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EFSA Journal - 2013 - Conclusion On The Peer Review of The Pesticide Risk Assessment of The Active Substances Beauveria

The EFSA report concludes the peer review of the pesticide risk assessment for Beauveria bassiana strains ATCC-74040 and GHA, focusing on their use as insecticides on various crops. The assessment identified several data gaps regarding the strains' toxicity, environmental impact, and the need for further studies on secondary metabolites. Overall, while low risks to certain non-target organisms were noted, comprehensive risk assessments cannot be finalized without addressing the identified gaps.

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EFSA Journal - 2013 - Conclusion On The Peer Review of The Pesticide Risk Assessment of The Active Substances Beauveria

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EFSA Journal 2013;11(1):3031

CONCLUSION ON PESTICIDE PEER REVIEW

Conclusion on the peer review of the pesticide risk assessment of the active
substances Beauveria bassiana strains ATCC-74040 and GHA1
European Food Safety Authority2

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT
The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk
assessments carried out by the competent authority of the rapporteur Member State Germany, for the pesticide
active substances Beauveria bassiana strains ATCC-74040 and GHA are reported. The context of the peer
review was that required by Commission Regulation (EC) No 2229/2004, as amended by Commission
Regulation (EC) No 1095/2007 and Commission Regulation (EU) No 114/2010. The conclusions were reached
on the basis of the evaluation of the representative uses of Beauveria bassiana strains ATCC-74040 and GHA as
an insecticide on tomatoes for strain ATCC-74040 and on tomatoes, cucumbers and ornamentals for strain GHA.
The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the
available studies and literature in the dossier peer reviewed, are presented. Missing information identified as
being required by the regulatory framework is listed. Concerns are identified.

© European Food Safety Authority, 2013

KEY WORDS
Beauveria bassiana strains ATCC-74040 and GHA, peer review, risk assessment, pesticide, insecticide

1
On request from the European Commission, Question No EFSA-Q-2009-00252 and EFSA-Q-2009-00251,
approved on 12 December 2012.
2
Correspondence: [email protected]

Suggested citation: European Food Safety Authority; Conclusion on the peer review of the pesticide risk assessment of the
active substances Beauveria bassiana strains ATCC-74040 and GHA. EFSA Journal 2013;11(1):3031. [44 pp.]
doi:10.2903/j.efsa.2013.3031. Available online: www.efsa.europa.eu/efsajournal

© European Food Safety Authority, 2013

18314732, 2013, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2013.3031 by CochraneUnitedArabEmirates, Wiley Online Library on [08/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Peer Review of the pesticide risk assessment of the active substance Beauveria bassiana
strains ATCC-74040 and GHA

SUMMARY
Beauveria bassiana strains ATCC-74040 and GHA is one of the 295 substances of the fourth stage of
the review programme covered by Commission Regulation (EC) No 2229/2004, as amended by
Commission Regulation (EC) No 1095/2007.

Beauveria bassiana strains ATCC-74040 and GHA were included in Annex I to Directive
91/414/EEC on 1 May 2009 pursuant to Article 24b of the Regulation (EC) No 2229/2004 (hereinafter
referred to as „the Regulation‟) and have subsequently been deemed to be approved under Regulation
(EC) No 1107/2009, in accordance with Commission Implementing Regulation (EU) No 540/2011, as
amended by Commission Implementing Regulation (EU) No 541/2011. In accordance with Article
25a of the Regulation, as amended by Commission Regulation (EU) No 114/2010, the European Food
Safety Authority (EFSA) is required to deliver by 31 December 2012 its view on the draft review
reports submitted by the European Commission in accordance with Article 25(1) of the Regulation.
This review report was established as a result of the initial evaluation provided by the designated
rapporteur Member State in the Draft Assessment Report (DAR). The EFSA therefore organised a
peer review of the DAR. The conclusions of the peer review are set out in this report.

Germany being the designated rapporteur Member State submitted the DARs on Beauveria bassiana
strains ATCC-74040 and GHA in accordance with the provisions of Article 22(1) of the Regulation,
which were received by the EFSA on 3 December 2007. The peer review was initiated on 22 April
2008 by dispatching the DARs to the notifiers (Intrachem Bio Italia S.p.A. for strain ATCC-74040 and
Mycotech Europe Ltd. for strain GHA) and on 11 June 2008 to the Member States for consultations
and comments. Following consideration of the comments received on the DARs, it was concluded that
EFSA should conduct a focused peer review in the area of ecotoxicology and deliver its conclusions
on Beauveria bassiana strains ATCC-74040 and GHA.

The conclusions laid down in this report were reached on the basis of the evaluation of the
representative uses of Beauveria bassiana strains ATCC-74040 and GHA as an insecticide on
tomatoes for strain ATCC-74040 and on tomatoes, cucumbers and ornamentals for strain GHA, as
proposed by the notifiers. Full details of the representative uses can be found in Appendix A to this
report.

In the area of identity of the microorganism, biological properties, physical and technical properties
and methods of analysis, data gaps were identified for both strains and both formulations.

The operator, worker and bystander risk assessment cannot be finalised due to the lack of information
on toxins/secondary metabolites production.

The consumer risk assessment cannot be finalised until the toxins/secondary metabolite issue is
addressed.

The information on fate and behaviour in the environment was not sufficient to characterise the
competitiveness/persistence of B. bassiana strains ATCC-74040 and GHA in soil in the context of the
representative uses assessed and therefore data gaps were identified. Satisfactory information is not
available to demonstrate that, under the conditions of use, any toxins/secondary metabolites produced
by Beauveria bassiana strains ATCC-74040 and GHA will not occur in the environmental
compartments in concentrations considerably higher than under natural conditions. Further data on the
persistence, transformation and mobility of these compounds might be needed in order to assess the
potential level of environmental exposure including groundwater.

Beauveria bassiana strain ATCC-74040: From infectivity and pathogenicity a low risk to birds,
mammals, fish, algae, earthworms, soil micro-organisms and non-target plants was concluded. A data
gap was identified to address the risk from infectivity and pathogenicity to aquatic invertebrates and
pollinators for both the field and greenhouse uses. Data gaps were concluded to address the risk to

EFSA Journal 2013;11(1):3031 2

honey bee brood and the risk to non-target arthropods infectivity and pathogenicity for the field use.
A data gap was concluded for information to address the risk to sewage treatment organisms for the
greenhouse use. Pending the data gaps for identification of secondary metabolites/toxins a risk
assessment for non-target organisms may be required for both the field and greenhouse uses.

Beauveria bassiana strain GHA: From infectivity and pathogenicity a low risk to birds, mammals,
fish, aquatic invertebrates, algae, and earthworms was concluded. Data gaps were identified to
address the risk to pollinators and sewage treatment organisms. Pending the data gap to finalise the
soil exposure assessment it may be necessary to address the risk to soil-dwelling non-target arthropods
and soil microorganisms. Pending the data gaps for identification of secondary metabolites/toxins a
risk assessment for non-target organisms may be required.

EFSA Journal 2013;11(1):3031 3

TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 2
Table of contents ...................................................................................................................................... 4
Background .............................................................................................................................................. 5
The active substance and the formulated product .................................................................................... 7
The identity of the microorganism and the properties of the formulated product .................................... 7
Conclusions of the evaluation .................................................................................................................. 7
1. Identity of the microorganism/biological properties/physical and technical properties and
methods of analysis. ................................................................................................................................. 7
2. Mammalian toxicity ......................................................................................................................... 7
3. Residues ........................................................................................................................................... 8
4. Environmental fate and behaviour ................................................................................................... 8
4.1. Fate and behaviour in the environment of the microorganism ............................................... 9
4.2. Fate and behaviour in the environment of any relevant metabolite formed by the
microorganism under relevant environmental conditions .................................................................. 10
5. Ecotoxicology ................................................................................................................................ 10
6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment
of effects data for the environmental compartments .............................................................................. 12
6.1. Soil ........................................................................................................................................ 12
6.2. Ground water ........................................................................................................................ 12
6.3. Surface water and sediment .................................................................................................. 12
6.4. Air ......................................................................................................................................... 13
7. List of studies to be generated, still ongoing or available but not peer reviewed .......................... 14
8. Particular conditions proposed to be taken into account to manage the risk(s) identified............. 15
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 ....................... 17
References .............................................................................................................................................. 19
Appendices ............................................................................................................................................. 20
Appendix A – list of end points for the active substance beauveria bassiana strain ATCC-74040
and the representative formulation ..................................................................................................... 20
Appendix B – list of end points for the active substance beauveria bassiana strain GHA and the
representative formulation ................................................................................................................. 31

EFSA Journal 2013;11(1):3031 4

BACKGROUND
Beauveria bassiana strains ATCC-74040 and GHA is one of the 295 substances of the fourth stage of
the review programme covered by Commission Regulation (EC) No 2229/20043, as amended by
Commission Regulation (EC) No 1095/20074.

Beauveria bassiana strains ATCC-74040 and GHA was included in Annex I to Directive 91/414/EEC5
on 1 May 2009 pursuant to Article 24b of the Regulation (EC) No 2229/2004 (hereinafter referred to
as „the Regulation‟) and has subsequently been deemed to be approved under Regulation (EC) No
1107/20096, in accordance with Commission Implementing Regulation (EU) No 540/20117, as
amended by Commission Implementing Regulation (EU) No 541/20118. In accordance with Article
25a of the Regulation, as amended by Commission Regulation (EU) No 114/20109 the European Food
Safety Authority (EFSA) is required to deliver by 31 December 2012 its view on the draft review
reports submitted by the European Commission in accordance with Article 25(1) of the Regulation
(European Commission, 2008a and European Commission, 2008b). This review report was established
as a result of the initial evaluation provided by the designated rapporteur Member State in the Draft
Assessment Report (DAR). The EFSA therefore organised a peer review of the DAR. The conclusions
of the peer review are set out in this report.

Germany being the designated rapporteur Member State submitted the DARs on Beauveria bassiana
strains ATCC-74040 and GHA in accordance with the provisions of Article 22(1) of the Regulation,
which were received by the EFSA on 3 December 2007 (Germany, 2007a and Germany, 2007b). The
peer review was initiated on 22 April 2008 by dispatching the DARs to the notifiers (Intrachem Bio
Italia S.p.A. for strain ATCC-74040 and Mycotech Europe Ltd. for strain GHA) and on 11 June 2008
to the Member States for consultations and comments. In addition, the EFSA conducted a public
consultation on the DAR. The comments received were collated by the EFSA and forwarded to the
RMS for compilation and evaluation in the format of a Reporting Table. The notifiers were invited to
respond to the comments in column 3 of the Reporting Table. The comments were evaluated by the
RMS in column 3 of the Reporting Table.

The scope of the peer review was considered in a telephone conference between the EFSA, the RMS,
and the European Commission on 16 December 2011. On the basis of the comments received and the
RMS‟ evaluation thereof it was concluded that the EFSA should organise a consultation with Member
State experts in the area of 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

3
Commission Regulation (EC) No 2229/2004 of 3 December 2004 laying down further detailed rules for the implementation
of the fourth stage of the programme of work referred to in Article 8(2) of Council Directive 91/414/EEC. OJ L 379,
24.12.2004, p.13-63.
4
Commission Regulation (EC) No 1095/2007 of 20 September 2007 amending Regulation (EC) No 1490/2002 laying down
further detailed rules for the implementation of the third stage of the programme of work referred to in Article 8(2) of
Council Directive 91/414/EEC and Regulation (EC) No 2229/2004 laying down further detailed rules for the implementation
of the fourth stage of the programme of work referred to in Article 8(2) of Council Directive 91/414/EEC. OJ L 246,
21.9.2007, p.19-28.
5
Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230,
19.8.1991, p. 1-32, as last amended.
6
Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing
of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309,
24.11.2009, p.1-50.
7
Commission Implementing Regulation (EU) No 540/2011 of 25 May 2011 implementing Regulation (EC) No 1107/2009 of
the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, 11.6.2011, p.1-186
8
Commission Implementing Regulation (EU) No 541/2011 of 1 June 2011 amending Implementing Regulation (EU) No
540/2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of
approved active substances. OJ L 153, 11.6.2011, p.187-188.
9
Commission Regulation (EU) No 114/2010 of 9 February 2010 amending Regulation (EC) No 2229/2004 as regards the
time period granted to EFSA for the delivery of its view on the draft review reports concerning the active substances for
which there are clear indications that they do not have any harmful effects. OJ L 37, 10.2.2010, p.12.

EFSA Journal 2013;11(1):3031 5

were identified as unresolved at the end of the comment evaluation phase and which required further
consideration, including those issues to be considered in consultation with Member State experts, and
additional information to be submitted by the notifiers, were compiled by the EFSA in the format of an
Evaluation Table.

The conclusions arising from the consideration by the EFSA, and as appropriate by the RMS, of the
points identified in the Evaluation Table, together with the outcome of the expert discussions where
these took place, were reported in the final column of the Evaluation Table.

A final consultation on the conclusions arising from the peer review of the risk assessment took place
with Member States via a written procedure in November 2012.

This conclusion report summarises the outcome of the peer review of the risk assessment on the active
substance and the representative formulation evaluated on the basis of the representative uses as an
insecticide on tomatoes for strain ATCC-74040 and on tomatoes, cucumbers and ornamentals for
strain GHA, as proposed by the notifiers. A list of the relevant end points for the active substance as
well as the formulation is provided in Appendix A. In addition, a key supporting document to this
conclusion is the Peer Review Report, which is a compilation of the documentation developed to
evaluate and address all issues raised in the peer review, from the initial commenting phase to the
conclusion. The Peer Review Report (EFSA, 2012) comprises the following documents, in which all
views expressed during the course of the peer review, including minority views, can be found:

• the comments received on the DAR,

• the Reporting Tables (13 December 2011),

• the Evaluation Tables (16 November 2012),

• the reports of the scientific consultation with Member State experts (where relevant),

• the comments received on the assessment of the points of clarification (where relevant),

• the comments received on the draft EFSA conclusion.

Given the importance of the DARs including their addenda (compiled versions of May 2012
containing all individually submitted addenda (Germany, 2012a and Germany, 2012b)) and the Peer
Review Report, both documents are considered respectively as background documents A and B to this
conclusion.

EFSA Journal 2013;11(1):3031 6

THE ACTIVE SUBSTANCE AND THE FORMULATED PRODUCT

THE IDENTITY OF THE MICROORGANISM AND THE PROPERTIES OF THE FORMULATED

PRODUCT
The strains under review are ATCC-74040 and GHA. Strain ATCC-74040 was originally isolated
from a boll weevil cadaver in Texas, USA. Strain GHA was originally isolated from the Southern corn
rootworm, on green beans in Oregon, USA.

The representative formulated products for the evaluation are „BotaniGard 22 WP‟ containing 4.4 x
1013 CFU/kg (strain GHA) and „Naturalis-L‟ containing 2.3 x 1010 CFU/L (strain ATCC-74040).

The representative uses evaluated are on tomatoes for strain ATCC-74040 and on tomatoes,
cucumbers and ornamentals for strain GHA. Full details of the GAP can be found in the lists of end
points in Appendix A.

CONCLUSIONS OF THE EVALUATION

1. Identity of the microorganism/biological properties/physical and technical properties

and methods of analysis.
Strain ATCC-74040 is maintained in the American Type Culture Collection. Strain GHA is also
maintained in this culture collection under the accession number ATCC 74250.

The strains are not human pathogens and are not related to known human pathogens. The strains are
not able to grow at or above 37 °C.

It is possible that these strains produce some toxins however insufficient information on this issue has
been provided. The content of contaminating microorganisms was not fully addressed and a data gap
has been identified. Validation of the biopotency method and other quantitative methods were not
available. Also methods for the unequivocal identification to strain level were not available. Methods
are not available for toxins and contaminating microorganisms.

For „BotaniGard 22 WP‟ containing strain GHA the following data gaps were identified: storage
stability, demonstrate that the poor wettability is not an issue, sprayability study to address
suspensibility and wet sieve test.

For „Naturalis-L‟ containing strain ATCC-74040 the following data gaps were identified: the proposed
storage regime should be given in detail and then compared to the storage stability data.

2. Mammalian toxicity
General data

B. bassiana can produce a number of metabolites. For strain ATCC-74040, the only available
information is about beauvericin suggesting that its concentration in the technical material is about 3 –
4 ppm. For the strain GHA it was shown that concentrations of approximately 50 ppm of beauvericin
and bassianolide in a batch employed in the acute studies for oral, dermal, intratracheal and
intraperitoneal toxicity did not exhibit adverse effects. Beauvericin, which was negative in an Ames
test, was therefore proposed as an indicator; however, all the other metabolites were not defined. For
both strains a data gap on strain specific data on the formation of toxins/secondary metabolites was
identified (see section 1). Being a fungus, Beauveria sp. is not expected to possess the potential for
transfer of genetic material.

EFSA Journal 2013;11(1):3031 7

Toxicity studies

Neither strain ATCC-74040 nor strain GHA showed any evidence of toxicity, infectivity or
pathogenicity, based on acute oral (GHA LD50 > 1 x 108 CFU/animal, ATCC-74040 LD50 > 1.9 x 108
CFU/animal), intratracheal (GHA LD50 > 1 x 108 CFU/animal, ATCC-74040 LD50 > 2.5 x 109
CFU/animal) and intraperitoneal (GHA LD50 > 1 x 107 CFU/animal, ATCC-74040 LD50 > 2.0 x 107
CFU/animal) studies. An Ames test performed with both strains showed negative results.

However, in the intratracheal study with strain ATCC-74040 body weight decrease was noted in
treated animals but not in the controls. Although the effect was clearly reversible, a data gap was
identified for addressing the toxicity after repeated doses via inhalation (the details of the submitted
published data on long-term toxicity do not allow for an independent assessment).

For both strains the warning phrase “Microorganisms may have the potential to provoke sensitising
reactions”, can be applied taking into account that hazard statements applicable to chemicals
(according to Regulation (EC) No 1272/200810) are not appropriate for microorganisms.

Reference values

Based on the 1st tier studies, for strain GHA no reference values are needed as the microorganism was
not shown to be pathogenic or infective based on the available data and studies. For strain ATCC-
74040 it is considered unlikely that the microorganism is pathogenic or infective based on the 1st tier
studies, not indicating the need of specific reference values; however, based on the submitted data, a
data gap was identified to further clarify repeated inhalation toxicity.

Exposure estimates

Since reference values are not necessary, no exposure estimates are required for the microorganism.
Due to the sensitisation potential of microorganisms observed in unprotected workers, the use of
adequate personal protective equipment for operators and re-entry workers should be further
considered for dermal and inhalatory exposure. Pending on the data gap for toxins/secondary
metabolites the risk assessment for the operator, worker and bystander cannot be finalised. It is noted
that bystander exposure is not relevant for the representative uses in greenhouse.

3. Residues
The microorganism is not pathogenic to humans. When applied to the plants it will only grow on/in
the host organism and if toxins are produced they will only be contained inside the host, the dead host
insect will either fall off or will be washed off. However, the consumer risk assessment cannot be
finalised until the issue of toxins/secondary metabolites is addressed for section 1.

4. Environmental fate and behaviour

No information has been provided in relation to potential interference of Beauveria bassiana strains
ATCC-74040 and GHA with the analytical systems for the control of the quality of drinking water
provided for in Directive 98/83/EC11 (see specific Annex VI decision making criteria in Directive
2005/25/EC12). However, as these methods require pathogenic bacteria to be identified and confirmed
as absent, it is probably unlikely that filamentous fungi or their conidia would interfere with
methodologies used for such determinations.

10
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.
11
Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. OJ L 330,
5.12.98, p.32-54
12
Council Directive 2005/25/EC of 14 March 2005 amending Annex VI to Directive 91/414/EEC as regards plant protection
products containing micro-organisms. OJ L 90, 8.4.2005, p.1-34

EFSA Journal 2013;11(1):3031 8

No information has been provided on the potential transfer of genetic material from Beauveria
bassiana strains ATCC-74040 and GHA to other organisms. Being a fungus, Beauveria sp. is not
expected to possess plasmids in their cytoplasm (only mitochondrial plasmids are known).
Consequently it is not expected to possess the potential for transfer of genetic material.

4.1. Fate and behaviour in the environment of the microorganism

Beauveria bassiana is naturally found throughout a wide range of habitats in temperate and tropical
climates. The level of natural occurrence of B. bassiana strains ATCC-74040 and GHA in the soil is
not precisely defined in the supplied literature. There are indications that B. bassiana strain GHA
multiplies in the environment only in insect hosts, where it sporulates and disperses only under
specific environmental conditions, particularly high humidity. In the absence of specific
environmental conditions, the organism dies with its target host. However, a data gap has been
identified for strain specific data on the environmental conditions required for dispersal and viability
of conidia for B. bassiana strain GHA.

Three studies on the persistence and multiplication in soil of B. bassiana strain ATCC-74040 were
submitted. However, uncertainties and deficiencies in terms of method of application, environmental
conditions and origin of soils were identified for these studies. Overall, the available information is
considered not sufficient to properly characterise the competitiveness/persistence of B. bassiana
strains ATCC-74040 and GHA in soil in the context of the representative uses assessed. Therefore, a
data gap has been identified. This data gap is also pertinent in respect of the representative use
assessed for B. bassiana strains ATCC-74040 and GHA for greenhouse and indoor uses, in particular
since what happens to the growth medium at the end of the production was not assessed in the dossier.
This route of environmental exposure should be taken into account in territories where spent growing
media can be spread on agricultural or horticultural land. The data gap is also relevant for production
in temporary greenhouses (e.g. polythene tunnels) for the soil in situ.

A data gap was also identified for information on viability/population dynamics in soil-less media for
the use of B. bassiana strains ATCC-74040 and GHA in greenhouse.

Little information has been provided on persistence and multiplication in water of B. bassiana
strains ATCC-74040 and GHA. A study on the effect of pH and metal ions on B. bassiana strain GHA
in aqueous suspension showed a loss in conidial viability in the different aqueous systems tested. In
another study on the determination of storage stability of B. bassiana strain ATCC-74040 it was
concluded that the microorganism is stable in water at pH 5, 7 and 9. No other information is given on
persistence and multiplication in water. However, it is known from scientific publications that
germination of conidia and therefore multiplication in water is not expected since B. bassiana is not an
aquatic fungus and is therefore not adapted to the conditions of the aqueous environment.

Conidia of B. bassiana can be easily transported through the air. Also transmission of fungal spores
through the air by insects is possible. On the basis of the information presented in the DAR and in
Addendum 2 (March 2012) for B. bassiana strain GHA, it could be shown that viability of B. bassiana
conidia in the air or epigeal habitats is mainly affected by the UV-B portion of the solar spectrum. On
the basis of persistence data, it may be concluded that significant degradation of B. bassiana conidia
on leaves under field conditions may occur within few days.

A study on mobility in soil showed that conidia of B. bassiana strain GHA are not very mobile in soil
and generally remain on the soil surface. It was seen that the movement of conidia vertically through
the soil profile was positively correlated with high infiltration rates in soil. Besides leaching,
movement in the soil in horizontal and vertical direction is possible by soil arthropods, especially
collembola. However, no groundwater risk assessment is necessary since B. bassiana is neither
pathogenic nor toxic to humans.

EFSA Journal 2013;11(1):3031 9

4.2. Fate and behaviour in the environment of any relevant metabolite formed by the
microorganism under relevant environmental conditions
A data gap has been identified for information on the production of toxins/secondary metabolites such
as beauvericin (see section 1). If, under the conditions of use, relevant metabolites are present in or
produced by the microorganism, data requirements and the corresponding risk assessment as outlined
under Annex II, Part A point 7 of Directive 91/414/EC need to be fulfilled if all the following
conditions are met:

- the relevant metabolite is stable outside the microorganism

- a toxic effect of the relevant metabolite is independent of the presence of the microorganism,
and

- the relevant metabolite is expected to occur in the environment in concentrations considerably

higher than under natural conditions.

This data gap is pertinent in respect of the representative uses in the field and greenhouse assessed for
B. bassiana strain ATCC-74040 and for strain GHA for both indoor and greenhouse uses and
following disposal of spent growing media, as what happens to the growth medium at the end of the
production was not assessed. This route of environmental exposure would need to be taken into
account in territories where spent growing media can be spread on agricultural or horticultural land.

5. Ecotoxicology
It is noted that a data gap was identified in relation to the formation of secondary metabolites/toxins,
such as beauvericin, in the treated crops and in relevant environmental compartments after application
of the product. Pending on the outcome of these data gaps, the potential risk of these secondary
metabolites/toxins to non-target organisms has to be addressed (see Section 1 and Section 4.2).

Beauveria bassiana strain ATCC-74040

A strain-specific study on Beauveria bassiana strain ATCC-74040 was submitted to address the
toxicity and pathogenicity to birds. The RMS noted a number of uncertainties in the study with regard
to the possibility to detect infectivity and pathogenicity. However, the study was specifically designed
to account for potential effects from infectivity and pathogenicity (5 days of exposure followed by 30
days observation period) and was considered to be useful for regulatory assessment. No indications of
infectivity or pathogenicity were observed in the study and therefore a low risk to birds from
infectivity and pathogenicity was concluded.

Beauveria bassiana strain ATCC-74040 was concluded not to be infectious or pathogenic to mammals
(see Section 2); therefore a low risk to wild populations of mammals from infectivity and
pathogenicity was concluded.

A study to address the toxicity, pathogenicity and infectiveness of B. bassiana strain ATCC-74040 on
fish was available. A study on algae was available with the plant protection product „Naturalis-L‟.
Overall, these studies were considered suitable to address the risk of infectiveness and pathogenicity,
which was indicated as low. A study with Daphnia magna indicated an effect on immobilisation. The
study was not considered sufficient for an assessment of infectivity and pathogenicity and therefore a
data gap was concluded.

A strain specific study on bees for B. bassiana strain ATCC-74040 was provided. The available data
submitted to address the effects on bees and pollinators were discussed at the Pesticides Peer Review
Teleconference TC 67. No adverse effects were observed on honey bees, however since reproductive
effects were observed for Typhlodromus pyri, experts agreed that the potential effects on bee brood are
not covered by the available data for the representative field use. Furthermore, experts agreed that

EFSA Journal 2013;11(1):3031 10

further data are needed to address the reproductive effects of B. bassiana strain ATCC-74040 on
pollinators for the representative field use. Therefore, data gaps were identified for these issues. This
data gap is also relevant for pollinators which may be used in greenhouses, although it was
acknowledged that bumble bees can be used to vector B. bassiana for insect control in greenhouses.

Some laboratory studies indicate that non-target arthropods may be susceptible to infection and
pathogenicity from B. bassiana strain ATCC-74040. Information suggests that humid conditions will
increase the likelihood of infection. With the information available, a high risk from infectivity and
pathogenicity to non-target arthropods cannot be excluded.

An acute earthworm toxicity study performed with the product „Naturalis-L‟ was available and
indicated no adverse effects. It is acknowledged that the study was not specifically designed to
investigate the potential for infectivity and pathogenicity; however, as the study was conducted for 14
days it is expected that there would have been some evidence of an effect. Therefore, on the basis of
the available study a low risk to earthworms from infectivity and pathogenicity was concluded.

The risk to soil microorganisms was considered to be low for all representative uses.

No data were available to address the effects on biological methods for sewage treatment plants. A
data gap was identified to address the risk for biological methods for sewage treatment plants for the
greenhouse use.

Beauveria bassiana strain GHA

Some strain-specific studies to address the pathogenicity and infectiveness of B. bassiana strain GHA
to fish and aquatic invertebrates were available. A study on algae with the plant protection product
„BotaniGard 22 WP‟ was also available. No indications of infectivity or pathogenicity were observed
in the available fish study and therefore a low risk from infectivity and pathogenicity could be
concluded. However, an adverse effect on the reproductive capacity was observed at the tested
concentration and therefore no NOEC could be derived. The study was not considered sufficient to
address the risk to fish from toxic effects and therefore further information is required (see data gap
regarding the secondary metabolites and toxins, Section 1 and Section 4.2). The available data for
aquatic invertebrates and algae indicated a low risk from infectivity and pathogenicity.

The risk assessment for pollinators which may be used in greenhouses was discussed at Pesticides
Peer Review Teleconference TC 67. It was agreed that further information was required to
demonstrate a low risk and therefore a data gap was identified.

A data gap was identified for information to complete the soil exposure assessment for Beauveria
bassiana strain GHA, following disposal of spent growing media from greenhouses (see Section 4).
Pending the outcome of the data gap it may be necessary to address the risk from infectivity and
pathogenicity to soil-dwelling non-target arthropods and soil microorganisms. This is therefore
identified as an assessment not finalised. A low risk to birds, mammals and earthworms from
infectivity and pathogenicity was concluded.

No studies were available to address the effects on biological methods for sewage treatment plants and
therefore a data gap was identified.

EFSA Journal 2013;11(1):3031 11

6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental
compartments

6.1. Soil
Compound
Persistence Ecotoxicology
(name and/or code)

Low risk for strain Beauveria bassiana strain ATCC-

74040.
Beauveria bassiana strain ATCC-74040 and GHA No data, data required
No data for soil microorganisms available for Beauveria
bassiana strain GHA.

Data gap pending on their identification and Data gap pending on their identification and
Relevant toxins or secondary metabolites
quantification quantification

6.2. Ground water

>0.1 μg/L 1m depth for
Compound the representative uses
Mobility in soil (at least one FOCUS Pesticidal activity Toxicological relevance Ecotoxicological activity
(name and/or code)
scenario or relevant
lysimeter)

Data gap pending on their Data gap pending on their Data gap pending on their Data gap pending on their
Relevant toxins or
identification and identification and - identification and identification and
secondary metabolites
quantification quantification quantification quantification

6.3. Surface water and sediment

Compound
Ecotoxicology
(name and/or code)

EFSA Journal 2013;11(1):3031 12

Beauveria bassiana strain ATCC-74040 – data gap to address the risk to aquatic invertebrates from infectivity and
pathogenicity.
Beauveria bassiana strain ATCC-74040 and GHA
Beauveria bassiana strain GHA – low risk from infectivity and pathogenicity.

Relevant toxins or secondary metabolites No data available. Data gap pending on their identification and quantification.

6.4. Air
Compound
Toxicology
(name and/or code)

Beauveria bassiana strain GHA Not acutely toxic after intratracheal administration

Beauveria bassiana strain ATCC-74040 Not acutely toxic after intratracheal administration; data gap for repeated dose inhalation toxicity

Relevant toxins or secondary metabolites (both strains

No data available
GHA and ATCC-74040)

EFSA Journal 2013;11(1):3031 13

7. List of studies to be generated, still ongoing or available but not peer reviewed
This is a complete list of the data gaps identified during the peer review process, including those areas
where a study may have been made available during the peer review process but not considered for
procedural reasons (without prejudice to the provisions of Article 7 of Directive 91/414/EEC
concerning information on potentially harmful effects).

Demonstrate that the level of microbial contamination complies with international standards. The
OECD issues paper (OECD, 2011) should be used as the reference (relevant for strain ATCC
74040; submission date proposed by the notifier: studies available but not evaluated or peer
reviewed; see section 1).

Demonstrate that the level of microbial contamination complies with international standards. The
OECD issue paper (OECD, 2011) should be used as the reference (relevant for strain GHA;
submission date proposed by the notifier: unknown; see section 1).

Validation of the biopotency method (relevant for all representative uses evaluated; submission
date proposed by the notifiers: unknown; see section 1)

Validation of the method to unequivocally identify the organism to strain level (relevant for all
representative uses evaluated; submission date proposed by the notifiers: unknown; see section 1).

Strain specific data on the formation of toxins/secondary metabolites. It is noted that some data are
available for the compound beauvericin (relevant for strain ATCC 74040; submission date
proposed by the notifier: unknown; see section 1 and 2).

Strain specific data on the formation of toxins/secondary metabolites (relevant for strain GHA;
submission date proposed by the notifier: unknown; see section 1 and 2).

Validated quantitative methods for the microorganism in the technical material and formulated
product (relevant for all representative uses evaluated; submission date proposed by the notifiers:
unknown; see section 1).

Validated methods for the microbial contaminants (relevant for strain ATCC 74040; submission
date proposed by the notifier: studies available but not evaluated or peer reviewed; see section 1).

Validated methods for the microbial contaminants (relevant for strain GHA; submission date
proposed by the notifier: unknown; see section 1).

Validated method of analysis for toxins (relevant for all representative uses of strain GHA
evaluated; submission date proposed by the notifier: unknown; see section 1).

The proposed storage conditions for the formulated product should be given in detail and it should
be checked if the storage stability data support the proposed conditions (relevant for all
representative uses of „Naturalis-L‟ evaluated; submission date proposed by the notifier: unknown;
see section 1).

Storage stability data; demonstration that during normal use the poor wettability does not cause a
problem; sprayability study to demonstrate that during normal use the poor suspensibility is not an
issue; wet sieve test (relevant for all representative uses of „BotaniGard 22 WP‟ evaluated;
submission date proposed by the notifier: unknown; see section 1).

Further clarification on the repeated inhalation toxicity of ATCC-74040 (relevant for all
representative uses of strain ATCC-74040; submission date proposed by the notifier: unknown;
see section 2).

EFSA Journal 2013;11(1):3031 14

Satisfactory information to demonstrate that Beauveria bassiana strains ATCC-74040 and GHA
will not persist in soil in concentrations higher than the natural background levels, taking into
account repeated applications over the years. Pending the outcome of the data gap, a risk
assessment for soil-dwelling non-target arthropods and soil microorganisms may be required
(relevant for all representative uses evaluated and in territories where spent growing media from
indoor/greenhouse uses can be spread on agricultural or horticultural land or in temporary
greenhouses (e.g. polythene tunnels) for the soil in situ; submission data proposed by the notifiers:
unknown; see section 4 and 5).

Satisfactory information to demonstrate that, under the conditions of use, any relevant
toxins/secondary metabolites produced by Beauveria bassiana strains ATCC-74040 and GHA
(which may include beauvericin) will not occur in the environmental compartments in
concentrations considerably higher than under natural conditions. Further data on the persistence,
transformation and mobility of these compounds may be needed in order to assess the potential
level of environmental exposure including groundwater (relevant for all representative uses
evaluated; submission date proposed by the notifiers: unknown; see section 4).

Strain specific data on the environmental conditions required for dispersal and viability of conidia
for B. bassiana strain GHA in the environment (relevant for all representative uses of strain GHA;
submission date proposed by the notifier: unknown; see section 4).

Information on viability/population dynamics in soil-less media for the use of B. bassiana strains
ATCC-74040 and GHA in greenhouse (relevant for greenhouse uses; submission date proposed by
the notifiers: unknown; see section 4).

Pending on the outcome of the data gaps identified for the secondary metabolites and toxins, the
risk to non-target organisms may need to be addressed (relevant for representative uses of B.
bassiana strains ATCC-74040 and GHA; submission date proposed by the notifier: unknown; see
section 5).

Further data to address the potential for infectivity and pathogenicity to aquatic invertebrates
(relevant for all representative uses of B. bassiana strain ATCC-74040; submission date proposed
by the notifier: unknown; see section 5).

Further data to address the potential effect on honey bee brood (relevant for representative field
use of B. bassiana strain ATCC-74040; submission date proposed by the notifier: unknown; see
section 5).

Further data to address the reproductive effect of B. bassiana strains ATCC-74040 and GHA to
pollinators (relevant for all representative uses of B. bassiana strains ATCC-74040 and GHA;
submission date proposed by the notifier: unknown; see section 5).

Further data to address the potential for infectivity and pathogenicity to non-target arthropods
(relevant for field representative uses of B. bassiana strain ATCC-74040; submission date
proposed by the notifier: unknown; see section 5).

Information to address the effects on biological methods for sewage treatment plants for the use of
B. bassiana strains ATCC-74040 and GHA in greenhouse (relevant for greenhouse uses;
submission date proposed by the notifier: unknown; see section 5).

8. Particular conditions proposed to be taken into account to manage the risk(s) identified
none

EFSA Journal 2013;11(1):3031 15

9. Concerns

9.1. Issues that could not be finalised

An issue is listed as an issue that could not be finalised where there is not enough information
available to perform an assessment, even at the lowest tier level, for the representative uses in line
with the Uniform Principles of Annex VI to Directive 91/414/EEC and where the issue is of such
importance that it could, when finalised, become a concern (which would also be listed as a critical
area of concern if it is of relevance to all representative uses).

1. The production of relevant toxins/secondary metabolites cannot be excluded and therefore the
risk assessment cannot be finalised for humans (including operators, workers, bystanders and
consumers) and the environment including the assessment of potential groundwater
contamination and the potential risk to non-target organisms.

2. Satisfactory information is not available to demonstrate that B. bassiana strains ATCC-74040 and
GHA will not persist in soil in concentrations higher than the natural background levels, taking
into account repeated applications over the years. Consequently, the level of exposure in soil of B.
bassiana strain ATCC-74040 cannot be reliably estimated for the field uses. This applies also to
the use of B. bassiana strains ATCC-74040 and GHA in temporary greenhouse systems (e.g.
polythene tunnels) or when spent growing media is spread on agricultural or horticultural land.
Pending the outcome of the data gap a risk assessment for soil-dwelling non-target arthropods and
soil microorganisms from B. bassiana strain GHA may be required.

3. The risk assessment for bees and other pollinators cannot be finalised with the available data for
the representative uses of B. bassiana strain ATCC-74040 and strain GHA.

9.2. Critical areas of concern

An issue is listed as a critical area of concern where there is enough information available to perform
an assessment for the representative uses in line with the Uniform Principles of Annex VI to Directive
91/414/EEC, and where this assessment does not permit to conclude that for at least one of the
representative uses it may be expected that a plant protection product containing the active substance
will not have any harmful effect on human or animal health or on groundwater or any unacceptable
influence on the environment.

An issue is also listed as a critical area of concern where the assessment at a higher tier level could not
be finalised due to a lack of information, and where the assessment performed at the lower tier level
does not permit to conclude that for at least one of the representative uses it may be expected that a
plant protection product containing the active substance will not have any harmful effect on human or
animal health or on groundwater or any unacceptable influence on the environment.

none

EFSA Journal 2013;11(1):3031 16

9.3. Overview of the concerns identified for each representative use considered
(If a particular condition proposed to be taken into account to manage an identified risk, as listed in
section 8, has been evaluated as being effective, then „risk identified‟ is not indicated in this table.)
Tomatoes,
Tomatoes Tomatoes Ornamentals
cucumber
Greenhouse Field Indoor
Representative use Greenhouse
ATCC-74040 ATCC-74040 GHA
GHA

Risk
identified
Operator risk
Assessment
not finalised
X1 X1 X1 X1
Risk
identified
Worker risk
Assessment
not finalised
X1 X1 X1 X1
Risk
identified
Bystander risk
Assessment
not finalised
X1
Risk
identified
Consumer risk
Assessment
not finalised
X1 X1 X1
Risk to wild Risk
non target identified
terrestrial Assessment
not finalised
X1 X1 X1 X1
vertebrates
Risk to wild Risk
identified
X
non target
terrestrial
organisms Assessment
not finalised
X1, 3 X1, 3 X1, 2, 3 X1, 2, 3
other than
vertebrates
Risk
Risk to aquatic identified
organisms Assessment
not finalised
X1 X1 X1 X1
Legal
parametric
Groundwater value
exposure active breached
substance Assessment
not finalised
Legal
parametric
value
breached
Groundwater
Parametric
exposure value of
metabolites 10µg/L(a)
breached
Assessment
not finalised
X1 X1 X1 X1
Comments/Remarks

EFSA Journal 2013;11(1):3031 17

The superscript numbers in this table relate to the numbered points indicated in sections 9.1 and 9.2. Where there is no
superscript number see sections 2 to 6 for further information.
(a): Value for non-relevant metabolites prescribed in SANCO/221/2000-rev 10-final, European Commission, 2003

EFSA Journal 2013;11(1):3031 18

REFERENCES
EFSA (European Food Safety Authority), 2012. Peer Review Report to the conclusion regarding the
peer review of the pesticide risk assessment of the active substance Beauveria bassiana strains
ATCC-74040 and GHA.
European Commission, 2003. Guidance Document on Assessment of the Relevance of Metabolites in
Groundwater of Substances Regulated under Council Directive 91/414/EEC. SANCO/221/2000-
rev. 10 - final, 25 February 2003.
European Commission, 2008a. Review Report for the active substance Beauveria bassiana strain
ATCC-74040 finalised in the Standing Committee on the Food Chain and Animal Health at its
meeting on 11 July 2008 in view of the inclusion of Beauveria bassiana strain ATCC-74040 in
Annex I of Directive 91/414/EEC. SANCO/1546/08 – rev. 3, 07 May 2008
European Commission, 2008b. Review Report for the active substance Beauveria bassiana strain
GHA finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on
11 July 2008 in view of the inclusion of Beauveria bassiana strain GHA in Annex I of Directive
91/414/EEC. SANCO/1547/08 – rev. 3, 07 May 2008.
FOCUS, 2008. “Pesticides in Air: Considerations for Exposure Assessment”. Report of the FOCUS
Working Group on Pesticides in Air, EC Document Reference SANCO/10553/2006 Rev 2 June
2008.
Germany, 2007a. Draft Assessment Report (DAR) on the active substance Beauveria bassiana strain
ATCC-74040 prepared by the rapporteur Member State Germany in the framework of Directive
91/414/EEC, November 2007.
Germany, 2007b. Draft Assessment Report (DAR) on the active substance Beauveria bassiana strain
GHA prepared by the rapporteur Member State Germany in the framework of Directive
91/414/EEC, November 2007.
Germany, 2012a. Final Addendum to Draft Assessment Report on Beauveria bassiana strain ATCC-
74040, compiled by EFSA, May 2012.
Germany, 2012b. Final Addendum to Draft Assessment Report on Beauveria bassiana strain GHA,
compiled by EFSA, May 2012.
OECD (Organisation for Economic Co-operation and Development), 2011. OECD Issue Paper on
Microbial Contaminant Limits for Microbial Pest Control Products, ENV/JM/MONO(2011)43.

EFSA Journal 2013;11(1):3031 19

APPENDICES

Appendix A – list of end points for the active substance Beauveria bassiana strain ATCC-74040
and the representative formulation
Chapter 1: Identity, Biological properties, Details of uses, Further information, and
Proposed Classification and Labelling

Active micro-organism: Beauveria bassiana (Balsamo) Vuillemin

Function (e.g. control of fungi): Control of insects

Rapporteur Member State Germany

Identity of the Microbial Pest control Agent / Active substance (OECD data point IIM 1)
Name of the organism: Beauveria bassiana (Balsamo) Vuillemin
Taxonomy: Phylum: Deuteromycota, Class: Hyphomycetes, Order:
Moniliales, Family: Moniliaceae, Genus: Beauveria
Species, subspecies, strain: B. bassiana strain ATCC 74040
Identification / detection: The organism has been identified according to the taxonomic
descriptions of de Hoog 1972 (in particular conidiospore
morphology) analysed by standard laboratory microscopy.
Open for specific method.
Culture collection: B. bassiana strain ATCC 74040 is maintained in the American
Culture Collection under ATCC 74040.
Minimum and maximum concentration 2 x 1010 CFU/g in the technical MPCA with a minimum of 1.6
of the MPCA used for manufacturing of x 1010 CFU/g.
the formulated product (viable granules;
g/kg):
Identity and content of relevant none
impurities, additives, contaminating
organisms in the technical grade of
MPCA:
Is the MCPA genetically modified; if so Not applicable
provide type of modification

Biological properties of the micro-organism (OECD data point IIM 2)

Origin and natural occurrence, Beauveria bassiana strain ATCC 74040 is an
Background level: entomopathogenic fungus which is a common pathogen of
many insect species. It is a ubiquitous fungus, found in a
variety of soils from a broad range of climatic conditions in
most countries.
Target organism(s): Whiteflies (Aleurodidade)
Mode of action: The conidia of B. bassiana strain ATCC 74040 adhere to the
insect cuticle by means of hydrophobic interaction between
the spore wall and epicuticle lipids. The conidia germinate,
EFSA Journal 2013;11(1):3031 20
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Peer Review of the pesticide risk assessment of the active substance Beauveria bassiana
strains ATCC-74040 and GHA

and the germ tube penetrates the cuticle, using a specific

series of enzymes, which in turn degrade the lipids, protein
and chitin in the insect cuticle. In the insect body, the fungus
multiplies in the haemocœl as a blastospore, or yeast-like
cell, and enzymes begin to destroy the internal structures of
the host insect causing morbidity within 36 - 72 hours.
Reduced feeding and immobility are rapidly evident, and the
insect dies within 4 to 10 days post-infection. The time to
death will depend on the insect species, age and conidial
dose. After death, the blastospores transform into mycelia,
which emerge through the cuticle and form spores. These
cover the cadaver as a characteristic white mould.
Sporulation occurs only in conditions of high humidity.
Host specificity: Infection mechanisms are specific to insects. Not pathogenic
to humans / plants.
Life cycle: The conidia adhere to the insect cuticle, germinate and
penetrate in the insect body, where they replicate as yeast-
like cells (blastospores) and destroy the internal structures,
causing morbidity within 36 - 72 hours. After death of the
insect, the blastospores transform into mycelia, which
emerge through the cuticle and form spores. B. bassiana
strain ATCC 74040 has no resting stages and no known
sexual stage.
Infectivity, dispersal and colonisation In general, B. bassiana grows in a wide temperature range
ability: from 5 to 35 °C. The optimal growth temperature for B.
bassiana is 23 to 28ºC. Maximum temperature for the
majority of analysed strains is below 35°C. Water activity
below 0.99 already leads to considerable reductions in
growth and no growth was observed at 0.88.
B. bassiana strain ATCC 74040 remained viable in aqueous
suspensions at pH 5, 7, and 9 for 24 hours in a laboratory test
system.
The germination rate of conidia decreased rapidly after
exposition to UV light, and short wave UV was more
effective in inactivation than long-wave UV. No viable
spores were detected after 32 min. of long-wave-UV
irradiation.
Relationships to known plant, animal Beauveria bassiana strain ATCC 74040 is not related to any
or human pathogens: known human or plant pathogen.
Genetic stability: During more than 10 years of commercial use and
production, no change has occurred in phenotypic
characteristics or RAPD analyses of Beauveria bassiana
Strain ATCC 74040.
Information on the production of Beauvericin: max content 5 mg/kg
relevant metabolites (especially Open for further studies.
toxins):
Resistance/ sensitivity to antibiotics / No specific information addressing this issue has been
anti-microbial agents used in human or submitted by the notifier or could be found in the open
veterinary medicine: literature. However, because spread of resistance (genes) to
antimycotic agents between fungal species is not of that
EFSA Journal 2013;11(1):3031 21
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Peer Review of the pesticide risk assessment of the active substance Beauveria bassiana
strains ATCC-74040 and GHA

concern as for bacteria with regard to antibiotics, and since

Beauveria bassiana itself is only an opportunistic pathogen
that has very rarely caused infections in humans and is not
related to any known human pathogen, this lack of
information is not considered a critical data gap.

EFSA Journal 2013;11(1):3031 22

Summary of uses supported by available data (Beauveria bassiana strain ATCC 74040)
Crop and/ Member Product F,G or Pests or PHI Remarks:
or situation State name I Group of (days)
(a) or (b) pests Formulation Application Application rate per treatment (m)
Country controlled MPCA
(c)
Type Conc. Method Growth Number Interval g as/hL water L/ha g as/ha
of as kind stage & min max between
season applications min max min max min max
(d-f) (i) (f-h) (j) (k)

Tomatoes Europe Naturalis-L G Whiteflies OD 0.0691 kg/L Spray - 3-5 5-7d 5.18 – (800) - 51.8 -69.1 NA Rate of
6.91 1000 MPCP:
(2.3 x 1010
(1.7 x 1010
CFU/L) 0.75-1 L/ha
(1.7 x 109 -
-
2.3 x 1010
9
2.3 x 10 CFU/ha)
CFU/hL)

Tomatoes Europe Naturalis-L F Whiteflies OD 0.0691 kg/L Spray - 3-5 5-7d 5.18 – (800) - 51.8 -69.1 NA Rate of
10 6.91 1000 MPCP:
(2.3 x 10
CFU/L) (1.7 x 1010 0.75-1 L/ha
(1.7 x 109
-
-
2.3 x 1010
2.3 x 109 CFU/ha)
CFU/hL)
(a) For crops, the EU and codex classifications (both) should be used; where relevant, the situation should be (h) Kind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plant - type
described (e.g. fumigation of a structure) of equipment used must be indicated
(b) Outdoor or field use (F), glasshouse application (G) or indoor application (I) (i) CFU/kg or CFU/L
(c) e.g. biting and sucking insects, soil born insects, foliar fungi, weeds (j) Growth stage at last treatment (BBCH Monograph, growth stages of Plants, 1997, Blackwell
(d) e.g. wettable powder (WP), emusifiable concentrate (EC), granule (GR) ISBN 3-8263-3152-4), including where relevant, information on season at time of application
(e) GCPF codes - Crop Life Technical Monograph No 2, 1989 (k) Indicate the minimum and maximum number of application possible under practical
(f) All abbreviations used must be explained conditions of use
(g) Method, e.g. high volume spraying, low volume spraying, spreading, dusting, drench (l) The volume should be given in g or kg whatever gives the more readable number ( eg. 200
kg/ha instead of 200000g/ha ) as well as in number of CFU
(m) PHI - minimum pre-harvest interval

EFSA Journal 2013;11(1):3031 23

Classification and proposed labelling (Symbol, Indication of danger, Risk phrases, Safety
phrases)
with regard to physical/chemical data: none
with regard to toxicological data R42 – May cause sensitisation by inhalation
R43 – May cause sensitisation by skin contact
with regard to fate and behaviour Not applicable
with regard to ecotoxicological data Not applicable

Chapter 2: Methods of analysis

Analytical methods for the micro-organism (OECD data point IIM 4.2, 4.3 and IIIM 5.3)
manufactured micro-organism (principal Analytical methods for the identification of the technical
of method): Beauveria bassiana strain ATCC 74040 include the following:
- Characterisation by light microscopic examination of the
visual morphology
- Random Amplified Polymorphic DNA (RAPD) analysis of
fractionated total genomic DNA.
- Discrimination of the strain from other strains of the same
species by microsatellites.
- Determination of fungal spore-count and spore viability by
haemocytometer counts and in vitro germination assay.
- Open for validation
Impurities and contaminating micro- Enumeration of microbial contaminants, determining the
organisms in manufactured material presence/absence of enteric bacteria such as Shigella,
(principal of method): Salmonella and Vibrio spp. by plating out on selective media
(Thiosulfate-Citrate-Bile-Sucrose and Salmonella-Shigella
agars).
The possible presence of impurities such as Aflatoxin and
Beauvericin is assessed by HPLC.
HPLC-UV (column switch) for beauvericin
Open for contaminating microorganisms
Microbial Pest control product (principle Analytical methods for the formulation are as used for the
of method): active substance.

Analytical methods for residues (viable and non-viable) in exposed compartments and
organisms (OECD data point IIM 4.5)
of the active micro-organism (principle Validated methods are not required. However, for water a
of method) method is available and validated with an LOQ of 6 x 107
CFU/L
Of relevant metabolites (principle of none relevant
method):

EFSA Journal 2013;11(1):3031 24

Chapter 3: Impact on Human and Animal Health

Medical data: No adverse effects in employees who had been in occupational
(including medical surveillance on contact with strain ATCC reported. For Beauveria bassiana in
manufacturing plant personnel) general, few cases of local (eye) and systemic infections were
(OECD data point IIM 5.1, 5.2) reported with the latter occurring only in immunocompromised
or severely ill patients. Evidence of allergenicity by respiratory
or skin contact.
Sensitisation: Microorganisms may have the potential to provoke sensitising
(OECD data point IIM 5.3.1 & IIIM reactions
7.1.6) For Beauveria bassiana in general, there is evidence of
allergenicity by inhalation in laboratory animals and humans
and indications of allergic skin reactions in humans. The
available supplementary M&K test with strain ATCC in guinea
pigs, although negative, was not sufficient to contravene these
findings.
Acute oral infectivity, toxicity and Rat: LD50 > 1.9 x 108 CFU/animal; no evidence of toxicity,
pathogenicity pathogenicity, or infectivity. Spores still detectable in faeces at
(OECD data point IIM 5.3.2 & IIIM end of study on day 14.
7.1.1)
Acute intratracheal/inhalation toxicity, Rat: LD50 > 2.5 x 109 CFU/animal; initial bw decrease, local
pathogenicity and infectivity: reversible signs of lung inflammation. No evidence of
(OECD data point IIM 5.3.3 & IIIM pathogenicity or infectivity. Clearance in lung tissue completed
7.1.3) by day 15 post-dosing.
Acute intravenous/intraperitoneal Rat: LD50 > 2.0 x 107 CFU; no evidence of toxicity (nasal
toxicity, pathogenicity and infectivity: secretion on day 1), pathogenicity or infectivity. Clearance in
(OECD data point IIM 5.3.4 & IIIM blood completed by day 2 post-dosing.
7.1.2)
Genotoxicity: Negative in the Ames test
(OECD data point IIM 5.3.5)
Cell culture study: No data – not required.
(OECD data point IIM 5.3.6)
Information on short-term toxicity and No data – not required.
pathogenicity:
(OECD data point IIM 5.3.7)
Dermal toxicity: Rabbit: LD50 > 1 x 1010 CFU/animal; no systemic effects,
(OECD data point IIIM 7.1.2) slightly irritating to the skin (no classification needed).

Specific-toxicity, pathogenicity and Rabbit: LD50 > 1 x 1010 CFU/animal; no systemic effects,
infectivity: slightly irritating to the skin (no classification needed).
(OECD data point IIM 5.5) Certain potential of the microorganism (strain not specified) to
damage the cornea confirmed in rabbits.
Long-term inhalative exposure to B. bassiana (strain not
specified) caused allergic lung reactions in rats and mice.
Data gap for specific repeated dose toxicity
Genotoxicity – in vivo studies in germ No data – not required.
cells:
(OECD data point IIM 5.5.3)

EFSA Journal 2013;11(1):3031 25

Additional studies with metabolites Toxicological studies with strain GHA including a beauvericin
and bassianolide contamination with approx. 50 mg/kg each
indicated no additional adverse effects after oral, dermal or ip
administration. Effects seen after intratracheal administration
are unlikely to be due to beauvericin and bassianolide.
Beauvericin was not mutagenic in an Ames test.
Exposure to (the sum of) the metabolites should stay below
1.5 µg/d (threshold of toxicological concern).

Reference values
AOEL Not established, not needed1
ARfD Not established, not needed1
ADI Not established, not needed1
1
based on the submitted data, a data gap was identified to further clarify repeated inhalation toxicity

Exposure (operator, workers, Operator

bystander, consumer): Not finalised (lack of information on toxin/metabolites
(OECD data point IIM 6.1, IIIM 7.2, 7.3 production)
and 8.0)
Bystander
Not finalised (lack of information on toxin/metabolites
production)
(For greenhouse applications, the bystander exposure is not
relevant).

Worker
Not finalised (lack of information on toxin/metabolites
production)

Chapter 4: Residues
Open for toxins/secondary metabolites

Chapter 5: Fate and Behaviour in the Environment (OECD IIM 7 & IIIM 9)
Persistence and multiplication

in soil: Data gap

The available data on measurement or observation
of persistence and multiplication in soil was
insufficient.
in water: In an aquatic environment spores will be subject to
sedimentation and can be expected to degrade
rapidly in natural water bodies.
Based on read across from strain GHA:
The conidia did not germinate in different tested
aqueous systems (distilled water, and aqueous

EFSA Journal 2013;11(1):3031 26

suspensions at pH 5, 7 and 9, or in metal ion

solutions in the presence of bacteria).
in air: Based on read across from strain GHA:
In the absence of a specific host insect, viability of
conidia of B. bassiana in the air or epigeal habitats
is mainly affected by the UV-B portion of the solar
spectrum. On the basis of the persistence data,
significant degradation of B. bassiana conidia on
leaves under field conditions may occur in less than
2 days.

Mobility: In soil:
Based on read across from strain GHA:
Conidia of B. bassiana are not very mobile in soil
and generally remain on the surface of the soil. No
groundwater risk assessment is necessary since B.
bassiana is neither pathogenic nor toxic to humans.
In water: The globose or ovoid shape of conidia is
not suitable for efficient transport in water.
In air:
Beauveria bassiana is dry, of small size and it is
produced in powdery clusters. Therefore, these
types of conidia can be easily transported through
the air. Also transmission of fungal spores through
the air by insects is possible.
However, in the absence of specific host insects,
conidia of Beauveria bassiana will not persist in the
sun-exposed phylloplane in significant amount for
more than 2 days.

EFSA Journal 2013;11(1):3031 27

Chapter 6: Effects on non-target organisms (OECD IIM 8 & IIIM 10)

Effects on birds (OECD IIM 8.1 & IIIM 10.1)
Application rate Crop Species Time-scale Toxicity, infectivity and
(kg MPCA/ha) pathogenicity
(endpoint, value or other
description of effects)
0.0691 Tomato, Bobwhite quail 5 days LD50 > 2667 mg/kg bw/day
(corresponding N/S- dosing / 25 corresponding to 6.85 x 1010
to 2.3 x 1010 Europe days CFU/kg bw/day
CFU/ha) (field) observatio (No observed external signs of
n pathogenicity. No findings of
the gross necropsy)

Effects on mammals
Application rate Crop Species Time-scale Toxicity, infectivity and
(kg MPCA/ha) pathogenicity
(endpoint, value or other
description of effects)
0.0691 Tomato, Rat Acute Rat: LD50 > 1.9 x 108
(corresponding N/S- CFU/animal; no evidence of
to 2.3 x 1010 Europe toxicity, pathogenicity, or
CFU/ha) (field) infectivity. Spores still
detectable in faeces at end of
study on day 14.

Invertebrate B. bassiana ATCC 74040 21 days Effects at lowest concentration

species: tested. (Mortality of adults)
Daphnia magna

Effects on algae: Pseudokirchneriella subcapitata; Naturalis-L; 3 d;

(growth, growth rate, capacity to ErC50: > 100 mg/L;
recover) EbC50: > 100 mg/L;
(OECD IIM 8.4 & IIIM 10.2) NOEbC: < 100 mg/L;
NOErC: > = 100 mg/L
100 mg corresponds to 2.3 x 106 CFU/L or 7.16 mg as/L
Effects on aquatic plants Not performed, not required
(OECD IIM 8.5 & IIIM 10.2)

EFSA Journal 2013;11(1):3031 28

Effects on bees (OECD IIM 8.7 & IIIM 10.3)

Application rate Crop Route Toxicity, infectivity and pathogenicity
(kg MPCA/ha) (endpoint, value or other description of
effects)
Laboratory Tests
Tomato, N/S- one time contact, Averaged survival rates:
Europe (field) 30-day continuous 92 % in the untreated control
dietary exposure 78 % in the attenuated control
78 % among treated honeybees
Laboratory Tests (bumblebees)
approx. 2.88 x 107 Tomato, N/S- dermal contact, LC50 > 3.45 x 107 CFU/L
CFU/L Europe (field) orally via sugar
water, orally via
pollen

Effects on terrestrial arthropods other than bees (OECD IIM 8.8 & IIIM 10.4)
Species Stage Test Dose Toxicity, infectivity and
Substance kg pathogenicity
MPCA/ha) (endpoint, value or other description
of effects)
Laboratory Tests
Aphidius adult Naturalis- 5.6, 11.6, LR50: 68.9 g MPCA/ha (0.997 L
rhopalosiphi L 24, 50 and Naturalis-L/ha)
104 g Corresponding to 2.3 x 1010 CFU/ha
a.s./ha
NOEC: 24 g MPCA/ha (0.347 L
(0.081,
Naturalis-L/ha)
0.168,
0.347, 0.724
and 1.505 L
product/ha)
Typhlodromus adult Naturalis- 50, 104, LR50: 402.1 g a.s./ha, equivalent to
pyri L 216, 450 5.819 L Naturalis-L/ha (Mortality)
and 934 g Corresponding to 1.34 x 1011 CFU/ha
a.s./ha,
ER50: 81 g a.s./ha; equivalent to 1.1
equivalent
L Naturalis-L/ha (Fecundity)
to 0.723,
1.505, Corresponding to 2.53 x 1010 CFU/ha
3.125, 6.512 NOEC: 50 g MPCA/ha (0.723 L
and 13.515 Naturalis-L/ha)
L Naturalis-
L/ha
Extended Laboratory Tests
Aphidius adult Naturalis- 50, 104, LR50: > 13.66 L/ha (> 1000 g
rhopalosiphi L 221, 470 MPCA/ha) (Mortality);
and 1000 g ER50: > 13.66 L/ha (> 1000 g
a.s./ha, MPCA./ha) (Reproduction)
correspondi
Corresponding to > 3.14 x 1011
ng to 0.698,
CFU/ha

EFSA Journal 2013;11(1):3031 29

1.453, NOEC: 13,66 L/ha (> 1000 g

3.087, 6.564 MPCA/ha)
and 13.966
L
product/ha

Effects on other terrestrial invertebrates (OECD IIM 8.9, 8.9.1, 8.9.2 & IIIM 10.5)
Toxicity, infectivity and LC50 (Mortality): > 1000 mg Naturalis-L/kg artificial soil
pathogenicity: > 2.3 x 107 CFU/kg artificial soil (Eisenia foetida)
(endpoint, value or other
description of effects)

Effects on soil microorganisms (OECD IIM 8.10 & IIIM 10.6)

1.29 and 12.87 mg Naturalis-L/kg soil dry weight, corresponding to 1 and 10 L Naturalis-L/ha or
0.092 and 0.92 mg as/kg or 2.98 x 104 and 29.8 x 104 CFU/kg, silty sand soil (pH 6.70)
short-term respiration:
Deviation from control after 28 days < 25% in both concentrations
nitrogen turnover:
Deviation from control in the lower concentration after 28 days: < 25%
Deviation from control at highest concentration after 28 days: > 25%
Deviation from control at highest concentration after 56 days: < 25

Additional studies (OECD IIM 8.11 & IIIM 10.7)

No data available for the biological methods of sewage treatment plants. Data gap was identified for
the biological methods of sewage treatment plants for the greenhouse use.

EFSA Journal 2013;11(1):3031 30

Appendix B – list of end points for the active substance Beauveria bassiana strain GHA and the
representative formulation
Chapter 1: Identity, Biological properties, Details of uses, Further information, and
Proposed Classification and Labelling

Active micro-organism: Beauveria bassiana (Balsamo) Vuillemin

Function (e.g. control of fungi): Control of insects

Rapporteur Member State Germany

Identity of the Microbial Pest control Agent / Active substance (OECD data point IIM 1)
Name of the organism: Beauveria bassiana (Balsamo) Vuillemin
Taxonomy: Phylum: Deuteromycota, Class: Hyphomycetes, Order:
Moniliales, Family: Moniliaceae, Genus: Beauveria
Species, subspecies, strain: B. bassiana strain GHA
Identification / detection: The organism has been identified according to the taxonomic
descriptions of de Hoog 1972 (in particular conidiospore
morphology) analysed by standard laboratory microscopy.
Open for specific method.
Culture collection: B. bassiana strain GHA is maintained in the American Culture
Collection under ATCC 74250.
Minimum and maximum concentration Nominal (mean) purity: 1.4 x 1011 viable CFU/g in the
of the MPCA used for manufacturing of technical material.
the formulated product (viable granules; Acceptable range: 1.24 x 1011 to 1.47 x 1011 viable CFU/g
g/kg):
Identity and content of relevant none
impurities, additives, contaminating
organisms in the technical grade of
MPCA:
Is the MCPA genetically modified; if so Not applicable
provide type of modification

Biological properties of the micro-organism (OECD data point IIM 2)

Origin and natural occurrence, Beauveria bassiana strain GHA is an entomopathogenic
Background level: fungus which is a common pathogen of many insect species.
It is a ubiquitous and cosmopolitan fungus, found in a variety
of soils from a broad range of climatic conditions in most
countries.
Target organism(s): Whiteflies (Aleurodidae), thrips (Thysanoptera) and aphids
(Aphididae).
Mode of action: The conidia of B. bassiana strain GHA adhere to the insect
cuticle by means of hydrophobic interaction between the
spore wall and epicuticle lipids. The conidia germinate, and
the germ tube penetrates the cuticle, using a specific series of
enzymes, which in turn degrade the lipids, protein and chitin
in the insect cuticle. In the insect body, the fungus multiplies

EFSA Journal 2013;11(1):3031 31

in the haemocœl as a blastospore, or yeast-like cell, and

enzymes begin to destroy the internal structures of the host
insect causing morbidity within 36 - 72 hours. Reduced
feeding and immobility are rapidly evident, and the insect
dies within between 4 to 10 days post-infection. The time to
death will depend on the insect species, age and conidial
dose. After death, the blastospores transform into mycelia,
which emerge through the cuticle and form spores. These
cover the cadaver as a characteristic white growth.
Sporulation occurs only in conditions of high humidity.
Host specificity: B. bassiana is specific to insects. Not pathogenic to humans
or plants.
Life cycle: The conidia adhere to the insect cuticle, germinate and
penetrate in the insect body, where they replicate as yeast-
like cells (blastospores) and destroy the internal structures,
causing morbidity within 36 - 72 hours. After death of the
insect, the blastospores transform into mycelia, which
emerge through the cuticle and form spores.
Infectivity, dispersal and colonisation B. bassiana strain GHA has an optimal growth temperature
ability: range between 23ºC and 28ºC. The conidia are not able to
germinate or grow at ≥36ºC. B. bassiana strain GHA is
stable in water at pH 5, 7 and 9 and has been shown to be
unaffected when exposed to metallic ions (Na, Ca, Fe, Mg).
Detectable effects have only been noted when the fungus is
exposed to copper, which is a known fungicide. Under
natural conditions, the conidia of B. bassiana germinate and
die in the absence of a suitable host insect within two days,
and in aqueous environments. The conidia survive naturally
in sheltered habitats and require specific environmental
conditions of moderate temperature, high humidity and high
insect population density for epizootic spread and dispersal.
Relationships to known plant, animal Beauveria bassiana strain GHA is not related to any known
or human pathogens: human or plant pathogen.
Genetic stability: During more than 10 years of commercial use and
production, no change has occurred in phenotypic
characteristics or RFLP cDNA fingerprint of B. bassiana
strain GHA.
Information on the production of Beauvericin: max 5 mg/kg
relevant metabolites (especially Open for further studies.
toxins):
Resistance/ sensitivity to antibiotics / No specific information addressing this issue has been
anti-microbial agents used in human or submitted by the notifier or could be found in the open
veterinary medicine: literature. However, because spread of resistance (genes) to
antimycotic agents between fungal species is not of that
concern as for bacteria with regard to antibiotics, and since
Beauveria bassiana itself is only an opportunistic pathogen
that has very rarely caused infections in humans and is not
related to any known human pathogen, this lack of
information is not considered a critical data gap.

EFSA Journal 2013;11(1):3031 32

Summary of uses supported by available data (Beauveria bassiana strain GHA)

Crop and/ Member Product F Pests or Group of Preparation Application Application rate per treatment PHI Remarks
or State or name G pests controlled (days)
Type Conc. of Method Growth Number Interval Kg water Kg MPCA/ha
situation Country or I
MPCA Kind stage & between MPCA/hL L/ha Viable
season min max applications granules
(min) MPCA/ha
(a) (b) (c) (l) (m)
(d-f) (i) (f-h) (j) (k) min min min max min max
Ornament Northern BotaniGar G, I Sucking insects WP 220 g/kg High Maturity 3-5 5-7 days 0.14 - 4.84 500 - 0.014 – 0 MPCP
als and d 22 WP volume (BBCH89) x 1012 2000 0.121 0.0625-
Southern 4.4 x 1013 Spraying CFU // 0.55
Europe CFU/kg 0.28-2.42 Kg/ha
13
x 10 CFU
Tomatoes, Northern BotaniGar G, I Sucking insects WP 220 g/kg High Ripeness 3-5 5-7 days 0.14 - 4.84 500 - 0.014 – 0 MPCP
Cucumber and d 22 WP volume (BBCH89) x 1012 2000 0.121 0.0625-
13
s Southern 4.4 x 10 Spraying CFU // 0.55
Europe CFU/kg 0.28-2.42 Kg/ha
x 1013 CFU

(a) For crops, the EU and codex classifications (both) should be used; where relevant, the situation should be (h) Kind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plant - type of equipment
described (e.g. fumigation of a structure) used must be indicated
(b) Outdoor or field use (F), glasshouse application (G) or indoor application (I) (i) viable granules = colony forming units and g/kg or g/L
(c) e.g. biting and sucking insects, soil born insects, foliar fungi, weeds (j) Growth stage at last treatment (BBCH Monograph, growth stages of Plants, 1997, Blackwell ISBN 3-
(d) e.g. wettable powder (WP), emusifiable concentrate (EC), granule (GR) 8263-3152-4), including where relevant, information on season at time of application
(e) GCPF codes - Crop Life Technical Monograph No 2, 1989 (k) Indicate the minimum and maximum number of application possible under practical conditions of use
(f) All abbreviations used must be explained (l) PHI - minimum pre-harvest interval
(g) Method, e.g. high volume spraying, spreading, dusting, drench (m) Remarks may include: Extent of use/economic importance/restrictions

EFSA Journal 2013;11(1):3031 33

Classification and proposed labelling (Symbol, Indication of danger, Risk phrases, Safety phrases)
with regard to physical/chemical data: none
with regard to toxicological data: R42 – May cause sensitisation by inhalation
R43 – May cause sensitisation by skin contact
with regard fate and behaviour: Not applicable
with regard to ecotoxicological data: Not applicable

Chapter 2: Methods of analysis

Analytical methods for the microorganism (OECD data point IIM 4.2, 4.3 and IIIM 5.3)
manufactured micro-organism (principal Analytical methods for the identification of the technical B.
of method): bassiana strain GHA include the following:
- Characterisation by microscopic examination of the visual
morphology
- Restriction Fragment Length Polymorphism (RFLP) profile
analysis of chromosomal DNA (cDNA) and Random
Amplified Polymorphic DNA analysis (RAPD)
Determination of fungal spore-count and spore viability by
haemocytometer counts and in vitro germination assay.
Open for validation
Impurities and contaminating micro- Enumeration of microbial contaminants (bacteria and fungi),
organisms in manufactured material determining the presence/absence of enteric bacteria such as
(principal of method): Shigella, Salmonella and Vibrio spp. by plating out on selective
media.
The method for beauvericin is not sufficiently validated.
Moisture analyser balance.
Open for contaminating microorganisms and toxins
Microbial Pest control product (principle Analytical methods for the formulation are as used for the
of method): active substance.

Analytical methods for residues (viable and non-viable) in exposed compartments and organisms
(OECD data point IIM 4.5)
of the active micro-organism (principle Validated methods are not required and not available.
of method) Nonetheless one method is validated for water with an LOQ of
1 x 106 CFU/L.
Of relevant metabolites (principle of None relevant
method):

EFSA Journal 2013;11(1):3031 34

Chapter 3: Impact on Human and Animal Health

Medical data: No adverse effects in employees who had been in occupational
(including medical surveillance on contact with strain GHA. For Beauveria bassiana in general,
manufacturing plant personnel) few cases of local (eye) and systemic infections were reported
(OECD data point IIM 5.1, 5.2) with the latter occurring only in immunocompromised or
severely ill patients. Evidence of allergenicity by respiratory or
skin contact.
Sensitisation: Microorganisms may have the potential to provoke sensitising
(OECD data point IIM 5.3.1 & IIIM reactions.
7.1.6) Evidence of allergenicity by inhalation in laboratory animals
and humans and indications of allergic skin reactions in
humans. Buehler test in Guinea pigs negative for the micro-
organism but positive for the formulation BotaniGard 22 WP.
Acute oral infectivity, toxicity and LD50 > 1 x 108 CFU/animal in rats; no evidence of toxicity,
pathogenicity: pathogenicity or infectivity; rapid clearance (by day 3 post-
(OECD data point IIM 5.3.2 & IIIM dosing)
7.1.1)
Acute intratracheal/inhalation toxicity, LD50 >1 x 108 CFU/animal in rats after intratracheal
pathogenicity and infectivity: application; no evidence of infectivity but local effects on the
(OECD data point IIM 5.3.3 & IIIM lungs (inflammation/immune reaction, organ weight increase),
7.1.3) transient reduction in body weight gain; clearance complete by
day 7 post-dosing
Acute intravenous/intraperitoneal LD50 > 1 x 107 CFU in rats; no evidence of toxicity,
toxicity, pathogenicity and infectivity: pathogenicity or infectivity; rapid clearance (by day 3 post-
(OECD data point IIM 5.3.4 & IIIM dosing)
7.1.2)
Genotoxicity: Negative in the Ames test
(OECD data point IIM 5.3.5)
Cell culture study: Not available, not required
(OECD data point IIM 5.3.6)
Information on short-term toxicity and Not available, not required
pathogenicity:
(OECD data point IIM 5.3.7)
Dermal toxicity: Acute dermal study in rabbits: LD50 > 1.6x 1011 CFU; no
(OECD data point IIIM 7.1.2) systemic effects but signs of slight but persisting local
irritation.
Specific-toxicity, pathogenicity and Acute dermal study in rabbits: LD50 > 1.6x 1011 CFU; no
infectivity: systemic effects but signs of slight but persisting local
(OECD data point IIM 5.5) irritation;
Certain potential of the micro-organism (strain not specified) to
damage the cornea confirmed in rabbits;
Long-term inhalative exposure to B. bassiana (strain not
specified) caused allergic lung reactions in rats and mice.

EFSA Journal 2013;11(1):3031 35

Genotoxicity – in vivo studies in germ Not available, not required

cells:
(OECD data point IIM 5.5.3)
Metabolites Toxicological studies with strain GHA including a beauvericin
and bassianolide contamination each with of approx. 50 mg/kg
indicated no additional adverse effects after oral, dermal or ip
administration. Effects seen after intratracheal administration
are unlikely to be due to beauvericin and bassianolide.
Beauvericin was not mutagenic in an Ames test.
Exposure to (the sum of) the metabolites should stay below
1.5 µg/d (threshold of toxicological concern).

Reference values
AOEL There is no need to set an AOEL as the
microorganism was not shown to be pathogenic
or infective based on the available data and
studies
ARfD There is no need to set an ARfD as the
microorganism was not shown to be pathogenic
or infective based on the available data and
studies
ADI There is no need to set an ADI as the
microorganism was not shown to be pathogenic
or infective based on the available data and
studies

Exposure (operator, workers, Operator

bystander, consumer): Not finalised (lack of information on toxin/metabolites
(OECD data point IIM 6.1, IIIM 7.2, 7.3 production)
and 8.0)
Bystander
Bystander exposure is not relevant for the representative uses
(greenhouse).

Worker
Not finalised (lack of information on toxin/metabolites
production).

EFSA Journal 2013;11(1):3031 36

Chapter 4: Residues
Open for toxins/secondary metabolites

Chapter 5: Fate and Behaviour in the Environment (OECD IIM 7 & IIIM 9)
Persistence and multiplication in soil, In Soil:
water and air: Data gap
The available data on measurement or observation of
persistence and multiplication in soil were insufficient.
In Water:
In an aquatic environment spores will be subject to
sedimentation and can be expected to degrade rapidly in natural
water bodies. The conidia did not germinate in different tested
aqueous systems (distilled water, and aqueous suspensions at
pH 5, 7 and 9, or in metal ion solutions in the presence of
bacteria).
In Air:
In the absence of a specific host insect, viability of conidia of
B. bassiana in the air or epigeal habitats is mainly affected by
the UV-B portion of the solar spectrum. On the basis of the
persistence data, significant degradation of B. bassiana conidia
on leaves under field conditions may occur in less than 2 days.
Mobility In soil:
Conidia of B. bassiana are not very mobile in soil and
generally remain on the surface of the soil. No groundwater
risk assessment is necessary since B. bassiana is neither
pathogenic nor toxic to humans.
In water:
The globose or ovoid shape of conidia is not suitable for
efficient transport in water.
In air:
Beauveria bassiana is dry, of small size and it is produced in
powdery clusters. Therefore, these types of conidia can be
easily transported through the air. Also transmission of fungal
spores through the air by insects is possible.
However, in the absence of specific host insects, conidia of
Beauveria bassiana will not persist in the sun-exposed
phylloplane in significant amount for more than 2 days.

EFSA Journal 2013;11(1):3031 37

Chapter 6: Effects on non-target organisms (OECD IIM 8 & IIIM 10)

Effects on birds (OECD IIM 8.1 & IIIM 10.1)
Application rate Crop Category Time-scale Toxicity, infectivity and
(kg MPCA/ha) (e.g. pathogenicity
insectivorous (endpoint, value or other
bird) description of effects)
0.121 Ornamentals Insectivorous 5 days LD50 (Falco sparverius) > 2.5 x
(corresponding , Tomatoes, bird dosing / 18 1010 conidia/kg body weight (no
to 2.42x1013 Cucumbers days evidence of pathogenicity)
CFU/ha) observation

Effects on Mammals
Application rate Crop Category Time-scale Toxicity, infectivity and
(kg MPCA/ha) (e.g. pathogenicity
insectivorous (endpoint, value or other
bird) description of effects)
tomatoes and ornamentals, rat acute LD50 > 5000 mg BotaniGard
cucumbers, tomatoes and 22WP/ kg bw corresponding to >
indoor cucumbers, 2.2 x 1014 CFU/ kg bw (product
indoor containing 4.4 x 1013 CFU/kg)
rat acute Beauveria bassiana strain GHA
was not toxic, pathogenic, or
infective to male and female rats at
1.05 x 108 CFU and 1 x 108 CFU
respectively.

Effects on aquatic organisms (OECD IIM 8.2, 8.3 & IIIM 10.2)
Group Test substance Time-scale Toxicity, infectivity and
pathogenicity
(endpoint, value or other
description of effects)
Laboratory tests
Fish species: Beauveria bassiana GHA 31 days NOEC < 7.5 x 108 CFU/L
Pimephales (effects on growth < 25 %; no
promelas evidence of infectivity or
pathogenicity)
Invertebrate species: Beauveria bassiana GHA 21 days NOEC = 4.7 x108 CFU/L
Daphnia magna (sublethal effects; no evidence of
infectivity or pathogenicity)

Effects on algae: Selenastrum capricornutum, 3 days, static, nominal

(growth, growth rate, capacity to EbC50 114000 µg/L (0.98 x 1013 CFU/L)
recover)
ErC50 237000 µg/L (2.04 x 1013 CFU/L)
(OECD IIM 8.4 & IIIM 10.2)
NOEC 75000 µg/L (2.64 x 1013 CFU/L) (all parameters)
Effects on aquatic plants not performed, not required
(OECD IIM 8.5 & IIIM 10.2)

EFSA Journal 2013;11(1):3031 38

Effects on bees (OECD IIM 8.7 & IIIM 10.3)

Application rate Crop Route Toxicity, infectivity and pathogenicity
(kg MPCA/ha) (endpoint, value or other description of
effects)
No data available
Study under practical conditions (honey bees)
Laboratory Test (bumblebees)

Effects on terrestrial arthropods other than bees (OECD IIM 8.8 & IIIM 10.4)
Species Stage Test Dose Toxicity, infectivity and pathogenicity
Substance kg (endpoint, value or other description of
MPCA/ha) effects)
Laboratory Tests
Aphidius adult BotaniGar 0.947 (1.33 Mortality: -5 %
rhopalosiphi d 22 WP x 1014 Reproduction: 20 %
CFU/ha) Mortality: -5 %
BotaniGar 1.695 Reproduction: 20 %
d ES
Typhlodromus pyri protonymp BotaniGar 0.947 (1.33 Mortality: 4 %
h d 22 WP x 1014 Reproduction: 43 %
CFU/ha) Mortality: -4 %
BotaniGar 1.695 Reproduction: 34 %
d ES
Orius laevigatus nymph BotaniGar 0.952 (1.33 Mortality: 3 %
d 22 WP x 1014 Reproduction: 23 %
CFU/ha) Hatching succ: 24 %
Mortality: 11 %
BotaniGar 1.693 Reproduction: -7 %
d ES Hatching succ: 29 %
Pardosa spec. adult BotaniGar No sufficient Mortality: 80 %
d 22 WP information

BotaniGar No sufficient Mortality: 45 %

d ES information
Further information
Xylocoris flavipes nymphs Beauveria Filter paper: 0, 16, 41 % infection after 10 days,
bassiana 2.5 x 1013, respectively.
GHA 2.5 x 1014,
2.5 x 1015
CFU/ha
Tenebrio molitor adult Beauveria 2 x 104 CFU, No significant mortality (100 %
bassiana 2.4 x 108 mortality of target organism
GHA CFU Melanoplus sanguinipes at 2 x 104
conidia/ha)

EFSA Journal 2013;11(1):3031 39

Effects on other terrestrial invertebrates (OECD IIM 8.9, 8.9.1 & IIIM 10.5)
Toxicity, infectivity and LC50 (Mortality) : > 1000 mg Beauveria bassiana strain GHA /kg
pathogenicity: artificial soil
(endpoint, value or other description > 8.6 x 1010 CFU/kg artificial soil (Eisenia foetida)
of effects)

Effects on non-target soil micro-organisms (OECD IIM 8.10 & IIIM 10.6)
No studies submitted

Additional studies (OECD IIM 8.11 & IIIM 10.7)

No data available for the biological methods of sewage treatment plants, data gap identified.

EFSA Journal 2013;11(1):3031 40

ABBREVIATIONS

1/n slope of Freundlich isotherm

λ wavelength
decadic molar extinction coefficient
°C degree Celsius (centigrade)
µg microgram
µm micrometer (micron)
a.s. active substance
AChE acetylcholinesterase
ADE actual dermal exposure
ADI acceptable daily intake
AF assessment factor
AOEL acceptable operator exposure level
AP alkaline phosphatase
AR applied radioactivity
ARfD acute reference dose
AST aspartate aminotransferase (SGOT)
AV avoidance factor
BCF bioconcentration factor
BUN blood urea nitrogen
bw body weight
CAS Chemical Abstracts Service
CFU colony forming units
ChE cholinesterase
CI confidence interval
CIPAC Collaborative International Pesticides Analytical Council Limited
CL confidence limits
cm centimetre
d day
DAA days after application
DAR draft assessment report
DAT days after treatment
DM dry matter
DT50 period required for 50 percent disappearance (define method of estimation)
DT90 period required for 90 percent disappearance (define method of estimation)
dw dry weight
EbC50 effective concentration (biomass)
EC50 effective concentration
ECHA European Chemical Agency
EEC European Economic Community
EINECS European Inventory of Existing Commercial Chemical Substances
ELINCS European List of New Chemical Substances
EMDI estimated maximum daily intake
ER50 emergence rate/effective rate, median
ErC50 effective concentration (growth rate)
EU European Union
EUROPOEM European Predictive Operator Exposure Model
f(twa) time weighted average factor
FAO Food and Agriculture Organisation of the United Nations
fd feed
FIR Food intake rate
FOB functional observation battery
FOCUS Forum for the Co-ordination of Pesticide Fate Models and their Use
g gram

EFSA Journal 2013;11(1):3031 41

GAP good agricultural practice

GC gas chromatography
GCPF Global Crop Protection Federation (formerly known as GIFAP)
GGT gamma glutamyl transferase
GM geometric mean
GS growth stage
GSH glutathion
h hour(s)
ha hectare
Hb haemoglobin
Hct haematocrit
hL hectolitre
HPLC high pressure liquid chromatography
or high performance liquid chromatography
HPLC-MS high pressure liquid chromatography – mass spectrometry
HQ hazard quotient
IEDI international estimated daily intake
IESTI international estimated short-term intake
ISO International Organisation for Standardisation
IUPAC International Union of Pure and Applied Chemistry
JMPR Joint Meeting on the FAO Panel of Experts on Pesticide Residues in Food and
the Environment and the WHO Expert Group on Pesticide Residues (Joint
Meeting on Pesticide Residues)
Kdoc organic carbon linear adsorption coefficient
kg kilogram
KFoc Freundlich organic carbon adsorption coefficient
L litre
LC liquid chromatography
LC50 lethal concentration, median
LC-MS liquid chromatography-mass spectrometry
LC-MS-MS liquid chromatography with tandem mass spectrometry
LD50 lethal dose, median; dosis letalis media
LDH lactate dehydrogenase
LOAEL lowest observable adverse effect level
LOD limit of detection
LOQ limit of quantification (determination)
m metre
M/L mixing and loading
MAF multiple application factor
MATC maximum allowable toxicant concentration
MCH mean corpuscular haemoglobin
MCHC mean corpuscular haemoglobin concentration
MCV mean corpuscular volume
mg milligram
mL millilitre
mm millimetre
mN milli-newton
MPCA microbial pest control agent
MPCP microbial pest control product
MRL maximum residue limit or level
MS mass spectrometry
MSDS material safety data sheet
MTD maximum tolerated dose
MWHC maximum water holding capacity
NESTI national estimated short-term intake

EFSA Journal 2013;11(1):3031 42

ng nanogram
NOAEC no observed adverse effect concentration
NOAEL no observed adverse effect level
NOEC no observed effect concentration
NOEL no observed effect level
OD oil dispersion
OECD Organisation for Economic Co-operation and Development
OM organic matter content
Pa pascal
PD proportion of different food types
PEC predicted environmental concentration
PECair predicted environmental concentration in air
PECgw predicted environmental concentration in ground water
PECsed predicted environmental concentration in sediment
PECsoil predicted environmental concentration in soil
PECsw predicted environmental concentration in surface water
pH pH-value
PHED pesticide handler's exposure data
PHI pre-harvest interval
PIE potential inhalation exposure
pKa negative logarithm (to the base 10) of the dissociation constant
Pow partition coefficient between n-octanol and water
PPE personal protective equipment
ppm parts per million (10-6)
ppp plant protection product
PT proportion of diet obtained in the treated area
PTT partial thromboplastin time
QSAR quantitative structure-activity relationship
r2 coefficient of determination
RAPD random amplification of polymorphic DNA
REACH Registration, Evaluation, Authorisation of CHemicals
RPE respiratory protective equipment
RUD residue per unit dose
SC suspension concentrate
SD standard deviation
SFO single first-order
SSD species sensitivity distribution
STMR supervised trials median residue
t1/2 half-life (define method of estimation)
TER toxicity exposure ratio
TERA toxicity exposure ratio for acute exposure
TERLT toxicity exposure ratio following chronic exposure
TERST toxicity exposure ratio following repeated exposure
TK technical concentrate
TLV threshold limit value
TMDI theoretical maximum daily intake
TRR total radioactive residue
TSH thyroid stimulating hormone (thyrotropin)
TWA time weighted average
UDS unscheduled DNA synthesis
UV ultraviolet
W/S water/sediment
w/v weight per volume
w/w weight per weight
WBC white blood cell

EFSA Journal 2013;11(1):3031 43

44
strains ATCC-74040 and GHA
Peer Review of the pesticide risk assessment of the active substance Beauveria bassiana

World Health Organisation

water dispersible granule

EFSA Journal 2013;11(1):3031

week
year
WHO
WG

wk
yr

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