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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviwed journal

Data source

Reference

Reference Type:

publication

Title:

The comet assay with 8 mouse organs: results with 39 currently used food additives

Author:

Yu F. Sasaki , Satomi Kawaguchi , Asako Kamayaa, Miyuki Ohshita , Kazumi Kabasawa , Kayoko Iwama, Kazuyuki Taniguchi , Shuji Tsuda

Year:

2002

Bibliographic source:

Mutation Research 519 (2002) 103–119

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: refer below principle

Principles of method if other than guideline:

An in vivo gene DNA damage and/or repair toxicity study of saccharin by using comet assay

GLP compliance:

not specified

Type of assay:

other: Single-cell gel electrophoresis

Test material

Test material information

Constituent 1

Reference substance name:

Saccharin

IUPAC Name:

Saccharin

Constituent 2

Reference substance name:

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

EC Number:

201-321-0

EC Name:

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

Cas Number:

81-07-2

Molecular formula:

C7H5NO3S

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): Saccharin
- Molecular formula (if other than submission substance): C7-H5-N-O3-S
- Molecular weight (if other than submission substance): 183.186 g/mole
- Substance type: Organic
- Physical state: No data available
- Purity: 98 +%
- Impurities (identity and concentrations): No data available

Test animals

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Japan SLC Co., Shizuoka, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): Commercial pellets MF, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24 °C
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12 h light–dark cycle.

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Saline
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0, 100, 1000 or 2000 mg/kg
- Amount of vehicle (if gavage or dermal): No data available
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available

Details on exposure:

No data available

Duration of treatment / exposure:

3 and 24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 1000 or 2000 mg/kg
Basis:
no data
for 3 hours

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
no data
for 24 hours

No. of animals per sex per dose:

Total: 20 males
0 mg/kg: 4 males
100 mg/kg: 4 males
1000 mg/kg: 4 males
2000 mg/kg (3 hr): 4 males
2000 mg/kg (24 hr): 4 males

Control animals:

yes

Positive control(s):

No data available

Examinations

Tissues and cell types examined:

Glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow nuclear DNA was examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were selected on the basis of limit test by determining LD50 > 2000 mg/kg

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): For 3 hours 100, 1000 and 2000 mg/kg, and for 24 hours 2000 mg/kg dose were used.

DETAILS OF SLIDE PREPARATION: Homogenized samples of glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow were collected and prepared accordingly.
Seventy-five microliters agarose GP-42 homogenizing solution was quickly layered on a slide coated with agarose GP-42 and covered with another slide. The slide sandwiches were placed horizontally to allow the agarose to solidify. The nucleus suspension was mixed 1:1 (v/v) with 2%, 45°C, agarose LGT, and 75 µl of the nucleus mixture was quickly layered in the same manner after removal of the covering slide. Finally, 75 µl of agarose GP-42 was quickly layered on again. Slides prepared from nuclei isolated by homogenization were placed in a chilled lysing solution (2.5M NaCl, 100mM Na2EDTA, 10mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) and kept at 0°C in the dark for about 1 night, then in chilled alkaline solution (300mM NaOH and 1mM Na2EDTA, pH 13) for 10 min in the dark at 0°C. Electrophoresis was conducted at 0°C in the dark for 15 min at 25V (0.96 V/cm) and approximately 250 mA. The slides were neutralized and then stained with 50 µl of 20g/ml ethidium bromide and examined.

METHOD OF ANALYSIS: 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope. The length of the whole comet (“length”) and the diameter of the head (“diameter”) were measured for 50 nuclei per organ per animal. Calculated migration as the difference between length and diameter for each of 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals.

When positive results were obtained in the comet assay, tissue sections stained by the hematoxylin–eosin and TUNEL methods were observed histopathologically.

OTHER: Animals were carefully observed for pharmacotoxic signs until just before they were killed.

Evaluation criteria:

The length of the whole comet (“length”) and the diameter of the head (“diameter”) were measured for 50 nuclei per organ per animal.

Statistics:

Statistical analysis is performed by using Dunnett test after one-way ANOVA. A P value less than 0.05 was considered statistically significant.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

positive

Remarks:

Colon

Toxicity:

not specified

Vehicle controls validity:

not valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

Glandular stomach, liver, kidney, bladder, lung, brain and bone marrow

Toxicity:

not specified

Vehicle controls validity:

not valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Solubility: No data available
- Clinical signs of toxicity in test animals: No mortality were observed .
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: LD50 were determined

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): No data available
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): ambiguous
Saccharin was considered to be a mutagenic-inducing chemical for colon, but a non-mutagenic chemical for glandular stomach, liver, kidney, bladder, lung, brain and bone marrow when ddY male mice treated with saccharin.

Executive summary:

In an in vivo gene DNA damage and/or repair toxicity study, male ddY mice were exposed to saccharin in the concentration of 100, 1000 or 2000 mg/kg for 3 hours and 2000 mg/kg for 24 hours. The choice of method for analysis was the comet assay. Significant migration of nuclear DNA was observed in colon at 3 hour after treatment with 1000 and 2000 mg/kg at 3 hour, and at 24 hours after treatment with 2000 mg/kg. It was also shown that non-nuclear DNA migrated in glandular stomach, liver, kidney, bladder, lung, brain and bone marrow at 3 and 24 hours. Therefore, saccharin was considered to be positive, i.e. have mutagenic effects, for colon, while being negative for all other investigated organs/tissues in male ddYmale mice after exposure tosaccharin for 3 and/or 24 hours.