(Verticillium) lecanii - REBECA

Comments on registration of microbial pesticides
W. Ravensberg, Koppert BV
REBECA meeting 18-22 September, Kiel
1. General
- see appendix I: statement on metabolites
- see appendix II: statement written in the context of RAFBCA concerning various aspects of
the procedure
2. Verticillium lecanii (Mycotal and Vertalec)
2.1
- many studies (acute package) have demonstrated that there are no harmful effects,
some studies were done with the MPCA, some with the MPCP. Some countries still
require a full set of studies with the MPCA as well as with the MPCP. What is the
rationale behind this? Also considering the food approved ingredients one set should
be enough.
- Even though countries follow the same EU directive re requirements different
countries require different tests. This should be harmonized and accepted from each
other.
2.2 metabolites
- In Rafbca it was found that V. lecanii under certain circumstances can produce
destruxins. This will be published soon. The quantity and the circumstance under with
the fungus makes these metabolites indicates that this is not of any significance
reklated to tox or the environment
2.3 Methods are available, but not in companies.
3. Trichoderma harzianum (Trianum)
It is questionable whether this trichoderma should be seen as a PPP. Its effects as very
close to effects shown by mycorrhizae.
3.1 Metabolites
- Trichoderma’s make numerous metabolites, but none of any significance related to
tox or environment. So identification and so on are non-relevant and should not be
required. Methods are not available.
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Appendix I.
RELEVANT METABOLITES
1) INTRODUCTION
In the registration process of plant protection products data requirements are not only limited
to the active ingredient and the product, but also include data of relevant metabolites. The
definition of a relevant metabolite has recently been refined for chemicals. The definition for
microbials has still not been defined. Differences in definitons used are listed below
2) CHEMICALS
Metabolite (Sanco/221/2000 –rev.10): all (biotic or abiotic) reaction or breakdown products
of an active substance of a plant protection product, formed in the environment.
Residues (91/414/EEG): substance(s) present in or on plant(product)s, edible animal
products or elsewhere in the environment and resulting from the use of a plant protection
product, including their metabolites and products resulting from their degradation or reaction.
Groundwater
Guidance document on the assessment of the relevance of metabolites in
groundwater of substances regulated under council directive 91/414/EEC
(Sanco/221/2000-rev.10)
Relevant metabolite: a metabolite for which there is reason to assume that
- it has comparable intrinsic properties as the active substance in terms of its biological target
activity,
- it has certain toxicological properties that are considered severe and unacceptable.
Such a metabolite is therefore treated like the parent active substance.
Metabolite of no concern: A metabolite which meets the following criteria:
- CO2 or an inorganic compound, without a heavy metal,
- organic compound of aliphatic structure (and chain length of 4 or less), consisting only of C,
H, N or O atoms without "alerting structures" such as epoxide, nitrosamine, nitrile or other
functional groups of known toxicological concern.
- known, non (eco)toxic substance, naturally occurring at much higher concentrations
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Non-relevant metabolite: a metabolite which does not meet the criteria provided for
“relevant metabolites” and “metabolites of no concern”. A non-relevant metabolite may be
subject, on a case-by-case basis, to an individual groundwater limit concentration
Drinking water directive (98/83/EC): concentrations of pesticides and their ‘relevant
metabolites’ in drinking water < 0.1μg/l
Sanco/221/2000-rev.10: stepwise scheme to determine ‘relevant metabolite’
Step 1: Exclusion of metabolite of no concern
Step 2: Quantification of potential groundwater contamination
If annual average concentration> 0.1 μg/l (using FOCUS-modelling) then:
Step 3: Hazard assessment: screening for biological activity
screening for genotoxicity
screening for toxicity
If none of the screenings positive then:
Step 4: Exposure assessment
If concentration < 0.75 μg/l then: non-relevant metabolite (inclusion Annex I possible):
Step 5: refined risk assessment.
Aquatic and terrestrial ecotoxicology
Guidance document on aquatic ecotoxicology (Sanco/3268/2001 rev 4)
Guidance document on terrestrial ecotoxicology (Sanco 10329/2002 rev 2)
Major metabolite: all metabolites that are formed in amounts of ≥10% of the
applied amount of active ingredient at any timepoint evaluated during the degradation
studies
Minor metabolite: all non-major metabolites
Ecotoxicologically relevant metabolite: a metabolite which poses a higher or
comparable risk to aquatic organisms as the active substance.
Minor metabolites only exceptionally included (when they give rise to particular concern) in
aquatic and terrestrial ecotoxicology evaluation.
3)MICROBIALS
Metabolites (2001/36/EU) products resulting from degradative and biosynthetic reactions
taking place within the micro-organism or other organisms used to produce the microorganism
of interest.
Relevant metabolites Metabolites that are of concern for human or animal health and/or the
Environment
Residues Viable micro-organisms and substances produced in significant quantities by
these micro-organisms which persist after the disappearance of the micro-organisms and are
of concern for human or animal health and/or the environment.
Impurities Any component (including contaminating micro-organisms and/or chemical
substances) other than the specified micro-organism, originating from the
manufacturing process or from degradation during storage
Relevant impurities Impurities, as defined above, that are of concern for human or animal
health and/or the environment
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Draft Guidance Document Criteria for evaluation and authorisation of plant protection
products containing micro-organisms (To become Annex 6 b of Directive 91/414/EEC)
(SANCO/1023/2001 rev. 4)
There are no precise descriptions available (no guidance documents) on quantities
(concentrations) of relevant metabolites for micro-organisms, although term is often used.
W. Ravensberg and R.W. Kolnaar
Koppert Beheer BV
16 June 2003
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Appendix II.
RAFBCA and EU 91/414 for micro-organisms
The current research under RAFBCA relates with the registration criteria of fungal biopesticides
in four areas, being resp. chapter 5 to 8: Effects on human health; Residues; Fate
and behaviour; and Non-target studies.
Below you will find an attempt to deal with unclearities in chapter 5 and to find solutions for
these issues from RAFBCA research.
Each chapter in the regulation needs a thorough study in order to expose the weak points
and to find solutions from RAFBCA that can help to make the criteria clearer and to develop
appropriate tests. Other chapters will be dealt with in a later stage and it needs to be
discussed to what extent RAFBCA research really contributes in a certain area.
In the first part we will expose the difficulties and inconsequence in the current guideline with
regard to Chapter 5. After that we will propose how to solve these problems.
1. Current regulations / criteria for fungal bio-pesticides
EU 91/414 Annex IIB : active substance = micro-organism
Chapter 5: EFFECTS ON HUMAN HEALTH
- CYTOTOX: no cytotoxity tests required
- instead whole animal (mammalian) tests (and medical data) required
- GENOTOX: Annex II 5.2.3 “genotoxicity testing” (no genotox required under
Annex IIIB for the PPP!!!)
TIER I
a) Metabolites and exotoxins present “If the micro-organism produces
exotoxins according to point 2.8, then these toxins and any other relevant
metabolites in the culture medium must also be tested for genotoxicity. Such tests on
toxins and metabolites should be performed using the purified chemical if possible.”
(italics: text out of EU guideline) (See also Annex I)
This question leaves a lot of space for various interpretations. Does the microorganism
need to be tested for genotoxicity? It seems not.
If exotoxins are produced, then these and all other relevant metabolites must
each be tested in pure chemical form.
A very critical word in this text is the word “relevant”. Does “relevant” only refer
to the metabolites, first of all, it should also refer to the exotoxins. The
information given in 2.8 (see below) will reveal whether or not a
toxin/metabolite is relevant. The routes of exposure add to the relevance of the
metabolite. The term “unacceptable effects on human health / and or the
environment” is used. This is quite essential. But based on this condition one
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would think that up to so far metabolite studies would not be required for any
existing fungal PPP!! But that is not the case.
b) no metabolites If basic studies do not indicate that toxic metabolites are
formed, studies on the micro-organism itself should be considered depending on
expert judgement on the relevance and validity of the basic data.
This question is unclear and does not give guidance to when these
are required or who the expert is.
The second part of the question is also very unclear: what is the set of basic
data in this case? As a result any regulator could demand these genotox
testing; it seems when no exotoxins are produced (not clear whether in situ, in
production or present in the PPP) also no metabolite testing is required.
Relevance does not seem to play a role here.
Above description of criteria is vague, not logic, without end point, not relevant
in all situations and can be interpreted in various ways. This should be
addressed on a case-by-case scenario, depending on the final product (PPP),
the application method, type of crop, and production of exotoxins in situ and
exposure in broad sense. So, 2.8 is very important for the decision about
genotox testing. But also in 2.8 the word relevant is not mentioned;
the mentioning of “unacceptable effects” is essential and should be clearly
defined in the Uniform Principles.
(2.8. Information on the production of metabolites (especially toxins) (In Chapter 2.
Biological properties of the micro-organism).
If other strains belonging to the same microbial species as the strain subject to the
application are known to produce metabolites (especially toxins) with unacceptable
effects on human health and/or the environment during or after application, the nature
and structure of this substance, its presence inside or outside the cell and its stability,
its mode of action (including external and internal factors of the micro-organism
necessary to action) as well as its effect on humans, animals or other non-target
species shall be provided.
The conditions under which the micro-organism produces the metabolite(s) (especially
toxin(s)) must be described.
Any available information on the mechanism by which the micro-organisms regulate
the production of the(se) metabolite(s) should be provided.
Any available information on the influence of the produced metabolites on the microorganism's
mode of action should be provided.)
The actuals tests: 5.2.3.1: in vitro studies. TIER I
a) micro-organism
three tests must be provided. Costs roughly € 40.000,--
b) micro-organism with exotoxins
three tests must be provided per metabolite/toxin
It is not clear from the guideline whether the micro-organism itself needs to be
tested also.
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Costs roughly; in case of m.o. + 1 toxin: 2x € 40.000,-- : € 80.000,--
More than 1 metabolite/toxin : € 120.000,--
or more
TIER II:
5.4. in vivo studies in somatic cells
“If all the results of the in vitro studies are negative further testing must be done with
consideration of other relevant information available. The test can be an in vivo study or an in
vitro study using a different metabolising system from that/those previously used.”
This refers back to the three in vitro studies of 5.2.3.1. So, if your organism shows no
genotoxicity, and that is what you wish, you still have to do a TIER II test. Very unclear
is the statement “with consideration of other relevant information available”. This needs
clearification. Than one can choose between an in vivo test and an in vitro test – must
be conducted. Unclear which one and how to decide on the basis of what?
5.4 Continues with: “If the in vitro cytogenetic test is positive, an in vivo test using somatic
cells (metaphase analysis in rodent bone marrow or micronucleus test in rodents) must
be conducted.”
Not clear which test is meant here, is this the clastogenicity test? But if it is positive,
again a new test must be conducted: not clear which one to choose based on what?
5.4 Continues with: “If either of the in vitro gene mutation tests are positive, an in vivo test to
investigate unscheduled DNA synthesis or a mouse spot test must be conducted.”
Not clear which one to choose based on what.
5.5. Genotoxicity – in vivo studies in germ cells
Looks like TIER II phase, following the test of 5.4. Text is clear, based on a case-bycase
study.
Comments based on Koppert’s experiences with 2 micro-organism
Our experience with these requirements is that any country could ask what they think is
needed and it varies by country. France asks one TIER I and one TIER II study, not
clear on the basis of what. To their opinion this is according to EU guideline. Our
opinion is that this is not the case, see above. On the otherhand hand it reduces costs.
Denmark just asks an Ames test, Nl, U.K. asked nothing on genotox (yet).
It is clear that guidelines are vague, not followed and wrongly interpreted and that
companies have no influence on this whatsoever.
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So, a strong need for harmonizing, simplification and clearer requirements, based on a
case-by-case.
In the Annex II/A (for chemical active substances) the test methods are explicitly
mentioned, this should be also done for micro-organisms. Also it gives a better
explanation of “the relevance of basic data”.
2. A proposal for the “Position of the RAFBCA on EU/91/414”
Concerning:
Cytotox studies: the goal is to develop alternative studies to test possible harmful
effects of the micro-organism (and its toxins / metabolites) on cell development and cell
proliferation. Currently various animal (mammalian) tests are required (oral, dermal,
pulmonary exposure, etc).
Industrial chemicals (30.000) need to be evaluated on their possible harmful effects and
for this purpose the industry and testing laboratories are working together to develop
alternative testing methods, such as cytotoxicity tests. We suggest to contact these
people in order to see where we can cooperate since our metabolites are also
chemicals. Perhaps their ideas can be supported by RAFBCA + IBMA.
Genotox studies: the goal is to simplify the current regulations (see before),
preferably with a clear set of decision making tools and when needed, with quick,
simple and cheaper tests and only when metabolites are relevant.
Relevance needs to be established, where possible, before any testing is done.
It depends on a number of aspects, such as (in order of importance):
• Mode of action
• Nature of the products - pure spores or
- spores + culture medium
• Exposure routes
• Toxicity of micro-organism (a.i.)
• Quantity of metabolites (crude extract of PPP)
• Toxicity testing of crude extract of PPP (cytotoxicity)
A decision model to determine the relevance of metabolites should be developed based
on these criteria. See flow chart. As a consequence we propose to test the possible
harmful effects of the m.o. and its toxin / metabolites by a crude extract of the PPP. So it
should be listed in Annex IIIB.
Characterization and quantification of each metabolite should not be necessary in case
no toxicity is found in animal tests / or cytotoxicity testing with the PPP.
Characterization and quantification of a metabolite should only be necessary if the
amount produced excesses a certain critical level. This level still needs to be determined
(based on RAFBCA research or other relevant mycotoxin research or guidelines (e.g.
Commission Regulations (EC) no. 257/2002 and no. 472/2002). We even could imagine
characterization is not needed at all.
A statement based on information on production of metabolites, nature of the product,
proposed use and exposure to humans etc. should warrant no performance of tests.
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Basically chapter 2 of the guidelines, particularly 2.8, should be that statement.
RAFBCA is studying various fungal micro-organisms and its metabolites. Our goal should
be to demonstrate by means of these studies with these model organisms how they
behave in terms of metabolite production and their fate, dispersal and risks. Based on the
results RAFBCA should propose a clearer set of decision tools whether or not certain
tests are needed. Our goal has to be that regulators realize that metabolites are less
relevant and less dangerous than they are perceived now and that less tests are
necessary.
In case testing is deemed necessary, new, alternative, simple and easy tests should be
used, as the ones that are being developed in RAFBCA at the moment.
RAFBCA should develop tests that substitute mammalian tests. We should be stressing
the fact that we are not developing “extra tests”. A meeting between genotox toxicologists
and metabolite experts (from fungal m.o.’s, so RAFBCA participants) should be
considered in order to discuss this further.
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Microorganism
strain
Other strain(s) of
species produces
exotoxins/
metabolites
relevant
Crude extract of plant
protection product (how
(many) extracted?)
Some indications
of genotoxicity
Species does not
produce exotoxins/
metabolites
Assessment of
relevance of
exotoxins/ metabolites
(statement - Point 2.8
Annex IIB)
Initial study for
genotoxicity (TIER I)
(which tests?)
Assessment of
risk level
(guidelines?)
Non-relevant
No genotoxicity
Low risk
relevant risk
CONTINUE
WITH
REGISTRATION
DOSSIER
No further testing
Further genotoxicity
testing (TIER II)
(which tests?)
mitigating
effects
Acceptable
risk
Assessment of
risk level
(guidelines?)
Decision flow chart
of genotoxicity
NO
REGISTRATION
Risk not
acceptable

Fate and behaviour
Residues
Non-target studies
These three chapters of the registration dossier need to be addressed also in
relation to
RAFBCA studies. This needs to be done in the near future. Also seperate flow
charts will
be developed for these tests.
Koppert BV
W.J. Ravensberg
R.W. Kolnaar
R&D Microbials
October 2003