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Submission Report

Submission Information

Submission ID :

25419404

Submission Name : 128449 - 1,2-Benzisothiazol-32H-one, 1,1-dioxide,

sodium salt

AR Number : 

Chemical Information

CAS Number : 128-

44-9

Ninth Collective Index Name : 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide,

sodium salt

Submitter Information

Submitter's Name : Orphan

Address : 

City : 

State : VA

ZIP/Postal Code: 

Country: 

Type of Submitter : Company

Technical Point of Contact Information

Technical Point of Contact : 

Title : 

Phone Number : 

Fax Number : 

E-Mail Address : 

Additional Technical Point of Contact Information

Technical Point of Contact : 

Title : 

Phone Number : 

Fax Number : 

E-Mail Address : 

Sponsor Information

Sponsor's Name : Orphan Chemical

Address : 

City : 

State : VA

ZIP/Postal Code : 

Country : US

Phone Number : 

Share
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Fax Number : 

E-Mail Address : 

Type of Sponsor : Company

Technical Point of Contact Information

Technical Point of Contact : 

Title : 

Phone Number : 

Fax Number : 

E-Mail Address : 

 Acute Toxicity to Aquatic Vertebrates

CAS Number : 128-44-9

Sponsored

Chemical Name :

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

Test Substance - Acute Toxicity To Aquatic Vertebrates

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium salt

Test Substance

Purity/Composition

and Other Test

Substance

Comments:

Sponsored

Chemical Result

Type:

Test Substance

Result Type:

Method - Acute Toxicity To Aquatic Vertebrates

Year Study

Performed:

1988

Method/Guideline

Followed:

Unknown

Other

Method/Guideline:

Deviations from

Method/Guideline:

Species: Pimephales promelas

GLP: Unknown

Analytical

Monitoring:

Yes

Test Type: Flow-through

Test Vessel:

Water Media Type:

Test

Concentrations:

Nominal and

Measured

Concentrations:

Not stated
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Exposure Period: 96 Hours

Vehicle Used:

Vehicle Name:

Vehicle Amount

and Units:

Alkalinity:

Dissolved Oxygen:

pH Value:

Test Temperature

and Units:

Photo

(Light/Dark):

Salinity:

TOC:

Water Hardness:

Method/Guideline

Test Conditions

Remarks:

Not stated

Limit Test:

Test Results - Acute Toxicity To Aquatic Vertebrates

NOEC Exposure

Duration:

NOEC:

LOEC Exposure

Duration:

LOEC:

NOELR Exposure

Duration:

NOELR:

LOELR Exposure

Duration:

LOELR:

LC/EC Mean Value : Exposure

Duration

Exposure

Units

LC/EC % Value

Description

Mean

Value

or

Lower

Mean

Value

Upper

Mean

Value

Units Effect

Observed

Basis for

Concentration

96 Hours LC 50 % = 18300 mg/L
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Results Remarks: Conclusions: 96h LC50 = 18,300 mg/L

Reliability/Data Quality - Acute Toxicity To Aquatic Vertebrates

Reliability: 3 - Not Valid

Reliability

Remarks:

This study is considered inadequate due to missing important study details.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity To Aquatic Vertebrates

Reference: ECOTOX Reference No. 12859. Geiger, D.L., D.J. Call, and L.T. Brooke. Acute

Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas) Volume

IV. Source: Ctr.for Lake Superior Environ.Stud., Volume 4, Univ.of WisconsinSuperior, Superior, WI. 355 pp. Publication Year: 1988.

 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Acute Toxicity

Route of Administration: Oral

Type of Exposure:

Species: Mouse

Mammalian Strain: Unknown

Gender:

Number of Animals per

Dose:

Dose: Not stated

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown
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GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated by the method of Miller & Tainter

(1944).

Single doses of sodium cyclamate, sodium saccharin or sodium

cyclamate/sodium saccharin (10:l) mixture were given to rats,

mice and rabbits by intragastric intubation or by intravenous

or intraperitoneal injection. The animals were observed for a

minimum of 5 days after dosing for mortality and signs of

toxicity. 

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 17.5 Other

Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total

):

Results Remarks: Endpoint value, oral: LD50 (mouse) = 17.5 g/kg

The acute oral toxicity varied with the species and strain of

animal. In Wistar rats the cyclamate/saccharin combination was

less toxic than either of the components, but this was not

the case with mice or mongrel rats. Dietary administration of

cyclamate or saccharin throughout gestation did not appear to

alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin

combination did cause some lessening of susceptibility,

although the differences were small and far less than one

order of magnitude.

Conclusion: The acute toxicity of the materials was shown to vary with

the strain of animal and the fat content of the diet. 

Reliability/Data Quality - Acute Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to lack of important

study details, non-standard method; limited number of test

substance concentrations tested; and non-standard strain

used.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp.

313-327.
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 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Acute Toxicity

Route of Administration: Intraperitoneal

Type of Exposure:

Species: Rat

Mammalian Strain: Other

Other Strain:

Gender:

Number of Animals per

Dose:

Dose: Not stated

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown

GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated by the method of Miller & Tainter

(1944).

Single doses of sodium cyclamate, sodium saccharin or sodium

cyclamate/sodium saccharin (10:l) mixture were given to rats,

mice and rabbits by intragastric intubation or by intravenous

or intraperitoneal injection. The animals were observed for a

minimum of 5 days after dosing for mortality and signs of

toxicity. 

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 7.1 Other
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Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total

):

Results Remarks: Endpoint value, intraperitoneal: LD50 (mongrel rats) = 7.1

g/kg

The acute oral toxicity varied with the species and strain of

animal. In Wistar rats the cyclamate/saccharin combination was

less toxic than either of the components, but this was not

the case with mice or mongrel rats. Dietary administration of

cyclamate or saccharin throughout gestation did not appear to

alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin

combination did cause some lessening of susceptibility,

although the differences were small and far less than one

order of magnitude.

Conclusion: The acute toxicity of the materials was shown to vary with

the strain of animal and the fat content of the diet. 

Reliability/Data Quality - Acute Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to lack of important

study details, non-standard method; limited number of test

substance concentrations tested; and non-standard strain

used.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp.

313-327.

 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:
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Method - Acute Toxicity

Route of Administration: Oral

Type of Exposure:

Species: Rat

Mammalian Strain: Wistar

Gender:

Number of Animals per

Dose:

Dose: Not stated

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown

GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated by the method of Miller & Tainter

(1944).

Single doses of sodium cyclamate, sodium saccharin or sodium

cyclamate/sodium saccharin (10:l) mixture were given to rats,

mice and rabbits by intragastric intubation or by intravenous

or intraperitoneal injection. The animals were observed for a

minimum of 5 days after dosing for mortality and signs of

toxicity. 

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 14.2 Other

Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total

):

Results Remarks: Endpoint value, oral: LD50 (Wistar rats) = 14.2 g/kg

The acute oral toxicity varied with the species and strain of

animal. In Wistar rats the cyclamate/saccharin combination was

less toxic than either of the components, but this was not

the case with mice or mongrel rats. Dietary administration of

cyclamate or saccharin throughout gestation did not appear to

alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin
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combination did cause some lessening of susceptibility,

although the differences were small and far less than one

order of magnitude.

Conclusion: The acute toxicity of the materials was shown to vary with

the strain of animal and the fat content of the diet. 

Reliability/Data Quality - Acute Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to lack of important

study details, non-standard method; limited number of test

substance concentrations tested; and non-standard strain

used.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp.

313-327.

 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Acute Toxicity

Route of Administration: Oral

Type of Exposure: Gavage

Species: Rat

Mammalian Strain: Wistar

Gender: Both M/F

Number of Animals per

Dose:

10

Dose: Not stated (at least 6 increasing dose levels)

Year Study Performed: 1989

Method/Guideline

Followed:

Unknown
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GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated using the Litchfield and Wilcoxon

method.

Clinical signs and symptoms were monitored during the

experimental period. Animals were given regular laboratory

chow 6 hours after a single administration and the number of

dead animals was counted until the 14th day. Anatomical

examination was performed immediately after death or at

terminal sacrifice on day 14.

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 1280 mg/kgbw

LD 50 % = 1580 mg/kgbw

Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total

):

Results Remarks: There were no acute toxic symptoms or change in tissues

observed.

Conclusion: The acute oral LD50 was 1280 mg/kg for male rats and 1580

mg/kg for female rats.

Reliability/Data Quality - Acute Toxicity

Reliability: 2 - Valid With Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Hasegawa et al. 1989. Acute Toxicity Tests on 113

Environmental Chemicals. Sci Rep Res Inst Tohoku Univ Ser C

Med: 36 (1-4): 10-16.

 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium
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salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Acute Toxicity

Route of Administration: Oral

Type of Exposure:

Species: Rat

Mammalian Strain: Other

Other Strain:

Gender:

Number of Animals per

Dose:

Dose: Not stated

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown

GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated by the method of Miller & Tainter

(1944).

Single doses of sodium cyclamate, sodium saccharin or sodium

cyclamate/sodium saccharin (10:l) mixture were given to rats,

mice and rabbits by intragastric intubation or by intravenous

or intraperitoneal injection. The animals were observed for a

minimum of 5 days after dosing for mortality and signs of

toxicity. 

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 17 Other

Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total
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):

Results Remarks: Endpoint value, oral: LD50 (mongrel rats) = 17.0 g/kg

The acute oral toxicity varied with the species and strain of

animal. In Wistar rats the cyclamate/saccharin combination was

less toxic than either of the components, but this was not

the case with mice or mongrel rats. Dietary administration of

cyclamate or saccharin throughout gestation did not appear to

alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin

combination did cause some lessening of susceptibility,

although the differences were small and far less than one

order of magnitude.

Conclusion: The acute toxicity of the materials was shown to vary with

the strain of animal and the fat content of the diet. 

Reliability/Data Quality - Acute Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to lack of important

study details, non-standard method; limited number of test

substance concentrations tested; and non-standard strain

used.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp.

313-327.

 Acute Toxicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Acute Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Acute Toxicity

Route of Administration: Intraperitoneal

Type of Exposure:

Species: Mouse

Mammalian Strain: Unknown

Gender:
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Number of Animals per

Dose:

Dose: Not stated

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown

GLP: Unknown

Method/Guideline and

Test Condition Remarks:

The LD50 was calculated by the method of Miller & Tainter

(1944).

Single doses of sodium cyclamate, sodium saccharin or sodium

cyclamate/sodium saccharin (10:l) mixture were given to rats,

mice and rabbits by intragastric intubation or by intravenous

or intraperitoneal injection. The animals were observed for a

minimum of 5 days after dosing for mortality and signs of

toxicity. 

Test Results - Acute Toxicity

Concentration (LC/LD) : LC/LD % Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

LD 50 % = 6.3 Other

Number of Deaths ( Male

):

Number of Deaths ( Female

):

Number of Deaths ( Total

):

Results Remarks: Endpoint value, intraperitoneal: LD50 (mouse) = 6.3 g/kg

The acute oral toxicity varied with the species and strain of

animal. In Wistar rats the cyclamate/saccharin combination was

less toxic than either of the components, but this was not

the case with mice or mongrel rats. Dietary administration of

cyclamate or saccharin throughout gestation did not appear to

alter the intraperitoneal toxicity of any of the three test

materials to the offspring. Feeding of the cyclamate/saccharin

combination did cause some lessening of susceptibility,

although the differences were small and far less than one

order of magnitude.

Conclusion: The acute toxicity of the materials was shown to vary with

the strain of animal and the fat content of the diet. 

Reliability/Data Quality - Acute Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to lack of important

study details, non-standard method; limited number of test

substance concentrations tested; and non-standard strain
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used.

Key Study Sponsor

Indicator:

Reference - Acute Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp.

313-327.

 Repeated-Dose Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Repeated-Dose Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Repeated-Dose Toxicity

Route of

Administration:

Oral

Type of Exposure: Gavage

Species: Dog

Mammalian Strain:

Gender:

Number of Animals per

Dose:

4

Dose: 0.065 g/kg

Year Study Performed: 1968

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period: 11 Months

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

Groups of four dogs received weekly 6 daily doses of sodium

saccharin (0.065 g/kg). A control group received the basic diet

only. The materials were administered by intragastric intubation

in equal volumes of water. The doses selected were equivalent in
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terms of "sweetness".

The dogs were observed daily for survival, general physical

condition, behavior, food and water consumption and character and

frequency of excreta. The animals were weighed weekly and

laboratory determinations, including renal and hepatic function

tests, were made initially and after 1, 2, 4, 9 and 11 months of

dosing, at which time the surviving dogs were killed off and

gross examinations were carried out.

Test Results - Repeated-Dose Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

Results Remarks: During the initial 6 months of the study, all dogs behaved

normally. General physical condition, appetite and response to

stimuli appeared to be normal throughout this period. During the

second half-year, however, one dog on the saccharin diet showed

marked anorexia with an ensuing general debilitation. This dog

steadily deteriorated and died from what appeared to be general

inanition. Post-mortem examination revealed no abnormal organ

pathology.

About month 10 of the study, all dogs developed soft, poorlyformed stools without frank diarrhea. This condition did not

appear to affect food or water consumption or general physical

appearance but was evident throughout the last 2 months of the

experiment. 

Bodyweight, hemoglobin, total leukocyte count, non-protein

nitrogen and phenol-sulphonphthalein retention were within normal

limits and total erythrocyte count, bilirubin and urinary

analyses were also unaffected. The gross and histological

appearance of the viscera at necropsy were within normal limits.

Conclusion: None stated.

Reliability/Data Quality - Repeated-Dose Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: Non-standard method; no LOAEL/NOAEL reported; limited number of

test substance concentrations tested; and non-standard species

was used.

Key Study Sponsor

Indicator:

Reference - Repeated-Dose Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp. 313-

327.
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 Repeated-Dose Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Repeated-Dose Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Repeated-Dose Toxicity

Route of

Administration:

Dietary

Type of Exposure:

Species: Rat

Mammalian Strain:

Gender: Both M/F

Number of Animals per

Dose:

14

Dose: 0.5% (~ 401 mg/kg/day)

Year Study Performed: 1967

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

Four groups of 14 male and 14 female individually-housed

rats, weighing 75-100 g, received diets containing no added

sweetener or 0.5 % sodium saccharin, sodium cyclamate or sodium

cyclamate/sodium saccharin (10:l) mixture. Food and water were

available ad libitum for 38 days. Survival, behavior, general

physical appearance and stool condition were noted daily. On day

39, the surviving animals were killed off and sections of renal

and hepatic tissues were fixed in formalin and stained with

haematoxylin eosin for microscopic examination.

Test Results - Repeated-Dose Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/ LOAEL/ Population Value Value/Lower Upper Units
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NOAEC) : LOAEC/

NOAEL/

NOAEC

Description Concentration Concentration

Results Remarks: It is evident that compared with the controls, there was a

marked reduction in growth rate and food intake in all three

test groups. Efficiency of food utilization was comparable among

the groups, as was survival. Despite the fact that some diarrhea

was noted in all groups, there were no overt differences in

behavior among the animals.

Gross and microscopic examination of representative survivors

revealed a number of inflammatory and hydropic lesions in the

liver and kidneys of the rats of the three test groups. These

may have been associated either with the physical effects of

diarrhea and water loss or with some endemic disease in these

rats. Three rats in each of the four groups had died during the

course of the study. Calculation of the amount of material

consumed daily indicated that intake of either saccharin or

cyclamate alone or of the combination was approximately 400

mg/kg/day.

Conclusion: Overall, it is evident that compared with the controls, there was

a marked reduction in growth rate and food intake in all three

test groups. Efficiency of food utilization was comparable among

the groups, as was survival. Despite the fact that some diarrhea

was noted in all groups, there were no overt differences in

behavior among the animals.

Reliability/Data Quality - Repeated-Dose Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: Non-standard method; no LOAEL/NOAEL reported; and limited number

of test substance concentrations tested.

Key Study Sponsor

Indicator:

Reference - Repeated-Dose Toxicity

Reference: Taylor, JD et al., 1967. Toxicological Studies with Sodium

Cyclamate and Saccharin. Fd. Cosmet. Toxicol. Vol. 6, pp. 313-

327.

 Genetic Toxicity in vivo

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Genetic Toxicity in vivo

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt
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Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Genetic Toxicity in vivo

Type of Study: Gene Mutation

Type of Test: Other

Route of Administration:

Type of Exposure:

Species: Mouse

Strain:

Gender:

Dose: 2000 mg/kg (i.p.), 5000 mg/kg (p.o.) (single dose); 5 x 400

mg/kg (i.p.), 

5 x 1000 mg/kg (p.o.) (repeated dose) 

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Duration of Treatment/

Exposure Period and

Units:

Frequency of Treatment:

Positive, Negative, and

Solvent

Control Substance(s):

Number of Animals per Sex

and Dose Group:

Method/Guideline and

Test Condition Remarks:

Statistical methods used: Chi-square test

Male mice were treated with a single dose of 2000 mg/kg

(i.p.) or 5000 mg/kg (p.o.) of sodium saccharin (S-Na), 250

mg/kg (i.p.) of ethyl methanesulfonate or 3.0 mg/kg (i.p.) of

mitomycin C, or repeated doses of 5 x 400 mg/kg (i.p.) or 5 x

1000 mg (p.o.) of S-Na at 12-h intervals. After treatment

with S-Na, EMS and MC, these males were mated to 4 females

per week for 6 consecutive weeks (single dose) or for 2

consecutive weeks (repeated dose). All females were autopsied

on day 12 of pregnancy and scored for corpora lutea,

implants, early and late dead embryos and live

fetuses. Results obtained were tested using ÷2-test and

calculated dominant lethality. 

Test Results - Genetic Toxicity in vivo

Systemic Toxicity:
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Genotoxic Effect: Positive

Results Remarks: In the S-Na i.p. group, a significant decrease (P < 0.01) in

the number of live fetuses per corpora lutea or implantations

was observed at 1-2 weeks after treatment. Furthermore, in

both groups of i.p. and p.o. repeated dose of S-Na,

implantations and live fetuses per corpora lutea decreased

with statistical significance (P < 0.001), while frequencies

of dominant lethality of EMS and MC agreed with previous

reports.

Conclusion: The test substance was considered to be mutagenic.

Reliability/Data Quality - Genetic Toxicity in vivo

Reliability: 2 - Valid With Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vivo

Reference: Masubuchi, M. et al. The mutagenicity of sodium saccharin

(S-Na). I. Dominant-lethal test. Mutation Research, 54

(1978) 218-219.

 Genetic Toxicity in vivo

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Genetic Toxicity in vivo

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Madhurin is the trade name of sodium saccharin manufactured

by M/s Sarabhai Chemicals Ltd.

Sponsored Chemical Result

Type:

Method - Genetic Toxicity in vivo

Type of Study: Gene Mutation

Type of Test:

Route of Administration:

Type of Exposure:

Species: Mouse

Strain: CBA

Gender: Both M/F
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Dose: 172 mg

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Duration of Treatment/

Exposure Period and

Units:

Frequency of Treatment:

Positive, Negative, and

Solvent

Control Substance(s):

Number of Animals per Sex

and Dose Group:

Method/Guideline and

Test Condition Remarks:

Male mice of CBA strain, 10-12 weeks old, with average body

weight of 25 g, were used for treatment and female mice of

101 strain of the same age group with body weight ranging

from 20-25 g were used for mating. Twelve tablets of

Madhurin weighing 172 mg were dissolved in 100 mL of

distilled water (1-72%) and each male mouse was allowed to

drink the water. All the males were tested for their litter

size (before allowing them to drink the water containing

sodium saccharin) which was found to be on an average

six. The treatment was continued for 30 days. Twenty-four

hours after the treatment ceased, each treated male was mated

with three normal females of 101 strain. The vaginal plugs

were examined every morning and the females which had mated

were replaced by fresh females. The matings were continued

for a period of four weeks. The mated females were dissected

on the fifteenth day of gestation and the number of live and

dead embryos were recorded. Control observations were made

on comparable females mated to males which were given

distilled water without sodium saccharin.

Test Results - Genetic Toxicity in vivo

Systemic Toxicity:

Genotoxic Effect: Positive

Results Remarks: The ratio of the number of dead embryos to that of the total

implants is expressed as percentage of dominant lethals. The

percentage of the dominant lethal was taken as the criterion

in assessing the damage caused to the male germ line by

sodium saccharin. The peak effect period seems to be the

fourth week followed by the third and second weeks of

mating. It is of interest to note that with each week, the

percentage of intrauterine deaths increased with the maximum

in the fourth week. Also, the dead embryos per female were

highest in the fourth week followed by third and second

weeks. The results obtained, when compared week-wise with

that of the control data were statistically significant. The

results obtained by Majumdar and Sanjeeva Rao et al (1971) in

Drosophila by feeding have clearly shown that all the premeiotic stages of germ plasm are highly susceptible to sodium

saccharin. 

Even though the results of chronic exposure (30 days) are

difficult to interpret, in terms of the susceptibility of the

stages of spermatogenesis, a comparison of the data obtained

in Drosophila and mice clearly indicated the mutagenic

potential of sodium saccharin in the pre-meiotic stages of
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germ plasm.

Sodium saccharin thus appears to be a potential mutagenic

agent and the results caution a more careful consideration of

its use in medicine and food industry. 

Conclusion: The test substance was considered to be mutagenic.

Reliability/Data Quality - Genetic Toxicity in vivo

Reliability: 1 - Valid Without Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vivo

Reference: Rao, M.S. and Qureshi, A.B. Induction of Dominant Lethals in

Mice by Sodium Saccharin. Indian Journal of Medical

Research, 60 (1972) 599-603.

 Genetic Toxicity in vivo

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Genetic Toxicity in vivo

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Genetic Toxicity in vivo

Type of Study: Bone Marrow Chromosomal Aberration

Type of Test: Chromosome aberration assay

Route of Administration:

Type of Exposure:

Species: Mouse

Strain:

Gender: Male

Dose: 4000 mg/kg (i.p.; single dose); 0-1600 mg/kg (i.p.; repeated

dose)

Year Study Performed:
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Method/Guideline

Followed:

Unknown

GLP: Unknown

Duration of Treatment/

Exposure Period and

Units:

Frequency of Treatment:

Positive, Negative, and

Solvent

Control Substance(s):

Number of Animals per Sex

and Dose Group:

Method/Guideline and

Test Condition Remarks:

After i.p. injection of sodium saccharin (S-Na; 4000 mg/kg),

metaphase plates were analyzed at 6, 24 or 48 h. Doseresponse relationship was studied after the 5 repeated i.p.

injections of various amounts of S-Na (0-1600 mg/kg) at

intervals of 12 h. 

Test Results - Genetic Toxicity in vivo

Systemic Toxicity:

Genotoxic Effect: Positive

Results Remarks: Frequencies of aberrant cells with breaks and/or gaps began

to increase at 6 h, reached a peak at 24 h and decreased

thereafter. At 24 h cells with breaks (3.6%, P < 0.01) and

gaps (3.3%) were scored. In the case of repeated dosing,

although aberrant cells increased as the dose was raised

(from 5 x 400 to 5 x 800 to 5 x 1600 mg/kg), a clear doseresponse relationship was not obtained.

Conclusion: High concentrations of S-Na induced chromosome aberrations in

vivo. 

Reliability/Data Quality - Genetic Toxicity in vivo

Reliability: 2 - Valid With Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vivo

Reference: Masubuchi, M. et al. The mutagenicity of sodium saccharin

(S-Na). II. Cytogenetic studies. Mutation Research, 54

(1978) 219.

 Genetic Toxicity in vitro

CAS Number : 128-44-9

Sponsored

Chemical Name :

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

Test Substance - Genetic Toxicity in vitro

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium salt

Test Substance
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Purity/Composition

and Other Test

Substance

Comments:

Sponsored

Chemical Result

Type:

Method - Genetic Toxicity in vitro

Type of Study: in vitro mammalian chromosome aberration test

Concentrations: 10, 15, 20, 25, 30 mg/L 

Year Study

Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Postive, Negative,

and Solvent

Control

Substance(s):

Method/Guideline

and

Test Condition

Remarks:

Chinese hamster cells were treated with 20 mg/mL of sodium saccharin

(S-Na) for 6, 24 or 48 h, and scored at metaphase. Dose-response

relationship was studied after treatment with various concentrations

of S-Na for 24 h. 

Test Results - Genetic Toxicity in vitro

Details on

Cytogenetic Assay:

Statistics:

Effect : Species Other

Species

Strain Other

Strain

Metabolic

Activation

Genotoxic

Effect

Conclusion

Mammalian

Cell Line

Mammalian

Cell Line:

Other

-

Chinese

hamster

cells

Unknown Positive

Results Remarks: At 24 and 48 h, cells showed a significant increase (P < 0.001) in

the rate of breaks and gaps (17.0-19.5% and 25.0-28.5%,

respectively). Aberrant cells increased as the dose was raised to

10, 15, 20, 25 and 30 mg/mL.

Conclusion: High concentrations of S-Na induced chromosome aberrations in

vitro. 

Reliability/Data Quality - Genetic Toxicity in vitro

Reliability: 2 - Valid With Restrictions

Reliability

Remarks:
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Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vitro

Reference: Masubuchi, M. et al. The mutagenicity of sodium saccharin (SNa). II. Cytogenetic studies. Mutation Research, 54 (1978) 219.

 Genetic Toxicity in vitro

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Genetic Toxicity in vitro

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Genetic Toxicity in vitro

Type of Study: in vitro mammalian chromosome aberration test

Concentrations: 10-20 mg/mL 

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Postive, Negative, and

Solvent

Control Substance(s):

Method/Guideline and

Test Condition

Remarks:

Cultured human embryo cells were exposed to the test substance.

Test Results - Genetic Toxicity in vitro

Details on Cytogenetic

Assay:

Statistics:

Effect : Species Other

Species

Strain Other

Strain

Metabolic

Activation

Genotoxic

Effect

Conclusion

Mammalian

Cell Line

Other -

Cultured

human

embryo

cells

Positive
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Results Remarks: Sodium saccharin was mutagenic.

Conclusion: High concentrations of S-Na induced chromosome aberrations in

vitro. 

Reliability/Data Quality - Genetic Toxicity in vitro

Reliability: 2 - Valid With Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vitro

Reference: Ashby, J. Mutagenicity of sodium saccharin to human cells in

vitro. Mutation Research, 222 (1989) 299-300.

 Genetic Toxicity in vitro

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Genetic Toxicity in vitro

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Genetic Toxicity in vitro

Type of Study: DNA damage or repair assay, unscheduled DNA synthesis in

mammalian cells in vitro

Concentrations: Not stated 

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Postive, Negative, and

Solvent

Control Substance(s):

Method/Guideline and

Test Condition

Remarks:

Sodium saccharin was tested for possible genetic activity in

cultured human cells by measuring the induction of chromosome

aberrations and unscheduled DNA synthesis. The fibroblast

cultures prepared from tissues of aborted fetuses were treated

with 0.1-10 mg/mL of sodium saccharin for 24 hours. 

Test Results - Genetic Toxicity in vitro
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Details on Cytogenetic

Assay:

Statistics:

Effect : Species Other

Species

Strain Other

Strain

Metabolic

Activation

Genotoxic

Effect

Conclusion

Mammalian

Cell Line

Other -

Cultured

human

cells

Positive

Results Remarks: There was a considerable increase in the rate of chromatid breaks

and gaps, but no significant amount of chromosome aberrations was

produced at the lower concentrations of 0.1 - 1 mg/mL. On the

other hand, sodium saccharin was effective at inducing

unscheduled DNA synthesis in cultured human cells. At 24 h of

incubation in ADM, cultured cells were exposed to sodium

saccharin dissolved in ADM for 1.5h. The level of UDS was

evaluated by counting the silver grains over slightly labeled

nuclei in the non-S cells. In control cultures, the mean number

of silver grains was 1.3 per nucleus, while it was 1.7, 2.1 and

3.5 per nucleus at concentrations of 1, 5, and 10 mg/mL

respectively. There was a marked dose-response relationship

between the level of UDS and the concentration of sodium

saccharin.

Conclusion: The test substance induced unscheduled DNA synthesis in this

assay.

Reliability/Data Quality - Genetic Toxicity in vitro

Reliability: 2 - Valid With Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Genetic Toxicity in vitro

Reference: Ochi, H. and Tonomura, A. Presence of unscheduled DNA synthesis

in cultured human cells after treatment with sodium saccharin.

Mutation Research 54 (1978), 208.

 Reproductive Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Reproductive Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium
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salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

99.2% pure. Obtained from PMC Specialties Group, Inc.

(Cincinnati, OH USA). Purity information was provided by the

supplier and confirmed by NMR. The impurities in saccharin from

this source have been characterized (Riggin and Kinzer, 1983) and

none of the impurities was found to be present at a level

exceeding 1 ppm of the saccharin.

Sponsored Chemical

Result Type:

Method - Reproductive Toxicity

Route of

Administration:

Oral

Type of Exposure: In Food

Species: Rat

Mammalian Strain: Sprague-Dawley

Gender: Both M/F

Number of Animals per

Dose:

Dose: 7.5% of diet

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

Animals and husbandry - 54 male and 90 female Sprague-Dawley

rats (Charles River Breeding Laboratories, Inc., Kingston, NY,

USA) were purchased when they were 4 weeks old. Additional rats

were purchased for health screening, which showed no

hematological, serological or histopathological

abnormalities. Breeding animals were housed individually in

polycarbonate cages with corncob bedding. Litters were housed

with their mothers until weaning. Pups were weaned at 21 days

of age, and thereafter housed by litter and sex in suspended

stainless-steel cages. The animal room was maintained at a

temperature of 71 +/- 5 °F and a humidity of 50 +/- 20%, and was

on a 12-hour light/dark schedule.

Diets and drinking water - Purina Rodent Chow No. 5002 (meal

form, Ralston-Purina Co., St. Louis, MO, USA) and distilled

water were provided ad libitum. Sodium saccharin (NaS) was mixed

in the diet at a level of 7.5% by weight. The dietary level of

saccharin was analysed by HPLC before each batch of test diet

was fed. The average measured concentration of NaS in the diet

was 7.3%.

Experimental design - The experimental conditions for this study

were designed to simulate a two-generation bioassay previously

conducted with NaS (Schoeing et al., 1985). After a 2-week
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adaptation period during which they were fed Purina Rodent Chow

No. 5002 ad libitum, 6-wk-old male and female F0 rats were

randomly assigned by weight to an experimental group (7.5% NaS,

group 1) or a control group (untreated diet, group 2) (Martin et

al., 1984). Mating was started after 4-6 weeks of test diet

feeding. Males were generally used for mating only until they

had successfully mated with two females. Our objective was to

obtain six litters per group, where each litter in group 1 was

matched with a litter in group 2 having the same birth date. To

that end, extra F0 rats were mated. The six matched litters

from each group were used for urine collection and analyses,

serum lipid and vitamin measurements, biochemical evaluation of

bladder epithelial cells and liver vitamin analyses. Litters not

selected for these primary analyses were used for other analyses

including hematology and serum clinical chemistries.

F0 males and non-mated females were killed, without autopsies,

at the end of the mating period or as soon as it was apparent

that the females were not pregnant. When possible, litters were

randomly culled to four males and four females at day 4 after

birth. Pups were weaned at 21 days of age and fed the same diet

as the parental rats. F0 female rats were killed after their

litters had been weaned. F1 rats were killed when 30 days old.

Urine and feces collection - 24-day-old pups from matched

litters were individually placed in metabolism cages with food

and water provided ad lib. After a 72-hour period of

acclimatization to the cages, urine was collected over three

consecutive 24-hour intervals. Urine was collected in 2 mL

toluene; glass wool was used above the collection tube to prevent

contamination of the urine by diet or feces. Urine specimens

were frozen at -20 °C until analysis. Analyses included volume

(by weight); osmalality (by freezing point depression), pH,

sodium, potassium, carbon dioxide and chloride, urea, creatinine,

calcium, magnesium, ammonium, phosphate and saccharin. Feces

were also collected and moisture content was determined from the

difference in weight after drying in an oven at 52 °C. Fecal

saccharin concentration was also measured. At the end of the

final collection period, after spending a total of 6 days in the

metabolism cages, the rats were killed.

Observations and measurements - The rats were observed twice

daily for evidence of overt toxicity and/or morbidity and

mortality. For F0 rats, food and water consumption were measured

over 7-day intervals. Body weight measurements and detailed

physical examinations were performed on the same days as food and

water consumption determinations. Food and water consumption

measurements for F0 rats began with a 3-day interval immediately

after weaning (day 21), followed by daily measurements while the

rats were in the metabolism cages. Pups in each litter were

weighed as a group by sex immediately after birth and at 4, 7

and 14 days after birth. The animals were then individually

weighed on days 21, 24, 27 and 30. The health status of the

pups was also determined on these days.

Biochemical analyses - Blood for hematology was collected from

the abdominal aorta of a number of non-mated females during the

mating period and from various dams of non-matched litters at

birth and on days 13 and 21 of lactation. Blood samples were

collected for hematology from some culled pups at 4 days postbirth. These females and pups were killed following blood

collection.

Milk was collected manually from several dams anesthetized with

Nembutal on day 13 of lactation. The dam and its litter were

killed after milk collection, and the milk was analyzed for

saccharin. No other analyses were performed on these animals.

At 30 days of age, F1 rats were killed under penthrane
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anesthesia. The pH of freshly voided urine was determined

immediately prior to anesthesia. Blood specimens were collected

at the time of killing from the abdominal aorta of pups in the

matched litters, for the measurement of serum levels of

cholesterol, triglycerides, high-density lipoprotein, vitamin C,

vitamin A and vitamin E, and folate. Blood from the pups in the

extra litters was used for hematology and clinical chemistries,

including blood urea nitrogen (BUN), creatinine, sodium,

potassium, chloride, carbon dioxide, calcium and phosphorus,

which were analyzed by the same methods as those used for urine.

The urinary bladder was removed at the time of killing and

inverted over a wood applicator stick, so that epithelial cells

could be scraped off using a No. 11 scalpel blade. The cells

were homogenized in phosphate buffered saline, using a motordriven Teflon pestle, and analyzed for DNA, cholesterol,

phospholipid and protein. Livers were weighed, homogenized and

analyzed for vitamin A, vitamin A palmitate, vitamin E and

folate. Caeca were weighed with and without contents.

Statistics - Statistical analyses were performed using a

generalized linear model. Duncan's multiple range test was used

for multiple comparisons of means. In addition to treatmentrelated effects, the data were also analyzed for inter-litter

variability and sex-related differences.

Pre-Mating Exposure /

Males:

Pre-Mating Exposure /

Females:

Test Results - Reproductive Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

Results:

Results Remarks: Results in F0 animals - Body-weight gain during the pre-mating

period was significantly depressed in both male and female rats

treated with 7.5% NaS: after 4 weeks, body weights were 10% less

than those of control rats. The mean body weight of NaS-treated

females remained significantly below that of control animals

throughout gestation and the first 2 weeks of

lactation. However, weight gain during pregnancy was equivalent

for the two groups and NaS-treated females actually gained more

weight than controls during lactation, so that there was no

difference between the groups on day 21 post-birth. Basal diet

consumption (g/rat/day) was significantly less in NaS-fed

animals, except during week 2 and 3 of gestation and week 1

post-birth when consumption was similar in the two groups. NaStreated rats consistently consumed more water (g/rat/day) than

control rats. NaS intake was approximately 1.0 g/rat/day in

females and 2.0 g/rat/day in males prior to mating and 2.0

g/rat/day in pregnant females. The NaS concentration in milk of

NaS-treated females was 2.9 mg/mL on day 13 of lactation. No
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NaS was detected in the milk of control rats.

It appeared that after giving birth, the females in both groups

were anemic, relative to the non-mated females, with decreases

in red blood cell count (RBC), hemoglobin (Hgb), and hematocrit

(HCT). There were no differences between the groups on the day

of birth. The control females recovered by day 13 post-birth,

but the NaS-fed females remained anemic until day 21.

Results in F1 animals: litter size and survival and pup weights

- Feeding 7.5% NaS did not adversely affect reproductive

performance. Both the control and test groups had high success

rates for matings and deliveries. There were no significant

differences between groups in the number of male and female pups

born per litter, few pups were stillborn in either group, and

body weights of pups at birth were similar for the two

groups. However, weight gain after birth was depressed in the

NaS-treated litters. NaS-treated male and female pups weighed

significantly less than control pups at 4 and 21 days postbirth. The study of survival was complicated by the fact that

some litters were killed at birth or on day 13 post-birth. The

litters were culled to four males and four females on day 4

post-birth as mentioned in the methods section. Survival

appeared to be slightly better in the control group than in the

NaS group.

Results in matched litters - body weights and food and water

consumption - Similar effects were generally observed in matched

and non-matched litters. After weaning, mean body weights of

NaS-treated pups continued to be significantly less than those

of control pups. When killed at 30 days post-birth, male NaS

treated rats weighed only 64% as much as control males and

female NaS treated rats weighed only 61% as much as control

females. During the first 3 days after weaning, NaS-treated

animals ate less than one-half as much diet as control animals

and also consumed less water. Both groups progressively showed

an increase in food consumption over the final 6 days, but NaStreated animals consistently consumed significantly less than

controls. However, when adjusted for body weight, the basal diet

consumption of the NaS-treated group on days 27 and 30 postbirth was equivalent to or greater than that of the control

group (on day 27: 200 and 183 g/kg body weight/day for NaStreated males and females, respectively, versus 172 and 167 g/kg

body weight/day for control males and females, respectively; on

day 30: 165 and 182 g/kg body weight/day for NaS-treated males

and females, respectively, versus 163 and 159 g/kg body

weight/day for control males and females, respectively). Between

days 25 and 30 post-birth, water consumption was increased in

NaS-treated females on day 30 post-birth (13.4 g/kg body

weight/day for males and 14.7 g/kg body weight/day for females).

Urine and feces analyses - There were numerous differences

between the groups. Urine analyses were performed separately on

days 28, 29 and 30. However, for statistical analysis the data

from the 3 days were pooled, and the differences between groups

were analysed by Duncan's multiple range test. Compared with the

controls, the urine of NaS-treated rats showed increases in

volume, sodium, magnesium, phosphate and ammonia. Urinary

potassium, calcium, creatinine, osmolality, urea, carbon dioxide,

chloride and pH were decreased. The pH of freshly voided urine,

obtained just prior to killing, was also decreased with NaS

treatment (male NaS = 6.5; male control = 7.2; female NaS = 6.4;

female control = 7.4).

There was little difference in the weight of dry feces between

the groups, but the feces of NaS-treated rats had a higher

moisture content: approx. 50% for NaS-treated rats and 30% for

the control rats. The caecal content weight of NaS-treated

animals was 2-2.5 times that of control animals. The absolute
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weight of the empty caecum was also increased from 0.51 g in

untreated males to 0.58 g in NaS-treated males, although there

was no difference with the females.

Bladder chemistry - NaS treatment had no significant effect on

the bladder content of cholesterol, phospholipid, DNA or

protein. Also no differences were observed between the groups

when the parameters were expressed in terms of amounts per μg

DNA or per μg protein.

Liver chemistry - Livers were collected from rats in the matched

litters. Each liver was weighed, and then the entire liver was

homogenized before aliquots were removed for the determination of

vitamin A, vitamin A palimitate, vitamin E and folate. Possible

variations associated with the distribution of the vitamins in

the liver were thereby avoided. In animals of both sexes, there

was a significant decrease in the hepatic folate concentration

associated with NaS treatment. In addition, the concentration of

vitamin E was significantly increased in NaS-treated females,

relative to controls, and the concentration of vitamin A

palmitate was significantly increased in NaS-treated males. Mean

liver weights of NaS-treated males and females were approximately

half those of control animals, and the liver weights in relation

to body weights were also significantly reduced by NaS

treatment. As a result, the total hepatic level of each of the

vitamins was significantly lower in NaS-treated animals than in

controls.

Serum vitamins and lipids - Compared with control levels, serum

vitamin A and folate in the NaS-treated rats were significantly

decreased and serum Vitamin E was increased. Vitamin C was

significantly decreased only in NaS-treated males. A 10-fold

increase in serum triglycerides was measured in NaS-treated

animals, and cholesterol was also significantly increased but to

a much less extent. The concentration of high-density

lipoproteins was significantly lower in NaS-treated rats. 

Hematology and serum chemistry in unmatched litters - Because the

volume of blood collected from animals in the matched litters was

only enough for the analysis of serum vitamins and lipids, blood

from rats in the other litters was used for hematology evaluation

and additional serum clinical chemistries. Hematocrit evaluated

in pups eliminated during the culling procedure on day 4 postbirth, was significantly lower in the NaS group than in the

control group. By day 30 post-birth the difference between the

groups was enhanced and significant decreases were also observed

in RBC and Hgb levels of NaS-treated rats.

There were significant differences between the groups for several

of the serum chemistry analyses. A considerable increase in BUN

was associated with NaS treatment, while creatinine was

decreased. Other changes were relatively minor. The serum NaS

concentration was approximately 0.20 mg/ml in both male and

female treated rats.

Differences between male and female F1 rats -There was no sex

difference in the number and survival of newborn pups and in pup

weights. Male and female rats did not differ in the amount of

food and water consumed. The NaS intake and excretion of male

and females in the treatment groups were also similar.

Similar urinalyses values were obtained from males and females of

each group. In controls, the urine of females had a

significantly smaller volume and a higher pH compared with

males. In the treated group, the urinary NaS concentration was

significantly lower in females than in males.

There were no sex-associated differences in any of the bladder

or liver chemistries. Female rats in the control group had
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significantly lower concentrations of serum vitamin C, vitamin A

and folate than control males. They also had significantly

higher levels of high-density lipoproteins than NaS-treated

males. NaS-treated females showed significantly higher serum

concentrations of BUN, sodium and chloride than NaS-treated

males. Hematology values were similar for males and females of

the same group. In general, relatively few sex-related

differences were observed in the treated and control groups.

Differences between F1 litters - For the various chemistry

parameters evaluated, analyses were performed to determine

whether significant differences existed between litters that

received the same treatment. The data for males and females were

analyzed separately. Inter-litter differences were numerous in

both sex and in both NaS-treated and control rats.

Conclusion: Overall, body weights were significantly depressed in NaS-treated

litters by 4 days after birth, and were 35% lower than controls

by 30 days when the animals were killed. NaS treatment of the

offspring was associated with an increase in fecal moisture

content and caecal content weight, changes in several urinary

analytes, a 50% increase in serum cholesterol, a 10-fold

increase in serum triglycerides and decreases in serum and

hepatic vitamins. In addition, NaS-treated dams and pups were

anemic. Relatively few differences between males and females

were noted, but significant inter-litter differences

existed. The numerous physiological changes indicate that 7.5%

dietary NaS exceeds the maximum tolerated dose for weanling

rats.

Reliability/Data Quality - Reproductive Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study is not adequate because only one dose-level was

tested; NOAEL/LOAEL not provided.

Key Study Sponsor

Indicator:

Reference - Reproductive Toxicity

Reference: Garland, E.M. et al. Effects of in utero and postnatal sodium

saccharin exposure of the nutritional status of the young

rat. I. Effects at 30 days post-birth. Food Chem Toxicology

29 (1991), 657-667.

 Reproductive Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Reproductive Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

99.2% pure. Obtained from PMC Specialties Group, Inc.

(Cincinnati, OH USA). Purity information was provided by the

supplier and confirmed by NMR. The impurities in saccharin from

this source have been characterized (Riggin and Kinzer, 1983) and

none of the impurities was found to be present at a level

exceeding 1 ppm of the saccharin.

Sponsored Chemical
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Result Type:

Method - Reproductive Toxicity

Route of

Administration:

Oral

Type of Exposure: In Food

Species: Rat

Mammalian Strain: Sprague-Dawley

Gender: Both M/F

Number of Animals per

Dose:

Dose: Number of animals/sex/dose: 22 females and 11 males per group in

groups 1-4; and 11 females and six males per group in groups 5

and 6.

Group 1 - F0 = Control, F1 = Control from conception until day

90 of age.

Group 2 - F0 = NA; F1 = 1% Sodium Saccharin (NaS) (from

conception until 90 days of age)

Group 3 - F0 = NA; F1 = 3% NaS (from conception until 90 days of

age)

Group 4 - F0 = 7.5% NaS; F1 = 7.5% NaS (from conception until 90

days of age)

Group 5 - F0 = 7.5% NaS; F1 = 7.5% NaS from conception until day

30 of age, Control from day 30 to day 90 of age.

Group 6 - F0 = Control, F1 = Control from conception until day

30 of age, 7.5% NaS from day 30 to day 90 of age.

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

Animals and husbandry - 56 male and 110 female Sprague Dawley

rats (Charles River Breeding Laboratories, Inc., Kingston, NY,

USA) were purchased when they were 4 weeks old. Additional rats

were purchased for health screening, which showed no

hematological, serological or histopathological

abnormalities. Breeding animals were housed individually in

polycarbonate cages with corncob bedding. Litters were housed

with their mothers until weaning. Pups were weaned at 21 days

of age, and thereafter housed by litter and sex in suspended

stainless-steel cages. The animal room was maintained at a

temperature of 71 +/- 5 °F and a humidity of 50 +/- 20%, and was

on a 12-hour light/dark schedule.

Diets and drinking water - Purina Rodent Chow No. 5002 (meal

form, Ralston-Purina Co., St. Louis, MO, USA) and distilled

water were provided ad libitum. Sodium saccharin (NaS) was mixed

in the diet at a level of 7.5% by weight. The dietary level of

saccharin was analyzed by HPLC before each batch of test diet
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was fed. The average measured concentration of NaS in the diet

was within +/- 6% of the nominal levels.

Experimental design - Male and female rats were randomly assigned

by weight to each experimental group: 22 females and 11 males

per group in groups 1-4 and 11 females and 6 males per group in

groups 5 and 6. They were placed on the appropriate test diet

at 6 weeks of age. Mating was started after 4-5 weeks of test

diet feeding. Litters were randomly culled on day 4 after birth

to four males and four females when possible. Pups were weaned

at 21 days of age and fed the same diet as the parental

rats. Parental males were killed at the end of the mating

period and female rats were killed after their litters had been

weaned. Seven litters from each group 1-4 were killed when they

were 30 days old to evaluate the dose-response effects of

NaS. Also at 30 days, the diet for group 5 was changed from

7.5% NaS to control, and the diet for group 6 was changed from

control to 7.5% NaS. Seven litters from each of groups 1, 4, 5

and 6 were then killed at 90 days of age to evaluate the

reversibility of the effects of NaS. The decision to kill a

litter at 30 or 90 days of age was based on its birth-date. An

attempt was made to kill litters from at least two groups, with

no more than three litters per group, on a given day.

Observations and measurements - The rats were observed twice

daily for evidence of overt toxicity and/or morbidity and

mortality. For F0 rats, food and water consumption were measured

for 1 week prior to mating and for 3 weeks during

gestation. All food consumption data were corrected for the NaS

content of the diet, and reflected the consumption of base diet

only. Body weight measurements and detailed physical

examinations were performed on the same days as food and water

consumption determinations and detailed physical examinations

were performed before mating. For rats of the second generation

(F1), food and water consumption were determined weekly after

weaning except during week 4 post-birth when some litters were

killed and others had their experimental diets changed. Pups in

each litter were weighed as a group by sex immediately after

birth and at 4, 7 and 14 days after birth and then individually

at weekly intervals until the time of killing. The health status

of the pups was also determined on these days.

Analyses - F1 rats were killed under penthrane anesthesia. Blood

specimens were collected in hematology (hematocrit, hemoglobin,

erythrocytes and leucocyte counts; mean corpuscular volume,

hemoglobin, and hemoglobin concentration). The measurement of

serum levels of cholesterol and triglycerides was carried out by

the enzymatic method of Spayd et al. using an Ektachem

700. Vitamins A and E were determined by a procedure similar to

those of Driskell et al. (1982) and Catignani and Bieri

(1983). A Quantaphase B-12/folate kit was used for folate

determination. Iron and iron-binding capacity were measured

according to Megraw et al. (1973) in pooled serum samples from

litters of groups 1 and 3; males killed at 30 days post-birth

and both males and females killed at 90 days post-birth were

evaluated. The adrenals, liver and full and emptied caecum were

removed at the time of killing and weighed.

The urinary bladder was inflated in situ with 2% glutaraldehyde

in 0.1 M-cacodylate buffer (pH 7.4) and then removed and

processed for light microscopy or for scanning electron

microscopy and classified. SEM evaluations were performed on

bladders from two males and two females per litter (from 14

males and 14 females per group) in groups 1 and 4 for both the

30- and 90-day sacrifice periods; from two males and two females

per litter in groups 5 and 6 killed at 90 days; and from one

male and one female per litter in groups 2 and 3 killed at 30

days. Bladders from one male and one female in each litter were

also examined by light microscopy.
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Livers were examined for lipids both histologically and

biochemically. For histological examination, a 3-mm3 piece of

the left lateral lobe of the liver was put into cold picric

acid-p-formaldehyde fixative, and the rest of the liver was

frozen in liquid nitrogen and then stored at 

-80 °C. The fixed liver tissue was dehydrated and embedded in

araldite 502 with DDSA and DMP resin in labeled gelatin

capsules. 1 - μm sections were stained with toluidine blue and

examined by light microscopy. In addition, thin sections were

stained with lead citrate and uranyl acetate and examined for

lipid with a Philips 300 electron microscope. For biochemical

determinations of cholesterol and triglyceride, the frozen liver

was weighed and homogenized (1:20, w/v) in chloroform-methanol

(2:1, v/v) using a motor-driven teflon pestle. Lipids were

extracted by the method of Folch et al. (1957). Separate

aliquots of the extract, each equivalent to 0.01 g liver tissue

were analysed for triglycerides and cholesterol. Triglycerides

were determined in fresh extracts, while cholesterol was measured

after storage of the extracts at 

-80 °C for approximately 1 month.

Statistics - Statistical analyses were peformed with a

generalized linear model procedure. Duncan's multiple range test

was use for multiple comparison of means. 

Pre-Mating Exposure /

Males:

Pre-Mating Exposure /

Females:

Test Results - Reproductive Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

Results:

Results Remarks: NaS treatment was associated with lower body-weight gains,

increased water consumption and decreased (in females) or

unchanged (in males) basal diet consumption. Significant

differences were noted only between the control and the highdose groups, with the exception of a significant increase in

water consumption by the females treated with 1% NaS (51.6

g/rat/day v. 42.1 g/rat/day by control females). In all the

groups, more than 80% of the females mated and at least 90% of

mated females gave birth. Litter weights at birth were similar

for all groups.

For male and female rats at week 4 post-birth, the values of

body weight and food consumption (basal diet) decreased as the

NaS concentration in the diet increased. Rats treated with 7.5%

NaS weighed about 60% as much as control rats, and males fed 3%

NaS weighed significantly less (approx. 10%) than

controls. Correspondingly, significant decreases in diet

consumption were also observed at 3 and 7.5% dietary NaS. Water
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consumption in NaS-treated groups did not differ significantly

from that of control animals.

At week 12 post-birth, body weights of 7.5% NaS-treated males

and females remained significantly lower than

controls. Subsequent to the change in diet at 30 days postbirth, body weights of rats in group 5 (NaS -> control)

approached control values, and rats in group 6 (control -> NaS)

gained less weight than control rats. All male rats fed NaS

including those switched from NaS to control diet consumed

significantly less diet than controls during wk 6-12 postbirth. Female rats in all NaS-treated groups consumed similar

amounts of diet as did controls. Water consumption during weeks

6-12 post-birth was significantly greater, compared with

controls in groups 4 (7.5% NaS) and 6 (control -> 7.5% NaS).

In male and female rats killed at 30 days post-birth, there was

a decrease in both absolute and relative liver weights related to

the dose of NaS. At 90 days, males on the 7.5% NaS diet (groups

4 and 6) had lower absolute and relative liver weights than

controls (group 1). Male rats switched from NaS to control diet

(group 5) also had lower absolute liver weights than control

rats. For female rats, liver weights were lower than those of

controls, on an absolute basis for those fed 7.5% NaS diet

throughout the study, and on an absolute and relative basis for

those switched from the control to NaS diet. Data collected for

the caeca and adrenals show that there were no differences at 30

days in the absolute weight of the empty caecum. At 90 days,

all animals exposed to 7.5% NaS, except females in group 5, had

increased caecal weights compared with controls. On an absolute

basis, adrenal weights from 7.5% NaS -fed animals tended to be

lower than those from control animals, but they were greater than

those of control animals when expressed relative to body weight.

At 30 days, rats exposed to NaS showed a dose response with

respect to hematology. Animals exposed to 7.5% NaS (group 4)

were anemic at 30 days post-birth, as indicated by the decrease

in RBC, Hgb and HCT. Mean values for mean corpuscular volume,

hemoglobin and hemoglobin concentration were consistent with a

diagnosis of iron deficiency. Rats of both sexes fed 1% NaS did

not show any signs of anemia, and those fed 3% NaS had

significantly decreased Hgb and HCT. At 90 days post-birth, the

values of these two parameters in rats exposed to 7.5% NaS

throughout the study were still significantly below control

values; however, the effect was less than that at 30 days postbirth. In addition, no difference was observed in hematological

parameters between the group switched from the 7.5% NaS to

control diet at 30 days post-birth and control group. Beginning

NaS treatment at 30 days post-birth did not produce anemia. The

effects in the female rats were similar to those in the males.

Compared with the control group, serum cholesterol, triglycerides

and vitamin E were increased and serum vitamin A and folate were

decreased in both males and females. Serum cholesterol,

triglycerides and vitamin E in the 1 and 3% groups were

slightly, and in some cases significantly, lower than in

controls. The decreases in serum vitamin A and folate were

dose-related: a significant decrease was observed in males and

females given NaS doses equal to or greater than 3 and 1%,

respectively. At 90 days post-birth, with continuous NaS

treatment (group 4), the changes in serum lipid concentrations

were reversed. Serum triglyceride levels in males and females

and serum cholesterol levels in males were lower than in

controls. The lipid values for groups 5 and 6 were also

significantly lower than or equivalent to those of the control

group. Serum folate in group 4 was similar to that in control

animals; elevated and reduced values were observed in groups 5

and 6, respectively. Serum vitamin A remained significantly

depressed in all male groups treated with 7.5% NaS. However,
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there were no differences between the mean serum vitamin A values

for the female groups. For both males and females in all groups

treated with NaS, serum vitamin E was significantly below the

control value. 

Iron and iron-binding capacity were measured in pooled serum

samples from litters in groups 1 (control) and 4 (7.5%

NaS). For male rats in group 4 killed at 30 days of age, the

serum iron level was significantly reduced to a level that was

approximately 20% of the control value and iron-binding capacity

was increased by 50% over the control value. Female rats could

not be evaluated at 30 days of age because insufficient serum

sample volume. At 90 days of age, the serum iron level of

control males was approximately half of that at 30 days, and NaS

treatment was associated with only a 30% decrease, relative to

the day-90 control value. Although iron-binding capacity was

greater at 90 days for male rats fed 7.5% NaS, the difference

was not statistically significant (p > 0.05). For female rats

killed at 90 days, there was no significant difference between

groups 1 and 3 in mean serum iron concentrations, but ironbinding capacity was significantly lower in group 4 than in group

1.

A significant increase was observed in the hepatic concentration

of triglycerides in males and females and cholesterol in females

treated with 7.5% NaS. Examination of the livers by light

microscopy showed an increase in the number and size of

microvesicular vacuoles in hepatocytes from group 4 in comparison

with group 1 at 30 days post-birth. Although not quantitative,

these findings are indicative of lipid accumulation. Examination

by electron microscopy confirmed the increased cytoplasmic lipid

in hepatocytes and also indicated the presence of decreased

glycogen in rats fed 7.5% NaS.

The microscopic effects of NaS treatment on the bladder were

negligible. Simple hyperplasia was noted in the bladders of some

7.5% NaS-treated rats at 90 days. Morphological changes in the

urothelium were also observed by SEM in these groups, but the

severity and incidence were low.

Conclusion: Overall, most physiological consequences of NaS treatment in the

weanling rat, including anemia and reductions in serum folate and

vitamin A concentrations were dose-dependent. Serum vitamin E,

cholesterol and triglyceride concentrations were decreased at the

two lower doses of NaS but were significantly increased with 7.5%

NaS. The no-effect level (NOEL) was similar for physiological

effects and for bladder tumor production in two-generation

studies (1% NaS in the diet). The reversibility of the effects

of 7.5% NaS was examined in 90-day-old rats. The increases in

lipids and vitamin E were reversible. Although values for

hematological parameters and serum vitamin A remained

significantly reduced at 90 days, changes were less severe than

at 30 days. Histological examinations revealed that the effects

of 7.5% dietary NaS on the bladder were negligible, indicating

that the physiological changes observed in the young rat are

probably not directly related to the production of bladder

tumors.

Reliability/Data Quality - Reproductive Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study is not adequate because reproductive measurements were

not provided and only one dose-level was tested.

Key Study Sponsor

Indicator:

Reference - Reproductive Toxicity
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Reference: Garland, E.M. et al. Effects of in utero and postnatal sodium

saccharin exposure of the nutritional status of the young

rat. II. Dose response and reversibility. Food Chem Toxicology

29 (1991), 669-679.

 Reproductive Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Reproductive Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

The two batches of sodium saccharin (Lot Nos. GS-1233 and S1022) used were manufactured by the Maumee process and contained

no detectable amount of o-TS (ortho-toluenesulfonamide; limit of

detection = 0.05 ppm). The total amount of organic soluble

impurities of Lot No. GS-1233 was approximately 5 ppm. This lot

of saccharin was fed for 25 weeks while the F0 generation was on

test and during the first week that the F1 animals were on

test. Lot No.S-1022, which was used during the remainder of the

study, contained a maximum of 13 ppm organic soluble

impurities. Both batches contained 40-50 ppm of water soluble

impurities. 

Sponsored Chemical

Result Type:

Method - Reproductive Toxicity

Route of

Administration:

Oral

Type of Exposure: In Food

Species: Rat

Mammalian Strain: Sprague-Dawley

Gender: Both M/F

Number of Animals per

Dose:

50

Dose: 5% in diet

Year Study Performed: 1979

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and The F0 or parent generation animals consisted of groups of 50
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Test Condition

Remarks:

male and 50 female (except where noted) Charles River, Caesaeanderived, Sprague-Dawley rats. The basal diet was Master

Laboratory cubes to which 4% corn oil (w/w) was added after the

cubes were ground. Analysis of several batches of feed during

the study identified background levels of heavy metals, an

average of 20 ppb of nitrosamines, trace amount of 10 halogenated

hydrocarbons ( total content 12 ppb), but no detectable amounts

of aflatoxin or aftlatoxin-like compounds. 

The study consisted of a treatment group administered 5% sodium

saccharin in the diet as well as a control group.

After 90 days on test, the F0 animals were mated on a one-to-one

basis within treatment groups for a period of 1 week. The liter

size was reduced to eight pups (approximately four males and four

females) at 4 days post partum. At 21 days of age, the pups

were weaned, placed in stock banks and fed their respective

parents' diet. At 28 days of age, animals were randomly selected

for either the F1 portion of the chronic feeding study or a

subacute study. Thus, the pups were exposed to the test

chemicals in utero, during lactation via the dams' milk, and for

the remainder of their lifetime via the diet. 

All animals were examined twice daily for general health status

and signs of toxicity. Individual body weight and feed

consumption were measured weekly, at which time a superficial

clinical examination was performed on each animal. Urine samples

were obtained from approximately 10 rats per group at 6-month

intervals from the control and treatment groups. Blood samples

were obtained from the tail aorta of approximately 10 rats per

group, under light anesthesia. The blood samples were obtained

from the F0 animals at 4.5 weeks (Day 0 on test), 21, 44.5, 57,

65, 69, 96 and 98 weeks of age; the F1 animals were sampled at

4.5, 18.5, 32.5, and 45.5 weeks of age. Where possible, the

same 10 rats were sampled for each observation. A complete

hematological examination was performed in duplicate, which

included: erythrocyte enumeration (RBC); hematocrit (HCT);

hemoglobin determination (Hb); mean corpuscular volume (MCV),

mean corpuscular hemoglobin concentration (MCHC); total leukocyte

enumeration (WBC); differential leukocyte count including

absolute values; and occasionally hematopoietic organs (bone

marrow, lymph node and spleen imprints) were examined in cases

where hematologic involvement was observed in moribund animals. 

Pre-Mating Exposure /

Males:

Pre-Mating Exposure /

Females:

Test Results - Reproductive Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

Results:
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Results Remarks: The general health of the animals throughout the study was

good. The only apparent treatment-related effect was a moist,

but well-formed fecal pellet excreted by rats consuming the diet

containing saccharin, a common observation when 5% sodium

saccharin is fed. 

Rats from both generations fed diets containing 5 % sodium

saccharin had decreased growth rates. There were no treatmentrelated effects upon reproduction, longevity, or hematological

parameters. The animals were free of the bladder parasite

Trichosomoides crassicauda. The incidence of bladder tumors in

male rats fed the 5 % saccharin diet was significantly increased

in both generations compared to their respective control

groups. An evaluation of individual feed consumption data for

animals fed saccharin-containing diets did not reveal any

statistical difference between the amount of feed consumed by

animals which had bladder tumors and those that did not. The

incidence of bladder tumors in the female rats fed 5% saccharin

diet was not significantly different from that in control

animals. 

Conclusion: The median time-to-death (time until 50% of the animals have

died) was 679 ± 22 and 731 ± 25 days for F0 males and females,

respectively, and 684 and 727 days for F1 males and females,

respectively.

Reliability/Data Quality - Reproductive Toxicity

Reliability: 3 - Not Valid

Reliability Remarks: This study was considered inadequate due to the testing of only

once test substance concentration (5% in diet), and a NOAEL/LOAEL

was not determined. 

Key Study Sponsor

Indicator:

Reference - Reproductive Toxicity

Reference: Arnold, DL et al., (1980). Long-Term Toxicity of orthoToluenesulfonamide and Sodium Saccharin in the Rat. Toxicol.

Appl. Pharmacol. 52:113-52.

 Reproductive Toxicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Reproductive Toxicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Reproductive Toxicity

Route of

Administration:

Oral
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Type of Exposure: In Food

Species: Rat

Mammalian Strain: Other

Other Strain: Charles River CD rat

Gender: Both M/F

Number of Animals per

Dose:

48

Dose: The dietary levels were 0, 0.01, 0.1, 1.0, 5.0 and 7.5% sodium

saccharin

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

The carcinogenic and reproductive effects of sodium saccharin

were studied in the Charles River CD rat in a combined chronic

and 3-generation reproduction study. For the chronic study, 48

male and 48 female F1 weanling offspring per test group were

selected and continued on the same test regiment as their

parents. The dietary levels were 0, 0.01, 0.1, 1.0, 5.0 and

7.5% sodium saccharin. The rats were mated to produce 2

litters. Serial sacrifices were performed at 14 and 18

months. The study was continued until the number of survivors in

a group fell to 20% of the starting number. The last rats were

killed approximately 28 months after the first weanlings were

selected for the chronic study. 

Pre-Mating Exposure /

Males:

Pre-Mating Exposure /

Females:

Test Results - Reproductive Toxicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

Results:
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Results Remarks: There were not significant differences between the test and

control groups in hematological values, organ weights or

survival. Average weaning weights were decreased in litters from

parents receiving 5 and 7.5% sodium saccharin; other reproductive

indices showed scattered variations but these were not consistent

for all generations. During the chronic study some of the

initial weaning weight depression was overcome, but rats on 5 and

7.5% sodium saccharin had lower average body weights throughout

the study. Histopathological examination of the urinary bladders

from rats on the chronic study longer than 18 months revealed an

increased incidence of transitional cell carcinomas in the 7.5%

sodium saccharin group.

Conclusion: None stated

Reliability/Data Quality - Reproductive Toxicity

Reliability: 4 - Not Assignable

Reliability Remarks: This study is not adequate because many details are missing,

including a description of all reproductive parameters measured,

and NOAEL/LOAEL values are not identified.

Results reported only as abstract.

Key Study Sponsor

Indicator:

Reference - Reproductive Toxicity

Reference: Taylor, J.M. et al. Combined chronic feeding and threegeneration reproduction study of sodium saccharin in the

rat. Toxicol. Appl. Pharmacol. 29 (1974), 154.

 Developmental Toxicity/Teratogenicity

CAS Number : 128-44-9

Sponsored Chemical

Name :

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

Test Substance - Developmental Toxicity/Teratogenicity

Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test

Substance Comments:

Sponsored Chemical

Result Type:

Method - Developmental Toxicity/Teratogenicity

Route of

Administration:

Oral

Type of Exposure: In Food

Species: Rat

Mammalian Strain: Wistar

Gender: Female
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Number of Animals per

Dose:

28

Dose: 0-4% 

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period: 20 Days

Frequency of

Treatment:

Post-Exposure Period:

Method/Guideline and

Test Condition

Remarks:

Groups of 26-28 female Wistar rats (obtained from Mus Rattus

Corp., Brunnthal, Munich and weighting 182-229 g) were mated

with 3- to 5-month-old males. The females were assumed to be

pregnant when spermatozoa were found in a vagina smear. The

pregnant rats were kept at an ambient temperature of 20-23 °C in

an atmosphere of 60% mean relative humidity, with a 12-hour

electric light/dark cycle. They were given Altromin-R powdered

feed (supplied by Altromin Ltd., Lage/Lippe) and tap-water ad

lib. From mating until day 20 of pregnancy, they were treated

with feed containing 0-4% sodium saccharin, complying in each

case with the standards of purity established in Germany for the

marketed product. The average amount of test substance ingested

daily was 98.8 mg sodium saccharin per rat. The mean daily

intake of a control group of similar size was 24.4 g. 

One half of the dams in each of the three groups were delivered

by caesarian section on day 20 of pregnancy. The other animals

were left to deliver their young spontaneously and rear them for

3 weeks.

For all the dams, appearance, behavior, deaths, body weight and

food consumption were recorded. Following surgical delivery, the

numbers of implantations, fetuses and resorbed embryos were

calculated, fetuses were inspected for external malformations,

and litter weights, mean fetal weights litter, numbers of runts

(fetuses < 3 g) and mean placental weights were recorded. Data

recorded for the rest of the dams in each group included the

duration of pregnancy, course of parturition, behavior during

lactation, numbers of implantation sites demonstrated at the end

of the experiment by the staining of Salewski (1964), and

pathological changes. For the offspring of these dams, records

were kept of weights and sex ratios at birth and after 1, 2 and

3 wk, and of deaths and behavior, time of eye-opening, formation

of fur, ability to run normally, macroscopic changes in the eye

and direct papillary reflexes in response to a flash-light in a

darkened room.

For the histological examination of the eyes, the fetuses and

young animals were killed with CO2, and the heads were fixed in

Bouin's fluid. For better penetration of the fixation fluid into

the area to be examined, the nose, lower jaw and back of the

head were removed, the decalcification of bone necessary in the

3-week-old animals was achieved using sodium

ehtylenediaminetetraacetate at 38 °C. The subjects for

examination were embedded, via methylbenzoate, in Paraplast, so

that the microtomy yielded fronto-parallel sections in the

cranio-caudal series. The ocular region, from fetuses and young

from each dam, was cut in series or states, regularly including
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both lenses and the insertion of the optic nerves. The sections

were about 3-5 μm thick and were alternately stained with

haemalum-eosin and Masson's trichrome.

Test Results - Developmental Toxicity/Teratogenicity

Concentration (LOAEL/

LOAEC/ NOAEL/

NOAEC) :

LOAEL/

LOAEC/

NOAEL/

NOAEC

Population Value

Description

Value/Lower

Concentration

Upper

Concentration

Units

NOAEL Female = 98.8 Other

Results Remarks: Endpoint value(s): Developmental NOAEL = 98.8 mg/day

The general study provided no indication of any primary

embyrotoxic effect of the treatment and the histological

examination showed that treatment with the sweetening agents

caused no ocular damage. The development of the eyes had

followed a normal course in all the fetuses and young animals

studied. No case of anophthalmia or microphthalmia

occurred. The retina showed no unusual folding, the ocular

lenses were of normal structure. In particular, no lytic,

necrotizing or vacuolating process of the ocular lenses were

present. The fibers of the lenses showed no increase in

circumference or disorientation. The epithelial cells of the

lenses had reached their regular position and showed normal

mitotic activity. The vitreous body and the implantation of the

optic nerves were normal. 

Conclusion: Administration of sodium saccharin to pregnant rats is tolerated

without embryotoxic effect.

Reliability/Data Quality - Developmental Toxicity/Teratogenicity

Reliability: 3 - Not Valid

Reliability Remarks: This study is not adequate for the developmental toxicity

endpoint because only one dose level of the test substance was

administered.

Key Study Sponsor

Indicator:

Reference - Developmental Toxicity/Teratogenicity

Reference: Luckhaus, G. & Machemer, L. Histological examination of

perinatal eye development in the rat after ingestion of sodium

cyclamate and sodium saccharin during pregnancy. Food Cosmet

Toxicol. 16 (1978), 7-11.

 Carcinogenicity

CAS Number : 128-44-9

Sponsored Chemical Name

:

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

Test Substance - Carcinogenicity
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Test Substance: (128-44-9) 1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide, sodium

salt

Test Substance

Purity/Composition

and Other Test Substance

Comments:

Sponsored Chemical Result

Type:

Method - Carcinogenicity

Route of Administration: Oral

Type of Exposure: In Food

Species: Rat

Mammalian Strain: Sprague-Dawley

Gender: Both M/F

Number of Animals per

Dose:

48

Dose: 0.01, 0.1, 1.0, 5.0 and 7.5% of the diet

Year Study Performed:

Method/Guideline

Followed:

Unknown

GLP: Unknown

Exposure Period:

Frequency of Treatment:

Animals at Interim

Sacrifice:

Interim Sacrifice Time:

Animals at Final Sacrifice:

Final Sacrifice Time:

Control Group Type:

Method/Guideline and

Test Condition Remarks:

Parental animals were weanling rats of the Charles River CD

(Sprague-Dawley) strain and were randomly distributed by

weight to the test groups. The chronic test rats were the

offspring of these treated parents. The parents were fed the

diets continuously from weaning through mating at

approximately 10 weeks of age, and through gestation to the

weaning of their litters. The offspring were selected

randomly from within a litter; the number selected from a

litter from the chronic study was designed to give as equal

representation of all available litters as possible and give

a total of 48 males and 48 females per test group. 

Rats were observed daily for changes in behavior. Body

weight and food consumption were measured weekly. The rats

were mated twice. Standard hematological determinations
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(hemoglobin, hematocrit, red and white cell count and

differential count) were made on 10 rats per sex per group

after 14, 18, and 23 months. Hemoglobin was determined on a

Fisher hemophotometer, hematocrit by the microhematocrit

method, red and white cell counts on a Coulter Counter, and

the differential count on a Wright's stained smear.

Interim sacrifices of 4/sex/group were made at 14 months and

5/sex/group at 18 months. The rats for interim sacrifice

were part of the original group. Treatment was continued

until the number of survivors in a test group fell to 20% of

the starting number. For this purpose, males and females

were considered separately. When survival in the control

group fell to 20% of the controls and all the remaining test

rats were killed. Rats scheduled for sacrifice, or found

moribund, were anesthetized with ether, exsanguinated and

subjected to thorough gross examination. The urinary bladder

was ligated at the neck and inflated with a small volume of

buffered formalin before excision. Other tissues taken for

histopathologic study included eyes, brain, pituitary gland,

salivary glands, thyroid, adrenals, trachea, lungs, heart,

liver, pancreas, esophagus, stomach, small and large

intestines, kidneys, testes, prostate, seminal vesicles (or

ovaries and uterus) spleen, bone marrow (sterna), skeletal

muscles, and skin. The liver, heart, spleen kidneys, and

testes were weighed before fixation. The stomach and

intestines were washed out before fixation. All tumor masses

were excised and fixed. Rats found dead were also necropsied

and tissue preserved unless severely autolyzed. The fixed

tissues were embedded in paraplast, sectioned at 6-7 um, and

stained routinely with hematoxylin and eosin. All the

tissues collected were examined microscopically. Statistical

comparisons between groups were madding using Student's ttest or chi square.

Test Results - Carcinogenicity

MTD Indicator:

Neoplastic Effect:

Male Survival Rate:

Female Survival Rate:

Total Survival Rate:

Clinical Observations:

Carcinogenic Effect: Yes

Results Remarks: General condition of rats was good throughout the study. 

There were a few significant differences in the mean

hematological values at each of the time periods, but no

dose- or time-related trend; the differences were not

considered to be biologically significant.

Deaths in the 0.01 and 0.1% sodium saccharin male groups

occurred earlier and these groups were terminated after 109-

113 weeks. Otherwise, treatment did not appear to affect

survival. The remaining males were killed after 114-121

weeks when survival in the control group fell to 20% of the

starting number. The number of control females killed

terminally exceeded 20% of the starting number, but the poor
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condition of several rats in this group necessitated

sacrifice of all surviving females after 117-121 weeks on

experiment. 

Females of the 7.5% sodium saccharin group had a number of

significantly decreased organ weights but these changes were

probably related to the decreased body weight rather than a

specific effect of the test substance. A few scattered

significant differences in absolute or relative organ weights

in the animals sacrificed serially at 15 and 18 months did

not show a dose- or time-related trend.

The most significant pathologic finding in the study was the

presence of 11 neoplasms of the urinary bladder. Nine

occurred in the 7.5% sodium saccharin group, one in the 5.0%

sodium saccharin group, and one in the control group. They

occurred primarily in males. The increased incidence of

tumors in the 7.5% sodium saccharin group in comparison with

control was significant. The earliest neoplasm was observed

in a 7.5% male found dead at 103 weeks.

The two neoplasms in the control and 5% groups were benign

and classified as transitional cell polyps. Four of nine

neoplasms at 7.5% were also benign, two as transitional cell

polyps and two as transitional cell papillomas. There were

five malignant tumors in the 7.5% group, all transitional

cell carcinomas. One showed focal squamous metaplasia; one

extensive squamous metaplasia. 

Incidences of hyperplasia were usually focal rather than

diffuse and occurred most commonly in the dome and midportion

of the urinary bladder. The lesion was composed of well

differentiated epithelial cells. There was a significant

increase in the incidence in the females of the 7.5% sodium

saccharin group in comparison with the control females. The

incidence in males of the 7.5% sodium saccharin group was

increased, but not significantly, in comparison to the

control males. 

The most frequently occurring non-neoplastic lesions were

chromic pulmonary disease with or without associated

broncheictasis or lung abscess which occurred in a majority

of animals on study longer than 18 months; chronic nephritis

which occurred in 53-82% of animals in the various groups and

calyceal polyposis, which occurred in 38-71% of the animals

in various groups. None of these were attributed to

treatment.

Conclusion: The test substance has the potential to be carcinogenic. 

Reliability/Data Quality - Carcinogenicity

Reliability: 1 - Valid Without Restrictions

Reliability Remarks:

Key Study Sponsor

Indicator:

Reference - Carcinogenicity

Reference: Taylor, J.M. et al. Chronic toxicity and carcinogenicity to

the urinary bladder of sodium saccharin in the in uteroexposed rat. Toxicology and Applied Pharmacology 54 (1980),

57-75.
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