bacillus amyloliquefaciens strain...

Regulation (EU) No 528/2012 concerning the making available on the market and

use of biocidal products

Evaluation of active substances

Assessment Report

Bacillus amyloliquefaciens strain ISB06

Product-type 03

(Veterinary hygiene)

October 2015

Germany

Bacillus amyloliquefaciens

strain ISB06

Product-type 03 December 2015

2

CONTENTS

1. STATEMENT OF SUBJECT MATTER AND PURPOSE ............................................... 3

1.1. Procedure followed ................................................................................................................................ 3

1.2. Purpose of the assessment report .................................................................................................. 3

2. OVERALL SUMMARY AND CONCLUSIONS ................................................................... 5

2.1. Presentation of the Active Substance ........................................................................................... 5 2.1.1. Identity, Biological Properties & Methods of Analysis ................................................................... 5

2.1.1.1. Identification of the active substance ......................................................................................... 5 2.1.1.2. Identification of the Biocidal Product .......................................................................................... 5 2.1.1.3. Biological Properties........................................................................................................................... 5 2.1.1.4. Methods of Analysis ........................................................................................................................... 7

2.1.2. Intended Uses and Efficacy ..................................................................................................................... 7 2.1.2.1. Details of uses ...................................................................................................................................... 7 2.1.2.2. Efficacy .................................................................................................................................................... 8

2.1.3. Classification and Labelling ..................................................................................................................... 8

2.2. Summary of the Risk Assessment ................................................................................................... 9 2.2.1. Human Health Risk Assessment............................................................................................................ 9

2.2.1.1. Effects assessment ............................................................................................................................. 9 2.2.1.2. Exposure assessment ...................................................................................................................... 11 2.2.1.3. Risk characterisation ....................................................................................................................... 12

2.2.2. Environmental Risk Assessment ......................................................................................................... 13 2.2.3. Fate and distribution in the environment ........................................................................................ 13

2.2.3.1. Persistence and Multiplication ...................................................................................................... 13 2.2.3.2. Mobility ................................................................................................................................................. 14 2.2.3.3. Conclusion ........................................................................................................................................... 15

2.2.4. Effects assessment ................................................................................................................................... 15 2.2.4.1. Effects on non-target Organisms ................................................................................................ 15 2.2.4.2. Conclusion ........................................................................................................................................... 19

2.2.5. PBT and POP assessment ...................................................................................................................... 19 2.2.6. Exposure assessment .............................................................................................................................. 19

2.2.6.1. Conclusion ........................................................................................................................................... 20 2.2.7. Risk characterisation ............................................................................................................................... 20 2.2.8. Assessment of endocrine disruptor properties .............................................................................. 21

2.3. Overall conclusions ...............................................................................................................................22

2.4. List of endpoints .....................................................................................................................................22

2.5. Requirement for further information related to reference product ..............................22

APPENDIX I: LIST OF ENDPOINTS ..................................................................................... 23

APPENDIX II : LIST OF INTENDED USES ....................................................................... 32

APPENDIX III: LIST OF STUDIES ........................................................................................ 33


strain ISB06


1. STATEMENT OF SUBJECT MATTER AND PURPOSE

1.1. Procedure followed

This assessment report has been established as a result of the evaluation of Bacillus

amyloliquefaciens strain ISB06 as product-type 3 (veterinary hygiene biocidal product),

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 528/2013, with a view to the

possible approval of this substance.

Bacillus subtilis was notified as an existing active substance, by COBIOTEX SNE,

hereafter referred to as the applicant, in product-type 3.

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, Germany 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 subtilis as an active substance in Product Type 3 was 31st July 2007, in

accordance with Annex V of Regulation (EC) No 1451/2007.

On 30th July 2007, the German competent authorities received a dossier from the

applicant. In November 2008 the identity of the micro-organism has been revised. Based

on the currently available information the micro-organism is Bacillus amyloliquefaciens

ISB06. The Rapporteur Member State accepted the dossier as complete for the purpose

of the evaluation on 17th March 2009.

On 23rd September 2014, 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 amyloliquefaciens strain ISB06 for

product-type 3, 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.

For the implementation of the common principles of Annex VI, the content and

conclusions of this report, which is available from the Agency web-site shall be taken into

account.

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


strain ISB06


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However, where conclusions of this 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.

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.


strain ISB06


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2. OVERALL SUMMARY AND CONCLUSIONS

2.1. Presentation of the Active Substance

2.1.1. Identity, Biological Properties & Methods of Analysis

2.1.1.1. Identification of the active substance

Bacillus amyloliquefaciens strain ISB06 is the biologically active ingredient of the product

Cobiotex 112 Biofilm+.

It has been isolated from an agricultural environment and is a wild-type, hence it has not

been modified genetically or in any other way.

Cells are gram-positive, mobile medium rods with rounded edges and subterminal

spores.

The species of ISB06 has been identified by means of physiological and molecular

methodologies, particularly with establishment of an API50 profile and DNA sequencing.

As a result, ISB06 has been identified as a strain of the species Bacillus

amyloliquefaciens. Furthermore it could be excluded that it belongs to closely related

species, e.g. Bacillus subtilis, as well as to facultative and obligate pathogens of the

genus Bacillus.

Independent production batches have been analysed for absence of toxins and

contaminants with pathogenic potential, i.e. Salmonella, Staphylococcus aureus,

coliforms, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolytiocus, Shigella,

Listeria monocytogenes, anaerobic spore-forming microorganisms, and moulds. All

batches were negative or within specified limits concerning the investigated

contaminants.

2.1.1.2. Identification of the Biocidal Product

COBIOTEX 112 Biofilm + is a white, odourless, suspensible powder. Its pH after aqueous

dilution is 7.89 ± 0.3. The results for particle size distribution are as follows: the

diameter d(0.1) is 16.902 µm, d(0.5) is 67.012 µm, diameter d(0.9) is 152.540 µm.

The composition of the biocidal product is confidential and contained in the confidential

section of Doc III B 1.4. The amount of active substance in the biocidal product is 0.05

% w/w. The minimal concentration of viable spores reported for the biocidal product is

105 cfu/g. The spore titres 3.08 + 2.13 x 1011 cfu/g (3 production batches) and 1.76 +

0.23 x 108 cfu/g (1 production batch) have been reported for batches of the technical

microbial active substance. Based on i) the minimal titre 105 cfu/g for the biocidal

product, ii) the investigated active substance titres, and iii) the weight percentage of

active substance in the biocidal product, a spore concentration of 106 cfu/g has been

approximated for the biocidal product. Maximal spore concentrations are 108 cfu/g BP, as

inferred from spore titres of the active substance.

For product registration, spore titres of biocidal product production batches are required.

2.1.1.3. Biological Properties

Bacillus amyloliquefaciens is an ubiquituous soil bacterium and has been described in

1943 (Fukomoto).


strain ISB06


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A close affinity of B. amyloliquefaciens with B. subtilis has long been recognized, and the

organism has been given subspecies status as “B. subtilis ssp. amyloliquefaciens” or has

been included in B. subtilis as a variant. B. amyloliquefaciens is closely related to B.

subtilis and the other species which compose of the B. subtilis groupcomplex: B.

licheniformis, and B. pumilus, B. vallismortis, B. tequilensis, B. mojavensis, B.

athrophaeus, B. sonorensis. These species share many physiological properties (Priest et

al., 1987; Rooney et al., 2009).

B. amyloliquefaciens has a long and safe history in the production of alpha-amylase for

starch liquefaction and detergents. The species is regarded as non-pathogenic and

granted QPS status by EFSA.

‘QPS approach can be taken to establish safety. It proposed that a safety assessment of

a defined taxonomic group (e.g. genus or group of related species) could be made based

on four pillars (establishing identity, body of knowledge, possible pathogenicity and end

use). If the taxonomic group did not raise safety concerns or, if safety concerns existed,

but could be defined and excluded (the qualification) the grouping could be granted QPS

status.’

Individual strains of B. amyloliquefaciens are employed as plant growth promoters due to

specific interaction with plant roots and due to antagonizing effects against pathogens.

Two such strains commercially employed in crop cultivation are B. amyloliquefaciens

FZB24 and FZB42. These strains are similar to ISB06 yet distinguishable from it due to

physiological and molecular differences. A close relationship between FZB42 and FZB24

has been proven by means of molecular methodologies (RiboPrinter analysis). In 2003,

the U.S. EPA has published an assessment of FZB24 considering this strain as Bacillus

subtilis var. amyloliquefaciens. Some of the experiences gained with FZB24 and FZB42

are considered in the following sections in order to extrapolate results to ISB06 where

data from the latter has not been available.

Another recently evaluated related Bacillus, B. amyloliquefaciens subsp. plantarum strain

D747 should be also mentioned in this context (SANCO/11391/2014 rev 1). B.

amyloliquefaciens subsp. plantarum strain D747 controls fungal plant pathogens by a

combination of different modes of action.

B. amyloliquefaciens FZB42 stimulates plant growth and produces secondary metabolites

that suppress soil-borne phytopathogens. The biocidal activity of B. amyloliquefaciens

ISB06 may rely on analogous principles. ISB06 antagonizes bacteria including potential

livestock pathogens via growth inhibition. Inhibition by ISB06 affects species of the

genera Enterococcus, Listeria, Staphylococcus, Escherichia, Pasteurella, Salmonella and

Yersinia and potentially others. Pseudomonas and Acinetobacter species have also been

tested but have not been impaired in growth. Hence, B. amyloliquefaciens ISB06 displays

specific rather than broad biocidal activity against microorganisms. In dedicated assays it

could be shown that ISB06 has no inhibitive or otherwise adverse effects on plants,

animals and human cell lines.

The mechanism of the biocidal activity of ISB06 is not fully clarified to date. The biocidal

effect may be dependent on several factors including competition with the target

microorganisms by nutritive competition and by competitive exclusion. Competitive

exclusion may be triggered by the synthesis of antibiotic compounds. The Bacillus-

subtilis-group including B. amyloliquefaciens is remarkably known for the production of a

wide and diverse range of antibiotics with antiviral, bactericidal or antifungal properties

or combinations of these properties. Lipopeptides like surfactin produced by B. subtilis

act by disruption of the plasma membrane via formation of small vesicles and

aggregation of intramembranous particles in microorganisms, e.g. yeast cells. Another

group of antibiotic compounds produced by Bacillus species are lantibiotics, i.e. peptide


strain ISB06


7

antibiotics which act on target cells by formation of membrane pores. Consequently,

cytoplasmic components are released from the target microorganism resulting in cell

death. Also in the case of B. amyloliquefaciens ISB06 there is evidence on the synthesis

of antibacterial compounds (Hyronimus et al., 2006 IIIA 2.2.1.).

Genetic stability of ISB06 has been demonstrated by analysis of physiological markers

and by PFGE across independent production batches. ISB06 is resistant to ampicillin

which is typical for strains of Bacillus amyloliquefaciens.

Spore preparations of ISB06 can be inactivated with heat (98 °C on wet material) and UV

radiation on wet material. Also chemical treatment with potassium peroxymonosulfate

(CAS 10361-76-9) based sanitizers is effective at 75 °C. Dry spores are resistant to heat

and UV radiation. Other sanitizers have been tested and shown to be ineffective in spore

inactivation at room temperature and at increased temperatures.

Bacillus amyloliquefaciens ISB06 is distantly related to the toxin-producing food-spoilage

bacterium Bacillus cereus as well as to the pathogen Bacillus anthracis, the causative

agent of anthrax. During identity investigation ISB06 could be firmly distinguished from

these Bacillus species on basis of physiological and molecular traits (2.1.1).

2.1.1.4. Methods of Analysis

Spore titres in the biocidal product are assayed according to ISO protocols. Purity from

Staphylococcus, coliforms, Salmonella and yeast is assessed according to ISO and NF

(norme française) protocols.

Further analyses are routinely performed during production and contained in confidential

sections.

2.1.2. Intended Uses and Efficacy

The biocidal product is designed to control potentially harmful bacteria in livestock

buildings and equipment of animal rearing facilities, e.g. for poultry and pig. The product

is intended to complement but not to substitute chemical disinfection measures as a

prophylactic treatment. The biocidal product is applied by spraying on abiotic surfaces.

2.1.2.1. Details of uses

The active substance is intended to be used by professionals only in control and

repression of potentially harmful bacteria in livestock buildings and on breeding

equipment under Product Type 3. The biocidal product is applied by spraying on abiotic

surfaces 24 to 48 hours after steps of cleaning-disinfection in order to avoid remanent

effects of disinfectants.

One kilogram of COBIOTEX 112 biofilm + product corresponding to about 109 cfu (diluted

in 100 liters of cold water) is sprayed for a livestock building of 1000 m2. The product

should be rehydrated from 30 minutes to 1 hour before application. A double application

is recommended. The spores adhere on abiotic surfaces of livestock buildings. In case of

favourable environmental conditions (e.g. temperature, humidity, nutrient, oxygen),

activation of “barrier flora” occurs via spore germination. If favourable conditions are not

met, the spores do not germinate and remain in a state of “dormancy”.


strain ISB06


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The product is applied in order to colonise the disinfected surfaces and to form a so-

called “positive biofilm” which leads and/or reduces the potential colonisation of the

abiotic surface by other commensal micro-organisms (in particular pathogen strains) and

thus reduces the microbial pressure in the local environment. Therefore, the product has

a prophylactic action but not a disinfecting one.

2.1.2.2. Efficacy

Target organisms of B. amyloliquefaciens ISB06 are bacteria including potential

pathogens. In test assays, ISB06 impaired growth of bacteria including the genera

Enterococcus, Listeria, Staphylococcus, Escherichia, Pasteurella, Salmonella and Yersinia,

while it had no effect on Pseudomonas and Acinetobacter.

The biocidal activity of ISB06 has been investigated by studies performed with both the

active substance and the biocidal product Cobiotex 112, the latter containing

approximately 106 cfu/g of the active substance.

The studies revealed reliable results for basic efficacy assessment. The following results

could be derived from the studies:

Absence of target organism growth caused by ISB06 at a concentration of 5x105 cfu/ml

(which equals approximately 5x107 cfu/m2) could be observed after 7 days of incubation.

In comparison, untreated controls of target organisms built up biofilms after 7 days of

incubation.

Absence of target organism growth caused by the biocidal product Cobiotex 112 at a

concentration of 0.05 g/ml corresponding to 5x104 cfu/ml of ISB06 (which equals

approximately 5x106 cfu/m2) could be observed after 7 days of incubation.

The claimed application rate is 0.01g of the product/ml corresponding to about 104

cfu/ml of the active substance ISB06. The proposed application rate to cover 1m2 is 1g of

the product (containing 106 cfu of the active substance) dissolved in 0.1 l water.

Compared to the claimed application rate the assays only provided support for the

efficacy at higher application rates. Data supporting efficacy at the claimed application

rate were not provided. However the data were accepted at the approval stage of the

active substance since it can be concluded from the performed risk assessment that even

much higher application rates of the product than the proposed rates will neither have

any adverse effects on human health nor on the environment.

The information provided is only sufficient to show a basic efficacy of ISB06. This is

accepted in the context of substance approval. For product authorisation, essentially

more information has to be provided: To support the claim “control of microorganisms”

further laboratory tests would be necessary. Additionally, further tests in the envisaged

area of use have to be provided.

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

No Classification and Labelling is necessary with regard to Regulation (EC) No

1272/2008. As in general a sensitising potential of micro-organisms is assumed, it is

appropriate to add the following labelling: “Micro-organisms may have the potential to

provoke sensitising reactions.”


strain ISB06


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It is further appropriate to specify on the product package and/or on specification and

usage leaflet(s) that the microbiological ingredients Bacillus amyloliquefaciens and lactic

acid bacteria comply with the QPS status provided by EFSA.

It should be noted that classification of microorganisms into risk groups may be subject

to national regulations. These national regulations may differ with regard to

microorganism range and classification from directive 2000/54/EC and should be taken

into consideration prior to international marketing of the active substance and the

biocidal product. Examples for national regulations are German and Swiss guidelines on

classification of microorganisms into risk groups (TRBA 466).

2.2. Summary of the Risk Assessment

2.2.1. Human Health Risk Assessment

2.2.1.1. Effects assessment

Absorption, Distribution, Excretion, and Metabolism

No studies available, no data requirement for micro-organisms.

Acute Toxicity

No acute oral study was carried out with B. amyloliquefaciens ISB06. An acute oral

toxicity study in rats with B. amyloliquefaciens FZB24 revealed that administration of

2.3x1010 cfu/kg bw (4.2x109 cfu/animal in males and 3.8x109 cfu/animal in females)

were neither toxic nor pathogenic. The LD50 is >2.3x1010 cfu/kg bw. No symptoms of

toxicity or pathogenicity were reported within the observation period of 14 days.

Clearance was not examined.

An acute intratracheal study in rats with strain ISB06 revealed no toxic or pathogenic

effects up to 22 days after administration. No mortalities were observed, the LD50 was

>1.0x109 cfu/kg bw (mortalities: 0/28). In some treated animals, low levels of the test

organism were found in heart (6/28), liver (10/28), kidneys (9/28), spleen (15/28),

mesenteric lymph nodes (9/28) and brain (7/28) but not in blood. Clearance from the

lungs was completed in 5/10 rats within 22 days while one female rat showed persistence

of high levels of B. amyloliquefaciens ISB06 in the lung (2.42x106 cfu/g lung tissue) with

ongoing clearance evident from the test organism present in caecum and faeces.

However, this was not associated with any sign of toxicity, pathogenicity or infection.

Intraperitoneal administration of a spore suspension of B. amyloliquefaciens ISB06

revealed no toxic or pathogenic effects up to 22 days after administration. The LD50 was

>1.0x109 cfu/kg bw (mortalities: 0/6). In none of the treated animals, strain ISB06 could

be detected in blood or brain. In most or all animals, low to moderate levels (1.2x101-

1.6x104 cfu/g tissue) were found in kidneys (6/6), spleen (6/6), liver (6/6), heart (4/6),

lungs (5/6) and mesenteric lymph nodes (6/6) at the end of the 22d observation period.

Short-term Toxicity

No short-term toxicity study with B. amyloliquefaciens ISB06 was submitted. In the

absence of toxic or pathogenic effects in acute studies no short-term studies are

necessary.


strain ISB06


10

Genotoxicity

No genotoxicity tests were submitted. It is not assumed that B. amyloliquefaciens ISB06

comprises a genotoxic potential since no indication for toxin production was found. (Data

requirement for exotoxin producing micro-organisms and viruses only)

Chronic Toxicity/ Carcinogenicity


Reproduction Toxicity


Neurotoxicity


Further Studies

Cytotoxicity testing of supernatants and cells of B. amyloliquefaciens strain ISB06 in

epithelial HeLa cells via trypan blue staining did not reveal cytotoxicity. Viability of cells

treated with ISB06 supernatant or cells were 97.8 % and 95 %, respectively. This was

comparable to the viability of HeLa cells treated with the negative control B. subtilis 168

culture supernatant (97.5 %) or cells (93.5 %). The positive control B. thuringiensis 407

exhibited a marked cytotoxicity (0 % viability). HeLa cells are considered a valid cell line

to identify emetic as well as diarrhoeic toxins of the Bacillus spp (SCAN, 2000; Ramarao

& Lereclus, 2006).

Medical Data

No medical data on B. amyloliquefaciens ISB06 or other strains of B. amyloliquefaciens

are available.

The US EPA (1997) which is still considering B. amyloliquefaciens as a subspecies of

Bacillus subtilis, concluded that Bacillus subtilis is not a frank human pathogen, nor is it

toxigenic like a few other members of the genus Bacillus.

No medical surveillance data for personnel involved in the production or processing of

B. amyloliquefaciens ISB06 are available.

Skin and respiratory sensitisation against B. subtilis and B. amyloliquefaciens enzymes

like proteases and amylases are frequently observed in workers in the enzyme industry.

Exposure to B. amyloliquefaciens ISB06 enzymes cannot be completely ruled out during

manufacturing and use and subsequent sensitation might occur.

Therefore, Bacillus amyloliquefaciens ISB06 has to be labelled regarding skin and

respiratory sensitisation.

No direct observations regarding pathogenicity of B. amyloliquefaciens strain ISB06 are

reported. B. amyloliquefaciens and close relatives, e.g. Bacillus subtilis, are regarded as

non-pathogenic micro-organisms and granted QPS status by the EFSA. Bacillus

amyloliquefaciens ISB06 does not possess genes encoding Bacillus enterotoxins or the

key gene implicated in the synthesis of emetic toxins, or otherwise not demonstrates

phenotypic characteristics of toxin production. However, some published reports are

available that suggest a limited opportunistic pathogenicity of this group. Since B.

amyloliquefaciens and B. subtilis were considered to be taxonomically identical at species

level until 1987, reports on pathogenicity of B. subtilis published before 1987 were taken

into consideration for the evaluation of B. amyloliquefaciens.


strain ISB06


11

2.2.1.2. Exposure assessment

Exposure of Professionals

The biocidal product COBIOTEX 112 BIOFILM is used for treatment of livestock buildings

and breeding equipment. The biocidal product acts as “barrier flora” or “positive biofilm”.

It is applied by spraying or misting. Prior to application the powdery biocidal product is

dissolved in water. The use concentration is 0.05 % active substance (approximately 106

cfu of Bacillus amyloliquefaciens/g b.p.).

The following intended uses are assessed by the rapporteur:

Spraying application in livestock buildings (scenario 1)

Misting application in livestock buildings (scenario 2)

Exposure assessment for different scenarios is based on approximate estimates and

reported here as colony-forming units (cfu) of B. amyloliquefaciens ISB06.

Before use of the biocidal product in scenario 1, the powdery biocidal product, COBIOTEX

112 BIOFILM + (0.05% a.s.), is dissolved in water to 0.0005% a.s. solution. During the

loading of the powder inhalation exposure to the dust of the biocidal product may occur.

The aqueous solution of the b.p. (containing 104 cfu of a.s./g b.p) is sprayed in livestock

buildings and inhalation exposure due to the released aerosols may occur during this

application phase. Inhalation exposure during post-application phase is not expected. The

resulting level of inhalation exposure to the microorganism is 1500 cfu for mixing &

loading phase and 1040 cfu/m³ for application phase.

Dermal exposure may occur at all stages of application. The total potential dermal

exposure is 297800 cfu/person/day.

For the scenario 2 also the powdery biocidal product, COBIOTEX 112 BIOFILM +, is

dissolved in water to 0.0005% a.s. solution. During the loading of the powder inhalation

exposure to dust of the biocidal product may occur. During the application phase the

treatment of livestock buildings and the equipment of breeding by misting is performed

using a cold fogging system. Inhalation exposure due to the formation of aerosols is

assessed for the application phase. Exposure during post-application phase is not

expected. The resulting level of inhalation exposure to the microorganism is 1500 cfu for

mixing & loading phase and 702 cfu/m³ for application phase.

Dermal exposure is expected during all phases of application. The total potential dermal

exposure is 89720 cfu/person/day.

In addition all microorganisms are considered as potential sensitizers. Therefore, PPE is

taken into account on a qualitative basis. The detailed exposure assessment is described

in Doc II B 9.3.

Exposure of Non-Professionals and the general public

According to Doc IIIB-7.3 the product is used solely by professional operators in

husbandry. Therefore exposure of a non-professional user is excluded. Exposure of the

general public is not expected since animal production facilities do not represent sites of

general public admittance.

Secondary Exposure of Livestock

Application, mixing and loading should be performed in absence of livestock.

After spray application an exposure of livestock to the biofilm might be expected in

animal housing facilities. However, depending on local micro-environmental conditions in

animal housing inoculation and biofilm formation after application of the product is

expected to vary considerably. Therefore, an exposure assessment is not possible.


strain ISB06


12

Furthermore, B. amyloliquefaciens is included in risk group I (i.e. least concern) of the

German and Swiss technical guideline on biological substances (TRBA 466), which

means, that this microorganism is unlikely to cause human, plant or animal disease. This

was confirmed by acute studies and cytotoxicity tests performed with B.

amyloliquefaciens strain ISB06 and FZB24 in which no signs of pathogenicity or toxicity

were observed. Taking also into account the high background exposure levels to

biological agents (e.g. moulds, bacteria etc.) in animal housing most often exceeding 106

cfu per m³ air an exposure assessment towards B. amyloliquefaciens strain ISB06 for

livestock is not necessary.

2.2.1.3. Risk characterisation

Risk Assessment for Professionals

In the “standard procedure” potential exposure and default assumptions on dermal and

inhalative absorption of an active substance are used for estimates of total internal body

burden. The resulting risk characterisation performed with the AEL approach compares

the total internal body burden with the derived reference dose.

In contrast to the above cited procedure, in this report a microbiological organism (B.

amyloliquefaciens strain ISB06) is assessed for which on one hand the described method

for risk characterisation is not applicable and on the other hand any sign of pathogenicity

or infection in the acute studies is missing. Moreover, the production of metabolites

(especially toxins) has been analysed and does not give rise concern to any cytotoxic

potential. Generally, metabolites include all intermediates and products of metabolism

and might have toxic effects. However, not all toxins originate from metabolic processes

e.g., the genetic information for toxins can also be encoded by plasmids.

Considering the absence of pathogenicity and toxicity in the studies a risk

characterisation has been regarded as unnecessary.

Based on the reasons outlined above the spraying application in livestock buildings

(scenario 1) and misting application in livestock buildings (scenario 2) is considered to be

of no concern. Relevant health risks to B. amyloliquefaciens are not expected to occur

under the specified conditions. The approval of B. amyloliquefaciens can be supported. It

is essential to indicate that this conclusion only applies to the active substance in the

biocidal product (and not to other ingredients).

Safety Measures for Professionals

For work with risk group 1 biological agents (as defined in the German technical technical

rule, TRBA 466) the principles of good occupational safety and hygiene should be

observed. Due to potential sensitisation for professional users, safe operational

procedures and appropriate organisational measures shall be established. Where

exposure cannot be reduced to an acceptable level by other means, products shall be

used with appropriate personal protective equipment (protective gloves, coverall

protecting from spray mist (type 4, EN 14605, respiratory protective equipment (particle

filter). In general, personal protective equipment (PPE) shall be replaced by technical

and/or organisational measures, if possible (according to the Chemical Agent Directive

98/24/EC, article 6, paragraph 2).

Risk Assessment for Non-Professionals and the general public

No health risk for non-professionals and the general public is expected from the intended

uses. Furthermore, B. amyloliquefaciens is included in risk group I (i.e. least concern) of


strain ISB06


13

the German and Swiss technical guideline on biological substances (TRBA 466), which

means, that this microorganism is unlikely to cause human, plant or animal disease.

However, all microorganisms may have the potential to provoke sensitising reactions.

Safety Measures for Non-Professionals and the general public

No special safety measures for non-professionals and general public are necessary.

2.2.2. Environmental Risk Assessment

For environmental risk assessment bridging was conducted and studies of B.

amyloliquefaciens FZB42 and B. subtilis FZB24 were extrapolated to B. amyloliquefaciens

ISB06. Read-across between the named strains has been considered applicable for the

reasons given in chapter 2.1.2.

Although the mode of action of B. amyloliquefaciens strain ISB 06 is still unclear (see Doc

IIA, section 2.4.) it might be triggered by the synthesis of antibiotics and anti-microbial

agents, such as surfactin and lantibiotics (see section 2.1.2). As the strain is a natural

occurring soil bacteria it can be assumed that the production of anti-microbial substances

is part of the survival strategy of the microorganism by improving its ability to compete

successfully for nutrients. The regulation of secondary metabolites, in turn, is generally

tightly coupled to the quantity and quality of available nutrients, determining the

microbial population size and metabolic activity. Additionally, their production takes

places only at specific phases during the growth cycle of the microorganism (Kinsella et

al., 2009). Therefore, the assessment of fate and behaviour of B. amyloliquefaciens

ISB06 in the different compartments inherently includes an assessment of possibly

produced antibiotics and anti-microbial agents. In conclusion, secondary metabolites of

B. amyloliquefaciens ISB06 are not expected to occur in the environment in

concentrations higher than under natural conditions. Particularly for the soil

compartment, no adverse environmental effects have been reported for the

taxonomically closely related Bacillus strain FZB24 which is commercially in use since

1999 (declaration of ABiTEP, 2010). Risk due to the synthesis and release of anti-

microbial compounds is therefore considered as low.

2.2.3. Fate and distribution in the environment

2.2.3.1. Persistence and Multiplication

Although bacterial spores are known to be highly resistant against various physical

impacts (wet and dry heat, UV, gamma radiation, extreme desiccation, oxidizing agents),

it was assumed that non-germinated endospores of B. amyloliquefaciens ISB06 do not

pose any environmental risk because of their inactive metabolism. The following

paragraphs therefore focus on the fate and behaviour of vegetative cells, including those

resulting from spore germination.

2.2.3.1.1. Soil

Bacillus species are the most common types of bacteria isolated from soil samples

(Hallmann et al. 1998). Natural abundances in the range of 102-105 cfu per g of soil have

been reported for Bacillus sp. and Bacillus subtilis for different locations (Cazorla et al.,

2007; Pandey & Palni, 1997; Siala, Hill and Gray, 1973), suggesting that the total


strain ISB06


14

number of these microorganisms varies depending on environmental factors, such as

oxygen supply, soil type, moisture content, plantation as well as type and amount of

applied fertilizers (van Veen et al., 1997).

Several studies on the persistence of Bacillus showed that, in general, the density of

introduced Bacilli populations declines more or less rapidly following their introduction

into a natural soil due to microbiostasis (Kilian et al., 72-93; van Veen et al., 1997). Thus

it has been shown that initially high levels of B. amyloliquefaciens FZB24 cells and spores

(1.5x103-1.1016cfu/g root mass) resulting from seed inoculation, declined to the normal

level after a maximum of 49 days (Kilian et al.2000; Presentation of ABiTEP, confidential

data, 1992).

Based on data from plant associated B. subtilis strains the US EPA (1997) concluded that

B. subtilis ssp. are generally safe for use in agriculture that is in the environment. It

should be noted that many strains authorised for agricultural uses under the taxonomic

designation of B. subtilis are now allocated to the species B. amyloliquefaciens.

Furthermore, analysis performed by ABiTEP on the commercially employed strains FZB24

and FZB42 which are closely related to ISB06 did not reveal evidence for concern

regarding persistence or multiplication of in soil, water or air.

Hence, risks associated with persistence or multiplication of ISB06 in soil can be

considered as low.

2.2.3.1.2. Water

B. amyloliquefaciens strains are used already as aquaculture probiotics and lake/pond

cleaner with no obvious adverse effects (Cao et al., 2011).

ABiTEP has not received any record or indication that FZB24 as active substance has

caused negative reactions in the environment, i.e. in the soil, water or air compartment

in connection with its use. FZB24 is closely related to ISB06 and is commercially

employed in agriculture since 1999. Furthermore, no adverse data with regard to

persistence or multiplication of commercially employed B. subtilis or B. amyloliquefaciens

strains in soil, water or air compartments (Declaration of ABiTEP, dated 9th April 2010)

are known.

Hence, risks due to persistence and multiplication of vegetative cells of ISB 06 in water

can be considered as low.

2.2.3.1.3. Air

Specific data which indicate the survivability of B. amyloliquefaciens in the atmosphere

after release have not been published. Survival of vegetative cells during aerosolisation is

typically limited due to stresses such as shear forces, desiccation, temperature, and UV

light exposure. However, its ability to survive in a broad habitat range and produce

endospores suggests that this organism may survive after release.

2.2.3.2. Mobility

Like other members of the family Bacillaceae, B. amyloliquefaciens is able to produce an

endospore that enables it to endure extreme conditions and to be dispersed in this stage.

Despite the mobility and survival of its endospores the possible spread of B.

amyloliquefaciens ISB06 in different environmental compartments is unlikely to create

any significant risk. Several studies on the persistence of B. subtilis ssp. in soil have

shown that, in general, the population sizes of introduced cultures decline more or less

rapidly following release into natural soils (Kilian et al., 72-93). When released into soil


strain ISB06


15

already containing a natural microbial flora, populations of exogenously introduced B.

subtilis strain are likely to decline to 1% or less of the population of indigenous B. subtilis

strains that the soil can support.

This is already considered by European and US authorities. Based on data from plant

associated Bacillus subtilis strains, the US EPA concluded that B. subtilis ssp. are

generally save for use in agriculture (US EPA, 1997). Noteworthy, many strains

authorised for agricultural uses under the taxonomic designation of B. subtilis are now

allocated to the species B. amyloliquefaciens. According to the European Food Safety

Authority (EFSA), B. amyloliquefaciens is given QPS status (Qualified Presumption of

Safety, EFSA, 2007) and is notified as farm animal feed supplement (Ecobiol®; EFSA,

2008). Additionally, in the recent EFSA opinion on the safety and efficacy of Bacillus

subtilis PB6 as a feed additive for laying hens and minor poultry species for laying no

evidence of toxigenic potential or of resistance to antibiotics of human and veterinary

importance was found as judged by the current guidelines. The strains of B. subtilis in

the additive are presumed safe for target animals, consumers and the environment

(EFSA, 2015).

Commercially available biocontrol rhizobacteria include B. amyloliquefaciens FZB42

(RhizoVital 42 li.; ABiTEP GmbH). No adverse environmental effects have been reported

for the strain FZB24 which is commercially in use since 1999 and closely related to ISB06

(declaration of ABiTEP, 2010).

The data document that dispersal of B. amyloliquefaciens ISB06 to different

environmental compartments is potentially possible but unlikely to be associated with

adverse accumulation in unintended sites as well as unlikely to be associated with any

significant risk.

2.2.3.3. Conclusion

Based on the data summarized above it can be concluded that ISB06 is unlikely to exert

adverse effects on the environment.

2.2.4. Effects assessment

2.2.4.1. Effects on non-target Organisms

As described in section 2.1.2, Bacillus amyloliquefaciens strain ISB06 is similar to the

commercially employed strains B. amyloliquefaciens FZB42 and B. amyloliquefaciens

FZB24. Therefore ecotoxicological studies conducted on B. amyloliquefaciens strain

FZB24 were considered in the present dossier and extrapolated to B. amyloliquefaciens

ISB06.

2.2.4.1.1. Effects on birds

The applicant provided a justification for the non-submission of a study on birds based

on the use of Bacillus amyloliquefaciens in the food and agricultural industries as well as

on the ubiquitous nature of these microorganisms.

B. amyloliquefaciens is an ubiquitous bacterium commonly recovered from water, soil,

air, and decomposing plant residue as well as from guts of animals and insects (Fossum

et al., 1986, Gatesoupe, 1999; Nicholson, 2002; Hong et al., 2005). It is not considered

pathogenic or toxic to humans, animals, or plants. Therefore the potential risk for birds

associated with the use of this bacterium is low.


strain ISB06


16

Furthermore a number of species from the Bacillus genus have been considered safe for

application in the food and agricultural industries. Compared with other bacteria that are

used for probiotic purposes, Bacillus spp. can be administered orally as cells or spores.

Bacillus spp. are being used as probiotics in animal nutrition and especially in broiler

breeding. Various studies showed that Bacillus genus and especially B. subtilis group has

no adverse effects on birds and may exert positive effects on health of the exposed

organisms (Hong et al., 2005). Although the evidence of the studies from the literature is

circumstantial, they indicate that the potential risk for birds associated with the use of

this bacterium is low.

2.2.4.1.2. Effects on aquatic organisms

For all standard trophic levels (fish, invertebrates and algae) acute toxicity tests with B.

amyloliquefaciens FZB24 were provided by the applicant.

2.2.4.1.3. Effects on fish

The test was conducted according to OECD guideline 203 with one concentration of

1x1010 cfu/L (nominal concentration). Rainbow trout (Oncorhynchus mykiss) was used as

test organism. The results of this study indicate no harmful effects of the test article

Bacillus amyloliquefaciens FZB24 on fish, neither when used as whole-cell nor as cell-free

preparations. Based on the similarity between B. amyloliquefaciens ISB06 and B.

amyloliquefaciens FZB24, the results of B. amyloliquefaciens FZB24 can be extrapolated

to B. amyloliquefaciens ISB06 and therefore no harmful effects are expected from B.

amyloliequefaciens ISB06..

2.2.4.1.4. Effects on freshwater invertebrates

A test was conducted according to OECD guideline 202 with Daphnia magna, Straus.

Based on the results of the preliminary test an EC50 (48h)-value of 4*108 cfu/L was

calculated using probit analysis whereas in the main test the daphnids were only slightly

immobilised (5 % at 1x1010 cfu/L) when using whole cells. However, immobilisation was

20 % and 30 % when using cell-free extracts equivalent to 1*1010 and 1*108 cfu/L. For

precautionary principle the results of the preliminary test were used for the further

assessment. Taking into account also the available data from literature no harmful effects

on invertebrates are expected.

2.2.4.1.5. Effects on algae growth

The test was conducted according to OECD guideline 201 with Scenedesmus subspicatus.

Inhibition on the algae growth was observed when using whole cells at a concentration

rate superior to 1.0*108 cfu/L of B. amyloliquefaciens FZB 24. However, no such

inhibition was noted when using cell-free extracts. Based on the results obtained an

EbC50-value of 1.21*109 cfu/L and an ErC50-value of 9.14*109 cfu/L were calculated.

Based on the similarity between B. amyloliquefaciens ISB06 and B. amyloliquefaciens

FZB24, B. amyloliquefaciens ISB06 should have no effect on algae growth when used

concentrations lower than 1.0*108 cfu/L. As the product is usually applied at lower

concentrations no unacceptable effects are expected for algae from the use of the

product.

2.2.4.1.6. Effects on plants other than algae

The applicant provided a justification for the non-submission of a study on plants other

than algae based on literature data.



strain ISB06


17

air, and decomposing plant residue (Gatesoupe, 1999; Nicholson, 2002; Hong et al.,

2005). The biological properties of this microorganism suggest that spores of B. subtilis

group are able to survive in aquatic ecosystems. However, no harm to aquatic organisms

are expected based on the absence of disease or other adverse effects in fish or other

aquatic organisms including plant related to B. amyloliquefaciens in published literature

despite this microorganism’s ubiquitous nature (Verschuere et al., 2000; Gatesoupe,

1999). Although the evidence of the studies from the literature is circumstantial they

support the results from the ecotoxicological studies which indicate no harmful effects of

the test article Bacillus amyloliquefaciens FZB24 on aquatic organisms.

2.2.4.1.7. Effects on bees

A published study reports a biological treatment of chalkbrood (Ascosphaera apis) in

honey bees (Apis mellifera) with a strain of B. amyloliquefaciens (Jacobsons, 2005). Oral

treatment of bee larvae was carried out with a spore solution of 1.0*104 cfu/ml of B.

amyloliquefaciens isolated from bees’ gut. This published study reported no adverse

effects on bee larvae in the tested concentrations.

2.2.4.1.8. Effects on arthropods other than bees

A test was carried out with Poecilus cupreus and mortality was determined by classifying

the beetles as alive, dead or burried. No adverse effects were detected in the tested

concentration of 6.25*1010cfu/L corresponding to an application rate of 2.57*1013 cfu/ha.

Bacillus amyloliquefaciens strain FZB24 appears to not to cause any acute toxicity

towards Poecilus cupreus. Based on the similarity between B. amyloliquefaciens ISB 06

and B. amyloliquefaciens FZB 24, we can extrapolate these results to B.

amyloliquefaciens ISB 06.

Moreover, members of the Bacillus group are frequently found in invertebrate’s gastro-

intestinal tract from insects, including aphids, mosquito larvae, cockroaches and in

certain arthropods. Bacillus species include B. licheniformis, B. cereus, B. sphaericus, B.

circulans, B. megaterium, B. alvei, B. pumilus, B. amyloliquefaciens and B. subtilis.

2.2.4.1.9. Effects on earthworms

Two acute toxicity studies on earthworms were conducted according to OECD 207

(artificial soil test) with earthworms (Eisenia fetida) using B. amyloliquefaciens FZB24 as

test material. No differences in biomass and mortality between the tested concentrations

and the control were observed. None of the exposed animals died. Only slight differences

in behaviour and habitus were observed. As no mortality occurred a LC50 value could not

be determined. The NOEC value was determined to be 1000 mg/kg dry weight soil

corresponding to 1*1011 cfu/kg dry weight soil in both tests. According to the studies on

effects on earthworms, B. amyloliquefaciens strain FZB24 appears not to cause any acute

toxicity towards earthworms in the tested range of concentrations. Based on the

similarity between B. amyloliquefaciens ISB06 and B. amyloliquefaciens FZB24, these

results can be extrapolated to B. amyloliquefaciens ISB06.

2.2.4.1.10. Effects on soil micro-organisms

The applicant provided a justification for the non-submission of a study on soil micro-

organisms based on the ubiquitous nature of Bacillus amyloliquefaciens and on data from

literature (Liang et al., 1982).

B. amyloliquefaciens is a ubiquitous soil microorganism that contributes to nutrient

cycling due to the various enzymes produced by individual strains of this species. The

spectrum of enzymatic activities is presented in Doc IIA-1.1.3. Several studies on the

persistence of B. subtilis group in soil have shown that, in general, the population sizes of


strain ISB06


18

introduced cultures decline more or less rapidly following introduction into natural soils.

When introduced into soils already containing a natural microbial flora, populations of the

introduced B. subtilis strain are likely to decline to 1% or less of the population of

indigenous B. subtilis strains that the soil can support. Sterile growth media, such as

those used in horticulture, may support higher levels of introduced B. subtilis strains but,

when such media are disposed of into a terrestrial environment, the population of the

introduced B. subtilis strain is likely to decline to the levels observed in natural soils. This

has been already demonstrated for B. amyloliquefaciens FZB24 in a study of Kilian et al.

(2000) and field soil experiments conducted by ABiTEP, in which the strain FZB24 was

used as PRGB (see above & Doc IIA, chapter 5.1.1.1). Therefore no elevated levels of the

microorganism are expected in the environment that could cause adverse effects on

other soil-microorganisms. It is noteworthy that before 1987 B. subtilis and B.

amyloliquefaciens were considered as synonymous. Furthermore, B. amyloliquefaciens is

a normal component of the soil, and the organism is not expected to affect

environmentally or economically important microbial species or microbiologically

mediated biogeochemical processes. In general, no adverse data are known with regard

to the persistence or multiplication of B. subtilis or B. amyloliquefaciens strains in soil,

water or air compartments (see 5.1.1, Declaration of ABiTEP, 2010). Particularly for the

soil compartment, no adverse environmental effects have been reported for the

taxonomically closely related Bacillus strain FZB24 which is commercially in use since

1999 (see 5.1.1, declaration of ABiTEP, 2010). The risk for adverse effects on soil-

microorganisms is therefore considered to be low.

Although the evidence of the studies from the literature is circumstantial, they indicate

that the potential risk for soil-microorganisms associated with the use of this bacterium

is low.

2.2.4.1.11. Further studies

2.2.4.1.11.1. Terrestrial plants

The applicant provided a justification for the non-submission of a study on terrestrial

plants based on the use of Bacillus amyloliquefaciens as plant growth-promoting

rhizobacteria (PGPR) and biological control agent.


air, and plants. Moreover, strains of B. amyloliquefaciens are commercially used as plant

growth-promoting rhizobacteria (PGPR) or biological control agent. Commercially

available biocontrol rhizobacteria include B. amyloliquefaciens FZB42 (RhizoVital® 42;

ABiTEP GmbH). Biological control refers to the purposeful utilisation of introduced or

resident living organisms to suppress the activities and populations of one or more plant

pathogens. These PGPR are antagonists of recognized root pathogens by colonizing plant

roots and stimulating plant growth and/or reduce the incidence of plant disease. A

mixture of B. subtilis strain GB122 and B. amyloliquefaciens strain GB99 (BioYield;

Gustafson) is used as PGPR against Arabidopsis’ pathogen Erwinia carotovora ssp.

carotovora but also Rhizoctonia solani, Pythium spp. Since B. amyloliquefaciens is used

as PGPR it is not considered to be a plant pathogen. Furthermore, no adverse

environmental effects have been reported for Bacillus amyloliquefaciens strain FZB24

which is commercially used since 1999 and closely related to B. amyloliquefaciens

(declaration of ABiTEP, 2010).

2.2.4.1.11.2. Mammals

The applicant provided a justification for the non-submission of a study on mammals

based on literature data.

In mammals, only a few reports on adverse effects can be found in published scientific

literature. Bacillus subtilis has been implicated in cases of bovine mastitis and


strain ISB06


19

reproductive disorders in goats (Fossum et al., 1986). However, very fewincidences of

mastitis associated with B. subtilis are reported compared to other microorganisms. It is

noteworthy that before 1987 B. amyloliquefaciens has been regarded as a subspecies or

variant of B. subtilis (Priest et al., 1987). No other reports of adverse effects were

reported in the literature. On the contrary different Bacillus-species are used as feed

supplements in animal husbandry for chickens, pigs or calves. B. amyloliquefaciens is

notified as farm animal feed supplement (Ecobiol®) by the EFSA (2008). Although the

evidence of the studies from the literature is circumstantial, they strengthen the

hypothesis, that the potential risk for mammals associated with the use of this bacterium

is low.

2.2.4.1.12. Other relevant species and processes

B. amyloliquefaciens is a ubiquitous bacterium commonly recovered from water, soil, air,

and decomposing plant residue. The submitted ecotoxicological studies and literature

data cover a wide range of species and processes. They are considered to be sufficient

for the assessment of the environmental risks associated with B. amyloliquefaciens

ISB06. Based on the currently available body of knowledge, these risks appear to be

neglibile.

2.2.4.2. Conclusion

According to literature data base and the ecotoxicological studies provided, it can be

assumed that Bacillus amyloliquefaciens ISB06 is a ubiquitous bacterium commonly

recovered from water, soil, air as well as decomposing plant residue and has no adverse

effects on aquatic and terrestrial organisms when used as specified for the product.

2.2.5. PBT and POP assessment

Not applicable.

2.2.6. Exposure assessment

The product Cobiotex 112 biofilm + is intended for indoor use only, for the treatment of

livestock buildings and breeding equipment by professionals. Therefore direct exposure of

the environment is not expected. However, application of manure and/or sewage sludge

(after releases to sewage treatment plants; STP) in agriculture will result in a potential

indirect exposure of soil, groundwater and, via run-off, also of surface water. Emissions

to surface water (recipient) may also occur after sewage treatment through the STP

effluent.

Emissions to manure and waste water following cleaning of the treated surface areas

seem to be the most relevant release pathways into environment after indoor use of

Cobiotex 112 biofilm +.

Emission to air is considered negligible, as Cobiotex112 biofilm + would only be used

indoors by spray application of an aqueous solution.

The fraction of the biocide reaching the manure storage system and/or the STP will

depend on the number of spores, which germinate after application, as the product

contains mainly the endospores of B. amyloliquefaciens strain ISB06. During application

these spores adhere to abiotic surfaces of livestock buildings. If environmental

conditions, e.g. temperature, humidity, nutrient, oxygen, allow it, there is activation of

“barrier flora” by germination of Bacilli spores. If conditions are unfavourable, the spores


strain ISB06


20

remain in a state of “dormancy”.

The vegetative cells of B. amyloliquefaciens strain ISB06, that develop from the spores

are obligatory aerobic and will not survive under the mostly anaerobic conditions in

manure and/or sewage sludge. Vegetative cells resulting from spore germination may

however proliferate at favourable conditions and subsequently yield new endospores.

Only endospores are able to endure extreme conditions and may survive after release

and germinate in the environment under favourable conditions.

A semi-quantitative environmental exposure assessment was done for the soil

compartment with the objective of comparing the predicted environmental concentration

(PEC) for the active substance with natural abundances reported for Bacillus sp. and

Bacillus subtilis, being in the range of 102-105 cfu per g of soil (see section 5.1.1.1). This

background level has been considered as realistic also for B. amyloliquefaciens, which

belongs to the B. subtilis group and is well known as plant- and soil-associated bacteria

(see section 5.1).

Cobiotex 112 biofilm + contains B. amyloliquefaciens strain ISB06 in a concentration of

0.05% w/w corresponding to 1*106 cfu /g product. One kilogram of the product is diluted

in 100 L water for the treatment of 1000 m2 of a life stock building. This corresponds to a

concentration of 1*107 cfu /L and to 1*106 cfu/m2 of treated surface. According to the

manufacturer, a double application is recommended.

Based on these data a PEC for soil was calculated. For PEC calculation a germination of

100% was assumed as worst case, though it is not possible to estimate or determine the

number of spores which will actually germinate after release, since spore germination at

a treated site strongly depends on conditions of moisture, nutrient availability,

temperatures and other factors. Furthermore, neither an increase nor a decline of the cell

number has been assumed in manure over the storage period, although it is very likely

that cell numbers will vary within the short-term in dependency from the presence of

easily available carbon and nutrients. PECsoil calculation was carried out according to the

approach described in Emission Scenario Documents (ESD) for Product Type 3:

Veterinary hygiene biocidal products (EUR 25116 EN, 2011) and applied only for veal

calves (i1 = 3) as worst case animal (sub)category based on the amount of active

ingredient released to soil via manure/slurry in dependence of the nitrogen immission

standard. From this a PECsoil of 102.59 cfu.kg-1 was calculated for grassland. Compared

to the natural abundance the number of cells and spores introduced into soil following

product application can be considered negligible.

2.2.6.1. Conclusion

Compared to the natural abundance of 102-105 cfu per g of soil the number of cells and

spores introduced into soil following product application can be considered negligible. It

might therefore be assumed that application of the product and subsequent

environmental exposure is unlikely to cause increased abundance of ISB06 in the

environment.

2.2.7. Risk characterisation

The risk for the environment is usually characterized by comparing the toxicity of the

substance (PNEC) with the exposure estimates (PEC). However, Cobiotex 112 biofilm + is

a microbial product with a very specific mode of action based on the endospores of

Bacillus amyloliquefaciens strain ISB06, who does not produce toxins and a quantitative

risk assessment is therefore not considered appropriate.

However, a semi-quantitative environmental exposure assessment for the soil compart-


strain ISB06


21

ment indicated that the number of cells and spores introduced into soil following product

application in animal housings and subsequent manure/slurry deposition on agricultural

land (grassland, arable land) can be considered negligible. A highest PECsoil of

102.59 cfu.kg-1 was calculated for grassland. Compared to the natural abundance of 102-

105 cfu per g of soil, it can be assumed that application of the product and subsequent

environmental exposure is unlikely to cause increased abundance of ISB06 in the

environment.

in addition, the availabel ecotoxicological studies implicate that no adverse effects will

occur after application of the product in the prescribed concentrations because effects on

exposed organisms occurred only in concentrations that were considerably higher than

levels of Bacillus spp. found after anthropogenic release.

From there, it can be concluded, that the potential indirect exposure of the environment

(soil, groundwater, surface water) resulting from the recommended indoor use of the

product, does not present any adverse impact on the environment.

Furthermore, organisms in the environment are naturally exposed to fluctuating levels of

B. amyloliquefaciens as it is a fairly ubiquitous bacterium commonly recovered from

water, soil, air, and decomposing plant residues. There are numerous reports of different

Bacillus species being isolated from fish and crustaceans, as well as shrimps, bivalves,

arthropods and insects. As these microorganisms are ubiquitous in the environment, they

will be found at the bottom of ponds, lakes and rivers. Organisms inhabiting these

environments like fish, crustaceans and shellfish will ingest Bacillus from the organic

matter. Even so, Bacillus species are recovered from the gastrointestinal tract (GIT) of

aquatic animals with remarkable ease and have been found in the microflora of the gills,

skin and intestinal tract of shrimps. Again, members of different Bacillus species are

frequently found in terrestrial invertebrates. They have been found in the gut of

numerous insects, including aphids, mosquito larvae and cockroaches as well as in the

gut of earthworms. No adverse effects resulting from the presence of the microorganism

were reported.

Also a number of species from the Bacillus genus have been considered safe for

application in the food and agricultural industries. Bacillus spp. are being intentionally

used as probiotics in animal nutrition and as plant growth-promoting rhizobacteria

(PGPR). Different studies in the published literature showed that the application has no

adverse effects on the exposed organisms.

Therefore, based on the literature data and the ecotoxicological studies it can be

concluded, that the potential indirect exposure of the environment resulting from the

recommended indoor use of the product, does not create any significant risk for the

environment when used as intended. Only the indoor scenario was covered by the risk

characterization, which includes that mixing and application of the product was only done

indoors by professionals and on impermeable ground to avoid further releases into the

environment.

2.2.8. Assessment of endocrine disruptor properties

Not applicable.


strain ISB06


22

2.3. Overall conclusions

The outcome of the assessment for B. amyloliquefaciens strain ISB06 in product-type 3 is

specified in the BPC opinion following discussions at the 13th meeting of the Biocidal

Products Committee (BPC). The BPC opinion is available from the ECHA website.

2.4. List of endpoints

The most important endpoints, as identified during the evaluation process, are listed in

Appendix I.

2.5. Requirement for further information related to reference product

The following information should be provided for product authorisation:

Data on microbial contaminants .

Information on storage stability, stability and shelf life


strain ISB06


23

Appendix I: List of endpoints

Chapter 1: Identity, Physical and Chemical Properties, Further Information, and

Proposed Classification and Labelling

Active substance Bacillus amyloliquefaciens strain ISB06

Function (e.g. fungicide) bactericide

Rapporteur Member State Germany

Identity (Annex IIIA, Section 1)

Common name Bacillus amyloliquefaciens strain ISB06

Taxonomic name Species Bacillus

amyloliquefaciens

(Fukomoto, 1943)

Strain ISB06

Genus Bacillus

Phylum Firmicutes

Collection and culture

reference number Confidential

Other substance No. Not applicable

Minimum purity of the active

substance as manufactured (g/kg or g/L)

Confidential

Identity of relevant impurities

and additives (substances of

concern) in the active

substance as manufactured (g/kg)

No relevant substances

Source and biological properties (Annex IIIA, Section 2 and 3)

Natural occurrence and

distribution

Ubiquituous species

Isolation methods Cultivation

Culture methods Confidential

Production methods Confidential

Composition of the micro-

organism

Confidential

Methods to preserve seed

stock

Confidential

Relationship to existing

pathogens

Related to but distinct from opportunistic and obligate

pathogens and other potentially harmful bacteria of the

genus Bacillus, in particular Bacillus licheniformis and the distantly related B. anthracis and B. cereus.

Effects on the target organism Growth inhibition by putative nutritive competition and

putative active disturbance of target cells via antimicrobial substances


strain ISB06


24

Transmissibility, infective dose

and mode of action and

information on the nature,

identity and stability of toxins

No concerns regarding human, animal and plant health

could be associated with ISB06 during investigations.

The EFSA QPS status takes into account that few

reports exist where the species has been proven or suspected to be implicated in clinical cases.

Dispersal and transmission from one organism to

another is not expected to occur via the infective route

but via the environmental route. Spore production

from vegetative cells is likely to facilitate

environmental dispersal. This is in line with the finding that the species is ubiquitous.

Toxins of human health concern could not be detected.

Determination of toxins and virulence factors of B.

amyloliquefaciens strain ISB 06 have been evaluated

following recommendations by the scientific committee

on animal nutrition on the safety of use of Bacillus

species in animal nutrition. The haemolytic and

cytotoxic activities as well as the genetic basis of the toxin production were determined.

Strain ISB06 does not display haemolytic activity on

blood agar plates and is devoid of B. cereus-like nhe

and hbl genes. Not the whole metabolite profile has been investigated as this is technically not feasible.

Substances with antimicrobial effects might be

produced and might be part of the biocidal mechanism of strain ISB06.

Infectivity and stability in use No concern. As a general precaution, ingestion,

inhalation, and introduction into wounds should be

avoided or appropriately treated. This holds particularly true for immunocompromised persons.

Genetic stability There are no signs of genetic instability as inferred

from physiological marker analysis and electrophoretic

genome profiling (PFGE) of independent production

batches.

Production is run from starter stocks. Quality controls of identity, purity and biocidal activity are performed.

Upon application, gain of genetic material as well as

loss of genetic material may occur according to natural

exchange mechanisms of genetic information among

microorganisms.

Resistance or sensitivity to

antibiotics

ISB06 is resistant to ampicillin.


strain ISB06


25

Classification and labelling (Annex IIA, point IX.)

with regard to biological properties none

with regard to toxicological data Micro-organisms may have the potential to provoke

sensitising reactions

with regard to fate and behaviour

data

none

with regard to ecotoxicological data none


strain ISB06


Chapter 2: Methods of Analysis

Analytical methods for the active substance

Technical active substance (principle of

method) (Annex IIA, point 4.1)

Physiological, microscopic and molecular

methods. Confidential details

Impurities in technical active substance

(principle of method) (Annex IIA, point 4.1)

Not relevant

Analytical methods for residues

Soil (principle of method and LOQ) (Annex

IIA, point 4.2)

Not necessary, no relevant residues expected

by CA.

Strain-specific detection is confidential.

Air (principle of method and LOQ) (Annex

IIA, point 4.2)

Not necessary. Due to the absence of any

evidence of toxicity, pathogenicity or infectivity

of Bacillus amyloliquefaciens during inhalation,

a limit of determination could not be derived by CA.


Water (principle of method and LOQ)

(Annex IIA, point 4.2)


by CA.


Body fluids and tissues (principle of

method and LOQ) (Annex IIA, point 4.2)

Not necessary, since not classified as toxic or

very toxic.

Food/feed of plant origin (principle of

method and LOQ for methods for

monitoring purposes) (Annex IIIA, point IV.1)


by CA.


Food/feed of animal origin (principle of

method and LOQ for methods for

monitoring purposes) (Annex IIIA, point

IV.1)


by CA.



strain ISB06


Chapter 3: Impact on Human Health

Medical data (including medical

surveillance on manufacturing plant personnel)

No adverse health effects reported with strain

ISB06; B. amyloliquefaciens enzymes may

cause allergic reactions. The closely related

species B. subtilis has been rarely reported to

be associated with food poisoning and infections in immuno-compromised patients.

Sensitisation Sensitiser (no studies available, labelling based

on general assumptions for micro-organisms

supported by positive findings in the

manufacturing of enzymes from B. amyloliquefaciens and B. subtilis)

Acute oral toxicity, pathogenicity and

infectivity

No data for strain ISB06,

LD50 (rat) for B. amyloliquefaciens FZB24:

> 3.8x109 cfu/animal, no evidence of adverse

effects, colonisation not tested

Acute inhalation toxicity, pathogenicity and

infectivity

Intratracheal LD50 (rat) for B. amyloliquefaciens ISB06: >1x109 cfu/kg bw,

no toxic or pathogenic effects observed,

complete clearance from the lung in 5/10 animals within 22 days

Intraperitoneal/Intravenous/Subcutaneous

single dose

Intraperitoneal LD50 (rat) for B.

amyloliquefaciens ISB06: >1x109 cfu/kg bw,

no toxic or pathogenic effects observed, no

complete clearance within 22 days

Genotoxicity No data – not required; no evidence of

genotoxic potential for secondary metabolites and toxins

Cell culture study HeLa cells: no cytotoxicity observed

Information on short-term toxicity and

pathogenicity

No data –not required

Specific toxicity, pathogenicity and

infectivity

Not skin irritating;

Eye irritation below classification threshold


strain ISB06


Exposure

Acceptable exposure scenarios (including method of calculation)

Professional users

Production of active substance: Not assessed by the rapporteur under the

requirements of the BPD

Formulation of biocidal product Not assessed by the rapporteur under the

requirements of the BPD

Intended uses Scenario 1

Spraying application in livestock buildings

Powdery b.p. dissolved in water are applied by

spraying or misting.

Powdery b.p. dissolved in water and applied by

spraying in livestock buildings. Potential dermal

and inhalation exposure is assumed. For work

with risk group 1 biological agents (as defined

in the German technical guideline TRBA 466)

the principles of good occupational safety and

hygiene should be observed. Due to potential

sensitisation for professional users PPE and RPE is recommended.

Intended uses Scenario 2

Misting application in livestock buildings

Powdery b.p. dissolved in water and applied by

misting in livestock buildings. Potential dermal

and inhalation exposure is assumed. For work

with risk group 1 biological agents (as defined

in the German technical guideline TRBA 466)

the principles of good occupational safety and

hygiene should be observed. Due to potential

sensitisation for professional users PPE and RPE is recommended.

Secondary exposure Primary exposure of non-professionals is not

expected.

Indirect exposure as a result of use (eg via

food or feed)

Primary exposure of non-professionals is not

expected.

Combined Exposure Residues on food or feeding stuffs are not expected at relevant concentrations.


strain ISB06


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)

B. amyloliquefaciens is ubiquituous in soil at species level and is an indigenous part of the

soil micro-flora community. However, it should be noted that specific strains like ISB06

might not be ubiquituously present. As ISB06 has been isolated from an agricultural

environment, the strain might not be indigenuous to locations where the biocide is

applied. Hence, the role of ISB06 as an allochthonous species should be taken into

consideration. However, B. amyloliquefaciens does not compete aggressively with other

soil micro-organisms and is an active member of the soil microbial community. Bacillus

species possess different mechanisms to establish themselves as part of the soil

microflora. Some strains of Bacillus amyloliquefaciens are employed in crop cultivation as

plant growth promoters. Their use is considered favourable for the environment as it

allows reducing amounts of chemical fertilizers. Alongside, the environmental impact of the strains themselves has not given rise to concerns so far.

B. amyloliquefaciens is not infectious and has a limited survival in the environment as B.

amyloliquefaciens vegetative cells are not persistent, not mobile in soil and not persistent

in water, resulting in limited spread of the organism. Degradation of Bacillus

amyloliquefaciens vegetative cells and poor germination of B. amyloliquefaciens spores in

soil indicate that the organism may not substantially multiply in soil. Airborne

concentrations of B. amyloliquefaciens are expected to be negligible following indoor

application.


strain ISB06


Chapter 5: Effects on Non-target Species

Toxicity data for aquatic species (most sensitive species of each

group)

(Annex IIA / IIIA , point 8.2)

Species Time-

scale

Endpoint Toxicity

Fish

Oncorhynchus mykiss

96 h LC0(96h) 2.4x109 cfu/L

Oncorhynchus mykiss

96 h LC0(96h) 2.0x105 cfu/L

(cell-free extract)

Invertebrates

Daphnia magna 48 h EC50(48h) 4x108 cfu/L

Algae

Scenedesmus subspicatus 72 h EbC50(72h)

ErC50(72h)

1.21x109 cfu/L

9.14x109 cfu/L

Microorganisms

not tested


strain ISB06


31

Effects on earthworms or other soil non-target organisms

Acute toxicity to earthworms.

(Annex IIA / IIIA, point 8.5) LC50(14d): >1x1011cfu/L, Eisenia fetida

Reproductive toxicity to …………………………

(Annex IIIA, point XIII.3.2) not tested

Acute toxicity to

arthropods………………..……..

(Annex IIA / IIIA, point 8.4)

LC50(14d): >6.25x1010 cfu/L; Poecilus

cupreus

LC50(14d): >2.5x1013 cfu/ha, Poecilus cupreus

Effects on soil micro-organisms (Annex IIA, point 7.4)

Nitrogen mineralization not tested

Carbon mineralization not tested

Effects on terrestrial vertebrates

Acute toxicity to mammals


Acute toxicity to birds


Dietary toxicity to birds


Reproductive toxicity to birds


Effects on honeybees (Annex IIIA, point XIII.3.1)

Acute oral toxicity not tested

Acute contact toxicity not tested

Effects on other beneficial arthropods (Annex IIIA, point XIII.3.1)

Acute oral toxicity not tested

Acute contact toxicity not tested

Acute toxicity to ………………………………….. not tested

Chapter 6: Other End Points


strain ISB06

Product-type 03 November2015

32

APPENDIX II : LIST OF INTENDED USES

Summary of intended uses

B. amyloliquefaciens is intended to be used in on abiotic surfaces of livestock buildings and

equipment of breeding in order to reduce the number of potentially harmful bacteria.

Object and/o

r situation

Member

State or Country

Product

name

Organisms

controlled

Formulati

on

Application

Applied amount

per treatment

Remarks:

(a)

(c)

Type

(d-f)

Conc

.

of as

(i)

meth

od

kind

(f-h)

numb

er

min max

(k)

interval

between applications (min)

g

as/L

min max

water

L/m2

min max

g

as/m2

min max

(m)

bactericide

France

COBIOTEX 112

BIOF

ILM +

Bacteria

Powder

> 1.0 105

CF

U /

g

Spraying

double applicatio

n is

recommended.

24 hours

1 kg of COBIOTEX 112 biofilm + product (diluted in 100 liters of cold

water) is

recommended for spraying indoor abiotic surfaces of a livestock building covering 1000 m2.

An application after each step of cleaning-

disinfection

(24 hours after disinfection stage in order to avoid remanence

effects of disinfectants) is recommended.

(c) GCPF Codes - GIFAP Technical Monograph No 2, 1989 ISBN 3-8263-3152-4); (d) All abbreviations

used must be explained

(e) g/kg or g/l;(f) Method, e.g. high volume spraying, low volume spraying, spreading, dusting, drench;

(g) Kind, e.g. overall, broadcast, aerial spraying, row, bait, crack and crevice equipment used must be

indicated;

(h) Indicate the minimum and maximum number of application possible under practical conditions of use;

(i) Remarks may include: Extent of use/e

conomic importance/restrictions


strain ISB06


33

Appendix III: List of studies

Data protection is claimed by the applicant in accordance with Article 60 of Regulation (EU)

No 528/2012.

Reference List according to Section Numbers

Section No / Reference No

Author(s) Year Title. Source (where different from company)

Company, Report No.

GLP (where relevant) / (Un)Published

Data Protection Claimed

(Yes/No)

Owner

DocI 2.1.2 EC 2014 Review report for the active substance Bacillus amyloliquefaciens subsp. plantarum strain D747

SANCO/11391/2014 rev 1

No Published

DocIIA 1.2.7 Chen X.-H., Koumoutsi A., Scholz R., Borriss R.

2009 More than anticipated - production of antibiotics and other secondary metabolites by Bacillus amyloliquefaciens FZB42. J Mol Microbiol

Biotechnol.;16(1-2):14-24

No

DocIIA 3.1.3 Flindt, M.L.H. 1969 Pulmonary disease due to inhalation of derivatives of

Bacillus subtilis containing proteolytic enzyme. Lancet

i:1177-1181

No N.A.

DocIIA 3.1.3 Johnsen, C.R., Sorensen, T.B., Larsen, A. I., Secher, A.B., Andreasen, E.,

Kofoed, G.S., Nielsen, L. F., Gyntelberg, F.

1997 Allergy risk in an enzyme producing plant: a retrospective follow up study. Occup. Environm. Med. 54:671-675

No N.A.

DocIIA 3.1.3 Larsen, A.I., Johnsen, C.R., Frickmann, J.,

Mikkelsen, S.

2007 Incidence of respiratory sensitisation and allergy to enzymes among employees in

an enzyme producing plant

and the relation to exposure and host factors. Occup. Environ. Med. 64:763-768

No

DocIIA 3.1.3 Pepys, J.,

Longbottom, J.L., Hargreave, F.E., Faux, J.

1969 Allergic reactions of the lungs

to enzymes of Bacillus subtilis. Lancet i: 1181-1184

No N.A.

Doc II B8 EC 2002 TNsG Human Exposure Technical Notes for Guidance in Support of Directive

98/8/EC of the European Parliament and the Council Concerning the Placing of Biocidal Products on the Market. Human Exposure to

Biocidal Products - Guidance

on Exposure Estimation [„Report 2002“

No Published


strain ISB06


34

Section No

/ Reference No

Author(s) Year Title.

Source (where different from company) Company, Report No. GLP (where relevant) / (Un)Published

Data

Protection Claimed (Yes/No)

Owner

http://ecb.jrc.it/biocides]

Doc II B8 Warren, N, Marquart, H., Christopher, Y.; Laitinen, J.; Van Hemmen, J.

2006 Task-based dermal exposure models for regulatory risk assessment, Ann. Occup. Hyg. Vol. 50, 491-503

No Published

DocIIIA 2.1.1 Alexander, M 1977

Introduction to soil

microbiology, J. Wiley & Sons, Inc., New York, in Final Risk Assessment of B. subtilis

No

DocIIIA 2.1.1

Burke SA,

Wright JD, Robinson MK, Bronk BV Warren RL

2004

Detection of molecular

diversity in Bacillus atrophaeus by Amplified Fragment Length Polymorphism Analysis, Appl Env Microbiol 70(5), 2786-90

No

DocIIIA 2.1.1 De Boer AS,

Diderichsen B 1991

On the safety of Bacillus subtilis and B. amyloliquefaciens: a review, Appl Microbiol Biotechnol

36(1), 1-4

No

DocIIIA 2.1.1

Fernando WGD,

Nakkeeran S, Zhang Y,

Savchuk S

2007

Biological control of Sclerotinia

sclerotiorum (Lib.) de Bary by

Pseudomonas and Bacillus species on canola petals, Crop Prot 26, 100-07

No

DocIIIA 2.1.1 Fritze, D. 2004

Taxonomy of the genus Bacillus and related genera: The aerobic endospore-forming

bacteria, Phytopathology 94, 1245-48

No

DocIIIA 2.1.1 Kokalis-Burelle

N, Kloepper JW, Reddy MS

2006

Plant growth-promoting

rhizobacteria as transplant amendments and their effects on indigenous rhizosphere microorganisms, Appl Soil

Ecology 31, 91-100

No

DocIIIA 2.1.1

Lechner S, Mayr

R, Francic KP, Prub BM, Kaplan T, Wieber-Gunkel E, Stewart GASB, Scherer S

1998

Bacillus weihenstephanensis

sp. Nov is a new psychrotolerant species of the Bacillus cereus group, Int J Sys Bacteriol 48, 1373-82

No

DocIIIA 2.1.1 Nakamura LK 1998 Bacillus pseudomycoides sp.

Nov., Int J Sys Bacteriol 48, 1031-35

No

DocIIIA 2.1.1 Nakamura LK,

Roberts MS, Cohan FM

1999

Relationship of Bacillus subtilis

clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus

subtilis subsp. spizizenii subsp.

nov., Int J Sys Bacteriol 49(3), 1211-15

No


strain ISB06


35

Section No

/ Reference No



Data


Owner

DocIIIA 2.2.1

Hyronimus B. N.A.

Determination of the target

organisms inhibited by B. amyloliquefaciens strain ISB 06

COBIOTEX

Unpublished

Yes

COBIOTEX

DocIIIA 2.2.1

Hyronimus, B.,

Le Marrec, C. and Urdaci, C.

1998

Coagulin, a pediocin-like

bacteriocin, produced by B. coagulans. Journal of Applied

Microbiology. 85, 42-50.

No N.A.

DocIIIA 2.2.2

Carrillo C., Teruel J., Aranda F., Ortiz

A.

2003

Molecular mechanism of

membrane permeabilization by the peptide antibiotic surfactin Biochimica et Biophysica Acta 1611 91–97

No N.A.

DocIIIA 2.2.2 Desai D. and Banat I.

1997

Microbial Production of Surfactants and Their Commercial Potential

Microbiology and molecular biology reviews

No N.A.

DocIIIA

2.2.2* Hyronimus B. N.A.

Competition by competitive

exclusion of B. amyloliquefaciens strain ISB 06

COBIOTEX

Unpublished

Yes

COBIOTEX

DocIIIA 2.2.2 Hyronimus B. N.A.

Determination of adhesion of B. amyloliquefaciens strain ISB 06 on different type of

supports

COBIOTEX

Unpublished

Yes

COBIOTEX

DocIIIA 2.2.2

Tremoulet F,

Duche O, Namane A,

Martinie B, Labadie JC,

2002

Comparison of protein

patterns of Listeria monocytogenes grown in biofilm or in planktonic mode

by proteomic analysis. Fems Microbiology Letters, 210 : 25-31.

No N.A.

DocIIIA 2.4 Hilbert DW,

Piggot PJ 2004

Compartmentalization of gene expression during Bacillus subtilis spore formation,

Microbiol Mol Biol Rev, 68(2), 234-62

No

DocIIIA 2.4 Nicholson WL 2002

Roles of Bacillus endospores in the environment, Cell Mol Life Sci 59, 410-16

No

DocIIIA 2.5 Logan NA 2011

Bacillus and relatives in foodborne illnesses, J Appl Microbiol 112(3), 417-29

No

DocIIIA 2.5 Nicholson WL,

Munakanata N, 2000

Resistance of Bacillus endospores to extreme

No


strain ISB06


36

Section No

/ Reference No



Data


Owner

Horneck G,

Melosh HJ, Setlow P

terrestrial and extraterrestrial

environments, Microbiol Mol Biol Rev 64(3), 548-72

DocIIIA 2.5

Weber, D.;

Susan M. Saviteer; William A.

Rutala; and Charlotte A. Thomann

1998

In Vitro Susceptibility of

Bacillus spp. to Selected Antimicrobial Agents Antimicrobial agents and

chemotherapy, p. 642-645 Vol. 32, No. 5

No N.A.

DocIIIA 2.6 Priest FG, Goodfellow M,

Todd C

1988 A numerical classification of the genus Bacillus, J Gen

Microbiol, 134(7), 1847-82

No

DocIIIA 2.6

Rooney AP,

Price NPJ, Ehrhardt C, Swezey JL, Bannan JD

2009

Phylogeny and molecular

taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov., Intl J Sys Evol Microbiol 59, 2429-36

No

DocIIIA 2.7

Duncan KE,

Istock CA, Graham JB, Ferguson N

1989

Genetic exchange between

Bacillus subtilis and Bacillus licheniformis: variable hybrid stability and the nature of bacterial species, Evolution 43(8), 1585-1609

No

DocIIIA 2.8

Christiansson A, Naidu AS, Nilsson I,

Wadström T, Pettersson HE

1989

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures (1989) Appl

Env Microbiol, 55(10), 2595-2600

No

DocIIIA 2.8

European

Commission Health & Consumer Protection

Directorate-

General Directorate B – Scientific Health Opinions Unit B3 – management of scientific

committees

2000

Opinion of the scientific

committee on animal nutrition on the safety of use of Bacillus species in animal nutrition (expressed on 17 february

2000)

No N.A.

DocIIIA 2.8

From C, Pukall R, Schumann P, Homazabal V,

Granum PE

2005

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group, Appl.

Env. Microbiol, 71(3), 1178-83

No

DocIIIA 2.8* Hyronimus, B. N.A.

Determination of cytotoxicity to Hela cells of B.

amyloliquefaciens strain ISB

06

COBIOTEX

Yes COBIOTEX


strain ISB06


37

Section No

/ Reference No



Data


Owner

Unpublished

DocIIIA 2.8* Hyronimus, B. N.A.

Determination of toxins and

haemolytic activity of B. amyloliquefaciens strain ISB 06

COBIOTEX

Unpublished

Yes COBIOTEX

DocIIIA 2.8

Matarante, A.;

Baruzzi, F.; Cocconcelli, P.; and Morea, M.

2004

Genotyping and Toxigenic

Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

Appl. Environ. Microbiol. 70: 5168-5176

No N.A.

DocIIIA 2.8 Ramarao N,

Lereclus D 2006

Adhesion and cytotoxicity of

Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively (2006) Microbes Inf, 8(6), 1483-91

No

DocIIIA 2.9 Daniel P. 2007

Determination of MICfrom B. amyloliquefaciens ISB 06

Genfast

Unpublished

Yes COBIOTEX

DocIIIA 2.9 Hyronimus, B. N.A.

Determination of MIC of B. amyloliquefaciens strain ISB06

COBIOTEX

Unpublished

Yes COBIOTEX

DocIIIA 2.9

Opinion of the Scientific Committee on Animal Nutrition

2003

Criteria for Assessing the

Safety of Micro-Organisms Resistant to Antibiotics of Human Clinical and Veterinary Importance

No N.A.

DocIIIA 2.10 Le Henaff M 2013

Quantification of Bacillus

amyloliquefaciens ISB 06 spore resistance to inactivation by (i) wet and dry

heat, (ii) UV, (iii) freeze-drying and (iv) four chemical agents

Yes Cobiotex

DocIIIA 2.10

Nicholson WL, Munakanata N, Horneck G, Melosh HJ,

Setlow P

2000

Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments, Microbiol Mol

Biol Rev 64(3), 548-72

No

DocIIIA 2.11

Jack RF,

Ringelberg DB,

White DC

1992

Differential corrosion rates of

carbon steel by combinations of Bacillus sp., Hafnia alvei and Desulfovibrio gigas

established by phospholipid analysis of electrode biofilm, Corr Sci 33(12), 1843-53

No


strain ISB06


38

Section No

/ Reference No



Data


Owner

DocIIIA 2.11 Racicot RJ, Crouch CD, Ranch ME

2007

Microbial influenced corrosion

studies of Bacillus licheniformis on AA 2024 aluminum alloys, Corr Rev 25(1-2), 97-106

No

DocIIIA 5.1.1

Directive 2000/54/EC of

the European Parlimant and of the Council of 18 September 2000

2000

On the protection of workers from risks related to exposure

to biological agents at work (seventh individual directive within the meaning of Article 16 (1) of Directive 89/391/EEC)

No N.A.

DocIIIA 5.1.1 Sietske de Boer,

A.; Diderichsen, B.

1991

On the safety of Bacillus subtilis and B.

amyloliquefaciens : a review Applied microbiology and biotechnology , vol. 36, pp 1-4

No N.A.

DocIIIA 5.1.1

Weber, D.;

Susan M. Saviteer,; William A. Rutala, and

Charlotte A. Thomann

1998

In Vitro Susceptibility of

Bacillus spp. to Selected Antimicrobial Agents Antimicrobial agents and chemotherapy, p. 642-645

Vol. 32, No. 5

No N.A.

DocIIIA 5.1.2 AMFEP N.A.

Position on SCAN report on

use of Bacillus species in animal nutrition (Opinion from SCAN expressed on 17 February 2000)

No N.A.

DocIIIA 5.1.2

Directive 2000/54/EC of

the European Parlimant and of the Council of 18 September 2000

2000

On the protection of workers from risks related to exposure

to biological agents at work (seventh individual directive within the meaning of Article 16 (1) of Directive 89/391/EEC)

No N.A.

DocIIIA 5.1.4

Weber, D.; Susan M. Saviteer,;

William A. Rutala, and Charlotte A. Thomann

1998

In Vitro Susceptibility of Bacillus spp. to Selected Antimicrobial Agents

Antimicrobial agents and chemotherapy, p. 642-645 Vol. 32, No. 5

No N.A.

DocIIIA

5.2.1* 2007

Acute dermal irritation in rabbits

Study No. 33284 TAL

Unpublished

Yes COBIOTEX

DocIIIA 5.2.1*

2007

Acute eye irritation in rabbits

Study No. 33285 TAL

Unpublished

Yes COBIOTEX

DocIIIA 5.2.2.1

Duc, H.; Huynh

A. Hong; Teresa M. Barbosa;

Adriano O.

2004

Characterization of Bacillus

Probiotics Available for Human Use Applied and

Environmental Microbiology, p.

No N.A.


strain ISB06


39

Section No

/ Reference No



Data


Owner

Henriques; and

Simon M.Cutting

2161–2171 Vol. 70, No. 4

DocIIIA 5.2.2.1* 1994

Final report : Acute Oral

Toxicity Test of “Bacillus subtilis FZB24” in Rats, IBR Forschungs GmbH, Project No.: 10-04-0552/00-93,

25/03/1994

Yes FZB

Biotechnik GmbH

DocIIIA 5.2.2.1

The EFSA Journal

2006

Opinion of the Scientific Panel on Additives and Products or

Substances used in Animal Feed on the safety and efficacy of the product “Calsporin”, a preparation of Bacillus subtilis, as a feed additive for chickens for fattening in accordance with

Regulation (EC) No 1831/2003 Adopted on 8 of March 2006 ; (2006) 336, 1-15

No N.A.

DocIIIA

5.2.2.1 Silley, P. 2006

Do bacteria need to be regulated ? Journal of Applied

Microbiology ISSN 1364-5072

No N.A.

DocIIIA 5.2.2.2.

Health Canada 2007 B. subtilis MBI 600 No N.A.

DocIIIA

5.2.2.2.

J.R. Saunders; T.N.K. Sebunya,

and A.D. Osborne

1983

Pulmonary Clearance of Bacillus subtilis Spores in Pigs

Can J Comp Med. 47(1): 43–47

No N.A.

DocIIIA

5.2.2.2.-01* 2010

Acute Pulmonary Toxicity And

Pathogenicity To The Rat; HLS study number : OAK 0001; Huntingdon Life Sciences /14.05.2010

Yes Cobiotex

DocIIIA

5.2.2.3* 1994

Final report : Acute Dermal

Toxicity Test of “Bacillus

subtilis FZB24” in Rats, IBR Forschungs GmbH, Project No.: 10-04-0553/00-93, 25 March 1994.

Yes FZB

Biotechnik

GmbH

DocIIIA

5.2.2.3.-01* 2010

Bacillus amyloliquefaciens ISB 06 : Acute Intraperitoneal Toxicity And Pathogenicity To

The Rat; HLS study number : OAK 0002; Huntingdon Life Sciences / 14.05.2010

Yes Cobiotex

DocIIIA 5.2.2.4

1994

Final report : Acute Dermal

Irritation/Corrosion Test of “Bacillus subtilis FZB24” in Rabbits, IBR Forschungs GmbH, Project No.: 10-03-

0554/00-93, 18/03/1994

Yes FZB

Biotechnik GmbH

DocIIIA

5.2.2.4 1994

Final report : Acute Eye

Irritation/Corrosion Test of

Yes FZB

Biotechnik


strain ISB06


40

Section No

/ Reference No



Data


Owner

“Bacillus subtilis FZB24” in

Rabbits, IBR Forschungs GmbH, Project No.: 10-03-0555/00-93, 05/04/1994.

GmbH

DocIIIA 5.2.3 The EFSA

Journal 2006

Opinion of the Scientific Panel on Additives and Products or Substances used in Animal

Feed on the safety and efficacy of the product

“Calsporin”, a preparation of Bacillus subtilis, as a feed additive for chickens for fattening in accordance with Regulation (EC) No 1831/2003 Adopted on 8 of March 2006 ;

(2006) 336, 1-15

No N.A.

DocIIIA

5.2.5.1

J.R. Saunders,

T.N.K. Sebunya, and A.D. Osborne

1983

Pulmonary Clearance of

Bacillus subtilis Spores in Pigs Can J Comp Med. 47(1): 43–47

No N.A.

DocIIIA 5.2.7

Weber, D.; Susan M.

Saviteer,; William A.

Rutala, and Charlotte A. Thomann

1998

In Vitro Susceptibility of Bacillus spp. to Selected

Antimicrobial Agents Antimicrobial agents and

chemotherapy, p. 642-645 Vol. 32, No. 5

No N.A.

DocIIIA 5.3 The EFSA

Journal 2007

Introduction of a Qualified

Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA1 Opinion of the Scientific Committee

(Question No EFSA-Q-2005-293) Adopted on 19 November

2007

No N.A

DocIIIA 7.1.1 ABiTEP 1992 Presentation FZB24 / ABiTEP

Confidential data / 1992 Yes

DocIIIA 7.1.1,

A 7.1.2, A 7.1.3, A 7.2

Beifort 2010 ABiTEP Declaration Effects FZB24, 2010

Yes

DocIIIA 7.1.1

Buensanteai N., Yuen G.Y. and Prathuangwong S.

2008 The Biocontrol Bacterium Bacillus amyloliquefaciens KPS46 Produces Auxin, Surfactin and Extracellular Proteins for Enhanced Growth of Soybean Plant Thai Journal

of Agricultural Science 2008,

41(3-4): 101-116

No

DocIIIA 7.1.1,

Fritze D. 2004 Taxonomy of the Genus Bacillus and Related Genera:

No


strain ISB06


41

Section No

/ Reference No



Data


Owner

A 7.1.2 The Aerobic Endospore-

Forming Bacteria 2004 Symposium The Nature and Application of Biocontrol Microbes: Bacillus spp.

DocIIIA 7.1.1

Hallmann J., Rodríguez-

Kábana R., Kloepper J.W.

1999 Chitin-mediated changes in bacterial communities of the

soil, rhizosphere and within roots of cotton in relation to nematode control Soil Biology and Biochemistry 31 (1999) 551-W

No

DocIIIA 7.1.1

Kilian M., Steiner U., Krebs B., Junge H., Schmiedeknecht G., Hain R.

2000 FZB24® Bacillus subtilis – mode of action of a microbial agent enhancing plant vitality, Pflanzenschutz-Nachrichten Bayer 1/00, 1 72–93

No

DocIIIA 7.1.1

Mc Spadden Gardener B. B.

2004 Ecology of Bacillus and Paenobacillus spp in agricultural system 2004 Symposium The Nature and

Application of Biocontrol Microbes: Bacillus spp.

No

DocIIIA 7.1.1

Palumbo J.D., Baker J.L. and Mahoney N.E.

2006 Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds, Microbial Ecology, Volume 52, 45–52 (2006)

No

DocIIIA 7.1.1,

A 7.1.2, A 7.1.3, A 7.2

Priest F.G., Goodfellow M., Shute A. and Berkeley r.C.W.

1987 Bacillus amyloliquefaciens sp. Nov. Norn. Rev. International journal of systematical bacteriolog 1987, 37(1), 69-71

No

DocIIIA 7.1.1,

A 7.1.2, DocIIIA

7.1.3, A 7.2

US EPA 1997 Risk assessment B. subtilis, 1997

No

DocIIIA 7.1.1

van Veen J.A., van Overbeek l.S. and van Elsas J.D.

1997 Fate and Activity of Microorganisms Introduced into Soil MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, June 1997, p. 121–135 Vol. 61, No. 2

No

DocIIIA 7.1.1

Wang L., Lee W., Tai C. and Kuo H.

2008 Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens; International Journal of Systematic and Evolutionary Microbiology (2008), 58, 671–

675

No

DocIIIA 7.1.2 Sass A.M., McKew B.A., Sass H., Fichtel

2008 Diversity of Bacillus-like organisms isolated from deep-sea hypersaline anoxic

No


strain ISB06


42

Section No

/ Reference No



Data


Owner

J., Timmis K.N.

and McGenity T.J.

sediments Saline Systems

2008 Page 1 of 11

DocIIIA 7.1.2

Saunders J.R. , Sebunya T.N.K. and Osborne A.D.

1983 Pulmonary Clearance of Bacillus subtilis Spores in Pigs, Can J Comp Med 1983; 47: 43-47.

No

DocIIIA 7.1.2, A 7.2

Siefert J.L., Larios-Sanz M., Nakamura L.K., Slepecky R.A., Paul J.H., Moore

E.R.B., Fox G.E., Jurtshuk P.Jr.

2000 Phylogeny of Marine Bacillus Isolates from the Gulf of Mexico CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 84–88

No

DocIIIA 7.1.2

Miranda C.A.C., Martins O.B., Mandetta C.M.

2008 Species-level identification of Bacillus strains isolates from marine sediments by

conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis Antonie van

Leeuwenhoek (2008) 93:297–304

No


ABiTEP 2007 Safety Data Sheet of RhizoVital 42 li.

Yes

DocIIIA 7.1.2 Alken-Murray Corporation

ALKEN CLEAR-FLO® 1006 (http://www.alken-

murray.com/Vibrio.htm)

No

DocIIIA 7.1.2 Bioway Technology

BON FEED / BON POND / BON WATER

No

DocIIIA 7.1.2

Earl A.M., Losick

R. and Kolter R.

2008 Ecology and genomics of

Bacillus subtilis Trends in Microbiology 2008 Vol.16 No.6

No

DocIIIA 7.1.2

EFSA 2008 Safety and efficacy of Ecobiol® (Bacillus amyloliquefaciens) as feed

additive for chickens for

fattening Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed The EFSA Journal (2008) 773, 1-13

No

DocIIIA 7.1.2

Gontang E.A., Fenical W. and Jensen P.R.

2007 Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2007, p. 3272–3282 Vol. 73, No. 10

No

DocIIIA 7.1.2

Haas D. and Defago G

2005 BIOLOGICAL CONTROL OF SOIL-BORNE PATHOGENS BY

FLUORESCENT PSEUDOMONADS 2005 doi:10.1038/nrmicro1129

No


strain ISB06


43

Section No

/ Reference No



Data


Owner

Published online 10 March

2005.GY

DocIIIA 7.1.2 A 8.1

A 8.2.3 A 8.2.4

Hong H.A., Duc L.H., Cutting S.M.

2005 The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29 (2005) 813–835

No

DocIIIA 7.1.2

Ivanova E.P.,

Vysotskii M.V., Svetashev V.I., Nedashkovskaya O.I., Gorshkova N.M., Mikhailov

V.V., Yumoto N., Shigeri Y., Taguchi T., Yoshikawa S.

1999 Characterization of Bacillus

strains of marine origin INTERNATL MICROBIOL (1999) 2:267–271

No


Kamala-Kannan S. and

Lee K. J.

2008 Metal Tolerance and Antibiotic Resistance of Bacillus species

isolated from Sunchon Bay Sediments, South Korea Biotechnology 7 (1) : 149-152, 2008

No

DocIIIA 7.1.2 Novozymes PondPlus® No

DocIIIA 7.2

EFSA 2007 Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA Opinion of the Scientific Committee

(Question No EFSA-Q-2005-293) Adopted on 19 November 2007 The EFSA Journal (2007) 587, 1-16

No

DocIIIA 8.1

Fossum K.,

Herikstad H., Binde M., Pettersen K.-E.

1986 Isolation of Bacillus subtilis in

Connection with Bovine Mastitis, Nord. Vet. Med. 1986, 38, 233-236

No N.A.

DocIIIA 8.1

A 8.2.2 A 8.2.3.

A 8.2.4

Gatesoupe F.J. 1999 The use of probiotics in

aquaculture. Aquaculture 180; 147–165

No N.A.

DocIIIA 8.1

A 8.2.4

Nicholson W. L. 2002 Roles of Bacillus endospores in the environment, CMLS, Cell. Mol. Life Sci. 59, 410- 416

No N.A.

DocIIIA 8.2.1 1994 Acute toxicity testing in fish,

test article: Bacillus subtilis FZB 24

Yes

DocIIIA 8.2.2 Jahnke M.

1994 Acute toxicity in Daphnia magna, test article: Bacillus subtilis FZB 24

Yes

DocIIIA 8.2.2

A 8.2.3

Verschuere L., Rombaut G., Sorgeloos P.,

Verstraete W.

2000 Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiology and Molecular

Biology Reviews, Dec. 2000, p. 655–671 Vol. 64, No. 4

No N.A.


strain ISB06


44

Section No

/ Reference No



Data


Owner

DocIIIA 8.2.3

Jahnke M.

1994 Algal growth inhibition test,

test article: Bacillus subtilis FZB 24

Yes

DocIIIA 8.3 Jakobsons B.

2005 Biological treatment of chalkbrood in honey bees

No N.A.

DocIIIA 8.4

Braunmiller 1996 Laufkäfertoxizitätsprüfung, Pflanzenschutzamt Berlin,

Report No. P15/95, 12/12/1996

Yes

DocIIIA 8.5-01

Braunmiller 1996 Regenwurmtoxizitätsprüfung – Vorprüfung,

Pflanzenschutzamt Berlin, Report No. E15.1/96, 12/12/1996

Yes

DocIIIA 8.5-02

Braunmiller 1996 Regenwurmtoxizitätsprüfung – Hauptprüfung, Pflanzenschutzamt Berlin,

Report No. E15.2/96, 20/12/1996

Yes

DocIIIA 8.7.1

Fernando W.G.D., Nakkeeran S.,

Zhang Y., Savchuk S.

2007 Biological control of Sclerotinia sclerotiorum (Lib.) de Bary by Pseudomonas and Bacillus

species on canola petals, Crop Protection 26 (2007), 100-107

No N.A.

DocIIIA 8.7.1

Kokalis-Burelle N., Kloepper J.W., Reddy M.S.

2006 Plant growth-promoting rhizobacteria as transplant amendments and their effects on indigenous rhizosphere

microorganisms, Applied Soil Ecology 31(2006), 91-100

No N.A.

DocIIIA 8.7.1

Liang L., N., Sinclair J., L., Mallory L., M.,

Alexander M.

1982 Fate in Model Ecosystems of Microbial Species of Potential Use in Genetic Engineering,

Appl. Environ. Microbiol., Sept. 1982, 708-714

No N.A.

DocIIIB 3.11

Bose B, Grossman AD

2011 Regulation of horizontal gene transfer in Bacillus subtilis by activation of a site-specific

protease, J Bac 193(1), 22-39

No

Butcher BG, Helmann JD

2006 Identification of Bacillus subtilis sigma-w-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by

Bacilli, Mol Microbiol 60(3), 765-82

No

Koonin EV, Makarova KS, Aravind L

2001 Horizontal gene transfer in prokaryotes: Quantification and classification, Annu Rev Microbiol 55, 709-42

No

DocIIIB 3.12

Hong H., Le Hong Duc, Simon M.

Cutting.

2005 The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29

813–835

No N.A.

Tannock G. 2004 A Special Fondness for No N.A.


strain ISB06


45

Section No

/ Reference No



Data


Owner

Lactobacilli Applied And

Environmental Microbiology, p. 3189–3194 Vol. 70, No. 6

DocIIIB 4.6.1

Kampf G, Kramer A

2004 Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs, Clin

Microbiol Rev 17(4), 863-93

No

DocIIIB 6.2.1

Hyronimus B. 21.11.2006 Surface tests in biofilm mode on nutritive medium with cobiotex 112 biofilm + COBIOTEX

Unpublished

Yes COBIOTEX

Tremoulet F, Duche O, Namane A, Martinie B, Labadie JC,

2002 Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis. Fems

Microbiology Letters, 210 : 25-31.

No N.A.

DocIIIB

6.2.2.

Choy H. 13.05.2003 Pre-conditioned surface tests in biofilm mode

COBIOTEX

Unpublished

Yes COBIOTEX

DocIIIB 6.3

Le Poule M. 23.12.2005 Trial conducted on Cobiotex 112 biofilm Virbac unpublished

Yes COBIOTEX

DocIIIB 7.1.2

J.R. Saunders, T.N.K. Sebunya, and A.D. Osborne

1983 Pulmonary Clearance of Bacillus subtilis Spores in Pigs Can J Comp Med. 47(1): 43–47

No N.A.

DocIIIB 7.2.1

2007 Acute dermal irritation in

rabbits Study No. 33286 TAL Unpublished

Yes COBIOTEX

DocIIIB 7.2.2 2007 Acute eye irritation in rabbits

Study No. 33287 TAL

Unpublished

Yes COBIOTEX

DocIIIB 7.3 Redebel S.A. 2010 Estimation of potential human

exposure to cobiotex 112 Yes COBIOTEX

DocIIIB 7.4 Cabo-sil N.A. Product specification sheet No N.A.

PSS Lactochem N.A. Product specification sheet No N.A.

* Key study