Conclusion on the peer review of the pesticide risk assessment of the active substance Streptomyces K61 (formerly Streptomyces griseoviridis) 1

Transcription

1 EFSA Journal 2013;11(1):3061 CONCLUSION ON PESTICIDE PEER REVIEW Conclusion on the peer review of the pesticide risk assessment of the active substance Streptomyces K61 (formerly Streptomyces griseoviridis) 1 ABSTRACT an Food Safety Authority 2 an Food Safety Authority (EFSA), Parma, Italy The conclusions of the an Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State, Estonia, for the pesticide active substance Streptomyces K61 are reported. The context of the peer review was that required by Commission Regulation (EC) No 2229/2004 as amended by Commission Regulation (EC) No 1095/2007 and Commission Regulation (EU) No 114/2010. The conclusions were reached on the basis of the evaluation of the representative uses of Streptomyces K61 as a fungicide on fruiting-, leaf- and brassica vegetables, fresh herbs, ornamentals, cut flowers, seedlings, bulbs, and vegetable, ornamental and herb seeds. The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the available studies and literature in the dossier peer reviewed, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified. an Food Safety Authority, 2013 KEY WORDS Streptomyces K61, Streptomyces griseoviridis, peer review, risk assessment, pesticide, fungicide 1 On request from the an Commission, Question No EFSA-Q , approved on 18 December Correspondence: pesticides.peerreview@efsa.europa.eu Suggested citation: an Food Safety Authority; Conclusion on the peer review of the pesticide risk assessment of the active substance Streptomyces K61 (formerly Streptomyces griseoviridis). EFSA Journal 2013;11(1):3061. [40 pp.] doi: /j.efsa Available online: an Food Safety Authority, 2013

2 SUMMARY Streptomyces K61 is one of the 295 substances of the fourth stage of the review programme covered by Commission Regulation (EC) No 2229/2004, as amended by Commission Regulation (EC) No 1095/2007, and Commission Regulation (EU) No 114/2010. Streptomyces K61 was included in Annex I to Directive 91/414/EEC on 1 May 2009 pursuant to Article 24b of the Regulation (EC) No 2229/2004 (hereinafter referred to as the Regulation ) and has subsequently been deemed to be approved under Regulation (EC) No 1107/2009, in accordance with Commission Implementing Regulation (EU) No 540/2011, as amended by Commission Implementing Regulation (EU) No 541/2011. In accordance with Article 25a of the Regulation, as amended by Commission Regulation (EU) No 114/2010, the an Food Safety Authority (EFSA) is required to deliver by 31 December 2012 its view on the draft review report submitted by the an Commission in accordance with Article 25(1) of the Regulation. This review report was established as a result of the initial evaluation provided by the designated rapporteur Member State in the Draft Assessment Report (DAR). The EFSA therefore organised a peer review of the DAR. The conclusions of the peer review are set out in this report. Estonia being the designated rapporteur Member State submitted the DAR on Streptomyces K61 in accordance with the provisions of Article 22(1) of the Regulation, which was received by the EFSA on 30 April The peer review was initiated on 25 April 2008 by dispatching the DAR to the notifier Verdera Oy, and on 17 March 2009 to the Member States for consultation and comments. Following consideration of the comments received on the DAR, it was concluded that there was no need to conduct an expert consultation and EFSA should deliver its conclusions on Streptomyces K61. The conclusions laid down in this report were reached on the basis of the evaluation of the representative uses of Streptomyces K61 as a fungicide on fruiting-, leaf- and brassica vegetables, fresh herbs, ornamentals, cut flowers, seedlings, bulbs, and vegetable, ornamental and herb seeds, as proposed by the notifier. Full details of the representative uses can be found in Appendix A to this report. In the areas of identity of the microorganism, biological properties, physical and technical properties and methods of analysis, the following data gaps were identified: identification of the species and its relationship to human pathogens; information to show compliance with international standards for microbial contamination; test for production of toxins/secondary metabolites in the manufacture and use of the microorganism; role of antibiotics in the mode of action; information to demonstrate that the methods of analysis are specific to strain level, and validation for the other methods; adhesion to and distribution on seed (for seed treatment use only). In the area of mammalian toxicology, data gaps were identified for the identification of secondary metabolites/toxins/antibiotics potentially present in the product and after application, and for a further literature search on the pathogenic potential of Streptomyces species. No signs of toxicity were observed in toxicity studies conducted by the oral route in rats, however high toxicity (mortality) upon intratracheal administration in rats and adverse effects upon inhalation in humans were observed, the cause of which is unknown (allergic reaction or inhalative toxicity, due to the microorganism itself or toxin/secondary metabolite). As no risk assessment could be performed, no conclusion could be reached on operator, worker or bystander exposure with regard to the representative uses of Streptomyces K61, and this was identified as a critical area of concern. For residues the issue of toxins/secondary metabolites production and pathogenicity needs further consideration and the risk assessment cannot be finalised. No information has been provided in relation to potential interference of Streptomyces K61 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC. No information has been provided on the potential transfer of genetic material from Streptomyces K61 EFSA Journal 2013;11(1):3061 2

3 to other organisms. Data or an assessment to demonstrate that transfer does not occur, or in case of occurrence will not lead to unacceptable effects on human and animal health, and on the environment is required. The information on fate and behaviour in the environment is sufficient to characterise the competitiveness / persistence of Streptomyces K61 in soil and water. However on the basis of the available information it is not possible to conclude on the potential contamination of groundwater by the relevant metabolites heptaene polyenes and indole-3-acetic acid with respect to the regulatory limit of 0.1 µg /L, or the potential persistence of heptaene polyenes and indole-3-acetic acid in soil, or confirm that they will not be formed above natural background levels in soil. The risk assessment for non-target organisms from toxins/secondary metabolites was not sufficiently addressed and a data gap was identified. For the representative field uses the risk from Streptomyces K61 to birds, mammals, aquatic organisms, bees, earthworms and biological methods of sewage treatment was concluded to be low, whereas the risk assessment could not be finalised on the basis of the available data for non-target arthropods, soil microorganisms, and non-target plants. A low risk was concluded for the representative glasshouse uses, with the exception of biological methods of sewage treatment, for which a data gap was identified. EFSA Journal 2013;11(1):3061 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background... 5 The active substance and the formulated product... 7 The identity of the microorganism and the properties of the formulated product... 7 Conclusions of the evaluation Identity of the microorganism/biological properties/physical and technical properties and methods of analysis Mammalian toxicity Residues Environmental fate and behaviour Fate and behaviour in the environment of the microorganism Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under relevant environmental conditions Ecotoxicology Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental compartments Soil round water Surface water and sediment Air List of studies to be generated, still ongoing or available but not peer reviewed Particular conditions proposed to be taken into account to manage the risk(s) identified Concerns Issues that could not be finalised Critical areas of concern Overview of the concerns identified for each representative use considered References Appendices Abbreviations EFSA Journal 2013;11(1):3061 4

5 BACKROUND Streptomyces K61 is one of the 295 substances of the fourth stage of the review programme covered by Commission Regulation (EC) No 2229/2004, 3 as amended by Commission Regulation (EC) No 1095/ and Commission Regulation (EU) No 114/ Streptomyces K61 was included in Annex I to Directive 91/414/EEC 6 on 1 May 2009 pursuant to Article 24b of the Regulation (EC) No 2229/2004 (hereinafter referred to as the Regulation ) and has subsequently been deemed to be approved under Regulation (EC) No 1107/2009, 7 in accordance with Commission Implementing Regulation (EU) No 540/2011, 8 as amended by Commission Implementing Regulation (EU) No 541/ In accordance with Article 25a of the Regulation, as amended by Commission Regulation (EU) No 114/2010 the an Food Safety Authority (EFSA) is required to deliver by 31 December 2012 its view on the draft review report submitted by the an Commission in accordance with Article 25(1) of the Regulation (an Commission, 2008). This review report was established as a result of the initial evaluation provided by the designated rapporteur Member State in the Draft Assessment Report (DAR). The EFSA therefore organised a peer review of the DAR. The conclusions of the peer review are set out in this report. Estonia being the designated rapporteur Member State submitted the DAR on Streptomyces K61 in accordance with the provisions of Article 22(1) of the Regulation, which was received by the EFSA on 30 April 2007 (Estonia, 2007). The peer review was initiated on 25 April 2008 by dispatching the DAR to the notifier Verdera Oy, and on 17 March 2009 to the Member States for consultation and comments. In addition, the EFSA conducted a public consultation on the DAR. The comments received were collated by the EFSA and forwarded to the RMS for compilation and evaluation in the format of a Reporting Table. The notifier was invited to respond to the comments in column 3 of the Reporting Table. The comments were evaluated by the RMS in column 3 of the Reporting Table. The scope of the peer review was considered in a telephone conference between the EFSA, the RMS, and the an Commission on 10 October On the basis of the comments received and the RMS evaluation thereof it was concluded that there was no need to conduct an expert consultation. The outcome of the telephone conference, together with EFSA s further consideration of the comments is reflected in the conclusions set out in column 4 of the Reporting Table. All points that were identified as unresolved at the end of the comment evaluation phase and which required further consideration, including the additional information to be submitted by the notifier, were compiled by the EFSA in the format of an Evaluation Table. 3 Commission Regulation (EC) No 2229/2004 of 3 December 2004 laying down further detailed rules for the implementation of the fourth stage of the programme of work referred to in Article 8(2) of Council Directive 91/414/EEC. OJ L 379, , p Commission Regulation (EC) No 1095/2007 of 20 September 2007 amending Regulation (EC) No 1/2002 laying down further detailed rules for the implementation of the third stage of the programme of work referred to in Article 8(2) of Council Directive 91/414/EEC and Regulation (EC) No 2229/2004 laying down further detailed rules for the implementation of the fourth stage of the programme of work referred to in Article 8(2) of Council Directive 91/414/EEC. OJ L 246, , p Commission Regulation (EU) No 114/2010 of 9 February 2010 amending Regulation (EC) No 2229/2004 as regards the time period granted to EFSA for the delivery of its view on the draft review reports concerning the active substances for which there are clear indications that they do not have any harmful effects. OJ L 37, , p Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230, , p. 1-32, as last amended. 7 Regulation (EC) No 1107/2009 of the an Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, , p Commission Implementing Regulation (EU) No 540/2011 of 25 May 2011 implementing Regulation (EC) No 1107/2009 of the an Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 541/2011 of 1 June 2011 amending Implementing Regulation (EU) No 540/2011 implementing Regulation (EC) No 1107/2009 of the an Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p EFSA Journal 2013;11(1):3061 5

6 The conclusions arising from the consideration by the EFSA, and as appropriate by the RMS, of the points identified in the Evaluation Table, together with the outcome of the expert discussions where these took place, were reported in the final column of the Evaluation Table. A final consultation on the conclusions arising from the peer review of the risk assessment took place with Member States via a written procedure in November This conclusion report summarises the outcome of the peer review of the risk assessment on the active substance and the representative formulation evaluated on the basis of the representative uses as a fungicide on fruiting-, leaf- and brassica vegetables, fresh herbs, ornamentals, cut flowers, seedlings, bulbs, and vegetable, ornamental and herb seeds, as proposed by the notifier. A list of the relevant end points for the active substance as well as the formulation is provided in Appendix A. In addition, a key supporting document to this conclusion is the Peer Review Report, which is a compilation of the documentation developed to evaluate and address all issues raised in the peer review, from the initial commenting phase to the conclusion. The Peer Review Report (EFSA, 2012) comprises the following documents, in which all views expressed during the course of the peer review, including minority views, can be found: the comments received on the DAR, the Reporting Table (17 October 2011), the Evaluation Table (17 December 2012), the comments received on the assessment of the points of clarification (where relevant), the comments received on the draft EFSA conclusion. iven the importance of the DAR including its addendum (compiled version of November 2012 containing all individually submitted addenda (Estonia, 2012)) and the Peer Review Report, both documents are considered respectively as background documents A and B to this conclusion. EFSA Journal 2013;11(1):3061 6

7 THE ACTIVE SUBSTANCE AND THE FORMULATED PRODUCT THE IDENTITY OF THE MICROORANISM AND THE PROPERTIES OF THE FORMULATED PRODUCT The strain under review is K61. This strain was originally thought to be Streptomyces griseoviridis however this was shown to be incorrect and the notifier considers that this is a new species and is different to all the currently named species. K61 was originally isolated from Sphagnum peat in Finland. The representative formulated product is, a wettable powder formulation () containing CFU/g (350- g/kg). The representative uses evaluated are as a fungicide on fruiting-, leaf- and brassica vegetables, fresh herbs, ornamentals, cut flowers, seedlings, bulbs, and vegetable, ornamental and herb seeds. Full details of the AP can be found in the list of end points in Appendix A. CONCLUSIONS OF THE EVALUATION 1. Identity of the microorganism/biological properties/physical and technical properties and methods of analysis. Strain K61 is lodged in the Deutsche Sammlung von Mikro-organismen und Zellkulturen mbh (DSMZ) with accession number DSM It is not known if this strain is a human pathogen. Some species of Streptomyces are human pathogens. The strain is able to grow at 37 C. A data gap to identify the strain and to consider the relationship with known human pathogens has been concluded. It is possible that this strain produces some toxins. The manufacturing method for this strain may result in the formation of toxins/secondary metabolites, and a data gap has been identified for a batch analysis study to identify and quantify these compounds. The content of contaminating microorganisms was not fully addressed and a data gap has been identified. For these reasons the specification for toxins and contaminating microorganisms is not finalised. It is clear from the information provided that antibiotics are involved in the mode of action but it is not clear to what extent. A data gap was identified to further investigate the role of antibiotics in the mode of action. For the representative use as a seed treatment, a data gap was identified with regard to adhesion to and distribution on treated seed. Several methods were proposed to identify the organism to strain level but it was not demonstrated that they would be capable of doing this and a data gap was identified. For all the other methods no validation was provided and this was also identified as a data gap. 2. Mammalian toxicity No detailed analysis of the batches used in the toxicological studies is available. The batches were stated to be compliant with the technical specification, which has not been finalised in section 1 (see data gaps in section 1). A data gap has been concluded on the identification of secondary metabolites/toxins/antibiotics potentially present in the product and after application of the product. Streptomycetes show pronounced antibiotic activity. Streptomyces K61 was found to produce an aromatic polyene with antibiotic activity in vitro. The metabolite has been characterised as an aromatic heptaene with a similar structure to candicidin, a macrolide antibiotic used in human medicine EFSA Journal 2013;11(1):3061 7

8 obtained from Streptomyces griseus. Many commercially available antibiotics for treatment of humans and animals have been isolated from Streptomyces species. No information has been provided on the potential transfer of genetic material from Streptomyces K61 to other organisms. This may be a concern since some species of Streptomyces are human or animal pathogens, and Streptomyces K61 appears to be a new species within the Streptomyces species and could not be identified to the strain level (see data gap in section 1). No clinically significant findings or symptoms were associated with the occupational exposure of dust in protected manufacturing personnel. Epidemiological data indicated the appearance of two instances of allergic reactions (in unprotected individuals), one in the laboratory of the notifier with symptoms of mild alveolar reaction in lungs, the other was recorded with respiratory symptoms while treating seeds with where symptoms disappeared within one hour. According to the notifier Streptomyces species are not known to cause infections in immune-competent humans, but may be opportunist in immuno-compromised individuals. As these assumptions have not been fully addressed and would clarify the potential for pathogenicity of the different Streptomyces species a data gap has been identified for a further literature search on this issue, with particular focus on the clinical conditions under which Streptomyces species have been isolated from infected humans or animals. The microorganism is capable of growing at human body temperature. Toxicological studies were carried out using the formulated product ( ) as its production is a continuous process and the technical grade of MPCA is only a hypothetical stage. Considering that hazard statements according to Regulation (EC) No. 1272/ are not appropriate for microorganisms, a warning phrase microorganisms may have the potential to provoke sensitising reactions should be applied. Furthermore, was found to be sensitising in a Magnusson & Kligman maximisation test. Low acute toxicity was found upon oral administration of to rats, raising no concern when the microorganism is ingested, however, mortality was observed when administered intratracheally (ca. 50% at 2-3 x 10 8 CFU/kg bw) or intraperitoneally (ca. 50% at 1.0 x 10 8 CFU/kg bw). Mortality observed upon intraperitoneal administration occurred with both high dose microorganism and autoclaved microorganism suggesting a toxic response rather than an infective process. However upon intratracheal administration (route of exposure possibly more relevant to human exposure), almost all mortality was related to the viable microorganism and not the heat-killed microorganism, indicating a high toxicity potential of the test substance by inhalation. Although some studies were considered inadequate for estimation of infectivity and pathogenicity, overall it was concluded that there were no indications of infectivity or pathogenicity related to administration to test animals. No adverse effects were observed in a 28-day oral toxicity study in rat up to the dose level of 5 x 10 8 CFU/kg bw per day. was found to be negative in a bacterial mutation assay. The crude extract and exotoxins were not tested for their mutagenic properties, and this may need to be re-considered if toxins/secondary metabolites are identified from the manufactured product or after application. Considering the concern raised by the acute intratracheal studies showing toxic effects in rats, and the uncertainties regarding the potential pathogenicity in humans, an exposure risk assessment is needed for operators, workers and bystanders. However, the data package was insufficient to derive reference values, and therefore the operator, worker and bystander exposure risk assessment could not be conducted. Therefore no conclusion could be reached on the risk related to exposure arising from the representative uses of Streptomyces K61, and this was identified as a critical area of concern. 10 Regulation (EC) No 1272/2008 of the an Parliament and of the Coucnil of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. OJ L 353, , p EFSA Journal 2013;11(1):3061 8

9 3. Residues Peer review of the pesticide risk assessment of the active substance Streptomyces K61 Streptomyces species are known to produce many secondary metabolites. In one residue study heptaenes were investigated but higher levels were found in the controls than in the treated crops. It is not clear why this was the case and the study is questionable as there should have been a sterile control. The consumer risk assessment cannot be finalised until the outstanding issues on toxins/secondary metabolites and the pathogenicity to humans are addressed and it is confirmed by the toxicological assessment that a quantitative consumer risk assessment is not necessary for the edible uses. 4. Environmental fate and behaviour 4.1. Fate and behaviour in the environment of the microorganism No information has been provided in relation to potential interferences of Streptomyces K61 with the analytical systems for the control of the quality of drinking water provided for in Council Directive 98/83/EC 11 (See specific Annex VI decision making criteria in Council Directive 2005/25/EC 12 ), and consequently a data gap was identified. No information has been provided on the potential transfer of genetic material from Streptomyces K61 to other organisms. However, actinomycetes are known to have the capacity to transfer genetic material through the exchange of plasmids (Ravel, J. et al (2000)) including plasmids coding for antibiotic production (Jorgensen, H. et al (2009)). Therefore, data or an assessment to demonstrate that this transfer does not occur, or in case of occurrence will not lead to unacceptable effects on the environment or human and animal health is required (See specific Annex VI decision making criteria in Council Directive 2005/25/EC), and a data gap was identified. Studies on persistence and multiplication in soil of Streptomyces K61 (laboratory incubations in five soils) indicated that viable cells of the strain decline in soil after application, with colony forming units of Streptomyces not being isolatable after 3 months when there is competition for nutrients from fungal species (Fusarium sp. were used in these investigations). In these experiments Streptomyces K61 was not competitive. Satisfactory information on the natural background levels of Streptomyces sp. or other actinomycetes in the upper layers of temperate soils (i.e. the primary data sources that report the original observations or measurements,) was not present in the notifier s dossier. Such information would be important to support some of the statements made by the notifier that use of the product as proposed would result in a maximum of 1% increase in the total Streptomycete soil population, and so would not significantly increase actinomycete amounts in the soil ecosystem. A data gap was identified for this information. Studies on persistence and multiplication in water of Streptomyces K61 indicated that viable cells of the strain survived for only 4-14 days in non-sterile tap water. In these experiments, where nutrient levels would be lower than in natural water systems, Streptomyces K61 was not competitive. With respect to the mobility of the microorganism, Streptomyces K61 moves by growing or when mechanically transported by water or arthropods. Transfer via air of spores may occur during spray application to soil before incorporation. The notifier considered that in air cells and spores will become rapidly non-viable as a consequence of exposure to solar irradiation, however, they did not present results of any test observations or measurements to demonstrate that Streptomyces K61 does not survive on the surface of leaves or flowers exposed to direct sunlight and other ambient environmental conditions. Consequently a data gap was identified. 11 Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. OJ L 330, , p Council Directive 2005/25/EC of 14 March 2005 amending Annex VI to Directive 91/414/EEC as regards plant protection products containing micro-organisms. OJ L 90, , p EFSA Journal 2013;11(1):3061 9

10 4.2. Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under relevant environmental conditions Studies where Streptomyces K61 was added to sterilised Sphagnum peat and incubated for 7 weeks resulted in antibiotic heptaene, polyenes, being identified. The heptaenes were detected at up to 1.2 IU g -1 substrate which was equated to 0.57mg candidicin equivalents/kg growing substrate or ca. 420 g/ha. (when an IU is mg of candicidin and incorporation over 5cm soil and soil bulk density of 1.5g/cm 3 is assumed). Heptaene, polyenes were not detected in the untreated controls. This level of environmental exposure of antibiotic compounds being potentially produced indicates that a more quantitative consideration for groundwater exposure assessment is needed for this class of compounds, which are expected to exhibit antifungal and possibly antibacterial activity. Data on the soil degradation rate and adsorption of heptaene, polyene compounds would appear to be necessary for such an assessment. Investigations on the level of heptaene, polyene compounds that may be produced in non-sterile growing media would also be important information for this assessment. Consequently a data gap was identified. Satisfactory information on the natural background levels of Streptomyces sp. or other actinomycetes in the upper layers of temperate soils was not available in the notifier s dossier. Such information would be important to support some of the statements made by the notifier that use of the product as proposed would not result in amounts of heptaene, polyene compounds above those that may be naturally present without application of Streptomyces K61 containing products. Consequently a data gap was identified for this information. A groundwater exposure assessment was not available for the plant growth regulator compound indole-3-acetic acid, which was demonstrated to have the potential to be formed by Streptomyces K61 in vitro. Some observation / measurement of levels that might be formed in soil / growing substrate / confirmation that formation is negligible in the situation of use, would be the first step in such an assessment. Consequently a data gap was identified for this information. Predicted environmental concentrations (PEC) in soil and surface water for Streptomyces K61 (considering aerial deposition as a result of spray drift for the surface water PEC) are presented in Appendix A. The available surface water PEC are expected to cover the exposure that will result from the other methods of application being assessed (seed treatment, drench, application via drip irrigation and bulb dipping / soaking). 5. Ecotoxicology The risk assessment was based on the following documents: an Commission (2002a, 2002b, 2002c) and SETAC (2001). The potential for production of secondary metabolites/toxins under the conditions of use, in soil and other growth media is open (see Sections 1 and 4). Therefore, the risk assessment for non-target organisms for secondary metabolites/toxins is also open. This is considered to be relevant for both the field and glasshouse representative uses (all groups of non-target organisms) where exposure to aquatic environments and soil cannot be excluded. For Streptomyces K61, toxicity and pathogenicity studies for birds were available and no signs of infection or pathogenicity were observed. For the representative drench, drip irrigation and incorporation uses a low risk to birds was concluded on the basis of the TER values. For the representative seed treatment uses the resulting TER values were not sufficient to demonstrate a low risk. However, given that no treatment-related mortality occurred in the avian studies, and the fact that there were no indications of infectivity and pathogenicity, overall, a low risk to birds was concluded. A risk assessment for the representative drench, drip and incorporation uses was sufficient to demonstrate a low risk to mammals. The risk assessment for the representative seed treatment was not sufficient to demonstrate a low risk to mammals. However, as no indications of infectivity or pathogenicity via oral exposure were observed in the available mammalian toxicology data (please EFSA Journal 2013;11(1):

11 refer to Section 2), overall a low risk to wild mammals was concluded. It should be noted that mortality occurred when Streptomyces K61 was administered intratracheally to rats, however, the cause of the mortality is unclear. The risk to wild terrestrial vertebrates via inhalation is not normally assessed in the ecotoxicological risk assessment. Furthermore, it is not known whether the intratracheal route of exposure is relevant in the field. Overall, for the representative uses, the risk from inhalation exposure is likely to be low. For Streptomyces K61, toxicity and pathogenicity studies for fish and Daphnia magna were available and no signs of infection or pathogenicity were observed. A toxicity study with algae was also available. A risk assessment for fish and aquatic invertebrates using the surface water PEC value calculated for the representative outdoor uses indicated a low risk to aquatic organisms. Overall, on the basis of the risk assessment and the lack of indications of infectivity or pathogenicity a low risk to aquatic organisms from Streptomyces K61 was concluded. For Streptomyces K61, a toxicity and pathogenicity study for bees was available and no signs of infection or pathogenicity were observed. In the oral toxicity study the amount of test substance consumed by the bees was not measured and therefore the resulting LD50 value is only an estimation. However, on the basis of low exposure to bees for the representative uses of Streptomyces K61 a low risk to bees was concluded. No non-target arthropod data were available and therefore a risk assessment could not be performed. The representative field uses of Streptomyces K61 (drip irrigation, drench and soil incorporation to field vegetables and ornamentals and as a seed treatment on vegetable, herb and ornamental seeds) will result in exposure to soil-dwelling arthropods, and therefore a risk assessment is required. A data gap was identified for further information on the natural background levels of Streptomyces K61 in soil (see Section 4). In the absence of reliable information demonstrating that the representative uses of Streptomyces K61 will not result in greater exposure to soil arthropods compared to natural background levels it was not possible to conclude a low risk to non-target arthropods. Furthermore, for the representative field uses via incorporation the product will be applied as a spray application. The spray application will lead to off-field exposure of soil-dwelling and foliar-dwelling arthropods. Therefore, a data gap is identified to address the risk to non-target arthropods for the representative field uses. The available earthworm data did not indicate any signs of infection or pathogenicity. A low risk to earthworms was concluded for Streptomyces K61. No soil microorganism data were available and therefore a risk assessment could not be performed. In the absence of reliable information demonstrating that the representative uses of Streptomyces K61 will not result in greater exposure to soil microorganisms compared to natural background levels it was not possible to conclude a low risk to soil microorganisms. Therefore, a data gap is identified to address the risk to soil microorganisms for the representative field uses. Reduced germination rates were reported in the available non-target plant data, however, details of the application rate which caused the reduced germination rates were not reported. For the representative field uses via incorporation the product will be applied as a spray application and therefore there is potential for exposure of non-target plants. Further consideration of the risk to non-target plants was considered necessary for the representative incorporation (spray) use, and a data gap was identified. For the representative field uses via drip irrigation, drench and seed treatment there is little potential for exposure of non-target plants and therefore a low risk can be concluded. For the representative field uses a low risk to biological sewage treatment processes was concluded. A low risk from Streptomyces K61 to birds, mammals, aquatic organisms, bees, non-target arthropods, earthworms, soil microorganisms and non-target plants was concluded for the representative glasshouse uses. No data were available for biological methods of sewage treatment. As the representative glasshouse uses may lead to exposure of biological methods of sewage treatment a data gap was identified. EFSA Journal 2013;11(1):

12 6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental compartments 6.1. Soil Compound (name and/or code) Streptomyces K61 indole-3-acetic acid heptaene, polyenes Persistence Evidence that viable cells of the strain decline in soil after application, with colony forming units of Streptomyces not being isolatable after 3 months. No information, data gap to demonstrate that amounts produced are within natural background levels. In this situation (within natural background levels) information on persistence is unnecessary. No information, data gap to demonstrate that amounts produced are within natural background levels. In this situation (within natural background levels) information on persistence is unnecessary. Ecotoxicology A low risk for earthworms was concluded. A data gap was identified to address the risk to soil microorganisms. No data were available and therefore a data gap was identified to address the risk to non-target organisms from secondary metabolites. No data were available and therefore a data gap was identified to address the risk to non-target organisms from secondary metabolites. EFSA Journal 2013;11(1):

13 6.2. round water Compound (name and/or code) Mobility in soil >0.1 μg/l 1m depth for the representative uses (at least one FOCUS scenario or relevant lysimeter) Pesticidal activity Toxicological relevance Ecotoxicological activity On the basis of the available data Streptomyces K61 is considered nonpathogenic and non-toxic to humans, when exposure is by the oral route. - - Yes No by the oral route but yes by inhalation A low risk to aquatic organisms from exposure via groundwater was concluded, as exposure via groundwater will be less than that resulting from spray drift, for which a low risk was concluded indole-3-acetic acid Data gap Data gap Yes No data No data heptaene, polyenes Data gap Data gap Yes No data, similar to antibiotic used in human medicine. No data 6.3. Surface water and sediment Compound (name and/or code) Streptomyces K61 indole-3-acetic acid heptaene, polyenes Ecotoxicology A low risk to aquatic organisms was concluded. No data were available and therefore a data gap was identified to address the risk to all non-target organisms from secondary metabolites. No data were available and therefore a data gap was identified to address the risk to all non-target organisms from secondary metabolites. EFSA Journal 2013;11(1):

14 6.4. Air Compound (name and/or code) Streptomyces K61 Toxicology Toxicity of concern by inhalation (intratracheal administration), allergic reactions from inhalation in humans; acute rat LC 50 = x 10 8 CFU/kg bw EFSA Journal 2013;11(1):

15 7. List of studies to be generated, still ongoing or available but not peer reviewed This is a complete list of the data gaps identified during the peer review process, including those areas where a study may have been made available during the peer review process but not considered for procedural reasons (without prejudice to the provisions of Article 7 of Directive 91/414/EEC concerning information on potentially harmful effects). The species must be identified and the relationship with known human pathogens must be addressed (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see sections 1 and 2). Demonstrate that the level of microbial contamination complies with international standards. The OECD issue paper (OECD, 2011) should be used as the reference (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 1). The strain must be tested for the production of toxins/secondary metabolites in the manufacturing process (5 batch analysis) and when used (see section 3) (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see sections 1 and 2). The role of antibiotics in the mode of action should be investigated further (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 1). Adhesion to and distribution on seed (relevant for the seed treatment use only; submission date proposed by the notifier: unknown; see section 1). Demonstrate that the identification methods are specific to strain level (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 1). Validation data for the methods of analysis (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 1). Literature search on infections in immune-competent and immuno-compromised individuals where Streptomyces species has been identified, with particular focus on the clinical conditions under which Streptomyces species have been isolated from humans or animals (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 2). No information has been provided in relation to potential interferences of Streptomyces K61 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC (relevant for all representative uses evaluated; submission date proposed by the notifier: no date proposed; see section 4). No information has been provided on the potential transfer of genetic material from Streptomyces K61 to other organisms (relevant for all representative uses evaluated; submission date proposed by the notifier: no date proposed; see sections 2 and 4). Satisfactory information on the natural background levels of Streptomyces sp. or other actinomycetes in the upper layers of temperate soils was not available in the notifier s dossier. Such information would be important to support some of the statements made by the notifier that use of the product as proposed would not significantly increase actinomycete amounts in the soil ecosystem or result in secondary metabolites being produced in amounts above natural background levels (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 4). EFSA Journal 2013;11(1):

16 A groundwater exposure assessment is not available for the antibiotic heptaene, polyene compounds demonstrated to have the potential to be formed in soil / growing substrate at levels that trigger the need to provide a groundwater exposure assessment (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 4). A groundwater exposure assessment is not available for the plant growth regulator compound indole-3-acetic acid, which was demonstrated to have the potential to be formed in vitro. Some observation / measurement of levels that might be formed in soil / growing substrate / confirmation that formation is negligible in the situation of use, would be the first step in such an assessment. Indole-3-acetic acid is a plant growth regulator and therefore categorised as a pesticide under Council Directive 98/83/EC, therefore an assessment to demonstrate that indole- 3-acetic acid that might the produced by Streptomyces K61 will not contaminate groundwater above 0.1µg/L as a consequence of the representative uses assessed is outstanding (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 4). The notifier has not presented results of any test observations or measurements to demonstrate that Streptomyces K61 does not survive on the surface of leaves or flowers exposed to direct sunlight and other ambient environmental conditions, to support their case as to why mobility via air might be considered insignificant (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 4). Information to address the risk to non-target organisms from potential secondary metabolites/toxins (relevant for all representative uses evaluated; submission date proposed by the notifier: unknown; see section 5). Information to address the risk to non-target arthropods (relevant for representative field uses; submission date proposed by the notifier: unknown; see section 5). Information to address the risk to soil microorganisms (relevant for representative field uses; submission date proposed by the notifier: unknown; see section 5). Information to address the risk to non-target plants for the representative field uses via incorporation (spray application) (submission date proposed by the notifier: unknown; see section 5). Information to address the risk to biological methods of sewage treatment for the representative glasshouse uses (submission date proposed by the notifier: unknown; see section 5). 8. Particular conditions proposed to be taken into account to manage the risk(s) identified None. 9. Concerns 9.1. Issues that could not be finalised An issue is listed as an issue that could not be finalised where there is not enough information available to perform an assessment, even at the lowest tier level, for the representative uses in line with the Uniform Principles of Annex VI to Directive 91/414/EEC and where the issue is of such importance that it could, when finalised, become a concern (which would also be listed as a critical area of concern if it is of relevance to all representative uses). EFSA Journal 2013;11(1):

17 1. The consumer risk assessment cannot be finalised in view of the outstanding issues on toxins and secondary metabolites, and pending confirmation from the toxicological assessment that a quantitative consumer risk assessment is not necessary for the edible uses. 2. No information has been provided in relation to potential interferences of Streptomyces K61 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC. 3. No information has been provided on the potential transfer of genetic material from Streptomyces K61 to other organisms. Therefore the assessment of potential transfer of genetic material and its effects on human and animal health including resistance to known therapeutic substances or the environment cannot be finalised. 4. Assessment of potential contamination of groundwater by heptaene polyenes and indole-3-acetic acid with respect to the regulatory limit of 0.1 µg /L is not available. 5. Assessment of the potential persistence of heptaene polyenes and indole-3-acetic acid and or information on natural background levels in soil is not available. 6. No information was available to address the risk to non-target organisms from secondary metabolites or toxins and therefore a data gap was identified to address the risk to all non-target organisms from secondary metabolites or toxins. 7. The available information was insufficient to address the risk to non-target arthropods and soil microbial processes for the field uses. 8. The available information was insufficient to address the risk to non-target plants for the representative field uses where there is incorporation (following spray application to the soil surface). 9. The available information was insufficient to address the risk to biological methods of sewage treatment for the representative glasshouse uses Critical areas of concern An issue is listed as a critical area of concern where there is enough information available to perform an assessment for the representative uses in line with the Uniform Principles of Annex VI to Directive 91/414/EEC, and where this assessment does not permit to conclude that for at least one of the representative uses it may be expected that a plant protection product containing the active substance will not have any harmful effect on human or animal health or on groundwater or any unacceptable influence on the environment. An issue is also listed as a critical area of concern where the assessment at a higher tier level could not be finalised due to a lack of information, and where the assessment performed at the lower tier level does not permit to conclude that for at least one of the representative uses it may be expected that a plant protection product containing the active substance will not have any harmful effect on human or animal health or on groundwater or any unacceptable influence on the environment. 10. High toxicity (mortality) following intratracheal administration of the viable microorganism in test animals, and adverse effects upon inhalation in humans were observed, the cause of which is unknown (allergic reaction or inhalative toxicity, due to the microorganism itself or toxin/secondary metabolite). Since a risk assessment could not be performed on the basis of the available data, no conclusion could be reached on the operator, worker or bystander exposure related to the representative uses of Streptomyces K61. Furthermore, uncertainty remains with regard to pathogenicity potential to humans. EFSA Journal 2013;11(1):

18 9.3. Overview of the concerns identified for each representative use considered (If a particular condition proposed to be taken into account to manage an identified risk, as listed in section 8, has been evaluated as being effective, then risk identified is not indicated in this table.) Representative use Edible crops glasshouse Edible crops field Ornamentals glasshouse Ornamentals field Operator risk Worker risk Bystander risk Consumer risk Risk to wild non target terrestrial vertebrates Risk to wild non target terrestrial organisms other than vertebrates Risk to aquatic organisms roundwater exposure active substance roundwater exposure metabolites Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Risk identified Assessment not finalised Legal parametric value breached Assessment not finalised Legal parametric value breached Parametric value of 10µg/L (a) breached X 10 X 10 X 10 X 10 X 10 X 10 X 10 X 10 X 10 X 10 X 1 X 1 X 6 X 6 X 6,9 X 6,7,8 X 6,9 X 6,7 X 6 X 6 X 6 X 6 EFSA Journal 2013;11(1):

19 Assessment not X 4 X 4 X 4 X 4 finalised Comments/Remarks X 2,3 X 2,3 X 2,3 X 2,3 The superscript numbers in this table relate to the numbered points indicated in sections 9.1 and 9.2. (a): Value for non-relevant metabolites prescribed in SANCO/221/2000-rev 10-final, an Commission, EFSA Journal 2013;11(1):

20 REFERENCES Estonia, Draft Assessment Report (DAR) on the active substance Streptomyces K61 prepared by the rapporteur Member State Estonia in the framework of Directive 91/414/EEC, April Estonia, Final Addendum to Draft Assessment Report on Streptomyces K61, compiled by EFSA, November EFSA (an Food Safety Authority), Peer Review Report to the conclusion regarding the peer review of the pesticide risk assessment of the active substance Streptomyces K61. an Commission, 2002a. uidance Document on Terrestrial Ecotoxicology Under Council Directive 91/414/EEC. SANCO/10329/2002 rev.2 final, 17 October an Commission, 2002b. uidance Document on Aquatic Ecotoxicology Under Council Directive 91/414/EEC. SANCO/3268/2001 rev 4 (final), 17 October an Commission, 2002c. uidance Document on Risk Assessment for Birds and Mammals Under Council Directive 91/414/EEC. SANCO/4145/2000. an Commission, uidance Document on Assessment of the Relevance of Metabolites in roundwater of Substances Regulated under Council Directive 91/414/EEC. SANCO/221/2000- rev final, 25 February an Commission, Review Report for the active substance Streptomyces K61 finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 11 July 2008 in view of the inclusion of Streptomyces K61 in Annex I of Directive 91/414/EEC. SANCO/1865/08 rev.3, 26 June Jorgensen, H., Fjærvik1, E., Hakvåg1, S., Bruheim1, P., Bredholt, H., Klinkenberg,., Ellingsen, T.E. & Zotchev1, S.B. (2009), Candicidin Biosynthesis ene Cluster is Widely Distributed among Streptomyces spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord, Norway, Applied Environmental Microbiology, 10 p OECD (Organisation for Economic Co-operation and Development), OECD Issue Paper on Microbial Contaminant Limits for Microbial Pest Control Products, ENV/JM/MONO(2011)43. Ravel, J. Wellington, E. M. H.& HILL, R. T. (2000), Interspecific Transfer of Streptomyces iant Linear Plasmids in Sterile Amended Soil Microcosms. Applied and Environmental Microbiology. p SETAC (Society of Environmental Toxicology and Chemistry), uidance Document on Regulatory Testing and Risk Assessment procedures for Plant Protection Products with Non- Target Arthropods. ESCORT 2. EFSA Journal 2013;11(1):

21 APPENDICES APPENDIX A LIST OF END POINTS FOR THE ACTIVE SUBSTANCE AND THE REPRESENTATIVE FORMULATION Chapter 1 Identity, Biological properties, Details of Uses, Further Information Active micro-organism Function (e.g. fungicide) Streptomyces K61 Fungicide Identity of the micro-organism (Annex IIM 1) Name of the organism: Taxonomy: Species, subspecies, strain: Identification: Streptomyces K61 enus: Streptomyces Family: Streptomycetaceae Bacterial order: Actinomycetales Strain K61, Open to identify the species Morphological characterization ISP (International Streptomyces Project) criteria 16S rdna sequencing DNA-DNA hybridization Open Culture collection: DSM 7206 Minimum and maximum concentration of the micro-organism used for manufacturing of the formulated product (cfu/g; cfu/l, etc.): Identity and content of relevant impurities in the technical grade micro-organism: Is the MCPA genetically modified; if so provide type of modification CFU/g 350- g/kg Aromatic heptaene polyenes (AHPs) 0.005% (w/w) Open Not genetically modified Biological properties of the micro-organism (Annex IIM) Origin and natural occurrence, background level: Target organism(s): Mode of action: Streptomycetes are naturally occurring soil microorganisms. Streptomyces K61 is originally isolated from Finnish Sphagnum peat. The background level for streptomycetes in soil is 10 6 microorganisms/g. is effective against Alternaria,. It also suppresses Botrytis and Rhizoctonia. Root colonisation and hyperparasitism Open for antibiotic issue EFSA Journal 2013;11(1):

22 Host specificity: Life cycle: Infectivity, dispersal and colonisation ability: Relationship to known pathogens: enetic stability: Production of relevant metabolites/toxins: Resistance/sensitivity to antibiotics/anti-microbial agents used in human or veterinary medicine: The mode of action of Streptomyces K61 is specific to different plant pathogenic fungi. The microorganism is not pathogenic to plants and animals. It is not known whether strain K61 is pathogenic to humans and this issue remains open. Streptomycetes exist for long periods as arthrospores or chlamydospores. They germinate in the presence of exogenous nutrients, lack of which prevents germination of most or all spores added to nonsterile soil. When localized organic substrates (e.g roots, fungi) are available, they are rapidly colonized by mycelial growth. This soon results in production of spores when nutrients are exhausted. The mean doubling time for streptomycetes was estimated to be 1.7 days. However, the spores of streptomycetes are not special resting spores nor can they tolerate extreme conditions. Streptomyces K61 is naturally occurring in soil. It is assumed that adding the microorganism to soil will not increase the levels of streptomycetes in the environment other than locally (0.01-1%) and for a limited time period. Streptomyces K61 is not infective. S. griseus (found in clinical material) has caused infections to humans. S. scabies is a plant pathogen, causing scab for potatoes. Neither of them is closely related to Streptomyces K61. S. galtieri has been shown to be pathogenic to guinea pigs and rabbits. Open when the species is identified this will need to be addressed. It is confirmed by RAPD-PCR and UP-PCR that the active organism K61 has been stable in use since 1987 Streptomyces K61 was found to produce small quantities of an aromatic heptaene polyene with antibiotic effect in vitro. Aromatic heptaene polyenes are antifungal secondary metabolites that are synthesized by many actinomycetes in soil. This metabolite is not considered to be toxicologically or environmentally relevant. Open for other toxins/secondary metabolites. Streptomyces K61 has proved to be sensitive to common bacterial antibiotics such as penicillin, kanamycin, ampicillin, tetracycline, streptomycin and rifampicin Classification and proposed labelling With regard to the micro-organism Microorganisms may have the potential to provoke sensitising reactions EFSA Journal 2013;11(1):

23 Summary of representative uses evaluated Crop and/ or situation Member State or Country Product name F or I Pests or roup of pests controlled Preparation Application Application rate per treatment PHI (days) Remarks (a) (b) (c) Fruiting vegetables Leaf vegetables and fresh herbs Brassica vegetables Northern Northern Northern and Central Northern and Central Northern and Central Northern and Central Type (d-f) Conc. of as cfu/g followed by g/kg (i) method kind (f-h) Drip irrigation Drench Drench growth stage & season (j) From transplanting From transplanting After emergence number min/ max (k) interval between applications (min) g as/l; cfu/l min max (l) 2 / / / 3 21 water L/ha min max * / / 5* * / / 5* g as/ha; cfu/ha min max (l) 100 / 157 2*10 10 / 3* (m) 50 / 250 1*10 10 / 5* / 500 2*10 10 / 1* Incorporation Before sowing Drench After emergence 1 / * / / 5* / 500 2*10 10 / 1* Incorporation Before sowing / 4 (min/max) g 1000 plants = 5*10 8 / 8*10 9 CFU/ 1000 plants. The rate as g/ha is based on the crop density of 4/m 2 EFSA Journal 2013;11(1):

24 Crop and/ or situation Member State or Country Product name F or I Pests or roup of pests controlled Preparation Application Application rate per treatment PHI (days) Remarks (a) (b) (c) Ornamental pot plants Cut flowers Ornamental cuttings Seedlings Vegetable, herb and Northern and Central Northern and Central Northern Northern, Central and Southern Northern and Central Northern, Central F, seed-borne fungi Type (d-f) Conc. of as cfu/g followed by g/kg (i) method kind (f-h) growth stage & season (j) number min/ max (k) Drench From potting 2 / 4 21 interval between applications (min) g as/l; cfu/l min max (l) water L/ha min max g as/ha; cfu/ha min max (l) *10 6 / *10 7 Incorporation Before potting Drench From sticking 2 / 4 21 Dipping Drench Dry seed treatment Rooted cuttings After emergence 1-1 / * / / 5* (m) 50 / 250 1* * *10 6 / * * / / 5* / 500 2*10 10 / 1*10 12 Before sowing / 2.5 g (min/max) 1000 plants = 2*10 8 / 5*10 9 CFU 1000 plants. The rate as g/ha depends on the crop density Application rate 2-8 g/kg of seeds. EFSA Journal 2013;11(1):

25 Crop and/ or situation Member State or Country Product name F or I Pests or roup of pests controlled Preparation Application Application rate per treatment PHI (days) Remarks (a) (b) (c) ornamentals seeds Bulbs Fruiting vegetables Cut flowers Fruiting vegetables and Southern Northern Central and Southern Central Central Central Southern Southern F, F, F, Type (d-f) Conc. of as cfu/g followed by g/kg (i) method kind (f-h) Soaking for 15 minutes Drip irrigation Drench growth stage & season (j) Before planting From transplanting From transplanting number min/ max (k) 1 - interval between applications (min) g as/l; cfu/l min max (l) 2 / / 4 21 Drench From sticking 2 / 4 21 Drip irrigation Drench From transplanting From transplanting water L/ha min max g as/ha; cfu/ha min max (l) (m) *10 6 / * * / / 5* * / / 5* / 4 21 days / 4 21 days 0.1 1*10 7 / 2500 / / 200 2*10 10 / 4* / 500 1*10 10 / 1* / 500 1* * / 400 4*10 10 / 8* / 500 5*10 10 / / 5.0 (min/max) g 1000 plants = 5*10 8 / 1*10 10 CFU 1000 plants. The rate as g/ha is based on the crop density of 4/m / 10 g (min/max) 1000 plants = 1*10 9 / 2*10 10 CFU plants. The rate as g/ha is based on the crop density of 4/m 2 EFSA Journal 2013;11(1):

26 Crop and/ or situation Member State or Country Product name F or I Pests or roup of pests controlled Preparation Application Application rate per treatment PHI (days) Remarks (a) (b) (c) Leaf vegetables and fresh herbs Brassica vegetables Ornamental pot plants Type (d-f) Conc. of as cfu/g followed by g/kg method kind (f-h) growth stage & season (j) EFSA Journal 2013;11(1): number min/ max (k) interval between applications (min) g as/l; cfu/l min max (l) water L/ha min max g as/ha; cfu/ha min max (l) (i) 1*10 8 1*10 12 Southern F, / 500 After Drench 1 / *10 emergence 2000 / / 2*10 5* *10 12 / 0 - Southern F, Incorporation Before sowing Southern F, / 500 After Drench 1 / * / / 2*10 10 emergence 5* *10 12 / 0 - Southern F, Incorporation Before sowing Southern F, Drench From potting 2 / 4 21 days Southern Incorporation Before potting (m) 2.5 / 5 g (min/max) 1000 plants = 5*10 8 / 1*10 10 CFU 1000 plants. The rate as g/ha depends on the crop density

27 Crop and/ or situation Member State or Country Product name F or I Pests or roup of pests controlled Preparation Application Application rate per treatment PHI (days) Remarks (a) (b) (c) Cut flowers Seedlings Seedlings Southern Southern Southern F, F, Type (d-f) Conc. of as cfu/g followed by g/kg (i) method kind (f-h) growth stage & season (j) number min/ max (k) interval between applications (min) Drench From sticking 2 / 4 21 days Drench Spraying After emergence After emergence, before transplanting 1 / / 2 21 g as/l; cfu/l min max (l) water L/ha min max * / / 1* * / / 5* *10 6 / 5* g as/ha; cfu/ha min max (l) (m) 250 / 500 5*10 10 / 1* / 500 2*10 10 / 1* *10 12 For uses where the column "Remarks" is marked in grey further consideration is necessary. Uses should be crossed out when the notifier no longer supports this use(s). (a) For crops, the EU and Codex classifications (both) should be taken into account; where relevant, the use situation should be described (e.g. fumigation of a structure) (b) Outdoor or field use (F), greenhouse application () or indoor application (I) (c) e.g. biting and suckling insects, soil born insects, foliar fungi, weeds (d) e.g. wettable powder (), emulsifiable concentrate (EC), granule (R) (e) CPF Codes - IFAP Technical Monograph No 2, 1989 (f) All abbreviations used must be explained (g) Method, e.g. high volume spraying, low volume spraying, spreading, dusting, drench (i) cfu/kg or cfu/l. (j) rowth stage at last treatment (BBCH Monograph, rowth Stages of Plants, 1997, Blackwell, ISBN ), including where relevant, information on season at time of application (k) Indicate the minimum and maximum number of application possible under practical conditions of use (l) The values should be given in g or kg whatever gives the more manageable number (e.g. 200 kg/ha instead of g/ha or 12.5 g/ha instead of kg/ha) as well as in number of cfu (m) PHI - minimum pre-harvest interval EFSA Journal 2013;11(1):

28 (h) Kind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plant- type of equipment used must be indicated EFSA Journal 2013;11(1):

29 Chapter 2 Analytical methods Analytical methods for the micro-organism (Annex IIM 4.2, 4.3; IIIM 5.4) Manufactured micro-organism (principle of method): Impurities and contaminating micro-organisms in manufactured material (principle of method): Microbial plant protection product (principle of method): The production of is a continuous process, and the technical grade of MPCA is only a hypothetical stage. The quantity of viable Streptomyces K61 bacteria is determined with a common microbial plate count technique. Open for validation For determination of contaminating microorganisms is used simple viability check, if the level of contaminating microorganisms is >1% the batch is discarded from the production. A molecular method for distinguishing Streptomyces K61 from other streptomycetes has been described. In this study, RAPD-PCR and UP-PCR methods were successfully used to produce strain specific banding profiles. Open for validation The quantity of viable Streptomyces K61 bacteria is determined with a common microbial plate count technique. Open for validation Analytical methods for residues (viable and non-viable) (Annex IIM 4.5) of the active micro-organism (principle of method): of relevant metabolites (principle of method): Viable residues of the microbe are monitored using microscope and a standard microbiological method of plate cultivation and cfu calculation. Open for validation For determinations of AHP s HPLC has been used. Open for validation EFSA Journal 2013;11(1):

30 Chapter 3 Impact on Human and Animal Health (Annex IIM 5; IIIM 7) Medical data, surveillance and observations Sensitisation (experience in humans and study results: type of study) Limited data: no adverse effects observed among researchers, production workers and field technicians except two instances of allergic reaction in unprotected personnel. Two instances of human respiratory allergic reaction. Magnusson-Kligman maximisation test positive result: moderate sensitiser Toxicity after acute oral exposure: after acute inhalation exposure: after acute intraperitoneal/subcutaneous exposure: No mortality, slight and transient clinical signs of toxicity. Rat LD 50 > 5000 mg /kg body weight (>10 8 CFU/kg bw) Intratracheal administration: 54% of the males and 48% of the females died within 2 days. Clinical signs: hunched posture, rough hair coat, tremors and convulsions; bw; changes in organ weight ( lungs, liver, spleen). ranulomatous and suppurative inflammation in lung tissue. ross lesions in the respiratory system, influx of reticulocytes at target site. Rat LC 50 (male) = 3.46 x 10 7 CFU/animal <> 25 mg /animal (bodyweight range g) <> mg /kg bw <> x 10 8 CFU/kg bw Rat LC 50 (female) = 3.02 x 10 7 CFU/animal = 20 mg /animal (bodyweight range g) <> mg /kg bw <> x 10 8 CFU/kg bw Intraperitoneal administration: Mortality in both heat-killed and viable microorganism within 2 days treatment. Adverse clinical signs returned to normal within 5 days in surviving mice, except cellular influx and enlarged spleens. Mouse LD 50 = 1000 mg /kg body weight <> 1.0 x 10 8 CFU/kg bw Infectivity after acute oral exposure: after acute inhalation exposure: after acute intraperitoneal/subcutaneous exposure: Limited data No indication of infectivity Limited data Pathogenicity after acute oral exposure: after acute inhalation exposure: Limited data No indication of pathogenicity EFSA Journal 2013;11(1):

31 after acute intraperitoneal/subcutaneous exposure: Limited data enotoxicity Cell culture study Short term toxicity/pathogenicity No evidence of mutagenic activity in an Ames test. Toxicological/genotoxicity data may be needed on metabolites/toxins if identified in the manufacturing process and when used. No data - not required 28-day oral, rat: No signs of toxicity were observed up to 5.0 x 10 8 (1000 mg /kg bw per day); microorganism clearance, infectivity and pathogenicity were not conducted Specific toxicity, pathogenicity and infectiveness studies No data ADI AOEL ARfD Based on submitted data it is not possible to establish ADI value Based on submitted data it is not possible to establish AOEL value Based on submitted data it is not possible to establish ARfD value Exposure scenarios (including method of calculation) Application method Operator Workers Drip irrigation, drench, incorporation (greenhouse and field use) and spraying (greenhouse applications) No conclusion could be reached: based on the submitted data, risk evaluation was not possible although concerns were raised due to sensitising potential and toxicity observed upon intratracheal administration. Furthermore, considering the data gap in section 1 for the identification of metabolites/toxins, an additional concern might be identified related to exposure to these potentially toxic compounds. No conclusion could be reached: based on the submitted data, risk evaluation was not possible although concerns were raised due to sensitising potential and toxicity observed upon intratracheal administration. Furthermore, considering the data gap in section 1 for the identification of metabolites/toxins, an additional concern might be identified related to exposure to these potentially toxic compounds. EFSA Journal 2013;11(1):

32 Bystanders Bystander exposure is not relevant to greenhouse applications. No conclusion could be reached for field scenarios: based on the submitted data, risk evaluation was not possible although concerns were raised due to sensitising potential and toxicity observed upon intratracheal administration. Furthermore, considering the data gap in section 1 for the identification of metabolites/toxins, an additional concern might be identified related to exposure to these potentially toxic compounds. EFSA Journal 2013;11(1):

33 Chapter 4 Residues Residues on Treated Products, Food and Feed (Annex IIM 6; IIIM 8) Non-viable residues: Viable residues: Open. Open subject to pathogenicity to humans. Chapter 5 Fate and behaviour in the environment (Annex IIM 7; IIIM 9) Persistence and multiplication in soil: in water: in air: Streptomycetes are common soil microorganisms. Streptomyces K61 multiplies in different soil compartments but the amount decreases to not being isolatable, in a few months. Data gap relating to total Streptomycete soil populations ranges, when there has been no product application. A worst case initial PECsoil was calculated as follows (assuming no degradation, no interception of the crop, a uniform distribution to 5 cm depth in soil and a bulk density of 1.5 g/cm 3 and use of four applications (4 x 500 g Streptomyces K61/ha/yr, which is equal to 4 x 1020 g /ha/yr)). PECsoil = 4.08/0.75 = 5.44 mg MPCP/kg soil (2.67 mg MPCA/kg soil) = 5.44* * 10 6 CFU/kg soil Streptomyces K61 does not persist in water. The only possible exposure for aquatic organisms is via run-off and drainage from soil after any outdoor uses, when is applied as a seed treatment or directly to soil as a drench or via drip irrigation systems. The following PECsw via drift is therefore expected to provide a conservative exposure estimate for these application methods and covers a spray application to the soil surface prior to incorporation. PECsw (assuming 4 x 500 g Streptomyces K61/ha/yr, which is equal to 4 x 1020 g /ha/yr, spray drift 2.77%) = µg MPCA/l (37.67 µg MPCP/l) = 37.67* *10 3 CFU/l water Data gap. Mobility: Streptomyces K61 The microorganism can be spread only by growing or mechanical transportation (water movement or arthropods). The issue of movement via air is open. EFSA Journal 2013;11(1):

34 Chapter 6 Effects on Non-target Organisms (Annex IIM 8; IIIM 10) Effects on terrestrial vertebrates Effects on mammals: Risk assessment for mammals: Effects on birds: Risk assessment for birds: Acute toxicity to mammals: Rat LD 50 > 5,000 mg /kg bw was not infective/pathogenic after oral exposure. Leafy crops scenario 4 applications at 1.02 kg MPCP/ha with a 21-day interval: TER a medium herbivorous mammal >147.3 Seed treatment scenario 8 g /kg seeds: TER a granivorous mammal >2.7 2 g /kg seeds: TER a granivorous mammal >10.9 Bobwhite Quail: 30-day oral LD 50 >2500 mg /kg bw (No treatment-related mortalities, clinical signs of toxicity or pathogenicity/infectivity were observed) Mallard Duck: 30-day oral LD 50 >2500 mg /kg bw (No treatment-related mortalities, clinical signs of toxicity or pathogenicity/infectivity were observed) Leafy crops scenario: 4 applications at 1.02 kg MPCP/ha with a 21-day interval TER a medium herbivorous bird >27.1 TER a small insectivorous bird >45.3 TER short-term medium herbivorous bird >62 TER short-term small insectivorous bird >81.3 Seed treatment scenario: 8 g /kg seeds: TER a granivorous bird >3.3 2 g /kg seeds: TER a granivorous bird >0.8 Effects on aquatic organisms Effects on fish: Effects on freshwater invertebrates: Rainbow Trout: 30-day static renewal LC mg /L NOEC 8.8 mg /l (assuming that tested powder, batch 41202, contains 5.7 x 10 9 cfu of Streptomyces K61 per gram) (Signs of toxicity included quiescence, surfacing, laboured respiration, discolouration, equilibrium loss. No signs of infectivity) Daphnia magna: 21-day static renewal EC mg /l EFSA Journal 2013;11(1):

35 LOEC 102 mg /l NOEC 10.2 mg /l (No treatment-related effects for reproduction and time to first brood. 100%, 25% and 0% mortality at 1000, 100 and 10 mg/l, respectively. 100% mortality in heatinactivated control.) Effects on algae: Risk assessment for aquatic organisms: did not inhibit algal growth. However, toxicity endpoints could not be established (EC 50, LOEC, NOEC) as the dose-response relationship could not be established with the produced data. PECsw µg MPCA/l (37.67 µg MPCP/l (calculated assuming that application rate: 2000g MPCA/ha (4x500g MPCA/ha), spray drift: 2.77 %). Fish: 30 day LC 50 = 12.5 mg MPCP/l, TER= 332; 30 day NOEC =8.8 mg MPCP/l, TER= 234 Daphnia: 21 day EC 50 =190 mg MPCP/l, TER=5044, 21 day NOEC = 10 mg MPCP/l, TER=265 Effects on arthropods Effects on bees: Risk assessment: Effects on terrestrial arthropods other than bees: Honey Bee: 5-day dietary LC 50 > 2400 mg /l of the diet (LD 50 > 48 g /bee/day) NOEC = 2400 mg /l of the diet The amount of test substance consumed by the bees was not quantified during the 5 day exposure period. The endpoint should be regarded as an estimation g /ha Oral HQ = 21 Data gap. Effects on soil organisms Effects on earthworms: Risk assessment: Effects on soil micro-organisms: Earthworm (Eisenia foetida): 14-day LC 50 > 1000 mg/kg NOEC = 1000 mg/kg (The tests were conducted in one-litre glass containers. No treatment related effects.) PEC = 5.44 mg/kg (assuming no degradation, no interception of the crop, a uniform distribution to 5 cm depth in soil and a bulk density of 1.5 g/cm 3 and use of four applications (4 x 500 g product/ha/yr). Acute TER >184 Data gap. EFSA Journal 2013;11(1):

36 Effects on terrestrial plants Effects on seed germination: Seeds of 35 vegetable species (including some herbs), 11 agronomic crop species, 2 fruit tree species and 42 ornamental plant species. Reduction in germination,, was observed in the following species: sweet corn (Zea mays), Dusty Miller (Centaurea cineraria) and cantaloupe melon (Cucumis melo), when seed treatment alone or a seed treatment followed by a soil drench was used. Phytotoxity was noted also in the case of gerbera (erbera jamesonii), lettuce (Lactuca sativa), sweet pepper (Capsicum annuum) and corn (Zea mays). Application rates were not specified. Data gap EFSA Journal 2013;11(1):

37 APPENDIX B USED COMPOUND CODES Code/Trivial name indole-3- acetic acid Chemical name 2-(1H- indol-3- yl)acetic acid Structural formula heptaene polyenes EFSA Journal 2013;11(1):