DECISION

14 January 2016 1. Summary

Substance MYCOTAL WG

Application code APP202247

Application type To import or manufacture for release any hazardous substance under Section 28 of the Hazardous Substances and New Organisms Act 1996 (“the Act”)

Applicant New Zealand Gourmet Limited

Purpose of the application To seek approval to import MYCOTAL WG, a microbial pest control agent containing the spores of the entomopathogenic fungus Lecanicillium muscarium 19-79 strain, for the control of whitefly in greenhouse crops

Date application received 13 April 2015

Consideration date 15 December 2015 Further information was requested from the applicant during the evaluation and review of the application in accordance with sections 52 and 58 of the Act and consequently the consideration was postponed in accordance with section 59 of the Act

Considered by The Chief Executive1 of the Environmental Protection Authority (“the EPA”)

Decision Approved with controls

Approval code HSR101089

Hazard classifications 6.5A, 6.5B, 9.1D

1 The Chief Executive of the EPA has made the decision on this application under delegated authority in accordance with section 19 of the Act.

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Decision on application for approval to import or manufacture Mycotal WG for release (APP202247) 2. Background

2.1. MYCOTAL WG is intended for use as a microbial pest control agent (MPCA) to control whitefly in greenhouse crops. It is a water dispersible granule (WDG) formulation containing spores of the fungus Lecanicillium muscarium 19-79 strain.

2.2. The applicant intends to import MYCOTAL WG into New Zealand fully formulated, packed and labelled in 500 g and 1 kg polyethylene bags in fibreboard containers.

2.3. MYCOTAL WG will be mixed with water at a rate of up to 1 g of MYCOTAL WG per litre of water and applied at application rates of up to 3 kg of MYCOTAL WG per hectare.

2.4. It is intended that the substance should be used completely i.e. until the container is emptied, to avoid a requirement for disposal.

3. Process, consultation and reasons for non-notification

3.1. The application was lodged pursuant to section 28 of the Act.

3.2. WorkSafe New Zealand, the Ministry for Primary Industries (ACVM group), and the Ministry for the Environment were advised of the application and invited to comment. No comments were received.

3.3. The applicant requested that the application be considered as an application for rapid assessment under section 28A of the Act.

3.4. Lecanicillium muscarium is not a new organism and therefore not regulated under the New Organisms provisions of the Act. However, the 19-79 strain of L. muscarium has not been used as a pesticide active ingredient in New Zealand previously. Other strains of L. muscarium already exist in New Zealand and similar substances containing other strains of this fungus have been approved for use as microbial pest control agents. It was considered that this substance should be evaluated by a full assessment and determined under section 29 of the Act, rather than by rapid assessment, in order to evaluate the risks of this new pesticide active ingredient.

3.5. It was considered that the application would not be of significant public interest as other strains of L. muscarium already exist in New Zealand, and similar substances containing other strains of this fungus have previously been approved for use as microbial pest control agents. Therefore the application was not publicly notified in accordance with section 53(2) of the Act.

4. Hazardous properties

4.1. The staff determined the hazard classification of MYCOTAL WG based on the information provided by the applicant and other available information as documented in Appendix B.

4.2. The classifications determined by the staff are different to those submitted by the applicant (Table 1). The classification determined by the applicant was based on advice provided to the applicant by the staff prior to this application; however, the formulation of MYCOTAL WG has changed since that advice was provided. The hazard classifications of the active ingredient have been reviewed based on

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the data submitted in this application, and as a result the staff determined that classification for respiratory sensitisation is appropriate for MYCOTAL WG.

Table 1 Hazard classifications of MYCOTAL WG as proposed by the applicant and the staff Hazard Endpoint Applicant classification EPA classification

Respiratory sensitisation - 6.5A Contact sensitisation 6.5B 6.5B Aquatic ecotoxicity 9.1D (from biocidal use) 9.1D (from biocidal use)

5. Assessment of risks, costs and benefits

Human health risk assessment

5.1. The currently available exposure models for pesticides are not appropriate for microorganisms, because the quality of the technical grade active ingredient is not related to the concentration or quantity of material that may be present for exposure assessment. This is due to the ability of microbial substances to reproduce very rapidly under favourable conditions. Additionally, L. muscarium 19-79 does not elicit any signs of toxicity, infectivity, or pathogenicity. As a result, a quantitative risk assessment of operator, worker and bystander exposure was not performed.

5.2. The staff have advised me that the level of risk from the use of MYCOTAL WG is expected to be negligible, for the following reasons:

 MYCOTAL WG exhibits a lack of toxicity, infectivity, and pathogenicity associated with L. muscarium 19-79.  Risks associated with sensitisation, through respiratory or contact routes, are considered to be negligible provided requirements imposed by the default HSNO controls are complied with. The default controls are discussed in section 6 below.

5.3. A qualitative assessment of the risks associated with the sensitisation hazards of MYCOTAL WG is set out in Table 2. Environmental risk assessment

5.4. As Mycotal WG contains a new active ingredient, L. muscarium 19-79, a full quantitative assessment of the ecotoxicological risks from the use of MYCOTAL WG was carried out. This quantitative risk assessment is detailed in Appendix F.

5.5. The staff identified several data gaps during the environmental risk assessment for Mycotal WG. No studies were provided with Mycotal WG. Studies were provided for a different formulation. In the reports provided by the applicant, information regarding the strain used, the formulation, and the test conditions were not able to be fully determined. There was insufficient information to conduct a thorough quantitative risk assessment of use the substance, in particular an assessment of

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environmental fate and behaviour, and effects to non-target species were not able to be quantitatively assessed.

5.6. The staff determined that for permanently covered structures, the risks to the environment are below the level of concern. However, for non-enclosed structures or structures that could be partly open during the application of Mycotal WG high risks were identified for birds.

5.7. The risks to non-target arthropods could not be fully assessed due to the lack of data and information.

5.8. Based on a qualitative assessment of the risks to the environment from the non-use phases of the lifecycle of MYCOTAL WG (import/manufacture, transport, storage, and disposal) the staff have advised me that risks during these lifecycle phases would be negligible due to the low hazard profile, and the very low likelihood of a significant environmental exposure. Relationship of Māori to the environment

5.9. The potential effects of MYCOTAL WG on the relationship of Māori to the environment have been assessed in accordance with sections 5(b), 6(d), and 8 of the Act. Under these sections all persons exercising functions, power and duties under this Act shall recognise and provide for the maintenance and enhancement of people and communities to provide for their cultural wellbeing; and take into account their culture and traditions with their ancestral lands, water, taonga and the principles of the Treaty of Waitangi (Te Tiriti o Waitangi).

5.10. The staff have advised me that MYCOTAL WG triggers a number of hazardous properties giving rise to the potential for cultural risk e.g. contact and respiratory sensitisation, and use as a biocide. Cultural risk includes the deterioration of the mauri of taonga flora and fauna species, the environment and the general health and well-being of individuals and the community.

5.11. In general, the introduction and use of hazardous substances has the potential to inhibit the ability of Māori to fulfil their role as kaitiaki. This is particularly in relation to the guardianship of waterways, given the ecotoxic nature of some substances to aquatic species, and potential risks to the mauri ora of human health under prolonged exposure to some substances.

5.12. The main concerns relating to this application for Māori are likely to be in relation to dangers to birds, as a result of the quantitative risk assessment showing the potential for non-negligible risks to birds. Maori traditional mātauranga systems value New Zealand’s native birds as taonga. Māori attribute cultural and spiritual values to birds, including their importance in folklore, art, ornamentation and textiles, in addition to some birds being considered traditionally cherished cuisine. Therefore there is the potential for cultural risk arising from harm to birds.

5.13. There is also uncertainty regarding the effects of Mycotal WG due to the data gaps in the information available for the ecotoxicological assessment, and therefore some potential for cultural risk arising from harm to species not fully assessed.

5.14. If MYCOTAL WG is applied in the prescribed manner, it is considered that it is not likely to breach the principles of the Treaty of Waitangi, in particular the principle of active protection.

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Assessment of risks to society and the community and the market economy

5.15. The staff did not identify any risks associated with the approval of MYCOTAL WG to society, communities or the market economy.

5.16. There are not expected to be any significant adverse impacts on the social environment with the controlled use of MYCOTAL WG, apart from the health effects and environmental effects already discussed. Consequently, I consider that this aspect of potential risk need not be considered further. New Zealand’s international obligations

5.17. The staff did not identify international obligations that may be impacted by the approval of MYCOTAL WG. Overall assessment of risks

5.18. I consider that the risks to human health and the environment from the non-use phases of the lifecycle of MYCOTAL WG are negligible.

5.19. I note that the operator exposures to MYCOTAL WG during mixing, loading, application and re-entry are considered to be acceptable, provided certain levels of PPE and RPE are used.

5.20. Having considered the results of the risk assessment of MYCOTAL WG, I note that there are a number of areas where there could be non-negligible risks. These areas of risk include:

 Risks of adverse effects to birds from use of MYCOTAL WG  Potential for cultural risk to Māori as a result of the risks to birds  Potential for other unmanaged risks given the data gaps and uncertainty in the risk assessment.

5.21. The controls applied to MYCOTAL WG must therefore address these risks.

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Table 2: Qualitative assessment of human health risks Lifecycle Description Likelihood Magnitude Risk Comment Residual according Risk to Matrix

Manufacture Contact and respiratory Highly Minor to Negligible Manufacturing and packaging facilities in New Zealand will be required Negligible and packaging sensitisation improbable moderate to meet the HSNO requirements for equipment, emergency depending management and Personal Protective Equipment (PPE). The need for on sensitivity compliance with HSNO information provisions for the constituent of individual components (e.g. labels, Safety Data Sheets (SDSs)), and WorkSafe New Zealand’s Health and Safety requirements will also apply. This means that workers handling the substance will need to be aware of the hazards and the measures that need to be undertaken to ensure their own safety. Provided these measures are taken, the level of risk is negligible.

Importation, Contact and respiratory Highly Minor to Negligible Workers and bystanders will only be exposed to the substance during Negligible transport, sensitisation improbable moderate this part of the lifecycle in isolated incidents where spillage occurs, storage depending therefore only risks from acute exposure are considered here. on sensitivity of individual Compliance with HSNO controls (e.g. labels, SDS) and adherence to the Land Transport Rule 45001, Civil Aviation Act 1990 and Maritime Transport Act 1994 (as applicable) is required.

Use Contact and respiratory Highly Minor to Negligible The substance will be labelled to identify its potential risks minimising Negligible sensitisation improbable moderate the opportunity for it to cause toxicity. It is considered that if the HSNO depending requirements for PPE, packaging, identification and emergency on sensitivity management requirements are complied with, the risks to operators of individual associated with sensitisation will be negligible. While the risk to re-entry workers is considered to be negligible, additional controls, such as PPE requirements and a re-entry interval after application, have been applied to ensure that the potential risk of sensitisation arising from exposure to Mycotal WG is adequately managed.

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Lifecycle Description Likelihood Magnitude Risk Comment Residual according Risk to Matrix It is not expected that bystanders will come into contact with the substance as it is intended for commercial use in greenhouses.

Disposal Contact and respiratory Highly Minor to Negligible The applicant indicates that the preferred disposal method is to use Negligible sensitisation improbable moderate the product according to label directions. Disposal of the substance and its used containers in New Zealand, if required, shall be in accordance with the requirements of the Hazardous Substances (Disposal) Regulations 2001. Compliance with these will reduce the opportunity for individuals to be exposed.

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Identification of benefits

5.22. According to the applicant, the approval of MYCOTAL WG will provide the following benefits:

 Higher crop yields from the control of whitefly  Use of MYCOTAL WG does not result in chemical residues in fruit and produce.  The different mode of action to chemical pesticides is useful in an integrated pest management programme to manage resistance.  The substance is intended as an alternative to replace an earlier approved substance that no longer has ACVM registration.  MYCOTAL WG has a lower chemical hazard profile than many chemical pesticides, which the applicant anticipates will reduce risks to workers in glasshouses.

The effects of the substance being unavailable

5.23. The staff have advised me that the likely effects of the substance being unavailable relate to less consumer choice resulting in less competitive pricing. This is because similar microbial pest control agents are available on the New Zealand market.

Overall assessment of benefits

5.24. I consider that MYCOTAL WG has a lower chemical hazard profile than many other approved pesticide substances, and therefore may present a lower overall level of risk to human health and the environment.

5.25. I am satisfied that the availability of MYCOTAL WG will provide beneficial economic effects for some businesses and individual farmers as well as flow-on effects to local communities and the New Zealand economy. 6. Controls Default controls

6.1. Based on the hazard classification determined for MYCOTAL WG as detailed above, a set of associated default controls specified by the regulations under the Act were identified by the staff as being applicable. These default requirements form the basis of the controls proposed by the staff as detailed in Appendix A.

6.2. Included in the default controls are requirements for the integrity of packaging, for personal protective equipment (PPE) to be worn, and identification requirements designed to make people aware of the hazards associated with the substance, as well as information about situations that could lead to harm, and how harm can be avoided.

6.3. In addition to the default controls prescribed by the HSNO regulations, I note that the transport, management, and use of MYCOTAL WG will also need to comply with other legislation such as the

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Land Transport Rule 45001, the Civil Aviation Act 1990, the Maritime Transport Act 1994 and Regional Plans set under the Resource Management Act 1991 (as applicable).

The setting of exposure limits

6.4. Tolerable Exposure Limits (TELs), Acceptable Daily Exposures (ADEs) and Potential Daily Exposures (PDEs) can be set to limit hazardous substances from entering the environment in quantities sufficient to present a risk to people. No TELs, ADEs or PDEs have been set for any component of MYCOTAL WG at this time as the risk of adverse effects to human health has been qualitatively assessed as negligible, provided users demonstrate compliance with the controls as set out in Appendix A.

6.5. Workplace exposure standards (WESs) can be set to limit exposure of people to toxic substances in places of work. The EPA typically adopts WES values listed in the WorkSafe New Zealand’s Workplace Exposure Standards document (effective from February 2013):

http://www.business.govt.nz/worksafe/information-guidance/all-guidance-items/workplace-exposure- standards-and-biological-exposure-indices/workplace-exposure-standards-and-biological-indices- 2013.pdf

The staff note that no WES values have been set for any component of MYCOTAL WG by WorkSafe New Zealand; therefore no WES values are applied to any component of MYCOTAL WG at this time.

6.6. Controls E1 and E2 relate to limiting exposure to ecotoxic substances through the setting of environmental exposure limits (EELs) and the setting of maximum application rates. As Mycotal WG is intended to be used in greenhouses only, significant environmental exposure is not anticipated. As a result, these controls are deleted.

Variation and deletion of controls

6.7. The staff note that the default controls do not address the risks associated with storage or use of the substances within stationary container systems (e.g. tanks). These risks include the failure of primary containment resulting in a large spill of the substance into the environment. In addition, the default controls do not allow for dispensation where it is unnecessary for any pipework associated with the stationary container systems to have secondary containment. Accordingly, controls addressing these risks are considered more effective than the default controls in terms of their effect on the management, use and risks of the substance. The revised controls are shown in Appendix A.

6.8. The default controls include restrictions on the carriage of toxic substances on passenger service vehicles (buses, trains etc.). The existing maximum quantity of class 6.5B solid substances (0.5 kg) has been reviewed for MYCOTAL WG and an increased maximum quantity of 3 kg is recommended for a 6.5 hazard classification. This ensures that carriage of this product on a public transport vehicle is practicable. These restrictions do not apply to transport of the substances within private vehicles.

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Additional controls

6.9. I consider that in order to address the areas of residual risk identified by the risk assessments it is necessary to impose additional controls on the substance, as these controls will be more more effective than the default controls in terms of their effects on the management, use and risks of the substance.

Application and operational restrictions 6.10. MYCOTAL WG is intended to be used in greenhouses. However, application of this substance outside of the use pattern intended by the applicant could lead to a significant environmental exposure. It is therefore necessary to put restrictions on application of MYCOTAL WG to mitigate the risk of death or adverse effects to aquatic and terrestrial organisms from use outside of greenhouses. Consequently, in order to mitigate the potential risks to the environment, the following additional control is applied to MYCOTAL WG:

 This substance must only be applied within an indoor horticultural facility, such as a greenhouse2. It must not be applied outdoors.

Restrictions on application rate and frequency of application 6.11. Application of MYCOTAL WG at rates, intervals and frequencies outside of the parameters assessed, could result in greater levels of risk than the assessment identified. Consequently a maximum application rate, a maximum number of applications, and a minimum interval between applications have been set as an additional control under section 77A of the Act. In o rder to mitigate potential risks to human health or to the environment, the following additional control is applied to MYCOTAL WG:

 The substance must not be applied at rates exceeding 2 kg of formulated product/ha per application (equivalent to 96 g active ingredient/ha); and  The substance must not be applied to the same area more than 12 times in any 365 day period; and  An interval of at least 7 full days must be observed before the substance is reapplied to the same area.

Restricted Entry Interval 6.12. The staff have advised me that there is the potential for exposure to cause sensitisation in users mixing, loading or applying the substance, and in workers re-entering the treated area. It is considered good practice to wear PPE when handling agrichemicals and appropriate PPE is likely to reduce the likelihood of exposure to MYCOTAL WG. The staff have advised me that it is appropriate to apply additional controls under section 77A of the Act to require appropriate full personal protective equipment for mixing, loading and application activities (including being in the application area during

2 Where greenhouse means a walk-in, permanent, enclosed space for crop production which reduces release of plant protection products into the environment.

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release of the substance). Furthermore, entry into a treated space immediately after application should only be allowed if appropriate PPE is worn to avoid contact with treated surfaces. A period of 4 hours after application is required to manage re-entry worker exposures. I consider it appropriate that the person in charge of the application area should be required to take all reasonable steps to ensure that persons authorised to be in the application area wear appropriate PPE during the restricted entry interval. The additional controls applying PPE requirements and a restricted entry interval are shown in appendix A.

Additional information requirements 6.13. The proposed additional controls set certain requirements or restrictions on users of the substance. I consider that an effective means of communicating this information to the user is through the substance label. Accordingly, I have applied additional requirements under section 77A of the Act requiring that information regarding the above additional controls be included on the label for MYCOTAL WG. This label information control is shown in Appendix A. Overall assessment of risks with controls

6.14. The staff have advised me that the controls applied to MYCOTAL WG, including additional controls applying restriction on application to indoor application in horticultural crop production, will reduce the likelihood of exposure of MYCOTAL WG to birds so that the level of risk is negligible. I consider that these controls also address the uncertainty arising from the data gaps in the risk assessment by reducing the likelihood of exposure to aquatic and terrestrial environments so that the level of risk to these environments will be negligible.

6.15. I consider that with controls in place, the risk to taonga bird and other species will be negligible. As a result, the potential cultural risk to Māori that could arise as a result of harm to taonga species will be mitigated.

6.16. Based on the information provided, with the controls in place, I consider that the risks to Māori culture or traditional relationships with ancestral lands, water, sites, wāhi tapu, valued flora and fauna or other taonga will be mitigated so that significant impacts are not anticipated.

Review of controls for cost-effectiveness

6.17. I consider that the controls listed in Appendix A are the most cost-effective means of managing the identified potential risks associated with this application. The applicant was given an opportunity to comment on the proposed controls as set out in this decision. The applicant indicated that they had no concerns with the proposed controls.

7. Conclusion 7.1. Taking into account the staff assessment of the potential risks and benefits associated with MYCOTAL WG, I consider that, with controls in place:

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 the risks to human health and the environment arising from the hazardous properties and the use of MYCOTAL WG are negligible  significant adverse impacts on the social or economic environment from the use of MYCOTAL WG are not anticipated  significant impacts on Māori culture or traditional relationships with ancestral lands, water, sites, wāhi tapu, valued flora and fauna or other taonga that will breach the principles of the Te Tiriti o Waitangi/Treaty of Waitangi are not anticipated  benefits will be derived for New Zealand by allowing the use of MYCOTAL WG

8. Decision

8.1. Pursuant to section 29 of the Act, I have considered this application to import a hazardous substance for release made under section 28 of the Act. In doing so, I have applied the relevant sections of the Act and clauses of the Hazardous Substances and New Organisms (Methodology) Order 1998.

8.2. I am satisfied with the hazard classifications identified by the staff in Table 1 and accordingly confer them on MYCOTAL WG.

8.3. I consider that, with controls in place, the risks to human health and to the environment are negligible, and there will be benefits associated with the release of MYCOTAL WG. Therefore, I consider that the application may be approved in accordance with clause 26, with the controls proposed by the staff and documented in Appendix A.

8.4. The importation of the hazardous substance, MYCOTAL WG, is thus approved with controls as listed in Appendix A. I am also satisfied that, as the manufacture of MYCOTAL WG would not impose any additional risks over the importation of the substance, this approval should apply to both importation and manufacture of MYCOTAL WG.

Dr Allan Freeth Date: 14 January 2016

Chief Executive, EPA

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Appendix A: Controls applying to MYCOTAL WG

Please refer to the Hazardous Substances Regulations3 for the requirements prescribed for each control and the modifications listed as set out in Section 6 of this document.

Table A1: Controls for MYCOTAL WG – codes, regulations and variations

Substances (Classes 6, 8, and 9 Controls) Regulations 2001

Code Regulation Description Variation

T1 Regs 11 – 27 Limiting exposure to toxic substances No TEL, ADE or PDE values are set for through the setting of TELs any component of the substance at this time

T2 Regs 29, 30 Controlling exposure in places of work No WES values are set for any through the setting of WESs. component of this substance at this time.

T4 Reg 7 Requirements for equipment used to handle substances

T5 Reg 8 Requirements for protective clothing and equipment

T7 Reg 10 Restrictions on the carriage of toxic or The maximum quantity of this substance corrosive substances on passenger that can be carried on a passenger service vehicles service vehicle is 3 kg per package

E6 Reg 7 Requirements for equipment used to handle substances

Hazardous Substances (Identification) Regulations 2001

Code Regulation Description Variation

I1 Regs 6, 7, 32 Identification requirements, duties of – 35, 36(1) – persons in charge, accessibility, (7) comprehensibility, clarity and durability

I9 Reg 18 Secondary identifiers for all hazardous substances

I11 Reg 20 Secondary identifiers for ecotoxic substances

I16 Reg 25 Secondary identifiers for toxic substances

I17 Reg 26 Use of generic names

I18 Reg 27 Requirements for using concentration ranges

3 The regulations can be found on the New Zealand Legislation website; http://www.legislation.co.nz

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Code Regulation Description Variation

I19 Regs 29 – 31 Additional information requirements, including situations where substances are in multiple packaging

I21 Regs 37 – 39, General documentation requirements 47 – 50

I28 Reg 46 Specific documentation requirements for toxic substances

I29 Regs 51, 52 Signage requirements

Hazardous Substances (Packaging) Regulations 2001

Code Regulation Description Variation

P1 Regs 5, 6, General packaging requirements 7(1), 8

P3 Reg 9 Criteria that allow substances to be packaged to a standard not meeting Packing Group I, II or III criteria

P13 Reg 19 Packaging requirements for toxic substances

PG3 Schedule 3 Packaging requirements equivalent to UN Packing Group III

PS4 Schedule 4 Packaging requirements as specified in Schedule 4

Hazardous Substances (Disposal) Regulations 2001

Code Regulation Description Variation

D4 Reg 8 Disposal requirements for toxic and corrosive substances

D5 Reg 9 Disposal requirements for ecotoxic substances

D6 Reg 10 Disposal requirements for packages

D7 Regs 11, 12 Information requirements for manufacturers, importers and suppliers, and persons in charge

D8 Regs 13, 14 Documentation requirements for manufacturers, importers and suppliers, and persons in charge

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Hazardous Substances (Emergency Management) Regulations 2001

Code Regulation Description Variation

EM1 Regs 6, 7, 9 – Level 1 information requirements for 11 suppliers and persons in charge

EM6 Reg 8(e) Information requirements for toxic substances

EM7 Reg 8(f) Information requirements for ecotoxic substances

EM8 Regs 12 – 16, Level 2 information requirements for 18 – 20 suppliers and persons in charge

EM11 Regs 25 – 34 Level 3 emergency management requirements: duties of person in charge, emergency response plans

EM13 Reg 42 Level 3 emergency management requirements: signage

Hazardous Substances (Tank Wagon and Transportable Containers) Regulations 2004

Code Regulation Description Variation

Tank Regs 4 to 43 Controls relating to tank wagons and Wagon as applicable transportable containers

Additional controls

Code Section of Control the Act

App 77A This substance must only be applied within an indoor horticultural facility, such as a method greenhouse. It must not be applied outdoors.

Application 77A The substance must not be applied at rates exceeding 2 kg of formulated product/ha Rate per application (equivalent to 96 g active ingredient/ha).

The substance must not be applied to the same area more than 12 times in any 365 day period.

An interval of at least 7 full days must be observed before the substance is reapplied to the same area.

PPE 77A Any person mixing, loading or applying this substance must wear full personal protective equipment and respiratory protection.

Re-entry 77A A re-entry interval of 4 hours after the end of the application of the substance must be observed at all times.

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Code Section of Control the Act The person in charge of the application area must take all reasonable steps to ensure that no person enters an area treated with the substance until the end of the re-entry interval unless they are wearing appropriate personal protective equipment to avoid contact with spray or residues of the substance.

Label 77A The label for this substance must indicate the following requirements: - The re-entry interval and associated requirements on the person in charge of an application - Appropriate personal protective equipment - The maximum application rate, maximum number of applications, and the minimum interval between applications for the substance - The restriction on application to within an indoor horticultural facility.

Interpretation

1. In these controls, unless otherwise specified below, a word has the same meaning as it is defined in the HSNO Act (if any).

2. Unless the context otherwise requires, the words/phrases listed below have the following meaning:

Term Description

The period after which the glasshouse or other indoor facility may be re-entered Re-entry interval after MYCOTAL WG has been applied.

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Appendix B: Staff classification of MYCOTAL WG

Formulation data were provided for some endpoints for MYCOTAL WG. Classification for endpoints where data were not provided was estimated using information on the effects of the individual components and mixture rules.

The mixture rules used for classifying substances can be found in the User Guide to Thresholds and Classifications4.

The classifications of MYCOTAL WG are shown in Table B1.

Data quality – overall evaluation

The EPA has adopted the Klimisch et al (1997)5 data reliability scoring system for evaluating data used in the hazard classification and risk assessment of chemicals.

The staff acknowledge that there are frequently data gaps in the hazard classification for chemicals which have been in use internationally for a long time. International programmes such as the OECD High Production Volume programme6, REACH7, and European Regulation 1107/2009/EC8 are progressively working towards filling these data gaps. As new information becomes available, staff will update the Hazardous Substances and New Organisms (HSNO) classifications for those substances. Hazard classifications of MYCOTAL WG

Table B1 Applicant and EPA Staff classifications of the mixture9

Method of Mixture classification classification

Hazard Class/Subclass Remarks Applicant’s Staff’s

classification classification 10

Mixture data Read across Mixture rules Expert Class 1 Explosiveness No ND statement instead of test

4 http://www.epa.govt.nz/Publications/ER-UG-03-2.pdf 5 Klimisch, H-J., Andrear, M., & U. Tillmann, 1997. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Reg. Toxicol. Pharmacol. 25, 1–5 (1997) 6 http://www.icca-chem.org/Home/ICCA-initiatives/High-production-volume-chemicals-initiative-HPV/ 7 http://ec.europa.eu/environment/chemicals/reach/reach_intro.htm 8 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:309:0001:0050:EN:PDF 9 NA: Not Applicable --> For instance when testing is technically not possible: testing for a specific endpoint may be omitted, if it is technically not possible to conduct the study as a consequence of the properties of the substance: e.g. very volatile, highly reactive or unstable substances cannot be used, mixing of the substance with water may cause danger of fire or explosion or the radio-labelling of the substance required in certain studies may not be possible. ND: No Data or poor quality data (according to Klimisch criteria ) --> There is lack of data. No: Not Classified based on actual relevant data available for the substance --> The data are conclusive and indicate the threshold for classification is not triggered. 10 Use of mixture rules may not adequately take into account interactions between different components in some circumstances and must be considered of lower reliability than substance (formulation) data.

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Expert Class 2, 3 & 4 Flammability No ND statement instead of test

Expert Class 5 Oxidisers/Organic No ND statement Peroxides instead of test

Expert Subclass 8.1 Metallic No ND statement corrosiveness instead of test

Subclass 6.1 Acute toxicity (oral) No ND

Mixture data provided are not Subclass 6.1Acute toxicity No ND reliable so (dermal) mixture rules applied

Read across of data from Subclass 6.1 Acute toxicity No No Mycotal was (inhalation) accepted by EPA staff.

Subclass 6.1 Aspiration hazard No ND

EPA Staff to into account the Subclass 6.3/8.2 Skin EFSA No No irritancy/corrosion conclusion to give No rather than ND.

EPA Staff to into account the Subclass 6.4/8.3 Eye EFSA No No irritancy/corrosion conclusion to give No rather than ND.

Lecanicillium muscarium 19- Subclass 6.5A Respiratory No 6.5A 79, based on sensitisation opinion on fungal protein.

Lecanicillium muscarium 19- Subclass 6.5B Contact 6.5B 6.5B 79, based on sensitisation opinion on fungal protein.

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Subclass 6.6 Mutagenicity No ND

Subclass 6.7 Carcinogenicity No ND

Subclass 6.8 Reproductive/ No ND developmental toxicity

Subclass 6.8 Reproductive/ developmental toxicity (via No ND lactation)

Subclass 6.9 Target organ No ND systemic toxicity

Subclass 9.1 Aquatic ecotoxicity 9.1D 9.1D Biocidal action

Subclass 9.2 Soil ecotoxicity No ND

Subclass 9.3 Terrestrial No ND vertebrate ecotoxicity

Subclass 9.4 Terrestrial No ND invertebrate ecotoxicity

Hazard classifications the active ingredient

Table B2 Applicant and Staff classifications of the active ingredient

Active ingredient Method of classification classification

Hazard Class/Subclass Remarks

Applicant’s classification Staff’s classification Test results Read across Expert statement Class 1 Explosiveness - No instead of test

Expert statement Class 2, 3 & 4 Flammability - No instead of test

Expert statement Class 5 Oxidisers/Organic Peroxides - No instead of test

Expert statement Subclass 8.1 Metallic corrosiveness - ND instead of test

Subclass 6.1 Acute toxicity (oral) - ND

Subclass 6.1Acute toxicity (dermal) - ND

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Subclass 6.1 Acute toxicity - ND (inhalation)

Subclass 6.1 Aspiration hazard - ND

Subclass 6.3/8.2 Skin - No irritancy/corrosion

Subclass 6.4/8.3 Eye - No irritancy/corrosion

Based on expert Subclass 6.5A Respiratory - 6.5A judgement relating sensitisation to fungal protein.

Based on expert Subclass 6.5B Contact sensitisation - 6.5B judgement relating to fungal protein.

Subclass 6.6 Mutagenicity - ND

Subclass 6.7 Carcinogenicity - ND

Subclass 6.8 Reproductive/ - ND developmental toxicity

Subclass 6.8 Reproductive/ - ND developmental toxicity (via lactation)

Subclass 6.9 Target organ systemic - ND toxicity

Subclass 9.1 Aquatic ecotoxicity - 9.1D Biocidal action

Subclass 9.2 Soil ecotoxicity - ND

Subclass 9.3 Terrestrial vertebrate - ND ecotoxicity

Subclass 9.4 Terrestrial invertebrate - ND ecotoxicity

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Appendix C: Active ingredient and metabolites

Identity

As this is the first full Part 5 application considered for this active ingredient, general information about Lecanicillium muscarium 19-79 is provided in the following tables.

Table C1 Identification of Lecanicillium muscarium Strain 19-79

Species Lecanicillium muscarium

Genus Lecanicillium

Family Clavicipitaceae

Order Hyphomycetes

Class Sordariomycetes

Phylum Deuteromycotina

Kingdom Fungi

CAS Number 67892-35-7

Purity -

Significant impurities/additives/metabolites (% - concentration)

Classified in the EU: Other international classification & R421: May cause sensitisation by inhalation. labelling R43: May cause sensitisation by skin contact.

Originally, the microbial active ingredient was classified as Verticillium lecanii, strain Ve6. In 2001 a revision of the taxonomy of the classification of the genus Verticillium was carried out (Zare and Gams, 2001) and the conclusion was that the entomopathogenic fungi Verticillium should be called as Lecanicillium. In the cited paper it was also concluded that the variable species Lecanicillium lecanii, divided into a number of strains, should be split along those strain lines into separate species. V. lecanii Ve6 was reclassified as Lecanicillium muscarium. The new species L. muscarium is conspecific with the old strain V. lecanii Ve6. The strain identifier “Ve6” was an internal number used at Tate & Lyle, and is no longer used. Therefore, the staff consider that the strain identifier “19-79” should be used instead of Ve6, since this refers to the sample in the IMI collection and to the original strain that was developed by Dr. R. Hall and later by Tate & Lyle, and still is in the product Mycotal. This reference is also mentioned by Zare and Gams.

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The applicant provided the following information about differences in strain coding: The strain code 19-79 was an original number given to the Mycotal strain by the author D. Chandler. Under this number the strain is mentioned in the Centraal bureau Schimmelcultures (CBS), Baarn, The Netherlands [CBS 102071]; and The Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF) USDA-ARS Plant Protection Research Unit, Ithaca, USA, [ARSEF 5128]. Some of the collections also refer to Mycotal. Number Ve-6 was an internal number used by Tate & Lyle from whom the strain was obtained by Koppert in 1980. The strain code Ve-6 was used also by Koppert for a certain time. The DAR (2007) indicates both these codes together with some others. The strain has been in Koppert’s possession for all these years. No changes in manufacturing location or material transfer had happened since its original registration in the UK and the Netherlands and strain’s approval on EU level in 2010. The Final Addendum to the DAR (2010) includes an explanation on why Koppert would like to use an original number 19-79. The Reporting Table in the EFSA Peer Review clearly indicated that both codes 19-79 and Ve6 are for the same strain as well.

Taking into account that the same strain has several codes including mostly used Ve6 and 19-79, and that the manufacturing site was not changed, and no material transfer happened from the moment of notification and following authorization, an equivalence of strain was not deemed required by the staff. Therefore, the explanation about the strain was considered acceptable by the staff.

Mode of action

The mechanism of action of Lecanicillium muscarium is via the infiltration of the fungus by growth in the arthropod host, and not through the production of toxins. Infiltration starts with breaching of the chitinous exoskeleton of insects by substrate-specific proteases; this same mechanism apparently also determines the effect of L. muscarium to some mildew types (Akary et al., 1997). There is a marked difference in proteases produced by entomopathogenic and phytopathogenic Lecanicillium species, which apparently is a major determinant of their host(-group) specificity (St Leger et al., 1997). Whether the fungus after infiltration will actually grow in the host depends on the host-specificity of the L. muscarium strain. Once in the host, L. muscarium forms blastospores1 which spread through the haemolymph of the arthropod host and lead to further infection. After 7–10 days, when a great number of hyphal bodies have been formed, the infected host dies. If relative humidity is high the fungus subsequently grows out through the cuticle and forms phialides. At the end of these phialides, conidia are formed in clusters (slime heads) (Schuler et al., 1991; Samson et al., 1985).

Histological section studies of whitefly killed and fixed after different exposure times indicate fungus penetration and invasion (destruction of the inner organs) of the tissues as the cause of death. Thrips are probably killed as a result of multiple lesions of the cuticle by enzymatic degradation, as no fungal material was found in the haemolymph of the insects at the time of death. In addition, L. muscarium has also been reported to secrete lytic enzymes that play a major role in penetrating the cyst wall of Heterodera schachtii.

In several publications Verticillium lecanii have been reported to produce a different combination of metabolites. Fungal metabolite production appeared to be dependent on the culture conditions and strain. Extracts from cultures grown under laboratory-scale still liquid conditions of Lecanicillium muscarium strain

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19-79 contained the so-called destruxins A, B and E. Destruxins are cyclic peptides that elicit a range of biological responses including cytotoxicity and inhibition of gene expression. Destruxins were not produced under conditions as practised in commercial situations (solid-state fermentation and in aerated (shaken) liquid fermentation); In addition, they were also not detected in the formulated product “Mycotal” or its non- formulated spores in greenhouse conditions 1 Fungal spore that is produced by budding and that acts as a resting spore or (as in yeasts) gives rise to another spore or a hypha.

Physico-chemical properties

Physico-chemical properties of Mycotal WG

Table C2 Physical and chemical properties of Mycotal WG

Test Klimisch Property Results Reference method Score (1-4)

Colour Beige 1

Physical state Solid (powder) 1

No characteristic Odour 1 odour HJ Knips (2000) 6.76 Determination of the pH appearance, pH, (1% aqueous CIPAC MT 75 1 (at 20 ±1 ºC) suspensability, wet dispersion) sieving and wettability Percentage before and after storage retention of at 2-6°C for 6 months of untreated Mycotal Mycotal. NOTOX B.V., in a 75 µm mesh S-Hertogenbosch, was 1.20% Netherlands. Project No Wet sieving CIPAC MT 59 1 290993 Percentage retention of treated Mycotal in a 75 µm mesh was 1.45%

J Franke (2005) Particle size distribution of Mycotal. Siemens AG Average mean Prozess-Sicherheit, Particle size distribution particle size = OECD 110 1 Industriepark Höchst, 64.2 µm C487 D-65926 Frankfurt am Main, Germany. Study No 20050730.01

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Physico-chemical properties of Leucanicillium muscarium 19-79

The staff did not receive any specific information regarding the physical-chemical properties of Leucanicillium muscarium 19-79. The information presented below is based on the SDS sent by the applicant.

Table C3 Physico-chemical properties of Leucanicillium muscarium 19-79

Klimisch Property Result Test method Reference Score (1-4)

Colour Beige - 1 SDS for L. muscarium 19-79

Physical state Solid (powder) - 1 SDS for L. muscarium 19-79

Odour - -

Oxidizing Expert Not oxidiser properties statement

Explosive Expert No explosive properties properties statement

Water Solubility Insoluble - 1 SDS for L. muscarium 19-79

Expert Flammability Not flammable statement

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Appendix D: Mammalian toxicology

Robust study summaries for the mixture (Mycotal WG)

The studies received mostly relate to Verticillium lecanii, which is sometimes identified as strain Ve6. As discussed above, Verticillium lecanii Ve6 is accepted as being the same strain as Lecanicillium muscarium 19-79 (see 3.1).

Reference to Mycotal is to a formulated product containing the same strain, but it is not considered the same formulation as Mycotal WG (water soluble granule), for which approval has been sought. EPA staff read across the data on Mycotal because, based on the limited information available, the components present other than the active ingredient are food materials similar to those in Mycotal WG. Therefore the results of these studies were taken into account in the classification of the active ingredient and Mycotal WG when appropriate. The only studies submitted for Mycotal were an acute dermal toxicity study, an acute inhalation study and a short term (28 day) inhalation study. The short term inhalation study is reviewed in the section on the active ingredient.

Acute toxicity [6.1]

Acute Oral Toxicity [6.1 (oral)]

No oral studies on Mycotal WG were provided for review.

Acute Dermal Toxicity [6.1 (dermal)]

Type of study Single dose dermal toxicity study in rabbits

Flag Disregarded study

Mycotal wettable powder, Lot No: 03M320. Verticillium lecanii, 1.05 x 1010 Test Substance spores/g

Endpoint Signs of toxicity, dermal irritation

8 Value LD50 >10 spores

Kyoko Barata; 1998. A Single Dermal Dose Toxicity Study of Mycotal Wettable Powder in Rabbits. Institute for Animal Reproduction Toxicology Reference Research Center. 1103, Fukaya, Kasumigaura-machi, Niihari-gun, Ibaraki 300-0134, Japan. Study No 497. [Translated by Izumi, H.]

Klimisch Score 3 (Not reliable)

Amendments/Deviations None

59-Nousan-No 3850 and 9-Nousan No 5092, Ministry of Agriculture, GLP Forestry and Fisheries, Japan (1984 and 1997).

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59-Nousan No. 4200(1985), 9-Agriculture No 5093 (1997) and Guidance Test Guidelines on Study Reports on Microbial Pesticides for the Safety Evaluations and Registration (9-Nousan No 5090, 1977).

Species Rabbits

Strain New Zealand White

No/Sex/Group 5 males and 5 females

108 spores per animal for 24 hours. The dose volume was indicated as 500 mg/animal. The substance was diluted to 0.2 x 109 spores/ml. The Dose Levels substance was diluted with sterile saline and applied at 108 spores/animal over a patch of circa 6 cm2 covered with gauze.

Exposure Type Dermal semi-occluded patch test.

No animal died during the study and there were no test substance-related effects observed during the clinical observations. There were no effects on body weight, nor any macropathological findings. There were no signs of Study Summary dermal irritation. The NOAEL for adverse toxicological and irritant effects via the dermal route in rabbits was 108 spores per animals.

EPA Staff note the dose applied expressed in mass is not provided. The US EPA OPPTS 885.3100 Acute dermal guideline for the use product states the dose should be 2 g/kg bw. [Not statement of the number of colony forming units administered is provided in this guideline.]

The formulation contains 1.05 x 1010 spores/gram, so 108 spores/animal represents a dose of circa 10 mg. In comparison to the dose recommended in the guideline above this is a very low dose. The male rabbits weighing Additional Comments 2.5 kg received a dose of approximately 10/2.5 = 4 mg/kg bw.

While there were no adverse findings identified, EPA staff consideration of dose level indicates that the appropriate classification should be “no data” because the dose was not close to the required limit dose for the formulated substance of 2000 mg of the test substance/kg bw. EPA Staff note that this conclusion is consistent with that of the EFSA review of this study (EFSA, 2008) which graded the study “not acceptable”.

The substance does not trigger classification for acute toxicity via the Conclusion dermal route, but the assigned conclusion is ND (no/insufficient data).

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Acute Inhalation Toxicity [6.1 (inhalation)]

Type of study 4 hour acute inhalation study

Flag Key study

Test Substance Mycotal (containing spores of Verticillium lecanii, 1.08 x 108 spores/g).

Endpoint LD50

Value > 8.93 mg/l

Arts J. H. E.; 1990. Acute (4 hour) Inhalation Toxicity Study with Mycotal in Rats. Department of Biological Toxicology, TNO-CIVO Toxicology and Nutrition Reference Institute, P. O. Box 360, 3700 AJ Zeist, The Netherlands. Report Number V 90.445/208221

Klimisch Score 2 (Reliable with restrictions)

No data for mass median aerodynamic diameter (MMAD) or Geometric Amendments/Deviations Standard Deviation (GSD) were presented.

GLP Yes

A protocol for the study was based on US EPA Guideline No 798.1150 (1985) Test Guideline/s and OECD No 403 (1981).

Species Rat

Strain Wistar. (Crl: WI (WU) BR)

No/Sex/Group 5

8.93 mg/l for 4 hours (reported as 893 mg/m3 in the study report). The Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviations Dose Levels (GSD) were not provided. 82% of the particles were within the range 1.8 – 3.8 µm.

Exposure Type Nose only inhalation

One male rat was found dead on Day 6 during the observation period. Wet head and fur and visually increased breathing frequency were observed during the exposure. An increased breathing frequency was also seen in male rats at Day 6 – 13 of the observation period. In general body weights were reduced seven days after exposure. During the Study summary second week of the observation period, the body weight of male rats had recovered. Female rats showed increased weights at Day 10, but slightly reduced weights at Day 14 of the observation period. Macroscopic examination of at the end of the observation period revealed red and/or pale discoloured and/or insufficiently collapsed lungs in most rats. The

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male that died on Day 6 was slightly autolytic and displayed intestines filled with water.

The 4 hours LC50 of the substance is higher than 8.93 mg/l

EPA staff note that the study largely followed guidelines existing at the time, but Additional Comments MMAD and GSD data were not provided. Nevertheless the aerosol was of respirable size. The study was considered acceptable by the EFSA review.

Conclusion The 4 hours LC50 of the substance is >8.93 mg/l

Skin Irritation [6.3/8.2]

No data were provided for the formulated product, Mycotal. EPA staff note that in its assessment the EU read across the data presented for skin irritation for the active ingredient to the formulated product, Mycotal, noting that it contains non-toxic, approved food additives or components that are commonly used in addition to the active ingredient. This is an assessment based on mixture rules not study data. Application of the mixture rules for Mycotal WG also indicates that no classification is required.

Eye Irritation [6.4/8.3]

No data were provided for the formulated product, Mycotal. EPA staff note that in its assessment the EU read across the data presented for eye irritation for the active ingredient to the formulated product, Mycotal, noting that it contains non-toxic, approved food additives or components that are commonly used in addition to the active ingredient. This is an assessment based on mixture rules not study data. Application of the mixture rules for Mycotal WG also indicates that no classification is required.

Contact Sensitisation [6.5]

No data were submitted relating to the formulated product, but some data with comments on the suitability of studies for microorganisms is discussed under tests on the active ingredient below.

Due to the nature of the substance (a microbial pesticide) application of a 6.5B contact sensitisation classification is appropriate based on the presence of bacterial proteins which are likely to stimulate an immune response in some individuals. EPA staff note the despite the negative findings in a sensitisation study with the active ingredient, the EFSA review noted that all micro-organisms are considered potential sensitisers.

General conclusion about acute toxicity classification EPA Staff note that limited data were presented relating to the acute toxicity profile of Mycotal WG, but the conclusion is that Mycotal WG is not toxic or infective when administered orally, dermally or by inhalation. No data were presented to address oral or dermal (percutaneous) toxicity for the formulation, but application of mixture rules does not trigger classification. EPA staff accepted read across of inhalation data for a similar formulation to support no classification for the inhalation route.

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Taking into account information on the active ingredient for skin and eye irritation and information on the other components of Mycotal WG no classification for skin or eye irritation is proposed.

In relation to contact and respiratory sensitivity, the contact sensitisation study with the active ingredient is considered of limited relevance. Classification of Mycotal WG for skin and respiratory sensitisation is proposed taking into account the presence of microbial protein components to which sensitisation may develop. EPA Staff note this conclusion is supported by some evidence of sensitisation in persons exposed to the substance (see “other studies” below), although the findings are mixed and the nature of the exposures uncertain. It is also consistent with the classification in the EU.

Mammalian toxicology - Robust study summaries for the active ingredient (Lecanicillium muscarium 19-79 strain DSM 14941)

Acute toxicity [6.1]

Acute Combined Toxicity [6.1 (oral, inhalation, intraperitonial)]

Type of study A combined intraperitoneal, oral and inhalation study in rats

Flag Supporting study

Verticillium lecanii spores [either Stabilised Spore Powder (SSP) or Test Substance Autoclaved Stabilised Spore Power (ASSP)]

Endpoint LD50/LC50, signs of toxicity/pathogenicity

8 For oral toxicity an LD50 of >3.0 x 10 spores/animal was established. Value No LD50 determined for the inhalation or intraperitoneal routes.

Hiscox, D.N.; et al.; 1982. Acute Toxicity and Infectivity Study in Rats Verticillium lecanii. Toxicol Laboratories Limited, Bromyard Road, Ledbury, Reference Herefordshire HR8 1LG, England. New Ref. No V-8. [Previously: Toxicol Report Ref. TAL/6/82]

2 (Acceptable with Restriction) – oral toxicity Klimisch Score 3 (Not reliable) – infectivity

Amendments/Deviations None

GLP Yes (US Code of Federal Regulations Title 21, Part 58 (1978)

Test Guidelines No information

Species Rat

Strain CD

5 males and 5 females in the IP groups (control - 0.9% w/v saline, 4% w/v No/ Group ASSP or 4% SSP), 2.0 ml/rat, but 1 ml/rat for the replacement animals.

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Two groups of 5 male and 5 female rats (10 males and females in total) receiving an oral dose of SSP.

One group (5 males and 5 females) received SSP by inhalation reported as the maximum practicable dose (but the estimated substance or spore concentrations were not provided).

IP groups received SSP or ASSP by intraperitoneal injection. A control group received normal saline. 2.0 ml 4% w/v suspension of the spores in sterile saline was given to each animal, except that this was reduced to 1ml for the replacement animals.

Oral dose: Two groups received oral dose of 2ml of a 10% w/v suspension of spores in sterile saline. This was equivalent to 2.1 x 109 spores/kg in males and 2.4 x 109 spores/kg in females. [EPA Staff note the number of spores is 3.0 x108/animal slightly above the current US EPA guideline Dose Levels recommendation (108/animal).]

The inhalation group was exposed for 4 hours. Appendix 32 of the report indicates the nominal test material concentration was 6.92 mg SSP/l. The measured concentrations were 0.18, 0.18 and 0.005 mg/L respectively for the >8 µm, > 1 µm, and 1 – 8 µm particle size distributions respectively. Researchers noted that only a small proportion of the particles could be considered suspended. [EPA staff note that the aerosol was not fine enough to be respirable.]

Exposure Type Intraperitoneal injection, oral (gavage), or inhalation (whole body exposure)

The consideration of the results of this study is divided according to the exposure route. Intraperitoneal study 5 males and 5 females in the IP groups (control - 0.9% w/v saline, 4% w/v ASSP or 4% SSP), 2.0 ml/rat, but 1 ml/rat for the replacement animals. The study plan had three separate groups scheduled for sacrifice on Day Study Summary 3, Day 8 and Day 15. (The Day 3 group were maintained till Day 30 due to mortalities in the other groups.)

Unscheduled death within 48 hours of intraperitoneal (IP) dosing resulted in modification to the intended study design, whereby animals scheduled for sacrifice on Day 2 were maintained for 28 days before sacrifice.

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Some replacement animals were dosed at a lower dose (1ml rather than 2ml) to replace the animals that died. The original study design had three groups of 5 male and 5 female rats dosed with SSP or ASSP (a total of 15 males and 15 females for each form of spores), scheduled for sacrifice in Day 3, Day 8 and Day 15. There were 9 deaths within the first two days in animals dosed by IP injection representing a morality rate of 9/30 in the combined sexes. The deaths occurred in three males and one female receiving ASSP and one male and four females receiving SSP. The clinical signs were reduced activity, hunched posture and piloerection. Findings at necropsy included distension of the gastrointestinal tract and adhesions in the abdominal cavity. Microscopic pathology revealed peritonitis in each decedent animal. There were no other deaths in the study. However, other animals showed the same clinical signs in the first 4 days after dosing. No clinical signs were seen thereafter. All the animals treated by IP injection lost weight over the first 2 days, but then gained weight slowly through the study. The saline control gained weight throughout the study. Reduced food consumption was seen in rats dosed with ASSP, SSP or carrier over the first 7 days after IP injection. After 7 days food consumption was similar or higher in treated groups compared to saline controls. Clinical pathology was only carried out for animals treated by IP injection. Key findings were a statistically significant increase in neutrophils (p <0.05) and a decrease in lymphocytes (p <0.05) in both sexes dosed with ASSP or SSP. Haematology did not reveal any changes at Day 29. Blood biochemistry revealed some changes in protein and blood urea nitrogen early in the study but these were unremarkable at Day 14. At Day 29 slightly reduced albumin values (p<0.01) were evident in the male SSP group and this gave a slightly decreased albumin/globulin ratio p<0.01). Macroscopic pathology revealed adhesions in the abdominal cavity involving the majority of organs (stomach, spleen, peritoneum, diaphragm, liver, gastrointestinal tract and or pancreas in the majority of rats dosed (by the IP route) with ASSP, SSP and the carrier material. Yellow/white nodules were seen associated with these adhesions in some animals. 7/10 dosed with the carrier, 21/36 of those dosed with ASSP and 20/35 of those dosed with SSP were effected. (These figures exclude decedents.)

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There were no apparent differences between male and females or between those animal dosed with ASSP or SSP. Fewer animals killed after 7 and 14 days were affected compared to the findings at 2 days, but those killed after 29 days were nearly all affected. Some animals had nodules present in the abdominal cavity no associated with any adhesions. This applied to 3/10 in the carrier, 11/36 with ASSP and 10/35 with SSP. A number of findings were seen in various organs examined at autopsy in IP exposed animals only. Degenerated cells characterised by the presence of degenerated cytoplasm and pyknotic or lysed nuclei were seen to occur in the lymphoid follicles and most frequently in the mesenteric lymph nodes, or spleen and other lymphoid tissue such as Peyer’s patches and in the cortex of the thymus. In the liver areas of necrosis were associated with treatment with SSP. Conclusion: Intraperitoneal phase. Extensive chronic peritonitis with formation of abscesses and granulomas was a result of intraperitoneal injection was seen both with SSP and ASSP. The similarity of the effects seen with both SSP and ASSP indicate no role for the viable organism, but were considered indicative of an immune response. The EFSA review concluded that the signs of peritonitis from the microorganism both in its viable (SSP) and inactivated form (ASSP), indicate that that the effects are not caused by the organism, but are immune-related. EFSA concluded the study is unacceptable for determining pathogenicity and infectiveness of the organism as the method of homogenisation of the organs was not described. The study was acceptable as supplementary information relating to the acute intraperitoneal toxicity of the organism.

Oral: Two groups receiving an oral dose were killed at 14 or 28 days. The animals administered the spore (SPP or ASSP) all survived and did not show any clinical signs of toxicity. Body weights were determined after 2, 7, 14, 21 and 28 days along with food consumption. The orally dosed animals all gained weight throughout the study. There was no influence of exposure to SSP on food consumption. No changes in organ weight, macroscopic or microscopic pathology were found. The fungus was not isolated from any organ. Conclusion: Oral phase

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EFSA concluded the study is unacceptable for determining pathogenicity and infectiveness of the organism as the method of homogenisation of the organs was not described. The study was acceptable as supplementary

information relating to the acute oral toxicity of the organism, and the LD50 is > 3.0 x 108 spores/animal.

Inhalation study: 5 males and 5 females received a whole body inhalation exposure for four hours and were sacrificed on Day 14. As noted above the particle size information was deficient.

Signs noted in the inhalation group following exposure to SSP were poor coat condition, particularly in males and slight hypoactivity in males only. These signs were not evident after 2 days following exposure. A reduction in body weight gain was seen in the group exposed to SSP by inhalation for the first 2 days, but thereafter a gradual increase in body weight was observed. Animals exposure to SSP by inhalation showed decreased food consumption which persisted throughout the 14 day observation period.

Conclusion: Inhalation phase The particle size is not in the respirable range and the dose is poorly defined. According to the study authors, an exacerbation of the base line pathological changes seen in the lungs of control animals was seen in the animals receiving SSP by inhalation. [EPA staff consider this conclusion of questionable validity. The response is said to be substance-related, but that seems to be variance with it being an exacerbation of a response to the exposure seen in control animals. EPA Staff note EFSA considered the study acceptable as supporting information for acute inhalation toxicity. However, EFSA concluded the study is unacceptable for determining pathogenicity and infectiveness of the organism as the method of homogenisation of the organs was not described.

Overall conclusion The deaths, clinical signs, macroscopic and microscopic findings in the intraperitoneal phase of the study with both SSP and ASSP are attributed to an immune response rather than signs of toxicity. Animals dosed by the oral or inhalation routes appeared unremarkable. The study is inadequate for provided conclusive determination of infectivity.

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The study suggests exposure to the substance (both SSP and ASSP) by injection caused nodules and peritonitis, which in some cases (at higher dose) is rapidly fatal. Noting that autoclaved spores also caused this effect it appears not to be a pathogenic infection response. The possibility of a role for the carrier in causing this is unable to be excluded (in part because no carrier control was included in the oral phase of the study). Additional Comments The number of animals in the control group for the oral phase were inadequate and the lack of a carrier and ASSP control is noted and considered a limitation in the study. The particle size of the aerosol in the inhalation phase is undesirably high (not respirable). No classification for the IP route is usually undertaken although this was the main focus of this investigation.

No data suitable for derivation of an LD50 value for the oral or inhalation Conclusion routes were available from this study.

Acute Oral Toxicity [6.1 (oral)]

Type of study Single dose oral toxicity in rat

Flag Supporting study

Test Substance Mycotal TGAI

Endpoint LD50, signs of toxicity, pathogenicity

Value LD50 >108 spores/animal (nominal value only)

Onishi, Y.; 1998. A Single Dose Toxicity Study of Mycotal TGAI Administered Orally to Rats. Mitsubishi Chemical Safety Institute Ltd, Reference Kashima Laboratory 14, Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, Japan. Study Number 7L621.

Klimisch Score 2 (Reliable with restrictions)

The low number of control animals is noted. The dilution of the test Amendments/Deviations material before administration is not consistent with current guidelines. EPA Staff do not consider these deviations to be critical.

GLP Yes

Guidance on Study Reports on Microbial Pesticides for their Safety Test Guidelines Evaluations and for Registration Applications (9-Nousan-No.5090, 1977)

Species Rat

Strain Crj; CD (SD) IGS

No/Sex/Group 14 male and female (test), 2 male and female rats for vehicle controls

Dose Levels 1.2 x 108 spores per animal

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Exposure Type Oral by gavage

No animal died (prior to terminal sacrifice) and no abnormalities were seen in clinical signs, body weight or at necropsy.

The test organism was not detected in faeces during the study.

Study Summary The test organism was not detected in kidneys, brain, liver, lungs, spleen, stomach, small intestine (duodenum), large intestine (colon) mesenteric lymph nodes or blood of any animals.

Mycotal TGAI was found to have no toxicity, infectivity or pathogenicity nor did it persist under the conditions of this study.

EPA Staff did a calculation to determine the administered dose. The Mycotal Technical Grade Active Ingredient (Verticillium lecanii KV01) contained 9.95 x 1010 spores/g. 127 – 143 g (males) and 106-121 g females. 0.12 w/v % of the test substance was suspended in phosphate buffer solution (PBS 0.01M, pH 6.8), based on a normal range of 8.5 – 10 x 1010 spores/g, so that the administered dose provided 108 spores/animal (as per the Guideline). Fasted animals were dosed with 1 ml of the mixture/animal and controls were given 1 ml of PBS. The administered dose was 1.2 x 108 spores/animal.

EPA staff note that the desired number of spores (108/animal) has been used (although this is the nominal dose). The US EPA guideline OPPTS 885.3050 (EPA, 1996) states: “One dose level of at least 108 units of the Additional Comments MPCA per test animal should be used. If a dose level of at least 108 units per test animal is not used, a justification/explanation must be provided. The test material should not be diluted to reach the limit dose at 108 units per test animal.”

EPA staff note the substance was diluted prior to administration. The guideline says: “Variability in test volume should be minimised”. EPA staff consider the nominal dose adequate, but note the EFSA comment on the actual dose below. The EFSA review noted a number limitations in this study making it supplementary information in particular: - Dose preparations were not analysed for viable spore content so

no LD50 could be deduced.

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- The method of homogenisation of organs is not described so no conclusion on infectivity/pathogenicity is possible.

- The study deviated from the US EPA OPPTS 885.3050 in not including a “shelf” control (2 animals cohoused with the treated animals).

No data suitable for derivation of an LD50 value for the oral route were Conclusion available from this study.

Acute Dermal Toxicity [6.1 (dermal)]

No dermal toxicity study was submitted for review.

Acute Inhalation Toxicity [6.1 (inhalation)]

No separate inhalation toxicity study was submitted, but the first study (see above) is a combined oral, inhalation and intraperitoneal injection study in mice.

Acute toxicity by other route (injection)

Type of study Acute toxicity/pathology after intraperitoneal injection in rats and mice with a 7 day observation period

Flag Supporting study.

Verticillium lecanii. This was described as Stabilised Spore Powder. Sample C631234 was described as a control as it was Autoclaved Test Substance Stabilised Spore Powder (ASSP) (Gp 3). A carrier control (238) was also used in mice. A saline control was used in both the mice and rats.

Endpoint Toxicity, pathogenicity.

Value No numerical value could be derived from these data.

Hiscox, D. N. et al.; 1982. Single Dose Intraperitoneal Injection Studies in Mice and Rats with 7 Day Observation Periods. Toxicol Laboratories Reference Limited, Bromyard Road, Ledbury, Herefordshire HR8 1LG, England. TAL/4/82 and TAL/5/82

Klimisch Score 2 (Reliable with Restrictions)

Amendments/Deviations None

GLP Yes (based on USA GLP regulations 1978)

Test Guidelines No information

Species Rats and mice

Strain CD-1 mice and CD rats.

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There were seven groups of female mice with 5 females in each group. No/ Group There were two groups of female rats in each group.

The mice were treated with 0.2 ml/animal of saline (control) (Gp1), Carrier 238 (control) (Gp 2), C631234 (control, autoclaved ASSP) (Gp 3), C631233 (Gp 4), C632235 (Gp 5), C234236 (Gp 6) or C237237 (Gp 7). Dose Levels The administered material consisted of a 4% w/v preparation with the exception of the saline control. The rats were treated with 1ml/animal of saline (control) or C631233.

Exposure Type Intraperitoneal injection

Background

The study report includes reference to previous work with Verticillium lecanii, strain Ve2, prepared by solid-substrate fermentation, which caused deaths associated with severe hepatocytic necrosis after sub-cutaneous dosing in rats. In a confirmatory study there were no deaths, but after sacrifice 34 days after dosing, liver sections were characterised by a marked increase in mitotic index. V lecanii, strains Ve2 and Ve6, prepared by submerged fermentation, had been administered parenterally to mice (both by sub-cutaneous and intraperitoneal injection) in studies 81/TYL 062/076 and TAL/3/82 without producing any deaths. Histopathological examination of the liver sections was conducted in both cases. The more detailed examinations conducted in TAL/3/82 revealed in animals sacrificed 7 days after dosing, occasional Study Summary single cell necrosis or focal necrosis and a slight increase in mitotic activity. The findings were similar with both live and autoclaved test material. The objective of the current study was to subject four batches of V. lecaniii (strains Ve2 and Ve6) to a 7-day intraperitoneal acute toxicity study in the mouse and compare the response in the rat for one batch (strain Ve6) using a similar study design.

Results

There were no deaths during the study. Body weight was only recorded at Day 0, 3 and 7.

Results for mice: The groups in mice consisted of:

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Group Sample Code Exposure Comment Group 1 N/A Control sterile saline Group 2 238 Control – carrier Group 3 C631234 Control - ASSP Group 4 C631233 SSP Group 5 C632235 SSP Group 6 C234236 SSP Group 7 C237237 SSP There was no mortality, nor any treatment-related signs. In the mice, all groups treated with the test substance and the carrier had lost weight at Day 3, but recovered to be close to the control weights by Day 7. Food consumption was reduced by more than 10% in all groups including carrier and autoclaved SSP, but not in two test groups, C631233 (Gp 4) and C237237 (Gp7). No water intake data were presented as no effect was suspected based on subjective observations. [EPA staff note the lack of rigour in this conclusion related to the age of the study.] Macroscopic pathology revealed adhesions in the abdominal cavity involving the diaphragm, spleen, liver, gastrointestinal tract, peritoneum and/or omentum in some animals in all treated groups (but not in the saline control). Incidence was 3/5 in Gp 3 (ASSP), 4/5 in Gp 4 (SSP), 2/5 in Gp5 (SSP), 2/5 in Gp 6 (SSP) and 1/5 in Gp 7 (SSP). White nodules were associated with the adhesions in some animals in all affected groups. Pale areas and/or nodules were noted in the liver and/or spleen of some mice in Groups 2, 3, 4, 6, and 7. Incidence was 2/5 (Gp 2, carrier), 2/5 (Gp 3, ASSP), 1/5 (Gp 4, SSP), 2/5 (Gp6, SSP) and 1/5 (Gp 7, SSP). [EPA Staff note that Group 2 is the carrier control and Group 3 is an autoclaved SSP] Test substance Group 5 (SSP) did not show these findings. [The lack of response in this SSP group appears surprising, but it relates to this particular finding, not to other pathological findings in general which were seen in this group.] Raised white areas/nodules were seen in the abdominal wall of some mice in Group 3, 5 and 6. White nodules were seen in the omentum of some mice in Groups 3, 4, 5, and 7. Nodules were seen in the peritoneum, stomach and/or diaphragm on some mice in Groups 4 and 6.

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Chronic/granulomatous peritonitis with some abscess formation was seen in Groups 3, 4, 5, 6, and 7. Chronic/granulomatous peritonitis was also seen in Groups 2 Carrier Control. A slight increase in the incidence of extramedullary haematopoiesis and minimal chronic inflammatory accumulations was seen in Groups 3, 4, 5, 6, and 7 of which some of the foci were slightly granulomatous and accompanied by single cell necrosis, the latter being most marked in Group 4. In the mice given Verticillium lecanii a very slight increase in the mitotic index was seen in Groups 3, 4 and 5.

Results for Rats: There were only two groups of five rats as follows:

Group Code No Exposure Comment Group 1 - Group 2 C631233 SSP

There were no deaths during the study. Observations were urinogenital staining on Day 4 and 5 post-treatment in 3/5 treated rats. There was a reduction in body weight of the treated group at Day 3 which largely recovered by Day 7. The controls gained weight throughout the 7 day study period. Food consumption in the test animals was reduced by 22% in comparison to controls. Macroscopic pathology revealed adhesions in the abdominal cavity involving the liver, spleen, stomach, kidney and/or gastrointestinal tract in all treated animals, with white nodules associated with this finding in 4 out of 5 rats. White nodules were seen on the omentum (1/5), broad ligament (/5) and on the gastrointestinal tract (3/5) of the treated animals.

A moderate to marked chronic/granulomatous peritonitis with abscess formation was seen in the test animals and a slight increase of minimal inflammatory sinusoidal/portal/perivascular accumulations accompanied by some single cell necrosis in all the animals. The report states (for rats), there was no alteration in the mitotic index was seen. [EFSA summary on the rat findings states a slight increase in mitotic index was seen in SSP

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treated animals only. EPA note the report is difficult to analyse as it reports results on all animals in some places and on mice and rats separately.]

The overall conclusion (called “discussion”) is that the response elicited in rats by i.p. injection of SSP is very similar to that in mice, differing only in the extent of the peritoneal response and the lack of extramedullary haematopoiesis and alteration in the mitotic index. [EPA Staff consider a weakness in the study is that the rat phase did not include control groups for the carrier or ASSP (autoclaved SSP) as for the mice. It is unclear why the rats were exposed to a small number of preparations if the purpose of the study was to document the rat response in comparison to that of mice. The assumption is the SSP preparations are equivalent, but rats were only exposed to one of these preparations, and mice did not react in the same way to all preparations.]

EPA staff note that the findings in this study are indicative of a response similar to that in other studies (by intraperitoneal injection) attributed to an immune process associated with the material (including the carrier) rather than the microbial active ingredient. Due to the limitations of the study this is supporting background information only. Additional Comments The EFSA conclusion was that since the viable spore content was not

determined no LD50 can be deduced. Under the circumstances of this study, signs of peritonitis were induced by viable spores, inactivated spores and carrier material (without spores) after intraperitoneal administration. The nominal dose was 6.9 x 106 spores/animal in mice and 3.4 x 107 spore/animal in rats.

No LD50 value following intraperitoneal injection could be determined from Conclusion this study.

Type of study Intraperitoneal injection study in mice with a 14 day observation period.

Flag Supporting information

Test Substance Verticillium lecanii Ve6. Spore content 5.6 x 109 spores/g

Endpoint LC50, signs of toxicity

Value No value determined

Hiscox D. N. and Paton, D.G.; 1982. Single intraperitoneal injection study Reference in mice with 14 day observation period.

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Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP No information

Test Guidelines Partially in accordance with EPA OPPTS guideline 885.3200

Species Mice

Strain CD-1

No/ Group 15 females

0.2 ml of 4% w/v per animal (equivalent to 4.5 x107 spores/animal) in 0.9% sterile saline. Separate groups were exposed to stabilised spore powder Dose Levels (SSP), homogenised SSP [note EFSA were unsure of the reason for the use of this group], autoclaved SSP and carrier SSP fermented broth (without spores).

Exposure Type Single intraperitoneal dose

No mortality was reported. There were no treatment–related signs of toxicity On day 2 a slight body weight loss was noted for animals treated with SSP, ASSP and homogenised SSP which was related to a decreased food consumption of the first 2 days. Body weight and food consumption was normalised thereafter. Pathology revealed adhesions in the abdominal cavity involving the gastro intestinal tract and/or the liver and/or the peritoneum after 7 and 14 days for mice treated with SSP and homogenised SSP, which was associated with white nodules or areas. Pale areas were noted in the liver in mice treated with SSP and ASSP after Study Summary 7 days and for one SSP treated mouse after 14 days. In the spleen, pale areas and/or nodules were noted for mice treated with SP and homogenised SSP after 14 days. After 7 days and enlarged spleen was noted for SSP mice.

Microscopic examination revealed after 2 days acute peritonitis with abscesses/granulomata, focal necrosis of the liver, slight increase in haematopoiesis and sinusoidal chronic inflammatory accumulations. After 7 days the same findings were noted in a greater number of animals. A slight increase in the hepatocyte mitotic index was noted for all treated groups except for the saline control. [EPA Staff note this means that mice treated with the carrier SSP (without spores) had these findings.] At day 7

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this was noted in mice treated with SSP, ASSP, clarified SSP [assumed by EPA staff to refer to carrier SSP] and homogenised SSP. Elevated mitotic index was still present on Day 14 except in the SSP- treated group.

EPA staff based its review on the EFSA DAR since the file provided was unreadable, but a letter of access was available. EFSA reported that no viable spore content was provided so a qualitative assessment only was Additional Comments possible. EPA staff note the findings are similar to other studies using intraperitoneal injection and that no effects specific to the viable spores were visible.

No LD50 value following intraperitoneal injection could be determined from Conclusion this study.

Type of study Acute toxicity by intravenous injection

Flag Supporting information

Mycotal TGAI (Verticillium lecanii KV01 3*. Lot No 160398 VL97115, 9.95 x Test Substance 1010 spore/g).

Endpoint LD50, signs of toxicity/pathogenicity

Value >1.2 x 107 spores/animal

Onishi, H.; 1998. A Single Dose Toxicity Study of Mycotal TGAI Administered Intravenously to Rats. Mitsubishi Chemical Safety Institute Reference Ltd, Kashima Laboratory 14, Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, Japan. Study Number 7L622 [Study translated by Smeshima H.; 2000]

Klimisch Score 1 (Reliable without restriction)

One analytical deviation is reported which is not considered to have Amendments/Deviations influenced the outcome of the study.

GLP Yes

Guidance on Study Reports on Microbial Pesticides for their Safety Test Guidelines Evaluations and for Registration Application (9-Nousan-No. 5090 (1977)

Species Rat

Strain Crj: CD (SD) IGS

17 males and 17 females, 2 male and 2 females used as controls No/ Group (administered phosphate buffered saline, 0.01M, pH 6.8 (PBS))

Dose Levels 1.2 x 107 spores/animal (administered suspended in PBS).

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Exposure Type Intravenous injection.

Mycotal TGAI was administered to rats intravenously. There was sequential sacrifice and necropsy of the animals. 3 males and 3 females immediately (within one hour) after administration and then at Day 3, Day 7, Day 14 and a final sacrifice of 5 males and 5 females at Day 21. The 2 males and 2 females control animals were sacrificed on Day 21. This enabled a time course for the detection of the test organism to be assessed.

Malt extract agar with streptomycin was used to detect the test microbe.

No abnormalities were noted in clinical signs, body weight or necropsy.

Study Summary Organs from the animals killed immediately (within 1 hour after administration) were examined for present of the organism. The test microbe was found in liver and spleens (105 CFU/g), kidney (103 – 104 CFU/g), brains, small intestines and large intestines 102 – 103 CFU/g), inguinal lymph nodes (103 CFU/g) and blood 102 – 103 CFU/ml). The test microbe decreased very rapidly. On Day 3 the spleen of one male had 7x 10 CFU/g and the blood of one female had 103 CFU/ml. No test microbe was detected on Day 7 or subsequently.

The conclusion was that Mycotal TGAI had no toxic or infective effect and caused no pathology, nor did it demonstrate internal persistence in rats following intravenous injection in this study.

EPA Staff note that the dose is in line with that recommended in the US EPA test guideline for acute injection toxicity/pathogenicity (OPPTS 885.3200). The EFSA review of this study while having similar limitation on the viability Additional Comments of the spore content to that in the oral study (above), concluded that Verticillium lecanii Ve6 was not acutely toxic after i.v. injection at a nominal dose of 1.2 x 107 spores/animal. EFSA concluded the study was able to show the test organism did not colonise nor was infective as the fungus was not isolated except immediately after dosing.

7 The LD50 of the substance via the intravenous route is > 1.2 x 10 Conclusion spore/animal

Skin Irritation [6.3/8.2]

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Type of study Skin irritation/corrosion test in rabbits

Flag Key study

Test Substance Verticillium lecanii SSP (stabilised spore power), Ref C631239

Endpoint Mean Draize Score

Value 0.0 for erythema and oedema [EPA notes scored at only 24 and 72 hours]

Haynes, G. 1982. Skin irritation study: Verticillium lecanii S.S.P. Toxicol Reference Laboratories Limited, Bromyard Road, Ledbury, Herefordshire HR8 1LG, England. Ref No. 96/8203

2 (Reliable with restrictions). Due to this being an early study without Klimisch Score compliance with GLP or international test guidelines and with scoring at only two time points.

The scoring was only reported at 24 and 72 hours. Since dosing was on Amendments/Deviations abraded skin on one side and normal skin on the other there were no control areas.

GLP No information.

Test Guidelines US Federal Register 1973, Vol 38, No 187, Section 1500: 41.

Species Rabbits

Strain New Zealand White

No/Sex/Group 6 Female

Dose Levels 0.5 g moistened with distilled water

Exposure Type Semi-occlusive patch test. (The area was abraded on one side).

There were no positive scores for erythema or oedema at any time point examined. Based on the data presented the mean Draize Scores for both erythema and Study Summary oedema are 0.0.

[EPA Staff note no microbiological investigation was reported in relation to the skin tests in parallel to those performed in the eye studies.]

Despite no observation being made at 48 hours, EPA staff consider these data can be accepted as indicating no classification is appropriate since no signs of irritation were visible at 24 or 72 hours. The study is so early it predated Additional Comments widespread compliance with GLP and international test guidelines.

The EU review identified that the study was partly compliant with OECD No 404 but had the following deviations from that methodology:

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- Shaving was performed 1 hour rather than 24 hours prior to dosing.

- Exposure was for 24 hours rather than 4 hours as in current guidelines.

- Scores were not obtained at 1 hour and 48 hours after patch removal.

Conclusion The mean Draize Score was 0.0 for erythema and oedema (at two time points).

Eye Irritation [6.4/8.3]

Type of study Eye irritation/corrosion in the rabbit

Flag Key study

Test Substance Verticillium lecanii SSP (stabilised spore power), Ref C631239

Endpoint Mean Draize Score

Conjunctival redness: 7/18 = 0.39 Value Conjunctival chemosis: 0/18 = 0.0 Cornea and iris: 0.0

Haynes, G. 1982. Eye irritation study: Verticillium lecanii S.S.P. Toxicol Reference Laboratories Limited, Bromyard Road, Ledbury, Herefordshire HR8 1LG, England. Ref No. 95/8203

2 (Reliable with restrictions). Due to this being an early study without Klimisch Score compliance to GLP or international test guidelines.

Amendments/Deviations None

GLP No information

Test Guidelines US Federal Register 1973, Vol 38, No 187, Section 1500: 42

Species Rabbit

Strain New Zealand White

No/Sex/Group 6 Female

Dose Levels 0.1 g/eye.

Exposure Type Instillation in the eye

The individual scores are provided and from the mean Draize Scores can be calculated.

Study Summary The score for conjunctival redness is: 7/18 = 0.39 The score for conjunctival chemosis is: 0/18 = 0.0

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The cornea and iris were zero at all time points so for these the score is 0.0 All the eyes were clear of any irritant effect at the 72 hour observation point.

The conclusion is that the substance does not trigger classification for eye irritancy or corrosivity.

Swabs from treated and untreated eyes were tested for microbial contamination using swab broths and plates. Positive findings were returned for three swabs in swab broths, two from treated eyes and one for an untreated eye. Verticillium sp were identified, the two treated eyes having V. lecanii Ve2 and the untreated eye V. lecanii Ve6. Researchers noted the morphological distinction of the colony types and considered the findings related to wild type organism contamination rather than growth from the test material. [EPA staff note this conclusion and consider it somewhat surprising, but also note there is no indication that the findings related to pathological findings.]

The study is so early it predated widespread compliance with GLP and international test guidelines. The EU review identified the study was partly compliant with OECD No 405 but had the following deviations from that methodology: - The eyes were not examined 1 hour post-dosing, and;

- The eyes were not rinsed (which is normal when a solid test substance is applied to the eye).

The lack of information on strain resulted in the EU deciding the conclusions on eye irritancy applied at the species level.

Additional Comments None

The Mean Draize Scores were: Conjunctival redness: 7/18 = 0.39 Conclusion Conjunctival chemosis: 0/18 = 0.0 Cornea and iris: 0.0

Contact Sensitisation [6.5]

Type of study Dermal sensitisation in the guinea pig (Guinea pig maximisation test - Magnusson & Kligman)

Flag Key study

Test Substance Verticillium lecanii SSP C631239

Endpoint Sensitisation response

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Value Negative

Teale, J.; 1982. Delayed Dermal Sensitisation Study in the Guinea Pig Reference Verticillium lecanii SSP C631239. Toxicol Laboratories Limited, Bromyard Road, Ledbury, Herefordshire HR8 1LG, England. Ref. No. 97/8203

Klimisch Score 2 (Reliable with restrictions)

Amendments/Deviations The study does not reference any positive controls.

GLP No information.

No information. [EPA staff note the method is relatively consistent with OECD Test Guidelines No 406 Skin Sensitisation Guinea Pig Maximisation test of Magnusson and Kligman.]

Species Guinea pig

Strain Dunkin Hartley

No/Sex/Group 20 animal test and control animals (females only).

Induction phase: Induction consisted of the induction by injection followed seven days later topical application over the injection site. A 5% concentration of the material in sterile distilled water for injection (with Dose Levels and without Freund’s Complete Adjuvant). A 25% concentration of the material in sterile distilled water for topical application. Challenge phase: A 25% and 12.5% concentration of the material in sterile distilled water.

Injection and topical application (single exposure) for induction. Topical Exposure Type challenge

A range-finding study to identify the maximum non-irritant concentration of the test substance was stated to have been conducted at concentrations of 100, 50, 25 and 12.5% of the test substance in sterile distilled water. The results do not report findings at 100 or 50% but do report findings at the other concentrations and also at 3.1 and 6.25%. Based on these findings 25% was considered the maximum non-irritant dose, but researchers used both 12.5% Study Summary and 25% in the challenge. [EPA Staff note the results of the tests at 100 and 50% are not included, so it is not entirely clear that a higher challenge concentration could not have been used. The congruence between what is reported in different parts of the report is poor.]

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In the conditions of this study no sensitisation reaction occurred when exposed to either 25% or 12.5% as a topical challenge.

No positive control data are presented to validate the laboratory’s methodology. Such studies are recommended by the OECD No. 406 at 6 monthly intervals. EPA Staff consider that the lack of documented positive controls does not Additional Comments invalidate the study. The EFSA review (EFSA, 2008) note that the spore content is not indicated and there is no indication of the strain, but they concluded the study was acceptable.

No skin reaction indicative of a sensitisation response occurred in this study Conclusion when challenged with the test substance at a concentration up to 25%.

General conclusion about acute toxicity classification and pathogenicity

The substance is not classified for acute toxicity by any route of exposure (although data for the dermal route were not presented). The substance (TGAI) does not trigger classification for skin and eye irritation or contact sensitisation based on the available study data. However EPA staff note that sensitisation studies with microorganisms are considered to be of limited value, and the assumption in the EU that all microbial organisms should be regarded as potential sensitisers (in contact with the skin and by inhalation). Therefore the staff consider that 6.5A and 6.5B classifications are appropriate.

Although data relating to the infectivity and pathology are somewhat deficient, taking into account the conclusion of the EFSA review EPA staff concluded that the organism is not infective or pathogenic in humans. The isolated case studies of infection from this organism in humans reported in the medical literature relate to special circumstances involving immune-compromised individuals.

Genotoxicity [6.6]

The EFSA review concludes that no genotoxicity testing for Verticillium lecanii Ve6 is required because the secondary metabolites which have been identified from the organism are not of toxicological concern. Destruxins A, B and E have been found but production of these is dependent on the production processes. “The Destruxins were only observed in extract from laboratory scale still liquid production, a type of process not used for commercial scale production), the amounts detected were low and a large variation between the batches was observed.” These metabolites were not detected in the formulated product, Mycotal, in the unformulated spores or in crops after application at 10 times the recommended application rate. The mechanism of action of the organism is not considered to be related to toxin production.

Nevertheless some genotoxicity data have been presented and are reviewed below.

In Vitro Studies

Study type/Test Guideline Result Reference

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Ames test and Vitotox test No reference to guideline and insufficient detail in the poster Kouvelis V.N. et al.; 2004. Ames and Vitotox presentation to assess compliance with tests: an ideal combination to determine OECD No 471 for the Ames test. Negative mutagenicity and genotoxicity. [The Vitotox assay appears to be a with and [Poster presented at the EC-RAFBCA-IBMA- non-guideline method. The EFSA without s9 IOBC Workshop: New insights into risk review notes this assay has advantages activation assessment and registration of biocontrol of automation (rapidity) and in being [but graded agents in Europe, Brussels, 30 September able to assess damage in the whole KS4 (not 2004. [This report is not suitable for suite of DNA in the genome of the assignable)] assessment in support of a substance Salmonella typhimurium test strain. The approval.] genotoxic compound initiate reaction that de-repress the promotor controlling the inserted luciferase gene.]

Typas et al, 2004. Final Report 01.11.01 – 31.10.04. Partner 8: University of Athens, Greece. EU-RAFBCA project: Risk Assessment of Fungal Biological Control Agents QLK1-2001-01391 Unpublished internal report.

Workpackage 2: Development of Tools and Negative Methodologies to Monitor Metabolites and with and Fungal BCAS in the Environment. Ames test without s9 Workpackage 4: Determine the Mode of Action No information on compliance with activation of Metabolites and Identify Target Sites and guideline but appears to be compliant [but graded Potential Risks with standard methodology KS4 (not assignable)] The only detailed data presented relate to a mutagenicity (Ames) test. These data are a more detailed presentation of the data presented above from Kouvelis, V. N.; et al.; 2004. In this study the destruxin A and Verticillium lecanii itself did not demonstrate mutagenic activity in this assay when tested up to 1000 µg/plate. (EPA Staff note the response curve appears to indicate a maximum test

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concentration of 100 µg V. lecanii /plate, but the figure may not include all endpoints.)

Nesslany, F.; 2002. Mutagenicity test on Salmonella typhimurium HIS- using B. N. Ames Technique with Verticillium lecanii. Institut Pasteur de Lille, Genetic Toxicology Laboratory, 1, rue du Professeur Calmette – BP. 245 F-59019 Lille Cedex. France. Report No: IPL-R 020805 Negative EPA Staff note that the maximum dose used with and 100 µg/plate is lower than the standard highest without s9 concentration 5000 µg/plate and no Ames test activation OECD No 471 (1997). explanation for this is given. [The KS 1 concentrations used were 1, 3, 10, 30 and 100 (reliable µg/plate of a Koppert B.V extract containing without 7.25 x 106 CFU/ml liquid culture.] The EFSA restriction) review considered this study acceptable and concluded that under the conditions of the study, the test organism did not induce point mutations in S. typhimurium. EFSA identified the strain used as Verticillium lecanii Ve6, although this was not documented in the study reported.

Conclusion See conclusion below

In Vivo Studies No in vivo genotoxicity studies were available to the EPA staff for review. The EFSA review included reference to an in vivo micronucleus test in the rat (Nesslany, 2004). Primarily due to the oral route of exposure the study was considered inconclusive and graded unacceptable. EFSA noted that since the first tier studies were negative no requirement for in vivo studies had been triggered.

General conclusion about genotoxicity classification

None of the studies carried out on Mycotal or the Verticillium lecanii spores provided evidence indicating any classification for genotoxicity is required. The studies were limited to in vitro techniques using bacterial test systems with and without s9 metabolic activation, of which the study by Nesslany, 2002, was most well documented. EPA staff note that EFSA did not consider genotoxicity testing is required for an organism not known to produce toxic metabolites.

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Carcinogenicity [6.7]

No studies for carcinogenicity of the active ingredient were provided.

General conclusion about carcinogenicity classification No data were presented, but in view of the whole dataset are not considered necessary.

Reproductive/Developmental Toxicity [6.8]

No studies for reproductive/developmental toxicity of the active ingredient were provided.

General conclusion about reproductive/developmental toxicity classification No data were presented, but in view of the whole dataset are not considered necessary.

Target Organ Systemic Toxicity [6.9]

No data for target organ systemic toxicity were provided for either the oral or dermal route for repeat administration.

Target Organ Systemic Toxicity [6.9] via the inhalation route Note that this study was carried out with the formulated product, Mycotal, rather than the active ingredient

Repeat dose inhalation study in rats

Type of study Sub-acute (28 day) inhalation toxicity of Mycotal in rats

Flag Key study

Mycotal, a microbial insecticide containing spores of the insect pathogen, Test Substance Verticillium lecanii. [Spore content 1.2 x 1010 spores/gram.]

LOAEC: 10 mg/m3 based on: rhinitis and epithelial hyperplasia in the nose; increased lymphocyte aggregation and accumulation of alveolar macrophages Endpoint in lungs; and lymphoid hyperplasia in the mediastinal lymph nodes.

NOAEC: 1 mg/m3 (1.08 x 107 spores/m3, 92.5% viable)

Lina, B.A.R. et al.; 1991. Sub-acute (28 day) inhalation toxicity study of Mycotal in Rats. Departments of Biological Toxicology and Experimental Biology, TNO- Reference Toxicology and Nutrition Institute and at the Department of General Microbiology, TNO-Biotechnology and Chemistry Institute, P. O. Box 360, 3700 AJ Zeist, The Netherlands. Report Number V 91.209

Klimisch Score 1 (Reliable without restriction)

Amendments/Deviations None considered to impact on the findings of the study

GLP Yes

Test Guidelines OECD No 412 (1987)

Species Rat

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Strain Wistar (Hsd/Cpb:WU)

5 for the main study with air control and three test groups. 3 for the satellite study with air control and high concentration groups. [EPA staff note that EFSA reported the group sizes were 8 per sex for high dose and control, and 5 per sex for the mid and low dose in their heading No/Sex/Group summary. This is incorrect (as identified in the EFSA analysis), as the additional 3 animals per sex for the control and high dose for determining spore loading were only exposed on a single day. (As stated in the footnote on p13 of the study.) The animals are not included in the evaluation of the toxic effects on the animals exposed for 28 days, for which the groups contained 5 animals/sex.]

0 (air control) 1, 10 or 100 mg Mycotal/m3, 6 hours per day, 5 days per week for 4 weeks

There were two additional groups, a clean air control and a high concentration group exposed for 6 hours on one day only to test lung for the presence of the spores.

The animals were exposed by nose only exposure in Battelle tubes used in an exposure unit (type 8132 P2 from ADG Developments Ltd, UK). The particle size distribution for the particles was not very well defined in the text. The report states “from the particle size distribution it appears that the Dose Levels aerodynamic diameter of most of the particles was smaller than 2.4 m”.

The size distributions from Table 3 for 100 mg/m3 as an example were: Date 10.01.91: > 4.2 µm = 1.1% < 1.0 µm = 29.1% Thus 69.8% of the particles are between 1 and 4.2 µm

These data are typical. On 19.01.91 the particle size was smaller, so the variation was not toward a less respirable mist. These data were similar to those for the 10 mg/m3 group, but the equivalent data for the 1 mg/m3 group were not provided.

The high dose group of 3 males and 3 females exposed (once) for 6 hours were found to have between 0.7 x 105 and 2.1 x 105 Colony Forming Units (CFUs) of Study Summary V. lecanii in the lungs. The total number of spores present was estimated to be at least a factor of 20 higher than the number of viable spores. This was based on the following comparison. The ratio of CFU to number of spores in the

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original test substance was a factor of 2-4, but the ratio in the in test atmosphere was much greater, a factor of 20. Researchers assumed the same ratio (20:1) to apply to the proportion of viable spores found in the lung (rather than by counting non-viable spores. Viable spore counts being determined by colony counting after incubation.)

Test atmospheres were generated by nebulising suspensions of the test substance in de-mineralised water (“demi water”)

The actual test concentrations were close to the target concentrations, and related to the concentration of the Mycotal formulation not the technical grade active ingredient. The grand mean (mean of daily means) and standard deviations for the three exposure groups were: Target 1 mg/m3: 1.2 ±0.2 mg/m3 Target 10 mg/m3: 10.9 ±0.9 mg/m3 Target 100 mg/m3: 102 ±7 mg/m3

No clinical symptoms or signs of systemic toxicity were observed during the study. All study animals survived to the scheduled sacrifice.

There were no statistically significant difference in mean body weights between the test groups and the controls. Mean body weights were slightly lower in females of the high concentration group through the study, but this difference was already present at the start of the study, and the growth rate was comparable to controls. [The authors note that computer randomisation of rats (as set out in the study protocol) was not possible due to the varied group size. EPA staff assume this may explain the difference in the group mean body weight at the start of the study for the high concentration female group.]

There were no statistically significant differences in the red blood cell variables or coagulation variables between the test groups and the controls.

Total white blood cell counts were decreased in males of the low and mid- concentration groups, but not in the high concentration group. As a result the absolute number of lymphocytes was decreased in the low and mid concentration groups as well. The researchers do not consider these changes treatment-related as there is no concentration-response relationship.

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The percentage of neutrophils was increased in males and females of the mid and high concentration groups, but the difference did not attain statistical significance. The absolute number of neutrophils was, however, statistically significantly increased in the high-concentration females (only).

A concentration-related increase in the absolute weight and weight relative to body weight of the lungs was observed in the mid and high concentration groups in both sexes and these changes were statistically significant (p < 0.01). Otherwise no treatment-related changes in organ weights were observed.

Lung weights of rats exposed to either control air or to 100 mg/m3 for six hours (on Day 15) and killed immediately afterward were comparable.

Pathology Gross examination at autopsy found treatment-related abnormalities in the lungs and mediastinal lymph nodes of several males and females of the mid and high concentration groups. The changes were grey discolouration of the lungs and a swollen and spongy appearance. The mediastinal lymph nodes of several male and females of the mid and high concentration group were enlarged, white discoloured and/or firm.

Gross examination of the rats exposed for 6 hours only to air or 100 mg/m3 and then immediately killed did not reveal any abnormalities except for red colour cervical lymph nodes in one male control rat.

Microscopic pathology Microscopic examination revealed treatment related changes in the nasal cavity, the lungs and mediastinal lymph nodes in all treatment groups, the changes being most pronounced in the mid and high concentration groups.

Nasal cavity changes included: - Increase in both the incidence and severity of respiratory epithelial hyperplasia in the mid and high concentration groups in both sexes. In females of the low concentration group the incidence of a very slight degree of this lesion was relatively high compared to controls.

- An increase in the incidence and severity of rhinitis in the high concentration group in both sexes.

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- Olfactory epithelial thinning and/or disarrangement in four males (4/5) and one female (1/5) of the high concentration group.

Lung changes comprised: - An increase in peribronchiolar, perivascular and alveolar aggregates of lymphocytes in male and female rats of all treatment groups, the severity of this condition increasing with concentration.

- Multifocal increased septal cellularity in the animals of the mid and high concentration groups.

- Diffuse accumulation of alveolar macrophages in most males and all females of the treatment groups.

Changes in the mediastinal lymph nodes comprised: - Paracortical and follicular lymphoid hyperplasia in males and females of the mid and high concentration groups, this condition was also found to a minimal degree in 3/5 females and 2/5 males of the low concentration groups as well as in 1/5 control male animals.

- Plasmacytic infiltration in several animals of the high-concentration group, this condition was also found in one female of the mid concentration group and one male and two females of the low concentration group.

EFSA considered these effects to represent a local immune reaction rather than a toxicological effect, but noted that since no control studies with inactivated spores were performed this cannot be fully substantiated. As a result, EFSA defined the NOAEC at 1 mg Mycotal/m3.

EPA staff note that minor effects were seen in the low dose animals and these represented a reduced severity of the findings seen in the mid and high dose animals. For 1mg/m3 to be accepted as the NOAEC, these effects must be regarded as not toxicologically significant.

Epithelial hyperplasia was only seen at a low degree in female rats in the low dose animals. Lymphocyte aggregation and alveolar macrophages were seen to a lower degree in the low dose animals.

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The effects on the mediastinal lymph nodes were primarily seen in mid and high dose animals.

The low severity of these findings, combined with the fact they occur at the site of entry should be taken into account in the assessment and resulted in EPA staff concluding these findings should not be considered a toxicologically significant systemic effect. Thus the NOAEC is established at 1 mg/m3.

EPA staff note that the study was carried out with the formulated product, Mycotal, rather than the active ingredient, but consider the study is suitable for assessment of toxicity and infectivity (as did EFSA, EFSA 2008). The testing was in accordance with an OECD guideline No 412 for testing of chemicals. EPA staff would normally expect a Mass Mean Aerodynamic Diameter and Geometric Standard Deviation for the mist in an inhalation study but these parameters were not provided. Nevertheless the appendix presenting data on particle size results for selected days demonstrates that the material is of respirable size. Additional Comments The EFSA review established 1 mg/m3 (1.08 x107 spores/m3) as the NOAEL (sic, should read NOAEC). EFSA notes that Mycotal contains 1.08 x 1010 spores/gram (92.5% viable). 12.3% of the Mycotal product is the spore power of V. lecanii 11.3% viable spores). The EFSA review notes that Mycotal contains a proteinaceous component at 20% which is known to be a respiratory sensitiser (EFSA refers to Annex C, but the formulation information is in Annex 1 to the report submitted to the EPA on p71). (Based on Annex A, the 20% proteinaceous respiratory sensitiser is skimmed milk powder. This is capable of causing sensitisation from human occupational exposure, but it is not commonly classified as a contact or respiratory sensitiser.)

Conclusion NOAEC of 1 mg Mycotal/m3

General conclusion about target organ systemic toxicity

No data for assessment of the toxicity or classification of the active ingredient for the oral or dermal routes after repeat administration were presented.

Relevant data for the active ingredient for inhalation following repeat administration in the form of a 28 day inhalation study were presented, but these were for the formulated product Mycotal.

The LOAEC of 10 mg/m3 (equivalent to 0.01 mg/L) could trigger classification of 6.9A for repeat systemic target organ toxicity by the inhalation route as a mist in comparison to the threshold of 0.02 mg/l for a 90 day study (EPA, 2008 User guide, p17-9). Note that if the classification were applied it would apply to the formulation Mycotal WG directly rather than to the TGAI.

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This requires the conclusion that the adverse findings at this concentration are systemic effects rather than site of entry effects, and that they are toxicologically significant. EPA staff have concluded that it is more appropriate to view these findings as site of entry effects (probably an immune response in the lung) in which case they would represent a normal physiological response rather than toxic effects. Consequently EPA staff concluded that no classification for target organ systemic toxicity by inhalation should apply.

Other studies - metabolites

Type of study Metabolites and manufacturing impurities

Flag Key study

Test Substance Review of active metabolites associated with Verticillium lecanii species.

LOAEL: N/A Endpoint NOAEL: N/A

Anonymous; 2005. Information on the production of metabolites (especially Reference toxins) by Verticillium lecanii. Koppert Biological Systems (Koppert Beheer BV)

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species N/A

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

A number of metabolites of Verticillium species are referred to in a review of open literature sources and references by Kopper Biological Systems:  Bassianolide, a cyclodepsipeptide, toxic to silk worms (Bombyx mori)

[Cyclodepsipeptide = A depsipeptide in which the amino and hydroxy carboxylic acid residues are connected in a ring.]  Dipicolinic acid (pyridine- 2,6 dicarboxylic acid) toxic to blowflies Study Summary (Calliphora erythrocephala).

 Phospholipids toxic to Bemisia tabaci (silverleaf whitefly) have been isolated from V lecanii isolated from Oxycarenus hyalinipennis (cotton seed bug) in Israel

 Vertilecanins toxic to Helicoverpa zea (corn ear worm).

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The report indicates that production of these metabolites has not been detected in strains V lecanii Ve2 and Ve6.

The EFSA review (EFSA, 2008) also concluded that: “There are no Additional Comments manufacturing impurities which are considered, on the basis of the information currently available, of toxicological or environmental concern.”

There are no manufacturing impurities or metabolites of Verticillium lecanii that Conclusion are known to be of toxicological concern.

Type of study Metabolites and manufacturing impurities

Flag Supporting information

Review of active metabolites from Metarhizium anisopliae V275. Individual Test Substance metabolites (destruxins A, B and E) and the complete crude extract from liquid cultures.

LOAEL: N/A Endpoint NOAEL: N/A

Skrobek, A. and Butt, T. M. 2005. Toxicity testing of destruxins and crude Reference extracts from the insect-pathogenic fungus Metarhizium anisopliae. FEMS Microbiology Letters Vol. 251, p 23 – 28

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guidelines None cited

Human leukemic cell line HL60 (Caucasian promyelotic) and the SF9 insect cell Species line (derived from Spodoptera frugiperda ovaries).

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxin A, B and E) or the complete crude extract from liquid cultures. Study Summary Studies were also done with four separate fractions of the crude extract, which were selected so they did not contain the main metabolites (the destruxins A, B and E). The conclusion was that testing of the crude extract offers an alternative approach to the time consuming and cost-intensive identification and

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toxicological assessment of each individual metabolite produced by a biological control agent (BCA). The pure destruxin A, B and E exhibited no effect on the cell viability of the human cell line at a concentration up to 500 ppm. There was not decrease in the number of live cells after either 4 or 24 hours. Similarly there were not effects from exposure to two or three of the destruxins in combination at 4 or 24 hours. Pure dextruxins or their mixture had no acute effect on SF9 cells up to 500 ppm, but destruxin A and its mixtures exhibited toxic activity after 24 hours of exposure with LC50 values ranging between 5 and 12 ppm. Destruxin B and E or their mixture had not effect on cell viability after 254 hour incubation. The other investigations with the crude extract not including the destruxins are not summarised in detail as they have limited relevance to an assessment of extracts from V. lecanii Ve6. Studies using high concentrations of the complete crude extract caused 50% mortality of HL60 cells after 24 hour incubation. The studies demonstrated that the HS60 cells were more sensitive to the crude extract than to the isolated destruxins. The study showed synergism and antagonism between components in the crude extract and suggested metabolites other than destruxins may be responsible for some of the toxic effects.

EPA staff note that Verticillium lecanii is known to produce destruxins and this is accepted by the EFSA review (EFSA 2008). The data from this study suggest destruxin A, B and E may not be cytotoxic to human cells, while they did show some toxicity in an insect cell line. On the other hand the crude extract from M. anisopliae V275 at high doses was toxic to the human cells, but the relevance Additional Comments of this finding to V. lecanii is uncertain. Since according to the EFSA review V. lecanii only produces destruxins when grown under specified conditions, and does not produce these metabolites under the conditions under which the Mycotal active ingredient is produced, there is no concern related to the destruxin metabolites.

No manufacturing impurities or metabolites of Verticillium lecanii have been Conclusion identified that are known to be of toxicological significance that are relevant to Mycotal WG.

Other studies - human exposure studies

Type of study Human industrial hygiene study

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Flag Supporting information

The relevant substances studies were V lecanii (Mycotal) and V lecanii Test Substance (Veralec)

LOAEL: N/A Endpoint NOAEL: N/A

Doekes, G; et al.; 2004. IgE Sensitization to Bacterial and Fungal Biopesticides Reference in a Cohort of Danish Greenhouse Workers: The BIOGART study. Am J Ind Med Vol 46, p404 -407.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans exposure

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

Preliminary finding. IgE sensitisation appears to have developed in 9-21% of Study Summary individuals following exposure to Verticillium lecanii, but the significance of this in relation to the health status of the affected individuals is unclear.

These data possibly support the default assignment of sensitisation to microbial pesticide active ingredients (MPAI). Additional Comments The EFSA DAR notes that “For a solid conclusion on the sensitisation- properties of Verticillium lecanii Ve6 a more specific IgE detection method is needed in combination with the assessment of work-related symptoms.”

Conclusion No conclusion based on this study is appropriate.

Type of study Occupational hygiene investigation

Flag Supporting information

Test Substance Exposure to wild fungi in cereal harvesting and processing activities.

LOAEL: N/A Endpoint NOAEL: N/A

Darke C. S.; et al.; 1976. Respiratory disease of workers harvesting grain. Reference Thorax, Vol 31, p294-302.

Klimisch Score 4 (Not assignable)

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Amendments/Deviations N/A

GLP N/A

Test Guidelines N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

Workers harvesting grain were investigated for respiratory symptoms on 16 farms between 1970 and 1976. A quarter of the cohort complained of respiratory distress after working on combine harvesters or near grain driers and elevators, with cough, wheezing and breathlessness sometimes so severe as to prevent work. The airborne dust around the combine harvesters contained up to 200 million fungal spores/m3. Verticiliium/Paecilomyces types spores, most from V. lecanii, Aphancladium album and Paecilomyces bacillosporus Study Summary were abundant in the dust. Extracts of these species produced immediate wheal reactions in skin tests, precipitin reactions with sera and rapid decreases in Forced Expiratory Volume

for 1 second (FEV1) when inhaled by affected workers. There were no delayed reactions. Results suggest type I immediate hypersensitivity to the spores although the physical effect of a heavy dust deposit could be important. Drivers could be protected by use of a cab ventilated with filtered air.

These data suggest classification for contact and respiratory sensitisation may be appropriate for microbial pesticides, but the study is old and non-specific in Additional Comments relation to strain for the Verticillium lecanii. EPA staff note there is no reference to EFSA evaluating this report (EFSA, 2008).

Conclusion No conclusion based on this report is appropriate.

Type of study Occupational hygiene investigation

Flag Supporting information

Test Substance Mycotal and Vertalec

LOAEL: Not applicable Endpoint NOAEL: Not applicable

Eaton K. K.; et al.; 1986. Verticillium lecanii. Allergological and Toxicological Reference Studies on Work Exposed Personnel. Ann Occ Hyg Vol 30 (2), p209 – 217.

Klimisch Score 4 (Not assignable)

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Amendments/Deviations None

GLP N/A

Test Guidelines N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

Staff from the institutions that developed V. lecanii strains were examined. These were from Tate and Lyle (T&L) and Glasshouse Crops Research Institute (GCRI).

The majority of the staff at T&L had a full medical examination, respiratory testing, chest X-ray and full blood profile. In total 141 staff were skin tested for allergy, 8 were positive to V. lecanii, four at each institute. Seven of these were allergic and one non-allergic, non-atopic. Sensitisation was not well correlated with “heavy exposure” [EPA staff note information on how “heavy exposure” was defined was not provided.] None of the subjects were aware of symptoms from handling V. lecanii and all Study Summary responses were less than to the histamine positive control (1:1000). Of the eight who responded to V. lecanii, five responded to V. lecanii (Ve2 alone and three to V. lecanii (Ve2 and Ve6). None responded to V. lecanii Ve6 alone. The testing of the two isolates of V. lecanii (Ve2 and Ve6) indicated that the major allergens were in Ve2. The 85 T&L staff completing medical tests (including 4 with positive skin prick tests) revealed no abnormalities. All X-rays were normal. The normal variability of respiratory function did not differ significantly between exposed and unexposed groups. There was no [detectable] toxic effect on blood parameters following exposure to V. lecanii.

These data suggest that a sensitisation response to V. lecanii Ve6 is less likely than from V. lecanii Ve2, although the response to the latter is also weak in Additional Comments comparison to a positive control. EPA staff do not consider these data support a classification of non-sensitising (6.5A and/or 6.5B) because the degree of exposure is unknown.

Conclusion No conclusion based on this report is appropriate.

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Type of study Occupational hygiene investigation

Flag Supporting information

Test Substance Verticillium lecanii (together with other microbiological pesticides)

LOAEL: N/A Endpoint NOAEL: N/A

Baelum J.; et al.; 2003. Sensitization and inflammatory lung disease among greenhouse workers exposed to microbiological pesticides. Department of Occupational and Environmental Medicine Odense, University Hospital, Denmark. Reference

Conference on Occupational Health Risks of Producing and Handling Organisms for Biological Control of Pests in Agriculture (eds. Madsen A. M. et al, 2003), pp13.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

This is the report of an occupational health investigation aimed at evaluating the risk of type I allergy and inflammatory lung diseases during occupational exposure to microbiological insecticides Bacillus thuringiensis and Verticillium lecanii and the fungicide Trichoderma harzianum.

The study included a cross section of 579 persons (32% male and 68% females) in 31 greenhouse companies. Of these 316 were re-examined in the Study Summary follow up 1 year later. Symptoms and working conditions were obtained by interview and annual examinations. Spirometry, bronchial challenge, and skin prick tests with standard inhalatory allergens were measured. The cohort was followed for three years.

The use of Verticillium was not related to any symptoms. Bacillus products were associated with increased prevalence and incidence of itching in the eyes at work and in highly exposed persons, chest tightness. Trichoderma in

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greenhouses was related to cough, difficulty in breathing and itching in the nose and more unspecific “annoyance”. The conclusion was the study showed a relatively high prevalence of symptoms among greenhouse workers. They found a significant but weak relation to estimates of exposure to Bacillus thuringiensis and Trichoderma harzianum, but not to Verticillium lecanii.

The study provides some evidence that use of V lecanii (strain not defined) may not be associated with allergic symptoms when used in greenhouses. EPA Staff consider the data inadequate to provide a basis to support no classification for Additional Comments 6.5A or 6.5B for Mycotal, due to the uncertainty about the level of exposure and the lack of information on the specific strain of V. lecanii workers were exposed to.

The study did not identify cases of sensitisation in greenhouse workers Conclusion presumed to be exposed to species of Verticillium lecanii.

Type of study Investigation in exposed humans

Flag Supporting information

Test Substance N/A

LOAEL: N/A Endpoint NOAEL: N/A

Eaton K. K. and Walport M; 1982. Investigation on human subjects exposed to Reference Verticillium lecanii. Tate and Lyle Group Research and Development, Pilot Plant Building, Deacon Way, Reading, UK.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

The report consists of two separate reports.

Study Summary Report of the investigation of subjects by Dr Eaton.

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Dr Eaton studied 6 individuals, all males, between the ages of 29 and 51. These are identified as atopic (2), allergic (1), non-atopic (1), and neither allergic nor atopic (2).

All subjects were tested using prick tests with Verticillium albo-atrum, Trachoderma and Cephalosporium acremonium] and all tested negative.

Use of Cephalosporium acremonium was due to a previous classification of Verticillium lecanii as a Cephalosporium. The individuals had been exposed to Verticillium lecanii but Dr Eaton was confident that cross reactivity between sub- species would occur.

All the lung function tests were clear. Patch tests had been used with other microorganisms for some subjects previously and results were negative.

The conclusion was that there was no evidence that exposure to Verticillium lecanii under pilot plant conditions resulted in sensitisation of any sort in these 6 subjects who were healthy and without symptoms.

A report on the Allergenic Potential of Verticillium lecanii. By Dr Walport.

Five individuals, all male, between the ages of 23 and 52, were examined. One of individual showed an allergic wheal response to Verticillium lecanii, when exposed at 10 mg/ml. The others gave a negative response. All five individuals had family or personal history indicating atopic status and responded to one or more other common allergens when challenged by Dr Walport. Dr Walport attributed the positive response to the Verticillium lecanii at 10 mg/ml to a response to polysaccharide and suggested this may be a cross reaction to an allergy to other fungi rather than being a specific response to Verticillium lecanii. EPA staff note that the responding individual had used the substance for 8 ½ years. He used it in the laboratory and sprayed it in a closed environment six times per year.

EPA staff note that these reports supports the view that the test microbe of interest is not commonly associated with allergic symptoms. Even among a group of 5 atopic individuals, there was a response in only one individual, which Additional Comments the researcher attributed to a cross reactivity rather than a specific response. EPA staff consider the response in one individual cannot be discounted from being related to the exposure to V. lecanii.

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The study does not provide data indicating that V. lecanii commonly causes Conclusion contact or respiratory allergenicity.

Type of study Clinical report

Flag Supporting information

Test Substance N/A

LOAEL: N/A Endpoint NOAEL: N/A

Das, D. K., et al.; 1997. Fine Needle Aspiration Cytology Diagnosis of a Fungal Reference Lesion of the Verticillium Species. Acta Cytologica Vol 41 (2) p577 – 582.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

The report is of a single clinical case. The patient was found to have an opportunistic infection of a Verticillium species. The species was not identified.

Study Summary The patient was being treated with immune suppressing medications following cancer surgery. The exposure route was assumed to be the result of contamination from his regular insulin injection combined with his immune suppressed status.

EPA staff note that the paper identifies that it is possible for Verticillium species to colonise and survive internally in humans, despite the reports that that the preferred temperature for culturing the organism is circa 22°C. The particularly Additional Comments vulnerable resistance status of the patient means the finding is likely to be very unusual, and of limited relevance. It is also noted that the lesion was successfully controlled by antifungal therapy.

The study showed that in rare circumstances infection with Verticillium species Conclusion is possible in humans.

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Type of study Clinical report

Flag Supporting information

Test Substance N/A

LOAEL: N/A Endpoint NOAEL: N/A

Grandesso, S.; et al; 1996. Fungal peritonitis in peritoneal dialysis: a critical Reference review of seven cases. Alpe Adria Microbiology Journal Vol 5, p15 - 21.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

In a group of seven patients with fungal peritonitis, one case was found to have a Verticillium species as the causative organism. All the patients had been Study Summary treated for bacterial peritonitis and were treated with intraperitoneal antibiotics in the previous two months. All cases were resolved after antifungal therapy.

EPA staff note that the paper identifies that it is possible for Verticillium species to colonise and survive internally in humans, despite the reports that that the preferred temperature for culturing the organism is circa 22°C. The particularly vulnerable resistance status of the patient means the finding is likely to be very Additional Comments unusual, and of limited relevance. It is also noted that the lesion was successfully controlled by antifungal therapy, and that the species and strain was not reported, so the relevance to the proposed microbial pesticides is unknown.

The study showed that in rare circumstances infection with Verticillium species Conclusion is possible in humans.

Type of study Clinical report

Flag Supporting information

Test Substance N/A

Endpoint LOAEL: N/A

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NOAEL: N/A

Shin, J-Y., et al; 2002. Keratitis caused by Verticillium species. Cornea, Vol 21, Reference p240 - 242.

Klimisch Score 4 (Not assignable)

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Species Humans

Strain N/A

No/Sex/Group N/A

Dose Levels N/A

The case of a 50 year old man suffering from infectious keratitis caused by a Study Summary Verticillium species without a history of trauma was described. The patient recovered after anti-fungal therapy.

The strain of Verticillium was not reported, so the relevance to the proposed Additional Comments microbial pesticides is unknown.

Conclusion Corneal infection from Verticillium species is possible in humans, but rare.

Summary of studies with NOAEL and LOAEL values and key effects.

Study type NOAEL LOAEL Key effect

Grey discolouration of the lungs and swollen, 28 day repeat dose spongey appearance at inhalation study with 1 mg (Mycotal)/m3 10 mg (Mycotal)/m3 autopsy. EPA staff note Mycotal (formulated that the toxicological product). significance of these findings are uncertain.

Toxicokinetics

No studies were submitted and the need for such studies was not considered necessary by EPA Staff for an organism not associated with toxic metabolites.

General conclusion about mammalian toxicology of active ingredient(s) and metabolite(s)

The study data do not indicate that the test organism Lecanicillium muscarium 19-79 exhibits toxic, infective or pathogenic effects in mammals. No microbial metabolites have been identified which are relevant to the technical grade active ingredient (TGAI) under the conditions of production.

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Appendix E: Ecotoxicology

Robust study summaries for the formulation (Mycotal WG)

The applicant provided studies with a formulation named Mycotal. The composition of this formulation was not identical to the composition of Mycotal WG. However, the staff consider that the results obtained with the substance “Mycotal” could to some extent be used for the assessment of Mycotal WG.

Aquatic toxicity

Fish Acute Toxicity (Freshwater Species)

Type of study Full test

Flag Key study

Test Substance Ve6-58 SSP (containing 10*1010 spores of V. lecanii per g)

Species Salmo gairdneri [former name of Oncorhynchus mykiss] (rainbow trout)

Type of exposure Static for 96 hours

Endpoint LC50

Value >97 mg a.i./L (> 6.2*109 cfu/L) [measured concentration]

MT Douglas & IB Pell (1983) The acute toxicity of VE6-58 SSP to rainbow trout Reference (Salmo gairdneri). Huntingdon Research Center, Huntingdon, Cambridgeshire. Study TTL 3/83415.

Klimisch Score 2

Amendments/Deviations None that have impacted the results

GLP Yes

Test Guideline/s Not mentioned

10 fish per replicate. 2 replicates for 1000 mg a.i./L; 1 replicate for 10 and 100 No/Group mg a.i./L and control

0 (control), 10, 100 and 1000 mg a.i./L (corresponding to 6.3*107 , 6.3*108 and Dose Levels 6.2*109 cfu/L)

Concentrations in water were verified by standard plate agar counts at 0, 1, 2, 3 Analytical measurements and 4 days after application.

The pH values in the test medium and in the control ranged from 8.24 to 8.37, the dissolved oxygen concentration was always higher than 60% and the temperature was in the range of 14 ± 1 ºC during the test period. Study Summary

The results from the analysis showed that the concentration of V. lecanii were in agreement with the nominal values (tanks prepared at 1000 mg/L of Ve6-58

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SSP which contained 8.8*109 spores/g should contain 107 spores/mL and the recorded values at time 0 were 3.8-7.9 x 106 spores/mL. The discrepancies between nominal and measured values seemed to have been caused by the formation of agglomerates on mixing the test material with water during formulation of the tank waters. The concentration of V. lecanii did not decrease significantly over the 96 hour period at any of the tested concentrations in the presence or absence of fish.

The test item had no toxic effect on the fish at the concentration used in this study. No signs of intoxication and abnormal behaviour were detected during the study. The residue left on the bottom of tanks with fish, when emptied at study termination, was of more granular appearance than that in the “no-fish” tank. Some white faeces were observed indicating that the fish had ingested some portion of the test substance. Fish were observed to “swallow” and then reject the residues at the bottom of the tank. There were no signs of pathogenicity/infectivity in the fish.

9 The 96h-LC50 was higher than 6.2*10 cfu/L (> 97 mg a.i./L).

The test was not performed with Mycotal WG but rather with a substance containing V. lecanii spores. The staff consider that the study provided Comment information about the absence of pathogenicity/infectivity to fish. The recovery of viable spores at the end of the study was very low, between 0.95-11%.

9 Conclusion The 96h-LC50 was higher than 6.2*10 cfu/L (> 97 mg a.i./L).

Fish Acute Toxicity (Marine Species)

No study provided.

Invertebrates Acute Toxicity (Freshwater Species)

Type of study Limit test

Flag Supporting study

Test Substance Mycotal

Species Daphnia sp.

Type of exposure Static for 24 h

Endpoint EC50

Value >6.0 mg/L (corresponding to 3.8*108 cfu/L) [actual concentration]

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RJ Quinlan (1983). Verticillium lecanii acute immobilisation of Daphnia test. Reference Huntingdon Research Center, Huntingdon, Cambridgeshire. Study TTL 3/83415.

Klimisch Score 3: see comments

Amendments/Deviations None

GLP No

Test Guideline/s Not provided

5 daphnids per replicate (4 replicates per concentration, i.e. 20 daphnids per No/Group concentration) Reference toxic: Potassium dichromate

0 (control), 62, 125, 250, 500 and 1000 mg a.i./L (nominal concentrations), Dose Levels corresponding to 3.9*109, 7.9*109, 1.6*1010 , 3.2*1010 , 6.3*1010 cfu/L.

Concentrations verified by standard malt extract plate agar counts 24 hours Analytical measurements after application.

The dissolved oxygen concentration was higher than 70% during the test. The temperature was maintained at 18-22 °C. The pH was 6.0 during the test. Actual concentrations of the active ingredient (a.i) were 0.29-0.66% of the nominal concentrations and constant per treatment. The mortality in the control was 10%. Mortality in treated animals was never greater than that in controls even at the maximum dose of 1 g/L. Mortality, Study Summary where present, appeared to be due to animals becoming trapped in residual cereal flour (filler in the formulation). Dead individual from the first test were washed in distilled water and removed onto malt extract agar. These plates were incubated at 20 °C for 5 days. V. lecanii was not found on any individual taken from treated beakers with Mycotal. Mycotal was considered not infective to Daphnia.

The strain used in the test couldn’t be verified. The time of exposure was only 24h, which is insufficient to evaluate the effects of the substance. The viability Comment of the spores at the end of the experiment was rather low (0.29-0.66% of the nominal concentrations). There was no information about the initial cfu of the product tested.

8 Conclusion 24hr-EC50 > 6 mg/L (corresponding to 3.8*10 cfu/L)

Invertebrates Acute Toxicity (Marine Species) No study provided.

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Algae Acute Toxicity (Freshwater Species) No study provided.

Algae Acute Toxicity (Marine Species) No study provided.

Aquatic Plants Acute Toxicity (Freshwater Species) No study provided.

Chronic toxicity studies No study provided.

Fish Chronic Toxicity (Freshwater Species) No study provided.

Invertebrate Chronic Toxicity (Freshwater Species) No study provided.

General conclusion about aquatic toxicity classification Mycotal WG does not trigger a classification based on the studies provided or as result of the application of mixture rules. However, Mycotal WG is classified as 9.1D (biocidal action) because according to the HSNO Thresholds and Classifications guideline, a substance should be classified for biocidal action if “tested for any of the specific ecotoxicity thresholds using the species identified in the acceptable test methodologies, it may not trigger any of the thresholds for aquatic, soil, terrestrial vertebrate and terrestrial invertebrate ecotoxicity. However, there is still potential for these substances to pose a risk to organisms in the environment.” In addition, a substance is designed for biocidal action if “the substance causes mortality, inhibited growth, or inhibited reproduction in an organism”.

Sediment toxicity (freshwater and/or marine)

No study provided.

Soil toxicity

Soil Macro-Invertebrates Acute Toxicity

No test provided.

Soil Macro-Invertebrate Chronic Toxicity

No test provided.

Non-Target Plant Toxicity

No test provided.

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Microbial respiration and Nitrogen transformation test – activated sludge

No study provided.

General conclusion about soil toxicity classification

Mycotal WG does not trigger a classification for soil toxicity.

Terrestrial vertebrate toxicity

For effects on terrestrial vertebrates other than birds, refer to the mammalian toxicity section.

Oral Acute Toxicity

No study provided.

Dietary Acute Toxicity

No study provided.

General conclusion about terrestrial vertebrate classification

Mycotal WG does not trigger a classification for vertebrate ecotoxicity. In addition, there was no toxicity in the tests with mammals.

Toxicity to terrestrial invertebrates

Bees - Laboratory Tests

No study provided.

Bumblebees – Brood test

Study type Brood test

Flag Supporting study: see comments

Test Substance Mycotal [1010 cfu of Lecanicillium muscarium per gram]

Species Bombus terrestris

Dusted spores direct onto bees and the brood nest and mixed spores through Type of exposure the food (pollen)

Endpoint Impact on bumblebees and brood

No effects to bumblebees and brood were observed after application of Value Mycotal.

A van Doorn (1998) Impact of the fungi Verticillium and Trichoderma on adult Reference bumblebees and bumblebee brood. Research Report R&D Natupol, Kopper Biological systems. [internal report, code no Pr980701]

Klimisch Score 4: originals not seen

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Amendments/Deviations -

GLP No

Test Guideline/s Not stated

15 colonies each with 20 workers No/Group 3 replicates for the Control, Verticillium dust, Trichoderma dust, Verticillium in pollen, Trichoderma in pollen

Dose Levels 0, 1 gram of product (1010 spores per gram)

Analytical measurements Not required

This study concerned the impact of the fungi Verticillium [former name of Lecanicillium muscarium] on adult bumblebees and bumblebee brood (larvae, pupae). Each colony received 1 gram of the product either dusted immediately onto the brood or mixed through the pollen (1 gram mixed through 80 grams of pollen). Bumblebees’ colonies were maintained in the laboratory at 28 °C, 65% Relative Humidity. Observations were made on the occurrence of fungal growth, on the mortality of bumblebees or brood and on colony development. Feeding of Verticillium did not cause any mortality of adults, larvae or pupae Study Summary over a period of 2½ weeks (pupal stage lasts 10 days). There was no obvious impact on the characteristics of colony development, expect for treated colonies (both dusted and fed through pollen) showing some tendency for an earlier production of young queens. It was not clarified if these results could be an artefact or not. The colonies (both adults and brood) were covered with a clearly visible layer of powder during some hours following treatment. The bees cleaned away most of this powder; a small part became integrated into the wax, making the wax more dark and stiff. No fungi were observed growing in the nest box, neither on the brood no on the adults. The conclusion was that Mycotal was safe for bumblebees.

The original report was not seen. There is not enough information to conclude Comment that 2.5 weeks is an adequate period of exposure to detect effects on target insects.

Conclusion Mycotal did not represent a risk for bumblebees and brood.

Non-target arthropods – Laboratory, Semi-field and Field

Type of study Laboratory, semi-field and field

Flag Key study

Test Substance Micro Germin (formulation containing Verticillium lecanii)

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Species Several species of parasitoids, mites and insects

Type of exposure Dried residues, direct exposure

Endpoint Initial toxicity (mortality), effects on reproduction

The formulation was considered harmless to parasitoids, predatory mites and Value insects in laboratory conditions and slightly harmful to Typhlodromus pyri in field conditions, at 4 kg product/ha.

G. Sterk et al (1999). Results of the seventh joint pesticide testing programme Reference carried out by the IOBC/WPRS-Working Group ‘Pesticides and Beneficials Organisms’. Biocontrol, 44:99-117.

Klimisch Score 4: originals not seen

Amendments/Deviations N/A

GLP N/A

Test Guideline/s Test methods mentioned in the IOBC/WPRD Working Group

No/Group Not mentioned

Dose Levels 4 kg/ha [not possible to determine the amount of spores (cfu) in the formulation]

Analytical measurements Not mentioned

In this study the effects of a formulation named Micro Germin containing Verticillium lecanii was tested in laboratory, semi-field and field conditions to several non-target arthropods.

Laboratory test The results showed that Micro Germin was harmless (< 30% effects) to three parasitoids Trichogramma cacoeciae, Encarsia formosa and Aphidius matricariae), two predatory mites (Phytoselulus persimilis, Typhlodromus pyri) and three predatory insects Chrysoperla carnea, Forficula auricularia, Study Summary Semiadalia11-notata).

Semi-field The results indicated that the product was harmless to C. carnea larvae exposed to dried residues on barley and bean leaves and for P. persimilis adults to dried residues at bean leaves.

Field Following spraying of 4 kg product/ha in an orchard the results were slightly harmless (25-50% effects) to T. pyri.

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It was not possible to determine the strain of L. muscarium in the product used in the tests or the amount of spores (cfu) in the formulation. There was no data Comment about the relative humidity or temperature. There is no indication if the exposure period is sufficient to observe adverse effects to non-target arthropods.

The formulation was considered harmless to parasitoids, predatory mites Conclusion and insects in laboratory conditions and slightly harmful to T. pyri in field conditions, at 4 kg product/ha.

General conclusion about toxicity to terrestrial invertebrates classification:

Mycotal WG does not trigger an invertebrate toxicity classification.

Environmental fate - Robust study summaries for the active ingredient and metabolites

For the benefit of consistency, the staff maintained the designation Verticillium lecanii in the test reports summarised in the following sections. In the general comments and in the conclusions about the environmental fate and behaviour the staff used the current designation Lecanicillium muscarium.

Geographic distribution and natural occurence

The entomopathogenic fungus Lecanicillium muscarium has a worldwide geographic distribution on many different substrates: as a soil pathogen (on other fungi), as a hyperparasite on rust fungi, and on plant material (Brady, 1979; Hall, 1981; Rombach and Gillepsie,1988). L. muscarium has also been found as a natural infestation of several greenhouse pests; whitefly on cucumber, chrysanthemum aphids (Ekbom, 1979; Hall, 1975), and has been described to decimate greenhouse populations of aphids and scales (Hall, 1981a). There is extensive literature that describes a broad spectrum of hosts/substrates for the fungus; Hanssler and Hermans (1981) describe the mode of action by which Lecanicillium invades/ attacks the sugar beet cyst-nematode Heterodera schachtii, Hill and Lacey (1983) described the colonisation of ripening grain, Lo and Chapman (1998) described the occurrence on scales. At present, it is not possible to provide an exhaustive analysis of the host range of Lecanicillium spp. The main reasons for this is that the identity to the species level remains unclear in most published studies, the host relationships have mainly been established for the organisms of economic importance (agricultural pests and plant diseases), and almost all studies on host suitability and pathogenicity have been conducted under laboratory and greenhouse conditions (Brodeur, 2012).

Water compartment

Expression in the aquatic environment

Study type Behaviour in water

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Flag Supplemental study

Conidiospores of Verticillium lecanii (with a concentration of approx. 7.5x1010 Test Substance spores/g)

Endpoint Behaviour in water

Half-life time of V. lecanii spores was about 3 days under sterile non aerated Value conditions. In aerated stirred water spores persisted more than 95% after 7 days.

R. Van der Pas (2000) Behaviour of Verticillium lecanii spores in water. Koppert Reference internal study [no study code provided]

Klimisch Score 3: see comments below

Amendments/Deviations N/A

GLP No

Test Guideline/s N/A

Dose Levels 7.5x1010 spores/g

From the non-stirred Erlenmeyer only spores that were settled to the bottom were sampled (direct germination test using agar plates). The spore sample Analytical measurements was diluted 10-100 times and the germination of the spores on the plates was assessed after 16-20 hours (indirect germination test).

Two gram conidiospores (an asexually produced fungal spore formed on a conidiophore) of Verticillium lecanii (with a concentration of approx.7.5x1010 spores/g) were suspended in 500 ml sterile demineralised water in a 1 litre Erlenmeyer. The pH of the water was approx. 7.5. The spores were suspended using a magnetic stirrer, for 30-45 minutes. One Erlenmeyer was kept on the magnetic stirrer constantly.

The spores that were kept on the magnetic stirrer permanently started to germinate after approx. 8 hours. Germination of all spores was accomplished Study Summary after approx. 20 hours. Newly formed blastospores were already formed at that time. After 2 days the germination tubes (from the conidiospores) started to disappear and only the freshly formed blastospores remain in the solution. When the spores were put on water agar they were still able to germinate, even when they were stirred for 7 days. The spores in the non-stirred Erlenmeyer did not germinate during the whole test period. When they were put on agar there was a clear decline in germination (35%) after 2 days with no germination at all after 6 days.

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This research indicated that spores of Verticillium lecanii couldn’t germinate when they were kept in a non-aerated situation. Only the spores that were attached to the glass surface were able to germinate and form a mycelium mat with sporulating fungus (no data was presented). The reason that still 20% germination was found in the non-stirred flask after 5 days (with the indirect germination test) was probably caused by spores from this mycelium mat when a sample from the sediment was taken.

It also seems that spores are not triggered to germinate anymore when the circumstances are not favourable or if there is a lack of nutrients. This is probably what happened in the stirred situation. The newly formed spores did not germinate as contrast with the initial spores that germinated very rapidly.

The half-life time of V. lecanii spores was about 3 days under sterile non aerated conditions. However, V. lecanii spores can survive for a longer time in soil or in an aerated liquid situation. Therefore, in the case of spores entering the surface water with the spray residues, only the spores that are on top of the surface or in an aerated or agitated situation would survive.

The strain used in the test couldn’t be verified. The test report is missing Comments information about the methodology used. The staff consider that the results are indicative of the degradation in water under sterile non aerated conditions.

The half-life time of spores was about 3 days under sterile non aerated Conclusion conditions. In aerated stirred water spores persisted more than 95% after 7 days.

Aqueous Photolysis

No study provided.

Water/sediment aerobic biodegradation

No study provided.

Water/sediment anaerobic biodegradation

No study provided.

Bioaccumulation potential

Bioconcentration test with fish

No study provided.

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Soil compartment

Expression in the soil compartment

Study type Degradation and persistence in soil

Flag Key study

Test Substance Verticillium lecanii [former name of Lecanicillium muscarium]

Endpoint Degradation in soil

Value V. lecanii is viable in soil after 32 days.

BS Hollingsworth (1983) Verticillium lecanii degradation and percolation in soil. Tate & Lyle PLC, Group Research and Development. Philip Lyle Memorial Reference Reserch Laboratory. PO Box 68, Whiteknights, Reading, RG6 2BX, England. [no study code provided]

Klimisch Score 2: see comments

Amendments/Deviations None that have impacted the results

GLP No

Test Guideline/s BBA No 36 - soil degradation

Dose Levels 100 mg a.i. (equivalent to 107 cfu/100 g of soil)

Analytical measurements Dilution platting on malt extract agar

Soil studies were carried out on strains Ve2 and Ve6 of the fungus Verticillium lecanii according to the procedures described in BBA No 36- soil degradation for 32 days. This protocol was modified for use on microbial products. The method of analysis was as follows: 1 gram soil was added to 9 ml solution. A dilution series was prepared. 0.1 ml aliquots of each dilution were plated on selective medium (rose Bengal chloramphenicol agar). Plates were incubated at ambient laboratory temperature, 22 ± 2 ºC. Plates were examined after 3, 5 and 7 days recording the number of V. lecanii colonies and calculating the total number per g soil. Method of analysis: shaking of soil with sterile tap water. The Study Summary solution was diluted and 0.1 ml aliquots were spread on selective agar plates. The V. lecanii colonies were counted after 7 and 10 days incubation at 25 ºC in the dark.

Properties of the soils used in the experiments

Parameter Standard Speyer Standard Speyer 2.2 2.3 Origin Speyer, Germany Speyer, Germany

Texture Loamy sand Sandy loam

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%OC 2.9 1.16

pH 6.1 6

Max WHC (%) 20 24

Following incubation of test soil samples for 4 days V. lecanii counts fell to 30- 40% of the initial level. After this decline the recovery was more or less stable between 26-45% after 32 days.

Only two soils were used, although these soils had different properties such as organic matter content. Around 26-45% of the initial level remained viable in the Comment soil after 32 days, therefore it was not possible to calculate a half-life for L. muscarium.

Conclusion V. lecanii is viable in soil after 32 days.

Percolation in soil

Study type Percolation in soil

Flag Key study

Test Substance Verticillium lecanii [former name of Lecanicillium muscarium]

Endpoint Percolation in soil

Value V. lecanii does not leach trough soil into ground water

BS Hollingsworth (1983) Verticillium lecanii degradation and percolation in soil. Tate & Lyle PLC, Group Research and Development. Philip Lyle Memorial Reference Reserch Laboratory. PO Box 68, Whiteknights, Reading, RG6 2BX, England. [no study code provided]

Klimisch Score 1

Amendments/Deviations None that have impacted the results

GLP No

Test Guideline/s BBA No 37-Soil Percolation.

Dose Levels 1 mg (± 1*107 cfu/column)

Analytical measurements Dilution platting on malt extract agar

Soil studies were carried out on strains Ve2 and Ve6 of the fungus Verticillium lecanii according to the procedures described in BBA No 37-Soil Percolation for Study Summary 28 days. This protocol was modified for use on microbial products. The method of analysis was as follows: 1 gram soil was added to 9 ml solution. A dilution series was prepared. 0.1 ml aliquots of each dilution were plated on selective

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medium (rose Bengal choramphenicol agar). Plates were incubated at ambient laboratory temperature, 22 ± 2 ºC. Plates were examined after 3, 5 and 7 days recording the number of V. lecanii colonies and calculating the total number per g soil. Glass percolation tubes were filled with washed soil to a depth of 30 cm. Soil columns were pre-saturated with water. The water flow was adjusted to 10-12 mL/hr (leaching by 200 mm rainfall). One mg (± 1*107 cfu/column) was added on top of the test soil. Filtrate was collected and sampled.

Properties of the soils used in the experiments

Parameter Standard Speyer Standard Speyer Standard 2.1 2.2 Speyer 2.3 Origin Speyer, Germany Speyer, Germany Speyer, Germany Texture Sand Loamy sand Sandy loam

%OC 0.66 2.9 1.16

pH 6.5 6.1 6

Max WHC (%) 12 20 24

After 4 days there was no evidence of V. lecanii present in the filtrates. Continued percolation until 28 days did not reveal the presence of V. lecanii in the filtrates either.

Conclusion V. lecanii does not leach trough soil into ground water.

Expression in the soil environment

Study type Behaviour in soil

Flag Key study

Test Substance Verticillium lecanii (Zimm.) Viegas strain 24

Endpoint Degradation in soil and mineral wool

In soils with low or intermediate water content (-0.8, to -10 MPa) the cfu decreased slowly. Constant high temperatures (> 30 °C) over longer periods Value reduced V. lecanii significantly. The survival in mineral wool was similar to the one observed in the soil.

P.U. Beyer et al (1996) The behaviour of the entomopathogenic fungus Reference Verticillium lecanii (Zimm.) Viegas in soil II. Longevity of V. lecanii in soil and

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mineral wool and the optimization of its survival by addition of promoting substances. Journal of Plant Diseases and Protection 104: 65-74.

Klimisch Score 2: see comments below

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Soil: 14*107, 7.0*105 , 14*104 spores / g soil. Dose Levels Mineral wool: 2.5*107 blastospores/mL

Analytical measurements Samples were taken after 5, 10, 14, 20, 28 and 40 days (mineral wool)

The viability of V. lecanii was investigated in two soils, mineral wool and under addition of promoting substances.

Table 1 - Properties of the soils used in the experiment

Parameter Garden Soil 1 Garden Soil 2

Origin Germany Germany

Dry soil density (g/L) 502 810

Humus content (% of 36 15 dry matter) pH 133 6.0

Max WHC (g water / 30 72 100 g dry soil)

Study Summary Soil incubation was carried out in 100 mL Erlenmeyer containing 10 g arid dried soil. Three vessels were used per treatment and time. One mL spore suspension was mixed into the soil.

In tests with addition of organic substances, 1.5 mL of stock solution of supplements (soy flour, glycerol or a combination of both) was added to obtain the final concentration and a water content of 20%.

Incubation in mineral wool was done in cylinders (3.8 cm diameter x height of 2.7 cm) and placed in glass beakers. Mineral wool was inoculated with 15 mL of a spore suspension (2.5 *107 blastospores/mL).

Table 2 - Water potential and % of Water Holding Capacity (WHC) at different soil content

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Soil water Soil water potential Percentage of content in % (g WHC water / 100 g wet soil *100)

Soil 1 20 -10 MPa 19

30 -1.5 MPa 33

44 -0.8 kPa 60

Soil 2 5 <-10MPa 7

20 -850 kPa 33

30 -0.5 kPa 60

Results - effects of initial concentration in soil on viability

The intensity of cfu reduction was dependent on the initial spore concentration. At high inoculum concentration (107 spores / g soil) only 0.2 to 1% of the initial concentration in the soil was observed after 40 days. At the lowest concentration (14*104 spores / g soil) 5-10% were recovered. Soil moisture represented an important factor in the decrease of V. lecanii.

Results - survival in different soils

The survival of V. lecanii was influenced by the soil organic content, since the survival was much higher in the soil with more humus content (see table below).

Incubation Garden Soil 1 Garden Soil 2 (days) (cfu / g soil) (cfu / g soil) 33% of 60% 33% of 60% WHC WHC WHC WHC

0 6 6 11 11

5 4 2 3 0.6

10 3 0.3 1.0 0.07

14 2 0.2 0.3 0.03

20 2 0.2 0.2 0.02

2 0.1 0.1 0.02 28

40 1 0.06 0.08 0.02

Results - addition of organic substances to the soil

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Until the 14th day the cfu increased nearly proportionally to the percentage of added soy flour (0.1, 0.25, 0.5 and 1%). After that the cfu dropped in the highest concentrations, whereas it remained constant in the other variants.

Results - survival in mineral wool

The cfu decreased with time in the same manner as in unsterile soil.

Overall, the main conclusions of this study were the following: a) Spore survival was greater in air-dried soil. In soils with low or intermediate water content (-0.8, to -10 MPa) the cfu decreased slowly; b) Constant high temperatures (> 30 °C) over longer periods reduced V. lecanii significantly; c) The survival in mineral wool was similar to the survival observed in the soil.

The study was not conducted with the strain used in Mycotal WG. The staff Comments consider that the results are indicative of the behaviour in soil and mineral wool of the strain used in the test.

In soils with low or intermediate water content (-0.8, to -10 MPa) the cfu decreased slowly. Constant high temperatures (> 30 °C) over longer Conclusion periods reduced V. lecanii significantly. The survival in mineral wool was similar to the one observed in the soil.

Expression in the soil environment

Study type Behaviour in soil

Flag Supplemental study

Test Substance Verticillium lecanii (Zimm.) Viegas strain 24

Endpoint Decrease in soil

Inoculums of 107 spores/g soil, 105 to 106 cfu/g soil will still remain viable after 2 Value to 3 weeks when soil moisture was 20 % (33% MHC)

P.U. Beyer et al (1996) The behaviour of the entomopathogenic fungus Reference Verticillium lecanii (Zimm.) Viegas in soil I. Viability in soil at different ecological conditions. Journal of Plant Diseases and Protection 104: 54-64.

Klimisch Score 2: see comments below

Amendments/Deviations N/A

GLP N/A

Test Guideline/s N/A

Dose Levels 10 mL spore suspension (3.2*107 spores/mL)

Analytical measurements Dilution on agar plates (at 25 °C in darkness)

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The viability of V. lecanii in soil was investigated under different ecological conditions. Conidiospores and blastospores were used in the test. A typical garden soil was used in the test.

Properties of the soil used in the experiment

Parameter Garden Soil

Origin Germany

Dry soil density (g/L) 810

Humus content (% of 15 dry matter) pH 6.0

Max WHC (g water / 72 100 g dry soil)

The influence of changing soil moisture was investigated in open 50 mL glass beakers containing 5 g dried soil inoculated with V. lecanii and moistened to 30% water content. After drying to a water content of 5 to 10% distilled water was added to 30% water content. The number of cfu was determined at the Study Summary beginning and at the end of each drying period.

Water potential and % of Water Holding Capacity (WHC) at different soil content

Soil water Soil water potential Percentage of content in % (g WHC water / 100 g wet soil *100) 5 <-10 MPa 7

20 -850 kPa 33

30 -0.5 kPa 60

40 0.0 kPa 98

The test with sterilised soil was undertaken in 500 mL glass beakers filled with 200 g soil of 30% water content. Sterilisation was carried out 3 times for 60 min at 121°C on 3 consecutive days. Each vessel (with the exception of the control) was inoculated with 10 mL spore suspension (3.2*107 spores/mL) by mixing in the soil. The influence of pH (3.5, 5, 6, 7 and 8.5) to the spore germination was evaluated in the colonies counted after 3, 5 and 7 days at 25 °C in darkness.

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Results – incubation at constant soil moisture

The decrease in cfu was faster in high soil moisture (40%), the degradation was 3 orders of magnitude (103) within 40 days. In contrast, cfu were only slightly decreased at 5% moisture.

Results – incubation at changing soil moisture

The decrease of cfu was considerable during the first and second period of drying. At a level of 10% of the inoculated number was reached and remained nearly constant until the end of the test. No differences were obtained by inoculation with conidiospores and blastospores. The decrease by changing moisture content was lower than at 20% constant moisture and higher in the dry moisture (5 % moisture).

Results – influence of the soil temperature

Incubation at 30 °C resulted in a decrease of the survival of V. lecanii compared to the incubation at 20 °C and 25 °C. The difference in the survival between the latter temperatures was small but significant from the 20th day. Soil incubation at fluctuating temperatures: in the first series the temperature was kept 12h at 20 °C and 12h at 30 °C; in the second series varied from the first series in the maximum day temperature that was set for 5h per day from 30 to 34°C. The decrease in the first series was similar to the decrease observed at the constant temperature of 25°C. In the second series the decrease was much more pronounced, this was attributed to the high temperature (above 30°C) during the incubation.

Results – influence of the pH

There was no difference in the optimum pH of ca 7 for the colony forming between conidio and blastospores. V. lecanii showed a broad tolerance between pH 5-9.

Results – effects of a soil sterilisation

Without sterilisation a general decrease of cfu was observed (two orders of magnitude during 20 days). In sterilised soil there was a cfu increase during 10 days about 50-fold and a subsequent decrease reaching the level of the starting point at 50 days. Measurements in the control vessels were sterile until 20 days, after that period contamination by bacteria and fungi was observed.

Overall, the results shown that V. lecanii can survive in soil over certain periods of time influenced by soil moisture and temperature. Using inoculums of 107 spores/g soil, 105 to 106 cfu/g soil will still remain viable after 2 to 3 weeks when

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soil moisture was 20 % (33% MHC). Unfavourable conditions for the survival were high soil moisture and temperatures equal or greater than 30 °C.

The study was not conducted with the strain used in Mycotal WG. The staff Comments consider that the results are indicative of the decrease pattern of the strain tested in the soil.

Inoculums of 107 spores/g soil, 105 to 106 cfu/g soil will still remain viable Conclusion after 2 to 3 weeks when soil moisture was 20 % (33% MHC).

Anaerobic Degradation in Soil (routes and rates) – Laboratory studies

No study provided

Soil photolysis

No study provided

Adsorption/desorption on soil particles

No study provided

Leaching Potential - Parent column leaching No study provided

Leaching Potential - Aged residue leaching No study provided

Leaching Potential No study provided

General conclusion about environmental fate For the evaluation of the fate and behaviour in the environment the staff evaluated the confidential study reports send by the applicant and various statements, scientific studies and reviews made with Verticillium lecanii (former name of Lecanicillium muscarium). In the study made by Hollingsworth (1983) there was not possible way to calculate a half-life for L. muscarium in soil although a loss of viability of almost 50% is achieved after 4 days in soil. From the same study it was possible to conclude that L. muscarium does not leach into groundwater. Degradation in water was evaluated by Van der Pas (2000), but it was not possible to calculate a half-life for L. muscarium. The mechanisms of spread of L. muscarium are not exactly known. It has been speculated that aphids take spores with them from the soil to the leaves, after which other insects can be infected too. Spores are not spread by air, and are not released from conidiophores without water contact. However, conidia released in this way have a short life span after drying up, preventing spread of infection by the air (Hall, 1981; Gardner et al., 1984). Passive spread can occur by means of splashing, and probably by mechanic transfer by other arthropods present in the greenhouse (Rombach & Gillespie, 1988). In surface water the spores will quickly

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sediment and lose their viability. Therefore, mobility of L. muscarium after application is not expected to occur. In a report sent by the applicant (Koppert, 2001), the effects of temperature and humidity demands of Mycotal were tested at a rate of 1 g product/ (1*107 spores/ml) in plates with water. Spores of strains of L. muscarium both germinate in a temperature range between 5 and 30°C, and the majority of spores germinated within 20 hours at temperatures between 13 and 30°C.

There is no data about the natural background levels in soil for this micro-organism in New Zealand. Therefore, it is impossible to determine if the application of Mycotal WG will or not lead to an increase in L. muscarium levels in the soil. As a result of all the considerations made above the staff consider that there is a data gap concerning the environmental fate and behaviour of L. muscarium in the soil and water compartment.

Ecotoxicity - Robust study summaries for Lecanicillium muscarium 19-79

Most of the studies evaluated by the staff were conducted using the former denomination Verticillium lecanii. The staff maintained that designation in the summaries below. For the general conclusions the staff used the current designation Lecanicillium muscarium.

Aquatic toxicity

Fish acute toxicity (Freshwater species)

No study provided.

Fish acute toxicity (Estuarine/Marine species)

No study provided.

Invertebrates acute toxicity (Freshwater species)

No study provided.

Invertebrates acute toxicity (Estuarine/Marine species)

No study provided.

Algae acute toxicity (Freshwater species)

No study was provided, however the applicant provided a statement about the toxic effects of L. muscarium to algae (Koppert, 2005) where it stated that it is “extremely unlikely that V. lecanii would be toxic to algae […] given the fact that V. lecanii is not toxic or pathogenic to mammals, including humans, is not phytopathogenic, is not harmful to fish and daphnids, and has a low persistence in water, it is extremely unlikely that V. lecanii would be toxic or infective to algae.”

The staff consider that a data gap exists regarding the toxic effects to algae. However, due to the proposed use pattern in greenhouses of Mycotal WG the effects to algae may be limited.

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Algae acute toxicity (Marine species) No study provided.

Aquatic plants acute toxicity (Freshwater species) No study provided.

Inhibition of the luminescent bacteria Vibrio fischeri

Fish chronic toxicity (Freshwater species) No study provided.

Fish chronic toxicity (Marine species) No study provided.

Invertebrates chronic toxicity (Freshwater species) No study provided.

Invertebrates chronic toxicity (Marine species) No study provided.

General conclusion about aquatic toxicity classification

Lecanicillium muscarium 19-79 is classified as 9.1D (biocidal action).

Sediment toxicity (freshwater and/or marine)

Invertebrates acute toxicity (sediment-dwelling species)

No study provided.

Invertebrates chronic toxicity (sediment-dwelling species)

No study provided.

General conclusion about sediment toxicity

There were no studies provided with sediment organisms.

Soil toxicity

Soil macro-invertebrates acute toxicity

Study type Acute earthworm test

Flag Key study

Test Substance Verticillium lecanii

Species Eisenia fetida

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Type of exposure Spiked soil for 14 days

Endpoint LC50

Value >1000 mg/kg dw (equivalent to 6.3 *1010 cfu/kg soil dw)

S. Wachter (2000) Acute toxicity of Verticillium lecanii on earthworms, Eisenia fetida using an artificial soil test. GAB Biotechnologie GmBH & IFU Reference Umweltanalytik GmBH, Niefern-Öschelbronn, Germany. Report No 20001292/01-NLEf

Klimisch Score 1

Amendments/Deviations None that have impacted the results

GLP Yes

Test Guideline/s OECD 207 (1984)

4 replicates with 10 worms for control and test concentrations. No/Group 2 replicates with 10 worms for the toxic standard.

0 (control), 100, 178, 316, 562 and 1000 mg/kg dw soil (mixed with soil). Dose Levels Toxic reference (positive control) = 2-chloroacetamide

Analytical measurements None

The test was carried out using the OECD artificial soil containing 10% sphagnum peat. Incubation of the test containers was carried out at 20 ± 2 °C under continuous artificial lightning (400-800 lux). The pH of the soil varied between 6.4 and 6.2. Earthworms were assessed for mortality and behavioural effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day Study Summary 14. After 14 days of exposure no mortality was recorded at the test concentrations of 100 and 562 mg/kg soil and in the control group. At the concentrations of 178, 316 and 1000 mg/kg soil 2.5% mortality was recorded. The biomass weight was not influenced in any of the test groups by exposure to

V. lecanii. The LC50 was higher than 1000 mg/kg soil, the LOEC (weight loss) was higher than 1000 mg/kg soil and the NOEC for weight loss was 1000 mg/kg soil.

10 Conclusion The LC50 was higher than 1000 mg/kg dw soil (6.3 *10 cfu/kg dw soil).

Other species acute toxicity

No study provided.

Soil macro-invertebrates chronic toxicity

No study provided.

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Other species chronic toxicity

No study provided.

Non-target plants toxicity

No study provided.

Microbial respiration and Nitrogen transformation test

No study provided.

General conclusion about soil toxicity classification

Lecanicillium muscarium does not trigger a soil toxicity classification.

Terrestrial vertebrate toxicity

For effects on terrestrial vertebrates other than birds, refer to the mammalian toxicity section.

Oral acute toxicity

Type of study Acute oral toxicity

Flag Key study

Test Substance Verticillium lecanii KV013

Species Coturnix coturnix japonica (Japonese quail)

Type of exposure 5 days of oral administration (gavage) + 25-day observation period

Endpoint LD50

Value > 19 mg/kg bw corresponding with 1.2*109 cfu/kg bw

Y Onishi (1998) An acute toxicity study of Mycotal TGAI administered orally to Reference Japanese quails. Mitsubishi Chemical Safety Institute Ltd., 1-30 Shiba 2- chome, Minato-ku, Tokyo, Japan. Report No 7L785

Klimisch Score 2: see comments

Yes: quarantine and acclimation period was 8 and 9 days instead of 7 days as Amendments/Deviations described in the protocol.

GLP Yes

Guidelines for Safety Evaluation of Microbial Pesticides - 9 Nousan No 5090 Test Guideline/s (1997)

No/sex/Group 3 replicates of 10 birds per treatments and control (5 males and 5 females)

Dose Levels 0 (control), 19 mg/kg bw for 5 days

Analytical measurements Malt extract agar media was used to detected the test microbe

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Mycotal TGAI was administered for 5 consecutive days, orally to 15 male and

8 15 female WE strain Japanese quails (28 days old) at a dose of 10

spore/animal/day. The studies were conducted separately as Experiment I, II and III which initiated 3 consecutive days. Five males and five females were used for dose group and control group in each Experiment. All birds at the termination of the study were necropsied and examined for Study Summary pathological changes. No animal died, and no abnormalities were observed in clinical signs body weight or necropsy. In faecal examination the test microbe was not detected in faeces during the study period. No test microbes were detected in kidneys, brain, liver, lungs, spleen, cecum and blood. There were no signs of toxicity/infectivity or pathogenicity during the study and no internal persistency under the study conditions.

The dose tested was not representative of the expected concentration after the Comments application of Mycotal WG. The staff consider that the results indicate that there was no infectivity/pathogenicity to birds.

9 Conclusion LD50 > 19 mg/kg bw (for both sexes), corresponding to 1.2*10 cfu/kg bw

Chronic toxicity (reproduction) No study provided.

General conclusion about terrestrial vertebrate classification

L. muscarium does not trigger a terrestrial vertebrate classification.

Ecotoxicity to terrestrial invertebrates

Bees - Laboratory tests (Oral and Acute Toxicity)

Study type Acute oral and contact

Flag Key study

Test Substance Verticillium lecanii [former name of Lecanicillium muscarium]

Species Apis mellifera

Type of exposure Oral, Contact for 96 hours

Endpoint LD50

Oral: > 112.32 µg/bee Value Contact: > 100.0 µg/bee

A Kling (2000) Assessment of side-effects of Verticillium lecanii to the Reference honeybee, Apis mellifera L. in the laboratory. GAB Biotechnologie GmBH & IFU

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Umweltanalytik GmBH, Niefern-Öschelbronn, Germany. Report No 20001292/01-BLUE

Klimisch Score 2 see comments

Amendments/Deviations None that impacted the results of the study

GLP Yes

Test Guideline/s EPPO 170 (1992)

No/Group 5 replicates of 10 individuals

Oral Test (actual intake): 0, 1.30, 2.80, 6.02, 13.44, 28.17 and 112.32 µg a.i./bee, Dose Levels Contact Test: 0, 100 µg a.i./bee Toxic standard: Dimethoate

Analytical measurements Not required

The objective of this study was to evaluate the toxicity and pathogenicity of the test items to the Honey bees (Apis mellifera). In the oral toxicity test up to the highest concentration (112.32 µg a.i./bee) the maximum corrected mortality was 11.6% after 96 hours. In the control the mortality was 14%. Study Summary In the contact toxicity test there was no corrected mortality was observed at the tested dose of 100.0 µg a.i./bee. In the control the mortality was 14%. The mortality was not significantly different between the test groups and there were no toxic symptoms observed in the test groups during the test period. The test solution consumption was not significantly different between the test groups.

In the test report, previous studies (considered invalid) are mentioned to justify the limit concentration for the contact test at 100.0 µg/bee. The staff couldn’t check the veracity of this statement. Comment The control mortality in this test is higher (> 10%) than what is considered acceptable for this kind of test according to OECD criteria. As the active ingredient is a biopesticide, the U.S. EPA OCSPP Guideline: 885.4380 is more appropriate as the test duration is up to 30 days.

LD50 (oral) was higher than 112.32 µg/bee Conclusion LD50 (contact) was higher than 100.0 µg/bee

Bees - Field Tests

No study provided.

Other non-target arthropods

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In the following studies, reduction of beneficial capacity (E) is calculated as follows: E (%) = 100-{(100-M)xR} with M = corrected mortality and R = reproductive ratio

The IOBC classification categories for laboratory tests is as follows: Reduction in beneficial capacity (E): 1. Harmless (E< 30%) 2. Slightly harmful (≥ 30 E ≤ 79%) 3. Moderately harmful (> 79 E ≤ 99%) 4. Harmful (E> 99%) The IOBC classification categories for extended laboratory tests is as follows: 1. Harmless (E< 25%); 2. Slightly harmful (25% ≤ E <50%) 3. Moderately harmful (50% ≤ E <75%) 4. Harmful (E ≥75%)

Non-target arthropods – Lab study

Type of study Lab study

Flag Supporting study

Test Substance Strains of V. lecanii (CBS 455.82, 456.82 and 457.82)

Aedes aegyptii, Blatella germanica, Encarsia formosa, Phytoseiulus persimilis Species and Pieris brassicae

Type of exposure Suspension applied by fine mist

Endpoint Toxicity/pathogenicity to non-target insects

Value Strains were considered non-hazardous for the species tested

RJ Quinlan and MA Chaudry [no date provided] Non-target insect test for Reference toxicity/pathogenicity. University of Reading, Department of Zoology, Citadel Building, Earley Gate, Reading, England. [no study code provided]

Klimisch Score 3

Amendments/Deviations N/A

GLP No

Test Guideline/s N/A

No/Group 10 adult insects

Dose Levels 2.5 g/L

Analytical measurements Not mentioned

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Samples of 3 strains (CBS 455.82, 456.82 and 457.82) of commercially manufactured and formulated Verticillium lecanii were tested against 5 insect species (Aedes, Blatella, Encarsia, Phytoseiulus and Pieris) for toxicity and Study Summary pathogenicity over 28 days. No infection was observed in Aedes, Blatella and Pieris. Slight infection of Encarsia and Phytoseiulus was observed above 90% of relative humidity (RH) but none below 70% RH. It was concluded that V. lecanii did not present a hazard to the insects at conventional humidity levels.

The study lacks information about the methodology (eg amount of cfu tested). Comment The strains used in the study were not the strain in Mycotal WG.

Conclusion Strains were considered non-hazardous for the species tested.

General conclusion about toxicity to terrestrial invertebrates classification

L. muscarium does not trigger a classification for terrestrial invertebrates.

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Appendix F: Ecotoxicological risk assessment

Summary of the data on the active ingredient and its metabolites

A summary of the environmental fate data is provided in Table F1.

Table F1: Summary of environmental fate data on Lecanicillium muscarium 19-79– Values in bold are used for the risk assessment. Test Lecanicillium muscarium 19-79

Conidiospores do not germinate in non-aerated Persistence in the conditions and remain viable only for 2-3 days, aquatic environment but in stirred water they persist for more than 95% after 7 days.

Spore counts fell to 30-40% following incubation Persistence in the for 4 days at 22°C and 40% maximum water terrestrial environment holding capacity.

Additional studies were provided but their validity was not good enough to be considered for further assessment.

Table F2: Summary of ecotoxicological data on Lecanicillium muscarium 19-79– Values in bold are used for the risk assessment. Lecanicillium Test Mycotal WG muscarium

Aquatic organisms

Oncorhynchus >97 mg a.i./L (> mykiss – rainbow - 3.2*109 cfu/L)* trout

Freshwater

invertebrates >6 mg a.i./L (> - 8 (Daphnia sp, 24hr- 3.8*10 cfu/L)*

EC50)

No studies Algae - submitted

Acute Earthworm LC > 1000 mg 50 - Eisenia fetida a.i./kg soil dw (>

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6.3*1010 cfu/kg soil dw)

No studies Terrestrial plants - provided

Coturnix coturnix

japonica (Japanese LC50 > 19 mg quail); 5 day oral a.i/kg bw (> - exposure + 3 days 1.2*109 cfu/kg bw observation

Oral: > 112 µg a.i./bee (> 7.1*106

Oral and Contact/ cfu/bee) - Honeybees Contact: > 100 µg a.i./bee (> 6.3*106 cfu/bee)

The formulation was considered harmless to parasitoids, predatory mites Non target and insects in arthropods laboratory (laboratory) conditions and slightly harmful to T. pyri in field conditions, at 4 kg product/ha *.

* test performed with a formulation different from Mycotal

- No data available

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Risk assessment Methodology

Methods used to assess environmental exposure and risk differ between environmental compartments.

Table F3 Reference documents for environmental exposure and risk assessments Environmental exposure Risk assessment

(GEN)eric (E)stimated Overview of the Ecological Risk Assessment (E)nvironmental (C)oncentration Process in the Office of Pesticide Programs, U.S. Aquatic organisms Model Version 2.0 – 01 August Environmental Protection Agency. Endangered 2002 and threatened Species Effects Determinations – AgDrift and EPA Software11 23 January 2004

Guidance on information requirements and Guidance on information requirements and Sediment chemical safety assessment, chemical safety assessment, Chapter R.10: organisms Chapter R.16: Environmental Characterisation of dose [concentration]-response Exposure Estimation, Version: 2 - for environment – May 2008 May 2010

Soil persistence models and EU Soil organisms, registration. The final report of the work of the Soil Modelling Work SANCO/10329/2002 rev 2 final. Guidance invertebrates group of FOCUS (FOrum for the Document on terrestrial ecotoxicology under (macro- Co-ordination of pesticide fate Council Directive 91/414/EEC- 17 October 2002 invertebrates) models and their USe) – 29 February 1997

Guidance for assessing pesticide risks to bees. US EPA, Health Canada Pest Bees Management Regulatory Agency, California Department of Pesticide Regulation, 19 June 2014

Guidance document on regulatory testing and risk assessment procedures for plant Terrestrial protection products with non-target arthropods. From ESCORT 2 Workshop – 21/23 organisms, March 2000

11 The Staff used two different models for assessing the EEC and associated risks:  Generic Estimated Environmental Concentration Model v2 (GENEEC2) surface water exposure model (USEPA, 2001) estimates the concentration of substance in surface water which may arise as a result of surface runoff and spraydrift.  To examine how buffer zones would reduce the active ingredient concentrations in receiving waters, the Staff used the AgDRIFT® model (developed under a cooperative Research and Development Agreement, CRADA, between the EPA, USDA, US Forest Service, and SDTF). AgDRIFT® incorporates a proposed overall method for evaluating off- site deposition of aerial, orchard or ground applied pesticides, and acts as a tool for evaluating the potential of buffer zones to protect sensitive aquatic and terrestrial habitats from undesired exposures. Calculations are made assuming the receiving water is a 30 cm deep pond. The model is used to estimate the buffer zone that would reduce exposure through spray drift to such a concentration that an acute risk quotient of 0.1 cannot be calculated. It is noted that unlike GENEEC2, AgDRIFT® model only considers transport by spray drift, input through runoff, volatilisation, etc will pose additional risks.

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invertebrates (non- target arthropods)

Guidance of EFSA. Risk assessment to birds and mammals – 17 December 2009. Terrestrial EFSA calculator tool - 200912 vertebrates (birds) SANCO/4145/2000 final. Guidance Document on risk assessment for birds and mammals under Council Directive 91/414/EEC- 25 September 2002

Technical Guidance Document on risk assessment in support of Commission Directive 93/67/EEC Guidance of EFSA. Risk assessment to birds and on Risk Assessment for new notified mammals – 17 December 2009 substances, Commission Secondary EFSA calculator tool - 2009 Regulation (EC) No 1488/94 on poisoning and Risk Assessment for existing SANCO/4145/2000 final. Guidance Document on biomagnification substances, Directive 98/8/EC of risk assessment for birds and mammals under the European Parliament and of the Council Directive 91/414/EEC- 25 September Council concerning the placing of 2002 biocidal products on the market – Part II - 2003

Consideration of threatened native species

No studies are requested to be conducted on native New Zealand species; the risk assessment is based on studies performed on standard surrogate species from Europe or North America. Uncertainty factors included in the risk assessment process encompass the possible susceptibility variations between the surrogate species and the native New Zealand species. However, these factors are designed to protect populations’ not individual organisms. EPA staff acknowledge that these factors may not be protective enough for threatened species for which the survival of the population could depend on the survival of each and every individual.

Therefore, the US EPA approach for risk assessment of endangered species has been implemented. Additional uncertainty factors are included, depending on the type of organisms. US EPA consider higher factors when organisms cannot escape the contaminated area (for aquatic organisms for instance) than for birds.

US EPA has not defined any additional factor for soil organisms except for plants, so EPA staff applied the same approach as for aquatic environment, considering that soil invertebrates won’t be able to escape from the contaminated area.

For the purpose of this risk assessment, the threatened species are those included in the following categories of the New Zealand Threat Classification System: threatened (Nationally critical, Nationally endangered, Nationally vulnerable) and at risk (declining, recovering, relict, naturally uncommon).

12 www.efsa.europa.eu/en/efsajournal/pub/1438.htm

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Aquatic risk assessment

For Class 9 substances, irrespective of the intrinsic hazard classification, the ecological risk can be assessed for a substance by calculating a Risk Quotient (RQ) based on an estimated exposure concentration. Such calculations incorporate toxicity values, exposure scenarios (including spray drift, leaching and run-off, application rates and frequencies), and the half-lives of the component(s) in water. For the aquatic environment, the calculations provide an Estimated Environmental Concentration (EEC) which, when divided by the L(E)C50 or a NOEC, gives a RQ acute or chronic.

퐸퐸퐶 퐴푐푢푡푒 푅푄 = 푠ℎ표푟푡−푡푒푟푚 퐿(퐸)퐶50

퐸퐸퐶 퐶ℎ푟표푛𝑖푐 푅푄 = 푙표푛𝑔−푡푒푟푚 푁푂퐸퐶

If the RQ exceeds a predefined level of concern, this suggests that it may be appropriate to refine the assessment or apply the approved handler control and/or other controls to ensure that appropriate matters are taken into account to minimize off-site movement of the substance. Conversely, if a worst-case scenario is used, and the level of concern is not exceeded, then in terms of the environment, there is a presumption of low risk which is able to be adequately managed by such things as label statements (warnings, disposal). The approved handler control can then be removed on a selective basis.

Levels Of Concern (LOC) developed by the USEPA (Urban and Cook, 1986) and adopted by EPA determine whether a substance poses an environmental risk (Table F4Table ).

Table F4 Levels of concern as adopted by EPA New Zealand Endpoint LOC Presumption

Aquatic (fish, invertebrates, algae, aquatic plants)

Acute RQ ≥ 0.5 High acute risk

Acute RQ 0.1 - 0.5 Risk can be mitigated through restricted use

Acute RQ < 0.1 Below the level of concern

Chronic RQ ≥ 1 High chronic risk

Aquatic threatened species

Acute RQ ≥ 0.05 High acute risk

Chronic RQ ≥ 0.1 High chronic risk

Plants (terrestrial)

≥ 1 calculated on the basis of

Acute RQ EC25 or ≥ 5 calculated on the High acute risk

basis of EC50

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Threatened plants species (terrestrial)

≥ 1 calculated on the basis of Acute RQ High acute risk the NOEC or EC05

Expected environmental concentrations modelling

Calculation of expected environmental concentrations

The parameters used for the expected environmental concentrations modelling are listed in Table F5.

Table F51 Input parameters for Expected Environmental Concentrations (EEC) in Surface Water Vegetables, Vegetables, Scenario Ornamental, Small Ornamental, Small fruits > 50 cm fruits > 50 cm

Application rate (g ai/ha) 1152 96

Application frequency 1 12

Application interval (days) - 7

Surface water body type and depth Pond, 30 cm Pond, 30 cm

Methods of application High Volume Spray High Volume Spray

Drift (%) [1] 8.02 6.26

[2] Water DT50 - -

[1] Guidance Document on Aquatic Ecotoxicology in the frame of the Directive 91/414/EEC' Sanco/3268/2001 pages 19-21

[2] It was not possible to determine a DT50 based on the tests provided by the applicant.

Since there is no information about aerobic soil degradation, water solubility, hydrolysis, aerobic aquatic degradation and aqueous photolysis of Lecanicillium muscarium 19-79 it was not possible to use GENEEC to calculate the amount in surface water. Therefore, the staff used an excel spreadsheet to calculate the estimated concentration in a standard water body of 1 m width and 30 cm depth using Ganzelmeier drift values (Ganzelmeier et al, 1995). This spreadsheet was obtained from the CRD website (http://www.pesticides.gov.uk/guidance/industries/pesticides/topics/pesticide-approvals/pesticides- registration/data-requirements-handbook/Environmental-Fate-Models). The staff followed a tiered approach for the determination of the EEC. As a first step a maximum loading was assumed based on the total amount of substance applied over the season/year and assuming that no degradation occurs. The maximum Estimated Environmental Concentration (EEC) for L. muscarium was estimated as 30.8 µg/L. This EEC was estimated assuming a drift value of 8.02%, the maximum drift value for the application on vegetables, ornamentals and small fruits in open field conditions. This approach was considered to be the worst-case estimation of the EEC in water. For permanent enclosed structures no drift is expected to occur, therefore no contamination of surface water is expected.

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Calculation of acute risk quotients using expected environmental concentrations

Table F6 gives calculated acute risk quotients for each trophic level considering EEC estimated and lowest relevant toxicity figures.

Table F6 Acute risk quotients derived from the model and toxicity data

LC50 or EC50 Trigger value / Species Peak EEC (mg/L) Acute RQ (mg/L) Presumption

Greenhouse crops – 96 g ai/ha, 12 applications at 7 days interval

Fish, <0.1 no concern Rainbow trout >97 < 0.00032 <0.05 no concern to threatened species 0.031 <0.1 no concern Daphnids >6.0 < 0.005 <0.05 no concern to threatened species

Calculation of chronic risk quotients using expected environmental concentrations

No chronic studies were provided with fish and/or aquatic invertebrates. Therefore, it was not possible to evaluate long-term effects to aquatic organisms. Due to the use pattern of Mycotal WG long-term effects cannot be excluded, thus the staff consider that a data gap exists.

Conclusion for the aquatic risk assessment Acute risks to aquatic organisms were below the level of concern.

Groundwater risk assessment

The staff adopted the EU approach regarding groundwater contamination in covered crops (EFSA, 2014). Leaching to groundwater from protected crop systems may occur, depending on environmental conditions, the construction technology of the system and the substance properties. The higher temperature (annual average) appeared to be the major factor in the lower leaching. Only closed walk-in systems are expected to have higher annual average temperatures. For open walk-in structures and low structures, the leaching is expected to be closer to the leaching from open field under otherwise comparable circumstances.

Conclusion for the groundwater risk assessment The presence of Verticillium lecanii [former name of Lecanicillium muscarium] in public drinking water in Germany was considered in a publication (Göttlich et al, 2002). The main conclusion was that L. muscarium did not belong to the fungal flora of groundwater-derived public drinking water. Taking into account the information about L. muscarium in soil, the staff consider that potential for groundwater contamination can be considered negligible in the conditions of the use pattern of Mycotal WG.

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Sediment risk assessment

No specific calculation is performed for sediment dwelling organisms because no studies were provided with sediment organisms.

Terrestrial risk assessment

For terrestrial organisms, Toxicity-Exposure Ratios (TERs) are used for earthworms and birds, Hazard Quotient (HQ) are used for terrestrial invertebrates and Risk Quotient (RQ) for bees. This convention results in concern arising if a risk quotient is less than the trigger value for earthworms and more than a trigger value for terrestrial invertebrates. LOC developed by the European Union and adopted by the Staff allowing to determine whether a substance poses an environmental risk are provided in the Table F7.

Table F7 Levels of concern as adopted by the Staff Level of Concern (LOC) Presumption

Earthworm/ Birds

Acute TER < 10 High risk

Chronic TER < 5 High risk

Threatened Bird species

Acute TER < 20 High risk

Chronic TER < 10 High risk

Threatened soil organisms species

Acute TER < 100 High risk

Chronic TER < 50 High risk

Bees

Acute RQ oral/contact > 0.4 High risk

Chronic RQ > 1 High risk

Terrestrial invertebrates

HQ in-field/off-field ≥2 High risk

For more details about the different factors used for calculating TER and RQ refer to the relevant reference documents listed in Table F3. Earthworm risk assessment

Soil Predicted Environmental Concentration (PEC) determination Both acute and reproductive earthworm tests are static tests where the test substance is applied to the system only once at the beginning. Therefore the nominal dose levels in the test match initial concentrations

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The concentration of active substance in the soil is calculated on the basis of the FOCUS (1997) document ‘Soil persistence models and EU registration’

푎푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 (kg a.i./ha) 푃퐸퐶 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 (mg/kg soil) = × 100 75 푘𝑔 푠표𝑖푙

Soil concentrations of the active ingredient are calculated by assuming the deposition would mix into the top 5 cm of soil, and this soil would have a bulk density of 1,500 kg/m3, i.e. the deposition expressed in mg/m2 would mix into 75 kg of soil.

In case of multiple applications, the following formula has to be used:

(1 − 푒−푛푘𝑖) 푃퐸퐶 푚푢푙푡𝑖푝푙푒 푎푝푝푙𝑖푐푎푡𝑖표푛푠 = 푃퐸퐶 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 × (1 − 푒−푘𝑖) where: n = number of applications

-1 k = ln2/DT50 (day ) i = interval between two consecutive applications (days)

o DT50 = half life in soil (days) Use only DT50 values of lab test done at 10-20 C and pH between 5 and 9. e = 2.718 (constant)

When there are DT50 values of several soils use GENEEC2 formula for determining the relevant DT50 to be used.

PEC calculation results are summarized for each scenario in Table and Table F9.

Calculation of TERs

퐿퐷 푇퐸푅푎푐푢푡푒 = 50 퐸푠푡𝑖푚푎푡푒푑 퐸푛푣𝑖푟표푛푚푒푛푡푎푙 퐶표푛푐푒푛푡푟푎푡𝑖표푛 푁푂퐸퐶 푇퐸푅푙표푛𝑔 − 푡푒푟푚 = 퐸푠푡𝑖푚푎푡푒푑 퐸푛푣𝑖푟표푛푚푒푛푡푎푙 퐶표푛푐푒푛푡푟푎푡𝑖표푛

Table F8 Acute in-field TER value for earthworms

Scenarios PEC (mg/kg LC50 Trigger value / TER acute soil) (mg/kg soil) Presumption

>10 / no concern Greenhouse crops - 12 applications of 96 g ai/ha at 7 1.5 >1000 > 653 >100 / no days interval concern for endangered species

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Table F9 Acute off-field TER value for earthworms

Scenarios PEC (mg/kg LC50 Trigger value / TER acute soil) * (mg/kg soil) Presumption

>10 / no Ornamentals, Strawberries < concern 50 cm - 12 applications of 96 g 0.023 >1000 >42946 >100 / v for ai/ha at 7 days interval endangered species

>10 / no concern Ornamentals, Strawberries > >100 / no 50 cm - 12 applications of 96 g 0.096 >1000 >10428 concern for ai/ha at 7 days interval endangered species *Basic drift values from more than 7 applications (Ganzelmeier et al, 1995).

For structures that can be considered non-permanent, that are limited in space and temporary in time the risk assessment for the soil compartment was performed using the same approach as for the open field. Therefore, the staff considered the basic drift values for more than 7 applications according to the drift values from open field scenarios. For permanent enclosed structures the staff considered that spray drift was of no concern and therefore no specific risk assessment was considered for off-field situations.

Conclusion for earthworm acute risk assessment

The risk to earthworms in field and off-field situations was considered to be below the level of concern.

Conclusion for earthworm chronic risk assessment

There were no studies on the chronic effects of Mycotal WG to earthworms or other soil organisms. The staff consider that a data gap exists, since chronic exposure cannot be excluded from the use pattern of Mycotal WG when applied in non-permanent structures.

Non-target plant risk assessment

Non target plants are non-crop plants located outside the treatment area. No studies were submitted concerning the effects to non-target plants. The staff considered that terrestrial plants are not among the natural target or host of L. muscarium and that the risks can be considered below the level of concern.

Conclusion for non-target plant risk assessment

The staff consider that the effects to non-target plants can be considered below the level of concern.

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Bird risk assessment

EPA uses EFSA’s Bird model and Excel© spreadsheets13 freely available on EFSA’s website to assess the risks to birds.

The methodology calculates TERs where exposure is calculated as the dose that a bird will receive when feeding in crops that have been sprayed. To avoid doing detailed evaluations for low risk scenarios, assessments are performed in tiers of increasing complexity.

The steps for the acute assessment are: (a) Screening assessment (b) Tier I assessment (c) Higher tier assessment

The steps for the reproductive assessment are: (d) Screening assessment (e) Phase-specific approach assessment (f) Higher tier assessment

Progression to the next tier is only made if the threshold for concern is exceeded at the previous tier.

Screening risk assessment (g) Determination of levels of exposure The principles underlying the exposure assessment are the same for all assessments other than higher tier assessments in which more specific field exposure data may be used. The dose that a bird receives (Daily Dietary Dose or DDD) is calculated from the application rate and a so-called ‘Shortcut value’ for the Residue per Unit Dose (RUD), reflecting the concentration on the bird’s food and the quantity of food consumed. Quantities consumed are based on a bird’s energy requirements, its energy assimilation and the energy content of its food (dry weight). Birds’ energy requirements are based on an algorithm based on bodyweight and bird type (e.g. passerine/non- passerine). For further details, refer to EFSA’ technical guidance document. Both screening step assessments (acute and reproduction) select from 6 ‘indicator species’ each applicable to a particular type of crop. They are not real species, but, by virtue of their size and feeding habits, their exposure is considered worst-case for birds in a particular crop type. For example, the representative species for orchards is described as a ‘small insectivorous bird’. It is assumed that the relevant indicator species feeds only on contaminated food and the concentration of pesticide on the food is not affected by the growth stage of the crop. Thus, the exposure assessment is expressed as follows depending on the number of applications:

13 different spreadsheets for spray application, granular application and seed treatment. For bait applications a spreadsheet with Daily Food Intake of NZ relevant species is available (Crocker et al., 2002).

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For acute test:

퐷퐷퐷 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 = 푎푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 (푘𝑔/ℎ푎) × 푠ℎ표푟푡푐푢푡 푣푎푙푢푒

퐷퐷퐷 푚푢푙푡𝑖푝푙푒 푎푝푝푙𝑖푐푎푡𝑖표푛푠 = 퐷퐷퐷 표푛푒 푎푝푝푙𝑖푐푎푡𝑖표푛 × 푀퐴퐹90

For reproduction test:

퐷퐷퐷 = 푎푝푝푙𝑖푐푎푡𝑖표푛 푟푎푡푒 (푘𝑔/ℎ푎) × 푠ℎ표푟푡푐푢푡 푣푎푙푢푒 × 푇푊퐴 ∗ × 푀퐴퐹푚푒푎푛

*if toxic effect is considered to be caused by long-term exposure, use TWA = 0.53 (estimates time-weighted exposure over 21 days assuming a default DT50 of 10 days).

The exposure to L. muscarium for bird acute, dietary and reproductive screening assessments is shown in Tables F10 and F11 respectively.

Table F10 Exposure of birds for acute screening assessment

Crop & BBCH Application Short-cut MAF Indicator No of class (where rate value (90th DDD species2 applications appropriate)1 (kg/ha) (90th%)3 %)4

Small omnivorous Strawberries 0.096 158 2.0 12 30.49 bird

Small Ornamentals insectivorous 0.096 46.8 2.0 12 8.99 and nursery bird

1 EFSA, 2009, Table 5 p27

2 EFSA, 2009, Table 6 p28

3 EFSA, 2009, Table 6 p28

4 EFSA, 2009, Table 7 p29

5The exposure assessment of the reproduction assessment uses time-weighted average (TWA) exposure estimates over 1, 2, 3 or 21 days for different phases of the assessment. 1 day = 1.0; 2 days = 0.93; 3 days = 0.9; 21 days = 0.53

Table F11 Exposure of birds for chronic screening assessment

Crop & BBCH Application Short-cut MAF Indicator No of class (where rate value (90th DDD species2 applications appropriate)1 (kg/ha) (90th%)3 %)4

Small omnivorous Strawberries 0.096 64.8 2.6 12 8.57 bird

Small Ornamentals insectivorous 0.096 18.2 2.6 12 2.41 and nursery bird

1 EFSA, 2009, Table 5 p27

2 EFSA, 2009, Table 6 p28

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3 EFSA, 2009, Table 6 p28

4 EFSA, 2009, Table 7 p29

5The exposure assessment of the reproduction assessment uses time-weighted average (TWA) exposure estimates over 1, 2, 3 or 21 days for different phases of the assessment. 1 day = 1.0; 2 days = 0.93; 3 days = 0.9; 21 days = 0.53

Table F12 Measures of exposure and toxicity used in the reproduction assessment Test endpoint used as Breeding phase Short-term exposure Long-term exposure surrogate

Pair formation/ 14 breeding site 0.1 x LD50 1 day DDD 21 day TWA DDD selection

NOAEL for the number of eggs Copulation and egg 1 day DDD 21 day TWA DDD laying (5 days pre- laid per hen laying through end of NOAEL for mean eggshell 1 day DDD 21 day TWA DDD laying thickness

0.1 x LD50 1 day DDD 21 day TWA DDD

NOAEL for proportion of viable Incubation and 1 day DDD 21 day TWA DDD eggs/eggs set/hen hatching NOAEL for proportion of 3 day TWA DDD 21 day TWA DDD hatchlings/viable eggs/hen

0.1 x LD50 (extrinsic adult) 2 day TWA DDD 21 day TWA DDD

1 day DDD based on 21 day TWA DDD based

0.1 x LD50 (extrinsic juvenile) chick shortcut values of on chick shortcut value Juvenile growth and 3.8 and 22.715 of 3.8 and 22.73 survival until fledging NOAEL for proportion of 14 day old juveniles/number of 3 day TWA DDD 21 day TWA DDD hatchlings/hen

1 day DDD based on 21 day TWA DDD based

0.1 x LD50 chick shortcut values of on chick shortcut value Post-fledging 3.8 and 22.73 of 3.8 and 22.73 survival NOAEL for 14 day old juvenile 3 day TWA DDD 21 day TWA DDD weights/hen

2 from acute study 3 The two values are to account for ground and foliar dwelling arthropods with mean residue unit doses of 3.5 and 21 respectively. Assessments are made with both values. If TER are exceeded with either value, then an assessment based on the actual composition of the diet of relevant species.

14 From acute study. 15 The two values are to account for ground and foliar dwelling arthropods with mean residue unit doses of 3.5 and 21 respectively. Assessments are made with both values. If TER are exceeded with either value, then an assessment based on the actual composition of the diet of relevant species.

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Calculation of TERs

TER calculations are detailed in Table F13Table .

Table F13 TER values for acute dietary risk assessment – Screening assessment Toxicity endpoint value Trigger value / Birds type DDD TER ratio (mg a.i./kg Presumption bw/d)

Strawberries - 12 applications of 96 g a.i./ha at 7 days interval

< 10 / High risk < 20 / High risk to Acute 30.49 LD50 > 19 > 0.6 threatened Small species omnivorous bird < 5 / High risk < 10 / High risk to Long-term 8.57 LD50/10 >1.9 > 0.2 threatened species

Ornamentals and Nursery plants - 12 applications of 96 g a.i./ha at 7 days interval

< 10 / High risk Acute < 20 / High risk to 8.99 LD50 > 19 > 2.1 threatened species Small insectivorous < 5 / High risk bird < 10 / High risk to Long-term 2.41 LD50/10 >1.9 > 0.8 threatened species

Conclusion for bird risk assessment (screening)

No reproductive studies were available; therefore the staff used the LD50/10 instead. High acute and chronic risks were identified for insectivorous and omnivorous birds following the application of Mycotal WG to strawberries and ornamentals. It should be pointed out that the LD50 was in fact a NOEC, so the conclusions are very conservative. The staff consider that for the use in enclosed permanent structures the risk to birds is below the level of concern.

First tier assessment

Table F14 TER values for acute dietary risk assessment – First tier assessment

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Toxicity BBCH crop Short-cut Trigger value / Birds type endpoint value TER ratio stage value Presumption (mg/kg bw/d)

Strawberries – 12 applications of 96 g a.i./ha at 7 days interval [BBCH > 20]

< 10 / High risk < 20 / High risk to Acute 25.2 LD50 > 19 > 3.9 threatened Small species insectivorous bird “wagtail" < 5 / High risk < 10 / High risk to Long-term 9.7 LD50/10 >1.9 > 1.5 threatened species

Strawberries – 12 applications of 96 g a.i./ha at 7 days interval [BBCH > 40]

> 10 / no concern < 20 / High risk to Acute 9.6 LD50 > 19 > 10.3 threatened Small species omnivorous bird “lark" < 5 / High risk < 10 / High risk to Long-term 4.4 LD50/10 >1.9 > 3.3 threatened species

Strawberries – 12 applications of 96 g a.i./ha at 7 days interval [BBCH 10-19]

< 10 / High risk < 20 / High risk to Acute 26.8 LD50 > 19 > 3.7 threatened Small species insectivorous bird “wagtail" < 5 / High risk < 10 / High risk to Long-term 11.3 LD50/10 >1.9 > 1.3 threatened species

Strawberries – 12 applications of 96 g a.i./ha at 7 days interval [BBCH 10-19]

< 10 / High risk < 20 / High risk to Acute 24.0 LD50 > 19 > 4.1 threatened Small species omnivorous bird “lark" < 5 / High risk < 10 / High risk to Long-term 10.9 LD50/10 >1.9 > 1.3 threatened species

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Toxicity BBCH crop Short-cut Trigger value / Birds type endpoint value TER ratio stage value Presumption (mg/kg bw/d)

Strawberries – 12 applications of 96 g a.i./ha at 7 days interval [Late flowering]

< 10 / High risk < 20 / High risk to Acute 27.0 LD50 > 19 > 3.7 threatened Frugivorous species birds “starling” < 5 / High risk < 10 / High risk to Long-term 13.4 LD50/10 >1.9 > 1.1 threatened species

Ornamentals - 12 applications of 96 g a.i./ha at 7 days interval [Application to plant]

< 10 / High risk < 20 / High risk to Acute 46.8 LD50 > 19 > 2.1 threatened Small species insectivorous bird “tit” < 5 / High risk < 10 / High risk to Long-term 18.2 LD50/10 >1.9 > 0.8 threatened species

Conclusion for bird risk assessment (first tier assessment)

There were high risks to birds for all crop stages modelled for the application to strawberries and ornamentals.

No reproductive studies with birds were provided, thus it was not possible to refine the risk assessment. For the chronic evaluation the default value of LD50/10 had to be used instead, therefore the calculations were conservative.

There were no signs of infectivity or pathogenicity in the short-term study to birds. However, due to the uncertainty about the effects to birds the staff consider that additional controls should be set for Mycotal WG.

Secondary poisoning

The staff consider that the risk of secondary poisoning can be considered below the level of concern due to the characteristics of L. muscarium.

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Bee risk assessment

The risk to bees is assessed as follows:

Tier 1- screening level risks If a reasonable potential for exposure to the pesticide is identified, a screening-level risk assessment is conducted. This step involves a comparison of Tier I estimated exposure concentrations (EECs) for contact and oral routes of exposure to adults and larvae to Tier I acute and chronic levels of effects to individual bees using laboratory-based studies. The conservatism of the Tier I screening-level risk quotient (RQ) value results primarily from the model-generated exposure estimates that, while intended to represent environmentally relevant exposure levels, are nonetheless considered high-end estimates. The resulting acute and chronic RQ values are then compared to the corresponding level of concern (LOC) values for acute and chronic risk (i.e., 0.4 and 1.0, respectively). Generally, if RQ values are below their respective LOCs, a presumption of minimal risk is made, since the Tier I risk estimation methods are designed to be conservative.

EEC are calculated as follows:

Measurement Exposure Exposure estimate Acute effect Chronic effect endpoint route (EEC)* endpoint endpoint#

Foliar application

Individual Application rate (kg Acute Contact None survival (adults) ai/ha) x 2.4 µg ai/bee contact LD50

Application rate (kg Chronic adult oral Individual Acute oral Diet ai/ha) x 98 µg ai/g x NOAEL (effects to survival (adults) LD50 0.292 g/day survival or longevity)

Application rate (kg Chronic larval oral Brood size and Diet ai/ha) x 98 µg ai/g x Larval LD50 NOAEL (effects to adult success 0.124 g/day emergence, survival)

Soil treatment

Chronic adult oral Individual Briggs EEC x 0.292 Acute oral Diet NOAEL (effects to survival (adults) g/day LD50 survival or longevity)

Chronic larval oral Brood size and Briggs EEC x 0.124 Diet Larval LD50 NOAEL (effects to adult success g/day emergence, survival)

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Seed treatment&

Chronic adult oral Individual Acute oral Diet 1 µg ai/g x 0.292 g/day NOAEL (effects to survival (adults) LD50 survival or longevity)

Chronic larval oral Brood size and Diet 1 µg ai/g x 0.124 g/day Larval LD50 NOAEL (effects to adult success emergence, survival)

Tree trunk application**

Chronic adult oral Individual µg ai applied to tree/g Acute oral Diet NOAEL (effects to survival (adults) foliage x 0.292 g/day LD50 survival or longevity)

Chronic larval oral Brood size and µg ai applied to tree/g Diet Larval LD50 NOAEL (effects to adult success foliage x 0.124 g/day emergence, survival)

* Based on food consumption rates for larvae (0.124 g/day) and adult (0.292 g/day) worker bees and concentration in pollen and nectar

** Note that concentration estimates for tree applications are specific to the type and age of the crop to which the chemical is applied.

# To calculate RQs for chronic effects, NOAEC can be used as the effect endpoint to compare with the exposure estimate in concentration

& Assume that pesticide concentration in pollen and nectar of seed treated crops is 1 mg a.i./kg (1 μg a.i./g). • No adjustment is made for application rate (Based on EPPO’s recommended screening value)

퐸퐸퐶 퐴푐푢푡푒 푅푄 = 퐿퐷50

퐸퐸퐶 퐶ℎ푟표푛𝑖푐 푅푄 = 푁푂퐴퐸퐿

Toxicity Application EEC (µg endpoint Trigger value Use scenario RQ rate (kg ai/ha) ai/bee) value (µg /Presumption ai/bee)

Greenhouses - 96 g a.i./ha, 12 applications at 7 days interval

0.4 / no Oral 0.096 2.75 >112 0.025 concern

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0.4 / no Contact 0.096 0.2304 >100 0.0023 concern

Acute effects on brood and chronic effects on adults and brood

No study presented

Non-target arthropod risk assessment

A study conducted in laboratory showed “harmless” effects (<30% effects) for parasitoids, predatory mites and predatory insects when exposed at a rate of 4 kg of a product containing Lecanicillium muscarium per ha. Slightly harmful effects (25-50% effects) to Typhlodromus pyri were observed in a field test with 4 kg of a product containing L. muscarium per ha. The information in those studies was insufficient to conclude about the test conditions (relative humidity, temperature) that influence the toxicity of L. muscarium.

Mortality was observed in Encarsia formosa (≤12.5 %) five days after a “mist” product containing L. muscarium spores was applied to bean plants. The mortality dropped to 5%, 28 days after the application. E. formosa is a beneficial arthropod used in IPM practices in greenhouses. Again, the staff consider that the test lacked some fundamental information to draw a conclusion about the detrimental effects.

The studies provided by the applicant and the open literature search made by the staff did not allow a thorough evaluation of the effects to non-target arthropods. In summary, the staff consider that effects to non-target arthropods are not sufficient addressed in the available body of proof.

Conclusion for non-target arthropod risk assessments The risks to bees were considered to be below the level of concern.

Due to the lack of data to conduct a risk assessment for non-target arthropods the staff consider that a maximum application rate should be set.

Summary and conclusions of the ecological risk assessment

The staff assessed the potential risk to be triggered by the use of Mycotal WG following the instructions captured in the proposed label and the GAP table. There was insufficient information (data gaps) to make a complete assessment of Mycotal WG. A risk assessment depends on the quality and quantity of data available. Due to the scarcity of tests conducted with Mycotal WG the staff consider that is advisable to take a precautionary approach and set additional controls for this substance. It should be stressed that the risk identification was the result of the assessment conducted with the relevant information gathered at this time.

Aquatic organisms

The risks to aquatic organisms were considered to be below the level of concern. However, due to the uncertainty regarding the available data on the effects to aquatic organisms the staff consider that the additional control should be applied: 1) Mycotal WG should not be applied onto or into water.

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Soil organisms

The risks to soil organisms were considered to be below the level of concern.

Terrestrial vertebrates (birds)

The first-tier assessment resulted in a high risks to birds. As a result, the staff consider that additional controls should be set for this substance limiting the use to enclosed greenhouses (permanent structures).

Terrestrial invertebrates (bees and other non-target arthropods)

The risks to bees were below the level of concern. However, there was insufficient information to assess the risks to non-target arthropods. The EPA staff consider that a maximum application rate should be set for this substance in order to mitigate the risks. 1) A maximum application rate of 96 g a.i./ha (corresponding with 2*1013 cfu per ha), 12 times at a 7 days interval per year should be set for this substance. 2) Mycotal WG should be applied using ground-based methods only.

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Appendix G: Staff risk and benefit assessment

The staff have evaluated the potential of MYCOTAL WG to cause adverse effects to people and/or the environment during all stages of the substance’s lifecycle. A qualitative risk assessment was carried out to assess these risks.

The process by which the risk assessment of substances is undertaken is specified in the Methodology16. Guidance on risk assessment is provided on the EPA website17.

To facilitate the assessment of risks the applicant and the staff identified the most common potential sources of risk to the environment and to human health and safety through release, spillage or exposure throughout the lifecycle of the substance. These are shown in Table G1.

Table G1: Potential sources of risks associated with hazardous substances

Lifecycle Activity Associated Source of Risk

Manufacture/Import An incident during the manufacture or importation of the substance resulting in a spill and subsequent exposure of people or the environment to the substance.

Packing An incident during the packing of the substance resulting in a spill and subsequent exposure of people or the environment to the substance.

Transport or storage An incident during the transport or storage of the substance resulting in a spill and subsequent exposure of people or the environment to the substance.

Use Application of the substance resulting in exposure of users or bystanders or the environment; or an incident during use resulting in a spill and subsequent exposure of users or the environment to the substance.

Disposal Disposal of the substance or packaging resulting in exposure of people or the environment to the substance.

16 http://www.legislation.co.nz/regulation/public/1998/0217/latest/DLM254556.html?src=qs 17 http://www.epa.govt.nz/Publications/ER-TG-05-02_03_09_Decision_Making.pdf

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Appendix H: Confidential Information

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Appendix I: Standard terms and abbreviations

ai active ingredient F field

ALD50 approximate median lethal dose, F0 parental generation 50% F1 filial generation, first AOEL acceptable operator exposure level F2 filial generation, second ARfD acute reference dose fp freezing point as active substance G glasshouse BCF bioconcentration factor GAP good agricultural practice bfa body fluid assay GC gas chromatography BOD biological oxygen demand GC-EC gas chromatography with electron BSAF biota-sediment accumulation factor capture detector

bw body weight GC-FID gas chromatography with flame ionization detector c centi- (x10-2) GC-MS gas chromatography-mass CA controlled atmosphere spectrometry CI confidence interval GC-MSD gas chromatography with mass- CL confidence limits selective detection

CNS central nervous system GLC gas liquid chromatography

COD chemical oxygen demand GLP good laboratory practice

DFR dislodgeable foliar residue GM geometric mean

DO dissolved oxygen H Henry’s Law constant (calculated as a unitless value) (see also K) DOC dissolved organic carbon ha hectare DT50 period required for 50 percent dissipation (define method of Hb haemoglobin estimation) HCG human chorionic gonadotropin DT90 period required for 90 percent Hct haematocrit dissipation (define method of estimation) HPLC high pressure liquid chromatography or high dw dry weight performance liquid chromatography ED50 median effective dose HPLC-MS high pressure liquid ERC environmentally relevant chromatography - mass concentration spectrometry

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I indoor LOAEL lowest observable adverse effect level I50 inhibitory dose, 50% LOD limit of detection IC50 median immobilization concentration or median inhibitory LOEC lowest observable effect concentration 6 concentration

ID ionization detector LOEL lowest observable effect level

Im intramuscular LOQ limit of quantification (determination) inh inhalation LPLC low pressure liquid chromatography ip intraperitoneal LSC liquid scintillation counting or IPM integrated pest management counter iv intravenous LSS liquid scintillation spectrometry

IVF in vitro fertilization LT lethal threshold

K Kelvin or Henry’s Law constant (in M molar atmospheres per cubic meter per mole) (see also H) μm micrometer (micron)

Kads adsorption constant MDL method detection limit

Kdes apparent desorption coefficient MFO mixed function oxidase

Koc organic carbon adsorption μg microgram coefficient MLT median lethal time Kom organic matter adsorption coefficient MLD median lethal dose kg kilogram mol Mole(s)

LC liquid chromatography MOS margin of safety

LC-MS liquid chromatography- mass mp melting point spectrometry MS mass spectrometry LC50 lethal concentration, median MSDS material safety data sheet LCA life cycle analysis NAEL no adverse effect level LC-MS-MS liquid chromatography with tandem nd not detected mass spectrometry NEL no effect level LD50 lethal dose, median; dosis letalis media ng nanogram

LDH lactate dehydrogenase nm nanometer

LOAEC lowest observable adverse effect NOAEC no observed adverse effect concentration concentration

NOAEL no observed adverse effect level

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NOEC no observed effect concentration RfD reference dose

NOEL no observed effect level RL50 median residual lifetime

NR not reported RP reversed phase

OC organic carbon content RRT relative retention time

ODP ozone-depleting potential RSD relative standard deviation

OM organic matter content sc subcutaneous

Pa pascal SD standard deviation

PEC predicted environmental se standard error concentration SF safety factor PECS predicted environmental SIMS secondary ion mass spectroscopy concentration in soil SOP standard operating procedures PECSW predicted environmental concentration in surface water sp species (only after a generic name)

PECGW predicted environmental SPE solid phase extraction concentration in ground water spp subspecies PHI pre-harvest interval SSD sulphur specific detector pKa negative logarithm (to the base 10) of the dissociation constant) STEL short term exposure limit

PNEC predicted no effect concentration t½ half-life (define method of estimation) POW partition coefficient between n- octanol and water TCLo toxic concentration, low ppb parts per billion (10-9) TER toxicity exposure ratio

PPE personal protective equipment TIFF tag image file format ppm parts per million (10-6) TOC total organic carbon ppp plant protection product TWA time weighted average ppq parts per quadrillion (10-24) UF uncertainty factor (safety factor) ppt parts per trillion (10-12) ULV ultra low volume

PTDI provisional tolerable daily intake UV ultraviolet r correlation coefficient v/v volume ratio (volume per volume) r2 coefficient of determination w/v weight per volume

REI restricted entry interval ww wet weight

Rf retardation factor w/w weight per weight

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December 2015