### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

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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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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.

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 

## 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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:

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] ):

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:

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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.

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 

## 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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) inDrosophila 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 werecollected 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 cellswere 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 weighedsignificantly 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 showedan 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 dietconsumption of the NaS-treated group on days 27 and 30 postbirth was equivalent to or greater than that of the controlgroup (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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 evaluatedin 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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.

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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. Significantdifferences 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 consumedsignificantly 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 controlvalues; 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,

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 betweengroups 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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:

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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:

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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

### High Production Volume Information System (HPVIS) | High Production Volume (HPV) Challenge | OPPT | US EPA

http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] (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. Statisticalcomparisons 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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http://iaspub.epa.gov/hpvis/ServSubmissionPrint [6/15/2011 10:24:19 AM] 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 tothe urinary bladder of sodium saccharin in the in uteroexposed rat. Toxicology and Applied Pharmacology 54 (1980),
57-75.

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