Developmental immunotoxicity of ethanol in an extended one-generation reproductive toxicity study.

The susceptibility of developing immune system to chemical disruption warrants the assessment of immune parameters in reproductive and developmental testing protocols. In this study, a wide range of immune endpoints was included in an extended one-generation reproduction toxicity study (EOGRTS) design to determine the relative sensitivity of immune and developmental parameters to ethanol (EtOH), a well-known developmental toxicant with immunomodulatory properties. Adult Wistar rats were exposed to EtOH via drinking water (0, 1.5, 4, 6.5, 9, 11.5 and 14 % (w/v EtOH)) during premating, mating, gestation and lactation and continuation of exposure of the F(1) from weaning until killed. Immune assessments were performed at postnatal days (PNDs) 21, 42 and 70. Keyhole limpet hemocyanin (KLH)-specific immune responses were evaluated following subcutaneous immunizations on PNDs 21 and 35. EtOH exposure affected innate as well as adaptive immune responses. The most sensitive immune parameters included white blood cell subpopulations, ConA-stimulated splenocyte proliferation, LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific immune responses. Most parameters showed recovery after cessation of EtOH exposure after weaning in the 14 % exposure group. However, effects on LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific parameters persisted until PND 70. The results demonstrate the relative sensitivity to EtOH of especially functional immune parameters and confirm the added value of immune parameters in the EOGRTS. Furthermore, this study identified an expanded KLH-specific parameter set and LPS-induced NO and TNF-α production by adherent splenocytes as valuable parameters that can provide additional information on functional immune effects.

Nasal passages of Göttingen minipigs from the neonatal period to young adult.

Histopathological examination of the nasal passages requires a standardized approach for recording lesion distribution patterns. Nasal diagrams provide guidance to map the lesions. Information on lesions exists for rodents, dogs, and monkeys, which all have been used in inhalation studies. Recently, minipigs have garnered interest as an inhalation model because minipigs resemble humans in many features of anatomy, physiology, and biochemistry and may be a good alternative to monkeys and dogs. The present work explored the microanatomy and histology of the nasal passages of Göttingen minipigs from postnatal day 1 until 6 months of age. Six nasal levels were selected, which allow examination of the squamous, transitional (nonciliated) and ciliated respiratory, and olfactory epithelia; the nasopharynx; and relevant structures such as the vomeronasal organ, olfactory bulb, and nasal/nasopharynx-associated lymphoid tissue.

A search for biomarkers as diagnostic tools for food allergy: a pilot study in peanut-allergic patients.

BACKGROUND: The impact of peanut allergy is large and accidental ingestion of peanut can lead to severe reactions. Currently used diagnostic tests, such as skin prick tests (SPT) and determination of specific immunoglobulins (IgE) have, however, limited sensitivity and specificity. Therefore, new tools have to be developed to improve the accuracy of the diagnostic work-up of food-allergic patients. Comprehensive metabolite analysis may provide biomarkers for diagnosing food allergy as metabolite levels reflect actual physiological conditions. We investigated whether metabolites can be found that discriminate between peanut-allergic patients and non-peanut-allergic subjects. Such metabolites may be used for future diagnostic purposes. METHODS: Plasma and saliva samples were obtained from 23 participants (12 peanut allergic and 11 peanut tolerant) prior to and after a peanut challenge and measured with (1)H nuclear magnetic resonance (NMR) spectroscopy with subsequent multivariate data analysis. RESULTS: Clear differences were observed between NMR spectra of peanut-allergic and peanut-tolerant subjects in plasma as well as saliva. Allergic patients already showed aberrant metabolite levels prior to peanut ingestion, thus before the onset of allergic reactions. CONCLUSION: This pilot study shows that aberrant metabolite levels as determined by NMR in combination with multivariate statistics may serve as novel biomarkers for food allergy.

Integration of efficacy, pharmacokinetic and safety assessment of interleukin-1 receptor antagonist in a preclinical model of arthritis.

Pharmacokinetic properties and safety profile of a drug are likely influenced by the disease state of a patient. In this study, we investigated the influence of arthritic processes on pharmacokinetics and immunotoxicity of interleukin-1 receptor antagonist (Anakinra) in the rat adjuvant arthritis model. Anakinra dose-dependently suppressed joint inflammation and degradation as demonstrated by reduced clinical arthritis score, paw thickness, synovial infiltration and bone degradation. In addition, plasma levels of chemokines MCP-1 and GRO/KC were reduced. Pharmacokinetic behaviour of Anakinra was influenced by disease state of the rats as judged from a decrease in C(max) and an increase of the MRT as the disease progressed at a dose of 24 and 72 mg Anakinra/kg body weight. The pharmacokinetic parameters increased dose-dependently, but non-proportionally with increasing dose. Low level anti-Anakinra antibody formation was observed at prolonged exposure to the biologic. Safety parameters, including haematology, splenic lymphocyte subset analysis, ex vivo stimulation of spleen cells and histopathology of immune system organs were affected by the disease itself to such extent that no additional effects of Anakinra could be observed. In conclusion, we demonstrated that pharmacokinetic behaviour of Anakinra was influenced by the arthritis background of the rats resulting in decreased internal exposure.

Safety evaluation of the food enzyme cellulase from the non-genetically modified Penicillium funiculosum strain DP-Lzc35.

The food enzyme cellulase (4-(1,3;1,4)-ß-d-glucan 4-glucanohydrolase; EC 3.2.1.4) is produced with the non-genetically modified Penicillium funiculosum strain Lzc35 by Danisco US Inc. The cellulase is intended to be used in distilled alcohol production, baking and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for baking and brewing processes. Based on the proposed maximum use levels, dietary exposure to the food enzyme-TOS was estimated to be up to 0.416 mg TOS/kg body weight (bw) per day. 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 84 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 200. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be 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.

Safety evaluation of the food enzyme α-amylase from the genetically modified Bacillus licheniformis strain DP-Dzb52.

The food enzyme α-amylase (1,4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP-Dzb52 by Danisco US Inc. The production strain contains multiple copies of an antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The α-amylase is intended to be used in starch processing for the production of glucose syrups, brewing processes and distilled alcohol production. Since residual amounts of the food enzyme are removed by the purification steps applied during the production of glucose syrups and distillation, no dietary exposure was calculated. Based on the maximum use levels recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the enzyme-total organic solids (TOS) was estimated to be up to 0.145 TOS/kg body weight per day in European populations. The toxicity studies were carried out with another α-amylase from B. licheniformis strain DP-Dzb54, considered by the Panel as a suitable substitute. Toxicological tests indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no observed adverse effect level was identified in rats which, compared with the dietary exposure, results in a margin of exposure of at least 750. A search for similarity of the amino acid sequence to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions can be excluded in distilled alcohol production and is considered low when the enzyme is used in starch processing and brewing. 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.

Safety evaluation of the food enzyme endo-1,3(4)-ß-glucanase from the genetically modified Bacillus subtilis strain DP-Ezm28.

The food enzyme endo-1,3(4)-ß-glucanase (3(or 4)-ß-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with a genetically modified Bacillus subtilis strain DP-Ezm28 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in distilled alcohol production and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for brewing processes. Based on the maximum use levels recommended for brewing processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-total organic solids was estimated to be up to 0.183 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 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 5464. Similarity of the amino acid sequence to those of known allergens was searched and two matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered 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.

Safety evaluation of the food enzyme alternansucrase from Leuconostoc citreum strain NRRL B-30894.

The food enzyme alternansucrase (sucrose:1,6(1,3)-α-d-glucan 6(3)-α-d-glucosyltransferase, EC 2.4.1.140) is produced with a non-genetically modified Leuconostoc citreum strain NRRL B-30894 by Cargill Incorporated. As a consequence of the absence of antimicrobial resistance genes identified in its genome, the production strain meets the criteria to qualify for the Qualified Presumption of Safety (QPS) approach to safety assessment. As no other concerns arising from the microbial source or from the manufacturing process have been identified, the Panel considers that toxicological tests are not needed for the assessment of this food enzyme. The alternansucrase food enzyme is intended to be used for the manufacture of α-d-glucan oligosaccharides as a sweetening agent. The purification processes applied to syrups produced from sucrose with alternansucrase are expected to largely remove the food enzyme. Any residual TOS remaining in the final product would consist of non-hazardous material. This is based on the QPS status of the production organism, the medium components and the identified material used in downstream processing. Consequently, the Panel decided that dietary exposure did not need to be calculated. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be 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.

Safety evaluation of the food enzyme with ß-glucanase and ß-xylanase activities from the Trichoderma reesei strain DP-Nya67.

The food enzyme with ß-glucanase and ß-xylanase (4-ß-d-xylan xylanohydrolase, EC 3.2.1.8) activities is produced with the non-genetically modified Trichoderma reesei (strain DP-Nya67) by DuPont. The food enzyme is intended to be used in brewing processes, grain treatment for the production of starch and gluten fractions, and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation and during grain treatment, dietary exposure was only calculated for brewing processes. Based on the maximum recommended use levels for brewing processes, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 4.585 mg TOS/kg body weight (bw) per day. Since the compositional data provided was insufficient to characterise the food enzyme batches used for toxicological testing, their suitability for use in the toxicological tests could not be established. As result, the toxicological studies provided were not further considered by the Panel. Similarities of the amino acid sequences to those of known allergens were searched and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. In the absence of compositional data sufficient to characterise the food enzyme batches used for toxicological testing, the Panel is unable to complete its assessment of the safety of the food enzyme.

Safety evaluation of the food enzyme endo-1,4-ß-xylanase and ß-glucanase from Disporotrichum dimorphosporum strain DXL.

The food enzyme with xylanases (4-ß-d-xylan xylanohydrolase, EC 3.2.1.8) and glucanases active against ß-1,4 linkages is produced with the non-genetically modified fungus Disporotrichum dimorphosporum strain DXL by DSM Food Specialities B.V. The food enzyme is intended to be used in brewing processes. Based on the maximum use level and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 0.167 mg TOS/kg body weight (bw) per day. 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 (NOAEL) at the highest dose of 199 mg TOS/kg bw per day that, compared with the estimated dietary exposure, results in a high Margin of Exposure of at least 1,100. Similarity of amino acid sequences of the identified xylanases and ß-glucanases to those of known allergens was searched. No matches were found for two endo-1,4-ß-glucanases and two endo-1,4-ß-xylanases. However, for a third endo-ß-1,4-glucanase the search resulted in matches with three mite protein sequences. While incidental cases of allergic reactions to endo-1,4-ß-xylanases and ß-glucanases have been reported after inhalation in respiratory sensitised individuals in the workplace, no allergic reactions to xylanases or ß-glucanases have been reported in the literature after oral exposure. The Panel considered that, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be 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.

Safety evaluation of the food enzyme beta-galactosidase from Bacillus sp. (strain M3-1).

The food enzyme ß-d-galactosidase galactohydrolase (EC 3.2.1.23) is produced with Bacillus sp. strain M3-1 by GenoFocus Inc. The food enzyme ß-galactosidase is intended to be used in the manufacture of galactooligosaccharides (GOS). Since residual amounts of total organic solids are removed by the purification steps applied during the production of GOS, toxicological studies were considered not necessary and no dietary exposure was calculated. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood of such reactions to occur is considered to be 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.

Safety evaluation of the food enzyme alpha-amylase from a genetically modified Bacillus subtilis (strain NBA).

The food enzyme alpha-amylase (4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with a genetically modified strain of Bacillus subtilis strain NBA by DSM Food Specialities B.V. This α-amylase is intended to be used in baking processes. The genetic modifications do not give rise to safety concerns and the food enzyme is free from viable cells of the production organism and recombinant DNA. The parental strain meets the required qualifications to be considered as a Qualified Presumption of Safety (QPS) organism and is therefore presumed to be safe. Since the production strain is not cytotoxic and since the introduced genetic modifications do not raise safety concerns, the presumption of safety made for the parental strain is extended to the production strain. The conclusions on safety of the food enzyme are made following the QPS approach in relation to the production strain, with additional consideration of the conditions of manufacture. However, the Panel considers no toxicological studies other than assessment of allergenicity necessary. This is based on the QPS status of the production strain and the absence of any hazards from the product and downstream processing. Based on the maximum use level recommended for the baking processes and individual data from the European Food Safety Authority (EFSA) Comprehensive European Food Consumption Database, dietary exposure was estimated to be up to 0.093 mg TOS/kg body weight per day in European populations. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme α-amylase from Bacillus licheniformis (strain DP-Dzb44).

The food enzyme α-amylase (1,4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP-Dzb44 by Danisco US Inc. The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The α-amylase is intended to be used in distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation, toxicological studies were not considered necessary and no dietary exposure was calculated. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions can be excluded. 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.

Safety evaluation of the food enzyme glucan 1,4-α-maltotetraohydrolase from Bacillus licheniformis (strain DP-Dzf24).

The food enzyme glucan 1,4-α-maltotetraohydrolase (EC 3.2.1.8) is produced with the genetically modified Bacillus licheniformis strain DP-Dzf24 by Danisco US Inc. The production strain contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in baking processes and starch processing for the production of glucose syrups. The residual amounts of the Total Organic Solids (TOS) in glucose syrups are removed by filtration and purification during starch processing. Consequently, dietary exposure was not calculated for this use. Based on the maximum use levels recommended for the baking processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 0.271 mg TOS/kg body weight per day in European populations. Toxicological tests with the food enzyme indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no-observed-adverse-effect level (NOAEL) was identified in rats, which, compared with the dietary exposure, results in a margin of exposure of at least 347. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that, under the intended conditions of use, the risk for allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the microbial source, genetic modifications, the manufacturing process, the compositional and biochemical data, the dietary exposure assessment and the findings in the toxicological studies, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme l-ascorbate oxidase from Cucurbita pepo L. and Cucurbita moschata Duchesne.

The food enzyme l-ascorbate: oxygen oxidoreductase (EC 1.10.3.3) is extracted from fruit peels of Cucurbita pepo L. and Cucurbita moschata Duchesne by Nagase (Europa) GmbH. This enzyme is intended to be used in baking and cereal-based processes. Based on maximum use levels recommended for the respective food processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 5.950 mg TOS/kg body weight per day in European populations. This exposure is in the same order of magnitude for infants and toddlers; but for children, adolescents, adults and the elderly it is one order of magnitude higher than the exposure to the fraction of the fruit peels comparable to the food enzyme-TOS. The Panel, while recognising the order of magnitude of difference in the exposure estimates, considers that any realistic exposure derived from the use of the food enzyme would be considerably lower and likely to be within the range of exposure through a typical diet. The Panel agreed that the requirements for exclusion of toxicological data were met. Amino acid sequence similarity to known allergens was searched and no match was found. The Panel considered that the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but are not expected to exceed the likelihood of the allergic reactions following consumption of pumpkin or zucchini per se, which is low. Based on the data provided and the origin of the food enzyme from edible parts of C. pepo L. and C. moschata Duchesne, the Panel considers that the food enzyme l-ascorbate oxidase does not raise safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme glucose isomerase from Streptomyces murinus (strain NZYM-GA).

The food enzyme is a glucose isomerase (d-xylose aldose-ketose-isomerase; EC 5.3.1.5) produced with a non-genetically modified Streptomyces murinus strain NZYM-GA by Novozymes A/S. The glucose isomerase is intended only to be used in an immobilised form in glucose isomerisation for the production of high fructose syrups. Residual amounts of total organic solids are removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme lysophospholipase from Trichoderma reesei (strain RF7206).

The food enzyme lysophospholipase (EC 3.1.1.5) is produced with the genetically modified Trichoderma reesei strain RF7206 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. The lysophospholipase food enzyme is intended to be used in starch processing for the production of glucose syrups. Residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, consequently, dietary exposure was not calculated. 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 at the highest dose tested of 927 mg TOS/kg body weight (bw) per day. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions to occur is considered to be low. Based on the data provided and the removal of TOS during the intended food production process, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme alpha-amylase from a genetically modified Trichoderma reesei (strain DP-Nzb48).

The food enzyme alpha-amylase (4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with a genetically modified strain of Trichoderma reesei by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. This α-amylase is intended to be used in distilled alcohol production and brewing processes. Residual amounts of total organic solids (TOS) are removed by distillation; consequently, dietary exposure was not calculated for this use. Based on the maximum use levels recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 1.701 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests with the food enzyme did not indicate a genotoxic 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 (NOAEL) at the highest dose of 230 mg TOS/kg bw per day. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the removal of residues of the food enzyme during distillation, the Panel concluded that the use of this enzyme in the distilled alcohol production is safe. When used in brewing processes, the margin of exposure calculated from the data provided is only (at least) 135, but no safety issues were identified.

Safety evaluation of the food enzyme pullulanase from a genetically modified Bacillus licheniformis (strain DP-Dzp39).

The food enzyme pullulanase (pullulan 6-α-glucanohydrolase; EC 3.2.1.41) is produced with a genetically modified Bacillus licheniformis (strain DP-Dzp39) by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. This pullulanase is intended to be used in brewing processes, starch processing for glucose syrups production and distilled alcohol production. Residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups, consequently, dietary exposure was not calculated for these food processes. For brewery products, based on the maximum use level recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 0.053 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests with the food enzyme did not raise 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 of 500 mg TOS/kg bw per day that, compared to the estimated dietary exposure, results in sufficiently high margin of exposure (at least 9,400). The amino acid sequence of the food enzyme did not match those of known allergens. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme α-amylase from Aspergillus oryzae (strain DP-Bzb41).

The food enzyme α-amylase (4-α-d-glucan glucanohydrolase, EC 3.2.1.1) is produced with a non-genetically modified Aspergillus oryzae (strain DP-Bzb41) by Danisco US Inc. (USA). The α-amylase food enzyme is intended to be used in baking, brewing, distilled alcohol production and starch processing for the glucose syrup production. Based on the maximum use levels for baking and brewing processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 2.59 mg TOS/kg body weight (bw) per day. Since residual amounts of TOS are removed during distilled alcohol production and by the purification steps applied during starch processing, dietary exposure for these processes was not calculated. 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 (NOAEL) of 1,000 mg TOS/kg bw per day, the highest dose tested. Comparison with the estimated dietary exposure, results in a margin of exposure of at least 386. Similarity of the amino acid sequence to those of known allergens was searched and one match to respiratory allergen was found (an amylase from another strain of A. oryzae). The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be 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.