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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-May-04 through 1998-Jun-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration 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): Acesulfame-K
- Physical state:solid, white
- Analytical purity: 99.9%
- Purity test date:08-Apr-1998
- Lot/batch No.:701NAAW27
- Expiration date of the lot/batch: 14-Sep-2002
- Stability under test conditions: stable
- Storage condition of test material: room temperature, closed container, light protection

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males: 31.0 ± 3.7 g, females: 26.9 ± 1.7 g
- Assigned to test groups randomly: yes, under following basis: body weight
- Fasting period before study: 8 h
- Housing: single, Makrolon Type 1 cages
- Diet: ad libitum, pelleted standard diet (Altromin, Lage, Germany)
- Bedding: granulated soft wood bedding (Altromin, Lage, Germany)
- Water: ad libitum (tap water)
- Acclimation period: at minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30% - 70%
- Photoperiod (hrs dark / hrs light): 12 h12 h

IN-LIFE DATES:
From: 1998-May-04
To: 1998-Jun-12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility and stability
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test artiele was dissolved in deionised water. All animals reccived a single standard volume of' 10 ml/kg bw.l

Duration of treatment / exposure:

Not applicable (single by gavage)

Frequency of treatment:

once

Post exposure period:

24 h and 48 h

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 60, 450, 1500, 2250 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): proven clastogenicity
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw dissolved in 0.9% NaCl solution

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Low acute and subacute toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 8 hours before. treatment with the test article the animals received no food but water ad libitum. Before the beginning of the treatment the animals were weighed and the individual volumes to be administered was adjusted to the animal’s body weight The animals received the test article orally once. Twelve animals, six males and six females, were treated per dose group

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with approximately 5 ml hypotonic potassium chloride solution (0.56 % w/v, prewarmed to 37 "C). The hypotonic cell suspension was then incubated for 20 min at 37 °C. The cells were sedimented by a brief centrifugation (1000 rpm), the hypotonic supernatant was discarded and the cell pellet was fixed with 3-1-1 absolute methanol-f-glacial acetic acid fixative for 60 min. Then the cell pellet was gently resuspended with fixative and stored overnight at 4 °C. Prior to making slides the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa_ Cover Slips were mounted with EUKITT (KINDLER, I)-791 10 Freiburg) One or more slides were made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, Fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. At least 100 well spread metaphases per animal were scored for cytogenetic damage and coded slides. The number of chromosome aberrations per metaphase was determined. Only metaphases with the characteristic chromosome number of 40 were included in the analysis. To describe a cytotoxic effect the mitotic Index (% cells in mitosis; 1000 cells were scored) was determined, in addition, per animal the number of polyploid cells (% polyploid metaphases) per 1000 metaphases were scored. Five animals per sex and group were evaluated as described.

Evaluation criteria:

According to OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test).

Statistics:

Non-parametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: no structural aberrations obsevred
- Appropriateness of dose levels and route: yes
- Statistical evaluation: negative by Mann-Whitney tests

Any other information on results incl. tables

Summary of Results:

 

Experimental group	Dose mg/kg bw	preparation hours post. administration	% aberrant incl.           excl. gaps          gaps		Mitotic index %#
Deionised water	0	18	0.2	0.2	6.78
Acesulfame-K	60	18	0.7	0.6	8.30
Acesulfame-K	450	18	0.7	0.6	7.59
Acesulfame-K	1500	18	0.3	0.3	6.15
Acesulfame-K	2250	18	0.8	0.8	7.21
Cyclophosphamide	20	18	10.9	10.3	6.15
# = number of cells scored = 1000; ## 0 number of cells scored = 10000

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Acesulfame potassium did not induce chromosome aberrations as determined by the chromosome aberration test with bone marrow cells of male and female mice.

Executive summary:

Acesulfame potassium was investigated for its potential to induce chromosome aberrations in bone marrow cells of the mouse.

 

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.

 

The test article was dissolved in deionized water. The volume administered orally was10 ml/kg bw. 18 h after a single administration of the test article the bone marrow cells were collected for chromosome aberration analysis. 10 animals (5 males, 5females) per group were evaluated for the occurrence of cytogenetic damage. Per animal100 well spread metaphases were scored for gaps, breaks, fragments, deletions, multiple aberrations, exchanges and chromosomal disintegrations. The test article was investigated in this cytogenetic assay at doses of 60, 450, 1500, and 2250 mg/kg bw.

 

The mitotic indices after treatment with the test substance were not decreased as compared to the value of the solvent control.

 

Treatment did not result in a statistically significant or biologically relevant enhancement of the aberration frequency at any dose as compared to the solvent control value.

 

Cyclophosphamide (20 m/kg bw.) was used as positive control and showed a statistically significant increase of induced aberration frequency.

 

Conclusion

Acesulfame potassium did not induce chromosome aberrations as determined by the chromosome aberration test with bone marrow cells of male and female mice.