A dietary embryo/fetal developmental toxicity study of arruva, an R,R-monatin salt isomer, in Crl:CD(SD) rats.

R,R-Monatin is an intensely sweet substance originally identified in the root bark of Sclerochiton ilicifolius. R,R-Monatin salt, commonly known as "arruva", has potential for use as a high-potency sweetener food ingredient. Previously, arruva was concluded to present no toxicologically relevant effects to Crl:CD(SD) rats and Crl:CD-1(ICR) mice fed up to 35,000 ppm arruva in the diet for 90 days. In the present study, groups of mated Sprague-Dawley rats (25 Crl:CD(SD) females/group) were exposed continuously to 0 (control), 15,000, 30,000, or 50,000 ppm arruva in the diet during gestation days 6-21. There were no fetal malformations or developmental variations that were attributable to arruva at any exposure level, nor were there any test article-related effects on intrauterine survival. Maternal toxicity, evidenced by lower mean body weights, body weight gains and feed efficiency, was observed at 50,000 ppm. A developmental effect, in the form of lower mean fetal body weight, was noted in the 50,000 ppm group in the presence of maternal toxicity. Therefore, the dietary no-observed-adverse-effect level (NOAEL) for maternal and embryo/fetal developmental toxicity of arruva in pregnant rats during gestation days 6-21 was 30,000 ppm (equivalent to 2564 mg/kg bw/day) based on reductions in maternal and fetal body weights.

A 90-day oral (dietary) toxicity study of arruva, an R, R-monatin salt isomer, in Crl:CD-1(ICR) mice.

R,R-Monatin [2R,4R- isomer of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid] is one of four natural constituent isomers in the root bark of Sclerochitin ilicifolius; and "arruva" is the common/usual name that is proposed to represent R,R-monatin salt forms, which have potential use as high potency sweetener food ingredients. In the present study, groups of male and female Crl:CD-1(ICR) mice were exposed to 0 (control), 5000, 10,000, 20,000, or 35,000ppm of arruva in the diet for 90days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower mean body weights and cumulative body weight gains were noted in the 35,000ppm group when compared to the control group. Mean body weights in the 35,000ppm group males and females were 9% and 7% less than the control group, respectively, at week 13. In the absence of observations associated with systemic toxicity and in consideration of the magnitude of body weight difference, these effects were not considered toxicologically significant. Based on the results of this study, the dietary no-observed-adverse-effect level (NOAEL) of arruva for 90days in male and female mice was 35,000ppm (equivalent to an exposure level of 5764 and 8013mg/kg bw/day, respectively).

A 90-day oral (dietary) toxicity study of the 2R,4R-isomer of monatin salt in Sprague-Dawley rats.

The root bark of Sclerochitin ilicifolius contains an intensely sweet substance analytically identified as isomers of 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid and generically coined "monatin." Groups of male and female Crl:CD(SD) rats were fed 0 (control), 5000, 10,000, 20,000 or 35,000 ppm R,R-monatin salt in the diet for 90 days. There were no toxicologically relevant clinical or histopathological findings in any of the test article-treated groups. Significantly lower cumulative body weight gains were noted in the 35,000 ppm group. Mean body weights in the 35,000 ppm group males and females were 7% and 12% lower, respectively, than the control group at study week 13. In the absence of other observations associated with systemic toxicity and lower food consumption, the magnitude of the body weight difference in the 35,000 ppm group females relative to the control group exceeded 10%, which indicated attainment of a maximum tolerated dose (MTD) level. Based on the results of this study, and conservatively assuming the body weight observations at the MTD to be indicative of an adverse effect, the dietary no-observed-adverse-effect level (NOAEL) of R,R-monatin salt for 90 days was 20,000 ppm in female rats (approximately 1544 mg/kg bw/day) and 35,000 ppm in male rats (approximately 2368 mg/kg bw/day).

The use of consumption data to assess exposure to biotechnology-derived foods and the feasibility of identifying effects on human health through post-market monitoring.

The pre-market safety assessment of foods derived through biotechnology provides a scientific basis for concluding reasonable certainty of no harm and ensuring safety. At a minimum, the outcome of such an assessment provides sufficient information to estimate the likelihood of adverse effects on consumers, generally precluding the need for post-market monitoring. Post-market monitoring (PMM) may be appropriate under certain conditions where a better estimate of dietary exposure and/or nutritional consequence of a biotechnology-derived food is required, when a potential safety issue, such as allergenicity, cannot be adequately addressed through pre-market studies, or to corroborate dietary intakes of a nutritionally improved food with beneficial effects on human health. Monitoring programs must be hypothesis-driven, and are dependent upon the availability of accurate consumption data. Exposure assessment methods include both deterministic and probabilistic estimates of intakes using food supply data, individual dietary surveys, household surveys, or total diet studies. In the development of a monitoring approach, resource allocation should be dependent upon both the desired level of conservatism and the endpoint of interest. However, the cost of monitoring varies substantially, and the potential to determine causation may be limited.

Risk assessment of packaging materials.

Risk assessment of packaging materials provides a unique challenge. Human exposure to packaging materials and/or their components occurs from migration into foods. There are various methods for determining migration into foods. Unlike most food additives, these exposures typically are very small. Because of this, and since complete toxicological data sets are not always available for packaging materials, the US Food and Drug Administration (FDA) has developed a process to make the evaluation of packaging materials more efficient, instead of the extensive review normally required for food additives. This process is used to determine 'when the likelihood or extent of migration to food of a substance used in a food-contact article is so trivial as not to require regulation of the substance as a food additive'. This trivial level, also known as the threshold of regulation, was based upon a large database of carcinogenic potencies and was determined to be 1.5 microg/person day(-1). This was determined to 'be low enough to ensure that the public health is protected, even in the event that a substance exempted from regulation as a food additive is later found to be a carcinogen'. Substances not having structural alerts, or that are not known carcinogens or potent toxins, based on existing toxicological information, and are below the threshold value, are considered by the FDA to be exempted from regulation as food additives. The threshold of regulation approach used by the FDA provides an excellent model by which to evaluate the majority of packaging materials.

A sandwich enzyme-linked immunosorbent assay for the detection of almonds in foods.

An enzyme-linked immunosorbent assay was developed to detect almonds as potential allergenic contaminants in food. Polyclonal antibodies directed against roasted almonds were partially purified from immunized sheep and rabbits and used as capture and secondary antibodies, respectively, in a sandwich-type, 96-well plate format. Food samples and almond-spiked samples were extracted 1:10 in phosphate-buffered saline at 60 degrees C for 2 h, centrifuged, and applied to wells coated with sheep anti-almond antibody. After incubation, washing, and the addition of rabbit anti-almond antibody, the amount of almond present was detected with the subsequent addition of goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. Plate absorbances were read at 410 nm, and standard curves were developed in all matrices to quantify unknowns. Antibodies developed were specific for almond; however, some cross-reactivity was observed with extracts of some tree nuts and sesame seeds. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that sheep anti-almond antibody recognized proteins extracted from black walnuts, Brazil nuts, cashews, hazelnuts, macadamia nuts, pistachios, and sesame seeds in addition to those from almond. The assay was optimized to detect less than 1 ppm of almond and was used successfully to determine almond residues in cereal and chocolate without cross-reacting interferences. A retail survey of 20 brands of cereal demonstrated that the assay produced statistically consistent results. This assay provides a useful quality control tool for the food industry for the protection of consumers allergic to almonds.

Identification of sunflower seed IgE-binding proteins.

BACKGROUND: Sunflower seed can cause severe anaphylactic reactions in some susceptible individuals. It is conceivable that the 2S sunflower seed protein is an allergen based on its high degree of homology (34%) with the allergenic mature 2S albumin protein of the Brazil nut. The first step in determining the allergenicity of sunflower seed proteins is to identify IgE-binding proteins. METHODS: Sera from sunflower seed-sensitive individuals were evaluated by radioallergosorbent test (RAST), isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with sunflower seed proteins. RESULTS: Positive RAST scores (>2) were observed in 3 individuals and immunoblotting demonstrated IgE-binding to 2-7 distinct proteins ranging in size from 10 to 50 kD. Two out of 3 sera recognized two proteins between 16 and 17 kD. The lower molecular weight protein (16 kD) approximates to the prepo region of the precursor methionine-rich 2S albumin protein found in sunflower seed (SFA-8/SSA). IEF followed by immunoblotting demonstrated several IgE-binding proteins, including two proteins with isoelectric points of 5.97 and 5.3, respectively, which are consistent with the mature and immature forms of the SFA-8/SSA region. CONCLUSIONS: Sunflower seed contains several IgE-binding proteins, including regions of the high-methionine 2S albumin SFA-8/SSA.

Occurrence of fumonisin B1 and B2 in beer.

A total of 29 nationally distributed brands of beer, representing 25 domestic US and four imported brands, were purchased in retail outlets in Lincoln, Nebraska and analysed for concentrations of fumonisin B1(FB1) and B2(FB2). Immunoaffinity column extraction and cleanup of fumonisins from the beer samples, coupled with detection and analysis by gradient high performance liquid chromatography (HPLC), provided a limit of quantitation for each toxin of 0.3 ng/ml. Of the brands of beer sampled, 86% were positive for FB1 and 41% were positive for FB2. No beer contained a detectable quantity of FB2 without a detectable quantity of FB1. The total fumonisin (FB1 + FB2) content of positive samples ranged from 0.3 to 12.7 ng/ml, with a mean concentration for all positive samples of 4.0 +/- 3.4 ng/ml (n = 25). Considering that the level of fumonisin contamination of corn in recent harvest years has been minimal, the results of this limited survey could represent levels associated with current agricultural and brewing practices.

The use of the chicken embryo screening test and brine shrimp (Artemia salina) bioassays to assess the toxicity of fumonisin B1 mycotoxin.

Chicken embryos and brine shrimp naulpii were utilized in short-term toxicity bioassays to assess their sensitivity to the mycotoxin fumonisin B1 (FB1). Fertile chicken eggs (Cobb x) were dosed with FB1 on day 2 of incubation by the injection of 100 microliters of aqueous solution into the air space of each egg. Eggs were incubated with mechanical rotation until hatch, at which time mortality was assessed. Probit transformation of the mortality data produced a linear line of best fit (P < 0.05), from which an LD50 of 52 micrograms FB1/egg, equivalent to a concentration of 1.3 microns hatched in artificial seawater and exposed to FB1 in an optimized 96-well plate assay with a 48 hr mortality endpoint. Probit transformation of the mortality data resulted in an LC50 of 1.7 microns FB1, or 1.2 micrograms FB1/ml. Thus, at the cellular level, both bioassays appeared sensitive to FB1; however, from the standpoint of use as a screening assay, the chicken embryo bioassay is limited by the relatively high dose of FB1 required per egg. It is anticipated that the design and simplicity of the brine shrimp bioassay will accommodate screening for FB1 toxicity in contaminated samples.

Effect of thermal processing on the stability of fumonisins.

Fumonisins, a group of mycotoxins produced by Fusarium moniliforme in corn, have been implicated in several animal and human diseases. F. moniliforme and the fumonisins are an area of incresing concern for corn producers and consumers. Consequently, there is interest in reducing human and animal exposure to these fungal toxins. Studies of the effects of biological, chemical, and physical treatments on the reduction of fumonisin levels in food have shown variable results. Work was conducted at the U.S. Food and Drug Administration, National Center for Food Safety and Technology, to determine the effects of thermal processing on fumonisins B1 (FB1) and B2 (FB2) in an aqueous buffer. Parameters that were studied included processing time (0-60 min), processing temperature (100-235 degrees C), and buffer pH (4, 7, and 10). The rate and extent of fumonisin decomposition increased with processing temperature. Less than 27% of FB1 and less than 20% of FB2 were lost when processing temperatures were less than or equal to 125 degrees C for 60 min. After 60 min at 150 degrees C, losses of FB1 and FB2 were 80-90% at pH 4, 18-30% at pH 7, and 40-52% at pH 10. At temperatures greater than or equal to 175 degrees C, more than 80% of FB1 and FB2 was lost after 60 min. These results indicate that foods reaching temperatures greater than 150 degrees C during processing may have lower fumonisin levels. More work is needed to quantitate the effects of different processing operations (baking, extrusion, frying) on the fumonisin content of corn-based foods.