regulation (eu) no 528/2012 concerning the making...

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

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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

Pythium Oligandrum M1 Product-type 10 January 2015

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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

animal

Material administered TL0 values

mg a.s./kg

bw

Observation

period

Examination

oral gavage

rat, mouse

active substance

in 12.5% suspension

single dose

>5000

14 days clinical,

necropsy

oral gavage

mouse

active substance,

sterilised, suspended

single dose

>20 000

10 hours mortality

behaviour

dermal

rat

active substance

moistened (1.5ml/g)

24 hour

semiocclusion

single dose

>5000

14 days weight, clinical,

local signs

necropsy

inhalation

rat

4 hour

exposure

active substance

5 mg/L air

GM 7.45 µm

single dose

>600

14 days weight, clinical signs,

behaviour,

necropsy,

histopathology of

respiratory system.

oral, in diet

rat, 70 days

active substance

0.8, 2.4, 4 % in diet

repeated

daily dose **

>4000

70 days weight, clinical,

behavioural **

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.


13

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.


14

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

micro-organism used for manufacturing of the

formulated product (cfu/g; cfu/L, etc.):

Beetween 15 to 20% of the TGAI is used to

manufacture the product.

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 10

6

Identity and content of relevant impurities in

thetechnical grade micro-organism:

approx. 95 % (w/w) of fermented remnants of cereal

based growing medium

Is the MCPA genetically modified; if so

provide type of modification

MPCA is not genetically modified

Biological properties of the micro-organism

.

Origin and natural occurrence, background

level:

Pythium oligandrum is a ubiquitous inhabitant of

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


15

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


16

expected that additional metabolites are produced.

Resistance/sensitivity to antibiotics/anti-

microbial agents used in human or veterinary

medicine:

The product is not intended to be mixed with other

fugicides or antibiotics. Pythium oligandrum Strain

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”


17

Chapter 2: Methods of Analysis

Analytical methods for the active substance

Manufactured micro-organism(principle of

method):

The content of oospores is enumareted under light

microscope in Buerkers chamber.

Impurities and contaminating micro-organisms in

manufactured material (principle of method):

The following control is performed on the input

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-

organism (principle of method):

The proposed use does not result any significant increase

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

metabolites (principle of method):

Pythium oligandrum Strain M1 Oligandrum produces no

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.


18

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

results; type of study):

No study has been performed.

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

exposure:

LD50

> 3.6 104oospores/animal

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

exposure:

Not possible to perform (see Infectiveness study,

below).

Pathogenicity

Pathogenicity after acute oral exposure Not pathogenic

Pathogenicity after acute inhalation exposure Not pathogenic

Pathogenicity after acute intraperitoneal/

subcutaneous exposure:

Not pathogenic

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


19

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.


20

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

relevant metabolites (DT50) (state pH

and temperature)

Not relevant.

Photolytic / photo-oxidative degradation

of active substance and resulting

relevant metabolites

Not relevant

Readily biodegradable (yes/no) Not relevant

Biodegradation in seawater Not relevant

Non-extractable residues Not relevant.

Distribution in water / sediment systems

(active substance)

Not relevant.

Distribution in water / sediment systems

(metabolites)

Not relevant

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,

with number of measurements, with

regression coefficient)

Not relevant

Field studies (state location, range or

median with number of measurements)

Not relevant

Anaerobic degradation Not relevant

Soil photolysis Not relevant

Non-extractable residues Not relevant

Relevant metabolites - name and/or

code, % of applied a.i. (range and

maximum)

Not relevant

Soil accumulation and plateau

concentration

Not relevant


21

Adsorption/desorption (Annex IIA, point XII.7.7; Annex IIIA, point XII.1.2)

Ka , Kd

Kaoc , Kdoc

pH dependence (yes / no) (if yes type of

dependence)

Not relevant

Not relevant

Not relevant

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

of study)

Not relevant

Ground water (indicate location and type

of study)

Not relevant

Air (indicate location and type of study) Not relevant


22

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-

scale

Endpoint Toxicity

Fish

Poecillia reticulata No risk can be

identified with the

proposed use.

-

Invertebrates

Waived No risk can be

identified with the

proposed use.

Algae


identified with the

proposed use.

Microorganisms


identified with the

proposed use.

Effects on earthworms or other soil non-target organisms

Acute toxicity to …………………………………..

(Annex IIIA, point XIII.3.2)

Waived

Reproductive toxicity to …………………………


Waived

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

use.

Acute toxicity to birds


Waived

Dietary toxicity to birds Waived


23


Reproductive toxicity to birds


Waived

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)

(DT90)

Not relevant.

Level of metabolites (%) in organisms

accounting for > 10 % of residues

Not relevant.


24

Appendix II: List of Intended Uses

Object

and/or

situation

Product

name

Organisms

controlled Formulation Application Applied amount per treatment

Re

marks:

Type

(d-f)

Conc.

of a.s.

(i)

method

kind

(f-h)

number

min max

interval

between

applications

(min)

g a.s./L

min max

water

L/m2

min

max

g a.s./m2

min max

*two

treatments

with different

doses and

concentrations

performed

Wall

(curative)

Biorepell Saprophytic

molds

powder 20% brushing 2 2

2 hours 0.02* 0.2* 1.5*

10*

0.04

0.04

Wall

(preventive)


molds

powder 20% brushing 1 1 n.a. 0.04 – 0.1 0.15

0.5

0.006

0.05

Wall

(preventive,

paint)


molds

powder 20% brushing 1 1 n.a. 0.12 – 0.12 0.072

0.1

0.00864

0.012

Pythium Oligandrum Product-type 10 January 2015

25

Appendix III: List of studies

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

Section

No /

Reference

No[1]

Author(s)[2] Year Title[3]

Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

IIA 2.1

IIIA 1.3.3 Jong SC, &

De Lucio, A.

1978 Re: Phytopath. Z. 90: 113-

115, 1977 Pythium

oligandrum a strain for

biological control of

damping off pathogens

GLP: No

Published: No

YES Biopreparáty,

spol. s r.o.

IIA 2.1

IIIA 1.3.3 Laichmanová M 2005 Záznam o deponování

kultury za účelem

bezpečného uložení, Czech

Collection of Micro-

organisms

GLP: No

Published: No

YES Biopreparáty,

spol. s r.o

IIA 2.3

IIA 2.4.3

IIA 5.1

IIIA 1.3.

IIIA 2.5

IIIA 2.6

IIIA 2.10

IIIA 4.1

Van der Plaats-

Niterink J

1981 MONOGRAPH OF THE

GENUS PYTHIUM , Studies

in Mycology, 21: 1-242

GLP: No

Published: Yes

NO Public

IIA 2.4.3

IIIA 1.3.4

IIIA 4.1

Godfrey SAC,

Monds, RD,

Lash DT

and Marshall JW

2003 Identification of

Pythium oligandrum

using species-specific

ITS rDNA PCR

oligonucleotides.

Mycological

research 107(7):790-

NO Public

[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.


26

Section

No /

Reference

No[1]


Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

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


27

Section

No /

Reference

No[1]


Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

IIIA 1.4.3 2014b

YES

IIIA 1.4.3 2014c

YES

IIIA 1.4.3 2014d

YES

IIIA 1.4.3 2014e

YES


28

Section

No /

Reference

No[1]


Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

IIIA 2.1.1 Martin FN, &

Hancock JG

1987 THE USE OF PYTHIUM

OLIGANDRUM FOR

BIOLOGICAL

CONTROL OF

PREEMERGENCE

DAMPING-OFF

CAUSED BY PYTHIUM

ULTIMUM,

Phytopathology, 87

(7): 1013-1020

GLP: No

Published: Yes

NO Public

IIIA 2.1.1 Veselý D 1977 POTENTIAL

BIOLOGICAL

CONTROL OF

DAMPING-OFF

PATHOGENS IN

EMERGING SUGAR

BEETS BY PYTHIUM

OLIGANDRUM

DRECHSLER.

Phytopath. Z. 90,

113-115 GLP: No

Published: Yes

NO Public

IIA 2.3

IIA 6

IIIA 2.2.2

Laing SAK, &

Deacon JW

1991 Video microscopical

comparison of

mycoparasitism by Pythium

oligandrum, P. nunn, and

an unnamed Pythium

species, Mycological

Research 95 (4): 469-479.

GLP: No

Published: Yes

NO Public

IIA 2.3

IIA 5.1

IIA 5.2.4

IIA 6

IIIA 2.2.2

Lewis K, Whipps

JM, & Cook RC

1989 Mechanisms of biological

disease control with special

reference to the case study

of Pythium oligandrum as

an antagonist. In:

Biotechnology of Fungi for

Improving Plant Growth.

British Mycological Society.

GLP: No

Published: Yes

NO Public

IIA 2.3

IIIA 2.3 Brožová J 2002 Exploitation of the

mycoparasitic fungus

Pythium oligandrum in

plant protection. Plant

NO Public


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

Publ ished: Yes

IIA 2.3 Deacon J 2006 Fungal biology, 4th edition NO Public

Blackwell Publishing Ltd

GLP:No

Publ ished: 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 AFRICA, NO Public

laprie C, Emerging lnfectuos

Guiard-Marigny Diseases, 11: 479-481

O, Bergeaud P, GLP:No Berthelemy M, &

Publ ished: Yes Gui llot 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

Publ ished: Yes

IIIA 2 .4 - 2003 YES

IIA 2.4.2 2013 YES

29

Pythium Oligandrum Product-type 10

Section No I Reference No£1l

Author( s) £2 l Year TitleC3 l Data Source Company Protection Report No. Claimed 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

January 2015

Owner


31

Section

No /

Reference

No[1]


Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

IIA 2.4.3

Vallance J 2009 Influence of Pythium

oligandrum Biocontrol

on Fungal and

Oomycete Population

Dynamics in the

Rhizosphere

GLP: No

Published: Yes

NO Public

IIA 2.4.3

Schroeder KL,

Martin FN,

de Cock

AWAM,

Lévesque CA,

Spies CFJ,

Okubara PA, &

Paulitz TC

2013 Molecular Detection

and Quantification of

Pythium Species:

Evolving Taxonomy,

New Tools, and

Challenges. Plant

Disease, January

2013, Volume 97,

Number 1 Pages 4 –

20

GLP: No

Published: Yes

NO Public

IIA 2.4.3

Lévesque CA,

& de Cock

AWAM

,

2003 Molecular phylogeny

and taxonomy of the

genus Pythium.

Mycol. Res.

108:1363–1383

GLP: No

Published: Yes

NO Public

Schurko AM,

Mendoza L,

Lévesque CA,

Désaulniers

NL,

De Cock

AWAM, &

Klassen GR

2003 A molecular

phylogeny of

Pythium, Mycol. Res.

107:537–544

GLP: No

Published: Yes

NO Public


32

Section

No /

Reference

No[1]


Source Company

Report No.

GLP (where

relevant)

(Un)Published

Data

Protection

Claimed

(Yes/No)

Owner

IIA 2.4.3

Allain-Boulé

N, Tweddell R,

Mazzola M,

Bélanger R, &

Lévesque CA

2004 Pythium

attrantheridium sp.

nov.: taxonomy and

comparison with

related species.

Mycol. Res. 108:795–

805

GLP: No

Published: Yes

NO Public

IIIA 2.6 Mendoza L,

Hernandez F, &

Ajello L

1993 Life Cycle of the Human

and Animal Oomycete

Pathogen Pythium

insidiosum, Journal of

Clinical Microbiology, vol.

31, no 11: 2967-73.

GLP: No

Published: Yes

NO Public

IIIA 2.8 Picard K, Ponchet

M, Blein JP, Tirilly

Y, & Benhamou N

2000 Oligandrin. A

Proteinaceous Molecule

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

NO Public

IIIA 2.8 Hase S, Shimizu A,

Nakaho K,

Takenaka S, &

Takahashi H

2006 Induction of transient

ethylene and reduction in

severity of tomato bacterial

wilt by Pythium

oligandrum, Plant

Pathology, 55, 537–543

GLP: No

Published: Yes

NO Public

IIIA 2.8 Le Floch G, Rey P,

Benizri E,

Benhamou N, &

Trilly Y

2003 Impact of auxin-

compounds produced by

the antagonistic fungus

Pythium oligandrum or the

minor pathogen Pythium

group F on plant growth,

Plant and soil, 104 (1): 1-

109.

GLP: No

Published: Yes

NO Public

IIA 4.2

IIIA 1979 Realisation of the

experiment – acute toxicity

YES Biopreparáty,

Pythium Oligandrum

Section No I Reference NoC1l

5.2.2 .1

IIA 4.2 IIIA 5.2.2.1

IIA 4 .2 IIIA 5.2.2.1

IIA 4.2 IIIA 5.2.2 .1

IIA 4.2.2 IIIA 5.2.2.2

IIA 4 .2.3 IIIA 5.2 .2 .3

IIA 4.3. 1 IIIA 5.2.2 .3

Author(s)c21

--

I

I-

Product-type 10

Year TitleC3 l

Source Company Report No. GLP (where relevant)

(Un)Published

GLP: No

Publ ished: No

1980 Realisation of the

experiment - acute toxicity

of given preparat ion

GLP:No

Publ ished: No

2001 Oospores of fungic

organism Pythium o/igandrum Drechsler -

Acute Oral Toxicity in Rats

Publ ished: No

2001 Oospores of f ungic

organism Pythium

o/igandrum Drechsler -

GLP: Yes

Publ ished: No

2002 Concentrate of Pythium o/igandrum Drechsler

oospores including ot her

propagules - Acute

Inhalation Toxicity - Llimit

GLP: Yes

Publ ished: No

2005 Propagules Pythium o/igandrum DV 74 - Acute

12003

GLP: Yes

Publ ished: No

Concentrate of Pythium o/igandrum Drechsler

oospores including ot her

33

Data Protection

Claimed (Yes/No)

YES

YES

YES

YES

YES

YES

January 2015

Owner

spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty, spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty, spol. s r.o



IIA 4.3 .1

IIA 4.2.1 IIIA 5.2.2 .4

IIIA 5.2.2.4

IIA 4 .5 IIIA 5.2 .5

IIA 5 .1 IIIA 7 .1

Author( s) £2 l Year TitleC3 l

Source Report GLP

Company No.

(where

Foley, MF, & Deacon, JW

2005

relevant)

(Un)Published

propagules Subcutaneous

Dermal Tolerance - Rabbit

GLP: Yes

Publ ished: No


oospores including other

propagules - Dermal Acute

GLP: Yes

Publ ished: No


GLP: Yes

Publ ished: No

1981 Realisation of experiment -

sub-acute toxicity of

biological preparat ion for

GLP: No

Publ ished: No

1985 Isolation of Pythium o/igandrum and other

necrotrophic mycoparasites

from soil. Trans.Br. Mycol.

Soc. 84(4):631-639

GLP: No

34

Data Protection

Claimed (Yes/No)

YES

YES

YES

YES

NO

January 2015

Owner

Biopreparaty,

spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty,

spol. s r.o

Biopreparaty,

spol. s r.o

Public

Pythium Oligandrum

Section Author(s)c21

No I Reference NoC1l

IIA 5 .1 IIIA 7 .1

IIIA 7 .1

IIA 5 .1 Al-Rawah i AK, & Hancock JG

IIA 5 .1.2 Madsen MA, & de

Neergaard E

IIA 5.1.2 Takenaka S,

Sekiguchi H,

Nakaho K, Tojo

M, Masunaka A,

& Takahashi H

IIIA 8 .2 .1 -

Product-type 10

Year TitleC3 l Data Source Company Protection Report No. Claimed GLP (where (Yes/No) relevant)

(Un)Published

Publ ished: Yes

2013a Horizontal mobility of YES

Pythium oligandrum in defined soil environment.

2013b Behaviour of Pythium YES

oligandrum on interface of sedimentary phase and water environment

1997 Rhizosphere NO

Competence of Pythium oligandrum. Phytopathology 87:951-959. GLP: No

Published: Yes 2004 Interactions Between the NO

Mycoparasite Pythium

o/igandrum and Sclerotia of

the Plant Pathogen

Sclerotinia sclerotiorum .

European Journal of Plant

Pathology, Vol. 105, Issue 8,

761-768).

2008 Colonization of Pythium NO

o/igandrum in the Tomato

Rhizosphere for Biological

Control of Bacterial Wi lt

Disease Analyzed by Real-

Time PCR and Confocal

Laser-Scanning Microscopy

Phytopathology. 2008,

Feb;98{2):187-95

2003 Concentrate of Pythium YES

o/igandrum Drechsler

oospores including other

propagules - Test of Acute

Toxicity (Poeci/ia reticu/ata)

35

January 2015

Owner

Biopreparaty,

spol. s r.o.

Biopreparaty,

spol. s r.o.

Public

Public

Public

Biopreparaty,

spol. s r.o.



IIIA 8 .3

Author( s) £2 l Year TitleC3 l

Source Report GLP

Company No.

(where

VeselyV

relevant)

(Un)Published

GLP: YES Published: No

1992 Classification of the preparation according to its effect on bees - binding expertise, Extract from the protocol No.330 BeeKeeping Research Institute, Doi, Laboratory report: 330 No. of decision 3953/92, report date: 11th and 12'h

December 1992

GLP: No Published: No

36

Data Protection

Claimed (Yes/No)

YES

January 2015

Owner

Biopreparaty, spol. s r.o.

regulation (eu) no 528/2012 concerning the making...

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