90-day Oral Toxicity Research Articles

Toxicologic evaluation of a novel, highly soluble biotin salt, magnesium biotinate

A novel, highly soluble biotin salt, magnesium biotinate (MgB), was assessed for general and genetic toxicity using several toxicologic tests. This battery of tests included in vitro bacterial reverse mutation test, in vitro mammalian micronucleus assay, and oral acute, 14-day, and 90-day repeat-dose toxicity in Sprague-Dawley (SD) rats. The results of the in vitro studies indicate that MgB is not mutagenic, clastogenic, or aneugenic. The acute oral toxicity study established an LD50 ≥ 5000 mg MgB/kg. In the 14-day oral toxicity study, doses of MgB up to 2500 mg MgB/kg/day produced no clinical signs or mortality. In the 90-day oral toxicity study, administration of 600 mg MgB/kg/day resulted in no clinical signs and was determined to be the no-observed-adverse-effect-level (NOAEL), which equates to 39 g biotin/day for a 70 kg human. Since MgB is composed of 93% biotin, the 600 mg NOAEL equates to approximately 1.3 million times the current recommended daily allowance of 30 μg biotin/day and 3900 times supplement levels of 10 mg biotin/day. Based on the toxicologic profile and lack of findings in various in vitro and in vivo studies, MgB may be considered safe for long-term human use.

Safety evaluation of Lactobacillus plantarum strain TWK10® isolated from Taiwan Pickled Cabbage

In recent years, probiotics have drawn increasing attention due to their ability to confer multiple health benefits. It is vital to evaluate the safety of a probiotic strain, to confirm it is safe and well-tolerated by human. Lactobacillus plantarum TWK10®, isolated from Taiwanese pickled cabbage, has been demonstrated to have beneficial effect on improving exercise performance and muscle mass in clinical studies. In this study, we evaluated the safety of TWK10 in a battery of animal and in vitro studies including oral toxicity, the Ames' bacterial reverse mutation assay, the mammalian micronucleus and chromosome aberration tests. A subacute repeated-dose 28-day toxicity study was conducted to determine the no observable adverse effect level (NOAEL) of TWK10. In Ames' test, the results showed no mutagenic effect of TWK10 with or without the addition of S9 mixture. The mammalian chromosomal aberration test conducted in Chinese hamster ovary cells (CHO-K1) showed TWK10 was not genotoxic. Additionally, TWK10 administration did not cause any significant change in reticulocyte ratio and micronucleus incidence in ICR mice suggesting that it is non-mutagenic. In the repeated dose 28-day subacute oral toxicity study, SD rats administered with TWK10 at 1.4 × 1010, 2.8 × 1010 and 5.6 × 1010 cfu/kg/day showed no clinical signs of toxicity or adverse effects. Based on these findings, the NOAEL of TWK10 was determined to be 5.6 × 1010 cfu/kg/day. These findings combined with the tolerability seen in human clinical studies suggests that TWK10 is safe for human consumption.

Safety evaluation of a food enzyme with glucan 1,4-α-glucosidase and α-amylase activities from the genetically modified Aspergillus niger strain NZYM-BX.

The food enzyme with glucan 1,4‐α‐glucosidase (EC 3.2.1.3) and α‐amylase (EC 3.2.1.1) activities is produced with the genetically modified strain of Aspergillus niger NZYM‐BX by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in starch processing for the production of glucose syrups and distilled alcohol. Since residual amounts of total organic solids are removed by distillation and by the purification steps applied during the production of glucose syrups, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The repeated dose 90‐day oral toxicity study in rats made with a substitute enzyme was not considered suitable. However, since no exposure was expected from the intended uses, this study was not considered necessary. Similarity of the amino acid sequence of the food enzyme 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 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.

A 90-Day Oral Toxicity of Hydroethanolic Root Extract of Carissa spinarum in Wistar Rats.

Background Herbal medication is a worldwide and ancient practice, mostly in developing countries, where a large part of the population is involved in this practice. Hence, studies must be conducted to evaluate their safety and efficiency to avoid or prevent toxicological risks due to their usage. In Togo, Carissa spinarum is a medicinal plant belonging to Apocynaceae family, used as an aphrodisiac or to heal some ailments including malaria, sickle cell anemia, hypertension, pain, and asthma. Notwithstanding its several ethnomedicinal benefits, just a few toxicological data associated with its chronic use are available. Objective Therefore, this study aims to assess the toxicity of an ethanolic root extract of Carissa spinarum in Wistar rats. Methods The 90-day oral toxicity process following OECD TG 408 guidelines is used. Male Wistar rats received Carissa spinarum root hydroethanolic extract at 500 and 1000 mg/kg for 90 days by oral gavage. Body weight changes, hematological and blood biochemical parameters, organ weight changes, malondialdehyde as a lipoperoxidation marker expressed according to tissue proteins, and histopathology of vital organs were assessed. Results No signs of toxicity or mortality were observed during the 90 days experiment. Hematological parameters have not shown any treatment-related abnormalities. According to biochemical parameters, an increase in the chloride ion level was observed at 1000 mg/kg (p < 0.01). There was no significant difference between the treated groups and the control group concerning the malondialdehyde concentration, body weight, and organ relative weight. No changes in necropsy and histopathology of vital organs associated with extract treatment were observed. Conclusion The results indicated that an ethanolic root extract of Carissa spinarum does not cause adverse effects, which can lead to Wistar rats' death after 90-day oral administration at 500 and 1000 mg.

Safety evaluation of the food enzyme preparation isomaltulose synthase from Serratia plymuthica strain Z12A

The food enzyme isomaltulose synthase (sucrose glucosylmutase; EC 5.4.99.11) is produced with Serratia plymuthica strain Z12A by BENEO‐Palatinit GmbH. The food enzyme is used only in the form of an immobilised preparation of non‐viable cells for the production of isomaltulose. Residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of isomaltulose consequently, dietary exposure was not calculated. Genotoxicity tests did not indicate 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 1,011 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence of the 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 use of an immobilised food enzyme and the removal of TOS during the production of isomaltulose the Panel concluded that this food enzyme preparation does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme triacylglycerol lipase from the genetically modified Aspergillus luchuensis strain FL100SC.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase EC 3.1.1.3) is produced with a genetically modified Aspergillus luchuensis strain FL100SC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The triacylglycerol lipase is intended to be used only in an immobilised form in the production of modified fats and oils by interesterification. Since residual amounts of total organic solids (TOS) are removed by filtration and purification steps applied during fats and oils processing for interesterification, no dietary exposure was 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 849 mg TOS/kg body weight (bw) per day, the highest dose tested. The similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and no match was found. The Panel considers 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, including the immobilisation process and the absence of TOS in the final product, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Toxicological evaluation of banana and milk combination as incompatible diet - An experimental exploration of Samyoga viruddha concept

BackgroundFood is the basic requirement and an essential part of all living beings which sustains life. A lot of emphasize has been given to food in the classics of Ayurveda and a detailed description of food which are both wholesome and unwholesome have been described. Viruddhahara (incompatible food) is a unique concept explained in Ayurveda which, in long run, may be harmful to the body. Objective(s)Intake of Kadaliphala (Musa paradisiaca. Linn, a variety of banana) and cow milk is an example of Samyoga Viruddha (incompatibility with reference to combination of things). This combination is routinely consumed by people. The present study was planned to evaluate the toxicological implication of their combination on wistar rats. Materials and methodsA subacute toxicity study was conducted on Wistar rats following the repeated dose 28-day oral toxicity study in rodents, 407 - OECD guidelines. Different haematological and biochemical parameters along with histopathology of important organs were carried out to assess the toxicological implication of the combination. ResultsRepeated administration of the combination of Kadaliphala (Banana) and cow milk showed statistically significant increase in SGOT & urea and statistically significant decrease in creatinine. Significant decrease was observed in the food intake, faecal wet weight and faecal water in the 7th day of study, food conversion ratio in the 14th day of study, in the food intake, faecal wet weight and faecal water in the 21st day of study and significant decrease in the food conversion ratio in the 28th day of study. In histopathological examination, the test drug administered group showed mild to moderate myocarditis in the sections from heart. In sections from liver of two rats of test group, diffused micro fatty changes were observed. In sections from spleen; mild to moderate increase in the white pulp portion was observed. ConclusionMarked variation in SGOT, urea and creatinine levels and alteration in sections of heart, liver and spleen are indicative of mild toxicological implications of the combination of banana and milk. Continuous intake of this incompatible combination may hence prove harmful to the body.

Elucidating the Relations between Gut Bacterial Composition and the Plasma and Fecal Metabolomes of Antibiotic Treated Wistar Rats

The gut microbiome is vital to the health and development of an organism, specifically in determining the host response to a chemical (drug) administration. To understand this, we investigated the effects of six antibiotic (AB) treatments (Streptomycin sulfate, Roxithromycin, Sparfloxacin, Vancomycin, Clindamycin and Lincomycin hydrochloride) and diet restriction (–20%) on the gut microbiota in 28-day oral toxicity studies on Wistar rats. The fecal microbiota was determined using 16S rDNA marker gene sequencing. AB-class specific alterations were observed in the bacterial composition, whereas restriction in diet caused no observable difference. These changes associated well with the changes in the LC–MS/MS- and GC–MS-based metabolome profiles, particularly of feces and to a lesser extent of plasma. Particularly strong and AB-specific metabolic alterations were observed for bile acids in both plasma and feces matrices. Although AB-group-specific plasma metabolome changes were observed, weaker associations between fecal and plasma metabolome suggest a profound barrier between them. Numerous correlations between the bacterial families and the fecal metabolites were established, providing a holistic overview of the gut microbial functionality. Strong correlations were observed between microbiota and bile acids, lipids and fatty acids, amino acids and related metabolites. These microbiome–metabolome correlations promote understanding of the functionality of the microbiome for its host.

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

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain NZYM‐KE by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The α‐amylase is intended to be used in starch processing for the production of glucose syrups and other starch hydrolysates, 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. 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 of 1,100 mg TOS/kg body weight (bw) per day. A search for similarity of the amino acid sequence of the food enzyme 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 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 endo-1,3(4)-β-glucanase from the genetically modified Bacillus subtilis strain DP-Ezm28.

The food enzyme endo‐1,3(4)‐β‐glucanase (3(or 4)‐β‐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.

Toxicological evaluations of betulinic acid and ursolic acid; common constituents of Houttuynia cordata used as an anthelmintic by the Naga tribes in North-east India

BackgroundBetulinic acid (BA) and ursolic acid (UA) are two major phytoconstituents of Houttuynia cordata Thunb., (Saururaceae) which is used as an anthelmintic in the traditional medicine system of the Nagas in Nagaland, India. This study evaluates their toxic potentials using rodent models (Swiss albino mice and Wistar rats) according to the OECD (Organization for Economic Cooperation and Development) guidelines. Acute and 28-day sub-acute oral toxicity studies were conducted, and evaluations were made based on biochemical, hematological, and histopathological observations.ResultsAcute oral toxicity study revealed the oral LD50 of both the test compounds to be > 2000 mg/kg in mice. Sub-acute administration of BA at 10 mg/kg body weight (b.w.) revealed a significant increase in serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), urea concentrations and eosinophil and lymphocyte counts in rats. Animals administered with 10 mg/kg b.w. UA revealed elevated neutrophil count, SGOT, ALP, and urea concentrations, whereas white blood cells (WBC), lymphocyte, and platelet counts were found to be low. Histopathological examinations of body organs revealed alterations in the architecture of the liver, kidney, and spleen tissues. Notably, all these alterations were recoverable as evident in the satellite group, indicating a recovering pattern from the toxic effects caused by the oral administration of these phytocompounds.ConclusionAlthough UA and BA possess several therapeutic properties, their long-term usage can cause mild toxicity in their users. This study also paves way for evaluating the optimum effective and safe dose of these phytocompounds.

Repeated dose 90-day oral toxicity study of Vitex megapotamica (Spreng.) Moldenke

Vitex megapotamica (Spreng.) Moldenke is a plant with medicinal properties popularly used in Brazil to treat diabetes and obesity. Despite the promising use of this plant, are still incipient toxicology studies on prolonged periods of treatment. The aim of this study was to evaluate the repeated dose 90-day oral toxicity study of V. megapotamica methanolic extract (VMME) in male and female Wistar rats. Different groups of rats (n = 8) were treated daily with three different doses of VMME (100, 300, and 900 mg/kg) or vehicle (filtered water). Body weight, water, and feed consumption, and clinical and behavioral changes were monitored daily. At the end of the experimental period, blood samples were obtained for hematological and biochemical analyzes. After euthanasia, the vital organs were removed for the determination of relative weight and for histopathological analysis. No clinical signs of toxicity or mortality were found during the experimental period. Treatment with VMME did not induce any change in body weight gain, eating patterns, and behavior. We found no statistically significant changes in the different hematological and biochemical parameters evaluated. The relative weight of the organs and histopathological analysis did not show any significant change when compared to animals treated with the vehicle. The data obtained in this study allow us to conclude that the VMME obtained from V. megapotamica is safe after a repeated-dose 90-day oral toxicity study in male and female Wistar rats.

Toxicity studies of highly bioavailable isoniazid loaded solid lipid nanoparticles as per Organisation for Economic Co-operation and Development (OECD) guidelines

Solid lipid nanoparticles (SLNs) are presently being promoted to improve bioavailability of encapsulated drugs. These are well tolerated in living systems, as they are made from biocompatible material. Despite finding extensive applicability, these systems have not been sufficiently investigated for the toxicity so far. We have reported use of SLNs to improve plasma bioavailability of isoniazid (INH), a hepatotoxic, antitubercular drug. Presently we evaluate acute and repeated (28-day) oral dose toxicity, with satellite group, of developed INH loaded COMBI-SLN. In addition to high bioavailability, the COMBI-SLN exhibited 3 times higher LD50 (2000 mg/kg BW) versus 650 mg/kg BW for free INH. Results were complemented with histopathological evidence in brain, sciatic nerve and liver tissue all of which indicated enhanced safety of INH upon incorporation into SLNs. In the repeated dose study at doses selected as per Organisation for Economic Co-operation and Development (OECD) guidelines, a series of behavioural and haematological tests, clinical biochemistry (kidney and liver function, lipid profile) and histopathological studies were performed to evaluate the effect of low (250 mg/kg BW), medium (500 mg/kg BW) and high oral dose (1000 mg/kg BW). Absence of adverse effects like hepatotoxicity and peripheral neuropathy observed in rats at an oral intake level of 500 and 1000 mg/kg BW of COMBI-SLN, that is 20–40 folds above the anticipated human intake levels (after normalizing the surface area correction for rats), supports the conclusion that SLN are an intrinsically safe nanocarrier system that improves both the efficacy and the safety of INH.

Genotoxicity and 28-day repeated dose oral toxicity study of ovatodiolide in rats.

Ovatodiolide is a bioactive cembrane-type diterpenoid isolated from Anisomeles indica (L.) Kuntze. It has been proven that ovatodiolide is anti-inflammatory, anti-tumorigenic, anti-melanogenic and attenuates asthma by regulating signaling pathways. The aim of this study was to evaluate the safety of ovatodiolide by conducting genotoxicity tests and 28-day oral toxicity tests in rats. Genotoxicity assays were conducted by using a bacterial reverse mutation test and mammalian chromosomal aberration test to assess whether ovatodiolide causes reverse mutations and mutagenicity with or without metabolism activation. For the in vivo mammalian erythrocyte micronucleus test, mice were administered a single dose of 0, 250, 500 or 1000 mg/kg b.w. ovatodiolide by single gavage. In the acute oral toxicity test, rats were given a single dose of ovatodiolide 1000 mg/kg b.w. by single gavage. In the 28-day oral toxicity test, groups were divided into a control, ovatodiolide 10, 25 and 50 mg/kg b.w. The results showed that there was no mutagenicity in the bacterial reverse mutation test or the mammalian chromosomal aberration test with or without S9 fraction. Ovatodiolide did not produce an increase in micronucleated reticulocytes in the micronucleus test. The results revealed that the acute oral toxicity of ovatodiolide is over 1000 mg/kg b.w. in rats. Moreover, 10, 25 and 50 mg/kg b.w. of ovatodiolide did not cause a significant effect in rats. According to the results of the genotoxicity and oral toxicity studies in rats, ovatodiolide did not produce any adverse effects, and the tested doses can serve as clinical references.

Safety assessment of β-fructofuranosidase from Aspergillus brunneoviolaceus

β-Fructofuranosidase (β-D-fructofuranoside fructohydrolase; EC 3.2.1.26) is used in the production of fructo-oligosaccharides that are commonly used by the food industry as prebiotics for their purported health benefits. The β-fructofuranosidase discussed herein is obtained from a novel source organism that is a non-genetically modified strain of Aspergillus brunneoviolaceus, which phylogenetically belongs to the Aspergillus section Nigri. The safety of β-fructofuranosidase was evaluated in a series of toxicology studies as prescribed by Tier 1 toxicity testing by the European Food Safety Authority, including an evaluation of the mutagenicity and genotoxicity potential using the in vitro bacterial reverse mutation and mammalian chromosomal aberration assays, as well as systemic toxicity in a 90-day oral subchronic toxicity study in Sprague-Dawley rats. β-Fructofuranosidase was demonstrated to lack mutagenic or genotoxic potential based on the results of the in vitro assays due to absence of increased revertant colonies in the bacterial reverse mutation test and incidence of chromosome aberrations in the chromosomal aberration assay. Administration of β-fructofuranosidase by gavage at doses up to 1200 mg total organic solids (TOS)/kg body weight/day for 90 days did not elicit any systemic toxic effects in rats based on a lack of adverse effect in any study parameter, and therefore the no-observed-adverse-effect level of β-fructofuranosidase was concluded to be 1200 mg TOS/kg body weight/day, the highest dose tested. The results of the toxicology studies on β-fructofuranosidase from A. brunneoviolaceus demonstrate this species to be a safe and suitable source of enzymes for use by the food industry.

A 90-day oral repeated-dose toxicity study of Monascus Color Y-001 in rats

Monascus Color Y-001, a natural food dye produced from Monascus purpureus fermentation, was administered orally by gavage to male and female SD rats for 90 days at doses of 0 (vehicle: 0.1% Tween 80, 10 mL/kg bw), 100, 300 and 1000 mg/kg/day. During the treatment period, there was no death, and test article effects on clinical signs were limited to reddish feces, soiled perineal region (reddish color) and salivation that were observed in both sexes at 300/1000 mg/kg/day. Prolongation in PT and APTT occurred in males at 1000 mg/kg/day, and the changes were without any evidence suggesting hemorrhage and/or hepatic dysfunction. Treatment-related histopathological findings were noted in thymus, liver and kidney, and were limited to the females at 1000 mg/kg/day. These findings included decreased cellularity in thymus with decreased thymus weights attributed to nonspecific stress, centrilobular hepatocellular hypertrophy with increase of liver weights attributed to adaptive change, and vacuolation of proximal tubules in kidneys accompanied with related parameter changes in urinalysis. From these results, the no-observed-adverse-effect level (NOAEL) was judged to be 300 mg/kg/day both in male and female rats.

A 90-day Sub-chronic Oral Toxicity Assessment of Mulberry Extract in Sprague Dawley Rats.

Mulberry extract from Fructus Mori contains an anthocyanin pigment and has been widely used as a food additive in China and other Eastern Asian countries. Only few research has been done on toxicological profiling of mulberry extract for its safety evaluation; however, the data is inconclusive. In the current study, mulberry extract of 4200, 1400, or 466 mg/kg were orally administrated to Sprague Dawley rats for 90 consecutive days followed by a recovery period of 28 days. No abnormalities were detected in body weights, food intake, ophthalmological, hematological, coagulation, clinical chemistry, and organ weights parameters. Discoloration of urine (red, purple, and brown) and feces (black), along with bedding material (purple) were observed in the 4200 mg/kg group. Further, microscopic examination revealed brown granules in the renal tubular cells for rats in 4200 and 1400 mg/kg groups. Since these changes were associated with excretory effect of the extract, the No Observed Adverse Effect Level was determined to be 4200 mg/kg, which was equivalent to the 1058.5 mg/kg of anthocyanin.

Subchronic safety evaluation of hot-water extract from thinned immature mangos (Mangifera indica 'Irwin'): 90-days oral toxicity study in rats.

Thinned immature fruit of the mango tree (Mangifera indica 'Irwin') are handled as waste. In this study, we conducted a 90-days toxicity study in male and female Sprague Dawley rats to evaluate the safety of a hot-water extract of thinned immature mango fruits (TIMEx) administered by oral gavage at doses of 500, 1000 and 2500 mg/kg body weight/day. Treatment did not result in death or changes in the behavior or external appearance of the animals. No alterations were observed in hematological or serum chemical parameters, urinalysis, food consumption, body weight gain or organ weights at the end of the treatment period, with the exception of higher mean corpuscular volume in male rats that received high doses and lower serum creatine phosphokinase levels in female rats that received medium doses. Under the conditions of this study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for TIMEx was 2500 mg/kg/day. The findings indicate that TIMEx is safe for consumption and should be investigated as a candidate food.

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)‐β‐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 Assessment of Cellulose Nanofibers Obtained from Soda Cooking Bamboo Pulp：Bacterial Reverse Mutation Test, Mouse Lymphoma Thymidine Kinase Assay, Micronucleus Test, and Repeated dose 90-day Oral Toxicity Study in Rats

Safety Assessment of Cellulose Nanofibers Obtained from Soda Cooking Bamboo Pulp：Bacterial Reverse Mutation Test, Mouse Lymphoma Thymidine Kinase Assay, Micronucleus Test, and Repeated dose 90-day Oral Toxicity Study in Rats