90-day Oral Toxicity Study Research Articles

Comprehensive Safety Assessment of Lacticaseibacillus paracasei subsp. paracasei NTU 101 Through Integrated Genotypic and Phenotypic Analysis

Probiotics, as defined by the World Health Organization, are live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host. Although some countries have approved specific probiotic species for use in food, safety concerns may still arise with individual strains. Lacticaseibacillus paracasei subsp. paracasei NTU 101 (NTU 101), isolated from the gut of healthy infants, has demonstrated various probiotic effects and shown safety in a prior 28-day animal feeding study. To further verify its safety and mitigate potential risks, we performed a comprehensive genotypic and phenotypic safety evaluation in accordance with the European Food Safety Authority guidelines for safety assessment through whole genome sequencing and related literature. In this research, minimum inhibitory concentration testing identified NTU 101’s resistance to chloramphenicol; however, subsequent gene analysis confirmed no associated risk of resistance. Assessments of safety, including biogenic amine content, hemolytic activity, mucin degradation, and D-lactic acid production, indicated a low level of risk. Additionally, a repeated-dose 90-day oral toxicity study in Sprague-Dawley rats revealed no toxicity at a dose of 2000 mg/kg body weight, further supporting the strain’s safety for consumption. Based on these comprehensive analyses, NTU 101 is considered safe for regular consumption as a health supplement.

Safety evaluation of the food enzyme glucan-1,4-α-glucosidase from the non-genetically modified Aspergillus niger strain DP-Azh100.

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 Aspergillus niger strain DP-Azh100 by Genencor International B.V. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in four food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the two remaining processes. It was estimated to be up to 1.390 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 1000 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 719. A search for the homology of the amino acid sequence of the food enzyme to known allergens was made and one match to a respiratory allergen was found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme 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.

Evaluating the performance of multi-omics integration: a thyroid toxicity case study.

Multi-omics data integration has been repeatedly discussed as the way forward to more comprehensively cover the molecular responses of cells or organisms to chemical exposure in systems toxicology and regulatory risk assessment. In Canzler etal. (Arch Toxicol 94(2):371-388. https://doi.org/10.1007/s00204-020-02656-y ), we reviewed the state of the art in applying multi-omics approaches in toxicological research and chemical risk assessment. We developed best practices for the experimental design of multi-omics studies, omics data acquisition, and subsequent omics data integration. We found that multi-omics data sets for toxicological research questions were generally rare, with no datasets comprising more than two omics layers adhering to these best practices. Due to these limitations, we could not fully assess the benefits of different data integration approaches or quantitatively evaluate the contribution of various omics layers for toxicological research questions. Here, we report on a multi-omics study on thyroid toxicity that we conducted in compliance with these best practices. We induced direct and indirect thyroid toxicity through Propylthiouracil (PTU) and Phenytoin, respectively, in a 28-day plus 14-day recovery oral rat toxicity study. We collected clinical and histopathological data and six omics layers, including the long and short transcriptome, proteome, phosphoproteome, and metabolome from plasma, thyroid, and liver. We demonstrate that the multi-omics approach is superior to single-omics in detecting responses at the regulatory pathway level. We also show how combining omics data with clinical and histopathological parameters facilitates the interpretation of the data. Furthermore, we illustrate how multi-omics integration can hint at the involvement of non-coding RNAs in post-transcriptional regulation. Also, we show that multi-omics facilitates grouping, and we assess how much information individual and combinations of omics layers contribute to this approach.

Subchronic Toxicity Evaluation of Dietary Administration of a Fungal Biomass From Rhizomucor pusillus.

Fungal-derived food products align with sustainable food supply principles and offer a sustainable and nutritious option for consumers. Rhizomucor pusillus strain CBS 143028 has emerged as a candidate food ingredient. Fermentation of R. pusillus CBS 143028 results in a mycelium biomass mainly comprising fungal proteins, cell wall components, and micronutrients. Although R. pusillus has a history of safe use in the production of food enzymes and in traditional fermented foods, the fungal biomass obtained after fermentation of R. pusillus CBS 143028 is considered a novel food and requires a thorough safety assessment. To this end, a 90-day oral toxicity study was conducted in which Wistar rats (10/sex/group) were provided diets containing 0, 100,000, 200,000, or 300,000 ppm of R. pusillus mycelium. Standard toxicity study parameters as given in OECD Test Guideline 408 were examined. The mean achieved dosages of R. pusillus mycelium were 6398, 12,738, or 19,668 mg/kg body weight/day for males and 7235, 14,949, or 22,461 mg/kg body weight/day for females in the low-, mid-, and high-dose groups, respectively. Although statistically significant differences were reported, these effects were not considered biologically relevant or test article-related due to atypical values in the control groups, the lack of a dose response relationship, or were attributed to normal biological variation. Thus, the no-observed-adverse-effect level (NOAEL) was established at 300,000 ppm (corresponding to 19,668 and 22,461 mg/kg body weight/day for males and females, respectively), the highest concentration tested.

Safety evaluation of the food enzyme endo-1,3(4)-β-glucanase from the non-genetically modified Talaromyces versatilis strain PF8.

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.

Safety evaluation of the food enzyme endonuclease from the non-genetically modified Penicillium citrinum strain NP 11-15.

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non-genetically modified Penicillium citrinum strain NP 11-15 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 the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.006 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 1010 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 168,333. 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 by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. 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 carboxypeptidase C from the genetically modified Aspergillus niger strain PEG.

The food enzyme carboxypeptidase C (EC 3.4.16.5) is produced with the genetically modified Aspergillus niger strain PEG by DSM Food Specialties B.V. 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. It is intended to be used in nine food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.053 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the carboxypeptidase to be used in the toxicological studies, because both strains were derived from the same recipient strain, the location of the inserts was comparable, no partial inserts were present and the production methods were essentially the same. 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 1850 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 901. A homology search for the amino acid sequence of the food enzyme to known allergens was made and one match with a wheat allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially in wheat-allergic individuals, 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.

Re-evaluation of the reduced heart weights in male rats in a 28-day oral repeated-dose toxicity study of tetramethylammonium hydroxide

We recently conducted a detailed hazard assessment of tetramethylammonium hydroxide (TMAH), a priority chemical substance under the Japan Chemical Substances Control Law. During this assessment, there was debate regarding the reduced heart weight observed in the treated male groups in the 28-day rat oral repeated-dose toxicity study. This finding was not observed in females in this study and in both sexes of oral toxicity studies for tetramethylammonium chloride (TMAC) or tetramethylammonium hydrogen phthalate (TMAHP). Unpublished individual data from the oral TMAH developmental and reproductive toxicity (DART) screening study were also obtained; no effect on heart weight was observed. In addition, background data on rat heart weight from six 28-day oral toxicity studies conducted in the same facility, year, strain, age, and breeder as the TMAH study were obtained from the Japan Existing Chemical Substances Database (JECDB). These investigations suggest that the statistically significant lower heart weight in the treated males in the 28-day toxicity study is likely caused by an incidental skewing of individuals with heavier heart weights toward control male groups and is not due to TMAH treatment. Thus, it is worthwhile to include as much relevant data as possible to confirm or refute unexpected findings in toxicity studies.

The effect of aging on the repeated-dose liver micronucleus assay using N-nitrosodipropylamine, quinoline, and carbendazim

The repeated dose liver micronucleus (RDLMN) assay has been sufficiently validated in terms of the numbers and types of chemicals studied. However, it remains unclear whether aging affects assay results. The OECD Test Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents) indicates that dosing should begin as soon as feasible after weaning and in any event before 9 weeks of age. Therefore, it is particularly important to determine whether there are age-related differences between 6 and 8 weeks of age at the start of dosing when considering the possibility of integrating this assay into a 4-week repeated dose general toxicity study. We evaluated the impact of the rats’ age on the RDLMN assay with three chemicals: N-nitrosodipropylamine, quinoline, and carbendazim. There were no significant age-related differences for the first two chemicals, whereas a markedly higher frequency of micronucleated hepatocytes (MNHEPs) was observed in younger rats for carbendazim. However, regardless of the age of animals, micronucleus induction was detected in all three chemicals. Combined with the previous reports on clofibrate and diethylnitrosamine, we concluded that animals of any age from 6 to 8 weeks could be used in the RDLMN assay.

Bacillus subtilis ZB423: 90-Day repeat dose oral (gavage) toxicity study in Wistar rats.

The novel genetically modified probiotic Bacillus subtilis ZB423 was assessed in a 90-day repeated-dose oral toxicity study adhering to Good Laboratory Practice (GLP) and Organization for Economic Cooperation and Development (OECD) guidelines. Spray-dried spores at a concentration of 1.1E12 CFU/g were administered at doses of 130, 260, and 519 mg/kg body weight/day correlating to 1.43 × 1011, 2.86 × 1011, and 5.71 × 1011 CFU/kg/day, respectively, by oral gavage to Wistar rats for a period of 90 consecutive days. Results showed no toxicologically relevant findings for B. subtilis ZB423 from measured parameters. The no observed adverse effect level (NOAEL) of B. subtilis ZB423 is 519 mg/kg body weight/day corresponding to 5.71 × 1011 CFU/kg/day for lyophilized B. subtilis ZB423 spores under the test conditions employed.

Fragile Guts Make Fragile Brains: Intestinal Epithelial Nrf2 Deficiency Exacerbates Neurotoxicity Induced by Polystyrene Nanoplastics.

Oral ingestion is the primary route for human exposure to nanoplastics, making the gastrointestinal tract one of the first and most impacted organs. Given the presence of the gut-brain axis, a crucial concern arises regarding the potential impact of intestinal damage on the neurotoxic effects of nanoplastics (NPs). The intricate mechanisms underlying NP-induced neurotoxicity through the microbiome-gut-brain axis necessitate further investigation. To address this, we used mice specifically engineered with nuclear factor erythroid-derived 2-related factor 2 (Nrf2) deficiency in their intestines, a strain whose intestines are particularly susceptible to polystyrene NPs (PS-NPs). We conducted a 28-day repeated-dose oral toxicity study with 2.5 and 250 mg/kg of 50 nm PS-NPs in these mice. Our study delineated how PS-NP exposure caused gut microbiota dysbiosis, characterized by Mycoplasma and Coriobacteriaceae proliferation, resulting in increased levels of interleukin 17C (IL-17C) production in the intestines. The surplus IL-17C permeated the brain via the bloodstream, triggering inflammation and brain damage. Our investigation elucidated a direct correlation between intestinal health and neurological outcomes in the context of PS-NP exposure. Susceptible mice with fragile guts exhibited heightened neurotoxicity induced by PS-NPs. This phenomenon was attributed to the elevated abundance of microbiota associated with IL-17C production in the intestines of these mice, such as Mesorhizobium and Lwoffii, provoked by PS-NPs. Neurotoxicity was alleviated by in vivo treatment with anti-IL-17C-neutralizing antibodies or antibiotics. These findings advanced our comprehension of the regulatory mechanisms governing the gut-brain axis in PS-NP-induced neurotoxicity and underscored the critical importance of maintaining intestinal health to mitigate the neurotoxic effects of PS-NPs.

90-day oral toxicity study in rats of a protein-rich powder derived from Xanthobacter sp. SoF1.

Xanthobacter sp. SoF1 (SoF1) is an autotrophic hydrogen-oxidizing bacteria that produces protein-rich biomass and has potential to be an alternative protein source that is more environmentally sustainable than animal and plant derived proteins. A protein-rich powder derived from SoF1 was the test material in a 90-day repeated-dose oral toxicity study to explore major toxic effects, demonstrate target organs, and provide an estimate of a no-observed-adverse-effect level (NOAEL). Daily doses of 0 (vehicle only), 375, 750, and 1500 mg/kg bw/day of the test material were administered by gavage to 10 Han:WIST rats/sex/group. An additional group was administered 1290 mg/kg bw/day whey protein concentrate as positive control. No treatment-related adverse effects were observed, and no target organs were determined after 90/91 days of consecutive administration of the test item. A NOAEL of 1500 mg/kg bw/day was determined.

Safety of glucosyl hesperidin as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on glucosyl hesperidin (GH) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF, which is produced from hesperidin and dextrin by enzymatic reactions, is a powder consisting mainly of monoglucosyl hesperidin (MGH) and unreacted hesperidin (flavonoid), which account in total for up to 92.8% (on dry basis) of the product. The applicant proposed to use the NF in specific drinks and food supplements leading to a maximum intake of up to 364 mg per day for adults. The target population is the general population, except for food supplements for which the proposed target population is children from 1 year onwards and adults. Taking into consideration the composition of the NF and the proposed uses, the consumption of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. Based on a 90-day oral toxicity study conducted with the NF, the Panel considers the NOAEL at the mid-dose group, i.e. ~ 1000 mg/kg body weight (bw) per day. By applying an uncertainty factor of 200, the resulting intake providing sufficient margin of exposure for humans would be 5 mg/kg bw per day. The available human intervention studies did not report clinically relevant changes in haematological or clinical chemistry parameters following the administration of GH/MGH at supplemental doses of up to 3 g/day for 12 weeks. Overall, the Panel considers that the margin of exposure (~ 200) between the intake of the NF at the proposed uses and use levels and the NOAEL from the 90-day study is sufficient. The Panel concludes that the NF, glucosyl hesperidin, is safe for the target population at the proposed uses and use levels.

Safety evaluation of Bifidobacterium animalis subsp. lactis BLa80 under in vitro, and in vivo conditions

Bifidobacterium animalis subsp. lactis BLa80 is a new probiotic strain with extensive applications in food products both domestically and internationally. Given the rising consumption of this probiotic, its safety assessment is increasingly crucial in the food industry. This study evaluates the safety of strain BLa80 using a combination of in vitro and in vivo assays along with genomic analysis. Methods included exposing the strain to artificial gastric and intestinal fluids, as well as a medium containing bile salts, to stimulate human digestive conditions. The strain showed high tolerance to gastric fluid at pH of 2.5 and to 0.3 % bile salts. It maintained a 99.92 % survival rate in intestinal fluid. Additional tests assessed hemolytic activity, antibiotic susceptibility (revealing sensitivity to 7 antibiotics), and biogenic amine production using HPLC-ELSD, confirming the absence of histamine, and other harmful amines. Bile salt hydrolase activity was demonstrated qualitatively, and metabolic byproducts were quantitatively analyzed using a D-/l-lactic acid assay kit, showing that BLa80 produces 1.48 mg/mL of l-lactic acid and no harmful d-lactic acid. Genomic analysis confirmed the absence of virulence or pathogenicity genes, and a 90-day oral toxicity study in rats confirmed no toxic effects at various doses. Overall, these findings support the safety classification of the strain BLa80.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus niger strain ASP.

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.

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Penicillium caseifulvum strain AE-LRF.

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.

Safety evaluation of the food enzyme β-glucosidase from the non-genetically modified Penicillium guanacastense strain AE-GLY.

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.

Safety evaluation of the food enzyme 3-phytase from the non-genetically modified Aspergillus niger strain PHY93-08.

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.

Safety evaluation of an extension of use of the food enzyme pullulanase from the non-genetically modified Pullulanibacillus naganoensis strain AE-PL.

The food enzyme pullulanase (pullulan 6-α-glucanohydrolase; EC 3.2.1.41) is produced with the non-genetically modified Pullulanibacillus naganoensis strain AE-PL 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 has requested to extend its use to include seven additional processes and to revise the previous use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eight food manufacturing processes. As the food enzyme-total organic solids (TOS) are not carried into the final foods in two food manufacturing processes, the dietary exposure was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.004 mg TOS/kg body weight (bw) per day in European populations. The Panel evaluated the repeated dose 90-day oral toxicity study in rats submitted in the previous application and identified a no observed adverse effect level of 643 mg TOS/kg bw per day, the highest dose tested. When compared with the calculated dietary exposure, this resulted in a margin of exposure of at least 160,750. 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.

Repeated dose, 28-day oral toxicity study of curcumin, anthocyanins, and sodium nitrite in Wistar rats

ObjectiveThe goal of the present study was to examine the repeated dose 28-day oral toxicity of curcumin, anthocyanins, and sodium nitrite in Wistar rats. MethodsFor this purpose, forty-eight male Wistar rats were randomly divided into 8 groups (n = 6 each), encompassing untreated controls and experimental groups treated with curcumin, anthocyanins, and sodium nitrite. Three rats from each group were sacrificed by cervical dislocation under di-ethyl ether anesthesia after 2 and 4 weeks of therapy, respectively. Blood samples were collected for serum chemistry. All of the animals' livers, hearts, and kidneys were removed and sent for histopathological examination. ResultsAfter two weeks of inquiry, certain groups displayed higher hematological values, while others had lower values compared to the control group. AST, CK, and LDH enzyme activity were higher in groups 2–8, but urea concentrations were higher in groups 6 and 8. After four weeks, the Hb, MCH, and MCHC values in group 4 were greater, as were the WBC levels in groups 4 and 6, whereas other groups had lower MCV and WBC values. The weekly body weight gain was insignificantly different between treatment groups. Throughout the experiment, none of the animals perished. Male rats' liver, kidney, and heart underwent histopathological changes after ingesting curcumin, sodium nitrite, and anthocyanin. ConclusionBased on the findings, rats were more detrimental when curcumin, sodium nitrite, and anthocyanin were ingested together than when they were consumed individually, as evidenced by histopathological abnormalities in the liver, kidneys, and heart.