Safety evaluation of pectin-derived acidic oligosaccharides (pAOS): genotoxicity and sub-chronic studies.

Pectin-derived acidic oligosaccharides (pAOS) are non-digestible carbohydrates to be used in infant formulae and medical nutrition. To support its safety, the genotoxic potential of pAOS was evaluated. pAOS was not mutagenic in the Ames test. Positive results were obtained in the chromosome aberration test only at highly cytotoxic concentrations. The effects obtained in the mouse lymphoma test were equivocal; pAOS was not mutagenic in vivo. A sub-chronic dietary study, preceded by 4-week parental and in utero exposure phase, investigated general safety. Administration of pAOS did not affect parental health nor pup characteristics. No effects specific for acidic oligosaccharides were observed in the subsequent sub-chronic study. Slight diffuse hyperplasia of epithelial layer of the urinary bladder was noted to result from concurrently elevated urinary sodium, due to high sodium in pAOS, and elevated urinary pH. This phenomenon was confirmed in a mechanistic (sub-chronic) study. In contrast, in rats fed pAOS in combination with NH(4)Cl, an acidifying agent, the induced low urinary pH completely prevented the development of urothelial hyperplasia. Hyperplasia induced by this mechanism in rats is considered not relevant to man. Based on the current knowledge we consider pAOS safe for human consumption under its intended use.

A sub-chronic (13 weeks) oral toxicity study in rats and an in vitro genotoxicity study with Korean pine nut oil (PinnoThin TG).

In this paper a sub-chronic (13 weeks) toxicity study in rats and an in vitro genotoxicity study with Korean pine (Pinus koraiensis Siebold & Zucc.) nut oil, KPNO (PinnoThin) are described. Both studies were performed in compliance with GLP, and in line with OECD guidelines applicable. In the sub-chronic toxicity study, no clinical signs, abnormalities in functional observation tests or ophthalmologic examinations or changes in body weight or food intake were noted at any of the doses of KPNO tested. Various changes in clinical biochemistry parameters were noted. Whilst these changes were not consistent in both sexes, and neither associated with any histopathological changes, nor dose-related, these were not considered to be toxicologically relevant. No toxicologically significant changes were noted in haematological parameters. There were a few histopathological observations such as a periportal vacuolation of the liver in all dose groups including the control, and renal tubular mineralisation in most females of the high dose group but also in all control female rats. These findings can be considered to be due to the high fat content of the diets, and are not related to the treatment with KPNO. Based on these findings a No Observable Adverse Effect Level (NOAEL) of 15% has been established for KPNO. This NOAEL corresponded to a mean of 8866 and 10,242 mg KPNO/kg bw/day for males and females, respectively. This dose level was the highest achievable oral dose for KPNO in rats. The in vitro reverse mutation test (Ames test), showed no significant dose-related increase in the number of revertants in two independently repeated mutation assays. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on these results it has been concluded that KPNO is not mutagenic in the Escherichia coli and Salmonella typhimurium reverse mutation assays. In conclusion, KPNO can be considered to be non-genotoxic in the AMES test. A NOAEL of 8866 and 10,242 mg KPNO/kg bw/day has been established for male and female rats, respectively. For both sexes, the NOAEL was achieved at the highest dose tested.

Toxins of cyanobacteria.

Blue-green algae are found in lakes, ponds, rivers and brackish waters throughout the world. In case of excessive growth such as bloom formation, these bacteria can produce inherent toxins in quantities causing toxicity in mammals, including humans. These cyanotoxins include cyclic peptides and alkaloids. Among the cyclic peptides are the microcystins and the nodularins. The alkaloids include anatoxin-a, anatoxin-a(S), cylindrospermopsin, saxitoxins (STXs), aplysiatoxins and lyngbyatoxin. Both biological and chemical methods are used to determine cyanotoxins. Bioassays and biochemical assays are nonspecific, so they can only be used as screening methods. HPLC has some good prospects. For the subsequent detection of these toxins different detectors may be used, ranging from simple UV-spectrometry via fluorescence detection to various types of MS. The main problem in the determination of cyanobacterial toxins is the lack of reference materials of all relevant toxins. In general, toxicity data on cyanotoxins are rather scarce. A majority of toxicity data are known to be of microcystin-LR. For nodularins, data from a few animal studies are available. For the alkaloids, limited toxicity data exist for anatoxin-a, cylindrospermopsin and STX. Risk assessment for acute exposure could be relevant for some types of exposure. Nevertheless, no acute reference doses have formally been derived thus far. For STX(s), many countries have established tolerance levels in bivalves, but these limits were set in view of STX(s) as biotoxins, accumulating in marine shellfish. Official regulations for other cyanotoxins have not been established, although some (provisional) guideline values have been derived for microcystins in drinking water by WHO and several countries.

Health implications of exposure to environmental nitrogenous compounds.

All living systems need nitrogen for the production of complex organic molecules, such as proteins, nucleic acids, vitamins, hormones and enzymes. Due to the intense use of synthetic nitrogen fertilisers and livestock manure in modern day agriculture, food (particularly vegetables) and drinking water may contain higher concentrations of nitrate than in the past. The mean intake of nitrate per person in Europe is about 50-140 mg/day and in the US about 40-100 mg/day. In the proximal small intestine, nitrate is rapidly and almost completely absorbed (bioavailability at least 92%). In humans, approximately, 25% of the nitrate ingested is secreted in saliva, where some 20% (about 5-8% of the nitrate intake) is converted to nitrite by commensal bacteria. The nitrite so formed is then absorbed primarily in the small intestine. Nitrate may also be synthesised endogenously from nitric oxide (especially in case of inflammation), which reacts to form nitrite. Normal healthy adults excrete in the urine approximately 62 mg nitrate ion/day from endogenous synthesis. Thus, when nitrate intake is low and there are no additional exogenous sources (e.g. gastrointestinal infections), the endogenous production of nitrate is more important than exogenous sources. Nitrate itself is generally regarded nontoxic. Toxicity is usually the result of the conversion of nitrate into the more toxic nitrite. There are two major toxicological concerns regarding nitrite. First, nitrite may induce methaemoglobinaemia, which can result in tissue hypoxia, and possibly death. Secondly, nitrite may interact with secondary or N-alkyl-amides to form N-nitroso carcinogens. However, epidemiological investigations and human toxicological studies have not shown an unequivocal relationship between nitrate intake and the risk of cancer. The Joint Expert Committee of the Food and Agriculture Organization of the United Nations/World Health Organization (JECFA) and the European Commission's Scientific Committee on Food have set an acceptable daily intake (ADI) for nitrate of 0-3.7 mg nitrate ion/kg bodyweight; this appears to be safe for healthy neonates, children and adults. The same is also true of the US Environmental Protection Agency (EPA) Reference Dose (RfD) for nitrate of 1.6 mg nitrate nitrogen/kg bodyweight per day (equivalent to about 7.0 mg nitrate ion/kg bodyweight per day). This opinion is supported by a recent human volunteer study in which a single dose of nitrite, equivalent to 15-20 times the ADI for nitrate, led to only mild methaemoglobinaemia (up to 12.2%), without other serious adverse effects. The JECFA has proposed an ADI for nitrite of 0-0.07 mg nitrite ion/kg bodyweight and the EPA has set an RfD of 0.1 mg nitrite nitrogen/kg bodyweight per day (equivalent to 0.33 mg nitrite ion/kg bodyweight per day). These values are again supported by human volunteer studies.