6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide, potassium salt

Endpoint summary

Additional information

Acesulfame potassium was studied in a series of genotoxicity studies using different test systems. In vitro as well as in vivo test systems were used.

Salmonella - liver microsome tests (Ames-tests)

Acesulfame potassium was tested in Salmonella typhimurium strains using levels of 0; 4; 20; 100; 500; 2500 and 5000 µg per plate with and without metabolic activation. The number of revertant colonies of all concentrations of Acesulfame potassium was in the same range as the corresponding negative controls. No significant increase and no dose dependent effect in the number of revertant colonies were obtained with any of the tester strains either in the absence or in the presence of the liver homogenate activation system.

 

Point mutation assay in Escherichia coli

The study was performed using the method described by Green with the tester strain E. coli WP2uvrA and dosage levels of 0; 4; 20; 100; 500; 2500 and 5000 µg per plate. No significant increase in the number of colonies was observed at any dose level of Acesulfame potassium.

 

Mammalian cell mutagenesis in vitro

The ability of Acesulfame potassium to induce mutations was examined in V79 Chinese hamster cells using the change from 8-azaguanine susceptibility to resistance as the marker.

Eleven different concentrations ranging from 10 µg/ml to 10000 µg/ml were studied. Acesulfame potassium did not cause an increase in 8-azaguanine-resistant colonies.

 

Malignant transformation in vitro

The M2 clone of mouse fibroblasts was used to determine the ability of Acesulfame potassium to induce malignant transformation. Concentrations ranging from 10 µg/ml up to 10000 µg/ml were studied. No signs of induced malignant transformation were seen, although reduced plating efficiency indicating cytotoxicity was observed at 10000 µg/ml and 5000 µg/ml.

 

Unscheduled DNA synthesis in primary rat hepatocytes

Acesulfame potassium was tested for unscheduled DNA synthesis in primary rat hepatocytes obtained from adult male Fischer 344 rats.

Owing to the fact that the concentration of 10000 µg/ml proved to be cytotoxic to the cells, eight concentrations in the range of 25 µg/ml to 5000 µg/ml were used in the test. None of the defined minimum criteria indicating unscheduled DNA synthesis was seen in any test concentration. The degree of nuclear labelling of the treatment groups remained even in the same range as the labelling of the negative control.

 

In vivo binding to DNA

In vivo binding of Acesulfame potassium to DNA was studied in Sprague-Dawley rats. The rats were pre-treated with a diet containing 3.0 % Acesulfame potassium for 7 days. 10 mg of the active compound was administered together with 240 mg inactive Acesulfame potassium as a single oral dose by gavage.

 

The covalent binding index as determined for highly purified DNA and chromatin protein obtained from liver and spleen was by magnitudes below values obtained for known mutagens and compounds of known genotoxicity. The data gave no indication of a covalent binding potential of Acesulfame potassium.

 

In vivo micronucleus test

A micronucleus test was performed in 10 male and 10 female NMRI mice. The animals received a volume of 20 ml/kg bw of an Acesulfame potassium solution at dose levels of 0, 450, 1500, and 4500 mg/kg bw. The doses were administered twice in an interval of 24 h by oral gavage.

 

No increase in the number of polychromatic erythrocytes with micronuclei compared to the number of micronuclei in control animals was found.

 

In vivo cytogenicity studies:

There were no signs of chromosome aberrations after administration of Acesulfame potassium at concentrations up to 4500 mg/kg bw. Five male and 5 female hamsters per group of an average age of 10 weeks received daily oral doses of 0; 450; 1500; and 4500 mg/kg bw on 5 consecutive days. Positive control animals were treated intraperitoneally with cyclophosphamide 6 and 30 h before sacrifice. Two hours before sacrifice the animals were injected intraperitoneally with colcemid. Six hours after the last treatment with Acesulfame potassium or cyclophosphamide the animals were killed by ether. The femurs were removed from the animals and the bone marrow cells were prepared for exanimation. Slides were coded to avoid investigator bias during evaluation and approximately 100 cells per animal were examined.

 

No treatment-related aberrations of any type were observed in this study.

 

In vivo cytogenetic studies in mice exposed to Acesulfame potassium (1997, A. Mukherjee and J. Chakrabarty, Food and Chemical Toxicology, 35, 1177-1179). Four male Swiss albino mice per dosage group, 8 — 10 weeks old, received oral doses of 0; 15; 30; 60; 450; 1500 and 2250 mg/kg bw. Animals were sacrificed 18 h after treatment. 90 min before sacrifice each animal received 4 mg/kg bw colchicine intraperitoneally. Femurs were removed and the bone marrow cells were prepared for examination. 50 well-spread metaphase plates per animal were scanned, i. e. 200 metaphases per dose group. All aberrations (chromatid gaps, isochromosome gaps, chromatid breaks, isochromosome breaks, rearrangements) were considered equal regardless the number of breakages involved. The rather linear shape of the dose-response curve would mark Acesulfame potassium as a very strong mutagen. In this case other tests for genotoxicity performed would have presented a positive result too.

 

Chromosome aberration assay in bone marrow cells of the mouse with Acesulfame potassium (1998). As contradicting results were obtained in studies on cytogenetic effects of Acesulfame potassium in mice and hamsters a study in mice was initiated which used the relevant concentrations of the 1997 study in mice to clarify whether a species-specific effect existed. Concentrations (except for the two lowest levels) and sampling time were the same as in the 1997 study but the number of test animals was increased, and male and female animals were used.

 

Six male and six female Swiss albino mice per group, approximately 8 — 10 weeks old, received 0; 60; 450; 1500 and 2250 mg/kg bw Acesulfame potassium as a single oral dose. The animals were sacrificed 18 h after administration of the test article. 2.5 h before sacrifice the animals were injected 2.0 mg/kg colcemid. Femurs were removed, and the bone marrow cells were prepared for examination.

 

The authors conclude that during the mutagenicity test described and under the experimental conditions reported, Acesulfame potassium did not induce chromosome mutations as determined by the chromosome aberration test with bone marrow cells of the mouse.

 

Conclusion

The chromosome aberration study in the Chinese hamster and the most recent study in the mouse did not show any signs of a possible potential of Acesulfame potassium to induce chromosome aberrations. They correspond with all other genotoxicity data on Acesulfame potassium (with the exception of the study published by Mukherjee and Chakrabarty in 1997) which give no indication for genotoxicity either.

 

The dose-response reported by Mukherjee and Chakrabarty is normally observed for compounds showing genotoxicity in other test system, too. The most recent mouse study which was performed under carefully controlled GLP conditions and used the same experimental design, i. e. the same mouse strain and the same top dose levels as Mukherjee and Chakrabarty, did not show any signs

 

The fact that it was not possible to repeat the results of Mukherjee and Chakrabarty and the general absence of signs of genotoxicity of Acesulfame potassium in all other studies suggest that their study is no appropriate basis to assume Acesulfame potassium genotoxicity.

 

Short description of key information:
Acesulfame potassium was non-mutagenic/genotoxic in various test systems and studies performed according to internationally accepted guideliens and/or under GLP conditions.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Acesulfame potassium was non-mutagenic/genotoxic in various test systems and studies performed according to internationally accepted guideliens and/or under GLP conditions.

The study results triggers the following classification/labelling:

EU Directive 1999/45/EC (as amended):        none

Regulation (EC) No 1272/2008 (CLP):           none

GHS (rev. 4) 2011:                                      unclassified