Safety assessment of a solid lipid curcumin particle preparation: In vitro and in vivo genotoxicity studies.

Curcumin, one of the three principal curcuminoids found within turmeric rhizomes, has long been associated with numerous physiologically beneficial effects; however, its efficacy is limited by its inherently low bioavailability. Several novel formulations of curcumin extracts have been prepared in recent years to increase the systemic availability of curcumin; Longvida®, a solid lipid curcumin particle preparation, is one such formulation that has shown enhanced bioavailability compared with standard curcuminoid extracts. As part of a safety assessment of Longvida® for use as a food ingredient, a bacterial reverse mutation test (OECD TG 471) and mammalian cell erythrocyte micronucleus test (OECD TG 474) were conducted to assess its genotoxic potential. In the bacterial reverse mutation test, Longvida® did not induce base-pair or frame-shift mutations at the histidine locus in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537, in the presence or absence of exogenous metabolic activation. Additionally, two gavage doses (24 h apart) of Longvida® to Swiss albino mice at 500, 1000, or 2000-mg/kg body weight/day did not cause structural or numerical chromosomal damage in somatic cells in the mammalian erythrocyte micronucleus test. It was therefore concluded that Longvida® is non-genotoxic.

Neonatal subchronic toxicity and in vitro genotoxicity studies of the human-identical milk oligosaccharide 3-fucosyllactose.

Human milk oligosaccharides, such as 3-fucosyllactose (3-FL), are bioactive components of breast milk associated with benefits for infant growth and development. Structurally identical compounds (human-identical milk oligosaccharides-HiMOs) can be produced using microbial fermentation, allowing their use in infant formula to increase its similarity with human milk. Toxicological studies are required to demonstrate safety of HiMOs and that of any impurities potentially carried over from the manufacturing process. Biotechnologically produced 3-FL was tested for potential genotoxicity (bacterial reverse mutation test and in vitro mammalian micronucleus test) and subchronic toxicity (90-day study with neonatal rats). In the 90-day study, 3-FL was administered by gavage to rats once daily from Day 7 of age, at doses up to 4000 mg/kg body weight (bw)/day (the maximum feasible dose), followed by a 4-week recovery period. Reference controls received 4000 mg/kg bw/day of oligofructose, an ingredient permitted for use in infant formula. Results for the genotoxicity studies were negative. In the 90-day study, there were no adverse effects of 3-FL on any of the parameters measured; thus, the no-observed-adverse-effect level (NOAEL) was 4000 mg/kg bw/day (the highest dose tested). These results support the safety of biotechnologically produced 3-FL for use in infant formula and other foods.

Reproductive and developmental toxicity screening study of an acetone extract of rosemary.

In 2017, JECFA requested reproductive and developmental toxicity studies to finalize an acceptable daily intake for solvent rosemary extracts. Thus, an OECD 421 reproductive/developmental toxicity study was conducted using an acetone rosemary extract that complied with JECFA and EFSA food additive specifications. Rosemary extract was provided to rats at dietary concentrations of 0 (control), 2100, 3600, or 5000 mg/kg, for 14 days before mating, during mating, and thereafter (throughout gestation and up to Lactation Day 13 for females) until necropsy. General toxicity (clinical signs, body weight, food consumption) and reproductive/developmental outcomes (fertility and mating performance, estrous cycles, anogenital distance, thyroid hormones, reproductive organ weights, thyroid histopathology) were assessed. There were no signs of general toxicity and no effects on reproduction; thus, the highest concentration tested (equivalent to mean daily intakes of 316 or 401 mg/kg bw/day [149 or 189 mg/kg bw/day carnosol and carnosic acid] for males and females, respectively) was established as the no-observed-adverse-effect level for general and reproductive toxicity. Dose-related reductions in T4 were observed for Day 13 pups (not seen on Day 4) but were not accompanied by thyroid weight changes or histopathological findings; further investigations are required to determine the biological relevance of these T4 reductions.

Toxicological safety evaluation of pasteurized Akkermansia muciniphila.

Gut microorganisms are vital for many aspects of human health, and the commensal bacterium Akkermansia muciniphila has repeatedly been identified as a key component of intestinal microbiota. Reductions in A. muciniphila abundance are associated with increased prevalence of metabolic disorders such as obesity and type 2 diabetes. It was recently discovered that administration of A. muciniphila has beneficial effects and that these are not diminished, but rather enhanced after pasteurization. Pasteurized A. muciniphila is proposed for use as a food ingredient, and was therefore subjected to a nonclinical safety assessment, comprising genotoxicity assays (bacterial reverse mutation and in vitro mammalian cell micronucleus tests) and a 90-day toxicity study. For the latter, Han Wistar rats were administered with the vehicle or pasteurized A. muciniphila at doses of 75, 375 or 1500 mg/kg body weight/day (equivalent to 4.8 × 109 , 2.4 × 1010 , or 9.6 × 1010 A. muciniphila cells/kg body weight/day) by oral gavage for 90 consecutive days. The study assessed potential effects on clinical observations (including detailed arena observations and a modified Irwin test), body weight, food and water consumption, clinical pathology, organ weights, and macroscopic and microscopic pathology. The results of both in vitro genotoxicity studies were negative. No test item-related adverse effects were observed in the 90-day study; therefore, 1500 mg/kg body weight/day (the highest dose tested, equivalent to 9.6 × 1010 A. muciniphila cells/kg body weight/day) was established as the no-observed-adverse-effect-level. These results support that pasteurized A. muciniphila is safe for use as a food ingredient.

Genotoxicity and subchronic toxicity studies of supercritical carbon dioxide and acetone extracts of rosemary.

Toxicology studies conducted with oil-soluble rosemary extracts to support authorization as a food additive (antioxidant) in the EU include an Ames test using a supercritical carbon dioxide extract (D74), a full 90-day study using D74 and an acetone extract (F62), and an investigative 90-day study with a 28-day recovery period (using D74 only). D74 was non-mutagenic in the Ames test. In the full 90-day study, where rats (20/sex/group) were either provided control diet or diets containing D74 (300, 600, or 2400 mg/kg) or F62 (3800 mg/kg), liver enlargement and hepatocellular hypertrophy were observed. To determine a mode of action and assess the reversibility of the hepatic effects, an investigative 90-day study was conducted using female rats (10/group receiving control diet or diet containing 2400 mg/kg D74). Liver enlargement was fully reversible after 28 days and microsomal enzyme analysis revealed reversible induction of cytochrome P450 enzymes (CYP2A1, CYP2A2, CYP2C11, CYP2E1, and CYP4A), demonstrating that the hepatic effects were adaptive and of no toxicological concern. Therefore, the highest dietary concentrations were established as the NOAELs. The investigative 90-day study NOAEL (providing 64 mg/kg bw/day carnosol and carnosic acid [the primary antioxidant components]) was used to establish a temporary ADI for rosemary extracts.

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.

Subchronic (90-day) toxicity assessment of Somacy-FP100, a lipopolysaccharide-containing fermented wheat flour extract from Pantoea agglomerans.

Pantoea agglomerans is a Gram-negative bacterium that is ubiquitous in the environment, colonizing animals, humans, and numerous plants, including cotton and wheat. A lipopolysaccharide-containing fermented wheat flour extract from P. agglomerans (Somacy-FP100) is proposed for use as a food ingredient for individuals seeking foods for healthy aging. Previously published genotoxicity studies with Somacy-FP100 reported its lack of genotoxicity in vitro, but a subchronic toxicity study has not yet been performed. Therefore, to demonstrate the safety of Somacy-FP100 for use as a food ingredient, a 90-day oral (gavage) toxicity study in rats was conducted. Male and female Han Wistar rats were administered vehicle (control) or Somacy-FP100 at 500, 1500, or 4500 mg/kg body weight/day at a dose volume of 10 mL/kg body weight, for at least 90 days. No test article-related adverse clinical signs or effects on body weight, food consumption, or clinical pathology were observed, and there were no macroscopic or microscopic findings related to the test article. Therefore, 4500 mg/kg body weight/day (the highest dose tested and highest feasible dose) was established as the no-observed-adverse-effect level. This absence of subchronic toxicity, in addition to the previously reported lack of genotoxicity, demonstrates the safety of Somacy-FP100 for use as a food ingredient.

Toxicological safety evaluation of a novel highly bioavailable turmeric extract formulation.

Turmeric (Curcuma longa L.) extracts have a long history of use worldwide, but a major limitation of these extracts is their extremely low oral bioavailability, caused by low absorption, rapid metabolism and rapid excretion following ingestion. Thus, a new highly bioavailable turmeric extract formulation (comprising turmeric extract, acacia gum, sunflower oil and quillaia extract) has been developed and is intended for use as a food ingredient. Safety of this novel extract was evaluated using the standard Tier 1 battery of in vitro genotoxicity tests (bacterial reverse mutation test and an in vitro mammalian cell micronucleus test) followed by repeated-dose 28- and 90-day oral toxicity studies in rats. In the 90-day study, male and female Sprague-Dawley rats were dosed once daily, by oral gavage, either with the vehicle or the test item at 500, 1500 or 3000 mg/kg body weight/day. Clinical examinations were conducted regularly, and body weights and food consumption were recorded weekly throughout the study. At the end of the study, blood samples were analyzed for clinical pathology parameters, before a macroscopic necropsy was conducted and a full list of tissues were examined histopathologically. There was no evidence of genotoxicity in vitro. No test item-related adverse effects were observed in the 28- or 90-day studies; therefore, 3000 mg/kg body weight/day (the maximum feasible dose and highest dose tested in rats) was established as the no-observed-adverse-effect level.

Toxicological safety evaluation of the human-identical milk oligosaccharide 6'-sialyllactose sodium salt.

Human milk oligosaccharides (HMOs) are abundant in breastmilk, but their presence in infant formula is negligible. Sialylated HMOs, such as 6'-sialyllactose, constitute a significant portion of the HMO fraction of human milk and are linked to important biological functions. To produce infant formula that is more comparable with human milk, biosynthesized sialyllactoses known as human-identical milk oligosaccharides (structurally identical counterparts to their respective naturally occurring HMOs in breastmilk) are proposed for use in infant formula and other functional foods for the general population. To support the safety of 6'-sialyllactose sodium salt (6'-SL), a 90-day oral (gavage) toxicity study and in vitro genotoxicity tests were conducted. The 90-day study is the first to be conducted with 6'-SL using neonatal rats (day 7 of age at the start of dosing), thus addressing safety of 6'-SL for consumption by the most sensitive age group (infants). In the 90-day study, neonatal rats received 6'-SL at doses up to 5000 mg/kg body weight (BW)/day and reference controls received 5000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in infant formula) for comparison with the high-dose 6'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. No test item-related adverse effects were observed on any parameter in the 90-day study, thus the high dose (5000 mg/kg BW/day) was established as the no-observed-adverse-effect level. These results confirm that 6'-SL is safe for use in formula milk for infants and in other functional foods for the general population.

Toxicological safety assessment of the human-identical milk oligosaccharide 3'-sialyllactose sodium salt.

Human breastmilk is a mixture of nutrients, hormones and bioactive molecules that are vital for infant growth and development. Infant formula (IF) lacks many of these compounds, most notably human milk oligosaccharides (HMOs), which are abundant in breastmilk but scarce in IF. Sialyllactoses, such as 3'-sialyllactose, constitute a large portion of the HMO fraction. To produce IF that matches breastmilk more closely, biosynthesized human-identical milk oligosaccharides (structurally identical to HMOs) such as 3'-sialyllactose sodium salt (3'-SL) are proposed for use in IF and foods for the general population. The safety assessment of 3'-SL comprised in vitro genotoxicity tests and a 90-day oral (gavage) toxicity study. This is the first 90-day study conducted with 3'-SL using neonatal rats (7 days old at the start of dosing-equivalent age to newborn human infants in terms of central nervous system and reproductive development), demonstrating the safety of 3'-SL for consumption by infants, the most sensitive age group. The neonatal rats received 3'-SL at doses up to 5,000 mg/kg body weight (BW)/day and reference controls received 5,000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in IF) for comparison with the high-dose 3'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. In the absence of any test item-related adverse effects in the 90-day study, the high dose (5,000 mg/kg BW/day) was established as the no-observed-adverse-effect level. This confirms the safety of 3'-SL for use in IF for infants, as well as in functional foods for the general population.

Preclinical safety evaluation of the human-identical milk oligosaccharide lacto-N-tetraose.

Lacto-N-tetraose (LNT) is one of the most abundant oligosaccharides that are endogenously present in human breast milk. To simulate the composition of human breast milk more closely, commercial infant formula can be supplemented with human-identical milk oligosaccharides, which are manufactured structurally identical versions of their naturally occurring counterparts. As part of the safety evaluation of LNT, in vitro genotoxicity tests and a subchronic oral gavage toxicity study (in neonatal Sprague-Dawley rats) were conducted. In the subchronic study, LNT was administered at dose levels of 0, 1,000, 2500 or 4000 mg/kg body weight (bw)/day, once daily for at least 90 days, followed by a 4-week treatment-free period. An identically comprised reference control group received fructooligosaccharides powder (a non-digestible oligosaccharide used in infant formula) at 4000 mg/kg bw/day, to allow for direct comparison against the high-dose LNT group. LNT was non-genotoxic in the in vitro tests. There were no compound-related adverse effects in the 90-day study; therefore, 4000 mg/kg bw/day (the highest feasible dose) was established as the no-observed-adverse-effect-level. These results support the safe use of LNT in infant formula and as a food ingredient, at levels not exceeding those found naturally in human breast milk.

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.