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Abstract Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Abstract Background Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Results Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Conclusions This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.

This study analyzes allelic expression bias in post-mortem brains of healthy individuals and those diagnosed with schizophrenia or bipolar disorder. The study shows that the number of imprinted genes is consistent with low estimates, and that allelic bias is independent of psychiatric disease status.

We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results.

A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s) that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil from the flowering tops of Thymbra capitata (L.) Cav. (Spanish type origanum oil) when used as a sensory additive in feed and in water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive under assessment is considered safe up to the maximum use level in complete feed of 15 mg/kg for poultry species, 30 mg/kg for pigs and horses, 20 mg/kg for ruminants, 25 mg/kg for rabbits, dogs, cats and ornamental fish, and 125 mg/kg for salmonids. These conclusions were extrapolated to other physiologically related species. For any other species, the additive is safe at 15 mg/kg complete feed. The FEEDAP Panel considered that the use level in water for drinking is safe provided that the total daily intake of the additive does not exceed the daily amount that is considered safe when consumed via feed. The use of the additive in animal feed under the proposed conditions of use is safe for the consumer and the environment. Regarding user safety, the essential oil under assessment should be considered as an irritant to skin and eyes and as a dermal and respiratory sensitiser. Since T. capitata and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of tea tree oil obtained from leaves and terminal branchlets of Melaleuca alternifolia (Maiden & Betche) Cheel when used as a sensory additive for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that tea tree oil was very unlikely to be of safety concern for long‐living and reproductive animals and is of no concern for target species for fattening at the following concentrations in complete feed: 1.1 mg/kg for chickens for fattening, 1.5 mg/kg for turkeys for fattening, 1.7 mg/kg for laying hens, 2.0 mg/kg for piglets, 2.4 mg/kg for pigs for fattening, 3.1 mg/kg for sows, 5.0 mg/kg for veal calves (milk replacer) and salmonids, 4.4 mg/kg for cattle for fattening, sheep/goats and horses, 2.9 mg/kg for dairy cows, 1.8 mg/kg for rabbits, 0.9 mg/kg for cats, 5.3 mg/kg for dogs, 6.6 for crustaceans and 15 mg/kg for ornamental fish. These conclusions were extrapolated to other physiologically related species. For any other species, the additive is very unlikely to be of safety concern at 1.1 mg/kg complete feed. No concerns for consumers and the environment were identified following the use of the additive up to the highest safe use level in feed. Regarding user safety, tea tree oil should be considered as an irritant to skin and eyes and as a dermal and respiratory sensitiser. It is classified as a reprotoxic substance (category 1B) following CLP criteria and should be handled accordingly. Since M. alternifolia and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Spanish sage oil from the leaves of Salvia officinalis ssp. lavandulifolia (Vahl) Gams (Spanish sage oil) when used as a sensory additive in feed and in water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive under assessment is considered safe up to the maximum use level of 14 mg/kg complete feed for all animal species. The FEEDAP Panel considered that the use of Spanish sage oil in water for drinking is safe provided that the total daily intake of the additive does not exceed the daily amount that is considered safe when consumed via feed. The use of Spanish sage oil in animal feed under the proposed conditions of use is safe for the consumer and the environment. Regarding user safety, the essential oil under assessment should be considered as an irritant to skin and eyes and as a dermal and respiratory sensitiser. Since the oil of the leaves of S. officinalis ssp. lavandulifolia (Vahl) Gams is recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Aspergillus tubingensis strain NL151 by Shin Nihon Chemical Co., Ltd. The food enzyme was free from viable cells of the production organism. It is intended to be used in six food manufacturing processes. Dietary exposure was estimated to be up to 0.278 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1669 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6004. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for the renewal of authorisation of a preparation containing a smoke flavouring extract for cats and dogs. The applicant provided data demonstrating that the additive currently on the market does not fully comply with the conditions of authorisation, but with newly proposed specifications based on different analytical methods. Considering that the additive under assessment contains benzofuran and styrene, for which a potential concern for genotoxicity has been identified, and that the whole mixture raises a potential concern for genotoxicity, additional data would be needed to complete the assessment. Therefore, the FEEDAP Panel is not in the position to conclude on the safety for cats and dogs. The additive is authorised for use in feed for cats and dogs, and therefore, there is no need to perform an assessment of the safety for the consumer and the environment. Regarding user safety, the additive should be considered as irritant to skin and eyes, and as a dermal and respiratory sensitiser. When handling the additive, exposure of unprotected users to potential genotoxic substances may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. There was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Abstract The food enzyme, a triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3), is produced with the non‐genetically modified Limtongozyma cylindracea strain AE‐LAYH (B) by Amano Enzyme Inc. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 0.315 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the quality presumption of safety (QPS) approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A homology search for the amino acid sequence of the food enzyme to those of known allergens was made and one match with a honeybee venom allergen was found. The Panel considered that a risk of allergic reactions by dietary exposure, particularly in individuals allergic to honey, cannot be excluded, but is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a Camellia oleifera C.Abel seed extract (Cosap®) as technological feed additive for all animal species except fin fish. In the absence of adequate tolerance studies in the target species or toxicological studies with the additive under assessment, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) could not conclude on the safety of C. oleifera extract for the target species. The use of the additive in animal nutrition is not expected to cause concern for consumer safety. The additive is considered as irritant to the eyes and mucous membranes. No conclusions can be reached on the potential of the additive to be irritant to the skin or to be a dermal sensitiser. The use of the additive under the proposed conditions of use is considered safe for the terrestrial compartment. However, no conclusion can be reached on the safety of the additive for the environment when used in feed of aquatic animals other than fin fish. The Panel concluded that the additive has the potential to be efficacious as an emulsifier when used according to proposed conditions of use.

Abstract The food enzyme endo‐1,3(4)‐β‐glucanase (3‐(1–3;1–4)‐β‐d‐glucan 3(4)‐glucanohydrolase; EC 3.2.1.6) is produced with the non‐genetically modified Talaromyces versatilis strain PF8 by Erbslöh Geisenheim AG. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.110 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2229 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure resulted in a margin of exposure of at least 20,264. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory or contact allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil from the fresh flowering tops of Lavandula angustifolia Mill. (lavender oil) when used as a sensory additive in feed and in water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive under assessment is considered safe up to the maximum proposed use levels in complete feed of 30 mg/kg for dogs and ornamental fish. For the other target species, the calculated safe concentrations were 9 mg/kg for chickens for fattening, 13 mg/kg for laying hens, 12 mg/kg for turkeys for fattening, 19 mg/kg for pigs for fattening, 16 mg/kg for piglets, 23 mg/kg for sows, 39 mg/kg for veal calves (milk replacer) and salmonids, 35 mg/kg for cattle for fattening, sheep/goats and horses, 22 mg/kg for dairy cows, 14 mg/kg for rabbits and 7 mg/kg for cats. These conclusions were extrapolated to other physiologically related species. For any other species, the additive is safe at 7 mg/kg complete feed. The use of lavender oil in water for drinking was considered safe provided that the total daily intake does not exceed the daily amount considered safe when consumed via feed. The use of lavender oil in animal feed under the proposed conditions of use is safe for the consumer and the environment. Regarding user safety, the essential oil under assessment should be considered as an irritant to skin and eyes and as a dermal and respiratory sensitiser. Since the oil of the flowering tops of L. angustifolia is recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Lietpak (EU register number RECYC327), which uses the EREMA MPR technology. The input material is hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are heated in a ■■■■■ reactor under vacuum. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migration of 0.1 μg/kg food, derived from the exposure scenario for infants, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long‐term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Ecopacking (EU register number RECYC324), which uses the EREMA Basic technology. The input material is ■■■■■ washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are heated in a ■■■■■ reactor ■■■■■ before being extruded. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migrations of 0.1 and 0.15 μg/kg food, derived from the exposure scenarios for infants and toddlers, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long‐term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.

Abstract The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the non‐genetically modified microorganism Bacillus licheniformis strain AE‐TA by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in eight food manufacturing processes. Subsequently, the applicant has requested to extend its use to include one additional process and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of nine food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining seven processes. Dietary exposure was calculated to be up to 0.382 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process KGL (EU register number RECYC326), which uses the EREMA Basic technology. The input material is hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are heated in a ■■■■■ reactor under vacuum before being extruded. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migrations of 0.1 and 0.15 μg/kg food, derived from the exposure scenarios for infants and toddlers, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long‐term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Penicillium caseifulvum strain AE‐LRF by Amano Enzyme Inc. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.013 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 69 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5308. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. However, the Panel noted that traces of ■■■■■, used in the manufacture of the triacylglycerol lipase, may be found in the food enzyme. The Panel considered that the risk of allergic reactions upon dietary exposure could not be excluded, particularly in individuals sensitised to fish. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme laccase (benzenediol:oxygen oxidoreductase, i.e. EC 1.10.3.2) is produced with the non‐genetically modified Trametes hirsuta strain AE‐OR by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in six food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of nine food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.030 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level previously reported (862 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 28,733. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Guolong (EU register number RECYC323), which uses the EREMA Basic technology. The input material is hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are heated in a ■■■■■ reactor under vacuum before being extruded. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migrations of 0.1 and 0.15 μg/kg food, derived from the exposure scenarios for infants and toddlers, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long‐term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.

Abstract In the context of entry into force of Regulation (EU) 2022/1616, EFSA updated the scientific guidance to assist applicants in the preparation of applications for the authorisation or for the modification of an existing authorisation of a ‘post‐consumer mechanical PET’ recycling process (as defined in Annex I of Regulation (EU) 2022/1616) intended to be used for manufacturing materials and articles intended to come into contact with food. This Guidance describes the evaluation criteria and the scientific evaluation approach that EFSA will apply to assess the decontamination capability of recycling processes, as well as the information required to be included in an application dossier. The principle of the scientific evaluation approach is to apply the decontamination efficiency of a recycling process, obtained from a challenge test with surrogate contaminants, to a reference contamination level for post‐consumer PET, set at 3 mg/kg PET for a contaminant resulting from possible misuse. The resulting residual concentration of each surrogate in recycled PET is then compared to a modelled concentration in PET that is calculated using generally recognised conservative migration models, such that the related migration does not give rise to a dietary exposure exceeding 0.0025 μg/kg body weight (bw) per day. This is the lowest threshold for toxicological concern (TTC) value, i.e. for potential genotoxicity, below which the risk to human health would be negligible. The information to be provided in the applications relates to: the recycling process (i.e. collection and pre‐processing of the input, decontamination process, post‐processing and intended use); the determination of the decontamination efficiency by the challenge test; the self‐evaluation of the recycling process. On the basis of the submitted data, EFSA will assess the safety of the mechanical PET recycling process.

Abstract The food enzyme β‐glucosidase (β‐D‐glucoside glucohydrolase; EC 3.2.1.21) is produced with the non‐genetically modified Penicillium guanacastense strain AE‐GLY by Amano Enzyme Inc. The food enzyme is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 4.054 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 943 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 233. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.792 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil obtained from the fruit of Carum carvi L. (caraway oil), when used as a sensory additive in feed and water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) Panel concluded that the use of caraway oil is of no concern up to the following concentrations in complete feed: 9 mg/kg for chickens for fattening, 13 mg/kg for laying hens, 12 mg/kg for turkeys for fattening, 16 mg/kg for piglets, 19 mg/kg for pigs for fattening, 24 mg/kg for sows, 35 mg/kg for veal calves (milk replacer), 11 mg/kg for cattle for fattening, 10 mg/kg for dairy cows, sheep, goats, horses and rabbits, 25 mg/kg for salmonids and dogs. These conclusions were extrapolated to other physiologically related species. For cats, ornamental fish and other species, no conclusion can be drawn. The use of caraway oil in animal feed under the proposed conditions of use is safe for the consumer and the environment. The additive under assessment should be considered as an irritant to skin and eyes, and as a respiratory and skin sensitiser. When handling the essential oil, exposure of unprotected users to perillaldehyde may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. Since C. carvi and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain AP‐01 by Nagase (Europa) GmbH. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining four food manufacturing processes. It was estimated to be up to 0.875 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme has the capacity to produce bacitracin and thus failed to meet the requirements of the Qualified Presumption of Safety approach. Bacitracin was detected in the industrial fermentation medium but not in the food enzyme itself. However, the limit of detection of the analytical method used for bacitracin was not sufficient to exclude the possible presence of bacitracin at a level representing a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and twenty‐eight matches with respiratory allergens, one match with a contact allergen and two matches with food allergens (melon and pomegranate) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon or pomegranate, cannot be excluded, but would not exceed the risk of consuming melon or pomegranate. Based on the data provided, the Panel could not exclude the presence of bacitracin, a medically important antimicrobial, and consequently the safety of this food enzyme could not be established.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil obtained from the fruit of Apium graveolens L. (celery seed oil), when used as a sensory additive in feed and water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) Panel concluded that the use of celery seed oil is of no concern up to the following concentrations in complete feed: 1.6 mg/kg for chickens for fattening, 2.3 mg/kg for laying hens, 2.1 mg/kg for turkeys for fattening, 2.8 mg/kg for piglets, 3.3 mg/kg for pigs for fattening, 4.1 mg/kg for sows, 6.5 mg/kg for veal calves (milk replacer), 6.2 mg/kg for cattle for fattening, sheep, goats and horses, 4.0 mg/kg for dairy cows, 2.5 mg/kg for rabbits, 6.8 mg/kg for salmonids and 7.2 mg/kg for dogs. These conclusions were extrapolated to other physiologically related species. For cats, ornamental fish and other species, no conclusion can be drawn. The use of celery seed oil in animals feed is not expected to pose concern for the consumers and for the environment. The additive under assessment should be considered as an irritant to skin and eyes, and as a respiratory and skin sensitiser. When handling the essential oil, exposure of unprotected users to perillaldehyde and bergapten may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. Since A. graveolens and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract The food enzyme glutaminase (l‐glutamine amidohydrolase; EC 3.5.1.2) is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐GT by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant requested to extend its use to thirteen additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eighteen food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining sixteen processes. Dietary exposure was calculated to be up to 0.678 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme α‐l‐rhamnosidase (α‐l‐rhamnoside rhamnohydrolase; EC 3.2.1.40) is produced with Penicillium adametzii strain AE‐HP by Amano Enzymes Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant has requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of four food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.022 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level reported in the previous opinion (300 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 13,636. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme 3‐phytase (myo‐inositol‐hexakisphosphate 3‐phosphohydrolase EC 3.1.3.8) is produced with the non‐genetically modified Aspergillus niger strain PHY93‐08 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in nine food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in two of the food manufacturing processes, dietary exposure was calculated only for the remaining seven processes. It was estimated to be up to 0.763 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2560 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 3355. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Fucine Film (EU register number RECYC322), which uses the Reifenhäuser technology. The input material consists of hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes mainly originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are extruded under vacuum into sheets. The recycled sheets are intended to be used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, excluded drinking water and beverages, for long‐term storage at room temperature, with or without hotfill. Based on the limited data available, the Panel concluded that the information submitted to EFSA was inadequate to demonstrate that the recycling process Fucine Film is able to reduce potential unknown contamination of the input PET flakes to a concentration that does not pose a risk to human health.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Palamidis (EU register number RECYC325), which uses the EREMA Basic technology. The input material is hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post‐consumer PET containers, including no more than 5% PET from non‐food consumer applications. The flakes are heated in a ■■■■■ reactor under vacuum before being extruded. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migrations of 0.1 and 0.15 μg/kg food, derived from the exposure scenarios for infants and toddlers, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long‐term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.

Abstract The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐NP by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in thirteen food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of fifteen food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining thirteen processes. Dietary exposure was calculated to be up to 35.251 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of Quillaja saponaria powder and Yucca schidigera powder (Magni‐Phi®) for all avian species (to slaughter age/weight, or to the point of lay) and ornamental birds, as a zootechnical additive (digestibility enhancer and other zootechnical additives). The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive is safe for chickens for fattening at the level of 250 mg/kg complete feed with a margin of safety of 20 assuming that the additive contains 3.58% of saponins. This conclusion was extrapolated to all growing poultry species and ornamental birds. The Panel concluded that the use of the feed additive in animal nutrition at 250 mg/kg complete feed is of no concern for the safety for the consumer and the environment. The Panel also concluded that the additive is not irritant to skin, but irritant to the eyes and to the respiratory system. Due to the lack of data, the FEEDAP Panel could not conclude on the skin sensitisation potential of the additive. The FEEDAP Panel was not in the position to conclude on the efficacy of the additive for all poultry species and ornamental birds.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a tincture from the dried flower bud of Syzygium aromaticum (L.) Merr. & L.M. Perry (clove tincture) when used as a sensory additive in feed and water for drinking for all animal species. The product is a ■■■■■) solution, with a dry matter content of ~ 1.66%. The product contains on average 0.511% phenolic acids (of which 0.0344% were flavonoids), 0.039% eugenol, 0.00019% methyleugenol and 0.00008% estragole. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the use of clove tincture is very unlikely to be of safety concern for the target species up to the maximum proposed use level of 50 mg clove tincture/kg complete feed for all animal species, except for horses, for which the proposed use level is 200 mg/kg complete feed. The FEEDAP Panel considers that the use in water for drinking alone or in combination with use in feed should not exceed the daily amount that is considered very unlikely to be of safety concern when consumed via feed alone. No safety concern would arise for the consumer and the environment from the use of clove tincture up to the maximum proposed use levels in feed. The additive under assessment should be considered as irritant to skin and eyes, and as a dermal and respiratory sensitiser. When handling the additive, exposure of unprotected users to methyleugenol and estragole may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. Since the flower buds of S. aromaticum and their preparations were recognised to flavour food and their function in feed would be essentially the same, no demonstration of efficacy was considered necessary.

Abstract The food enzyme inulinase (1‐β‐d‐fructan fructanohydrolase; EC 3.2.1.7) is produced with the non‐genetically modified Aspergillus welwitschiae strain NZYM‐KF by Novozymes A/S. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of fructo‐polysaccharides for the production of fructo‐oligosaccharides. Since residual amounts of total organic solids (TOS) are removed during the food manufacturing process, toxicological studies other than allergenicity were considered unnecessary and dietary exposure was not calculated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with tomato allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded, but is expected not to exceed that of tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme sucrose phosphorylase (sucrose: phosphate α‐d‐glucosyltransferase; EC 2.4.1.7) is produced with the genetically modified Escherichia coli strain LE1B109‐pPB129 by c‐LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was free from viable cells of the production organism. It is intended to be used in combination with a cellobiose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of food enzyme–total organic solids are removed by the downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of tinctures obtained from the dried leaves of Ginkgo biloba L. (ginkgo tinctures) when used as sensory additives. The tinctures are water/ethanol solutions with a dry matter content of 5.7% (tincture A) and 3.0% (tincture B). The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additives under assessment are safe for the target species at the following concentrations in complete feed: (i) ginkgo tincture A at 240 mg/kg for horses and 750 mg/kg for dogs; (ii) ginkgo tincture B at 600 mg/kg for horses and 50 mg/kg for all other animal species. No safety concern would arise for the consumer from the use of ginkgo tinctures up to the maximum proposed use level in feed for the target species. The tinctures should be considered as irritants to skin and eyes, and as dermal and respiratory sensitisers. The use of ginkgo tinctures at the proposed use levels in feed for the target species is not considered to be a risk to the environment. While the available data indicate that Ginkgo preparations have a distinctive flavour profile, there is no evidence that ginkgo tinctures would impart flavour to a food or feed matrix. Therefore, the FEEDAP Panel cannot conclude on the efficacy of the additives.

Abstract The food enzyme β‐amylase (4‐α‐d‐glucan maltohydrolase, EC 3.2.1.2) is produced with the non‐genetically modified Bacillus flexus strain AE‐BAF by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to four additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme‐total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme‐TOS was estimated only for the remaining six processes. The dietary exposure was estimated to be up to 0.247 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the dietary exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of citronella oil obtained from the leaves of Cymbopogon nardus (L.) Rendle, when used as a sensory additive for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that citronella oil from C. nardus is of low concern for long‐living and reproductive animals at the use levels in complete feed of 3.5 mg/kg for laying hens and rabbits, 6 mg/kg for sows and dairy cows, 9.5 mg/kg for sheep/goats and horses, 2.0 mg/kg for cats and 10 mg/kg for dogs. For short‐living animals (species for fattening), the additive was considered of no concern at concentrations of 18 mg/kg in chickens for fattening, 24 mg/kg in turkeys for fattening, 20 mg/kg for piglets, pigs for fattening, veal calves (milk replacer), cattle for fattening, sheep/goats for meat production, horses for meat production and rabbits for meat production, and 30 mg/kg for salmonids. The conclusions were extrapolated to physiologically related minor species. For any other species, the additive is considered of low concern at 2.0 mg/kg complete feed. The use of citronella oil in animal feed is expected to be of no concern for the consumers and for the environment. The essential oil under assessment should be considered as irritant to skin and eyes and as a dermal sensitiser. When handling the essential oil, exposure of unprotected users to methyleugenol may occur. Therefore, to reduce the risk, the exposure of the users should be minimised. Since the leaves of C. nardus and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

Abstract The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of amines, di‐C14‐C18‐alkyl, oxidised, renamed by the Panel as amines, di‐C14‐C20‐alkyl, oxidised, from hydrogenated vegetable oil. The substance amines, bis(hydrogenated tallow alkyl) oxidised, consisting of the same components, but originating from tallow, is currently authorised as FCM substance No 768. The vegetable‐sourced substance is intended to be used at up to 0.1% w/w as antioxidant and/or stabiliser in the manufacture of polyolefin food contact materials (FCM) and articles intended for contact with dry, aqueous and acidic foods. The substance is a mixture consisting of linear N,N‐dialkyl hydroxylamines and their corresponding amine, nitrone and oxime derivatives, as well as further components: tert‐N‐oxides, secondary amides and carboxylic acids. Specific migration was tested from polyethylene samples in 10% ethanol and 3% acetic acid for 2 h at 100°C followed by 10 days at 60°C. None of the non‐authorised components were detected to migrate at detection limits (LoD) in the range 0.003–0.029 mg/kg. The LoD of authorised carboxylic acids was 0.35 mg/kg. The Panel reassessed the genotoxicity studies carried out on FCM No 768 and evaluated two new bacterial reverse mutation tests on the nitrone and oxime derivatives as well as new (qualitative/quantitative) structure–activity relationship (Q)SAR analyses on other components. The Panel concluded that the substance did not raise a concern for genotoxicity. The Panel concluded that the substance is not of safety concern for the consumers if it is used as an additive at 0.1% w/w in the manufacture of polyolefin FCM intended to be in contact with foods simulated by food simulants A, B, C and E, except for infant formula and human milk, for storage above 6 months at room temperature and below, including hot‐fill conditions and heating up to 100°C for 2 h.

Abstract The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.2.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐G by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant requested to extend its use to nine additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of 10 food manufacturing processes. As the food enzyme–total organic solids (TOS) is removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining eight processes. Dietary exposure was up to 0.424 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1868 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 4406. Based on the data provided for the previous evaluation and the margin of exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.