A 21-day safety evaluation of biotechnologically produced 3-fucosyllactose (3-FL) in neonatal farm piglets to support use in infant formulas.

3-Fucosyllactose (3-FL) is one of the most abundant fucosylated oligosaccharides in human breast milk and is an approved infant formula ingredient world-wide. 3-FL functions as a prebiotic to promote early microbial colonization of the gut, increase pathogen resistance and modulate immune responses. To investigate safety and potential gut microbiota effects, 3-FL was fed for 21-days to farm piglets beginning on Postnatal Day (PND) 2. Fructooligosaccharide (FOS), an approved infant formula ingredient, was used as a reference control. Standard toxicological endpoints were evaluated, and the gut microbiota were assessed. Neither 3-FL (245.77 and 489.72 mg/kg/day for males and 246.57 and 494.18 mg/kg/day for females) nor FOS (489.44 and 496.33 mg/kg/day males and females, respectively) produced any adverse differences in growth, food intake or efficiency, clinical observations, or clinical or anatomic pathology changes. Differences in the gut microbiota after 3-FL consumption (versus control and FOS groups) included the absence of Bifidobacterium species from the piglets, enrichment of Prevotellamassilia timonensis, Blautia species, Mediterranea massiliensis, Lachnospiraceae incertae sedis, and Eubacterium coprostanoligens and lower relative abundance of Allisonella histaminiformans and Roseburia inulinivorans. This study further supports the safe use of 3-FL produced using biotechnology as a nutritional ingredient in foods.

Genotoxicity and neonatal subchronic toxicity assessment of a novel mixture of the human-identical milk oligosaccharides lacto-N-fucopentaose I and 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) are a complex group of bioactive molecules largely observed in human breast milk but also occurring in limited amounts in other mammalian milks. Advances in biotechnology have enabled production of human-identical milk oligosaccharides (HiMOs), structurally identical molecules to HMOs found naturally in human milk, intended for addition to infant formula to more closely replicate breast milk. Biosynthesis of a novel mixture of two major HMOs, lacto-N-fucopentaose I and 2'-fucosyllactose (LNFP-I/2'-FL), recently became possible. To support the safety of LNFP-I/2'-FL for use in infant formula and other foods, it was subject to a safety assessment comprising a bacterial reverse mutation test, an in vitro mammalian cell micronucleus test, and a 90-day oral gavage study in neonatal rats. In the 90-day study (the first HiMO study to include the new endocrine-sensitive endpoints described in the 2018 version of OECD Test Guideline 408), LNFP-I/2'-FL was administered by oral gavage to neonatal rats once daily (from Day 7 of age) for 90 consecutive days, at doses up to 5000 mg/kg bw/day, followed by a 4-week recovery period. Concurrent reference controls received 5000 mg/kg bw/day of the approved infant formula ingredient oligofructose. LNFP-I/2'-FL was nongenotoxic in vitro. The highest dose tested (5000 mg/kg bw/day) was established as the no-observed-adverse-effect level in the 90-day study, as there were no test article-related adverse effects on clinical observations, body weight, food consumption, clinical pathology, and organ weights nor any noteworthy macroscopic or microscopic findings. This supports the safety of LNFP-I/2'-FL for its intended uses in food.

Safety evaluation of a mixture of the human-identical milk oligosaccharides 2'-fucosyllactose and difucosyllactose.

Human milk oligosaccharides (HMOs) are endogenous indigestible carbohydrates representing the largest compositional difference between human breastmilk and infant formula (IF). Two major HMOs in human breastmilk are 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL); commercial IF can be supplemented with manufactured structurally identical versions of HMOs [known as human-identical milk oligosaccharides (HiMOs)] to better replicate the composition of human milk. As 2'-FL and DFL are always found together in human milk, a mixture of these HiMOs (2'-FL/DFL) has been proposed for use in IF and as a food ingredient. Safety assessment of 2'-FL/DFL included conduct of in vitro genotoxicity tests and a subchronic oral toxicity study. In the subchronic study, 2'-FL/DFL (8:1 ratio) was administered to neonatal rats at doses up to 5000 mg/kg body weight (bw)/day, once daily for 90 days, followed by a 4-week recovery period. A concurrent reference control group received 5000 mg/kg bw/day of an oligosaccharide already used in IF (fructooligosaccharide), for direct comparison with the high-dose 2'-FL/DFL group. No evidence of genotoxicity was observed. In the absence of compound-related adverse effects in the 90-day study, 5000 mg/kg bw/day was established as the no-observed-adverse-effect-level. These results support the use of 2'-FL/DFL in IF and as a food ingredient.

A 3-week pre-clinical study of 2'-fucosyllactose in farm piglets.

One of the most abundant oligosaccharides found in human milk is 2'-fucosyllactose, a trisaccharide composed of fucose and lactose, and multiple studies have demonstrated a health benefit to this compound. Recent advances have allowed for the large-scale production of oligosaccharides via fermentation, including 2'-fucosyllactose. A neonatal piglet model was used to evaluate the tolerability of 2'-fucosyllactose, produced through this process, in order to demonstrate the suitability of this compound for human infants under 12 weeks of age. Crossbred farm piglets, at lactation day 2, were assigned to one of four treatment groups receiving a liquid diet containing 0, 200, 500 or 2000 mg/L of 2'-fucosyllactose. The calculated consumption of 2'-fucosyllactose corresponded to dose levels of 29.37, 72.22 and 291.74 mg/kg/day, respectively, in males and 29.30, 74.31, and 298.99 mg/kg/day, respectively in females. Piglets were administered diet for 3 weeks; and there were no test article-related effects on growth and development (clinical observations, body weight and food consumption), clinical pathology parameters (hematology, clinical chemistry, coagulation and urinalysis), or any histopathologic changes. Therefore, dietary exposure to 2'-fucosyllactose at concentrations up to 2000 mg/L was well tolerated by neonatal farm piglets and did not result in adverse health effects or impact piglet growth.

Pre-clinical safety evaluation of the synthetic human milk, nature-identical, oligosaccharide 2'-O-Fucosyllactose (2'FL).

In order to match the composition of human breast milk more closely, it is now possible to supplement commercial infant formula (IF) with synthesised oligosaccharides that are chemically identical to human milk oligosaccharides. The safety data generated on a new human-identical milk oligosaccharide (HiMO), 2'-O-Fucosyllactose (2'FL), are presented. Standard in vitro genotoxicity tests were performed. To investigate the toxicological profile in a model representative of the intended target population, 2'FL was administered via gavage in a juvenile adapted sub-chronic rat study at dose levels of 0, 2000, 5000 and 6000 mg/kgbw/day. Fructooligosaccharide (FOS), currently acknowledged as safe and approved for use in IF, was used as a reference high-dose control at 6000 mg/kgbw/day. 2'FL was non-mutagenic in the in vitro assays. Oral administration up to 5000 mg/kgbw/day to rats over 90 days was not associated with any adverse effects based on clinical observations, body weight gain, food consumption, ophthalmoscopy, clinical pathology, organ weights and histopathology findings. Based on this 90-day study, a No Observed Adverse Effect Level (NOAEL) of 5000 mg/kgbw/day for both male and female rats was established for 2'FL. These findings support the safety of synthetic 2'FL for possible use in infant food.

Membrane lipid-modulated mechanism of action and non-cytotoxicity of novel fungicide aminoglycoside FG08.

A novel aminoglycoside, FG08, that differs from kanamycin B only by a C8 alkyl chain at the 4″-O position, was previously reported. Unlike kanamycin B, FG08 shows broad-spectrum fungicidal but not anti-bacterial activities. To understand its specificity for fungi, the mechanism of action of FG08 was studied using intact cells of the yeast Saccharomyces cerevisiae and small unilamellar membrane vesicles. With exposure to FG08 (30 µg mL(-1)), 8-fold more cells were stained with fluorescein isothiocyanate, cells had 4 to 6-fold higher K(+) efflux rates, and 18-fold more cells were stained with SYTOX Green in comparison to exposure to kanamycin B (30 µg mL(-1)). Yeast mutants with aberrant membrane sphingolipids (no sphingoid base C4 hydroxyl group, truncated very long fatty acid chain, or lacking the terminal phosphorylinositol group of mannosyl-diinositolphosphorylphytoceramide were 4 to 8-fold less susceptible to growth inhibition with FG08 and showed 2 to 10-fold lower SYTOX Green dye uptake rates than did the isogenic wild-type strain. FG08 caused leakage of pre-loaded calcein from 50% of small unilamellar vesicles with glycerophospholipid and sterol compositions that mimic the compositions of fungal plasma membranes. Less than 5 and 10% of vesicles with glycerophospholipid and sterol compositions that mimic bacterial and mammalian cell plasma membranes, respectively, showed calcein leakage. In tetrazolium dye cytotoxicity tests, mammalian cell lines NIH3T3 and C8161.9 showed FG08 toxicity at concentrations that were 10 to 20-fold higher than fungicidal minimal inhibitory concentrations. It is concluded that FG08's growth inhibitory specificity for fungi lie in plasma membrane permeability changes involving mechanisms that are modulated by membrane lipid composition.

Influence of fourth generation poly(propyleneimine) dendrimers on blood cells.

Dendrimers provide many exciting opportunities for potential biomedical applications. However, owing to their positively charged surfaces, poly(propyleneimine) (PPI) dendrimers show toxic and haemolytic activities. One of the methods for masking the peripheral cationic groups is to modify them using carbohydrate residues. In this study, three types of the fourth generation PPI dendrimers-uncoated (PPI-g4), approximately 35% maltotriose (Mal-III)-coated (PPI-g4-OS), and approximately 90% Mal-III-coated (PPI-g4-DS) were investigated by assessing their effects on red blood cell (RBC) haemolysis in samples of pure RBCs, RBCs in the presence of human serum albumin (HSA) or human plasma, and RBCs in whole blood. Lymphocyte proliferation and platelet (PLT) aggregation were also studied in the presence of various concentrations of dendrimers. Although all dendrimers examined affected all the blood cells studied, the unmodified PPI-g4 had the most damaging effect. It caused high RBC haemolysis rates and PLT aggregation and greatly inhibited lymphocyte proliferation. These effects were caused by the cationic surface of this polymer. The modification of PPI-g4 with Mal-III reduced the effect of the dendrimer on all blood cells. The presence of HSA or plasma in the buffer containing the RBCs or RBC in whole blood significantly decreased the extent of dendrimer-driven haemolysis.

Antibacterial to antifungal conversion of neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs into agrofungicides.

Many Actinomycetes aminoglycosides are widely used antibiotics. Although mainly antibacterials, a few known aminoglycosides also inhibit yeasts, protozoans and important crop pathogenic fungal oomycetes. Here we show that attachment of a C8 alkyl chain to ring III of a neamine-based aminoglycoside specifically at the 4″-o position yields a broad-spectrum fungicide (FG08) without the antibacterial properties typical for aminoglycosides. Leaf infection assays and greenhouse studies show that FG08 is capable of suppressing wheat fungal infections by Fusarium graminearum-the causative agent of Fusarium head blight-at concentrations that are minimally phytotoxic. Unlike typical aminoglycoside action of ribosomal protein translation miscoding, FG08's antifungal action involves perturbation of the plasma membrane. This antibacterial to antifungal transformation could pave the way for the development of a new class of aminoglycoside-based fungicides suitable for use in crop disease applications. In addition, this strategy is an example of reviving a clinically obsolete drug by simple chemical modification to yield a new application.

Safety of a novel galacto-oligosaccharide: Genotoxicity and repeated oral dose studies.

A series of safety tests were undertaken on a novel galacto-oligosaccharide (GOS) produced from lactose by a two-step enzymatic process involving Sporobolomyces singularis and Kluyveromyces lactis. Bacterial reverse mutation and chromosomal aberration tests, with or without metabolic activation, were performed. These tests showed no mutagenesis in the Ames assay or in Escherichia coli WP2uvrA, and no chromosomal aberrations in cultured fibroblast cells from Chinese hamster lungs (CHL/IU). Micronuclei were not induced in the reticulocytes of mouse peripheral blood following oral administration of GOS. In a 90-day repeated oral dose toxicity study in rats, GOS was administered at 0, 500, 1000 and 2000 mg/kg to male and female Sprague-Dawley rats. There were no GOS-related changes in clinical signs, body weight, water intake, feed intake, urinalysis, ophthalmology, haematology, blood chemistry, organ weights, gross pathology or histopathology in any of the treatment groups compared to the control group. The no observed adverse effect level (NOAEL) of GOS was at least 2000 mg/kg/day in both males and females.

Structure-toxicity relationship of aminoglycosides: correlation of 2'-amine basicity with acute toxicity in pseudo-disaccharide scaffolds.

A new pseudo-disaccharide NB23 with a 3',4'-methylidene protection was designed and its properties were evaluated in comparison to other two structurally related pseudo-disaccharides. The basicity of the 2'-amine was found to be well correlated to acute toxicity data in mice: the increase in the basicity is associated with the toxicity increase. Based on these data, a new pseudo-trisaccharide NB45 was constructed. NB45 exhibited significant antibacterial activity while at the same time retained low acute toxicity.

Acarbose alone or in combination with ethanol potentiates the hepatotoxicity of carbon tetrachloride and acetaminophen in rats.

Acarbose reduces the absorption of monosaccharides derived from dietary carbohydrates, which play an important role in the metabolism and toxicity of some chemical compounds. We studied the effects of acarbose on the hepatotoxicity of carbon tetrachloride (CCl4) and acetaminophen (AP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 2E1 (CYP2E1). Male Sprague-Dawley rats were kept on a daily ration (20 g) of powdered chow diet containing 0, 20, 40, or 80 mg/100 g of acarbose, with drinking water containing 0% or 10% of ethanol (vol/vol). Three weeks later, the rats were either killed for an in vitro metabolism study or challenged with 0.50 g/kg CCl4 orally or 0. 75 g/kg AP intraperitoneally. The ethanol increased the hepatic microsomal CYP2E1 level and the rate of dimethylnitrosamine (DMN) demethylation. The 40- or 80-mg/100 g acarbose diet, which alone increased the CYP2E1 level and the rate of DMN demethylation, augmented the enzyme induction by ethanol. The 40- or 80-mg/100 g acarbose diet alone potentiated CCl4 and AP hepatotoxicity, as evidenced by significantly increased levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma of rats pretreated with acarbose. Ethanol alone also potentiated the toxicity of both chemicals. When the 40- or 80-mg/100 g acarbose diet was combined with ethanol, the ethanol-induced potentiation of CCl4 and AP hepatotoxicity was augmented. Our study demonstrated that high doses of acarbose, alone or in combination with ethanol, can potentiate CCl4 and AP hepatotoxicity in rats by inducing hepatic CYP2E1.

In vitro cariogenicity of trans-galactosyl-oligosaccharides.

Trans-galactosyl-oligosaccharides (TOS) are a class of oligosaccharides produced by transgalactosylation of lactose. TOS are used as bifidogenic factors in human and animal nutrition. TOS can be present in the oral cavity and form a risk of caries. All oral bacteria tested were able to degrade and ferment both TOS and galactosyllactose (GLL), one of its components. Growth was improved compared with carbohydrate-free media and acid was produced after 24 h incubation of the bacteria with TOS and GLL. Degradation patterns, using HPAEC, showed degradation of most components. GLL was degraded only partially. Rapid acidification was only observed for Streptococcus mutans, resulting in a pH of 5.4 within 30 min. All other strains fermented TOS and GLL only slowly. Plaque formation could not be detected on both substrates. It can be concluded that TOS and GLL present only a small risk of caries formation, unless proven otherwise in animal studies.

Safety profile of acarbose, an alpha-glucosidase inhibitor.

Acarbose, an alpha-glucosidase inhibitor, delays absorption of carbohydrate in the gut, thereby lowering postprandial glucose levels. Safety data on this drug have been gathered in a series of studies on animals and in extensive clinical trials in humans. Although an initial long term feeding study in rats showed an excess of renal tumours at very high dosages of acarbose (up to 300 mg/kg bodyweight daily), further evaluation with similar studies in rats, hamsters, and dogs indicated that the problem was related to carbohydrate malabsorption. With adequate glucose intake and in gavage studies, no difference in tumour incidence between placebo- and acarbose-treated groups was seen. From 1976 to 1989, safety data on acarbose were obtained in approximately 8800 patients in 2 separate groups of clinical trials, the Bayer International Clinical Data Pool and the American phase III trials. Almost all adverse experiences, as reported by 56 to 76% of patients on acarbose vs 32 to 37% of patients on placebo, were related to the digestive system and included diarrhoea, flatulence, bloating and nausea. Most symptoms were of mild to moderate intensity and tended to improve with time. In the American trials a small but significant increase in liver transaminases was seen, 3.8% in acarbose-treated patients vs 0.9% in controls together with a 1% increase in anaemia in the acarbose group. Overall, acarbose was well tolerated and the adverse experience profile was clinically acceptable.

Lysosomal storage of glycogen as a sequel of alpha-glucosidase inhibition by the absorbed deoxynojirimycin derivative emiglitate (BAYo1248). A drug-induced pattern of hepatic glycogen storage mimicking Pompe's disease (glycogenosis type II).

Effects of the two absorbable alpha-glucosidase inhibitors miglitol (BAYm1099) and emiglitate (BAYo1248) on hepatic and muscular glycogen concentrations were investigated in the rat after 3, 7, and 28 days. Both compounds were (orally) administered at very high doses (5-50-500 mg/kg b.wt.). In a second experiment, glycogen storage after oral administration of acarbose (1000 mg/kg b.wt.) was studied after 7 days. In a third protocol, hepatic glycogen concentrations were investigated in the fed rat after 7 days of either inhibitor at the respective highest dosage. In fasted rats, emiglitate induced a significant, dose-dependent increase of hepatic glycogen concentrations, which--at the dose of 500 mg/kg b.wt.--were present after 3, 7, and 28 days, but resulted in a significant increase of the liver weight after 28 days only. Light and electron microscopy proved that the increase in hepatic glycogen was due to lysosomal storage of glycogen only. Emiglitate in the amount of 5 mg/kg b.wt. did not induce significant changes either of glycogen concentrations or at the EM-level. While emiglitate also increased hepatic glycogen at a dosage of 50 mg/kg b.wt., miglitol led to significant storage of hepatic glycogen after 3, 7, or 28 days at the highest dose only. With miglitol (500 mg/kg b.wt.), only insignificant lysosomal storage of glycogen could be detected by electron and light microscopy, and liver weight was essentially unaffected. Both compounds displayed a dose-dependent tendency towards higher glycogen concentrations in the soleus muscle, which was significant with the highest dosage of either inhibitor. At an oral dose of o.i.d. 1000 mg/kg b.wt., the almost unabsorbable alpha-glucosidase inhibitor acarbose induced significantly increased glycogen concentrations both in the liver and in the soleus muscle after 7 days. With respect to an enormous enlargement of the lysosomes (EM) and in the absence of cytoplasmatic alpha-glycogen, this accumulation of glycogen must be attributed to lysosomal storage. In fed rats, all alpha-glucosidase inhibitors investigated significantly decreased postprandial hepatic glycogen concentrations (emiglitate greater than miglitol greater than acarbose), thereby reflecting the modulation of absorption. It is concluded that in the rat acarbose at approximately 1000 x ED50 may penetrate the intestinal mucosa at amounts significant enough to induce lysosomal storage of glycogen. Miglitol may cause some hepatocellular, lysosomal glycogen storage at a dose of 500 mg/kg b.wt., but no glycogen storage could be proven up to 100 x ED50 over 28 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Fusion of storage lysosomes in experimental lipidosis and glycogenosis.

This ultrastructural investigation on renal collecting duct cells and hepatocytes of rats deals with the question of whether or not lipid-storage lysosomes as induced by cationic amphiphilic compounds retain their ability to fuse with autophagosomes/autolysosomes. These were recognized by their glycogen content which was made to persist by means of acarbose, an inhibitor of lysosomal alpha-glucosidase. To induce lipidosis, rats were pretreated for several weeks with chloroquine or chlorphentermine; they then received combined treatment with the lipidosis-inducing drug plus acarbose. In renal collecting duct cells, mixed storage lysosomes displaying the features of both lipidosis and glycogenosis were found to predominate, indicating that fusion between lipid-laden lysosomes and glycogen-containing autophagosomes/autolysosomes was efficient. Hepatocytes also displayed some mixed storage lysosomes; these were, however, regularly accompanied, within a given hepatocyte, by greater numbers of pure lipidosis-related inclusions and pure glycogen vacuoles. This observation indicates that in hepatocytes lipid-storage lysosomes were rather reluctant to fuse, thus displaying a feature of telolysosomes which are no longer capable of participating in cellular digestion.