An investigation of the genotoxic potential of a well-characterized yerba mate extract.

Yerba Mate (Ilex paraguariensis) is historically used as a beverage and its extracts are considered traditional medicine in South America. Extract use has been expanding to North American and European markets and the currently available genetic toxicology literature indicate discrepancies in genotoxicity findings for yerba mate. As botanical extract use expands, assumption in safety should be made with caution assuring a good understanding of the test material characterization. Authoritative agencies suggest a two-step paradigm to investigate genotoxicity, and this was implemented to evaluate the safety of yerba mate hydroxycinnamic acid extract. Four OECD compliant assays were employed: bacterial reverse mutation, in vitro micronucleus and a parallel in vivo micronucleus, and comet assay. No evidence of mutagenicity was observed in the in vitro Ames assay, but the results of an in vitro micronucleus study were inconclusive. However, oral gavage treatment of rats for the in vivo micronucleus and comet assays demonstrated negative findings. The results from this battery of tests, supports that this yerba mate hydroxycinnamic acid extract is not anticipated to pose genotoxicity concerns. A high-level comparison of results to other available genotoxicity literature on yerba mate is presented with emphasis on the importance of identity when drawing conclusions on botanicals.

Lack of potential carcinogenicity for steviol glycosides - Systematic evaluation and integration of mechanistic data into the totality of evidence.

Steviol glycosides are present in the leaves of the Stevia rebaudiana plant, have a sweet taste, and have been used as a sweetener for centuries. To build on previous authoritative safety assessments of steviol glycosides, a systematic assessment of mechanistic data related to key characteristics of carcinogens (KCCs) was conducted. Over 900 KCC-relevant endpoints from peer-reviewed literature and high-throughput screening data (ToxCast/Tox21) were identified across individual steviol glycosides and derivatives, metabolites, and whole leaf extracts. Most data (both in vivo and in vitro, including human cells), showed inactivity. Studies were weighted according to quality and relevance. Although data were available for eight of the ten KCC, genotoxicity, oxidative stress, inflammation, and cell proliferation/cell death represent the KCCs with the most data. The data for these KCC primarily show beneficial activity (anti-inflammatory, antioxidant, and anti-proliferative). Following integration across all data, and accounting for study quality and relevance, the totality of the evidence demonstrated an overall lack of genotoxic and carcinogenic activity for steviol glycosides. This is in agreement with previous regulatory decisions, and is consistent with the lack of tumor response in two-year rodent cancer bioassays. The findings support prior conclusions that steviol glycosides are unlikely to be carcinogenic in humans.

Lack of potential carcinogenicity for acesulfame potassium - Systematic evaluation and integration of mechanistic data into the totality of the evidence.

The safety of low- and no-calorie sweeteners remains a topic of general interest. Substantial evidence exists demonstrating a lack of carcinogenicity of the no-calorie sweetener acesulfame potassium (Ace K). The objective of this evaluation was to conduct a systematic assessment of available mechanistic data using a framework that quantitatively integrates proposed key characteristics of carcinogens (KCCs) into the totality of the evidence. Over 800 KCC-relevant endpoints from a variety of in vitro and in vivo assays were assessed for quality, relevance, and activity, and integrated to determine the overall strength of the evidence for plausibility that Ace K acts through the KCC. Overall, there was a lack of activity across the KCCs (overall integrated score <0 and no "strong" categorization for evidence of activity) in which data were identified. Together with the absence of treatment-related tumor effects in rodent bioassays, these results support the conclusion that Ace K is unlikely to induce a carcinogenic response. This assessment employed a weight of the evidence analysis that includes the consideration of factors such as reliability, strength of the model system, activity, and dose in a complex and heterogeneous dataset, and the ultimate integration of multiple data streams in the cancer hazard evaluation.

Lack of potential carcinogenicity for aspartame - Systematic evaluation and integration of mechanistic data into the totality of the evidence.

Despite repeated confirmation of aspartame safety in a variety of foods and beverages, there continues to be interest in researching the potential carcinogenic risk associated with its consumption. The objective of this evaluation was to conduct a systematic assessment of available mechanistic data using a framework for quantitatively integrating the key characteristics of carcinogens (KCCs). For aspartame, 1332 endpoints were appraised for quality and relevance and quantitatively integrated using an algorithm to determine the potential for individual KCC activity based on all available evidence, and subsequently assessed in the context of human and animal evidence streams. An overall lack of activity (integrated scores <0 and no "strong" categorizations) was observed for all KCCs except oxidative stressor (#5), for which activity was determined to be unlikely to be related to a carcinogenic response. Overall, the KCC-based analysis, together with the lack of consistent evidence of carcinogenicity in experimental animals, continue to support lack of carcinogenicity from aspartame consumption. This comprehensive evaluation of available mechanistic data demonstrates the need for a systematic approach to identify and appraise all avaialble data as part of weight-of-evidence determinations related use of KCC in evaluations of potential human carcinogenicity.

Lack of potential carcinogenicity for sucralose - Systematic evaluation and integration of mechanistic data into the totality of the evidence.

Sucralose is widely used as a sugar substitute. Many studies and authoritative reviews have concluded that sucralose is non-carcinogenic, based primarily on animal cancer bioassays and genotoxicity data. To add to the body of knowledge on the potential carcinogenicity of sucralose, a systematic assessment of mechanistic data was conducted. This entailed using a framework developed for the quantitative integration of data related to the proposed key characteristics of carcinogens (KCCs). Data from peer-reviewed literature and the ToxCast/Tox21 database were evaluated using an algorithm that weights data for quality and relevance. The resulting integration demonstrated an overall lack of activity for sucralose across the KCCs, with no "strong" activity observed for any KCC. Almost all data collected demonstrated inactivity, including those conducted in human models. The overall lack of activity in mechanistic data is consistent with findings from animal cancer bioassays. The few instances of activity across the KCC were generally accompanied by limitations in study design in the context of either quality and/or dose and model relevance, highlighted upon integration of the totality of the evidence. The findings from this comprehensive and integrative evaluation of mechanistic data support prior conclusions that sucralose is unlikely to be carcinogenic in humans.

Lack of induction of micronuclei in human peripheral blood lymphocytes treated with hydroquinone.

Hydroquinone (HQ) has been reported to produce chromosomal effects in some in vivo and in vitro animal models. Its potential for inducing similar effects in human lymphocytes is less clear. The purpose of this study was to examine human lymphocytes treated with HQ for the presence of chromosomal anomalies, using an accepted assay for micronuclei. In addition, the stability of HQ in culture medium was determined to verify exposures. Lymphocyte cultures were obtained from eight donors so that variable responses amongst individuals could be assessed. The micronucleus assays utilized were a common 72 h assay with no wash, as well as two assay variations to maximize cell division. Assay variations consisted of either cell washing at 44 h or allowing unwashed cultures an extra 24 h recovery period before harvest. In all assays treatment was at 24 h post-mitogenic stimulation and cytochalasin B was added to stop dividing cells from undergoing cytokinesis. Thus, cells that were scored had undergone one division in the presence of the chemical. Stability results showed that while HQ was detectable in cultures at least for 15 h, it was considerably more stable at 25 than at 100 or 250 microM treatment levels. Results generated using any of the three micronucleus assay variations showed no significant increase in micronuclei in cultures treated with 12.5-200 microM HQ. Colchicine, the positive control and a known spindle disrupter, produced elevated levels of micronuclei. At certain HQ concentrations, a block in cell division was observed, as evidenced by a decrease in percent binucleated cells and replicative index end-points. By varying the assay conditions, cell cultures overcame this block in division and divided at HQ concentrations up to 200 microM, depending on the donor. The reversible block in cell division observed may be a protective response, allowing cells to recover without gross chromosomal damage. This study has substantially expanded the database with regard to the effects of HQ treatment on lymphocytes.

Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5).

MCL-5 cells are Epstein Barr virus-transformed human lymphoblasts which have been genetically engineered for use in mutagenicity testing. We have examined the modal chromosome number, karyotype and spontaneous micronucleus (MN) and sister chromatid exchange (SCE) frequencies of the cell line. Replicate experiments were conducted on two different shipments purchased from Gentest Corp. Although the modal chromosome number was 48 (range 40-54, n = 400 metaphases) for both cell shipments, the second stock showed greater variation in chromosome number than the first. A total of 60 G-banded metaphase cells was analyzed and seven karyotypes were prepared. Consistent structural abnormalities (translocations, deletions and isochromosomes) were found involving the X chromosome and seven autosomes (1-3, 5, 6, 9 and 11). The karyotype typical of this cell line was: 48,der(X)t(X;?)(p22.3;?)Y,t(1;2)(q23;p23),del(3)(q12q21), + i(3q),t(5;6) (q31;p23),+i(9p),der(11)t(11;13)(q23;q12). The mean MN frequency was 41.8 MN/1000 binucleate cells (n = 5000). When compared with our historical controls for primary lymphocyte cultures this number (41.8) is significantly (8.4-fold) higher. The mean SCE frequency was 7.3 per metaphase (n = 100). We observed a hyperdiploid chromosome number of 48 in the majority of metaphase spreads, indicating a significant deviation from the normal diploid number characteristic of the parent cells (RPMI 1788) established in 1969. The variation in chromosome number distribution observed between shipments suggests the potential for further changes. The elevated MN frequency suggests that evaluating mutagenicity using this cytogenetic end-point may require excessive dosing to produce a significant response over background. We conclude that careful interpretation of cytogenetic end-points is necessary when using MCL-5 cells in the light of the possibility of clonal evolution presented here.