CONCLUSION ON PESTICIDES PEER REVIEW

APPROVED: 5 December 2016

doi: 10.2903/j.efsa.2017.4668

Peer review of the pesticide risk assessment of the active

substance Pseudomonas chlororaphis strain MA 342
European Food Safety Authority (EFSA)

Abstract
The conclusions of EFSA following the peer review of the initial risk assessments carried out by the
competent authorities of the rapporteur Member State, the Netherlands, and co-rapporteur Member
State, Denmark, for the pesticide active substance Pseudomonas chlororaphis strain MA 342 are
reported. The context of the peer review was that required by Commission Implementing Regulation
(EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative
uses of Pseudomonas chlororaphis strain MA 342 as a seed treatment in cereals (wheat, rye and
triticale), carrots and peas against seed-borne diseases and foliar application in cereals against foliar
and ear pathogens. The reliable end points, appropriate for use in regulatory risk assessment, are
presented. Missing information identified as being required by the regulatory framework is listed.
Concerns are identified.
© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf
of European Food Safety Authority.

Keywords: Pseudomonas chlororaphis strain MA 342, peer review, risk assessment, pesticide,
fungicide

Requestor: European Commission

Question number: EFSA-Q-2015-00814
Correspondence: [email protected]

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Suggested citation: EFSA (European Food Safety Authority), 2017. Conclusion on the peer review of
the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342. EFSA
Journal 2017;15(1):4668, 21 pp. doi:10.2903/j.efsa.2017.4668
ISSN: 1831-4732
© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf
of European Food Safety Authority.
This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited and no
modifications or adaptations are made.

The EFSA Journal is a publication of the European Food

Safety Authority, an agency of the European Union.

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Peer review of the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342

Summary
Commission Implementing Regulation (EU) No 844/2012 (hereinafter referred to as ‘the
Regulation’) lays down the procedure for the renewal of the approval of active substances submitted
under Article 14 of Regulation (EC) No 1107/2009. The list of those substances is established in
Commission Implementing Regulation (EU) No 686/2012. Pseudomonas chlororaphis strain MA 342 is
one of the active substances listed in Regulation (EU) No 686/2012.
In accordance with Article 1 of the Regulation, the rapporteur Member State (RMS), the
Netherlands, and co-rapporteur Member State (co-RMS), Denmark, received an application from
Lantma €nnen BioAgri AB for the renewal of approval of the active substance Pseudomonas chlororaphis
strain MA 342. Complying with Article 8 of the Regulation, the RMS checked the completeness of the
dossier and informed the applicant, the co-RMS (Denmark), the European Commission and the
European Food Safety Authority (EFSA) about the admissibility.
The RMS provided its initial evaluation of the dossier on P. chlororaphis strain MA 342 in the
renewal assessment report (RAR), which was received by EFSA on 18 December 2015. In accordance
with Article 12 of the Regulation, EFSA distributed the RAR to the Member States and the applicant,
Lantma €nnen BioAgri AB, for comments on 9 February 2016. EFSA also provided comments. In
addition, EFSA conducted a public consultation on the RAR. EFSA collated and forwarded all comments
received to the European Commission on 13 April 2016.
Following consideration of the comments received on the RAR, it was concluded that additional
information should be requested from the applicant, and that EFSA should conduct an expert
consultation in the areas of mammalian toxicology, environmental fate and behaviour, and
ecotoxicology.
In accordance with Article 13(1) of the Regulation, EFSA should adopt a conclusion on whether
P. chlororaphis strain MA 342 can be expected to meet the approval criteria provided for in Article 4 of
Regulation (EC) No 1107/2009 of the European Parliament and of the Council.
The conclusions laid down in this report were reached on the basis of the evaluation of the
representative uses of P. chlororaphis strain MA 342 as a seed treatment in cereals (wheat, rye and
triticale), carrots and peas against seed-borne diseases and foliar application in cereals against foliar
and ear pathogens, as proposed by the applicant. Full details of the representative uses can be found
in Appendix A of this report.
Data were submitted to conclude that the representative uses proposed for P. chlororaphis strain
MA 342 at the European Union (EU) level result in a sufficient fungicidal efficacy against seed-borne
diseases in cereals, carrots and peas, and against foliar and ear pathogens in cereals.
With regard to the review of the scientific peer-reviewed open literature on the active substance
and its relevant metabolites, a data gap was identified for an updated search dealing with side effects
on non-target species.
Data gaps were identified for the suspensibility determination of the formulation for the
determination of the growth temperatures of P. chlororaphis strain MA 342 and to prove that besides
2,3-deepoxy-2,3-didehydro-rhizoxin (DDR) no relevant secondary metabolites/toxins are produced.
In the mammalian toxicology, several data gaps were identified: information to show that a
potential transfer of genetic material will not lead to unacceptable effects on human and animal
health; a new acute oral toxicity study addressing the infectivity and the clearance and further
considerations on the derivation of reference values for the genotoxic metabolite DDR. The risk
assessment for workers and residents related to exposure to DDR and unknown secondary
metabolites/toxins could not be finalised.
In residues, in relation to the use of P. chlororaphis strain MA 342, the relevance of viable residues
for consumers is pending finalisation of the assessment of effects on human health. Moreover, non-
viable residues were found to be relevant since a metabolite DDR with mutagenic properties was
identified. On the basis of the currently available data and information, a refinement of the consumer
risk assessment is not possible. In a first tier assessment using the threshold of toxicological concern
(TTC) approach, consumer safety could not be demonstrated (critical area of concern) and specific
toxicological reference values should be derived for metabolite DDR.
A number of data gaps have been identified with respect to the fate and behaviour of the
microorganism into the environment: information regarding viability/population dynamics, information
in relation to the mobility of the microorganism in the environment, information to show that a
potential transfer of genetic material will not lead to unacceptable effects on the environment and
information in relation to potential interference with the analytical systems for the control of the

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quality of drinking water (issue not finalised). Another issue not finalised was identified regarding the
assessment of potential groundwater exposure by toxins/secondary metabolites.
In the area of ecotoxicology, data gaps were identified for further information to address the risk
from P. chlororaphis strain MA 342 and metabolite DDR to birds and wild mammals, aquatic organisms,
honeybees, non-target arthropods and soil macro- and microorganisms.

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Table of contents

Abstract.................................................................................................................................................. 1
Summary................................................................................................................................................ 3
Background ............................................................................................................................................ 6
The identity of the microorganism and the properties of the formulated product.......................................... 8
Conclusions of the evaluation ................................................................................................................... 8
1. Identity of the microorganism/biological properties/physical and technical properties and methods of
analysis ........................................................................................................................................... 8
2. Mammalian toxicity........................................................................................................................... 9
3. Residues.......................................................................................................................................... 10
4. Environmental fate and behaviour ..................................................................................................... 10
4.1. Fate and behaviour in the environment of the microorganism .............................................................. 11
4.2. Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under
relevant environmental conditions...................................................................................................... 11
5. Ecotoxicology ................................................................................................................................... 12
6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of
effects data for the environmental compartments (Tables 1–4) ............................................................ 14
7. Data gaps........................................................................................................................................ 15
8. Particular conditions proposed to be taken into account to manage the risk(s) identified ....................... 16
9. Concerns ......................................................................................................................................... 16
9.1. Issues that could not be finalised ...................................................................................................... 16
9.2. Critical areas of concern.................................................................................................................... 16
9.3. Overview of the concerns identified for each representative use considered (Table 5)............................ 17
References.............................................................................................................................................. 17
Abbreviations .......................................................................................................................................... 18
Appendix A – List of end points for the active substance and the representative formulation......................... 20
Appendix B – Used compound codes ........................................................................................................ 21

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Background
Commission Implementing Regulation (EU) No 844/20121 (hereinafter referred to as ‘the
Regulation’) lays down the provisions for the procedure of the renewal of the approval of active
substances, submitted under Article 14 of Regulation (EC) No 1107/20092. This regulates for the
European Food Safety Authority (EFSA) the procedure for organising the consultation of the Member
States, the applicant(s) and the public on the initial evaluation provided by the rapporteur Member
State (RMS) and/or co-rapporteur Member State (co-RMS) in the renewal assessment report (RAR),
and the organisation of an expert consultation where appropriate.
In accordance with Article 13 of the Regulation, unless formally informed by the European
Commission that a conclusion is not necessary, EFSA is required to adopt a conclusion on whether the
active substance can be expected to meet the approval criteria provided for in Article 4 of Regulation
(EC) No 1107/2009 within 5 months from the end of the period provided for the submission of written
comments, subject to an extension of up to 3 months where additional information is required to be
submitted by the applicant(s) in accordance with Article 13(3).
In accordance with Article 1 of the Regulation, the RMS the Netherlands and co-RMS Denmark
received an application from Lantma €nnen BioAgri AB for the renewal of approval of the active
substance Pseudomonas chlororaphis strain MA 342. Complying with Article 8 of the Regulation, the
RMS checked the completeness of the dossier and informed the applicant, the co-RMS (Denmark), the
European Commission and EFSA about the admissibility.
The RMS provided its initial evaluation of the dossier on P. chlororaphis strain MA 342 in the RAR,
which was received by EFSA on 18 December 2015 (Netherlands, 2015).
In accordance with Article 12 of the Regulation, EFSA distributed the RAR to the Member States
and the applicant, Lantma €nnen BioAgri AB, for consultation and comments on 9 February 2016. EFSA
also provided comments. In addition, EFSA conducted a public consultation on the RAR. EFSA collated
and forwarded all comments received to the European Commission on 13 April 2016. At the same
time, the collated comments were forwarded to the RMS for compilation and evaluation in the format
of a reporting table. The applicant was invited to respond to the comments in column 3 of the
reporting table. The comments and the applicant’s response were evaluated by the RMS in column 3.
The need for expert consultation and the necessity for additional information to be submitted by
the applicant in accordance with Article 13(3) of the Regulation were considered in a telephone
conference between EFSA and the RMS on 26 May 2016. On the basis of the comments received, the
applicant’s response to the comments and the RMS’s evaluation thereof, it was concluded that
additional information should be requested from the applicant, and that EFSA should conduct an
expert consultation in the areas of mammalian toxicology, environmental fate and behaviour, and
ecotoxicology.
The outcome of the telephone conference, together with EFSA’s further consideration of the
comments, is reflected in the conclusions set out in column 4 of the reporting table. All points that
were identified as unresolved at the end of the comment evaluation phase and which required further
consideration, including those issues to be considered in an expert consultation, were compiled by
EFSA in the format of an evaluation table.
The conclusions arising from the consideration by EFSA, and as appropriate by the RMS, of the
points identified in the evaluation table, together with the outcome of the expert consultation and the
written consultation on the assessment of additional information, where these took place, were
reported in the final column of the evaluation table.
A final consultation on the conclusions arising from the peer review of the risk assessment took
place with the Member States via a written procedure in November 2016.
This conclusion report summarises the outcome of the peer review of the risk assessment of the
active substance and the representative formulation, evaluated on the basis of the representative uses
of P. chlororaphis strain MA 342 as a seed treatment in cereals (wheat, rye and triticale), carrots and
peas against seed-borne diseases and foliar application in cereals against foliar and ear pathogens, as

1
Commission Implementing Regulation (EU) No 844/2012 of 18 September 2012 setting out the provisions necessary for the
implementation of the renewal procedure for active substances, as provided for in Regulation (EC) No 1107/2009 of the
European Parliament and of the Council concerning the placing of plant protection products on the market. OJ L 252,
19.9.2012, p. 26–32.
2
Regulation (EC) No 1107/2009 of 21 October 2009 of the European Parliament and of the Council concerning the placing of
plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, 24.11.2009,
p. 1–50.

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proposed by the applicant. A list of the relevant end points for the active substance and the
formulation is provided in Appendix A.
In addition, a key supporting document to this conclusion is the peer review report (EFSA, 2016),
which is a compilation of the documentation developed to evaluate and address all issues raised in the
peer review, from the initial commenting phase to the conclusion. The peer review report comprises
the following documents, in which all views expressed during the course of the peer review, including
minority views, where applicable, can be found:
• the comments received on the RAR;
• the reporting table (30 May 2016);
• the evaluation table (5 December 2016);
• the report(s) of the scientific consultation with the Member State experts (where relevant);
• the comments received on the assessment of the additional information (where relevant);
• the comments received on the draft EFSA conclusion.
Given the importance of the RAR, including its revisions (Netherlands, 2016), and the peer review
report, both documents are considered as background documents to this conclusion and thus are
made publicly available.
It is recommended that this conclusion report and its background documents would not be
accepted to support any registration outside the European Union (EU) for which the applicant has not
demonstrated that it has regulatory access to the information on which this conclusion report is based.

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The identity of the microorganism and the properties of the formulated

product
Pseudomonas chlororaphis strain MA 342 bacterium is deposited at the culture collection of the UK
National Collection of Industrial and Marine Bacteria Ltd (NCIMB) under the accession number NCIMB
40616. It is a wild-type strain isolated from roots of wild crowberry (Empetrum nigrum L.) in Sweden.
The representative microbial pest control product (MPCP) for the evaluation was ‘Cerall’, a flowable
concentrate for seed treatment (FS) containing 5 9 1012 colony-forming units (CFU)/kg (200 g/kg,
min. 1 9 1012 CFU/kg, max. 1 9 1013 CFU/kg) P. chlororaphis strain MA 342.
The representative uses evaluated comprise applications as a fungicide as seed treatment in cereals
(wheat, rye and triticale), carrots and peas against seed-borne diseases and foliar application in
cereals against foliar and ear pathogens. Full details of the Good Agricultural Practices (GAPs) can be
found in the list of end points in Appendix A.
Data were submitted to conclude that the representative uses proposed for P. chlororaphis strain
MA 342 at the EU level result in a sufficient fungicidal efficacy against seed-borne diseases in cereals,
carrots and peas, and against foliar and ear pathogens in cereals, following the guidance document
SANCO/10054/2013-rev. 3 (European Commission, 2013).
A data gap has been identified for an updated search, including more detailed assessment, of the
scientific peer-reviewed open literature on the active substance and its relevant metabolites, dealing
with side effects on non-target species and published within the last 10 years before the date of
submission of dossier, to be conducted and reported in accordance with the Guidance of EFSA on the
submission of scientific peer-reviewed open literature for the approval of pesticide active substances
under Regulation (EC) No 1107/2009 (EFSA, 2011).
P. chlororaphis strain MA 342 was discussed at the Pesticides Peer Review TC 139 experts’
teleconference on mammalian toxicology, environmental fate and behaviour, and ecotoxicology
(September 2016).

Conclusions of the evaluation

1. Identity of the microorganism/biological properties/physical and
technical properties and methods of analysis
The following guidance documents were followed in the production of this conclusion: SANCO/
3029/99-rev. 4 (European Commission, 2000a), SANCO/3030/99-rev. 4 (European Commission,
2000b), SANCO/825/00-rev. 8.1 (European Commission, 2010) and OECD Issue Paper on Microbial
Contaminant Limits for Microbial Pest Control Products (OECD, 2011).
The content of viable cells of P. chlororaphis strain MA 342 in the active agent of the microbial pest
control product (MPCA) used to produce the plant protection product should be between 1.5 and
3 9 1013 CFU/L. 2,3-deepoxy-2,3-didehydro-rhizoxin (DDR) is considered a relevant impurity with a
maximum content of 2 mg/L.
P. chlororaphis strain MA 342 can be distinguished from bacterial isolates by restriction fragment
length polymorphism (RFLP) analysis and random amplification of polymorphic DNA (RAPD). It should
be noted, however, that currently there is no method available to distinguish the P. chlororaphis strain
MA 342 from all soil bacterial isolates which show similar morphological and biochemical properties
and have the same restriction patterns.
Pseudomonas strains are known to produce different potentially relevant metabolites (phenazines,
2R- + 3R-butanediol, 4-ACPA, HCN, Prn, HPR, HHL, fluorescens insecticidal toxin (Fit)). The absence of
Fit gene from P. chlororaphis strain MA 342 has been confirmed by polymerase chain reaction (PCR).
No information is available on production of the other above-mentioned metabolites by P. chlororaphis
strain MA 342, as a consequence a data gap was identified to prove that this strain is not producing
other (eco)toxicologically relevant secondary metabolites/toxins besides DDR. There is no evidence of
direct relationships of P. chlororaphis strain MA 342 to known plant, animal or human pathogens. A
data gap was identified for the determination of the growth temperatures of P. chlororaphis strain
MA 342 performed under good laboratory practice (GLP). No growth of the strain was shown below pH
4 or above 10.
The assessment of the data package revealed no issues that need to be included as critical areas of
concern with respect to the identity, physical and technical properties of the active substance or the

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representative formulation; however, a data gap was identified for the determination of the
suspensibility of the formulation.
Acceptable methods are available for the determination of the microorganism in the technical
material and formulated product and for the determination of the content of contaminating
microorganisms. A high-pressure liquid chromatography–diode array detection (HPLC–DAD) method
exists for the determination of DDR on wheat seeds and on plant parts grown from treated barley
seeds with a limit of quantification (LOQ) of 1 lg/kg.

2. Mammalian toxicity
In the technical product, the metabolite DDR, having genotoxic properties, has to be considered as
a toxicologically relevant impurity.
Since 1996, there has been no report on any adverse effect to health related to research,
development, handling or production of this strain. In addition to this, no adverse effects have been
reported for workers in seed production or farmers who have worked with P. chlororaphis strain
MA 342 containing products since their introduction in Sweden in 1998.
In the literature, a case of human infection with P. chlororaphis was identified in relationship with
the presence of a gene responsible of antibioresistance. It should be further investigated if a potential
transfer of genetic material will not lead to unacceptable effects on human and animal health,
including resistance to known therapeutic substances (data gap, not supported by the RMS).
Several toxicity studies were performed with the microorganism. In a skin sensitisation study
(modified Buhler test), negative results were observed; however, considering the limitations of such a
study for a microbial and the lack of investigation of the sensitisation by inhalation, the following
warning phrase is proposed: ‘Pseudomonas chlororaphis strain MA 342 may have the potential to
provoke sensitising reactions’.
The lack of investigations of the infectivity (clearance) in the acute oral toxicity studies was
discussed by the experts. No signs of toxicity or pathogenicity were observed in a first acute oral study
with rat (LD50 > 2 x 1010 bacteria/kg body weight (bw)). Clearance was investigated in a second study
and could not be concluded upon due to the limitations of the study (enumeration method and
bacterial infection which may have overgrown any P. chlororaphis strain MA 342). The experts agreed
that a new oral acute toxicity study should be provided to address the infectivity and clearance using
validated analytical methods (data gap). Pending on the results of this study and on the confirmation
of the growth temperature (see Section 1), further consideration will have to be given to the possible
need for a repeated dose toxicity study. It was also agreed that an intraperitoneal study should not be
required because it would represent an unrealistic exposure route. After intratracheal administration
(1 9 108 CFU/animal), neither the microorganism nor the formulations were shown to be toxic,
pathogenic or infective, and clearance was demonstrated.
The toxicity profile of the metabolite DDR was also discussed by the experts. In a first genotoxicity
study (Onfeld, 2000, in Netherlands, 2015; non-GLP and not guideline compliant), DDR was
demonstrated as being a specific and efficient microtubule inhibitor. In a second in vivo micronucleus
assay (Abramsson-Zetterberg, 2000, in Netherlands, 2015; non-GLP), a significant increase in the
incidence of micronucleated erythrocytes was observed at the high dose only, with high mean DNA
content, indicating an aneugenic effect. The experts considered that there was uncertainty in the no
observed effect level (NOEL) proposed in the micronucleus assay since an aneugenic effect due to
non-disjunction (as shown in the first study) can be induced at lower concentrations than an
aneugenic effect by chromosome loss. As a consequence, a threshold for this genotoxic effect could
not be derived on the basis of the available data. It was agreed to propose classification3 for
genotoxicity (Mutagen Category 1B). After the meeting, EFSA noted that, following the European
Chemicals Agency (ECHA) guidance on the application of the classification, labelling and packaging
(CLP) Criteria (ECHA, 2015), positive results in at least one in vivo valid mammalian somatic cell
genotoxicity test supported by positive in vitro mutagenicity results would trigger the classification in
category 2. Accordingly, the proposed classification for DDR is Mutagen Category 2 H341 ‘Suspected of

3
It should be noted that harmonised classification and labelling is formally proposed and decided in accordance with Regulation
(EC) No 1272/2008. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on
classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/
45/EC, and amending Regulation (EC) No 1907/2006. OJ L 353, 31.12.2008, p. 1–1355.

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causing genetic defects’. In order to perform the risk assessment for consumers, derivation of specific
reference values should be further considered (data gap, not supported by the RMS).
Other metabolites are known to be produced by P. chlororaphis (see Section 1) but no assessment
of their toxicological properties was available (data gap, not supported by the RMS).
For the representative formulated product (‘Cerall’), it was considered that the majority of tests
performed on the microorganism can be extrapolated to the formulation. An acute pulmonary
toxicity/pathogenicity study performed with another formulation (‘Cedomon’) was also considered
relevant for ‘Cerall’ and showed similar results to those obtained with the microorganism.
For the exposure considerations related to DDR, the experts agreed to use the threshold of
toxicological concern (TTC) value for genotoxic compounds and to perform a risk characterisation for
operators and bystanders considering exposure levels at the LOQ. The resulting exposure estimates
were below the TTC value. For workers (and residents), considering that there is uncertainty in
assuming that DDR residues would always be below the LOQ on plants over the period between
application and harvest, EFSA considers that the risk assessment cannot be finalised.
Pending on further investigations on toxins/secondary metabolites produced after application,
further considerations will have to be given to their potential toxicity in order to conclude on the risk
assessment for workers and residents (data gap, not supported by the RMS).

3. Residues
As it concerns viable residues, the consumer risk assessment for the uses of P. chlororaphis strain
MA 342 cannot be finalised as long as a conclusion that P. chlororaphis strain MA 342 will not lead to
unacceptable effects on human health is pending (see Section 2).
Moreover, in relation to the use of P. chlororaphis strain MA 342, non-viable residues were found to
be relevant since a metabolite DDR with mutagenic properties was identified. Studies where levels of
the DDR metabolite are investigated in cereals at harvest following foliar application of P. chlororaphis
strain MA 342 according to the supported GAP were not available in sufficient detail to assess the
reliability of the trial results. The information is crucial to assess the consumer exposure potential for
the metabolite DDR following foliar application (data gap).
Seed treatment uses are assumed less critical taking into account the much longer period between
sowing of treated seeds and harvest of the mature crops as well as growth dilution of potential
residues of DDR. This assumption can only hold true if it can be conclusively demonstrated that
P. chlororaphis strain MA 342 does not have the capacity to translocate to plant parts after seed
emergence and to proliferate, which is currently not the case (see Section 4).
For the seed treatment uses, an indicative assessment was conducted using a TTCgenotox of
0.0025 lg/kg bw, assuming residues in the mature cereal crops, peas and carrots growing from
treated seeds will contain at the maximum, residue levels at the LOQ of 1 lg/kg applied in the
available trials with seed treated cereals. For both the acute and chronic scenario defined in the EFSA
PRIMo, the TTCgenotox will be exceeded (452% TTCgenotox in the chronic scenario with wheat being the
main contributor and up to 2,540% TTCgenotox in the acute scenario with carrots being the critical
commodity). The consumer risk assessment could not be conducted for the foliar application use
(more critical use than seed treatment use).
On the basis of the currently available data and information, a refinement of the consumer risk
assessment is not possible, and specific toxicological reference values should be derived for the
genotoxic metabolite DDR. A critical area of concern was identified as a first tier assessment using the
TTC approach failed to demonstrate consumer safety.

4. Environmental fate and behaviour

No information has been provided in relation to potential interference of P. chlororaphis strain
MA 342 with the analytical systems for the control of the quality of drinking water provided for in
Directive 98/83/EC4 (as is needed by the decision-making criteria in Part B of Commission Regulation
(EU) No 546/20115). Therefore, a data gap has been identified for evidence to demonstrate that no

4
Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. OJ L 330,
5.12.1998, p. 32–54.
5
Commission Regulation (EU) No 546/2011 of 10 June 2011 implementing Regulation (EC) No 1107/2009 of the European
Parliament and of the Council as regards uniform principles for evaluation and authorisation of plant protection products. OJ L
155, 11.6.2011, p. 127–175.

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interferences will be produced with methods for the determination of microbiological contamination of
water given in Directive 98/83/EC, in particular with the method for the analysis of Pseudomonas
aeruginosa in drinking water.
Potential transfer of genetic material from P. chlororaphis strain MA 342 to other bacteria is
considered possible. Information to show that this transfer will not lead to unacceptable effects on the
environment would need to be provided (data gap).
EFSA considers a number of statements presented in the old dossier as unreliable (details can be
found in the background documents to the EFSA conclusion (EFSA, 2016)). The RMS does not agree
on the EFSA conclusion regarding the reliability of non-GLP data.

4.1. Fate and behaviour in the environment of the microorganism

Information on persistence and mobility of P. chlororaphis strain MA 342 in soil, available in the
peer-reviewed scientific literature, indicates certain capacity to proliferate in soil after application in
carrots and onions (Bennett and Whipps, 2008). This capacity to proliferate has also been observed for
other strains of P. chlororaphis (Kozdroj et al., 2004; Das et al., 2008). Therefore, a data gap has been
identified to provide information on viability/population dynamics in several cultivated and uncultivated
soils representative of soils typical of the various EU regions where use exists or is anticipated. The
provisions on choice of soil and its collection and handling have to be followed. As indicated in
Regulation (EU) No 283/2013, test guidelines for chemicals can be adapted for microorganisms as
appropriate or other recognised guidance can be used (e.g. US EPA). In this case, for example, the US
EPA OPPTS 885.5200 test guideline (US EPA, 1996a) can be used.
In two reports, not accepted as reliable by EFSA and proposed to be considered as supporting
information by the RMS, it is claimed that P. chlororaphis strain MA 342 is able to translocate to plant
parts, especially to the coleoptile. In a GLP study, analysis of wheat grains produced from seeds
treated with P. chlororaphis strain MA 342 shows that, at the time of harvest, no bacteria is found in
the grains above the LOQ of 1 lg/kg. However, fully reliable studies investigating the presence of
P. chlororaphis strain MA 342 in other plant parts and different growth stages are not available.
Therefore, it is still uncertain to which extent P. chlororaphis strain MA 342 can translocate from the
roots to other plant parts. A data gap has been identified to provide information in relation to the
mobility of the microorganism in the environment.
In relation to the fate and behaviour of P. chlororaphis strain MA 342 in water, the experiments
available cannot be considered representative of natural water/sediment systems. Therefore, a data
gap has been identified to provide information or studies on viability/population dynamics in natural
sediment/water systems under both dark and illuminated conditions. As indicated in Regulation (EU)
No 283/2013, test guidelines for chemicals can be adapted for microorganisms as appropriate or other
recognised guidance can be used (e.g. US EPA). In this case, for example, the OPPTS 885.5300 test
guideline (US EPA, 1996b) can be used.
According to a report provided in the dossier, not accepted as reliable by EFSA and proposed to be
considered as supporting information by the RMS, it cannot be excluded that, at least in the closed
environments where the seeds are treated, P. chlororaphis strain MA 342 can be transported through
air.
A data gap has been identified for a reliable study to investigate the range of temperatures where
growth of P. chlororaphis strain MA 342 may occur (see Section 1).

4.2. Fate and behaviour in the environment of any relevant metabolite

formed by the microorganism under relevant environmental
conditions
In two peer-reviewed studies performed with different strains (P. chlororaphis strain SRB127;
P. chlororaphis strain P34), the key role of the metabolites (volatile and non-volatile antibiotics) in the
antifungal activity is demonstrated even in the absence of the live bacteria (Das et al., 2008;
€rnkranz et al., 2012). No equivalent study is available for the specific strain P. chlororaphis strain
Fu
MA 342 but a similar mode of action can be assumed. Therefore, a data gap has been identified for
the identification and assessment of the toxicological and ecotoxicological relevance of the
P. chlororaphis strain MA 342 metabolites produced in the environment, including the assessment of
potential groundwater exposure (see Section 1).

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5. Ecotoxicology
Suitable studies demonstrating that P. chlororaphis strain MA 342 is neither infectious nor
pathogenic to birds and wild mammals were not available. A study demonstrating that
P. chlororaphis strain MA 342 does not grow at temperatures above 33°C was proposed as a waiver for
vertebrates (birds and wild mammals) studies. This study was considered as not valid by the experts
at the Pesticides Peer Review teleconference 139. Considering that exposure to P. chlororaphis strain
MA 342 for birds and mammals could not be excluded for all the representative uses assessed, a data
gap was identified for further information to address the infectivity and pathogenicity to birds and wild
mammals for P. chlororaphis strain MA 342. In the view of reducing testing on vertebrates, as a first
step, it may be possible to address this data gap with a new study on the growth temperature of
P. chlororaphis strain MA 342 (see Section 1).
The potential for exposure to aquatic organisms following the use of P. chlororaphis strain
MA 342 in seed dressing is deemed to be low; a low risk for this use for aquatic organisms could be
concluded.
An acute fish study was available in the dossier; this study was considered as not sufficient to
address the infectivity and pathogenicity to fish due to its short duration. Literature studies were
available demonstrating the natural presence of P. chlororaphis in the fish intestine and demonstrating
that the use of P. chlororaphis as a biocontrol agent did not affect fish. This information was not
specific to the strain under evaluation and to the intended uses and was considered as not sufficient to
address the risk to fish from P. chlororaphis strain MA 342 (data gap).
Regarding aquatic invertebrates, the available 21-day study on Daphnia magna was discussed
during the Pesticides Peer Review teleconference 139. Considering that i) reproductive effects were not
derived from the study; ii) 44% mortality occurred in the tested item group; iii) daphnids in the test
item group were reported to be paler and smaller; iv) analytical measurements were performed on
fresh samples only, the experts agreed that a data gap should be identified for a new 21-day study
investigating toxicity (including reproductive effects), pathogenicity and infectivity of P. chlororaphis
strain MA 342 to D. magna. In any repeated test, appropriate consideration should be given to the
influence of the food quality on daphnids’ fitness; in this respect the provisions of the available
guideline should be followed. On the basis of the available data a low risk to algae for P. chlororaphis
strain MA 342 was concluded for all representative uses.
An acute (oral and contact) toxicity study for P. chlororaphis strain MA 342 on honeybees was
available. In the study signs of toxicity, infectivity or pathogenicity were not observed. However, the
duration of the study (48 h) was considered as not sufficient to address the infectivity and
pathogenicity of P. chlororaphis strain MA 342 to honeybees. The insecticidal activity of P. chlororaphis
strain MA 342 was discussed during the Pesticides Peer Review teleconference 139. From the available
information, the presence of the genes associated with the insecticidal activity in strain MA 342 cannot
be excluded. Also effects of P. chlororaphis on Lepidoptera larvae were reported in the literature (Flury
et al., 2016). Several literature studies were available in the RAR indicating that Pseudomonas spp. are
found living in the gut of arthropods including some bee species. None of these studies were specific
to P. chlororaphis. Therefore, a data gap was identified to further address the risk to honeybee adults
and brood for all representative uses of P. chlororaphis strain MA 342. The RMS considered the
potential for exposure to bee larvae as questionable and therefore disagreed with this conclusion.
Regarding non-target arthropods, as reported above, the presence of the genes associated with
the insecticidal activity in strain MA 342 cannot be excluded. In the absence of additional information
to address the toxicity, infectivity and pathogenicity of P. chlororaphis strain MA 342, a high risk to
non-target arthropods could not be excluded for all representative uses and a data gap is, therefore,
identified.
Suitable studies addressing the toxicity, infectivity and pathogenicity of P. chlororaphis strain
MA 342 to earthworms and other soil macroorganisms and the effects on soil microorganisms
were not available (data gap, not supported by the RMS). P. chlororaphis strain MA 342 is a ubiquitous
soil bacterium, however, with the available information it cannot be confirmed whether the uses
foreseen would trigger a significant increase in the background soil levels of P. chlororaphis strain
MA 342.
Regarding the secondary metabolite DDR, suitable toxicological studies on non-target organisms
were not available. Considering that exposure cannot be excluded, data gaps were identified to further
address the risk for this metabolite to aquatic organisms (relevant for the foliar application use only),

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to honeybees (adults and brood), to birds and wild mammals (via dietary exposure and via
consumption of contaminated water), to non-target arthropods, and to soil macro- and
microorganisms (relevant for all representative uses).
From the available information, it cannot be ruled out whether secondary metabolites/toxins, other
than DDR, are produced by the P. chlororaphis strain MA 342 (see Section 1).

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 Peer review of the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342

6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of
effects data for the environmental compartments (Tables 1–4)
Table 1: Soil
Compound
Persistence Ecotoxicology
(name and/or code)
Pseudomonas chlororaphis strain MA 342 Data gap Data gap
Toxins/secondary metabolites Data gap Data gap

Table 2: Groundwater
Compound
> 0.1 lg/L at 1 m depth for Pesticidal
(name and/or Mobility in soil Toxicological relevance
the representative uses(a) activity
code)
Toxins/secondary Data gap pending on their Data gap pending on their – DDR: genotoxic
metabolites identification and quantification identification and quantification Other potential metabolites: data gap pending on their
identification and quantification
DDR: 2,3-deepoxy-2,3-didehydro-rhizoxin.
(a): At least one FOCUS scenario or relevant lysimeter.

Table 3: Surface water and sediment

Compound
Ecotoxicology
(name and/or code)
Pseudomonas chlororaphis strain MA 342 Data gap
Metabolites produced in the environment Data gap

Table 4: Air
Compound
Toxicology
(name and/or code)
Pseudomonas chlororaphis strain MA 342 Not toxic, pathogenic or infective (NOAEL > 1 9 108 CFU/animal)
NOAEL: no observed adverse effect level; CFU: colony-forming units.

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7. Data gaps
This is a list of data gaps identified during the peer review process, including those areas in which
a study may have been made available during the peer review process but not considered for
procedural reasons (without prejudice to the provisions of Article 56 of Regulation (EC) No 1107/2009
concerning information on potentially harmful effects).
• An updated search of the scientific peer-reviewed open literature on the active substance and
its relevant metabolites, dealing with side effects on non-target species and published within
the last 10 years before the date of submission of the dossier, to be conducted and reported in
accordance with EFSA guidance on the submission of scientific peer-reviewed open literature
for the approval of pesticide active substances under Regulation (EC) No 1107/2009 (EFSA,
2011). It should be ensured that all relevant studies are included and a more detailed
assessment of the literature search is presented (relevant for all representative uses evaluated;
submission date proposed by the applicant unknown).
• Suspensibility of the formulation (relevant for uses evaluated as foliar applications; submission
date proposed by the applicant: unknown, see Section 1).
• Applicant to provide a study on the determination of the growth temperatures of
P. chlororaphis strain MA 342 (in order the study can be used also for human health evaluation
it would need to be performed under GLP) (relevant for all representative uses evaluated;
submission date proposed by the applicant: unknown; see Sections 1, 2, 4 and 5).
• Applicant to submit laboratory and/or field studies or reliable scientific information where levels
of DDR and other potential secondary metabolites/toxins are investigated (including an
assessment of their toxicological and ecotoxicological relevance), using appropriately validated
analytical methods, in soil and plants following applications of P. chlororaphis strain MA 342
according to the supported GAPs (relevant for all representative uses evaluated; submission
date proposed by the applicant: unknown; see Sections 1, 2, 4 and 5).
• Applicant to provide information to show that potential transfer of genetic material from
P. chlororaphis strain MA 342 will not lead to adverse effects on human and animal health,
including resistance to known therapeutics, and will not lead to unacceptable effects on the
environment (relevant for all representative uses evaluated; submission date proposed by the
applicant: unknown; see Sections 2 and 4).
• Acute oral toxicity study addressing the infectivity and clearance of P. chlororaphis strain
MA 342 after administration using validated analytical methods (relevant for all representative
uses evaluated; study ongoing, submission date proposed by the applicant: study ongoing and
expected in 2017; see Section 2).
• Derivation of specific reference values for the metabolite DDR should be further considered for
the consumers’ risk assessment (relevant for all representative uses evaluated; submission
date proposed by the applicant: unknown; see Sections 2 and 3).
• Applicant to submit full details on residue trials where levels of the DDR metabolite are
investigated in cereals at harvest following foliar application of P. chlororaphis strain MA 342
according to the supported GAP (relevant for uses evaluated as foliar applications; submission
date proposed by the applicant: unknown, see Section 3).
• Applicant to provide information in relation to potential interferences of P. chlororaphis strain
MA 342 with the analytical systems for the control of the quality of drinking water in particular
with the method for the analysis of P. aeruginosa in drinking water (relevant for all
representative uses evaluated; submission date proposed by the applicant: unknown; see
Section 4).
• Applicant to provide information or studies regarding viability/population dynamics in several
cultivated and uncultivated soils representative of soils typical of the various EU regions where
use exists or is anticipated, and in natural sediment/water systems under both dark and
illuminated conditions. Test guidelines for chemicals can be adapted for microorganisms as
appropriate or other recognised guidance can be used (e.g. US EPA). In this case, the US EPA
OPPTS 885.5200 and OPPTS 885.5300 test guidelines could be used (relevant for all
representative uses evaluated; submission date proposed by the applicant: unknown; see
Section 4).

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• Applicant to provide information in relation to the mobility of the microorganism in the

environment (relevant for all representative uses evaluated; submission date proposed by the
applicant: unknown; see Section 4).
• Further information to address the risk to birds and wild mammals from P. chlororaphis strain
MA 342. In the view of reducing testing on vertebrates, as a first step, it may be possible to
address this data gap with a new study on the growth temperature of P. chlororaphis strain
MA 342 (relevant for all representative uses evaluated; submission date proposed by the
applicant: unknown; see Sections 1 and 5).
• Further information to address risk to fish and aquatic invertebrates (relevant for the use as
foliar application) and to honeybees (adult and brood) and other non-target arthropods, soil
macro- and microorganisms for P. chlororaphis strain MA 342 (relevant for all representative
uses evaluated; submission date proposed by the applicant: unknown; see Section 5).
• Further information to address the risk from the secondary metabolite DDR to aquatic
organisms (relevant for the foliar application use), to birds and wild mammals (via dietary
exposure and via consumption of contaminated water), to honeybees and other non-target
arthropods, and to soil macro- and microorganisms (relevant for all representative uses
evaluated; submission date proposed by the applicant: unknown; see Section 5).

8. Particular conditions proposed to be taken into account to manage

the risk(s) identified
No particular conditions are proposed for the representative uses evaluated.

9. Concerns
9.1. Issues that could not be finalised
An issue is listed as ‘could not be finalised’ if there is not enough information available to perform
an assessment, even at the lowest tier level, for the representative uses in line with the uniform
principles in accordance with Article 29(6) of Regulation (EC) No 1107/2009 and as set out in
Commission Regulation (EU) No 546/2011 and if the issue is of such importance that it could, when
finalised, become a concern (which would also be listed as a critical area of concern if it is of relevance
to all representative uses).
An issue is also listed as ‘could not be finalised’ if the available information is considered insufficient
to conclude on whether the active substance can be expected to meet the approval criteria provided
for in Article 4 of Regulation (EC) No 1107/2009.
1) The assessment of potential transfer of genetic material (e.g. responsible of
antibioresistance) from P. chlororaphis strain MA 342 to other organisms could not be
finalised.
2) The production of toxins/secondary metabolites cannot be excluded. Therefore, the risk
assessment could not be finalised for workers, residents, consumers and the environment
including the assessment of potential groundwater exposure.
3) Risk assessment for workers and residents exposed to the genotoxic metabolite DDR could
not be concluded on the basis of the available data.
4) Potential interferences of P. chlororaphis strain MA 342 with the analytical systems for the
control of the quality of drinking water could not be excluded.
5) A high risk to non-target arthropods following the exposure to P. chlororaphis strain MA 342
and its secondary metabolite DDR could not be excluded.

9.2. Critical areas of concern

An issue is listed as a critical area of concern if there is enough information available to perform an
assessment for the representative uses in line with the uniform principles in accordance with Article 29
(6) of Regulation (EC) No 1107/2009 and as set out in Commission Regulation (EU) No 546/2011, and
if this assessment does not permit the conclusion that, for at least one of the representative uses, it
may be expected that a plant protection product containing the active substance will not have any
harmful effect on human or animal health or on groundwater, or any unacceptable influence on the
environment.

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An issue is also listed as a critical area of concern if the assessment at the higher tier level could
not be finalised due to lack of information, and if the assessment performed at the lower tier level
does not permit the conclusion that, for at least one of the representative uses, it may be expected
that a plant protection product containing the active substance will not have any harmful effect on
human or animal health or on groundwater, or any unacceptable influence on the environment.
An issue is also listed as a critical area of concern if, in the light of current scientific and technical
knowledge using guidance documents available at the time of application, the active substance is not
expected to meet the approval criteria provided for in Article 4 of Regulation (EC) No 1107/2009.
6) A refinement of the consumer risk assessment is currently not possible in the absence of
specific toxicological reference values for the genotoxic metabolite DDR. A first tier
assessment using the TTC approach failed to demonstrate consumer safety.

9.3. Overview of the concerns identified for each representative use

considered (Table 5)
(If a particular condition proposed to be taken into account to manage an identified risk, as listed in
Section 8, has been evaluated as being effective, then ‘risk identified’ is not indicated in Table 5.)
Table 5: Overview of concerns
Cereals Carrots Peas Cereals
Representative use Seed Seed Seed Foliar
treatment treatment treatment application
Operator risk Risk identified
Assessment not finalised
Worker risk Risk identified
Assessment not finalised X2,3 X2,3 X2,3 X2,3
Resident/ Risk identified
bystander risk Assessment not finalised X2,3 X2,3 X2,3 X2,3
Consumer risk Risk identified X6 X6 X6 X6
Assessment not finalised X2 X2 X2 X2
Risk to wild non- Risk identified
target terrestrial Assessment not finalised
vertebrates
Risk to wild non- Risk identified
target terrestrial Assessment not finalised X5 X5 X5 X5
organisms other
than vertebrates
Risk to aquatic Risk identified
organisms Assessment not finalised
Groundwater Legal parametric value breached
exposure to active Assessment not finalised
substance
Groundwater Legal parametric value breached
exposure to Parametric value of 10 lg/L
metabolites breached
Assessment not finalised X2 X2 X2 X2
Columns are grey if no safe use can be identified. The superscript numbers relate to the numbered points indicated in
Sections 9.1 and 9.2. Where there is no superscript number, see Sections 2–6 for further information.

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MA 342 prepared by the rapporteur Member State the Netherlands, in the framework of Commission
Implementing Regulation (EU) No 844/2012, December 2015. Available online: www.efsa.europa.eu
Netherlands, 2016. Revised Renewal Assessment Report (RAR) on the active substance Pseudomonas chlororaphis
strain MA 342 prepared by the rapporteur Member State the Netherlands in the framework of Commission
Implementing Regulation (EU) No 844/2012, August 2016. Available online: www.efsa.europa.eu
OECD (Organisation for Economic Co-Operation and Development), 2011. Issue Paper on Microbial Contaminant
Limits for Microbial Pest Control Products, Series on Pesticides No. 65, ENV/JM/MONO(2011)43, 12 October
2011.
US EPA (United States of America Environmental Protection Agency), 1996a. Microbial Pesticide Test Guidelines
OPPTS 885.5200 Expression in a Terrestrial Environment, 1996.
US EPA (United States of America Environmental Protection Agency), 1996b. Microbial Pesticide Test Guidelines
OPPTS 885.5300 Expression in a Freshwater Environment, 1996.

Abbreviations
bw body weight
CLP classification, labelling and packaging
CFU colony-forming units
DDR 2,3-deepoxy-2,3-didehydro-rhizoxin
ECHA European Chemicals Agency
EEC European Economic Community
EFSA PRIMo EFSA Pesticide Residue Intake Model
FAO Food and Agriculture Organization of the United Nations
Fit fluorescens insecticidal toxin
FOCUS Forum for the Co-ordination of Pesticide Fate Models and their Use
FS flowable concentrate for seed treatment
GAP Good Agricultural Practice
GLP good laboratory practice
HPLC-DAD high-pressure liquid chromatography-diode array detection
LD50 lethal dose, median; dosis letalis media

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Peer review of the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342

LOQ limit of quantification

MPCA active agent of the microbial pest control product
MPCP microbial pest control product
NCIMB UK National Collection of Industrial and Marine Bacteria Ltd
NOAEL no observed adverse effect level
NOEL no observed effect level
OECD Organisation for Economic Co-operation and Development
PCR polymerase chain reaction
RAPD random amplification of polymorphic DNA
RAR renewal assessment report
RFLP restriction fragment length polymorphism
SMILES simplified molecular-input line-entry system
TTC threshold of toxicological concern
WHO World Health Organization
US EPA US Environmental Protection Agency

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Peer review of the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342

Appendix A – List of end points for the active substance and the
representative formulation
Appendix A can be found in the online version of this output (‘Supporting information’ section):
https://doi.org/10.2903/j.efsa.2017.4668.

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Peer review of the pesticide risk assessment of the active substance Pseudomonas chlororaphis strain MA 342

Appendix B – Used compound codes

Code/trivial name Chemical name/SMILES notation Structural formula

DDR (1R,2R,3E,5R,7R,8S,10S,13E,16R)-8-Hydroxy- CH
CH33
HH
OO OO
2,3-Deepoxy-2, 10-[(2S,3R,4E,6E,8E)-3-methoxy-4,8-dimethyl-
3-didehydro-rhizoxin 9-(2-methyl-1,3-oxazol-4-yl)nona-4,6,8-trien-2-
yl]-2,7-dimethyl-6,11,19-trioxatricyclo OO
HHOO
[14.3.1.05,7]icosa-3,13-diene-12,18-dione HH3CC CH
CH33
3 HH
Cc1occ(n1)\C=C(/C)\C=C\C=C(/C)[C@H](OC) OO
NN CH
CH33 CH
CH33
[C@@H](C)[C@H]3OC(=O)C=CC[C@H]2CC(=O) OO
OO
O[C@H](C2)[C@H](C)C=C[C@H]4O[C@]4(C) CH
CH33
[C@@H](O)C3 HH3CC
OO
3

4-ACPA 4-Carbamoylphenyl acetate O

O=C(C)Oc1ccc(cc1)C(N)=O O CH3
O
H2N

HCN Hydrocyanic acid H N

Hydrogen cyanide N#C

2R-3R-Butanediol (2R,3R)-Butane-2,3-diol OH

C[C@@H](O)[C@@H](C)O CH3
H3C

OH

Prn 3-Chloro-4-(3-chloro-2-nitrophenyl)-1H-pyrrole H
N
Pyrrolnitrin Clc2cncc2c1cccc(Cl)c1[N+]([O-])=O O
–

+
O N
Cl
Cl

HPR 2-Hexyl-5-propylbenzene-1,3-diol OH
2-Hexyl-5-propyl- Oc1cc(cc(O)c1CCCCCC)CCC
resorcinol H3C

HO CH3

HHL N-[(3S)-2-Oxotetrahydrofuran-3-yl]hexanamide O
N-Hexanoyl O=C1OCC[C@@H]1NC(=O)CCCCC NH CH3
homoserine lactone O
O

SMILES: simplified molecular-input line-entry system.

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