Peer review of the pesticide risk assessment of the active substance Bacillus thuringiensis ssp. israelensis (serotype H-14) strain AM65-52.

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Sweden and co-rapporteur Member State Spain for the pesticide active substance Bacillus thuringiensis ssp. israelensis (serotype H-14) strain AM65-52 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 use of Bacillus thuringiensis ssp. israelensis (serotype H-14) strain AM65-52 as an insecticide on ornamental plants (permanent greenhouse production). 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 ssp. israelensis (serotype H‐14) strain AM65‐52 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), Sweden, and co‐rapporteur Member State (co‐RMS), Spain, received an application from Sumitomo Chemical Agro Europe S.A.S. for the renewal of approval of the active substance Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52.

An initial evaluation of the dossier on Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 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 ssp. israelensis (serotype H‐14) strain AM65‐52 according to the representative uses as an insecticide on ornamental plants (permanent greenhouse production), as proposed at the European Union (EU) level result in a sufficient insecticidal efficacy against the target fungus gnats.

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, three data gaps were identified: one related to the lack of information on the occupational health surveillance programme during manufacture of Gnatrol SC, and two others related to potential adverse effects after repeated exposure by inhalation to Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 and to potential genotoxic effect of Cry proteins via non‐dietary exposure. On this basis, the risk assessment for workers, residents and bystanders cannot be concluded (issue not finalised).

The only representative use for this microorganism is on ornamental plants which are not intended for human consumption. The consumer risk assessment is finalised provided the planting of edible succeeding crops on treated soil is restricted.

An inclusion into Annex IV of Regulation (EC) 396/2005 is not recommended.

Adequate information was not available in relation to potential interference of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 with the analytical systems for the control of the quality of drinking water. Therefore, the assessment of the potential for Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 to interfere with the analytical systems for the control of the quality of drinking water could not be finalised.

In the area of ecotoxicology, a low risk to terrestrial vertebrates (birds and mammals), aquatic organisms, wild bees, non–target arthropods other than bees and soil organisms was concluded.

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(s) and the public on the initial evaluation provided by the rapporteur Member State (RMS) and/or co‐rapporteur Member State (co‐RMS) in the renewal assessment report (RAR), and the organisation of an expert consultation where appropriate.

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

In accordance with Article 1 of the Regulation, the RMS Sweden and co‐RMS Spain received an application from Sumitomo Chemical Agro Europe S.A.S. for the renewal of approval of the active substance Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52. Complying with Article 8 of the Regulation, the RMS checked the completeness of the dossier and informed the applicant, the co‐RMS (Spain), the European Commission and EFSA about the admissibility.

The RMS provided its initial evaluation of the dossier on Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in the RAR, which was received by EFSA on 27 July 2018 (Sweden, 2018).

In accordance with Article 12 of the Regulation, EFSA distributed the RAR to the Member States and the applicant, Sumitomo Chemical Agro Europe S.A.S., for consultation and comments on 9 April 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 9 June 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 8 November 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, environmental fate and behaviour, and ecotoxicology.

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

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

A final consultation on the conclusions arising from the peer review of the risk assessment took place with Member States via a written procedure in September‐October 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 use of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 as an insecticide on ornamental plants (permanent greenhouse production), 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 (8 November 2019);

the evaluation table (28 October 2020);

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

the comments received on the draft EFSA conclusion.

Given the importance of the RAR, including its revisions (Sweden, 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 European Union (EU) for which the applicant has not demonstrated that it has regulatory access to the information on which this conclusion report is based.

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

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is a bacterium deposited at the American Type Culture Collection (ATCC), USA, under the deposit number ATCC‐1276. Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is a naturally occurring, indigenous wild‐type bacterium, initially isolated from Culex pipiens larvae in Israel (Negev Desert).

The representative formulated product for the evaluation was ‘Gnatrol SC’, a suspension concentrate (SC) containing 123 g/L of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 (1.2 × 1013 colony forming units (CFU)/L, minimum and maximum content 5.55 × 1012 to 2.5 × 1013 CFU/L) with a biopotency of 1,200 ITU/mg.

The representative uses evaluated were indoor (greenhouse, permanent structures) applications by drenching (via a sprayer or irrigation) against fungus gnats on ornamental plants in the EU. Full details of the good agricultural practices (GAPs) can be found in the list of end points in Appendix A.

Data were submitted to conclude that the use of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 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).

A data gap has been identified for a search of the scientific peer‐reviewed open literature on the active substance and its relevant metabolites, dealing with side effects on health, the environment and non‐target species and published within the 10 years before the date of submission of the dossier, to be conducted and reported in accordance with EFSA guidance on the submission of scientific peer‐reviewed open literature for the approval of pesticide active substances under Regulation (EC) No 1107/2009 (EFSA, 2011).

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

The following guidance documents were followed in the production of this conclusion: SANCO/12116/2012–rev. 0 (European Commission, 2012) and Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance (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 (MPCP). As a consequence, the specification is given only for the end‐use formulated product ‘Gnatrol SC’ of minimum content of 5.55 × 1012 CFU/L (nominal: 1.2 × 1013 CFU/L, and maximum 2.5 × 1013 CFU/L) with a biopotency of 1,200 ITU/mg. A data gap was identified for the address(es) of the manufacturing plant(s) of the MPCP.

Whole genome sequencing can be used for unequivocal identification of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 at the strain level; however, a data gap was identified for a study confirming this.

Based on the scientific progress on the taxonomy of the Bacillus cereus group in general and that of Bacillus thuringiensis in particular, it was proposed that Bacillus cereus sensu stricto and Bacillus thuringiensis are in fact one and the same species which differ only in some phenotypic traits. Nevertheless, a proposal has been put forward which maintains the current species assignment. Accordingly, both the phenotypic assignment ‘Bacillus thuringiensis serovar israelensis and the phylogenetic assignment ‘Bacillus cereus sensu stricto serovar israelensis biovar Thuringiensis’ would be valid for strain AM65‐52. For taxonomic conservation, ‘ssp.’ is used throughout this conclusion.

The active organism produces the δ‐endotoxins Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba and Cyt1Ca. Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 has the ability to produce enterotoxins, but the levels were low compared to Bacillus cereus. The absence of enterotoxin shown under manufacturing process indicated that the enterotoxin genes are only expressed under certain conditions (See Section 2). It was shown that Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 cannot produce cereulide and highly cytotoxic Cyt K1. However, it can produce haemolysin BL, non‐haemolytic enterotoxin and Cyt K2. It was demonstrated by using of a non‐validated method that haemolysin and non‐haemolytic enterotoxin are not detectable in a final product (different from the representative formulation). The content of Cyt K2 was not measured. The absence of β‐exotoxins in a theoretical MPCA was demonstrated. Considering the intended use (on ornamentals) no additional information for the content of these enterotoxins was deemed necessary. The lack of strain specific information regarding production of vegetative insecticidal proteins and secreted insecticidal proteins (vip/sip proteins) was also considered a data gap.

The optimal growth temperature range of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is between 25°C and 35°C. At temperatures lower than 30°C and up to 45°C, a high pH promoted the growth. At temperatures greater than 45°C, high pH becomes a limiting factor of the growth. Data gaps for additional information on the life cycle of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 specifically addressing generation time and environmental requirements (temperature, pH, humidity, nutrition requirements, etc.) for survival, reproduction, colonisation, damage (including human tissues) and effectiveness of the microorganism were set.

Antimicrobial susceptibility of the Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 was tested against 15 antibiotics: gentamicin, kanamycin, streptomycin, neomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, vancomycin, quinupristin and dalfopristin combination, linezolid, trimethoprim, ciprofloxacin and rifampicin. The strain was susceptible to all the antibiotics tested except trimethoprim. It should be noted however, that literature data showed resistance of B. thuringiensis ssp. to the β‐lactams: penicillin, ampicillin and cephalothin.

The main data regarding the identity of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 and its biological properties are given in Appendix A. Data on suspensibility at minimum used concentration and suspensibility and wet sieve test after storage of the MPCP at low temperature were identified as data gaps.

Acceptable methods are available for the determination of the microorganism in the MPCP and for the determination of the content of contaminating microorganisms. However, the content of microbial contaminants in MPCP was not properly addressed according to the Working Document on Microbial Contaminant Limits (European Commission, 2012), as a consequence a data gap was identified for additional information.

The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) method can be used to measure the crystal protein content in Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52.

Post‐registration monitoring methods are not needed since maximum residue levels (MRLs) in food/feed of plant origin and in animal products were not set and residue definitions for monitoring in the environmental compartments were not proposed.

Mammalian toxicity

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 was discussed at the Pesticide Peer Review Meeting Teleconference 25 in March 2020.

General data

From the medical data, no adverse reactions have been documented or reported in individuals as a result of contact with Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 during its development, manufacture, preparation or field application. However, the report of the manufacturing laboratories does not contain detailed information on the design of the occupational health surveillance program (data gap).

Allergenicity observations: The results indicate that production of specific immunoglobulin E (IgE) can occur in workers exposed to products containing Bacillus thuringiensis ssp. israelensis, but do not show any effects on the incidence or prevalence of respiratory symptoms, lung function or bronchial responsiveness. The product Vectobac Technical Powder (equivalent to Gnatrol SC) gave positive results in a skin sensitisation test (Buehler technique).

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, Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 may have the potential to provoke sensitising reactions. Additionally, the technical grade material also contains 1,2‐benzisothiazol‐3(2H)‐one, which may produce an allergenic reaction at the proposed levels.

In acute oral toxicity studies with rats, Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 showed no signs of toxicity or pathogenicity and sufficient evidence of clearance was observed among the three available studies.

In acute inhalation toxicity studies with rats, no significant toxic or pathogenic effect were observed, while a complete clearance was not demonstrated (and has been further investigated in repeat dose toxicity studies, see below). In mice, clinical signs were observed after intratracheal administration of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM‐65‐52 live vegetative cells, while a closely related strain induced mortality with lung lesions possibly caused by haemolysin after intranasal instillation. No adverse effects were observed after acute intravenous and acute intraperitoneal administration.

No acute dermal toxicity, and mild skin or eye irritation were observed in rabbits exposed to Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in a number of non‐Good Laboratory Practice (GLP) studies.

In repeat dose toxicity studies (dietary exposure in dogs, inhalation exposure in rats and mice, only one dose investigated), no adverse effect or signs of infectivity were observed in dogs and rats while interstitial lung inflammation was observed in mice at 70 days. The potential concern for serious health effects after repeated exposure by inhalation could not be excluded on the basis of the available data4 (data gap).

Secondary metabolites/toxins

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 has been shown to be able to produce enterotoxins (inactivated at low pH therefore preformed enterotoxins are 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) was concluded as applicable to cover the risk of food‐borne poisonings caused by the B. cereus group of microorganisms.5

Several studies investigating the toxic properties of the crystal proteins/endotoxins were identified in the literature. In a mouse micronucleus study with intraperitoneal administration, positive results were observed with the spore‐crystal complex containing Cry1Aa and Cry1Ac. These results were considered equivocal especially regarding whether the Cry‐proteins had been solubilised/activated prior to administration or not (data gap). It was concluded that genotoxicity is not a concern for dietary exposure but it was not possible to conclude for non‐dietary exposure.

Reference values and exposure

For the representative use on ornamentals, no toxicological reference values are triggered for the consumers in the absence of dietary exposure. 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). In the absence of a quantitative risk assessment, the use of respiratory protective equipment for the operators and workers might be considered to reduce the exposure via inhalation.

Residues

The only representative use for Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 on ornamental plants represents three soil applications with soil bound systems in permanent greenhouses at all growth stages. Therefore, a consumer risk assessment is not considered necessary in the frame of the renewal assessment unless succeeding crops are planted on the treated soil used for ornamental plant cultivation.

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is applied to horticultural seed plugs and pots in permanent greenhouses. Before application, its content in soil is expected to be negligible. Immediately after application, the concentration in soil can be estimated considering that three applications would be equivalent to 3 × 1014 CFU/ha soil. Subsequently, the viable counts of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in soil will be diluted by incorporation of fresh untreated soil since the plug is planted on.

The applied amounts of colony forming units according to the GAP at the time of treatments in soil were provided; however, the actual viable counts following each of the three applications were not quantified experimentally.

A quantification of viable counts in soil after treatment is still needed to characterise and to conclude on the residue behaviour of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 (data gap in case the restriction to not use the treated soil for the cultivation of edible crops does not apply, see below) noting that edible crops may be grown in rotation on treated soil and the relevance of a threshold level of 1 × 105 CFU/g of viable residues of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 on edible commodities (see Section 2). It is to be noted that the RMS does not agree that recycling of soil or subsequent planting of edible crops in soil previously treated with Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 poses a consumer risk. The RMS outlined that even though the background levels of Bacillus in soil might show a slight predominance of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 after treatment, considering the fact that enterotoxin production of the strain is low and uptake via plant is considered minimal compared to spraying directly on leaves this is not anticipated to result in a consumer risk.

Given that currently the studies on different strains of Bacillus thuringiensis in soil were considered insufficient to conclude on the likely competitiveness, persistence and multiplication of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in soil (see Section 4), rotation of ornamentals with edible crops and recycling of treated soil to grow edible crops should be restricted to allow finalisation of the consumer risk assessment for the representative use on non‐edible ornamental plants (see Section 8).

Bacillus thuringiensis spp. israelensis (serotype H‐14) strain AM65‐52 cannot be recommended for inclusion into Annex IV of Regulation (EC) No 396/2005 considering the issues raised in the toxicology section (see Section 2) and in the absence of a consumer risk assessment for edible commodities.

Environmental fate and behaviour

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 was discussed at the Pesticide Peer Review Meeting Teleconference 25 in March 2020.

Satisfactory information was not provided in relation to potential interference of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC6 (see specific Annex VI decision making criteria in Part II Commission Regulation (EU) No 546/20117). Therefore, it cannot be excluded that Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 would interfere with the methodologies used for such determinations. Consequently, this resulted in a data gap and assessment that could not be finalised.

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is a ‘wild type’ and there are no marker genes in the strain which would permit 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 is not different for Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 than for other naturally occurring Bacillus thuringiensis strains, transfer of genetic material by Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 after application is possible (the strain has plasmids), so could not be excluded based on the information in the dossier.

Fate and behaviour in the environment of the microorganism

Information was mainly derived from published literature on different strains of Bacillus thuringiensis in relation to its persistence and multiplication in soil. There was a specific study available on Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 which provided limited information on the population dynamics in soil as there was no correlation between the application rate and the abundance of the microbial determined in soil. The studies on different strains of Bacillus thuringiensis in soil were considered insufficient to conclude on the likely competitiveness, persistence and multiplication of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in soil. 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 soil in concentrations considerably higher than the natural background levels, taking into account repeated applications over the years. However for the intended use of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in soil‐bound cropping systems in permanent greenhouses, it was concluded that an assessment in soil is not essential for the strain when considering the criterion in the EFSA guidance on protected crops (EFSA, 2014) of DT90 needing to be above 1 year.

With respect to the persistence and multiplication in surface water published studies were available providing information on the persistence of Bacillus thuringiensis in water. There was a specific study available for Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 which indicated that the strain would constitute a very small percentage of the natural bacteria present in surface water and sediment directly after application. However, a published paper on the occurrence of Bacillus thuringiensis in surface water showed that the species of Bacillus thuringiensis is present and that it is likely that this species is capable of growing in freshwater environments under nutrient/oxygen‐rich conditions. 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. The information on the persistence/multiplication/germination of the strain in natural surface water was considered insufficient to demonstrate that Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 is likely to decline in surface water. For the representative use by drench via a sprayer or irrigation on soil bound systems in permanent greenhouse the exposure to surface water is expected to be negligible.

Limited information was provided on the occurrence and behaviour of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 in air. A study on strain AM65‐52 indicated that the viability may be reduced due to UV radiation.

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 species Bacillus thuringiensis ssp. israelensis is able to produce secondary metabolites, which are crystalline proteins, e.g. contain the δ‐endotoxins, Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba, and Cyt1Ca. These crystalline proteins constitute components in the formulated product within and outside spores and together with spore germination and proliferation of the vegetative cell into the haemocoel of the target organism are responsible for the insecticidal mode of action of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52.

It is not known to what extent Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 will produce crystalline proteins following its application. However, as the concentrations of the crystalline 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 (Pesticide Peer Review Meeting Teleconference 25). For the crystal proteins, the experts agreed it would be appropriate to read across degradation and adsorption end points between the different crystal proteins from the available data set that contains measured end points from only a subset of these different δ‐endotoxins and/or crystal proteins. Full details of the available experimental end points and which δ‐endotoxins or crystalline protein test material they were derived from can be found in Appendix A. As these end points were not available for all the δ‐endotoxins present in Bacillus thuringiensis ssp. israelensis, 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. An assessment in soil for crops grown in permanent greenhouse structures is not needed according to the EFSA protected crops guidance (EFSA, 2014) as the DT90 of crystalline proteins is indicated to be less than 1 year. Satisfactory calculations were provided for an environmental exposure assessment of the crystalline proteins in surface water, sediment and groundwater for the representative uses. The FOCUS surface water Step 1 and 2 calculator (v3.2) for the surrogate crop bulb vegetable was used for surface water and sediment calculations (FOCUS, 2001). For groundwater calculations PEARL 4.4.4 and PELMO 5.5.3 were used for the surrogate crop onions (European Commission, 2014a)8 (see Appendix A). It was concluded that the potential for leaching of the crystalline 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.

Ecotoxicology

Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 was discussed at the Pesticide Peer Review Meeting Teleconference 25 in March 2020.

The risk assessment was based on the following documents: European Commission (2002a,b), EFSA (2009), and SETAC (2001).

Toxicity data and several publications from the scientific literature were available to assess the risk to non–target organisms. As the representative uses are restricted to permanent greenhouses by drench via a sprayer or irrigation, negligible exposure to terrestrial vertebrates (birds and mammals), aquatic organisms, wild bees,non‐target arthropods other than bees and soil organisms is anticipated. Consequently, a low risk to these groups of non–target organisms can be concluded. A similar conclusion can be drawn for the crystalline proteins. As part of integrated pest management practices, further data may be requested at EU Member State level of Bacillus thuringiensis ssp. israelensis or beneficial organisms introduced.

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).

Data gaps identified for the representative uses evaluated

A search of the scientific peer‐reviewed open literature on the active substance and its relevant metabolites, dealing with side effects on health, the environment and non‐target species and published within the 10 years before the date of submission of the dossier, to be conducted and reported in accordance with EFSA guidance on the submission of scientific peer‐reviewed open literature for the approval of pesticide active substances under Regulation (EC) No 1107/2009 (EFSA, 2011) (relevant for the representative use evaluated and Sections 1, 2, 3, 4 and 5).

The address(es) of the manufacturing plant(s) of MPCP (relevant for the representative use evaluated; see Section 1).

Method providing unequivocally identification of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 down to the strain level (relevant for the representative use evaluated; see Section 1).

Additional information showing that MPCP complies with requirements of the Working Document on Microbial Contaminant Limits (European Commission, 2012; relevant for the representative use evaluated; see Section 1).

Additional information related with capability of the Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 to produce vip/sip proteins (relevant for the representative use evaluated; see Section 1).

Additional information on the life cycle of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 specifically addressing generation time (relevant for the representative use evaluated; see Section 1).

Additional information on environmental requirements (temperature, pH, humidity, nutrition requirements, etc.) for survival, reproduction, colonisation, damage (including human tissues) and effectiveness of the microorganism (relevant for the representative use evaluated; see Section 1).

Data on suspensibility at minimum used concentration (relevant for the representative use evaluated; see Section 1).

Data on suspensibility and wet sieve test after storage of the MPCP at low temperature (relevant for the representative use evaluated; see Section 1).

The content of microbial contaminants in MPCP was not properly addressed according the Working Document on Microbial Contaminant Limits (relevant for the representative use evaluated; see Section 1).

More detailed information on the occupational health surveillance program during manufacture of Gnatrol SC should be provided (relevant for the representative use evaluated; see Section 2).

Further assessment of potential health effects after repeated exposure by inhalation to Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 should be provided (relevant for the representative use evaluated; see Section 2).

Further assessment of the genotoxic potential of the Cry proteins by non‐dietary exposure should be provided (relevant for the representative use evaluated; see Section 2).

Magnitude of colony forming units in soil following treatments in case that edible crops are grown on treated soil for ornamental cultivation (relevant for the representative use evaluated; see Section 3).

Adequate information was not available in relation to potential interference of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC (see specific Annex VI decision making criteria in Part II Commission Regulation (EU) No 546/2011; relevant for the representative use evaluated; see Section 4).

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

In the absence of a quantitative risk assessment, the use of respiratory protective equipment for the operators and workers might be considered to reduce the exposure by inhalation (see Section 2).

Planting of edible crops succeeding to ornamental crops or recycling of treated soil to grow edible crops should be restricted (see Section 3).

Concerns

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 546/20119 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.

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 (relevant for the representative use, see Section 2).

Adequate information was not available in relation to potential interference of Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 with the analytical systems for the control of the quality of drinking water provided for in Directive 98/83/EC6 (see specific Annex VI decision making criteria in Part II Commission Regulation (EU) No 546/20117 Therefore, the potential for Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52 to interfere with the analytical systems for the control of the quality of drinking water could not be finalised (relevant for all representative uses evaluated; see Section 4).

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 were identified.

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

Representative use		Ornamental plants (permanent greenhouse use)
Operator risk	Risk identified
	Assessment not finalised
Worker risk	Risk identified
	Assessment not finalised
Resident/bystander risk	Risk identified
	Assessment not finalised	X1
Consumer risk	Risk identified
	Assessment not finalised
Risk to wild non‐target terrestrial vertebrates	Risk identified
	Assessment not finalised
Risk to wild non‐target terrestrial organisms other than vertebrates	Risk identified
	Assessment not finalised
Risk to aquatic organisms	Risk identified
	Assessment not finalised
Groundwater exposure to active substance	Legal parametric value breached
	Assessment not finalised
Groundwater exposure to metabolites	Legal parametric value breacheda
	Parametric value of 10 μg/Lb breached
	Assessment not finalised

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

When the consideration for classification made in the context of this evaluation under Regulation (EC) No 1107/2009 is confirmed under Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008.

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

Abbreviations

American type culture collection

colony forming units

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

period required for 90% dissipation (define method of estimation)

European Economic Community

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

Good Agricultural Practice

good laboratory practice

immunoglobulin E

organic carbon linear adsorption coefficient

microbial pest control agent

microbial pest control product

maximum residue level

Renewal Assessment Report

rapporteur Member State

suspension concentrate

secreted insecticidal protein

vegetative insecticidal protein

ultraviolet

Appendix A can be found in the online version of this output (‘Supporting information’ section): https://doi.org/10.2903/j.efsa.2020.6317

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

Click here for additional data file.

Table 1

Soil

Compound (name and/or code)	Persistence	Ecotoxicology
Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52	Open regarding strain specific information but considered not needed when uses are in permanent greenhouses	Low risk
Toxins/secondary metabolites such as crystalline proteins, Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba and Cyt1Ca	An assessment in soil crops grown in permanent greenhouse structures is not needed according to the EFSA protected crops guidance (EFSA, 2014) as the DT90 is less than 1 year for the crystalline proteins	Low risk

DT90: period required for 90% dissipation.

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 crystalline proteins, Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba and Cyt1Ca	The mobility of the crystalline proteins in soil is low	No	Yes	No (for dietary exposure) Data gap (for non‐dietary exposure)

FOCUS scenarios or a relevant lysimeter.

Table 3

Surface water and sediment

Compound (name and/or code)	Ecotoxicology
Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52	Low risk
Toxins/secondary metabolites such as crystalline proteins, Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba and Cyt1Ca	Low risk

Table 4

Air

Compound (name and/or code)	Toxicology
Bacillus thuringiensis ssp. israelensis (serotype H‐14) strain AM65‐52	Data gap
Toxins/secondary metabolites such as crystalline proteins, Cry4Aa, Cry4Ba, Cry10A, Cry11A, Cyt1Aa, Cyt2Ba and Cyt1Ca	No data