Bacillus sphaericus 2362 - Serotype H5a5b - Strain ABTS1743dissemination.echa.europa.eu/Biocides/ActiveSubstances/1244-18/1244-18... · 2362, Serotype H5a5b, Strain ABTS1743 as product-type

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

Bs2362 Product-type 18 July 2014

1

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


2

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


3

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.


4

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


5

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.


6

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


7

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


8

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.


9

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.


10

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


11

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


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.


13

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.


14

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.


15

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


16

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).


17

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)


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


18

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.


19

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)


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.


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


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)


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]


23


Following the step-2 approach, in case of one application:

EEDlocal, influent = 2.8x105 [CFU/L]

EEDlocal, sw = 2.8x104 [CFU/L]


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.


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


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.


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.


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


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


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


Air (principle of method and LOQ) Not applicable

Water (principle of method and LOQ) For viable residues enumeration of colony



30




Body fluids and tissues (principle of

method and LOQ)

For viable residues enumeration of colony





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


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.


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


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)


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


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)


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


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.