William J. Waddell

Aspartame: A Safety Evaluation Based on Current Use Levels, Regulations, and Toxicological and Epidemiological Studies

Aspartame is a methyl ester of a dipeptide used as a synthetic nonnutritive sweetener in over 90 countries worldwide in over 6000 products. The purpose of this investigation was to review the scientific literature on the absorption and metabolism, the current consumption levels worldwide, the toxicology, and recent epidemiological studies on aspartame. Current use levels of aspartame, even by high users in special subgroups, remains well below the U.S. Food and Drug Administration and European Food Safety Authority established acceptable daily intake levels of 50 and 40 mg/kg bw/day, respectively. Consumption of large doses of aspartame in a single bolus dose will have an effect on some biochemical parameters, including plasma amino acid levels and brain neurotransmitter levels. The rise in plasma levels of phenylalanine and aspartic acid following administration of aspartame at doses less than or equal to 50 mg/kg bw do not exceed those observed postprandially. Acute, subacute and chronic toxicity studies with aspartame, and its decomposition products, conducted in mice, rats, hamsters and dogs have consistently found no adverse effect of aspartame with doses up to at least 4000 mg/kg bw/day. Critical review of all carcinogenicity studies conducted on aspartame found no credible evidence that aspartame is carcinogenic. The data from the extensive investigations into the possibility of neurotoxic effects of aspartame, in general, do not support the hypothesis that aspartame in the human diet will affect nervous system function, learning or behavior. Epidemiological studies on aspartame include several case-control studies and one well-conducted prospective epidemiological study with a large cohort, in which the consumption of aspartame was measured. The studies provide no evidence to support an association between aspartame and cancer in any tissue. The weight of existing evidence is that aspartame is safe at current levels of consumption as a nonnutritive sweetener.

Tissue and cellular disposition of paraquat in mice

Abstract Male C57B1/6J mice were injected intravenously with [ methyl - 14 C]paraquat dichloride and frozen at 1, 3, 9, and 24 hr for whole-body autoradiography or with [ methyl - 3 H]paraquat dichloride and the lungs were removed at 3, 24, and 48 hr for cellular autoradiography. The methods do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. The whole-body autoradiographs revealed localization of radioactivity at all time intervals in melanin, lung, choroid plexus, muscle, and excretory pathways such as proximal tubules of kidney, urine, liver, gallbladder, and intestinal contents. The radioactivity in lung was much higher in certain discrete, unidentified areas at all time intervals except at 1 hr. The concentration was high in myocardium at 1 and 3 hr. Cellular resolution autoradiography revealed that the radioactivity within the lung was confined almost entirely to cells having the distribution of alveolar type II cells at the three time intervals studied. The radioactivity in these cells was easily washed away indicating that an active transport process was probably involved instead of binding to a cellular constituent. The localization suggested that choline might be antidotal to paraquat toxicity. However, there was not a significant increase in survival of mice given 100 mg/kg choline chloride simultaneously to or following treatment with 50 mg/kg paraquat chloride.

The distribution of [14C]acrylamide in male and pregnant Swiss-Webster mice studied by whole-body autoradiography☆☆☆

Male and 13.5- and 17.5-day pregnant Swiss-Webster mice were administered 120 mg/kg [2,3-14C]acrylamide orally. The male mice were frozen 0.33, 1, 3, 9, 24, 72, and 216 hr later, and the pregnant mice at each gestational period were frozen at 3 and 24 hr. Whole-body autoradiographs from the male mice at early time intervals revealed accumulation of radioactivity in the contents of the gastrointestinal tract, liver, pancreas, testis, brain and gallbladder, and epithelia of oral cavity, esophagus, and bronchi. The distribution appears to be similar in the male and pregnant mice. Absorption from the stomach was virtually complete by 3 hr; renal and hepatic elimination was essentially complete at 24 hr. Radioactivity in the male reproductive tract appeared in the parenchyma of the testis at 1 hr, moved to the seminiferous tubules and head of the epididymis at 9 hr, and by 9 days remained only in the tail of the epididymis and the crypts of the epithelium of the glans penis. This movement parallels that of spermatids. The 13.5-day fetuses were uniformly labeled except for a slightly increased uptake in fetal brain. The distribution of radioactivity in the 17.5-day fetal tissues resembled that in maternal tissues; the remarkable exception was an intense accumulation in fetal skin. This study indicates that acrylamide is efficiently absorbed from the stomach and eliminated by the liver, kidney, and possibly the pancreas. A previously unrecognized affinity of acrylamide or a metabolic product was demonstrated for fetal skin in late gestation and for adult epithelia of oral cavity, esophagus, forestomach, and bronchi. Also, acrylamide or a metabolite appears to bind to spermatids at a specific stage near maturation.

Potential Human Cancer Risks from Exposure to PCBs: A Tale of Two Evaluations

In 1999 the Agency for Toxic Substances and Disease Registry (ATSDR) released a Draft Toxicological Profile for Polychlorinated Biphenyls (PCBs). In reviewing the potential human carcinogenicity of PCBs, ATSDR (1999) concluded that “The weight of evidence does not support a causal association for PCBs and human cancer at this time.” Just 1 year later, in an updated Toxicological Profile for Polychlorinated Biphenyls (PCBs), the conclusions of another analysis (ATSDR, 2000) on whether exposure to PCBs might represent a carcinogenic risk to humans had dramatically changed to “Overall, the human studies provide some evidence that PCBs are carcinogenic” and “some of these studies provide meaningful evidence that PCBs are carcinogenic in humans.” Because this is a substantially different conclusion than that reached only one year previously, it raises a number of questions that must be considered particularly since “weight of evidence” has a precise meaning in the context of evaluating a body of epidemiological data. The present review addresses the additional scientific data that became available between the ATSDR 1999 and 2000 evaluations that was of a magnitude to offset the weight of evidence from numerous epidemiological studies that exposure to PCBs was not causally associated with human cancer to a conclusion only 1 year later that there was now “meaningful evidence” that PCBs posed a carcinogenic risk to humans. Also of interest are the criteria upon which this conclusion is based and the distinction between “weight of evidence” and the newer descriptors of “some evidence” and “meaningful evidence.” However, as shown in this review, only one relevant study was published between the ATSDR 1999 and 2000 evaluations and the results of this study were unequivocally supportive of the 1999 conclusion. Because of the continuing controversy surrounding this issue, in this review, all relevant epidemiological data on PCBs are summarized and subjected to another weight of evidence evaluation. This critical review is based on the most recent guidelines (U.S. EPA, 1999a, 2003) for conducting weight-of-evidence evaluations on a body of epidemiological data. Applying a weight-of-evidence evaluation to the PCB epidemiological studies can only lead to the conclusion that there is no causal relationship between PCB exposure and any form of cancer, thereby confirming the conclusions of ATSDR (1999). Also considered is the methodology and logic used by ATSDR (2000) that resulted in overturning the weight of evidence conclusions concerning the human carcinogenicity of PCBs in ATSDR (1999). This issue may have public health and policy implications. It seems appropriate that unbiased evaluations of a body of data, even of controversial issues such as the potential human carcinogenicity of PCBs, be conducted in a transparent manner following applicable guidelines. The dramatic differences between the conclusions of ATSDR (1999) and ATSDR (2000) do not appear to be consistent with this process.

The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons used as flavor ingredients

This publication is the thirteenth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2600 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic potential.

Critique of dose response in carcinogenesis.

A few landmarks in the development of dose response in toxicology are presented, with an explanation of why dose should only be considered on a logarithmic scale. Examples are shown, illustrating that the current practice of labeling dose-response curves for carcinogenesis as supralinear, linear or sublinear, is meaningless unless the dose-response scales are defined. Since many reports labeling such curves as supralinear, linear, or sublinear are carried out with dose on a linear scale, the scientific significance of the shape of the curve is obscured. Examples of dose-response curves for carcinogenesis from 2-acetylaminofluorene, N-nitrosodiethylamine, aflatoxins, and radium are shown. In addition, more than 500 National Toxicology Program Technical Reports (NTP-TR) on carcinogenicity were examined; from this database, three groups of studies were selected. The first group consisted of those studies in which the lowest dose produced no tumors and the study had a positive dose response. The second group consisted of those studies with three or more doses, with a positive dose response producing tumors, but in which there were no tumors in the control group. The third group of more than 50 studies was from NTP-TR-00 to NTP-TR-52 that had only two data points with a positive dose response. These studies were all evaluated on the Rozman et al. scale, since it conforms to the laws of nature and allows evaluation of all doses. It was observed that virtually all of these NTP-TR carcinogenicity studies show a linear response when dose is on this logarithmic scale; a clear threshold for carcinogenicity is typical for nearly all of these chemicals. An exponential dose-response curve was a better fit for a few, but experimental error could account for this deviation from linearity. It is pointed out that there is strong experimental evidence that the mere presence of DNA adducts does not necessarily lead to tumor production. Hormesis probably applies to carcino-genesis and proof of this will require abandoning the no threshold concept. Experiments showing that cumulative dose is a better metric than daily dose may require reevaluating almost all carcinogenicity studies.

Threshold for carcinogenicity of N-nitrosodiethylamine for esophageal tumors in rats.

In a study on 4080 rats by Peto et al. [Cancer Res. 51 (1991) 6415], esophageal neoplasms induced by N-nitrosodiethylamine (NDEA) were the tumors most clearly demonstrated to have a dose response from administration of the compound to male rats. However, they were unable to predict the shape of the dose-response curve at low doses. These data were reanalyzed in the present study for dose response on a logarithmic scale for dose. In contrast to the conclusion of Peto et al., the reanalysis shows a convincingly sharp threshold at 10(17.1) molecules/kg/day. This unequivocal threshold has substantial implications for risk assessment of chemical carcinogens.

Comparison of human exposures to selected chemicals with thresholds from NTP carcinogenicity studies in rodents

The National Toxicology Program (NTP) Technical Reports online database was reviewed to find chemicals that were reported to show clear evidence of carcinogenicity in the NTP rodent studies and for which data on human exposure could be found. Six representative compounds were selected. Three volatile compounds: ethyl benzene, perchloroethylene, and methylene chloride; two drugs in current use: phenytoin and primidone; and one naturally occurring, widely used, flavor: allyl isothiocyanate, were selected. The carcinogenicity data from each of the NTP Technical Reports were plotted using the Rozman scale to determine the threshold for carcinogenicity from the rodent studies. The human exposures for each chemical were calculated and compared with that threshold. The thresholds for carcinogenicity of the three volatile compounds were several orders of magnitude above the levels present in ambient air in the USA. The Threshold Limit Value (TLV) of the American Conference of Governmental Industrial Hygienists (ACGIH) for these three compounds varied between several orders of magnitude below the carcinogenicity threshold to being at the threshold. The maximum recommended doses of both drugs were at the carcinogenicity threshold. The estimated mean daily human consumption of the natural flavor was less than 100 × below the carcinogenicity threshold. This method of comparison between human exposure and animal carcinogenicity studies is more objective and informative than those in current use.

Inhibition of the localization of urethane in mouse tissues by ethanol

[ethyl-1-14C]Urethane in water or in 12% ethanol was administered orally to male A/JAX mice and 1 hr later the mice were frozen and processed for whole-body autoradiography to identify sites of localization of radioactivity. When the [14C]urethane was administered in water, radioactivity was localized in the liver and bile, the salivary, seromucous and Harderian glands, the bone marrow and pancreas and the stomach and intestinal epithelia. When the labelled urethane was administered in 12% ethanol, localization of radioactivity in each of these sites was almost completely inhibited; radioactivity was still seen within the lumen of the stomach and intestine. Using a defined chemical system, no transesterification was observed between urethane and 12% aqueous [2H6]ethanol at pH 1.5 in 80 min. The inhibition of the localization of radioactivity in the tissues appears to be due most probably to blocking of the metabolism of urethane in tissues. This suggests that ethanol may inhibit the carcinogenicity of urethane in mice.

Autoradiographic disposition of [1-methyl-14C]- and [2-14C]caffeine in mice

Male, C57B1/6J mice received either [1-methyl-14C]caffeine or [2-14C]caffeine via the tail vein at a dose of 0.7 or 11 mg/kg, respectively. At 0.1, 0.33, 1, 3, 9, and 24 hr after treatment, the mice were anesthetized with ether and frozen by immersion in dry ice/hexane. The mice were processed for whole-body autoradiography by the Ullberg technique; this procedure does not allow thawing or contact with solvents. All autoradiographs revealed some retention of radioactivity at early time intervals in the lacrimal glands, seminal vesicle fluid, nasal and olfactory epithelium, and retinal melanocytes. The remaining portion of the animal was densitometrically uniform except for the lower levels noted in the CNS and adipose tissues. Excretion of radioactivity by the liver and kidneys seems to be the major routes of elimination. Localization in the liver at late time intervals was confined principally to the centrilobular region. Late sites of retention, observed only after [1-methyl-14C]caffeine administration, included the pancreas, minor and major salivary glands, splenic red pulp, thymal cortex, bone marrow, and gastrointestinal epithelium. Sites of localization present in both studies included the olfactory epithelium, lacrimal glands, hair follicles, and retinal melanocytes. Further studies are needed to determine whether the localization at these various sites is due to metabolic degradation, active transport, or possibly a specific receptor interaction.