The potential developmental neurotoxicity of calcium cyclamate in CD rats.

The developmental neurotoxicity of calcium cyclamate was evaluated in Sprague Dawley [Crl:CD(SD)] rats, administered in drinking water, in comparison to a concurrent control group (water) and a positive control group given propylthiouracil (PTU). Calcium cyclamate was administered to F0 females for 4 weeks prior to pairing, throughout mating, gestation and lactation and to F1 offspring from weaning to 12 weeks of age, PTU was administered by gavage to F0 females from Day 6 of gestation up to Day 20 of lactation. Target calcium cyclamate doses were 0, 250, 500 and 1,000 mg/kg bw/day, while the PTU dose was 0.5 mg/kg bw/day. No treatment-related effects of cyclamate were observed in either the F0 or F1 generations on reproductive performance or neurobehavioral development. In comparison, PTU exposure resulted in developmental delays, memory impairment and a number of neuropathological and morphometric outcomes. The results from the unique developmental neurotoxicity study design, corroborate the absence of hyperactivity and any other neurotoxic effects following cyclamate administration at levels up to 878 mg/kg bw/day in F0 females and 784 mg/kg bw/day in F1 animals. This demonstrates the suitability of PTU as a positive control and confirms the safe use of cyclamate as a no-calorie sweetener.

Toxicity of food sweetener-sodium cyclamate on osteoblasts cells.

In this paper, the effect of commonly used food sweetener (sodium cyclamate) on the proliferation and differentiation of osteoblasts has been researched. The morophology change of osteoblasts was investigated by confocal laser scanning microscopy. Cell viability was studied by MTT analysis. BMP2 expression was analyzed by western blot and immunofluorescence. Mineralization ability of osteoblasts was researched by using alizarin red staining method. The results indicate that a very low concentration (0.06 µM) of sodium cyclamate can curle and fold microfilament and microtubule of osteoblasts. The increase addition of sodium cyclamate resulted significantly decrease of cells viability. The expression of bone morphogenetic protein-2 (BMP2) was seriously suppressed by sodium cyclamate. Alizarin Red staining experiment revealed that sodium cyclamate decreased the mineralization ability of osteoblasts. The present results suggest that sodium cyclamate can seriously inhibit the proliferation and differentiation of osteoblasts.

The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells.

CONTEXT: Artificial sweeteners (AS) have been associated with tumor development (including colon cancer) in both animals and humans although evidence has been conflicting. OBJECTIVES: Additional research was thus conducted by studying the effects of 5 AS on the morphology, cell proliferation and DNA in cells by utilizing Caco-2, HT-29 (colon) and HEK-293 (kidney) cell lines. MATERIALS AND METHODS: Cells were exposed to sodium cyclamate, sodium saccharin, sucralose and acesulfame-K (0-50 mM) and aspartame (0-35 mM) over 24, 48 and 72 hours. Morphological changes were presented photographically and % cell viability was determined by using the MTT cell viability assay. Possible DNA damage (comet assay) induced by the AS (0.1, 1 and 10 mM, treated for 24, 48 and 72 hours) was studied. The appearance of "comets" was scored from no damage to severe damage (0-4). RESULTS: Cells became flatter and less well defined at higher AS concentrations (>10 mM). At concentrations >10 mM, decreased cell viability was noted with both increasing concentration and increasing incubation time for all cell lines tested. In general, HEK-293 cells seemed to be less affected then the colon cancer cells. Sucralose and sodium saccharin seemed to elicit the greatest degree of DNA fragmentation of all the sweeteners tested in all the cell lines used. DISCUSSION: Morphological cell alterations, cell viability and DNA fragmentation seemed to be more in the colon cancer cells. CONCLUSIONS: Further studies have to be performed to clarify mechanisms involved causing these alterations in mammalian cells.

Effects of sodium cyclamate in kidneys of rats fetuses: a morphometric study

El ciclamato es usado como edulcorante no calórico en muchos alimentos y bebidas y es 30 veces mas dulce que la sacarosa, sin el gusto amargo de la sacarina. Aparecen en su composición, los productos como ciclamato de sodio, ciclamato de calcio y acido ciclamico. El objetivo del trabajo fue evaluar los efectos del ciclamato de sodio en ri¤ones de fetos de ratas, considerandose las alteraciones morfometricas en glomerulo, túbulos contornados proximal y distal y conducto coletor. Fueron utilizadas 10 ratas adultas (Rattus norvegicus) variedad Wistar, con peso medio de 238 g, 5 ratas para el grupo control y 5 ratas tratadas con ciclamato de sodio. Entre el 10 y 14 dia de la pre¤ez, 5 ratas recibieron una inyección diaria intraperitoneal de 60mg/Kg/dia de ciclamato de sodio durante 5 dias. En el 20 dia, los animales fueron sacrificados y los fetos fijados en solución de Alfac, incluidos en parafina, cortados y te¤idos com H-E. El método utilizado fue la morfometría por la técnica cariométrica. Hubo disminución significativa en los pesos de los fetos y de la placenta en el grupo tratado con ciclamato de sodio (p= 0,004) comparado con el grupo control. En el volumen glomerular y tama¤o nuclear de las celulas de los túbulos contornados proximal, distal y conducto colector del ri¤ón fetal de las ratas tratadas con ciclamato de sodio, hubo aumento estadisticamente significativo. Los resultados mostraron que el uso del ciclamato de sodio produjo la reducción del peso de los fetos, placenta y longitud del cordón umbilical. Hubo aumento significativo en el volumen glomerular y en el tama¤o nuclear de las células de los túbulos contornados proximal, distal y conducto colector, sugerente de nefrotoxicidad.

Effects of a saccharin and cyclamate mixture on rat embryos.

Sodium saccharin (NaS) and calcium cyclamate (CaC) are artificial sweeteners widely used in food and drink. To evaluate their toxicological effects on preimplantation mammalian embryos, pregnant rats were gavaged with 1.65 mg NaS/kg bw + 3.85 mg CaC/kg bw (DI) or 6.6 mg NaS/kg bw + 15.4 mg CaC/kg bw (D2) on days 1, 2, 3 and 4 of pregnancy (positive vaginal smear = day 1). The female rats were killed on day 5 of the pregnancy (GD 5), maternal organs weighed, and the blastocysts collected, counted and evaluated for gross morphology, cell number and mitotic index. There was no alteration in maternal organ weights, but there was an increase of the cell number/embryo in the dams treated with that NaS + CaC mixtures (D1 = 37.20 +/- 7.96; D2 = 37.26 +/- 10.90) compared to control group (32.24 +/- 6.73). Embryos whose dams were exposed to NaS + CaC may have adapted for implantation into the uterus but more studies are needed to demonstrate this mechanism of action.

Long-term toxicity and carcinogenicity study of cyclamate in nonhuman primates.

Twenty-one monkeys (cynomolgus, rhesus, African green) were fed cyclamate (100 mg/kg and 500 mg/kg) in the diet five times per week from a few days after birth and continuing for up to 24 years. Malignant tumors were diagnosed in three 24-year-old cyclamate monkeys; these were metastatic colon carcinoma (rhesus; 500 mg/kg), metastatic hepatocellular carcinoma (cynomolgus; 500 mg/kg), and a small, well differentiated adenocarcinoma of the prostate (cynomolgus; 100 mg/kg). Benign tumors were found at necropsy in three females; these were adenoma of the thyroid gland (rhesus; 100 mg/kg) and two cases of leiomyoma of the uterus (rhesus; 100 mg/kg and 500 mg/kg). No tumors were detected in an age-matched control group of 16 monkeys. Examination of the testes revealed complete testicular atrophy in one of the old cyclamate monkeys, and focal germ cell aplasia (Sertoli-only tubules) in two other cyclamate monkeys. Focal spermatogenic interruption (maturation arrest) at various germ cell levels mixed with normal spermatogenesis was observed in both the cyclamate-treated and the control monkeys, all of which were over 20 years old. Measurements of terminal cyclohexylamine concentrations showed that three of the males dosed with cyclamate at 500 mg/kg were high converters, with plasma concentrations comparable to the levels that produce testicular atrophy in rats. However, only one of the three high converters showed histologic evidence of irregular spermatogenesis. The overall conclusion is that the testicular abnormalities and the sporadic cases of different malignancies found after more than 20 years of dosing do not provide clear evidence of a toxic or carcinogenic effect of sodium cyclamate in monkeys.

[Changes in cytomembrane and surface cells of the large intestine resulting from the action of sweetening agents]. / Alteraciones en la citomembrana y células de superficie del intestino grueso por acción de los edulcorantes.

Saccharin and cyclamates have been proved to cause organic damage. This work attempt to describe drug-induced changes brought about by drugs in 1/1000 saccharin and cyclamate-fed rats for 90 days. The ultrastructural findings show: microvilli hypertrophy; membranous mitochondrial increase in absorptive cells; and secretion changes in calciform cells. Such changes are cell-response phenomena to interference or mutagenic action on nuclear DNA or on cytoplasmatic metabolism.

Alteraciones en la citomembrana y celulas de superficie del intestino grueso por accion de los edulcorantes / Changes of cytomembrane and surface cells of the large intestine by sweetening agents action

Se ha demostrado que la sacarina y los ciclamatos provocan lesiones en el organismo. Describimos en este trabajo las alteraciones producidas por estas drogas en ratones alimentados con una solución de sacarina y ciclamato al 1/1000, durante 90 días. El estudio ultraestructural revela: Hipertrofia microvellositaria e incremento membranoso mitocondrial en las células absortivas y alteraciones en la secreción de células calciformes. Estas modificaciones son fenómenos de respuesta celular por interferencia o acción mutágena sobre el ADN nuclear o metabolismo citoplasmático. Los autores agradecen a la señora Carolina S. de Santolaya por el apoyo técnico realizado. Esta investigación fue apoyada financieramente por el Consejo de Investigación de la Unviersidad Nacional de Tucumán

Alteraciones en la citomembrana y celulas de superficie del intestino grueso por accion de los edulcorantes / Changes of cytomembrane and surface cells of the large intestine by sweetening agents action

Se ha demostrado que la sacarina y los ciclamatos provocan lesiones en el organismo. Describimos en este trabajo las alteraciones producidas por estas drogas en ratones alimentados con una solución de sacarina y ciclamato al 1/1000, durante 90 días. El estudio ultraestructural revela: Hipertrofia microvellositaria e incremento membranoso mitocondrial en las células absortivas y alteraciones en la secreción de células calciformes. Estas modificaciones son fenómenos de respuesta celular por interferencia o acción mutágena sobre el ADN nuclear o metabolismo citoplasmático. Los autores agradecen a la señora Carolina S. de Santolaya por el apoyo técnico realizado. Esta investigación fue apoyada financieramente por el Consejo de Investigación de la Unviersidad Nacional de Tucumán (AU)

Fetal mouse salivary glands and applications for determining teratogenic mechanisms in vitro.

Culture systems offer advantages for studying the actions of chemicals on mechanisms of development. Growth, [3H]thymidine incorporation, and 14C-amino acid incorporation were measured to determine culture conditions and the response of glands to three chemicals. Eagle's MEM supported the best growth and the best time for double isotope presence was the second 24 h of culture. Cyclamate had no effect on growth or isotope incorporation but cycloheximide disturbed 14C more than 3H. 5-Azacytidine was toxic but the incorporation of both isotopes exhibited the same pattern.

Assessment of the carcinogenicity of the nonnutritive sweetener cyclamate.

The weight of the evidence from metabolic studies, short-term tests, animal bioassays, and epidemiological studies indicates that cyclamate (CHS) is not carcinogenic by itself; however, there is evidence from in vitro and in vivo studies in animals that implies it may have cancer-promoting or cocarcinogenic activity. Epidemiological studies indicate that the use of nonnutritive sweeteners (CHS and saccharin) has not resulted in a measurable overall increase in the risk of bladder cancer in individuals who have ever used these products. No epidemiological information exists on the possible associations of these sweeteners and cancers other than those of the urinary tract. It is recommended that (1) no further studies on the metabolism of CHS to evaluate its carcinogenicity are required since no potentially hazardous metabolites have been appreciably detected in humans; (2) no further animal bioassays to test for the carcinogenicity of CHS by itself are necessary; (3) the studies in rodents that suggest a promotional or cocarcinogenic effect of CHS should be repeated because they cannot be ruled out; (4) because the significance to human health of a positive outcome of such studies is uncertain, additional research aimed at understanding the predictive value for human health of such results and more generic studies to develop well-validated systems that can be relied on in the assessment of cancer-promoting agents are recommended; (5) in populations where CHS continues to be used, epidemiological monitoring should be continued to determine whether there is an increased risk of cancer in humans who are heavy or long-term users or for those observed long after first exposure. In such monitoring, other cancer sites--in addition to the bladder--should be considered.

Acceptable daily intake and the regulation of intense sweeteners.

At the present time there are four intense sweeteners that are available in a number of countries: acesulfame-K, aspartame, cyclamate and saccharin. Extensive toxicity databases are available on each sweetener and these have been assessed by both national and international regulatory authorities. This review considers briefly the critical toxicity of each sweetener that is the basis for establishing the no adverse effect level in animal studies. The calculation of an acceptable daily intake (ADI) for human intake employs a large safety factor applied to the no-effect level. The magnitude of the safety factor for each sweetener is discussed in relation to the ADI values recommended by the Scientific Committee for Food in 1985.

The pharmacokinetics and tissue concentrations of cyclohexylamine in rats and mice.

Cyclohexylamine showed dose-dependent kinetics after administration of single oral doses up to 500 mg/kg in rats, with a reduction in plasma clearance, an increase in apparent half-life, and an increased area under the testicular concentration-time curve. Cyclohexylamine was absorbed and eliminated more rapidly by mice. Saturation of cyclohexylamine uptake by rat renal cortical slices in vitro and of renal tubular secretion in vivo occurred at concentrations and doses comparable to the oral dose studies. During chronic dietary administration the concentrations of cyclohexylamine in the plasma and testes showed a pronounced diurnal variation in rats which was not detected in mice. The steady-state plasma clearance in rats was approximately one-half that in mice. The concentrations of cyclohexylamine in the plasma and testes of rats, but not mice, showed a nonlinear relationship to dietary intake. Elevated concentrations were found at intakes greater than 200 mg/kg/day. The pharmacokinetics of cyclohexylamine make an important contribution to the difference in sensitivity to testicular atrophy in rats and mice and the dose-response relationship for this toxicity in rats.

Assessment of the genotoxicity of calcium cyclamate and cyclohexylamine.

Calcium cyclamate and its major metabolite cyclohexylamine have been subjected to numerous evaluations for genetic activity. With the exception of studies for chromosome damage, the results have been negative. Results from a wide range of in vitro and in vivo cytogenetic assays ranged from clearly negative to various degrees of clastogenicity. Interpretation of the cytogenetic studies has been complicated by the conflicting responses, although some of the positive effects seem to be the consequence of secondary effects produced by high ion levels and excessive toxicity. In the studies presented here calcium cyclamate and cyclohexylamine were tested for mutagenic activity using an in vitro mammalian cells assay for gene mutation and an in vitro unscheduled DNA synthesis assay in rat hepatocytes with the Drosophila sex-linked recessive lethal assay. Calcium cyclamate was not genetically active in any of the three assays when tested to the maximum possible concentrations. The compound was largely nontoxic but did show some evidence of cytotoxicity in rat hepatocytes at concentrations of 1 mg/ml and higher. Cyclohexylamine was also negative in the three assays, but was considerably more cytotoxic at the concentrations used. The results from the three studies conducted in this evaluation are in general agreement with the majority of published genetic toxicology data for these two chemicals and indicated that the calcium cyclamate and cyclohexylamine have no direct, intrinsic genotoxicity of the type measured by these assays.

No evidence found for induction of dominant lethal mutations and heritable translocations in male mice by calcium cyclamate.

Calcium cyclamate, an artificial sweetener, was studied for its effectiveness in inducing transmissible chromosomal aberrations in germ cells of male mice. Both the dominant-lethal and the heritable translocation tests were carried out following daily treatment (on weekdays) of males by oral intubation with the maximum tolerated dose for 6 weeks. Calcium cyclamate is negative in both tests; therefore, there is no evidence of induced chromosome breakage and exchange.

Myocardial effects of calcium cyclamate in the BIO F1D Alexander Syrian golden hamster.

In a subchronic 90-day oral toxicity study, 5 groups (20 males, 20 females each) of F2-descendants of BIO F1D Alexander hamsters received 0, 275, 550, 1,100 and 2,200 mg/kg b.w./day doses, respectively, of calcium cyclamate in the drinking water. These doses caused no adverse health effects in any treated animal and the multifocal myocardial calcifications, detectable at a 100% incidence in the male and female control hamsters (20 males, 20 females), were not exacerbated by the sweetener. Instead, the myocardial lesions exhibited a striking negative dose-response relationship with a 0% incidence in the males and a 5% incidence in the female hamsters of the highest dose group.

Effects of treatment with N-methyl-N-nitrosourea, artificial sweeteners, and cyclophosphamide on adult rat urinary bladder in vitro.

The effect of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and cyclophosphamide on rat bladder explants in vitro was studied. MNU administered as a single dose or in multiple treatments induced concentration-dependent changes in urothelial ultrastructure and cell surface topography. In a single treatment protocol, extensive cytotoxicity was observed in both the urothelium and stroma at concentrations of 500 to 1000 micrograms/ml, establishing a toxic threshold within this range. In a multiple treatment protocol, repeated doses of low concentrations of carcinogen (7 or 8 x 50 micrograms/ml, 6 x 100 micrograms/ml) induced hyperplastic and dysplastic changes in the urothelium with no cytotoxicity, but cytotoxic effects were observed following treatments of 4 x 200 micrograms/ml or 2 x 400 micrograms/ml. Sodium saccharin, sodium cyclamate, and cyclophosphamide induced changes in urothelial cell surface topography consistent with hyperplasia and preneoplasia. Prolonged exposure to saccharin or cyclamate followed by a single dose of MNU elicited more extensive abnormalities in the urothelium than either saccharin or cyclamate alone, suggesting that these artificial sweeteners have initiating activity in a multistage process. The ultrastructural changes induced by in vitro treatment showed a good correlation with the pathological changes observed in vivo in rats treated with MNU or fed either with saccharin or cyclamate.