Peer review of the pesticide risk assessment of the active substance Bacillus thuringiensis subsp. kurstaki strain SA‐12

Abstract

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Denmark, and co‐rapporteur Member State, the Netherlands, for the pesticide active substance Bacillus thuringiensis subsp. kurstaki strain SA‐12 and the considerations as regards the inclusion of the substance in Annex IV of Regulation (EC) No 396/2005 are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of Bacillus thuringiensis subsp. kurstaki strain SA‐12 as an insecticide on pome fruits (field use), protected tomato (including permanent greenhouses and walk‐in tunnels) and ornamentals (field use). The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

Summary

Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659, lays down the procedure for the renewal of the approval of active substances submitted under Article 14 of Regulation (EC) No 1107/2009. The list of those substances is established in Commission Implementing Regulation (EU) No 686/2012. Bacillus thuringiensis subsp. kurstaki strain SA‐12 is one of the active substances listed in Regulation (EU) No 686/2012.

In accordance with Article 1 of Regulation (EU) No 844/2012, the rapporteur Member State (RMS), Denmark, and co‐rapporteur Member State (co‐RMS), the Netherlands, received an application from Certis USA LLC for the renewal of approval of the active substance Bacillus thuringiensis subsp. kurstaki strain SA‐12.

An initial evaluation of the dossier on Bacillus thuringiensis subsp. kurstaki strain SA‐12 was provided by the RMS in the renewal assessment report (RAR) and subsequently, a peer review of the pesticide risk assessment on the RMS evaluation was conducted by EFSA in accordance with Article 13 of Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The following conclusions are derived.

The uses of Bacillus thuringiensis subsp. kurstaki strain SA‐12 according to the representative uses as an insecticide on pome fruits (field use), tomato (including permanent greenhouses and walk‐in tunnels) and ornamentals (field use), as proposed at EU level result in a sufficient insecticidal efficacy against the target lepidopteran pests.

The assessment of the data package revealed no issues that could not be finalised or that need to be included as critical areas of concern with respect to identity, biological properties of the active substance and physical and technical properties of the representative formulation.

With respect to mammalian toxicology, two data gaps are identified in relationship to potential adverse effects after repeated exposure by inhalation to Bacillus thuringiensis subsp. kurstaki strain SA‐12 and to potential genotoxic effect of the insecticidal proteins by non‐dietary exposure. On this basis, the risk assessment for residents and bystanders cannot be concluded (issue not finalised).

In the area of residues, a data gap was identified related to the proposed threshold of 1 × 10⁵ colony‐forming units (CFU)/g for viable residues on edible plant commodities at the time of harvest, where quantification of viable counts linked to specific preharvest intervals (PHIs) is requested to finalise the consumer risk assessment.

Bacillus thuringiensis subsp. kurstaki strain SA‐12 is not proposed to be included into Annex IV of Regulation (EC) No 396/2005.

In the area of environmental fate and behaviour, the available information was considered sufficient to complete the necessary environmental exposure assessments.

Satisfactory information was not provided leading to issues not being finalised for the potential effects to honeybee larvae, for the potential for infectivity and pathogenicity to bees and non‐target arthropods for representative field and walk‐in tunnel uses; for the potential for toxicity, infectivity and pathogenicity to earthworms and potential adverse effects to soil microorganisms for representative field and walk‐in tunnel uses and for a hazard characterisation and an assessment of the risk to non‐target organisms from toxins/secondary metabolites such as crystal proteins present after the application of the product.

Background

Commission Implementing Regulation (EU) No 844/20121 as amended by Commission Implementing Regulation (EU) No 2018/16592 (hereinafter referred to as ‘the Regulation’), lays down the provisions for the procedure of the renewal of the approval of active substances, submitted under Article 14 of Regulation (EC) No 1107/20093. This regulates for the European Food Safety Authority (EFSA) the procedure for organising the consultation of Member States, the applicant and the public on the initial evaluation provided by the rapporteur Member State (RMS) and/or co‐rapporteur Member State (co‐RMS) in the renewal assessment report (RAR), and the organisation of an expert consultation where appropriate.

In accordance with Article 13 of the Regulation, unless formally informed by the European Commission that a conclusion is not necessary, EFSA is required to adopt a conclusion on whether the active substance can be expected to meet the approval criteria provided for in Article 4 of Regulation (EC) No 1107/2009 within 5 months from the end of the period provided for the submission of written comments, subject to an extension of an additional 3 months where additional information is required to be submitted by the applicant in accordance with Article 13(3).

In accordance with Article 1 of the Regulation, the RMS, Denmark, and co‐RMS, the Netherlands, received an application from Certis USA LLC for the renewal of approval of the active substance Bacillus thuringiensis subsp. kurstaki strain SA‐12. Complying with Article 8 of the Regulation, the RMS checked the completeness of the dossier and informed the applicant, the co‐RMS (the Netherlands), the European Commission and EFSA about the admissibility.

The RMS provided its initial evaluation of the dossier on Bacillus thuringiensis subsp. kurstaki strain SA‐12 in the RAR, which was received by EFSA on 2 April 2019 (Denmark, 2019).

In accordance with Article 12 of the Regulation, EFSA distributed the RAR to the Member States and the applicant, Certis USA LLC, for consultation and comments on 14 May 2019. EFSA also provided comments. In addition, EFSA conducted a public consultation on the RAR. EFSA collated and forwarded all comments received to the European Commission on 14 July 2019. At the same time, the collated comments were forwarded to the RMS for compilation and evaluation in the format of a reporting table. The applicant was invited to respond to the comments in column 3 of the reporting table. The comments and the applicant's response were evaluated by the RMS in column 3.

The need for expert consultation and the necessity for additional information to be submitted by the applicant in accordance with Article 13(3) of the Regulation were considered in a telephone conference between EFSA, the RMS on 10 October 2019. On the basis of the comments received, the applicant's response to the comments and the RMS's evaluation thereof, it was concluded that additional information should be requested from the applicant, and that EFSA should conduct an expert consultation in the areas of mammalian toxicology, residues and environmental fate and behaviour.

The outcome of the telephone conference, together with EFSA's further consideration of the comments, is reflected in the conclusions set out in column 4 of the reporting table. All points that were identified as unresolved at the end of the comment evaluation phase and which required further consideration, including those issues to be considered in an expert consultation, were compiled by EFSA in the format of an evaluation table.

The conclusions arising from the consideration by EFSA, and as appropriate by the RMS, of the points identified in the evaluation table, together with the outcome of the expert consultation and the written consultation on the assessment of additional information, where these took place, were reported in the final column of the evaluation table.

A final consultation on the conclusions arising from the peer review of the risk assessment took place with Member States via a written procedure in August–September 2020.

This conclusion report summarises the outcome of the peer review of the risk assessment of the active substance and the representative formulation evaluated on the basis of the representative uses of Bacillus thuringiensis subsp. kurstaki strain SA‐12 as an insecticide on pome fruits (field use), protected tomato (including permanent greenhouses and walk‐in tunnels) and ornamentals (field use), as proposed by the applicant. In accordance with Article 12(2) of Regulation (EC) No 1107/2009, risk mitigation options identified in the RAR and considered during the peer review are presented in the conclusion. A list of the relevant end points for the active substance and the formulation is provided in Appendix A.

In addition, a key supporting document to this conclusion is the peer review report (EFSA, 2020), which is a compilation of the documentation developed to evaluate and address all issues raised in the peer review, from the initial commenting phase to the conclusion. The peer review report comprises the following documents, in which all views expressed during the course of the peer review, including minority views, where applicable, can be found:

• the comments received on the RAR;

• the reporting table (17 October 2019);

• the evaluation table (14 September 2020);

• the report(s) of the scientific consultation with Member State experts (where relevant);

• the comments received on the assessment of the additional information (where relevant);

• the comments received on the draft EFSA conclusion.

Given the importance of the RAR, including its revisions (Denmark, 2020), and the peer review report, both documents are considered as background documents to this conclusion and thus are made publicly available.

It is recommended that this conclusion report and its background documents would not be accepted to support any registration outside the EU for which the applicant has not demonstrated that it has regulatory access to the information on which this conclusion report is based.

The identity of the microorganism and the properties of the formulated product

Bacillus thuringiensis subsp. kurstaki strain SA‐12 is a bacterium deposited at the ARS Culture Collection (Northern Regional Research Laboratory; NRRL), at the Microbial Properties Research Unit, National Centre for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture Peoria, Illinois, USA, under the deposit number NRRL B‐30791. Bacillus thuringiensis subsp. kurstaki strain SA‐12 is a naturally occurring, indigenous wild‐type bacterium, initially isolated from the insect Ephestia cantella.

The representative formulated product for the evaluation was ‘CoStar WG’, a water‐dispersible granule (WG) containing 850 g/kg of Bacillus thuringiensis subsp. kurstaki strain SA‐12 (specified minimum content 8.5 × 10¹² colony‐forming units (CFU)/kg and maximum 5.7 × 10¹³ CFU/kg) with a minimum target bio‐potency of 90,000 IU/mg.

The representative uses evaluated were field spray applications for the biological control of insect pests of the order of Lepidoptera on pome fruits and ornamentals and on protected tomato (including permanent greenhouses and walk‐in tunnels). Full details of the good agriculture practices (GAPs) can be found in the list of end points in Appendix A.

Data were submitted to conclude that the use of Bacillus thuringiensis subsp. kurstaki strain SA‐12 according to the representative uses proposed at EU level results in a sufficient insecticidal efficacy against the target organisms, following the guidance document SANCO/2012/11251‐rev. 4 (European Commission, 2014b).

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

The following guidance documents were followed in the production of this conclusion: European Commission, 2012 and EFSA FEEDAP Panel, 2018.

The technical grade microbial pest control agent (MPCA) is only a hypothetical stage in the continuous production process of the end‐use product (microbial pest control product; MPCP). As a consequence, the specification is given only for the formulated product ‘CoStar WG’ of minimum content of 8.5 × 10¹² CFU/kg (maximum 5.7 × 10¹³ CFU/kg) with a minimum bio‐potency of 90,000 IU/mg.

Bacillus thuringiensis subsp. kurstaki strain SA‐12 is characterised by serotyping, plasmid profiling, activity spectrum, fatty acid analysis, DNA fingerprinting (amplified fragment length polymorphism; AFLP) and cry toxin analysis. To allow an unequivocal identification of strain SA‐12, strain‐specific primers based on the sequences of the whole genome and plasmids of strain SA‐12 were developed. The developed markers were tested for specificity with a set of Bacillus thuringiensis subsp. kurstaki, aizawai, israelensis and tenebrionis, as well as with potentially pathogenic Bacillus cereus strains. Two specific primer pairs were unique for Bacillus thuringiensis subsp. kurstaki strain SA‐12 and can be used for identification at strain level.

Bacillus thuringiensis subsp. kurstaki strain SA‐12 contains genes encoding for potential production of cytotoxin type K₂ (CytK₂), six crystal insecticidal proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab), two Cry‐like proteins and one vegetative insecticidal protein (VIP 3Aa10). Furthermore, Bacillus thuringiensis subsp. kurstaki strain SA‐12 has the potential to form a non‐haemolytic (Nhe) and haemolytic (Hbl) enterotoxin complex (which also includes CytK₂). The genes for the highly cytotoxic Cyt K₁, for the cytotoxic variant of haemolysin II (HlyII), for cereulide and for ß‐exotoxins are missing in the strain. In addition, it has been demonstrated that compared to a pathogenic Bacillus cereus strain (whose group it is member), the enterotoxins Nhe and Hbl are produced at significantly lower levels under optimal growth conditions in enriched substrate favouring enterotoxin production.

The content of microbial contaminants of the MPCP was below the limits defined in the SANCO/12116/2012 working document (European Commission, 2012). Bacillus thuringiensis spores can remain viable for years in soil, but applied as a spray, the δ‐endotoxins are rapidly degradable and endospores are rapidly inactivated when exposed to ultraviolet (UV) radiation.

As a member of the Bacillus cereus‐group, Bacillus thuringiensis subsp. kurstaki is closely related to Bacillus anthracis and Bacillus cereus. Bacillus thuringiensis strains are, however, distinguishable from Bacillus cereus and Bacillus anthracis.

Bacillus thuringiensis subsp. kurstaki strain SA‐12 was shown to be sensitive to all relevant antibiotics as provided in EFSA FEEDAP Panel Guidance (2018): chloramphenicol, tetracycline, streptomycin, clindamycin, erythromycin, kanamycin, gentamicin and vancomycin.

The main data regarding the identity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 and its biological properties are given in Appendix A.

Acceptable methods for CFU counts of Bacillus thuringiensis subsp. kurstaki strain SA‐12 in the formulation for the determination of the microorganism in the MPCP and for the determination of the content of contaminating microorganisms are available.

Methods for the determination and quantification of residues are currently not required as no residue definition applies to the microorganism and no maximum residue level (MRL) was set for any of the intended uses. However, it is noted that a validated enumeration method in high water commodities (lettuce) was provided with a limit of quantification (LOQ) of 1.3 × 10³ CFU/g, and for unambiguous identification of Bacillus thuringiensis subsp. kurstaki strain SA‐12, an AFLP method with two highly specific primer pairs is available. The strain‐specific molecular markers can be used for monitoring of the strain upon field application.

Methods of analysis for viable residues in the environment are not required.

Quantification of Cry1Ab in soil can be done with commercial enzyme‐linked immunosorbent assay (ELISA) kit with an LOQ of 0.25 μg/L. Determination of Cry1Ab in water can be done with ELISA with a detection limit of 2.1 ng/L.

2. Mammalian toxicity

Bacillus thuringiensis subsp. kurstaki strain SA‐12 was discussed at the Pesticides Peer Review Meeting Teleconference 25 in March 2020.

General data

From the medical data, no adverse reactions in individuals as a result of contact with Bacillus thuringiensis subsp. kurstaki strain SA‐12 during its development, manufacture, preparation or field application have been documented or reported. The results of allergenicity observations indicate that increased IgE levels can occur in greenhouses workers exposed to products containing Bacillus thuringiensis subsp. kurstaki, but no effect on the occurrence of respiratory symptoms or lung function was observed.

Bacillus thuringiensis is not recommended for the Qualified Presumption of Safety list (EFSA BIOHAZ Panel, 2020).

Toxicity/Infectivity/Pathogenicity studies

As the available methods for testing dermal sensitisation are not suitable for testing microorganisms and there are no validated test methods for sensitisation by inhalation, the following warning phrase is proposed: ‘Microorganisms may have the potential to provoke sensitising reactions’.

Laboratory studies on mammalian toxicity testing an appropriate concentration of a liquid formulation of Bacillus thuringiensis subsp. kurstaki strain SA‐12 (Thuricide SC) have been conducted in rats upon oral, respiratory (intranasal) and intravenous acute single doses. Adverse effects and signs of infectivity or pathogenicity were not observed. Clearance occurred within 2 weeks.

A dermal toxicity and a skin irritation study conducted with formulated Bacillus thuringiensis subsp. kurstaki strain SA‐11 (Delfin WG, considered similar to CoStar WG) did not reveal any toxic or irritating effects. In an eye irritation study conducted with Bacillus thuringiensis subsp. kurstaki strain SA‐12, only transient slight conjunctival irritation was observed.

Concerning repeated exposure toxicity, a lung interstitial inflammation was observed in mice after subchronic (14‐day) inhalation exposure to an appropriate concentration of formulated Bacillus thuringiensis subsp. kurstaki (strain HD‐1 in Dipel) and still present 70 days after the exposure. The potential concern for serious health effects after repeated exposure by inhalation could not be excluded on the basis of the available data (data gap).4 RMS and co‐RMS disagreed.

Secondary metabolites/toxins

Bacillus thuringiensis subsp. kurstaki strain SA‐12 has been shown to have the genes that would enable it to produce certain enterotoxins (see Section 1). Enterotoxins are inactivated at low pH; therefore, preformed enterotoxins are considered not relevant by oral exposure. Based on the available evidence, the peer review concluded that only the spores are able to survive the stomach passage and to germinate and produce enterotoxins in the intestinal tract (potentially leading to diarrhoeal‐associated food‐borne disease in humans). Considering the available evidence and uncertainties, the threshold of 10⁵ CFU/g food as determined by the BIOHAZ Panel Opinion (EFSA BIOHAZ Panel, 2016) to cover the risk of food‐borne poisonings caused by the Bacillus cereus group of microorganisms was concluded as applicable for the group of Bacillus thuringiensis by the majority of experts.5 The RMS and the co‐RMS disagreed.

In a mouse micronucleus study with intraperitoneal administration, positive results were observed with the spore‐crystal complex containing Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa. These results were considered equivocal, especially regarding whether the Cry‐proteins had been solubilised/activated prior to administration or not (data gap). The majority of the experts concluded that genotoxicity of the insecticidal proteins is not a concern for dietary exposure but a possible concern for non‐dietary exposure could not be excluded.6

Reference values and exposure

As mentioned previously, the threshold of 10⁵ CFU/g food as determined by the BIOHAZ Panel Opinion (EFSA BIOHAZ Panel, 2016) is considered applicable to the group of Bacillus thuringiensis to cover the risk of food‐borne poisonings caused by the Bacillus cereus group of microorganisms. With regard to non‐dietary exposure, since toxicity/infectivity after repeated exposure by inhalation could not be concluded, and a genotoxic potential of the Cry proteins could not be excluded by non‐dietary exposure, the risk assessment by inhalation for residents and bystanders cannot be concluded (issue not finalised) while the use of respiratory protective equipment is recommended for operators and workers.

3. Residues

Bacillus thuringiensis subsp. kurstaki strain SA‐12 was discussed at the Pesticides Peer Review Meeting Teleconference 25 in March 2020.

Considering the available evidence and uncertainties, the threshold of 10⁵ CFU/g plant commodity at the time of harvest as determined by the BIOHAZ Panel Opinion (EFSA BIOHAZ Panel, 2016) is considered applicable to all Bacillus thuringiensis strains to cover the risk of food‐borne poisonings (caused by the Bacillus cereus group of microorganisms; see Section 2). Non‐viable residues are not of concern for the dietary consumption (see Section 2). The co‐RMS wishes to inform that work is ongoing on the development of a new diagnostic tool to discriminate Bacillus thuringiensis biocontrol strains from Bacillus cereus sensu lato species and strains. The expectation is that a tool will be soon available to distinguish between Bacillus cereus senso lato strains and commercial Bacillus thuringiensis strains and to ensure correct conclusions and decisions can be taken with regard to the origin of food borne outbreaks.

Therefore, only information on viable residues i.e. CFU per g or kg plant commodities at harvest is needed to demonstrate that the threshold of 10⁵ CFU/g edible plant commodity is not exceeded.

To ensure this, the setting of a PHI may be needed because available experimental data in lettuce (up to 2.2 × 10⁵ CFU/g after treatment) and estimated counts on harvested pome fruits (up to 2.2 × 10⁷ CFU/g) considering representative uses demonstrate that counts at harvest exceed this threshold. Since samples in the lettuce study were stored at ‐18 °C prior to analysis, a storage stability of Bacillus thuringiensis subsp. kurstaki strain SA‐12 in high‐water commodities is still required (data gap).

Viable counts of commercial Bacillus thuringiensis strains including Bacillus thuringiensis subsp. kurstaki strain SA‐12 were demonstrated in the scientific literature and by supporting experimental evidence to decline following application and not to persist or multiply on edible plant commodities (fruiting vegetable and leafy crops). Furthermore, in the literature a body of evidence supports inactivation and decline of viable spores by environmental factors such as solar radiation, rainfall, plant growth and temperature. Based on the available data in the RAR, a half‐life of viable spores of around one day or 24 h can reasonably be estimated.

Considering the above‐mentioned threshold, a half‐life time of 24 h and estimated viable counts on pome fruits at harvest, a PHI of at least 8 days would be required. It is, however, to be noted that the representative uses on pome fruits and on tomatoes are indicating a BBCH of up to 89 which corresponds to the ripe fruits ready for consumption and a PHI was not specified and it can be assumed that the PHI is less than 8 days if this growth stage of application is maintained. Therefore, quantification of viable counts linked to specific PHIs is requested (data gap).

This would still need to be addressed before a consumer risk assessment can be finalised.

Because of the above‐mentioned data gaps and issue not finalised, Bacillus thuringiensis subsp. kurstaki strain SA‐12 is not proposed to be included into Annex IV of Regulation (EC) No 396/20057.

4. Environmental fate and behaviour

Bacillus thuringiensis subsp. kurstaki strain SA‐12 was discussed at the Pesticides Peer Review Meeting Teleconference 25 in March 2020.

Satisfactory information was provided in relation to potential interference of Bacillus thuringiensis subsp. kurstaki strain SA‐12 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC8 (see specific Annex VI decision‐making criteria in Part II Commission Regulation (EU) No 546/20119). It was concluded that Bacillus thuringiensis subsp. kurstaki strain SA‐12 is unlikely to interfere with the methodologies routinely used for such determinations.

Bacillus thuringiensis subsp. kurstaki strain SA‐12 is a ‘wild type’ and there are no marker genes in the strain which would permit the analysis of a frequency of genetic exchange. As the genetic diversity and drift in the wild‐type population has not been ascertained, it would not be possible to distinguish any genetic drift from that in the wild population based on the information provided. Though it is acknowledged that the possibility and effects of transfer of genetic material are not different for Bacillus thuringiensis subsp. kurstaki strain SA‐12 than for other naturally occurring Bacillus thuringiensis strains, transfer of genetic material by Bacillus thuringiensis subsp. kurstaki strain SA‐12 after application is possible (the strain has plasmids), so could not be excluded based on the information in the dossier. Information in the dossier confirms that plasmid exchange between vegetative cells of different strains of the species can be measured when applications were made to leaf surfaces. Note the applied material in the product is spores and not vegetative cells.

Specific environmental exposure estimates for greenhouse uses were not provided. The applicant chose to address the representative use on protected tomato by stating that greenhouse uses are covered by the exposure assessments provided for the field uses.

4.1. Fate and behaviour in the environment of the microorganism

Information was derived from published literature on different strains of Bacillus thuringiensis in relation to its persistence and multiplication in soil. Information specific to strain SA‐12 was not available. Information on subsp. kurstaki demonstrated that spores remain viable for many years (more than 7). The species has been reported to have spores that can germinate in the rhizosphere of some plants. Based on a weight of evidence, it appears that germination of spores does not occur in the bulk soil where nutrient levels are generally more limited than in the rhizosphere. Overall, it is considered that repeated use over the years would result in the accumulation of Bacillus thuringiensis subsp. kurstaki strain SA‐12 spores in the soil environment. Consequently, EFSA concluded that the information is sufficient to address the uniform principles criterion. The spores of the strain are expected to persist and be present above natural background levels in soil, taking into account repeated applications over the years, but multiplication in bulk soil will not occur. PEC soil covering the intended uses has been calculated (see Appendix A).

With respect to the persistence and multiplication in surface water, information specific to strain SA‐12 was not available. Information on subsp. kurstaki demonstrated that in a flowing water catchment levels of CFU declined after applications were made, but the authors attributed this to the dilution and removal effect of the flowing water. The available literature indicates the species Bacillus thuringiensis is present in surface water and that it is likely that the species is capable of growing in freshwater environments under nutrient/oxygen‐rich conditions. Overall, it is concluded that the information available on the persistence/multiplication/germination of the strain in natural surface water was insufficient to demonstrate that Bacillus thuringiensis subsp. kurstaki strain SA‐12 is likely to decline in surface water. Consequently, EFSA concluded that the information is insufficient to address the uniform principles criterion of the strain not being expected to persist and multiply in surface water in concentrations considerably higher than the natural background levels, taking into account repeated applications over the years. This conclusion identifies a data gap in this respect (see Section 7). PEC surface water for the intended uses on pome fruit and ornamentals (use patterns with greatest potential for spray drift exposure) have been calculated (see Appendix A).

Information was provided on the occurrence and behaviour of Bacillus thuringiensis subsp. kurstaki spores in air. Re‐aerolisation of applied spores occurred, but spore transport distances were limited being up to 30 m. Spores rapidly lost viability following release to air.

4.2. Fate and behaviour in the environment of any relevant metabolite formed by the microorganism under relevant environmental conditions

According to scientific papers from the literature search, the subspecies Bacillus thuringiensis kurstaki is able to produce secondary metabolites, which are crystal proteins e.g. contain the δ‐endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa and Cry2Ab. These crystal proteins constitute components in the formulated product within and outside spores and are responsible for the insecticidal mode of action of Bacillus thuringiensis subsp. kurstaki strain SA‐12. Genes encoding two Cry‐like proteins and VIP 3Aa10 protein are also present in strain SA‐12.

It is not known to what extent Bacillus thuringiensis subsp. kurstaki strain SA‐12 will produce crystal proteins following its application. However, as the concentrations of the crystal proteins in the formulated product is known it was considered appropriate to complete an exposure assessment for them for surface water and groundwater based on their content in the product (Pesticides Peer Review Meeting Teleconference 25). For the crystal proteins, the experts agreed it would be appropriate to read across degradation and adsorption endpoints between the different crystal proteins from the available dataset that contains measured endpoints from only a subset of these different δ‐endotoxins and/or crystal proteins. Full details of the available experimental endpoints and which δ‐endotoxins or crystal protein test material they were derived from can be found in Appendix A. As these endpoints were not available for all the δ‐endotoxins present in Bacillus thuringiensis subsp. Kurstaki, the experts agreed that the most conservative values available should be selected and used in the exposure calculations. These values were a DT₅₀ soil of 41.3 days, Kdoc estimated at 1,000 mL/g and DT₅₀ water system of 28 days. It can also be considered that these properties might be read across to the two Cry‐like proteins and the VIP 3Aa10 protein should they be produced by strain SA‐12. Satisfactory calculations were provided for an environmental exposure assessment of the crystal proteins in soil, surface water, sediment and groundwater covering the representative uses. Soil exposure was calculated for the use on ornamentals. The FOCUS surface water Step 1 and 2 calculator (v3.2) for the FOCUS crop pome/stone fruit (FOCUS crop with greatest potential for spray drift exposure) was used to calculate surface water and sediment exposure for the intended uses on ornamentals and pome fruits (FOCUS, 2001). For groundwater calculations, PEARL 4.4.4 was used for the use on ornamentals using the FOCUS crop pome fruits as a surrogate for ornamentals (European Commission, 2014a)10 (see Appendix A). It was concluded that the potential for leaching of the crystal proteins to groundwater above the parametric drinking water limit of 0.1 μg/L is low for the representative uses assessed in geoclimatic situations represented by the FOCUS groundwater scenarios.

5. Ecotoxicology

A study on the toxicity and pathogenicity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 to birds was available and did not indicate any adverse effects. Investigation of infectivity was not performed in the study. Based on the lack of toxicity and pathogenicity in the available study, a low risk to birds was concluded (relevant for all representative uses).

As concluded in Section 2, sufficient information is available to finalise the assessment of infectivity and pathogenicity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 in mammals. A low risk to wild mammals is concluded (relevant for all representative uses).

Adequate studies were available with aquatic organisms showing no toxicity and pathogenicity to aquatic organisms from Bacillus thuringiensis subsp. kurstaki strain SA‐12. In the study with Daphnia infectivity was not assessed. A high margin of safety was observed when comparing the endpoints with expected spore concentrations in the environment after entry into surface water from the intended field uses. Based on the lack of toxicity and pathogenicity in the available studies, a low risk to aquatic organisms was concluded for all representative uses.

Insufficient data were available to address potential effects to honeybee larvae, infectivity and pathogenicity to honeybee from Bacillus thuringiensis subsp. kurstaki strain SA‐12. Consequently, a data gap leading to an assessment not finalised was identified for the potential effects to honeybee larvae and for the infectivity and pathogenicity to honeybee from Bacillus thuringiensis subsp. kurstaki strain SA‐12 for the intended uses in open field and walk‐in tunnels. The co‐RMS disagreed with the data gap and issue not finalised identified for honeybees as the information available in the RAR was considered of sufficient length to conclude on the lack of infectivity and pathogenicity to honeybees. Low risk identified for representative uses in permanent greenhouses as the exposure to honeybees is expected to be negligible.

Insufficient data were available to address infectivity and pathogenicity to non‐target arthropods from Bacillus thuringiensis subsp. kurstaki strain SA‐12. Consequently, a data gap leading to an assessment not finalised was identified for the infectivity and pathogenicity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 to non‐target arthropods for the intended uses in open field and walk‐in tunnels. For representative uses in permanent greenhouses, the risk is low as the exposure to non‐target arthropods is expected to be negligible.

For representative uses in permanent greenhouses, an assessment to the soil compartment is not requested according to EFSA (EFSA, 2014). Insufficient data were available on earthworms to indicate if Bacillus thuringiensis subsp. kurstaki strain SA‐12 would be toxic, infectious or pathogenic to earthworms. Consequently, a data gap leading to an assessment not finalised was identified for the toxicity, infectivity, pathogenicity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 to earthworms for the intended uses in field and walk‐in tunnel. Insufficient data were available on soil microorganisms to indicate if Bacillus thuringiensis subsp. kurstaki strain SA‐12 would cause adverse effects to soil microorganisms for the representative field uses. Consequently, a data gap leading to an assessment not finalised was identified for the toxicity of Bacillus thuringiensis subsp. kurstaki strain SA‐12 to soil microorganism for the intended use in open field and walk‐in tunnels. The RMS disagreed with the data gap and issue not finalised identified for earthworms and soil microorganisms and rather support a waiver.

Adequate data were available and indicated that Bacillus thuringiensis subsp. kurstaki strain SA‐12 is unlikely to cause adverse effects on non‐target plants, and therefore, a low risk to non‐target plants is concluded for all representative uses.

The risk assessment of toxins/secondary metabolites such as crystal proteins could not be finalised for terrestrial‐ and aquatic non‐target organisms for the representative field and walk‐in tunnel uses. Toxicity data were not available to perform a hazard characterisation (resulting in data gap and issue not finalised). For non‐target aquatic organisms, exposure to surface water cannot be excluded for the representative use in permanent greenhouse (resulting in a data gap and issue not finalised).

6. Overview of the risk assessment of compounds listed in residue definitions triggering assessment of effects data for the environmental compartments (Tables 1, 2, 3–4)

Table 1.

Soil

Compound (name and/or code)	Persistence	Ecotoxicology
Bacillus thuringiensis subsp. kurstaki strain SA‐12	Spores remain viable for many years (more than 7) multiplication in bulk soil will not occur	Data gap for all representative uses in open field and walk‐in tunnels
Toxins/secondary metabolites such as crystal proteins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, 2 Cry‐like proteins and Vip 3Aa10 protein	Very low to moderate persistence Soil DT50 0.02–41 days	Data gap for all representative uses in open field and walk‐in tunnels

Table 2.

Groundwater

Compound (name and/or code)	Mobility in soil	> 0.1 μg/L at 1 m depth for the representative usesa	Pesticidal activity	Toxicological relevance
Toxins/secondary metabolites such as crystal proteins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, 2 Cry‐like proteins and VIP 3Aa10 protein	The mobility of the crystal proteins in soil is low	No	Yes	Not triggered (for dietary exposure) Data gap (for non‐dietary exposure)

^aFOCUS scenarios or relevant lysimeter.

Table 3.

Surface water and sediment

Compound (name and/or code)	Ecotoxicology
Bacillus thuringiensis subsp. kurstaki strain SA‐12	Low risk for the strain for all representative uses
Toxins/secondary metabolites such as crystal proteins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, 2 Cry‐like proteins and VIP 3Aa10 protein	Data gap

Table 4.

Air

Compound (name and/or code)	Toxicology
Bacillus thuringiensis subsp. kurstaki strain SA‐12	Data gap
Toxins/secondary metabolites such as crystal proteins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, 2 Cry‐like proteins and VIP 3Aa10 protein	No data

7. Data gaps

This is a list of data gaps identified during the peer review process, including those areas in which a study may have been made available during the peer review process but not considered for procedural reasons (without prejudice to the provisions of Article 56 of Regulation (EC) No 1107/2009 concerning information on potentially harmful effects).

• Further assessment of potential health effects after repeated exposure by inhalation to Bacillus thuringiensis subsp. kurstaki strain SA‐12 should be provided (relevant for all representative uses, see Section 2).

• Further assessment of the genotoxic potential of the insecticidal proteins by non‐dietary exposure should be provided (relevant for all representative uses, see Section 2).

• A storage stability of Bacillus thuringiensis subsp. kurstaki strain SA‐12 in high‐water plant commodities should be provided (relevant for pome fruits and tomato; see Section 3).

• Quantification of viable counts linked to specific PHIs is requested for the GAPs of the representative uses to demonstrate that the threshold of 10⁵ CFU/g is respected at harvest (relevant for pome fruits and tomato; see Section 3).

• Adequate information to address the uniform principles criterion of the strain not being expected to persist and multiply in surface water in concentrations considerably higher than the natural background levels, provided that repeated applications over the years were not available (relevant for all representative uses evaluated; see Section 4).

• Further data to address the potential effects to honeybee larvae and the infectivity and pathogenicity to bees and non‐target arthropods (relevant for representative field and walk‐in tunnel uses; see Section 5).

• Further data to address the potential toxicity, infectivity and pathogenicity to earthworms and potential adverse effects to soil microorganisms (relevant for representative field and walk‐in tunnel uses; see Section 5).

• Further hazard characterisation and assessment of the risk to terrestrial‐ and aquatic non‐target organisms from toxins/secondary metabolites such as crystal proteins present after the application of the product (relevant for representative field and walk‐in tunnel uses; see Section 5).

• Further assessment of the risk to non‐target aquatic organisms from toxins/secondary metabolites such as crystal proteins exposure in surface water (relevant for the representative use in permanent greenhouse; see Section 5).

8. Particular conditions proposed to be taken into account to manage the risk(s) identified

• Use of respiratory protective equipment is recommended for workers and operators in order to avoid potential adverse effects following exposure by inhalation (see Section 2).

9. Concerns

9.1. Issues that could not be finalised

An issue is listed as ‘could not be finalised’ if there is not enough information available to perform an assessment, even at the lowest tier level, for the representative uses in line with the uniform principles in accordance with Article 29(6) of Regulation (EC) No 1107/2009 and as set out in Commission Regulation (EU) No 546/201111 and if the issue is of such importance that it could, when finalised, become a concern (which would also be listed as a critical area of concern if it is of relevance to all representative uses).

An issue is also listed as ‘could not be finalised’ if the available information is considered insufficient to conclude on whether the active substance can be expected to meet the approval criteria provided for in Article 4 of Regulation (EC) No 1107/2009.

1. Since adverse effects after repeated exposure by inhalation could not be excluded and a genotoxic potential of the Cry proteins could not be excluded by non‐dietary exposure, the risk assessment by inhalation for residents and bystanders cannot be finalised (relevant for all representative uses, see Section 2).

2. Since it is not demonstrated that the threshold of 10⁵ CFU/g is respected at harvest (quantification of viable counts linked to specific PHIs is requested) for the representative uses on edible plant commodities, the consumer risk assessment cannot be finalised (relevant for representative uses on pome fruits and tomatoes, see Section 3).

3. The risk assessment to bees and non‐target arthropods cannot be finalised as satisfactory information for the potential effects to honeybee larvae and for infectivity and pathogenicity to bees and non‐target arthropods was not provided (relevant for representative field and walk‐in tunnel uses; see Section 5).

4. The risk assessment to earthworms and soil microorganisms cannot be finalised as satisfactory information for the potential for toxicity, infectivity and pathogenicity to earthworms and potential adverse effects to soil microorganisms was not provided (relevant for representative field and walk‐in tunnel uses; see Section 5).

5. Satisfactory information was not provided for a hazard characterisation and an assessment of the risk to terrestrial and aquatic non‐target organisms from toxins/secondary metabolites such as crystal proteins present after the application of the product (relevant for representative field and walk‐in tunnel uses; see Section 5).

6. Satisfactory information was not provided for a hazard characterisation and an assessment of the risk to non‐target aquatic organisms from toxins/secondary metabolites such as crystal proteins exposure in surface water (relevant for the representative use in permanent greenhouse; see Section 5).

9.2. Critical areas of concern

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

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

An issue is also listed as a critical area of concern if, in the light of current scientific and technical knowledge using guidance documents available at the time of application, the active substance is not expected to meet the approval criteria provided for in Article 4 of Regulation (EC) No 1107/2009.

• No critical areas of concern identified.

9.3. Overview of the concerns identified for each representative use considered

(If a particular condition proposed to be taken into account to manage an identified risk, as listed in Section 8, has been evaluated as being effective, then ‘risk identified’ is not indicated in Table 5.)

Table 5.

Overview of concerns

			Tomato		Ornamentals
Representative use		Pome fruit	Permanent greenhouse	Walk‐in tunnel	Field grown
Operator risk	Risk identified
	Assessment not finalised
Worker risk	Risk identified
	Assessment not finalised
Resident/bystander risk	Risk identified
	Assessment not finalised	X1	X1	X1	X1
Consumer risk	Risk identified
	Assessment not finalised	X2	X2	X2
Risk to wild non‐target terrestrial vertebrates	Risk identified
	Assessment not finalised	X5		X5	X5
Risk to wild non‐target terrestrial organisms other than vertebrates	Risk identified
	Assessment not finalised	X3,4,5		X3,4,5	X3,4,5
Risk to aquatic organisms	Risk identified
	Assessment not finalised	X5	X6	X5	X5
Groundwater exposure to active substance	Legal parametric value breached
	Assessment not finalised
Groundwater exposure to metabolites	Legal parametric value breached
	Parametric value of 10 μg/La breached
	Assessment not finalised

Columns are grey if no safe use can be identified. The superscript numbers relate to the numbered points indicated in Sections 9.1 and 9.2. Where there is no superscript number, see Sections 2–6 for further information.

^aValue for non‐relevant metabolites prescribed in SANCO/221/2000‐rev. 10 final, European Commission (2003).

Abbreviations

AF

assessment factor

AFLP

amplified fragment length polymorphism

CFU

colony‐forming units

CI

confidence interval

DT₅₀

period required for 50% dissipation (define method of estimation)

ELISA

enzyme‐linked immunosorbent assay

FOCUS

Forum for the Co‐ordination of Pesticide Fate Models and their Use

GAP

Good Agricultural Practice

HR

hazard rate

ISO

International Organization for Standardization

IU

international unit

iv

intravenous

K_doc

organic carbon linear adsorption coefficient

LOQ

limit of quantification

M/L

mixing and loading

mm

millimetre (also used for mean measured concentrations)

MPCA

microbial pest control agent

MPCP

microbial pest control product

MRL

maximum residue level

NRRL

Northern Regional Research Laboratory

OECD

Organisation for Economic Co‐operation and Development

PEC

predicted environmental concentration

PHI

preharvest interval

RAR

Renewal Assessment Report

RBC

red blood cells

REACH

Registration, Evaluation, Authorisation of Chemicals Regulation

SC

suspension concentrate

SNP

single‐nucleotide polymorphism

UV

ultraviolet

W/S

water/sediment

w/v

weight per unit volume

w/w

weight per unit weight

WBC

white blood cell

WG

water‐dispersible granule

WHO

World Health Organization

Appendix A – List of end points for the active substance and the representative formulation

1.

Appendix A can be found in the online version of this output (‘Supporting information’ section): https://doi.org/10.2903/j.efsa.2020.6262

Supporting information

List of end points for the active substance and the representative formulation

Suggested citation: EFSA (European Food Safety Authority) , Anastassiadou M, Arena M, Auteri D, Brancato A, Bura L, Carrasco Cabrera L, Chaideftou E, Chiusolo A, Crivellente F, De Lentdecker C, Egsmose M, Fait G, Greco L, Ippolito A, Istace F, Jarrah S, Kardassi D, Leuschner R, Lostia A, Lythgo C, Magrans O, Mangas I, Miron I, Molnar T, Padovani L, Parra Morte JM, Pedersen R, Reich H, Santos M, Sharp R, Szentes C, Terron A, Tiramani M, Vagenende B and Villamar‐Bouza L, 2020. Conclusion on the peer review of the pesticide risk assessment of the active substance Bacillus thuringiensis subsp. kurstaki strain SA‐12. EFSA Journal 2020;18(10):6262, 19 pp. 10.2903/j.efsa.2020.6262