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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-Jul-15 through 1986-Sep-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

IUPAC Name:

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

Constituent 2

Reference substance name:

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

EC Number:

259-715-3

EC Name:

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

Cas Number:

55589-62-3

Molecular formula:

C4H5NO4S.K

IUPAC Name:

potassium 6-methyl-2,2,4-trioxo-3,4-dihydro-1,2λ⁶,3-oxathiazin-3-ide

Constituent 3

Reference substance name:

Acesulfame potassium

IUPAC Name:

Acesulfame potassium

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Sunett
- Physical state: colourless-white crystals
- Analytical purity: 99%
- Impurities (identity and concentrations): Fluorid: <3 ppm; Arsen: <3 ppm, Selen: <3 ppm, heavy metals: < 10 ppm
- Purity test date: 1986-05-06
- Lot/batch No.: 126 1955
- Stability under test conditions: stable
- Storage condition of test material: dark at 22 °C

Method

Target gene:

In the Ames test with Salmonella typhimurium strains the effect of the test compound upon the number of back mutations to histidine prototrophy using histidine auxotrophic mutants is investigated. Using Escherichia ca1i WP2uvrA, a tryptophan dependent auxotroph strain, mutagenicity is based an reversion to tryptophan independence.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver homogenate from male Sprague-Dawöley rats

Test concentrations with justification for top dose:

1st experiment: all strains: 0, 4, 20, 100, 500, 2500, 10000 µg/plate (with/without metabolic activation)
2nd experiment: all strains: 0, 4, 20, 100, 500, 2500, 5000 µg/plate (with/without metabolic activation)
3rd experiment: TA 100: 0, 4, 20, 100, 500, 2500, 5000 µg/plate (with/without metabolic activation)

Vehicle / solvent:

Vehicle:
- Vehicle: bi-distilled waterMSO
- Justification for choice of solvent/vehicle: non toxicity and solubility

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, N-methyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxi¬city
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 – 72 h at 37 °C
- Expression time (cells in growth medium): 48 – 72 h
- Selection time (if incubation with a selection agent): 48 – 72 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 – 72 h

SELECTION AGENT: Histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF EXPERIMENTS: 2 (strains: TA 1535, TA 1537, TA 1538, TA 98, WP2uvrA), 3 (strain TA 100)

DETERMINATION OF CYTOTOXICITY
- Method: reduced rate of spontaneous occurring colonies

Evaluation criteria:

According to OECD 471

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: No

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

   

Results: Experiment 1: Number of mean revertant colonies per plate in bacteria strains

 

	S. typhimurium					E. coli
Dose µg/plate	TA100	TA1535	1537	1538	98	WP2uvrA
	Without metabolic activation
0	132	14	7	12	27	34
4	143	14	7	15	28	34
20	143	16	6	13	26	36
100	148	14	7	12	27	32
500	148	11	8	8	25	32
2500	151	13	5	13	23	31
10000	155	19	6	10	26	35
	With metabolic activation
0	128	13	6	18	35	30
4	146	11	6	21	34	38
20	129	14	8	16	35	39
100	148	9	4	23	28	37
500	137	11	4	21	29	33
2500	123	14	6	21	36	40
10000	145	12	8	20	34	26

 

Results: Experiment 2: Number of mean revertant colonies per plate in bacteria strains

 

	S. typhimurium					E. coli
Dose µg/plate	TA100	TA1535	1537	1538	98	WP2uvrA
	Without metabolic activation
0	208	13	9	15	24	41
4	205	16	9	12	24	48
20	221	16	7	13	22	37
100	227	15	7	15	25	45
500	239	14	6	18	23	37
2500	258	20	7	14	26	38
5000	263	19	7	14	24	39
	With metabolic activation
0	200	16	8	17	29	42
4	230	18	6	22	26	40
20	206	17	8	21	23	37
100	217	16	6	17	27	33
500	241	16	9	20	33	39
2500	225	16	6	15	37	41
5000	237	13	10	19	34	38

 

Results: Experiment 3: Number of mean revertant colonies per plate in S. typhimurium strain TA100 only

 

	S. typhimurium
Dose µg/plate	TA100
	Without metabolic activation
0	179
4	173
20	172
100	174
500	168
2500	172
5000	146
	With metabolic activation
0	177
4	173
20	178
100	179
500	172
2500	174
5000	170

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with/wihtout metabolic activation

Acesulfame potassium was not mutagenic in the in vitro reverse gene mutation assay in bacteria with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA in the presence and absence of metabolic activation.

Executive summary:

Acesulfame potassium was investigated for its potential mutagenic activity in thein vitroreverse gene mutation assay in bacteria with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 ofSalmonella typhimuriumandEscherichia coliWP2uvrA. The test was performed in the absence and presence of a metabolizing system derived from rat liver homogenate (male Sprague-Dawley rat liver S9-mix induced by Aroclor 1245). The test substance (batch: 126 1955 dated 06 May 1986, crystalline, purity: 99%) was dissolved in bi-distilled water and up to 3 independent experiments with 3 replicate each were performed. A range-finding study to investigate bacteriotoxicity was performed. The solvent served also as negative control and appropriate positive controls were used.

This study is assessed as appropriate and valid since it was performed according to internationally accepted OECD and EU testing guidelines and according to GLP. Reporting, assessment and data presentation in the study report was considered as appropriate.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds led to the expected increase in the number of revertant colonies.

 

Acesulfame potassium was not toxic to the bacterial strains up to the highest concentration of each 5000 or 10000 µg/plate in the presence or absence of metabolic activation.

 

In the presence and absence of a metabolic activation, Acesulfame potassium did not led to any relevant increase in the number of revertant colonies in the bacterial tester strains.

 

Conclusion:

Acesulfame potassium was not mutagenic in thein vitroreverse gene mutation assay in bacteria with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 ofSalmonella typhimuriumandEscherichia coliWP2uvrA in the presence and absence of metabolic activation.