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.

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.

Genotoxicity of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™).

The genotoxic potential of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) was evaluated in a battery of genotoxicity tests. The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in 2 separate in vitro chromosomal aberration test in Chinese hamster lung (CHL) fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity of PQQ was noted. In the in vivo micronucleus assay in mice, PQQ at doses up to 2,000 mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. The weak responses in the in vitro test CHL cells were considered of little relevance under conditions of likely human exposure. PQQ disodium was concluded to have no genotoxic activity in vivo.

Genotoxicity and subchronic oral toxicity of L-ornithine monohydrochloride.

L-Ornithine monohydrochloride was evaluated in two in vitro genotoxicity assays and a rat 90-day oral toxicity study. No evidence of genotoxicity was observed in the reverse bacterial mutation assay or the chromosome aberration test at doses of up to 5000 µg/plate or 1686 µg/mL, respectively, both in the presence and absence of metabolic activation. Rats were administered L-ornithine monohydrochloride at dietary concentrations of 0 (basal diet), 1.25%, 2.5%, or 5.0% for 90 days. No changes in body weight, food consumption, ophthalmoscopy, or hematology were observed. Transient increases in water intake and urinary volume, and a decrease in specific gravity were observed in males receiving 5.0% L-ornithine monohydrochloride; however, these were likely attributable to the central role of ornithine in the urea cycle and the consequent increase in urea production. A decrease in serum chloride concentration and an increase in urinary chloride excretion were observed; however, these were likely attributable to administration of the hydrochloride salt of ornithine and were not considered to be of any toxicological significance. No remarkable findings were noted at necropsy. Based on the results of the study, a no-observed-adverse effect level (NOAEL) of 3445 and 3986 mg/kg body weight/day was established for male and female rats.

In utero and acute exposure to benzene: investigation of DNA double-strand breaks and DNA recombination in mice.

Benzene, a ubiquitous pollutant, has been identified as a human leukemogen and early exposure to environmental carcinogens such as benzene has been linked to childhood leukemia. It is known that genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which can initiate DNA recombinational repair mechanisms. In this study we investigated the induction of micronuclei, the formation of gamma-H2A.X as a marker of DNA DSBs, and the induction of somatic DNA recombination events in hematopoietic tissue from pKZ1 transgenic mice exposed acutely or in utero to benzene. Adult male C57Bl/6N mice were treated with a single i.p. injection of benzene, and timed-pregnant females pKZ1 were treated with daily i.p. injections of 200 mg/kg or 400 mg/kg benzene through gestational days 7-15. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice exposed in utero. Immunoblotting techniques did not detect benzene-induced increases in the formation of gamma-H2A.X in bone marrow cells of adult male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells after specific time-point exposures. Finally, no recombination events were detected in adult pKZ1 mouse tissue; however, in post-natal day 9 pups in utero exposure to 400 mg/kg of benzene caused a trend towards increasing recombination frequency although this did not reach statistical significance. These results demonstrate that in utero exposure increases the frequency of micronuclei and DNA recombination events in hematopoietic tissue of fetal and post-natal mice and may be an initiating event in the etiology of childhood leukemias. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias.

Chewing gum consumption in the United States among children, adolescents and adults.

Data regarding chewing gum consumption habits and attitudes were collected for 963 children and adolescents (aged 6-14) and 3150 adolescents and adults (aged 13 years+) in the United States (U.S.) using a dedicated online food frequency questionnaire. A total of 79.6% of children/adolescents reported using chewing gum in the last 3 months, whereas 61.8% of adolescents/adults chewed gum in the previous 6 months. The mean and 90th percentile of consumption among children/adolescents aged 6-14 were 1.95 and 4.71 g day-1, equivalent to 0.75 and 1.78 pieces per day, respectively. Stratification by gender and age brackets revealed that the consumption of chewing gum was more heavily reported in boys compared to girls. Among adolescents/adults aged 13 years and older, the mean and 90th percentile of consumption of chewing gum was estimated to be 2.98 and 7.67 g day-1, or 1.05 and 3.00 pieces per day, respectively. Stratification by gender and by age brackets in this cohort did not reveal any marked patterns although it was noted that there was a decrease in the percent consuming with age (from 87.0 to 34.5%). The most commonly reported chew frequency among all ages was 'two or three times a week' (23.7 to 26.8%). The average and high-level estimates reported herein provide up-to-date estimates of chewing gum consumption in the U.S. Comparisons of the calculated intake values with those reported from the National Health and Nutrition Examination Survey dataset suggests that national nutrition surveys may result in an under-reporting of the percent consumers, but similar estimates for the daily intakes in g day-1.

Temporal patterns of caffeine intake in the United States.

To investigate whether caffeine intake among adolescents and adults in the U.S. varies across the week or throughout the day, data from a 7-day online beverage consumption survey (2010-2011) were analyzed. Mean (206.8-213.0 mg/day) and 90th percentile (437.4-452.6 mg/day) daily caffeine intakes among consumers 13 years and older were relatively constant across the week with no marked difference among weekdays versus weekend days. Percent consumers of caffeinated beverages likewise remained stable across the week. Mean daily caffeine intake for coffee and energy drink consumers 13 years and older was higher than contributions for tea and carbonated soft drink consumers. Caffeinated beverage consumers (13 + yrs) consumed most of their caffeine in the morning (61% versus 21% and 18% in the afternoon and evening) which was driven by coffee. Caffeinated beverage consumption patterns among adolescents (13-17 yrs) - who typically consume less daily caffeine - were more evenly distributed throughout the day. These findings provide insight into U.S. temporal caffeine consumption patterns among specific caffeinated beverage consumers and different age brackets. These data suggest that while caffeine intakes do not vary from day-to-day, mornings generally drive the daily caffeine intake of adults and is predominantly attributed to coffee.

Benzoates intakes from non-alcoholic beverages in Brazil, Canada, Mexico and the United States.

Food consumption data from national dietary surveys were combined with brand-specific-use levels reported by beverage manufacturers to calculate the exposure to benzoic acid and its salts (INS Nos 210-213) from non-alcoholic beverages in Brazil, Canada, Mexico and the United States. These four jurisdictions were identified as having some of the most prevalent use of benzoates in beverages globally. Use levels were weighted according to the brand's market volume share in the respective countries. Benzoates were reported to be used primarily in 'water-based flavoured drinks' (Codex General Standard for Food Additives (GSFA) category 14.1.4). As such, the assessments focused only on intakes from these beverage types. Two different models were established to determine exposure: probabilistic (representing non-brand loyal consumers) and distributional (representing brand-loyal consumers). All reported-use levels were incorporated into both models, including those above the Codex interim maximum benzoate use level (250 mg kg-1). The exception to this was in the brand-loyal models for consumers of regular carbonated soft drinks (brand loyal category) which used (1) the interim maximum use level for beverages with a pH ≤ 3.5 and (2) all reported use levels for beverages pH > 3.5 (up to 438 mg kg-1). The estimated exposure levels using both models were significantly lower than the ADI established for benzoates at the mean level of intake (4-40% ADI) and lower than - or at the ADI only for toddlers/children - at the 95th percentile (23-110% ADI). The results rendered in the models do not indicate a safety concern in these jurisdictions, and as such provide support for maintaining the current Codex interim maximum benzoate level of 250 mg kg-1 in water-based beverages.

Chewing gum intake in Europe: a survey of intakes in France, Germany, Italy, Spain and the UK.

A limited number of studies have examined chewing gum intakes in Europe. In the current study, chewing gum intakes were estimated in children, adolescents and adults via customised online questionnaires developed to collect data on patterns of chewing gum consumption (including usage, duration and gum variety) in September and October 2011. A total sample of chewing gum consumers composed of 1001 children and 5186 adolescents and adults from five European countries was included in the survey, with 79% and 66% of children and adolescents/adults reporting chewing gum in the previous 3 and 6 months, respectively. The intake of chewing gum on a per g basis was estimated by establishing the mean weights of each form of gum, and using factors for the frequency of chewing gum intake obtained through the online survey. Intakes of chewing gum at the mean, 90th and 95th percentile in children were 1.87, 3.79 and 6.59 g day(-1) (equivalent to 0.75, 1.67 and 2.40 pieces/day), respectively. In adolescents and adults, intakes at the mean, 90th and 95th percentile were 1.87, 5.40 and 8.00 g day(-1) (equivalent to 0.98, 3.00 and 4.00 pieces/day), respectively. Intakes were similar across the five countries. Chewing gum use was significantly higher in older compared with younger children and was significantly higher in adolescents compared with older adults. The data reported herein provide a reliable resource for chewing gum intakes in Europe and will be important for estimating exposure to food additives and flavourings intended for use in chewing gum.

Subchronic and reproductive/developmental (screening level) toxicity of complexation products of iron trichloride and sodium tartrate (FemTA).

A complexation/reaction product, termed FemTA, of sodium tartrate [D(-)- and L(+)-tartaric acid and mesotartaric acid], sodium hydroxide, and iron trichloride may have use as an anticaking agent in salt preparations. FemTA is composed of about 4% sodium tartrate, approximately 10% mesotartaric acid, approximately 7% chloride, approximately 4% iron, approximately 7% sodium, approximately 0.3% sodium oxalate, and approximately 65% water. FemTA was tested in a 90-d oral toxicity study, which included a screening level reproductive/developmental toxicity phase, in Harlan Wistar rats. FemTA was administered by oral gavage at 500, 1000, and 2000 mg/kg body weight/d prior to and during mating, or about 20, 40, or 80 mg of iron/kg body weight/d, such that males received 90/91 d of treatment and females 104 to 109 d. Treatment was associated with inflammatory lesions of the lower GI tract at the mid- and high-dose levels, increased liver and kidney weights, increased serum bile acids and blood urea nitrogen, decreased chloride, and changes to hematological parameters consistent with inflammation. The effects were considered the result of iron overload. There were no effects on reproductive/developmental toxicity parameters. The no-observed-adverse-effect level (NOAEL), based on gastrointestinal tract effects was 500 mg/kg body weight/d. The NOAEL for reproductive/developmental toxicity was 2000 mg/kg body weight/d, the highest dose tested.

Subchronic and reproductive toxicity of whole dried Hoodia parviflora aerial parts in the rat.

Hoodia parviflora is being developed commercially for use in weight loss food and dietary supplement products. As part of the safety assessment process for H. parviflora, a freeze dried powder preparation was tested in a 90-day oral toxicity study with reproductive/recovery component in rats. Groups of 10 male and female Sprague-Dawley rats were administered H. parviflora dried powder at doses of 0, 100, 250, and 350 mg/kg body weight/day by gavage for an 11-week pre-mating period and a 14-day co-habitation period, and for females, through lactation day 4. An additional 5 rats/sex/group received 0 or 350 mg/kg bw/day for 90 days and were sacrificed 28 days after cessation of treatment. Statistically significant, non-adverse reductions in body weight, body weight gain, food consumption and food efficiency were observed at 250 and 350 mg/kg/day in females. Food consumption was reduced in high-dose males. There were no adverse effects on hematological, blood biochemical, coagulation or urinalysis parameters or on the results of the functional observational battery and histopathological examinations. No evidence of any effect was noted on reproductive or developmental parameters. The NOAEL for dried H. parviflora powder was 350 mg/kg bw/day, the highest permissible dose tested, for both male and female rats.

Genotoxicity of dried Hoodia parviflora aerial parts.

Hoodia parviflora is being developed commercially for use in weight loss food and dietary supplement products. Its effects are ascribed to a number of glycosides that have been shown to be present in plant extracts from several Hoodia species, the best known of which is H. gordonii. H. parviflora has been identified as an alternative to H. gordonii, and, as part of the process to develop H. parviflora, in vitro genotoxicity tests, as recommended by recent European Food Safety Authority guidance, were conducted on a dried powder preparation of H. parviflora aerial parts. The preparation was tested for reverse mutation at doses up to 5,000µg/plate in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and in Escherichia coli WP2 uvrA TA, both in the presence and in the absence of an exogenous source of metabolic activation (rat liver S9). In addition, the dried powder was evaluated in an in vitro cytotoxicity chromosome aberration assay using human lymphocytes. Test conditions included both a 4 (up to 2500µg/mg) and 44-h exposure period (up to 1000µg/mg) and the incorporation of an exogenous source of metabolic activation (4-h exposure only). H. parviflora dried powder was non-genotoxic in both in vitro assays.