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 Product-type 03 December 2015 strain ISB06
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
2 Bacillus amyloliquefaciens Product-type 03 December 2015 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
Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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.
4 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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).
5 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
6 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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”.
7 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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.”
8 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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) 10 were neither toxic nor pathogenic. The LD50 is >2.3x10 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.
9 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
No studies available, no data requirement for micro-organisms.
Reproduction Toxicity
No studies available, no data requirement for micro-organisms.
Neurotoxicity
No studies available, no data requirement for micro-organisms.
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.
10 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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.
11 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
12 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
13 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
14 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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.
15 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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. 8 Based on the results of the preliminary test an EC50 (48h)-value of 4*10 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 9 9 EbC50-value of 1.21*10 cfu/L and an ErC50-value of 9.14*10 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.
B. amyloliquefaciens is an ubiquitous bacterium commonly recovered from water, soil,
16 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
17 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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. B. amyloliquefaciens is an ubiquitous bacterium commonly recovered from water, soil, 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
18 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
19 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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-
20 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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.
21 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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
22 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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 Confidential reference number Other substance No. Not applicable Minimum purity of the active Confidential substance as manufactured (g/kg or g/L) Identity of relevant impurities No relevant substances and additives (substances of concern) in the active substance as manufactured (g/kg)
Source and biological properties (Annex IIIA, Section 2 and 3) Natural occurrence and Ubiquituous species distribution Isolation methods Cultivation Culture methods Confidential Production methods Confidential Composition of the micro- Confidential organism Methods to preserve seed Confidential stock Relationship to existing Related to but distinct from opportunistic and obligate pathogens 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
23 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
Transmissibility, infective dose No concerns regarding human, animal and plant health and mode of action and could be associated with ISB06 during investigations. information on the nature, The EFSA QPS status takes into account that few identity and stability of toxins 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 ISB06 is resistant to ampicillin. antibiotics
24 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
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 none data with regard to ecotoxicological data none
25 Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
Chapter 2: Methods of Analysis
Analytical methods for the active substance Technical active substance (principle of Physiological, microscopic and molecular method) (Annex IIA, point 4.1) methods. Confidential details Impurities in technical active substance Not relevant (principle of method) (Annex IIA, point 4.1) Analytical methods for residues Soil (principle of method and LOQ) (Annex Not necessary, no relevant residues expected IIA, point 4.2) by CA. Strain-specific detection is confidential. Air (principle of method and LOQ) (Annex Not necessary. Due to the absence of any IIA, point 4.2) evidence of toxicity, pathogenicity or infectivity of Bacillus amyloliquefaciens during inhalation, a limit of determination could not be derived by CA. Strain-specific detection is confidential. Water (principle of method and LOQ) Not necessary, no relevant residues expected (Annex IIA, point 4.2) by CA. Strain-specific detection is confidential. Body fluids and tissues (principle of Not necessary, since not classified as toxic or method and LOQ) (Annex IIA, point 4.2) very toxic. Food/feed of plant origin (principle of Not necessary, no relevant residues expected method and LOQ for methods for by CA. monitoring purposes) (Annex IIIA, point Strain-specific detection is confidential. IV.1) Food/feed of animal origin (principle of Not necessary, no relevant residues expected method and LOQ for methods for by CA. monitoring purposes) (Annex IIIA, point Strain-specific detection is confidential. IV.1)
Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
Chapter 3: Impact on Human Health
Medical data (including medical No adverse health effects reported with strain surveillance on manufacturing plant ISB06; B. amyloliquefaciens enzymes may personnel) 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 No data for strain ISB06, infectivity 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 No data –not required pathogenicity Specific toxicity, pathogenicity and Not skin irritating; infectivity Eye irritation below classification threshold
Bacillus amyloliquefaciens Product-type 03 December 2015 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 Powdery b.p. dissolved in water are applied by Spraying application in livestock buildings 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 Powdery b.p. dissolved in water and applied by Misting application in livestock buildings 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 Primary exposure of non-professionals is not food or feed) expected. Combined Exposure Residues on food or feeding stuffs are not expected at relevant concentrations.
Bacillus amyloliquefaciens Product-type 03 December 2015 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.
Bacillus amyloliquefaciens Product-type 03 December 2015 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- Endpoint Toxicity scale Fish 9 Oncorhynchus mykiss 96 h LC0(96h) 2.4x10 cfu/L
5 Oncorhynchus mykiss 96 h LC0(96h) 2.0x10 cfu/L (cell-free extract) Invertebrates
8 Daphnia magna 48 h EC50(48h) 4x10 cfu/L Algae
9 Scenedesmus subspicatus 72 h EbC50(72h) 1.21x10 cfu/L 9 ErC50(72h) 9.14x10 cfu/L Microorganisms not tested
Bacillus amyloliquefaciens Product-type 03 December 2015 strain ISB06
Effects on earthworms or other soil non-target organisms Acute toxicity to earthworms. LC50(14d): >1x1011cfu/L, Eisenia fetida (Annex IIA / IIIA, point 8.5) Reproductive toxicity to ………………………… not tested (Annex IIIA, point XIII.3.2) Acute toxicity to LC50(14d): >6.25x1010 cfu/L; Poecilus arthropods………………..…….. cupreus (Annex IIA / IIIA, point 8.4) 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 not tested (Annex IIIA, point XIII.3.3) Acute toxicity to birds not tested (Annex IIIA, point XIII.1.1) Dietary toxicity to birds not tested (Annex IIIA, point XIII.1.2) Reproductive toxicity to birds not tested (Annex IIIA, point XIII.1.3)
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
31 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
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 Memb Prod Organi and/o er uct sms Formulati Application Applied amount Remarks: r State nam control on per treatment situati or e led on Count
ry Type Conc meth numb interval g water g 2 2 (a) (c) (d- . od er between as/L L/m as/m (m) f) of as kind min applicati min min min ons (i) (f-h) max max max max (min) (k)
bacter Franc COB Bacteri Po > Spra doub 24 1 kg of COBIOTEX An application icide e IOTE a wd 1.0 ying le hours 112 biofilm + after each 5 product (diluted in X er 10 appli step of 100 liters of cold 112 CF catio cleaning- water) is BIOF U / n is disinfection recommended for ILM g reco (24 hours + mme spraying indoor after nded abiotic surfaces of disinfection . a livestock building stage in order 2 covering 1000 m . 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
32
Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
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 Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published DocI 2.1.2 EC 2014 Review report for the active No Published substance Bacillus amyloliquefaciens subsp. plantarum strain D747 SANCO/11391/2014 rev 1 DocIIA 1.2.7 Chen X.-H., 2009 More than anticipated - No Koumoutsi A., production of antibiotics and Scholz R., other secondary metabolites Borriss R. by Bacillus amyloliquefaciens FZB42. J Mol Microbiol Biotechnol.;16(1-2):14-24 DocIIA 3.1.3 Flindt, M.L.H. 1969 Pulmonary disease due to No N.A. inhalation of derivatives of Bacillus subtilis containing proteolytic enzyme. Lancet i:1177-1181 DocIIA 3.1.3 Johnsen, C.R., 1997 Allergy risk in an enzyme No N.A. Sorensen, T.B., producing plant: a Larsen, A. I., retrospective follow up study. Secher, A.B., Occup. Environm. Med. Andreasen, E., 54:671-675 Kofoed, G.S., Nielsen, L. F., Gyntelberg, F. DocIIA 3.1.3 Larsen, A.I., 2007 Incidence of respiratory No Johnsen, C.R., sensitisation and allergy to Frickmann, J., enzymes among employees in Mikkelsen, S. an enzyme producing plant and the relation to exposure and host factors. Occup. Environ. Med. 64:763-768 DocIIA 3.1.3 Pepys, J., 1969 Allergic reactions of the lungs No N.A. Longbottom, to enzymes of Bacillus subtilis. J.L., Hargreave, Lancet i: 1181-1184 F.E., Faux, J. Doc II B8 EC 2002 TNsG Human Exposure No Published 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“
33 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published http://ecb.jrc.it/biocides] Doc II B8 Warren, N, 2006 Task-based dermal exposure No Published Marquart, H., models for regulatory risk Christopher, Y.; assessment, Ann. Occup. Hyg. Laitinen, J.; Van Vol. 50, 491-503 Hemmen, J. Introduction to soil No microbiology, J. Wiley & Sons, DocIIIA 2.1.1 Alexander, M 1977 Inc., New York, in Final Risk Assessment of B. subtilis Burke SA, Detection of molecular No Wright JD, diversity in Bacillus atrophaeus DocIIIA 2.1.1 Robinson MK, 2004 by Amplified Fragment Length Bronk BV Polymorphism Analysis, Appl Warren RL Env Microbiol 70(5), 2786-90 On the safety of Bacillus No subtilis and B. De Boer AS, DocIIIA 2.1.1 1991 amyloliquefaciens: a review, Diderichsen B Appl Microbiol Biotechnol 36(1), 1-4 Biological control of Sclerotinia No Fernando WGD, sclerotiorum (Lib.) de Bary by Nakkeeran S, DocIIIA 2.1.1 2007 Pseudomonas and Bacillus Zhang Y, species on canola petals, Crop Savchuk S Prot 26, 100-07 Taxonomy of the genus No Bacillus and related genera: DocIIIA 2.1.1 Fritze, D. 2004 The aerobic endospore-forming bacteria, Phytopathology 94, 1245-48 Plant growth-promoting No rhizobacteria as transplant Kokalis-Burelle amendments and their effects DocIIIA 2.1.1 N, Kloepper JW, 2006 on indigenous rhizosphere Reddy MS microorganisms, Appl Soil Ecology 31, 91-100 Lechner S, Mayr Bacillus weihenstephanensis No R, Francic KP, sp. Nov is a new Prub BM, Kaplan psychrotolerant species of the DocIIIA 2.1.1 T, Wieber- 1998 Bacillus cereus group, Int J Sys Gunkel E, Bacteriol 48, 1373-82 Stewart GASB, Scherer S Bacillus pseudomycoides sp. No DocIIIA 2.1.1 Nakamura LK 1998 Nov., Int J Sys Bacteriol 48, 1031-35 Relationship of Bacillus subtilis No clades associated with strains 168 and W23: a proposal for Nakamura LK, Bacillus subtilis subsp. subtilis DocIIIA 2.1.1 Roberts MS, 1999 subsp. nov. and Bacillus Cohan FM subtilis subsp. spizizenii subsp. nov., Int J Sys Bacteriol 49(3), 1211-15
34 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Determination of the target Yes COBIOTEX organisms inhibited by B. DocIIIA 2.2.1 amyloliquefaciens strain ISB Hyronimus B. N.A. 06 COBIOTEX Unpublished Coagulin, a pediocin-like No N.A. Hyronimus, B., DocIIIA 2.2.1 bacteriocin, produced by B. Le Marrec, C. 1998 coagulans. Journal of Applied and Urdaci, C. Microbiology. 85, 42-50. Molecular mechanism of No N.A. Carrillo C., membrane permeabilization Teruel J., DocIIIA 2.2.2 2003 by the peptide antibiotic Aranda F., Ortiz surfactin Biochimica et A. Biophysica Acta 1611 91–97 Microbial Production of No N.A. Surfactants and Their Desai D. and DocIIIA 2.2.2 1997 Commercial Potential Banat I. Microbiology and molecular biology reviews Competition by competitive Yes COBIOTEX exclusion of B. DocIIIA amyloliquefaciens strain ISB Hyronimus B. N.A. 2.2.2* 06 COBIOTEX Unpublished Determination of adhesion of Yes COBIOTEX B. amyloliquefaciens strain ISB 06 on different type of DocIIIA 2.2.2 Hyronimus B. N.A. supports COBIOTEX Unpublished Comparison of protein No N.A. Tremoulet F, patterns of Listeria Duche O, monocytogenes grown in DocIIIA 2.2.2 Namane A, 2002 biofilm or in planktonic mode Martinie B, by proteomic analysis. Fems Labadie JC, Microbiology Letters, 210 : 25- 31. Compartmentalization of gene No expression during Bacillus Hilbert DW, DocIIIA 2.4 2004 subtilis spore formation, Piggot PJ Microbiol Mol Biol Rev, 68(2), 234-62
Roles of Bacillus endospores in No DocIIIA 2.4 Nicholson WL 2002 the environment, Cell Mol Life Sci 59, 410-16
Bacillus and relatives in No DocIIIA 2.5 Logan NA 2011 foodborne illnesses, J Appl Microbiol 112(3), 417-29
Nicholson WL, Resistance of Bacillus No DocIIIA 2.5 2000 Munakanata N, endospores to extreme
35 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Horneck G, terrestrial and extraterrestrial Melosh HJ, environments, Microbiol Mol Setlow P Biol Rev 64(3), 548-72
Weber, D.; In Vitro Susceptibility of No N.A. Susan M. Bacillus spp. to Selected Saviteer; Antimicrobial Agents DocIIIA 2.5 William A. 1998 Antimicrobial agents and Rutala; and chemotherapy, p. 642-645 Charlotte A. Vol. 32, No. 5 Thomann Priest FG, A numerical classification of No DocIIIA 2.6 Goodfellow M, 1988 the genus Bacillus, J Gen Todd C Microbiol, 134(7), 1847-82 Phylogeny and molecular No Rooney AP, taxonomy of the Bacillus Price NPJ, subtilis species complex and DocIIIA 2.6 Ehrhardt C, 2009 description of Bacillus subtilis Swezey JL, subsp. inaquosorum subsp. Bannan JD nov., Intl J Sys Evol Microbiol 59, 2429-36 Genetic exchange between No Duncan KE, Bacillus subtilis and Bacillus Istock CA, licheniformis: variable hybrid DocIIIA 2.7 1989 Graham JB, stability and the nature of Ferguson N bacterial species, Evolution 43(8), 1585-1609 Christiansson A, Toxin production by Bacillus No Naidu AS, cereus dairy isolates in milk at DocIIIA 2.8 Nilsson I, 1989 low temperatures (1989) Appl Wadström T, Env Microbiol, 55(10), 2595- Pettersson HE 2600 European Opinion of the scientific No N.A. Commission committee on animal nutrition Health & on the safety of use of Bacillus Consumer species in animal nutrition Protection (expressed on 17 february Directorate- 2000) DocIIIA 2.8 General 2000 Directorate B – Scientific Health Opinions Unit B3 – management of scientific committees Toxin-producing ability among No From C, Pukall Bacillus spp. outside the R, Schumann P, DocIIIA 2.8 2005 Bacillus cereus group, Appl. Homazabal V, Env. Microbiol, 71(3), 1178-83 Granum PE
Determination of cytotoxicity Yes COBIOTEX to Hela cells of B. DocIIIA 2.8* Hyronimus, B. N.A. amyloliquefaciens strain ISB 06 COBIOTEX
36 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Unpublished Determination of toxins and Yes COBIOTEX haemolytic activity of B. amyloliquefaciens strain ISB DocIIIA 2.8* Hyronimus, B. N.A. 06 COBIOTEX Unpublished Genotyping and Toxigenic No N.A. Potential of Bacillus subtilis Matarante, A.; and Bacillus pumilus Strains Baruzzi, F.; DocIIIA 2.8 2004 Occurring in Industrial and Cocconcelli, P.; Artisanal Cured Sausages and Morea, M. Appl. Environ. Microbiol. 70: 5168-5176 Adhesion and cytotoxicity of No Bacillus cereus and Bacillus thuringiensis to epithelial cells Ramarao N, DocIIIA 2.8 2006 are FlhA and PlcR dependent, Lereclus D respectively (2006) Microbes Inf, 8(6), 1483-91
Determination of MICfrom B. Yes COBIOTEX amyloliquefaciens ISB 06 DocIIIA 2.9 Daniel P. 2007 Genfast Unpublished Determination of MIC of B. Yes COBIOTEX amyloliquefaciens strain ISB06 DocIIIA 2.9 Hyronimus, B. N.A. COBIOTEX Unpublished Criteria for Assessing the No N.A. Opinion of the Safety of Micro-Organisms Scientific DocIIIA 2.9 2003 Resistant to Antibiotics of Committee on Human Clinical and Veterinary Animal Nutrition Importance Quantification of Bacillus Yes Cobiotex amyloliquefaciens ISB 06 spore resistance to DocIIIA 2.10 Le Henaff M 2013 inactivation by (i) wet and dry heat, (ii) UV, (iii) freeze- drying and (iv) four chemical agents Nicholson WL, Resistance of Bacillus No Munakanata N, endospores to extreme DocIIIA 2.10 Horneck G, 2000 terrestrial and extraterrestrial Melosh HJ, environments, Microbiol Mol Setlow P Biol Rev 64(3), 548-72 Differential corrosion rates of No carbon steel by combinations Jack RF, of Bacillus sp., Hafnia alvei DocIIIA 2.11 Ringelberg DB, 1992 and Desulfovibrio gigas White DC established by phospholipid analysis of electrode biofilm, Corr Sci 33(12), 1843-53
37 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Microbial influenced corrosion No Racicot RJ, studies of Bacillus DocIIIA 2.11 Crouch CD, 2007 licheniformis on AA 2024 Ranch ME aluminum alloys, Corr Rev 25(1-2), 97-106 Directive On the protection of workers No N.A. 2000/54/EC of from risks related to exposure the European to biological agents at work DocIIIA 5.1.1 Parlimant and of 2000 (seventh individual directive the Council of within the meaning of Article 18 September 16 (1) of Directive 2000 89/391/EEC) On the safety of Bacillus No N.A. Sietske de Boer, subtilis and B. DocIIIA 5.1.1 A.; Diderichsen, 1991 amyloliquefaciens : a review B. Applied microbiology and biotechnology , vol. 36, pp 1-4 Weber, D.; In Vitro Susceptibility of No N.A. Susan M. Bacillus spp. to Selected Saviteer,; Antimicrobial Agents DocIIIA 5.1.1 William A. 1998 Antimicrobial agents and Rutala, and chemotherapy, p. 642-645 Charlotte A. Vol. 32, No. 5 Thomann Position on SCAN report on No N.A. use of Bacillus species in DocIIIA 5.1.2 AMFEP N.A. animal nutrition (Opinion from SCAN expressed on 17 February 2000) Directive On the protection of workers No N.A. 2000/54/EC of from risks related to exposure the European to biological agents at work DocIIIA 5.1.2 Parlimant and of 2000 (seventh individual directive
the Council of within the meaning of Article 18 September 16 (1) of Directive 2000 89/391/EEC) Weber, D.; In Vitro Susceptibility of No N.A. Susan M. Bacillus spp. to Selected Saviteer,; Antimicrobial Agents DocIIIA 5.1.4 William A. 1998 Antimicrobial agents and Rutala, and chemotherapy, p. 642-645 Charlotte A. Vol. 32, No. 5 Thomann Acute dermal irritation in Yes COBIOTEX DocIIIA rabbits 2007 5.2.1* Study No. 33284 TAL Unpublished Acute eye irritation in rabbits Yes COBIOTEX DocIIIA 2007 Study No. 33285 TAL 5.2.1* Unpublished Duc, H.; Huynh Characterization of Bacillus No N.A. DocIIIA A. Hong; Teresa Probiotics Available for Human 2004 5.2.2.1 M. Barbosa; Use Applied and Adriano O. Environmental Microbiology, p.
38 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Henriques; and 2161–2171 Vol. 70, No. 4 Simon M.Cutting Final report : Acute Oral Yes FZB Toxicity Test of “Bacillus Biotechnik DocIIIA subtilis FZB24” in Rats, IBR GmbH 1994 5.2.2.1* Forschungs GmbH, Project No.: 10-04-0552/00-93, 25/03/1994 Opinion of the Scientific Panel No N.A. on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the product DocIIIA The EFSA “Calsporin”, a preparation of 2006 5.2.2.1 Journal 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 Do bacteria need to be No N.A. DocIIIA Silley, P. 2006 regulated ? Journal of Applied 5.2.2.1 Microbiology ISSN 1364-5072 DocIIIA B. subtilis MBI 600 No N.A. Health Canada 2007 5.2.2.2. J.R. Saunders; Pulmonary Clearance of No N.A. DocIIIA T.N.K. Sebunya, Bacillus subtilis Spores in Pigs 1983 5.2.2.2. and A.D. Can J Comp Med. 47(1): 43– Osborne 47 Acute Pulmonary Toxicity And Yes Cobiotex Pathogenicity To The Rat; HLS DocIIIA 2010 study number : OAK 0001; 5.2.2.2.-01* Huntingdon Life Sciences /14.05.2010 Final report : Acute Dermal Yes FZB Toxicity Test of “Bacillus Biotechnik DocIIIA subtilis FZB24” in Rats, IBR GmbH 1994 5.2.2.3* Forschungs GmbH, Project No.: 10-04-0553/00-93, 25 March 1994. Bacillus amyloliquefaciens ISB Yes Cobiotex 06 : Acute Intraperitoneal DocIIIA Toxicity And Pathogenicity To 2010 5.2.2.3.-01* The Rat; HLS study number : OAK 0002; Huntingdon Life Sciences / 14.05.2010 Final report : Acute Dermal Yes FZB Irritation/Corrosion Test of Biotechnik DocIIIA “Bacillus subtilis FZB24” in GmbH 1994 5.2.2.4 Rabbits, IBR Forschungs GmbH, Project No.: 10-03- 0554/00-93, 18/03/1994 DocIIIA Final report : Acute Eye Yes FZB 1994 5.2.2.4 Irritation/Corrosion Test of Biotechnik
39 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published “Bacillus subtilis FZB24” in GmbH Rabbits, IBR Forschungs GmbH, Project No.: 10-03- 0555/00-93, 05/04/1994. Opinion of the Scientific Panel No N.A. on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the product The EFSA “Calsporin”, a preparation of DocIIIA 5.2.3 2006 Journal 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 J.R. Saunders, Pulmonary Clearance of No N.A. DocIIIA T.N.K. Sebunya, Bacillus subtilis Spores in Pigs 1983 5.2.5.1 and A.D. Can J Comp Med. 47(1): 43– Osborne 47 Weber, D.; In Vitro Susceptibility of No N.A. Susan M. Bacillus spp. to Selected Saviteer,; Antimicrobial Agents DocIIIA 5.2.7 William A. 1998 Antimicrobial agents and Rutala, and chemotherapy, p. 642-645 Charlotte A. Vol. 32, No. 5 Thomann Introduction of a Qualified No N.A Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA1 The EFSA DocIIIA 5.3 2007 Opinion of the Scientific Journal Committee (Question No EFSA-Q-2005- 293) Adopted on 19 November 2007 ABiTEP 1992 Presentation FZB24 / ABiTEP Yes DocIIIA 7.1.1 Confidential data / 1992 DocIIIA Beifort 2010 ABiTEP Declaration Effects Yes 7.1.1, FZB24, 2010 A 7.1.2, A 7.1.3, A 7.2 Buensanteai N., 2008 The Biocontrol Bacterium No Yuen G.Y. and Bacillus amyloliquefaciens Prathuangwong KPS46 Produces Auxin, S. Surfactin and Extracellular DocIIIA 7.1.1 Proteins for Enhanced Growth of Soybean Plant Thai Journal of Agricultural Science 2008, 41(3-4): 101-116 DocIIIA Fritze D. 2004 Taxonomy of the Genus No 7.1.1, Bacillus and Related Genera:
40 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published A 7.1.2 The Aerobic Endospore- Forming Bacteria 2004 Symposium The Nature and Application of Biocontrol Microbes: Bacillus spp. Hallmann J., 1999 Chitin-mediated changes in No Rodríguez- bacterial communities of the Kábana R., soil, rhizosphere and within DocIIIA 7.1.1 Kloepper J.W. roots of cotton in relation to nematode control Soil Biology and Biochemistry 31 (1999) 551-W Kilian M., 2000 FZB24® Bacillus subtilis – No Steiner U., mode of action of a microbial Krebs B., agent enhancing plant vitality, DocIIIA 7.1.1 Junge H., Pflanzenschutz-Nachrichten Schmiedeknecht Bayer 1/00, 1 72–93 G., Hain R. Mc Spadden 2004 Ecology of Bacillus and No Gardener B. B. Paenobacillus spp in agricultural system 2004 DocIIIA 7.1.1 Symposium The Nature and Application of Biocontrol Microbes: Bacillus spp. Palumbo J.D., 2006 Isolation of Bacterial No Baker J.L. and Antagonists of Aspergillus DocIIIA 7.1.1 Mahoney N.E. flavus from Almonds, Microbial Ecology, Volume 52, 45–52 (2006) DocIIIA Priest F.G., 1987 Bacillus amyloliquefaciens sp. No 7.1.1, Goodfellow M., Nov. Norn. Rev. International A 7.1.2, Shute A. and journal of systematical A 7.1.3, Berkeley r.C.W. bacteriolog 1987, 37(1), 69- A 7.2 71 DocIIIA US EPA 1997 Risk assessment B. subtilis, No 7.1.1, 1997 A 7.1.2, DocIIIA 7.1.3, A 7.2 van Veen J.A., 1997 Fate and Activity of No van Overbeek Microorganisms Introduced l.S. and into Soil MICROBIOLOGY AND DocIIIA 7.1.1 van Elsas J.D. MOLECULAR BIOLOGY REVIEWS, June 1997, p. 121– 135 Vol. 61, No. 2 Wang L., 2008 Bacillus velezensis is a later No Lee W., Tai C. heterotypic synonym of and Bacillus amyloliquefaciens; DocIIIA 7.1.1 Kuo H. International Journal of Systematic and Evolutionary Microbiology (2008), 58, 671– 675 Sass A.M., 2008 Diversity of Bacillus-like No DocIIIA 7.1.2 McKew B.A., organisms isolated from deep- Sass H., Fichtel sea hypersaline anoxic
41 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published J., Timmis K.N. sediments Saline Systems and 2008 Page 1 of 11 McGenity T.J. Saunders J.R. , 1983 Pulmonary Clearance of No Sebunya T.N.K. Bacillus subtilis Spores in Pigs, DocIIIA 7.1.2 and Can J Comp Med 1983; 47: Osborne A.D. 43-47. Siefert J.L., 2000 Phylogeny of Marine Bacillus No Larios-Sanz M., Isolates from the Gulf of Nakamura L.K., Mexico CURRENT DocIIIA Slepecky R.A., MICROBIOLOGY Vol. 41 7.1.2, Paul J.H., Moore (2000), pp. 84–88 A 7.2 E.R.B., Fox G.E., Jurtshuk P.Jr. Miranda C.A.C., 2008 Species-level identification of No Martins O.B., Bacillus strains isolates from Mandetta C.M. marine sediments by conventional biochemical, 16S DocIIIA 7.1.2 rRNA gene sequencing and inter-tRNA gene sequence lengths analysis Antonie van Leeuwenhoek (2008) 93:297– 304 DocIIIA ABiTEP 2007 Safety Data Sheet of Yes 7.1.2, RhizoVital 42 li. A 7.2 Alken-Murray ALKEN CLEAR-FLO® 1006 No DocIIIA 7.1.2 Corporation (http://www.alken- murray.com/Vibrio.htm) Bioway BON FEED / BON POND / BON No DocIIIA 7.1.2 Technology WATER Earl A.M., Losick 2008 Ecology and genomics of No DocIIIA 7.1.2 R. and Kolter R. Bacillus subtilis Trends in Microbiology 2008 Vol.16 No.6 EFSA 2008 Safety and efficacy of No Ecobiol® (Bacillus amyloliquefaciens) as feed additive for chickens for DocIIIA 7.1.2 fattening Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed The EFSA Journal (2008) 773, 1-13 Gontang E.A., 2007 Phylogenetic Diversity of No Fenical W. and Gram-Positive Bacteria Jensen P.R. Cultured from Marine DocIIIA 7.1.2 Sediments APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2007, p. 3272–3282 Vol. 73, No. 10 Haas D. and 2005 BIOLOGICAL CONTROL OF No Defago G SOIL-BORNE PATHOGENS BY DocIIIA 7.1.2 FLUORESCENT PSEUDOMONADS 2005 doi:10.1038/nrmicro1129
42 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Published online 10 March 2005.GY DocIIIA 7.1.2 Hong H.A., 2005 The use of bacterial spore No A 8.1 Duc L.H., formers as probiotics. FEMS A 8.2.3 Cutting S.M. Microbiology Reviews 29 A 8.2.4 (2005) 813–835 Ivanova E.P., 1999 Characterization of Bacillus No Vysotskii M.V., strains of marine origin Svetashev V.I., INTERNATL MICROBIOL Nedashkovskaya (1999) 2:267–271 O.I., Gorshkova DocIIIA 7.1.2 N.M., Mikhailov V.V., Yumoto N., Shigeri Y., Taguchi T., Yoshikawa S. Kamala-Kannan 2008 Metal Tolerance and Antibiotic No S. and Resistance of Bacillus species DocIIIA Lee K. J. isolated from Sunchon Bay 7.1.2, Sediments, South Korea A 7.2 Biotechnology 7 (1) : 149- 152, 2008 DocIIIA 7.1.2 Novozymes PondPlus® No EFSA 2007 Introduction of a Qualified No Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA Opinion of DocIIIA 7.2 the Scientific Committee (Question No EFSA-Q-2005- 293) Adopted on 19 November 2007 The EFSA Journal (2007) 587, 1-16 Fossum K., 1986 Isolation of Bacillus subtilis in No N.A. Herikstad H., Connection with Bovine DocIIIA 8.1 Binde M., Mastitis, Nord. Vet. Med. Pettersen K.-E. 1986, 38, 233-236 DocIIIA 8.1 Gatesoupe F.J. 1999 The use of probiotics in No N.A. A 8.2.2 aquaculture. Aquaculture 180; A 8.2.3. 147–165 A 8.2.4 Nicholson W. L. 2002 Roles of Bacillus endospores in No N.A. DocIIIA 8.1 the environment, CMLS, Cell. A 8.2.4 Mol. Life Sci. 59, 410- 416 1994 Acute toxicity testing in fish, Yes DocIIIA 8.2.1 test article: Bacillus subtilis FZB 24 Jahnke M. 1994 Acute toxicity in Daphnia Yes DocIIIA 8.2.2 magna, test article: Bacillus subtilis FZB 24 Verschuere L., 2000 Probiotic Bacteria as Biological No N.A. Rombaut G., Control Agents in Aquaculture. DocIIIA 8.2.2 Sorgeloos P., Microbiology and Molecular A 8.2.3 Verstraete W. Biology Reviews, Dec. 2000, p. 655–671 Vol. 64, No. 4
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Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Jahnke M. 1994 Algal growth inhibition test, Yes DocIIIA 8.2.3 test article: Bacillus subtilis FZB 24 Jakobsons B. 2005 Biological treatment of No N.A. DocIIIA 8.3 chalkbrood in honey bees Braunmiller 1996 Laufkäfertoxizitätsprüfung, Yes Pflanzenschutzamt Berlin, DocIIIA 8.4 Report No. P15/95, 12/12/1996 Braunmiller 1996 Regenwurmtoxizitätsprüfung – Yes Vorprüfung, DocIIIA 8.5- Pflanzenschutzamt Berlin, 01 Report No. E15.1/96, 12/12/1996 Braunmiller 1996 Regenwurmtoxizitätsprüfung – Yes Hauptprüfung, DocIIIA 8.5- Pflanzenschutzamt Berlin, 02 Report No. E15.2/96, 20/12/1996 Fernando 2007 Biological control of Sclerotinia No N.A. W.G.D., sclerotiorum (Lib.) de Bary by DocIIIA 8.7.1 Nakkeeran S., Pseudomonas and Bacillus Zhang Y., species on canola petals, Crop Savchuk S. Protection 26 (2007), 100-107 Kokalis-Burelle 2006 Plant growth-promoting No N.A. N., rhizobacteria as transplant Kloepper J.W., amendments and their effects DocIIIA 8.7.1 Reddy M.S. on indigenous rhizosphere microorganisms, Applied Soil Ecology 31(2006), 91-100 Liang L., N., 1982 Fate in Model Ecosystems of No N.A. Sinclair J., L., Microbial Species of Potential DocIIIA 8.7.1 Mallory L., M., Use in Genetic Engineering, Alexander M. Appl. Environ. Microbiol., Sept. 1982, 708-714 Bose B, 2011 Regulation of horizontal gene No Grossman AD transfer in Bacillus subtilis by activation of a site-specific protease, J Bac 193(1), 22-39 Butcher BG, 2006 Identification of Bacillus No Helmann JD subtilis sigma-w-dependent genes that provide intrinsic DocIIIB 3.11 resistance to antimicrobial compounds produced by Bacilli, Mol Microbiol 60(3), 765-82 Koonin EV, 2001 Horizontal gene transfer in No Makarova KS, prokaryotes: Quantification Aravind L and classification, Annu Rev Microbiol 55, 709-42 Hong H., Le 2005 The use of bacterial spore No N.A. Hong Duc, formers as probiotics.
Simon M. FEMS Microbiology Reviews 29 DocIIIB 3.12 Cutting. 813–835 Tannock G. 2004 A Special Fondness for No N.A.
44 Bacillus amyloliquefaciens Product-type 03 November2015 strain ISB06
Section No Author(s) Year Title. Data Owner / Reference Source (where different Protection No from company) Claimed Company, Report No. (Yes/No) GLP (where relevant) / (Un)Published Lactobacilli Applied And Environmental Microbiology, p. 3189–3194 Vol. 70, No. 6 Kampf G, 2004 Epidemiologic background of No Kramer A hand hygiene and evaluation DocIIIB 4.6.1 of the most important agents for scrubs and rubs, Clin Microbiol Rev 17(4), 863-93 Hyronimus B. 21.11.2006 Surface tests in biofilm mode Yes COBIOTEX on nutritive medium with cobiotex 112 biofilm + COBIOTEX Unpublished DocIIIB 6.2.1 Tremoulet F, 2002 Comparison of protein No N.A. Duche O, patterns of Listeria Namane A, monocytogenes grown in Martinie B, biofilm or in planktonic mode Labadie JC, by proteomic analysis. Fems Microbiology Letters, 210 : 25- 31. Choy H. 13.05.2003 Pre-conditioned surface tests Yes COBIOTEX DocIIIB in biofilm mode 6.2.2. COBIOTEX Unpublished Le Poule M. 23.12.2005 Trial conducted on Cobiotex Yes COBIOTEX 112 biofilm DocIIIB 6.3 Virbac unpublished J.R. Saunders, 1983 Pulmonary Clearance of No N.A. T.N.K. Sebunya, Bacillus subtilis Spores in Pigs DocIIIB 7.1.2 and A.D. Can J Comp Med. 47(1): 43– Osborne 47 2007 Acute dermal irritation in Yes COBIOTEX rabbits DocIIIB 7.2.1 Study No. 33286 TAL Unpublished 2007 Acute eye irritation in rabbits Yes COBIOTEX DocIIIB 7.2.2 Study No. 33287 TAL Unpublished Redebel S.A. 2010 Estimation of potential human Yes COBIOTEX DocIIIB 7.3 exposure to cobiotex 112 Cabo-sil N.A. Product specification sheet No N.A. DocIIIB 7.4 PSS Lactochem N.A. Product specification sheet No N.A.
* Key study
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