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The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case-by-case assessment is necessary., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

EFSA's Panel on Biological Hazards (BIOHAZ Panel) deals with questions on biological hazards relating to food safety and food-borne diseases. This covers food-borne zoonoses, transmissible spongiform encephalopathies, antimicrobial resistance, food microbiology, food hygiene, animal-by products, and associated waste management issues. The scientific assessments are diverse and frequently the development of new methodological approaches is required to deal with a mandate. Among the many risk factors, product characteristics (pH, water activity etc.), time and temperature of processing and storage along the food supply chain are highly relevant for assessing the biological risks. Therefore, predictive microbiology becomes an essential element of the assessments. Uncertainty analysis is incorporated in all BIOHAZ scientific assessments, to meet the general requirement for transparency. Assessments should clearly and unambiguously state what sources of uncertainty have been identified and their impact on the conclusions of the assessment. Four recent BIOHAZ Scientific Opinions are presented to illustrate the use of predictive modelling and quantitative microbial risk assessment principles in regulatory science. The Scientific Opinion on the guidance on date marking and related food information, gives a general overview on the use of predictive microbiology for shelf-life assessment. The Scientific Opinion on the efficacy and safety of high-pressure processing of food provides an example of inactivation modelling and compliance with performance criteria. The Scientific Opinion on the use of the so-called 'superchilling' technique for the transport of fresh fishery products illustrates the combination of heat transfer and microbial growth modelling. Finally, the Scientific Opinion on the delayed post-mortem inspection in ungulates, shows how variability and uncertainty, were quantitatively embedded in assessing the probability of Salmonella detection on carcasses, via stochastic modelling and expert knowledge elicitation., Competing Interests: Declaration of competing interest The authors declare that they have no known conflict of financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A quantitative microbiological spoilage risk assessment model (QMSRA) of aerobically stored fresh poultry fillets was developed based on pseudomonads growth and metabolic activity. Simultaneous microbiological and sensory analyses were performed in poultry fillets to evaluate the relation between pseudomonads concentration and sensory rejection due to spoilage. The analysis showed no organoleptic rejection at pseudomonads concentrations less than 6.08 log CFU/cm 2 . For higher concentrations, a "spoilage-response" relationship was developed using a beta-Poisson model. The above relationship was combined with a stochastic modeling approach for pseudomonads growth by taking into account both variability and uncertainty of factors affecting spoilage. To enhance the reliability of the developed QMSRA model, uncertainty was quantified and separated from variability using a second order Monte Carlo simulation. For a batch of 10,000 units, the QMSRA model predicted a median number of 11, 80, 295, 733 and 1,389 spoiled units for retail storage times of 6,7, 8, 9 and 10 days, respectively, while no spoiled units were predicted for storage time of up to 5 days at retail. Scenario analysis showed that a reduction of 1 log in the pseudomonads concentration at the time of packaging or 1 °C in retail storage temperature results in up to 90% reduction of the spoiled units while the combination of the above interventions can reduce the risk of spoilage by up to 99%, depending on the storage time. The poultry industry can utilize the QMSRA model as a transparent scientific basis to support food quality management decisions in determining appropriate expiration dates which maximize the utilization of the product's "true" shelf life while minimize the risk of spoilage to an acceptable level. Furthermore, the scenario analysis can provide the necessary components for an effective cost-benefit analysis, enabling the identification and comparison of appropriate strategies for extending the shelf life of fresh poultry products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 38 microorganisms notified to EFSA between October 2022 and March 2023 (inclusive) (28 as feed additives, 5 as food enzymes, food additives and flavourings, 5 as novel foods), 34 were not evaluated because: 8 were filamentous fungi, 4 were Enterococcus faecium and 2 were Escherichia coli (taxonomic units that are excluded from the QPS evaluation) and 20 were taxonomic units (TUs) that already have a QPS status. Three of the other four TUs notified within this period were evaluated for the first time for a possible QPS status: Anaerobutyricum soehngenii , Stutzerimonas stutzeri (former Pseudomonas stutzeri ) and Nannochloropsis oculata . Microorganism strain DSM 11798 has also been notified in 2015 and as its taxonomic unit is notified as a strain not a species, it is not suitable for the QPS approach. A. soehngenii and N. oculata are not recommended for the QPS status due to a limited body of knowledge of its use in the food and feed chains. S. stutzeri is not recommended for inclusion in the QPS list based on safety concerns and limited information about the exposure of animals and humans through the food and feed chains., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

An assessment was conducted on the level of inactivation of relevant pathogens that could be present in processed animal protein of porcine origin intended to feed poultry and aquaculture animals when methods 2 to 5 and method 7, as detailed in Regulation (EU) No 142/2011, are applied. Five approved scenarios were selected for method 7. Salmonella Senftenberg, Enterococcus faecalis , spores of Clostridium perfringens and parvoviruses were shortlisted as target indicators. Inactivation parameters for these indicators were extracted from extensive literature search and a recent EFSA scientific opinion. An adapted Bigelow model was fitted to retrieved data to estimate the probability that methods 2 to 5, in coincidental and consecutive modes, and the five scenarios of method 7 are able to achieve a 5 log 10 and a 3 log 10 reduction of bacterial indicators and parvoviruses, respectively. Spores of C. perfringens were the indicator with the lowest probability of achieving the target reduction by methods 2 to 5, in coincidental and consecutive mode, and by the five considered scenarios of method 7. An expert knowledge elicitation was conducted to estimate the certainty of achieving a 5 log 10 reduction of spores of C. perfringens considering the results of the model and additional evidence. A 5 log 10 reduction of C. perfringens spores was judged: 99-100% certain for methods 2 and 3 in coincidental mode; 98-100% certain for method 7 scenario 3; 80-99% certain for method 5 in coincidental mode; 66-100% certain for method 4 in coincidental mode and for method 7 scenarios 4 and 5; 25-75% certain for method 7 scenario 2; and 0-5% certain for method 7 scenario 1. Higher certainty is expected for methods 2 to 5 in consecutive mode compared to coincidental mode., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Anoxybacillus flavithermus and Bacillus licheniformis are among the predominant spore-formers of heat-processed foods. To our knowledge, no systematic analysis of growth kinetic data of A. flavithermus or B. licheniformis is currently available. In the present study, the growth kinetics of A. flavithermus and B. licheniformis in broth at various temperature and pH conditions were studied. Cardinal models were used to model the effect of the above-mentioned factors on the growth rates. The estimated values for the cardinal parameters T min ,T opt ,T max ,pH min and pH 1/2 for A. flavithermus were 28.70 ± 0.26, 61.23 ± 0.16 and 71.52 ± 0.32 °C, 5.52 ± 0.01 and 5.73 ± 0.01, respectively, while for B. licheniformis they were 11.68 ± 0.03, 48.05 ± 0.15, 57.14 ± 0.01 °C, 4.71 ± 0.01 and 5.670 ± 0.08, respectively. The growth behaviour of these spoilers was also investigated in a pea beverage at 62 and 49 °C, respectively, to adjust the models to this product. The adjusted models were further validated at static and dynamic conditions and demonstrated good performance with 85.7 and 97.4% of predicted populations for A. flavithermus and B. licheniformis, respectively, being within the -10%-10% relative error (RE) zone. The developed models can be useful tools in assessing the potential of spoilage of heat-processed foods including plant-based milk alternatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017-28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease-associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high-risk target groups for surveillance should be revised, and 'road kill' removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene ( PRNP ) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high-risk target groups, sustained over time, using sampling units and a data-driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data-driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double-strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Geobacillus stearothermophilus is one of the predominant spoilers of UHT-treated food products, due to its extremely heat-resistant spores. However, the surviving spores should be exposed to temperature higher than their minimum growth temperature for a certain time to germinate and grow to spoilage levels. Considering the projected temperature increase due to climate change, the events of non-sterility during distribution and transportation are expected to escalate. Hence, the aim of this study was to build a quantitative microbial spoilage risk assessment (QMRSA) model to quantify the risk of spoilage of plant-based milk alternatives within Europe. The model consists of four main steps: 1. Initial contamination of raw materials 2. Heat inactivation of spores during UHT treatment 3. Partitioning 4. Germination and outgrowth of spores during distribution and storage. The risk of spoilage was defined as the probability of G. stearothermophilus to reach its maximum concentration (N max  = 10 7.5 CFU/mL) at the time of consumption. The assessment was performed for North (Poland) and South (Greece) Europe, and the risk of spoilage was estimated for the current climatic conditions and a climate change scenario. Based on the results, the risk of spoilage was negligible for the North European region, while the risk of spoilage in South Europe was 6.2 × 10 -3 95% CI (2.3 × 10 -3 ;1.1 × 10 -2 ) under the current climatic conditions. The risk of spoilage was increased for both tested countries under climate change scenario; from zero to 1.0 × 10 -4 in North Europe, risk multiplied 2 or 3 in South Europe depending on air conditioning implementation at consumer's place. Therefore, the heat treatment intensity and the use of insulated trucks during distribution were investigated as mitigation strategies and led to significant reduction of the risk. Overall, the QMRSA model developed in this study can support risk management decisions of these products by quantify the potential risk under current climatic conditions and climate change scenarios., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, new information was found leading to the withdrawal of the qualification 'absence of aminoglycoside production ability' for Bacillus velezensis. The qualification for Bacillus paralicheniformis was changed to 'absence of bacitracin production ability'. For the other TUs, no new information was found that would change the status of previously recommended QPS TUs. Of 52 microorganisms notified to EFSA between April and September 2022 (inclusive), 48 were not evaluated because: 7 were filamentous fungi, 3 were Enterococcus faecium , 2 were Escherichia coli , 1 was Streptomyces spp., and 35 were taxonomic units (TUs) that already have a QPS status. The other four TUs notified within this period, and one notified previously as a different species, which was recently reclassified, were evaluated for the first time for a possible QPS status: Xanthobacter spp. could not be assessed because it was not identified to the species level; Geobacillus thermodenitrificans is recommended for QPS status with the qualification 'absence of toxigenic activity'. Streptoccus oralis is not recommended for QPS status. Ogataea polymorpha is proposed for QPS status with the qualification 'for production purposes only'. Lactiplantibacillus argentoratensis (new species) is included in the QPS list., (© 2023 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The impact of dry-ageing of beef and wet-ageing of beef, pork and lamb on microbiological hazards and spoilage bacteria was examined and current practices are described. As 'standard fresh' and wet-aged meat use similar processes these were differentiated based on duration. In addition to a description of the different stages, data were collated on key parameters (time, temperature, pH and a w ) using a literature survey and questionnaires. The microbiological hazards that may be present in all aged meats included Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., Staphylococcus aureus , Listeria monocytogenes , enterotoxigenic Yersinia spp., Campylobacter spp. and Clostridium spp. Moulds, such as Aspergillus spp. and Penicillium spp., may produce mycotoxins when conditions are favourable but may be prevented by ensuring a meat surface temperature of -0.5 to 3.0°C, with a relative humidity (RH) of 75-85% and an airflow of 0.2-0.5 m/s for up to 35 days. The main meat spoilage bacteria include Pseudomonas spp . , Lactobacillus spp. Enterococcus spp., Weissella spp., Brochothrix spp., Leuconostoc spp., Lactobacillus spp., Shewanella spp. and Clostridium spp. Under current practices, the ageing of meat may have an impact on the load of microbiological hazards and spoilage bacteria as compared to standard fresh meat preparation. Ageing under defined and controlled conditions can achieve the same or lower loads of microbiological hazards and spoilage bacteria than the variable log 10 increases predicted during standard fresh meat preparation. An approach was used to establish the conditions of time and temperature that would achieve similar or lower levels of L. monocytogenes and Yersinia enterocolitica (pork only) and lactic acid bacteria (representing spoilage bacteria) as compared to standard fresh meat. Finally, additional control activities were identified that would further assure the microbial safety of dry-aged beef, based on recommended best practice and the outputs of the equivalence assessment., (© 2023 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

Copper is an essential micronutrient and also a regulated product used in organic and in conventional farming pest management. Both deficiency and excessive exposure to copper can have adverse health effects. In this Scientific Opinion, the EFSA 2021 harmonised approach for establishing health-based guidance values (HBGVs) for substances that are regulated products and also nutrients was used to resolve the divergent existing HBGVs for copper. The tightly regulated homeostasis prevents toxicity manifestation in the short term, but the development of chronic copper toxicity is dependent on copper homeostasis and its tissue retention. Evidence from Wilson disease suggests that hepatic retention is indicative of potential future and possibly sudden onset of copper toxicity under conditions of continuous intake. Hence, emphasis was placed on copper retention as an early marker of potential adverse effects. The relationships between (a) chronic copper exposure and its retention in the body, particularly the liver, and (b) hepatic copper concentrations and evidence of toxicity were examined. The Scientific Committee (SC) concludes that no retention of copper is expected to occur with intake of 5 mg/day and established an Acceptable Daily Intake (ADI) of 0.07 mg/kg bw. A refined dietary exposure assessment was performed, assessing contribution from dietary and non-dietary sources. Background copper levels are a significant source of copper. The contribution of copper from its use as plant protection product (PPP), food and feed additives or fertilisers is negligible. The use of copper in fertilisers or PPPs contributes to copper accumulation in soil. Infant formula and follow-on formula are important contributors to dietary exposure of copper in infants and toddlers. Contribution from non-oral sources is negligible. Dietary exposure to total copper does not exceed the HBGV in adolescents, adults, elderly and the very elderly. Neither hepatic copper retention nor adverse effects are expected to occur from the estimated copper exposure in children due to higher nutrient requirements related to growth., (© 2023 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

Individual-cell heterogeneity is a major source of variability in biological systems affecting importantly, among others, microbial behavior. Characterization of cell populations of pathogenic bacterial strains in their entirety, ignoring the phenotypic variability of single cells, may result in erroneous safety risk estimates. The objective of the present study was the evaluation and comparison of the heterogeneity in the individual-cell growth dynamics of different strains of Salmonella enterica. The stochasticity in the growth of single cells of five S. enterica ser. Typhimurium strains was quantitatively described using time-lapse microscopy, and the existence of a strain effect was statistically assessed. In total, 831 growing microcolonies originating from single cells were monitored and analyzed, and the growth kinetic parameters of lag time (λ) and maximum specific growth rate (μ max ) for each one of them were estimated. An extensive heterogeneity in individual-cell growth kinetics was recorded, while significant inter-strain differences in their heterogeneity were evident based on simultaneous Bonferroni confidence intervals and Levene's tests. The Logistic and LogLogistic probability distribution provided the best fitting for μ max and λ data, respectively for all the tested strains. The strain effect on the above distributions was also demonstrated with pairwise comparisons of the decile differences. The impact of strain-dependent heterogeneity on microbial growth was visualized by comparing stochastic growth curves of different strains using Monte Carlo simulation. In conclusion, the individual-cell growth dynamics of S. enterica are heterogeneous, with the magnitude of the observed heterogeneity appearing to be an inherent characteristic of bacterial strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

An alternative method for the production of renewable fuels from rendered animal fats (pretreated using methods 1-5 or method 7 as described in Annex IV of Commission Regulation (EC) No 2011/142) and used cooking oils, derived from Category 3 animal by-products, was assessed. The method is based on a catalytic co-processing hydrotreatment using a middle distillate followed by a stripping step. The materials must be submitted to a pressure of at least 60 bars and a temperature of at least 270°C for at least 4.7 min. The application focuses on the demonstration of the level of reduction of spores from non-pathogenic spore-forming indicator bacterial species ( Bacillus subtilis and Desulfotomaculum kuznetsovii ), based on a non-systematic review of published data and additional extrapolation analyses. The EFSA BIOHAZ Panel considers that the application and supporting literature contain sufficient evidence that the proposed alternative method can achieve a reduction of at least 5 log 10 in the spores of B. subtilis and a 12 log 10 reduction in the spores of C. botulinum . The alternative method under evaluation is considered at least equivalent to the processing methods currently approved in the Commission Regulation (EU) No 2011/142., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The transmission of antimicrobial resistance (AMR) between food-producing animals (poultry, cattle and pigs) during short journeys (< 8 h) and long journeys (> 8 h) directed to other farms or to the slaughterhouse lairage (directly or with intermediate stops at assembly centres or control posts, mainly transported by road) was assessed. Among the identified risk factors contributing to the probability of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), the ones considered more important are the resistance status (presence of ARB/ARGs) of the animals pre-transport, increased faecal shedding, hygiene of the areas and vehicles, exposure to other animals carrying and/or shedding ARB/ARGs (especially between animals of different AMR loads and/or ARB/ARG types), exposure to contaminated lairage areas and duration of transport. There are nevertheless no data whereby differences between journeys shorter or longer than 8 h can be assessed. Strategies that would reduce the probability of AMR transmission, for all animal categories include minimising the duration of transport, proper cleaning and disinfection, appropriate transport planning, organising the transport in relation to AMR criteria (transport logistics), improving animal health and welfare and/or biosecurity immediately prior to and during transport, ensuring the thermal comfort of the animals and animal segregation. Most of the aforementioned measures have similar validity if applied at lairage, assembly centres and control posts. Data gaps relating to the risk factors and the effectiveness of mitigation measures have been identified, with consequent research needs in both the short and longer term listed. Quantification of the impact of animal transportation compared to the contribution of other stages of the food-production chain, and the interplay of duration with all risk factors on the transmission of ARB/ARGs during transport and journey breaks, were identified as urgent research needs., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The Scientific Committee (SC) reconfirms that the benchmark dose (BMD) approach is a scientifically more advanced method compared to the no-observed-adverse-effect-level (NOAEL) approach for deriving a Reference Point (RP). The major change compared to the previous Guidance (EFSA SC, 2017) concerns the Section 2.5, in which a change from the frequentist to the Bayesian paradigm is recommended. In the former, uncertainty about the unknown parameters is measured by confidence and significance levels, interpreted and calibrated under hypothetical repetition, while probability distributions are attached to the unknown parameters in the Bayesian approach, and the notion of probability is extended to reflect uncertainty of knowledge. In addition, the Bayesian approach can mimic a learning process and reflects the accumulation of knowledge over time. Model averaging is again recommended as the preferred method for estimating the BMD and calculating its credible interval. The set of default models to be used for BMD analysis has been reviewed and amended so that there is now a single set of models for quantal and continuous data. The flow chart guiding the reader step-by-step when performing a BMD analysis has also been updated, and a chapter comparing the frequentist to the Bayesian paradigm inserted. Also, when using Bayesian BMD modelling, the lower bound (BMDL) is to be considered as potential RP, and the upper bound (BMDU) is needed for establishing the BMDU/BMDL ratio reflecting the uncertainty in the BMD estimate. This updated guidance does not call for a general re-evaluation of previous assessments where the NOAEL approach or the BMD approach as described in the 2009 or 2017 Guidance was used, in particular when the exposure is clearly lower (e.g. more than one order of magnitude) than the health-based guidance value. Finally, the SC firmly reiterates to reconsider test guidelines given the wide application of the BMD approach., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

EFSA was asked by the European Commission to evaluate synthetic biology (SynBio) developments for agri-food use in the near future and to determine whether or not they are expected to constitute potential new hazards/risks. Moreover, EFSA was requested to evaluate the adequacy of existing guidelines for risk assessment of SynBio and if updated guidance is needed. The scope of this Opinion covers food and feed risk assessment, the variety of microorganisms that can be used in the food/feed chain and the whole spectrum of techniques used in SynBio. This Opinion complements a previously adopted Opinion with the evaluation of existing guidelines for the microbial characterisation and environmental risk assessment of microorganisms obtained through SynBio. The present Opinion confirms that microbial SynBio applications for food and feed use, with the exception of xenobionts, could be ready in the European Union in the next decade. New hazards were identified related to the use or production of unusual and/or new-to-nature components. Fifteen cases were selected for evaluating the adequacy of existing guidelines. These were generally adequate for assessing the product, the production process, nutritional and toxicological safety, allergenicity, exposure and post-market monitoring. The comparative approach and a safety assessment per se could be applied depending on the degree of familiarity of the SynBio organism/product with the non-genetically modified counterparts. Updated guidance is recommended for: (i) bacteriophages, protists/microalgae, (ii) exposure to plant protection products and biostimulants, (iii) xenobionts and (iv) feed additives for insects as target species. Development of risk assessment tools is recommended for assessing nutritional value of biomasses, influence of microorganisms on the gut microbiome and the gut function, allergenic potential of new-to-nature proteins, impact of horizontal gene transfer and potential risks of living cell intake. A further development towards a strain-driven risk assessment approach is recommended., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The growth of naturally contaminated pseudomonads on fresh breast and thigh poultry fillets during aerobic storage was studied and modeled as a function of temperature (0-30°C). A statistical comparison of the models for breast and thigh fillets showed that muscle type does not significantly affect the temperature dependence of pseudomonads growth kinetics. A unified model for breast and thigh was developed and validated against pseudomonads growth rate data under isothermal conditions extracted from literature and experimental data under dynamic temperature conditions. The validation results showed a satisfactory performance of the model with the bias and accuracy factors ranging from 0.85 to 1.09 and 1.02 to 1.21, respectively. The model was further used to predict the shelf life of fresh poultry as the time required by pseudomonads to reach the spoilage level for various scenarios of temperature, initial contamination level, and physiological state of pseudomonads demonstrating its application in a risk-based shelf-life assessment of fresh poultry products., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Of the 50 microorganisms notified to EFSA in October 2021 to March 2022 (inclusive), 41 were not evaluated: 10 filamentous fungi, 1 Enterococcus faecium , 1 Clostridium butyricum, 3 Escherichia coli and 1 Streptomyces spp. because are excluded from QPS evaluation, and 25 TUs that have already a QPS status. Nine notifications, corresponding to seven TUs were evaluated: four of these, Streptococcus salivarius, Companilactobacillus formosensis, Pseudonocardia autotrophica and Papiliotrema terrestris , being evaluated for the first time. The other three, Microbacterium foliorum, Pseudomonas fluorescens and Ensifer adhaerens were re-assessed. None of these TUs were recommended for QPS status: Ensifer adhaerens, Microbacterium foliorum, Companilactobacillus formosensis and Papiliotrema terrestris due to a limited body of knowledge , Streptococcus salivarius due to its ability to cause bacteraemia and systemic infection that results in a variety of morbidities, Pseudonocardia autotrophica due to lack of body of knowledge and uncertainty on the safety of biologically active compounds which can be produced, and Pseudomonas fluorescens due to possible safety concerns ., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

High-pressure processing (HPP) is a non-thermal treatment in which, for microbial inactivation, foods are subjected to isostatic pressures (P) of 400-600 MPa with common holding times (t) from 1.5 to 6 min. The main factors that influence the efficacy (log 10 reduction of vegetative microorganisms) of HPP when applied to foodstuffs are intrinsic (e.g. water activity and pH), extrinsic (P and t) and microorganism-related (type, taxonomic unit, strain and physiological state). It was concluded that HPP of food will not present any additional microbial or chemical food safety concerns when compared to other routinely applied treatments (e.g. pasteurisation). Pathogen reductions in milk/colostrum caused by the current HPP conditions applied by the industry are lower than those achieved by the legal requirements for thermal pasteurisation. However, HPP minimum requirements (P/t combinations) could be identified to achieve specific log 10 reductions of relevant hazards based on performance criteria (PC) proposed by international standard agencies (5-8 log 10 reductions). The most stringent HPP conditions used industrially (600 MPa, 6 min) would achieve the above-mentioned PC, except for Staphylococcus aureus . Alkaline phosphatase (ALP), the endogenous milk enzyme that is widely used to verify adequate thermal pasteurisation of cows' milk, is relatively pressure resistant and its use would be limited to that of an overprocessing indicator. Current data are not robust enough to support the proposal of an appropriate indicator to verify the efficacy of HPP under the current HPP conditions applied by the industry. Minimum HPP requirements to reduce Listeria monocytogenes levels by specific log 10 reductions could be identified when HPP is applied to ready-to-eat (RTE) cooked meat products, but not for other types of RTE foods. These identified minimum requirements would result in the inactivation of other relevant pathogens ( Salmonella and Escherichia coli ) in these RTE foods to a similar or higher extent., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The qualified presumption of safety (QPS) approach was developed to provide a generic pre-evaluation of the safety of biological agents. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. The QPS list was updated in relation to the revised taxonomy of the genus Bacillus , to synonyms of yeast species and for the qualifications 'absence of resistance to antimycotics' and 'only for production purposes'. Lactobacillus cellobiosus has been reclassified as Limosilactobacillus fermentum . In the period covered by this statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TU)s. Of the 70 microorganisms notified to EFSA, 64 were not evaluated: 11 filamentous fungi, one oomycete, one Clostridium butyricum , one Enterococcus faecium , five Escherichia coli , one Streptomyces sp., one Bacillus nakamurai and 43 TUs that already had a QPS status. Six notifications, corresponding to six TUs were evaluated: Paenibacillus lentus was reassessed because an update was requested for the current mandate. Enterococcus lactis synonym Enterococcus xinjiangensis , Aurantiochytrium mangrovei synonym Schizochytrium mangrovei , Schizochytrium aggregatum , Chlamydomonas reinhardtii synonym Chlamydomonas smithii and Haematococcus lacustris synonym Haematococcus pluvialis were assessed for the first time. The following TUs were not recommended for QPS status: P. lentus due to a limited body of knowledge, E. lactis synonym E. xinjiangensis due to potential safety concerns, A. mangrovei synonym S. mangrovei , S. aggregatum and C. reinhardtii synonym C. smithii , due to lack of a body of knowledge on its occurrence in the food and feed chain. H. lacustris synonym H. pluvialis is recommended for QPS status with the qualification 'for production purposes only'., (© 2022 Wiley‐VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.)

The European Commission requested EFSA to assess if different thermal processes achieve a 5 log 10 reduction in Enterococcus faecalis or Salmonella Senftenberg (775W) and (if relevant) a 3 log 10 reduction in thermoresistant viruses (e.g. Parvovirus ) as well as if different chemical processes achieve a 3 log 10 reduction of eggs of Ascaris sp., in eight groups of Category 2 and 3 derived products and animal by-products (ABP). These included (1) ash derived from incineration, co-incineration and combustion; (2) glycerine derived from the production of biodiesel and renewable fuels; (3) other materials derived from the production of biodiesel and renewable fuels; (4) hides and skins; (5) wool and hair; (6) feathers and down; (7) pig bristles; and (8) horns, horn products, hooves and hoof products. Data on the presence of viral hazards and on thermal and chemical inactivation of the targeted indicator microorganisms and biological hazards under relevant processing conditions were extracted via extensive literature searches. The evidence was assessed via expert knowledge elicitation. The certainty that the required log 10 reductions in the most resistant indicator microorganisms or biological hazards will be achieved for each of the eight groups of materials mentioned above by the thermal and/or chemical processes was (1) 99-100% for the two processes assessed; (2) 98-100% in Category 2 ABP, at least 90-99% in Category 3 ABP; (3) 90-99% in Category 2 ABP; at least 66-90% in Category 3 ABP; (4) 10-66% and 33-66%; (5) 1-33% and 10-50%; (6) 66-90%; (7) 33-66% and 50-95%; (8) 66-95%, respectively. Data generation on the occurrence and reduction of biological hazards by thermal and/or chemical methods in these materials and on the characterisation of the usage pathways of ABP as organic fertilisers/soil improvers is recommended., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A cardinal model (CM) for the effects of temperature (range: 32-59 °C), pH (range: 5.0-8.5) and water activity (a w ) (range: 0.980-0.995) on Bacillus coagulans DSM 1 growth rate was developed in brain heart infusion broth (BHI), using the Bioscreen C method and further validated in selected food products. The estimated values for the cardinal parameters T min , T opt , T max , pH min , pH opt , pH max , [Formula: see text] and [Formula: see text] were 23.77 ± 0.19 °C, 52.89 ± 0.01 °C, 59.37 ± 0.07 °C, 4.70 ± 0.02, 6.43 ± 0.02, 8.56 ± 0.01, 0.969 ± 0.0007 and 0.998 ± 0.0011, respectively. The growth behaviour of B. coagulans was studied in five commercial non-refrigerated ready-to-eat food products under static conditions at 53 °C in order to estimate the optimum specific growth rate for each tested food product. The developed models were validated in the five selected food products under four different dynamic temperature profiles by comparing predicted and observed growth behaviour of B. coagulans. The validation results indicated a good performance of the model for all tested products with the overall Bias factor (B f ) and Accuracy factor (A f ) estimated at 1.00 and 1.12, respectively. The developed model can be considered an effective tool in predicting B. coagulans growth and spoilage risks of non-refrigerated ready-to-eat food products during distribution and storage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The European Commission requested EFSA to assess, in collaboration with EMA, the specific concentrations of antimicrobials resulting from cross-contamination in non-target feed for food-producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health, as well as the levels of the antimicrobials which could have a growth promotion/increase yield effect. The assessment was performed for 24 antimicrobial active substances, as specified in the mandate. This scientific opinion describes the methodology used, and the main associated data gaps and uncertainties. To estimate the antimicrobial levels in the non-target feed that would not result in emergence of, and/or selection for, resistance, a model was developed. This 'Feed Antimicrobial Resistance Selection Concentration' (FARSC) model is based on the minimal selective concentration (MSC), or the predicted MSC (PMSC) if MSC for the most susceptible bacterial species is unavailable, the fraction of antimicrobial dose available for exposure to microorganisms in the large intestine or rumen (considering pharmacokinetic parameters), the daily faecal output or rumen volume and the daily feed intake. Currently, lack of data prevents the establishment of PMSC and/or FARSC for several antimicrobials and animal species. To address growth promotion, data from an extensive literature search were used. Specific assessments of the different substances grouped by antimicrobial classes are addressed in separate scientific opinions. General conclusions and recommendations were made., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of colistin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of colistin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of tilmicosin, tylosin and tylvalosin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tilmicosin and tylosin, whilst for tylvalosin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these three antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of tiamulin and valnemulin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tiamulin, while for valnemulin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of lincomycin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of lincomycin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for lincomycin., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of tetracycline, chlortetracycline, oxytetracycline and doxycycline in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for these four tetracyclines was estimated. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tetracycline, chlortetracycline, oxytetracycline, whilst for doxycycline no suitable data for the assessment were available. Uncertainties and data gaps associated with the levels reported were addressed. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for these antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of florfenicol and thiamphenicol in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for florfenicol was estimated. However, due to the lack of data, the calculation of the FARSC for thiamphenicol was not possible until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for florfenicol, whilst for thiamphenicol no suitable data for the assessment were available. Uncertainties and data gaps associated to the levels reported were addressed. For florfenicol, it was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC, whereas for thiamphenicol, the recommendation was to generate the data required to fill the gaps which prevented the FARSC calculation., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of sulfonamides in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were identified for three sulfonamides: sulfamethazine, sulfathiazole and sulfamerazine. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of amoxicillin and penicillin V in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for amoxicillin, whilst for penicillin V no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of flumequine and oxolinic acid in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of trimethoprim in non-target feed for food-producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for trimethoprim was estimated. Uncertainties and data gaps associated to the levels reported were addressed. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for trimethoprim., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of apramycin, paromomycin, neomycin and spectinomycin in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for these antimicrobials, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for apramycin and neomycin, whilst for paromomycin and spectinomycin, no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these four antimicrobials., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The specific concentrations of amprolium in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for amprolium, it was not possible to conclude the assessment. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of amprolium in feed that showed to have an effect on growth promotion/increased yield were reported. The lack of antibacterial activity at clinically relevant concentrations for amprolium suggests that further studies relating to bacterial resistance are not a priority., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

This Opinion assesses the biological relevance of the non-monotonic dose responses (NMDR) identified in a previous EFSA External Report (Beausoleil et al., 2016) produced under GP/EFSA/SCER/2014/01 and the follow-up probabilistic assessment (Chevillotte et al., 2017a,b), focusing on the in vivo data sets fulfilling most of the checkpoints of the visual/statistical-based analysis identified in Beausoleil et al. (2016). The evaluation was completed with cases discussed in EFSA assessments and the update of the scientific literature. Observations of NMDR were confirmed in certain studies and are particularly relevant for receptor-mediated effects. Based on the results of the evaluation, the Opinion proposes an approach to be applied during the risk assessment process when apparent non-monotonicity is observed, also providing advice on specific elements to be considered to facilitate the assessment of NMDR in EFSA risk assessments. The proposed approach was applied to two case studies, Bisphenol A and bis(2-ethylhexyl phthalate (DEHP) and these evaluations are reported in dedicated annexes. Considering the potential impact of NMDRs in regulatory risk assessment, the Scientific Committee recommends a concerted international effort on developing internationally agreed guidance and harmonised frameworks for identifying and addressing NMDRs in the risk assessment process., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A quantitative microbial risk assessment (QMRA) model predicting the listeriosis risk related to the consumption of Ready- To- Eat (RTE) cooked meat products sliced at retail stores in Greece was developed. The probability of illness per serving assessed for 87 products available in the Greek market was found highly related to the nitrite concentration; products having a lower concentration showed a higher risk per serving. The predicted 95 th percentiles of the annual listeriosis cases totaled 33 of which 13 cases were <65 years old and 20 cases ≥65 years old. The highest number of cases was predicted for mortadella, smoked turkey, boiled turkey and parizer, which were the most frequently consumed product categories. Two scenarios for assessing potential interventions to reduce the risk were tested: setting a use-by date of 14 days (these products have no use-by date based on current European Union legislation) and improving the temperature control during domestic storage. The two scenarios resulted in a decrease of the 95 th and 99 th percentiles of the total annual cases by 97% and 88%, respectively., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo . A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo . If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health-based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The EFSA has updated the Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain, human and animal health. It covers the application areas within EFSA's remit, including novel foods, food contact materials, food/feed additives and pesticides. The updated guidance, now Scientific Committee Guidance on nano risk assessment (SC Guidance on Nano-RA), has taken account of relevant scientific studies that provide insights to physico-chemical properties, exposure assessment and hazard characterisation of nanomaterials and areas of applicability. Together with the accompanying Guidance on Technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (Guidance on Particle-TR), the SC Guidance on Nano-RA specifically elaborates on physico-chemical characterisation, key parameters that should be measured, methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. The SC Guidance on Nano-RA also details aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vitro/in vivo toxicological studies are discussed and a tiered framework for toxicological testing is outlined. Furthermore, in vitro degradation, toxicokinetics, genotoxicity, local and systemic toxicity as well as general issues relating to testing of nanomaterials are described. Depending on the initial tier results, additional studies may be needed to investigate reproductive and developmental toxicity, chronic toxicity and carcinogenicity, immunotoxicity and allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read-across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes or mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Following a mandate from the European Commission, EFSA has developed a Guidance on Technical Requirements (Guidance on Particle-TR), defining the criteria for assessing the presence of a fraction of small particles, and setting out information requirements for applications in the regulated food and feed product areas (e.g. novel food, food/feed additives, food contact materials and pesticides). These requirements apply to particles requiring specific assessment at the nanoscale in conventional materials that do not meet the definition of engineered nanomaterial as set out in the Novel Food Regulation (EU) 2015/2283. The guidance outlines appraisal criteria grouped in three sections, to confirm whether or not the conventional risk assessment should be complemented with nanospecific considerations. The first group addresses solubility and dissolution rate as key physicochemical properties to assess whether consumers will be exposed to particles. The second group establishes the information requirements for assessing whether the conventional material contains a fraction or consists of small particles, and its characterisation. The third group describes the information to be presented for existing safety studies to demonstrate that the fraction of small particles, including particles at the nanoscale, has been properly evaluated. In addition, in order to guide the appraisal of existing safety studies, recommendations for closing the data gaps while minimising the need for conducting new animal studies are provided. This Guidance on Particle-TR complements the Guidance on risk assessment of nanomaterials to be applied in the food and feed chain, human and animal health updated by the EFSA Scientific Committee as co-published with this Guidance. Applicants are advised to consult both guidance documents before conducting new studies., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of biological agents, intended for addition to food or feed, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Schizochytrium limacinum , which is a synonym for Aurantiochytrium limacinum , was added to the QPS list. Of the 78 microorganisms notified to EFSA between October 2020 and March 2021, 71 were excluded; 16 filamentous fungi, 1 Dyella spp., 1 Enterococcus faecium , 7 Escherichia coli , 1 Streptomyces spp., 1 Schizochytrium spp. and 44 TUs that had been previously evaluated. Seven TUs were evaluated: Corynebacterium stationis and Kodamaea ohmeri were re-assessed because an update was requested for the current mandate. Anoxybacillus caldiproteolyticus , Bacillus paralicheniformis , Enterobacter hormaechei, Eremothecium ashbyi and Lactococcus garvieae were assessed for the first time. The following TUs were not recommended for QPS status: A. caldiproteolyticus due to the lack of a body of knowledge in relation to its use in the food or feed chain, E. hormaechei, L. garvieae and K. ohmeri due to their pathogenic potential , E. ashbyi and C. stationis due to a lack of body of knowledge on their occurrence in the food and feed chain and to their pathogenic potential. B. paralicheniformis was recommended for the QPS status with the qualification 'absence of toxigenic activity' and 'absence of genetic information to synthesize bacitracin'., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The cardinal model for the effect of temperature on Geobacillus stearothermophilus ATCC 7953 growth developed by Kakagianni, Gougouli, & Koutsoumanis, 2016 was expanded for the effect of pH and water activity (a w ). The effect of pH (range: 5.7-8.5) and a w (range: 0.985-0.999) on G. stearothermophilus growth rate was studied in tryptone soy broth (TSB) using the Bioscreen C method and further modelled using a Cardinal Model (CM). The estimated values for the cardinal parameters [Formula: see text] , and [Formula: see text] were 5.65 ± 0.14, 6.74 ± 0.03, 8.71 ± 0.03, 0.984 ± 0.007 and 0.998 ± 0.001, respectively. The growth behaviour of G. stearothermophilus was investigated in 7 commercial non-refrigerated plant-based milk alternatives under static conditions (62 °C) and the estimated maximum specific growth rates were used to determine the optimum growth rate for each product. The developed model was validated against observed growth of G. stearothermophilus in the 7 products during storage at non-isothermal conditions (testing 4 different temperature profiles). The validation results showed a good performance of the model with overall Bias factor (B f ) = 1.06 and Accuracy factor (A f ) = 1.12. The developed model can be used as an effective tool by the food industry in predicting spoilage of plant-based milk alternatives during distribution and storage at retail and domestic levels., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The role of food-producing environments in the emergence and spread of antimicrobial resistance (AMR) in EU plant-based food production, terrestrial animals (poultry, cattle and pigs) and aquaculture was assessed. Among the various sources and transmission routes identified, fertilisers of faecal origin, irrigation and surface water for plant-based food and water for aquaculture were considered of major importance. For terrestrial animal production, potential sources consist of feed, humans, water, air/dust, soil, wildlife, rodents, arthropods and equipment. Among those, evidence was found for introduction with feed and humans, for the other sources, the importance could not be assessed. Several ARB of highest priority for public health, such as carbapenem or extended-spectrum cephalosporin and/or fluoroquinolone-resistant Enterobacterales (including Salmonella enterica ), fluoroquinolone-resistant Campylobacter spp., methicillin-resistant Staphylococcus aureus and glycopeptide-resistant Enterococcus faecium and E. faecalis were identified. Among highest priority ARGs bla CTX -M , bla VIM , bla NDM , bla OXA -48-like , bla OXA -23 , mcr , armA , vanA , cfr and optrA were reported. These highest priority bacteria and genes were identified in different sources, at primary and post-harvest level, particularly faeces/manure, soil and water. For all sectors, reducing the occurrence of faecal microbial contamination of fertilisers, water, feed and the production environment and minimising persistence/recycling of ARB within animal production facilities is a priority. Proper implementation of good hygiene practices, biosecurity and food safety management systems is very important. Potential AMR-specific interventions are in the early stages of development. Many data gaps relating to sources and relevance of transmission routes, diversity of ARB and ARGs, effectiveness of mitigation measures were identified. Representative epidemiological and attribution studies on AMR and its effective control in food production environments at EU level, linked to One Health and environmental initiatives, are urgently required., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The European Parliament requested EFSA to develop a holistic risk assessment of multiple stressors in honey bees. To this end, a systems-based approach that is composed of two core components: a monitoring system and a modelling system are put forward with honey bees taken as a showcase. Key developments in the current scientific opinion (including systematic data collection from sentinel beehives and an agent-based simulation) have the potential to substantially contribute to future development of environmental risk assessments of multiple stressors at larger spatial and temporal scales. For the monitoring, sentinel hives would be placed across representative climatic zones and landscapes in the EU and connected to a platform for data storage and analysis. Data on bee health status, chemical residues and the immediate or broader landscape around the hives would be collected in a harmonised and standardised manner, and would be used to inform stakeholders, and the modelling system, ApisRAM, which simulates as accurately as possible a honey bee colony. ApisRAM would be calibrated and continuously updated with incoming monitoring data and emerging scientific knowledge from research. It will be a supportive tool for beekeeping, farming, research, risk assessment and risk management, and it will benefit the wider society. A societal outlook on the proposed approach is included and this was conducted with targeted social science research with 64 beekeepers from eight EU Member States and with members of the EU Bee Partnership. Gaps and opportunities are identified to further implement the approach. Conclusions and recommendations are made on a way forward, both for the application of the approach and its use in a broader context., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A risk-based approach was used to develop guidance to be followed by food business operators (FBOs) when deciding on food information relating to storage conditions and/or time limits for consumption after opening a food package and thawing of frozen foods. After opening the package, contamination may occur, introducing new pathogens into the food and the intrinsic (e.g. pH and a w ), extrinsic (e.g. temperature and gas atmosphere) and implicit (e.g. interactions with competing background microbiota) factors may change, affecting microbiological food safety. Setting a time limit for consumption after opening the package (secondary shelf-life) is complex in view of the many influencing factors and information gaps. A decision tree (DT) was developed to assist FBOs in deciding whether the time limit for consumption after opening, due to safety reasons, is potentially shorter than the initial 'best before' or 'use by' date of the product in its unopened package. For products where opening the package leads to a change of the type of pathogenic microorganisms present in the food and/or factors increasing their growth compared to the unopened product, a shorter time limit for consumption after opening would be appropriate. Freezing prevents the growth of pathogens, however, most pathogenic microorganisms may survive frozen storage, recover during thawing and then grow and/or produce toxins in the food, if conditions are favourable. Moreover, additional contamination may occur from hands, contact surfaces or contamination from other foods and utensils. Good practices for thawing should, from a food safety point of view, minimise growth of and contamination by pathogens between the food being thawed and other foods and/or contact surfaces, especially when removing the food from the package during thawing. Best practices for thawing foods are presented to support FBOs., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A new alternative method for the production of biodiesel from rendered fat, including animal by-product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10-15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end-product, biodiesel and the co-product, glycerine. Prions (PrP S c ), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrP S c , of at least 6 log 10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log 10 reduction in detectable PrP S c , by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrP S c from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log 10 reduction in PrP S c . Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

This Statement presents a proposal for harmonising the establishment of Health-Based Guidance Values (HBGVs) for regulated products that are also nutrients. This is a recurrent issue for food additives and pesticides, and may occasionally occur for other regulated products. The Statement describes the specific considerations that should be followed for establishing the HBGVs during the assessment of a regulated product that is also a nutrient. It also addresses the elements to be considered in the intake assessment; and proposes a decision tree for ensuring a harmonised process for the risk characterisation of regulated products that are also nutrients. The Scientific Committee recommends the involvement of the relevant EFSA Panels and units, in order to ensure an integrated and harmonised approach for the hazard and risk characterisation of regulated products that are also nutrients, considering the intake from all relevant sources., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A predictive mathematical model describing the effect of temperature on the inactivation of Legionella pneumophila in water was developed. Thermal inactivation of L. pneumophila was monitored under isothermal conditions (51 - 61°C). A primary log-linear model was fitted to the inactivation data and the estimated D values ranged from 0.23 to 25.31 min for water temperatures from 61 to 51°C, respectively. The effect of temperature on L. pneumophila inactivation was described using a secondary model, and the model parameters z value and D ref (D-value at 55°C) were estimated at 5.54°C and 3.47 min, respectively. The developed model was further validated under dynamic temperature conditions mimicking various conditions of water thermal disinfection in plumbing systems. The results indicated that the model can satisfactorily predict thermal inactivation of the pathogen at dynamic temperature environments and effectively translate water temperature profiles to cell number reduction. The application of the model in combination with effective temperature monitoring could provide the basis of an integrated preventive approach for the effective control of L. pneumophila in plumbing systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Superchilling entails lowering the fish temperature to between the initial freezing point of the fish and about 1-2°C lower. The temperature of superchilled fresh fishery products (SFFP) in boxes without ice was compared to that of products subject to the currently authorised practice in boxes with ice (CFFP) under the same conditions of on-land storage and/or transport. A heat transfer model was developed and made available as a tool to identify under which initial configurations of SFFP the fish temperature, at any time of storage/transport, is lower or equal to CFFP. A minimum degree of superchilling, corresponding to an ice fraction in the fish matrix of SFFP equal or higher than the proportion of ice added per mass of fish in CFFP, will ensure with 99-100% certainty (almost certain) that the fish temperature of SFFP and the consequent increase of relevant hazards will be lower or equal to that of CFFP. In practice, the degree of superchilling can be estimated using the fish temperature after superchilling and its initial freezing point, which are subject to uncertainties. The tool can be used as part of 'safety-by-design' approach, with the reliability of its outcome being dependent on the accuracy of the input data. An evaluation of methods capable of detecting whether a previously frozen fish is commercially presented as 'superchilled' was carried out based on, amongst others, their applicability for different fish species, ability to differentiate fresh fish from fish frozen at different temperatures, use as a stand-alone method, ease of use and classification performance. The methods that were considered 'fit for purpose' are Hydroxyacyl-coenzyme A dehydrogenase (HADH) test, α-glucosidase test, histology, ultraviolet-visible-near-infrared (UV-VIS/NIR) spectroscopy and hyperspectral imaging. These methods would benefit from standardisation, including the establishment of threshold values or classification algorithms to provide a practical routine test., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of biological agents, intended for addition to food or feed, to support the work of EFSA's Scientific Panels. It is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of knowledge, safety concerns and antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at strain or product level, and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Of the 36 microorganisms notified to EFSA between April and September 2020, 33 were excluded; seven filamentous fungi (including Aureobasidium pullulans based on recent taxonomic insights), one Clostridium butyricum , one Enterococcus faecium , three Escherichia coli , one Streptomyces spp. and 20 TUs that had been previously evaluated. Three TUs were evaluated; Methylorubrum extorquens and Mycobacterium aurum for the first time and Bacillus circulans was re-assessed because an update was requested in relation to a new mandate. M. extorquens and M. aurum are not recommended for QPS status due to the lack of a body of knowledge in relation to use in the food or feed chain and M. aurum, due to uncertainty concerning its pathogenicity potential. B. circulans was recommended for QPS status with the qualifications for 'production purposes only' and 'absence of cytotoxic activity'., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)