Steviol glycoside safety: is the genotoxicity database sufficient?

The safety of steviol glycoside sweeteners has been extensively reviewed in the literature. National and international food safety agencies and approximately 20 expert panels have concluded that steviol glycosides, including the widely used sweeteners stevioside and rebaudioside A, are not genotoxic. However, concern has been expressed in recent publications that steviol glycosides may be mutagenic based on select studies representing a small fraction of the overall database, and it has been suggested that further in vivo genotoxicity studies are required to complete their safety profiles. To address the utility of conducting additional in vivo genotoxicity studies, this review evaluates the specific genotoxicity studies that are the sources of concern, and evaluates the adequacy of the database including more recent genotoxicity data not mentioned in those publications. The current database of in vitro and in vivo studies for steviol glycosides is robust and does not indicate that either stevioside or rebaudioside A are genotoxic. This, combined with a lack of evidence for neoplasm development in rat bioassays, establish the safety of all steviol glycosides with respect to their genotoxic/carcinogenic potential.

The absence of genotoxicity of sucralose.

Sucralose is a non-nutritive sweetener that is approximately 600 times sweeter than table sugar. It is currently approved for use in over 80 countries. Evidence from chronic studies demonstrates that this compound is not carcinogenic. This report summarizes the results of genotoxicity studies that were part of the original safety assessment of sucralose-conducted early in the safety investigation and shared with regulatory agencies around the world. Studies included the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli pol A+/A- test, an in vitro chromosome damage assay in human lymphocytes, mutation in TK +/- mouse lymphoma cells, an in vivo chromosome aberration test in rats and two separate micronucleus tests in mice. All results were evaluated as negative. These results support the overall conclusion by regulatory and heath agencies that sucralose is safe for its intended use.

Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages.

Rebaudioside A is a sweet tasting steviol glycoside extracted and purified from Stevia rebaudiana (Bertoni). Steviol glycosides can currently be used as a food ingredient in only a handful of countries. Questions on specifications, safety and special population effects have prevented steviol glycosides from obtaining a legal status permitting their use as a sweetener in most countries. A set of papers reporting results of research studies and reviews has been compiled in this Supplement to definitively answer unresolved questions. Specifically, recently completed studies on the general and reproductive toxicity of rebaudioside A corroborate studies carried out with purified steviol glycosides demonstrating safety at high dietary intake levels. Comparative metabolism studies provide further affirmation of the common metabolic pathway for all steviol glycosides and the common metabolism between rats and humans. Finally, clinical studies provide further evidence that purified rebaudioside A has no effect on either blood pressure or glucose homeostasis. This paper summarizes the information used to conclude that high purity rebaudioside A (rebiana) produced to food-grade specifications and according to Good Manufacturing Practices is safe for human consumption under its intended conditions of use as a general purpose sweetener.

A critical review of the genetic toxicity of steviol and steviol glycosides.

Extracts of the leaves of the stevia plant (Stevia rebaudiana Bertoni) are used to sweeten food and beverages in South America, Japan and China. The components responsible for the sweet properties of the plant are glycosides of steviol, primary stevioside (ent-13-hydroxykaur-16-en-18-oic acid), which is 250-300 times sweeter than sucrose and rebaudiosides A and C. Stevioside and steviol have been subjected to extensive genetic testing. The majority of the findings show no evidence of genotoxic activity. Neither stevioside nor its aglycone steviol have been shown to react directly with DNA or demonstrate genotoxic damage in assays relevant to human risk. The mutagenic activity of steviol and some of its derivatives, exhibited in strain TM677, was not reproduced in the same bacteria having normal DNA repair processes. The single positive in vivo study measuring single-strand DNA breaks in Wistar rat tissues by stevioside, was not confirmed in experiments in mice and appears to be measuring processes other than direct DNA damage. Neither stevioside nor steviol-induced clastogenic effects at extremely high dose levels in vivo. Application of a Weight-of-Evidence approach to assess the genetic toxicology database concludes that these substances do not pose a risk of genetic damage following human consumption.

Reevaluation of the cancer potency factor of toxaphene: recommendations from a peer review panel.

This reevaluation of the current U.S. EPA cancer potency factor for toxaphene is based upon a review of toxaphene carcinogenesis bioassays in mice conducted by Litton Bionetics (unpublished report, 1978) and the National Cancer Institute (NCI) (Technical Report Series No. 37, conducted by Gulf South Research Institute, 1979). The mechanistic data available for toxaphene, including consideration of the potential of the compound to induce genotoxicity, was examined with an emphasis on whether this information supports a change in the cancer potency factor. If a quantitative dose-response assessment for toxaphene is to be performed, the data from both the NCI and Litton cancer bioassays should be used. Additionally, liver tumor results from female mice, rather than male mice, should be used for estimating potential human cancer risk because the background rate of liver tumors in females is lower and less variable than that exhibited by males. An ED(10) was estimated as the point of departure. The mechanistic data were not sufficient to fully support a margin of exposure approach. Therefore, we believe that applying a linear extrapolation from the ED(10) to the origin is an appropriate means to estimate risk at low doses. This is a highly conservative approach and, when it is applied, we conclude that the current EPA cancer potency factor should be reduced from 1.1 (mg/kg/day)(-1) to 0.1 (mg/kg/day)(-1).

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Genotoxicity of radiofrequency radiation. DNA/Genetox Expert Panel.

During the past several years, concerns have been raised regarding the potential adverse effects of exposures to nonionizing radiation, particularly in the extremely low frequency (ELF) range (50 to 60 MHz) and radiofrequency radiation (RFR) with frequencies ranging from 30 KHz to 30,000 MHz. One focus of concern has been potential DNA interactions. Publications reviewing the genotoxicity of ELF radiation [McCann et al. (1993): Mutat Res 297(1):61-95; Murphy et al. (1993): Mutat Res 296:221-240; NAS (1997)], have been uniform in concluding that the weight of evidence does not indicate any genotoxic risk from exposure to this type of radiation. Concern that RFR may be associated with adverse biological effects [WHO, 1993], including recent allegations that they may be involved in the production of brain tumors in humans [Elmer-Dewit (1993): Time, February 8:42], has resulted in the production of a large number of publications describing the effects of RFR on the integrity of nucleic acids. Data from studies conducted in a frequency range from 800 to 3,000 MHz were reviewed and subjected to a weight-of-evidence evaluation. The evaluation focused on direct toxicological effects of RFR as well as on studies addressing basic biological responses to RFR at the cellular and molecular level. The data from over 100 studies suggest that RFR is not directly mutagenic and that adverse effects from exposure of organisms to high frequencies and high power intensities of RFR are predominantly the result of hyperthermia; however, there may be some subtle indirect effects on the replication and/or transcription of genes under relatively restricted exposure conditions.

Assessment of the genotoxic risk from laxative senna products.

Laxative senna products and several of their specific components have been submitted to a large number of genetic tests. While most studies gave negative responses, results from some of the studies suggest that components of senna products, particularly emodin and aloe-emodin, have genotoxic activity. Assessment of the genotoxicity profile of these substances, in light of other data from animal and human metabolism or kinetic studies, human clinical trials and rodent carcinogenicity studies do not support concerns that senna laxatives pose a genotoxic risk to humans when consumed under prescribed use conditions.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Genetic risk extrapolation from animal data to human disease. A Taskgroup Report. No. 00105.

This report describes a model for producing quantitative genetic risk assessments for human populations. The model is patterned after current methods used in cancer risk analysis. The risk to humans is expressed as the number of additional dominant genetic diseases added to the existing genetic burden, in the offspring of the exposed individuals.

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk.

An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per 10(8) offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings.

An assessment of the genetic toxicity of atrazine: relevance to human health and environmental effects.

The genetic toxicity of atrazine, a member of the s-triazine herbicides, was reviewed with the objective of classifying the chemical. Atrazine has been subjected to a broad range of genetic tests with predominantly negative results. Some publications, specifically those measuring dominant lethality in mice and bone marrow clastogenicity in rodents, reported conflicting results across two or more independent tests. Two approaches were employed to evaluate and interpret the results. The first approach attempts to classify each type of genetic endpoint as positive or negative and resolve test conflicts by critical assessment of the study and detailed data. This is the more traditional "expert judgement" approach to hazard assessment. The second approach employs a computer-assisted weight-of-evidence method of data analysis. This approach does not require resolution of conflicts but uses all data sets to arrive at a classification of hazard. The first approach was able to resolve some conflicts but not all. Use of the "expert judgement" results in an equivocal conclusion and classification. Use of the weight-of-evidence method resulted in a conclusion that atrazine does not pose a mutagenic hazard. The weight-of-evidence scheme is proposed to be a more practical and relevant approach for assessing complex data sets.

Personal thoughts on the future of the Environmental Mutagen Society.

The Environmental Mutagen Society (EMS) was one of the first professional scientific societies organized to respond to an environmental concern. The threat of environmental pollution stimulated the formation of the organization in 1969. The Society's mission was to create a forum for discussion of methods and strategies to deal with mutagenic agents formed and/or released into the environment. During the past 25 years, EMS has provided a forum for innovation and scientific discussions. The Environmental Mutagen Society, and, in particular, its applied role in genetic toxicology, has had a profound positive impact on many disciplines in toxicology and safety assessment (i.e., carcinogenesis and in vitro alternatives.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

Correlation of carcinogenic potency with mouse-skin 32P-postlabeling and muta-Rmouse lac Z- mutation data for DMBA and its K-region sulphur isostere: comparison with activities observed in standard genotoxicity assays.

The genotoxicities in vitro and in vivo of the mouse-skin carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) have been compared with those of its weakly carcinogenic 4,5-sulphur analogue, 6,11-dimethylbenzo[b]naphtho-[2,3-d]thiophene (S-DMBA). The only datasets that correlated with the relative carcinogenicity of these agents to the skin were those conducted using topically exposed mouse skin. Thus, both chemicals induced lacZ- mutations in the skin of lacZ+ transgenic mice, and both produced DNA adducts on mouse-skin DNA as assessed using the 32P-postlabeling technique. In each case, DMBA gave a stronger response than did S-DMBA. In contrast to these responses, only DMBA was active in the mouse bone-marrow micronucleus assay and in the C3H10T1/2 in vitro cell transformation assay. Both chemicals were mutagenic to Salmonella and of approximately equal potency. The molecular geometry of DMBA and S-DMBA are compared, and divergent CASE predictions of activity in the Salmonella assay and skin-painting bioassay are discussed. The importance of conducting predictive genotoxicity assays in systems close to those in which carcinogenicity is to be assessed is emphasized by these data.

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.

Detection of chemical mutagens using Muta Mouse: a transgenic mouse model.

A transgenic mouse strain with a high copy number of rescuable lacZ sequences was evaluated for its effectiveness in detecting lacZ- mutations in selected tissues. Procarbazine, cyclophosphamide, ethylnitrosourea, 7,12-dimethylbenz[a]anthracene (DMBA), acrylamide and chlorambucil were tested following either single or repeated dosing regimens. Bone marrow, liver, skin and testis tissues were selected to assess as target sites for mutation. Bone marrow, liver and testis tissues were examined for mutation following exposures to ethylnitrosourea and chlorambucil. Increased mutant frequencies were found for both chemicals in all three tissues. Bone marrow tissue was examined for mutation following procarbazine, cyclophosphamide and acrylamide exposures, and skin was examined for mutation following dermal application of DMBA. Mutation induction was observed in all cases. The results obtained from this investigation demonstrate the applicability of this transgenic mouse as an effective model to detect and analyze gene mutation in selected organs including germinal tissues. Studies of organotrophic chemical mutagens and carcinogens are possible with this model as are studies of the susceptibility of germinal tissues to mutagen exposures.