Pythium Oligandrum Strain M1 Product-Type 10 (Masonry Preservative) January 2015

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Regulation (EU) No 528/2012 concerning the making available on the market and use of biocidal products

Evaluation of active substances

Assessment Report

Pythium oligandrum Strain M1 Product-type 10 (Masonry preservative) January 2015

The Czech Republic

Pythium oligandrum M1 Product-type 10 January 2015

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...... 4

2.1. Presentation of the Active Substance ...... 4 2.1.1. Identity, Physico-Chemical Properties & Methods of Analysis ...... 4 2.1.2. Intended Uses and Efficacy ...... 4 2.1.3. Classification and Labelling ...... 5

2.2. Summary of the Risk Assessment ...... 5 2.2.1. Human Health Risk Assessment ...... 5 2.2.1.1. Hazard identification ...... 5 2.2.1.2. Effects assessment ...... 5 2.2.1.3. Exposure assessment ...... 6 2.2.1.4. Risk characterisation ...... 7 2.2.2. Environmental Risk Assessment ...... 9 2.2.2.1. Fate and distribution in the environment ...... 9 2.2.2.2. Effects assessment ...... 10 2.2.2.3. PBT and POP assessment ...... 11 2.2.2.4. Exposure assessment ...... 12 2.2.2.5. Risk characterisation ...... 12 2.2.3. Assessment of endocrine disruptor properties ...... 12

2.3. Overall conclusions ...... 13

2.4. List of endpoints ...... 13

APPENDIX I: LIST OF ENDPOINTS ...... 14

APPENDIX II: LIST OF INTENDED USES ...... 24

APPENDIX III: LIST OF STUDIES ...... 25

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1. STATEMENT OF SUBJECT MATTER AND PURPOSE

1.1. Procedure followed

This assessment report has been established as a result of the evaluation of the active substance Pythium oligandrum Strain M1 as product-type 10, carried out in the context of the work programme for the review of existing active substances provided for in Article 89 of Regulation (EU) No 528/2012, with a view to the possible approval of this substance.

On 12 July 2005, the CZ competent authorities received a dossier from the applicant. The Rapporteur Member State accepted the dossier as complete for the purpose of the evaluation on 4 August 2010.

On 8 November 2011 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 Pythium oligandrum M1 for product-type 10, 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 assessment report, which is available from the Agency web-site shall be taken into account.

However, where conclusions of this assessment report are based on data protected under the provisions of Regulation (EU) No 528/2012, such conclusions may not be used to the benefit of another applicant, unless access to these data for that purpose has been granted to that applicant.

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2. OVERALL SUMMARY AND CONCLUSIONS

2.1. Presentation of the Active Substance

2.1.1. Identity, Biological and Physico-Chemical Properties and Methods of Analysis

Active substance is concentrate of spores of Pythium oligandrumStrain M1 (Technical grade Pythium oligandrum) consisting of the remnants of the growing medium (about 95%), mycelium and spores of Pythium oligandrum (2.5 x 106 – 10 x 106 oospores/g per a.s.). Pythium oligandrum belongs to the Oomycota class classified as a part of newly created kingdom Stramenopila (syn. Chromista). Pythium oligandrum is a living organism, its dormant spores can survive temperatures under 60°C, but do not grow at temperatures > 37°C in tissues of animal origin. Pythium oligandrum is naturally occurring in soil where it colonizes the rhizosphere of plants. It requires a humid environment and has a minimum growth temperature of 7ºC, optimum around 30 ºC. The M1 strain of Pythium oligandrum was deposited in American Type Culture Collection (ATCC) under reference number ATCC 38472 and in Czech Collection of micro-organisms under mark M1. Technical grade Pythium oligandrum (further on tgPO) is fine whitish powder, dispersible in water. Quality management of the manufacturing process ensures that no toxins or pathogens relevant for human health are present in the technical grade active ingredient.

Analysis The micro-organism could be identified by light microscopy – it has well distinguishable spiny- walled oospores- and can also be identified using PCR. Oospores are counted in Buerker's chamber. PCR enables differentiation between different Pythium oligandrum Strain M1 strains. Coupling PCR with other methods provides a battery of methods enabling identification of Pythium oligandrum Strain M1 at strain level. The methods are listed and described in DOC IIA and their detailed descriptions are given in the corresponding DOC III section or annex I to DOC IIA. Strain level characterisation can be done by a combination of PCR, HPLC MS/MS and methods based on biological characteristics (e.g. multiplication rate under specific conditions).

2.1.2. Intended Uses and Efficacy

Pythium oligandrum is a mycoparasite. With its hyphae, Pythium oligandrum penetrates cells of the target organisms (mould or yeast), drawing from it necessary substances for its nourishment. As soon as the nourishment source is exhausted, the microorganism will transform into a spore stage and wait for more favourable conditions.

The assessment of the biocidal activity of the active substance demonstrates that it has a sufficient level of efficacy against moulds on masonry and the evaluation of the summary data provided in support of the efficacy of the accompanying product, establishes that the product may be expected to be efficacious.

Efficacy tests were conducted with the product Biorepel: walls heavily infected by saprophytic molds, monoculture of Aspergillus terreus or mixture of A. niger, A. terreus and A. auranogriseum were sucessfuly cleansed in 4 weeks by a single treatment with suspension containing 200mg TGAI/L water, one litre of suspension per 5 m2. Moulds on wooden carriers were not eliminated even after 6 weeks.

The field of use envisaged is indoor application against undesirable moulds on walls and plasters, both as curative and preventative treatment by brushing/rolling. 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.

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2.1.3. Classification and Labelling

The substance is a microorganism and hence is covered neither by Directive 67/548/EEC nor by the CLP regulation. It produces no metabolite classified as hazardous according to Directive 67/548/EE or the CLP regulation. The classification and labelling for Pythium oligandrum Strain M1 according to Regulation (EC) No 1272/2008 (CLP Regulation) is not required as the active substance is a microorganism not covered by this regulation. The classification according to Directive 67/548/EE or the CLP regulation could only concern chemical substances produced by the Pythium oligandrum Strain M1 or already contained in it. No substance that could be classified as hazardous was identified and the toxicological studies provided no indication of toxicity. Considering that all microbials should be regarded as potential sensitisers, the agreed warning phrase on the product label is: “Microorganisms may have the potential to provoke sensitising reactions”.

2.2. Summary of the Risk Assessment

2.2.1. Human Health Risk Assessment

It is noted that EFSA in their expert opinion on Pythium oligandrum Strain M1 published in 2013 concluded that a derivation of reference values is not necessary and no quantitative risk assessment is required. In the light of this and the conclusions from the data on which this report is based measures protecting humans, animals and the environment following form the hazard identification has been preferred in this report. The human exposure calculations and their comparison to reference values are provided in this report for information only.

Toxicokinetics and metabolism

Pythium oligandrum produces several substances that play a role in the organism’s mode of action. None of these substances are considered to be of toxicological concern.

Dermal absorption of particulate material is not expected.

2.2.1.1. Hazard identification

No systemic effects have been observed in any of the toxicological studies provided by the applicant. Two studies on skin irritation and standard test of acute eye irritation provide evidence of slight irritating properties of the TGAI.

2.2.1.2. Effects assessment

Systemic effects As no systemic effects have been observed after neither single nor repeated dosing, the top doses of the technical active substance are indicated as toxicity level 0 (TL0, NOAEL) values (Table 2.1). Due to the lack of toxicity derivation of reference values and hence, a quantitative risk assessment is not required. It is noted that the same conclusion has been reached by the pesticide peer review on Pythium oligandrum Strain M1 M1 published in 20131. Pythium oligandrum does not produce any known metabolites considered to be of toxicological concern: genotoxicity, carcinogenicity and reproductive toxicity testing of specific metabolites therefore has not been conducted.

1 EFSA Journal 2013;11(1):3034

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Table 2.1 TL0 values Route Material administered TL0 values Observation Examination animal mg a.s./kg period bw oral gavage active substance single dose 14 days clinical, rat, mouse in 12.5% suspension >5000 necropsy oral gavage active substance, single dose 10 hours mortality mouse sterilised, suspended >20 000 behaviour dermal active substance single dose 14 days weight, clinical, rat moistened (1.5ml/g) >5000 local signs 24 hour necropsy semiocclusion inhalation active substance single dose 14 days weight, clinical signs, rat 5 mg/L air >600 behaviour, 4 hour GM 7.45 µm necropsy, exposure histopathology of respiratory system. oral, in diet active substance repeated 70 days weight, clinical, rat, 70 days 0.8, 2.4, 4 % in diet daily dose ** behavioural ** >4000 haematology, necropsy, organ weights, histopathology

* active suspension has been filtered, resulting count of propagules in the ip dose = 1.8 x 105 /kg bw, corresponding to active substance dose of 360 mg/kg bw. ** for an average daily consumption of diet 100 g/kg bw; increased body weight in exposed animals and differences in some behavioural tests are not indicative of toxicity.

Infectivity and pathogenicity The active substance is not considered as infective or pathogenic. Maximum tolerated temperature of 37 ºC, determined in the in-vitro study on growth of Pythium oligandrum in tissues of animal origins, prevents deep infections in tissues of mammals.

Local effects Two studies on the skin irritation and standard test of acute eye irritation provide evidence of slight irritating properties of the neat a.s. No immediate or delayed reactions were observed after 24 hour exposure of 1/10 of skin surface (cca 10 cm2) to concentrated suspension of 5000 mg of the active substance. Local concentration of the active substance is estimated to have been at least 500 mg/cm2. Human data: No clinical cases or sensitisation / allergic reactions were noted in the facilities of the applicant.

2.2.1.3. Exposure assessment

The human exposure calculations and their comparison to reference values are provided only for information. Exposure to a concentrated product occurs during industrial manufacture of biocidal product, which takes place once a year, and during mixing of the active suspension by professional operators or amateur users. Exposure is possible only in the phase of manual pouring of the active substance or of mixed product (max. 20% a.s.). Inhalation and dermal deposition of dust are the relevant routes of exposure.

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Exposure to an active suspension occurs during application of the suspension (100 – 200 mg a.s./L) by manual tools (brushing). Inhalation and dermal deposition of the liquid aerosol are the relevant routes of exposure.

The exposure calculations and the resultant values are provided in section 3.2 of DOC IIB. The exposure values are provided only for information.

2.2.1.4. Risk characterisation

Due to the lack of toxicity, derivation of reference values and human exposure calculation are considered unnecessary. Consequently, a quantitative risk assessment is not considered necessary and is included only for information in the CAR. The conclusions on measures to protect man, animals and the environment follow from consideration of the relevant routes of exposure and the hazard identification.

Critical endpoints and routes of exposure The toxicologically relevant endpoints for the active substance are summarised in Document IIA, Chapter 3.

Dermal absorption is highly unlikely due to the nature of the active substance. Maximum tolerated temperature of 37 ºC prevents deep infections in tissues of mammals. No systemic toxic effects or signs of infectivity were observed in the toxicity tests. Sensitisation has not been observed in exposed workers but in the absence of a reliable test on sensitisation, as for other microorganisms, it has not been ruled out.

Relevant exposure routes are inhalation and dermal exposure to the powder of active substance or product, and to liquid aerosol generated e.g. in the brushing or mixing/loading phase.

Industrial formulation of the active substance

This operation is conducted once per year by professionals: exposure is considered to be accidental. The estimated doses are compared with the acute NOAELs for information only.

Inhalation and dermal exposure to powder during manual loading of mixing and packaging machines is estimated to result in the total daily (external) dose of 6.52 mg a.s./kg bw, MOE = 5000/6.52 = 767. The estimate of dermal deposition is 0.44 mg of a.s./cm2 , < 1/1000 of no-effect local concentration of 500 mg/cm2.

With respect to possible sensitising potency of the active substance, both inhalation and dermal exposures of workers not using PPE may be too high during manual pouring of the active substance and of the product. Thus, when performing this task appropriate PPE including gloves and RPE should be worn.

Application of the biocidal product

Total daily exposure is estimated in the first tier calculation to be ≤ 0.084 mg a.s./kg bw for professional operators. Using subacute TL0 (oral) of 4000 mg/kg bw as a measure, MOE = 4000/0.084 > 4x 104. Margins over exposure are even higher for non-professional users (>105 ) for the first tier calculation.

Since Pythium oligandrum Strain M1 is a microorganism the risk of sensitizing effects during mixing and loading and its application is assessed. The duration of 150 minutes proposed in User guidance p.47 for professional brush-painting of wooden objects provides a conservative estimate of exposure duration of the proposed use. The non-professional exposure is assumed to be of shorter duration, usually not exceeding 60 minutes.

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For non-professionals, the risk of exposure to the undiluted product at the loading/mixing stage can be mitigated by using water soluble packaging. This type of packaging is considered as a necessary risk mitigation measure for the non-professionals, unless reliable data or scientifically sound justification showing that the exposure to the undiluted product poses no unacceptable risk are provided at the product authorisation stage.

For the professional users an alternative to water soluble packaging is the use of appropriate personal protective equipment (PPE) including gloves, long sleeved coveralls and respiratory protective equipment (RPE, dust mask).

Dermal exposure during the application phase is considered as posing no unacceptable risk either to professionals or non-professionals due to the high dilution rate of the working solution This conclusion is supported by the fact that the representative product contains only 20% of technical grade active ingredient (TGAI). TGAI contains only 5% of oo-spores and mycelium (the rest are remnants of nutrients and agar from its manufacture). Thus, in total the biocidal product contains only 1% of “pure” Pythium oligandrum. In reality the exposure of both professionals and non-professionals takes place only during the brushing phase when the product is diluted with water provided that the exposure during the mixing phase is prevented water soluble packaging. The worst case application solution then contains 1g of Biorepel product in 1 liter of water. The concentration of the “pure” a.i. in the application solution is therefore 0.001% which is considered sufficiently low. This, combined with the fact that Pythium oligandrum Strain M1 does not grow in animal tissue at 37oC and cannot therefore elicit an allergic reaction by continually producing chemical substances during its growth, adds weight to the conclusion that the brushing application does not pose unacceptable risk to either non-professionals or professional users.

The by-stander inhalation exposure to Pythium oligandrum Strain M1 from the plant protection product (PPP), Polyversum application was compared to the exposures of users and bystanders during biocidal product application by brushing. According to the PPP assessment report (RMS SE), by-standers were considered to be exposed for 30 minutes to spray drift when walking next to a field being treated, considered to be 10 m from the sprayer. According to the assessment, the estimated inhaled dose was 0.000021 mg/kg bw of the active. The exposure of professional and non-professional users, as well as by-standers during the brushing application is considered to be even lower, as the in-use concentration is more than fourfold lower, and exposure from a spray drift is a worst case compared to brushing. Consequently, the exposure via inhalation to Pythium oligandrum Strain M1 during the brushing application is considered as safe both for professionals and non-professionals as well as for by-standers.

In summary, considering the hazard profile and the exposure, even without the use of PPE the brushing application does not pose unacceptable risk to either by-standers, non-professional or professional users.

Indirect exposure

Indirect exposure can accidentally take place by skin contact with freshly treated wall and/or inhalation of the particles of active substance released from the wall after drying. The estimate of exposure due to contact with the freshly treated wall is lower than that due to hand-brush application by non-professional users.

Regarding the inhalation of the active particles released from the wall after it has dried the factors influencing its release to the air have been considered. It is assumed that the exposure via inhalation is possible only if the active substance is released form the wall surface due to a physical disturbance and is kept in the air by a sufficiently intense air movement. These factors are regarded as negligible for the Pythium Oligandrum Strain M1 intended use – indoor - leading to negligible exposure compared to the inhalatory exposure to Pythium oligandrum naturally present in the soil dust.

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Conclusion

The quantitative risk assessment was provided only for information as the toxicological studies identified no hazard to human health. As a precautionary measure applied to microorganisms in general, it has to be considered that Pythium oligandrum Strain M1 may cause a sensitization reaction. Therefore, significant exposure to the technical grade active ingredient or the undiluted product by industrial workers and/or professionals should be prevented by the use of appropriate PPE including RPE, or other RMM. Exposure of professionals and non- professionals to the aqueous suspension of the biocidal product during application is considered to be acceptable without the use of PPE.

Indirect exposure due to the proposed use is considered to be acceptable. The combined exposure is considered to be acceptable for both professionals and non-professionals.

This risk assessment for the proposed use of the technical grade Pythium oligandrum Strain M1 in the Product Type 10 has demonstrated that there are no concerns regarding human health.

2.2.2. Environmental Risk Assessment

2.2.2.1. Fate and distribution in the environment

Pythium oligandrum Strain M1 is intended for preventive and /or curative treatment of walls against molds indoors only. Mostly, the applied Pythium oligandrum Strain M1 is left on the wall even after the mould has been destroyed. Should the conditions under which humidity and hence moulds re-occur the a.s. will “wake up” and feed on the moulds as described in the section 1.3. For this reason the place affected shall not be rinsed after the application, hence the emissions of Pythium oligandrum Strain M1 to the environment will be negligible. The emissions to the STP are semi-quantified in DOC IIB with the conclusion that the STP exposure to Pythium oligandrum Strain M1 due to application of the biocidal product Biorepel is negligible compared to its exposure to Pythium oligandrum Strain M1 washed to STP from soil during rain.

Biotic degradation Pythium oligandrum Strain M1 is a micro-organism naturally occurring in the environment, mainly in soil. It is not biodegradable in the meaning of degradation known in organic chemical substances. Pythium oligandrum Strain M1 occurs in soil where it colonizes the rhizosphere of plants. Its concentration in soil depends on a number of factors primarily including pH, temperature and occurrence of fungi it can feed on. The optimal conditions for Pythium oligandrum Strain M1 abundance in soil are provided in section 4.1. of DOC IIA.

Abiotic degradation Hydrolysis: Pythium oligandrum Strain M1 is a micro-organism, hydrolysis is not possible. Photolysis: Pythium oligandrum Strain M1 is a micro-organism, photolysis is not possible. Phototransformation in air: In case of Pythium oligandum Strain M1, air is a transport medium for oospores, a growth itself does not occur in air.

Distribution Pythium oligandrum Strain M1 is a naturally occurring soil micro-organism. The proposed indoor application of products based on Pythium oligandrum Strain M1 can have significant effects on its fate, behavior and distribution in the environment. Pythium oligandrum Strain M1 occurs in relatively high densities in many agricultural soils “feeding on” many soil fungi. Pythium oligandrum occurs mostly in rhizosphaere of soils of disturbed sites (agricultural land).

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It was observed that Pythium oligandrum population decreased with time after being transplanted in sterile soil. Takenaka et all (2008). Madsen (2004) reported concentration between 4 and 26 oospores/g soil in Danish soils, Al-Rawahi and Hancock (1997) reported 89- 151 oospores of Pythium oligandrum/g of raw field soil in Pakistan. Pythium oligandrum Strain M1 does not occur in aquatic environment which is considered as unfavorable for its growth (Foley and Deacon (1985), Klaban (2013).

Adsorption onto/desorption from soil No adsorption or desorption, as known in organic chemical substances, occur in Pythium oligandum Strain M1.

Accumulation Pythium oligandrum Strain M1 is originally a soil micro-organism and it does not accumulate nor multiplicates in living organisms. With its hyphae, the fungal organism Pythium oligandrum penetrates cells of the parasite (mould or yeast), drawing from it necessary substances for its nourishment. As soon as the nourishment source is exhausted, the fungus will change into the spore stage. It does not reproduce in haematothermal organisms; temperatures above 37 °C do not suit it (Kratochvílová, 2006).

2.2.2.2. Effects assessment

Acute and chronic toxicity to fish No effects on fish were observed in the acute toxicity fish study. Therefore the LC50 value could not be calculated. No fish deaths or morphological changes were observed at concentration 100 mg active substance per litre (top dose). Therefore, the acute NOEC could be considered as > 100 mg/L. Acute and chronic toxicity to invertebrates No adverse effects on invertebrates were identified by a literature research. Since direct release of the active substance to the aquatic environment is not expected to occur, a test on invertebrates was waived. Growth inhibition on algae Pythium oligandrum Strain M1 is ubiquitous as a naturally-occurring soil colonizer whose modes of action that is not consistent with toxicity or pathogenicity to algae. This is confirmed by an extensive literature search where no adverse effects on algae were identified. For more details and references supporting these arguments see section 4.1 of Document IIA. Since direct release of the active substance to the aquatic environment is not expected to occur, a test on algae was waived.

Inhibition of activated sludge respiration rate Pythium oligandrum Strain M1 is ubiquitous as a naturally-occurring soil colonizer whose level in the STP will not significantly increase with the use of products that contain this strain. For information, the added STP concentration is quantified in DOC IIB using an emission scenario for indoor painting. In the calculation, parameters selected by expert judgement were used as no harmonized guidance is available. The thus obtained number of oospores is significantly lower than the number of oospores assumed to enter the STP with soil due to commonly performed anthropogenic activities. It is further noted that Pythium oligandrum Strain M1 has modes of action that are not consistent with toxicity or pathogenicity to bacteria occurring in the STP sludge. This is confirmed by an extensive literature search where no adverse effect on aquatic microorganisms was identified. Furthermore, water is not the environment, where Pythium oligandrum could multiply (for more details and references supporting these arguments see section 4.1 in DOC IIA). Thus, no unacceptable risk to STP is envisaged due to the proposed use of Pythium oligandrum Strain M1.

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Atmosphere Air might be a transport medium for oospores but probably not an environment for growth of the Pythium oligandrum Strain M1.

Terrestrial compartment Pythium oligandrum Strain M1 naturally occurs in various soil types in which it may persist. The conditions for Pythium oligandrum Strain M1 occurrence in the terrestrial compartment are provided in detail in section 4.1 of DOC IIA. Its mobility and persistence in soil is primarily dependent on the presence of fungus which, in turn, are dependent on the presence of plants. The conditions for Pythium oligandrum Strain M1 occurrence in the terrestrial compartment are provided in greater detail in DOC IIA.

It has no dangerous effects on bees and other arthropods, and soil organisms (earthworms are in contact with the active substance, which is naturally occurring in soil, for their whole lifecycle without any adverse effects).

The risk for plants is provided in the assessment report elaborated in support of Pythium oligandrum Strain M1 inclusion to Annex I to Directive 91/414 EC. The active substance is not harmful to plants.

Determination of predicted no effect concentrations for aquatic compartment Due to the substance nature, no data for PNEC calculation are available. If used as specified, the active substance shall not enter the aquatic environment.

Determination of predicted no effect concentrations for terrestrial compartment Due to the substance nature, no data for PNEC calculation are available.

Determination of predicted no effect concentrations for STP micro-organism Due to the substance nature, no data for PNEC calculation are available. If used as specified, the active substance shall not enter sewage treatment plants.

2.2.2.3. PBT and POP assessment

P assessment Pythium oligandrum Strain M1 is a microorganism and therefore a PBT assessment, as it is normally performed for chemical substances, is irrelevant. However, according to Addendum to TNsG on Data Requirements for microorganisms appropriate information on the persistence of the microorganism in all the relevant compartments has to be given, unless it can be justified that exposure of the particular environmental compartment to the micro-organism is unlikely to occur. Since Pythium oligandrum Strain M1 is intended for indoor use only, direct emissions to any environmental compartment are unlikely to occur. The exposure of STP is semi- quantified in DOCIIB with the conclusion that it is negligible. It follows that indirect exposure of surface water or soil is also negligible. For more information on possible Pythium oligandrum Strain M1 survival or multiplication in these environmental compartments (see section 4.1. of DOC IIA). In the light of this, it can be concluded that persistence or multiplication of Pythium oligandrum Strain M1 in the environment due to the proposed use is of no concern and no further studies are needed on this endpoint.

B assessment Pythium oligandum Strain M1 is a micro-organism, it does not meet criteria for being classified as bioaccumulative. The strain M1 has been shown not to be able to grow at 37 ºC (see B.6.2.2) ( ), thus preventing multiplication in mammals.

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T assessment Pythium oligandum Strain M1 is a micro-organism and thus cannot be toxic per se. Regarding chemical substances Pythium oligandrum Strain M1 contains or produces, their toxicity was tested in toxicological studies (see section 3.10. of DOC IIA). These studies show that Pythium oligandrum Strain M1 contains no substances resulting in it being classified as toxic and T classification is therefore excluded.

Overall assessment Pythium oligandum Strain M1 is neither a PBT/vPvB nor is it POP.

2.2.2.4. Exposure assessment

Aqueous medium The proposed use as a fungicide intended for the treatment of walls indoors ensures that aquatic environment exposure is considered to be negligible. For more information including semi-quantitative assessment of Pythium oligandrum Strain M1 emissions to STP on its application see section 3.3. in DOC IIB.

Terrestrial medium The proposed use as a fungicide intended for the treatment of walls indoors ensures that there are no direct emissions to soil, and neither direct or indirect emissions to STPs. Theoretically, Pythium oligandrum Strain M1 could enter the terrestrial compartment when the STP sludge is applied on soil. However, only negligible amount of Pythium oligandrum Strain M1 can get to the STP following Biorepel application (for semi-quantitative assessment of Pythium oligandrum Strain M1 emissions to STP on its application see section 3.3. in DOC IIB). Furthermore STP sludge is treated befeore it is applied onto the soil thus preventing any viable Pythium oligandrum Strain M1 oospores from getting to the soil (see section 4.1 in DOC IIA for details).

Air There is no direct emission to the atmosphere due to Pythium oligandrum Strain M1 application.

Exposure scenarios Pythium oligandum is part of the environment. If used as specified there are no significant emissions to the environment. However semi quantitative assessment of Pythium oligandrum Strain M1 emissions to STP is provided in section 3.3. in DOC II B with the conclusion that the emissions to STP due to Biorepel application are negligible and significantly lower than the emissions due to rain.

2.2.2.5. Risk characterisation

Pythium oligandum Strain M1 is a natural part of the environment; no dangerous properties for the environment were identified during the risk assessment. Thus, if used as specified in this Pythium oligandrum Strain M1 poses no unacceptable risk to the environment.

2.2.3. Assessment of endocrine disruptor properties

Pythium oligandrum Strain M1 or substances it produces are not endocrine disruptors. No toxicity due to mechanism disrupting the endocrine signalling or glands was observed in the submitted toxicological studies nor any evidence exists suggesting a potential for endocrine disruption by Pythium oligandrum Strain M1 in humans following long term manufacture and usage.

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2.3. Overall conclusions

The outcome of the assessment for Pythium oligandrum Strain M1 in product-type 10 is specified in the BPC opinion following discussions at the BPC 8 meeting of the Biocidal Products Committee (BPC). The BPC opinion is available from the ECHA web-site.

2.4. List of endpoints

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

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Appendix I: List of endpoints

Chapter 1: Identity, Biological Properties, Details of Uses, Further Information, and Proposed Classification and Labelling

Active micro-organism Pythium oligandrum, M1 strain, Technical grade Function (e.g. fungicide) fungicide

Rapporteur Member State Czech Republic

Identity (Annex IIA, point II.) Name of the organism: Pythium oligandrum Taxonomy: Species: Pythium oligandrum; genus: Pythium; family: Pythiaceae; order: Perensporales; class: Oomycetes. Species, subspecies, strain: Pythium oligandrum, M1 identification: Pythium oligandrum is identified using microscopic taxonomic analysis of species-characteristic spiny- walled oospores and oogonia. This method cannot be used for identification at strain level, but is sufficient to distinguish Pythium oligandrum from other Pythium species, and most significantly Pythium insidiosum. Pythium oligandrum can be identified at strain level by a battery of methods including PCR, HPLC MS MS and multiplication at approprtiate medium. The PCR on its own has been used to differentiate between different strains of Pythium oligandrum.

Culture collection:. ATCC 38472 Minimum and maximum concentration of the Beetween 15 to 20% of the TGAI is used to micro-organism used for manufacturing of the manufacture the product. formulated product (cfu/g; cfu/L, etc.): TGAI conatins approx. 5 % (w/w) of spores of Pythium oligandrum with remnants of lifeless mycelium, the number of oospores per g TGAI ranges from 2.5x 106 – 10 x 106 Identity and content of relevant impurities in approx. 95 % (w/w) of fermented remnants of cereal thetechnical grade micro-organism: based growing medium Is the MCPA genetically modified; if so MPCA is not genetically modified provide type of modification

Biological properties of the micro-organism .

Origin and natural occurrence, background Pythium oligandrum is a ubiquitous inhabitant of level: the soil where it colonizes the rhizosphere and rhizoplane of plants and parasitizes other fungi. Pythium oligandrum has been recorded from soil and plants in several countries in Europe, Africa, Australia and North andSouth America. The background level of Pythium oligandrum Strain

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M1 in Danish soil was quantified to be 4 and 26 oospores. g–1 soil (Madsen, 2004). Al-Rawahi (1997) reported 89-151/g soil. Target organisms.: Saphrophytic moulds occurring on walls and plastrons Mode of action: Mycoparasitism, mediated by intimate hyphal interactions and space competition Host specifity: Pythium oligandrum is a hyperparazitic micromycet parasiting more than 20 genera of fungal organisms. Life cycle: Pythium oligandrum has two cycles of reproduction, one sexual and one asexual, with the sexual accounting for around 20 % of the total reproduction.

Infectivity, dispersal and colonisation ability: Pythium oligandrum is naturally occurring in soil where is colonizes the rhizosphere of plants. It is assumed that possible leakage of the micro- organism to soil will not increase the levels of Pythium oligandrum in the environment other than locally and for a limited time period. Pythium oligandrum M1 was shown not to be able to grow on animal tissues and it never grows at 37 O C and more. Thus infectivity to humans is not expected. Relationship to known pathogens: Several Pythium species have been reported to cause disease in fish and plants (Rivierre et al, 2005). The only species in the genus that infects mammals is Pythium insidiosum, known to cause life-threatening infections in humans and animals. P. insidiosum differs from all known. Pythium species by the septation of the main hyphae, as produced in particular on many agar media, and by the formation of conspicious, thick-walled fertilization tubes. It has filamentous, non-inflated sporangia, large antheridia and high optimum temperature with a peak growth rate at 37°C and inhibited growth at 40°C. Genetic stability: Stock cultures of lyophilised samples of original cultures are used for manufacturing of the active substance, thus preventing genetic drift. In the available literature there is no indication that Pythium oligandrum is able to exchange genetic material with other organisms. Production of relevant metabolites/toxins: Pythium oligandrum produces several substances each of which plays a role in the organism’s mode of action. These substances include oligandrin, ethylene, cell wall hydrolytic enzymes such as cellulases, chitinases,tryphtanime (TNH2) and two types of cell wall protein fractions, D-type containing two major proteins with molecular mass of ~28 kDa, and S- type containing one major protein with molecular mass of ~27 kDa. None ofthese substances are considered to be of toxicological concern. As the knowledge about the mode of action of Pythium oligandrum is still limited, it could be

15 Pythium Oligandrum M1 Product-type 10 January 2015

expected that additional metabolites are produced. Resistance/sensitivity to antibiotics/anti- The product is not intended to be mixed with other microbial agents used in human or veterinary fugicides or antibiotics. Pythium oligandrum Strain medicine: M1 is rather sensitive to some fungicides or antibiotics. This is supported by Foley & Deacon (1985) who reported that pentachloronitrobenzene, chloramphenicol, gallic acid and aureomycin were moderately or markedly inhibitory to both mycoparasitic and non-mycoparasitic Pythium spp. Also, in the applicant´s experience Pythium oligandrum Strain M1 M1 is sensitive to fungicides used in agriculture (sulphur, tebuconazol, chlorthalonil, fenhexamid, mancozeb, folpet, prothioconazole, fenbuconazole etc.) On the hand, antibiotics are used in selective agars serving for isolation of Pythium oligandrum from soils. Al- Rawahi and Hancock (1997) used for this purpose semi-selective medium based on cornmeal agar containing 400 mg of saponin, 25 mg of pimaricin, 50 mg of penicillin G, 50 mg of polymyxin B, 60 mg of streptomycin sulfate, and 20 mg of rose Bengal per liter. This implies that Pythium oligandrum Strain M1 is rather resistant against these antibiotics in the given concentrations.

Classification and proposed labelling with regard to micro-organism As a micro-organism Pythium oligandrum Strain M1 does not fall under the CLP regulation or Directive 67/548/EE. However, as other microorganisms it is considered as a potential sensitizer and its label shall contain the following phrase:

”Microorganisms may have the potential to provoke sensitising reactions”

16 Pythium oligandrum M1 Product-type 10 January 2015

Chapter 2: Methods of Analysis

Analytical methods for the active substance Manufactured micro-organism(principle of The content of oospores is enumareted under light method): microscope in Buerkers chamber. Impurities and contaminating micro-organisms in The following control is performed on the input manufactured material (principle of method): inoculum. 1 cm2 of pure culture of Pythium oligandrum is put in cultivation bouillon, cultivated for 3-4 days at room temperature and spread on non-selective agar medium. In case of contamination, the culture of P. oligandrum excluded from the next technology steps. The laboratory batches are macroscopically observed each day of the fermentation process and contaminated batches are excluded from the production The same method for quantification of oospores is used as that used for the active substance. For each batch, the viability of the oospores is checked in the following way: 1 g of the preparation is homogenized in 50 mL of sterile demineralised water and one drop of Tween 80 is added. The suspension number of germinating oospores is counted using microscope after 1, 24, 48, 72, 96 and 120 hours. Enumeration is carried our four times at each time interval and the median is calculated.

Analytical methods for residues (viable and non-viable) Analytical methods for residues of the active micro- The proposed use does not result any significant increase organism (principle of method): of M1 strain in any of the environmental comprtments. Thuspost approval monitoring on a regular basis is not justified. In case of accident Pythium oligandrum can be identified by microscopic taxonomic analysis. Quantifying P. oligandrum oospores can be made by diluting the sample and counting the oospores in Bürker’s chamber. The strain M1 can be differentiated from other strains using PCR method utilizing differences in ITS regions and sequences of of cytochromoxidase genes (Cox I and Cox II) between differnet Pythium oligandrum Strain M1 strains (

This method can be complemented by differentiation of Pythium oligandrum Strain M1 strains from each other by sequencing the internal transcribed spacer 1 (ITS- 1,complete sequence, ITS-2(partial sequence) and 5.8S gene (complete sequence) using FastDNA spin kit. For determination of the Pythium sequences the primers described by Schurko and coworkers (2003) and the protocol described by Allain-Boule and coworkers (2004) can be utilized. Analytical methods for residues of relevant Pythium oligandrum Strain M1 Oligandrum produces no metabolites (principle of method): toxicologically relevant metabolite . The metabolites produced by Pythium oligandrum Strain M1 can be determined by LC MS/MS method combining digestion of the proteins with trypsin, LC-MS/MS separation and fragmentation of peptides and identification of proteins by means of database searching with obtained MS/MS spectra.

17 Pythium oligandrum M1 Product-type 10 January 2015

Chapter 3: Impact on Human Health

Medical data, surveillance and observations: No literature reports or any other reports of adverse effects due to Pythium oligandrum. Sensitisation (experience in humans and study No study has been performed. results; type of study): Pythium oligandrum M1 should be considered as a potential sensitizer.

Acute toxicity Toxicity after acute oral exposure: LD50 > 5 000 mg kg-1 bw, corresponding to 2.5 x 107 oospores kg-1 bw Toxicity after acute inhalation exposure: LC50 >5 mg/L corresponding to 1 – 1.5 x 104 oospores L-1 Toxicity after acute intraperitoneal/ subcutaneous LD50 > 3.6 104oospores/animal exposure:

Infectivity (Annex IIA, point 6.3) Infectivity after acute oral exposure Not possible to perform (see Infectiveness study, below). Infectivity after acute inhalation exposure Not possible to perform (see Infectiveness study, below). Infectivity after acute intraperitoneal/ subcutaneous Not possible to perform (see Infectiveness study, exposure: below).

Pathogenicity Pathogenicity after acute oral exposure Not pathogenic Pathogenicity after acute inhalation exposure Not pathogenic Pathogenicity after acute intraperitoneal/ Not pathogenic subcutaneous exposure:

Genotoxicity Pythium oligandrum does not produce any known metabolites considered to be of toxicological concern. Genotoxicity testing of specific metabolites is therefore not required. There are no validated methods for genotoxicity testing of whole cell extracts: false positive and false negative results can both be anticipated and there is a lack of relevant positive and negative controls, making the results of any such test difficult to interpret. Until suitable methods are developed, genotoxicity testing of cell extracts is not required.

Cell culture study: Not applicable since Pythium oligandrum is an oomycete without any known mechanism for intracellular replication.

Short term toxicity/pathogenicity: Waived

18 Pythium oligandrum M1 Product-type 10 January 2015

Specific toxicity, pathogenicity and infectiveness studies Dermal toxicity: LD50 > 5 000 mg kg-1 bw, corresponding to approximately 1 x 107 oospores kg-1 bw. Neurotixicity: NOAEL = 4g .kg -1 bw day -1 corresponding to 4 x 106 oospores kg -1 bw day -1 Infectiveness study: Propagules were detected in blood and caecum samples of all animals at 0 hours after administration. At 1, 3, 6, and 24 hours, no propagules were found. Homogenization had a lethal effect on the oospores. On the basis of the results of this study, performing a combined toxicity/pathogenicity test on Pythium oligandrum appeared irrelevant Growth ability study: Slight growth of P. oligandrum on potato extract agar at 26°C and 35°C but no growth at 37°C and 40°C or at any temperature on sheep’s blood agar. Fructification was not noted at any temperature. Skin irritation Not irritating Eye irritation: Not irritating

AOEL: Not relevant due to lack of significant adverse effects in relevant studies. ADI: Not relevant due to lack of significant adverse effects in relevant studies.

Acceptable exposure scenarios Professional users Brush and roller application. Non-professional users Brush and roller application. Indirect exposure as a result of use Improbable scenario.

19 Pythium oligandrum M1 Product-type 10 January 2015

Chapter 4: Fate and Behaviour in the Environment

Route and rate of degradation in water (Annex IIA, point 7.6, IIIA, point XII.2.1, 2.2) Hydrolysis of active substance and Not relevant. relevant metabolites (DT50) (state pH and temperature) Photolytic / photo-oxidative degradation Not relevant of active substance and resulting relevant metabolites Readily biodegradable (yes/no) Not relevant Biodegradation in seawater Not relevant Non-extractable residues Not relevant. Distribution in water / sediment systems Not relevant. (active substance) Distribution in water / sediment systems Not relevant (metabolites)

Route and rate of degradation in soil (Annex IIIA, point VII.4, XII.1.1, XII.1.4; Annex VI, para. 85) Mineralization (aerobic) Not relevant Laboratory studies (range or median, Not relevant with number of measurements, with regression coefficient) Field studies (state location, range or Not relevant median with number of measurements) Anaerobic degradation Not relevant Soil photolysis Not relevant Non-extractable residues Not relevant Relevant metabolites - name and/or Not relevant code, % of applied a.i. (range and maximum) Soil accumulation and plateau Not relevant concentration

20 Pythium oligandrum M1 Product-type 10 January 2015

Adsorption/desorption (Annex IIA, point XII.7.7; Annex IIIA, point XII.1.2) Ka , Kd Not relevant

Kaoc , Kdoc Not relevant pH dependence (yes / no) (if yes type of Not relevant dependence)

Fate and behaviour in air (Annex IIIA, point VII.3, VII.5) Direct photolysis in air Not relevant Quantum yield of direct photolysis Not relevant Photo-oxidative degradation in air Not relevant Volatilization Not relevant

Monitoring data, if available (Annex VI, para. 44) Soil (indicate location and type of study) Not relevant Surface water (indicate location and type Not relevant of study) Ground water (indicate location and type Not relevant of study) Air (indicate location and type of study) Not relevant

21 Pythium oligandrum M1 Product-type 10 January 2015

Chapter 5: Effects on Non-target Species

Toxicity data for aquatic species (most sensitive species of each group) (Annex IIA, point 8.2, Annex IIIA, point 10.2) Species Time- Endpoint Toxicity scale Fish Poecillia reticulata No risk can be - identified with the proposed use.

Invertebrates Waived No risk can be identified with the proposed use.

Algae Waived No risk can be identified with the proposed use.

Microorganisms Waived No risk can be identified with the proposed use.

Effects on earthworms or other soil non-target organisms Waived Acute toxicity to ………………………………….. (Annex IIIA, point XIII.3.2) Waived Reproductive toxicity to ………………………… (Annex IIIA, point XIII.3.2)

Effects on soil micro-organisms (Annex IIA, point 7.4) Nitrogen mineralization Not relavant. Carbon mineralization Not relevant.

Effects on terrestrial vertebrates Acute toxicity to mammals No risk can be identified with the proposed (Annex IIIA, point XIII.3.3) use. Acute toxicity to birds Waived (Annex IIIA, point XIII.1.1) Dietary toxicity to birds Waived

22 Pythium oligandrum M1 Product-type 10 January 2015

(Annex IIIA, point XIII.1.2) Reproductive toxicity to birds Waived (Annex IIIA, point XIII.1.3)

Effects on honeybees (Annex IIIA, point XIII.3.1) Acute oral toxicity - Acute contact toxicity Indications that there might not be any effects with approximately 103 oospores per bee.

Effects on other beneficial arthropods (Annex IIIA, point XIII.3.1) Acute oral toxicity - Acute contact toxicity The indicative test on bees can also be valid for other arthropods, in which there is an indication of no effect at an exposure rate of approximately 103 oospores per bee, which means that one bee need to be sprayed with 1 l. - Acute toxicity to …………………………………..

Bioconcentration (Annex IIA, point 7.5) Bioconcentration factor (BCF) Not relevant.

Depration time (DT50) Not relevant.

(DT90) Level of metabolites (%) in organisms Not relevant. accounting for > 10 % of residues

23 Pythium oligandrum M1 Product-type 10 January 2015

Appendix II: List of Intended Uses

Object Product Organisms Re and/or Formulation Application Applied amount per treatment name controlled marks: situation Type Conc. method number interval g a.s./L water g a.s./m2 *two (d-f) of a.s. kind min max between min max L/m2 min max treatments (i) (f-h) applications min with different (min) doses and max concentrations performed

Wall Biorepell Saprophytic powder 20% brushing 2 2 2 hours 0.02* 0.2* 1.5* 0.04 (curative) molds 10* 0.04 Wall Biorepell Saprophytic powder 20% brushing 1 1 n.a. 0.04 – 0.1 0.15 0.006 (preventive) molds 0.5 0.05

Wall Biorepell Saprophytic powder 20% brushing 1 1 n.a. 0.12 – 0.12 0.072 0.00864 (preventive, molds paint) 0.1 0.012

24 Pythium Oligandrum Product-type 10 January 2015

Appendix III: List of studies

Data protection is claimed by the applicant in accordance with Article 60 of Regulation (EU) No 528/2012.

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published

IIA 2.1 Jong SC, & 1978 Re: Phytopath. Z. 90: 113- YES Biopreparáty, 115, 1977 Pythium spol. s r.o. IIIA 1.3.3 De Lucio, A. oligandrum a strain for biological control of damping off pathogens GLP: No Published: No

IIA 2.1 Laichmanová M 2005 Záznam o deponování YES Biopreparáty, IIIA 1.3.3 kultury za účelem spol. s r.o bezpečného uložení, Czech Collection of Micro- organisms GLP: No Published: No IIA 2.3 Van der Plaats- 1981 MONOGRAPH OF THE NO Public IIA 2.4.3 Niterink J GENUS PYTHIUM , Studies IIA 5.1 in Mycology, 21: 1-242 IIIA 1.3. GLP: No IIIA 2.5 Published: Yes IIIA 2.6 IIIA 2.10 IIIA 4.1 IIA 2.4.3 Godfrey SAC, 2003 Identification of NO Public IIIA 1.3.4 Monds, RD, Pythium oligandrum IIIA 4.1 Lash DT using species-specific and Marshall JW ITS rDNA PCR oligonucleotides. Mycological research 107(7):790-

[1] Section Number/Reference Number should refer to the section number in Doc III-A or III-B. If the study is non-key, and hence not summarised in Doc III but mentioned in Doc II, it should be included in the reference list alongside related references and its location in Doc II indicated in brackets. (If there is a need to include a cross-reference to PPP references then an additional column can be inserted). [2] Author’s Name should include the author’s surname before initial (s) to enable the column to be sorted alphabetically. If the Human Rights Charter prevents author’s surnames on unpublished references being included in non-confidential documents, then it will be necessary to consider including ‘Unpublished [number/year & letter] ’ in Doc II, and both ‘ Unpublished [number/year & letter]’ and the ‘Authors Name’ in the reference list’. This may necessitate the need for an additional column to state whether a reference is unpublished which can then be sorted. [3] Title, Source (where different from company), Company, Report No., GLP (where relevant), (Un)Published should contain information relevant to each item (ideally on separate lines within the table cell for clarity). If useful, the name of the electronic file containing the specific study/reference could be added in brackets.

25 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published 796. GLP: No Published: Yes IIIA 1.4.3 2002 YES

IIIA 1.4.3 2006 YES

IIIA 1.4.3 2014 YES

IIIA 1.4.3 2014a YES

26 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published

IIIA 1.4.3 2014b YES

IIIA 1.4.3 2014c YES

IIIA 1.4.3 2014d YES

IIIA 1.4.3 2014e YES

27 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published

IIIA 2.1.1 Martin FN, & 1987 THE USE OF PYTHIUM NO Public Hancock JG OLIGANDRUM FOR BIOLOGICAL CONTROL OF PREEMERGENCE DAMPING-OFF CAUSED BY PYTHIUM ULTIMUM, Phytopathology, 87 (7): 1013-1020 GLP: No Published: Yes IIIA 2.1.1 Veselý D 1977 POTENTIAL NO Public BIOLOGICAL CONTROL OF DAMPING-OFF PATHOGENS IN EMERGING SUGAR BEETS BY PYTHIUM OLIGANDRUM DRECHSLER. Phytopath. Z. 90, 113-115 GLP: No Published: Yes IIA 2.3 Laing SAK, & 1991 Video microscopical NO Public comparison of IIA 6 Deacon JW IIIA 2.2.2 mycoparasitism by Pythium oligandrum, P. nunn, and an unnamed Pythium species, Mycological Research 95 (4): 469-479. GLP: No Published: Yes IIA 2.3 Lewis K, Whipps 1989 Mechanisms of biological NO Public IIA 5.1 JM, & Cook RC disease control with special IIA 5.2.4 reference to the case study IIA 6 of Pythium oligandrum as an antagonist. In: IIIA 2.2.2 Biotechnology of Fungi for Improving Plant Growth. British Mycological Society. GLP: No Published: Yes IIA 2.3 Brožová J 2002 Exploitation of the NO Public IIIA 2.3 mycoparasitic fungus Pythium oligandrum in plant protection. Plant

28 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)c21 Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed NoC1l GLP (where (Yes/No) relevant) (Un)Published

Protection Science. 38: 29- 35 GLP:No Published: Yes

IIA 2.3 Deacon J 2006 Fungal biology, 4th edition NO Public Blackwell Publishing Ltd GLP:No Published: Yes IIA 2.3 Kwon-Chung 1994 Phylogenetic NO Public KJ Spectrum of Fungi That Are Pathogenic to Humans, Clinical Infectious Diseases, 19 (Suppl 1): 1-7.

IIA 2.3 2006 YES IIA 5.1 IIA 5.2.4

llA 2.3 Rivierre C, 2005 PYTHIOSIS IN AFR ICA, NO Public laprie C, Emerging lnfectuos Guiard-Marigny Diseases, 11: 479-481 O, Be rgeaud P, GLP:No Berthelemy M, & Published: Yes Guillot J IIIA 2.3 Fletcher JT, 1990 PYTHJUM OLIGANORUM NO Public Smewin BJ, & ASSOCIATED WITH A O'Brien A CROPPING DISORDER OF AGAR/CVS BJSPORUS. Plant Pathology, 39, 603-605. GLP: No Published: Yes IIIA 2.4 - 2003 YES

IIA 2.4.2 2013 YES

29 Pythium Oligandrum Product-type 10 January 2015

Section Author( s) £2l Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed No£1l GLP (where (Yes/No) relevant) (Un)Published

IIA 2.4.2 2013a YES

IIA 2.4.3 2013b YES

IIA 2.4.3 2013 YES

IIA 2.4.3 2013a YES

30 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published

IIA 2.4.3 Vallance J 2009 Inﬂuence of Pythium NO Public oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere GLP: No

Published: Yes IIA 2.4.3 Schroeder KL, 2013 Molecular Detection NO Public Martin FN, and Quantification of de Cock Pythium Species: AWAM, Evolving Taxonomy, Lévesque CA, New Tools, and Spies CFJ, Challenges. Plant Okubara PA, & Disease, January Paulitz TC 2013, Volume 97, Number 1 Pages 4 – 20 GLP: No

Published: Yes IIA 2.4.3 Lévesque CA, 2003 Molecular phylogeny NO Public & de Cock and taxonomy of the AWAM genus Pythium. , Mycol. Res. 108:1363–1383 GLP: No

Published: Yes Schurko AM, 2003 A molecular NO Public Mendoza L, phylogeny of Lévesque CA, Pythium, Mycol. Res. Désaulniers 107:537–544 NL, GLP: No De Cock AWAM, & Published: Yes Klassen GR

31 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)[2] Year Title[3] Data Owner No / Source Company Protection Reference Report No. Claimed No[1] GLP (where (Yes/No) relevant) (Un)Published IIA 2.4.3 Allain-Boulé 2004 Pythium NO Public N, Tweddell R, attrantheridium sp. Mazzola M, nov.: taxonomy and Bélanger R, & comparison with Lévesque CA related species. Mycol. Res. 108:795– 805 GLP: No

Published: Yes

IIIA 2.6 Mendoza L, 1993 Life Cycle of the Human NO Public Hernandez F, & and Animal Oomycete Ajello L Pathogen Pythium insidiosum, Journal of Clinical Microbiology, vol. 31, no 11: 2967-73. GLP: No Published: Yes IIIA 2.8 Picard K, Ponchet 2000 Oligandrin. A NO Public M, Blein JP, Tirilly Proteinaceous Molecule Y, & Benhamou N Produced by the Mycoparasite Pythium oligandrum Induces Resistance to Phytophthora parasitica Infection in Tomato Plants, Plant Physiology 124 (1): 379- 395. GLP: No Published: Yes IIIA 2.8 Hase S, Shimizu A, 2006 Induction of transient NO Public Nakaho K, ethylene and reduction in Takenaka S, & severity of tomato bacterial Takahashi H wilt by Pythium oligandrum, Plant Pathology, 55, 537–543 GLP: No Published: Yes IIIA 2.8 Le Floch G, Rey P, 2003 Impact of auxin- NO Public Benizri E, compounds produced by Benhamou N, & the antagonistic fungus Trilly Y Pythium oligandrum or the minor pathogen Pythium group F on plant growth, Plant and soil, 104 (1): 1- 109. GLP: No Published: Yes IIA 4.2 1979 Realisation of the YES Biopreparáty, IIIA experiment – acute toxicity

32 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)c21 Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed NoC1l GLP (where (Yes/No) relevant) (Un)Published 5.2.2.1 spol. s r.o

GLP: No Published: No

IIA 4.2 1980 Realisation of the YES Biopreparaty, IIIA experiment - acute toxicity spol. s r.o 5.2.2.1 - of given preparat ion

GLP:No Published: No

IIA 4 .2 2001 Oospores of fungic YES Biopreparaty, IIIA organism Pythium spol. s r.o 5.2.2.1 o/igandrum Drechsler - - Acute Oral Toxicity in Rats

Published: No

IIA 4.2 2001 Oospores of f ungic YES Biopreparaty, IIIA organism Pythium spol. s r.o 5.2.2.1 o/igandrum Drechsler -

GLP: Yes Published: No IIA 4.2.2 2002 Concentrate of Pythium YES Biopreparaty, IIIA o/igandrum Drechsler spol. s r.o 5.2.2.2 oospores including ot her propagules - Acute Inhalation Toxicity - Lli mit

GLP: Yes Published: No

IIA 4 .2.3 2005 Propagules Pythium YES Biopreparaty, IIIA o/igandrum DV 74 - Acute spol. s r.o 5.2.2.3 I

GLP: Yes Published: No Concentrate of IIA 4.3.1 12003 Pythium YES Biopreparaty, IIIA o/igandrum Drechsler spol. s r.o 5.2.2.3 I- oospores including ot her

33 Pythium Oligandrum Product-type 10 January 2015

Section Author( s) £2 l Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed No£1l GLP (where (Yes/No) relevant) (Un)Published

propagules Subcutaneous Dermal Tolerance - Rabbit

IIA 4.3.1 2005 YES Biopreparaty, spol. s r.o

GLP: Yes Published: No IIA 4.2.1 2001 Concentrate of Pythium YES Biopreparaty, IIIA o/igandrum Drechsler spol. s r.o 5.2.2.4 oospores including other propagules - Dermal Acute

GLP: Yes Published: No IIIA 2002 Concentrate of Pythium YES Biopreparaty, 5.2.2.4 o/igandrum Drechsler spol. s r.o

GLP: Yes Published: No IIA 4 .5 1981 Realisation of experiment - YES Biopreparaty, IIIA 5.2.5 sub-acute toxicity of spol. s r.o biological preparation for

GLP: No Published: No

IIA 5.1 Foley, MF, & 1985 Isolation of Pythium NO Public IIIA 7.1 Deacon, JW o/igandrum and other necrotrophic mycoparasites from soil. Trans.Br. Mycol. Soc. 84(4):631-639 GLP: No

34 Pythium Oligandrum Product-type 10 January 2015

Section Author(s)c21 Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed NoC1l GLP (where (Yes/No) relevant) (Un)Published

Published: Yes IIA 5.1 2013a Horizontal mobility of YES Biopreparaty, IIIA 7.1 Pythium oligandrum spol. s r.o. in defined soil environment.

IIIA 7.1 2013b Behaviour of Pythium YES Biopreparaty, oligandrum on spol. s r.o. interface of sedimentary phase and water environment

IIA 5.1 Al - Rawah i 1997 Rhizosphere NO Public AK, Competence of & Hancock JG Pythium oligandrum. Phytopathology 87:951-959. GLP: No Published: Yes IIA 5.1.2 Madsen MA, & de 2004 Interactions Between the NO Public Neergaard E Mycoparasite Pythium o/igandrum and Sclerotia of the Plant Pathogen Sclerotinia sclerotiorum . Eu ropean Journal of Plant Pathology, Vol. 105, Issue 8, 761-768). IIA 5.1.2 Takenaka S, 2008 Colonization of Pythium NO Public Sekiguchi H, o/igandrum in the Tomato Nakaho K, Tojo Rh izosphere for Biological M, Masunaka A, Control of Bacterial Wilt & Takahashi H Disease Analyzed by Rea l- Time PCR and Confocal Laser-Scanning Microscopy Phytopathology. 2008, Feb;98{2):187-95 IIIA 8.2.1 2003 Concentrate of Pythium YES Biopreparaty, o/igandrum Drechsler spol. s r.o. - oospores including other propagules -Test of Acute Toxicity (Poeci/ia reticu/ata)

35 Pythium Oligandrum Product-type 10 January 2015

Section Author( s) £2 l Year TitleC3 l Data Owner No I Source Company Protection Reference Report No. Claimed No£1l GLP (where (Yes/No) relevant) (Un)Published

GLP : YES Published: No IIIA 8.3 VeselyV 1992 Classification of the YES Biopreparaty, preparation according to its spol. s r.o. effect on bees - binding expertise, Extract from the protocol No.330 Bee Keeping Research Institute, Doi, Laboratory report: 330 No. of decision 3953/92, report date: 11th and 12'h December 1992 GLP: No Published: No