’ WG is used for the control of mosquitoes larvae (principally Culex and
Anopheles species while it is not effective enough on Aedes subgenus Stegomyia species)
in a range of aquatic breeding habitats, such as stagnant and standing ponds, flood and
irrigation water, ditches, storm water retention areas, tidal water and salt marshes,
sewerage settling ponds and water with moderate to high organic content. The product is
not for use in rice fields during the last month before harvest.
3. Application rate
‘VectoLex’ WG is applied at a rate of 0.5 to 1.5 kg/ha.
4. Method of application
‘VectoLex’ WG is applied as a spray by terrestrial or boat spray, after dispersion of the
granules in a quantity of water suitable for the equipment used. Aerial application should
be left up to each Member State dependent on the critical habitats to be treated
5. Number and timing of applications
A maximum of 5 treatments per season per site with 7 day interval.