1,2-benzisothiazol-3(2H)-one 1,1-dioxide, sodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Van Went-De Vries and Kragten

Year:

1975

Bibliographic source:

Fd Cosmetic. Toxicol

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vivo chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2-benzisothiazol-3(2H)-one 1,1-dioxide, sodium salt

EC Number:

204-886-1

EC Name:

1,2-benzisothiazol-3(2H)-one 1,1-dioxide, sodium salt

Cas Number:

128-44-9

Molecular formula:

C7H5NO3S.Na

IUPAC Name:

sodium 1,1,3-trioxo-2,3-dihydro-1H-1λ⁶,2-benzothiazol-2-ide

Details on test material:

- Name of test material: Saccharin sodium
- Molecular formula: C7H4NO3S.Na
- Molecular weight: 205.169 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): The saccharin contained six or seven impurities, the most important of which (comprising 0.5% of the material) was identified as o-toluenesulphonamide. The other impurities have not yet been identified.

Test animals

Species:

hamster, Chinese

Strain:

not specified

Details on species / strain selection:

No data

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: 2-3 months
- Weight at study initiation: 20 g
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
- Concentration of test material in vehicle: 0 or 1.5 mg/Kg bw/day
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in water to give a dose level of 0 or 1.5 mg/Kg bw/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

Duration of exposure: 3 days
Duration of treatment: 8 days

Frequency of treatment:

3 days

Post exposure period:

5 days

Doses / concentrations

Remarks:

0 or 1.5 mg/Kg bw/day

No. of animals per sex per dose:

Total: 40
0 mg/Kg bw/day: 20
1.5 mg/kg bw/day: 20

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The absence of data on the acute toxicity of saccharin in the Chinese hamster, an LD50 equal to that in the mouse was assumed, and on this basis the dose level chosen was relatively high being 10% of the LD50 for the mouse.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: No data

METHOD OF ANALYSIS: Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases.

OTHER: No data

Evaluation criteria:

The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted

Statistics:

It was assumed that the observed frequencies would follow approximately a Poisson distribution. In order to obtain a normal distribution, the data were transformed according to the transformation of Freeman and Tukey. A student’s t test was applied to the transformed data. Because we were not interested in the possible decrease in chromosome aberrations by saccharin, only the probability of an increase in abnormalities by chance was tested. For this reason a one tailed test was used.

Results and discussion

Test results

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No data
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay): No data
- Ratio of PCE/NCE (for Micronucleus assay): No data
- Appropriateness of dose levels and route: No data
- Statistical evaluation: No statistically significant differences were found between the control and treated groups.

Any other information on results incl. tables

Table: Number of polyploidy cells on 50 metaphases in the bone marrow of control and the test chemical treated Chinese hamsters

Polyploid cells*		No. of hamsters
X	Y	Controls	Saccharin treated
0	1	4	5
1	2.41	4	2
2	3.15	4	3
3	3.73	4	3
4	4.24	4	1
5	4.69	0	2
6	5.10	0	1
7	5.47	0	1
8	5.83	0	1
12	7.07	0	1

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of polyploidy cells were not significant: t: 0.9830 (38df); 0.10<P_R<0.25

Table: Number of anueploid cells on 50 metaphases in the bone marrow of control and treated Chinese hamsters

Anueploid cells*		No. of hamsters
X	Y	Controls	Saccharin treated
0	1	7	6
1	2.41	4	1
2	3.15	4	3
3	3.73	2	3
4	4.24	3	3
5	4.69	0	4

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of anueploid cells were not significant: t: 1.2797 (38df); 0.10<P_R<0.25

 

Table: Number of structural chromosome abnormalities in 50 metaphases in the bone marrow of control and treated Chinese hamsters

Structural abnormalities*		No. of hamsters
X	Y	Controls	Saccharin treated
0	1	6	7
1	2.41	3	7
2	3.15	8	3
3	3.73	0	2
4	4.24	1	1
5	4.69	2	0

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of structural chromosome abnormalities were not significant: t: 0.9576 (38df); P_R<0.75

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce a statistical significant increase in the chromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.

Executive summary:

In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical. Chinese hamsters were treated with the test chemical at dose levels of 0 or 1.5 mg/Kg bw/day. Water was used as control. Animals in both groups were given an ip injection of pertussis vaccine, each animal receiving 0.25 ml of a suspension containing 16 x 10⁹bacteria/ml. On days 2, 3 and 4 after the pertussis injection, water or saccharin dissolved in water was administered by gastric intubation to animals of the control and test group, respectively. Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases. The test chemical did not induce a statistical significant increase in thechromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.